Innovations

in Food Analysis

Table of contents

Video Introduction Laura Bush

Welcome

LC-MS Francesco Cacciola, Paola Donato, Marco Beccaria, Paola Dugo and Luigi Mondello

Advances in LCMS for Food Analysis

GC-MS Peter Quinto Tranchida, Paola Dugo and Luigi Mondello

Advances in GCMS for Food Analysis

HPLC Manpreet Kaur, Ashok Kumar Malik and Baldev Singh

Determination of Phenylurea Herbicides in Tap Water and Soft Drink Samples by HPLCUV and Solid-Phase Extraction

Data Handling Hans Mol, Arjen Lommen, Paul Zomer, Henk van der Kamp, Martijn van der Lee and Arjen Gerssen

Data Handling & Validation in Automated Detection of Food Toxicants Using Full Scan GCMS and LCMS

CONTENTS

TOC Innovations in Food Analysis

INTRODUCTION

Welcome

Advances in LCMS for Food Analysis

By F  rancesco Cacciola, Paola Donato, Marco Beccaria, Paola Dugo and Luigi Mondello

Within the wide range of hyphenated techniques, liquid chromatographymass spectrometry (LCMS) has recently emerged to a central role in different fields, including food analysis. In this review, the most recent LCMS approaches are discussed, as well as the technical requirements for linking an LC system to a mass spectrometer. The advantages of on-line two-dimensional liquid chromatography (2DLC) in the comprehensive mode are also illustrated and selected applications for the analysis of common foodstuffs, such as triacylglycerols, carotenoids, and polyphenols, are described. Finally, future trends for LCMS in food analysis are reported.

ood products are complex mixtures containing both organic and inorganic constituents. The analysis of food products is generally directed towards the assessment of food safety and authenticity, the control of a technological process, the determination of nutritional values and the detection of molecules with a possible beneficial or toxic effect on human health.

Consequently, one of the most stringent demands of food chemistry is directed towards the continuous improvement and development of powerful analytical techniques (1) to analyse the major and minor components of food samples. Liquid chromatographymass spectrometry (LCMS) is an increasingly valuable tool in food analysis and has been widely applied to the analysis of many food products (2). Recent achievements in instrumentation and data processing have allowed LCMS to play a central role in food-related analysis. However, when dealing with the extreme complexity of many real-world samples, one-dimensional chromatography may not provide sufficient analytical results. As a consequence, considerable research has recently been devoted to the development of multidimensional LC techniques (MDLC), with enhanced resolving power (3).

LC-MS

The purpose of this review is to acquaint the reader with some of the existing recent applications of LCMS-based techniques in food analysis. Topics covered will include MS analysis of LCamenable food compounds, namely triacylglycerols, carotenoids and polyphenols. Technical sections will briefly introduce both theoretical and practical concerns of this hyphenated technique, also in the multidimensional comprehensive mode (LCLCMS).

Sponsored Detection of Pharmaceuticals in Water by LC-MS-MS

http://www.learnpharmascience. com/tablet-apps/food-issue1/ article1/detection_pharma_ in_water.pdf
Application Note: 466

Liquid ChromatographyMass Spectrometry (LCMS)

Detection of Pharmaceuticals, Personal Care Products, and Pesticides in Water Resources by APCI-LC-MS/MS
1

Liza Viglino1, Khadija Aboulfald1, Michle Prvost2, and Sbastien Sauv1 Department of Chemistry, Universit de Montral, Montral, QC, Canada; 2Dpartement of Civil, Geological, and Mining Engineering, cole Polytechnique de Montral, Montral, QC, Canada

Introduction
Key Words TSQ Quantum Ultra Water Analysis Solid Phase Extraction Pharmaceuticals (PhACs), personal care product compounds (PCPs), and endocrine disruptors (EDCs), such as pesticides, detected in surface and drinking waters are an issue of increasing international attention due to potential environmental impacts1,2. These compounds are distributed widely in surface waters from human and animal urine, as well as improper disposal, posing a potential health concern to humans via the consumption of drinking water. This presents a major challenge to water treatment facilities. Collectively referred to as organic wastewater contaminants (OWCs), the distribution of these emerging contaminants near sewage treatment plants (STP) is currently an area of investigation in Canada and elsewhere3,4. More specifically, some of these compounds have been detected in most effluent-receiving rivers of Ontario and Qubec5,6. However, it is not clear whether contamination is localized to areas a few meters from STP discharges or whether these compounds are distributed widely in surface waters, potentially contaminating sources of drinking water. A research project at the University of Montreals Chemistry Department and Civil, Geological, and Mining Engineering Department was undertaken to establish the occurrence and identify the major sources of these compounds in drinking water intakes in surface waters in the Montreal region. The identification and quantification of PhACs, PCPs, and EDCs is critical to determine the need for advanced processes such as ozonation and adsorption in treatment upgrades. The establishment of occurrence data is challenging because of: (1) the large number and chemical diversity of the compounds of interest; (2) the need to quantify low levels in an organic matrix; and (3) the complexity of sample concentration techniques. To address these issues, scientists traditionally use a solid phase extraction (SPE) method to concentrate the analytes and remove matrix components. After extraction, several different analytical techniques may perform the actual detection such as GC-MS/MS and more recently, LC-MS/MS7,8. Another analytical challenge resides in the different physicochemical characteristics and wide polarity range of organic compounds making simultaneous preconcentration, chromatography separation, and determination difficult. Analytical

methods capable of detecting multiple classes of emerging contaminants would be very useful to any environmental monitoring program. However, up to now, it has often been a necessity to employ a combination of multiple analytical techniques in order to cover a wide range of trace contaminants9. This can add significant costs to analyses, including equipment, labor, and time investments.

Goals
To develop a simple method for the simultaneous determination of trace levels of compounds from a diverse group of pharmaceuticals, pesticides, and personal care products using SPE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Determine which selected substances are present in significant quantities in the water resources around the Montreal region.

Materials and Method

Analyte selection

Compounds were selected from a list of the mostfrequently encountered OWCs in Canada4-6 (Figure 1).
Sample collection

Raw water samples were taken from the Mille Iles, des Prairies, and St-Laurent rivers. Three samples were collected at the same time from each river in pre-cleaned, four-liter glass bottles and kept on ice while being transported to the laboratory. These water sources vary widely due to wastewater contamination and sewer overflow discharges. All samples were acidified with H2SO4 for sample preservation and stored in the dark at 4 C. Immediately before analysis, samples were filtered using 0.7 m poresize fiberglass filters followed by 0.45 m pore size mixedcellulose membranes (Millipore, MA, USA). Samples were extracted within 24 hours of collection.

The potential benefits arising from the hyphenation of LC to MS become clear if the limitations of the two independent techniques are considered, and to what extent such a combination may alleviate them. Peak overlapping sometimes precludes unambiguous identification, even if disposing of reference standard material. Even the most widely used ultraviolet (UV) detector can rarely provide unambiguous data on the separated analytes, regardless of the degree of chromatographic separation obtained; the situation is further complicated if quantitative determination is also desired. On the other hand, mass spectral data are, in many instances, specific enough to support positive identification and in discriminating nonisobaric compounds, providing the analyst with structural information, in addition to the molecular weight. MS systems can also be used with non-UV absorbing analytes and can be operated in the full scan mode, viz total ion current (TIC), or, more specifically, in tandem MS (MSMS) experiments or in the selected ion monitoring (SIM) mode. SIM operation is preferred for the development of selective and sensitive quantitative assays, while tandem MS data, generated by using soft ionization techniques, provide structural information which can help in the identification of unknown analytes. Mass spectrometry allows for quantitative determination to be performed accurately, precisely and with high sensitivity (at the picogram level), using isotopicallylabeled compounds as internal standards (ISs). Whenever the quantification, or even the detection of a target trace component, in SIM mode, is hampered by the presence of high background ions with the same m/z values, constant neutral loss or precursor ion scanning techniques help in distinguishing the ions of interest from unspecific matrix components. The so-called selected reaction monitoring (SRM) mode enhances selectivity and lowers detection limits, therefore reducing sample consumption, analysis times and the need for clean-up procedures (4).

LCMS plays a central role in both basic and applied research because of significant advances in interface technology and ionization techniques and it also has a broad range of applicability and high sensitivity for the analysis of high-polar and highmolecular mass compounds. In addition, the replacement of the older sector machines with ion trapping instruments (IT), quadrupoles (Q), timeof-flight (ToF) systems and a variety of hybrid instruments characterized by high resolution, enhanced sensitivity, as well as increased mass accuracy over a wide dynamic range. Among these are the ion mobility time-of-flight (IM-ToF), quadrupole ToF (Q-ToF), ion trapToF (IT-ToF) and linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR). Ultimate generation singlequadrupoles allow for high speed scanning (up to 15,000 amu/sec) and ultrafast polarity switching; the small size and the possibility to perform tandem MS make them ideal for benchtop LCMS. On the other hand, ToF instruments present a number of advantages: high speed (up to 20,000 Hz), high resolution (using a reflectron), virtually no limit on mass range, femtogram-level sensitivity, sub-ppm mass accuracy, improved in-spectrum dynamic range without loss in sensitivity, high mass resolution and feasibility to use as a second stage in tandem MS experiments, in combination with either an IT-ToF or a Q-ToF. From the quantitative standpoint, the linear dynamic range depends on the type of source employed; electrospray ionization (ESI) is characterized by a dynamic range over 23 orders of magnitude, and currently represents the most common choice for routine LCMS analysis. However, atmospheric-pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) techniques offer greater sensitivity and a wider dynamic range (45 orders of magnitude), though their use for large bio-molecules is precluded (5). Liquid chromatography nanoelectrospray ionization (LCnano-ESI) operation has become feasible in recent years (6), boosting the sensitivity of LCMS techniques. The newly developed interfaces are suitable for linkage with capillary-type LC columns, operated in the L-to-nL flow range; current configurations using goldcoated capillaries or automated chips allow analyte detection down to the femtomole level.

LCMS Applications for Triacylglycerol Analysis

Plant oils, animal fats and fish oils are natural sources of triacylglycerols (TAGs) in the human diet. Since they may contain hundreds of different TAGs, which are characterized by the total carbon number (CN), the number, position and configuration (cis/trans) of double bonds (DBs) in fatty acids (FA) acyl chains and the stereospecific position of FAs on the glycerol skeleton (sn -1, 2 or 3), tremendously complex mixtures may arise (7). Two chromatographic techniques are more widespread in the analysis of TAGs in natural samples, namely nonaqueous reversed-phase liquid chromatography (NARP-LC) and silverion chromatography (SIC).

LLL LnLnLn LnLnLn OLLn LLM OLLn LLM LnLnLn+LnLSt LnLnLn+LnLSt LnLP LnLP LnLn Ln+ LnLn LnLSt Ln+LnLSt LnLnLn Ln Ln LLM0 LLM0 StStP StStP LLC15:0 LLC15:0 LnLLn LnLLn C20:2LL C20:2LL LnLnLLn OLL OLL LLSt LLSt LnLLnLnL LnLnL LnOStLnOSt LLP LLP StLnP St LnP LnOSt LLLn LLLn StLnP StLnP LnOSt + PLnP 0 OLM0 LL Ln+LnOLn LLLn+LnOLn PLnP+OLM LnOLn LnO Ln LLMaGLL LLMaGLL LnOLn LnOOLSt+LnLnP Ln OLSt+LnLnP LnLnP+SLnSt LnLnP+SLnSt LnLnP+StLP LnLnP+StLP OLO OLO LLL LLL SLL SLL OLLn SLnSt SLnSt OL Ln OLOLLn Ln LnLP LnLP PLP C19:0LL PLP C19:0LL OLP OLP LnLP+OOSt LnLP+OOSt SLnLn+SLSt+StOP SLnLn+SLSt+StOP SLnLn+LLC15:0 SLnLn+LLC15:0 OLMa GLO GLO OLMa OLL OLL OLLn OLLn OOO OOO OLLn OL Ln LLP LLP SLLn+LnOP SLO ALL SLO ALL SLLn+LnOP Ln+SL LnOP Ln+LnOP SOSt SL SOSt SLP OOP SLP OOP PLnP+LLMa PLnP+LLMa POP C21:0LL POP C21:0LL GLL OLO GLL OLO OOMa OOMa GOLn+SLL GOLn+SLL PPP PPP GOO GOO SOLn OLP SOLn OLP GLS+BLL GLS+BLL OLMaGLO OLMa SO Ln+PLP SOLn+PLP GLO SOO ALO SOO ALO GLO GLO ALP+SLS ALP+SLS OOO OOO SOP SOP ALL+SLO ALL+SLO SPP SPP SLP SLP OOP OOP LgLL LgLL POP POP BLO BLO PPP+GOO PPP+GOO ALO ALO AOO AOO SOO SOO ALS ALS SLS SLS SOP SOP AOP+SOS AOP+SOS AOO AOO SOS SOS AOS AOS

3.0 2.0

1.0

1.0

2.0 1.0

0.5

0.5

1.0 0.0

60

65

70

75

80

85

90

95

0.0

Intens 3 0.0 x10 Intens 3 x10 2.5

60

65

70

75

80

85

90

95

Intens 0.0 3 x10 Intens x103 0.8

2.5 2.0

0.8 0.6

2.0 1.5

LnLnStLnLnSt LnLnSt LnLnSt

1.5 1.0

0.6 0.4

LLL LLL LLPo LLPo PoLPo+OLLn PoLPo+OLLn LnOPo LnOPo LnLP LnLP PLnPo PLnPo LnLLn LnLLn OLL OLL OLPo OLPo OOLn+PoOPo OOLn+PoOPo LLP LLP PLPo PLPo LLLn LLLn LnLLn LnLLn PLnP LnOP+PPoPo PLnP LnOP+PPoPo LnLLn LnLLn LLL LLL OLO OLO OOPoOLP OOPo OLLn OLLn OLP GLnLn+ GLn LnLP Ln+LnLP POPo POPo PLP PPoP PLP SLnLnSLnLn GLLn GLLn PPoP MoOP MoOP OLL OLL GLO GLO LLP+OO LLP+OO Ln Ln SLLn+SL LnOP Ln+LnOP OOP GLL OLO PLnP PGLL LnPOLO C22:1L C22:1L Ln Ln POP POP GOLn+SLL GOLn+SLL OOMa OOMa OLP OLP PPP PPP GLnP+AL GLnP+AL Ln Ln GOO GOO SO Ln+PLP SO Ln+PLP SLnP C22:1LL SLnP C22:1LL GOP GOP SOO SOO GLO C24:1L GLO C24:1L Ln+OOO Ln+OOO SOP SOP SLO GLP+ALL SLO GLP+ALL SLPPOP SLP OOP+C22:1 LnP+BL LnP+BL Ln+G LnS Ln+GLnS POP OOP+C22:1 S LnS+C24:1LL S LnS+C24:1LL BLO BLO GLG+C22:1LO GLG+C22:1LO Ln LnAOO AOO GOO+C24:10 GOO+C24:10 LnP+SOO LnP+SOO ALO+GOP+C24:1 ALO+GOP+C24:1 GLS+BLL GLS+BLL AOP+SOS SOP ALP+SLS SOP ALP+SLSBLnP+C22:1 BLnP+C22:1 LnC22:1+C241LO LnC22:1+C241LOAOP+SOS C22L100 C22L100 C24:1LP+LgLL C24:1LP+LgLL LgLO LgLO C22:1OP+C241 C22:1OP+C241 LnS+GOS LnS+GOS AOO AOO BOO BOO BLP+ALS BLP+ALS AOP+SOS AOP+SOS C25:OLO C25:OLO C22:10G+C24:100 C22:10G+C24:100 C24:1LS C24:1LS C24:10P+C22:1OS+LgLO C24:10P+C22:1OS+LgLO BOO BOO LgOO LgOO LgLP+BLS LgLP+BLS BOP+AOS BOP+AOS LgOP LgOP C22:10C22:1+C24:10G C22:10C22:1+C24:10G C24L1OS C24L1OS C25:0OO C25:0OO LgLS LgOO LgLS LgOO C26:0OO C26:0OO LgOP+BOS LgOP+BOS
65 70 75 80 85 90 95 100 65 70 75 80 85 90 95 100

As shown in Figure 1, in NARP-LC, TAG retention times increase with the increasing partition number (PN) (8). In SIC, TAG separation is governed mainly by the number of DBs. Double bond positional isomers, cis-/trans -isomers or regioisomers (R1R1R 2 vs. R1R 2R1) can be also separated (9). APCI-MS coupled to LC represents the most powerful tool for TAG identification, because of the full compatibility with common NARP-LC conditions, easy ionization of nonpolar TAGs and the attainment of both protonated molecules [M+H]+ and fragment ions [M+HRiCOOH]+; in addition, ESI or matrix-assisted laser desorption/ionization (MALDI) have also been used (10,11). LCMS offers possibilities for a better determination of minor compounds whose signals might otherwise be suppressed; in terms of Figure 1: NARP-LCAPCI-MS analysis of plant oils: (a) grape seed-red (Vitis vinifera) and mass spectrometric analysers, TAG analysis has been developed and implemented (b) avocado (Persea americana), (c) redcurrant (Ribes rubrum) and (d) borage (Borago successfully with tandem quadrupoles and linear ion traps. Tandem mass spectrometry officinalis). Adapted and reprinted from Journal of Chromatography A, 11981199, 115130 (2008): M. Lisa and (MSMS) has proved to be an essential tool for unambiguous structural characterization M. Holcapek, Triacylglycerols Profiling in Plant Oils Important in Food Industry, Dietetics and Cosmetics using Highperformance Liquid Chromatographyatmospheric Pressure Chemical Ionization Mass spectrometry. Copyright 2008, for mixtures of isobaric species, yielding product ions from both positive and negative with permission from Elsevier. fragmentation processes. Later on, the triple quadrupole mass spectrometer was found to be well suited for TAG analysis through MSMS operation, including product ion (a) (b) scanning and selected reaction monitoring (SRM). High resolution mass analysis of (a) (b) molecular ion species and product ions after collision-induced dissociation (CID) became routinely possible with the second generation ToF analysers, FT-ICR and orbitrap mass Time (min) spectrometer. Hybrid mass spectrometers such as the Q-ToF afford superior spectral resolution and mass accuracy, also overcoming duty cycle issues typically associated with other scanning instruments; the so-called MSE acquisition mode actually allows for many precursor and neutral loss acquisitions within a single experimental run. From the LC standpoint, recent advances in column technology (sub-2 m and shellparticles) and hardware (allowing operating pressures up to 15,000psi) have arrived to meet the Time (min) (d) (c) expected performance in terms of resolution, speed and sensitivity with respect to (d) (c) conventional LC analysis. UHPLCMS platform combining ultra high performance liquid chromatography with an orthogonal-accelerated timeofflight (oa-ToF) spectrometer offer high-throughput sample analysis, providing narrow chromatographic peaks (< 3 sec) with good ion statistics from accurate mass measurement (<2ppm) (12).
3.0 1.5

LLLn

LLLn

OOO OOP

Intens x103

Intens 1.5 x103

OOO

LLL

Intens x103

Intens x103

0.4 0.2

StLnSt StLnSt

1.0 0.5

0.5 0.0

0.2 0.0

LnLn Ln LnLnLn

LCMS Applications for Carotenoid Analysis

40

50

60

70 Time (min) 70 Time (min)

80

90

50

60

70

80 Time (min) 80 Time (min)

90

100

0.0

40

50

60

80

90

0.0

50

60

70

90

100

Carotenoids are a class of naturally occurring compounds in foods and food products, usually characterized by a C40tetraterpenoid structure, with a centrally located, extended conjugated double bond system. They are usually divided into two groups: hydrocarbon (carotenes) and oxygenated carotenoids (xanthophylls). The latter can be found in either a free form or in a more stable, fatty acid esterified form.

Several works have highlighted the preventive effect of these molecules against serious health disorders such as cancer, heart disease and macular degeneration (13). Most of the studies performed so far have been directed to the analysis of free carotenoids, usually after a saponification step, to reduce sample complexity. Major drawbacks of such a strategy consist of the strong conditions used for hydrolysis, which may lead to carotenoid loss, as well as isomerization. C18 and C30 stationary phases have been extensively used to achieve the separation of carotenoids differing in hydrophobicity within Figure 2: Identification of carotenoid -Cryptoxanthin by LCMSIT-TOF (APCI+): high mass accuracy and resolution is achieved, independent of MS mode (unpublished data a given structural class (14).
property of the authors).
-Cryptoxanthin, exact mass: 553.4404 Event#:1 MS (APCI+)
inten.(X1,000,000) 2.0

HO
536.1642 519.1407 533.1626 550.1742 500.0 525.0 550.0 575.0 600.0 625.0 650.0 675.0 m/z

553.4410

[M+H]+

1.0 417.1384 415.0413 0.0 400.0 425.0

445.1092 459.1327 473.3743 450.0 475.0

Selected precursor ion: 553.4410 Event#:2 MS/MS (APCI+)

1.00 0.75 0.50 0.25 530.0 532.5 535.0 537.5 m/z inten.(X100,000)

[M+H-H2O]+

535.4320

MASS ACCURACY Expected Found 553.4410 535.4320 269.2194 Error (ppm)

Although widely used for carotenoid identification, photodiode array (PDA) detection nevertheless fails in the situation of analytes that exhibit similar, or even identical, spectra. To circumvent such an issue, many researchers have complemented carotenoid identification by using other detection methods (15). Among these, mass detection turned out to be a great aid for the analysis of these substances, allowing structure elucidation on the basis of both molecular mass and fragmentation pattern. Several ionization methods have been reported for MS analysis of carotenoids, including electron impact (EI), fast atom bombardment (FAB), MALDI, ESI, APCI and, more recently, APPI and atmospheric pressure solids analysis probe (ASAP). The latter can be used for the direct analysis of samples, without the need for sample preparation or chromatographic separation, thus allowing for a fast detection screen of multiple analytes. Sometimes, LCMSMS or MS n can be advantageously applied to carotenoid analysis, through the use of specific transitions and daughter ions for the identification of analytes through precursor ion selection, with high mass accuracy (eventually allowing to discriminate between carotenoids having equal molecular masses, but different fragmentation patterns); an example is shown in Figure 2. Further advantages are represented by minimal sample clean-up, leading to a decrease in overall analysis time and a higher sample throughput (16).

Selected precursor ion: 535.4320 Event#:3 MS (APCI+)

3
inten.(X100,000)

MS

553.4404

1.08 +3.17 9.28

MS/MS 535.4303 MS3 269.2169

3.0 2.0 1.0 0.0 265.0

269.2194

[M+H-H2O-266]+
270.0 275.0 280.0 m/z

LCMS Applications for Polyphenol Analysis

Occurring in many food products with enormous structural variability (5000 derivatives are known today), phenols and flavonoids are receiving special interest because of their broad spectrum of pharmacological effects (17). The identification of these polyphenol derivatives in food samples is a difficult task as a result of the complexity of the structures and the limited standards commercially available. For this reason, the most common separation techniques used to determine these kinds of bioactive compounds in such samples have been capillary electrophoresis (CE), GC and LC.

Figure 3: Comparison of a (a) UV-chromatogram and (b) a MS-chromatogram of the same sample (UV: at 330 nm; inset at 210 nm). Adapted and reprinted from Journal of Chromatography
B, 879(24), 24592464 (2011): B.F. Zimmermann, S.G. Walch. L.N Tinzoh, W. Sthlinger and D. W. Lachenmeier, Rapid UHPLC Determination of Polyphenols in Aqueous Infusions of Salvia officinalls L. (sage tea). Copyright 2011, with permission from Elsevier.
(a)
5.5e-1 5.0e-1 4.5e-1 41 Luteolin-Glucuronide 4.0e-1 3.5e-1 29 Lutedin-Diglucurenide 3.0e-1 2.5e-1 2.0e-1 1.5e-1 1.0e-1 5.0e-2
5.42

45 Rcemarinic Acid

53,54,55 Rosmancl-lsomers

UV330nm

13.16 1.45e-1 1.4e-1 1.35e-1 1.3e-1 1.25e-1 1.2e-1 1.15e-1 1.1e-1 1.05e-1 1.0e-1 9.5e-2 9.0e-2 15.31 18.00

UV210nm

27.02

52 Camosol-lsomer

19.75

446 Apigarin-Glucuronide 50 Salviandic Acid K

56,57 Camosol-lsomers

31 Hydroxyluteol-Glucuronid

20.73

18.13

58 Camosic-Acid-lsomer

AU

CE provides high efficiencies in short migration times with small amounts of reagents and sample volumes needed (18), although its main disadvantage is the low concentration sensitivity. GC is the least used technique for this purpose because a derivatization step is necessary. LC in combination with MS proved to be by far the most useful analytical approach for identification, structural characterization and quantitative analysis of these compounds. The sensitivity of the MS response is clearly dependent on the interface technology employed. As ionization interfaces, APCI and ESI, under positive and negative ionization modes, are usually adopted. In general, polyphenolic components are detected with a greater sensitivity as negative ions (protonated or not), while significant fragments are obtained in the positive ionization mode; in this regard, the two modes complement each other to provide useful information for identification purposes. In contrast, API typically yields only a single intense ion, thus hampering analyte accurate identification. Often, spectral data from single-stage MS are combined with UV absorbance to afford positive identification of polyphenolic compounds, with the support of commercial standards and/or reference data when available (Figure 3) (19). The employment of MSMS and MS n achievable by ion trap or triple quadruple MS is a very powerful tool since it discriminates between positional isomers, as well as elucidates the aglycone and sugar moiety (20).

11.76

AU

4.02

24.80

22 Saponarin

26.80 18.25 18.92 22.41

9.48 10.22 11.47

6.76 7.56

0.0

20.00

22.00

24.00

26.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

Time ( min)

58 Camosic Acid

(b)

MS TIC 53,54,55 Rosmanol-lsomers

59 Camosic Acid

38 Luteolin-Glycoside 40 Luteolin-Rutinoside lsomer 41 Luteolin-7-O-Glucuronide

42.44 Apigenin-Rutinoside lsomers 45 Rosmarinic Acid 46 Apigenin-Glucuronide 48 Salvianolic Acid B 50 Salvianolic Acid K

56,57 RCamosol-lsomers

29 Luteclin-Digluronide 30 Enoctrin 31 Hydroxyluteolin-Glucuronid

58 Camosic Acid-lsomer

AU

Comprehensive TwoDimensional Liquid ChromatographyMass Spectrometry (LCLCMS)

The enormous complexity of realworld samples, including foodstuffs,poses high demand both in terms of separation power and sensitivity of detection. In the last few decades, much effort has been directed to the development of multidimensional LC (MDLC) systems, especially in the comprehensive mode (LCLC), aimed at increased resolution through careful selection of independent (orthogonal) separation modes. Hyphenation to mass spectrometry represents a third, added dimension to an LCLC system, generating the most powerful analytical tool today for non-volatile analytes. Moreover, tandem MS techniques can afford structure elucidation through characteristic fragmentation pattern; each MS stage representing an added dimension to the LC or 2DLC system in terms of isolation, selectivity or structural information. Additional degrees of freedom can be obtained by the unprecedented mass accuracy (< 1 ppm) and resolution (> 100,000) of modern ToF, triple quadrupole and FT-ICR analyzers. These high- and ultra-high resolution mass spectrometers, used in conjunction with soft ionization methods, can help

10 Monohydroxybenzoic Acid 10 Chlorogenic Acid 11 Coumarayl-Apiosyl-Glucose 17 Caffeic Acid 22 Saponain 24 Salvianolic Acid F-lsomer

1 Danshensu 2,3 Protocatechuoyl-Hexoses

28 Salvianolic Acid I-lsomer

2.00

4.00

8 Comaroyl-Hexoses

6.00

8.00

10.00

12.00

14.00 Time ( min)

16.00

18.00

52 SCamosol-lsomer 20.00

22.00

24.00

26.00

Sponsored Measurement of Chloramphenicol in Honey with LC-MS-MS

http://www.learnpharmascience. com/tablet-apps/food-issue1/ article1/measure_chlora.pdf

to resolve highly complex samples (21,22). An increased number of spectra and improved mass resolution make ToF analysers the optimum choice for detection in 2DLC. Technical requirements for linkage of an LCLC system to an MS one include: the choice of the mobile phase (buffer and salts), flow rate (splitting), type of ionization (interface) and coupling mode (off-line and on-line). The choice of column sets, spatial resolution, mass resolving power, mass accuracy and tandem-MS capabilities are also crucial, both from the LC and the MS standpoint. Selectivity can be further tuned by adjusting experimental parameters, such as: mobile phase composition and pH value, ion-pairing agents, elution (either isocratic or gradient), flow rate and temperature. When designing an on-line LCLCMS technique, the detector response and the limited dynamic range of the instrument must be considered also. Tubing and valves used may cause significant dilution and negatively affect the MS response, with the overall dilution factor being multiplicative of the dilution factors in each dimension. The use of trapping columns for fraction collection may alleviate such an issue, since analyte re-concentration occurs (23). Requirements for the second dimension ( 2 D), which represent the back-end separation prior to the MS system, are the same as those for a one-dimensional LCMS analysis; reversed phase (RP) chromatography is the most common choice, since typical mobile phases at the end of an RP separation contain a high percentage of organic solvents and volatile additives, which are beneficial for MS ionization. With regards to column dimension, miniaturization (use of a microbore, 1.0 mm i.d. or capillary, 0.10.5 mm i.d. column) is also beneficial as far as dilution and sensitivity are concerned; in addition, reduced mobile-phase flow rates (L to the nL range) avoids flow splitting prior to the MS source. In LCLCMS platforms, a fast MS acquisition rate is often necessary, since the 2 D separation can be very rapid and peak widths characterized by a few seconds or less are not unusual. To adequately sample the 2 D effluent, the mass analyser should be capable of acquiring at least 610 data points per peak. Maximizing the resolution is beneficial for subsequent MS detection, since it alleviates ion suppression effects due to insufficient separation, which may cause high abundant species to obscure the detection of the less abundant. On the other hand, the potential spreading of minor peaks over too many fractions must be considered, since high sampling rates may reduce the concentration of low abundant components and, therefore, hamper their detection.

Application Note: 473

Measurement of Chloramphenicol in Honey Using Automated Sample Preparation with LC-MS/MS

Catherine Lafontaine, Yang Shi, Francois Espourteille, Thermo Fisher Scientic, Franklin, MA USA

Introduction
Key Words Aria TLX-1 TurboFlow technology TSQ Vantage mass spectrometer Food safety Chloramphenicol (CAP) (Figure 1) is a bacteriostatic antimicrobial previously used in veterinary medicine. CAP has been found to be potentially carcinogenic, which makes it an unacceptable substance for use with any foodproducing animals, including honey bees. The United States, Canada, and the European Union (EU), as well as many other countries, have completely banned the usage of CAP in the production of food. The EU has set a minimum required performance level (MRPL) for CAP in food of animal origin at a level of 0.3 g/kg1. Currently sample preparation for the detection of CAP in honey by liquid chromatography-mass spectrometry (LC-MS/MS) involves complex offline extraction methods such as solid phase extraction, QuEChERS, or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are timeconsuming, often taking 2 hours or more per sample, and are more vulnerable to variability due to errors in manual preparation. To offer a high sensitivity (low ppbs) CAP detection method and timely, automated analysis of multiple samples, our approach is to use the Thermo Scientific Aria TLX-1 system powered by TurboFlow automated sample preparation technology coupled to the detection capabilities of a high-sensitivity Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer.

Goal
Develop a quick, automated sample preparation, LC-MS/MS method for chloramphenicol (CAP) in honey by negative ion heated electrospray ionization (H-ESI) using a deuterated internal standard (CAP-d5).

Experimental
Sample Preparation Organic wildflower honey used in this analysis for the preparation of blanks, QCs, and standards was obtained from a local supermarket. CAP was obtained from SigmaAldrich, US (Fluka) and CAP-d5 (100 g/mL in acetonitrile) from Cambridge Isotope Labs, Inc. (Andover, MA, USA). A CAP working solution was prepared in 1:1 methanol/water at 100 ng/mL. The honey was diluted by adding 30 mL of purified water to 10 g of honey (1:3 w/v). CAP standards and QC standards were serially diluted to the target concentrations with 1:3 honey/water containing 250 pg/mL CAP-d5 as an internal standard. Target standard concentrations ranged from 0.024 g/kg to 1.5 g/kg. Four samples of honey obtained internationally and one sample obtained from a local grocery store were analyzed as samples and prepared by dissolving 5 g of honey in 15 mL of purified water. The internal standard was added to a final concentration of 250 pg/mL. The injection volume was 25 L. Method The honey extract clean-up was accomplished using the Thermo Scientific TurboFlow method run on an Aria TLX-1 LC system using a TurboFlow Cyclone polymerbased extraction column. Simple sugars were un-retained and moved to waste during the loading step, while the analyte of interest was retained on the extraction column. This was followed by organic elution to a Thermo Scientific Hypersil GOLD end-capped silica-based C18 reversed-phase analytical column and gradient elution to a TSQ Vantage triple stage quadrupole MS with a H-ESI source. CAP precursor m/z 321 257, 152, and 194 high resolution selective reaction monitoring (H-SRM) transitions were monitored in the negative ionization mode. The 257 m/z product ion for CAP was used for quantitation and the 152 and 194 m/z product ions were used as confirmation. Precursor 326 m/z 157 m/z and 262 m/z H-SRM transitions were monitored for CAP-d5. The total LC-MS/MS method run time was about 5 minutes.

Figure 1: Chemical structure of chloramphenicol

LCLCMS Applications for Triacylglycerol Analysis

In the last few years, comprehensive two-dimensional systems in combination with mass spectrometry have been investigated for TAG separation in various food samples, namely

Section 2: LC-MS

Figure 4: Contour plots of a corn oil sample constructed on the basis of the TIC
chromatogram. (a) total contour plot and (b) expansion of the contour plot in (a). Adapted
and reprinted from Journal of Chomatography A, 1178(12), 4355 (2008). E.J.C. van der Klift, G. Vivo-Truyols, F.W. Claassen, F.L. van Holthoon and T.A. van Beek, Comprehensive Two-dimensional Liquid Chromatography with Ultraviolet, Evaporative Light Scattering and Mass Spectrometric Detection of Triacylglycerols in Corn Oil. Copyright 2008, with permission from Elsevier.

rice oil (24), soybean and linseed oils (25), donkey milk (26) and corn oil (27), using SIC- in the first dimension (1D) and NARP-LC in 2 D, respectively. The two separation modes are based on different retention mechanisms, and hence, the column combination can be considered as entirely orthogonal. For 1D separation, either a microbore (1 mmi.d.) or a narrow-bore column (2.1 mm i.d.) were employed, both lab-silvered using a Nucleosil 5-SA (strong cation exchange) column. In most cases (2426), n -hexane modified with a small amount of acetonitrile was used in 1D, whereas isopropanol and acetonitrile, in a gradient programme, were used in 2 D. The issues related to solvent incompatibility and analyte-focusing were solved by using a microbore column with a very low flow rate in the 1D and by decreasing the percentage of the weaker solvent (acetonitrile) in the 2 D. The 2 D chromatograms were characterized by the formation of group-type patterns, with TAGs located in characteristic positions in relation to their PN and DB values. With regards to MS conditions, a data acquisition rate of 5 Hz and a mass scan range of 400900 amu, allowed the acquisition of approximately 10 spectra for peaks of approximately 2 sec duration; the spectral production frequency was sufficient for reliable peak identification. An innovative LCLC system has recently been investigated by van der Klift et al. (27) for the analysis of a corn oil sample. In this work, TAGs could be rapidly and efficiently separated with a methanolbased solvent on an Ag-coated ion exchanger, avoiding the use of hexane and enabling peak focusing at the head of the 2 D column. In terms of detection, the authors compared UV, evaporative light scattering detector (ELSD) and APCI-MS with the aim of improving the accuracy and precision of TAG quantitation. APCI-MS TIC data were processed both manually and automatically from the untransformed LCLC chromatograms (Figure 4). After correction with literaturederived response factors, the quantitative values obtained were compared with values previously reported for corn oil TAGs by GCFID analysis. The main trend coincided, but significant deviations were observed for individual components. This was probably caused by the use of correction factors obtained under different experimental conditions (both chromatographic and MS parameters). However, in terms of peak overlap, the developed LCLCAPCI-MS system showed a remarkable increase in peak capacity, with respect to onedimensional techniques, and was of great help in the qualitative and quantitative determination of the TAG fraction in the complex food sample tested.

LCLCMS Applications for Carotenoid Analysis

Different LCLCPDAAPCI-MS systems were investigated to elucidate the carotenoid composition of complex food samples, either on free carotenoids attained after a saponification step, or on the native forms (2831). In the first approach, a silica microbore column, operated under normal phase (NP) conditions, was coupled to a C18 monolithic

column, under RP conditions (28). In the second set-up, a cyano microbore column, operated under NP conditions, was coupled to either a C18 monolithic (29,30) or shell-packed column (31), under RP conditions. In the 1D, n-hexane/butylacetate/acetone 80:15:5 (v/v/v ) and n -hexane were used, whereas 2-propanol and 20% water in acetonitrile (v/v ) were employed in the 2 D. Under NP-LC conditions, free carotenoids are separated into groups of different polarity, from the non-polar carotenes up to the polar polyols. In the RP-LC mode, carotenoids are eluted according to their increasing hydrophobicity and decreasing polarity. In all cases, the use of two detection systems, namely PDA and MS, proved to be a mandatory tool for carotenoid identification. Concerning MS conditions, in three out of four setups tested, a quadrupole system in the mass range of 2501300 m/z was employed, while for the most recent application, an IT-TOF mass spectrometer, in the mass range of 2001200 m/z , both equipped with an APCI interface. In the most recent work, special attention was devoted to the elucidation of the epoxycarotenoid fraction, whose composition could be a useful parameter to evaluate juice age and freshness (31). In fact, re-arrangements from 5,6(violaxanthin, antheraxanthin) to 5,8-epoxides (luteoxanthin, mutatoxanthin) can occur with time, partially due to the natural acidity of the juice, which can affect the juice freshness. The attained MS and UV spectra were purer, as a result of the higher LCLC separation power, with respect to conventional LC, thus highlighting the power of the LCLCAPCI-MS approach (31).

LCLCMS Applications for Polyphenol Analysis

Polyphenol content in many food samples is high and highly variable; for this reason, conventional LC techniques are often insufficient for complete characterization. Thus, LCLC MS techniques were considered for the study of such compounds in beer (32), wine and juices (33,34) , as well as apple and cocoa extracts (35). Hajek et al. optimized an LCLC method for the analysis of polyphenols, using UV, electrochemical coulometric and MS detection (32). A polyethylene glycol (PEG) column was used in the 1D, and different C18 and C8 stationary phases were tested in the 2 D. The combination of the columns tested, under matching gradient profiles, provided a high degree of orthogonality for 27 natural antioxidants. A similar setup, in combination with PDA and MS (ESI-ITTOF) detection, has been investigated (33) for the separation of polyphenolic antioxidants in a commercial red wine. A microphenyl column in the 1D, while a partially porous C18 and a monolithic C18 column of identical dimensions (30 4.6 mm; 2.7 m) were compared for the 2 D separation. The high resolution and accuracy of the IT-TOF-MS was beneficial for identification, with an ESI source operated under both positive and negative ionization conditions; the general MS

Figure 5: Identification of dimeric and trimeric procyanidins by alignment of RP-LCMS

extracted ion chromatograms with the relevant section of the fluorescence contour plot.
Adapted and reprinted from Journal of Chromatography A, 1216(35), 62746284 (2009): K.M. Kalili and A. de Villiers, Off-line comprehensive 2-dimensional hydrophilic interactionreversed phase liquid chromatography analysis of procyanidins. Copyright 2009, with permission from Elsevier.
21 18 15 12 9 9.02 10.69 865.5 12.02 13.69

100

864.5 577.3 461.3 865.5

865.5

m/z = 865.5

577.3 9.00 10.00 11.00 12.00 13.00 577.3 14.00

accuracy was lower than 3.1 ppm. A novel RPLCRPLC system was developed (34) for the quantification of polyphenolic antioxidants in wine and juice. In the configuration described, the well separated components in the 1D were directed to the detector, while the more complex part of the sample was diverted to the 2 D via a ten-port valve. Two C18 columns, the second with an ion-pair reagent (tetrapentylammonium bromide), were used. Compound identification was performed using LCESI-TOF-MS for primarycolumn analytes, since the ionpair reagent used in the 2 D was not compatible with MS. A comprehensive HILICRPLC approach was investigated by Kalili and de Villiers for the analysis of procyanidins in cocoa and apple extracts (35). Depending on the degree of polymerization, these structures, composed of flavan3ol monomeric units, can be very complex and their separation challenging. Oligomeric procyanidins were separated according to molecular weight using a HILIC and RP-LC columns, respectively, in the 1D and 2 D. The combination of the two separation modes provided very high orthogonality, and a significant improvement in the resolution of oligomeric procyanidin isomers ( Figure5 ). Positive confirmation was then achieved by the combination of both MS and fluorescence data, dramatically decreasing the probability of false identification.

737.5

Time

100

577.3

m/z = 577.3

Conclusions

577.3 407.3 1153.7 9.00 10.00 1153.7 11.00 1153.7 12.00 13.00 1153.7 14.00

In recent years, there has been a notable increase in the amount of literature pertaining to LCMS analysis of several food products. Several methodologies have been developed to detect, identify and quantify various foodrelated naturally occurring substances. Instrumentation incorporating ESI sources has recently come Time to dominate many areas of MS. Predictably, both ESI-MS and MALDIMS will continue to have expanding roles in the future. It is expected that the automation of the entire LCMS system (benchtop instrumentation inclusive of on-line sampling treatment) will favour its diffusion for routine analysis. In this scenario, scientists involved in producing new analytical instrumentation and developing new analytical methods play a crucial role in order to give a correct answer to these new needs and expectations.

Francesco Cacciola1,2, Paola Donato3,1, Marco Beccaria1, Paola Dugo1,3 and Luigi Mondello1,3
1 Dipartimento 2 Chromaleont

Farmaco-chimico, Universit degli Studi di Messina, Messina, Italy, s.r.l. A spin-off of the University of Messina, c/o Dipartimento Farmaco-chimico, Universit degli Studi di Messina, Messina, Italy, 3 Centro Integrato di Ricerca (C.I.R.), Universit Campus Bio-Medico, Roma, Italy.

How to Cite this Article

10

F. Cacciola, P. Donato, M. Beccaria, P. Dugo, and L. Mondello, Advances in LC-MS for Food Analysis, LCGC Europe 25(s5), Advances in Food Analysis supplement, 1524 (2012).

References
H.-D. Belitz and W. Grosch, Food Chemistry, Springer-Verlag, Berlin, (1999). M. Careri, F. Bianchi and C. Corradini, J. Chromatogr. A, 970(12), 364 (2002). P. Dugo, F. Cacciola, T. Kumm, G. Dugo and L. Mondello, J. Chromatogr. A, 1184(12), 353368 (2008). S.H. Hoke II, K.L. Morand, K.D. Greis, T.R. Baker, K.L. Harbol and R.L.M. Dobson, Int. J. Mass Spectrom., 212(13), 135196 (2001). (5) S.S. Cai, K.A. Hanold and J.A. Syage, Anal. Chem., 79(6), 24912498 (2007). (6) M. Wilm and M. Mann, Anal. Chem., 68, 18 (1996). (7) W.C. Byrdwell, E.A. Emken, W.E. Neff and R.O. Adlof, Lipids, 31(9), 919935 (1996). (8) M. Lisa and M. Holcapek, J. Chromatogr. A, 11981199, 115130 (2008). (9) M. Lisa, H. Velinska and M. Holcapek, Anal. Chem., 81(10), 39033910 (2009). (10) N.L. Leveque, S. Heron and A. Tchapla, J. Mass Spectrom., 45(3), 284296 (2010). (11) M. Malone and J.J. Evans, Lipids, 39(3), 273284 (2004). (12) J.M. Castro-Perez, J. Kamphorst, J. DeGroot, F. Lafeber, J. Goshawk, K. Yu, J.P. Shockcor, R.J. Vreeken and T. Hankemeier, J. Proteome Res., in press (10.1021/pr901094j). (13) C.E. Scott and A.L. Eldridge, J. Food Comp. Anal ., 18(6), 551559 (2005). (14) F. Khachik, Analysis of carotenoids in nutritional studies in Carotenoids. Nutrition and Health (Vol. 5), G. Britton, S. LiaaenJensen and H. Pfander, Eds. (Basel, Boston, Berlin: Birkhauser Verlag), 744 (2009). (15) Q. Su, K.G. Rowley and N.D.H. Balazs, J. Chromatogr. B , 781(12), 393418 (2002). (16) S.M. Rivera and R. Canela-Garayoa, J. Chromatogr. A, 1224, 110 (2012). (17) M. Ganzera, Electrophoresis, 29(17), 34893503 (2008). (18) V. Garca-Canas and A. Cifuentes, Electrophoresis, 29(1), 294309 (2008). (19) B.F. Zimmermann, S.G. Walch, L.N. Tinzoh, W. Sthlinger and D.W. Lachenmeier, J. Chromatogr. B , 879(24), 2459 2464 (2011). (20) P. Dugo, F. Cacciola, P. Donato, R. Assis Jacques, E. Bastos Caramo and L. Mondello, J. Chromatogr. A , 1216(43), 72137221 (2009). (21) F.W. McLafferty, Int. J. Mass Spectrom., 212(13), 8187 (2001). (22) R.W. Kondrat, Int. J. Mass Spectrom., 212(13), 8995 (2001). (23) K. Horvath, J. Fairchild and G. Guiochon, J. Chromatogr. A, 1216(12), 25112518 (2009). (24) L. Mondello, P.Q. Tranchida, V. Stanek, P. Jandera, G. Dugo and P. Dugo, J. Chromatogr. A , 1086(12), 9198 (2005). (25) P. Dugo, T. Kumm, M.L. Crupi, A. Cotroneo and L. Mondello, J. Chromatogr. A, 1112(12), 269275 (2006). (26) P. Dugo, T. Kumm, B. Chiofalo, A. Cotroneo and L. Mondello, J. Sep. Sci., 29(8), 11461154 (2006). (27) E.J.C. van der Klift, G. Vivo-Truyols, F.W Claassen, F.L. van Holthoon and T.A. van Beek, J. Chromatogr. A, 1178(12), 4355 (2008). (1) (2) (3) (4)

11

(28) (29) (30) (31) (32) (33) (34) (35)

P. Dugo, V. Skerikova, T. Kumm, A. Trozzi, P. Jandera and L. Mondello, Anal. Chem., 78(22), 77437750 (2006). P. Dugo, M. Herrero, T. Kumm, D. Giuffrida, G. Dugo and L. Mondello, J. Chromatogr. A , 1189(12), 196206 (2008). P. Dugo, M. Herrero, D. Giuffrida, T. Kumm and L. Mondello, J. Agric. Food Chem., 56(10), 34783485 (2008). P. Dugo, D. Giuffrida, M. Herrero, P. Donato and L. Mondello, J. Sep. Sci., 32(7), 973980 (2009). T. Hajek, V. Skerikova, P. Cesla, K. Vynuchalova and P. Jandera, J. Sep. Sci., 31(19), 33093328 (2008). P. Dugo, F. Cacciola, M. Herrero, P. Donato and L. Mondello, J. Sep. Sci., 31(19), 32973308 (2008). M. Kivilompolo and T. Hyotylainen, J. Chromatogr. A, 1145(12), 155164 (2007). K.M. Kalili and A. de Villiers, J. Chromatogr. A, 1216(35), 62746284 (2009).

12

Advances in GCMS for Food Analysis

By P  eter Quinto Tranchida, Paola Dugo and Luigi Mondello

An overview of important gas chromatographymass spectrometry (GCMS) techniques currently used in food analysis is described. Considerable attention is devoted to the use of the mass spectrometer, in relation to its potential for separation and identification. The importance of comprehensive two-dimensional GC (GCGC) is also discussed.

ood products are usually of a highly complex nature and are composed of organic material (fats, sugars, proteins and vitamins) and inorganic material (water and minerals). Apart from natural constituents, foods can contain xenobiotic compounds deriving from a variety of sources, including the environment, packaging, agrochemical treatments, etc. Many xenobiotic compounds can have a profound negative effect on human health even at trace concentration levels. A gas chromatography-mass spectrometry (GCMS) analysis of a food can vary in scope. For example, a GCMS method can be used for the qualitative/quantitative analysis of untargeted volatiles (for example, elucidation of an aroma profile) or targeted ones (such as pesticides). Furthermore, a GCMS method can also be exploited for the generation of a chromatography profile (fingerprinting), with the aim of distinguishing between food samples of the same type (for example, to determine geographical origin). In such studies, the exploitation of statistical methods is almost obligatory. However, for almost any purpose, a GCMS technique must be both sensitive and selective, as well as possessing a decent separation power and speed. The extent to which one or more of the aforementioned features prevails is dependent on the initial analytical objective.

GC-MS

Sponsored Pesticide Analysis in Green Tea with QuEChERS and GC-MSn

Technical Note: 10295

Multi-residue Pesticide Analysis in Green Tea by a Modied QuEChERS Extraction and Ion Trap GC/MSn Analysis
David Steiniger, Guiping Lu, Jessie Butler, Eric Phillips, Yolanda Fintschenko, Thermo Fisher Scientic, Austin, TX, USA

Introduction
Key Words ITQ 700 Food Safety GC/MSn Green Tea Pesticide Residues QuEChERS Recently formulated pesticides are quite different in their physical properties from their predecessors such as 4,4'-DDT. Most of these newer pesticides are smaller in molecular weight and were designed to break down rapidly in the environment. Therefore, to successfully identify and quantify these compounds in foods, more careful consideration must be placed on the sample preparation for extraction and the instrument parameters for analysis. This study will cover the preparation of extracts and the optimization of the analytical parameters of the splitless injection, separation, and detection. The determination of pesticides in fruits, vegetables, grains and herbs has been simplied by a new sample preparation method, QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), published recently as AOAC Method 2007.01.1 The sample preparation is simplied by using a single step buffered acetonitrile (MeCN) extraction and liquid-liquid partitioning from water in the sample by salting out with sodium acetate and magnesium sulfate (MgSO4).1 QuEChERS can be used to prepare green tea samples for analysis by gas chromatography/tandem mass spectrometry (GC/MSn) on the Thermo Scientic ITQ 700 GC-ion trap mass spectrometer. The study was performed to determine the linear ranges, quantitation limits and detection limits for a partial list of pesticides that are commonly used on green tea crops, prepared in matrix using the QuEChERS sample preparation guidelines. A splitless injection of 22 pesticides was made in a single injection with detection in electron ionization (EI) MS/MS. Since the extracts were prepared in MeCN, a solvent exchange was made to hexane/acetone (9:1) prior to conventional splitless injection.2 Once the calibration curve was constructed, multiple matrix spikes were analyzed at levels of 37.5, 75, 150, 225, 600, or 1200 ng/g (ppb) and low level spikes of 7.5, 15, 37.5, 75, or 300 ng/g (ppb) to verify the precision and accuracy of the analytical method. These concentrations were chosen based on the requirements of various regulatory agencies.

preparation and analysis were rigorously tested (Table 1). A list of the pesticides to be studied was created that would address all of the various functional groups and different physical properties of most pesticides. MSn parameters were optimized with the use of variable buffer gas, the testing of the isolation efciency, and adjustment of the Collision Induced Dissociation (CID) voltage. A surge splitless injection was made into a Thermo Scientic TRACE TR-Pesticide III 35% diphenyl/65% dimethyl polysiloxane column, (0.25 mm x 30 meter, and a lm thickness of 0.25 m with a 5 m guard column).
Item Descriptions
TRACE TR-Pesticide III 35% diphenyl/65% dimethyl polysiloxane column, 0.25 mm x 30 meter, 0.25 m w/ 5 m guard column 5 mm ID x 105 mm liner (pk of 5) 10 L syringe Septa (pk of 50) Liner graphite seal (pk of 10) Ion volume, EI open Ion volume holder Graphite ferrule 0.1-0.25 mm (pk of 10) Ferrule 0.4 mm ID 1/16 G/V (pk of 10) Blank vespel ferrule for MS interface (pk of 10) 2 mL amber glass vial, silanized glass, with write-on patch (pk of 100) Blue cap with ivory PTFE/red rubber seal (pk of 100) Acetonitrile analytical grade (4L) Hexane GC Resolv* (4L) Acetone GC Resolv* (4L) Organic bottle top dispenser HPLC grade glacial acetic acid 50 mL Nalgene FEP centrifuge tubes (pk of 2) Clean up tube:15 mL tube ENVIRO 900 mg MgSO4, 300 mg PSA 150 mg C18 (pk of 50) 50 mL PP Tubes 6 g MgSO4, 1.5 g CH3CHOONa (anhydrous) (pk of 250) Clean up tube: 2 mL tubes 150 mg MgSO4, 50 mg PSA, 50 mg C18 (pk of 100) Table 1: Consumables for QuEChERS sample preparation and GC/MS analysis

Current one-dimensional GC approaches are generally based on the use of a 30 m 0.25 mm0.25m column, which generates peak capacities in the 400600 range, and are the most commonly exploited tools for the separation of food volatiles. One can expect to fully-resolve around 80100 analytes, using such a capillary column (compound more, compound less). However, because food samples are generally of moderate-to-high complexity, then the occurrence of solute co-elution at the column outlet is common, leading to difficulties or possible errors in the identification and quantification of specific components. In the GCMS field, most analysts are located in one of two groups. On the one hand, there are the many separation scientists who are mainly GC specialists and devote their time almost entirely to the optimization of the separation step, and tend to treat the MS instrument as a simple detector. Such an approach is fine if the ion source receives totallyisolated solutes, identified commonly by using dedicated MS databases. Problems arise when peak overlapping occurs, hence demanding a deeper exploitation of the MS step (for example, by using peak deconvolution methodologies, extracted ions or knowledge of MS fragmentation processes). On the other hand, several MS specialists pay little attention to the GC process, and prefer to circumvent a poor GC separation by exploiting a mass-analysing second dimension: multi-compound bands are transformed into a bunch of ions that are resolved and detected on a mass basis. It is obvious, however, that in the case of extensive co-elution, the reliability of the qualitative/quantitative results can be hampered. In truth, both analytical dimensions are complementary, and should be pushed to their full capacities.

Experimental Conditions
The sample preparation involves careful homogenization of the sample. Extraction solvents must be buffered and the powdered reagents measured at appropriate amounts for the size of sample prepared. Some reagents cause an exothermic reaction when mixed with water, which can adversely affect the recoveries of target compounds. The recommended consumables required for sample

http://www.learnpharmascience.com/ tablet-apps/food-issue1/article2/ residue_tea.pdf

One-Dimensional GC-Based Processes

In this section, a series of recent GCMS food applications will be described, in which different types of MS systems have been used. Emphasis will be directed to the potential of the MS analytical step, which is often exploited to a greater extent in the presence of a single GC dimension. Cajka et al. used a high resolution time-of-flight mass spectrometer (HR ToF MS), connected to a 10 m 0.53mm0.5m 5% phenyl column for the target analysis of 111 pesticides in baby foods (1). The short mega-bore column was used to exploit the low pressure (LP) conditions created by the mass spectrometer, increasing the optimum gas linear velocity. A QuEChERS (quick, easy, cheap, effective, rugged and safe) method was used for sample preparation, while a programmed temperature vaporizor (PTV) was employed as injection system. The GC step was a fast (10.75 min total run time) and low resolution one, hence higher demands were put on the high resolution MS process. The HR ToF MS instrument used, operated under electron-ionization (EI) conditions, was reported to have a mass resolution of approximately

Relative response (%)

7,000 and was operated at an acquisition frequency of 4 Hz, which was considered sufficient for quantification purposes. With only a few exceptions, the limits of quantification were 0.01mg/ Kg. Pesticide identification was performed exploiting mass spectral deconvolution, while quantification was performed by using extracted ions with a 0.02 Da mass window. A Figure 1a-b: GC separation of isomers of HCH (m/z 180.938), DDD and DDT (m/z disadvantage of the proposed approach appeared in the low resolving power of the 235.008) and difenoconazole (m/z 323.024) at a concentration of 0.05 mg/kg under capillary column (circa 20,000 N); as can be observed in Figure 1(a), which reports (a) LP-GC (b) conventional GC conditions. A mass window of 0.02 Da was used in the extracted-ion chromatogram segments relative to the separations of (m/z = 180.938) HCH experiment. Adapted and reprinted from Journal of Chromatography A, 1186(12), 281294 (2008): T. Cajka, J. (hexachlorocyclohexane), (m/z = 235.008) DDD (dichlorodiphenyldichloroethane)/DDT Hasjlova, O. Lacina, K. Mastovska and S. Lehotay, Rapid Analysis of Multiple Pesticide Residues in Fruit-based Baby Food using Programmed Temperature Vaporiser Injection-low-pressure Gas Chromatographyhigh-resolution (dichlorodiphenyltrichloroethane) and (m/z = 323.024) difenoconazole isomers, a series Time-of-flight Mass Spectrometry. Copyright 2009, with permission from Elsevier. of co-elutions do occur. The use of a conventional GC column (circa 120,000 N), with the (a) 3.74 4.92 2.79 2.91 sacrifice of speed, is probably a better,if slower, option [Figure 1(b)]. 100 100 Difenoconazole I 100
-HCH 2.89 -HCH -HCH 3.01 -HCH 3.63 . -DDD . -DDD + . -DDT Difenoconazole II 3.88 . -DDT

2.70

2.80

2.90

Time (min)

3.60

3.70

3.80

Time (min)

Relative response (%)

100

(b)

9.76 -HCH 10.27 -HCH

100 10.50 -HCH 11.15 -HCH 16.49

17.41 . -DDD

. -DDD

17.46 18.15 . -DDT . -DDT

9.50

10.00

10.50

Time (min)

16.50

17.00

17.50

Time (min)

Triple quadrupole MS instrumentation (MSMS analysis) can provide greater selectivity and sensitivity than fullscan systems, with the requisite of prior knowledge on what youre 0 looking for. An MSMS analysis is comparable to a selectedionmonitoring (SIM) one, 4.70 4.80 4.90 5.00 Time (min) performed by using a single quadrupole MS system, but with higher selectivity. Koesukwiwat 23.06 et al. performed an LP-GCMS-MS analysis, using a 5% phenyl mega-bore capillary (10 m 100 Difeno23.16 0.53 mm 1m), on 150 relevant pesticides in four vegetable foods (2). The GC retention time conazole I Difenoconazole II window ranged from 2.9 min to 6.2 min, and thus the occurrence of compound overlapping, at the lowresolution column outlet, was inevitable. Again, high demands were put on the 0 MS process. Twenty-six multiple reaction monitoring (MRM) segments (60 transitions for each 22.80 23.00 23.20 Time (min) segment) were set across a brief time space (2.66.7 min) to cover all the target analytes. Two ion transitions were monitored for each of the 150 pesticides, with the most intense ion exploited for quantification purposes and the other for qualitative ones. The choice of the precursor ion, a process which required a substantial amount of preliminary work, privileged higher mass ions. The triple quadrupole MS system was operated at a cycle time of about 208 msec (4.8 Hz), and a dwell time of 2.5 msec was used for each transition. The sensitivity of the method was generally sufficient for the requirement of food pesticide analysis, with LCL (lowest calibration level) values down to the 5 ppb level. A fast GCMS method was developed by Scandinaro et al. for the construction of a novel MS database, named EI-MS F&F (electronimpact-MS flavour & fragrance) (3). The authors reported the use of a micro-bore apolar GC column (10m 0.10mm0.10m), and a rapid-scanning quadrupole mass spectrometer (qMS). The qMS system employed was characterized by a scan speed of 10,000 amu/s, and a 25Hz data acquisition frequency under a normal mass range (for example, m/z = 40360). Such instrumental characteristics are sufficient for the requirements of qualitative/quantitative fast GC analysis. Single quadrupole MS instruments are the most commonly used in the GC field, combining a relatively low cost with robustness and reduced

dimensions. Furthermore, MS databases are normally constructed using qMS spectra and, thus, peak assignment, through database matching, is quite reliable. To reach sufficient degrees of sensitivity, extracted-ion chromatograms must be used or, even better (in sensitivity terms), the SIM mode. It is clear that, in the latter case, full-scan spectral information is lost. A disadvantage of qMS instrumentation can be mass spectral skewing, an effect which can be observed particularly in fast GCMS analysis. Skewing is related to the change of analyte concentration in the ion source during a scan, causing the generation of inconsistent spectral profiles across a peak. During the experiment performed by Scandinaro et al., the authors subjected 200 essential oils to fast GC-qMS analysis. After, a single spectrum was derived from each chromatogram by averaging the spectra relative to all peaks in the chromatogram (apart from the solvent) and was added to the database. In principle, each spectrum attained can be considered in the same manner as a direct MS injection. The EI-MS F&F database was found to be an effective tool to give a reliable name to an unknown essential oil. After, the GCMS chromatogram can be used for compound-to-compound identification. Mass spectrometric systems can be considered, in their own right, as a second separative dimension. However, such an analytical characteristic is often masked when using the most common ionization mode, namely EI. In fact, when using such an ionization procedure, a great number of fragments are generated in the ion source for each compound, thus creating complex spectral profiles. On the other hand, if a soft ionization method is used [for example, chemical ionization (CI), field ionization (FI) or photoionization (PI)], generating a low degree of fragmentation, then the separation potential of the mass analyser is exalted. Hejazi et al. analysed fatty acid methyl ester (FAME) mixtures by using a highly polar cyanopropyl 60m0.25mm 0.25 m column, with the latter entering the ion source of an HR-ToF MS instrument (4). Instead of using EI, the authors applied FI, a soft ionization method with very little or no fragmentation (the TIC is essentially a molecular-ion chromatogram). In the first dimension, FAMEs were separated on the basis of increasing polarity, while in the second MS dimension separation was dominated by molecular weight.

Figure 2: Bidimensional GC-FI ToF MS data derived from an analysis on fish oil. Fragmentation under EI conditions, for two positional isomers (C18:3), is shown on the left-hand side. Adapted and reprinted from Analytical Chemistry, 81(4),14501458 (2009): L. Hejazi, D. Ebrahimi,
M. Guilhaus and D.B. Hibbert, Determination of the Composition of Fatty Acid Mixtures using GCFI-MS: A Comprehensive Two-Dimensional Separation Approach. Copyright 2009, with permission from American Chemical Society.

20 Elution time relative to saturated FAMEs

CO1Mo

CO1Mo

22:6 ion, 208 16:4 18:4 18:3 16:3 20:3 16:2 16:1 20:4 20:5 21:5 22:5

ion, 108

ion, 150

16

ion, 236

12

OH

18:2 20:2

108 150 208 236 m/z

108 150 208 236 m/z

17:1

Anti-oxidant
14:0 15:0 16:0 17:0

18:1

20:1

22:1 22:0

24:1

8:0

12:0

18:0

20:0

Relative elution time of unbranched saturated FAMEs

Branched saturated FAMEs

150

200

250 Molecular m/z

300

350

The GC-FI ToF MS data was represented on a 2D plane, with m/z values located along an x-axis and elution times along a y-axis. The GC elution times were manipulated so that the saturated FAMEs were shifted onto a horizontal line; relative retention times, with respect to the saturated FAME line, were then derived for the other FAMEs. The 2D plane derived from the analysis of fish oil is illustrated in Figure 2. As can be seen, analyte distribution is highly organized: FAMEs with the same C number are located in specific zones, while the same can be affirmed for analytes with the same number of double bonds. A great amount of information was

obtained through the GC-FI ToF MS experiment (exact masses + 2D plane locations); however, the GC-FI ToF MS data was not sufficient to distinguish positional isomers (that is, C18:3 3 and C18:3 6). The method proposed by the authors was abbreviated as GCFI MS, to emphasize the similarity with comprehensive two-dimensional gas chromatography (GCGC). However, GCGC would appear to be a more powerful method for the identification of FAMEs as a result of the formation of highly organized chemical-class patterns (5). Isotope discrimination during plant biosynthesis can be exploited to evaluate geographic origin and adulteration of natural plant-derived foods. For such aims, isotope ratio mass Figure 3: Organic strawberry 13C/12C authenticity range, along with values derived spectrometry (IRMS), combined with gas chromatography, is a useful analytical tool (6). for commercial strawberries and strawberry-flavoured foods. For compound identification IRMS enables the measurement of deviations of isotope abundance ratios, from an agreed see (8). Adapted and reprinted from Journal of Chromatography A, 1218(42), 74817486 (2011): L. Schipilliti, P. Dugo, standard, by only a few parts per thousand for C, as well as for other atoms such as H, N, O I. Bonaccorsi and L. Mondello, Headspace-solid-phase microextraction coupled to Gas Chromatography-combustionand S. A requirement for IRMS analysis is that each element must be converted into a gas isotope ratio Mass Spectrometer and to Enantioselective Gas Chromatography for Strawberry-flavoured food quality control. Copyright 2011, with permission from Elsevier. before entrance to the ion source. In particular, the determination of the 13C/12C ratio is now rather established, and is obtained by converting the C atoms of a specific analyte into CO2 -14 and then by comparing the C isotope ratio of that constituent to that of a known standard. A dimensionless quantity () is used to express the isotope ratio value of a specific solute, in Min organic relation to the standard and is expressed in % (7). strawberry
-19
Max organic strawberry yogurt 1 yogurt 2

-24

lolly ice candles

-29

comm. strawberry

-34
1 2 3 4 5 6 7 8 9 10 11 12

Schipilliti et al. used solid-phase microextraction (SPME) to extract volatiles from the headspace of (organic) strawberries (as well as from other fruits such as pineapple and peach) (8). The extracted compounds were then subjected to GC analysis, on an apolar 30m0.25mm0.25m capillary. IRMS analysis was carried out for twelve characteristic strawberry aroma constituents and an authenticity range was constructed (Figure 3). Moreover, SPME-GC-IRMS applications were carried out on non-organic strawberries and on strawberryflavoured foods (yogurt, sweets and ice lollies). As can be seen from the graph presented in Figure 3, the 13C/12C values for non-organic strawberries were within the authenticity range, while the values relative to the other food commodities indicated that real strawberry extracts had not been employed for flavouring.

13C

compounds

In general, GC-IRMS is a very useful technique for unveiling adulterations in food analysis. However, it should be added that the series of connections from the GC column outlet (for example, the combustion chamber) to the ion source can cause substantial band broadening and, ultimately, resolution losses. Consequently, whenever the complexity of a food sample exceeds a certain level, the use of a heart-cutting multidimensional GCIRMS system is advisable. One way to circumvent the insufficient resolution/selectivity observed in one-dimensional GC is to analyse the same sample on two columns with a different selectivity. Such an approach was

Figure 4: (a) Full-scan GCMS chromatogram, (c, d) expansions derived from extracted-ion GCMS chromatograms, and (b) a GC-PFPD chromatogram. For compound identification see (9). Adapted and reprinted from Talanta, 72(5), 16371643, (2007): K. Sasamoto, N.Ochial and H. Kanda, Dual low
thermal mass gas chromatographymass spectrometry for fast dual-column separation of pesticides in complex sample. Copyright 2007 , with permission from Elsevier.
DB-5
(c) 26 8 Intensity / x104 a.u. 6 4 2 0
5.22 5.26 5.30 5.34

DB-17
(d) 28 Intensity / x104 a.u. 27 8 6 4 2 0
5.38 13.10 13.14 13.18 13.22 13.26

26

28

27 25 33 28 32 31

31 28 33 32

25

Retention time (min)

16

Retention time (min)

Intensity / x105 a.u.

14 12 10 8 6 4 2 0 5 4 3 2 1 0 3.00

(a)

exploited by Sasamoto et al. to analyse selected pesticides in a brewed green tea (9). The authors achieved a rapid twincolumn GCMS experiment using dual low thermal mass (LTM) modules mounted on a gas chromatograph, equipped with a quadrupole MS and a pulsed flame photometric detector (PFPD). The two columns used were of equivalent dimensions (10 m0.18mm 0.18m), with a different stationary phase (5% phenyl and 50% phenyl), and were attached to a single injector. The column outlets were connected to the detectors via a cross union. Independent applications were performed using differential temperature programmes. Such an approach is rather interesting because two TIC traces, relative to two different capillaries, can be stored as a single GCMS chromatogram. Figure 4 illustrates the TIC trace relative to a mixture of 82 standard pesticides, separated on each column. Expansions derived from extracted-ion chromatograms [Figure 4(c) and 4(d)] highlight a series of co-elutions, and, ultimately, the insufficient overall GC resolving power.

Intensity / x108 a.u.

(b)

Comprehensive TwoDimensional GC-Based Processes

5.00 7.00 9.00 11.00 13.00 15.00 17.00

Retention time (min)

If a multidimensional GC (MDGC) instrument is used in food analysis, then ideally totally-isolated compounds should be delivered to the mass spectrometer. Although such a positive outcome is not always the case, it is also true that in most MDGCMS applications, an EI unit-mass resolution MS system [either single quadrupole or low-resolution (LR) ToF] is generally used. The reason for such a choice is obvious, being related to the highresolution and selective nature of the GC step, hence decreasing the need for a powerful MS process (for example, HR ToF MS or MSMS). An MDGC set-up usually consists of two columns connected in series and characterized by a differing selectivity (for example, apolar-polar, polar-chiral, etc.). MDGC methods are classified into two large groups, namely heart-cutting and comprehensive. Approaches belonging to the former class are characterized by the transfer of a limited number of chromatography bands from the first to the second column. In comprehensive two-dimensional GC, the entire initial sample is analysed in both dimensions. GCGC experiments are normally performed using a conventional primary and a short secondary microbore column (12 m); the latter receives first-dimension cuts in a continuous and sequential manner. The GCGC transfer device, defined as modulator (usually cryogenic), enables the rapid accumulation and re-injection of chromatography bands from the first to the second column. Seconddimension separations are very fast, normally completed within 58 s. The time between sequential second-dimension injections is defined as modulation period, and is equal to the analysis time on the secondary capillary. Each seconddimension analysis is characterized by solutes with the same first-dimension elution time (expressed in min) and different second-dimension retention times [expressed in seconds (s)]. If a 2,000s GCGC application with a 5-s modulation period is considered, then 400 5-s second-dimension traces, stacked side-by-side, will form a (monodimensional) comprehensive 2D GC chromatogram (only one detector is used). Dedicated

Sponsored Fast GC Columns to Analyze FAMEs http://www. learnpharmascience. com/tablet-apps/ food-issue1/article2/ fast_gc_columns.pdf
White Paper: DSGSCFASTGC 0509

Thermo Scientic FAST GC Columns

Reduce Analysis Times
Rob Bunn, Thermo Fisher Scientic, Runcorn, Cheshire, UK

Key Words TRACE GC Columns FAST GC Higher Throughput Reduced Run Times Screening

Thermo Scientic FAST GC columns will save you time and money by utilizing state-of-the-art technology available in modern GCs.

Why Use FAST GC Columns?

The major advantage of FAST GC columns is their ability to deliver equivalent resolution compared with conventional length and diameter columns in shorter analysis times. Often run times can be reduced by 50% using these columns. These columns are not ideally suited for use with quadrupole and ion trap mass spectrometers. The peak width with these columns can be as fast as half a second. These fast peaks place a heavier demand on the system, as fast sampling rates are required. This new range of columns is especially suited to modern gas chromatographs which have high pressure (up to 100 psi) electronic pressure control and detectors with fast sampling rates. Detectors such as the FID, ECD and EPD all have fast sampling rates and can handle the very narrow peaks that FAST GC columns provide. Additionally, the very latest GCs can handle very fast oven temperature program rates and programmed pressure proles which are advantageous for these columns.

Applications for FAST GC Columns

FAST GC columns are especially suited for screening applications. For example, screening of water and soil samples for environmental pollutants or drug screening in human and animal samples. This application note presents a number of examples demonstrating applications of Thermo Scientic FAST GC capillary columns. Analysis times with FAST GC columns can be reduced even further with temperature programs higher than 30 C/min. Conditions used in these applications are also attainable with most GCs. PAH analysis is one of the most routine methods used in environmental laboratories throughout the world.

What Liner Should I Use with FAST GC Columns?

For FAST GC column applications, a liner with a smaller internal diameter and a small volume is suitable. Most conventional liners have an internal diameter of about 4 or 5 mm. But with FAST GC columns it is recommended to use a liner of approximately 2 mm ID. In narrow bore capillary chromatography the band broadening that occurs within the column is minimal. But, the low carrier gas ow rates associated with the technique can exaggerate the band broadening that occurs in the injection port. In some cases the sample band in the liner could expand faster than it is drawn into the column, causing incomplete sample transfer in splitless and band broadening in split injections. For this reason it is very important to use inlet liners with small internal diameters to increase the velocity of the carrier gas through the liner. This results in much higher sensitivity and allows you to take full advantage of the higher efciency associated with FAST GC columns.

software is necessary to generate a bidimensional GC space: each high-speed chromatogram is positioned at 90 to an x-axis, while the compounds separated in the second dimension are aligned along a y -axis and are characterized by an oval shape. With regards to quantification issues, it is necessary to sum the peak areas relative to the same compound in each high-speed seconddimension trace, again by using dedicated software. For more details on GCGC the reader is directed to the literature (10). A common characteristic of all GCGC separations is that very narrow GC peaks (200500 msec) are generated. For this reason, high-speed (up to 500 Hz) LR ToF MS systems have dominated the GCGC market over the past decade. The reason for such a supremacy is mainly related to quantification: at least 810 data points/peak are necessary for correct peak reconstruction.
Figure 5: SPME-GCGC-LR ToF MS chromatogram relative to the headspace of marine salt, with indication of the chemical-class patterns. For compound identification see (11). Adapted and reprinted from Journal of Chromatography A, 1217(34), 55115521 (2010): I. Silva, S.M. Rocha,
M. A. Colmbra and P .J. Marriot, Headspace Solid-phase Microextraction with Comprehensive Two-dimensional Gas Chromatography Time-of-flight Mass Spectrometry for the Determination of Volatile Compounds from Marine Salt. Copyright 2010, with permission from Elsevier.

2nd Dimension retention time(s)

127

Lactones Terpenoids Aliphatic Alcohols Aliphatic Aldehydes Aliphatic Hydrocarbons

58 3 13 26 73 105 78

96 102 109 107 142 155 119

Silva et al. reported an SPME-GCGC-LR ToF MS investigation, on the headspace of marine salt (11). The ToF mass spectrometer was operated at a 100 Hz spectral acquisition frequency, using a 41415 m/z mass range. Prior to entrance in the ion source, the analytes were separated on an apolar-polar column combination. The GCGC-LR ToF MS instrument was equipped with dedicated software for instrumental control and data processing. Automated data processing was used to tentatively identify peaks with a signal-tonoise threshold > 100. The SPMEGCGCLR ToF MS result for the most complex (101 identified compounds) salt sample is shown in Figure 5. As can be observed, the bidimensional chromatogram is characterized both by unsuspected complexity and by the formation of chemical-class patterns. The investigation of Silva et al. highlighted a series of positive features of GCGC, namely, increased separation power, enhanced sensitivity (due to cryogenic modulation) and the generation of structured chromatograms.

19

C9

C10 C11

C12 C13

C14

C15

C16

C17

C18

C19

C20

300

800

1300 1st Dimension retention time(s)

1800

Recently, Purcaro et al. evaluated the performance of a novel rapidscanning (scan speed: 20,000 amu/s) qMS system in the GCGC analysis of pesticides contained in water (12). The analytes were extracted by using direct solid-phase microextraction and then separated on a 5% diphenyl 30m0.25mm0.25m primary column (SLB-5ms), and on a mediumpolarity ionic liquid 1m0.10mm 0.08 m secondary one (SLBIL59). The MS system was operated using a wide 50450 m/z mass range (which is necessary for heavier weight components) and a 33Hz spectral production rate, a frequency which was found sufficient for reliable quantification. The authors reported that the time required to achieve a scan was 19.5 msec, accompanied by an interscan delay of less than 11msec. Heptachlor, namely the fastest peak generated (base width of circa 300 msec), was reconstructed with ten data points. Spectra derived at different peak points showed good spectral consistency. Under a more common GC mass range (50340 m/z), the qMS system has been capable of producing 50 spectra/s (13).

Conclusions

A brief overview of some of the current possibilities in the field of gas chromatographymass spectrometry analysis of foods has been discussed. The number of instrumental options is high and the present contribution can, only in part, direct the food analyst to the most appropriate choice, on the basis of the initial analytical objective. INTERVIEW Interview with author Luigi Mondello In the case of target analysis, then a single GC column, combined with an appropriate MS approach (MSMS, SIM, EIC, deconvolution methods, etc.), can do the job fine. If the entire chemical profile of a low-to-medium complexity food sample needs to be unravelled, then straightforward GC, with unit-mass resolution MS, is a still a good choice. In the case of a highly complex sample, then the need for a powerful GC step is, in many cases, required. Obviously, a method such as GCGC is suitable for the analyses of unknowns, as well as for target analysis.

FrancescoCacciola1,2, PaolaDonato3,1, MarcoBeccaria1, PaolaDugo1,3 andLuigiMondello1,3

1 DipartimentoFarmaco

chimico,UniversitdegliStudidiMessina,Messina,Italy theUniversityofMessina,c/oDipartimentoFarmacochimico,UniversitdegliStudidiMessina,Messina,Italy 3 CentroIntegratodiRicerca(C.I.R.),UniversitCampusBio-Medico,Roma,Italy.

2 Chromaleonts.r.l.Aspin-offof

How to Cite this Article P.Q. Tranchida, P. Dugo, and L. Mondello, Advances in GC-MS for Food Analysis, LCGC Europe 25(s5), Advances in Food Analysis supplement, 2530 (2012).

(1) T. Cajka, J. Hajslova, O. Lacina, K. Mastovska and S.J. Lehotay, J. Chromatogr. A , 1186(12), 281294 (2008). (2) U. Koesukwiwat, S.J. Lehotay and N. Leepipatpiboon, J. Chromatogr. A , 1218(39), 70397050 (2010). (3) M. Scandinaro, P.Q. Tranchida, R. Costa, P. Dugo, G. Dugo and L. Mondello, LCGC Europe , 23(9), 456464 (2010). (4) L. Hejazi, D. Ebrahimi, M. Guilhaus and D.B. Hibbert, Anal. Chem., 81(4), 14501458 (2009). (5) P.Q. Tranchida, R. Costa, P. Donato, D. Sciarrone, C. Ragonese, P. Dugo, G. Dugo and L. Mondello, J. Sep. Sci., 31(19), 33473351 (2008). (6) A. Mosandl, in: R.G. Berger (Ed.), Flavours and Fragrances Chemistry, Bioprocessing and Sustainability, Springer, p. 379, (2007). (7) J.T. Brenna, T.N. Corso, H.J. Tobias and R.J. Caimi, Mass Spectrom. Rev., 16(5), 227258 (1997). (8) L. Schipilliti, P. Dugo, I. Bonaccorsi and L. Mondello, J. Chromatogr. A , 1218(42), 74817486 (2011). (9) K. Sasamoto, N. Ochiai and H. Kanda, Talanta , 72(5), 16371643 (2007). (10) M. Adahchour, J. Beens and U.A. Th. Brinkman, J. Chromatogr. A , 1186(12), 67108 (2008). (11) I. Silva, S.M. Rocha, M.A. Coimbra and P.J. Marriott, J. Chromatogr. A , 1217(34), 55115521 (2010). (12) G. Purcaro, P.Q. Tranchida, L. Conte, A. Obiedziska, P. Dugo, G. Dugo and L. Mondello, J. Sep. Sci., 34(18), 24112417 (2011). (13) G. Purcaro, P.Q. Tranchida, C. Ragonese, L. Conte, P. Dugo, G. Dugo and L. Mondello, Anal. Chem. , 82(20), 85838590 (2010).

References

Determination of Phenylurea Herbicides in Tap Water and Soft Drink Samples by HPLCUV and Solid-Phase Extraction
By M  anpreet Kaur, Ashok Kumar Malik and Baldev Singh

A simple and sensitive high performance liquid chromatography (HPLC) UV method has been developed for the analysis of phenylurea herbicides, namely, monuron, diuron, linuron, metazachlor, and metoxuron, that involves a preconcentration step using solid-phase extraction. The mobile phase used was acetonitrilewater at a flow rate of 1 mL/min with direct UV absorbance detection at 210 nm. Separation of analytes was studied on a C18 column. The method was applied successfully to the analysis of the herbicides in three soft drink brands and tap water. Good linearity and repeatability were observed for all the pesticides studied.

henylurea herbicides are used widely in a broad range of herbicide formulations as well as for nonagricultural use; consequently, their residues frequently are detected as major water contaminants in areas where these are used extensively (1). Diuron and linuron are substituted urea compounds that are soluble in water and can migrate in soil and enter the food chain (2). These herbicides are of significant toxicological risk to humans and wildlife. Diuron, which is used in cotton growing areas and with fruit crops, is rated as the third most hazardous pesticide for groundwater resources. These herbicides also are applied on railway tracks to maintain quality and provide a safer working environment (3), but this may lead to groundwater contamination as their leaching potential is significant. Phenylureas enter the environment through pathways such as spray drift, runoff from treated fields, and leaching into groundwater. Most of the excess material penetrates into the soil where it is subjected to the action of microorganisms (4) and degradation as studied by Canonica and colleagues (5). Phenylureas are unstable photochemically, as discussed by Khodja and colleagues (6), but these can persist in water

HPLC

Sponsored Phenolic Acids in Red Wine by SPE and HPLC http://www. learnpharmascience. com/tablet-apps/ food-issue1/article3/ herbicides_wineV3.pdf

for several days or weeks depending on the temperature and pH. Cases of incidental pesticide pollution of water reservoirs (24,713) have become more numerous in recent years. Phenylurea residues can be found in water sources, processed products, and on the crops where these are applied. In India, most of the soft drink bottling plants use surface water from canals and rivers, which have a high risk of pesticide contamination. The water treatment measures used are insufficient for complete removal of these pesticide residues, which have been found to be above permissible limits. The evidence for the abovestated facts was provided in a 2003 Centre for Science and Environment (CSE, New Delhi, India) report that found several pesticide residues in many soft drink samples of leading international brands procured from all over India. The CSE findings were affirmed further by a Joint Parliamentary Committee (JPC) created to verify the facts. In 2006, CSE conducted another round of tests and found pesticides yet again in soft drink samples. Keeping this in mind, the present work has great importance, as it involves the determination of phenyl urea herbicides in soft drink samples and tap water. Therefore, it is imperative that sensitive, selective, and efficient methods for herbicide analysis be designed. The common analytical methods used are high performance liquid chromatography (HPLC)UV (24,79), solid-phase microextraction (SPME)HPLC (10), diode array (11), immunosorbent trace enrichment and HPLC (12,14), LCmass spectrometry (MS) (15,16), gas chromatography (GC)MS (13), capillary electrophoresis (17,18, 19), photochemically induced fluorescence (20,21), and derivative spectrophotometry (22). A useful review is presented by Sherma (23) on the use of thin-layer chromatography (TLC) and its modified versions for the analysis of these herbicides. Solid-phase extraction (SPE) of phenylurea herbicides has been reported in literature by several workers (2429). The SPE of soft drinks has been reported extensively (3036). As the use of polar and degradable pesticides becomes widespread, it is urgent that more sensitive analytical methods be developed for their residual analysis in various matrices. HPLC has several advantages over GC, as it can be used for simultaneous analysis of thermally unstable, nonvolatile, polar, and neutral species without a derivative step. Because of the thermally unstable nature of phenylurea herbicides, the direct application of GC to these compounds is not possible and derivatization prior to the detection is needed. For this reason, HPLC with UV absorption or fluorescence detection (710) is preferred over GC. As a result, HPLC is gaining popularity and preference as a pesticide analyzing technique. The present work describes a simple and sensitive HPLCUV method for the analysis of phenyl urea herbicides (namely, monuron, diuron, linuron, metazachlor, and metoxuron) and it involves a single-step preconcentration by SPE.

Materials and Methods

O CH3 N C N H CH3 O O CI CH2 H3C C N CH2 N CH3 N CH3 N C N H Linuron CI CI CH3 N O O C N H CI CI
The HPLC system used included a P680 HPLC pump (Dionex, Sunnyvale, California), a 250 mm 4.6 mm, 5-m Acclaim C18 RP analytical column (Dionex), and a UVD 170U detector operated at a wavelength of 210 nm coupled to a Chromeleon computer program for the acquisition of data (Dionex). Monuron, diuron, linuron, metoxuron, and metazachlor (Figure 1) pesticide standards were obtained from Riedel-de-Haen (Seelze, Germany). HPLC-grade acetonitrile and methanol were obtained from J.T. Baker (Phillipsburg, New Jersey). All the solvents were filtered through nylon 6.6 membrane filters (Rankem, New Delhi, India) using a filtration assembly (Perfit, India) and sonicated before use. Triple-distilled water was used for all purposes.

CH3

Diuron

CI

CH3

Standard Preparation
Stock solutions were prepared in a mixture of 50:50 methanolwater. All the solutions were stored under refrigeration below 4 C.

Monuron Metazachlor CI O CH3O NH C N CH3 Metoxuron

Figure 1: Structures of phenylurea herbicides

Sample Preparation
CH3
The SPE of the tap water and soft drink samples was performed using a Visiprep SPE vacuum manifold (Supelco, Bellefonte, Pennsylvania) and C18 cartridges from J.T. Baker. The SPE cartridges were attached to the solvent-recovery assembly and connected to a vacuum pump. The conditioning was done with 1 mL each of acetonitrile, methanol, and tripledistilled water. Soft drink samples: The presence of phenylurea herbicides was studied in three different types of locally purchased soft drinks (Coke, Mirinda, and Limca). These were filtered with nylon 6.6 membrane filters and degassed by sonicating for 30 min. The samples were spiked with the metoxuron, monuron, diuron, metazachlor, and linuron at a concentration of 5 ng/mL. A 20-mL volume of these samples was passed through the C18 SPE cartridges under vacuum, and the analytes were eluted with 1.5 mL of acetonitrile. The eluants were further used for the HPLCUV analysis. The sample blanks also were prepared similarly. Tap water sample: The tap water sample was taken from the laboratory. It was filtered and then degassed with an ultrasonic bath. The sample was spiked with metoxuron, monuron, diuron, metazachlor, and linuron at a concentration of 5 ng/mL each. A 50-mL sample of the tap

1 0.8
Absorbance (mAU)

0.6 0.4 0.2 0 -1 -0.2 -0.4 1 2 3 4 5

water containing the mixture of herbicides was preconcentrated using C18 SPE cartridges. A 1.5-mL volume of acetonitrile was used for the elution, and the eluant was subjected to HPLCUV analysis. The sample blanks were prepared by the same method.

Procedure
14

Retention time (min)

Aliquots of the mixture of five herbicides were taken, having concentrations of 5500 ppb. These mixtures were analyzed at an optimum wavelength of 210 nm. The mobile phase is an important factor in HPLC analysis, as it interacts with solute species of the sample. Hence, the composition of the mobile phase was selected carefully as 60:40 acetonitrilewater, and the flow rate was set at 1 mL/min. All measurements were taken at ambient temperature. The calibration curves for all five herbicides were prepared and the curves were linear in the range studied.

Figure 2: HPLCUV chromatogram of mixture containing 5 ppb each of the phenylurea herbicides: 1 = metoxuron, 2 = monuron, 3 = diuron, 4 = metazachlor,

Results and Discussion

HPLCUV studies: The separation of these herbicides was studied using direct injection of samples, and parameters such as the effect of flow rate, selection of suitable wavelength, and composition of mobile phase were optimized. The composition of the mobile phase was 60:40 acetonitrilewater. At higher flow rates than 1.0 mL/min, the separations were not up to the baseline, and with lower flow rates, peak tailing was observed, so the flow rate was optimized to 1.0 mL/min. The wavelength for detection was selected from the UV absorption spectra of the five herbicides as 210 nm. Preparation of calibration curves: The calibration curves were constructed for the detection of monuron, linuron, diuron, metoxuron, and metazachlor in the range of 5500 ppb under the optimized conditions using the HPLC with UV detection. The calibration curves were linear over this range. Various characteristics of HPLCUV, including regression equation, working range, and RSD, are summarized in Table I. The LODs of the phenylurea herbicides were calculated using 3.3 S /m (S = standard deviation, m = slope of calibration curve), and they were found to be in the range 0.821.29 ng/mL. Characteristic chromatograms with HPLCUV detection at 210 nm are shown in Figures 2 and 3 for the separation of these herbicides.

3
Absorbance (mAU)

2.5 2 1.5 1 0.5 0 3 5

(a)

(b)

(c) (d)

11

13

Retention time (min)

Figure 3: HPLCUV chromatograms of (a) tap water, (b) Coke, (c) Limca, and (d) Mirinda spiked with a mixture of phenylurea herbicides containing 5 ppb of each, obtained after preconcentration by SPE

Table I: Analytical figures of merit obtained under optimum conditions

Characteristic
Regression equation R
2

Metoxuron
0.0016x + 0.0712 0.992 4.3 5500 0.92 2.76 81.0 (2.4)

Monuron
0.0014x + 0.0308 0.994 4.9 5500 0.82 2.46 85.4 (3)

Diuron
0.0035x + 0.128 0.992 7.25 5500 0.93 2.79 91.1 (3)

Metazachlor
0.0017x + 0.083 0.992 8.68 5500 1.28 3.84 88.2 (3.2)

Linuron
0.002x + 0.1664 0.993 12.4 5500 1.29 3.87 92.3 (5)

Retention time (min) Linear range (ng/mL) LOD = 3.3 S/m (ng/mL) LOQ = 10 S/m (ng/mL) Recovery % (RSD)

* Amount of phenylurea herbicides taken 5 ng/mL each (n = 5)

Table II: Analytical figures of merit obtained using various samples

Samples tested
Tap water Linear range (ng/mL) LOD (ng/mL) LOQ (ng/mL) Recovery* % (RSD) Limca Linear range (ng/mL) LOD (ng/mL) LOQ (ng/mL) Recovery* % (RSD) Coke Linear range (ng/mL) LOD (ng/mL) LOQ (ng/mL) Recovery* % (RSD) Mirinda Linear range (ng/mL) LOD (ng/mL) LOQ (ng/mL) Recovery* % (RSD) 5500 0.96 2.88 81.1 (3.4) 5500 0.89 2.67 83.4 (3.2) 5500 0.99 2.97 87.6 (4) 5500 1.37 4.11 85.1 (4.3) 5500 1.42 4.26 77.3 (5.3) 5500 0.95 2.85 77.5 (4.6) 5500 0.90 2.70 80.2 (4.7) 5500 1.0 3.0 88.6 (5) 5500 1.37 4.11 85.3 (5) 5500 1.40 4.20 77.3 (4.8) 5500 0.95 2.85 79.4 (4) 5500 0.89 2.67 83.1 (4) 5500 0.99 2.97 87.8 (4.2) 5500 1.39 4.17 87.8 (4.5) 5500 1.41 4.23 75.4 (5.1) 5500 0.92 2.76 80.6 (4) 5500 0.84 2.52 84.2 (3.1) 5500 0.91 2.73 90.1 (4) 5500 1.30 3.90 87.1 (4) 5500 1.35 4.05 76.3 (5) Phenylurea Herbicide

Metoxuron

Monuron

Diuron

Metazachlor

Linuron

Recoveries, repeatability, and LODs: The method detection limits were calculated for these herbicides per the ICH Harmonized Tripartite Guidelines (www.ich.org/ LOB/media/MEDIA417.pdf). The method LOQs can be calculated by using 10 S /m . The accuracy (% recovery) and precision (%RSD) of the HPLCUV method were evaluated for each analyte by analyzing a standard of known concentration (5 ng/mL) five times and quantifying it using the calibration curves. Method optimization and validation parameters are presented in Tables I and II. Good linearity and repeatability were observed for all the compounds studied (with correlation coefficient >0.99). The method gives satisfactory results when used to quantify these herbicides in soft drink and tap water samples (Table II) with percentage recoveries ranging from 75% to 90.1%.

Applications
The phenylurea herbicides were studied in various soft drink and tap water samples and no interfering peaks appeared at the retention times of these herbicides in the spiked samples. The tap water, Coke, Mirinda, and Limca (Figure 3) samples were spiked with metoxuron, monuron, diuron, metazachlor, and linuron at a concentration of 5 ng/mL. The analytical validation for the simultaneous quantification of metoxuron, monuron, diuron, metazachlor, and linuron has been performed with good recovery. The recoveries obtained are very good in all cases. Thus, this method can be used to detect the presence of these harmful herbicides in the soft drink and water samples.

* Samples spiked at 5 ng/mL, n = 5

Sponsored High-Pressure IC for Beverage Analysis webcast

Conclusions
The objective of the current study is to develop a simple, isocratic, reproducible, specific, and highly sensitive method for quantitative and qualitative determination of phenylurea herbicides. In the present method the analysis time is 13 min (linuron t R 12.4 min), which is rapid in comparison to some of the other reported methods, like Patsias and colleagues (37) (linuron t R 18.88 min.), Gerecke and colleagues (38) (linuron t R 17.58 min), and Mughari and colleagues (39) (linuron t R 15 min). The proposed method can determine phenylurea herbicides at very low concentrations. The present paper describes the application of HPLC to the separation and quantitative determination of five phenylurea herbicides, and the feasibility of the method developed was tested by simultaneous determination of these herbicides in different brands of soft drinks and in tap water samples. Good linearity and repeatability were observed for all the compounds studied (with correlation coefficient >0.99). It is hoped that the results of the present study contribute to increased scientific knowledge in the field of pesticide residue analysis in various food and environmental samples.

Manpreet Kaur, Ashok Kumar Malik, and Baldev Singh are with the Department of Chemistry,
Punjabi University, Punjab, India. How to Cite this Article M. Kaur, A.K. Malik, and B. Singh, Determination of Phenylurea Herbicides in Tap Water and Soft Drink Samples by HPLC UV and Solid-Phase Extraction, LCGC North America 29(4), 338347 (2011).

References
(1) S.R. Sorensen, C.N. Albers, and J. Aamand, Appl. Environ. Microbiol. 74, 23322340 (2008). (2) G.M.F Pinto and I.C.S.F. Jardim, J. Liq. Chrom. and Rel. Technol. 23, 13531363 (2000). (3) H. Cederlund, E. Brjesson, K. nneby, and J. Stenstrm, Soil Biology and Biochemistry 39, 473484 (2007). (4) E. Van-der-Heeft, E. Dijkman, R.A. Baumann, and E.A. Hogendorn, J. Chromatogr. A 879, 3950 (2000). (5) S. Canonica and H.U. Laubscher, Photochem. Photobiol. Sci. 7, 547551 (2008). (6) A.A. Khodja, B. Laverdine, C. Richard, and T. Sehili, Int. J. Photoenergy, 4, 147151 (2002). (7) L.E. Sojo, D.S. Gamble, and D.W. Gutzman, J. Agric. Food Chem. 45, 36343641 (1997). (8) J. F. Lawerence, C. Menard, M.C. Hennion, V. Pichon, F. LeGoffic, and N Durand, J. Chromatogr. A 732, 147154 (1996). (9) Organonitrogen pesticides Method: 5601, NIOSH manual of analytical methods, 121 (1998). (10) H. Berrada, G. Font and J.C. Molto, J.Chromatogr. A 1042, 914 (2004). (11) R. Jeannot, H. Sabik, and E. Genin, J. Chromatogr. A 879, 5571 (2000). (12) S. Herrera, A. Martin Esteban, P. Fernandez, D. Stevenson, and C. Camara Fresinius J. Anal. Chem. 362, 547551 (1998). (13) Fast multi-residue pesticide analysis in soil and vegetable samples, application note, mass spectrometry, www.appliedbiosystems.com.

(14) A. Martin-Esteban, P. Fernandez, D. Stevenson, and C. Camara, Analyst 122, 11131117 (1997). (15) I. Ferrer and D. Barcelo, Analusis Magazine 26, 118122 (1998). (16) T. Yarita, K. Sugino, T. Ihara, and A. Nomura, Analytical communications 35, 9192 (1998). (17) M.S. Barroso, L.N. Konda, and G. Morovjan, J. High Resol. Chromatogr. 22, 171176 (1999). (18) S. Batista, E. Silva, S. Galhardo, P. Viana, and M.J. Cerejeira, Int. J. Env. Anal. Chem. 82, 601609 (2002). (19) M. Chicharro, E. Bermejo, A. Sanchez, A. Zapardiel, A. Fernandez-Gutierrez, and D. Arraez, Anal. Bioanal. Chem. 382, 519526 (2005). (20) A. Bautista, J.J. Aaron, M.C. Mahedero, and A. Munoz de La Pena, Analusis 27, 857863 (1999). (21) M.D. Gil-Garca, M. Martinez-Galera, P. Parrilla-Vzquez, A.R. Mughari, and I.M. Ortiz-Rodrguez, Journal of Fluorescence 18, 365373 (2008). (22) I. Baranowiska and C. Pieszko, Anal. Letters 35, 413486 (2002). (23) J. Sherma, Acta. Chromatographia 15, 530 (2005). (24) M.M.C. de la Pea and A. Bautista-Snchez, Talanta 13, 279285 (2003). (25) I. Ferrer, V. Pichon, M.C. Hennion, and D. Barcel, Journal of Chromatography A 1, 9198 (1997). (26) F. Li, D. Martens, and A. Kettrup, Se Pu 19, 534537 (2001). (27) T. Cserhati, E. Forgcs, Z. Deyl, I. Miksik , and A. Eckhardt, Biomedical Chromatography 18, 350359 (2004). (28) M.J.I. Mattina, Journal of Chromatography A 549, 237245 (1991). (29) M. Hamada and R. Wintersteiger, Journal of Planar Chromatography-Modern TLC 15, 1118 (2002). (30) J.F. Garca-Reyes, B. Gilbert-Lpez, and A. Molina-Daz, Anal. Chem. 30, 89668974 (2002). (31) M.A. Mumin, K.F. Akhter, and M.Z. Abedin, Malaysian Journal of Chemistry 8, 4551 (2008). (32) X. L. Cao, J. Corriveau, and S. Popovic, J. Agric. Food Chem. 57, 13071311 (2009). (33) Z. Pan, L. Wang, W. Mo, C. Wang, W. Hu, and J. Zhang, Anal. Chim. Acta. 545, 218223 (2005). (34) R. Lucena, S. Cardenas, M. Gallego, and M. Valcarcel, Anal. Chim. Acta. 530, 283289 (2005). (35) E. Papadopoulou-Mourkidou, J. Patsias, E. Papadakis, and A. Koukourikou, Fresenius J. Anal. Chem. 371, 491496 (2001). (36) N. Yoshioka and K. Ichihashi, Talanta 74, 14081413 (2008). (37) J. Patsias and E. Papadopoulou-Mourkidou, JAOAC International 82, 968981 (1999). (38) A. C. Gerecke, C. Tixier, T. Bartels, R.P. Schwarzenbach, and S.R. Mller, J. chromatography A. 930, 919 (2001). (39) A.R. Mughari, P. Parrilla Vzquez, and M. M. Galera, Anal. Chimica Acta. 593, 157163 (2007).

Data Handling & Validation in Automated Detection of Food Toxicants Using Full Scan
By H  ans Mol, Arjen Lommen, Paul Zomer, Henk van der Kamp, Martijn van der Lee and Arjen Gerssen

GCMS and LCMS

Generic methods based on chromatography with full scan MS detection are maturing. Progress has been made in the development of software for automated detection or identification of the analytes, but this still is the bottleneck inhibiting implementation for routine analysis. Validation of qualitative wide-scope screening is another hurdle to be taken before application. An overview of current chromatography-based food toxicant screening is presented.
uring production, processing, storage and transport of food and feed, a variety of potentially hazardous compounds may enter the food chain. These include residues left after treatment of crops and animals with pesticides and veterinary drugs, natural toxins produced by fungi (mycotoxins) and plants, environmental contaminants (persistent pollutants) and processing contaminants. The potential presence of these compounds is an important issue in the field of food and feed safety. Consequently, extensive legislation has been established to protect consumers from unnecessary or excessive exposure to these substances. Analytical chemists are facing a huge challenge when it comes to efficient verification of compliance of a wide variety of products with this legislation and, preferably at the same time, to provide occurrence data for new contaminants which are not yet regulated. In short, hundreds of different products need to be analysed for thousands of known contaminants and more. Within each class of contaminants, the current gold standard to deal with this challenge is the use of multi-analyte methods based on LC or GC with tandem MS detection. Such methods typically cover tens to several hundreds of analytes and are well established in the field of pesticides,13 with other fields following the same concept (e.g., mycotoxins 4,5 and

Data Handling

KEY POINTS
Full scan acquisition is more straightforward than SIM and tandem MS and enables the detection of many more analytes without the need for knowing a priori. Although the time spent on sample preparation and set up of instrumental acquisition methods is declining, the opposite is true for optimization of automated detection and finding the fit-for-purpose balance between false positives and false negatives. Systematic validation studies of fully automated chromatography-based screening methods are still lacking. The next challenge, after developing generic screening methods, is the development of generic software tools for data evaluation and data mining

veterinary drugs 6,7). The instrumental methods involve targeted acquisition where detection of each compound is individually optimized during instrumental method development. The methods are typically extensively validated with respect to quantitative performance and data handling usually involves a manual review of extracted ion chromatograms to verify peak assignment and integration. A logical next step is to combine multi-methods beyond their contaminant class. The feasibility of this approach has recently been demonstrated 8 by simultaneous determination of pesticides, veterinary drugs, mycotoxins and plant toxins in a variety of food and feed commodities. Sample preparation for such integrated methods is very generic and straightforward (as simple as a single extraction/dilution step). Because the anticipated number of analytes to be covered in this approach is beyond what can easily be accommodated by tandem MS, full scan MS is the detection method of choice. Here the measurement is non-targeted, which makes the instrumental analysis much more straightforward (i.e., no application-specific instrument adjustments or optimization needed, no acquisition-time windows). In principle, the scope of the method is unlimited. As long as analytes elute from the column and are ionized in the source, they can be detected. The moment of setting the scope of the method shifts from before, to after the instrumental analysis. The conclusion from the trend described above is that both sample preparation and instrumental analysis are becoming more and more generic and, to a certain extent, independent of the analyte of current or future interest. This also means that the main effort in terms of development, optimization and validation is clearly moving from sample preparation and instrumental analysis towards data processing to ensure reliable detection of the compounds of interest. This paper will provide a brief overview of the full scan MS options currently available for chromatography-based wide-scope screening of food toxicants. Aspects related to automated detection and identification will be discussed based on literature and experiences within the authors laboratory, with emphasis on data handling. An in-house developed software package (MetAlign), which features instrument independent and uniform data preprocessing and automated identification, will be presented.

General Considerations on Automated Detection/ Identification After Full Scan MS Measurement

The raw data files obtained after GC or LC with full scan MS detection, are typically 20500Mb and contain information on retention time (1 or 2 dimensional), m/z = 501000 (nominal or accurate mass) and abundance (from noise to saturation). Getting meaningful results out of this huge amount of

information in an efficient manner is not a trivial task. In principle two approaches can be pursued: non-targeted and targeted data evaluation. With non-targeted data analysis, the raw data file is processed to obtain a list of all individual peaks present in the entire chromatographic run. The number of peaks can be in the 10 000s, which rules out a manual identification. Without any a priori information, one could restrict the evaluation to the major peaks, but these are usually originating from non-toxic endogenous compounds, rather than contaminants, which are mostly present at low levels. Another option is to filter out contaminants by comparing overall LCMS or GCMS profiles of samples, with profiles obtained after analysis of the corresponding non-contaminated product. For this, extensive databases for the different food commodities would be required, which still need to be generated. Consequently, a more feasible option for the time being is to perform a targeted data evaluation based on libraries containing information on the target compounds. All individual peaks assigned after data (pre)processing can then be matched against the information in the library. Alternatively, the software could use the target library as a starting point and restrict the search to compound-specific retention time windows and ion traces from the raw data file. Either way, some sort of match between experimental data and library data is obtained. Depending on the MS device used, this match can be based on a combination of retention time(s), ions ratios, mass spectra, isotope patterns and/or mass accuracy. Criteria will have to be set for each of the match parameters in such a way that the number of so-called false negatives and false positives are within acceptable limits. False negatives are compounds known to be present in the sample but missed by the automated screening method, false positives are compounds known to be absent but appearing on the list of (provisionally) detected compounds.

GC-Full Scan MS
Sponsored Screening and Quantitating Pesticides in Water with LC-MS http:// learnpharmascience. com/tablet-apps/ food-issue1/article4/ pesticides.pdf Various options for full scan MS detection are available for GC. Single quadrupole and ion trap instruments have been commonly applied for food analysis since the 1990s, especially for the determination of pesticide residues in vegetables and fruits. Sensitivity limitations encountered in the past when using full scan acquisition have been overcome by large volume injection using PTV injectors 9 and improvements in MS devices. A big advantage of GCMS is that the MS spectra obtained after electron ionization are highly characteristic and, to a large extent, are instrument independent. This means that spectra generated elsewhere can be used and that libraries can be purchased. Despite the screening potential, the instruments were and still are mainly used for quantitative analysis rather than for screening. One reason for this is that the spectra of the analytes in the sample often contain interfering ions from other compounds, which complicates automated matching against library spectra. To a certain extent, the quality of sample extraction can be improved by software alogorithms, such as deconvolution, that resolve spectra from overlapping peaks and background. Deconvolution software tools are available both commercially and as free downloads (for example, AMDIS from NIST10). A second reason for the limited

Figure 1: Three-dimensional GCGCTOFMS image obtained after a 10 L injection of a standard solution containing >200 pesticides (for more details see reference 19).

exploitation of the screening potential is the lack of dedicated sofware for automatic detection. The standard sofware that comes with the GCMS instrument is usually able to perform searches of sample spectra against libraries, but is often not really suited and not user-friendly with respect to automated identification and report generation in routine practice. This has caused users to develop in-house solutions without11,12 or with deconvolution.13,14 For the user, deconvolution tools that are integrated into the instruments data analysis software are most convenient. Some instrument manufacturers offer this as default15 or as an optional package combined with dedicated libraries that not only include the mass spectra but also retention times for prescribed GC conditions.16 For extracts of increasing complexity and/or in case of lower analyte levels, GC with full scan quadrupole or ion trap MS lacks selectivity, even when applying preprocessing algorithms such as deconvolution. Currently, there are two options to improve selectivity in GC with full scan MS. The first is the use of high resolution/accurate mass MS detectors (GChrTOFMS). The higher selectivity arises from the ability to separate ions that have the same nominal mass but differ in their exact mass. A main disadvantage is that, to take full advantage of this, exact mass library spectra are needed. Because these are not (yet) available, they would need to be generated by the user. In addition, the current mass resolving power (6000) and dynamic range are not adequate for all applications. The second option to improve selectivity is through enhanced chromatographic resolution. The current-state-of-the-art here is comprehensive GC (GCGC). Compounds are typically first separated by volatility on a regular GC column and then, by polarity, on a second short narrow-bore column. A visualization of the resulting separation is shown in Figure1. For full scan MS detection a high scan speed is required (100250 Hz) in order to have sufficient data points across the narrow (100 ms) chromatographic peaks. TOFMS detectors allowing scan speeds up to 500 Hz are available (nominal resolution only). Cleaner spectra are obtained in the first place due to the enhanced GC separation and also here data preprocessing for peak purification can be performed. Given the comprehensiveness of this type of analysis, its main application lies in profiling and classification of samples in various areas including metabolomics, petrochemicals, food and environmental,17 but several applications for qualitative and quantitative determination of residues and contaminants have been reported.1820 Due to the complexity and the amount of data obtained after comprehensive GC analysis, data handling is a major challenge. Both proprietary software and in-house developed software tools for data handling have been described. They have been excellently reviewed by Pierce et al.21 At the moment, no general ready-to-use solution is available for automated detection. Consequently, in our laboratory data handling after GCGCTOF-MS analysis is performed using a combination of the instrument software for initial processing and deconvolution and an Excel macro for matching retention times, data reduction and generation of a list of detected compounds. Currently, an in-house developed alternative for preprocessing/resolving overlapping peaks called MetAlgin is being evaluated.

Figure 2: Effect of resolving power on mass assignment of residues and contaminants (0.025 mg/kg) in a complex compound feed matrix (for details see reference 22).
Horse feed 25 ng/g
< 2 ppm 100 90 80 % of 150 analytes 70 60 50 40 30 20 10 0 10,000 25,000 50,000 100,000 Resolution 2-5 ppm 5-10 ppm 10-25 ppm > 25 ppm/ND

LC-Full Scan MS
For full scan measurement of low levels of toxicants in food and feed after LC separation, high resolution/accurate mass MS detectors are required. During ionization, little or no fragmentation occurs and compounds are detected through the accurate mass of their (de)protonated molecule or adduct. TOF-MS has been used in most investigations. Especially over the past few years, resolution, mass accuracy, dynamic range, scan speed and sensitivity have greatly improved. In addition, a single stage Orbitrap-MS has been introduced, making a resolving power up to 100 0 00 an affordable option for food toxicant analysis. For selective and efficient automated detection, a reliable high mass accuracy is essential. The instrument specifications are typically within 5 ppm or even better. However, in practice this mass accuracy can only be achieved when co-eluting compounds with the same nominal mass can be mass spectrometrically resolved. Consequently, for real-life samples the resolving power of the MS is an important parameter as well. This has been shown recently by Kellmann et al.22 The resolving power required depends on the application, that is, on the complexity of the extract (sample complexity, sample preparation), the analyte (sensitivity) and its concentration. For generic extracts of honey, a resolving power of 25 0 00 was sufficient for obtaining a mass accuracy of <2ppm for pesticides, natural toxins and veterinary drugs, down to the 0.01 mg/kg level. For a much more complex animal feed matrix 100 0 00 was needed. The effect of resolving power on assigned mass accuracy is illustrated in Figure 2.

Figure 3(a): Effect of retention time tolerance on the number of provisionally detected compounds in non-contaminated samples (low response threshold). For sample preparation details, see reference 8; for LC-MS analysis, see reference 22.
140 120 100 # of Suspect Hits 80 60 40 20 0 0.5 0.4 0.3 0.2 0.1 0.05
Rt Window ( min) {1905 compounds, Mass Accuracy 5 ppm}

Retention Time Window

Solvent

Compound Feed

Mass Accuracy
200

140 120 100 # of Suspect Hits 80 60 40

Retention Time Window

20 Figures 3(b) and 3(c): Effect of (b) mass accuracy tolerance and 0 (c) number of entries on the number of provisionally 0.5 0.4 0.3 0.2 0.1 detected compounds in non-contaminated samples (low 0.05 Rt Window ( min) {1905 compounds, Mass Accuracy 5 ppm} response threshold). For sample preparation details, see Solvent Compound Feed reference 8; for LC-MS analysis, see reference 22.

Mass Accuracy
200

# of Suspect Hits

150

100

50

0 25 10 5 2.5 1 0.5
Mass Accuracy ( ppm) {Rt 0.5 min, 1905 compounds}

Solvent

Compound Feed

# Compounds in Database
100 90 80 # of Suspect Hits 70 60 50 40 30 20 10 0 1905 1500 1200 900 600 300
# of Compounds in Database {Rt 0.5 min, M ass Accuracy 5 ppm}

While the hardware to enable screening is becoming more and more fit-for-purpose, development of software and databases for automated detection is still in full progress, with major improvements being made in the past two years. In its simplest form, analytes can automatically be detected in the raw data through matching the accurate mass of peaks found against a list of target compounds with their exact mass and retention time. However, accurate mass and retention time alone are often not sufficiently unique for selective automatic identification. Depending on the tolerances set for matching retention time and accurate mass, the response threshold, the complexity of the matrix and the number of compounds in the target database, the number of potential detections triggering further confirmatory (data) analysis may be too high for screening purposes. This is illustrated in Figure 3(a) and Figures 3(b) & 3(c). To increase specificity, isotope patterns can be used. Like the exact mass, they can be calculated and no prior experimental determination is required. However, for small molecules the isotope ions (except chlorine and bromine isotopes) have substantially lower abundance, thereby increasing the limit of identification. Another option to improve specificity in automated detection is through analyte fragments. Such fragments can be induced during ionization (so-called in-source collision induced ionization,IS-CID) or in a collision cell (e.g., a quadrupole of a QTOF) with the remark that no precursor ion selection is performed and fragmentation is done using fixed generic fragmentation conditions. The formation of fragment ions, to some extent, but certainly their relative abundance, depends on the instrument and conditions applied. Therefore, in contrast to GCMS spectral databases, fragmentation patterns cannot easily be extrapolated and used in other laboratories and will need to be generated by the user (or vendor) for each instrument.

Solvent

Compound Feed

Figure 4: LCTOF-MS data file (a) before and (b) after preprocessing using MetAlign (see reference 23).

Toward Generic Data Processing and Data Mining

While methods for generic extraction and subsequent full scan instrumental analysis are maturing, universal approaches for data (pre)processing, detection of toxicants and formats for archiving preprocessed result files hardly exist. This means that the data files containing comprehensive information on a wide variety of food and feed samples and the (un)known toxicants they may contain, can only be (further) explored by the laboratory that generated the data. That laboratory might only be interested in pesticides (and just perform a targeted data analysis limited to that), while others might be interested in natural toxins in the same commodity. Furthermore, libraries used for automated detection may differ in scope, which affects the output. The issue, as such, is not unique for food toxicant analysis but, unlike other areas such as metabolomics, has hardly been addressed so far. In our institute, as a spin-off from development of data handling software for application in the field of metabolomics, preprocessing tools are being applied and dedicated search tools have been developed for food toxicant screening. The preprocessing tool, called MetAlign, is freely available and described in detail elsewhere.23 One of the main features of MetAlign is that it can handle, either direct or after automated conversion, data from different vendors covering a broad range of GCMS and LCMS instruments, both with nominal and accurate mass. Data preprocessing involves baseline correction, noise elimination and peak picking. The software also deals with detector saturation and brand and instrument specific artifacts. The output is a 50500 reduced data file in a uniform format which can be exported to Excel, or formats allowing visual review through proprietary instrument software already available in the laboratory (see Figure 4). Data alignment can be performed as well, which can be of interest in searching for truly unknowns through comparison of profiles of the same products. The resulting files can be searched against target databases using dedicated modules for GCMS, GCGCMS (application to be published elsewhere) andLCMS. Due to the strongly reduced size, files can be easily stored and searching is fast. A comparison of the automated detection performance after GCGCTOF-MS between the instruments software and MetAlign/GCGCMS_ search showed that in feed samples spiked with 106 analytes, more compounds (32% vs 62% at the 0.0025 mg/kg level) were found using the MetAlign software, without increase in the number of false positives. A generic approach for data preprocessing can facilitate data sharing and data mining, thereby making much more efficient use of (existing) information available at different laboratories (Figure 5).

Figure 5: Data (pre)processing: MetAlign as interface between instrument raw data and a uniform database for data mining.23

Sponsored Detection of Mycotoxins in Corn Meal with LC-MS-MS http://www. learnpharmascience. com/tablet-apps/ food-issue1/article4/ mycotoxins.pdf

Validation of Chromatography-Based (Qualitative) Screening Methods

Once automated detection and reporting have been optimized, the overall method (i.e., sample preparation + instrumental analysis + automated data processing and reporting) has to be validated before it can be applied in routine practice. This essentially means that for a certain matrix (or group of matrices), at the anticipated reporting level, the level of confidence of detection of the target analytes needs to be established. False negatives need to be minimized, for obvious reasons. False positives are undesirable because they will trigger further manual data evaluation and confirmatory analyses which takes time and effort. Up to now, only explorative evaluation of screening performance has been done using limited numbers of spiked samples or by comparison of the detection rate of the automated screening method with (earlier) results from established quantitative. Lee et al. tested automated identification using a test set of 106 pesticides and contaminants spiked to a complex compound feed sample at various levels, using GCGCTOF-MS.19 Detection rates were clearly concentration dependent and varied from 100% to 73% to 17% for 0.10, 0.01 and 0.001 mg/kg, respectively. Mezcua et al.24 investigated the potential of LCTOF-MS based screening for pesticides in vegetables and fruits. Automated detection was done using an in-house compiled database and instrument software. Most pesticides found by the conventional LCMSMS method were also automatically found by the LCTOF-MS screening method. Very recently, two groups did a more extensive verification of automated detection of pesticides using GCMS (single quadrupole) analysis combined with Agilents Deconvolution Reporting Software tool. Mezcua et al.25 spiked 95 pesticides to eight vegetable and fruit samples, at levels between 0.02 and 0.10 mg/kg. The evaluation of Norli et al.26 involved a test set of 177 pesticides spiked in triplicate to 3 matrices at 0.02 and 0.1 mg/kg. The studies revealed that the detectability is, as expected, compound, matrix and concentration dependent and that, even in cases of repeated analysis of the same extract, inconsistencies occurred with respect to automated detection. In all studies mentioned, the data generated were too limited to derive a confidence level for detectability of the target analytes in a certain matrix (group). On the other hand, through analysis of real samples, the studies did show that, compared to the conventional methods, more pesticides could be found in less time, clearly demonstrating the potential of the approach. This has been encouraging enough to apply the methods as an extension to the established quantitative multi-methods because any additional toxicant automatically found can be considered worthwhile. To summarize the above, systematic validation studies of the qualitative screening methods are still lacking. The limited availability of appropriate software and/or target compound libraries up

Figure 6: Reliability of automated detection of residues and contaminants in feed commodities analysed with GCGCTOF-MS. Data processing and automatic detection: ChromaToF 4.1 + Excel macro.

to now might be one reason for this. Another reason might be that, in contrast to quantitative methods, validation procedures and criteria for qualitative chromatography-based methods were not very well addressed in guidance documents for residues/contaminant analysis. For veterinary drugs, EU directive 657/2002 states that screening methods should be validated and that the false compliant rate (false negatives) should be <5% at the level of interest,28 without providing much detail on how to establish this. Fortunately, beginning this year both the veterinary drug 27 and the pesticide29 community in the EU came up with supplemental and updated guidance documents addressing this issue. Essentially, both documents prescribe that in an initial validation, at least 20 samples spiked at the anticipated screening reporting level (< MRL) need to be analysed and the target analyte(s) need to be detectable in at least 19 out of 20 samples (corresponding to the <5% false negative rate). The initial validation needs to be continuously supplemented by analysis of spiked samples during routine analysis and periodical re-assessment of the data needs to be performed to demonstrate validity based on the extended data set. With the recent guidelines in mind, a first retrospective evaluation of automatic detection of pesticides and contaminants in feed commodities after GCGCTOF-MS analysis was done in the authors laboratory. The evaluation was based on spiked samples concurrently analysed with routine samples over a period of 9 months. The results for 100 target compounds at the 0.05 mg/ kg level are summarized in Figure 6. It shows that, at the software detection settings applied (which were not yet exhaustively optimized) >90% of the target compounds are detected with a confidence level >70%. In a retrospective validation exercise, for wheat the validation criterion was met for 70% of the analytes from the test set. Across different feed commodities, this percentage was substantially lower, although it should be mentioned that this type of application is one of the most challenging in food/feed toxicant analysis.

Conclusions
Chromatography combined with advanced full scan mass spectrometry is developing into a universal tool for screening (and quantitative determination) of a wide variety of food toxicants. Time spent on sample preparation and the set-up of instrumental acquisition methods is declining. The opposite is true for data processing, optimization of software parameters for automated detection and finding the fit-for-purpose balance between false positives and false negatives; but progress is being made. The screening methods have already proven their added value. In the foreseeable future, such methods are likely to become more dominating, thereby enabling more efficient and effective control of food safety and providing more comprehensive, and more rapidly available, data for risk assessment.

Hans Mol is a senior scientist and heads the group of National Toxins and Pesticides at RIKILT. He has over 15 years experience in residue and contaminant analysis in the food chain using chromatography with a range of mass spectrometric techniques. Paul Zomer (BSc) is a research chemist in chromatography with various mass spectrometric detection
techniques, applied to pesticide residues and mycotoxins in feed and food matrices.

Arjen Lommen is a senior scientist focusing on the development of data preprocessing and alignment

software and adaption of this software for various applications ranging from metabolomics, profiling to targeted screening. Other areas of expertise include NMR.

Henk van der Kamp (BSc) is a research chemist using gas chromatography, mainly focusing on GCGC TOF-MS analysis of food and feed with an emphasis on data evaluation and reporting. Martin van der Lee is a junior scientist with extensive experience in GCGCTOF-MS analysis of pesticides
and environmental contaminants. His current work also focuses on elemental speciation analysis using chromatography with ICP-MS.

Arjen Gerssen is finishing his PhD on the analysis of marine biotoxins. As well as his continued involvement
in this area, his current research topics also include nano-LCMS and the development and the application of software tools for data evaluation in chromatographic screening methods and data mining. How to Cite this Article H. Mol, A. Lommen, P. Zomer, H. van der Kamp, M. van der Lee, and A. Gerssen, Data Handling and Validation in Automated Detection of Food Toxicants Using Full Scan GCMS and LCMS, LCGC Europe, 23(4), 200210 (2010).

References
(1) H.G.J. Mol et al., Anal. Bioanal. Chem., 389, 17151754 (2007). (2) P. Pay et al., Anal. Bioanal. Chem., 389, 16971714 (2007). (3) S.J. Lehotay et al., J. AOAC Int., 88(2), 595614 (2005). (4) M. Spanjer, P.M. Rensen and J.M. Scholten, Food Additives and Contaminants, 25(4), 472489 (2008). (5) V. Vishwanath et al., Anal. Bioanal. Chem., 395, 13551372 (2009). (6) S. Bogialli and A. Di Corcia, Anal. Bioanal. Chem., 395(4), 947966 (2009). (7) G. Stubbings and T. Bigwood, Anal. Chim. Acta., 637(12), 6878 (2009). (8) H.G.J. Mol et al., Anal. Chem., 80, 94509459 (2008). (9) E. Hoh and K. Mastovska, J. Chromatogr. A, 1186(12), 215 (2008) (10)  N ationa l Institute of Standards and Technolog y, Automated Mass Spectra l Deconvolution and Identification System (AMDIS). http://chemdata.nist.gov/mass-spc/amdis (11) H.J. Stan, J. Chromatogr. A, 892 , 347377 (2000).

10

(12) K. Kadokami et al., J.Chromatogr. A, 1089, 219226 (2005). (13) A. Robbat, J. AOAC int., 91, 14671477 (2008). (14) W. Zhang, P. Wu and C. Li, Rapid Commun. Mass Spectrom., 20, 1563-1568 (2006). (15) S. de Konig et al., J. Chromagr. A, 1008 , 247252 (2003). (16) P.L. Wylie, Screening for 926 pesticides and endocrine disruptors by GC/MS with Deconvolution Reporting Software and a new pesticide library, Agilent Application note 5989-5076EN (2006). (17) H.J. Cortes et al., J. Sep. Sci., 32(56), 883904 (2009). (18) L. Mondello et al., Anal. Bioanal. Chem., 389, 17551763 (2007). (19) M.K. van der Lee et al., J. Chromatogr. A, 1186 , 325339 (2008). (20) S.H. Patil et al., J. Chromatogr. A, 1217, 20562064 (2009) (21) K.M. Pierce et al., J. Chromatogr. A, 1184, 341352 (2008). (22) M. Kellmann et al., J. Am. Soc. Mass Spectrom., 20 (8), 14641476 (2009). (23) A. Lommen, Anal. Chem., 81(8), 30793086 (2009). (24) M. Mezcua et al., Anal. Chem., 81(3), 913929 (2009). (25) M. Mezcua et al., J. AOAC Int., 92(6), 17901806 (2009). (26) H.R. Norli, A. Christiansen and B. Hole, J. Chromatogr. A , 1217, 20562064 (2010). (27)  2002/657/EC, Official Journal of the European Communities L 221/8-36, Commission decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. (28)  Community Reference Laboratories Residues (CRLs), Guidelines for the validation of screening methods for residues of veterinary medicines (initial validation and transfer), http://ec.europa.eu/food/food/chemicalsafety/residues/Guideline_Validation_Screening_en.pdf (29) SANCO/10684/2009, Method validation and quality control procedures for pesticides residues analysis in food and feed , http:// ec.europa.eu/food/plant/protection/resources/qualcontrol_en.pdf

11

ResouRces
CHROMacademys Interactive HPLC Troubleshooter - Try it now at:

http://www.chromacademy.com/hplc_troubleshooting.asp
sponsoRed by

CHROMacademys Interactive GC Troubleshooter

http://www.chromacademy.com/gc_troubleshooting.asp
sponsoRed by

Chromatography Applications Library

http://www.thermoscientific.com/applicationslibrary