11 Deriva

Chemical Reagents and Derivatization Procedures in Drug Analysis
Neil D. Danielson, Patricia A. Gallagher, and James J. Bao

in Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) pp. 70427076 John Wiley & Sons Ltd, Chichester, 2000
CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS
Chemical Reagents and Derivatization Procedures in Drug Analysis

Neil D. Danielson and Patricia A. Gallagher Miami University, Oxford, USA James J. Bao Procter and Gamble Pharmaceuticals, Mason, USA
1 Introduction 2 Gas Chromatography 2.1 Alkylation 2.2 Acylation 2.3 Silylation 2.4 Sample Handling 3 High-performance Liquid Chromatography 3.1 Alkaloids 3.2 Amines 3.3 Antibiotics 3.4 Barbiturates 3.5 Carbonyl Compounds and Carboxylic Acids 3.6 Catecholamines 3.7 Hydroxy Compounds 3.8 Steroids 3.9 Sulfur Compounds 4 Capillary Electrophoresis 4.1 Derivatization for Ultraviolet Detection 4.2 Derivatization for Fluorescence Detection 4.3 Special Applications 4.4 Derivatization Modes 5 Conclusion Abbreviations and Acronyms Related Articles References
1 2 2 3 5 7 8 8 10 13 14 14 16 16 17 18 19 19 20 21 22 23 23 23 24
to enhance the volatility, temperature stability, and/or detectability. The GC section considers derivatization of drugs by reagent class: alkylation, acylation, and silylation. The GC sample handling section describes an approach which combines the extraction and derivatization steps together. Both pre- and postcolumn derivatization are common approaches for high-performance liquid chromatography (HPLC) and many of these methods have been adapted for CE. Derivatization for HPLC is often directed toward aliphatic amines, carboxylic acids, or alcohols that are difcult to detect at low levels by absorbance, luminescence, or electrochemical means. In addition, small hydrophilic molecules upon prederivatization are often converted into larger more hydrophobic compounds, making reversed-phase HPLC easier or even feasible. The HPLC section considers derivatization of drugs based on type: alkaloids, amines, antibiotics, barbiturates, carbonyl compounds and carboxylic acids, catecholamines, hydroxy compounds, steroids, and sulfur compounds. For the GC and HPLC sections, tables are included giving the structures of the more important derivatizing agents, the analytes, and the corresponding reaction products. A brief rationale for derivatization chemistry with CE concludes this article. For CE, derivatization is often done to improve detectability since the path length for absorbance detection is very short and uorescence can be effective using laser-based systems.
1 INTRODUCTION
Derivatization in conjunction with chromatography or CE can be done either in an off-line mode, often prior to the separation step (prederivatization), or in an online mode (postcolumn derivatization). Generally, only prederivatization is used in GC, principally to enhance the volatility, temperature stability, and/or detectability. Both pre- and postcolumn derivatization are common approaches for HPLC and many of these methods have been adapted for CE. Derivatization for HPLC is often directed toward aliphatic amines, carboxylic acids, or alcohols that are difcult to detect at low levels by absorbance, luminescence, or electrochemical means. In addition, small hydrophilic molecules upon prederivatization are often converted into larger more hydrophobic compounds, making reversed-phase HPLC easier or feasible. For CE, derivatization is often done to improve detectability since the path length for absorbance detection is very short and uorescence can be effective using-laser based systems. The desirable conditions to be met for precolumn derivatization are (1) the reaction stoichiometry and product structure should be known, (2) the reaction
This article describes both pre- and postcolumn derivatization chemistry used in conjunction with either chromatography or capillary electrophoresis (CE) to facilitate the determination of drugs. Generally, only prederivatization is used in gas chromatography (GC), principally Encyclopedia of Analytical Chemistry R.A. Meyers (Ed.) Copyright John Wiley & Sons Ltd
2
should be reasonably fast and proceed quantitatively (or at least reproducibly), and (3) the derivative should be stable and readily separable and distinguishable from the starting material. One advantage of the prederivatization approach is that simple equipment is commercially available to allow for the reaction chemistry to be done in the batch mode. Subsequent sample analysis using an autoinjector and a standard unmodied chromatograph is straightforward. Alternatively, the use of a robot to carry out the entire prederivatization chemistry and an HPLC instrument equipped with an autosampler means that the sample throughput will usually be limited by the chromatographic step. Because postcolumn derivatization involves mixing the column efuent with a reagent or passing it through a reactor to form the derivative before it is detected, the automation step is built in. Sample handling before analysis is minimal, reducing the chance of error from sample loss. The conditions desirable to be met for postcolumn derivatization are (1) although the reaction must be rapid, within about 2 min, and be reproducible, the reaction products do not have to be very stable, (2) the reagent itself must have no or a very low detection response, and (3) the reactor volume must be small to minimize dilution and band broadening. The remainder of the article is divided into three major sections, GC, HPLC, and CE. The GC section considers derivatization of drugs by reagent class: alkylation, acylation, and silylation. In addition, a short discussion of sample handling is included. The HPLC section considers the derivatization of drugs based on type: alkaloids, amines, antibiotics, barbiturates, carbonyl compounds and carboxylic acids, catecholamines, hydroxy compounds, steroids, and sulfur compounds. A brief rationale for derivatization chemistry with CE concludes this article.
PHARMACEUTICALS AND DRUGS
Alkylation, acylation, and silylation reaction chemistries are the most common approaches to derivatization for GC and will be the primary focus in this article. A specic example and an overview for each of these reaction classes will be described below. 2.1 Alkylation Alkylation represents the replacement of an active hydrogen, found in an organic acid or amine, by an aliphatic or aliphatic aromatic (e.g. benzyl) group. Alkylation reactions can also be used to prepare ethers from alcohols such as phenol, thioethers from sulfur compounds, and N-alkylamines, amides, and sulfonamides from amines. One principal chromatographic use is the conversion of carboxylic acids into esters that are stable and volatile with good chromatographic characteristics. Esterication of carboxylic acids has traditionally been done through reaction with an alcohol in the presence of an acid catalyst. Generally, a 1 2-mg sample of the acid is heated with 100 L of the alcohol (methanol or ethanol) containing 3 M HCl for 30 min at 70 C. The alcohol is evaporated, leaving the ester as a residue. A faster reaction in only about 2 min using a boiling water-bath is possible with a BF3 methanol solution (Table 1, reagent 1). Generally the esters are extracted into n-heptane followed by evaporation of the solvent. 4 DMFDA is a rapid methylating agent which provides quantitative yields upon mixing in a nonaqueous solvent with the sample of interest (Table 1, reagent 2). Carboxylic acids are converted to methyl esters, thiols to thioethers, and phenols to methyl ethers. Primary amines are converted into their N,N-dimethylaminoethylene derivatives and amino acids are simultaneously modied as this derivative at the amino group and as the methyl ester at the carboxyl group. Aliphatic hydroxyl groups are not methylated. Ethyl, propyl, n-butyl, and t-butyl acetals may be used analogously. Pyrolysis of a quaternary ammonium salt (QUAT) in the presence of the organic analyte can be a convenient methylation procedure. 5 Upon injection of the QUAT mixed with the drug having a reactive amino, hydroxyl, or carboxyl group, into the hot injection port (250 300 C), formation of the appropriate methyl derivative will occur (Table 1, reagent 4). The derivative is immediately swept onto the analytical column for separation and detection. TMAH is the most common reagent used but trimethyl (a,a,a-triuoro-m-tolyl)ammonium hydroxide can be used at a lower injection temperature, which is important for labile unsaturated fatty acids. This method is particularly good for barbiturates, sedatives, xanthine bases, phenolic alkaloids, and dilantin. PFB-Br can convert carboxylic acids, phenols, sulfonamides, and thiols into halogenated derivatives that can
2 GAS CHROMATOGRAPHY
Prior to analysis by GC, compounds containing functional groups with active hydrogens such as COOH, OH, NH, and SH need to be protected. Compounds with these functional groups tend to form intermolecular hydrogen bonds, reducing volatility. They are also thermally unstable and can interact with either fused silica or the stationary phase, causing peak broadening. Several good review sources describing derivatization reagents, reactions, and applications for GC are available. At least one commercial company provides a good overview of the commercial reagents and the apparatus available. 1 Two other references are fairly comprehensive in describing many of the published methods for GC derivatization either by reaction class 2 or type of organic compound. 3
Table 1 Common GC derivatives alkylation

No. 1. Reagenta
F H F B: O CH3 F (BF3methanol) O CH3 (CH3)2N C H O CH3 (DMFDA) F F F F CH2Br F (PFB-Br)
Compounds
R COOH
Derivatives
R COOCH3
2.
R COOH R NH2 R OH R SH R COOH R OH
R COOCH3 R NH (CH2)2 N(CH3)2 R O CH3 R S CH3 O R C O (PFB) R O (PFB) (PFB) HN R S (PFB) R O CH3 R NH CH3 R COOCH3 SO2NHR
3.
Sulfonamides
R SH
4.
CH3 N+ OH H3C CH3 (TMAH)
R OH R NH2 R COOH
DMFDA, N,N-dimethylformamide dimethylacetal; PFB-Br, pentauorobenzyl bromide; TMAH, trimethylanilinium hydroxide.
be easily detected by electron capture (EC) (Table 1, reagent 3). For carboxylic acids, the reaction is run in an organic solvent in the presence of a base such as a tertiary amine for 5 min at 40 C. Interestingly, tertiary amines have been derivatized with pentauorobenzyl chloroformate prior to GC with EC detection. 6 The analysis of cannabidiol (CBD) and the internal standard tetrahydrocannabidiol in blood plasma involved derivatization with PFB-Br followed by purication on a Florisil column. 7 The derivatization procedure involved reuxing a hexane extract with PFB-Br overnight with stirring. Acetone washings of this solution were evaporated to dryness and the residue was dissolved in hexane. A portion of this hexane solution was injected into a gas chromatograph equipped with an EC detector. A typical chromatogram taken on a packed OV-225 glass column is shown in Figure 1. Although a packed column has limited separation capability, it can handle larger amounts of sample. A detection limit of 50 ng mL 1 of plasma was possible. 2.2 Acylation Acylation is the use of a carboxylic acid or carboxylic acid derivative to convert compounds that have active hydrogens (OH, SH, NH) into esters, thioesters, and amides, respectively. Acyl derivatives tend to direct the mass
2 1
inj. 0 4 8 12 16 24 28
Time (min)
Figure 1 Typical chromatogram from a plasma sample containing 2.0 g of the internal standard tetrahydrocannabidiol (1) and calculated to contain 1.5 g of the CBD (2). [Reproduced with permission from Jones et al. 7 ]
4
spectrometry (MS) fragmentation patterns of compounds providing useful structural information. Peruoroacylimidazoles such as triuoroacetylimidazole (TFAI), pentauoropropionylimidazole (PFAI), or heptauorobutylimidazole (HFBI) can react with hydroxyl groups and primary or secondary amines to generate the halogenated derivative and the relatively inert by-product imidazole (Table 2, reagent 1). Generally the reaction times are 1 3 h at 75 C. Indoleamines and indole alcohols, which are acid-sensitive compounds, have been modied by this procedure. Metoclopromide, an antispasmodic agent related to procainamide, has been derivatized with HFBI for GC with EC detection. 8 These reagents are often used in bifunctional derivatization schemes involving silylation chemistry. For example, phenolic amines can be silylated with N-trimethylsilylimidazole (TMSI) to protect the hydroxyl group and acylated to protect the amine group. Other halogenated imidazoles such as p-bromobenzyl- and p-chlorobenzylimidazole have been used because the detectability of the EC detector is enhanced. However, the volatility and stability of the derivatives under GC conditions tend to be lower compared with the uorinated types. Table 2 Common GC derivatives acylation
No. 1. Reagenta
N O N C R
MBTFA, shown in Table 2 as reagent 3, triuoroacetylates primary and secondary amines and carbohydrates under mild nonacidic conditions. 9 For carbohydrates, a single derivative for mono-, di-, tri-, and tetrasaccharides was obtained. The N-methyltriuoroacetamide byproduct is stable and volatile, being amenable to GC. Peruoro acid anhydrides [R(CDO) O (CDO)R], such as TFAA, PFAA and HFAA, react readily with alcohols, phenols, and urine for the preparation of peruoroacyl derivatives, which are particularly useful for EC detection (Table 2, reagent 2). Triethylamine is added as part of the reaction mixture to neutralize the acidic by-products and drive the reaction to completion. Drugs of abuse are often derivatized in this way before gas chromatography/mass spectrometry (GC/MS) conrmation. A GC/MS example of derivatization of amphetamine and methamphetamine by HFAA 10 is shown in Figure 2(a) and (b). The splitting of the peaks
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00 2 10.00
R OH R2 R1 NH R NH2
O R O C R R1 R2 O N C R
(a) 550 000 500 000 450 000 400 000 350 000 300 000 250 000 200 000 150 000 100 000 50 000 0 5.50 6.00 6.50
Time (min)
R=CF3 (TFAI), C2F5 (PFAI), or C3H7 (HFBI)
Abundance
O R NH C R O R O C R O O C R R2 O R1 N C R O R NH C CF3 R2 O R1 N C CF3 O R O C CF3 O R S C CF3
2.
O O R C O C R R=CF3 (TFAA), C2F5 (PFAA), or C3H7 (HFAA)
R OH OH R2 R1 NH
7.00
7.50
8.00
8.50
9.00
9.50
3.
O O F3C C N C CF3 CH3 (MBTFA)
R NH2 R2 R1 NH R OH R SH
(b)
Time (min)
TFAA, triuoroacetic anhydride; PFAA, pentauoropropionic anhydride; HFAA, heptauorobutyric anhydride; MBTFA, N-methylbistriuoroacetamide.
Figure 2 (a) Total ion current chromatogram of HFAA-derivatized amphetamine (1) and methamphetamine (2). The overloaded peak at about 5.6 min is due to excess HFAA. (b) Total ion current chromatogram of HFAA derivatized amphetamine (1) and methamphetamine (2) after extraction of the excess derivatizing agent. The baseline level in this chromatogram is only about 10% of that in (a). The splitting of the drug peaks in both chromatograms is due to the presence of the deuterated internal standards (amphetamine-d5 and methamphetamine-d8 ). [Both gures reproduced with permission from Jones and Mell. 10 ]
10.50
10.50
Compounds
Derivatives
400 000 350 000 300 000 250 000 200 000 150 000 100 000 50 000 0
Abundance
2 1
is due to the presence of deuterated internal standards. Excess HFAA, which causes a high background level, column contamination and eventual degradation of the stationary phase, was removed by the procedure outlined below. The drugs of interest were extracted from 2 mL of urine by solid-phase extraction (SPE) columns with a 10% 2-propanol in chloroform elution solvent. Sodium periodate oxidation of interfering drugs was carried out if necessary. The extract was acidied with 10% HCl in methanol and the solvent evaporated before reconstitution of the residue in hexane with 0.1 M trimethylamine. HFAA was added and the mixture was heated in a sealed tube for 1 h at 70 C. After cooling, deionized water was added to the tube, the tube was shaken, and then the aqueous phase was discarded. A 4% ammonia solution was shaken with the remaining hexane volume and then this organic layer was removed for injection into the GC/MS system. The limit of quantication and limit of detection were both of the order of 50 ng mL 1 and the linearity was observed up to at least 4000 ng mL 1 . 2.3 Silylation Silylation is probably the most widely used derivatization scheme for GC. Active hydrogens from acid, alcohol, thiol, amine, and amide groups can all be protected, usually with trimethylsilyl (TMS) groups. The ease of silylation of these functional groups follows the order alcohol > phenol > carboxylic acid > amine > amide. Steric hindrance is also a factor within a class of organic compounds. For alcohols, the reactivity order is primary > secondary > tertiary, and, for amines, primary > secondary. In general, silylation reagents are unstable and must be protected from moisture. Trimethylchlorosilane (TMCS) is the simplest reagent that can be used, often to derivatize carboxylic acids (Table 3, reagent 1). TMCS is often used in conjunction with HMDS (Table 3, reagent 2) to improve the silylation of sugars and related compounds. GC of silyl derivatives is generally straightforward, with only one major concern, the presence of excess silylating agent. Often an excess of the silylating agent is used in the derivatization reaction to minimize the problem of moisture or other acidic components in the sample. If possible, it is recommended that the excess silylation reagent be evaporated from the sample using a stream of nitrogen before injection into the GC column. This avoids several problems such as large reagent blank peaks in the chromatogram and fouling of the ame ionization detector by SiO2 deposits. A polar stationary phase such as poly(ethylene glycol) (Carbowax 20 M) and free fatty acid phase (FFAP) will be derivatized by excess silylation reagent and cannot be used.
Silylacetamides are represented in the most popular group of silylation agents, owing to their ability to react quickly and quantitatively under mild conditions. BSA is a highly reactive TMS donor (Table 3, reagent 3). BSTFA (Table 3, reagent 4) has the advantage of giving a more uorinated by-product than BSA. This permits derivatization of lower molecular weight analytes without potential overlap of the triuoroacetamide by-product. The addition of TMCS to BSTFA will promote the derivatization of amides, secondary amines, and hindered hydroxyl groups. A comprehensive article on drug detection methodology describes the determination of cocaine, heroin, and metabolites in hair, plasma, saliva, and urine samples after isolation by SPE and subsequent derivatization with BSTFA with TMCS before analysis by GC/MS. 11 After spiking with internal standards, plasma, saliva, and hair extracts were passed through SPE columns and the drugs eluted with a methylene chloride 2 propanol ammonia solvent. The eluate was evaporated to dryness, reconstituted with acetonitrile, and allowed to react with a BSTFA TMCS mixture at 60 C for 30 min before analysis by GC/MS. Representative chromatograms are shown in Figure 3(a c). Separations were effected on a relatively short 12 m 0.2 mm ID capillary column. For urine, saliva, and plasma, the limit of detection was about 1 ng mL 1 and for hair 0.1 ng mg 1 . MSTFA has similar donor strength to BSA and BSTFA but generates an even more volatile by-product, N-methyltriuoroacetamide (Table 3, reagent 5). Both excess MSTFA and the by-product often elute with the solvent peak, eliminating the presence of extra peaks in the chromatogram. Cocaine and its metabolites benzoylecgonine and ecgonine methyl ester were sequentially derivatized with thyliodide to obtain the ester derivative and then MSTFA to form the o-TMS derivatives. 12 The derivatized compounds were separated by a GC instrument with a methylphenylsilicone column and nitrogen phosphorus detection. Again TMCS can be added to MSTFA to promote the derivatization of amides and hindered amines and hydroxyl groups. The strongest reagent for silylation of hydroxyl groups is TMSI (Table 3, reagent 6). It does not react with amines or amides and is effective for most steroids, including those with unhindered and highly hindered OH groups. It also reacts quickly and smoothly with carboxyl groups. Silyl compounds other than those that derivatize with a TMS group have also been used in conjunction with GC. Methyltestosterone was derivatized using either the dimethylethylsilyl (DMES)imidazole or dimethylisopropylsilyl (DMIPS)-imidazole to the corresponding silyl ether derivative. 13 Reaction conditions involve heating at 70 C for 1 h before removal of the excess reagent by N2 and reconstitution
6
Table 3 Common GC derivatives silylation
No. 1. Reagenta
CH3 H3C Si CH3 Cl (TMCS) CH3 CH3 H3C Si N Si CH3 H CH3 CH3 (HMDS)
Compounds
R COOH
Derivatives
2.
R OH
R O Si(CH3)3 O R C O Si(CH3)3
3.
CH3 CH3 H3C Si O C N Si CH3 CH3 CH3 CH3 (BSA)
R OH R NH2 R1 NH R2 O R C NH2 R COOH
R O Si(CH3)3 R N Si(CH3)3 Si(CH3)3 R1 N Si(CH3)3 R2 O Si(CH3)3 R C N Si(CH3)3 O R C O Si(CH3)3
4.
CH3 CH3 H3C Si O C N Si CH3 CH3 CF3 CH3 (BSTFA)
Same as BSA, leaving group

O H F3C C N Si (CH3)3
more volatile than that of BSA Same as BSA, leaving group

O H F3C C N CH3
5.
CH3 O F3C C N Si CH3 H3C CH3 (MSTFA)
is very volatile
R OH (steroids) R COOH R O Si(CH3)3 O R C O Si(CH3)3
6.
CH3 N Si CH3 CH3 (TMSI)
No reaction with amines

a
HMDS, hexamethyldisilazane; BSA, N,O-bis(trimethylsilyl)acetamide; BSTFA, N,O-bis (trimethylsilyl)triuoroacetamide; MSTFA, N-methyl-N-trimethylsilyltriuoroacetamide.
of the residue in cyclohexane. Because the desired application was the assay of methyltestosterone in bulk powder and tablets, separation on a packed column was adequate in the concentration range 0.1 1.5 mg mL 1 (Figure 4a c). Alternatively, N-methylN-(t-butyldimethylsilyl)triuoroacetamide (MTBSTFA) will derivatize hydroxyl, carboxyl, thiol, primary and secondary amines by adding t-butyldimethylsilyl (TBDMS) groups. These TBDMS derivatives are more stable to hydrolysis than TMS compounds and, when analyzed by GC/MS, a strong M 57 fragment is noted which
can be used to determine the original compounds molecular weight. These advantages have been demonstrated in the determination of short- and long-chain carboxylic acids. 14 Pentauorophenyldimethylsilyl (ophemesyl) derivatives are often generated from steroids by reaction with ophemesylamine at room temperature for 15 min. This is a selective reaction with only primary and secondary hydroxyl groups in steroids reacting in the presence of unprotected ketone groups. GC with EC detection provides detection limits in the nanograms picograms range. Flophemesyl derivatives also have favorable
7
R2 R1 N OCH 3 R1 R2
advantages for GC/MS, producing diagnostic ions which carry more of the current than TMS derivatives. 15 2.4 Sample Handling Generally, drugs are found in aqueous matrices such as urine and plasma; extraction with an organic solvent is
3 4 1 2 7 5 13 6 8 11 12 14 17 16 18 9 10 15 20 19 21 23 22
2770 2840 1 2
(1) CH2 CH3 (2) H C 2H 5
(a)
24
2880
(a) 2 5 7 9
2960 2
14 16 19 20
(b)
23
2980
(b) 75 2 1 5 1 5 75 10 14 6 9 7 11 8 12 3 4 5 15 16 15
1 3060 2
17 20 19 21 24 23
2 (c)
Time (min)
4.0 (c)
5.0
6.0
7.0
8.0
9.0
10.0
11.0
Time (min)
Figure 3 Single ion monitoring recordings of extracts from

(a) standard cocaine/opiate hair, (b) drug-free control hair, and (c) a hair sample collected from a heroin user. Analytes are identied as follows: anhydroecgonine methyl ester (1), [2 H-3]ecgonine methyl ester (2), ecgonine methyl ester (3), ecgonine ethyl ester (4), [2 H-3]cocaine (5), cocaine (6), [2 H-3]cocaethylene (7), cocaethylene (8), [1 H-3]benzoylecgonine (9), benzoylecgonine (10), norcocaine (11), norcoceathylene (12), benzoylnorecgonine (13), [2 H-3]codeine (14), codeine (15), [2 H-3]morphine (16), morphine (17), norcodeine (18), [2 H-6]-6-acetylmorphine (19), [2 H-3]-6-acetylmorphine (20), 6-acetylmorphine (21), normorphine (22), [2 H-9]heroin (23), heroin (24). [Reproduced with permission from Wang et al. 11 ]
Figure 4 Typical gas chromatograms of (1) methyltestosterone and (2) norethandrolone as (a) TMS, DMCS (b) and (c) DMIPS derivatives. Note the bulkier derivatives are shifted to a cleaner area in the GC trace. [Reproduced with permission from Zakhari et al. 13 ] required not only to isolate the drug in a nonaqueous solvent which is compatible with most derivatization reagents but also to remove inorganic salts which are not compatible with GC. An alternative approach called direct derivatization combines the extraction and derivatization steps together. Direct derivatization of drugs in untreated biological samples for GC analysis has the primary advantages of improved extractability of
8
a derivatized polar compound and avoidance of stability problems. Direct derivatization of drugs in untreated biological samples can mean not only derivatization in the sample matrix followed by extraction, but also a twophase reaction where the derivatization takes place in the organic phase while extraction of the analyte is continuing from the aqueous phase. 16 Extractive alkylation has been applied to valproic acid and ketoprofen, in which the tetrabutylammonium ion acted as the anion-pairing agent to pull the compound into the organic phase, where alkylation to generate the methyl or phenacyl derivative was possible. Phenolic compounds such as elioquinol have been determined in an analogous fashion. Acylation involving peruorinated anhydrides and chloroformates has also been used for the determination of drugs such as metanephrine and normetanephrine. Chloroformates can de-alkylate tertiary amines to form stable carbamates.
3 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

The variety of chemistries that can be adopted for either pre- or postcolumn derivatization in conjunction with HPLC is large in part because either aqueous or organic solvent-compatible reactions are possible. Most of the books reviewing fundamental reactions for pre- and postcolumn derivatization chemistry in conjunction with HPLC were published in the 1980s, which appears to have been the most active time for research in this eld. 17 23 Many review articles have focused on postcolumn derivatization with HPLC. 24 31 Some specic review articles on derivatization chemistry of pharmaceutical compounds with HPLC have also been published. 32 36 Although the other articles do not try to be comprehensive, Ahuja 35 gave a virtually complete review of the topic up to 1979. Danielson et al. 36 published another fairly complete review covering the period 1979 87, which is the basis for this part of this article. Again, a specic example and an overview for each major class of pharmaceutical compounds will be given. 3.1 Alkaloids Separation of atropine and ergotamine by normal-phase liquid chromatography (LC) followed by postcolumn ion-pair extraction with an aqueous solution of 9,10dimethoxyanthracene-2-sulfonate has been reported. 37 The organic mobile phase was monitored uorimetrically with detection limits of 40 100 ng. Conversely, methadone, phencyclidine, and their metabolites were separated by reversed-phase HPLC using 9,10-dimethoxyanthracene-2-sulfonic acid in the mobile phase. 38 The resultant ion pairs (Table 4, reagent 3)
were extracted with chloroform postcolumn on-line and detected uorimetrically with detection limits of 1 6 ng mL 1 in plasma. A precolumn ion-pair derivatization method for atropine, hyoscyamine, scopolamine, and ergotamine, involving picric acid and normal-phase chromatography, provided ultraviolet (UV) detection limits of about 200 ng. 39,40 Morphine has been detected uorimetrically after postcolumn oxidation with alkaline potassium hexacyanoferrate(II) to form the dimer pseudomorphine. 41 Morphine can be oxidized to the uorescent dimer pseudomorphine using alkaline hexacyanoferrate(III). Other opiates, such as normorphine, naborphine, codeine, norcodeine, and others, were also reactive. The excitation and emission wavelengths were 323 and 432 nm, respectively. A comparison of the UV and uorescence response was made using the instrumentation shown in Figure 5. The mobile phase, a 12.5 : 87.5 methanol 0.1 M KBr solution, was propelled through a C18 reversed-phase column. The derivatizing agent was 50 mg of K3 Fe(CN)6 in 250 mL of 4 M ammonia solution delivered at 0.4 mL min 1 . The presence of 4% of the nonionic surfactant Triton X100 at the micellar level in the derivatization reagent solution increased the signal about twofold. The reason for the improvement was credited to an increase in the actual dimer formation yield. Morphine could be determined at the 2 30 g mL 1 level in biological samples. The selectivity advantage of the uorescence detector for the determination of morphine in a urine extract was shown and urine and serum samples were analyzed with detection limits of about 10 ng. Fluorescence detection of morphine and related opiates was improved after postcolumn derivatization with alkaline hexacyanoferrate(III) and micelle formation with Triton X. 42 A detection limit of 0.2 pmol was possible for morphine after precolumn dansylation. 43 The heroin metabolite 6-acetylmorphine has been determined in urine by reversed-phase HPLC with uorescence detection after automated precolumn oxidation with hexacyanoferrate(III). 44 A detection limit of 1 ppb was reported. Reserpine, an antihypertensive agent, was detected uorimetrically after postcolumn reaction with nitrous acid and UV irradiation. 45,46 Using this UV photochemical reaction, a 20-fold increase in signal over the native uorescence of reserpine was found. Physostigmine, an acetylcholinesterase inhibitor, was separated from its degradation products and reacted with coulometrically generated bromine. 47 Electrochemical detection of unreacted bromine was inversely proportional to the amount of drug, and a detection limit of 0.5 ng could be attained. An on-line photochemical reaction detector caused decreased uorescence of ergot alkaloids, permitting the identication of these compounds in complex chromatograms. 48 A similar system has been shown to convert cannabinol into a uorescent
Table 4 Common uorescent (F) and electrochemical (E) derivatives for HPLC
No. Compounds Reagent(s)
CHO CHO , RSH
Derivative
S R N R
1.
R NH2 (F)
CN
2.
RNH2 (F)
CHO , CN CHO OCH3 OCH3
N R
3.
R3NH (F)
OCH3 O O N N CH3 CH3 H3C RSH2C O RCHN OCH3 O N N O CH3
SO3
SO3 +NHR3
4.
RSH (F)
H3C BrH2C
5.
RCOOH (E)
H2N
OH
OH
6.
Steroid (E)
O
H2NHN
NO2
NHN
NO2
HO
OH
Mobile phase reservoir
Pump I
Injector Column
UV detector
3-Way valve Waste Tee Dual-pen recorder Reaction coil Derivatization reagent reservoir Pump II Gauge
Waste Fluorescence detector
Figure 5 HPLC system for morphine using postcolumn derivatization and uorescence detection. [Reproduced with permission
from Nelson et al. 41 ]
10
derivative providing detection limits of less than 1 ng in urine. 49 Post-column photochemical derivatization has the advantage of no sample handling and without the problems of pumping a reagent or detection background from a reagent. On-line reaction of cannabinoids with Fast Blue Salt B produced colored derivatives for detection at 490 nm. 50 Pilocarpine was quaternized with p-nitrobenzyl bromide before reversed-phase HPLC separation and detection at 254 nm. 51 3.2 Amines Primarily uorescent methods have been developed for amino acids and peptides. The o-phthalaldehyde (OPA), mercaptoethanol (or other thiol) reaction (Table 4, reagent 1) has been well studied in both pre- and postcolumn modes. Specic articles describing common amino acids are cited in general references. 17 20 Other amino acids such as s-carboxymethyl-L-cysteine, 52 baclofen, 53,54 and melphalan 55 have also been derivatized with OPA. A more recent modication of this method is to use naphthalene-2,3-dicarboxyaldehyde with cyanide ion (Table 4, reagent 2). 56 Detection limits down to 200 fmol with improved stability of the derivatives were stated advantages. g-Aminobutyric acid was modied with dansyl chloride before reversed-phase HPLC and uorescent detection. 57 Detection at 360 nm was possible for N-acetylcysteine after reaction with 2,4-dinitro-11,2-Diamino-4,5-dimethoxybenzene uorobenzene. 58 was used to form a uorescent derivative of phydroxybestatin. 59 The peptide leupeptin was reacted through the guanidino moiety with benzoin to form a uorescent derivative. 60 Felypressin, a nonapeptide, was derivatized with uorescamine, providing detection limits of 0.3 ng. 61 A dinitrophthalic anhydride reaction of peptides permitted either electrochemical or absorbance detection. 62 An ion-pair detection technique has been applied to hydrophobic amino acids and peptides. 63 A variety of other uorescent methods have also been reported for specic drugs. Tranexamic acid has been detected using OPA. 64 Biogenic amines such as tyramine, tryptamine, and serotonin were reacted with OPA in a post- 65 or precolumn 66 mode. The OPA precolumn uorescent reaction has also been applied to L-buthionine-(S,R)-sulfoximine in plasma 67 and mexiletine. 68 Fluorescent derivatization with uorescamine has been applied to tocainide 69 and in the postcolumn mode for sulfapyridine. 70 Enantiomers of mexiletine have also been resolved. 71 After conversion of the drug panthenol to aminopropanol, the uorescamine reaction provided detection limits of 0.4 g. 72 The determination of debrisoquine and its hydroxy metabolites was possible by reaction through the guanidine moiety to form uorescent compounds. 73
A postcolumn photochemical reactor caused the cleavage of methotrexate to form the highly uorescent 2,4-diaminopteridine-6-carboxyaldehyde. 74 Indolethylamines were condensed with an aldehyde or a-keto acid to form uorescent carboline derivatives. 75 Thiamine (vitamin B1 ) was oxidized with hexacyanoferrate(III) in base to give the uorescent thiochrome in either the precolumn 76 or postcolumn 77 mode. Riboavin, already uorescent, and thiamine, after precolumn oxidation to thiochrome using hexacyanoferrate(III), were determined by HPLC in a variety of food powders such as our, milk, and beans. 78 Hydrazine compounds, such as isoniazid, were reacted with m-uorobenzyl chloride before reversed-phase separation and detection at 220 nm. 79 A precolumn coumarin reaction permitted uorescent detection of 5-uoro-20 -deoxyuridine. 80 Fluorescein isothiocyanate (FITC) modied a bronchodilator before HPLC uorescence analysis. 81 Some UV methods have been developed for aliphatic amines. Phenyl isothiocyanate has been used as a precolumn derivatizing agent for amino acids in conjunction with reversed-phase HPLC. Both primary and secondary amines can react (Table 5, reagent 1B) and the aromatic tag provides good retention for the smaller amino acids. 82 Both primary and secondary amines were derivatized with 9-uorenylmethyl chloroformate (FMOC) before separation by reversed-phase HPLC with UV detection at 254 nm. After SPE of a urine sample, b-phenethylamine was determined with a detection limit of 0.1 ng mL 1 . 83 Amphetamine and methamphetamine have been reacted with 4-methoxybenzoyl chloride (Table 5, reagent 1A) and other acid chlorides for subsequent UV detection. 84 A comparison of methods for amphetamines has been made. 85 The cardiotonic drug heptaminol has been derivatized with aminoazobenzene-4-isothiocyanate for UV detection at 420 nm 86 or OPA for electrochemical detection. 87 Secondary amines, such as piperazines, were converted into UV derivatives with m-toluoylacyl chloride. 88 Enantiomers of metoprolol have been separated after reaction with the chiral reagent (S)-( )-phenylethyl isocyanate. 89 The determination of isophenindamine in the presence of phenindamine tartrate has been achieved by forming charge transfer complexes using AgNO3 and reversedphase HPLC. 90 Antihistamines and other pharmaceuticals with a tertiary amine moiety have been derivatized to provide for luminescence detection. An ion-pair extraction detector using dimethoxyanthracene sulfonate (Table 4, reagent 3) has permitted the uorescent determination of tertiary amines such as bromopheniramine and chlorpheniramine, 91,92 ephedrine, 93 and hyoscyamine. 94,95 Detection limits of 200 500 pg were possible. Precolumn reaction of chlorpheniramine with
11
Table 5 Common UV derivatives for HPLC

No. 1. Compounds RNH2
(A) Cl
Reagent(s)
O C OCH3 (B) NCS (B) (A) R O C
Derivative
N H
OCH3 NHCSNHR
2.
R2NH
SO2Cl
SO2NR2
3.
R3N
(A) O H C C Cl O CH2 N H (B) C C N C O H 2 H2 O C O C
O C
NHR2
4.
RCOOH
CH2Br Br
R O C C H2 O
Br
5.
RSH
O
Cl C H2C C C2H5
Cl OCH2COOH
Cl O C RS H2C CH C2H5
Cl OCH2COOH
6.
Penicillin
R C O H N N O S CH3 CH3 COOH NaOH, HgCl2, EDTA R C O H N N H S CH3 CH3 COO
OOC
benzyl chloroformate gives a uorescent derivative with a detection limit of 0.1 ng mL 1 . 96 Antihistamines, such as diphenhydramine, were converted through the tertiary amine group into uorescent derivatives using 2-naphthyl chloroformate. 97 Aliphatic tertiary amines can be determined at the picomole level by postcolumn chemiluminescent detection, using tris(bipyridyl)ruthenium(III). 98 Antihistamines in urine have also been separated by HPLC and detected with good selectivity using this ruthenium metal complex (Figure 6a c). 99 Tamoxifen and its metabolites in human serum can be UV photochemically activated to form uorescent phenanthrenes. 100 Postcolumn UV irradiation of tamoxifen and its derivatives
caused rearrangement to a substituted phenanthrene, permitting uorescent detection with 0.1 ng mL 1 detection limits. 101 Derivatives of nicotine, cotinine, and other metabolites can be detected at 530 nm in urine after HPLC, 102 by using diethylthiobarbituric acid as a color-forming agent. Racemic phenothiazines were N-demethylated with vinyl chloroformate to form their secondary amines, which could then be reacted with (R)-(C)-1-phenylethyl isocyanate (Table 5, reagent 3), and these compounds were separated by reversed-phase HPLC. 103 Tertiary tetrahydroisoquinolines, such as diclofensine, were oxidized before photochemical conversion to uorescent
12
Pump 1

Analytical column
Pump 2 K 2Cr2O 7 (0.1 mL min 1)
Mobile phase (0.7 mL min 1)
t1
A
Detector t2
= 290 nm
Pump 3
0 (a)
12
18
24 (b)
12
18
24
NaHSO 3 (0.1 mL min 1)
Figure 7 Schematic representation of the Pt HSO3 reaction

detector for cisplatin-type compounds. [Reproduced with permission from Marsh et al. 106 ]
1
0.0025 au
0.05 au
2 3
B C
0 (c)
12
18
24
Time (min)
1 1
1 2 3
Figure 6 Chromatograms of an undiluted urine sample spiked
pheniramine, (B) 0.26 g mL browith (A) 0.15 g mL mopheniramine, and (C) 0.29 g mL 1 diphenhydramine taken with (a) UV (214 nm), (b) UV (254 nm), and (c) Ru(bpy)3 3C chemiluminescence detection. [Reproduced with permission from Holeman et al. 99 ]
Absorbance (290 nm)
Absorbance (300 nm)
0 (b)
10
isoquinolinium derivatives. After postcolumn ion-pair extraction of secoverine, peroxylate chemiluminescence detection was carried out. 105 Postcolumn detection of platinum(II) antineoplastic agents such as cisplatin is possible, as shown in Figure 7. The cisplatin-derived species are rst reacted with a oxidant such as dichromate to form an activated species, which then combines rapidly with bisulfate to give a UVabsorbing complex. Using knitted open-tubular reactors, delay times of 26 s for the dichromate reaction and 4.7 min for the bisulfate reaction were found to be optimal. Figure 8(a) and (b) shows the improvement in response over conventional UV detection using the derivatization
104
(a)
Time (min)
Time (min)
Figure 8 Comparison of UV detection (300 nm) after HPLC separation (a) and reactor response (290 nm) to derivatized platinum (b) for a mixture of cis-dichloroplatinum complexes: (1) cis-dichloro(1,2-diaminocyclohexane)platinum(II), (2) cis-dichloro(ethylenediamine)platinum(II), and (3) cis-dichlorodiammineplatinum(II). [Reproduced with permission from Marsh et al. 106 ] scheme. Detection limits of 5 10 g mL 1 were possible. Cisplatin was also determined in a plasma ultraltrate sample at the 5-ng level. 106
13
3.3 Antibiotics This class of pharmaceuticals has received major attention for both pre- and postcolumn derivatization. Fluorescent derivatization of the primary amine group of many antibiotics with OPA and a sulfhydryl compound, often mercaptoethanol, has been commonly performed (Table 4, reagent 1). Gentamicin, 107,108 penicillin V after enzymatic conversion to 6-aminopenicillanic acid, 108 penicillin N, 109 cephalosporin C, 110 cycloserine, 111 and udalanine 112 were assayed by using an OPA postcolumn reactor. Spectinomycin after postcolumn oxidation with hypochlorite could also be derivatized with OPA in a second reaction coil. 113 Reversedphase separation was employed for all these separations because the OPA reaction is carried out in an alkaline buffer solution. Precolumn reaction with OPA for gentamicin, 114 sisomicin, 115 phosphinothricin and its alanine analog 116,117 before reversed-phase separation, have all been reported. Amikacin isomers were rst adsorbed on a silica gel column before reaction with OPA; the derivatives were eluted with ethanol before separation by reversed-phase HPLC. 118 A comparison of pre- and postcolumn OPA reaction conditions for gentamycin has been made. 119 For all these OPA uorescence methods, detection limits are about 1 g mL 1 . For example, b-lactams found in microbial fermentation broths were separated on a C18 HPLC column and detected uorimetrically with excitation at 350 nm and emission at 450 nm after reaction with OPA and mercaptoethanol. Cephamycin, penicillin N, cephalosporin C, and 6-aminopenicillanic acid were separated in about 12 min at a ow rate of 1.5 mL min 1 with postcolumn conditions involving the OPA reagent at pH 12, a reaction coil of 12 m, a temperature of 90 C, and a reagent ow rate of 0.8 mL min 1 . Detection limits of less than 0.5 and 1.0 g mL 1 were achieved for penicillin N and cephalosporin C, respectively. 120 Numerous other postcolumn UV, uorimetric, and electrochemical methods have been reported for tetracycline-type antibiotics and related compounds. Formation of a mercuric mercaptide of penicillins permits UV detection at 310 nm with detection limits of 10 ng. 121 Sulfonamides in egg, milk, and meat samples have been detected at 450 nm after derivatization with pdimethylaminobenzaldehyde. 122 Monensin, narasin, and salinomycin in animal feeds have been reacted with vanillin to give products detectable at 520 nm. 123 Penicillins, such as amoxicillin, ampicillin, and others, can be separated by reversed-phase HPLC with alkaline degradation in the presence of mercuric chloride 124 (Table 5, reagent 6). Methanol promotes this reaction, forming the ring-cleaved product, which absorbs at 274 nm, giving a detection limit of 50 ng mL 1 . It was
discovered later that sodium hypochlorite could replace the mercuric chloride reagent while maintaining a 1-min hydrolysis time. 125 Application of this latter method to penicillins in biological samples has been made. 126,127 Fluorescent detection of streptomycin reacted through the guanidino groups with naphthoquinone-4-sulfonate in alkaline solution has been reported in serum samples. 128 Photothermal derivatization of ciprooxacin and its metabolites permits uorescent detection throughout a linear range of about 2 1000 ng mL 1 . 129 Photolysis with electrochemical detection of penicillins and cefoperazone has provided detection limits of about 6 ng. 130 Electrochemically generated bromine was used as an oxidizing agent for cephalosporins and their decomposition products. 131 The excess bromine was detected at 0.4 V using a glassy carbon electrode. A comparison of this method with UV detection and postcolumn uorescamine derivatization has recently been summarized. 132 Acidication of a serum sample sometimes improved recovery of cephalosporins when micellar chromatography was used with sodium dodecyl sulfate (SDS) in the mobile phase. 133 Fluorescamine has been used in an automated system for amoxycillin in biological uids. 134 Chemiluminescence detection of clindamycin phosphate using tris(bipyridyl)ruthenium(III) 135 gave detection limits of 8 ppb compared with 970 ppb using UV detection at 214 nm. A variety of reagents are also available for precolumn derivatization of antibiotics and UV or uorimetric detection. Phenacyl esters of some natural penicillins were prepared using dibromoacetophenone before reversedphase HPLC with UV detection. 136 Nitrobenzene derivatives of neomycin B and C can be separated by normal-phase HPLC and detected at 350 nm. 137 Similar methods using 1-uoro-2,4-dinitrobenzene to form 2,4-dinitrophenyl derivatives of neomycin B and C 138,139 and amikacin 140,141 have also been published. Neomycin and other aminoglycosides have been published. Neomycin and other aminoglycosides have been converted to benzoyl derivatives before UV detection at 230 nm. 142 Aminoglyosides have also been reacted with 2,4,6-trinitrobenzenesulfonic acid, permitting UV detection at 350 nm. 143 Ion-pair formation of methscopolamine bromide with an aromatic anion during reversedphase HPLC permitted UV detection. 144 The secondary amine group of spectinomycin was derivatized with 2naphthalenesulfonyl chloride (Table 5, reagent 2) before normal-phase HPLC and UV detection at 254 nm. 145 The reagent FMOC formed uorescent derivatives of natural penicillins, cephalosporins, and their precursors in biological samples. 146 b-Lactams were prederivatized with FMOC for 5 min at 20 C in borate buffer at pH 7.7 before injection of the derivatives on to a C18 reversed-phase column. Excitation and emission
14
B D 100
3.4 Barbiturates Precolumn methods primarily include reaction with various alkylating reagents. The highly reactive chlorine of N-chloromethylphthalimides permits derivatization of OH and NH functional groups to form UV-absorbing compounds. 154 Detection limits of 5 ng for phenobarbital have been reported. 154,155 2-Naphthacyl bromide forms strongly absorbing derivatives of barbiturates at 254 nm with detection in plasma or serum below the therapeutic range. 156 An online solid-phase anion-exchange extraction provided enhanced chromatographic selectivity of barbiturates in urine. 157 Postcolumn UV detection of barbiturates can be conveniently enhanced by mixing with a pH 10 borate buffer. 158,159 A wavelength shift for maximum absorbance from 220 to 240 nm provides for detection limits of about 6 g for butabarbital. The UV detection of barbiturates can be signicantly enhanced through postcolumn photochemical derivatization. Barbiturates have no signicant absorbance above 230 nm and detection in the 200 220-nm range can be complicated by the presence of interfering peaks, particularly in biological samples such as serum. Using a 25 m 0.25 mm ID Teon knitted open tubular reactor mounted around a mercury lamp, which provided an irradiation time of about 190 s, excellent signal enhancement at 270 nm was possible for barbiturates (Figure 10a and b). It was determined by HPLC that de-alkylation at the 5-position to give ethylbarbituric acid was the mechanism of the photochemical reaction. 160 3.5 Carbonyl Compounds and Carboxylic Acids Most of these methods refer to carboxylic acids; short-chain carboxylic acids were modied using pbromophenacyl bromide (Table 5, reagent 4) to form esters that could be easily detected by UV absorbance. 161 However, rst a short summary of the methods involving the reduction of quinone compounds to form uorescent hydroxy derivatives will be given. Vitamin K1 in human plasma was electrochemically reduced at 0.4 V and the uorescent derivative detected at levels as low as 25 50 pg. 162 Comparison of this method with UV and electrochemical oxidation detection systems has been made. 163 Danthron (1,8-dihydroxyanthraquinone) has been reduced with dithionite and determined by either ow injection analysis in Modane tablets or HPLC in urine. 164 A comparison of UV and uorescence chromatograms for danthron spiked in a urine sample showed the selectivity advantage of uorescence (Figure 11a and b). Dansylhydrazine formed uorescent derivatives of tetraphenylacetone isomers before separation. 165 5-Bromomethyluorescein was studied for the prederivatization of carboxylic acids for detection by either
Relative fluorescence
10
20
30
40
50
Retention time (min)

Figure 9 Reversed phase HPLC of FMOC derivatives of
cephalosporins (5 g mL 1 ) in a fermentation broth using a borate buffer acetonitrile gradient mobile phase. (A) Deacetylcephalosporin C; (B) Deacetoxycephalosporin C; (C) cephalosporin C; (D) ampicillin. [Reproduced with permission from Shah and Adlard. 146 ]
wavelengths for the uorescent detection were 260 and 313 nm, respectively. A typical chromatogram is given in Figure 9 showing the modest acetonitrile gradient until after elution of ampicillin in which the acetonitrile is taken to 100%. Detection limits were 0.01 and 0.05 g mL 1 for 6-aminopenicillanic acid and isopenicillin N, respectively. Application of the method to fermentation broths was made over a range of 0.05 100 g mL 1 . 147 A maleimide reagent gave a uorescent derivative for penicillamine. 148 An imidazole mercuric chloride reagent can convert penicillins into mercury-stabilized penicillinic acids after reaction at 50 C for 50 min prior to UV detection at 325 nm. 149 A comparison of this method with the postcolumn OPA uorescence method has been made. 110 A similar method using 1,2,4-triazole and Hg(II) for ampicillin, amoxicillin, and other antibiotics has been studied. 150 152 Two precolumn derivatization studies with UV detection were checked with real samples. Tobramycin plus impurities neamine and kanamycin and also the degradation product nebramine were derivatized with 2,4-dinitrouorobenzene for 20 min at 70 C in 0.8 mM sulfuric acid. Separation of the derivatives on a C18 column with detection at 365 nm was carried out and the stability of tobramycin in ophthalmic solutions was determined. 153
Acetonitrile (%)
15
1 2 5 4 0.04 AUFS UV 0.15% Relative fluorescence Danthron
Lamp off
0 (a)
10
0 (b)
Lamp on
10
Time (min)
Time (min)
0 2 4 6 8 (a)
0 (b)
Time (min)
Time (min)
Figure 10 Chromatogram of a standard sample of barbiturates

detected at 270 nm, (a) without and (b) with on-line photochemical reaction. (1) Aprobarbital; (2) butethal; (3) pentobarbital; (4) mephobarbital; (5) secobarbital. [Reproduced with permission from Wolf and Schmid. 106 ]
Figure 11 Separation of danthron spiked in urine (2 ng mL 1 ).

(a) UV detection (254 nm). Column, 250 4.6 mm C18 ; mobile phase, 60 : 40 methanol water at 1.2 mL min 1 . (b) Fluorescence detection (388 nm excitation, 510 nm emission). A solution of 90 mM dithionite in 2% borate buffer was pumped into the efuent at 0.4 mL min 1 . [Reproduced with permission from Miller and Danielson. 164 ]
UV absorbance or uorescence (standard and laser induced). Model analytes included prostaglandins (unsaturated carboxylic acids) and the drug cefuroxime, and also standard aliphatic and aromatic carboxylic acids. Dicarboxylic acids did not react with bromomethyluorescein, possibly owing to solubility problems in the organic reaction medium. 166 Fatty acids and prostaglandins were converted into p-hydroxanilides using p-aminophenol (Table 4, reagent 5). These hydroxy derivatives were then oxidized through electrochemical detection after reversed-phase HPLC. 167 A UV-absorbing naphthacyl ester of the prostaglandin carboprost has been formed before normal-phase HPLC. 168 Fluorescent derivatives of prostaglandins using 9-anthryldiazomethane provided detection limits of 100 pg after reversed-phase HPLC. 169 The prostaglandin arbaprotstil was derivatized with panacyl bromide before column switching using uorescent derivatization. 170,171 Prostaglandins have also been derivatized with p-(9-anthroyloxyl)phenacyl bromide. 172 Oxidation of prostaglandins to the corresponding 15-oxo derivatives using pyridinium dichromate
permitted UV detection at 228 nm and picomole detection limits. 173,174 Prostaglandins have been labeled with the uorescent reagent 9-anthoyldiazomethane through the carboxyl group. This esterication reaction can be carried out under mild conditions such as 40 C for 30 min. Five prostaglandin derivatives were separated by reversedphase HPLC with uorescent detection with excitation at 365 nm and emission at 418 nm at the 8-ng level. 175 Ascorbic acid from 5 to 800 mg L 1 was detected by chemiluminescence after postcolumn reaction with lucigenin. 176 After derivatization with 1,2-phenylenediamine both ascorbic acid and dehydroascorbic acid were separated by reversed-phase ion-pair HPLC. 177 The same two compounds were separated by reversed-phase HPLC, and the dehydroascorbic acid was reduced to ascorbic acid with dithiothreitol for UV detection at 267 nm. 178 UV detection of bis(dinitrophenyl)hydrazine derivatives of ascorbic and dehydroascorbic acid was
16
possible at 497 nm. 179 Four forms of ascorbic acid, the previous two plus isoascorbic acid and its dehydro form, were detected postcolumn using benzamidine and uorescence. 180 Total ascorbic acid was determined uorimetrically after reaction with diaminodimethoxybenzene. 181 Finally, a variety of methods directed toward pain relievers and other compounds have been published. Indomethacin formed a uorescent derivative after postcolumn alkaline hydrolysis, giving a detection limit of 5 pg. 182 Postcolumn alkaline hydrolysis has also been applied to aspirin in plasma in order to form the uorescent salicylic acid. 183 The enantiomeric composition of ibuprofen in human plasma has been resolved after prederivatization with (S)-( )-1-(naphthenyl)ethylamine 184 or ethyl chloroformate leucinamide. 185 Flunoxaprofen enantiomers have been separated after reaction with (S)( )-1-phenylethylamine. 186 Artesunic acid was determined after derivatization with o,p-nitrobenzyl-N,N 0 -diisopropylisourea by HPLC with UV detection. 187 Choline, betaine, and related compounds have been reacted to form either 40 -bromophenacyl esters 188 or p-nitrobenzyl oxines 189 to permit UV detection at 254 nm. A new acridinium sulfonylamide label permits the chemiluminescent detection of carboxylic acids such as the test compound ibuprofan. This alkylation reaction takes place in dry acetonitrile for 20 min at 50 C and separation of the derivative is possible on a C18 column with an acetonitrile water tetrahydrofuran mobile phase with the ion-pairing agent tetrabutylammonium ion. Chemiluminescence detection of the acridinium label is possible by postcolumn addition of potassium hydroxide solution. A detection limit of 3 pg of derivatized ibuprofen was found. 190 3.6 Catecholamines Precolumn uorescent derivatization of catecholamines with OPA and a thiol has been well established (Table 4, reagent 1). Norepinephrine, dopamine, and normetanephrine have been measured at the low picogram level after reversed-phase HPLC. 191,192 An electrochemical cell placed between the injector and column was used for oxidation of adrenaline and levodopa to the corresponding quinones for detection in the visible region. 193 Resolution of norephedrine enantiomers after derivatization with either acetylglycosyl isothiocyanates 194 or 4-methyl-5-phenyl-2-oxazolidone 195 has been reported. Fluorescent catecholamine derivatives have been generated using 1,2-diphenylethylenediamine, providing detection limits in the femtomole range. 196 Postcolumn uorescent reaction of norepinephrine and epinephrine with trihydroxyindole provided 1-pg detection limits. 197 This method was compared with the
OPA thiol reaction in the postcolumn mode. 198,199 The same two catecholamines can be converted into uorescent products by heating in an alkaline borate buffer after ion-exchange chromatography. 200 Dopamine isomers in serum and urine have been separated before hydrolysis and reaction with p-aminobenzoic acid to form uorescent products. 201 Glycylglycine as an alternative postcolumn reagent to glycinamide increased the rate of formation of uorescent derivatives after ion-exchange HPLC. 202,203 Catecholamines catalyze the reaction between formaldehyde and o-dintrobenzene and permit their detection at 560 nm. 204 3.7 Hydroxy Compounds Most precolumn methods focus on enhancing UV detection. Isotachysterol derivatives of vitamin D improved detection at 290 nm after normal-phase HPLC. 205 Oncolumn periodate oxidation of ephedrine sulfate, a nasal decongestant, to form benzyl alcohol has been carried out in a seven-laboratory study. 206 Indirect photometric detection using n-heptyl p-aminobenzoate in the mobile phase and a C18 column has been applied to menthol. 207 Enantiomers of propranolol and 4-hydroxypropranolol were formed upon derivatization with (C)-1-phenylethyl isocyanate 208 or (C)tetraacetyl b-D-glucopyranosyl isothiocyanate 209,210 and separated by reversed-phase HPLC. Enantiomers of derivatized or underivatized propranolol have been separated. 211 Diastereomeric derivatization of 1-methyl3-pyrolidinol 212 and proxyphylline 213 for UV detection has been accomplished. Qinghaosu, an antimalaria component in a Chinese herb, can be converted into a UV-absorbing compound using a sodium hydroxide solution before HPLC separation. 214 A derivative of qinghaosu has been esteried with diacetyldihydrouorescein before HPLC and UV detection. 215 Cholic acid and its derivatives have been labeled with 1-anthroylnitrile before HPLC and uorescent detection at the femtomole level. 216 Trospium has been converted into the corresponding spiro alcohol before uorescent derivatization with benoxaprofen chloride and reversed-phase HPLC. 217 Derivatization can be an effective method to facilitate the chiral separation of pharmaceuticals. Nadolol diastereomers were derivatized with (R)-( )-1-(1naphthyl)ethyl isothiocyanate to chiral urea derivatives through the secondary amine group by reaction for 5 min at 45 C. Separation of the RS, SR, RR, and SS diastereomers was straightforward on a C18 column using a 60 : 40 water acetonitrile mobile phase (Figure 12a c). Fluorescence detection with excitation at 285 nm and emission at 340 nm provided selectivity for the determination of nadolol in plasma samples. The limit of detection
17
200 180 160 140 120 100 80 60 40 20 (a) 0 200 180 160 140 120 100 80 60 40 20 (b) 0 200 180 160 140 120 100 80 60 40 20 0 0 (c) 5 10 15 20 25
Cyclodextrins in biological uids were assayed by negative colorimetric detection after postcolumn complexation with phenolphthalein. 223 Vitamin B6 (pyridoxine) has been postcolumn derivatized with 2,6dibromoquinone-4-chlorimide to form a colored product with a maximum absorbance at 650 nm. 224 3.8 Steroids Esteried estrogens were converted into their free phenolic forms by acid hydrolysis, and separation could be achieved by reversed-phase HPLC. 225 The chromatographic behavior of estrogen carbonyls, such as equilin, equilin, and estrone, was improved upon reduction to the 17-a-hydroxy compounds using sodium borohydride. 226 Prederivatization of conjugated estrogens with dansyl chloride permitted uorescent detection after separation by normal-phase HPLC (Table 4). 227 Ketosteroids, such as androsterone, dehydroepiandrosterone, epiandosterone, and etiocholanolone, were derivatized with p-nitrophenylhydrazine (Table 4, reaction 6) and detected electrochemically at levels as low as 200 pg. 228 Isonicotinoyl hydrazine was used to tag steroids, such as corticosterone, to permit uorescent detection after normal-phase HPLC. 229 Further work using this method with reversed-phase HPLC and optimized reaction conditions permitted detection limits as low as 7 10 ng. 230,231 17-Oxosteroids were labelled with dansylhydrazine and then chromatographed on a silica column with subsequent uorescent detection from 60 to 100 pg. 232 A similar method for this class of compounds using 3-chloroformyl-7-methoxycoumarin and reversedphase HPLC has been published. 233 Hydroxysteroids were derivatized with anthroylnitrile, showing the feasibility of the reaction for uorescent detection after HPLC. 234 Six corticosteroids were separated within 25 min and reacted with a lucigenin KOH solution for chemiluminescence detection. 235 The reactive site is an a-hydroxycarbonyl group, and detection limits of 2 pg were comparable to those with precolumn uorescent methods. Corticosteroids were also postcolumn derivatized with glycinamide in the presence of hexacyanoferrateIII) before uorimetric detection at the 5-ng level. 236 Digoxin, a widely used cardiac glycoside, has been detected uorimetrically by postcolumn reaction with a solution of ascorbic acid, peroxide, and HCl. 237,238 A detection limit less than 1 ng was attained. Digoxin and its metabolites, such as digoxigenin, were derivatized with 1-naphthoyl chloride before separation and uorescent detection 239 and 5-ng amounts could be determined in urine or feces. A similar method using 4-nitrobenzoyl chloride has been reported. 240 Dihydrodigoxin, a major metabolite of digoxin, has been
2 3 4
1 2 3 4
30
35
40
45
Time (min)
Figure 12 Typical chromatograms of (a) blank control dog

plasma, (b) plasma spiked with 50 ng mL 1 of each diasteromer and (c) plasma obtained 2 h after oral administration of 1 mg kg 1 of racemic nadolol. Peaks: 1 D SR -nadolol; 2 D RS -nadolol; 3 D RR -nadolol; 4 D S, S -nadolol. [Reproduced with permission from Hoshino et al. 218 ]
was 2.5 ng mL 1 , representing 50 pg injected. This chiral derivatization method has been used for the determination of enantiomers of other b-blocker drugs. 218 A few of the post-column approaches are outlined below. Using a photochemical reactor, diethylstilbestrol (DES) has been converted into a uorescent derivative and determined at the low parts per billion level in biological matrixes. 219,220 Anabolic stilbenes, such as DES, have been measured in urine by HPLC and an offline chemiluminescence immunochemical assay. 221 The antihypertensive agent fenoldopam was formed using a postcolumn enzyme reactor from the corresponding glucuronide, and detected electrochemically. 222
18
separated from digoxin with dual UV and postcolumn uorescent derivatization detection. 241 The photoreduction of anthroquinone-2,6-disulfonate to the uorescent 9,10-dihydroxyanthracene-2,6-disulfonate occurs only in the presence of hydrogen atom-donating substrates, such as alcohols, aldehydes, amines, ethers, and saccharides. Using a knitted Teon reactor, cardiac glycosides, such as digoxin, digoxigenin, and diginatin, have been determined in the range 50 500 ng. 242 3.9 Sulfur Compounds Most methods employ a precolumn reaction to form uorescent or UV derivatives. Dansylaziridine 243 was used to determine cysteine and other thiols. Biological thiols, such as glutathione and ergothioneine, were reacted with monobromobimane (Table 4, reagent 4) before ion-exchange chromatography. 244 This method was also adopted for the determination of dithiols, such as 2,3-dimercaptopropane-1-sulfonic acid, in urine at levels down to 10 pmol. 245 Fluorescent derivatization of 2-mercaptopropionylglycine using N-(7dimethylamino-4-methyl-3-coumarinyl)maleimide could probably be extended to other thiol compounds. 246 Resolution of the optical isomers of diltiazem was accomplished using UV detection after derivatization with optically pure 2-naphthylsulfonyl-2-pyrollidinecarbonyl chloride. 247 Three N-substituted maleimides were compared for precolumn derivatization of thiols such as penicillamine before electrochemical detection at the picogram level. 248 Ethacrynic acid (Table 5, reagent 5) forms UV-detectable derivatives of thiols, such as captopril, N-acetyl-L-cysteine, and mercaptopropionylglycine with detection limits down to 0.5 g mL 1 . 249 Postcolumn derivatization of thioethers, such as ampicillin and ranitidine, was accomplished using on-line generated bromine and electrochemical detection of the excess bromine. 250 An analogous method has been reported for phenothiazines, such as thioridazine; however, the resultant products from the oxidation reaction with bromine are detected uorimetrically. 251 Photochemical activation of phenothiazines and demoxepam in 2 min and subsequent uorescence detection provided detection limits a factor of 10 better than UV detection. 252 Applications of photochemical reactors to a variety of pharmaceuticals, such as phenothiazines, have been summarized. 253,254 A precolumn derivatization method for phenothiazine involves desulfurization with Raney nickel to produce diphenylamine, which is electrochemically active. 255 A detection limit of 10 pg was found. The reagent pyrenemaleimide provided derivatization of N-acetylcysteine with a detection limit of 10 pmol. 256 Postcolumn complexation of disulram and two of its metabolites using Cu2C allowed colorimetric
Waste
AS
CO
P(A)
MP (A)
VAL 1 MP (B)
P(B) C1
C2
DET (A) VAL 2 MP (C) DET (B) C3
Waste
P(C)
Waste
Figure 13 Schematic diagram of the HPLC system. P(A),

P(B), and P(C) D pumps; AS D autosampler; VAL-1 and VAL-2 D six-port valves 1 and 2; C0 D cleanup column; Cl, C2, and C3 D columns 1, 2, and 3; DET(A) and DET B D UV detectors A and B; MP(A), MP(B), and MP C D mobile phases A, B, and C. The solid and dotted lines in the six-port valves indicate valve positions 0 and 1, respectively. [Reproduced with permission from Funakoshi et al. 259 ]
detection at 435 nm with detection limits in the low parts per billion range. 257 Quenched peroxalate chemiluminescence has been employed using immobilized reagents in a postcolumn reactor for thioridazin, sulforidazine, and methimazole. 258 On-line precolumn derivatization to improve reproducibility has been demonstrated for the determination of busulfan in human serum. A schematic diagram of the HPLC system is shown in Figure 13. All three columns C1, C2, and C3 were C18 types with different lengths and/or diameters. After extraction of busulfan from serum, the residue reconstituted in water was injected on to C1, where it was derivatized with diethyldithiocarbamate (DCC) in mobile phase A for 5 min. Then the busulfan DCC derivative from C1 was backushed on to C2, where it was separated using mobile phase B. Because the background interference from mostly excess DCC overlapped completely the busulfan DCC derivative peak, valve 2 was used to take a heart cut of this peak of interest and inject it on to C3. The resulting separation using mobile phase C is shown in Figure 14(a) and (b). Detection was at 278 nm and the lower limit of quantitation in serum was 10 ng mL 1 . The total time for the derivatization and separation was 33 min. 259
19
Busul fan/16.150
the detectability. Since UV/VIS and LIF detection are the most common detection methods used in CE for pharmaceutical analysis, we shall discuss the most recent work related to this area.
0.0006 a.u. 24
4.1 Derivatization for Ultraviolet Detection Although most organic compounds have UV/VIS chromophores, derivatization is still important, and sometimes necessary, for UV/VIS detection in CE. Very often, UV/VIS detection of the chromophores does not give a satisfactory response owing to the very short light pathlength (50 100 m) in CE. For example, the anticancer drug prospidin is a piperazinium derivative with chloroxypropyl groups but no UV/VIS chromophore. Derivatization with DCC at 37 C for 90 min in a basic solution, with loss of HCl, generates a product that absorbs light at 254 nm. Separation of the desired product from excess derivatizing agent is possible in 10 min by CE with a detection limit of 1 ng L 1 . 265 Rimantadine is a synthetic analog of amantadine. Both are antiviral agents used for prophylaxis and treatment of inuenza A. Because rimantadine is almost transparent in the UV/VIS range, either indirect detection or derivatization has to be used to detect this compound. The indirect detection method used 5 mM 4-methylbenzylamine in 1 : 4 methanol water as absorbing background electrolyte for detection at 210 nm. The derivatization method used rimantadine to react with 1,2-naphthoquinone-4-sulfonic acid in alkaline medium. CE determination of the derivative at 280 nm was performed in an uncoated capillary (44 cm 75 m ID) using a 40 mM tetraborate buffer at pH 9.2. The detection limits were 0.1 and 2 mg L 1 for indirect detection and derivatization methods, respectively. The methods were used to determine rimantadine in pharmaceutical products and for dissolution testing of Flumadin tablets. 266 Most of the naturally occurring amino acids do not have proper UV/VIS chromophores for CE analysis. Derivatization is a necessity for the determination of amino acids at reasonable concentrations. By using dansyl chloride to derivatize the amino acids, the enantiomeric forms of novel depsipeptide antitumor antibiotic BMY-45012, and its analogs, were determined by CE with UV/VIS detection. 267 The compounds were subjected to total hydrolysis in a vacuum hydrolysis tube with 6 M HCl at 110 C for 24 h. The hydrolyzed residue (about 7.8 mg) was dissolved in water acetonitrile solvent. For subsequent derivatization, the solution was further diluted to about 1.7 mg mL 1 with water. Dansyl chloride dissolved in acetonitrile (3.0 mg mL 1 ) was utilized for the derivatization of standard native amino acids and those present in the hydrolyzate. A 50-L sample solution was mixed with 50 L of dansyl chloride solution and an aliquot of
Inj.
0 (a)
16
24
Inj. 0 (b)
16
Time (min)
Time (min)
Figure 14 Typical chromatograms of (a) drug-free serum and

(b) serum spiked with busulfan (100 ng mL 1 ) obtained with columns Cl, C2, and C3. The arrow indicates the retention time of the busulfan derivative. [Reproduced with permission from Funakoshi et al. 259 ]
4 CAPILLARY ELECTROPHORESIS
CE has developed into a versatile separation technique well suited for the determination of pharmaceutical and biomedical samples. The major advantages of CE over chromatographic separation techniques are its simplicity and efciency. However, CE suffers from poor sensitivity in detection owing to the extremely small sample volume involved. One way to improve the sensitivity of CE detection is to derivatize the analytes with more favorable detection characteristics by adding either an ultraviolet/visible (UV/VIS) chromophore or a uorophore. Derivatization in conjuction with GC and HPLC is a well-developed eld. With the advent of CE, derivatization chemistry previously developed for HPLC is often applicable to CE. 260 At least one comprehensive review specically focused on the derivatization in CE has appeared. 261 Several other review articles dealt with more specic topics, such as postcolumn derivatization, 262 diastereomer derivatization, 263 and dyes used to derivatize molecules for CE with laserinduced uorescence (LIF) detection in drug analysis. 264 The pros and cons of the various modes of derivatization and a detailed comparison of this topic between LC and CE can be found in the literature. 261 Instead of listing the derivatization reagents suited for labeling amino, aldehyde, keto, carboxyl, hydroxyl, and sulfhydryl groups, we intend to focus on the purpose of derivatization in CE from a practical point of view, i.e. enhancing
20
borate buffer at pH 9.08. The mixture was held at room temperature for 2 h and used directly for injection. The aminocychitol antibiotic amikacin can be derivatized with 1-methoxycarbonylindolizine-3,5-dicarbaldehyde at room temperature for 15 min before determination in plasma by micellar CE with UV detection at 280 nm and standard uorescence detection (excitation at 414 nm, emission at 482 nm). 268 However, there was only a twofold gain in sensitivity on switching from UV to uorescence detection of the amikacin derivative. If laser excitation can be used (as discussed in the next section), the gain in sensitivity should be signicantly higher. 4.2 Derivatization for Fluorescence Detection In principle, almost any detection mode can be combined with a derivatization procedure. In practice, uorescence monitoring is favored in most cases. The principles of uorescence and the prerequisites for a good uorophore, including the potential of using diode lasers in combination with a labeling procedure, have been reviewed previously. 261 LIF detection is now commercially available for CE, and more and more industrial scientists are using LIF detection to detect trace amounts of analytes in complex matrices. LIF detection has several advantages. First, it offers excellent sensitivity; under optimized conditions, a level of 10 10 M is feasible. 269 This is critical to pharmaceutical applications because sensitivity is often the most important parameter to consider when analyzing biological samples. Second, LIF offers a certain selectivity because, unlike UV absorbance, LIF selectively detects compounds that are uorescent at certain wavelengths, and most organic compounds do not uoresce. On the other hand, the fact that most organic compounds do not uoresce is also a major limitation of LIF detection. Since the available excitation wavelengths of various lasers are limited, derivatization is often required when using CE with LIF detection. The following examples demonstrate the applicability of derivatization in CE with LIF detection for pharmaceutical analysis. Captopril, an antihypertensive agent, was derivatized through the thiol group with a dicarbocyamine label to give a uorescent derivative with a wavelength maximum at 675 nm. 270 This wavelength was compatible with a semiconductor laser at 670 nm, which was used for LIF detection in conjunction with CE. Using a methanol borate running electrolyte, CE also separated other labeled thiols such as cysteine and reduced glutathione from derivatized captopril (Figure 15). The detection limit of 2.5 10 8 M for captopril is limited by dilution due to the derivatization reaction. Amines can easily be derivatized with FITC isomer I and analyzed by CE using alkaline buffers with or
H3C
C CH2
+
H3C C
CH3
X O
(CH2 )4
N CH3
SO3 CY5.3a.IA: X = NH H HS
CH3 N
O C HO O Captopril 1
7 5
6 4 3
40
30
20
10
Migration time (min)

Figure 15 Electropherogram of a number of thiols labeled
with CY5.3a.IA: (1) unreacted label; (2) captopril; (3) DL-homocysteine; (4) L-cysteine; (5) 3-mercaptopropionic acid; (6) 2-mercaptoacetic acid; (7) reduced glutathione. [Reproduced with permission from Couderc et al. 264 ]
without dodecyl sulfate micelles. Ramseier et al. reported the determination of FITC-derivatized amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine and b-phenylethylamine in human urine by CE using chip-based and fused-silica capillary instrumentation with LIF detection. 271 The results obtained via direct labeling of fortied urine were compared with those generated after FITC labeling of urinary extracts that were prepared by SPE. Using 5 mL of urine with a spiked amine to FITC ratio of 1 : 250, the SPE extract had a sensitivity of 200 ng mL 1 urine. That value is relevant for toxicology drug screening and conrmation. In contrast, with direct labeling of 10 L of urine that had been alkalinized and diluted for derivatization, the limit of identication was 10 g mL 1 , a value that is too high for practical purposes. Compared with fused-silica capillaries, electrophoresis in microstructures is shown to provide faster separation and higher efciencies without loss of accuracy and precision.
Relative peak height
21
4.3 Special Applications 4.3.1 Chiral Conrmation With proper derivatization, CE has been used for the chiral separation of enantiomeric forms of derivatized amino acids. Liu et al. reported the determination of enantiomeric forms of amino acids derived from the novel depsipeptide antitumor antibiotic BMY-45012, and its analogs, the proposed structure of which is shown as (1) [reproduced with permission from Liu et al. 267 ]. Amino acids were analyzed by complete hydrolysis and the hydrolyzate was derivatized with either dansyl chloride for UV absorbance detection or FITC for LIF detection in CE. For uorescence detection, the uorogenic reagent FITC was dissolved in acetone (0.01 M) as a stock solution. Amino acids were derivatized with FITC-derivatizing solution (5.0 10 4 M) under basic conditions (borate buffer, pH 9.08). The reaction was allowed to proceed for 2 4 h in the dark at room temperature and then stored at 20 C prior to use. Both a metal chelate chiral CE method and a cyclodextrin-mediated host guest interaction approach in micellar electrokinetic chromatography (MEKC) with LIF detection conrmed the presence of several chiral amino acids, such as D-serine and L-b-hydroxyl-Nmethylvaline, and the nonchiral amino acid sarcosine in the proposed structure. These methodologies provide a quick and sensitive approach for the determination of amino acid racemization in pharmaceutical natural products and have proven to be useful for structural elucidation renement. 4.3.2 Oligosaccharide Determination The derivatization of the pseudo-oligosaccharide acarbose (2) and its main metabolite (3) with
7-aminonaphthalene-1,3-disulfonic acid (ANDS) (Scheme 1) in human urine allowed the on-column LIF detection of the pseudo-oligosaccharides in human urine in the nanomolar range. 272 Before derivatization, 0.5mL samples of urine or spiked urine were evaporated to dryness. A 100-L volume of 0.08 M ANDS solution in acetic acid water (3 : 17 v/v) and a 50-L volume of 0.9 M NaCNBH3 solution in dimethyl sulfoxide (DMSO) were added to each sample residue. The reagent solutions were freshly prepared before derivatization. The reaction tubes were vortex mixed and then incubated overnight at 40 C. The efcient separation of these derivatives by CE using 100 mM triethylammonium phosphate buffer at pH 1.5 allowed the quantitation of acarbose and (3) in human urine after application of 300 mg of acarbose. 4.3.3 Derivatization of Protein Samples With the increased interest in the therapeutic use of the recombinant monoclonal antibody (rMAb) technique, a generic analytical approach for the analysis of size-based rMAb variants is desired. CE/LIF with proper derivatization has been shown to be promising as a general method. The rMAb can be derivatized with a neutral uorophore, e.g. 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRSE) for CE/LIF analysis. 273 Samples containing 2.5 mg of rMAb were buffer exchanged into 800 L of 0.1 M sodium hydrogencarbonate (pH 8.3) using a NAP-5 column. A 10-L volume of 5-TAMRSE (1.4 mg mL 1 ) dissolved in DMSO was then added to 190 L of rMAb solution and the resultant mixture incubated for 2 h at 30 C. After incubation, 190 L of the antibody dye conjugate was loaded on to a second NAP-5 column and collected in 700 L of 0.1 M sodium hydrogencarbonate (pH 8.3). The labeled sample, after incubating with
CH3O
N N O O O H3C HO H N
O N N O
H N
O N O CH OR1 3 O N N
CH3 CH3 OH N CH3 O O N H O N N OCH3
N CH3 CH3
R2O CH3 O N O
O N H O
(1)
Compound BMY45012 Analog-G439B Analog-G451 Analog-G435 Analog-G439A R1 CH(CH3)2 CH2CH3 C(CH3)3 C(CH3)3 CH(CH3)2 R2 CH(CH3)2 CH2CH3 C(CH3)3 CH(CH3)2 CH3
22
CH2OH HO HO CH3 HO O HN CH2OH HO HO O O CH2OH HO HO O O OH HO HO
(2)
CH2OH HO HO CH3 HO O HN CH2OH HO HO O O OH HO HO
of the bulk manufacture of a protein pharmaceutical and in providing a size-based separation of product-related variants and also nonproduct impurities. The CE/LIF with derivatization demonstrated comparable resolution and sensitivity to silver-stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) but offered the advantages of enhanced precision and robustness, speed, ease of use, and on-line detection. CE was used for the study of palmitoyl derivatization of interferon-a2b (p-IFN-a). 274 The derivative was prepared by covalent attachment of the fatty acid to lysine residues in the protein through a reaction with Nhydroxysuccinimide palmitate ester (Scheme 2). The rst step involved the preparation of an intermediate (NHSP), which in turn was reacted with IFN-a. CE was able to study the effect of reaction time and reagent/protein ratio. 4.4 Derivatization Modes
(3)
CH2OH O CH2OH OH O HO C O HO H
SO3H H2N SO3H
O HO
OH HO Carbohydrate
In general, derivatization in CE can be accomplished by either pre- or postcolumn derivatization. The precolumn derivatization is similar to methods used for derivatization in HPLC. Since CE has a much superior separation power, the interference from the derivatization by-products may be less of a problem than in LC. However, CE is very sensitive to the ionic strength of the sample. Additional sample preparation may be needed if the mixture in the derivatization reaction has a high ionic strength.
O R COOH + HO N
DCC
O HO
CH2OH OH C N HO H Schiff base
SO3H
O R O N + DCU
SO3H
NaCNCH3
O Active ester (isolated) + NH2 IFN

pH 7.2
CH2OH OH O CH2 NH HO HO
SO3H
O R C N IFN H
SO3H
Scheme 2 Synthetic process for the fatty acrylation of IFN-a.

R COOH palmitic acid. [Reproduced with permission from Foldvari et al. 274 ]
Scheme 1 Reaction of (2) and (3) for derivatization with ANDS by reductive amination. [Reproduced with permission from Rethfeld and Blaschke. 272 ] SDS, can be separated by CE using a hydrophilic polymer as a sieving matrix. Using these precolumn labeling conditions, the detection of rMAb at a low-nanomolar concentration (9 ng mL 1 ) is obtained with no apparent decrease in resolution or changes to the distribution of rMAb analyte species in comparison with an unlabeled sample. This assay can be used in monitoring consistency
Postcolumn detection strategies have also been developed for CE. An overview of the advantages and limitations of postcolumn derivatization for CE can be found in a recent paper. 261 The details about the instrumental developments and applications of post-column derivatization in CE can also be found in the literature. 262 Various systems to merge the reagent solution with the separation medium have been developed, including coaxial capillary reactors, gap reactors, and free-solution or end-column systems. For all reactor types, the geometry of the system,
23
and the method used to propel the reaction mixture (by pressure or by voltage) appear to be critical to preserve the separation efciency. To minimize peak broadening, careful design in terms of connecting a reagent capillary to form a tee is necessary. Because of the small (50 75 m) ID of the capillaries used, normal mixing through a diffusion process is sufcient. The most frequently applied reactors are (1) co-axial, (2) gap, (3) free-solution, and (4) sheath ow. With proper design, plate numbers of over 100 000 could be realized. The strict requirements on the reaction rate in postcolumn derivatization in CE limit the number of different reagents that have been used. For LIF detection, mainly OPA and its naphthalene analog have been used. With careful instrument design, the detection limits of postcolumn derivatization can be comparable to those of precolumn derivatization.
FFAP FITC FMOC GC GC/MS HFAA HFBI HMDS HPLC LC LIF MBTFA MEKC MS MSTFA
5 CONCLUSION
It can be argued that the advances made in MS detection for GC, HPLC, and CE have diminished the importance of chemical derivatization for these techniques. However, chemical derivatization of drugs is critical for GC because these samples, which often contain multiple polar substituents, are simply not volatile or thermally stable. MS detection for HPLC and CE is still expensive and requires considerable operator expertise. Chemical derivatization with HPLC to permit uorescence detection is certainly one of the more desirable methods for routine use to solve selectivity and detectability problems for drug samples not amenable to GC. As the cost of laser technology comes down, the same statement will eventually be true for CE also.
MTBSTFA OPA PFAA PFAI PFB-Br QUAT rMAb SDS SDS/PAGE SPE TBDMS TFAA TFAI TMAH TMCS TMS TMSI UV UV/VIS 5-TAMRSE
ABBREVIATIONS AND ACRONYMS

ANDS BSA BSTFA CBD CE DCC DES DMES DMFDA DMIPS DMSO EC 7-Aminonaphthalene-1,3-disulfonic Acid N,O-Bis(trimethylsilyl)acetamide N,O-Bis(trimethylsilyl)triuoroacetamide Cannabidiol Capillary Electrophoresis Diethyldithiocarbamate Diethylstilbestrol Dimethylethylsilyl N,N-Dimethylformamide Dimethylacetal Dimethylisopropylsilyl Dimethyl Sulfoxide Electron Capture
Free Fatty Acid Phase Fluorescein Isothiocyanate 9-Fluorenylmethyl Chloroformate Gas Chromatography Gas Chromatography/Mass Spectrometry Heptauorobutyric Anhydride Heptauorobutylimidazole Hexamethyldisilazane High-performance Liquid Chromatography Liquid Chromatography Laser-induced Fluorescence N-Methylbistriuoroacetamide Micellar Electrokinetic Chromatography Mass Spectrometry N-Methyl-N-trimethylsilyltriuoroacetamide N-Methyl-N-(t-butyldimethylsilyl)triuoroacetamide o-Phthalaldehyde Pentauoropropionic Anhydride Pentauoropropionylimidazole Pentauorobenzyl Bromide Quaternary Ammonium Salt Recombinant Monoclonal Antibody Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Solid-phase Extraction t-Butyldimethylsilyl Triuoroacetic Anhydride Triuoroacetylimidazole Trimethylanilinium Hydroxide Trimethylchlorosilane Trimethylsilyl N-Trimethylsilylimidazole Ultraviolet Ultraviolet/Visible 5-Carboxytetramethylrhodamine Succinimidyl Ester
RELATED ARTICLES
Pharmaceuticals and Drugs (Volume 8) Eluent Additives and the Optimization of Highperformance Liquid Chromatography Procedures Gas and Liquid Chromatography, Column Selection for, in Drug Analysis Planar Chromatography in Pharmaceutical Analysis Solid-phase Extraction and Clean-up Procedures in Pharmaceutical Analysis
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Gas Chromatography (Volume 12) Gas Chromatography: Introduction Column Technology in Gas Chromatography Sample Preparation for Gas Chromatography Liquid Chromatography (Volume 13) Liquid Chromatography: Introduction Capillary Electrophoresis Column Theory and Resolution in Liquid Chromatography Normal-phase Liquid Chromatography Reversed Phase Liquid Chromatography
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Chemical Reagents and Derivatization Procedures in Drug Analysis

Neil D. Danielson, Patricia A. Gallagher, and James J. Bao

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

Chemical Reagents and Derivatization Procedures in Drug Analysis

PHARMACEUTICALS AND DRUGS

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

Table 1 Common GC derivatives alkylation

R COOH R NH2 R OH R SH R COOH R OH

CH3 N+ OH H3C CH3 (TMAH)

DMFDA, N,N-dimethylformamide dimethylacetal; PFB-Br, pentauorobenzyl bromide; TMAH, trimethylanilinium hydroxide.

PHARMACEUTICALS AND DRUGS

R=CF3 (TFAI), C2F5 (PFAI), or C3H7 (HFBI)

O R NH C R O R O C R O O C R R2 O R1 N C R O R NH C CF3 R2 O R1 N C CF3 O R O C CF3 O R S C CF3

O O R C O C R R=CF3 (TFAA), C2F5 (PFAA), or C3H7 (HFAA)

O O F3C C N C CF3 CH3 (MBTFA)

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

PHARMACEUTICALS AND DRUGS

CH3 CH3 H3C Si O C N Si CH3 CH3 CH3 CH3 (BSA)

R OH R NH2 R1 NH R2 O R C NH2 R COOH

R O Si(CH3)3 R N Si(CH3)3 Si(CH3)3 R1 N Si(CH3)3 R2 O Si(CH3)3 R C N Si(CH3)3 O R C O Si(CH3)3

CH3 CH3 H3C Si O C N Si CH3 CH3 CF3 CH3 (BSTFA)

Same as BSA, leaving group

more volatile than that of BSA Same as BSA, leaving group

CH3 O F3C C N Si CH3 H3C CH3 (MSTFA)

CH3 N Si CH3 CH3 (TMSI)

No reaction with amines

HMDS, hexamethyldisilazane; BSA, N,O-bis(trimethylsilyl)acetamide; BSTFA, N,O-bis (trimethylsilyl)triuoroacetamide; MSTFA, N-methyl-N-trimethylsilyltriuoroacetamide.

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

(1) CH2 CH3 (2) H C 2H 5

Figure 3 Single ion monitoring recordings of extracts from

PHARMACEUTICALS AND DRUGS

3 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

CHO , CN CHO OCH3 OCH3

Mobile phase reservoir

Waste Fluorescence detector

PHARMACEUTICALS AND DRUGS

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

Table 5 Common UV derivatives for HPLC

(A) O H C C Cl O CH2 N H (B) C C N C O H 2 H2 O C O C

PHARMACEUTICALS AND DRUGS

Pump 2 K 2Cr2O 7 (0.1 mL min 1)

Mobile phase (0.7 mL min 1)

NaHSO 3 (0.1 mL min 1)

Figure 7 Schematic representation of the Pt HSO3 reaction

Figure 6 Chromatograms of an undiluted urine sample spiked

Absorbance (290 nm)

Absorbance (300 nm)

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

PHARMACEUTICALS AND DRUGS

Retention time (min)

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

1 2 5 4 0.04 AUFS UV 0.15% Relative fluorescence Danthron

Figure 10 Chromatogram of a standard sample of barbiturates

Figure 11 Separation of danthron spiked in urine (2 ng mL 1 ).

PHARMACEUTICALS AND DRUGS

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS

Figure 12 Typical chromatograms of (a) blank control dog

PHARMACEUTICALS AND DRUGS

DET (A) VAL 2 MP (C) DET (B) C3

Figure 13 Schematic diagram of the HPLC system. P(A),

CHEMICAL REAGENTS AND DERIVATIZATION PROCEDURES IN DRUG ANALYSIS