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To cite this article: Susanna Eerola, Iciar Otegui, Leena Saari & Aldo Rizzo (1998): Application of liquid
chromatographyatmospheric pressure chemical ionization mass spectrometry and tandem mass spectrometry to the
determination of volatile nitrosamines in dry sausages, Food Additives and Contaminants, 15:3, 270-279
To link to this article: http://dx.doi.org/10.1080/02652039809374641
nitrosodimethylamine; NDPA, ./V-nitrosodipropylamine; NPIP, N-nitrosopiperidine; NPYR, Af-nitrosopyrrolidine; SIM, selected ion monitoring; TEA,
thermo energy analyser, TIC, total ion current.
Introduction
(Received 24 March 1997, revised 27 June 1997; accepted 27
June 1997)
nitrosamines,
dry
sausages,
111
Sample preparation
Samples were purchased from retail markets and
frozen at -20C before analysis. The principles of
the procedure for sample preparation and clean-up
were described by Pensabene et al. (1992). Ten g of
sample was weighted into a 250 screw-cap Erlenmeyer
flask, and 300 mg propyl gallate was added directly to
the sample. The reagent blank sample was prepared
similarly. The sample was spiked with 100 ul internal
standard (NDPA-solution 1 ug/ml). Fifteen g anhydrous Na2SO4, 15 g Celite and 50 ml DCM was
added. The flask was shaken for 15min. The sample
was poured through folded filter paper into a round
250 ml evaporator flask. The Erlenmeyer flask was
rinsed with 3 x 10 ml DCM and the rinsings were
added to the sample through the same filter. The
DCM extract was concentrated to ca 5 ml in a rotavapor apparatus without heating. After evaporation
1 ml of H-pentane was added to the flask.
A solid phase column was prepared by adding 4-0 g
silica between polyethylene frits into an empty reservoir and rinsing the column with 10 ml each of DCM
and -pentane. The sample from the evaporator flask
was added over the silica layer and the flask was
rinsed with an addition 1 ml of w-pentane and then
with 5 ml of washing solvent, 25% DCM in npentane. After the sample, together with the rinsing
solvents, had passed through the column, the sample
272
S. Eerola et al.
HPLC conditions
The LC-system was a Hewlett Packard HP 1090
Series M LC (Waldbronn, Germany), including an
HP 1090 gradient module, binary DR5 pumps, an
autoinjector system with a 200(al loop and a diodearray detector. A Spherisorb ODS2 (5 urn)
124 x 4 mm column (Merck, Darmstadt, Germany)
with a C-18 precolumn was operated with a waterMeOH gradient at 0-5ml/min increasing from 30%
MeOH to 90% MeOH in 9 min. The analyte signals
were monitored at 230-4 and 254-4 nm in the initial
studies for optimizing the resolution of analytes and
the flow rate of eluents.
Standards and samples were prepared in 50% MeOH.
Injection volumes used were 50 and 100 ul. The run
time was 12 min, with 8 min post time before the next
run. The first injection was with 50% MeOH and
possible trace effects were monitored with eluent runs
between samples and standards. Standard dilutions
used for calibration were in the range of 100-0-1 ng/
ml.
LC-APCI-MS conditions
A Finnigan MAT LCQMT instrument (San Jose,
USA) was employed, equipped with a Finnigan atmospheric pressure chemical ionization/LC interface.
The instrument was operated in the positive ionization model. Optimization of APCI conditions was
performed with standard solutions of NDMA, NPYR
and NDPA (lOug/ml) using flow injections into
water-MeOH (50:50) at a flow-rate of 0-5 ml/min.
The source parameters were: corona current 5 mA,
Statistics
Calculations of correlation and regression analysis
between the contents of biogenic amines and NPYR
were performed using the Microsoft EXCEL 4.0
Analysis Tools.
MH +
CID energy
Product ion
(m/z)
(%)
(m/z)
75-2
101-1
103-1
115-1
131-1
8-0
90
100
90
55-2
75-2
691
89-2
273
(b)
5.76
(C)
8.53
(d)
6.34
7.27
9.94
(e)
1fl.45
9.21
\
7
8
Time (min)
TO
Vi
12
1*3
14
15
Figure 1. TIC of the sample blank for spiked sample. See figure 4 for the elution order. The concentration of NPYR was
3-1 \ig/kg, other analytes were below the limits of determination: NDMA < 0-5 \ig/kg, NDEA andNPIP < 0-2 \i-gjkg. The
recovery of NDPA was 28%.
274
5. Eerola et al.
2.87
100^
(a)
50i
0J5 1.Q2 1.6.3 2.28
3.94
Figure 2. TIC of the spiked sample (10\ig/kg). See figure 4 for the elution order. The recoveries were 59% for
98% for NPYR, 62% for NDEA, 60% for NPIP and 35% for NDPA.
2.21
10O;
NDMA,
(a)
.35
0.62
1.46
A
i
O1
100^
3.54
(b)
5(>i
4.57
5.36
100^
6.00
50T
4.81
(c)
JL
100^
6.68
(d)
78g
50z
9.87
IOO7
(e)
50;
8.99
w
11
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I I I I I I I I I I I I I
/ V ig43
1 1 i 1 1 1 1 1 1 1 1 1 1 i
7
8
Time (min)
1D
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
11
12
13
14
15
Figure 3. TIC of the sample containing nitrosamines below the limit of determinations. See figure 4 for the elution order.
The recovery of NDPA was 42%.
275
100^
(a)
5oi
0,29
1.15
2.2.5
3.99
loo;
(b)
50^
5.72
10&7
50^
5.03
6.72
10O;
(d)
50i
- 19
8.12 8.74
9.89
10O;
50z
9.Q1_ 9-59
7
8
Time (min)
*l9
10
11
12
13
lU
i's
Figure 4. Total ion current (TIC) of the standard mixture containing 2ng of each compound: (a) NDMA, (b) NPYR,
(c) NDEA, (d) NPIP and (e) NDPA.
Contents of volatile nitrosamines in fermented
sausages
Using the method described, four different nitrosamines were determined in 27 dry sausage samples.
The results are presented in table 2. It is interesting to
note that NPYR was detected in concentrations higher than the other nitrosamines. NPYR is frequently
found in cooked or fried bacon at concentrations up
to 200ug/kg (Walker 1990) whereas many studies do
not report detectable amouns of NPYR in dry
sausages (table 3). However, high levels of NPYR
have been confirmed in fermented sausages. Some of
these findings were considered to be caused by spice-
Positive
samples
Mean
Range
NDMA
NPYR
NDEA
NPIP
15/27
23/27
2/27
3/27
1-2
7
0-30
0-31
0-51-3-0
2-1-22
0-24-0-36
0-24-0-35
The levels of NDMA, NDEA and NPIP are comparable to those reported in other surveys. Two samples
276
S. Eerola et al.
Sample
Salami
Metwurst
Salami
Smoked pepperone
Dry sausage
Dry sausage
Fermented sausage
Salami
Salami
Sausage
Salami
Sausage
Sausage
No. of
samples
NDMA
range
NPYR
range
NDEA
range
NPIP
range
Reference
25
9
64
1
21
16
1
10
4
194
12
14
71
nd-10
nd-35
nd
nd-3-5
nd-tr
0-2
nd-6-3
0-59-7-76
nd-7-4
01-0-9
0-5-1-8
nd-9-3
nd-105
nd-105
nd
nd
nd-tr
nd
nd
nd
nd-14
nd
nd
nd
nd
nd-3-9
nd-O-38
nd-60
nd-25
nd
nd
nd
nd-29-5
nd-4-04
nd-6-7
nd-0-5
Warthesen et al. (1975), putrescine caused the formation of NPYR in buffered system when treated with
nitrite at 22C for 6 days. The proposed pathway
involves a cyclization resulting in secondary amines
and further nitrosation to nitroso compounds. Nitroso compounds are thus not only formed in heated
conditions but also during ripening of sausages by
reaction betweeen residual nitrite and amines formed
during the fermentation process.
However, it has been demonstrated that the limiting
factor in volatile nitrosamine formation is not the
availability of amine precursors but the amount of
nitrosating agent applied (Mclntyre and Scanlan
1993). Residual nitrite and nitrate were not analysed
in the present study, but the reference values for
Finnish salami samples are 3-5-14-0 mg/kg nitrite
and 7-272 mg/kg nitrate (Penttila et al. 1990). In the
study of Dethmers et al. (1975), residual nitrite levels
of 7-14 mg/kg did not lead to the formation of
detectable amounts of nitrosamines in Thuringer
sausages whereas Groenen et al. (1982) detected
0-1-0-7 ug/kg NDMA from smoked sausages after
2h incubation at pH3 with added 5-7mg/l nitrite.
The fact is that there is no alternative to nitrite as a
preservative agent, and it is also necessary for colour
and flavour formation in sausage products. Much is
already known about the inhibition of production of
nitrosamines, e.g. by ascorbic acid. The manufacture
of fermented sausages by taking into account the
inhibition of biogenic amines may also affect the
formation of nitrosamines.
277
20
ii
15
'.
NPYR ug/kg
10
i1
'
100
200
300
400
500
600
Putrescine mg/kg
25
20
15
1
NPYR ug/kg
10
]1
>
200
400
600
800
1000
1200
1400
Figure 5. The correlation figures between the concentrations of NPYR and putrescine, and between the concentrations of
NPYR and total amines in dry sausages. Biogenic amine results were obtained from the report of Eerola et al. (1997).
Conclusion
In conclusion, a new method for analysing volatile
nitrosamines is presented. The procedure is rapid and
reliable, with low interference from the sample matrix. Application of the LC-APCI-MS technique is
easy to perform and could be further developed to
analyse other iV-nitroso compounds, non-volatile
278
S. Eerola et al.
Acknowledgements
This study was supported by the Academy of Finland, the Finnish Centre for International Mobility
(CIMO) and the Spanish Foreign Affairs Ministry.
under simulated gastric condition. N-nitroso Compounds: Occurrence and Biological Effects, IARC Scientific Publication No.
41, edited by H. Bartsch, M. Castegnaro and I. K. Griciute
(Lyon: International Agency for Research on Cancer), pp. 321331.
HAVERY, D. C., KLINE, D. A., MILETTA, E. M., JOE, F. L., and F A -
ZIO, T., 1976, Survey of food products for voltile N-nitrosamines. Journal of the Association of Official Analytical Chemists,
59, 540-546.
HOTCHKISS, J. H., LIBBEY, J. F . , BARBOUR, J. F . , and SCANLAN,
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