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Application of liquid chromatographyatmospheric

pressure chemical ionization mass spectrometry and
tandem mass spectrometry to the determination of
volatile nitrosamines in dry sausages
a

Susanna Eerola , Iciar Otegui , Leena Saari & Aldo Rizzo

Department of Chemistry, National Veterinary and Food Research Institute, PO Box

368 (Hmeentie 57), Helsinki, 00231, Finland
b

Department of Nutrition and Food Science, Faculty of Pharmacy, University of Pais

Vasco, Paseo de la Universidad 6, Vitoria, 01006, Spain
Version of record first published: 10 Jan 2009

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
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Food Additives and Contaminants, 1998, Vol. 15, No. 3, 270-279

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Application of liquid chromatography-atmospheric pressure

chemical ionization mass spectrometry and tandem mass
spectrometry to the determination of volatile nitrosamines
in dry sausages
Susanna Eerola, Iciar Otegui, Leena Saari and
Aldo Rizzo
National Veterinary and Food Research Institute, Department of
Chemistry, PO Box 368 (Hmeentie 57), 00231 Helsinki, Finland;
University of Pais Vasco, Department of Nutrition and Food
Science, Faculty of Pharmacy, Paseo de la Universidad 6, 01006
Vitoria, Spain

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)

A high-performance liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric

method was developed for the determination of volatile
nitrosamines in dry sausages. Tandem mass spectrometry was applied for the detection of N-nitrosopyrrolidine, N-nitrosodiethylamine and N-nitrosopiperidine.
N-nitrosomethylamine was detected by using the selected ion monitoring mode. The occurrence of the four
different nitrosamines was monitored in 27 dry sausage
samples and a correlation was observed between Nnitrosopyrrolidine and biogenic amines. Nitroso compounds are thus not only formed in heated conditions
but formation can also occur during ripening of dry
sausages by reaction between residual nitrite and
amines formed during the fermentation process.
Keywords: volatile
HPLC-APCI-MS

nitrosamines,

dry

sausages,

Abbreviations: APCI, atmospheric pressure chemical

ionization; CID, collision-induced dissociation;
DCM, dichloromethane; GC, gas chromatography;
HPLC, high performance liquid chromatography;
LC, liquid chromatography; MeOH, methanol; MS,
mass spectrometry; MS/MS, tandem mass spectrometry; NDEA, N-nitrosodiethylamine; NDMA, N-

Nitrosamine compounds have been found to occur in

meat products preserved with nitrite. Among the
simple volatile compounds of this type detected are
NDMA, NPIP and NPYR, which have been shown
to be carcinogenic in laboratory animals (Preussmann
and Stewart 1984). In meat products, it has been
suggested that NDMA is derived from creatine
through its breakdown to sarcosine, and possible
precursors of NPYR and NPIP include proline and
lysine, respectively. Secondary amines formed from
these precursors, dimethylamine, pyrrolidine and
piperidine, are known to be converted into their Nnitroso derivatives by the reaction with nitrous acid
(Walters 1992). However, some primary amines frequently found in fermented sausages and in stored
meat, e.g. tyramine, putrescine and cadaverine, have
been proposed to be potential precursors for nitrosamine formation (Lijinsky and Epstein 1970, Bills et al.
1973, Warthesen et al. 1975). These findings have
been reported as one of the risk factors of biogenic
amines (Askar and Treptow 1986).
Either aqueous (Sen et al. 1987, Shahidi et al. 1995) or
mineral oil distillation methods (Hotchkiss et al. 1980,
Oliveira et al. 1995) have been employed for sample
clean-up before chromatographic separation. The
distillation method can be replaced with solvent
extraction (Bellec et al. 1996), solid phase extraction
(Pensabene et al. 1992) or super critical fluid extraction (Fiddler and Pensabene 1996).

0265-203X/98 $12.00 1998 Taylor & Francis Ltd

Determination of volatile nitrosamines in dry sausages

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The trace analysis of volatile nitrosamines in food

samples is most commonly performed with GC interfaced to a nitrosamine-specific chemiluminescence
detector (TEA). The principle of this detection mode
was published by Fine et al. (1975). Recently, the
detection limit for the GC-TEA method was determined to be 1-Oug/kg (Pensabene and Fiddler 1994)
and confirmation was obtained with MS (10 ng injection was needed for confirmation of spectra.
HPLC-TEA methods are applicable to the determination of both volatile and non-volatile nitrosamines
(Massey et al. 1982, Riihl and Reush 1985, Conboy
and Hotchkiss 1989). These techniques require the use
of cold traps to remove organic solvent vapours prior
to detection and they are unworkable with aqueous
mobile phases because of freeze-up and blockage of
cold traps. HPLC methods for indirect determination
of nitrosamines after acidic denitrosation using precolumn (Wang et al. 1992) and postcolumn derivatization (Fu et al. 1993) with dansyl chloride have been
reported. In these methods, sample pretreatment must
include the elimination of interfering amines. Another
precolumn derivatization method using quinolizinocoumarin has been reported to detect nitrosamine
traces at concentrations lower than 0-1 ug/kg (Zhou
et al. 1994). However, no pretreatment procedure for
food samples has been described. Recently, a reversed
phase HPLC and post-column photohydrolysis-colorimetric method was introduced as an alternative to
the GC-TEA technique (Bellec et al. 1996).
MS provides a valuable tool for the identification and
quantitation of trace levels of nitrosamines in food
samples. Hitherto, GC-MS has been more widely
used than the HPLC-MS technique mainly because
of the commercial availability of advanced instrumentation. In this study an HPLC-APCI-MS technique
with solid phase extraction sample pretreatment is
described. APCI is a soft ionization technique, in
which a high voltage creates a corona discharge that
forms chemical ionization reagent ions in the gas
phase. These reagent ions then react with gas phase
analyte molecules to form analyte ions. This proposed
method has been applied to assess the quantities of
NDMA, NPYR, NDEA and NPIP in dry sausages.
The samples which were chosen for this study had
been previously analysed for their biogenic amine
content. The aim was to determine whether there is
a relationship between biogenic amines and nitrosamine formation.

111

Materials and methods

Chemicals and materials
Chemicals and solvents were of analytical or HPLCgrade. Ultra-pure water was obtained from a Milli-Q
UF water purification system (Millipore Co., Bedford, USA). NPYR, NPIP, NDEA, NDPA were
obtained from Sigma (St Louis, USA) and NDMA
from Supelco Inc. (Beliefonte, USA). A mixed solution was prepared by dissolving 1 ug/ml of each
compound in MeOH.
Empty reservoirs (25 ml) and polyethylene frits
(20 um pore size) used for solid-phase extraction were
purchased from Varian (Harbor City, USA) and
syringe-tip filter units (0-45 um) for sample filtration
were purchased from Millipore (Millipore Co., Bedford, USA).

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.

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was washed with 25 ml of washing solvent and the

analytes were eluted with 30 ml of eluent containing
30% diethylether in DCM directly to a 100 ml
evaporator flask. The sample eluent was evaporated
in a rotavapor apparatus at 25C and the residue was
dissolved in lml 50% MeOH. The sample was
transferred to a 3-5 ml screw-cap vial and the evaporator flask was rinsed with an addition 1 ml of 50%
MeOH which was added to the vial. The vial was
thoroughly shaken with a vortex mixer and possible
fat residue and silica particles were separated from the
aqueous phase by centrifugation at 2000 rpm for
5 min. The sample was filtered into a LC vial.

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,

source octapole 1 offset voltage 3-50 to 4-24 V,

source octapole 2 offset voltage -7-50 to -11-00V,
vaporizer temperature 450C, capillary temperature
150C, capillary voltage 3-00 to 4-00 V. Nitrogen was
used as sheath gas at 49-67 (arbitrary units) and
helium as the auxiliary gas at 48 (arbitrary units).
Total microscans were 3 s and the maximum injection
time was 300 ms.
NDMA was detected using MH + as an SIM detection mode because it has a product ion smaller than
50 m/z. CID product ion spectra were determined for
other analytes under the conditions described. Product ions monitored were losses of the nitroso group
or the alkyl group from the parent compound.
Product ion spectra obtained from MH+ were recorded in MS/MS mode and collision offset voltages
were adjusted to induce total dissociation to the main
product ion with its highest intensity. MH + , CID
energies and product ions are listed in table 1. The
MS was programmed to monitor product ions in
separate time-segments for each analyte according
to retention times and resolutions.

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.

Results and discussion

Sample preparation
The modifications made to the original method
(Pensabene et al. 1992) were the lower amounts of
Table 1. MH + ions, CID-energies and product ions in
MS-detection
Compound
NDMA
NPYR
NDEA
NPIP
NDPA

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

Determination of volatile nitrosamines in dry sausages

NDMA was detected in the SIM mode because of its

low molecular weight and the other analytes by MS/
MS technique. Figure 4 shows a typical TIC of a
standard mixture solution. The peak areas of these
compounds in the chromatograms showed a linear
relationship (r > 0-995) within the range of 0-1-20 ng
injected. Quantification was made with an external
standard method, although the internal standard
NDPA was added to the samples. The recovery of
internal standard was not comparable to that of the
analytes and the recovery factor was not used for
correction. The recoveries from spiked samples also
were not used for correction because most of the
positive findings were NPYR, which had almost
100% mean recovery.

Downloaded by [University of Arizona] at 10:38 04 August 2012

organic solvent used and the concentration steps with

vacuum evaporation. Propyl gallate was added prior
to sample pretreatment in order to avoid artefact
nitrosation. The artefact formation of nitrosamines
was studied in the original method and none were
detected.
Recovery experiments were carried out by addition of
mixture solution to the sample at the level of 10 ug/kg
before the extraction step. The recoveries were
NDMA 53% 9; NPYR 96% 17; NDEA
59% 12; NPIP 60% 10 and NDPA 25% 6
(n = 10). The recovery trial was carried out in conjunction with each analysis series and the values
presented above were averaged from different analyses. Examples of blank and spiked samples are
presented in figures 1 and 2. Figure 3 shows a TIC
of negative sample.

The reproducibilities of quantification as relative

standard deviation (RSD) for five replicate analyses
of 5ng injected and 1-25 ng injected were 3-2 and
2-1% for NDMA, 6-6 and 8-1% for NPYR, 8-9 and
8-7% for NDEA, 4-7 and 4-3% for NPIP and 2-7 and
2-2% for NDPA, respectively. The limits of the
determinations were defined from signal to noise ratio
of standard dilutions and they were 0-5 ug/kg
(NDMA), 1-0 ug/kg (NPYR) and 0-2 ug/kg (NDEA,
NPIP).

Identification, calibration and precision

Identification of analytes was based on retention
times and product ions in the optimized conditions.

(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

Downloaded by [University of Arizona] at 10:38 04 August 2012

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

Determination of volatile nitrosamines in dry sausages

2.86

100^

(a)

5oi
0,29

1.15

2.2.5

3.99

loo;

(b)

50^
5.72

10&7

50^
5.03
6.72

Downloaded by [University of Arizona] at 10:38 04 August 2012

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

curing mixtures containing nitrosamines. NPYR and

NPIP also occurred at high concentrations in spices in
the study of Tricker et al. (1991).

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-

When the total ./V-nitroso compounds of different

cured meat products were studied, fermented salami
was found to contain higher levels of these compounds than ham or bacon samples (Fiddler et al.
1995). The report cited did not present data on
individual nitrosamines but it was also suggested that
50% of the total 7V-nitroso compound content was
associated with an insoluble protein fraction in the
adipose tissue, known to be responsible for NPYR in
fried bacon (Tricker et al. 1985). This may indicate
that the NPYR findings in this study were not
artifacts formed during sample pretreatment. The
reagent blank samples, which were performed regularly during the survey, and eluent runs between
samples in each analysis time also failed to show
any determinable trace effects. Furthermore, Fiddler
and Pensabene (1996) suggested that non-distillation
methods are more effective in isolating NPYR than
conventional distillation methods mostly used in
sample pretreatment.

Table 2. The occurrence of volatile nitrosamines in

studied dry sausages (\i.gjkgj.
Compound

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.

Table 3. Volatile nitrosamine levels in fermented sausages (\i-gjkg).

Downloaded by [University of Arizona] at 10:38 04 August 2012

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

Panalaks et al. 1973

Sen et al. 1973
Panalaks et al. 1974
Havery et al. 1976
Kotter et al. 1976
Anonymous 1980
Osterdahl and Olsson 1983
Yamamoto et al. 1984
Gavinelli et al. 1988
Song and Hu 1988
Penttila' et al. 1990
Tricker et al. 1991
Biaudet et al. 1994

nd = not detected, tr= l-5ng/kg, = not determined.

contained NDEA and three samples contained NPIP.

The amounts reported are low when considered as
single doses related to dry sausage consumption. A
level of 5-10ug/kg has been reported as a tolerable
dose of low molecular weight nitrosamines (Preussmann 1973). Therefore, this level of concentration
must be detectable in chemical analyses. There are
no safe levels of these compounds, however, and
nitrosamines are known to be more effective as
carcinogens in experimental animals when applied in
repeated small doses than when presented in larger
single applications (Walters 1992).

Correlation to biogenic amines

When comparing the results of this study with previously studied biogenic amine contents in the same
sausages (Eerola et al. 1997), there are apparent
correlations between putrescine and NPYR (r =
0-72) as well as between total amines (histamine +
tyramine + cadaverine + putrescine) and NPYR (r =
0-74) (figure 5). Two samples contained >5ug/kg of
NPYR without high levels of amines, and no relationship with biogenic amines was found in these samples.
In connection with the prevention of nitrosamine
formation, only the reduction of nitrite additions
and total prohibition of nitrate have been extensively
discussed. The present study also demonstrates the
importance of inhibiting the formation of possible
precursors, biogenic amines. In the earlier studies of

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

Determination of volatile nitrosamines in dry sausages

25 T

20
ii

15

'.

NPYR ug/kg

10

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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

Tyramine + Histamine + Putrescine + Cadaverine mg/kg

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

nitrosamines and nitrosamides from food samples.

The occurrence of four different nitrosamines was
reported and a comparison was made between NPYR
and biogenic amines. It is proposed that the occurrence of biogenic amines in dry sausages is not only a
sign of deteriorated quality and of the presence of
agents which can cause acute food intoxications, but
that they are also potential precursors of carcinogenic
nitrosamines.

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,

References

Downloaded by [University of Arizona] at 10:38 04 August 2012

ANONYMOUS, 1980, A survey of nitrosamines in sausages and drycured meat products. Food Technology, July, 45-53, 103.
ASKAR, A., and TREPTOW, H., 1986, Amine als Vorlufer von

R. A., 1980, Combination of a GC-TEA and GC-MS-data

system for the mg/kg estimation and confirmation of volatile Nnitrosamines in foods. Nitroso Compounds: IARC Scientific
Publications Analysis, Formation and Occurrence, No. 31, edited
by E. A. Walker, L. Griciute, M. Castegnaro and M. Borzonyi
(Lyon: International Agency for Research on Cancer), pp. 361373.
KOTTER, L., FISCHER, A., and SCHMIDT, H., 1976, Zum Vorkommen

Nitrosaminen. Biogene Amine in Lebensmitteln: Vorkommen,

Bedeutung und Bestimmung (Stuttgart, Germany: Eugen Ulmer
GmbH & Co.), pp. 107-116.

Von Nitrosaminen in Fleischerzeugnissen und Untersuchungen

an schnittfesten Rohwrsten bei unterschiedlichen Zustzen.
Die Fleischwirtschaft, 7, 997-1007.

BELLEC, G., CAUVIN, J. M., SALAUN, M. C., L E CALVE, K., DREANO,

Y., GOUEROU, H., MENEZ, J. F., and BERTHOU, F., 1996,

LIJINSKY, W., and EPSTEIN, S. S., 1970, Nitrosamines as environ-

Analysis of N-nitrosamines by high-performance liquid chromatography with post-column photohydrolysis and colorimetric detection. Journal of Chromatography A, 727, 83-92.

MCINTYRE, T., and SCANLAN, R. A., 1993, Nitrosamines produced in

BIAUDET, H., MAVELLE, T., and DEBRY, G., 1994, Mean daily intake

MASSEY, R. C., CREWS, C., MCWEENY, D. J., and KNOWLES, M. E.,

of N-nitrosodimethylamine from foods and beverages in France

in 1987-1992. Food and Chemical Toxicology, 32, 417-421.

1982, Analysis of a model ionic nitrosamine by microbore highperformance liquid chromatography using a thermal energy
analyser chemiluminescence detector. Journal of Chromatography, 236, 527-529.

BILLS, D. D., HILDRUM, K. I., SCANLAN, R. A., and LIBBEY, L. M.,

1973, Potential precursors of N-nitrosopyrrolidine in bacon and

other fried foods. Journal of Agricultural and Food Chemistry,
21, 876-877.
CONBOY, J. J., and HOTCHKISS, J. H., 1989, Photolythic interface for

high-performance liquid chromatography chemiluminescence

detection of non-volatile nitroso compounds. Analyst, 144,
155-159.
DETHMERS, A. E., ROCK, H., FAZIO, T., and JOHNSTON, R. W., 1975,

mental carcinogens. Nature, 225, 21-23.

selected foods under extreme nitrosation conditions. Journal of
Agricultural and Food Chemistry, 41, 101-102.

OLIVEIRA, C. P., GLORIA, M. B. A., BARBOUR, J. F., and SCANLAN,

R. A., 1995, Nitrate, nitrite, and volatile nitrosamines in wheycontaining food products. Journal of Agricultural and Food
Chemistry, 43, 967-969.
STERDAHL, B.-G., and OLSSON, I.-M., 1983, Flyktiga nitrosaminer i
bacon, sidflsk och andra charkuterivaror. Vr Fda, 35, 440450.

Effect of added sodium nitrite and sodium nitrate on sensory

quality and nitrosamine formation in Thuringer sausage.
Journal of Food Science, 40, 491-495.

PANALAKS, T., IYENGAR, J. R., and SEN, N . P., 1973, Nitrate, nitrite

EEROLA, S., ROIG-SAGUS, A.-X., and HIRVI, T., 1997, Biogenic

PANALAKS, T., IYENGAR, J. R., DONALDSON, B. A., MILES, W. R., and

amines in Finnish dry sausages. Journal of the Science of Food

and Agriculture (in press).
FIDDLER, W., and PENSABENE, J. W., 1996, Supercritical fluid extraction of volatile N-nitrosamines in fried bacon and its drippings:
method comparison. Journal of the Associaton of Official
Analytical Chemists, 19, 895-901.
FIDDLER, W., PENSABENE, J. W., DOERR, R. C., and GATES, R. A.,

1995, Determination of extractable, apparent total N-nitroso

compounds in cured-meat products. Journal of the Association
of Official Analytical Chemists International, 78, 1435-1439.
FINE, D. J., LIEB, D., and RUFEH, F . , 1975, Principle of the operation

of the thermal energy analyser for trace analysis of volatile and

non-volatile N-nitrosamines. Journal of Chromatography, 24,
980-984.
Fu, C., Xu, H., and WANG, Z., 1993, Sensitive assay system for
nitrosamines utilizing high-performance liquid chromatography
with peroxyoxalate chemiluminescence detection. Journal of
Chromatography, 634, 221-227.
GAVINELLI, M., FANELLI, R., BONFANTI, M., DAVOLI, E., and AIROL-

DI, L., 1988, Volatile nitrosamines in foods and beverages:

preliminary survey of the Italian market. Bulletin of Environmental Contamination and Toxicology, 40, 41-46.
GROENEN, P. J., LUTEN, J. B., D H O N T , J. H., D E COCK-BETHBEDER,
M. W., PRINS, L. A., and VRREKEN, J. W., 1982, Formation

of volatile JV-nitrosamines from food products, especially fish,

and dimethylnitrosamine in cured meat products. Journal of the

Association of Official Analytical Chemists, 56, 621-625.
SEN, N. P., 1974, Further survey of cured meat products for
volatile N-nitrosamines. Journal of the Association of Official
Analytical Chemists, 57, 806-812.
PENSABENE, J. W., and FIDDLER, W., 1994, Gas chromatographic/

thermal energy analyzer method for N-nitrosodibenzylamine in

hams processed in elastic rubber nettings. Journal of the
Association of Official Analytical Chemists International, 11,
981-984.
PENSABENE, J. W., FIDDLER, W., and GATES, R. A., 1992, Solid-phase

extraction method for volatile JV-nitrosamines in hams processed with elastic rubber netting. Journal of the Association of
Official Analytical Chemists International, 75, 438-442.
PENTTILA, P.-L., RSNEN, L., and KIMPPA, S., 1990, Nitrate, nitrite

and N-nitroso compounds in Finnish foods and estimate of

dietary intake. Zeitschrift fr Lebensmittel-Untersuchung und
Forschung, 190, 336-340.
PREUSSMAN, R., 1973, Toxicity of nitrite and N-nitroso compounds.
Proceedings of the International Symposium on Nitrite in Meat
Products, Zeist, the Netherlands, September 10-14, edited by B.
Krol and B. J. Tinberg (Wageningen: Centre for Agricultural
Publishing and Documentation), pp. 217-225.
PREUSSMANN, R., and STEWART, S. W., 1984, N-nitrosocarcinogens.

ACS-monographs, 182, 643-823.

RHL, C., and REUSCH, J., 1985, Analysis of volatile nitrosamines by
microbore high-performance liquid chromatography and ther-

Determination of volatile nitrosamines in dry sausages

mal energy analyser detection. Journal of Chromatography, 328,
362-366.
SEN, N. P., MILES, W. F., DONALDSON, B., PANALAKS, T., and IYEN-

GAR, J. R., 1973, Formation of nitrosamines in a meat curing

mixture. Nature, 245, 104-105.
SEN, N . P., BADDOO, P. A., and SEAMAN, S. W.,

1987, Volatile

nitrosamines in cured meats packaged in elastic rubber nettings.

Journal of Agricultural and Food Chemistry, 35, 346-350.
SHAHIDI, F., SYNOWIECKI, J., and SEN, N . , 1995, N-Nitrosamines

in

nitrite-cured chicken-seal salami. Journal of Food Production,

58, 4 4 6 - 4 8 .
SONG, P. J., and Hu, J. F., 1988, N-nitrosamines in Chinese foods.
Food and Chemical Toxicology, 26, 205-208.

Downloaded by [University of Arizona] at 10:38 04 August 2012

TRICKER, A. R., PERKINS, M. J., MASSEY, R. C., and MCWEENEY,

279

WALKER, R., 1990, Nitrate, nitrite and N-nitrosocompounds: a review

of the occurrence in food and diet and the toxicological
implications. Food Additives and Contaminants, 7, 717-768.
WALTERS, C. L., 1992, Reactions of nitrate and nitrite in foods with
special reference to the determination of N-nitroso compounds.
Food Additives and Contaminants, 9, 441-447.
WANG, Z., X U , H., and CHENGGUANG, F., 1992, Sensitive fluorescence

detection of some nitrosamines by precolumn derivatization

with dansyl chloride and high-performance liquid chromatography. Journal of Chromatography, 589, 349-352.
WARTHESEN, J. J., SCANLAN, R. A., BILLS, D . D . , and LIBBEY, L. M.,

1975, Formation of heterocyclic N-nitrosamines from the

reaction of nitrite and selected primary diamines and amino
acids. Journal of Agricultural and Food Chemistry, 23, 898-902.

D. J., 1985, Some nitrosamino acids in bacon adipose tissue

and their contribution to the total N-nitroso compound concentration. Zeitschrift fiir Lebensmittel-Untersuchung und
Forschung, 180, 379-383.

YAMAMOTO, M., IWATA, R., ISHIWA, H., YAMADA, T., and TANI-

TRICKER, A. R., PFUNDSTEIN, B., THEOBALD, E., PREUSSMANN, R.,

ZHOU, Z. Y., DAUPHIN, C., PROGNON, P., and HAMON, M., 1994,

and SPIEGELHANDLER, B., 1991, Mean daily intake of volatile

N-nitrosamines in West-Germany in 1989-1990. Food and
Chemical Toxicology, 29, 729-732.

Luminarin 9: a new fluorescent reagent for the precolumn derivatization of main volatile and non-volatile N-nitrosamines in
RP-HPLC. Chromatographia, 39, 185-191.

MURA, A., 1984, Determination of volatile nitrosamine levels

in foods and estimation of their daily intake in Japan. Food and
Chemical Toxicology, 22, 61-64.