EUROPEAN COMMISSION

[JOINT
RESEARCH
CENTRE

Environment Institute
Consumer Protection & Food Unit
21020 - Ispra (VA) Italy

A Review on Analytical Methods to Determine

Nitrosamines in Food
D. Wiltschko, M. Lipp, E. Anklam

1998

EUR 18053 EN

EUROPEAN COMMISSION

IJOINT
RESEARCH
I CENTRE
Environment Institute
Consumer Protection & Food Unit
21020 - Ispra (VA) Italy

A Review on Analytical Methods to Determine

Nitrosamines in Food
D. Wiltschko, M. Lipp, E. Anklam

1998

EUR 18053 EN

LEGAL NOTICE
Neither the European Commission nor any person
acting on behalf of the Commission is responsible for the use which
might be made of the following information.

EUR 18053 EN
European Commission, 1998
Printed in Italy

Content
page
EXECUTIVE SUMMARY
1.

INTRODUCTION

2.

GENERAL ASPECTS

2.1.

Chemistry, Substances

2.2.

Formation in food

2.2.1.

2.3.

General formation

2.2.2. In vivo

Occurrence in food

2.3.1.

General

2.3.2. Examples of N-nitrosamine occurrence in food

2.4.

Intake

11

2.5.

Toxicity and Precautions

12

3.

ANALYTICAL METHODS

15

3.1.

Classification: volatile and non-volatile N-nitrosamines

3.1.1.

3.2.
3.3.

Volatile N-nitrosamines

15
16

3.1.2. Non volatile N-nitrosamines

17

3.1.3.

18

Total N-nitrosamines

Clean up/Extraction methods

Identification and Quantification

19
20

3.3.1.

Gas Chromatography

20

3.3.2.

High Performance Liquid Chromatography

28

3.5.

Mass spectrometry for confirmation

31

4.

CONCLUSION

32

5.

ACKNOWLEDGEMENT

32

6.

GLOSSARY

33

7.

REFERENCES

34

EXECUTIVE SUMMARY
Nitrosamines have shown the potential

of being carcinogenic

to some

animal species and are likely to be related to human cancer. Therefore, there is a
strong consumer concern about the occurrence of nitrosamines in food products.
Nitrosamines can be found in processed food especially in those where nitrate and
nitrite are used (e.g. in meat and fish products) or special techniques are used for
processing (e.g. drying, baking, food packaging). They can be classified into volatile
and non-volatile nitrosamines leading to different technical approaches for analysis.
The main analytical

methods to determine nitrosamines in food are based on

chromatographic qualification

and quantification

after extraction from the food

matrices.
In this review paper, examples of nitrosamine occurrence and its toxic
effects are listed in addition to dietary intake values over a period of the last 20
years.

The

various

analytical

techniques

(extraction

from

the

food,

gas

chromatography (GC) and high performance liquid chromatography (HPLC) ) are

described in detail.
A special emphasis is taken on the analytical conditions in respect to various
food matrices and on the use of various detectors. While most of the common
chromatographic methods use the thermal energy analyser (TEA) as a selective
detector,

mass spectrometry

(MS)

is the most satisfactory

method

for

the

confirmation of nitrosamines. Especially GC-MS has been applied extensively for

this purpose. Although the GC-TEA approach is a highly sensitive detection method
for nitrosamines, analysis by HPLC offers some advantages: volatile and non-volatile
nitrosamines can be determined equally and other sensitive detectors such as
fluorescence, chemiluminescence or electrochemical detecors can be applied.
Very often the lack of information on the identity of nitrosamines has to be
encountered. For this reason, several attempts have been made to determine the
amount of 'total' nitroso groups that will estimate the sum of the volatile and non
volatile nitrosamine contents of food products.

1. INTRODUCTION
There

is an

increasing

concern

about

the

occurrence

of

N-nitroso

compounds (NOCs) in food, because these compounds have been demonstrated to

be carcinogenic in several species of animals and are therefore likely to be related
to human cancer [Takatsuki et al., 1990].
This, together with the fact that humans are exposed to trace amounts of Nnitroso compounds

from several sources has initiated

the research on their

occurrence, formation and biological activity. NOCs can also be formed inside the
body and diet can influence the quantity and type of the endogenic formed NOCs
[Hotchkiss, 1988],
Research carried out during the past 30 years has provided

a better

understanding of the mechanism of formation of these compounds, and changes in

processing have reduced the levels of nitrosamines in certain foodstuffs [Sen et al.,
1990].
The toxicity of some NOCs has been manifested even at ug/kg (ppb) levels.
Therefore,

sensitive and

selective

methods

for the

determination

of these

nitrosamines at trace levels are essential [Kataoka et al., 1995].

This review reports the analytical methods used for the determination
NOCs in foodstuffs presently and during the last years.

of

2. GENERAL ASPECTS
2.1. Chemistry, Substances
N-Nitrosocompounds can be described by the general formula:
R, R2 N-N=0
They can be classified by two main groups: nitrosamines and nitrosamides.
In nitrosamines, Ri and R2 can be either alkyl or aryl groups or may form part of a
cyclic ring. Ri and R2 may also contain other substituents such as hydroxy and
carboxy groups. Chemical formulae and abbreviations of nitrosamines detected in
food are shown in Figure 1.
In nitrosamides, the groups may be alkyl or aryl, one of which contains an
acyl functional group. Hence the proper generic name for these should be N-acyl-Nnitroso compounds. An example of an nitrosamide is N-nitroso-N-methylurea (NMU),
its structure is shown in Figure 2. Thus far, no nitrosamides have been detected in
foods, but they might be formed in vivo in the stomach due to the interaction of
foodborne amides and ingested or salivary nitrite [Sen et al., 1992],

Figure 2:

N-Nitroso-N-methylurea (NMU)

NH2

\
C=0
/
N-N=0
/
CH3

Figurei : Some N-Nitrosocompounds analyze d in food

Me

/'

NON

OH

\cOOH
'N

NO

N-Nitrosodimethylamine
(NDMA)
e
e
N-Nitroscpip ridin
(NPIP)

NO
N-Nitroso-4-hydroxyproline(NHPRO)

Me
NO
Et
N-Nitrosthylmethylamine
(NEMA)

NO

M
\

\.

N-Nitrosothiazolidine
(NT HZ)

HOCH2

/ - C O OH

NO

NO

Et

N-Nitrosodiethylamine
(NDEA)

2methyi propylamine
(NMAMPA)

Et

NNitrosoN(1m ethyl

acetoryl)-

N-Nitrosothiazolidine4carboxylc acid (NTCA)

OOH

Me
N

NO
NNitroso2methylthiazolidine
4carboxyiicacid(NMTCA)

HOCH21"C

Bu

"N*

NO

NO

Bu
N-Nitrosodibutylamine
(NDBA)

N-Nitroscmorpholine
(NMOR)
Me

CH2COOH

NO
N-Nitrosopyrrolidine
(NPYR)

N-Nitrosa2-hydroxymethylthiazolidine-4-caboxylic acid
(NHMTCA)

Me

COOH

NO-

N-Nitrososarcosine
(NSAR)

\^

NO
N-Nitroso-5-methyloxazolidine4-carboxylic acid (NMOCA)

VcOOH

^ N '

I
NO
N-N itroso-3-hy cr oxypyrolidine (NHPYR)

N-Nitrosoproline (NPRO)

VCOO
H

NO

NO
N-Nitroso-2-hydroxymethylthiazolidine(NHMTHZ)

N-Nitroso-N(1-methylacetonyl)3-methylbutylamine
(NM AMBA)

NNitrosa
o)azolidine4
carboxylicacid
(NOCA)

2.2. Formation in food

2.2.1. General formation
Nitrosamines are formed by the reaction of secondary or tertiary amines with
suitable nitrosating species. The nitrite ion by itself or nitrous acid are poor
nitrosating agents. However, under acidic conditions the above two species are
converted to nitrous anhydride, as shown in Figure 3, which then reacts with a
secondary amine to produce a nitrosamine:

Figure 3:

Formation of nitrosamines

N0 2-+ H+

<-

HNO2

2HN02

<->

N2O3 + H2O

RiR2NH + N 2 0 3

**

RiR2N-N=0 + HN0 2

Reaction rate = K1[RiR2NH][HN02]2

The reaction rate is of first order with respect to the concentration of the
unprotonated secondary amine and second order with respect to the concentration
of nitrite, as two molecules

are needed to form one molecule

of N203. The

concentration of the unprotonated amine depends on its basicity (pKa), the higher
the basicity, the lower the concentration of the unprotonated amine and the lower
the rate of nitrosation.
For the commonly occuring foodborne secondary amines, most of which are
highly basic (e.g. dimethylamine,

pyrrolidine, piperidine), the optimum

nitrosation lies between 3 - 3.5. Tertiary amines, such as trimethylamine,

pH for
which

occurs in fish in fairly high levels, can also form nitrosamines, but their nitrosation
rate is much slower than that of the secondary amines.
On the other hand, the amides do not have a pH optimum for nitrosation.
Between pH 1 to 3, the rate of nitrosation of amides seems to increase by 10-fold for
a drop of one pH unit and to be linear dependent on the concentration of nitrite.

In general, the nitrosation rates for various amines and amides increase in
the following order (strongly basic amines having the slowest rates) as shown in
Figure 4.
Figure 4: Nitrosation rates of various amines and amides:

Strong basic amines (e.g. dimethylamine, piperidine), N-alkylamides,

N-aikylguanidines

I
Weak basic amines (e.g. morpholine, dibenzylamine), certain amino acids
(sarcosine, hydroxyproline)

i
N-alkylcarbamates (e.g. ethyl-N-methylcarbamate)

i
N-alkylureas ( e.g. N-methylurea)

i
Alicyclic ureas (e.g. ethylene ureas)

Within the same series of compounds (e.g. amines or amides), these

nitrosation rates can vary over several orders of magnitudes. For example, Nmethylaniline

and morpholine,

respectively,

undergo nitrosation

147.000 and 250 times faster than dimethylamine.

approximately

The extent of nitrosamine

formation depends on the nature of the amines involved, on the pH, concentration
of the reactants (both the amines and nitrite), as well as on the presence of catalysts
(e.g. SCN", I', Br", CI") or inhibitors (e.g. ascorbic acid, -tocopherol) [Sen et al.,
1992].

2.2.2. Formation in vivo

Nitrosamines can be formed 'chemically' in the acidic milieu of the gastric
juice

from amines

and

nitrosating

compounds

like

N0 2 ,

N0 X ,

N203

nitrosylhalides. This nitrosating reaction can be hampered by ascorbic acid.

and

The intake of cured meat products and other food (e.g. vegetables) puts the
human under strain of high amounts of nitrite. In addition, food can be a big source
of nitrate intake to the human body (e.g. vegetables, processed cheese, tap water).
Nitrates can be easily reduced to nitrites in the mouth, the gastro-intestinal
tract and bacterial infected urethral path ways. Bacteria (e.g. E. coli, Proteus vulg.)
and

macrophages

catalyse

the

reduction

of

nitrate

and

the

formation

of

nitrosamines from nitrite and amines.

Persons with chronic

infections of the urethral pathways are therefore

predestinated to form tumors in the urethral pathways caused by nitrosamines. The

strain of endogen formed nitrosamines is difficult to judge because of the great
significance of the individual parameters [Reichl, 1997].

2.3. Occurrence in food

2.3.1. General
The first conclusive evidence of the occurrence of nitrosamines in food or
animal feed was manifested by the analysis of nitrite-preserved herring meal used for
animal consumption. The fact that N-nitrosodimethylamine (NDMA) could be found
in sufficient concentrations to cause acute

hepatoxicity

and sometimes lethal

effects in animals caused a great deal of concern [Sen et al., 1991], This led to a
worldwide

search for nitrosamines

in

food,

especially

those

preserved

with

nitrate/nitrite.
NOCs may be present in food because of their:

formation as a result of the use of nitrate/nitrite additives

formation during processing

contamination from secondary sources such as packaging materials or other

ingredients (e.g. waxes) [Sen et al., 1992].
After bacterial degradation of meat and adding nitrite salt especially under

strong heating conditions (open fire, barbecue) there is evidence that high amounts
of the released amines can be nitrosated.
Fish proteins deteriorate very easily. Cured fish products can be critical

for

human consumption regarding their amount of nitrosamines.

Some literature references for the occurrence of N-nitrosamines in food and
cosmetics are listed in Table 1.

Table 1: References of N-nitrosamine-occurrence in food (and cosmetics):

Matrix

References

Food and beverages

(in general)

Biaudet et al., 1994; Cornee J et al., 1992; Dich et al., 1996; GarciaRoche et al., 1990; Hotchkiss et al., 1988; Mavelle et al., 1991; Ohta et
al., 1990; Penttil et al., 1990; Satoh et al., 1985; Tutelyan et al., 1990

Bacon and edible fats

Canas et al., 1986; Fiddler et al., 1996; Gloria et al., 1997; Greenfield et
al., 1982; Hotchkiss et al., 1985; Ikins et al., 1986; Massey et al., 1991;
sterdahl et ai., 1990; Sen et al., 1985; Sen et al., 1991; Veccio et al.,
1986

Cheese, whey

Dellisanti et al., 1996; Oliveira et al., 1995

Malt, beer

Am. Soc. Brew. Chem., 1985; Izquierdo et al. 1995; Longo, et al., 1995;
Righezza et al., 1987; Sen et al., 1996

Cured meat

Fiddler et al., 1995; Greenfield et al., 1982; Pensabene et al., 1990;

Shahidi etat, 1994

Sausages

Fiddler et al., 1993; Maxwell et al., 1993; Pensabene et al., 1990 and
1991

Fish, seafood

Gomez-Guillen et al., 1991; Groenen et al., 1982; Lintas et al., 1990;

Sen et al., 1985; Takatsuki et al., 1990; Tozawa et al., 1991

Food contact
nettings,

Marsden et al., 1993; Pensabene et al., 1992 and 1995; Sen et al., 1993

Smoked food

Sen et al., 1993

Cosmetics

Billedau et al., 1994; Challis et al., 1995; Meyer et al., 1991

2.3.2. Examples of N-nitrosamine occurrence in food

Beer:
Beer has contributed in the past to a major part of the daily intake of NDMA
through the diet. Malt was found to be the main source of NDMA contamination in
beer, and it was shown to be formed during direct drying of malt using hot flue
gasses - a practice that was common prior to 1980 [Sen et al., 1996].
Burning of sulphur with the fuel or the introduction of S 0 2 gas into the hot
flue gas, especially during the first 8-10 h of malt kilning, greatly reduced NDMA
formation. Only negligible amounts of NDMA are formed in malt dried by indirect
heating (where it does not come into physical contact with the hot flue gas), or dried
by using an electric heating source.
Following these discoveries, maltsters in various countries started adopting indirect
malt drying techniques. However, some still continued

using the direct

drying

method with sulphur-burnig cycles extended from 25 to 50 g of sulphur/100 kg of

malt [Am. Soc. Brew. Chem., 1985].
Microwave, effect of cooking:
Bacon was analysed for volatile nitrosamines after microwave cooking, and
the results were compared with those obtained

after frying bacon

in a pan.

Microwave-cooked bacon contains statistically significant (p<0,01) lower levels of

the

volatile

nitrosamines

NDMA,

NPIP

(N-nitrosopiperidine)

and

NPYR

(N-

nitrosopyrolidine) than bacon fried in a pan. This is probably due to the low cooking
temperature ( up to about 100"C) and short exposure to the heat with microwave
cooking [sterdahl et al., 1990].
The effect of cooking on N-nitroso compounds formation was studied in samples
cooked based on traditional Italian recipes (stewing, boiling, deep-frying, grilling).
The results of the nitrosation experiments indicate that there was a noticeable
increase of NDMA formation especially using stewing or boiling as cooking methods
[Lintas et al., 1990].
The effects of bacon composition (fatty acid composition) and processing (effect of
frying atmosphere, effect of smoking) and the inhibition of nitrosamine formation has
also been investigated [Skrypec at al., 1985].
Elastic rubber nettings :
Previous research has shown that traces to fairly high levels of certain Nnitrosamines can be detected in cured pork products packaged in elastic rubber
nettings. The nitrosamines are formed due to the interaction of the additive nitrite in
the meat and amine additives in the rubber that are used as accelerators in the
curing of rubber. The highest levels of N-nitrosamines were found in smoked pork
cottage rolls and smoked pork shoulders [Sen et al., 1993], It is not clear whether this
is due to the type of netting used or to special curing and smoking conditions (e.g.
temperature and duration of smoking and curing, nitrite concentration) employed in
the preparation of such products. Further research on this aspect would be desirable
[Sen et al., 1993].
Smoked mutton:
N-nitroso-N-methylaniline and other nitrosamines were detected in Icelandic
smoked mutton. N-nitroso-N-methylaniline

is probably produced by the interaction

of nitrite and smoke generated by burning sheep dung, the traditional source of fuel
used for smoking such products [Sen et al., 1990].

IO

2.4. Intake
Dietary

intake of

N-nitrosamines

were estimated

based

on

available

information about their contents in different foods. These contents have been
reported in several studies during recent years. Since considerably more information
is available for NDMA than for other N-nitrosocompounds found in food the dietary
intake of this compound was estimated [Dich et al., 1996].
Table 2:

Estimates of daily intake (pg/person) of N-nitrosamine (NDMA) in

different countries [Dich et al., 1996].

Nat.

NDMA intake

Major NDMA source in the diet

Year

Cured meats (35%)

1978

Beer (71%)

1980

in 9/3

UK

0,14a

NL

0,38b
b

1990

<0,10
D

1,1 (men)

Beer (64%), Cured meats (10%)

1980

0,57 (women)

Cured meats (10%)

1980

0,53 (men)

Beer (40%)

1983

0,35 (women)

Cured meats (18%)

1983

0,29 (men)

Cured meats (36%), Beer (32%), Fish products

1991

(15%)
0,18 (women)

Cured meats (44%), Beer (16%), Fish products

1991

(19%)
F

0,26

Dairy products including cheese (42%),

1992

Cured meats (25%), Fish products (20%)

Japan

1,8

Dried fish (91%)

1980

0,12

Cured meats (61%), Beer (32%), Smoked fish (1%)

1988

FIN

0,08

Beer (75%), Smoked fish (25%)

1990

0,12:

Beer (58%)

1996

0,18 (men)

Salted and smoked fish (22%)

0,06 (women)

Cured meats (20%)

ll

It has been shown that the exogenous

nitrosamines (NVA)
modifications

exposure

to

certain

volatile

and non volatile nitrosamines (NVNA) is decreasing due to

in processing of food and the reduction of nitrate/nitrite

levels

permitted as additives in cured food products. The total daily exposure to VNA is
generally in the order of 0.3 - 2.0 pg/day in western countries [Tricker et al., 1992].

Table 3:

References for the intake (humans) or concentration in the diet of

nitrosamines in different countries:

Intake or Content

Country

Reference

Concentration

Cuba

Garcia-Roche et al., 1990

Concentration & Intake

Finland

Penttilae et al., 1990

Intake

Finland

Dich et al., 1996

Concentration

France

Mavelle et al., 199.1

Concentration

France

Cornee et al., 1992

Intake

France

Biaudet et al., 1994

Intake

Japan

Satoh e t a t , 1985

Concentration

Japan

Otah et al., 1990

Intake

USA

Hotchkiss et al., 1988

Concentration

USSR

Tutelyan et al., 1990

2.5. Toxicity
As a class of chemical carcinogens, NOCs are unique in the sense that there
are a very few classes of chemicals, if any, that can induce cancer in so many
organs of so many species. The target organ may vary depending on many factors,
predominant among which are the chemical structure of the compound and the
animal

species. Various dose-response studies suggest that

nitrosamines

are

carcinogenic to laboratory animals already at extremly low doses [Sen et al., 1992].
Many nitrosamines are also potent mutagenes, but they need metabolic
activation in bacteria, yeast, or fungal assay systems [Sen et al., 1992]. N-Nitroso
compounds (NOCs) have a high significance as environmental carcinogens. The

12

intake can be oral, cutan or through inhalation. Various NOCs can also be formed
endogenously.
Some drugs, e.g nitroso Cimetidin, the nitrosated form of the endogastric
therapeuticum
Aminophenazon

Cimetidin

were proved

is metabolized

to

to

the

be

mutagenic

highly

and

carcerogenic

carcinogenic.
compound

N-

nitrosodimethylamine (NDMA) and therefore not used anymore [Sen et al., 1992],

Effecf
About 90% of more than 300 N-nitrosocompounds

tested in laboratory

animals have been found to be carcinogenic. Tumors caused by N-nitrosamines

occurr preferentially in the oesophagus, stomach, liver, kidney and urethral path
ways. In high doses, nitrosamines have been shown to be cytotoxic and to cause
necroses. After acute poisoning a delayed damage of the medulla parent cells with
extreme leukopenie and severe haemorhagie and ulzerations in the gastro-intestinal
tract are characteristic.

Mechanism
Biochemical

studies

suggest

that

most

N-nitrosocompounds

require

metabolic activation before effecting their toxic and biological properties. The first
step in this metabolic activation process is the hydroxylation of the a-C-atom (with
respect to the N-N=0 group) mediated by cytochrom P450.
The generated instable -hydroxy nitrosamin decays to formaldehyde and
methyldiazohydroxyd

which

splits up into a diazonium

ion or a carbenium

intermediate (Figure 5). These compounds can alkylate DNA, RNA and proteins and
are the ultimate carcinogens [Reichl, 1997].

Figure 5:

Biotransformation of N-nitrosodimethylamin [Reichel, 1997]:

(H3C)2N-N=0

^Cytochrom P-450^

(H0-CH2)(H3C)N-N=0

-Hydroxynitrosamine (instable)

NDMA

(HO-CH2)(H3C)N N=0

-Hydroxy nitrosamine

(H3C)N=N-OH

H2C=0
Formaldehyd

Methyldiazohydroxide

(H3C)N=N-OH

(N2, OH), H3C+ -

Methyldiazo-

Carbenium-

hydroxide

intermediat

Reactions with proteins, DNA, RNA

Safety precautions to be taken for nitrosamine analysis

The following safety precautions are required strictly for the analysis of
nitrosamines:

Nitrosamines are considered to be potent carcinogens. EXTREME CARE must be

exercised in handling nitrosamines or solutions of nitrosamines. Skin contact
must be avoided.

Mechanical pipetting aids should be used for all pipetting procedures.

All samples containing nitrosamines should be properly labeled as 'carcinogenic'

or 'spiked with nitrosamines'or with other adequate warnings.

All glassware used for nitrosamine analysis should be thoroughly and routinely
cleaned with 'Chromerge' (or equivalent) and thoroughly rinsed with distilled
water and dichloromethane.

Some nitrosamines degrade

upon exposure to ultraviolet

light.

Prolonged

exposure to fluorescent lights should be avoided unless lights are covered with
yellow translucent shields to filter out ultraviolet

light. Alternatively

containers can be covered with foil or other suitable material

sample

to provide

protection from light.

Store standards and extracts in a freezer, in amber bottles or foil-covered

containers [Am. Soc. Brew. Chem., 1985].

14

3. ANALYTICAL METHODS

3.1. General Aspects

3.1.1. Classification: volatile and non-volatile N-nitrosocompounds
N-nitroso compounds can be divided into two functional categories:
* Volatile N-nitrosamines (VNA)
* Non-volatile N-nitrosamines (NVNA)
Volatile N-nitrosamines (VNA) are generally considered to be N-nitrosated
drivtes of simple, low molecular weight dialkylamines and cyclic

compounds

which can be isolated in good yields (>70%) from food matrices by distillation
(steam, mineral oil, atmospheric
chromatography

or vacuum). They can be analysed by gas

(GC) without derivatisation. The most commonly

encountered

examples being N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and Nnitrosopyrroiidine (NPYR). Studies of the occurrence of N-nitrosocompounds and
their precursors in the environment have mainly concentrated on volatile N-nitroso
compounds (VNA). They have been reviewed by several authors as listed in Table 4.
Non-volatile

N-nitrosamines

(NVNA) are not amenable

to isolation

via

distillation techniques without prior chemical derivatisation. The most commonly

encountered examples of NVNA are simple hydroxylated compounds such as Nnitrosohydroxypyrrolidine

(NHPYR), N-nitrosated drivtes of amino

acids (e.g.

hydroxypyroline, proline and sarcosine), amino acid drivtes and condensation

products of amine acids with aldehydes. The references of studies on NVNAs are
also listed in Table 4.

15

Table 4:

Volatile

References of volatile, non-volatile and total appearent N-nitrosamines

Biaudet et al., 1983; Canas et al., 1986; Cornee et al., 1992; Delibanti et al., 1996;
Dich et al., 1996; Fiddler et al., 1993, 1996; Frassantino et al., 1994; Garcia-Roche et
al., 1990; Gloria et al., 1997; Groenen et al., 1982; Hotchkiss et al., 1985; Izquierdo
Pulido et al., 1995; Kataoka et al., 1996; Kuehne et al., 1981; Lintas et al., 1990;
Longo et al., 1995; Marsden et al., 1993; Mavelle et al., 1991; Maxwell et al., 1993;
Oliveira et al., 1995; Pensabene et al., 1990, 1992, 1995; Pentill et al., 1990; Pylypiw
et al., 1985; Righezza et al., 1987; Satoh et al., 1985; Sen et al., 1982, 1996; Shahidi
et al., 1994; Takasuki et al., 1990; Veccio et al., 1986
Billedau et al., 1994; Cox et al., 1995; Pensabene et al., 1990, 1991; Tutelyan et al.,

Non

1990

volatile
Bellec et al., 1996; Fiddler et al., 1995; Ikins et al., 1986; Massey et al., 1991; Sen et
al., 1985, 1990, 1991,1993, 1996; Walters et al., 1992; Zhou et al., 1994
Total

3.1.2. Analysis of volatile N-Nitrosamines:

Volatile N-Nitrosamines, e.g. NDMA, NPYR, are easily analysed by gas
chromatography (GC) coupled with a thermal energy analyser (TEA). There are,
however, a large number of polar nitroso compounds not generally amenable to
direct analysis by GC, either because of ther low volatility or their thermal instability.
Reversed phase high performance liquid chromatography (HPLC) seems to be the
method of choice for the analysis of apolar and polar NOCs to-date. Attempts to
combine HPLC with a chemiluminescence detector gave inconsistent results with
respect to both, sensitivity and resolution. However, an efficient photolytic interface
between HPLC and TEA detector has been described [Bellec et al., 1996].

16

3.1.3. Analysis of non-volatile N-Nitrosamines:

GC with the TEA as a selective and sensitive detector can be used for some
non-volatile N-nitrosamines, such as N-nitrosamino acids after their methylation or
silylation. Attempts have also been made to link the TEA to HPLC for other nonvolatile N-nitroso compounds. Here, the eluent is passed into the pyrolyser of the
TEA at high temperature in which

it is vaporised and the N-N bond of the

nitrosamines can be cleaved. The vaporised mobile phase can then be condensed
in cold traps prior to detection of the nitric oxide by its chemiluminescence

[Walters

et al., 1992].
A HPLC-TEA interface was described using a particle beam (PB) type of
instrumentation developed initially for interfacing HPLC to mass spectrometry (MS)
[Billedeau et al., 1994]. The high solvent removal efficiency of this PB interface has
made the HPLC-TEA analysis possible with reversed-phase solvents (e.g. methanol,
water, acetonitrile)

without the need for solvent venting or cryogenic

trapping

techniques, currently being used in alternative HPLC-TEA interfaces [Billedau et

al., 1994].

Analysis of N-nitrosamino acids (NNA):

The majority of published methods apply an extraction method, solvent
partitioning, and centrifugation of the sample prior to quantitation of the NNAs
directly

by

liquid

chromatography

or

after

derivatisation

prior

to

gas

chromatography. These methods are inherently slow, suffer often from emulsion
problems due to a content of fat in the samples, and are limited in the number and
type of N AAs that can be simultaneously separated on a chromatographic column.
Methods for volatile

N-nitrosamines

in food

based on solid-phase

extraction

techniques have been developed and seem to be very versatile [Pensabene et al.,
1990].
To ensure the destruction of residual nitrite, and to prevent artifactual NNA
formation, sulphamic acid was added to the sample matrix together with ascorbic
acid. The latter is a proven nitrosation inhibitor. Sulphamic acid was also used as
an acidulant to faciliate NNA extraction.
There are many methods described for the derivatisation of NAAs. However,
the most frequently used involve some form of methylation or silylation. Preparing
volatile GC drivtes of the simple NAAs works well using either of these two
methods, whereas complete derivatisation of the hydroxylated NAAs has always
been less favourable. It has been reported that the GC analysis of the methyl esters
of N-nitroso-4-hydroxyproline (NHPRO), N-nitrosdo-5-hydroxypiperolic acid (NHPIC)

17

and N-nitroso-2-(hydroxymethyl)thiazolidine-4-carboxylic

acid (NHMTHZCL) exhibit

a low TEA response due to peak broadening caused by non specific adsorption and
the free hydroxyl group. This was studied intensively and it has been found that
acylation

with acetic

anhydride-pyridine was the best derivatisation

agent for

hydroxylated nitrosamines. In a mixture of both, simple and hydroxylated NNAs,

methylation followed by acylation

proved to be the most reliable

method for

preparing volatile GC drivtes [Pensabene et al., 1990].

For separation and subsequent quantitation of the NNAs by GC-TEA, most
published methods have been shown to be similar [Pensabene et al., 1990].

3.1.4. Analysis of total N-nitrosocompounds:

Techniques

for

the

determination

of

pg/kg

(ppb)

levels

of

volatile

nitrosamines in food are now well established. The development of analytical

methods for non-volatile
primarly to nitrosamino

nitrosamines, however, has been slow and

acids, nitrosamines

containing

restricted

hydroxyl groups, or a

combination of those. Volatile derivatives of non volatile nitrosamines are generally

prepared in order to permit direct analysis by
detection

(TEA)

detection

of

nitric

oxide

GC

with

and

chemiluminescence

confirmation

by

gas

chromatography/mass spectrometry (GC/MS). Despite the ability to interface a liquid

Chromatograph to a TEA, this technique has not been widely adopted for analysis of
non-volatile nitrosamines in food products because of problems with aqueous
mobile phases. The lack of information on the identity of nitrosamines has also to be
encountered. For this reason, several attempts have been made to determine the
amount of 'total' nitroso groups that will estimate the sum of the volatile and nonvolatile nitrosamine contents of food products. The use of UV light has been chosen
to selectively cleave the N-NO bond and to liberate nitrite, which can be detected
colorimetrically after addition of the Griess reagents [Daiber et al., 1964].
An improved detection method for determination of nonvolatile nitrosamines
was developed [Havery et al., 1990]. This method uses a postcolumn system to
denitrosate the nitrosamine with hydrogen iodide in an aqueous mobile phase. The
technique

can

be

used without

an LC column

to

estimate

the

total

N-

nitrosocompound (TNC) content of samples. Re-analysis with another column can

help to identify the compounds responsible for the nitric oxide response.
This method was modified [Fiddler et al., 1995] and less noisy signals and
higher responses were achieved by adding Kl before rather than after injection with
acid. This method has a number of advantages for the determination of total N-

18

nitroso compounds: detection of thermally labile compounds, minimal loss of acidlabile nitrosamines, and high sample throughput [Fiddler et al., 1995].

3.2. Clean-up/Extraction methods

The following extraction and clean-up methods have been employed:
Supercritical fluid extraction (SFE) using supercritical carbon dioxide
Solid phase extraction (SPE)
Mineral oil distillation (MOD)*
low-temperature vacuum distillation (LTVD)
Mineral oil distillation: Fine et al 1975 described a vacuum distillation procedure which has
been widly accepted. Distillaten was carried out by placing the sample with an equal amount of
mineral oil and sodium hydroxide. Vacuum was applied and the mixture was slowly heated to
110C. The distillate was trapped in vapor traps immersed in liquid nitrogen. After the specified
temperaure was reached, the vacuum was discontinued and the distillate was thawed, extracted
and concentrated [Hotchkiss, 1981].

Table 5: References of the different extraction methods

SFE

SPE

MOD

LTVD

Maxwell et al., 1993;

Pensabene et al.,

Canas et al., 1986;

Oliveira et al., 1995;

Pensabene et al.,

1990, 1991, 1992;

Greenfield et al.,

sterdahl et al., 1990;

1995

Plypiw etat, 1985

1982; Hotchkiss et

Sen et al., 1993;

al., 1985; Ikins et al.,

Shahidi et al., 1994,

1986; Veccio et al.,

1995

1986

Comparison of extraction methods

Samples of fried bacon were treated by SFE, SPE, MOD and LTVD methods
for the determination of PNYR and NDMA, using the same GC-chemiluminescence
detections (TEA) conditions [Fiddler et al., 1996]. The range of values for SFE was
0.7 to 20 ppb for NPYR and 0 to 2.4 ppb for NDMA. Analysis of variance of the
NPYR data showed a significant difference (p<0.05) between SFE and SPE results
and significant differences between these and those obtained by MOD and LTVD.
Overall, SFE was superior to the other methods with the highest recoveries, best
repeatibility, speed of extraction, and solvent characteristics. Similar results were

19

obtained for SFE after comparison with distillation

and SPE

for determining

nitrosamines in fried bacon drippings [Fiddler et al., 1996].

Solvents
Extraction of N-nitroso compounds should, whenever possible be carried out
with a water-immiscible solvent. Ethyiacetate extraction resulted in highly variable
recoveries, but acetonitrile was effective for the nitrosamines studied. Only a small
amount of residual nitrite was taken up by acetonitrile [Walters et al., 1984].
Even though acetonitrile and water are miscible in the absence of the sample, two
layers are formed when sulphuric acid-sulphamic acid solution is added [Fiddler et
al., 1995].
The necessity of removing residual nitrite in the present in the samples
before analysis was stated because it interferes with quantitation of apparent total Nnitroso compounds (ATNC) by giving a false-positive response. The rapid and mild
conditions, used for nitrite removal, described in the Fiddler method, did not
compromise the acid-labile nitrosamines. Because of the formation of two layers,
the nitrosamines in the upper acetonitrile layer had only limited exposure to the
acid at the interface [Fiddler et al., 1995].

3.3. Identification and Quantification:

3.3.1. Gas Chromatography (GC)
In most GC methods, N-nitrosamines are determined directly in their free form using
a thermal energy analyser (TEA), based on the detection of the chemiluminescence
emitted from a reaction between released NO radicals and ozone after thermal
cleavage of the N-NO bond in N-nitrosocompounds [Kataoka et al., 1996].
Although GC-TEA is sensitive and specific for N-nitroso compounds, it is very
expensive. Therefore, nitrogen-phosphorus detection (NDP) has been used for the
determination of N-nitrosodimethylamine

(NDMA) in its free form. However, the

application of the GC-TEA and GC-NDP methods to the free forms of N-nitrosamines
required

time-consuming

chromatography

prior to

clean
GC

up

analysis.

by

aluminia
Further,

decomposed by acid or peroxide produced

or

silica

N-nitrosamines

gel
tend

column
to

be

in the solvents during these pre-

treatments [Takatsuki et al., 1990].

20

GC with electron-capture detection (ECD) based on the conversion of Nnitrosamines into their corresponding N-nitramine analogues by pertrifluoroacetic
acid

oxidation

has been reported [Copper et al., 1987]. However, this method

requires purification

of the derivatives by adsorption chromatography

on

basic

aluminia prior GC analysis. A method based on the denitrosation of N-nitrosamines

to secondary amines and subsequent conversion of the secondary amines into Ndiethylthiophosphoryl

(DETP)

derivatives

and

gas

chromatography-flame

photometric detection (GC-FPD) analysis was developed by [Kataoka, et al., 1996],

In the following, the experimental conditions are listed:

3.3.1.1. Gas chromatography-thermal enegy analysis (GC-TEA):

Table 6:

GC-TEA: NOCs in meat, cured

Ref.

Subst.

GC-TEA parameters

Extraction

Kuehne et
al., 1981

NDMA,
NPYR,
NPIP

vacuum
distillation

Fiddler et
al., 1995

NPro,
NTHZC
NSar,
NHPro
NMU,
NMA
NNMG
NSAR,
NMAPA,
NAZETC,
NPRO
NHPIC,
NTHZC
NINA,
NHPRO
NHPIC,
NHMTHZ
C
NDMA

Stainless steel column 3mx1/8" o.d., 12% Carbowax

20M with terephtalic acid on Gaschrom Q 100/120
mesh, carrier gas: He 30mL/min, temperature 150C
or stainless steel column 3mx1/8" o.d., 10%
diethylenglycol-succinate (DEGS) on Chromosorb WAW 60/80 mesh, carrier gas He 30ml/min,
temperature: 130C
An Altrex pump was interfaced to a TEA
chemiluminiscence detector. The operating conditions,
the Teflon tubing reaction coil system, the solvent
trapping system, and the fitting adapted to aspirate
the sulfuric-acetic acid were the same as described by
[Harvey DJ, 1990, J.Anal.Toxicol. 14,181-185]
30mx0,527mm DB-5 fused silica capillary column, He
carrier gas at 25 mL/min, column oven programmed
from 80 to 200C at 4/min; injector port 200C; TEA
furnace 485C; TEA vacuum 1,0 mm; liquid nitrogen
cold trap.

solid phase
extraction

Column 6ftx1/8in. o.d., Ni tubing packed with 20%

Carbowax 20M and 2% NAOH on 80-100 mesh acidwashed Chromosorb P; carrier gas Ar, 30 mL/min; GC
oven temperature 170C, TEA vacuum chamber
pressure 1 mm Hg; TEA cold trap, stainless steel trap
immersed 1/3 in. liquid nitrogen, recorder 1mV span.

lowtemperature
vacuum
distillation

Pensabene et al.,
1990

Shahidi et
al., 1994

extraction
with
acetonitrile

21

Table 7:

GCTEA of NOCs in bacon and its fat

Ref.

Subst.

GC-TEA parameters

Extraction

Canas et
al., 1986

NDMA,
NDPa,
NPIP,
NPYR,
NMOR

mineral oil
distillation

Fiddler et
al., 1996

NKMA,
NMEA,
NDEA,
NDPA,
NAZET,
NDBA,
NPIP,
NPyR,
NMOR,
NHMI
NDMA,
NDEA,
NDBA,
NPIP,
NPYR,
NTHZ

Column: 2.7x4mm glass column packed with 10 %

Carbowax 1540 and 5 % KOH of 100120 mesh
Chrmosorb WH P; carrier gas argon 40 mL/min;
injector temperature: 200 C, column temperature
programmed from 100 to 180 C at 4 C/min, TEA
furnace temperature: 450 C; pressure 1.2 torr; liquid
nitrogen cold trap
GC method as described by Pnesabne, Fiddler, 1992

GCTEA as described by H otchkiss et al. (1980) with

the following differences :
column: 2m 1.6 mm i.d. packed with 15% Carbowax
20Mterephtalic acid (TPA) on Chromosorb 6080
mesh; Column temperature: 120C for 10 min,
programmed to 180C at 4C/min, and maintained at
180C for 30 min; Injection port temperature: 190C;
Carrier gas: H e 30 mL/min; GC column was interfaced
to a TEA. The TEA trap was maintained at 150C with
liquid nitrogen and isopentane, the pyrolysis chamber
was kept at 475C, and the vacuum was maintained at
1,0 torr. Identification and quantification of the
nitrosamines were accomplished by injecting known
amounts of nitrosamine standard solutions. NDPA was
used as an internal standard.
Screening procedure: Of the many analytical methods
developed for the detection of volatile nitrosamines
the Mineral oil distillation method in conjunction with
the TEA reported by Fine DH et al. 1978 seemed to
be the most feasible

vacuum
mineral oil
distillation

Column: 3mx0.32mm i.d. SS, 10% Carbowax 20M on

80100 Chromosorb WH P, H e: 25 mL/min, injector
190C, column 150C; TEA; interface 175C,
pyrolizer 525C, trap 150C, pressure 2.2 mm H g.
Glass column packed with a mixed phase, 1% OV210
and 2% OV17, an 80/120mesh Chromosorb W
(Supelco Inc., Bellefonte, PA), GC temperature
programmed from 100 to 180C at 8C/min.

mineral oil
distillation

Glass column: 1.8mx1.9mm i.d., containing 20%

Carbowax 20M and 25% KOH on Chromosorb W
interfaced with a TEA, GLCTEA conditions:
column temperature: 160C ; injector temperature:
210C, H e carrier gas flow about 27 ml/min, furnace
temperature 475C, oxygen flow about 5 ml/min,
vacuum pressure about 1mm H g, CTR gas stream
filter
GCTEA was used for the analysis of volatile
nitrosamines. GLC conditions: column, 6ftx1/8 in o.d.,
Ni tubing packed with 20% Carbowax 20 M and 2%
NaOH on 80100 mesh Chromosorb P, acid washed;

vacuum
distillation

Gloria et
al., 1997

Greenfield
et al.,
1982

Hotchkiss
et al.,
1985

NDMA,
NDEA,
NDBA,
NPIP
NPYR,
NMOR
NDMA,
NPYR,
NDPA,
NDMM

Ikins et
al., 1986

NDMA,
NPYR,
NTHZ

sterdahl
et al.,
1990

NPYR,
NDMA
NPIP

Sen et al.,
1982

NDMA,
NDEA
NDPA,
NDBA

method
comparison

mineral oil
vacuum
distillation

mineral oil
distillation

rapid liquid
liquid
extraction

22

NAZET
NPYR,
NPIP
NMOR
Veccio et
al., 1986

Table 8:

NDMA,
NPIP,
NPYR,
NTHZ

carrier gas Ar, 30 mL/min; GLC oven temperature

170C; injector port temperature 220C; TEA furnace
temperature 450C, TEA vacuum chamber pressure,
1mm; TEA cold trap, stainless steel trap, immersed
one-third in liquid nitrogen.
Column 3mx0.32cm o.d. Ni packed with 10%
Carbowax 20M on 80/100 Chromosorb WHP; carrier
gas He 25mL/min, temperatures: injector190C,
column 150C, interface175C, pyrolyzer 525C, Trap
-150C, TEA pressure 2.2mm Hg.

vacuum
mineral oil
distillation

GC-TEA: NOCs in "Frankfurter" sausages:

Ref.

Subst.

GC-TEA parameters

Extract.

Maxwell et
al., 1993

NDMA,
NMEA
NDEA,
NDPA
NDBA,
NAZET
NPYR,
NPIP
NHMI,
NMOR
NDMA,
NPYR,
NMOR,
NAZET

2,7mx2,6mm glass column packed with 15% Carbowax

20M-TPA on 60-80 mesh Gas Chrom , He carrier gas
35 mL/min, injector 180C, TEA furnace 475C, TEA
vacuum 0,4 mm, liquid nitrogen cold trap, and a
temperature programme from 120 to 220C at 4C/min.

supercriti
cal fluid
extraction

3mx2,6 mm i.d. glass column packed with 15%

Carbowax 20M-TPA on 60-80 mesh GasChrom P; He
carrier gas 35 mL/min: temperature programmed from
120 to 220C at 4/min; injector 200C; TEA furnace
450C; TEA vacuum 1,0 mm; liquid N2 cold trap
NTHZC determined as by [Pensabene, Fiddler 1990].
NTHZ was determined by [Pensabene, Fiddler, 1982].
During the initial extraction procedure, two separate
columns were used instead of one, as described for
analysis of NDMA [Pensabene, Fiddler, 1988].

solid
phase
extraction

Pensabe
ne et al.,
1990

Pensabe
ne et al.,
1991

NTHZC
NTHZ

solid
phase
extraction

Table 9:

GC-TEA: NOCs in other meat products:

Ref.
Shahidi et
al., 1994

Subst
NDMA

Sen et al.,
1990

NDMA,
NPYR

Pylypiw et
al., 1985

NDMA,
NMOR,
NDPA

Table 10:

GC-TEA parameters
Column 6ftx1/8in. o.d., Ni tubing packed with 20%
Carbowax 20M and 2% NAOH on 80-100 mesh acidwashed Chromosorb P; carrier gas Ar, 30 mL/min;
GC oven temperature 170C, TEA vacuum chamber
pressure 1 mm Hg; TEA cold trap, stainless steel
trap immersed 1/3 in. liquid nitrogen.
Carbowax 20M column with added alkali was used,
wheras a coiled glass column packed with Carbowax
20M without any added alkali or a DB-wax megabore
column (30 mx0,53 mm i.d., 1-um coating), was
used for the analysis of NMA and NTHZ. The
operating conditions for the megabore column were
as follows: carrier gas (He) flow, 8ml_/min, injector
temperature 65C, GLC temperature 80C for 2 min,
then increased to 135C at 6C/min with a hold for 5
min at 135C, and then increased to 180C at
10C/min, held for 10 min.
Column 6ftx2mm i.d. glass, packed with 10%
Carbowax 20M +2% KOH on Chromosorb W AW,
80/100 mesh; programmed from 120 to 190C at
5.0C/min; final hold time 1.0 min, total run time
15.2 min; carrier gas He, flow rate 12mL/min, on
column injection 250C, interface line 1/8in o.d.
glass-lined stainless steel, 250C, TEA furnace
525C, TEA cold trap -151C, TEA detector pressure
1.4 torr.

Extraction
lowtemperature
vacuum
distillation

low
temperature
alkaline
vacuum
distillation

distillationextraction
apparatus

GC-TEA: NOCs in food packed in nettings

Ref.

Substance

GC-TEA parameters

Extract.

Marsden
et al.,
1993

NDMA, NDEA,
NDBA,

no further details

ethanol
extraction

Pensabe
ne et al.,
1992

NDMA, NMEA
NDEA, NDPA,
NAZET, NDBA
NPIP, NPYR
NMOR, NHMI

solid phase
extraction

Pensabe
ne et al.,
1995

NDMA, NMEA
NDEA, NDPA,
NAZET, NDBA
NPIP, NPYR
NMOR, NHMI
NDMA, NDEA,
NDPA, NDBA
NPIP, NPYR
NMOR, NDBzA
NDBZA, NDBA,
NDEA

glass column 2.7mx2.6mm packed with 15%

Carbowax 20M-TPA on 60-80 mesh Gas Chrom
; He carrier gas 35ml_/min; injector 180C,
TEA furnace 475C, TEA vacuum 0.4 mm;
liquid nitrogen cold trap; column programmed
from 120 to 200C at 4C/min
GC-TEA described at [Pensabene et al., 1992
and 1994]

GC-TEA described at [Pensabene et al., 1994]

supercritical
fluid
extraction

volatile N-nitrosamines and NDBZA analyzed

as described at [Sen et al., 1987 and 1988]
(GC-TEA and GC/MS)

distillation

Pensabene et al.,
1995
Sen et
al., 1992

solid-phase
extraction

24

Table 11:

GCTEA: NOCs in malt and beer

Ref.

Subst.

GC-TEA parameters

Extraction

Am. Soc.
of Brewing
Chemists,
1985

NDMA

A) celite
column
extraction
B) hot
aqueous
extraction

Izquierdo
Pulido et
al., 1995

NDMA

Column: 6 ftx6mm i.d. glass packed with 10%

carbowax 20M + 5% KOH on Anakrom AB, 100120
mesh; Column temperature: 145C, Injection port
temperature: 200C, Carrier gas: H e at 35 ml/min
TEA: furnace temperature: 475C, vacuum with
oxygen: 1,0 torr, Trap temperature: 120 to 130C
Beers were anaslysed for volatile Nnitrosamines
according to the Celite column procedure described by
[Hotchkiss et al. 1981 and Marinelli et al., 1981] the
sample size was increased from 25,0 to 50,0 g.
Detection was by GCTEA [Marinelli et al.,1981].

Table 12:

no details
given

GC-TEA: NOCs in cheese and wheycontaining food

Ref.

Subst.

GC-TEA parameters

Extract.

Dellisanti
et al.,
1996

NDMA,
NDEA,
NDPA,
NPYR,
NMOR,
NBPA

Analysis as described by [Gavinelli et al. 1986 and

1988] modifying it slightly to fit the fatty matrix of
cheese better. Injector Temperature: 200C;
wallcoated fused silica capillary column, CP WAX 52
CB, 25 m 0,32 mm i.d., 1,2 mm film thickness
heated from 60C to 160C at a rate of 25/min,
staying at 130C for 2 min. The GCTEA interface and
pyrolyzer temperatures were 250 and 500C.

simultane
ous
distillation
extraction
procedure

Oliveira et
al., 1995

NDMA,
NDEA
NDPA,
NDBA
NPIP,
NPYR

column: 6ftx6mm, i.d. packed with 15% Carbowax 20M

terephtalic acid (TPA) on Chrom 6080 mesh;
column temperature, 120C/10 min increase to 180C
at 4C/min, and keep at 180C for 30 min; injection
port temperature, 190C; carrier gas H e at 30mL/min;
TEA furnace temperature 475C vacuum with oxygen,
1,0 Torr; trap temperature, 150C.

vacuum
distillation

Table 13:

GCTEA: NOCs in fish and seafood

Ref.

Subst.

GC-TEA parameters

Extraction

Untas et
al., 1990

NDMA

For detection and quantification, portions of the

samples were analysed against an external standard
by injection into a stainless steel column
(1,8mx1,9mm i.d.) packed with 10% Carbowax 20M
and 2% KOH on Chromosorb W (80100) mesh. The
GC was operated sothermally at 100C with a He
carrier gas flow of 25 ml/min. The TEA analyser oven
temperature was 350C.
GCTEA for the analysis of volatile nitrosamines.
Details see [Sen et al 1982].
For NTH Z analysis a GLC column without any added
alkali was used.

extraction
with
methylene
chloride

Sen et al.,
1985

25

Table 14:

GC-TEA : NOCs in food and beverages

Ref.

Subst.

GC-TEA parameters

Extraction

Biaudet
et al 1993

NDMA

Stainless steel column (4.5 mx1/8 in.) packed with

10% (w/w) Carbowax 20M on Chromosorb WAW
(80-100 mesh); Carrier gas: Argon; injection port
temperature: 220C, oven temperature: 180C

mineral oil dist.

(solid samples),
dichloromethane extr.
ChemElut
cartrige (liquid
samples)

Table 15:

GC-TEA: NOCs in cosmetics

Ref.

Subst.

GC-TEA parameters

Extraction

Challis et
al., 1994

NDPA,
NMOR,
NPIP

Capillary GC/TEA isothermaily at 110C on a

BP20 (SGE, 12m 0.33mm i.d.) silica column

dichloromethane

3.3.12. . Gas chromatography-mass spectrometry (GC-MS):

Table 16:

GC-MS: for NOCs in various matrices

Ref.

Subst

Matrix

GC-MS parameters

Extract.

Frassanito
et al.,
1994

NDMA,
NDEA,
NDPA,
NDBA

pesti
cides

SPE

Longo et
al., 1995

NDMA

beer

MS: coupled directly with a GC, eqipped with a

split-splitless injector. El electron energy 70
eV; Capillary column: CP Wax 57CB, 26
mx0,22 mm i.d., film thickness 0,22 urn
Chromatographic conditions: injection
temperature 150 C, oven temperature
program: 50C maintained for 5 min, then
from 50C to 150C, 15C/min, final
temperature maintained for 2 min, carrier gas,
He, head pressure 61 kPa.
GC coupled to an quadrupole MS, equipped
with a high-energy detector (HED) and
operated in the positive-ion CI mode. Samples
were injected via an on-column injector onto a
CPWax 52CB fused-silica capillary column
(25mx0,25mm i.d., 0,2um film thickness)
connected to a deactivated fused-silica tube
(1,5mx0,32mm i.d.) used as a precolumn.
Carrier gas: He, head pressure: 50 kPa.
Initial oven temperature was 35CC for 1 min
followed by a programme rate of 70C/min to

distillation
and
subsequ.
extraction
with
dichloro
methane

26

55DC, a 7min isothermal step, a programme

rate of 3C/min to 70C, then a programme
rate of 20C/min to 180C. The source

Shahidi et
al., 1994

NDMA

nitritefree
cured
muscle
foods

Sen NP et
al., 1985

NDELA,
NHPYR
NSAR,
NPRO
NPIC,
NTCA
NTHZ,
NBHBA
NDMA,
NPYR

fish

Sen NP et
al., 1990

Iceland,
smoked
mutton

temperature was 200C and the filament

emission current and electron energy were
300uA and 150 eV, respectively. Methane was
used as the reagent gas (1.0 torr source
pressure) for chemical ionization.
Quantification was performed by selected-ion
monitoring (SIM) of the [M+H]+ on of NDMA
(m/z = 75,0) and [2H6] NDMA (m/z =81,0).
MS equipped with an electron-impact ionization
source and coupled to a GC
The GC column was similar to that used for
GC-TEA analysis. The MS was operated in the
selective ion monitoring mode for the
molecular ion of NDMA at a resolution of
5000-7000. Operating conditions:
source temperature 250C, emission current
2mA, electron voltage 71 eV; accelerating
voltage 3kV. The GC was operated under
isothermal conditions (115C).
MS equipped with an electron-impact ionization
source and coupled to a GC was used for the
MS confirmation. The GLC column was similar
to that used for GLC-TEA analysis [Sen and
Seaman, 1982].

Both the selected on monitoring technique

(resolution 1 in 8000) and for the identification
of NDMA and NPYR. a repetitive exponential
scanning (full-scan MS) The GLC conditions
and MS parameters used were similar to those
described preciously [Sen et al., 1989]. For
GLC-MS confirmation of NMA in smoked
meat, the selected ion monitoring (SIM)
technique using two fragment ions, namely,
the molecular ion at m/z 136.0637 and the ion
at m/z 106.0657, at a resolution of 8000 (10%
valley definition) and tandem mass
spectrometry (MS/MS) were used. In the
MS/MS mode, the instrument was operated in
the configuration EBQQ (E = electric sector, B
= magnetic sector, and q= quadrupole) [Weber
et al., 1988, Sen et al., 1990]. Argon (at a
total analyzer pressure of 4x10"7 Torr) was
used as the collison gas, and the collison
energy was set at 19 eV. The reaction
monitored were m/z 136->106 and m/z
136->77. The identity of NDMA in one sample
was confirmed by the SIM technique using the
molecular ion at m/z 74.0480.

low
temperature
vacuum
distillation

low
tempterature
vacuum
distillation,
different
extracton
methods
low
temperature
alkaline
vacuum
distillation

27

3.3.1.3. . Gas chromatography - flame photometric detection (GC-FPD):

Table 17:

GC-FPD for NOCs in cigarettes smokes:

Ref.

Subst.

Matrix

GC-FPD parameters

Extraction

Kataoka H
et al.,
1995

NDMA,
NDEA
NPYR,
NPIP
NMOR
NDBA

cigarette
smokes

N-nitrosamines were derivatised as

secondary amines (after denitrosation with
hydrobromic acid) with
diethylchlorothiophosphate to produce the
corresponding Ndiethylthiophosphorylamines.
GC equipped with a flame photometric
detector (P-filter); fused-silica capillary
column (15m 0,53 mm i.d.,1,0 urn film
thickness) of crosslinked DB-1701
Column temperature: programmed from
100 to 260C at 10 min/min;
Injection and detector temperatures:280C
nitrogen flow rate: 10 ml/min

derivatisa
tion of Nnitrosamines

3.3.2. High Performance Liquid Chromatography

Numerous N-nitrosamines were identified on the basis of their retention times
after photohydrolysis followed by the specific detection by Griess reagent [Bellec et
al.,

1995]. The

HPLC-photohydrolysis-colorimetric

method

offers a

powerful

analytical tool for trace analysis of N-nitrosamines that are found in body fluids and
food extracts. Although

the TEA is a highly

sensitive detection

method

for

nitrosamines separated by GC, the HPLC procedure allows a highly specific analysis
of polar N-nitrosamines with good sensitivity. [Bellec et al., 1995]. The HPLC
presents the possibilities of coupling to other detectors such as fluorescence [Sen et
al., 1995], chemiluminiscence [Fu et al., 1992] or electrochemical

[Righezza et

al., 1987] for N-nitrosamines analysis.

28

Table 18:

HPLC parameters for NOCs in meat:

Ref.

NOCs

Injector

Column

Mobile Phase

Detector

Sen et
al. 1990

NMA

Rheo
dyne
sample
loop 20
or 50uL

25x4.1mm i.d.
stainless steel
, LiChrosorb Si
100 (5um)

1% acetone in n
hexane

Sen et
al.1991

HMNTCA,
HMNTHZ

Sen et al
1990

Sen et al 1990

Sen et al 1990

Sen et
al. 1993

HMNTCA,
HMNTHZ,

Sen at al
1992

Sen at al 1992

Sen at al 1992

TEA
furnace
temperature:
550C, cold trap,
mixture of dry ice
and acetone
TEA
derivatisation of
the methyl ester
(treatment with
diazomethane) with
heptaflourobutyric
anhydride
TEA (methyl ester)

Table 19: H PLC parameters for NOCs in beer, wine and gastric juice
Ref.

NOCs

Bellec et
al. 1995

NDMA, NMEA,
NDEA, NMPA,
NMBA, NMtBA,
, NEBA,
NPBA, NDBA,
NDiBA, NMAA,
NDAA, NMBzA,
NDPhA,
NMPheTA,
NPIP, NMOR,
NSAR, NNor,
NPiperaz,
NPYR, NPRO,
NMGua, NNK
NDMA

Righezza
et al. 1987

Sen et
al.1995

NTHCCA.NMT
HCCA

Injector

Column

Mobile Ph.

Detector

Ultraspher ODS
15x0.46cm;
Lichrosher C18,
250x0.4cm;
Nucleosil C18,
25x0.46 cm all
with 5um particle
diameter

variable
mixture of
water with 1 %

photodetector, in
which a knittet
teflon tubig coil
of 6mx0.30mm
i.d. is placed
around a low
pressure UV
lamp emitting at
254nm, 1.2W
capacity, NO
detected at
546nm (Griess*)

Rheodyn
e 7125
injector
with a
20ul
sample
loop

Column:125x
4mm I.D. RP18
(3um);
Hibar column
250x4mm i.d.
LiChrosorb RP
18 (5um)

aqueous
Sodiumhydro
genphosphat
e/acetonitrile

Rheodyn
e injector
sample
loop 20
or 50 ul

stainless steel
column
25cmx46mm i.d.
with a guard
column
Supercosil LC18
phase (5um)

variing
mixture of
acetonitrile,
trifluoroacetic
acid,

(V/V) H 3PO4

or glacial acid
and 5% water
containing
1 % acid

electrochemical
detector:
PTFE tubing
(100cmx 5mm
i.d.) coiled
around a 40W
mercury lamp
connected to an
Eldec 201
fluorescence
detector
exc: 270nm,
emi: 340nm

*Griess reagent:
consists one part 0.1% (w/v) naphtyl ethylene diamine dihydrochloride in distilled water plus one
part 1 % sulfanilamide in 5% concentrated orthophosphoric acid, the two parts being mixed
together each day

29

Table 20:

HPLC parameters for NOCs in cosmetics

Ref.

NOCs

Injector

Column

Mobile Phase

Detector

Billedau et
al. 1994

NDELA,
NMPABAO

50ul loop

cosmetics: Meta
Chem C-8, 5um,
250x4,6m;
NDELA:
Supercosil LC-18,
5um,
250x4.6mm;
NMPABAO
SynChropak
SCD-100
250x4.6mm

NDELA:
5%MeOH-water
(0.05M
ammonium
acetate);
NMPABAO
mixture of
ammonium
acetate,
acetonitrile-water

TEA
with a particle
beam (PB)
interface
operated at 1.5
torr vacuum,
550C pyrolysis
tube

Table 21:

HPLC parameters for NOCs in aqueous or organic solutions

Ref.

NOCs

Injector

Column

Mobile Phase

Detector

Zhou et
al.1994

NDMA,
NPYR,
NDELA,
NPRO

20ul
Rheodyne
loop

5um Nucleosil
C18 column (
250x4.6 mm I.D.

acetonitrile-water
(52:48, v:v)

Fu et al.
1992

NDMA,
NPYR,
NDEA,
NPIP,
NDPA,
NDBA

Rheodyne
injection
valve 20
ul loop

C18 (3um)
analytical column
(83mmx4.6mm
i.d.)

acetonitrile/water
(63.5: 36.5, v:v)
with imidazole
added (3.0 mmol/l)

Gorski et
al.1994

NDMA,
NDPA,
NNDPhA,
NDEAn

Rheodyne
injection
valve

150x4.6mm
Shim pack CLCODS (5um)
100um sample
loop

0.1 M phosphate
buffer, pH1

Lumarin9
derivatization :
Flourescence
detector; exc:
399nm emi:
489nm
chemiluminescence
detector: 40ul
quartz worm
pipe micro flow
cell,
photomultiplier
tube, high
voltage supply,
weak signal
amplifier;
dansyl
drivtes
detected
at 298nm
electrochemical
detector: BAS
three electrode
flow cell, with a
MF 1000 glassy
carbon indicator
(0.07 cm2)

30

3.4. Mass spectrometry for confirmation

While most of the common analytical

methods for nitrosamines use the

thermal energy analyser (TEA) as a selective detector, a subcommittee

of the

International Agency for Research on Cancer (IARC) has concluded that mass
spectrometry

is the most satisfactory

method

available

for the

unequivocal

confirmation of the presence of nitrosamine impurities [Frassantino et al., 1994].

GC-MS has been applied extensively in order to confirm the presence of
NOCs in samples previously tested by means of other techniques. Methods based on
GC-MS have sometimes been used for the quantification

of NOCs on various

matrices. Higher levels of sensitivity to NDMA were achievable

by chemical

ionization (CI) MS in comparison with electron impact ionization [Gaffield et al.,

1976]. Actually, most of the ion current in CI is generally carried by the protonated
molecular ion [M+Hf and few fragment ions are observed. [Longo et al., 1995]. A
method alternative to TEA detection based of GC-isotope dilution (ID) CI-MS has
been described [Longo et al., 1995], Quantification was performed by selected-ion
monitoring (SIM) of the [M+H]+ ion of NDMA and its deuterated analogue.
A fast and sensitive method for routine analysis of nitrosamine impurities,
based of a simple clean-up procedure and on a rapid detection and quantification
by capillary gas chromatography-selected ion recording mass spectrometry with
deuteriom labelled

NDPA has been developed

[Frassanito et al., 1994]. The

sensitivity achieved by this method is lower than 2 ppb. This method positively
compares with the most popular method that use TEA detectors, and provide better
selectivity, since the ion traces are related to the single nitrosamines.

31

4. CONCLUSION
Since the discovery of carcinogenicity

of N-nitrosodimethylamine (NDMA),

extensive work has been performed to determine the role of nitrosamines in causing
human cancer. As a result, nitrosamines have been analysed extensively in food
and beverages. Techniques for the determination of parts per billion levels (ppb =
pg/kg) of volatile nitrosamines in food are well established to-date.
Methods for the determination of NDMA and other volatile nitrosamines are
mainly based on gas chromatography (GC) coupled with a thermal energy analysis
(TEA) detector. The TEA detector is a highly sensitive and selective technique for
NOCs, even though

it also responds to some other nitroso and other

nitro

compounds. Unfortunately, owning to its relatively high costs and limited versatility,
a TEA detector is not available
application

in many laboratories. On the other hand, the

of mass spectrometry (MS) as a detection technique

for GC has

expanded widely in recent years.

Development of analytical methods for nonvolatile nitrosamines, however,
has been slow and restricted primarly to nitrosamino acids, nitrosamines containing
hydroxyl groups, or a combination of those.
Derivatisation of nonvolatile nitrosamines allows the direct analysis by GC
with TEA detection of nitric oxide and confirmation by GC/MS.
Despite the ability to interface a liquid

Chromatograph

to a TEA, this

technique has not been widely adopted for analysis of non volatile nitrosamines in
food products because of problems with aqueous mobile phases.
Very often the lack of information on the identity of nitrosamines has to be
encountered. For this reason, several attempts have been made to determine the
amount of 'total' nitroso groups that will estimate the sum of the volatile and non
volatile nitrosamine contents of food products.

5. ACKNOWLEDGEMENT
The authors are grateful to the librarians Mrs. Unna Cullinan and Mr. Jens Christian
Olesen for the help in bibliographic search.

32

6. GLOSSARY

General:
NOC
VNA
NVNA
SFE:
SPE:
MOD:
LTVD

N-Nitrosocomponds
Volatile N-Nitrosamines
Non Volatile N-Nitrosamines
Supercritical Fluid Extraction
Solid Phase Extraction
Mineral Oil Distillation
Low Temperature Vacuum Distillation

N-Nitrosamines:
HMFuNTZ:
HMNTCA:
HMNTHZ:
MeNTZ:
MNNG:
NAZET:
NAZET:
NAZETC
NBHBA;
NBPA:
NDEA:
NDEAn:
NDELA:
NDBA:
NDiBA:
NDMA:
NDMM:
NDPA:
NDPhA:
NEBA:
:
NG
NGC
NHMI:
NHMTCA
NHMTHZ:
NHMTHZC:
NHPIC
NHPRO:
NHPYR:
NINA:
N MAA:
NMEA:
NMOR:
NMA:
NMA:
NMAMBA:
NMAMP:
NMAPA:
NMBA:
NMtBA:
NMBzA:
NMGua:
NMOCA
NMPA

2-(5-Hydroxymethylfuryl)-N-nitrosothiazolidine
2-(Hydroxymethyl)-N-nitrosothiazolidine-4-carboxylic acid
2-(Hydroxymethyi)-N- nitrosothiazolidine
2-Methyl-N-nitrosothiazolidine
N-Methyl-N-nitro-N-nitrosoguanidine
N-Nitrosoazetidine-2-carboxylic acid
N-Nitrosoazetidine
N-Nitrosoazetidine-2-carboxylic acid
N-Nitrosobutyl-(4-hydroxybutyl)amine
N-Nitrosobutylpropy lamine
N-Nitrosodiethy lamine
4-Nitroso-N-N-diethylaniline
N-Nitrosodiethanolamine
N-Nitrosodibuty lamine
N-Nitrosodiisobuty lamine
N-Nitrosodimethy lamine
N-Nitroso-2,6-dimethylmorpholine
N-Nitrosodipropy lamine
N-Nitrosodipheny lamine
N-Nitrosoethylbuty lamine
N-Nitrosoethylpropy lamine
N-Nitrosoguvacine
N-Nitrosoguvacoline
N-Nitrosohexamethyleneimine
N-Nitroso-2-(hydroxymethyl)thiazolidine-4-carboxylic acid
2-hydroxymethyl-N-nitrosothiazolidine
N-Nitroso-2-(hydroxymethyl) thiazolidine-4-carboxylic acid
N-nitroso-5-hydroxypiperolic acid
N-Nitroso-4-hydroxy proline
N-Nitroso-3-hydroxy pyrrolidine
N-nitrosoisonipecotic acid
N-Nitrosodiamy lamine
N-Nitrosomethylethy lamn
N-Nitrosomorpholine
N-Methylaniline
N-Nitrosomethylacetamide
N-Nitroso-N-methylacetonly-N-3-methylbuty lamine
N-Nitroso-N-methylacetonly-N-2-methylproy lamine
3-(N-Nitroso-N-methylamino) propionic acid
N-N itrosomethylbuty lamine
N-N itrosomethyl-t-buty lamine
N-Nitrosomethylbenzy lamine
N-Nitroso-N-methyl-N-guanidine
N-Nitroso-5-methyloxazolidine-4-carboxylic acid
3-(Methylnitrosamino)propionaldehyde

NMPN
NMTCA
NMTHZ
NMPA:
NMPABAO:
NMPhEtA:
NMTHCCA:
NMU:
NNor:
NNDPhA:
NNK:
NNMG:
NOCA:
NPBA:
NPIC:
NPIP:
NPiperaz:
NPRO:
NPYR:
NSAR:
NTCA:
NTHCCA:
NTHZ:
NTHZC or NTCA:

3-(Methylnitrosamino)propionitrile
N-Nitroso-2-methylthiazolidine-4-carboxylic acid
N-Nitroso-2-methylthiazolidine
N-Nitrosomethylpropy lamine
N-Nirosomethyl-p-amino-2-ethylhexylbenzoate
N-Nitroso-N-methyl-N-phenethy lamine
1 -Methyl-1,2,3,4-tetrahydro--carboline-3-carboxylic acid
N-Nirosomethyiurea
N-Nitrosonomicotine
N-Nitrosodipheny lamine
N-Nitroso-N-methyl-N-[1 -(3-Pyridyl)-1 -butanone]amine
N-Methyl-N'-nitro-N-nitrosoguanidine
N-Nitrosooxazolidine-4-carboxylic acid
N-Nitrosopropylbuty lamine
N-Nitrosopipecolic acid
N-Nitrosopiperidine
N-Nitrosopiperazine
N-Nitrosoproline
N-Nitrosopyrrolidine
N-Nitrososarcosine
N-Nitrosothiazolidine-4-carboxylic acid
2-Nitroso-1,2,3,4-tetrahydro--carboline-3-carboxylic acid
N-Nitrosothiazolidine
N-Nitrosothiazolidine-4-carboxylic acid

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