EUROPEAN COMMISSION [JOINT RESEARCH

Í 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 3

2. GENERAL ASPECTS 4

2.1. Chemistry, Substances 4 2.2. Formation in food 6

2.2.1. General formation 6

2.2.2. In vivo 7

2.3. Occurrence in food 8

2.3.1. General 8

2.3.2. Examples of N-nitrosamine occurrence in food 9

2.4. Intake 11 2.5. Toxicity and Precautions 12

3. ANALYTICAL METHODS 15 3.1. Classification: volatile and non-volatile N-nitrosamines 15

3.1.1. Volatile N-nitrosamines 16

3.1.2. Non volatile N-nitrosamines 17

3.1.3. Total N-nitrosamines 18

3.2. Clean up/Extraction methods 19 3.3. Identification and Quantification 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 N-

nitroso 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 of

NOCs in foodstuffs presently and during the last years.

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-N-

nitroso 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 analyzed in food

NO—N / '

\

Me

O NO

N-Nitrosodimethylamine

(NDMA) N-Nitroscpiperidine

(NPIP)

N O — Ν

Me

Et

N-Nitrosœthyl-

methylamine (NEMA)

N O — Ν /

M

\

Et

Et

N-Nitrosodiethylamine (NDEA)

N O — Ν

Bu

Bu

N-Nitrosodibutylamine

(NDBA)

O NO

N-Nitrosopyrrolidine (NPYR)

N-N itroso-3-hy cr oxy-

pyrolidine (NHPYR)

O NO

N-Nitrosothiazolidine (NT HZ)

HOCH2 Ό I NO

N-Nitroso-2-hydroxymethyl-thiazolidine(NHMTHZ)

N-Nitroscmorpholine (NMOR)

NO-\

Me

CH2COOH

N-Nitrososarcosine (NSAR)

\ ^ VcOOH ^ N '

I

NO

N-Nitrosoproline (NPRO)

OH

( \cOOH ' N

NO N-Nitroso-4-hydroxy-proline(NHPRO)

\ . / - C O OH

NO

N-Nitrosothiazolidine-4­­carboxyl¡c acid (NTCA)

Me

N­Nitroso­N­(1­m ethyl acetoryl)-2­methyi propylamine (NMAMPA)

C V­COOH

Ν

NO

OOH

N

I NO

N­Nitroso­2­methylthiazolidine­4­carboxyiicacid(NMTCA)

N­Nitrosa o)azolidine­4­carboxylicacid (NOCA)

«­"C / Ό Ο Ο Η HOCH21

"N* I

NO

N-Nitrosa2-hydroxymethyl-thiazolidine-4-caboxylic acid (NHMTCA)

Me COOH

NO

N-Nitroso-5-methyloxazolidine-4-carboxylic acid (NMOCA)

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

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

N02 -+ H+

2HN0 2

RiR2NH + N203

Reaction rate

<-» HNO2

<-> N2O3 + H2O

* * RiR2N-N=0 + HN02

= 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 pH for

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

methylaniline and morpholine, respectively, undergo nitrosation approximately

147.000 and 250 times faster than dimethylamine. 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, α-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 N02, N0X, N203 and

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

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

Food and beverages (in general)

Bacon and edible fats

Cheese, whey

Malt, beer

Cured meat

Sausages

Fish, seafood

Food contact nettings,

Smoked food

Cosmetics

References

Biaudet et al., 1994; Cornee J et al., 1992; Dich et al., 1996; Garcia-Roche et al., 1990; Hotchkiss et al., 1988; Mavelle et al., 1991; Ohta et al., 1990; Penttilä et al., 1990; Satoh et al., 1985; Tutelyan et al., 1990

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; Österdahl et ai., 1990; Sen et al., 1985; Sen et al., 1991; Veccio et al., 1986

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

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

Fiddler et al., 1995; Greenfield et al., 1982; Pensabene et al., 1990; Shahidi etat , 1994

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

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

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

Sen et al., 1993

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 S0 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 [Österdahl 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 N-

nitrosamines 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.

UK

NL

D

F

Japan

S

FIN

NDMA intake in μ9/ρβΓ3οη

0,14a

0,38b

<0,10b

1,1 (men) 0,57 (women) 0,53 (men) 0,35 (women) 0,29 (men)

0,18 (women)

0,26

1,8

0,12

0,08 0,12: 0,18 (men) 0,06 (women)

Major NDMA source in the diet

Cured meats (35%)

Beer (71%)

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

Beer (40%)

Cured meats (18%)

Cured meats (36%), Beer (32%), Fish products (15%)

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

(19%)

Dairy products including cheese (42%),

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

Dried fish (91%)

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

Beer (75%), Smoked fish (25%) Beer (58%)

Salted and smoked fish (22%) Cured meats (20%)

Year

1978

1980 1990

1980 1980 1983 1983 1991

1991

1992

1980

1988

1990

1996

l l

It has been shown that the exogenous exposure to certain volatile

nitrosamines (NVA) and non volatile nitrosamines (NVNA) is decreasing due to

modifications 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

Concentration

Concentration & Intake

Intake

Concentration

Concentration

Intake

Intake

Concentration

Intake

Concentration

Country

Cuba

Finland

Finland

France

France

France

Japan

Japan

USA

USSR

Reference

Garcia-Roche et al., 1990

Penttilae et al., 1990 Dich et al., 1996

Mavelle et al., 199.1

Cornee et al., 1992

Biaudet et al., 1994

Satoh e ta t , 1985

Otah et al., 1990

Hotchkiss et al., 1988

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 Cimetidin were proved to be mutagenic and carcinogenic.

Aminophenazon is metabolized to the highly carcerogenic 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 α-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 NDMA

(HO-CH2)(H3C)N· N=0

^Cytochrom P-450^ ( H 0 - C H 2 ) ( H 3 C ) N - N = 0

α-Hydroxynitrosamine (instable)

-» (H3C)N=N-OH

α-Hydroxy nitrosamine Methyldiazo-

(H3C)N=N-OH Methyldiazo-hydroxide

—»

hydroxide

(N2, OH), H3C+ -» Carbenium-

intermediat

+ H2C=0 Formaldehyd

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 sample

containers can be covered with foil or other suitable material 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

dérivâtes 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 or vacuum). They can be analysed by gas

chromatography (GC) without derivatisation. The most commonly encountered

examples being N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-

nitrosopyrroiidine (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 N-

nitrosohydroxypyrrolidine (NHPYR), N-nitrosated dérivâtes of amino acids (e.g.

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

products of amine acids with aldehydes. The references of studies on NVNAs are

also listed in Table 4.

15

Table 4: References of volatile, non-volatile and total appearent N-nitrosamines

Volatile

Non volatile

Total

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; Pentillä 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., 1990

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

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 non­

volatile 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 dérivâtes 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 dérivâtes [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 nitrosamines, however, has been slow and restricted

primarly to nitrosamino acids, nitrosamines containing hydroxyl groups, or a

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

prepared in order to permit direct analysis by GC with chemiluminescence

detection (TEA) detection of nitric oxide and 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 non­

volatile 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 acid-

labile 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 110°C. 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

Maxwell et al., 1993;

Pensabene et al.,

1995

SPE

Pensabene et al.,

1990, 1991, 1992;

Plypiw etat , 1985

MOD

Canas et al., 1986;

Greenfield et al.,

1982; Hotchkiss et

al., 1985; Ikins et al.,

1986; Veccio et al., 1986

LTVD

Oliveira et al., 1995;

Österdahl et al., 1990;

Sen et al., 1993; Shahidi et al., 1994,

1995

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 N-

nitroso 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 clean up by aluminia or silica gel column

chromatography prior to GC analysis. Further, N-nitrosamines tend to be

decomposed by acid or peroxide produced in the solvents during these pre-

treatments [Takatsuki et al., 1990].

20

GC with electron-capture detection (ECD) based on the conversion of N-

nitrosamines 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 N-

diethylthiophosphoryl (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.

Kuehne et al., 1981

Fiddler et al., 1995

Pensabe­ne et al., 1990

Shahidi et al., 1994

Subst.

NDMA, NPYR, NPIP

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

GC-TEA parameters

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 150°C or stainless steel column 3mx1/8" o.d., 10% diethylenglycol-succinate (DEGS) on Chromosorb W-AW 60/80 mesh, carrier gas He 30ml/min, temperature: 130°C 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 200°C at 4°/min; injector port 200°C; TEA furnace 485°C; TEA vacuum 1,0 mm; liquid nitrogen cold trap.

Column 6ftx1/8in. o.d., Ni tubing packed with 20% Carbowax 20M and 2% NAOH on 80-100 mesh acid-washed Chromosorb P; carrier gas Ar, 30 mL/min; GC oven temperature 170°C, TEA vacuum chamber pressure 1 mm Hg; TEA cold trap, stainless steel trap immersed 1/3 in. liquid nitrogen, recorder 1mV span.

Extraction

vacuum distillation

extraction with acetonitrile

solid phase extraction

low-temperature vacuum distillation

21

Table 7: GC­TEA of NOCs in bacon and its fat

Ref.

Canas et al., 1986

Fiddler et al., 1996

Gloria et al., 1997

Greenfield et al., 1982

Hotchkiss et al., 1985

Ikins et al., 1986

Österdahl et al., 1990

Sen et al., 1982

Subst.

NDMA, NDPa, NPIP, NPYR, NMOR

NKMA, NMEA, NDEA, NDPA, NAZET, NDBA, NPIP, NPyR, NMOR, NHMI

NDMA, NDEA, NDBA, NPIP, NPYR, NTHZ

NDMA, NDEA, NDBA, NPIP NPYR, NMOR

NDMA, NPYR, NDPA, NDMM

NDMA, NPYR, NTHZ

NPYR, NDMA NPIP

NDMA, NDEA NDPA, NDBA

GC-TEA parameters

Column: 2.7x4mm glass column packed with 10 % Carbowax 1540 and 5 % KOH of 100­120 mesh Chrmosorb WHP; 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 Hotchkiss et al. (1980) with the following differences :

column: 2m χ 1.6 mm i.d. packed with 15% Carbowax

20M­terephtalic acid (TPA) on Chromosorb Ρ 60­80

mesh; Column temperature: 120°C for 10 min,

programmed to 180°C at 4°C/min, and maintained at

180°C for 30 min; Injection port temperature: 190°C;

Carrier gas: He 30 mL/min; GC column was interfaced

to a TEA. The TEA trap was maintained at ­150°C with

liquid nitrogen and isopentane, the pyrolysis chamber

was kept at 475°C, 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

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

80­100 Chromosorb WHP, He: 25 mL/min, injector

190°C, column ­150°C; TEA; interface 175°C,

pyrolizer 525°C, trap 150°C, pressure 2.2 mm Hg.

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 180°C at 8°C/min.

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: 160°C ; injector temperature:

210°C, He carrier gas flow about 27 ml/min, furnace

temperature 475°C, oxygen flow about 5 ml/min,

vacuum pressure about 1mm Hg, 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;

Extraction

mineral oil distillation

method comparison

vacuum mineral oil distillation

mineral oil vacuum distillation

mineral oil distillation

mineral oil distillation

vacuum distillation

rapid liquid­liquid extraction

22

Veccio et al., 1986

NAZET NPYR, NPIP NMOR

NDMA, NPIP, NPYR, NTHZ

carrier gas Ar, 30 mL/min; GLC oven temperature 170°C; injector port temperature 220°C; TEA furnace temperature 450°C, 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: injector190°C, column 150°C, interface175°C, pyrolyzer 525°C, Trap -150°C, TEA pressure 2.2mm Hg.

vacuum mineral oil distillation

Table 8: GC-TEA: NOCs in "Frankfurter" sausages:

Ref.

Maxwell et al., 1993

Pensabe­ne et al., 1990

Pensabe­ne et al., 1991

Subst.

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

NTHZC NTHZ

GC-TEA parameters

2,7mx2,6mm glass column packed with 15% Carbowax 20M-TPA on 60-80 mesh Gas Chrom Ρ, He carrier gas 35 mL/min, injector 180°C, TEA furnace 475°C, TEA vacuum 0,4 mm, liquid nitrogen cold trap, and a temperature programme from 120 to 220°C at 4°C/min.

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 220°C at 4°/min; injector 200°C; TEA furnace 450°C; 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].

Extract.

supercriti­cal fluid extraction

solid phase extraction

solid phase extraction

Table 9: GC-TEA: NOCs in other meat products:

Ref. Shahidi et al., 1994

Sen et al., 1990

Pylypiw et al., 1985

Subst NDMA

NDMA, NPYR

NDMA, NMOR, NDPA

GC-TEA parameters Column 6ftx1/8in. o.d., Ni tubing packed with 20% Carbowax 20M and 2% NAOH on 80-100 mesh acid-washed Chromosorb P; carrier gas Ar, 30 mL/min; GC oven temperature 170°C, 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 65°C, GLC temperature 80°C for 2 min, then increased to 135°C at 6°C/min with a hold for 5 min at 135°C, and then increased to 180°C at 10°C/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 190°C at 5.0°C/min; final hold time 1.0 min, total run time 15.2 min; carrier gas He, flow rate 12mL/min, on column injection 250°C, interface line 1/8in o.d. glass-lined stainless steel, 250°C, TEA furnace 525°C, TEA cold trap -151°C, TEA detector pressure 1.4 torr.

Extraction low-temperature vacuum distillation

low temperature alkaline vacuum distillation

distillation-extraction apparatus

Table 10:

Ref.

Marsden et al., 1993

Pensabe­ne et al., 1992

Pensabe­ne et al., 1995

Pensabe-ne et al., 1995

Sen et al., 1992

GC-TEA: NOCs in food packed in nettings

Substance

NDMA, NDEA, NDBA,

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

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

GC-TEA parameters

no further details

glass column 2.7mx2.6mm packed with 15% Carbowax 20M-TPA on 60-80 mesh Gas Chrom Ρ; He carrier gas 35ml_/min; injector 180°C, TEA furnace 475°C, TEA vacuum 0.4 mm; liquid nitrogen cold trap; column programmed from 120 to 200°C at 4°C/min GC-TEA described at [Pensabene et al., 1992 and 1994]

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

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

Extract.

ethanol extraction

solid phase extraction

solid-phase extraction

supercritical fluid extraction

distillation

24

Table 11: GC­TEA: NOCs in malt and beer

Ref.

Am. Soc. of Brewing Chemists, 1985

Izquierdo­Pulido et al., 1995

Subst.

NDMA

NDMA

GC-TEA parameters

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

carbowax 20M + 5% KOH on Anakrom AB, 100­120

mesh; Column temperature: 145°C, Injection port

temperature: 200°C, Carrier gas: He at 35 ml/min

TEA: furnace temperature: 475°C, vacuum with

oxygen: 1,0 torr, Trap temperature: ­120° to ­130°C

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].

Extraction

A) celite column extraction B) hot aqueous extraction

no details given

Table 12: GC-TEA: NOCs in cheese and whey­containing food

Ref.

Dellisanti et al., 1996

Oliveira et al., 1995

Subst.

NDMA, NDEA, NDPA, NPYR, NMOR, NBPA

NDMA, NDEA NDPA, NDBA NPIP, NPYR

GC-TEA parameters

Analysis as described by [Gavinelli et al. 1986 and

1988] modifying it slightly to fit the fatty matrix of

cheese better. Injector Temperature: 200°C;

wallcoated fused silica capillary column, CP WAX 52

CB, 25 m χ 0,32 mm i.d., 1,2 mm film thickness

heated from 60°C to 160°C at a rate of 25°/min,

staying at 130°C for 2 min. The GC­TEA interface and

pyrolyzer temperatures were 250 and 500°C.

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

terephtalic acid (TPA) on Chrom Ρ 60­80 mesh;

column temperature, 120°C/10 min increase to 180°C

at 4°C/min, and keep at 180°C for 30 min; injection

port temperature, 190C; carrier gas He at 30mL/min;

TEA furnace temperature 475°C vacuum with oxygen,

1,0 Torr; trap temperature, ­150°C.

Extract.

simultane­ous distillation­extraction procedure

vacuum distillation

Table 13: GC­TEA: NOCs in fish and seafood

Ref.

Untas et al., 1990

Sen et al., 1985

Subst.

NDMA

GC-TEA parameters

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 100°C with a He

carrier gas flow of 25 ml/min. The TEA analyser oven

temperature was 350°C.

GC­TEA for the analysis of volatile nitrosamines. Details see [Sen et al 1982]. For NTHZ analysis a GLC column without any added alkali was used.

Extraction

extraction with methylene chloride

25

Table 14:

Ref.

Biaudet et al 1993

GC-TEA

Subst.

NDMA

: NOCs in food and beverages

GC-TEA parameters

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: 220°C, oven temperature: 180°C

Extraction

mineral oil dist. (solid samples), dichlorometha-ne extr. ChemElut cartrige (liquid samples)

Table 15: GC-TEA: NOCs in cosmetics

Ref.

Challis et al., 1994

Subst.

NDPA, NMOR, NPIP

GC-TEA parameters

Capillary GC/TEA isothermaily at 110°C on a BP20 (SGE, 12m χ 0.33mm i.d.) silica column

Extraction

dichloromethane

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

Table 16: GC-MS: for NOCs in various matrices

Ref.

Frassanito et al., 1994

Longo et al., 1995

Subst

NDMA, NDEA, NDPA, NDBA

NDMA

Matrix

pesti­cides

beer

GC-MS parameters

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: 50°C maintained for 5 min, then from 50°C to 150°C, 15°C/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 70°C/min to

Extract.

SPE

distillation and subsequ. extraction with dichloro­methane

26

Shahidi et al., 1994

Sen NP et al., 1985

Sen NP et al., 1990

NDMA

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

nitrite-free cured muscle foods

fish

Iceland, smoked mutton

55DC, a 7min isothermal step, a programme rate of 3°C/min to 70°C, then a programme rate of 20°C/min to 180°C. The source temperature was 200°C 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 250°C, emission current 2mA, electron voltage 71 eV; accelerating voltage 3kV. The GC was operated under isothermal conditions (115°C). 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 temptera-ture vacuum distillation, different extracton methods low tempera­ture alkaline vacuum distillation

27

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

Table 17:

Ref.

Kataoka H et al., 1995

GC-FPD for NOCs in cigarettes smokes:

Subst.

NDMA, NDEA NPYR, NPIP NMOR NDBA

Matrix

cigarette smokes

GC-FPD parameters

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

Extraction

derivatisa­tion of N-nitrosamines

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.

Sen et al. 1990

Sen et al.1991

Sen et al. 1993

NOCs

NMA

HMNTCA, HMNTHZ

HMNTCA, HMNTHZ,

Injector

Rheo­dyne sample loop 20 or 50uL

Sen et al 1990

Sen at al 1992

Column

25x4.1mm i.d. stainless steel , LiChrosorb Si­100 (5um)

Sen et al 1990

Sen at al 1992

Mobile Phase

1% acetone in n­hexane

Sen et al 1990

Sen at al 1992

Detector

TEA

furnace

temperature:

550°C, 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: HPLC parameters for NOCs in beer, wine and gastric juice

Ref.

Bellec et al. 1995

Righezza et al. 1987

Sen et al.1995

NOCs

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

NDMA

NTHßCCA.NMT

HßCCA

Injector

Rheodyn e 7125 injector with a 20ul sample loop

Rheodyn e injector sample loop 20 or 50 ul

Column

Ultraspher ODS

15x0.46cm;

Lichrosher C­18,

250x0.4cm;

Nucleosil C­18,

25x0.46 cm all

with 5um particle

diameter

Column:125x

4mm I.D. RP­18

(3um);

Hibar column

250x4mm i.d.

LiChrosorb RP­

18 (5um)

stainless steel column

25cmx46mm i.d. with a guard column

Supercosil LC­18 phase (5um)

Mobile Ph.

variable mixture of water with 1 %

(V/V) H3PO4

or glacial acid and 5% water containing 1 % acid

aqueous Sodiumhydro genphosphat e/acetonitrile

variing mixture of acetonitrile, trifluoroacetic acid,

Detector

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

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, 550°C pyrolysis tube

Table 21:

Ref.

Zhou et al.1994

Fu et al. 1992

Gorski et al.1994

HPLC parameters for NOCs in aqueous or organic solutions

NOCs

NDMA, NPYR, NDELA, NPRO

NDMA, NPYR, NDEA, NPIP, NDPA, NDBA

NDMA, NDPA, NNDPhA, NDEAn

Injector

20ul Rheodyne loop

Rheodyne injection valve 20 ul loop

Rheodyne injection valve

Column

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

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

150x4.6mm Shim pack CLC-ODS (5um) 100um sample loop

Mobile Phase

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

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

0.1 M phosphate buffer, pH1

Detector

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 dérivâtes 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 in many laboratories. On the other hand, the

application 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: ΝΕΡΑ: 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 lamín 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: NMTHßCCA: NMU: NNor: NNDPhA: NNK: NNMG: NOCA: NPBA: NPIC: NPIP: NPiperaz: NPRO: NPYR: NSAR: NTCA: NTHßCCA: 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-ß-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-ß-carboline-3-carboxylic acid N-Nitrosothiazolidine N-Nitrosothiazolidine-4-carboxylic acid

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38