Analysis of volatile N-nitrosamines in alcoholic beverages

Research Section ANALYSIS OF VOLATILE N-NITROSAMINES IN ALCOHOLIC BEVERAGES E. U. Nnv

Et~qhd

Imriturejor

Lijii

and D. H.

GOFF

Scirt7ces.

125 Secor7d

(Received

FINE Aver7ur.

Wulrhan7,

MA

02154,

USA

4 Ju17e 1979)

Abstract-A rapid extraction procedure for the analysis of volatile N-nitrosamines in alcoholic beverages is described. The technique involves liquid-liquid extraction, using dichloromethane, on a Preptube followed by concentration of the eluate and analysis on a gas chromatograph interfaced to a Thermal Energy Analyser. Detection limits, based on a 15-ml sample, vary from 0.2 &itre for Nnitrosodimethylamine (NDMA) to 05 &litre for N-nitrosopyrrolidine. Volatile nitrosamines were not found in any of the wines, sherries, liqueurs, brandies, gins, vodkas and rums that were analysed. All I8

brands of domestic and imported beer contained NDMA, at levels rangmg from 0.4 to 7.0 &litre and six out of seven brands of Scotch whisky were also shown to contain NDMA, at levels between 0.3 and 2.0 pg/litre. INTRODUCllON

There have been numerous reports suggesting an association between the consumption of alcoholic beverages and cancer in man (McGlashan, 1969; Rothman, 1975; Williams & Horm, 1977). The strongest associations are for cancer of the mouth, pharynx and larynx (Pell & D’Alonzo, 1973; Schmidt & delint. 1972), oesophagus (Gsell & Loffler, 1962; Wildner & Gibel, 1966; Wynder & Bross, 1961), and liver (Leevy, Gellene & Ning, 1964; Lee, 1966). The possibility that the causative agent is a trace contaminant has been considered by many workers (Bogovski. Walker. Castegnaro & Pignatelli, 1974; Rothman, 1975). N-Nitrosamines are of special interest in this respect since a number of them are known to induce cancer of the oesophagus in animals. In 1968. using polarography and thin-layer chromatography, McGlashan, Walters & McLean claimed to have found hi-nitrosodimethylamine (NDMA)

at levels

up to 2000 pg/litre

in home-brewed

Zambian spirits. Subsequently McGlashan, Patterson & Williams (1970) showed that furfural would have interfered in the method and the findings were shown to be false by gas chromatography-mass spectrometry (GC-MS). Using an improved GC-MS method, sensitive at the 100 pg/litre level, Collis, Cook, Foreman & Palframan (1971) found no evidence of the presence of volatile nitrosamines in similar spirits from East Africa. Using thin-layer chromatography and a GC method capable of detecting 25 pg/litre, Sen & Dalpe (1972) could find no evidence of N-nitrosamines in a number dizing

of beers, whiskies, the N-nitrosamine

sherries to the

or rums. N-nitramine,

By

oxiand

then using GC-electron capture. Castegnaro, Pignatelli & Walker (1974) developed a method sensitive to I pg nitrosamine/htre and using this NDMA was detected in 14 out of 30 farm-produced apple cider distillates at levels between 1 and 10 pg/litre (Bogovski et ul. 1974). Using the clean-up procedure of Sen & Dalpe (1972), followed by GC-MS Bassir & Maduagwu (1978) found 0.5 pg NDMA/litre in a sample of Nigerian palm-wine. They also confirmed the finding by GC-MS. 569

Spiegelhalder, Eisenbrand & Preussmann (1979) distilled beer samples from mineral oil (Fine, Rounbehler & Oettinger, 1975) and then, using GC interfaced to a Thermal Energy Analyser (TEA), showed that I I I out of 158 German beer samples were contaminated with NDMA at mean levels of between 0.2 and I I.2 pg/litre. Their results were confirmed by GC-MS. We report here on a much simplified analytical technique involving a single step liquid-liquid extraction on a Preptube, followed by concentration and analysis by GC-TEA. The method, sensitive at the 0,2pg/litre level for NDMA. was used to screen a wide variety of popular US and imported alcoholic drinks. EXPERIMENTAL Materiuls. Liqueurs, wines, whiskies, brandies, beers, etc., were purchased from liquor stores in Massachusetts and at state liquor stores in New Hampshire. The seals on all bottles were broken just before analysis. Standard nitrosamine solutions and Preptubes were obtained from the Analytical Services Laboratory of Therm0 Electron (Waltham, MA). The organic solvents were glass-distilled and were obtained from Burdick and Jackson (Muskegon, MI). Appuratus. Gas chromatographic analyses were carried out using an isothermal GC (Model 661, Therm0 Electron Corp., Waltham, MA), interfaced to a TEA analyser (Model 502LC, Therm0 Electron Corp.). The GC column was a 14-ft stainless steel tube (outer diameter l/8 in.), packed with 10% Carbowax 20 M containing 0.5% KOH on Chromosorb WHP SO/l00 (Analabs, New Haven, CT). The GC was operated at I25 and 15O”C, with a helium carrier-gas flow of 50ml/min. The TEA analyser oven temperature was 450°C. The high-pressure liquid chromatographic (HPLC) analyses were carried out using a Varian 8500 LC pump (Varian Instrument Division, Palo Alto, CA) interfaced to a TEA-502. The LC cohnnn (3.2 x 250 mm) was 10~ Lichrosorb Si60 (Altex Corp., Berkeley, CA). The solvent system was acetoneisooctane (7:93, V/v).

570

E. U. Table 1.

Defection limits nitrosqmines

(signal 10 noise recovered from

N-Nitrosamine

FINE

ratio = 3:/) of sewn alcoholic beverages

uolutile

N-

Detection limit (dlitre)

Recovery efficiency %

0.2 02 0.4 0.5 0.5 0.5 0.5

45-62 47-68 57-77 61-81 67-82 62-83 69-97

N-Nitrosodimethylamine N-Nitrosodiethvlamine N-Nitrosodiprdpylamine N-Nitrosodibutylamine N-Nitrosopiperidine N-Nitrosopyrrolidine N-Nitrosomorpholine Procedure. Samples containing more than 20% (v/v) alcohol, were diluted with distilled water before analysis. Dilution was necessary to retain the alcohol on the Preptube. Samples with less than 20% (v/v) alcohol were analysed without prior dilution. The Preptube was pre-wetted with 10 ml dichloromethane and 15ml aliquots of the samples were loaded onto it. The samples were eluted with 5 x 10 ml dichloromethane and the eluates were collected in a KudernaDanish evaporator, to which 0.3ml isooctane was added as a keeper, and the volume was reduced to approximately I ml in a water bath at 55°C. Aliquots (2Od) of each sample were then analysed by GCTEA. NDMA-positive samples were confirmed using two methods. Firstly, the retention time for NDMA was confirmed using HPLC. Secondly, CC-high resolution MS was used with the magnetic analyser set to resolve the exact mass of NDMA (C,H,N,a74.0480; resolution 10,000, peak matched against a perfluoroalkane standard), from other background materials. Detection of an exact mass fitting the composition for the molecular-ion of NDMA, combined with the coincident retention time, confirmed the presence of NDMA (Spiegelhalder et al. 1979). Recovery studies were conducted by spiking the liquor samples with 5 pg/litre of each of the following N-nitrosamines: NDMA; N-nitrosodiethyl-

Table

D. H.

and

GOFF

amine (NDEA); N-nitrosodipropylamine (NDPA); N-nitrosodibutylamine (NDBA); N-nitrosopiperidine (NPIP); N-nitrosopyrrolidine (NPYR); N-nitrosomorpholine (NMOR). RESULTS

AND

DISCUSSION

The range of recoveries and the detection limits (at a 3 : I signal to noise ratio) for the seven nitrosamines in the spiked liquor samples are listed in Table I. The GC-TEA chromatogram of a wine sample to which a mixture of these nitrosamines (5 pg/litre of each) had been added is shown in Fig. 1. The 71 different brands of alcoholic beverages which were analysed are listed in Table 2. NDMA was present in all the beers at levels from 0.4 to 7,0pg/litre, and in six out of the seven Scottish whiskies at levels from 0.3 to 2.Opg/ litre. Typical CC-TEA chromatograms of a Scotch whisky and a beer sample are shown in Figs 2 and 3, respectively. Volatile nitrosamines were not found in any of the wines, sherries, liqueurs, brandies, gins, vodkas and rums that were analysed. To ensure the NDMA was not being formed as an artefact during extraction and/or analysis, three experiments were carried out. Firstly, when excess sulphamic acid and a-tochopherol were added to the beer and whisky samples, no decrease in the measured

2. N-Nitrosodimethylamine

content

of alcoholic

beverages

NDMA pg/litre Type

Country of origin

Alcohol content (%)

GC-TEA

HPLC-TEA

Wine

Pink White Sauterne Dry white table Red Chianti Rose Fruity Rose White Rose Dessert White dinner White White Rose Red Rose White sparkling Red Bordeaux White Burgundy

USA CA, USA CA, USA CA, USA NY, USA CA. USA CA, USA CA, USA NY, USA CA, USA Ontario. Canada W. Germany W. Germany Italy Italy Portugal Portugal France France NY,

13.7 12.5 Il.5 12.5 12 I1 11.5 12 20 19 13 IO IO 9 9 12 12 II.5

12

ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND (Continued

on next

page)

N-Nitrosamines in alcoholic beverages

571

Table 2. (Continued)

Type

Country of origin

Alcohol content (%)

NDMA pg/litre GC-TEA

HPLC-TEA

Sherry

Dry Pale dry Orange (dessert wine)

Spain NY, USA CA, USA

Coffee Orange Orange peel Orange

Jamaica USA MA, USA France

Dry Dry Dry

Gin MA, USA London, England London, England

Grape Cognac Grape Blackberry

CA, USA France Greece Holland

Grain Grain Grain

PA. USA CT; USA CT, USA

Light, dry Dark heavy bodied Light

Puerto Rico Virgin Islands Virgin Islands

Blended Blended Sour mash Blended Sour mash Blended Blended Blended Blended Blended Blended Blended Blended Blended

IL. USA IN, USA TN, USA CA, USA TN, USA Canada Ireland Scotland Scotland Scotland Scotland Scotland Scotland Scotland

Light Light Light Light Light Light Light Dark Dark Dark Lager Dark ale Light Light Light Light Dark Dark

France Philippines Japan Greece Holland Holland Holland W. Germany Mexico Ireland Australia United Kingdom USA USA USA USA USA USA

20 18 I9

ND ND ND

32.5 30 26 40

ND ND ND ND

40 47 47.3

ND ND ND

40 40 42 35

ND ND ND ND

40 50 40

ND ND ND

40 40 40

ND ND ND

45 40 45 43 45 43.4 43 43.5 43 43 43 43 43 43.4

ND ND ND ND ND ND ND 0.4 0.3 2.0 1.2 1-2 1.7 ND

ND ND 2.8 I .o I.3 1.7 ND

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

0.6 3.4 3.7 0.5 5.2 3.1 0.7 5.3 0.4 0.6 1.8 6.4 7.0 I.8 0.9 4.4 1.4 3.1

0.6 3.6 3.7 ND 4.7 3.2 0.8 4.8 ND I.0 I 23 7.0 6.1 1.7 04 4.0 0.9 3.0

Liqueur

Brandy

Vodka

Rum

Whisky

Beer

NDMA = N-Nitrosodimethylamine ND = Not detected (less than 0,2pg/litre) NC = Not calculated GC-TEA = Gas chromatography-Thermal Energy Analysis HPLC-TEA = High-pressure liquid chromatography-Thermal Energy Analysis

512

E. U. GOFF and D. H. FINE

f

(bl

Fig. 1. GC-TEA chromatogram of a sample of wine to which 5 pg/litre each of NDMA. NDEA. NDPA. NDBA. NPIP, NPYR and NMOR had been added. NDMA levels was observed, indicating that nitrosation was not occurring during analysis. Secondly, the addition of excess piperidine to the beer and whisky samples did not lead to the production of NPIP. Thirdly. synthetic beverage solutions (3% and 20%

1 lb1

-e-r+-+ T,me.mln

Fig. 3. (a) GC-TEA chromatogram of a 2.3 ng NDMA standard. (b) Typical GC-TEA chromatogram of a beer sample, showing the presence of 7 pg NDMA/litre. ethanol in distilled water) did not contain NDMA (at a detection limit of @2 pg/litre). The levels of NDMA in beer detected in this study are similar to those reported by Spiegelhalder et al. (1979) in German beers. Similar results were also obtained by E. A. Walker (personal communication 1979) in samples of US and European beers. The presence of NDMA in Scotch whisky has not previously been reported, but since both beer and Scotch whisky are made from barley. this finding is not surprising. False-negative results using the GC-TEA have not been reported (Fine, 1979; Fine & Rounbehler, 1975; Cough, Webb, Pringeur & Wood, 1977; Havery, Fazio & Howard, 1978; Krull & Fine, 1979). It is therefore concluded that in wines, sherries, liqueurs, brandies, gins, vodkas and rums, volatile N-nitrosamines cannot be considered as possible causative agents in alcohol-related cancers.

Ac~rlo,~,ledynflenrs-We

--b -Tkb Time,

mn

Fig. 2. (a) CC-TEA chromatogrom of a 1.5 ng NDMA standard. (b) Typical CC-TEA chrornatogram of a Scotch whisky sample. showing the presence of I.7 erg NDMA ‘litre.

thank

Ernest

Walker

of

IARC.

Lyon. France and Gerhard Eisenbrand. Bertold Spiegelbalder and Rolf Preussmann of the lnstitut fiir Toxikologie und Chemotherapie am Deutschen Krebsforschungszentrum, Heidelberg, Federal Republic of Germany for access to their data prior to publication. We also thank Jim Coombs for technical assistance. The GC-high resolution MS was performed by Bertold Spiegelhalder. This work was supported by the National Science Foundation under Grant No. ENV75-20802. Any opinions. findings. conclusions and recommendations expressed are those or the authors and do not necessarily reflect the \~ews of the National Science Foundation.

N-Nitrosamines

in alcoholic

REFERENCES

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