International proficiency testing of analytical laboratories for foods and feeds from 1990 to 1996: the experiences of the United Kingdom Food Analysis Performance Assessment Scheme.

The Food Analysis Performance Assessment Scheme (FAPAS) organized by a Secretariat of the UK Ministry of Agriculture, Fisheries, and Food has checked the proficiency of analytical laboratories for foods and feeds from 1990 to 1996. FAPAS was started for UK laboratories but was expanded worldwide at the request of analysts in other countries who did not have a home-based scheme. Thirteen thousand homogeneity-checked test materials were issued, covering a very wide range of analytes, including pesticides, toxins, veterinary drug residues, trace and nutritional elements, food colors, preservatives, sweeteners, alcohol congeners, fatty acids, nitrate, and proximate analysis. Participants returned 85% of requested data, and 47,000 z-score proficiency assessments were made, of which 81% were satisfactory. Evidence is presented of improvements in overall analytical ability with increased participation in proficiency testing in the areas of proximate analysis; organochlorine pesticide analysis; and lead, mercury, and acesulfame-K analyses. Little improvement was shown in other analytical areas such as calcium analysis. Overall accuracies for analysis of specific pesticides and specific trace elements in the circulated test materials were compared.

Analytical quality assurance for the WHO GEMS/Food-EURO programme--results of 1993/94 laboratory proficiency testing.

As a means of assessing the performance of European laboratories who contribute analytical data on food contamination to the World Health Organization (WHO) Global Environmental Monitoring Scheme (GEMS), a series of five proficiency testing exercises were carried out during 1993 and 1994. In total 136 laboratories from 21 different countries took part in one or more of the exercises which covered the analysis of trace elements (lead, cadmium and mercury) in milk powder, pesticides (organochlorine, organophosphorus and pyrethroid) in spinach powder, nitrate in spinach powder, aflatoxins in nut-based animal feed and patulin in apple juices. The proficiency testing was carried out according to the ISO/IUPAC/AOAC INTERNATIONAL Harmonized Protocol and laboratories were awarded z-scores signifying their analytical capability based on their reported results for each of the respective exercises. Overall 60% of laboratories were satisfactory for accuracy for trace element analysis, 41% for pesticides, 43% for nitrate, 88% for aflatoxins and 53% for patulin. These results gave an overall poorer performance (68%) than the average for other similar schemes (79%), indicating the need for care in collating data for such programmes as GEMS and the need for remedial measures to assist in improving performance.

Volatile, non-volatile and total N-nitroso compounds in bacon.

Twenty-five smoked and unsmoked fried bacon samples have been analysed by a group selective procedure to measure the concentration of apparent total N-nitroso compounds (ATNC). The levels of a range of individual N-nitroso compounds, including simple volatile N-nitrosamines, N-nitrosothiazolidines, N-nitrosamino acids and N-nitrosothiazolidine carboxylic acids have also been examined. Concentrations of ATNC varied from 430 to 6800 micrograms(N-NO)/kg with a mean of 2700 micrograms(N-NO)/kg. Protein-bound N-nitrosoproline was the most abundant compound detected in unsmoked bacon, mean 260 micrograms/kg, and on average accounted for 4% of the ATNC concentration. For smoked bacon, bound N-nitrosoproline was detected in levels of up to 890 micrograms/kg and contributed 5% to the ATNC total. The most abundant compound present in smoked bacon was N-nitrosothiazolidine-4-carboxylic acid, mean 660 micrograms/kg, and this accounted for 6% of the ATNC. N-Nitrosothiazolidine, mean 340 micrograms/kg, and 2-(hydroxymethyl)-3-nitrosothiazolidine-4-carboxylic acid, mean 180 micrograms/kg, were the next most prominent compounds detected in smoked bacon. The combined sum of all the individual N-nitroso compounds measured accounted for, on average, 16% of the total ATNC. The identities of the N-nitroso compounds comprising the majority of the ATNC in bacon remain unknown.

Endogenous N-nitrosation in man assessed by measurement of apparent total N-nitroso compounds in faeces.

The faecal concentration of substances responding to the chemical test for N-nitroso compounds (apparent total N-nitroso compounds, ATNC) was investigated in human subjects consuming their normal free-choice diet. Concentrations ranged from 40 to 590 micrograms (N-NO)/kg faeces. To ascertain the likely relative contributions of endogenous ATNC formation and preformed, dietary ATNC, the subjects consumed a diet low in nitrate and ATNC for 8 days. At the end of this period, ATNC had decreased substantially with concentrations ranging from below the 40 micrograms (N-NO)/kg detection limit up to 143 micrograms (N-NO)/kg, mean 82 micrograms (N-NO)/kg. On supplementing this diet with 300 mg nitrate/day, faecal ATNC levels increased markedly. On the third day of this regime, values were in the range 73-714 micrograms (N-NO)/kg with a mean of 307 micrograms (N-NO)/kg. The results, together with the known limited occurrence of ATNC in the majority of foodstuffs so far tested, generally non-detectable or less than 100 micrograms (N-NO)/kg, suggest that endogenous formation via species derived from dietary nitrate is likely to be an important source of ATNC in human faeces.

An investigation of the levels of N-nitrosodimethylamine, apparent total N-nitroso compounds and nitrate in beer.

Over 170 retail samples of beer have been analysed for N-nitrosodimethylamine (NDMA), apparent total N-nitroso compounds (ATNC) and nitrate. Levels of NDMA ranged from below 0.1 up to 1.2 micrograms/kg with a mean of 0.2 micrograms/kg. ATNC was detected in 42% of the samples in concentrations of up to 569 micrograms (N-NO)/kg. The levels of nitrate ranged from less than 0.2 up to 143 mg/kg with a mean of 16.8 mg/kg. There was no correlation between the amounts of NDMA and ATNC found in the retail beers. Samples taken during the course of fermentation showed that NDMA was unaffected by the bacterial reduction of nitrate which causes ATNC formation. HPLC studies using a photolysis/chemiluminescence detector revealed that the ATNC in beer are highly polar species of as yet unknown identity.

The determination of nitrate and nitrite in cured meat by HPLC/UV.

A rapid strong anion exchange HPLC/UV procedure has been developed for the determination of nitrate and nitrite in a wide variety of cured meats. The accuracy of this technique has been confirmed by the good agreement achieved with the existing British Standard colorimetric method. The applicability and repeatability of the procedure has been established in a survey of over 200 samples. The agreement between duplicate determinations and their respective means averaged +/- 3.4% for nitrite and +/- 4.3% for nitrate as defined by the term [(a - b)/(a + b)] X 100% where a and b are the repeat determination values.

Examination of some fermented foods for the presence of apparent total N-nitroso compounds.

The fermented foods yoghurt, bread and cheese were analysed for the presence of apparent total N-nitroso compounds (ATNC) by a group-selective procedure involving direct chemical denitrosation and chemiluminescence detection of the released nitric oxide. The levels of ATNC were below the 20 micrograms(N-NO)/kg detection limit in all 20 yoghurts, 23 of the 24 bread samples and 28 out of 31 different varieties of cheese analysed. ATNC were detected in most of those cheese samples manufactured with added nitrate, including Edam, Gouda and Havarti, in concentrations ranging from 30 to 210 micrograms(N-NO)/kg.

Investigation of ethyl carbamate levels in some fermented foods and alcoholic beverages.

An analytical procedure has been developed for the determination of trace amounts of ethyl carbamate in fermented foodstuffs and alcoholic beverages. Concentrations were generally below the 1-5 micrograms/kg detection limit in bread, cheese, yoghurt, beer, gin and vodka. Higher concentrations were found in the other alcoholic beverages examined, which included whisky, fruit brandy, liqueur, wine, sherry and port.

An investigation of the endogenous formation of apparent total N-nitroso compounds in conventional microflora and germ-free rats.

The endogenous formation of apparent total N-nitroso compounds (ATNC) has been investigated in germ-free (GF) and conventional (CV) microflora rats as a function of the drinking-water nitrate concentration. ATNC levels were below the 40 micrograms (N-NO)/kg detection limit in the blood, liver, kidney, spleen and small intestine of all CV and GF rats. For the CV rats ATNC were detected in concentrations of up to 370 micrograms (N-NO)/kg in the large intestine and up to 50 micrograms (N-NO)/kg in the stomach and there was a significant positive correlation between ATNC formation and the drinking-water nitrate level. Comparison of these results with those from GF rats showed that the ATNC in the stomach and large intestine of the CV animals were formed by microbial action, most probably involving bacterial nitrate-reductase activity.

An investigation of apparent total N-nitroso compounds in beer.

The concentration of apparent total N-nitroso compounds (ATNC) in beer has been investigated using a group-selective procedure based on chemical denitrosation with hydrogen bromide and chemiluminescence detection of the released nitric oxide. In a survey of samples of 40 brands of beer and lager, detectable levels of ATNC were present in 17 samples at concentrations of 20-100 micrograms N-NO/kg in 11 and 100-500 micrograms N-NO/kg in six. To determine the origin of ATNC in beer the production of a commercial batch was examined in detail. ATNC levels were below the detection limit in the sweet wort (aqueous extract of malt), bitter wort (malt extract boiled with hops) and also at the start of fermentation, but during the course of fermentation the concentration of ATNC increased appreciably and that of inorganic nitrate decreased; detectable, though transitory, levels of inorganic nitrite were observed. None of the brewing ingredients contained sufficiently high enough levels of ATNC to account for the concentration of these compounds present in the beer after fermentation. These findings suggest that the presence of detectable levels of ATNC in some beers is a result of N-nitrosation reactions occurring in the fermenting wort with the nitrosating species derived from reduction of nitrate, due probably to the presence of microbial species with nitrate reductase activity.

Total N-nitroso group analysis of foods. II. Further studies on the precision and sensitivity of the assay.

The total N-nitroso content of foods can be measured by chemical denitrosation with hydrogen bromide and chemiluminescence detection of the cleaved nitric oxide radical. The denitrosation reagent itself causes a significant detector response which has limited the application of the technique to trace analysis. A procedure is described in which the errors associated with this interference are minimized. Application of this method to the trace analysis of aqueous and solid samples is reported together with an investigation of the effects of sample size on the accuracy and sensitivity of the assay as applied to aqueous analytes. The magnitude and significance of the false-positive response from nitrate is discussed in relation to the analysis of cured meats.

Incidence of some non-volatile N-nitroso compounds in cured meats.

Procedures are described for the use of high-performance liquid chromatography and gas chromatography with a chemiluminescence detector in the analysis of N-nitrosoamino acids, N-nitrosothiazolidine-4-carboxylic acid, N-nitroso-oxazolidine-4-carboxylic acids and N-nitroso dipeptides N-terminal in N-nitrosoproline in cured meat products. The detection limit is around 5-10 micrograms/kg. Evidence is presented for the presence of all these species except the N-nitrosated dipeptides.

The application of a chemical denitrosation and chemiluminescence detection procedure for estimation of the apparent concentration of total N-nitroso compounds in foods and beverages.

The apparent total N-nitroso content of foods can be measured by a procedure based on chemical denitrosation and chemiluminescent detection of the eliminated nitric oxide. Procedures have been established which substantially reduce the 'apparatus blank' response to the denitrosating agent and allow total nitroso contents down to 10 micrograms (N-NO)/kg to be measured reproducibly on a 1-g sample. Typically, duplicate analyses of samples containing 10-1000 micrograms (N-NO)/kg differ by less than 15% of their mean. Potentially the method can be subject to some interference from compounds other than N-nitroso compounds, but at least in some commodities these interfering compounds do not exist in measurable amounts.

N-nitrosamine analysis in foods: N-nitrosoamino acids by high-performance liquid chromatography/thermal energy analysis and total N-nitroso compounds by chemical denitrosation/thermal energy analysis.

The total N-nitroso content of foods can be measured by chemical denitrosation and chemiluminescent detection of the eliminated nitric oxide. Appropriate procedures substantially reduce the 'system response' to the denitrosating agent, so that N-nitroso group contents down to 10 micrograms/kg can be measured on a one-gram sample. Using N-nitrosamine standards added to beer, the coefficients of variation are approximately 10% and 5% at N-nitroso contents of 19 and 94 micrograms/kg, respectively. In cured meats, the coefficient of variation for unidentified N-nitroso compounds is 26% for a 0.3-g sample containing 600 micrograms/kg. Some interference from non-nitroso compounds is possible, but, in some commodities at least, these interfering compounds are not detectable. Conditions have been established that allow measurement of N-nitrosoamino acids in foods using a high-pressure liquid chromatograph interfaced to a Thermal Energy Analyzer, without the need for prior derivatization. After extraction of lipids with hexane, nitrosoamino acids are extracted with ethyl acetate and subjected to appropriate clean-up stages prior to high pressure liquid chromatography on Microbondapak CN with a hexane:ethanol:acetic acid mobile phase and Thermal Energy Analyzer detection. Recoveries from cured meat are in the 55-75% range for N-nitrososarcosine, N-nitrosoproline and N-nitrosohydroxyproline; elution is complete within seven minutes.