A flow-injection spectrophotometric method for nitrate and nitrite determination through nitric oxide generation

A flow injection method with novel spectrophotometric detection for the determination of nitrite and nitrate in foodstuffs is presented. The method is based on the reduction of nitrite and nitrate to nitric oxide, with subsequent reaction with iron (II) and thiocyanate in an acid medium, forming FeSCNNO⁺. The absorbance of the complex, with a maximum at 460 nm, is proportional to the nitrite and nitrate concentrations. The NO is generated in two stages: (1) reduction of nitrate to nitrite in a cadmium copper reductor column and (2) reduction of the nitrite to NO in a sulfuric acid medium. The influence of reagent concentrations and manifold parameters were evaluated. Nitrite and nitrate can be determined in the range of 0.30–3.00 and 1.00–10.00 mg l⁻¹, respectively. The sampling rate of analyses was 30–40 h⁻¹ and, considering a sample of 5.0 g, the determination limit of the method was 20 and 13 mg kg⁻¹ of nitrate and nitrite, respectively. Nitrite and nitrate were determined in vegetables and meat products by the proposed method. The precision and accuracy of the proposed method were comparable to those of the reference spectrophotometric method (official AOAC reference method for the determination of nitrate in foodstuffs).     

Time-dependent depletion of nitrite in pork/beef and chicken meat products and its effect on nitrite intake estimation

The food additive nitrite (E249, E250) is commonly used in meat curing as a food preservation method. Because of potential negative health effects of nitrite, its use is strictly regulated. In an earlier study we have shown that the calculated intake of nitrite in children can exceed the acceptable daily intake (ADI) when conversion from dietary nitrate to nitrite is included. This study examined time-dependent changes in nitrite levels in four Swedish meat products frequently eaten by children: pork/beef sausage, liver paté and two types of chicken sausage, and how the production process, storage and also boiling (e.g., simmering in salted water) and frying affect the initial added nitrite level. The results showed a steep decrease in nitrite level between the point of addition to the product and the first sampling of the product 24 h later. After this time, residual nitrite levels continued to decrease, but much more slowly, until the recommended use-by date. Interestingly, this continuing decrease in nitrite was much smaller in the chicken products than in the pork/beef products. In a pilot study on pork/beef sausage, we found no effects of boiling on residual nitrite levels, but frying decreased nitrite levels by 50%. In scenarios of time-dependent depletion of nitrite using the data obtained for sausages to represent all cured meat products and including conversion from dietary nitrate, calculated nitrite intake in 4-year-old children generally exceeded the ADI. Moreover, the actual intake of nitrite from cured meat is dependent on the type of meat source, with a higher residual nitrite levels in chicken products compared with pork/beef products. This may result in increased nitrite exposure among consumers shifting their consumption pattern of processed meats from red to white meat products.     

Effect of adding smoke-flavouring to frankfurters on nitrite and nitrate levels

Different concentrations of sodium nitrite, potassium nitrate or both together were used to prepare four standard solutions and four frankfurter formulations. These were elaborated with and without the addition of 2 g/kg of a commercial solid smoke-flavouring preparation. Monitoring of nitrite and nitrate residual levels in standard solutions stored at 3 °C showed that smoke-flavouring addition results in a depletion of nitrite levels and/or reduction of nitrate to nitrite. In a much more complex system such as meat products, nitrite and nitrate, incorporated in the formulation separately or combined, were also rapidly depleted when smoke-flavouring was added.     

Modification of the ion exchange HPLC procedure for the detection of nitrate and nitrite in dairy products

Existing ion exchange HPLC methodology for nitrate and nitrite analysis in cured meat products suffers from high analyte variability at low concentrations and also chromatographic interference by artifacts in some other foods, such as dairy products. An investigation into the sources of variability has shown that both the cyclohexyl solid phase extraction cartridge and the glass fibre filter used in the original method can introduce artifacts which interfere with the determination of the nitrate in foodstuffs. We have also found that the use of a graphitized solid phase extraction cartridge used in tandem with the cyclohexyl solid phase extraction cartridge removed the artifacts from the chromatograph of dairy products that co-eluted with nitrite and nitrate. Values for the nitrite and nitrate content of dairy products were obtained by the HPLC procedure using these two solid phase extraction cartridges and the values obtained were in close agreement with those obtained by cadmium column reduction and colorimetry.     

Simultaneous Determination of Nitrite and Nitrate in Milk Samples by Ion Chromatography Method and Estimation of Dietary Intake

The presence of nitrate and nitrite in foods may be considered hazardous after ingestion in the gastrointestinal tract due to their reaction with naturally occurred secondary amines to form potentially carcinogenic nitrosamines. Due to this fact, a new method was developed in this study for the simultaneous determination of nitrite and nitrate in milk samples using by ion chromatography. Proposed mobile phase composed of sodium hydrogen carbonate and sodium carbonate (1.0 and 3.2 mmol/L) with a flow rate of 0.7 ml/min. The average recoveries for nitrate and nitrite were higher than 86 and 88%, respectively. The limit of detection for nitrate and nitrite were 0.24 and 0.09 mg/L, respectively. The results of 102 real milk samples showed nitrate was found in all of the samples (100%) with a mean of 34 ± 11 mg/L, while nitrite was found in none of the samples. The mean intake of nitrate in all age groups was lower than World Health Organization guideline. The present assessment concludes that the maximum contaminant level was equal to 82.8 mg/L nitrate. This method was fast, sensitive and accurate and is capable of being an alternative method in food control laboratories for investigation of nitrite and nitrate content. This is the first study of the determination and survey of nitrite and nitrate and exposure assessment of the Iranian population to nitrite and nitrate level in milk, which was widely used in infants and adolescents as one of the basic food components.     

Determination of nitrate and nitrite in dairy samples by sequential injection using an in-line cadmium-reducing column

A sequential injection analysis (SIA) system for the spectrophotometric determination of nitrate in dairy samples was developed. A test portion was aspirated into a carrier solution containing ethylendiaminetetracetic acid (EDTA) and ammonium buffer, which flowed into a copperized cadmium reduction column installed in-line for the determination of the nitrate plus nitrite contents of samples. For the nitrite determination, another test portion of sample was aspirated and directly sent to the detector without reduction. Nitrate content was calculated from the difference between nitrate plus nitrite (expressed as nitrite) and nitrite content. The spectrophotometric determination is based on the Griess reaction. The proposed method was used to test several dairy samples (ultrapasteurized milk with 1.7% milk fat, whey, raw bovine milk and several cheese varieties). Results were statistically in good agreement with those provided by the reference procedure, with a detection limit of 0.15 mg L⁻¹. A sampling rate of 21 determinations per hour can be achieved with this procedure.     

Corn leaf nitrate reductase--a nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h(-1), sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite bythe enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete overthe entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously bythe two methods was not significantly different from zero at the p = 0.05 level.     

Metabolism of nitrate in fermented meats: the characteristic feature of a specific group of fermented foods

Within the universe of food fermentation processes the multi-purpose use of nitrate and/or nitrite is a unique characteristic of meat fermentations. These curing agents play a decisive role in obtaining the specific sensory properties, stability and hygienic safety of products such as fermented sausages, ham and, more recently, emulsion type of sausages. The use of nitrate is the traditional method in curing processes and requires its reduction to reactive nitrite. Thus, nitrate reduction is the key event that is exclusively performed by microorganisms. Under controlled fermentation conditions starter cultures are used that contain staphylococci and/or Kocuria varians, which in addition to strongly affecting sensory properties exhibit efficient nitrate reductase activity. To obtain clean label products some plant sources of nitrate have been in use. When producing thermally treated sausages (e.g. of emulsion type), starter cultures are used that form nitrite before cooking takes place. Staphylococci reduce nitrite to ammonia after nitrate has been consumed. K. varians is devoid of nitrite reductase activity. Nitrate and nitrite reductases are also present in certain strains of lactobacilli. It was shown that their application as starter cultures warrants efficient activity in sausages made with either nitrate or nitrite. NO is formed from nitrite in numerous chemical reactions among which disproportionation and reaction with reductants either added or endogenous in meat are of practical importance. Numerous nitrosation and nitrosylation reactions take place in the meat matrix among which the formation of nitrosomyoglobin is of major sensory importance.Safety considerations in meat fermentation relate to the safe nature of the starter organisms and to the use of nitrate/nitrite. Staphylococci (“micrococci”) in fermented meat have a long tradition in food use but have not received the QPS status from the EFSA. They require, therefore, thorough assessment with regard to toxigenicity and pathogenicity determinants as well as presence of transferable antibiotic resistance. Nitrate and nitrite are still considered basically undesired in food. The main objections are based on their potential to form nitrosamines with carcinogenic potential. In view of new results from intensive research of NO, potential risks are opposed by positive effects on human health.     

The oral nitrate and nitrite intake in The Netherlands: evaluation of the results obtained by HPIC analysis of duplicate 24-hour diet samples collected in 1994.

In spring and autumn of 1994 duplicates of 24-h diets were collected from 123 respondents. One of the goals of this study was to determine the amount of nitrite and nitrate in the duplicates of 24-h diets to establish the oral daily intake of these analytes. For this purpose an HPIC/UV method for the determination of nitrate and nitrite in duplicate diets was developed and validated. The sample preparation procedure was derived from the in-house method used for the determination of nitrate and nitrite in human blood plasma. The sample is diluted with water, deproteinized with Carrez reagent, followed by chromatographic clean-up on an SPE C18-column. Both the nitrate and the nitrite results are quantitative. The recovery for nitrite was on average 104% (n = 21, spiking levels: 0.84-95 mg/kg) and for nitrate on average 103% (N = 21, spiking levels: 1.8-404 mg/kg). Samples of duplicates of 24-h diets were analysed according to the method developed. The median intake of nitrite calculated from the samples collected in spring 1994 was 0.6 mg/person day (range < 0.1-6.1 mg/person/day). For the samples collected in autumn 1994 these figures were < 0.2 mg/person/day (range < 0.1-16 mg/person/day). The mean intake of nitrate was 73 mg/person/day (range 7-322 mg/person/day) in spring 1994 and 87 mg/person/day (range 1-310 mg/person/day) in autumn 1994. The overall mean intake of nitrate in 1994 was 80 mg/person/day. The daily intake for nitrate was higher than that found in the duplicate diet study carried out in 1984/1985, when an average daily intake of 52 mg/person was measured. The intake of nitrite was also higher than found in the duplicate diets collected in 1984/1985. The findings of the study are discussed in the context of the ADI for nitrate and nitrite as well as the outcome of other recent European intake studies.     

离子色谱法同时测定烟草中的硝酸盐与亚硝酸盐

采用离子色谱建立一种烟草及烟草制品中硝酸盐和亚硝酸盐的同时测定方法。在超声条件下,用水萃取试样中的硝酸盐和亚硝酸盐离子,萃取液经阴离子交换色谱柱分离,用电导检测器检测,外标法定量。结果发现:该方法在测试范围内线性良好;硝酸盐的检出限为1.06 mg/kg,回收率在96.3%～99.0%之间,亚硝酸盐的检出限为1.23 mg/kg,回收率在96.1%～101.6%之间;相对标准偏差均小于5%;且操作简单、高效、重复性好、检出限低、样品回收率高,能满足当前烟草中硝酸盐和亚硝酸盐的同时快速检测要求。      

烤烟中硝酸盐和亚硝酸盐的测定

本文提出了一种使用紫外分光光度计测定硝酸盐和亚硝酸盐的方法。主要过程包括将烟样在弱碱性条件下热水中萃取,亚硝酸盐标准曲线的绘制,亚硝酸盐含量的测定,硝酸盐的还原,硝酸盐含量的测定,回收率的测定。该方法具有简单、高效、重复性好、不需专用仪器设备等特点,适用于各种烟草样品中硝酸盐和亚硝酸盐含量的测定。      

食品加工处理对银耳中硝酸盐和亚硝酸盐含量的影响

采用盐酸萘乙二胺比色法检测了银耳中硝酸盐和亚硝酸盐的含量,同时也比较了不同储存温度下不同处理方法对硝酸盐和亚硝酸盐的影响。结果表明,室温和4 ℃条件下储存48 h后,银耳所含的亚硝酸盐含量分别由25.43 μg/g增加至128.11 μg/g和98.64 μg/g,硝酸盐含量分别由458.24 μg/g降低至364.64 μg/g和394.11μg/g;而经巴氏灭菌后,亚硝酸盐增加至39.76 μg/g和43.59 μg/g,硝酸盐则降低至430.52 μg/g和438.17 μg/g。煮沸30 min后,硝酸盐和亚硝酸盐分别由458.24 μg/g、25.43 μg/g降低至89.09 μg/g、6.50 μg/g。食品加工处理可以降低银耳中硝酸盐和亚硝酸盐的含量。     

Exposure estimates of nitrite and nitrate from consumption of cured meat products by the U.S. population

The dietary exposures of nitrite and nitrate from consumption of cured meat products were estimated for the U.S. population aged 2 years and older, and children aged 2 to 5 years, using both 2-day food consumption data from the publicly available combined 2009–2012 National Health and Nutrition Examination Survey (NHANES) and 10–14-day food consumption data from the 2009 and 2012 NPD Group, Inc. National Eating Trends-Nutrient Intake database (NPD NET-NID), and residual nitrite and nitrate levels in cured meat products available from the recent American Meat Institute Foundation/National Pork Board (AMIF/NPB) national market survey of the nitrite and nitrate levels in cured meat products in the U.S.A. The dietary exposure for consumers of cured meat products (eaters-only) was estimated at the mean and 90th percentile for three exposure scenarios: low exposure, average exposure, and high exposure, to account for the range in the amount of nitrite and nitrate in a given cured meat product category. In addition, a cumulative exposure that takes into account all cured meat product categories containing nitrite and nitrate was determined, and the relative percent contribution of each cured meat product category to the cumulative exposure was estimated. Cured, cooked sausages and whole-muscle brine-cured products were the two major contributing categories to dietary exposure of nitrite and nitrate for both U.S. population aged 2 years and older and children aged 2–5 years.     

Determination of nitrates in milk by ion-chromatography

A method is described for the determination of nitrates in cow milk, human milk, milk powder or milk-based infant formulae using liquid chromatography on Spheron DEAE and a direct photometric detection (205 nm). The influence of removing proteins by precipitation with Carrez reagent on the accuracy of determination was studied. The proposed method gives identical results with the reference method (photometry after reduction of nitrate to nitrite) but is more rapid. Its limit of determination is 0.5 mg NO3-/l of milk, its reproducibility is 4% (relative standard deviation).     

Nitrate and nitrite quantification from cured meat and vegetables and their estimated dietary intake in Australians

High dietary nitrate and nitrite intake may increase the risk of gastro-intestinal cancers due to the in vivo formation of carcinogenic chemicals known as N-nitroso compounds. Water and leafy vegetables are natural sources of dietary nitrate, whereas cured meats are the major sources of dietary nitrite. This paper describes a simple and fast analytical method for determining nitrate and nitrite contents in vegetables and meat, using reversed-phase HPLC-UV. The linearity R² value was >0.998 for the anions. The limits of quantification for nitrite and nitrate were 5.0 and 2.5 mg/kg, respectively. This method is applicable for both leafy vegetable and meat samples. A range of vegetables was tested, which contained <23 mg/kg nitrite, but as much as 5000 mg/kg of nitrate. In cured and fresh meat samples, nitrate content ranged from 3.7 to 139.5 mg/kg, and nitrite content ranged from 3.7 to 86.7 mg/kg. These were below the regulatory limits set by food standards Australia and New Zealand (FSANZ). Based on the average consumption of these vegetables and cured meat in Australia, the estimated dietary intake for nitrate and nitrite for Australians were 267 and 5.3 mg/adult/day, respectively.     

Factors controlling the growth of Clostridium botulinum types A and B in pasteurized cured meats VI. Nitrite monitoring during storage of pasteurized pork slurries

Residual nitrite levels were monitored during storage for up to 6 months, in a model pork slurry system used to study the relative effects of curing ingredients and additives used in pasteurized cured meats to control the growth of Clostridium botulinum.
In 'low' pH slurries the rate of loss of nitrite fell with reducing storage temperature. Less residual nitrite remained after HIGH heat treatment but the rate of loss of that residual nitrite was slower during storage than nitrite remainly after LOW heat treatment. Inclusion of nitrate resulted in higher residual nitrite levels, particularly after HIGH heat and if stored below 20°C. If isoascorbate was added nitrite became undetectable within circa 30 days, even when nitrate had been added. The rate of loss of nitrite was slower in 'high' pH slurries (pH 6.3–6.8).
Monitoring levels of nitrite in the product soon after production would detect its accidental overuse but monitoring nitrite in the product during distribution or at retail, without knowledge of the composition and prior history of the product, gives little indication of the amount used at manufacture. The level of residual nitrite was not directly related to the ability of the curing mixture to control the growth of CI. botulinum types A and B. Some slurries in which C1. botulinum grew least during 6 months' storage contained no residual nitrite because isoascorbate was also present.     

A Micro-Flow-Batch Analyzer Using an In-line Cadmium Sponge Microcolumn for the Photometric Determination of Nitrate and Nitrite in Dairy Samples

In this study, a micro-flow-batch analyzer (μFBA) using an in-line cadmium reduction microcolumn for the photometric determination of nitrate and nitrite in dairy samples is described. The method is based on the Griess-Llosvay reaction and measuring of the absorbance at 540 nm using a green LED integrated into the μFBA built in the urethane-acrylate resin. Initially, the nitrite content of the dairy sample is analyzed in the mixing chamber, while the nitrate is reduced to nitrite in cadmium sponge microcolumn coupled to the microsystem. Nitrate content was calculated from the difference between nitrate plus nitrite (expressed as nitrite) and nitrite content. The analytical curve for nitrate and nitrite was linear in the work range of 10.0–100.0 μg L^?1 with a correlation coefficient of 0.992 and 0.998, respectively. The limit of detection and relative standard deviation were estimated at 0.39 μg L^?1 and <1.7 % (n?=?5) for nitrite and 0.41 μg L^?1 and <1.3 % (n?=?5) for nitrate. Comparing with the reference methods, no statistically significant differences were observed when applying the paired t test at a 95 % confidence level. The accuracy was assessed through recovery test (97.7 to 102.9 %). The proposed microsystem-employed in-line cadmium sponge microcolumn presented satisfactory portability, robustness, flexibility, low-cost device, and reduced chemicals consumption compared to recent methods. Thus, μFBA is potentially useful as an alternative for other automatic determinations using in-line pretreatment steps.     

Validation of EN ISO standard methods 6888 Part 1 and Part 2: 1999--enumeration of coagulase-positive staphylococci in foods

The methods of European and International Organisations for Standardization for the enumeration of coagulase-positive staphylococci (CPS, Staphylococcus aureus and other species) described in EN ISO 6888 Part 1 and Part 2: 1999 were validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). EN ISO 6888-1 prescribes the use of Baird-Parker (BP) agar whereas EN ISO 6888-2 prescribes the use of Rabbit Plasma Fibrinogen Agar (RPFA). The objective was to determine the precision of each method in terms of repeatability (r) and reproducibility (R) using three different food types inoculated with various levels of S. aureus and a typical background flora. The results are intended for publication in the associated standards. Cheese, meat and dried egg powder were examined by 24 laboratories from 16 countries in Europe. Each participant received eight test materials per food type: blind duplicates at four inoculum levels (0, 10(3), 10(4) to 10(5), 10(5) to 10(6) cfu/g). In addition, two reference materials (RM) (capsules containing milk powder inoculated with S. aureus) were included in the study. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. Two statistical methods were used to calculate the precision parameters. Draft EN ISO 16140: 2000 method appeared more appropriate to the case of microbiological data than ISO 5725-2: 1994 method and was retained to calculate the precision data.Concerning EN ISO 6888-1, overall values for repeatability (r) when used with food test materials was r=log(10) 0.28 (expressed as an absolute difference between log(10)-transformed test results). For the reference materials, r=log(10) 0.19. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.43. For the reference materials, R=log(10) 0.39.Concerning EN ISO 6888-2, overall values for repeatability (r) when used with food test materials were r=log(10) 0.22. For the reference materials, r=log(10) 0.17. Overall values for reproducibility (R) when used with food test materials were R=log(10) 0.33. For the reference materials, R=log(10) 0.31.These results were presented to the ISO technical committee and to the Comité Européen de Normalisation (CEN). Both committees agreed to incorporate the precision data obtained with food materials as two amendments to EN ISO 6888-1 and -2, and to give an equal status to each part of the standard.     

Survival of Listeria innocua in dry fermented sausages and changes in the typical microbiota and volatile profile as affected by the concentration of nitrate and nitrite

The involvement of nitrate and nitrite in the formation of N-nitrosamines in foods is a matter of great concern. This situation has led to revise the real amount of nitrate and nitrite needed in meat products to exert proper technological and safety activities, and also to extensive research to find alternatives to their use. The present study addresses the possibility of reducing the ingoing amounts of these additives below the legal limits established by the current European regulations. Different concentrations of nitrate and nitrite were tested on Spanish salchichón-type dry fermented sausages concerning their role in the microbiota and volatile profile. Sausages were manufactured with the maximum ingoing amounts established by the EU regulations (150 ppm NaNO₃ and 150 ppm NaNO₂), a 25% reduction and a 50% reduction; control sausages with no nitrate/nitrite addition were also prepared. The mixtures were inoculated with 5 log cfu/g of Listeria innocua as a surrogate for Listeria monocytogenes. L. innocua numbers in the final product were approximately 1.5 log cfu/g lower in the batch with the maximum nitrate/nitrite concentration when compared to 25 and 50% reduced batches, and about 2 log cfu/g in comparison to the control sausages. The final numbers of catalase-positive cocci were 1 log cfu/g higher in the 50% nitrate/nitrite reduced batch and 2 log cfu/g higher in the control sausages, compared to products manufactured with the maximum nitrate/nitrite concentration. This increase was related to a higher amount of volatile compounds derived from carbohydrate fermentation and amino acid degradation. Sausages with no addition of nitrate/nitrite showed higher amount of volatiles from lipid oxidation. Enterobacteriaceae counts reached detectable values (1-2 log cfu/g) in both nitrate/nitrite reduced sausages and in the control batch, while these organisms were not detected in the batch with the maximum ingoing amount. Nitrate and nitrite exerted a significant effect on the typical microbiota of dry fermented sausages and effectively contributed to control Listeria. These considerations should be taken into account in view of a future restriction in the use of these curing additives.     

A rapid method for the determination of nitrate and nitrite by chemiluminescence

Nitrite and nitrate + nitrite can be determined by selective chemical reduction to nitric oxide which is measured using a chemiluminescence analyser. The reducing agents are sodium iodide in acetic acid for nitrite and ferrous ammonium sulphate-ammonium molybdate for nitrate + nitrite. The concentrations of the reducing agents have been optimized to obtain the maximum yield of nitric oxide and the minimum coefficient of variation. Under these conditions, it is possible to inject repeated samples into the refluxing reducing agents and to obtain rapid evolutions of nitric oxide from which the determinations can be made. Nitric oxide has also been produced using the nitrite reagents from organic nitrites, a S-nitrosothiol, a pseudonitrole and N-nitrosamines. Similarly, an organic nitrate and some C-nitroso compounds respond to the method for nitrate but only to the extent of a yield of nitric oxide of about 10% of the theoretical. Very low or zero responses were evident from aliphatic and aromatic C-nitro compounds but not omega-N-nitroarginine which gave a large yield of nitric oxide using the reagents for nitrate. In general, however, concentrations of nitrate will be in considerable excess of those of related compounds which would interfere with the determinations. Nitrate can be determined either by difference in its mixtures with nitrite or by prior removal of the nitrite using ascorbic acid provided oxygen and nitric oxide are removed by degassing with nitrogen.