132 Analyst, February, 1967, Vol. 92, $$. 132-136 The Automated Analysis of Nitrite and Nitrate in Blood BY M. H. LITCHFIELD (Imfierial Chemical Industries Ltd., Industrial Hygiene Research Laboratories, Alderley Park, Cheshire) A method is presented in which inorganic nitrite and nitrate are deter- mined automatically in blood. The dialysis unit in the automated system separates nitrite and nitrate from blood protein and thus eliminates any pre- liminary manual de-proteinisation procedure. The diazotisation and coupling reactions involving sulphanilic acid and N- ( 1 -naphthyl) ethylenediamine form the basis of the method. Nitrate is reduced to nitrite by a zinc column incor- porated into the system, and this feature enables a rapid change from nitrite to nitrate analysis and vice ~XYSGC.The method is especially suitable for the analysis of a large number of samples. Recovery from blood is reproducible and almost 100 per cent. The limit of detection is 0-1 rug per ml for nitrite, and 0.2pg per ml for nitrate, and less than 1.0ml of blood is needed for complete analysis. Range expansion can substantially increase the sensitivity if needed. THE main difficulties encountered during tlie determination of nitrite and nitrate in blood arise from the initial procedure for removing protein. Several m e t h o d ~ l ~ ~ J ~ * have been suggested for de-proteinising blood before determining the nitrite and nitrate by colorimetric or polarographic procedures. A critical survey of these methods was undertaken in these laboratories in connection with a programme on the metabolism of organic nitrates by experimental animals. It was found that all of the methods suffered drawbacks, such as poor and erratic recoveries and interference by the de-proteinising reagents on metallic columns, when these were used for the reduction of nitrate to nitrite.Moreover, these methods substantially increased the time required for analysis, and the dilutions involved decreased the sensitivity of detection. The Technicon AutoAnalyzer has been used for determining nitrite and nitrate in water,5,6 and it seemed likely that this instrument would be especially suited for the analysis of these components in blood. The dialysis unit enables separation of the nitrite and nitrate from blood protein without any prior complicating procedure, while reduction of nitrate 15-mm flow cell Recorder Fig.1. Flow diagram for the automatic analysis of nitrite and nitrate in blood ILITCHFIELD 133 can be strictly controlled in the automatic system. It was found that a zinc column reduction leads to a simpler manifold with fewer reagents and a more rapid change-over from nitrate to nitrite analysis than a system involving liquid reductants, such as hydrazine and sodium hydroxide. On these considerations the apparatus was set up as described in the following sectim, based upon the well known diazotisation and coupling reactions with sulphanilic acid and N-( l-naphthyl)ethylenediamine.7 METHOD GENERAL DESCRIPTION- The flow diagram for the AutoAnalyzer system is shown in Fig. 1. The .Ampling rate is 40 per hour with a water wash between samples.The modules shown are s-andard, apart from the system containing the zinc reduction column with a by-pass (Fig. 2), which is- described in greater detail later. REAGENTS- All reagents should be of analytical-reagent grade. Sodium chloride solution-A 0.9 per cent. w/v solution of sod :um chloride in distilled water is used. Sodium acetate huger solution-Dissolve 34 g of soYLium acetate trihydrate, CH3COONa.3H,0, in distilled water and make up to 1 litre. .idd 10 ml of K hydrochloric acid and mix. Check that the pH is 6.0, and adjust if neczssary with hydrochloric acid or sodium hydroxide solutions. Sulphanilic acid solution-Dissolve 10 g of sulphanil;,: acid in 2 N hydrochloric acid. Warm to dissolve, make up to 1 litre with 2 N hydroc1,loric acid and filter.Store in an amber-glass reagent bottle. N-( 1-Naphthy1)ethyZenediamine solution-Dissolve '.d g of N-( 1-naphthy1)ethylenediamine dihydrochloride in distilled water and make up to 1 1:tre. Filter and store in an amber-glass reagent bottle. Stock nitrite solution-Dissolve 16000 g of sodium nitrite in distilled water and make up to 1 litre. This is equivalent to 1000 pg per ml of nitrite as NO,-. Preserve with 1 ml of chloroform and keep the solution in the re'rigerator. Working standard nitrate solutions, wh/,n required, are made up as follows: 1-0, 2.0, 3.0, 4.0 and 5.0ml of stock solution are each diluted to 500 ml with distilled water and are equivalent to 2-0, 4-0, 6.0, 8.0 and lG-Opg per ml of nitrite, respectively.Stock nitrate solution-Dissolve 1.63Vd g of potassium nitrate in distilled water and make up to 1 litre. This is equivalent to 1cdOpg per ml of nitrate as NO,-. Working standard nitrate solutins, when required, are made up as follows: 1.0, 2-0, 3.0, 4.0 and 5-0 ml of stock solutic,i are each diluted to 200ml with distilled water, and are equivalent to 5.0, 10.0, 15.0, LO.0 and 25.0 pg per ml of nitrate, respectively. PROCEDURE- Whole or diluted blood is 3ampled directly from the cups on the sample plate and pumped to the dialyser module togFcher with the diluting saline stream. Nitrite and nitrate pass into the receptor stream CL acetate buffer of pH 6.0, and then to the zinc column by-pass system (Fig. 2) of two 2 way taps joined on one leg with transmission tubing, and on the other leg with transmi;sion tubing filled with zinc clippings, the preparation of which is described later.To de- bu b bler Fig. 2. Zinc reduction column incorporating a by-pass134 LITCHFIELD : AUTOMATED ANALYSIS [Artalyst, VOl. 92 L5’hen nitrite is to be determined, the receptor stream from the dialyser by-passes the zinc reduction column, and is then de-bubbled and pumped back through the manifold, re-aerated, and then mixed with sulphanilic acid for the diazotisation step, followed by the introduction of N-( l-naphthy1)ethylenediamine for the coupling reaction. The colour pro- duced is measured in the 15-mm tubular flow-cell with the 550-mp filter. When nitrate is determined, the taps on the zinc column reduction system are turned so that the stream from the dialyser is passed through the zinc column.Here nitrate is reduced to nitrite, while any nitrite originally present passes through unaffected. The stream is then de-bubbled, thus removing any hydrogen formed in the reduction step, and, as described above, it is pumped back through the manifold and then on for diazotisation and coupling. The colour produced is equivalent to the sum of nitrite plus nitrate. Nitrate can, therefore, be determined by subtraction of the nitrite response recorded previously. Each sample of blood is thus sampled twice if both nitrite and nitrate are to be determined. The zinc column is prepared by cutting AnalaR zinc metal (granulated) into pieces small enough to be inserted into a 30-cm length of transmission tubing.The first clipping is pushed to the centre of the tube by a thin piece of wire or by vibration, and other clippings are added from each end until the tube is filled. The column must be conditioned before use by inserting it into the zinc column reduction system and running the reagents through it for half an hour before any sample is passed. The efficiency of the column remains stable throughout a day’s working, although it is always advisable to include standards among the samples to check this. At the end of a day’s run the zinc column is removed and inserted in the line after the de-bubbler, and reagents are pumped through for 5 minutes until all of the air is removed from the column. The column is then stored, filled with this solution and capped off each end to prevent access of air.If this procedure is followed, little loss of efficiency results before the next day’s run. The column should be conditioned for half an hour, as described previously, before each day’s run. RESULTS AND DISCUSSION RECOVERY OF NITRITE AND NITRATE FROM BLOOD- Standard solutions of nitrite or nitrate were added to whole rat blood, the blood itself never being diluted by more than 10 per cent. with the standard solution. Ten determinations were carried out on a bulk sample at each dilution, and theresults of the analyses, corrected for the dilution of the standard solutions, are tabulated (Table I). TABLE I THE RECOVERY OF NITRITE AND NITRATE FROM BLOOD Nitrite or nitrate added, 0.20 0.50 1.00 2.00 5-00 10.00 25.00 r-lg Per ml Nitrite -A- 7 Recovered, Recovery r-lg Per ml range, mean f S.D.per cent. 80 to 110 88 to 102 92 to 100 92 to 100 93 to 101 94 to 101 0.19 f 0.02 0-48 -_t 0.02 0.96 f 0.02 1.92 f 0.04 4.85 f 0.10 9.80 f 0.21 Nitrate 7 - p -7 Recovered, Recovery r-lg Per ml range, mean f S.D. per cent. 78 to 110 85 to 105 89 to 102 92 to 103 92 to 102 93 to 100 - - 0-47 & 0.06 0.96 f 0.06 1.90 f 0.08 4.85 f 0.18 9.70 f 0-30 24.00 f 0-40 LIMIT OF DETECTION- The limits of detection are 0-1 pg per ml and 0.2 pg per ml for nitrite and nitrate, respectively. The control nitrite content of rat blood could not be detected by this method, i.e., it is less than 0-1 pg per ml. The control nitrate value varied between 0-5 and 1.0 pg per ml. The sensitivity of the method was adequate for the analyses carried out in these labora- tories.The use of the range-expander module was investigated, however, and it was found that even on the times 10 range the base-line was steady, and an over-all increase in sensitivity of some 8-fold could be achieved.February, 19671 OF NITRITE AND NITRATE I N BLOOD 135 Fig. 3 shows the reproducibility of peak heights even when lower concentrations follow higher ones. Fig. 3. Recording of peak heights showing reproducibility of nitrite determinations with standard solutions COLLECTION AND PRESERVATION OF SAMPLES- It should be emphasised that adequate precautions must be taken during collection and storage of specimens of blood for nitrite and nitrate analysis. Nitrite can be rapidly oxidised in blood in vitro, as was shown when rat blood, initially containing 52.5 pg per ml of added nitrite and 1.0 pg per ml of nitrate, was incubated at 37" C.After 1 hour the nitrite had fallen to 6.0 pg per ml and the nitrate value had risen to 64pg per ml, an increase in proportion to the loss of 46-5 pg per ml of nitrite. Storage at 4" to 5" C minimises this oxidation, the loss being less than 10 per cent. of nitrite in 1 hour. We have found oxalate - fluoride anti-coagulant (2 mg of potassium oxalate and 1 mg of sodium fluoride per ml of blood) tends to prevent this oxidation rather better than heparin. In any circumstances blood samples taken for nitrite and nitrate determination should preferably be analysed within the shortest possible time. INTERFERENCE- Organic nitrates are reduced on the zinc column to give positive interference in the nitrate determination.It is necessary to remove free organic nitrates from blood samples when their presence is suspected. Fortunately, tney can be easily removed by extraction with ether. Nitroglycerin, ethylene glycol dinitrate and 1,2 propylene glycol dinitrate can be removed completely in concentrations up to 50 pg per ml by one extraction with 10 ml of ether per ml of blood, while ethylene glycol mononitrate requires two such extractions for complete removal. To check that inorganic nitrate was not removed during these extractions the following experiment was carried out. Nitrate was added to rat blood in a concentration of 50.0 pg per ml, and portions of the blood were taken by two operators, one singly extracting with ether, and the other doubly extracting.Duplicate determinations were carried out by TABLE I1 RESIDUAL NITRATE AFTER ETHER EXTRACTION OF RAT BLOOD CONTAINING 50 pg PER ml OF NITRATE Nitrate, p g per ml Single extraction Double extraction A f \ Operator 1 . . . . 4s.5, 47.5 48.0, 48.0 Operator 2 . . . . 48.0, 45.5 48.5, 48.5136 LITCHFIELD each operator who then changed procedures. The residual extracted samples of blood from each extraction were then analysed for nitrate, and the results are shown in Table 11, from which it will be seen that the loss of inorganic nitrate is within the experimental error of the test. CONCLUSIONS The method described has been used for the analysis of nitrite and nitrate in the blood of experimental animals during metabolism studies of organic nitrates. The analyses of large numbers of samples in the minimum of time has been achieved on small amounts of blood containing a wide range of nitrite and nitrate concentrations. Mr. T. Green provided expert technical assistance in preparing the automatic analytical system. REFERENCES 1. 2. 3. 4. 5. 6. 7. Whelan, M., J . Biol. Chem., 1930, 86, 189. Scott, E. W., and Bambach, K., Ind. Engng Chem. Analyt. Edn, 1942, 14, 136. Hasegawa, H., Sato, &I., Yoshikawa, H., Sakabe, H., Jamaguchi, M., and Hotta, K., Bull. Nat. Cass, L. J., Frederik, W. S., and de Lucia, H., Angiology, 1962, 13, 469. Britt, R. D., jun., Analyt. Chem., 1962, 34, 1728. O’Brien, J. E., and Fiore, J., Wastes Engng, 1962, 33, 128. Shinn, -12. B., I n d . Engng Chern. Analyt. Edn, 1941, 13, 33. Inst. Indust. Health, 1962, 8, 10. Received August 23~d, 1966