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A study was made of 82 possible interferences in the spectrophotometric determination of nitrite by the diazotizationcoupling technique using three composite reagents [sulphanilamide and N-( 1 -naphthy1)ethylenediamine (NED); sulphanilic acid and NED; and 4-nitroaniline and NED]. The effects of interferences when using these three reagents differ considerably in many instances. Large amounts of inert salts, such as alkali metal and ammonium sulphates, chlorides and nitrates cause high results with sulphanilic acid -NED reagent and high or low results with sulphanilamide -NED and 4-nitroaniline -NED reagents owing to the Bronsted salt effect. Carbonate, hydrogen carbonate, phosphate, pyrophosphate, tetraborate, acetate, tartrate and oxalate salts can interfere by changing the acidity of the solution or by their buffering action. This interference is less with the 4-nitroaniline -NED method than the other two methods. Silver, mercury(l), bismuth, antimony, lead, barium, strontium and calcium can interfere by precipitating as chloride, oxychloride, oxysulphate or sulphate. Chromium(lll), nickel and cobalt interfere by reason of the colour of their ions.Oxidising and reducing agents usually produce low results. Aluminium, manganese, zinc, cadmium, tin(lV) mercury(ll), titanium(lV), zirconium(lV), iron(lll), copper(ll), molybdenum(Vl), tungsten(Vl1, arsenic(Vl), uranium(Vl), thallium(l), gold(lll), platinum(lV), selenium(lV), bromide, iodide, thiocyanate, cyanide, cyanate, hexacyanoferrate(lI1) and -(ll), fluoride, perchlorate and silicate can interfere in diverse ways.

The problem of the interference of ascorbic and isoascorbic acids in the spectrophotometric determination of nitrite by the diazotisationcoupling technique is important because these substances are frequently added to cured meats containing nitrite. The chemistry of the reaction of ascorbic and isoascorbic acids with nitrite is discussed. The effect of pH on the reaction at room temperature and on digestion at 80°C was investigated. Two composite diazotisationcoupling reagents [sulphanilamide and N-( 1-naphthy1)ethylenediamine;4-nitroaniline and N-(1-naphthy1)ethylenediaminel were used in this investigation. At room temperature 1 mg of ascorbic or isoascorbic acid (per 50 ml) interfered only slightly or moderately at pH 10 and 12 but interfered very significantly at pH 8 and below. When solutions at pH 8, 10 and 12 were heated at 80°C for 1 h, satisfactory recovery was obtained in the presence of 2.5 mg of ascorbic or isoascorbic acid (per 50 ml). A study of the stability of pure nitrite solutions indicated that solutions at pH 6.5, 8, 10 and 12 show no decomposition after standing at room temperature for 24 h. The solutions at pH 8, 10 and 12 showed no significant decomposition on heating at 80 "C for 2 h. A tentative method is proposed for the determination of nitrite in cured meats containing ascorbic or isoascorbic acid, whereby a slurry of 5 g of the sample in 0.01 M sodium hydroxide is digested at 80°C for 90 min, zinc sulphate is added as a clarifying agent, the volume is made up to 250 ml, the solution is filtered and nitrite in an aliquot of the filtrate is determined.

1083V L = 0.02 ml. of a 0.2% solution = 0.04 mg. Substit,uting: where these are significantly greater than the 2 to 3% variation attributable to the disks themselves. 811 1As the degree of purity of the original enzyme preparation 22 4 30 0 04 ultimately determines the activity of the disks, preparations I ' = -X -X -= 3.01 micromoles/minute/mg. Table I11 summarizes the specific activities as calculated for each of the methods above together with the standard deviation to be expected for each met,hod of assay. The variability found for each method as calculated from the ratio of the standard deviations t o the mean specific activit,y is wit'hin a few per cent in every instance.STABILITY O F CHOLISEBTER.4SE DISKS UPON STOR.4GE. The stabilit,y of the disks prepared in the various media was studied by determining the activity of freshly made disks by one cr more of the methods previously described; t'hen st,oring similar diska under the conditions and for the time noted in Table IV followed by a det,ermination of their activity.Inspection of Table I T T shorn that optimal retention of initial activity i.5 achieved by storing in the cold over a desiccant. Disks decrease t o 62% of t,heir initial act,ivity in 22 days when exposed t o an atmosphere of 100% humidity a t room temperature.The effect of the stabilization inediuni is shown by a coniparison of the disks prepared in the bovine albumin n-ith those prepared in the gelatin medium and stored under identical conditions. The disks prepared in the 47, bovine albumin had lost virtually no activity in 6 months, whereas those prepared in the gelatin medium had lost one fourt,h of t,heir initial activity. DISCUSSIONThe conditions used in the four methods for measuring the cholinesterase activity of the disks differ in salt concentration, presence of protein stabilizer, substrate concentration, and period of time used in measurement,. Because these factors all affect the stability and activity of the enzyme, it is to be expected that the specific cholinesterase act,ivit)-of a given preparation, meaeured by different procedures but expressed in the same units, will vary from one method to the nest. However, the variability of 2 to 3% found for the activity of the disks by any of the procedures described here permits their use in calibrating the activity measured bjone met,hod in terms of another, provided that a given set of conditions is consistently followed. I n addition, the disks may serve to point out sources of experimental variations other than the one used in this study will yield different specific activities. Standardization of different techniques may nevertheless be achieved with any such preparation.Under the conditions specified in this report the titrimetric procedure gave the highest specific activity, followed in turn by the colorimetric, manometric, and electrometric procedures.The last named method has the advantage of requiring the fewest reagents, and permitting inany samples t o be measured with a minimum of manual operations, thus minimizing the ...

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