The method of Venkataraman et al. (1968) for the direct estimation of acetylisoniazid in

urine has been modified to make it suitable for application to urine specimens containing

sugar. The urine is first extracted with a mixture of chloroform and n-butanol, the

organic phase re-extracted with dilute sulphuric acid, and the original method then

applied to the acid extract. With the modified method, recoveries of acetylisoniazid were

quantitative, both in the presence and the absence of glucose. Interference due to

hydrazones and isonicotinyl glycine was substantially reduced by the modification, while

that due to isoniazid remained the same. There was little or no interference from

isonicotinic acid, with either the original or the modified method

Immanuel, Chandra

Kailasam, S

Kannapiran, M

Nair, N G K

Radhakrishna, S

Raghupati Sarma, G

NIRT Institutional Repository

Indian J Med Res 62, 6, June 1974, pp 945-952A Modified Method for the Estimation ofAcetylisoniazid in UrineG. Raghupati Sarma, Chandra Immanuel, S. Kailasam, M. Kannapiran,N.G.K. Nair and S. RadhakrishnaTuberculosis Chemotherapy Centre, Madras-600031.Received for publication, January 24, 1974.The method of Venkataraman et al. (1968) for the direct estimation of acetylisoniazid inurine has been modified to make it suitable for application to urine specimens contain-ing sugar.The urine is first extracted with a mixture of chloroform and n-butanol, theorganic phase re-extracted with dilute sulphuric acid, and the original method thenapplied to the acid extract.With the modified method, recoveries of acetylisoniazid werequantitative, both in the presence and the absence of glucose. Interference due tohydrazones and isonicotinyl glycine was substantially reduced by the modification, whilethat due to isoniazid remained the same. There was little or no interference fromisonicotinic acid, with either the original or the modified method.With the advent of intermittent regimens in the chemotherapy of tuberculosis,the rate of inactivation of isoniazid (INH) has attained considerable prognosticimportance (Tuberculosis Chemotherapy. Centre, Madras, 1970; 1973). Simplemethods for classifying subjects as slow or rapid inactivators of isoniazid, based on theratio of urinary excretion of acetylisoniazid (Ac. INH) to INH, have been described(Eidus et al., 1971; Venkataraman et al., 1972; Ellard et al., 1973a, b). While thereare several methods for estimating INH in urine, the only known direct methods for esti- mating Ac. INH in urine are those described by Venkataraman et al. (1968) and Ellardet al. (1972).In the course of routine estimations of Ac. INH at this Centre by the directmethod of Venkataraman et al. (1968), it was noticed that a few urine specimens pro-duced a yellow colour instead of the expected pink colour.Preliminary investigationsshowed that these. specimens had been collected from diabetic patients, and that thepresence of glucose ( > 2.5 mg./ml.) in urine significantly interfered with the estimationof Ac. INH. To resolve this difficulty, it was decided to first obtain an acid extractfrom urine (as for the estimation of free INH), and then employ the direct method ofVenkataraman et al. (1968). The findings with the modified method (extractionmethod), both in the presence and the absence of glucose, are described in this paper;information on possible interference from other substances such as INH, isonico-tinic acid (INA), isonicotinyl glycine (INAG), monoacetylhydrazine (MAH), diacetyl-hydrazine (DAH), acid labile pyruvic acid isonicotinyl hydrazone (H-INH-PA) and12 Estimation of Acetylisoniazid in Urinea -ketoglutaric acid isonicotinyl hydrazone (H-INH-KA) is also presented for thismethod, as well as for the direct method.Material and MethodsPure INH and INA were purchased from Bayer & Co. (West Germany) andL. Light & Co. (U.K.), respectively.Ac INH (Fox and Gibas, 1953), H-INH-KA(Dymond and Russell, 1970), and DAH (Turner, 1947) were synthesized as described.MAH was prepared employing a general method for the preparation of acid hydra-zides (Vogel, 1971). H-INH-PA and INAG were gifts from Dr. G.A. Ellard. Allthe other chemicals used were of analytical grade.Direct method :The method described by Venkataraman et al. (1968), withoutprior oxidation using potassium permanganate and without the reverse blank pro-cedure, was employed; however, with a view to extend the range of estimation,0.6 ml. of urine diluted to 1.0 ml. with water was used, instead of 1.0 ml. of urine.Extraction method :The method of Rao et al. (1971) was employed for obtainingan acid extract from urine.Thus, to 3 ml. of urine, 1 ml. of 0.1N sodium hydroxidewas added, followed by 3.2 g. of ammonium sulphate and 30 ml.of a mixture of chloro-form and n-butanol (7 : 3).The contents were shaken on a rotary shaker for 30 min.(the organic extract was clarified, if necessary, with the addition of more ammoniumsulphate), and filtered through cotton wool.Next, 20 ml. of this extract were shakenwith 2.0 ml. of 0.1N sulphuric acid for 15 min. 0.4 ml. of 0.1 N sodium hydroxide wasadded to 0.6 ml. of the acid extract, giving a pH of 5.8±0.2; the Ac. INH contentwas then estimated by the direct method.For routine classification of patients as slow or rapid inactivators of isoniazida larger volume of this extract was required since both Ac. INH and INHhad to be estimated.Consequently, some modifications were introduced in the aboveprocedure.Thus, to 5 ml. of urine, 1.7 ml. of 0.1N sodium hydroxide were added,followed by 5.2 g. of ammonium sulphate and 30 ml. of a mixture of chloroform andn-butanol (7 : 3). The contents were shaken on a rotary shaker for 30 min. Next,20 ml. of this extract (clarified, if necessary) were shaken with 6.0 ml. of 0.1N sulphuricacid for 15 min. and the Ac.INH content determined as described in the aboveparagraph.ResultsRecovery of acetylisoniazid and interference due to glucose :On each of 10 days, AC.INH was set up in concentrations of 0, 5, 10, 20, 30, 40, 50 and 60 µg./ml. in normalurine (from volunteers), and in urine containing a high concentration of glucose(50 mg./ml.) After randomization, the specimens were processed by both the extrac-tion and direct methods and the optical densities determined. Water standardswere also set up in parallel. The geometric means are presented in Table 1.With Ac. INH concentrations of 5 to 60 µg./ml. the findings with the extractionand direct methods were similar in normal urine, and closely resembled those with theG. Raghupati Sarma et al. 3Table I. Optical density readings by direct and extraction methods.Mean optical density* with the followingconcentration of acetylisoniazid (µg./ml.)Meanopticaldensity with5 10 20 30 40 50 60 blanks0.091 0.179 0.359 0.531 0.708 0.919 1.096 0.030Extraction method  0.087 0.173 0.356 0.526 0.716 0.914 1.081 0.032Direct method 0.084 0.172 0.352 0.533 0.711 0.906 1.091 0.070Extraction method   0.085 0.179 0.355 0.532 0.716 0.895 1.074 0.031water standards, indicating that the recovery of Ac. INH was quantitative by both themethods.With blank specimens the mean optical density with the extraction methodwas very similar to that with the water standard, but that with the direct method wasconsiderably higher.The presence of glucose (50 mg./ml.) did not affect the findings with the extrac-tion method. However, with the direct method, the blanks had a mean opticaldensity of 0.250 in the presence of glucose as compared with 0.070 in the absence ofglucose (P<10-4). Further, the mean optical densities (unadjusted for blanks) in thepresence of glucose were 0.258, 0.266, 0.296, 0.315, 0.330, 0.364 and 0.390, respecti-vely, for Ac. INH concentrations of 5, 10, 20, 30, 40, 50 and 60 µg./ml.; thecorresponding values in the absence of glucose were 0.154, 0.242, 0.422, 0.603, 0.781,0.976 and 1.161, respectively.Thus, the presence of glucose resulted in a failure ofthe direct method; this failure persisted even when the reverse blank procedure ofVenkataraman et al. (1968) was employed (unpublished data).Sensitivity : Blank urine specimens from 59 patients were processed by the extractionmethod; all the specimens had yielded a negative result for Ac. INH by the qualitativetest of Eidus and Hamilton (1964).The geometric mean optical density for the waterblanks set up in parallel was 0.034.The corresponding mean for the blank urine speci-mens was 0.035 and the 99 per cent confidence limits were 0.018 and 0.070.The upperlimit corresponds to a concentration of 2 µg./ml., which may therefore be regardedas the sensitivity of the test.Reproducibility : On each of 3 days, Ac. INH was set up in concentrations of 0, 5, 10,20, 40 and 60 ug./ml. in normal urine from volunteers. Four aliquots of each concen-tration were made, all the aliquots randomized and processed by the extraction method,and the optical densities determined.Beer’s law was followed in this range of concen-trations, the correlation between concentration and mean optical density being 0.9997.The coefficient of variation for replicate estimations was 4-6 per cent for concentrationsof 5-10 µg./ml., and 2-3 per cent for concentrations of 20-60 µg./ml.Interference due to various compounds :On each of 5 days (4 only in the case of INH),known concentrations of Ac. INH were added to aliquots of normal urine (from volun-4 Estimation of Acetylisoniazid in Urineteers), together with known concentrations of one of several other metabolites ofisoniazid (Table II).The aliquots were randomized, processed by both the direct andextraction methods, and the optical densities determined.Next, the differences betweenthe actual Ac. INH concentrations added and the observed Ac. INH concentrations inthe presence of different concentrations of each compound were obtained.A po ledregression analysis of ‘the difference’ on ‘the concentration of the compound’ was thenundertaken, to provide an estimate of the proportion of the compound that wasestimated as Ac. INH.This proportion has been termed as ‘Interference’ due to thecompound, and has also been expressed on a molar basis (Table II).Table II. Interference due to various compounds in the estimation of acetylisoniazid by direct&dextraction methods.Compound and concentrations(µg./ml.)Method of Interference,estimating Interference* expressed onAc. INH a molar basis* Expressed as the percentage of the compound that is estimated as Ac. INH.** Concentrations expressed as INH equivalent.++ Concentrations expressed as INA equivalent.The interference due to INH was 7.3 per cent by the direct method and 6.2 percent by the extraction method.Considering next the interference due to hydrazones,the presence of H-INH-KA or H-INH-PA, even in a concentration of 10 µg./ml. (INHequivalent), resulted in significant overestimation of the Ac. INH content by the directmethod.It was computed that 56 per cent of H-INH-KA and 41 per dent of H-INH-PAwere estimated as Ac. INH by this method.With the extraction method, the inter-ference from the hydrazones might be expected to be considerably less because theywould be converted to INH, the interference due to which is relatively small.In theevent, an investigation with H-INH-KA showed that the interference was 6.6 per cent(H-INH-PA could not be investigated due to paucity of the compound).The interference due to INAG was considerably less with the extraction method(3.8 per cent) than with the direct method (13 per cent).As regards INA, the inter-G. Raghupati Sarma et al. 5ference was slight with the direct method (1.1 per cent) and nil with the extractionmethod.There was no interference from MAH or DAH, either with the direct or withthe extraction method.Classification of patients as slow or rapid inactivators of isoniazid : The ratioof Ac. INH to INH in urine collected over the period 3-4 h. following an intra-muscular dose of INH 3 mg./kg. body-weight was determined for 458 patients.AC.INH was estimated by the extraction method, and INH by the method of Rao et al.(1971). The graph illustrates the findings on a logarithmic scale, the logarithmic trans-formation having been employed to obtain approximately the same error variance(that is, the variance between duplicate estimations) for rapid and slow inactivatorsof isoniazid.The distribution is bimodal and suggests that the patients with a ratioof 1.25 or more are rapid inactivators and those with a ratio of 1.24 or less areslow inactivators.Employing this criterion 182 (40 per cent) of the 458 patientswere classified as rapid, and 276 (60 per cent) as slow inactivators.Distribution of 458 patients according to the ratio of acetylisoniazid to isoniazid in urinefor the period 3-4 h. after an intramuscular dose of isoniazid 3 mg./kg. body-weight.Ratio of acetylisoniazid to isoniazidIn a sub-sample of 142 consecutive patients, the Ac. INH was also estimated by thedirect method, and the patients classified as rapid or slow inactivators, the criterion fora rapid inactivator being a ratio of 2.00 or more (Venkataraman et l., 1972). In 141of the 142 patients, the classification was identical by the extraction and directmethods. The lone exception had a ratio of 1.26 by the extraction method (rapidinactivator) and 1.63 by the direct method (slow inactivator); at 4½ h. after theintramuscular test dose, this patient’s serum INH concentration, estimated by the6 Estimation of Acetylisoniazid in Urinespectrophotometric method of Rao et al. (1971), was 0.63 µg./ml. (rapid inactivator).In the 141 patients with an identical classification by the two methods, thediscrimination, assessed as the ratio of the difference in means between rapid andslow inactivators to its standard error, was slightly higher with the extraction method(26.0) than with the direct method (23.7).DiscussionUntil the publication of Ellard et al. (1972), the only method available for thedirect estimation of Ac. INH in urine was that of Venkataraman et al. (1968). Ourobservations have shown that the presence of glucose ( > 2.5 mg./ml.) in urine resultsin the development of a yellow colour with the latter method instead of the expectedpink colour, this possibly being due to a reaction of glucose with the cyanogen chlorideliberated.The presence of a very high concentration of ascorbic acid (10 mg./ml.) hasalso been found to result in a yellow colour (unpublished data).Obviously, in suchcircumstances, estimates of Ac.INH content by this method will not be accurate. Withthe extraction method described in this paper, there was no interference from eitherascorbic acid (unpublished data) or glucose, because neither is extracted into the chloro-form-butanol layer.The recoveries were quantitative both in the presence and absenceof sugar, and Beer’s law was followed over the range 5-60 µg./ml.Finally, the blankreadings were low and similar to those obtained with water, while those with the directmethod were considerably higher.Interference from INH is about the same with the direct and the extraction method,and is of the order of 6-7 per cent.As regards INA, there is little or no interferencewith either method.One method of reducing the interference due to INH, suggestedby Venkataraman et al. (1968), is to oxidize the INH to INA by employing potassiumpermanganate.However, this reaction is highly sensitive, and it is difficult to deter-mine the exact endpoint for the addition of potassium permanganate.Also, thereis the possibility that long exposure to potassium permanganate may oxidize theAc. INH also to INA.The presence of hydrazones in urine leads to considerable over-estimation ofAc. INH by the direct method, as 56 per cent of H-INH-KA and 41 per cent of H-INH-PA are also estimated as Ac. INH.n contrast, the interference is slight (about7 per cent) with the extraction method, as the hydrazones are hydrolysed to INHin the test procedure and the interference due to INH itself is small. The hydra-zones could also be converted to INH by prior acid treatment of the urine, and thedirect method then employed.The presence of INAG in urine also leads to over-estimation of the Ac. INHcontent.However, the proportion of INAG that is estimated as Ac. INH is consider-ably less with the extraction method (4 per cent) than with the direct method (13 percent) probably because of the incomplete extraction of the INAG or because of thehydrolysis of INAG to INA and glycine, neither of which affect the estimation of AC.INH.G. Raghupati Sarma et al. 7It may be concluded that the extraction method is more specific than the directmethod for the estimation of Ac. INH in urine.How ver, it involves an extra step–namely, obtaining the acid extract.In practice, it is not uncommon to estimate thecontent of both Ac. INH and INH for the same urine specimen (for instance, whendetermining the isoniazid inactivation rate from urinary excretions), and to obtain anacid extract for the estimation of INH. In such situations, the extraction methodwould obviously involve no extra labour.Two other methods for the direct estimation of Ac. INH in urine, which can beemployed even in the presence of glucose, have been described by Ellard et al. (1972).One is a fluorimetric method which is highly sensitive (0.2 µg./ml.) but slightly compli-cated, and requires special equipment.The other is a less sensitive (1 µg./ml.) butsimpler calorimetric method, which involves the coupling of the hydrazine side chainwith p-dimethylaminobenzaldehyde and suffers an interference of 43 per cent fromDAH. The extraction method described in this paper depends on the cleavage of thepyridine ring, and consequently suffers no interference from DAH.It involves fewersteps than the calorimetric method of Ellard et a , (1972) but is less sensitive (2 µg./ml). Roth methods,, however, are adequate for purposes of phenotyping patients asrapid or slow inactivators of isoniazid.AcknowledgmentWe are grateful to Dr. S.P. Tripathy, Mr. P.R. Somasundaram, and Dr. G.A.Ellard for valuable suggestions in the preparation of the report, to Mrs. F. William fortechnical assistance, and to Mr. A.S.L. Narayana and Mr. A.M. Ramanathan forassistance with the computations.ReferencesDymond, L.C. and Russell, D.W. 1970. Rapid determination of isonicotinic acidhydrazide inwhole blood with 2,4,6-trinitrobezene sulphonic acid. Clin Chim Acta 27, 513.Eidus, L. and Hamilton, E.J. 1964. A new method for the determination of N-acetylisoniazid inurine of ambulatory patients.Am Rev Resp Dis 89, 587.Eidus, L., Harnanansingh, A.M.T. and Jessamine, A.G.inactivators.Am Rev Resp Dis 184, 587.1971. Urine test for phenotyping isoniazidEllard, G.A., Gammon, P.T., Polansky, F., Viznerova, A., Havlik, I. and Fox, W. 1973a. Furtherstudies on the pharmacology of a slow-release matrix preparation of isoniazid (Smith andNephew HS 82) of potential use in the intermittent treatment of tuberculosis.Tubercle 54, 57Ellard, G.A., Gammon, P.T. and Tiitinen, H. 1973b. Determination of the acetylator phenotypefrom the ratio of the urinary excretion of acetylisoniazid to acid-labile isoniazid.Tubercle 54,201.Ellard, G.A., Gammon, P.T., and Wallace, S.M.acetyisoniazid,1972 The determination of isoniazid and its metabolitesmonoacetylhydrazine, diacetylhydrazme, isonicotine acid andisonicotinylglycine, in serum and urine. Biochem J 126, 449.Fox, H.H and Gibas, J.T. 1953. Synthetic tuberculostats.VIII. Acetyl derivatives of isonicotinylhydrazine. J Org Chem 18, 1375.Rao, K.V.N., Kailasam, S., Menon, N.K. and Radhakrishna, S. 1971. Inactivation of isoniazidby condensation in a syrup preparation.Bull WHO 45, 625, and Indian J Med Res 89,1343.8Tuberculosis Chemotherapy Centre, Madras 1970.A controlled comparison of a twice-weekly andthree once-weekly regimens in the initial treatment of pulmonary tuberculosis.B ll WHO 43, 1431973. A controlled comparison of two fully supervisedonce-weekly regimens in the treatment of newly diagnosed pulmonary tuberculosis.Tubercle 54,23.Turner, R.A. 1947. Reactions of semicarbazones. J Am Chem Soc 69, 875.Venkataraman, P., Eidus, L. and Tripathy, S.P.1968. Method for the estimation of acetylisoniazidin urine. Indian J Med Res 56, 895, and Tubercle 49, 210.Venkataraman, P., Menon, N.K., Nair, N.G.K., Radhakrishna, S., Chandra Ross and Tripathy.S.P. 1972. Classification of subjects as slow or rapid inactivators of isoniazid, based on theratio of the urinary excretion of acetylisoniazid to isoniazid.Indian J Med Res 60, 685, andTubercle 53, 84.Vogel, LA. 1971. A text-book of practical organic chemistry, p. 395, 3rd edition, London:English Language Rook Society and Longman Group Limited.

A Modified Method for the Estimation of Acetylisoniazid in Urine

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A Modified Method for the Estimation of Acetylisoniazid in Urine

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