zinoic

acid in urine using an anion-exchange resin (Dowex-1) have been described.

Recoveries were quantitative, and the sensitivity was 5 μg/ml for ail 3 estimations.

In serum, pyrazinoic acid at a concentration of 50 μg/ml did not interfere with the

estimation of pyrazinamide. In urine, pyrazinamide and pyrazinoic acid could

be separated from each other and estimated even when the 2 compounds were

present together at concentrations of 2000 μg/ml each

Gurumurthy, Prema

Nair, N G K

Raghupati Sarma, G

NIRT Institutional Repository

Indian J Med Res 71, January 1980 pp 129-134Methods for the estimation of pyrazinamide andpyrazinoic acid in body fluidsPrema Gurumurthy, N.G.K. Nair* and G. Raghupati SarmaTuberculosis Research Centre, Madras.Received March 19, 1979; revised article received June 13, 1979.Methods for the estimation of pyrazinamide in serum and of pyrazinamide and pyra-zinoic acid in urine using an anion-exchange resin (Dowex-1) have been described.Recoveries were quantitative, and the sensitivity was 5 µg/ml for ail 3 estimations.In serum, pyrazinoic acid at a concentration of 50 µg/ml did not interfere with theestimation of pyrazinamide. In urine, pyrazinamide and pyrazinoic acid couldbe separated from each other and estimated even when the 2 compounds werepresent together at concentrations of 2000 µg/ml each.Studies on the pharmacology ofpyrazinamide have gained importancein view of the suitability of this drug forshort-course chemotherapy of tubercu-losis1. Pyrazinamide is usually estimatedin body fluids by reaction with alkalinenitroprusside. However, pyrazinoic acid–the principal metabolite of pyrazinamide,also reacts with nitroprusside under theseconditions.For separation of pyrazina-mide from pyrazinoic acid in body fluids,Ellard2 used a solvent extraction proce-dure while Subbammal and associates3used an anion-exchange resin. In thelatter method, described from our Centrefor the estimation of pyrazinamide inserum, the entire eluate is used, and if arepetition becomes necessary, the entireprocedure starting from serum has to berepeated. To overcome this difficulty,we have modified the procedure slightlyand have also extended it to the estima-ti n of pyrazinamide and pyrazinoic acidin urine.Material and MethodsEstimation of pyrazinamide in serum : To3.5 ml of serum was added 1.8 ml of 10per cent trichloroacetic acid and thecontents were stirred and centrifuged.T re  ml of the protein-free supernatewas applied to a column (75 x 6 mm) ofDowex 1- x 8 (Cl-form, 200-400 mesh)and the column was washed with smallmounts of water till 10 ml of the eluatewas collected.Three ml of this eluate wastreat d with 0.5 ml of a freshly preparedsolution of 0.2 per cent sodium nitro-prusside in water followed by 0.5 ml of2N sodium hydroxide solution. The tubeswere left at room temperature for 15 minand the optical densities were recordedat 495 nm in one cm cells in a UnicamSP 600 spectrophotometer.*Present address : Institute for Research in Medical Statistics (Madras Chapter).129130 Pyrazinamide and pyrazinoic acid estimation in body fluidsEstimation of pyrazinamide and pyrazinoicacid in urine : Three ml of urine were app-lied to the column of the anion-exchangeresin and pyrazinamide was eluted fromthe column by washing it with smallamounts of water till 15 ml of the eluatewas collected. For pyrazinoic acid, thecolumn was first washed with 15 ml ofwater followed by small amounts of 0.5Msodium chloride solution till 15 ml of theeluate was collected. Three ml of therespective eluates were treated withalkaline nitroprusside and the opticaldensities recorded as described for serum.ResultsRecovery of pyrazinamide from serum :On each of 5 occasions, pyrazinamidewas set up, in duplicate, in concentra-tions of 0, 2.5, 5, 10, 25, 50 and 100 µg/mlin horse serum. The serum sampleswere randomised and the optical densi-ties were recorded as described before.Another set of pyrazinamide concentra-tions (as above), in duplicate, in horseserum was processed directly, i.e., 3 ml ofthe protein-free supernate (after a 3 in10 dilution) was directly reacted withalkaline nitroprusside solution and theoptical densities recorded. Waterstandards in the concentrations mention-ed were also set up concurrently on eachoccasion and processed directly. Thegeometric mean optical densities arepresented in Table I.Results showed that with the ion-exchange method, the optical density wasproportional to the concentration ofpyrazinamide over the range tested.Comparison with serum and water stan-dards processed directly showed that re-covery of pyrazinamide from serum wasquantitative. The coefficient of varia-tion for replicate estimations with theion-exchange method was 19.5 per centfor a concentration of 2.5 µg/ml and3-5 per cent for concentrations of5-100 µg/ml.Readings obtained with water blankswere much higher than those obtainedwith horse serum, the mean opticaldensities being 0.137 and 0.044, respec-tively.Blank sera from different patients : Bloodwas collected from 15 patients not recei-ving pyrazinamide, serum separated andthe serum samples processed for estima-tion of pyrazinamide. The blank read-ings for the different specimens rangedfrom 0.090 to 0.098, but the differenceswer  not significant statistically (P>0.2).The mean optical density for the 15patients was 0.094, which was consider-ably lower than the mean optical densitywith water standards (0.137) and consi-derably higher than that with horsese um (0.044). This suggests that it isessential to employ human serum blanksfor estimating serum pyrazinamide con-centrations in human subjects. As theblank readings from different patientswer  similar, pooled human serum canbe employed for setting up blanks.Recovery of pyrazinamide and pyrazinoicacid from urine : On each of 5 occasions,pyrazinamide and pyrazinoic acid wereset up, in duplicate, in concentrations of0, 5, 10, 25, 50 and 100 µg/ml in poolednormal urine (from volunteers). Opticaldensities were determined by the ion-exchange method described above as wellas by the direct processing method.The geometric mean optical densitiesre presented in Table II. The resultsindicate that with both pyrazinamide andpyrazinoic acid, Beer’s law is followedGurumurthy et al. 131Table I. Optical density readings with pyrazinamide in horse serum and waterMethodMean optical density* with the following concentrationsof pyrazinamide (µg/ml)2.5 5 10 25 50 100MeaopticaldensitywithblanksHorseserum Ion-exchange 0.020 0.036 0.064 0.139 0.284 0.558 0.046Direct 0.020 0.038 0.068 0.142 0.288 0.555 0.044Water Direct 0.015 0.033 0.062 0.141 0.286 0.562 0.137*After subtracting values obtained with blanks.Table II. Optical density readings with pyrazinamide and pyrazinoic acid in urineMethodMean optical density* with the following concentrationsMeaof pyrazinamide and pyrazinoic acid (µg/ml)opticaldensitywith5 10 25 50 100 blanksPyrazinamide Ion-exchange0.024 0.047 0.111 0.222 0.441 0.015Direct 0.023 0.045 0.108 0.223 0.439 0.033Pyrazinoicacid Ion-exchange 0.022 0.044 0.107 0.210 0.413 0.019Direct 0.022 0.044 0.105 0.211 0.413 0.031*After subtracting values obtained with blanks.over the range 5-100 µg/ml. Comparisonwith urine standards processed directlyindicates a quantitative recovery of boththe compounds from urine. With theion-exchange method, the coefficient ofvariation for replicate estimations at aconcentration of 5 µg/ml was 5.7 per centfor pyrazinamide and 4.8 per cent forpyrazinoic acid,and 3-6 per cent forboth in concentrations of 10-100 µg/ml.Sensitivity of the methods : Sensitivityof the method has been regarded as thelowest concentration where the co-efficient of variation for replicate esti-ma ions is 10 per cent or less. In theestimation of pyrazinamide in serum, thecoefficient of variation was 19.5 per centa a concentration of 2.5 µg/ml and4.8 per cent at 5 µg/ml; in urine, theco fficients of variation at 5 µg/ml were5.7 per cent for pyrazinamide and 4.8 percent for pyrazinoic acid.The sensitivityof all the three methods is, therefore,5 µg/ml.The sensitivity of the method forestimation of pyrazinamide in serum(5 µg/ml) was not a serious limitation asthis concentration is well below theminimum inhibitory concentration ofpyrazinamide, reported to be between9 and 16 µg/ml. in a Tween-free highcitrate medium (pH 5.6)4,5.Interference studiesPyrazinamide in serum : On eachof 3 occasions, pyrazinoic acid in con-centrations of 0 and 50 µg/ml was addedto pyrazinamide concentrations of 0, 10,25 and 50 µg/ml set up in horse serum,and the optical densities determined afterrandomisation of the specimens.Theblank readings for pyrazinamide werethe same both in the absence and in thepresence of pyrazinoic acid (viz.,0.087).The mean optical densities were 0.060,0.142 and 0.274 with pyrazinamide con-centrations of 10, 25 and 50 µg/ml in theabsence of pyrazinoic acid, and 0.070,0.149 and 0.274 respectively, in the pre-sence of pyrazinoic acid 50 µg/ml,demonstrating that pyrazinoic acid didnot interfere with the estimation of pyra-zinamide. However, if a 30 per centsolution of trichloroacetic acid is used,instead of 10 per cent solution emplo-yed here, exchange of pyrazinoic acidisdiminished considerably (unpublishedobservation) as the stronger trichloroace-tic acid is exchanged in preference topyrazinoic acid and the latter is elutedout with water itself.Pyrazinamide and pyrazinoic acid inurine : On each of 4 occasions, pyrazi-noic acid 0 and 2000 µg/ml was addedto pyrazinamide concentrations of 0 and2000 µg/ml in pooled normal urine andthe optical densities determined for boththe compounds after a 3 in 50 dilutionof the respective eluates. Concurrently,dards of 2000 µg/ml in urine were pro-cessed directly after a 3 in 50 dilutionof the samples. The mean optical den-siti s for pyrazinamide 2000 µg/ml aloneand in the presence of pyrazinoic acid2000 µg/ml were 0.592 and 0.590, respec-tively. On direct reaction, pyrazinamide2000 µg/ml gave a reading of 0.593. Themean optical densities for pyrazinoic acid2000 µg/ml alone and in the presence ofpyrazinamide 2000 µg/ml were 0.562 and0.561, respectively.On direct reaction,pyrazinoic acid 2000 µg/ml gave a read-ing of 0.555.These findings demonstratethat in urine, effective separation ofpyrazinamide from pyrazinoic acid wasachieved even when these two com-pounds were present together at concen-trations of 2000 µg/ml each, and alsothat the elution of both was quantitativeeven at these high concentrations.Interference due to some anti-tuberculosis drugs : Streptomycin, p-aminosalicylic acid, thioacetazone, rifam-picin, ethambutol,ethionamide andcycloserine in concentrations up to 500µg/ml did not interfere with the estima-tion of either pyrazinamide or pyrazinoicacid (0, 10 and 25 µg/ml) in urine.Neither did isoniazid, but acetylisonia-zid caused a significant over-estimationof pyrazinamide. For example, acetyl-isoniazid 100µg/ml gave an opticaldensity reading equivalent to that obtain-ed with pyrazinamide 7.5 µg/ml; the esti-mation of pyrazinoic acid was, however,not affected. It is, therefore, necessaryto withhold isoniazid for at least 48 hprior to any pharmacological investiga-tio s with pyrazinamide.DiscussionThe methods described here for the132 Pyrazinamide and pyrazinoic acid estimation in body fluidspyrazinamide and pyrazinoic acid stan-estimation of pyrazinamide and pyrazi-Gurumurthy et al. 133noic acid are fairly simple and quitespecific, as prior separation of the twois achieved with the use of an anion-exchange resin. When an aqueous solu-tion containing a mixture of pyrazinamideand pyrazinoic acid is passed through ananion-exchange resin, pyrazinoic acid isretained on the column and is eluted onlywith a neutral salt solution (0.5 Msodium chloride solution was used in ourstudies).This procedure can also be used toestimate pyrazinoic acid in serum. Pyra-zinoic acid can be eluted from the columnwith 10 ml of 0.5 M sodium chloridesolution following removal of pyrazina-mide with water.The method will haveonly a limited applicability as the sensi-tivity is likely to be 5 µg/ml, and Ellard2has shown that peak serum concentra-tions of pyrazinoic acid did not exceed10 µg/ml in African patients receivingpyrazinamide 3 g.In the estimation of pyrazinamideand pyrazinoic acid reported here, waterblanks gave higher readings than horseserum, human serum or urine blanks.(The lower readings obtained with horseand human sera and urine may be due toan inhibition of the nitroprusside reac-tion due to increasing concentrations ofammonia present in these samples2).Dilution of the samples (or the eluates)with water would therefore result inhigher optical density readings. Whensuch a dilution becomes necessary (forsamples containing high concentrationsof pyrazinamide or pyrazinoic acid), itis essential to dilute the blanks alsosimilarly.In the estimation of pyrazinamideand pyrazinoic acid in urine, it has beenobs rved that a column containing theanion-exchange resin can be re-used atleast 10 times (unpublished observation)a  the resin is regenerated at the time ofelution of pyrazinoic acid from thecolumn with 0.5 M sodium chloridesolution. In the case of the estimation ofpyrazinamide in serum, however, it will benecessary to regenerate the resin by wash-ing it with 0.5 M sodium chloride solutionfollowing elution of pyrazinamide withwater. In routine practice, it is advisableto wash the column with 15 to 20 ml ofwater, after treatment with sodium chlo-ride solution, prior to application of thefresh sample.The solvent extraction proceduredescribed by Ellard2 for estimation ofpyrazinamide in serum is more sensitive(1 µg/ml) than that reported here. Thetwo methods are of the same sensitivityfor estimation of pyrazinamide andpyrazinoic acid in urine. In the proceduredescribed by Ellard2, a slight interferenceof 3.2 per cent due to pyrazinamide inthe estimation of pyrazinoic acid hasbeen reported; also, care has to be takento avoid prolonged contact of pyrazina-mide with alkali. The methods describedby us here have fewer steps. Further,pyrazinamide and pyrazinoic acid do notinterfere with the estimation of eachother in urine; nor does pyrazinoic acidinterfere with the estimation of pyrazina-mide in serum.AcknowledgmentThe authors wish to thank ShriM. Maswood for technical assistance andShri A.S.L. Narayana for computationalassistance.134 Pyrazinamide and pyrazinoic acid estimation in body fluidsReferences1. Fox, W. and Mitchison, D.A. Short-coursechemotherapy for pulmonary tuberculosis.Am Rev Respir Dis 111 (1975) 325.2. Ellard, G.A. Absorption, metabolism andexcretion of pyrazinamide in man. Tubercle50 (1969) 144.3. Subbammal, S., Krishnamurthy, D.V.,Tripathy, S.P. and Venkataraman, P. Con-centrations of pyraxinamide attained in serumwith different doses of the drug. Bull WHO39 (1968) 771.4. Dickinson, J.M. and Mitchison, D.A. Ob-servations in vitro on the suitability of pyra-zinamide for intermittent chemotherapy oftuberculosis. Tubercle 51 (1970) 389.5. Carrada Bravo, T., El lard, G.A. andMitchison, D.A. Reappraisal of the activityof morphozinamide against M. uberculosis.Tubercle 56 (1975) 211.Reprint. requests :The Director, Tuberculosis Research Centre, Spur Tank Road, Chetput,Madras-600031.

Methods for the estimation of pyrazinamide and

pyrazinoic acid in body fluids

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Methods for the estimation of pyrazinamide and pyrazinoic acid in body fluids

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