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Rapid determination of isonicotinic acid hydrazide in whole blood with 2,4,6-trinitrobenzenesulphonic acid
CLINICA CHIMICA ACTA
513
RAPID DETERMINATION OF ISONICOTINIC ACID HYDRAZIDE IN WHOLE BLOOD WITH 2,4,6-TRINITROBENZENESULPHONIC ACID
L. C. D Y M O N D ^,~D D. W. R U S S E L L
Department of Biochemistry, Dalhousie University, Halifax, N.S. (Canada) (Received November I I, J969)
SUMMARY
(I) A spectrophotometric method for tile quantitative determination of isonicotitfic acid hydrazide (INH) in whole blood is described. (2) The method involves reaction of INH with 2,4,6-trinitrobenzenesulphonic acid and extraction of the coloured product into methyl isobutyl ketone. The absorbance of the extract at 500 nm is proportional to INH concentration up to 30 /~g/mi. (3) Streptomycin and known metabolites of INH do not react, but p-aminosalicylic acid should be absent.
INTRODUCTION
Isonicotinic acid hydrazide (INH), seventeen years after its introduction into medicine, is still one of the most useful among a handful of effective antitubercular drugs. Its metabolism in the human body is unusual; among the population at large there is a bimodal distribution of the rate of INH disappearance from the circulation, so that individuals may be classed as slow or fast inactivators. IN H inactivator status, which is genetically determined t, has implications for IN H therapy. Thus, slow inactivators might be more likely to suffer from the effects of pyridoxine deficiency, caused by reaction of INH with pyridoxal phosphate. Again, the fast inactivator nfight be more likely to develop resistant strains of tile tuberculosis organism. Finally, the therapeutic efficacy of a given dosage regimen might vary according to the rate at which the drug disappears from the circulation t. These possibilities have all been investigated. Polyneuritis associated with INH therapy is more common among slow than fast inactivatorsl-L but there is no evidence that the two groups differ in the development of INH-resistant bacilli during treatment ~. The relationship between inactivator status and therapeutic response to INH remains unclear. In the study by Evans et al.L INH inactivator phenotype did not appear to influence the outcome of tuberculosis treatment by standard treatment schedules which included INH. However, fast inactivators responded more slowly to a non-standard dosage regimen than did slow inactivators 5. Clin. Chim. Acta, 27 (I97 o) 513-.520
514
DYMOND, RUSSELL
Thus the question of differential response related to inactivator status appears still to be open, and could be pertinent to the introduction of new dosage regimens, such as intermittent INH therapy 6. The possibility of differential development of drugresistant organisms in different inactivator phenotypes may also require reassessment in relation to such regimens. These considerations point to the need of a method for determining inactivator statu,.~ which is simple enough for routine use. Recent work 7 suggests that a single iniection of INH (5 mg/kg), followed by a single determination of INH blood concentration 3 h later, can differentiate fast from slow inactivators in most cases. The mean serum INH level of 143 fast inactivators was 2.1 pg/ml, and of 198 slow inactivators was 5.0/,g/ml. Urine levels being a less reliable indicator than blood levels 7, it seems unlikely that the procedure for obtaining a sample for inactivator status determinations can be simplified much further. However, there appears to be room for a very simple method for determining the concentration of INH in blood samples. Such a method is described in the present paper. MATERIAl S
Isonicotinic acid hydrazide was obtained from J. T. Baker Chemical Co., Phillipsburg, N.J., and recrystallized from 80% v/v methanol to constant m.p. 17o-171°. Methyl isobutyl ketone from various sources was satisfactory without further purification. 2,4,6-Trinitrobenzenesulphonic acid (TNBS) 8 from Eastman Organic Chemicals, Rochester, N.Y., was twice recrystallized from 3 N HCI. Sodium p-aminosalicylate was prepared from the commercially available free acid, and purified by recrystallization from 95% ethanol. It conformed to specifications for the pharmaceutical grade compound 9. Metabolites of INH were either purchased and purified, or synthesized by published methods t°, except for the two ketoacid hydrazones. Pyruvic acid isonicotinylhydrazone was prepared as its sodium salt. Thus INH (5.0 g) and sodium pyruvate (5.0 g) were boiled in 2-propanol (125 ml) water (30 ml) for 15 rain; decolorizing charcoal (I g) was added, the mixture was boiled for a further 5 min and filtered. The product which separated on cooling the filtrate was dissolved in water (25 ml), the solution was diluted with 2-propanol (IOO ml) and chilled thoroughly. Sodium pyruvate isonicotinylhydrazone (4-9 g) crystallized as a colourless somewhat hygroscopic solid which darkened on heating and decomposed at 262-263 ° (ref. IO, m.p.: 258-26o ° decomp.). For analysis it was once more recrystallized, and dried in vacuo at IOO° over phosphorus pentoxide. Found: INH released on hydrolysis, 59.6%; CgHsN303Na requires 59.8%. Sodium hydrogen 0t-ketoglutarate isonicotinylhydrazone was similarly prepared. Thus sodium hydrogen g-ketoglutarate (2.5 g) and INH (1.6 g) were dissolved in water (33 ml). Rapid precipitation of the hydrazone ensued. The reaction was completed by addition of 2-propanol (66 ml) and boiling under reflux for 15 rain. The salt which separated on cooling the solution was recrystallized from water-2-propanol 1:2 v/v (60 ml) and the product was dried in vacuo over phosphorus pentoxide. The crystalline hydrazone (1.3 g) was a colourless solid which melted with decomposition at 258260 °. For analysis it was once more recrystallized, and dried in vacuo at IOO° over phosphorus pente-xide. Found: !NI{ re!cased on hydrolysis, 47.7%; CnHl0N30~Na requires 47.7%. Clin. Chzm. Acta, 27 (t97 o) 513-52o
ISONIAZIE DETERMINATION IN BLOOD
515
Blood from healthy volunteers was mixed immediately with one-sixth of its volume of acid citrate-dextrose solution~L and stored at 4 °. METHODS
Reagents x. Isonicotinic acid hydrazide stock solution: 200 mg INH, previously dried in vacuo at xoo ° over phosphorus pentoxide, is dissolved in sufficient distilled water to produce I 1. This solution (2oo btg/ml) is stable at room temperature for at 1,~ast several weeks. 2. Isonicotinic acid hydrazide standard solutions: the stock solution is diluted with distilled water to give standard solutions containing 3, 6, IO, 2o and 30/~g/ml. 3. Phosphate solution: 17o g K2HPO4 is dissolved in 500 ml distilled water. 4. 2,4,6-Trinitr°benzenesulp h°nic acid solution: 0.25 g TNBS is dissolved in 5° mi distilled water immediately before use. Procedures Preparation of calibration curve. One ml of INH standard solution Is i h c e d in a stoppered centrifuge tube of approx, x3 ml capacity, and 5 ml of phosphate solution is added. After brief mixing, the solution is treated with 1.5 ml of TNBS solution, and the contents of the tube are thoroughly mixed. Methyl isobutyl ketone (4 ml) is added without mixing, and the reaction is allowed to proceed in the dark at room temperature for x5 m~n. The IN H--TNBS reaction product is then extracted into the upper phase by thorough agitation, and the phases are separated by centrifugation; 5 rain at Iooo x g is suitable. The absorbance a of the upper phase is determined at 50o nm in a I-cm cuvette with distilled water as reference. The absorbance b of a reagent blank, prepared using distilled water in place of INH standard solution, is subtracted from a to give the corrected absorbance. Determination of I N H in whole citrated blood. One ml of blood is treated exactly as described for preparation of the calibration curve. In addition, a "blood blank" is prepared by diluting x ml of the blood sample with 5 ml of phosphate solution, adding x.5 ml of distilled water in place of the TNBS solution, and completing the procedure as before. If the absorbance of the "blood blank" is c then the corrected absorbance due to INH in the blood sample is a--(b+c). The concentration of INH in the citrated blood is determined irom the calibration curve, the validity of which is checked by simultaneous determinations on two INH standard solutions, conveniently of 3 and Io/~g/ml respectively. Determination of I N H in its acid-labile derivatives. Solutions (; ml) of the isonicotinylhydrazones of sodium pyruvate or sodium hydrogen a-ketoglutarate, containing about 25/~g/ml, were acidified with I drop of 6 N HCI. The liberated INH was determined as before, using INH standard solutions containing the same nominal amount of INH and similarly acidified. Determination of apparent I N H in its metabolites. Solutions (I r,fi) of the metabolites, containing IOO/~g/ml, were treated as described for the preparation of the calibration curve, and the apparent INH content was determined by reference to that curve. Recovery of I N H from blood. A solution (0.025 ml) of INH containing either Clin. Chim. Acta, 27 (I97 o) 513-52o
DYMOND, RUSSELL
516
4 ° or 400/~g/ml was added to I ml of blood; after thorough mixing the determination was completed as described. RESULTS
2,4,6.Trinitrobenzenesulphonic acid Tile acid prepared as described is almost colour!ess, but solutions in glassdistilled water turn yellow in a few hours at room tem oecature. Although freshly prepared and one-day old TNBS solutions gave identical results in the present procedure, solutions were freshly prepared as a matter of good analytical practice. The reagent is readily soluble at the concentration used.
The chromogenic reaction
Our unpublished results have shown that in alkaline solution simple acyl hydrazides react with TNBS to form i:x coloured complexes, ).m~x 503 nm, similar to the amino acid-TNBS complexes studied by Goldfarb '2. Between pH values of 8 and 9.5, INH reacts with TNBS to give a stable product with 2ma~ 432 nm. At higher pH, a second product is formed with 2max 495 nm. Accordingly, the chemistry of the chromogenic reaction is more complex than is the case with simple acyl hydrazides, and further work is necessary to establish the nature of the coloured products. At pH 9, formation of the product with 2max 432 nm is very rapid, tile velocity falling with decreasing pH. This, and the instability of the colour above pH 9.5, determined the choice of reaction medium. For determining INH in aqueous solution, borate is an ideal buffer (pKt = 9.2), but low recoveries were obtained from blood. Phosphate is a poor buffer at pH 9, but this deficiency is outweighed by its greater solubility. Thus, reaction mixtures prepared as described had pH's of 9.2 (water) and 9.0 (blood). In the presence of blood the chromogenic reaction was complete 0,9"
0.8" 0.7°r- 0.6o 13 0
o.s.
~ 0.40.30.2 0.1
460
4~o
560
~0
Wavelength (nm) Fig. I. Spectra of reaction product formed by isonicotinic acid hydrazide (22.5/~g/ml) and 2,4,6trinitrobenzenesulphonic acid. A. Aqueous reaction mixture (total volume 7.5 ml). B. Methyl isobutyl ketone extract of reaction mixture (total volume 4 ml). The reference cell in each case contained a reagent blank. The path length was I cm. The absorbances of tile reagent blanks were: A, 0.20 at 432 nm; B, o.oo at 480 and 500 nm. Clin. Chim. Acta, 27 (t97 o) 513-52o
ISONIAZID DETERMINATION IN BLOOD
517
in less than I min at room temperature (23°). The amount of extractable colour remained constant for at least I h. The spectrum of the coioured complex in aqueous solution is shown in Fig. I.
Extraction of the coloured complex In order to use the chromogenic reaction for estimating INH in blood it was clearly necessary to extract the reaction product into an immiscible solvent. Such a solvent had to satisfy three requirements: x. It should be transparent at the wavelength used to measure absorbance. 2. The partition coefficient of the INH-TNBS complex between water and the solvent should be large. 3- The solvent should not extract, from reaction mixtures containing blood, any color other than that produced by the complex. Since the complex absorbed in the visible region, the first requirement was readily met by most water-immiscible organic liquids. The second was met by a wide variety of moderately polar solvents, including low molecular weight alcohols, esters and ketones. The third requirement proved troublesome° BL.,~pMcontains very many compounds in which amino groups are present, and which accordingly react with TNBS in alkaline solution s. At least part of the products of such reaction was extracted by most of tile solvents wlfich satisfied requirement 2. These pr~duct~ are fortunately considerably more polar than the INH-TNBS complex, so that selectivity in favour of the latter could be achieved by lowering the polarity of the solvent. Thus, the selectivity of methyl ethyl ketone was increased by mixing it with diisobutyl ketone, and a 2:3 v/v mixture of these solvents gave excellent results, satisfying all three requirements. Mixed solvents suffer from the disadvantage that careless storage may alter their composition because of volatility differences, and methyl isobutyl ketone, which has the same degree of selectivity, was more satisfactory. It has the added advantage.~ o: cheapness and availability in the requisite state of purity. It extracts very small amounts of blood pigments, which necessitates the use of a "blood blank". The absorbance of such a blank does not exceed 0.020, and is generally much lower. Reagent blank values are less than 0.005 absorbance units. Care is needed in the extraction step if reliable results are to be obtained. The two phases have very different densities, so that thorough agitation is required for complete equilibration, the aim being to produce a coarse emulsion of the upper phase in the lower. Fortunately this is nmch more readily achieved in mixtures containing blood, presumably because of the presence of surface-active protein. Thus a shaking technique which gives reproducible results with standard INH solutions will give reliable results with blood. We recommend vigorous shaking for at least I min. The spectrum of the coloured complex in methyl isobutyl ketone is shown in Fig. I. Extraction results in a shift of 2ma, from 432 to 478 nm and, because a smaller volume of solvent is used, the extract has a higher absorbance at its Area,. Recovery of INH added to blood was slightly low when readings were made at 478 nm. The reason appears to be that at this wavelength a small proportion of the colour is contributed by end-absorption of the TNBS anion. Some TNBS is removed by reaction with blood components, resulting in an apparent loss of INH. For this Clin. Chim. Acta, 27 (I97o) 5 t 3 - 5 2 o
5I 8
DYMOND, RUSSELL
reason, readings are made at 500 nm where TNBS makes no contribution to the absorbance. Both reagent blanks and samples increase slowly in absorbance on standing, but the difference, a--(b+c), remains constant for at least t8 h.
Evaluation of the method When the method was applied to INH standard solutions, the absorbance was proportional to INH concentration up to 30/~g/ml (Fig. 2). Recovery experiments were performed in order to determine the accuracy and reproducibility of the method. The results obtained for recoveries of x and xo #g/ ml are given in Table I. 1.1 1.0 0.90.80.7-
g o.ei.
~ o.s-
~ 0.40.3" 0.215.1 I
I
l
I
I
I
5 10 1~i 2 0 : 2 5 30 iNH concentration (jug/rnl)
Fig. 2. C a l i b r a t i o n curve for i s o n i c o t i n i c acid h y d r a z i d e ( I N H ) d e t e r m i n a t i o n u s i n g 2,4,6-trinitrob e n z e n e s u l p h o n i c acid. TABLE
I
RECOVERY OF
INH
A D D E D TO I
ml
C1TRATED BLOOD
1NH added pg
Number of samples
I N H recovered, lag Highest value Lowest value
mean
5.D.*
S.E.*
to t
3t 35
IO.33 0.80
xo.ol I .oo
o.x661 o. I I
0.0298 o.o2
9.7 ° x .25
* S.D. is t h e s t a n d a r d d e v i a t i o n of a single result, S.E. is t h e s t a n d a r d e r r o r of t h e mean.
Specificity of the method All the known metabolites of INH were synthesized, and analysed by the proposed method. The major metabolites t0, namely isonicotinic acid, isonicotinamide, N-isonicotinylglycine and NLacetyl-N~-isonicotinylhydrazine, gave less than o.1% of the colour produced by the same weight of INH, as did the disputed t° metaboiite, Nt,N~-di-isonicotinylhydrazine. Values given by the minor metabolites t0, isonicotinylhydrazones of D-glucose, pyruvic acid and ~-ketoglutaric acid, were less than 1%. It is concluded that the method is specific for INH in that the products of its metal)Clin. Chim. Ac,a ~7 (197 o) 5 x 3 - 5 2 o
5X9
I S O NI A Z I D D E T E R M I N A T I O N IN BLOOD
olism are not estimated. Free hydrazine interferes in the determination, but has not been detected in the blood of patients receiving the drug ~3. Streptomycin (too /~/ml) gave a colour equivalent to less than 0.03/~g/m" of apparent INH. In general, however, any drug or metabolite with a free amino group is a potential source of interference. In particular, p-aminosalicylic acid gave a colour in the method described, too/~g]ml being equivalent to approximately x / ~ / m l of apparent INH. DISCUSSION
After reviewing the methods then available for determining INH in urine, Peters 1° concluded that no method of absolute specificity had been described. This observation is equally relevant to the problem of determining I NH in blood. Two new methods have since been reportedt*, 15, but the behaviour of INH metabolites in these procedures was not examined. 2,4,6-Trinitrobenzenesulphonic acid was introduced as an analytical reagent by Satake", and has since been used to determine a variety of compounds containing primary amino groups. No precise structural requirements for this reaction have so far been formulated, but in general the presence of an uncharged primary or secondary nitrogen appears to be necessary. Substituents which decrease the nucleophilicity of the nitrogen must be absent; thus, acid amides do not react. Acyi hydrazides, in which the -NHm group is further removed from the carbonyl function, would be expected to react readily, and we have found this to be so. It could also be predicted that modification of the -NHa group by acylation or condensation with carbonyl compounds would prevent reaction. These considerations led us to explore the use of TNBS as a reagent for determining INH in biological fluids. The present method is highly specific for INH in blood, since it does not measure any of the known metabolites of the drug. It is sufficiently sensitive for clinical and many other purposes, permitting measurement of I/~g/ml with reasonable accuracy (see Table I). Since INH is reported not to enter erythrocytes ~6, this sensitivity may be approximately doubled by the use of plasma rather than whole blood. This, however, involves some sacrifice of the simplicity which we regard as an important feature of the method. From the clinical chemist's point of view the specificity is not absolute because of interference from PAS. Although the colour yield from this compound in the present procedure is only about 1% of that given by INH, significant interference will occur in practice, because PAS is employed in very high dosage. Thus, its concentration in blood 2 h after a dose of 4 g may exceed IOO/~g/ml". Thus, the method in its present form cannot be used while PAS is being administered. This is the only known practical limitation. The other standard antitubercular drug, streptomycin, does not react, presumably because its guanidino groups are fully protonated at pH 9. However, any other drug present in the sample to be examined should first be tested as a possible source of interference. The method as it stands is not applicable to determination of INH in urine. Unlike blood, urine contains a substance or substances, most likely ammonia, which react with TNBS to give an extractable colour. A simple and specific method for determining free urinary INH would be useful '°, and efforts are being made to adapt the present procedure to that end. Clin. Chim. .4eta, 27
(I97o) 513-52o
~20
DYMOND, RUSSELL
ACKNOWLEDGEMENTS
We thank Mrs. Elizabeth Mason for painstaking technical assistance. This investigation was supported by grants from the Medical Research Council of Canada (MA 2480) and the Bunting Research Foundation. Part of the work described forms the substance of a thesis presented by one of us (L. C. D.) in partial fulfilment of tile requirements for the degree of Master of Science in Daihousie University. REFERENCES I D. A. P. EvaNs, K. A. MANLEY AND V. A. McKuSlCK, Brit. Med. J., ii (I96o) 485 . 2 5. DEVADa'rTA, P. R. J. GANGADHARAM, R. H. ANDREWS, W . Fox, C. V. RAMAKRISHNAN, J. B. SELKON AND 5. VELU, 13ull. World Health Organ., 23 (i96o) 587 . 3 D. A. P. E v a N s AND C. A. CLARKE, Brit. Med. Bull., 17 (196I) 234. 4 J. P. BII~HL, in Trans. r6th Conf. Chemotherapy of Tuberculosis, February 1r-r4, x957, St. Louis Medical Society Auditorium, U.S. Veterans Bureau, Washington, p. xo8. 5 R. 5. MITCHELL, D. K. RmMENSNIDER, J. R. HARSCH AND J. C. BELL, in 7"va~. z7th Conf. Chemotherapy of Tuberculosis, February 3-6, t958, Memphis, Tennessee, U. S. Veterans Bureau, Washington, p. 77. 6 J. M. DICKINSO,~, in The Chemotherapy of Tuberculosis in Developing Count;ies, Supplement to Tubercle, 49 (1968) 66-85. 7 H. TIIT]NEN, Scand. J. Respirat. Diseases, 5° (1969) I1o. 8 K. SaTAKF~, T. OKUY^MA, M. OIlasHI AND T. SHINODA, J. Biochem. (Tokyo), 47 (196o) 654. 9 British PhaJ'macopoeia z968, General Medical Council, London, I968, p. 89I. Iv J. H. PETERS, K. S. MILLER AND P. BROWN, Anal. Biochem., Iz (I965) 379. II W. R. DF,CESARE, J. R. B o r e ^ND F. G. EBAUGH, Transfusion, 4 (1964) 237. tz A. R. GOLDFAnn, Biochemistry, 5 (I966) 2570. 13 W. F. J. CUTHBERTSON, D, M. IRELAND, W. WOLFF aND 5. W. A. KVPER, Brit. Med. J., i (1954) 609. x4 K. B. BJORNESJ6 AND B. JARNULF, Scand. J. Clin. Lab. Invest., 20 (1967) 39. 15 M. H. HaSHML A. S. ADIL, F. R. MALIK AND A. I. AJ~AL, Mikrochim. Acta, (I969) 772. 16 E. I. SHORT, Lancet, i (x954) 656. 17 A. C. COHF.,~, The Drug Treatment of Tube~'culosis, Charles C. Thomas, Springfield, I11., 1966, p. 42.
Clin. Chim. ,4cta, 27 (i97 o) 513-52o
513
RAPID DETERMINATION OF ISONICOTINIC ACID HYDRAZIDE IN WHOLE BLOOD WITH 2,4,6-TRINITROBENZENESULPHONIC ACID
L. C. D Y M O N D ^,~D D. W. R U S S E L L
Department of Biochemistry, Dalhousie University, Halifax, N.S. (Canada) (Received November I I, J969)
SUMMARY
(I) A spectrophotometric method for tile quantitative determination of isonicotitfic acid hydrazide (INH) in whole blood is described. (2) The method involves reaction of INH with 2,4,6-trinitrobenzenesulphonic acid and extraction of the coloured product into methyl isobutyl ketone. The absorbance of the extract at 500 nm is proportional to INH concentration up to 30 /~g/mi. (3) Streptomycin and known metabolites of INH do not react, but p-aminosalicylic acid should be absent.
INTRODUCTION
Isonicotinic acid hydrazide (INH), seventeen years after its introduction into medicine, is still one of the most useful among a handful of effective antitubercular drugs. Its metabolism in the human body is unusual; among the population at large there is a bimodal distribution of the rate of INH disappearance from the circulation, so that individuals may be classed as slow or fast inactivators. IN H inactivator status, which is genetically determined t, has implications for IN H therapy. Thus, slow inactivators might be more likely to suffer from the effects of pyridoxine deficiency, caused by reaction of INH with pyridoxal phosphate. Again, the fast inactivator nfight be more likely to develop resistant strains of tile tuberculosis organism. Finally, the therapeutic efficacy of a given dosage regimen might vary according to the rate at which the drug disappears from the circulation t. These possibilities have all been investigated. Polyneuritis associated with INH therapy is more common among slow than fast inactivatorsl-L but there is no evidence that the two groups differ in the development of INH-resistant bacilli during treatment ~. The relationship between inactivator status and therapeutic response to INH remains unclear. In the study by Evans et al.L INH inactivator phenotype did not appear to influence the outcome of tuberculosis treatment by standard treatment schedules which included INH. However, fast inactivators responded more slowly to a non-standard dosage regimen than did slow inactivators 5. Clin. Chim. Acta, 27 (I97 o) 513-.520
514
DYMOND, RUSSELL
Thus the question of differential response related to inactivator status appears still to be open, and could be pertinent to the introduction of new dosage regimens, such as intermittent INH therapy 6. The possibility of differential development of drugresistant organisms in different inactivator phenotypes may also require reassessment in relation to such regimens. These considerations point to the need of a method for determining inactivator statu,.~ which is simple enough for routine use. Recent work 7 suggests that a single iniection of INH (5 mg/kg), followed by a single determination of INH blood concentration 3 h later, can differentiate fast from slow inactivators in most cases. The mean serum INH level of 143 fast inactivators was 2.1 pg/ml, and of 198 slow inactivators was 5.0/,g/ml. Urine levels being a less reliable indicator than blood levels 7, it seems unlikely that the procedure for obtaining a sample for inactivator status determinations can be simplified much further. However, there appears to be room for a very simple method for determining the concentration of INH in blood samples. Such a method is described in the present paper. MATERIAl S
Isonicotinic acid hydrazide was obtained from J. T. Baker Chemical Co., Phillipsburg, N.J., and recrystallized from 80% v/v methanol to constant m.p. 17o-171°. Methyl isobutyl ketone from various sources was satisfactory without further purification. 2,4,6-Trinitrobenzenesulphonic acid (TNBS) 8 from Eastman Organic Chemicals, Rochester, N.Y., was twice recrystallized from 3 N HCI. Sodium p-aminosalicylate was prepared from the commercially available free acid, and purified by recrystallization from 95% ethanol. It conformed to specifications for the pharmaceutical grade compound 9. Metabolites of INH were either purchased and purified, or synthesized by published methods t°, except for the two ketoacid hydrazones. Pyruvic acid isonicotinylhydrazone was prepared as its sodium salt. Thus INH (5.0 g) and sodium pyruvate (5.0 g) were boiled in 2-propanol (125 ml) water (30 ml) for 15 rain; decolorizing charcoal (I g) was added, the mixture was boiled for a further 5 min and filtered. The product which separated on cooling the filtrate was dissolved in water (25 ml), the solution was diluted with 2-propanol (IOO ml) and chilled thoroughly. Sodium pyruvate isonicotinylhydrazone (4-9 g) crystallized as a colourless somewhat hygroscopic solid which darkened on heating and decomposed at 262-263 ° (ref. IO, m.p.: 258-26o ° decomp.). For analysis it was once more recrystallized, and dried in vacuo at IOO° over phosphorus pentoxide. Found: INH released on hydrolysis, 59.6%; CgHsN303Na requires 59.8%. Sodium hydrogen 0t-ketoglutarate isonicotinylhydrazone was similarly prepared. Thus sodium hydrogen g-ketoglutarate (2.5 g) and INH (1.6 g) were dissolved in water (33 ml). Rapid precipitation of the hydrazone ensued. The reaction was completed by addition of 2-propanol (66 ml) and boiling under reflux for 15 rain. The salt which separated on cooling the solution was recrystallized from water-2-propanol 1:2 v/v (60 ml) and the product was dried in vacuo over phosphorus pentoxide. The crystalline hydrazone (1.3 g) was a colourless solid which melted with decomposition at 258260 °. For analysis it was once more recrystallized, and dried in vacuo at IOO° over phosphorus pente-xide. Found: !NI{ re!cased on hydrolysis, 47.7%; CnHl0N30~Na requires 47.7%. Clin. Chzm. Acta, 27 (t97 o) 513-52o
ISONIAZIE DETERMINATION IN BLOOD
515
Blood from healthy volunteers was mixed immediately with one-sixth of its volume of acid citrate-dextrose solution~L and stored at 4 °. METHODS
Reagents x. Isonicotinic acid hydrazide stock solution: 200 mg INH, previously dried in vacuo at xoo ° over phosphorus pentoxide, is dissolved in sufficient distilled water to produce I 1. This solution (2oo btg/ml) is stable at room temperature for at 1,~ast several weeks. 2. Isonicotinic acid hydrazide standard solutions: the stock solution is diluted with distilled water to give standard solutions containing 3, 6, IO, 2o and 30/~g/ml. 3. Phosphate solution: 17o g K2HPO4 is dissolved in 500 ml distilled water. 4. 2,4,6-Trinitr°benzenesulp h°nic acid solution: 0.25 g TNBS is dissolved in 5° mi distilled water immediately before use. Procedures Preparation of calibration curve. One ml of INH standard solution Is i h c e d in a stoppered centrifuge tube of approx, x3 ml capacity, and 5 ml of phosphate solution is added. After brief mixing, the solution is treated with 1.5 ml of TNBS solution, and the contents of the tube are thoroughly mixed. Methyl isobutyl ketone (4 ml) is added without mixing, and the reaction is allowed to proceed in the dark at room temperature for x5 m~n. The IN H--TNBS reaction product is then extracted into the upper phase by thorough agitation, and the phases are separated by centrifugation; 5 rain at Iooo x g is suitable. The absorbance a of the upper phase is determined at 50o nm in a I-cm cuvette with distilled water as reference. The absorbance b of a reagent blank, prepared using distilled water in place of INH standard solution, is subtracted from a to give the corrected absorbance. Determination of I N H in whole citrated blood. One ml of blood is treated exactly as described for preparation of the calibration curve. In addition, a "blood blank" is prepared by diluting x ml of the blood sample with 5 ml of phosphate solution, adding x.5 ml of distilled water in place of the TNBS solution, and completing the procedure as before. If the absorbance of the "blood blank" is c then the corrected absorbance due to INH in the blood sample is a--(b+c). The concentration of INH in the citrated blood is determined irom the calibration curve, the validity of which is checked by simultaneous determinations on two INH standard solutions, conveniently of 3 and Io/~g/ml respectively. Determination of I N H in its acid-labile derivatives. Solutions (; ml) of the isonicotinylhydrazones of sodium pyruvate or sodium hydrogen a-ketoglutarate, containing about 25/~g/ml, were acidified with I drop of 6 N HCI. The liberated INH was determined as before, using INH standard solutions containing the same nominal amount of INH and similarly acidified. Determination of apparent I N H in its metabolites. Solutions (I r,fi) of the metabolites, containing IOO/~g/ml, were treated as described for the preparation of the calibration curve, and the apparent INH content was determined by reference to that curve. Recovery of I N H from blood. A solution (0.025 ml) of INH containing either Clin. Chim. Acta, 27 (I97 o) 513-52o
DYMOND, RUSSELL
516
4 ° or 400/~g/ml was added to I ml of blood; after thorough mixing the determination was completed as described. RESULTS
2,4,6.Trinitrobenzenesulphonic acid Tile acid prepared as described is almost colour!ess, but solutions in glassdistilled water turn yellow in a few hours at room tem oecature. Although freshly prepared and one-day old TNBS solutions gave identical results in the present procedure, solutions were freshly prepared as a matter of good analytical practice. The reagent is readily soluble at the concentration used.
The chromogenic reaction
Our unpublished results have shown that in alkaline solution simple acyl hydrazides react with TNBS to form i:x coloured complexes, ).m~x 503 nm, similar to the amino acid-TNBS complexes studied by Goldfarb '2. Between pH values of 8 and 9.5, INH reacts with TNBS to give a stable product with 2ma~ 432 nm. At higher pH, a second product is formed with 2max 495 nm. Accordingly, the chemistry of the chromogenic reaction is more complex than is the case with simple acyl hydrazides, and further work is necessary to establish the nature of the coloured products. At pH 9, formation of the product with 2max 432 nm is very rapid, tile velocity falling with decreasing pH. This, and the instability of the colour above pH 9.5, determined the choice of reaction medium. For determining INH in aqueous solution, borate is an ideal buffer (pKt = 9.2), but low recoveries were obtained from blood. Phosphate is a poor buffer at pH 9, but this deficiency is outweighed by its greater solubility. Thus, reaction mixtures prepared as described had pH's of 9.2 (water) and 9.0 (blood). In the presence of blood the chromogenic reaction was complete 0,9"
0.8" 0.7°r- 0.6o 13 0
o.s.
~ 0.40.30.2 0.1
460
4~o
560
~0
Wavelength (nm) Fig. I. Spectra of reaction product formed by isonicotinic acid hydrazide (22.5/~g/ml) and 2,4,6trinitrobenzenesulphonic acid. A. Aqueous reaction mixture (total volume 7.5 ml). B. Methyl isobutyl ketone extract of reaction mixture (total volume 4 ml). The reference cell in each case contained a reagent blank. The path length was I cm. The absorbances of tile reagent blanks were: A, 0.20 at 432 nm; B, o.oo at 480 and 500 nm. Clin. Chim. Acta, 27 (t97 o) 513-52o
ISONIAZID DETERMINATION IN BLOOD
517
in less than I min at room temperature (23°). The amount of extractable colour remained constant for at least I h. The spectrum of the coioured complex in aqueous solution is shown in Fig. I.
Extraction of the coloured complex In order to use the chromogenic reaction for estimating INH in blood it was clearly necessary to extract the reaction product into an immiscible solvent. Such a solvent had to satisfy three requirements: x. It should be transparent at the wavelength used to measure absorbance. 2. The partition coefficient of the INH-TNBS complex between water and the solvent should be large. 3- The solvent should not extract, from reaction mixtures containing blood, any color other than that produced by the complex. Since the complex absorbed in the visible region, the first requirement was readily met by most water-immiscible organic liquids. The second was met by a wide variety of moderately polar solvents, including low molecular weight alcohols, esters and ketones. The third requirement proved troublesome° BL.,~pMcontains very many compounds in which amino groups are present, and which accordingly react with TNBS in alkaline solution s. At least part of the products of such reaction was extracted by most of tile solvents wlfich satisfied requirement 2. These pr~duct~ are fortunately considerably more polar than the INH-TNBS complex, so that selectivity in favour of the latter could be achieved by lowering the polarity of the solvent. Thus, the selectivity of methyl ethyl ketone was increased by mixing it with diisobutyl ketone, and a 2:3 v/v mixture of these solvents gave excellent results, satisfying all three requirements. Mixed solvents suffer from the disadvantage that careless storage may alter their composition because of volatility differences, and methyl isobutyl ketone, which has the same degree of selectivity, was more satisfactory. It has the added advantage.~ o: cheapness and availability in the requisite state of purity. It extracts very small amounts of blood pigments, which necessitates the use of a "blood blank". The absorbance of such a blank does not exceed 0.020, and is generally much lower. Reagent blank values are less than 0.005 absorbance units. Care is needed in the extraction step if reliable results are to be obtained. The two phases have very different densities, so that thorough agitation is required for complete equilibration, the aim being to produce a coarse emulsion of the upper phase in the lower. Fortunately this is nmch more readily achieved in mixtures containing blood, presumably because of the presence of surface-active protein. Thus a shaking technique which gives reproducible results with standard INH solutions will give reliable results with blood. We recommend vigorous shaking for at least I min. The spectrum of the coloured complex in methyl isobutyl ketone is shown in Fig. I. Extraction results in a shift of 2ma, from 432 to 478 nm and, because a smaller volume of solvent is used, the extract has a higher absorbance at its Area,. Recovery of INH added to blood was slightly low when readings were made at 478 nm. The reason appears to be that at this wavelength a small proportion of the colour is contributed by end-absorption of the TNBS anion. Some TNBS is removed by reaction with blood components, resulting in an apparent loss of INH. For this Clin. Chim. Acta, 27 (I97o) 5 t 3 - 5 2 o
5I 8
DYMOND, RUSSELL
reason, readings are made at 500 nm where TNBS makes no contribution to the absorbance. Both reagent blanks and samples increase slowly in absorbance on standing, but the difference, a--(b+c), remains constant for at least t8 h.
Evaluation of the method When the method was applied to INH standard solutions, the absorbance was proportional to INH concentration up to 30/~g/ml (Fig. 2). Recovery experiments were performed in order to determine the accuracy and reproducibility of the method. The results obtained for recoveries of x and xo #g/ ml are given in Table I. 1.1 1.0 0.90.80.7-
g o.ei.
~ o.s-
~ 0.40.3" 0.215.1 I
I
l
I
I
I
5 10 1~i 2 0 : 2 5 30 iNH concentration (jug/rnl)
Fig. 2. C a l i b r a t i o n curve for i s o n i c o t i n i c acid h y d r a z i d e ( I N H ) d e t e r m i n a t i o n u s i n g 2,4,6-trinitrob e n z e n e s u l p h o n i c acid. TABLE
I
RECOVERY OF
INH
A D D E D TO I
ml
C1TRATED BLOOD
1NH added pg
Number of samples
I N H recovered, lag Highest value Lowest value
mean
5.D.*
S.E.*
to t
3t 35
IO.33 0.80
xo.ol I .oo
o.x661 o. I I
0.0298 o.o2
9.7 ° x .25
* S.D. is t h e s t a n d a r d d e v i a t i o n of a single result, S.E. is t h e s t a n d a r d e r r o r of t h e mean.
Specificity of the method All the known metabolites of INH were synthesized, and analysed by the proposed method. The major metabolites t0, namely isonicotinic acid, isonicotinamide, N-isonicotinylglycine and NLacetyl-N~-isonicotinylhydrazine, gave less than o.1% of the colour produced by the same weight of INH, as did the disputed t° metaboiite, Nt,N~-di-isonicotinylhydrazine. Values given by the minor metabolites t0, isonicotinylhydrazones of D-glucose, pyruvic acid and ~-ketoglutaric acid, were less than 1%. It is concluded that the method is specific for INH in that the products of its metal)Clin. Chim. Ac,a ~7 (197 o) 5 x 3 - 5 2 o
5X9
I S O NI A Z I D D E T E R M I N A T I O N IN BLOOD
olism are not estimated. Free hydrazine interferes in the determination, but has not been detected in the blood of patients receiving the drug ~3. Streptomycin (too /~/ml) gave a colour equivalent to less than 0.03/~g/m" of apparent INH. In general, however, any drug or metabolite with a free amino group is a potential source of interference. In particular, p-aminosalicylic acid gave a colour in the method described, too/~g]ml being equivalent to approximately x / ~ / m l of apparent INH. DISCUSSION
After reviewing the methods then available for determining INH in urine, Peters 1° concluded that no method of absolute specificity had been described. This observation is equally relevant to the problem of determining I NH in blood. Two new methods have since been reportedt*, 15, but the behaviour of INH metabolites in these procedures was not examined. 2,4,6-Trinitrobenzenesulphonic acid was introduced as an analytical reagent by Satake", and has since been used to determine a variety of compounds containing primary amino groups. No precise structural requirements for this reaction have so far been formulated, but in general the presence of an uncharged primary or secondary nitrogen appears to be necessary. Substituents which decrease the nucleophilicity of the nitrogen must be absent; thus, acid amides do not react. Acyi hydrazides, in which the -NHm group is further removed from the carbonyl function, would be expected to react readily, and we have found this to be so. It could also be predicted that modification of the -NHa group by acylation or condensation with carbonyl compounds would prevent reaction. These considerations led us to explore the use of TNBS as a reagent for determining INH in biological fluids. The present method is highly specific for INH in blood, since it does not measure any of the known metabolites of the drug. It is sufficiently sensitive for clinical and many other purposes, permitting measurement of I/~g/ml with reasonable accuracy (see Table I). Since INH is reported not to enter erythrocytes ~6, this sensitivity may be approximately doubled by the use of plasma rather than whole blood. This, however, involves some sacrifice of the simplicity which we regard as an important feature of the method. From the clinical chemist's point of view the specificity is not absolute because of interference from PAS. Although the colour yield from this compound in the present procedure is only about 1% of that given by INH, significant interference will occur in practice, because PAS is employed in very high dosage. Thus, its concentration in blood 2 h after a dose of 4 g may exceed IOO/~g/ml". Thus, the method in its present form cannot be used while PAS is being administered. This is the only known practical limitation. The other standard antitubercular drug, streptomycin, does not react, presumably because its guanidino groups are fully protonated at pH 9. However, any other drug present in the sample to be examined should first be tested as a possible source of interference. The method as it stands is not applicable to determination of INH in urine. Unlike blood, urine contains a substance or substances, most likely ammonia, which react with TNBS to give an extractable colour. A simple and specific method for determining free urinary INH would be useful '°, and efforts are being made to adapt the present procedure to that end. Clin. Chim. .4eta, 27
(I97o) 513-52o
~20
DYMOND, RUSSELL
ACKNOWLEDGEMENTS
We thank Mrs. Elizabeth Mason for painstaking technical assistance. This investigation was supported by grants from the Medical Research Council of Canada (MA 2480) and the Bunting Research Foundation. Part of the work described forms the substance of a thesis presented by one of us (L. C. D.) in partial fulfilment of tile requirements for the degree of Master of Science in Daihousie University. REFERENCES I D. A. P. EvaNs, K. A. MANLEY AND V. A. McKuSlCK, Brit. Med. J., ii (I96o) 485 . 2 5. DEVADa'rTA, P. R. J. GANGADHARAM, R. H. ANDREWS, W . Fox, C. V. RAMAKRISHNAN, J. B. SELKON AND 5. VELU, 13ull. World Health Organ., 23 (i96o) 587 . 3 D. A. P. E v a N s AND C. A. CLARKE, Brit. Med. Bull., 17 (196I) 234. 4 J. P. BII~HL, in Trans. r6th Conf. Chemotherapy of Tuberculosis, February 1r-r4, x957, St. Louis Medical Society Auditorium, U.S. Veterans Bureau, Washington, p. xo8. 5 R. 5. MITCHELL, D. K. RmMENSNIDER, J. R. HARSCH AND J. C. BELL, in 7"va~. z7th Conf. Chemotherapy of Tuberculosis, February 3-6, t958, Memphis, Tennessee, U. S. Veterans Bureau, Washington, p. 77. 6 J. M. DICKINSO,~, in The Chemotherapy of Tuberculosis in Developing Count;ies, Supplement to Tubercle, 49 (1968) 66-85. 7 H. TIIT]NEN, Scand. J. Respirat. Diseases, 5° (1969) I1o. 8 K. SaTAKF~, T. OKUY^MA, M. OIlasHI AND T. SHINODA, J. Biochem. (Tokyo), 47 (196o) 654. 9 British PhaJ'macopoeia z968, General Medical Council, London, I968, p. 89I. Iv J. H. PETERS, K. S. MILLER AND P. BROWN, Anal. Biochem., Iz (I965) 379. II W. R. DF,CESARE, J. R. B o r e ^ND F. G. EBAUGH, Transfusion, 4 (1964) 237. tz A. R. GOLDFAnn, Biochemistry, 5 (I966) 2570. 13 W. F. J. CUTHBERTSON, D, M. IRELAND, W. WOLFF aND 5. W. A. KVPER, Brit. Med. J., i (1954) 609. x4 K. B. BJORNESJ6 AND B. JARNULF, Scand. J. Clin. Lab. Invest., 20 (1967) 39. 15 M. H. HaSHML A. S. ADIL, F. R. MALIK AND A. I. AJ~AL, Mikrochim. Acta, (I969) 772. 16 E. I. SHORT, Lancet, i (x954) 656. 17 A. C. COHF.,~, The Drug Treatment of Tube~'culosis, Charles C. Thomas, Springfield, I11., 1966, p. 42.
Clin. Chim. ,4cta, 27 (i97 o) 513-52o