Pharmacology

Chemotherapy 1975;21:1-10

Determination of Isonicotinic Acid Hydrazide in Serum H. P.

Bartels Spring

Department of Internal Medicine, University of Basel, Kantonsspital, Basel Key Words Isoniazid Determination Automatic method Metal complex Pharmacokinetics

Abstract A method for the determination of isonicotinic acid hydrazide is given. The hydrazide is complexed with cupric ions and condensated with an aromatic aldehyde substituted in ortho-position with a hydroxyl group. The method is adapted to the AutoAnalyzer equipment of first and second generation. Pilot measurements on the pharmacokinetic behaviour of isonicotinic acid hydrazide are presented. Request reprints from: H. Bartels, PhD, Ciba-Geigy Ltd, CH-4000 Basel (Switzerland)

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In the treatment of tuberculosis, isonicotinic acid hydrazide (INH) is the most frequently used agent. Due to the nature of this disease, the drug has to be administered regularly for months. Repeated administration always bears the risk of drug accumulation in the organism, the extent of which is -for a given dosage scheme – only determined by the rate of elimination. Fortunately, the rate of elimination varies only insignificantly for most drugs from one individual to another so that a ‘fixed’ dosage scheme can be applied to all patients. Problems arise, however, when the rate of drug elimination is not constant. Such a variation is highly probable in the case of INH due to the following: in humans, INH is partly eliminated by the kidneys [1]. Therefore, it has to be expected that the rate of elimination is decreased in patients with impaired renal function. To an even higher percentage, INH is eliminated by metabolic transformations. It is known that there are genetically determined individual rates of metabolism, with a polymodal frequency distribution [2]. ‘Fast inactivators’ with a mean elimination half-life of about 1 h can be distinguished from ‘slow inactivators’ with a mean half-life of about 3 h. If the same ‘fixed’ dosage scheme is used for all patients, there is the risk of therapeutic inefficiency for the ‘fast inactivators’ and the 1 Aided by the Swiss National Research Foundation. 2 Bartels/Spring Me+ +H H I Fig. 1. Proposed reaction scheme for the INH determination. Me=Metal ion; S = substituent. risk of toxicity for the ‘slow inactivators’. In contrast to most other chemo-therapeutic drugs, the quantitative relation between the half-life and the degree of kidney impairment is not known for INH. Moreover, there is no way to distinguish ‘fast inactivators’ from ‘slow inactivators’. Thus,

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to design individual dosage schemes, a method for the determination on INH in serum is needed in order to determine the individual elimination rate of a given patient. A quantitative determination of INH may be obtained by the reaction of bromocyan with the pyridine ring [3] or by the reaction of the basic hydrazide group with an aromatic aldehyde [4]. The latter has the advantage that those metabolites of INH, originating from hydrolysis or carboxylation, are excluded from the indicating reaction. Nevertheless, even this reaction mechanism is not very specific, for it just indicates an amine so that one must reckon with a false quantitative analysis. For this reason, the basic INH has to be extracted from biological material; as it is only slightly soluble in organic solvents, the extraction becomes a difficult procedure. This is why the existing methods for the quantitation of INH are tedious and unreliable. They are rarely used. We decided to look for a specific test for aromatic hydrazides. Already in 1958, Fallab and Erlenmeyer [5] showed that hydrazides are able to form metal complexes, whereby an enolisation of the C = 0 bond occurs. This reaction mechanism is highly suitable for the INH determination, especially after the INH is condensed with an aromatic aldehyde. By enolisation, the π-electron system of the aromatic aldehyde would come into conjugation with the pyridine ring. Besides the high specificity, Determination of Isonicotinic Acid Hydrazide in Serum 3 one would expect a bathochromic shift of the absorption band. Experiments with several aromatic aldehydes showed that the corresponding complex can only be formed if an OH group is substituted in ortho-position. The reason for the necessity of a further co-ordination is the low basicity of the now double-bonded nitrogen atom of the hydrazone. The reaction to be studied is the one in figure 1. Reagents for the Determination Several aromatic aldehydes with ortho-substituted hydroxyl groups have been studied in order to find a simple technique and a high extinction coefficient of the reaction product. Table I gives the values for the several substituted salicylic aldehydes. If the latter were not sufficiently soluble in water, we substituted an ethanolic solution. However, this cannot be recommended for routine work because of the air bubbles forming after mixing. Metal Salt Several metal ions are suitable for the enolisation of the C = O-double bond. Preferably, only one complex should form. Iron and cobalt can form a 1:1 as well as a 1:2 complex. Copper, however, can with a tridented ligand only form a 1:1 complex as it only has four coordination sites. Furthermore, the copper complex is expected to have a high stability. Cupric ions enhance the condensation considerably, immediately after mixing, the reaction product is formed. The corresponding complexes have an absorption maximum between 420 and 440 nm, depending on the substitution of the aldehyde. Measurement is possible at the mercury emission lines 405 and 436 nm with a sufficiently high precision. Influence of pH As hydrogen ions participate in condensation reactions and as the degree of complex formation is determined by the free hydrazide group, a pH dependence has to be considered. At a high pH, the condensation rate is slow, whereas at low pH the rate of complex formation decreases. For 2hydroxy-l-naphthaldehyde-4-sulfonic acid and copper ions we found the optimal pH to be 2.5. Deproteinization The deproteinization has to be performed in a way that the protein-bound INH is dissociated first; low pH, addition of detergents and high

4 Bartels/Spring Table I. Extinction coefficients (E) for salicylate aldehydes substituted with different radicals (R) R E, 103 H 6.0 4-OH 10.2 3-OCH3 6.1 4-N(CH3)2 7.0 2-Hydroxy-l-naphthaldehyde 8.7 2-Hydroxy-l-naphthaldehyde 4-sulfonic acid 10.5 degree of dilution are possible. As the reaction is carried through in an acid solution, it is advisable to use the deproteinization by addition of acid. Specificity The observable change of absorption due to condensation and complex formation are specific for hydrazides. Acylated hydrazides do not react, in particular this could be shown for acetylated INH. Method The reagents were purchased from Fluka, Buchs (Switzerland); p-dimethylamino-salicylic aldehyde was synthesized by the method of Baird and Shriner [6] and 2-hydroxy-lnaphthaldehyde-4-sulfonic acid by a Reimer-Tiemann reaction according to a Geigy patent [7]. The acetyl derivative of INH was synthesized by addition of acetic anhydride and recrystallisation from aqueous ethanol. Synthesis of2-Hydroxy-Naphthaldehyde-4-Sulfonic Acid 20 g 8-naphthol-4-sulfonic acid is dissolved in 100 ml of 30% (w/w) NaOH – for example, in a three-necked flask equipped with a stirring device, cooler and thermometer. This solution is heated to 60 °C. 30 ml chloroform is added by a dropping funnel at a rate maintaining the temperature (about 1 h). Concentrated HCl is added to obtain a precipitation of the desired aldehyde at about pH 3. The precipitate can either be re-crystallized from aqueous ethanol or just be suspended in absolute ethanol, filtered and washed with ethyl ether. Reagents for the Manual Procedure (1) Sodium tungstate 10%: 10 g of this compound is dissolved in 100 ml of distilled water. (2) Sulphuric acid ⅝ n: 66 ml 1 n H2SO4 ad 100 ml disDetermination of Isonicotinic Acid Hydrazide in Serum 5 Dialyser OMC ~_JW\WΓ

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040 0.1N HCl 056 Sample 045 Air 081 Colour 045 AiΓ 030 Colour 073 Waste Sampler \y hzzh Colorimeter Expander Recorder Fig. 2. Manifold for the INH determination with AutoAnalyzer I equipment. Diameters of pump tubes in inches [see Technical Manual of Technicon Corporation, Tarry-town, N.Y.]. tilled water. (3) Aldehyde: 100 mg of 2-hydroxy-l-naphthaldehyde-4-sulfonic acid is dissolved in 100 ml of distilled water. (4) Copper sulphate 1 m: 249 g C11SO4 · 5 H2O is made up to 1 litre with distilled water. All reagents are stable for at least 2 months. 2 ml serum or standard respectively is diluted with 3 ml of distilled water. The protein is precepitated by addition of 1 ml Na tungstate 10% and 2 ml % N H2SO4. After shaking for several times, the mixture is centrifuged. To 4 ml of the supernatant 1 ml aldehyde 100 mg% is added. The solution is transfered into semi-micro cuvettes with 4 cm light path. The absorption is read at 436 nm (Ei). 0.2 ml Q1SO4 1 m is added, mixed, and the extinction is read again (E2). From a standard curve, the difference E2-E1 is converted to concentration. The coefficient of variation within the series is 3%, Beer Lambert’s law is obeyed up to at least 5 mg INH/litre serum. For large series, as we had to face them, this technique was still too complicated. Therefore, we decided to automate the method. The Auto-Analyzer seemed to be suitable. As we do not like to change the manifolds on the pump, we just modified the manifold for the serum iron determination after Young and Hicks [8]. Now it is possible to analyse for INH or serum iron by just changing the reagents. The manifold used is given in figure 2. As INH has a low dialysance, we had to use an expander in order to obtain suitable peak heights. If an expander is not at hand, a sufficient expansion (factor 2) can be obtained by modifying the recorder as shown in figure 3. Bartels/Spring Blue \ Green Fig. 3. Expanding device for AutoAnalyzer I. Sampler Waste o 0.035 Sample

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0.025 Acid Air Acid Colour Fig. 4. Manifold for the INH determination with AutoAnalyzer II equipment. Diameters of pump tubes in inches. The blue wire of contact ‘L’ at the back of the door carrying the paper role is connected with a 1,500-Ohm potentiometer. Later experiments were carried out with an instrument of the second generation analyzer. The corresponding manifold is given in figure 4. Determination of Isonicotinic Acid Hydrazide in Serum 7 Reagents for the Automated Procedure Diluting reagent: 0.1 n HC1, 2% Brij (a detergent specified by Technicon Corporation). This reagent at the same time dissociates the INH from the proteins. Colour reagent: 500 mg 2-hydroxy-l-naphthaldehyde-4-sulfonic acid is dissolved in 50 ml 1 n HC1, diluted with 25 ml 1 n Q1SO4 and made up to 500 ml with distilled water. 30–60 samples can be run on the AutoAnalyzer. Standards are made up by adding concentrated INH solutions to pool serum. The reproduceability of INH determinations with range from 0 to 10 mg/l was characterized by the standard deviation determined by ™”(Wia-Wi)2 _ ΣWi. Σ Σ ~~ = 0.075 mg/l, Wt = j Y m (n-1) n for m=160 specimens and n = 3 determinations/specimen. The recovery of added INH is 97– 103%. INH in serum is not stable at room temperature. After 3 days, 30–40 % is decomposed. The degree of decomposition is less if the serum is kept in the refrigerator. The INH is even more stable in the freezer (fig. 5). It is, therefore, safest to analyze the serum samples on the day they are drawn. This is possible as no extra manifold is used and the reagents are stable. Besides the experiments with added INH, the results were compared with the ones obtained with a manual method, isolating the INH by extraction [9]. The coefficient of correlation was r = 0.96. Determination of Half-Live The suitability of the method was demonstrated with two patients. Patient A was a 83-year-old man with normal kidney function (endogenous creati-nine clearance Vcr=176 ml/min). Patient B was a 61-year-old man with severe kidney insufficiency and subacute glomerulonephritis (Vcr=1.5 ml/ min). Both patients obtained a intravenous injection of a single dose of INH. At suitable time intervals, several samples of venous blood (5 ml) were drawn for the determination

of INH in the serum. Corresponding to our stability experiments, the serum, if not analyzed on the same day, was stored at -30°C. Table II shows the results. S 60 20 »C 40 20 20 10 0 4°C In ID l∏ ⅜H Ill :ι -20°C -to. ⅜L_⅜L JJ IP Time, days Fig. 5. In vitro stability of INH at different temperatures. For the pharmacokinetic evaluation of the experiments, the following equation describing a onecompartment first-order process was assumed to be valid: D In c = In Δ·G – kt = In Co–kt, where D = dose (mg), Δ= relative volume of distribution (1/kg), G = body weight (kg), k = constant of elimination (h_1), c = INH concentration in serum (mg/l) at the time t after application of a dose, c0 = apparent initial concentration of INH (mg/l). The regression line was calculated from the values in table II, and from this the elimination constant k as well as the relative distribution volume Determination of Isonicotinic Acid Hydrazine in Serum 9 Table II. Plasma concentration (c) of INH at different times (t) after the administration of one single dose to a patient with normal (A) and to a patient with severely impaired (B) kidney function (see fig. 6)

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Table III. Pharmacokinetic parameters of INH calculated from the results of the experiments in two patients described in table II Patient A Patient B D, mg 300.000 400.000 G, kg 55.800 66.500 Vcr, ml/min 176.000 1.500 Co, mg/l 10.000 9.900 A, 1/kg 0.538 0.608 k, h-1 0.590 0.108 r 0.999 0.998

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tι/2, h 1.180 6.410 using the least squares of error. The half-life was obtained from tι/2 = In 2/k. The pharmacokinetic constants are listed in table III. The high coefficients of correlation confirms the validity of the equation (fig. 6). Patient A with a half-life ti/s≈ 1.18 h is a typical ‘fast inactivator’. 10 Bartels/Spring Dose 10 8 64I 21 0.8H 0.6T–I–I–I–I–I–I–I–I–I–I–I–I–I–I–I–I–I–I–1–I–I–I0 4 8 12 16 20 24 Time, h Fig. 6. Semi-logarithmic plasma concentration-time curve of INH after the administration on one single dose in a patient with normal (A) and in a patient with severely impaired (B) kidney function. For further explanation, see text. The half-life of patient B (tι/2 – 6.4 h) is five times as long. A value that high is uncommon for a ‘slow inactivator’; the kidney insufficiency, at least in part, is the reason for such a long half-life. References Hughes, H. B.: On the metabolic rate of isoniazid. J. Pharmacol, exp. Ther. 109: 444–452 (1953). Evans, D. A. P.; Manley, K. A., and McKusick, V. A.: Genetic control of isoniazid metabolism in man. Brit. med. J. ii: 485–491 (1960). Rubin, S. H.; Drektor, L.; Scheiner, J., and De Ritter, E.: Determination of blood plasma levels of hydrazine derivatives of isonicotinic acid. Dis. Chest 21: 439 (1952). Maher, J. R.; Whitney, J. M.; Chambers, J. S., and Stanonis, D. J.: The quantitative determination of isoniazid and / > -amino-salicylic acid in body fluids. Amer. Rev. Tuberc. 76: 852 (1957). Fallab, S. and Erlenmeyer, H.: Isonicotinic acid hydrazide copper complex. Ex-perientia 8: 298 (1952). Baird, W. C. and Shriner, R. L.: The synthesis and properties of 7-dimethylamino flavylium salts. Amer. chem. Soc. 86: 3142 (1964). Geigy Patent: Synthesis of oxy-naphthaldehyde sulfonic acids; DRP 97934 (1897), in Frdl. J: 140 (1900). Young, D. S. and Hicks, J. M.: Automatic determination of serum iron. J. clin. Path. 18: 98 (1965).

Determination of isonicotinic acid hydrazide in serum.

Pharmacology Chemotherapy 1975;21:1-10 Determination of Isonicotinic Acid Hydrazide in Serum H. P. Bartels Spring Department of Internal Medicine,...
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