High-performance liquid chromatographic determination of isoniazid, acetylisoniazid and hydrazine in biological fluids

The basic principle of derivatization of a hydrazide moiety with an aldehyde as applied in the method developed by Lacroix et al. [J. Chromatogr., 307 (1984) 137–144] for the quantitation of isoniazid and acetylisoniazid was imppoved by modification, standardization and extension to allow quantitation of hydrazine in patient samples. It could be shown that 40 μl of 1% methanonic cinnamaldehyde per 200 μl of deproteinized analysate gave maximal chromophoric isoniazid-cinnamaldehyde conjugate, read at 340 nm. The hydrolytic loss of isoniazid, crucial to the quantitation of acetylisoniazid, could be compensated for by introduction of an appropriate set of calibration curves. Although the method described here allows quantitation of monoacetylhydrazie and diacetylhydrazine, in addition to hydrazine, in mono-spiked samples, the method cannot be used for the quantitation of the acetylated metabolites of hydrazine in patient samples because of a lack of specificity. Linear calibration curves in the range 1–25 μg/ml for isoniazid and acetylisoniazid, 10–400 ng/ml for hydrazine and 50–1000 ng/ml for mono-acetylhydrazine and diacetylhydrazine, could be constructed; analyte recoveries approaching 100% could be achieved in all instances.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Elevation of Brain GABA Content by Chronic Low-Dosage Administration of Hydrazine, a Metabolite of Isoniazid

Abstract: When γ-aminobutyric acid aminotransferase (GABA-T) activity was measured in vitro in rat brain, neither isoniazid (INH) nor four of its known metabolites (isonicotinic acid, acetylisoniazid, acetylhydrazine, diacetylhydrazine) inhibited the enzyme in concentrations (5 mM) far higher than those likely to be achieved when INH is administered to man. In contrast, hydrazine (5 μM) caused a 50% inhibition of GABA-T without inhibiting glutamic acid decarboxylase (GAD). Rats were injected daily for 109 days with hydrazine (0.08 or 0.16 mmol/kg/day), after which amino acid contents and enzyme activities were measured in their brains. Both hydrazine doses caused significant elevations of whole brain GABA content and reductions of GABA-T activity, but did not affect GAD activity. Chronic administration of hydrazine at thee doses did not reduce weight gain or alter rat behavior, nor did it produce any irreversible pathologic changes in liver or alterations in hepatic aryl hydrocarbon hydroxylase activity. However, hydrazine treatment caused changes in the contents of many brain amino acids besides GABA, and markedly increased concentrations of ornithine, tyrosine, and α-aminoadipic acid in rat plasma. Inhibition of GABA-T activity and the other biochemical alterations observed in patients given high doses of INH probably result from hydrazine formed in the metabolic degradation of INH. Thus administration of hydrazine might be a more direct means of elevating brain GABA content in patients where this seems indicated, and might not entail a greater risk of adverse effects.     

Effect of rifampin, phenobarbital pretreatment, and acetylator phenotype on acetylisoniazid metabolism in the rabbit

The metabolism of [14C]acetylisoniazid was studied in male New Zealand White rabbits. Pretreatment of the rabbits with the microsomal enzyme inducers rifampin and phenobarbital had little effect on acetylisoniazid metabolism. Rifampin appears to produce some inhibition of acetylation of the metabolite acetylhydrazine to diacetylhydrazine. Acetylation phenotype was an important factor. Covalent binding of 14C to hepatic protein increased as the acetylation rate decreased. In plasma and urine acetylhydrazine levels were negatively correlated with acetylation rate and diacetylhydrazine levels were positively correlated as one would expect. It was concluded that in the rabbit covalent binding to hepatic protein was more dependent on the acetylation rate than on induction of microsomal oxidase.     

Comprehensive assay for pyrazinamide, rifampicin and isoniazid with its hydrazine metabolites in human plasma by column liquid chromatography

A comprehensive assay for determination of pyrazinamide (PZA), rifampicin (RIF), isoniazid (INH) and hydrazine metabolites is described. The method involves organic solvent extraction of PZA and RIF, followed by derivatization of INH, monoacetylhydrazine (mHYD) and hydrazine (HYD) with salicylaldehyde and extraction with diethyl ether. Acetylisoniazid (acINH) and diacetylhydrazine (dHYD) were hydrolyzed to INH and mHYD, respectively, and processed as above. Using a gradient solvent programmer, PZA and RIF were analyzed on a C₈ (5 μm) column at 248 nm, while INH and metabolites were analyzed on a C₁₈ (5 μm) ODS2 column at 280 nm.     

Determination of rifampicin, desacetylrifampicin, isoniazid and acetylisoniazid by high-performance liquid chromatography: Application to human serum extracts, polymorphonucleocytes and alveolar macrophages

A method for the determination of rifampicin, desacetylrifampicin, isoniazid, and acetylisoniazid by high-performance liquid chromatography and using the same extract of the same sample is reported. After protein precipitation and extraction of these antituberculous drugs, two reversed-phase chromatographies were necessary. The technique was applied to serum extracts, polymorphonucleocytes and alveolar macrophages from patients treated for tuberculosis.     

High-performance liquid chromatography with UV detection and diode-array UV confirmation of isonicotinic acid hydrazide in cattle milk

A method for the determination of isonicotinic acid hydrazide (isoniazid) in milk was developed. Milk was deproteinized with trichloroacetic acid. Isoniazid was condensed with cinnamaldehyde and assayed on a reversed-phase HPLC system, with good sensitivity and accuracy (10 μg/l) with UV detection at 330 nm. Use of solid-phase extraction with a C₁₈ cartridge allows the detection limit to be lowered to 0.1 μg/l with UV detection and confirmation of isoniazid hydrazone from the diode-array UV spectrum.     

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37°C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4 v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Simultaneous determination of trimethoprim,sulphamethoxazole and N4-acetylsulphamethoxazole in serum and urine by high-performance liquid chromatography

The simultaneous determination of trimethoprim, sulphamethoxazole and N⁴-acetyl-sulphamethoxazole in serum and urine by high-performance liquid chromatography using sulphafurazole as internal standard is described. The separation was achieved on a reversed-phase column employing acetic acid—methanol as the mobile phase with spectrophotometric detection at 230 nm. Precise simultaneous quantitative analysis of the relative components has been achieved at levels of 0.1 μg/ml for trimethoprim and 1.0 μg/ml for both sulphamethoxazole and its N⁴-acetyl metabolize using 1 ml of serum or urine.     

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.     

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig.

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.     

Bioassay for Hamycin and Amphotericin B in Serum and Other Biological Fluids

A bioassay suitable for measuring concentrations of the polyene antifungal agents hamycin and amphotericin B in biological fluids is described. By using Paecilomyces varioti as the indicator organism, sensitivity of the bioassay was found to be in the range of 0.01 to 0.02 mug/ml. A linear dose-response curve was obtained with amphotericin B; the curve for hamycin was curvilinear. In a series of assays, hamycin serum levels in the range of 0.01 to 3.5 mug/ml were measured; with amphotericin B, serum levels in the range of 0.015 to 0.175 mug/ml were measured in patients receiving orthodox intravenous medication and as high as 9.0 mug/ml in one patient treated with extraordinarily high doses of the drug.     

Quantification of isoniazid and acetylisoniazid in rat plasma and alveolar macrophages by liquid chromatography-tandem mass spectrometry with on-line extraction

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC-MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1-250ng/ml of INH and 0.05-50ng/ml of AcINH (r(2)>0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.     

Direct analysis of salicylic acid,salicyl acyl glucuronide,salicyluric acid and gentisic acid in human plasma and urine by high-performance liquid chromatography

A method for the simultaneous direct determination of salicylate (SA), its labile, reactive metabolite, salicyl acyl glucuronide (SAG), and two other major metabolites, salicyluric acid and gentisic acid in plasma and urine is described. Isocratic reversed-phase high performance liquid chromatography (HPLC) employed a 15-cm C₁₈ column using methanol-acetonitrile-25 mM acetic acid as the mobile phase, resulting in HPLC analysis time of less than 20 min. Ultraviolet detection at 310 nm permitted analysis of SAG in plasma, but did not provide sensitivity for measurement of salicyl phenol glucuronide. Plasma or urine samples are stabilized immediately upon collection by adjustment of pH to 3–4 to prevent degradation of the labile acyl glucuronide metabolite. Plasma is then deproteinated with acetonitrile, dried and reconstituted for injection, whereas urine samples are simply diluted prior to injection on HPLC. m-Hydroxybenzoic acid served as the internal standard. Recoveries from plasma were greater than 85% for all four compounds over a range of 0.2–20 μg/ml and linearity was observed from 0.1–200 μg/ml and 5–2000 μg/ml for SA in plasma and urine, respectively. The method was validated to 0.2 μg/ml, thus allowing accurate measurement of SA, and three major metabolites in plasma and urine of subjects and small animals administered salicylates. The method is unique by allowing quantitation of reactive SAG in plasma at levels well below 1% that of the parent compound, SA, as is observed in patients administered salicylates.     

Gas chromatographic determination of low concentrations of benzoic acid in human plasma and urine

A method for the determination of benzoic acid down to concentrations of 10 ng/ml in plasma or urine is described. After addition of an internal standard, benzoic acid is extracted at acid pH into diethyl ether. Both compounds are derivatized with pentafluorobenzyl bromide. The derivatives are determined by gas chromatography using a ⁴³Ni electron-capture detector. Hippuric acid is hydrolysed in plasma and urine and total benzoic acid is determined by the same technique.     

Survival and Macromolecular Synthesis During Incubation of Escherichia coli in Limiting Thymine

Survival and the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured during incubation of a thymine auxotroph of Escherichia coli in a series of media containing thymine concentrations below the optimal level of 2 mug/ml. The rate of increase in viable count gradually diminishes to no net growth with 0.2 mug/ml. With lower concentrations of thymine, the rate of cell death gradually increases, resulting in a typical thymineless death curve with 0.02 mug/ml. Both the rate of cell growth and the rate of cell inactivation vary linearly with the thymine concentration. Thirty minutes of incubation in media containing limiting concentrations of thymine before a shift to complete thymine starvation results in a progressive decrease in the length of the lag period preceding thymineless death. These data suggest that only one type of cellular damage occurs during the various degrees of thymine limitation. Prolonged preincubation in media containing 0.1 to 0.2 mug/ml of thymine results in an immunity to thymineless death. This immunity differs from that observed with amino acid-starved cells in its kinetics; ultraviolet irradiation of preincubated cells indicates that the cells are inactivated at the same rate as log-phase cells. These results suggest that the immunity is not associated with chromosome alignment. Thymine concentrations between 2 mug/ml and 0.2 mug/ml permit essentially the same amount of protein and RNA synthesis. The total amount of synthesis then decreases linearly to 40 to 50% of the control level with further reduction in the amount of thymine present. Protein and RNA synthesis are first affected at the same thymine concentration at which lethality is first detectable, and this correlation suggests that the synthesis of these macromolecules is involved in the mechanism of thymineless death. DNA synthesis, on the other hand, is directly dependent on the thymine concentration for levels of 0.5 mug/ml or less. There are no critical changes in DNA synthesis associated with lethality, and DNA synthesis is still occurring under conditions of thymine limitation which result in immunity. These observations suggest that DNA synthesis is not directly involved in thymineless death.     

Mutagenicity induced by lyophilization or storage of urine from isoniazid-treated rats.

Urine collected during 24 h after treatment of rats with 90--550 mg/kg isonicotinic acid hydrazide (isoniazid, INH) was after lyophilization, mutagenic for Salmonella typhimurium TA1535. Urine collected directly from bladders of INH-treated rats was not mutagenic, and solutions of INH in water or urine became mutagenic only after lyophilization. In the absence of lyophilization, sterile urine from INH-treated rats became mutagenic after 8--14 days' storage at room temperature.