Acetylisoniazid In Urine Research Articles

A validated liquid chromatography-tandem mass spectrometry assay for the analysis of isoniazid and its metabolite acetyl-isoniazid in urine

IntroductionIsoniazid (INH) is one of the most effective and potent first-line anti-tubercular drug. INH is also effectively administered as a preventative monotherapy and has been shown to significantly reduce TB incidence. INH is primarily metabolised to acetyl-isoniazid (AcINH) in the liver. AcINH is mainly excreted in urine presenting as a target for monitoring adherence to INH therapy. ObjectiveThe study aimed to develop and fully validate a bioanalytical method using liquid chromatography-tandem mass spectrometry for the quantification of INH and AcINH in human urine. MethodsThe samples were prepared using solid phase extraction, with the internal standards isoniazid-d4 and acetyl-isoniazid-d4 being used. The extracts were chromatographed on an Atlantis T3 analytical column with an isocratic mobile phase. For detection, a AB Sciex™ API 5500 triple quadrupole mass spectrometer was used at unit resolution in the multiple reaction monitoring mode, following positive electrospray ionization. ResultsThe analytical method demonstrated sufficient sensitivity, as indicated by average signal-to-noise ratios of 7.07 and 6.23 at the lower limit of quantification for INH and AcINH, respectively. Validation was performed over three consecutive batches, demonstrating accuracy, precision, and overall robustness based on peak area ratios within the analytical range of 0.234–30.0 µg/mL for both INH and AcINH. All required validation experiments were assessed and met the acceptance criteria guidelines of the US Food and Drug Administration and European Medicines Agency. The validated method was utilized to measure concentrations of AcINH in urine as a means of assessing adherence to the intake of isoniazid in order to prevent TB infection during a phase III open-label multicenter trial. ConclusionA bioanalytical method was developed and fully validated for quantifying isoniazid (INH) and acetyl-isoniazid (AcINH) in 100 µL of human urine.

Simple and rapid method for simultaneous determination of isoniazid and acetyl isoniazid in urine by HPLC

A high performance liquid chromatographic method for simultaneous determination of isoniazid and acetyl isoniazid in urine was developed. Urine samples were filtered through Whatman number 1 filter paper and diluted filtrate was directly injected into C8 column (250mm). The mobile phase comprised of water and methanol in the ratio of 85:15. The analytes were detected at a wavelength of 274nm. The assay was specific for isoniazid and acetyl isoniazid and linear from 1.25 to 40.0μg/ml for isoniazid and 3.125 to 100μg/ml for acetyl isoniazid. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of isoniazid and acetyl isoniazid from Urine was 100% and 95% respectively. Due to its simplicity, the assay can be used for determination of isoniazid acetylator status using urine and to check patient adherence to tuberculosis treatment.

A specific rapid gaschromatographic assay for the determination of isoniazid N-acetylation: Observation in rats with induced constant urine flow

An assay for determination of the degree of enzymatic N-acetylation in rats was presented. A simple method for inducing a continuous urine flow from rats was applied to measure the degree of acetylation at definite intervals. A rapid gaschromatographic method for the simultaneous quantitative determination of isoniazid as the substrate for polymorphic N-acetyltransferase and its metabolite, acetylisoniazid in urine was described. Isoniazid and acetylisoniazid samples were derivatized with a silylating reagent. The gaschromatographic species were identified by GC-mass spectrometry as di-trimethylsilyl derivatives of isoniazid and acetylisoniazid. Significant dose-dependent and age-dependent changes in the degree of acetylation were observed in female rats. For example, relatively more acetylisoniazid is excreted after a 50 mg/kg isoniazid dose than after treatment with 100 mg/kg isoniazid. Young rats of 6–7 weeks acetylate the same dose to a much higher degree than 8-month-old rats.

Mass Phenotyping of Isoniazid Inactivators by Automated Determination of Acetylisoniazid in Urine

Abstract An automated procedure is described for phenotyping isoniazid inactivators. The method is based on the color reaction produced by N-acetylisoniazid in aqueous solution, as described by Eidus and Hamilton [Amer. Rev. Resp. Dis. 89, 587 (1964)]. Maximum sampling rate is 60 samples per hour. Results of this procedure correlated well (r = 0.987) with those of a manual phenotyping method.

Method for the estimation of acetylisoniazid in urine

A method for the estimation of acetylisoniazid in urine is described. It is simple, sensitive and reliable and follows Beer's law between 10 μg./ml. and 40 μg./ml. It yields complete recovery of acetylisoniazid from specimens of urine to which the compound has been added in known concentrations. In this procedure, the drug-containing urine itself serves as the blank if the reagents are mixed first and then the urine added. Isoniazid in high concentrations produces a colour reaction in this method. This interference is eliminated by potassium permanganate which destroys isoniazid but not acetylisoniazid under the conditions of the estimation.

Sodium nitroprusside test for the detection of isoniazid and acetylisoniazid in urine

A test, using alkaline sodium nitroprusside and accitic acid as reagents, is described for detecting the presence of isoniazid and acetylisoniazid in urine. The test result is positive in all urine specimens collected at eight hours after administration of 200 mg. or 400 mg. of isoniazid, and in about 90% of the specimens collected between 10 and 12 hours. The test appears to be slightly more sensitive than the paper test of Gangadharam, Nair & Subbaiah (1962) for free isoniazid, but is appreciably less sensitive than the acetylisoniazid test of Eidus & Hamilton (1964) . It does not suffer any interference from streptomycin, PAS and thiacetazone (thioacetazone) at conventional dosages used in clinical practice, but is affected by pyrazinamide, cycloserine and ethionamide. Un test utilisant une solution alcaline de nitroprussiate de soude et l'acide acétique comme réactifs, est décrit pour déceler la présence d'isoniazide libre et acétylé dans l'urine. Le résultat de ce test est positif pour tous les spécimens d'urine recueillis 8 heures après administration de 200 ou de 400 mg, d'isoniazide, et pour environ 90% des spécimens recueillis entre 10 et 12 heures. Ce test semble légèrement plus sensible que le test au papier de Gangadharam, Nair et Subbaiah (1962) pour l'isoniazide libre, mais il est nettement moins sensible que le test de l'acétylisoniazide de Eidus et Hamilton (1964) . Il n'existe aucune interférence avec la streptomycine, le PAS et la thiacetazone (thioacétazone) aux doses habituelles utilisées en clinique, mais les résultats sont affectés par le pyrazinamide, la cyclosérine et l'éthionamide. Se describe un test para detectar la presencia de isoniazida y acetil-isoniazida en orina usando nitroprusiato alcalino de sodio y ácido acético. El resultado del test es positivo en todas las muestras de orina recogidas 8 horas despnes de administrar 200 mg. ó 400 mg. de isoniazida, y en el 90% de las muestras recogidas entre 10 y 12 horas. Este test parece ser algo mas sensible que el test de Gangadharam, Nair y Subbaiah (1962) para isoniazida libre, pero mucho menos sensible que el test de acetil-isoniazida de Eidus y Hamilton (1964) . No se interfiere con el uso de estreptomicina, PAS o thiacetazona en dosis corrientes, pero está influido por el uso de pirazinamida, cicloserina y ethionamida.