Development and validation of a simple LC-MS/MS method for simultaneous determination of moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol in human plasma.

Treatment of multidrug-resistant tuberculosis (MDR-TB) is challenging due to high treatment failure rate and adverse drug events. This study aimed to develop and validate a simple LC-MS/MS method for simultaneous measurement of five TB drugs in human plasma and to facilitate therapeutic drug monitoring (TDM) in MDR-TB treatment to increase efficacy and reduce toxicity. Moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were prepared in blank plasma from healthy volunteers and extracted using protein precipitation reagent containing trichloroacetic acid. Separation was achieved on an Atlantis T3 column with gradient of 0.1% formic acid in water and acetonitrile. Drug concentrations were determined by dynamic multiple reaction monitoring in positive ion mode on a LC-MS/MS system. The method was validated according to the United States' Food and Drug Administration (FDA) guideline for bioanalytical method validation. The calibration curves for moxifloxacin, levofloxacin, prothionamide, pyrazinamide and ethambutol were linear, with the correlation coefficient values above 0.993, over a range of 0.1-5, 0.4-40, 0.2-10, 2-100 and 0.2-10 mg/L, respectively. Validation showed the method to be accurate and precise with bias from 6.5% to 18.3% for lower limit of quantification and -5.8% to 14.6% for LOW, medium (MED) and HIGH drug levels, and with coefficient of variations within 11.4% for all levels. Regarding dilution integrity, the bias was within 7.2% and the coefficient of variation was within 14.9%. Matrix effect (95.7%-112.5%) and recovery (91.4%-109.7%) for all drugs could be well compensated by their isotope-labelled internal standards. A benchtop stability test showed that the degradation of prothionamide was over 15% after placement at room temperature for 72 h. Clinical samples (n = 224) from a cohort study were analyzed and all concentrations were within the analytical range. The signal of prothionamide was suppressed in samples with hemolysis which was solved by sample dilution. As the method is robust and sample preparation is simple, it can easily be implemented to facilitate TDM in programmatic MDR-TB treatment.

Low Serum Concentrations of Moxifloxacin, Prothionamide, and Cycloserine on Sputum Conversion in Multi-Drug Resistant TB.

PURPOSE: Low serum concentrations of drugs used to treat multi-drug resistant tuberculosis (MDR-TB) have occasionally been associated with treatment failure. We determined the frequencies of low serum concentrations of anti-MDR-TB drugs, and assessed the effects of these concentrations on 2-month sputum conversion. MATERIALS AND METHODS: The serum levels of moxifloxacin (MF), prothionamide (PTH), and cycloserine (CS) were determined for 89 serum samples by high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: Low serum concentrations of MF, PTH, and CS below the minimal levels of the normal ranges were 83.3% (20/24), 59.2% (29/49), and 71.2% (47/66), respectively. There were no significant differences between the 2-month sputum conversion group (n=25) and the 2-month sputum non-conversion group (n=4) in median drug concentrations (µg/mL) of MF (1.46 vs. 1.60), PTH (0.91 vs. 0.70), and CS (14.90 vs. 14.90). However, a poor compliance rate was significantly greater in the 2-month sputum non-conversion group (75.0%, 3/4) than in the 2-month sputum conversion group (0%, 0/25) (p=0.001). CONCLUSION: The frequency of low serum concentrations of anti-MDR-TB drugs was substantial and might not affect the 2-month sputum conversion rate. Larger prospective studies with timely sampling are needed to investigate the role of therapeutic drug monitoring in MDR-TB.

Low Serum Concentrations of Moxifloxacin, Prothionamide, and Cycloserine on Sputum Conversion in Multi-Drug Resistant TB

PURPOSE: Low serum concentrations of drugs used to treat multi-drug resistant tuberculosis (MDR-TB) have occasionally been associated with treatment failure. We determined the frequencies of low serum concentrations of anti-MDR-TB drugs, and assessed the effects of these concentrations on 2-month sputum conversion. MATERIALS AND METHODS: The serum levels of moxifloxacin (MF), prothionamide (PTH), and cycloserine (CS) were determined for 89 serum samples by high-pressure liquid chromatography-tandem mass spectrometry. RESULTS: Low serum concentrations of MF, PTH, and CS below the minimal levels of the normal ranges were 83.3% (20/24), 59.2% (29/49), and 71.2% (47/66), respectively. There were no significant differences between the 2-month sputum conversion group (n=25) and the 2-month sputum non-conversion group (n=4) in median drug concentrations (microg/mL) of MF (1.46 vs. 1.60), PTH (0.91 vs. 0.70), and CS (14.90 vs. 14.90). However, a poor compliance rate was significantly greater in the 2-month sputum non-conversion group (75.0%, 3/4) than in the 2-month sputum conversion group (0%, 0/25) (p=0.001). CONCLUSION: The frequency of low serum concentrations of anti-MDR-TB drugs was substantial and might not affect the 2-month sputum conversion rate. Larger prospective studies with timely sampling are needed to investigate the role of therapeutic drug monitoring in MDR-TB.

Ex vivo conversion of prodrug prothionamide to its metabolite prothionamide sulfoxide with different extraction techniques and their estimation in human plasma by LC-MS/MS.

BACKGROUND: The present work reports an ex vivo conversion study of prothionamide to its active metabolite prothionamide sulfoxide in human plasma during sample preparation by three conventional extraction techniques. RESULTS: The chromatography was done on a Hypersil™ Gold C18 (50 × 2.1 mm, 3.0 µm) column using 0.1% acetic acid and acetonitrile (20:80, v/v) as the mobile phase. Quantitation of the analytes was done by MS/MS in the positive ionization mode. The method was validated over a wide concentration range of 30 to 6000 ng/ml for prothionamide and 50 to 10,000 ng/ml for prothionamide sulfoxide. The recovery for both the analytes was greater than 89%. Stability was extensively validated under different storage conditions. CONCLUSION: The extraction protocol was optimized using acetonitrile as protein precipitant for their simultaneous determination in human plasma by LC-MS/MS. The method was applied to a bioequivalence study of 250 mg prothionamide tablet formulation in 14 healthy Indian subjects.

Pharmacokinetics of prothionamide in patients with multidrug-resistant tuberculosis.

SETTING: National Masan Tuberculosis Hospital, Masan, South Korea. OBJECTIVE: To evaluate the pharmacokinetics of prothionamide (PTH) in South Korean patients with multidrug-resistant tuberculosis (MDR-TB) and to investigate whether differences in body mass index (BMI) could explain observed differences in PTH disposition. DESIGN: Seventeen patients participated in the study; all had MDR-TB and had received combination anti-tuberculosis treatment, including PTH, cycloserine, ofloxacin, para-aminosalicylic acid and streptomycin or kanamycin, for at least 2 weeks. The patients were divided into two groups based on BMI: Group A (18.5 < or = BMI<23), and Group B (BMI<18.5). Serum samples were collected over 24 h, and the plasma PTH concentration was determined by a validated high-performance liquid chromatography assay. RESULTS: After steady-state administration of PTH, the mean area under the plasma concentration-time curve from time 0 to 12 h (AUC(0-12h)) was 11.0 +/- 3.7 microg h/ml. The mean T(max) and t(1/2) were respectively 3.6 h and 2.7 h. No significant difference in PTH disposition was observed between groups A and B, except for ke and t(1/2). CONCLUSION: In the pharmacokinetic parameter estimates for PTH in MDR-TB patients during routine treatment, the pharmacokinetics of PTH did not appear to correlate with extent of emaciation in MDR-TB patients.

Simple, rapid and sensitive determination of protionamide in human serum by high-performance liquid chromatography.

A simple high-performance liquid chromatography method has been developed that allows the sensitive determination of protionamide (2-n-propyl-pyridine-4-carboxylic acid thioamide, PTH) in human serum. After pretreatment of the serum with trichloroacetic acid (TCA) and centrifugation the supernatants were neutralized using NaHCO3. PTH was separated on a Kromasil 100 C4 column (acetonitrile-sodium tetraborate buffer pH 8-dibutylamine) and determined photometrically at 291 nm. The lower limit of quantification for 300 microl serum precipitated with 60 microl TCA and injection of 50 microl was 27 microg/l and linearity was observed up to 15 mg/l.

A comparison of the blood levels and urinary excretion of ethionamide and prothionamide in man.

The blood levels and urinary excretion of the anti-mycobacterial drugs ethionamide and prothionamide have been compared after oral dosage in man. High pressure liquid chromatographic methods were used to determine the two closely related thioamides and their microbiologically active sulphoxide metabolites after the ingestion of both single and combined doses of the two drugs. Both drugs were rapidly eliminated from the body, the half-life for the urinary excretion and removal from the plasma of prothionamide being slightly less than that of ethionamide. Less than 0.1% of the orally administered doses were excreted unchanged in the faeces. Plasma concentrations of ethionamide and its sulphoxide metabolite were substantially higher than those of prothionamide and prothionamide sulphoxide. The implications of these findings for the use of ethionamide or prothionamide in the treatment of lepromatous leprosy are discussed.