Isoniazid (INH) is highly effective for the management of tuberculosis. However, it can cause liver injury and even liver failure. INH metabolism has been thought to be associated with INH-induced liver injury. This review summarized the metabolic pathways of INH and discussed their associations with INH-induced liver injury.

This is the latest of a series of reviews focused on the analysis of genotoxic impurities. This review summarises the analytical approaches reported in the literature relating to hydrazine, hydrazines, hydrazides and hydrazones. It is intended to provide guidance for analysts needing to develop procedures to control such impurities, particularly where this is due to concerns relating to their potential genotoxicity. Of particular note is the wide variety of techniques employed, both chromatographic and spectroscopic, with most involving derivatisation. Such a wide variety of options allow the analyst a real choice in terms of selecting the most appropriate technique specific to their requirements. Several generic methodologies, covering the three main analytical approaches; i.e. HPLC (high performance liquid chromatography), GC (gas chromatography) and IC (ion chromatography), are also described.Copyright © 2010 Elsevier B.V. All rights reserved.

Today the determination of hydrazines is an important application in analytical chemistry. This review shows the current state-of-the-art analyses and discusses the merits of the direct chromatographic methods for the determination of hydrazines such as ion-, ion-exclusion, ion-pair and hydrophilic interaction chromatography. The methodological aspects of the separation and detection of hydrazines are considered for these methods. Examples of hydrazine determination in real samples are presented.Copyright © 2012 Elsevier B.V. All rights reserved.

Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels. Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous quantitative analysis of hydrazine and acetylhydrazine in human plasma based on the strategy of p-tolualdehyde derivatization. The derivatization reactions were easily realized by ultrasonic manipulation for 40min. Good separation of the derivatization products was achieved using a C column by gradient elution. The optimized mass transition ion-pairs (m/z) monitored for the two hydrazine derivatives were m/z 237.1≫>119.9 and m/z 176.9≫>117.8, respectively. The limit of detection (LOD) and limit of quantification (LOQ) for hydrazine were 0.002 and 0.005ngmL separately. And they were 0.03 and 0.05ngmL for acetylhydrazine, respectively. The linear range was 0.005-50ngmL for hydrazine and 0.05-500ngmL for acetylhydrazine with R greater than 0.999. The recovery range was determined to be 95.38-108.12% with the relative standard deviation (RSD) in the range of 1.24-14.89%. The method was successfully applied to detect 30 clinical plasma samples of pulmonary tuberculosis patients treated with isoniazid. The concentrations were from 0.04-1.99ngmL for hydrazine and 0.06-142.43ngmL for acetylhydrazine. The results indicated that our developed method had the potential for the detection of hydrazine toxicology in complex biological samples. Furthermore, the method has an important significance to clinical treatment with drugs.Copyright © 2017. Published by Elsevier B.V.

Isoniazid and its metabolites are potentially associated with hepatotoxicity and treatment outcomes in patients who receive antituberculosis (TB) therapy. To further understand the pharmacokinetic profiles of these molecules, a method based on LC-MS/MS was developed to determine the concentration of these compounds in human plasma. Isoniazid, acetylisoniazid, and isonicotinic acid were directly analyzed, whereas hydrazine and acetylhydrazine were determined after derivatization using p-tolualdehyde. Chromatographic separation was conducted on reversed-phase C18 columns with gradient elution, and detection was carried out in multiple reaction monitoring mode. The calibration curves were linear with correlation coefficients (r) greater than 0.9947 for all analytes. The intra- and inter-day precision was less than 13.43%, and the accuracy ranged between 91.63 and 114.00%. The recovery and matrix effect of the analytes were also consistent (coefficient of variation was less than 9.36%). The developed method successfully quantified isoniazid and its metabolites in TB patients. The method has broad applications in clinical research, including isoniazid one-point-based therapeutic drug monitoring, genotype–phenotype association studies of isoniazid metabolic profile and isoniazid-induced hepatotoxicity, and the initial dose prediction of isoniazid using population pharmacokinetic modeling.

We describe the development and validation of a new high performance liquid chromatography (HPLC) method for analysis of a combination of the first-line anti-tubercular drugs isoniazid, pyrazinamide, and rifampicin together with clofazimine. This is a unique challenge since clofazimine and rifampicin are relatively highly lipophilic drugs, whereas isoniazid and pyrazinamide are considerably more hydrophilic. Thus, clear separation of peaks and quantification of four individual drugs can present difficulties during the development of an analytical method. Detection was established at two wavelengths—254 nm for isoniazid and pyrazinamide and 320 nm for clofazimine and rifampicin. Gradient elution was employed using 0.1% aqueous formic acid (A) and acetonitrile (B); clear separation of the four drugs was achieved within 10 min. A linear relationship was indicated by a correlation coefficient (r2) of 0.9999 for each anti-tubercular drug, respectively. The limit of detection (LOD) for the individual drugs was 0.70 µg/mL (isoniazid), 0.30 µg/mL (pyrazinamide), 0.20 µg/mL (rifampicin) and 0.20 µg/mL (clofazimine). Precision experiments rendered a mean recovery percentage of 101.25% (isoniazid), 98.70% (pyrazinamide), 99.68% (rifampicin) and 97.14% (clofazimine). This HPLC method was validated and is reliable, repeatable, and accurate for the purpose of conducting simultaneous HPLC analyses of the four anti-tubercular drugs.

In recent years, liquid chromatography with tandem mass spectrometry (LC–MS/MS) has become a fundamental technology in clinical practice. In Japan, the LC–MS/MS system is used in many large hospitals. It has become popular among pharmacists and laboratory technicians. LC–MS/MS has some advantages in terms of accuracy, speed, and comprehensiveness compared to conventional automated chemical testing equipment. However, LC–MS/MS is by no means a universal method, and it is necessary to understand its characteristics before using it. In the field of therapeutic drug monitoring (TDM), there is an issue with linearity in comprehensive measurement; however, ion‐abundance adjustment methods, such as in‐source collision‐induced dissociation, have been proposed as a solution to this problem. The development of a biomarker analysis includes search, identification, and quantification, and it is necessary to select an appropriate mass spectrometric method for each step. In this paper, we review cutting‐edge technologies that can expand the performance of LC–MS/MS in the clinical field and consider current issues and future prospects.

Fungal infections, especially invasive types, have become a serious healthcare problem as the immunocompromised population increases. There are five main classes of antifungal drugs: polyenes, flucytosine, allylamines, azoles, and echinocandins. Therapeutic drug monitoring (TDM) is justified for flucytosine and triazoles due to their large inter- and intra-individual pharmacokinetic variability and their high tendency for drug-drug interactions. Available methods for measuring these drugs include bioassay, liquid chromatography and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The LC-MS/MS approach is preferred due to its superior analytic sensitivity and specificity. In this review, we highlight TDM methods by LC-MS/MS for these antifungal drugs searchable in PubMed by December 1, 2018. LC-MS/MS methods that were developed for other purposes such as pharmacokinetics or toxicokinetics were also included. We have critically analyzed these methods with an emphasis on sensitivity, specificity, simplicity, throughput and robustness.Copyright © 2019 Elsevier B.V. All rights reserved.

In the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen enormous growth in routine toxicology laboratories. LC-MS/MS offers significant advantages over other traditional testing, such as immunoassay and gas chromatography-mass spectrometry methodologies. Major strengths of LC-MS/MS include improvement in specificity, flexibility, and sample throughput when compared with other technologies. Here, the basic principles of LC-MS/MS technology are reviewed, followed by advantages and disadvantages of this technology compared with other traditional techniques. In addition, toxicology applications of LC-MS/MS for simultaneous detection of large panels of analytes are presented.Copyright © 2016 Elsevier Inc. All rights reserved.

Idiosyncratic drug‐induced liver injury (DILI) is a significant adverse effect of antitubercular therapy with isoniazid (INH). Although the drug has been used for many decades, the underlying mode of action (both patient‐specific and drug‐specific mechanisms) leading to DILI are poorly understood. Among the patient‐specific determinants of susceptibility to INH‐associated DILI, the importance of HLA genetic variants has been increasingly recognized, whereas the role of polymorphisms of drug‐metabolizing enzymes (NAT2 and CYP2E1) has become less important and remains controversial. However, these polymorphisms are merely correlative, and other molecular determinants of susceptibility have remained largely unknown. Regarding the drug‐specific mechanisms underlying INH‐induced liver injury, novel concepts have been emerging. Among these are covalent protein adduct formation via novel reactive intermediates, leading to hapten formation and a potential immune response, and interference with endogenous metabolism. Furthermore, INH and/or INH metabolites (e.g. hydrazine) can cause mitochondrial injury, which can lead to mitochondrial oxidant stress and impairment of energy homeostasis. Recent studies have revealed that underlying impairment of complex I function can trigger massive hepatocellular injury induced by otherwise nontoxic concentrations of INH superimposed on these mitochondrial deficiencies. This review discusses these emerging new paradigms of INH‐induced DILI and highlights recent insights into the mechanisms, as well as points to the existing large gaps in our understanding of the pathogenesis.

Monitoring of anti‐tuberculosis drug concentrations and dose adjustment can be helpful in cases that show poor response to treatment. Here, we describe a method that can rapidly and simultaneously measure the blood concentrations of four anti‐tuberculosis drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) and two major metabolic ratios (acetylisoniazid/isoniazid and 25‐desacetylrifampicin/rifampicin) using high‐performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). A C18 reversed‐phase column and gradients of methanol in 0.3% formic acid and water were used for HPLC separation. The drug concentrations were determined by multiple reaction monitoring in positive ion mode and the assay performance was evaluated. We determined peak concentration ranges for each drug and acetylisoniazid/isoniazid and 25‐desacetylrifampicin/rifampicin ratios by analyzing 2‐h post‐dose samples in patients treated with standard dosing as a first‐line treatment. The preparation of 20 samples including two steps of deproteinization with 50% and 100% methanol was performed within 20 min and chromatographic separation was achieved within 4 min/sample. Interassay calibration variability data obtained over concentrations of 0–8 µg/mL for isoniazid and ethambutol and 0–80 µg/mL for rifampicin and pyrazinamide showed a linear and reproducible curve. Within‐run and between‐run imprecision (CVs) were 1.9–5.5% and 3.5–10.5% and the lower limits of detection and quantification were 0.01–0.5 µg/mL and 0.05–1.0 µg/mL, respectively. The isoniazid concentration was found to be inversely correlated to the acetylisoniazid/isoniazid ratio (R = −0.739, P < 0.001). The devised method allows for the simple, rapid, sensitive and reproducible quantification of isoniazid, rifampicin, pyrazinamide, ethambutol and their two metabolic ratios and should be helpful for therapeutic drug monitoring in tuberculosis patients. Copyright © 2007 John Wiley & Sons, Ltd.

A new type of deep eutectic solvents (DESs) have been prepared and used as extraction solvents for ultrasound assisted-deep eutectic solvent based emulsification liquid phase microextraction method (UA-DES-ELPME) for the determination and speciation of total chromium, chromium(III) and chromium(VI). The chromium concentration in DES rich phase (extraction phase) was determined by using microsample injection flame atomic absorption spectrometer (FAAS). The detection limit (LOD), the quantification limit (LOQ), preconcentration factor and relative standard deviation were found as 5.5µgL(-1), 18.2µgL(-1), 20 and 6%, respectively. The accuracy of the developed method was evaluated by the analysis of water the certified reference materials (TMDA-53.3 Fortified environmental water and TMDA-54.4 Fortified Lake Water) and addition-recovery tests for water samples. Copyright © 2016 Elsevier B.V. All rights reserved.

When choosing an analog internal standard (IS) in a quantitative LC-MS/MS assay, careful selection and thorough verification are important for developing an accurate quantitative assay. The IS is a critical component in quantitative mass spectrometry because it is used to normalize results by compensating for variations in sample preparation and instrument performance. Here we present the results of our investigation in the selection process for a structural analog IS (SA-IS) to be used in the quantification of 6-methylmercaptopurine (6-MMP) in cytolysed red blood cell (RBC).A cocktail solution of 9 SA-ISs including the isotopically labeled structural isomer and the 6-MMP stable isotope-labeled IS (SIL-IS) was spiked into cytolysed RBC controls and patient samples. Linearity, accuracy, sensitivity, precision, run stability, method comparison, and reinjection reproducibility experiments were performed. Ion suppression was also assessed by T-infusing the cocktail solution.All analogs were linear from 100 to 1200 ng/mL 6-MMP with acceptable precision and sensitivity by use of a spiked blank lysate. Method comparison plots of 6-MMP concentrations in patient samples had excellent agreement for 2 of the SA-ISs (i.e., the isotopically labeled structural isomer and an SA-IS with an added methyl group) when compared to the SIL-IS. Halogen-substituted analogs (i.e., Cl and Br) also met the criteria as an acceptable IS. However, 2 of the selected SA-ISs having substituted amine moieties showed unacceptable performance, with ≥15% bias when compared to the SIL-IS.There are many parameters to consider when determining if an analog will be a good IS choice, and the approaches highlighted in this article can be applied to the selection of SA-IS in the development of other LC-MS/MS assays.© 2018 American Association for Clinical Chemistry.

Patients with tuberculosis (TB) coinfected with HIV are more likely to have low blood concentrations of the first‐line anti‐TB drugs (associated with poor outcomes). Therapeutic drug monitoring (TDM) is recommended for certain patient populations with TB at increased risk for a poor outcome. Our objective was to estimate the diagnostic accuracy of a 2‐hour TDM serum sample for the first‐line anti‐TB drugs among patients with HIV/TB and evaluate the information gained by an additional 6‐hour sample. We created a virtual (n = 1000) HIV/TB patient population and performed pharmacokinetic simulations using published population models for isoniazid, rifampin, pyrazinamide, and ethambutol. We performed receiver operating characteristic analysis to compare the diagnostic performance of a single 2‐hour serum sample with samples obtained at 2 and 6 hours after dosing. The sensitivity of a single 2‐hour serum concentration to identify patients with HIV/TB with adequate serum exposures was lowest for rifampin (54.9%; 95%CI, 50.79%‐59.41%) and highest for ethambutol (70.8%; 95%CI, 66.06%‐72.61%) for maximum concentration (Cmax) targets. Diagnostic accuracy of a single 2‐hour serum sample for the area under the concentration‐time curve (AUC) from time 0 to 24 hours target was highest for isoniazid (93%; 95%CI, 90.9%‐94.1%) and lowest for pyrazinamide (66.3%; 95%CI, 62.6%‐70.0%). In summary, the diagnostic performance of TDM for Cmax and AUC from time 0 to 24 hours targets demonstrated variability across the first‐line anti‐TB drugs. The addition of a 6‐hour serum sample led to the highest statistically significant improvement (P <.001) and highest increase in diagnostic accuracy (area under the receiver operating characteristic curve) for rifampin for Cmax and AUC. The other first‐line drugs had modest/negligible increases in diagnostic accuracy.

Tuberculosis (TB) remains one of the world's deadliest communicable diseases. Although cure rates of the standard four-drug (rifampicin, isoniazid, pyrazinamide, ethambutol) treatment schedule can be as high as 95-98 % under clinical trial conditions, success rates may be much lower in less well resourced countries. Unsuccessful treatment with these first-line anti-TB drugs may lead to the development of multidrug resistant and extensively drug resistant TB. The intrinsic interindividual variability in the pharmacokinetics (PK) of the first-line anti-TB drugs is further exacerbated by co-morbidities such as HIV infection and diabetes.Therapeutic drug monitoring has been proposed in an attempt to optimize treatment outcome and reduce the development of drug resistance. Several studies have shown that maximum plasma concentrations (C max), especially of rifampicin and isoniazid, are well below the proposed target C max concentrations in a substantial fraction of patients being treated with the standard four-drug treatment schedule, even though treatment's success rate in these studies was typically at least 85 %.The proposed target C max concentrations are based on the concentrations of these agents achieved in healthy volunteers and patients receiving the standard doses. Estimation of C max based on one or two sampling times may not have the necessary accuracy since absorption rate, especially for rifampicin, may be highly variable. In addition, minimum inhibitory concentration (MIC) variability should be taken into account to set clinically meaningful susceptibility breakpoints. Clearly, there is a need to better define the key target PK and pharmacodynamic (PD) parameters for therapeutic drug monitoring (TDM) of the first-line anti-TB drugs to be efficacious, C max (or area under the curve (AUC)) and C max/MIC (or AUC/MIC).Although TDM of first-line anti-TB drugs has been successfully used in a limited number of specialized centers to improve treatment outcome in slow responders, a better characterization of the target PK and/or PK/PD parameters is in our opinion necessary to make it cost-effective.