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Tuberculosis (TB) remains a leading cause of death worldwide. Lipid rich, phenotypically antibiotic tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse and the need for long-term TB treatment. We present a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis. We then perform optical analysis in the form of wavelength modulated Raman spectroscopy (WMRS) on the trapped M. smegmatis for up to eight hours, and also in the presence of isoniazid (INH). The Raman fingerprints of M. smegmatis exposed to INH change substantially in comparison to the unstressed condition. Our work provides a real-time assessment of the impact of INH on the increase of lipids in these mycobacteria, which could render the cells more tolerant to antibiotics. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to different conditions and stimuli.

Among the physicochemical properties, pKa and LogP values help us in studying drug parameters like ADME and could be predicted to some extent. With this view, here we wish to predict these two properties of our previously synthesized biologically active derivatives of isoniazid using on-line available program Marvin, a Java-based chemical software application frequently used for chemical modeling. According to Marvin, pKa values predicted 99.99% unionized states of INH and some derivatives at physiological pH 7.4. Marvin calculated LogP values estimated good oral absorption for all the synthesized compounds. Therefore it can be said that the findings of the study emerged in an ideal region that permits the formulation of these derivatives. Since this was just a theoretical study, it demands more experimentation to determine accurate situation.

Thanks to operational simplicity, speediness, possibility of miniaturization and real-time nature, electrochemical sensing is a supreme alternative for non-electrochemical methodologies in drug quantification. This review, highlights different nanotech-based sensory designs for electroanalysis of isoniazid and rifampicin, the most important medicines for patients with tuberculosis. We first, concisely mention analyses with bare electrodes, associated impediments and inspected possible strategies and then critically review the last two decades works with focus on different nano-scaled electrode modifiers. We organized and described the materials engaged in several categories: Surfactants modifiers, polymeric modifiers, metallic nanomaterials, carbon based nano-modifiers (reduced graphene oxide, multi-walled carbon nanotubes, ordered mesoporous carbon) and a large class of multifarious nano composites-based sensors and biosensors. The main drawbacks and superiorities associated with each array as well as the current trend in the areas is attempted to discuss. Summary of 79 employed electrochemical approaches for analysis of isoniazid and rifampicin has also been presented., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We measured hair and plasma concentrations of isoniazid among sixteen children with tuberculosis who underwent personal or video-assisted directly observed therapy and thus had 100% adherence. This study therefore defined typical isoniazid exposure parameters after two months of treatment among fully-adherent patients in both hair and plasma (plasma area under the concentration-time curve, AUC, estimated using pharmacokinetic data collected 0, 2, 4, and 6 hours after drug administration). We found that INH levels in hair among highly-adherent individuals did not correlate well with plasma AUC or trough concentrations, suggesting that each measure may provide incremental and complementary information regarding drug exposure in the context of TB treatment.

Aim: To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH)., Materials & Methods: The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples' cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS., Results: INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism., Conclusion: The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.

Assessing treatment adherence and quantifying exposure to anti-tuberculosis drugs among children is challenging. We undertook a 'proof of concept' study to assess the drug concentrations of isoniazid (INH) in hair as a therapeutic drug monitoring tool. Children aged <12 years initiated on a thrice-weekly treatment regimen including INH (10 mg/kg) for newly diagnosed tuberculosis were enrolled. INH concentrations in hair were measured using liquid chromatography-tandem mass spectrometry at 1, 2, 4 and 6 months after initiating anti-tuberculosis treatment. We found that INH hair concentrations in all children on thrice-weekly INH were detectable and displayed variability across a dynamic range.

Background: Tuberculosis (TB) is the leading cause of death from an infectious pathogen worldwide and the most prevalent opportunistic infection in people living with HIV. Isoniazid preventive therapy (IPT) reduces the incidence of active TB and reduces morbidity and mortality in HIV-infected patients independently of antiretroviral therapy. However, treatment of latent or active TB is lengthy and inter-patient variability in pharmacokinetics and adherence common. Current methods of assessing adherence to TB treatment using drug levels in plasma or urine assess short-term exposure and pose logistical challenges. Drug concentrations in hair assess long-term exposure and have demonstrated pharmacodynamic relevance in HIV., Methods: A large hair sample from a patient with active TB was obtained for assay development. Methods to pulverize hair and extract isoniazid were optimized and then the drug detected by liquid chromatography/ tandem mass spectrometry (LC/MS-MS). The method was validated for specificity, accuracy, precision, recovery, linearity and stability to establish the assay's suitability for therapeutic drug monitoring (TDM). Hair samples from patients on directly-observe isoniazid-based latent or active TB therapy from the San Francisco Department of Public Health TB clinic were then tested., Results: Our LC/MS-MS-based assay detected isoniazid in quantities as low as 0.02ng/mg using 10-25 strands hair. Concentrations in spiked samples demonstrated linearity from 0.05-50ng/mg. Assay precision and accuracy for spiked quality-control samples were high, with an overall recovery rate of 79.5%. In 18 patients with latent or active TB on treatment, isoniazid was detected across a wide linear dynamic range., Conclusions: An LC-MS/MS-based assay to quantify isoniazid levels in hair with performance characteristics suitable for TDM was developed and validated. Hair concentrations of isoniazid assess long-term exposure and may be useful for monitoring adherence to latent or active TB treatment in the setting of HIV.

In this study, a multisyringe chromatography system (MSC) using a C18 monolithic column was proposed for the on-line monitoring of the photocatalytic degradation of isoniazid (INH, 10 mg L(-1)) and pyrazinamide (PYRA, 5mgL(-1)) mixtures in aqueous solution using a small sample volume (200 μL) with an on-line filtration device in a fully automated approach. During the photocatalytic oxidation using TiO2 or ZnO semiconductor materials, total organic carbon (TOC) and the formed intermediates were analyzed off-line using ion chromatography, ion exclusion HPLC, and ESI-MS/MS. The results showed that TiO2 exhibits a better photocatalytic activity than ZnO under UV irradiation (365 nm) for the degradation of INH and PYRA mixtures, generating 97% and 92% degradation, respectively. The optimal oxidation conditions were identified as pH 7 and 1.0 g L(-1) of TiO2 as catalyst. The mineralization of the initial organic compounds was confirmed by the regular decrease in TOC, which indicated 63% mineralization, and the quantitative release of nitrate and nitrite ions, which represent 33% of the nitrogen in these compounds. The major intermediates of INH degradation included isonicotinamide, isonicotinic acid, and pyridine, while the ESI-MS/MS analysis of PYRA aqueous solution after photocatalytic treatment showed the formation of pyrazin-2-ylmethanol, pyrazin-2-ol, and pyrazine. Three low-molecular weight compounds, acetamide, acetic acid and formic acid, were detected during INH and PYRA decomposition. PYRA was more resistant to photocatalytic degradation due to the presence of the pyrazine ring, which provides greater stability against OH attack., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Setting: In most developing countries, paediatric tuberculosis is treated with split tablets leading to potential inaccuracy in the dose delivery and drug exposure. There is no data on the quality of first-line drugs content in split fixed-dose combination tablets., Objective: To determine Isoniazid, Pyrazinamide and Rifampicin content uniformity in split FDC tablets used in the treatment of childhood tuberculosis., Design: Drug contents of 15 whole tablets, 30 half tablets and 36 third tablets were analysed by high performance liquid chromatography. The content uniformity was assessed by comparing drug content measured in split portions with their expected amounts and the quality of split portions was assessed applying qualitative specifications for whole tablets., Results: All whole tablets measurements fell into the USP proxy for the three drugs. But a significant number of half and third portions was found outside the tolerated variation range and the split formulation failed the requirements for content uniformity. To correct for the inaccuracy of splitting the tablets into equal portions, a weight-adjustment strategy was used but this did not improve the findings., Conclusion: In split tablets the content of the three drugs is non-uniform and exceeded the USP recommendations. There is an absolute need to make child-friendly formulations available for the treatment of childhood tuberculosis.

Objective: To determine the content of certain antituberculosis (TB) drugs supplied at TB treatment centres of the Revised National TB Control Programme (RNTCP) in the state of Tamil Nadu, India., Methods: Eight districts across the state were selected, and the following drugs were collected from five settings (District TB centre, TB unit, designated microscopy centres, DOT providers) in each district: rifampicin (150 and 450 mg), isoniazid (300 mg), pyrazinamide (500 and 750 mg), ethambutol (400 and 600 mg), ethionamide (250 mg), levofloxacin (500 mg) and cycloserine (250 mg). A maximum of 10 tablets/capsules were collected from each setting. The drugs were coded prior to analysis. All drugs were assayed by validated spectrophotometric methods. The acceptable limits for drug content were taken as 90-110% of the stated content., Results: More than 90% of tablets of rifampicin 450 mg, isoniazid 300 mg, pyrazinamide 500 and 750 mg, ethambutol 400 and 600 mg and ethionamide 250 mg were within acceptable limits. Eighty per cent of rifampicin 150 mg, 21% of cycloserine 250 mg and 87% of levofloxacin 500 mg were within acceptable limits. The mean cycloserine content was below the acceptable limit in all districts, the mean drug content being 200 mg (range: 108-245 mg)., Conclusion: This systematic study showed that the stated drug content of cycloserine was not reached in all districts. Deterioration of cycloserine could be minimised by storing the drug in refrigerators. The geographical location of the districts had no influence on the drug content., (© 2013 John Wiley & Sons Ltd.)

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, infects approximately one third of the current world population. Isoniazid is one of the most frequently used first-line anti-TB drugs. In this study, we developed a sensitive cation-selective exhaustive injection-sweeping-micellar electrokinetic chromatography method (CSEI-Sweep-MEKC) for analyzing isoniazid in human plasma. Parameters including acetonitrile (ACN) percentage in the separation buffer; the injection time, and concentration of the high-conductivity buffer; sodium dodecyl sulfate (SDS) concentration; phosphate concentration in the sample matrix; and the sample injection time were all optimized to obtain the best analytical performance. The optimal background electrolyte comprised 50 mM phosphate buffer, 100 mM SDS, and 15% ACN. Non-micelle background electrolyte, containing 75 mM phosphate buffer and 15% ACN, was first injected into the capillary, followed by a short plug of 200 mM phosphate (high-conductivity buffer). Run-to-run repeatability (n=3) and intermediate precision (n=3) of peak area ratios were found to be lower than 8.7% and 11.4% RSD, respectively. The accuracy of the method was within 98.1-106.9%. The limit of detection of isoniazod in human plasma was 9 ng mL(-1). Compared with conventional MEKC, the enhancement factor of the CSEI-Sweep-MEKC method was 85 in plasma samples. The developed method was successfully used to determine isoniazid concentration in patient plasma. The results demonstrated that CSEI-Sweep-MEKC has the potential to analyze isoniazid in human plasma for therapeutic drug monitoring and clinical research., (© Springer-Verlag 2011)

Ethyl chloroformate was examined as a precolumn derivatizing reagent for the gas chromatographic (GC) determination of isoniazid (INH) and hydrazine (HZ). Phenylhydrazine (PHZ) was used as an internal standard. GC separation was carried out on an HP-5 column (30 m x 0.32 mm i.d.) with flame ionization detection. The elution was carried out at an initial column temperature of 150 degrees C for 1 min at a heating rate of 10 degrees C/min up to 250 degrees C, nitrogen flow rate of 4 ml/min and a split ratio of 10:1, v/v. The linear calibration ranges for INH and HZ were observed between 3.5-37.5 and 3.5-35 microg/ml with corresponding detection limits of 0.18 and 0.17 ng reaching the detector. The method was subsequently applied to the determination of INH and HZ in pharmaceutical preparations, achieving a relative standard deviation (RSD) of 3.8-5.8%. The recovery percentage of INH from isoniazid syrup was 98% with an RSD of 5.2%.

Three of the most frequent antitubercular agents employed against Mycobacterium tuberculosis are: Rifampicin, Isoniazid and Pyrazinamide. It has been proven that the use of these antitubercular agents together, shortens the treatment period from 12-18 months to 6 months [1]. In this work we use a new Density Functional Theory chemistry model called CHIH-DFT (Chihuahua-Heterocycles-Density Functional Theory) that reflects the mixture of Hartree Fock exchange and DFT exchange, according to a mixing parameter based on empirical rules suited for heterocyclic systems. This new chemistry model was used to calculate the molecular structure of these antitubercular compounds, as well as their infrared, UV spectra, chemical reactivity and electronic properties. The UV and infrared spectra were obtained by experimental techniques. The calculated molecular structure, UV and IR spectra values from CHIH-DFT were compared with experimentally obtained values and theoretical studies. These results are in good agreement with experimental and theoretical studies. We also predicted using the relative electrophilicity and relative nucleophilicity concepts as defined by Roy et al. [2] the chemical active sites for the three antitubercular compounds as well as their electronegativity, ionization potential, electron affinity, hardness, dipole moment, E(HOMO)-E(LUMO) gap energy, etc.

In this paper a continuous-flow chemiluminescence (CL) system with artificial neural network calibration is proposed for simultaneous determination of rifampicin and isoniazid. This method is based on the different kinetic spectra of the analytes in their CL reaction with alkaline N-bromosuccinimide as oxidant. The CL intensity was measured and recorded every second from 1 to 300 s. The data obtained were processed chemometrically by use of an artificial neural network. The experimental calibration set was 20 sample solutions. The relative standard errors of prediction for both analytes were approximately 5%. The proposed method was successfully applied to the simultaneous determination of rifampicin and isoniazid in a combined pharmaceutical formulation.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

In this work new strategies were exploited to implement multi-pumping flow systems relying on the utilisation of multiple devices that act simultaneously as sample-insertion, reagent-introduction, and solution-propelling units. The solenoid micro-pumps that were initially used as the only active elements of multi-pumping systems, and which were able to produce pulses of 3 to 25 microL, were replaced by syringe pumps with the aim of producing pulses between 1 and 4 microL. The performance of the developed flow system was assessed by using distinct sample-insertion strategies like single sample volume, merging zones, and binary sampling in the spectrophotometric determination of isoniazid in pharmaceutical formulations upon reaction with 1,2-naphthoquinone-4-sulfonate, in alkaline medium. The results obtained showed that enhanced sample/reagent mixing could be obtained with binary sampling and by using a 1 microL per step pump, even in limited dispersion conditions. Moreover, syringe pumps produce very reproducible flowing streams and are easily manipulated and controlled by a computer program, which is greatly simplified since they are the only active manifold component. Linear calibration plots up to 18.0 microg mL(-1), with a relative standard deviation of less than 1.48% (n=10) and a throughput of about 20 samples per hour, were obtained.

A method for the determination of isoniazid (ISO), pyrazinamide (PYR) and rifampicin (RIF) in pharmaceutical products, by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection is described. The influence of pH, concentration of surfactants, buffer and organic solvents, over the separation were studied as experimental variables. The optimal separation was carried out at 30 degrees C and 20 kV, using a 40 mM borate buffer and 100 mM sodium dodecylsulphate (SDS) adjusted to pH 8.5. Under these conditions, the analysis is accomplished in about 8 min. The method was applied to the determination of these compounds in different pharmaceuticals with good results when compared with a reference liquid chromatographic (LC) method.

Based on a new electrogenerated chemiluminescence (ECL) analytical idea, this paper explains a sensitive and selective flow-injection ECL method using luminol for the determination of isoniazid, based on the sensitizing effect of isoniazid for the weak ECL emission of electrochemically oxidized luminol. Under the optimum experimental conditions, the relative ECL intensity was linear with isoniazid concentration in the range of 4.0 x 10(-8) mol/L to 8.0 x 10(-6) mol/L and with a detecting limit of 2.8 x 10(-8) mol/L.

Coupling of isoniazid with polysuccinimide afforded a water-insoluble polymeric pro-drug; by reaction with ethanolamine it was chemically transformed in a water-soluble adduct. The in vitro release of isoniazid from the drug-polymer adduct was studied by using an artificial stomach wall lipid membrane. The transfer rate constant from simulated gastric juice to simulated plasma was defined and compared with that of an equivalent dose of pure drug.

1. A solvent system was devised for the extraction of isoniazid and its metabolites acetylisoniazid, monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine from serum and urine. 2. Specific chemical and fluorimetric methods were developed for the determination of the extracted isoniazid and acetylisoniazid, and chemical methods for the determination of monoacetylhydrazine, diacetylhydrazine, isonicotinic acid and isonicotinylglycine. 3. When applied to serum, these methods were capable of measuring concentrations of down to about 0.005mug of isoniazid/ml, 0.05mug of acetylisoniazid/ml, 0.2mug of monoacetylhydrazine/ml, 0.2mug of diacetylhydrazine/ml, 0.02mug of isonicotinic acid/ml and 0.1mug of isonicotinylglycine/ml. 4. In urine, these methods were capable of measuring concentrations of down to about 0.05mug of isoniazid/ml, 0.2mug of acetylisoniazid/ml, 1mug of diacetylhydrazine/ml, 0.1mug of isonicotinic acid/ml and 0.2mug of isonicotinylglycine/ml. 5. The stability of these compounds was studied in serum and urine and a method devised to decrease their decomposition in serum.