Simultaneous Determination of First-Line 4-FDC Antituberculosis Drugs by UHPLC-UV and HPLC-UV: A Comparative Study.

Tuberculosis is the second most deadly infectious disease, surpassed only by HIV/AIDS, and has resulted in over 1 billion deaths in the last 200 years. The World Health Organization estimates that in 2014, 9.6 million people were infected by this disease and 1.5 million had died. First-choice treatment consists of fixed-dose combination tablets containing rifampicin, isoniazid, pyrazinamide, and ethambutol hydrochloride (4-FDC). There are pharmacopeial protocols available to test 4-FDC, but they are prolonged, two-step methods. One single-step method in the literature performs the simultaneous determination by HPLC, but requires a long acquisition time. In this context, an ultra-HPLC (UHPLC) method was developed based on the HPLC method with the objective of reducing analysis time. A C18 column (1.9 µm particle size) was used with UV-diode-array detection at 238 and 282 nm. The method was found to be selective, linear, exact, precise, and robust. Samples from two batches were analyzed and the results compared with those obtained by the HPLC method, with no statistically significant differences observed (P > 0.05). This UHPLC method reduced the analysis time from 17 to 4 min, with a more than 90% reduction in sample and reagent consumption and a financial economy of almost 50-fold.

Potential of multisyringe chromatography for the on-line monitoring of the photocatalytic degradation of antituberculosis drugs in aqueous solution.

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.

Method for simultaneously determining the Rifampin, Isoniazid, Pyrazinamide and Ethambutol in the combined fixed-dose tablets / Determinação simultânea de Rifampicina, Isoniazida, Pirazinamida e Etambutol em comprimidos com doses fixas combinadas

Tablets containing isoniazid, pyrazinamide, ethambutol and rifampicin are used for tuberculosis treatment, and theyare freely distributed by the Brazilian National Health System. The official analytical methods for testing those substances in fixed-dose combined tablet are described in the United States Pharmacopeia and theInternational Pharmacopoeia. None of these official compendiums refers to the methodologies forconducting the simultaneous analysis of these four drugs. This study aimedatdeveloping an analytical methodology to determine simultaneously allof four drugs in tablets for tuberculosis treatment, bymeans ofhigh performance liquid chromatography with ultraviolet-visible detector. The developed method used a chromatographic column with octadecylsilane stationary phase (250 mm x 4.6 mm x 5 m particle size). The mobile phase was aqueous (85 % ammonium formate buffer pH 5, 15 % methanol and 250 mg CuSO4.5H2O), and organic phase (methanol, 0.1 % triethylamine and 0.2 % formic acid). The flow was 1.0 mL/min, at a wavelength of 265 nm or, when the equipment allowed, a wavelength of 265 nm for isoniazid, pyrazinamide and ethambutol and 335 nm for rifampicin. The developed methodology showed satisfactory results regarding the precision parameter, with relative standard deviation lower than 2.0 % for the studied drugs.(AU)

Os comprimidos utilizados no tratamento da tuberculose possuem quatro fármacos associados, isoniazida, pirazinamida, etambutol e rifampicina, e são distribuídos gratuitamente pelo Sistema Único de Saúde. Os métodos analíticos oficiais para analisar este medicamento estão especificados na Farmacopeia Americana 36a edição e na Farmacopeia Internacional 4a edição. Porém, estes compêndios oficiais não possuem monografias para análise simultânea dos quatro fármacos. O objetivo deste estudo foi desenvolver uma metodologia para determinar simultaneamente os princípios ativos em comprimidos dose fixa combinada, utilizando-se cromatografia a líquido de alta eficiência com detector de ultravioleta-visível, pois é de grande importância para o controle da qualidade do medicamento. O método desenvolvido utilizou coluna cromatográfica C18 (250 x 4,6) mm e 5 m, fase móvel constituída de fase aquosa (85 % tampão formiato de amônio 0,3 mol/L pH 5, 15 % metanol e 0,005 mol/L de Cu2+ ou 250 mg/L de CuSO4.5H2O) e fase orgânica (metanol, 0,1 % de trietilamina e 0,2 % de ácido fórmico). O fluxo foi de 1,0 mL/min e comprimento de ondade 265 nm para isoniazida, pirazinamida e o etambutol e de 335 nm para rifampicina. Este método apresentou desvio padrão relativo inferior a 2,0 % na precisão e linearidade para os quatro fármacos estudados.(AU)

CHIH-DFT determination of the molecular structure infrared spectra, UV spectra and chemical reactivity of three antitubercular compounds: Rifampicin, Isoniazid and Pyrazinamide.

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.

Simultaneous determination of rifampicin, isoniazid and pyrazinamide in tablet preparations by multivariate spectrophotometric calibration.

The use of multivariate spectrophotometric calibration is presented for the simultaneous determination of the active components of tablets used in the treatment of pulmonary tuberculosis. The resolution of ternary mixtures of rifampicin, isoniazid and pyrazinamide has been accomplished by using partial least squares (PLS-1) regression analysis. Although the components show an important degree of spectral overlap, they have been simultaneously determined with high accuracy and precision, rapidly and with no need of nonaqueous solvents for dissolving the samples. No interference has been observed from the tablet excipients. A comparison is presented with the related multivariate method of classical least squares (CLS) analysis, which is shown to yield less reliable results due to the severe spectral overlap among the studied compounds. This is highlighted in the case of isoniazid, due to the small absorbances measured for this component.