ABSTRACT
This study describes the development and validation of a novel 96-microwell-based high throughput spectrophotometric assay for pharmaceutical quality control of crizotinib (CZT), a novel drug for the treatment of non-small cell lung cancer. We examined the reaction between CZT and 1,2-naphthoquinone-4-sulphonate, a chromogenic reagent. A red-colored product showing a maximum absorption peak (λmax) at 490 nm was produced in an alkaline medium (pH 9). We examined stoichiometry of the reaction and postulated the reaction mechanism. To our knowledge, this is the first study to describe a color-developing reaction for the proposed assay. The reaction was performed in a 96-microwell plate, and the absorbance of the colored product was measured using an absorbance reader at 490 nm. Under optimized reaction conditions, Beer's law, which shows a correlation between absorbance and CZT concentration, was obeyed in the range of 4-50 µg/well with an appropriate correlation coefficient (0.999). The limits of detection and quantification were 1.73 and 5.23 µg/well, respectively. The assay showed high precision and accuracy. The proposed assay was applied successfully for the determination of CZT in capsules. Thus, the assay proposed in this study is practical and valuable for routine application in pharmaceutical quality control laboratories...
Este estudo descreve o desenvolvimento e a validação de um novo ensaio espectrofotométrico em larga escala em 96 micropoços para o controle farmacêutico de crizotinibe (CZT), novo fármaco para o tratamento de câncer de pulmão de células não pequenas. Examinamos a reação entre o CZT e o 4-sulfonato de 1,2-naftoquinona, um reagente cromogênico. Obteve-se, em meio alcalino (pH 9), produto vermelho, com absorção máxima (λmax) em 490 nm. Examinamos a estequiometria da reação e propusemos mecanismo de reação. Este, segundo nosso conhecimento, é o primeiro estudo para descrever reação de desenvolvimento de cor para o ensaio proposto. A reação foi realizada em placas de 96 micropoços e mediu-se a absorbância do produto colorido utilizando-se leitor de absorbância a 490 nm. Sob condições otimizadas de reação, a lei de Beer, que mostra a correlação entre a absorbância e a concentração de CZT, foi obedecida na faixa de 4-50 µg/poço, com coeficiente de correlação apropriado (0,999). Os limites de detecção e de quantificação foram, respectivamente, 1,73 e 5,23 µg/poço. O ensaio mostrou alta precisão e exatidão. O ensaio proposto foi aplicado com sucesso para a determinação de CZT em cápsulas e é prático e válido para a aplicação de rotina em laboratórios de controle farmacêutico...
Subject(s)
Humans , Spectrum Analysis/analysis , Spectrum Analysis/methods , Protein-Tyrosine Kinases , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/pharmacology , Carcinoma, Non-Small-Cell Lung , Quality Control/methods , Laboratory Chemicals/pharmacologyABSTRACT
For therapeutic monitoring and pharmacokinetic studies of the potent hypocholesterolaemic agent fluvastatin (FLV), a specific antibody was required for the development of a sensitive enzyme-linked immunosorbent assay (ELISA) for the accurate determination of FLV in plasma. In this study, a highly specific polyclonal antibody against FLV has been prepared. FLV was coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) using carbodiimide reagent. FLV-KLH conjugate was used as an immunogen. Female 8-weeks old New Zealand white rabbits were immunized with an emulsion of FLV-KLH with Freund's adjuvant. The immune response of the rabbits was monitored by direct ELISA using FLV-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to FLV was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on a protein A column. The specificity of the purified antibody for FLV was evaluated by indirect competitive ELISA using various competitors from the FLV-structural analogues and therapeutic agents used with FLV in a combination therapy. The high affinity of the antibody (IC50 = 150 pg ml-1) enabled the determination of FLV in plasma at concentrations as low as 20 pg ml-1.
