Liberato Brum Junior

Validation of an RP‐LC Method and Assessment of rhG‐CSF in Pharmaceutical Formulations by Liquid Chromatography and Biological Assay

Abstract Gradient reversed‐phase liquid chromatography (RP‐LC) was validated for the analysis of rhG‐CSF in pharmaceutical formulations. The LC method was carried out on a Jupiter C4 column (250 mm×4.6 mm I.D.), the mobile phase A consisted of water:acetonitrile (90:10, v/v) with 0.1% TFA and the mobile phase B was water:acetonitrile (20:80, v/v) with 0.1% TFA, run at a flow rate of 0.5 mL/min and detection at 280 nm. Validation parameters were evaluated and the method was linear in the range of 10–300 µg/mL. The dimers, high molecular mass forms, sulphoxides, and deamidates were analysed by the LC methods and then subjected to independent neutropenia mouse bioassay, giving overall biological activities within 13.47% and 15.63%. The pharmaceutical samples were analysed by the chromatographic methods and compared to the bioassay, showing mean difference between the estimated potency of 2.04% lower for the RP‐LC, and 4.03% lower for the SE‐LC, with significant correlation (P>0.05). Due to the bioactivity of the rhG‐CSF‐related proteins, the SE‐LC is proposed in combination with the RP‐LC as an alternative to the bioassay for the potency assessment, improving the quality control of rhG‐CSF in pharmaceutical dosage forms.

Validation of an LC‐Tandem MS/MS Method for the Determination of Etoricoxib in Human Plasma and Pharmaceutical Formulations

Abstract An analytical method based on liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was developed and validated for the determination of etoricoxib in spiked human plasma and pharmaceutical dosage forms. Etoricoxib and piroxicam (internal standard) were extracted from the plasma by liquid–liquid extraction using tert‐butyl methyl ether as extraction solvent and separated on a C18 analytical column (50 mm×3.0 mm I.D.). The mobile phase consisted of acetonitrile:water (95:5)/0.1% acetic acid (90:10, v/v). Detection was carried out by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode. The chromatographic separation was obtained within 2.0 min and was linear in the concentration range of 1–5000 ng/mL. The mean extraction recoveries of etoricoxib and piroxicam from plasma were 96.09 and 95.54%, respectively. Method validation investigated parameters such as the linearity, precision, accuracy, specificity, and stability, giving results within the acceptable range. Moreover, the proposed method was successfully applied for routine quality control analysis of pharmaceutical products and the results compared with those obtained by the RP‐HPLC method, showing significant correlation (P>0.05).

Determination of etoricoxib in human plasma using automated on-line solid-phase extraction coupled with LC-APCI/MS/MS

A liquid chromatography-tandem mass spectrometry method with atmospheric pressure chemical ionization (LC-APCI/MS/MS) was validated for the determination of etoricoxib in human plasma using antipyrin as internal standard, followed by on-line solid-phase extraction. The method was performed on a Luna C18 column and the mobile phase consisted of acetonitrile:water (95:5, v/v)/ammonium acetate (pH 4.0; 10 mM), run at a flow rate of 0.6 mL/min. The method was linear in the range of 1-5000 ng/mL (r2>0.99). The lower limit of quantitation was 1 ng/mL. The recoveries were within 93.72-96.18%. Moreover, method validation demonstrated acceptable results for the precision, accuracy and stability studies.

Avaliação comparativa da atividade biológica de heparinas não-fracionadas em produtos farmacêuticos

Realizou-se a avaliacao biologica de potencia de heparinas convencionais contra o 5o Padrao Internacional de heparina suina pelos ensaios preconizados da inibicao da coagulacao do plasma ovino (ICPO), tempo de tromboplastina parcial ativada (TTPA) e anti-fator Xa. Correlacionaram-se os resultados, demonstrando que o ICPO fornece potencias em media 10,72%, significativamente superiores aos outros procedimentos. Analisou-se a influencia de diferentes lotes de plasma sobre o resultado do ensaio do ICPO observando-se variacao de ate 7,32%. Como alternativa, padronizou-se o ensaio do anti-fator IIa e efetuou-se a determinacao de potencia das amostras obtendo-se valores reprodutiveis, comparaveis ao metodo do anti-fator Xa, que demonstram a viabilidade da inclusao como ensaio harmonizado para o controle da qualidade. Os resultados de potencia, fornecidos lote a lote, em geral, cumprem as especificacoes farmacopeicas e demonstram a qualidade que garante a seguranca e eficacia clinica dos medicamentos.

Evaluation of the changes on hemostatic parameters induced by valdecoxib in male Wistar rats

3The effects of the cyclooxygenase (COX)-2 selective inhibitor, valdecoxib, on blood coagulation parameters were evaluated, along with aspirin in male Wistar rats. Groups of animals were administered a daily oral dose of 10 mg/kg rat of valdecoxib, 100 mg/kg rat of aspirin and the vehicle alone during 4 weeks. Blood samples were collected at the end of 1, 2, 3 and 4 weeks of administration period and the plasma concentrations of valdecoxib were determined by RP-HPLC giving mean values of 101.1, 113.5, 164.0 and 184.6 ng/mL, respectively. The same plasma samples were used for the analysis of hematological parameters and the results compared to the controls. Valdecoxib induced significant activated partial thromboplastin time reduction (18%) after 2 weeks and prothrombin time reduction (12.2%) after 3 weeks (P<0.05). There were no significant changes in the platelet count and fibrinogen levels. The antifactor Xa and anti-factor IIa activities showed slight reductions, but only significant for the anti-factor Xa on the 3rd week (6.7%). The results showed that valdecoxib at the dose tested with the plasmatic concentrations induced some changes in the hemostatic function of rats, which can be helpful to understand the side effects and the safe use of the drug. Rev. bras. hematol. hemoter. 2006;28(1):28-32.

Biological potency evaluation and characterization of rhG-CSF in pharmaceutical products

The identification of rhG-CSF was carried out in pharmaceutical preparations by non-reducing polyacrylamide gel electrophoresis and western blotting with specific antibodies, showing a single band in the 19 kDa region. The potency was assessed by the neutropenia mouse bioassay giving values between 88.4 and 122.4% of the stated potency. The precision index expressed by weight was between 141 and 432 for the independent assays. Batch-to-batch, the samples met the requirements for the safety test and bacterial endotoxins test. The biological and immunological results showed the quality of the products in clinical use and the specifications established contribute to assuring the safety and efficacy of biological medicines.

DESAFIOS DO DESENVOLVIMENTO DOS DOSSIÊS DE REGISTRO DE MEDICAMENTOS FITOTERÁPICOS

Os beneficios da utilizacao de plantas medicinais sao amplamente discutidos no âmbito academico atraves de pesquisa basica e pela populacao em geral, baseado no ainda presente uso tradicional. Porem, e evidente a baixa demanda por registro no orgao sanitario competente (ANVISA) de produtos considerados medicamentos fitoterapicos ou produto tradicional fitoterapico. A ANVISA tem implementado requisitos visando garantir a qualidade, seguranca e eficacia destes produtos, a luz do que se exige aos medicamentos classificados como sinteticos. Nesta revisao, aspectos relacionados a pesquisa e desenvolvimento, em linhas gerais, de um medicamento fitoterapico sao relacionados com o arcabouco regulatorio que normatiza o registro de tais produtos. Cada etapa de desenvolvimento relaciona-se a uma normativa em especifico, de tal forma, que a execucao de qualquer experimento de forma diversa da preconizada, impossibilita sua utilizacao na documentacao de registro do produto. Este link e essencial para que se obtenha resultados satisfatorios no sentido de viabilizar-se o registro e futura comercializacao dos produtos desenvolvidos. O aproveitamento dos estudos realizados, a qualidade da documentacao gerada e a aderencia aos requisitos regulatorios, permitem a submissao de dossies de registro, que uma vez analisados, serao aprovados pelo orgao competente. A aplicabilidade das politicas de atencao basica a saude que preconizam a utilizacao de fitoterapicos, depende do correto desenvolvimento destes produtos, aprovacao do orgao regulador para que somente entao a populacao possa ter acesso.