Sergio Luiz Dalmora

A simple RP-HPLC method was developed and validated for the determination of rimonabant in a pharmaceutical dosage form. The separation was performed on a C18 column (150 x 4.6 mm id, 5 microm) with acetonitrile-water (75 + 25, v/v) mobile phase. The detection was achieved with a diode array detector at 215 nm. The method was linear in the concentration range of 0.5-50 microg/mL (r = 1) with an LOQ of 0.24 microg/mL. The specificity and stability-indicating capability of the method were proved through forced degradation studies, and it was shown that there was no increase of the cytotoxicity. Rimonabant was exposed to hydrolytic, oxidative, and photolytic stress conditions, and the samples were analyzed by the proposed method. Under optimized conditions, rimonabant was successfully separated from its degradation products within 10 min, and the resolution was found to be greater than 2. The RSD values for intraday and interday precision were always less than 2%. Interday accuracy ranged from 98.1 to 101.7% (RSD = 1.0%). Moreover, method validation demonstrated acceptable results for sensitivity and robustness. The method was applied for the quantitative analysis of rimonabant in a tablet dosage form to demonstrate its use for improving the QC of pharmaceuticals containing this drug.

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Reversed-phase liquid chromatography (LC) and LC/tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the determination of etoricoxib in pharmaceutical dosage forms. The LC method was performed by reversed-phase chromatography on a Synergi fusion C18 column (150 × 4.6 mm id) maintained at ambient temperature. The mobile phase consisted of 0.01 M phosphoric acid, pH 3.0acetonitrile (62 + 38, v/v) at a flow rate of 1.0 mL/min, and photodiode array detection at 234 nm was used. The chromatographic separation was obtained within 7.0 min, and calibration curves were linear in the concentration range of 0.02-150 μg/mL. The LC/MS/MS method was performed on a Luna C18 column (50 × 3.0 mm id). The mobile phase consisted of acetonitrilewater (95 + 5)0.1% acetic acid (90 + 10, v/v). Detection was performed by positive electrospray ionization in the multiple reaction monitoring mode, monitoring the transitions 359.3 > 280.0 and 332.0 > 95.0 for etoricoxib and piroxicam (internal standard), respectively. The chromatographic separation was obtained within 2.0 min, and calibration curves were linear in the concentration range of 15000 ng/mL. Validation parameters, such as specificity, linearity, precision, accuracy, and robustness, were evaluated, which gave results within the acceptable range for both methods. Moreover, the proposed methods were successfully applied for routine quality control analysis of pharmaceutical products and showed significant correlation (r = 0.9999) of the results.

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.

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).

The low-molecular weight heparin nadroparin calcium is used clinically for the prevention and treatment of venous and arterial thrombosis. The antifactor Xa and antifactor IIa assays were validated by investigating the parameters of range, linearity (r² 0.9905 and r² 0.9914, respectively) precision, accuracy, and robustness. The 2 methods incorporated a chromogenic endpoint and detection at 405 nm, yielding good results with detection limits of 0.004 and 0.01 IU/mL and quantitation limits of 0.01 and 0.03 IU/mL, respectively, for the antifactor Xa and antifactor IIa assays. Nadroparin calcium pharmaceutical products were evaluated by the antifactor Xa assay and the antifactor IIa assay, giving potencies between 93.86 and 109.88%, with an antifactor Xa/antifactor IIa ratio between 3.2 and 3.7. The results demonstrated the validity of the assays that are useful methodologies for the routine quality control of nadroparin in pharmaceutical formulations.

Enoxaparin is a low-molecular weight heparin used clinically for the prevention and treatment of venous and arterial thrombosis. An anti-factor Xa assay was used to evaluate the potency of the final drug preparation. Method validation investigated parameters such as the range, linearity (r² = 0.9971), precision, accuracy, and robustness; the biological assay incorporated a chromogenic endpoint and detection at 405 nm. The method yielded good results with a quantitation limit of 0.037 IU/mL and a detection limit of 0.011 IU/mL. The results demonstrated the validity of the anti-factor Xa assay for the determination of enoxaparin.