Lucélia Magalhães da Silva

Assessment of rhEPO in Pharmaceutical Formulations by a Reversed‐Phase Liquid Chromatography Method and Bioassay

Abstract A gradient reversed‐phase liquid chromatography (RP‐LC) was developed for the analysis of alpha and beta rhEPO in pharmaceutical formulations. The RP‐LC method was carried out on a Jupiter C4 column (250 mm×4.6 mm I.D., with a pore size of 300 Å). The elution was performed by a gradient at a constant flow rate of 0.5 mL/min. Mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.08% TFA:acetonitrile (30∶70, v/v), using a photodiode array (PDA) detection at 280 nm. The chromatographic separation was obtained within 60 min and was linear in the concentration range of 10–150 µg/mL. The parameters validated, such as the specificity, precision, accuracy, and robustness gave results within the acceptable range. The pharmaceutical samples were analysed by the chromatographic method and compared to the normocythaemic mice bioassay, showing the mean difference between the estimated potencies of 11.2%±1.8 higher for the RP‐LC, with significant correlation (r=0.9799) as calculated by the Pearsons coefficient. The proposed RP‐LC method represents an alternative to the bioassay that can be applied for the potency assessment, improving the quality control of rhEPO in pharmaceutical dosage forms.

Determination of rupatadine in pharmaceutical formulations by a validated stability-indicating MEKC method

A stability-indicating MEKC was developed and validated for the analysis of rupatadine in tablet dosage forms, using nimesulide as internal standard. The MEKC method was performed on a fused-silica capillary (50 microm id; effective length, 40 cm). The BGE consisted of 15 mM borate buffer and 25 mM anionic detergent SDS solution at pH 10. The capillary temperature was maintained at 35 degrees C and the applied voltage was 25 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 5 s, with detection by photodiode array detector set at 205 nm. The method was linear in the range of 0.5-150 microg/mL (r2=0.9996). The specificity and stability-indicating capability of the method were proven through degradation studies inclusive by MS, and showing also that there was no interference of the excipients and no increase of the cytotoxicity. The accuracy was 99.98% with bias lower than 1.06%. The LOD and LOQ were 0.1 and 0.5 microg/mL, respectively. The proposed method was successfully applied for the quantitative analysis of rupatadine in pharmaceutical formulations, and the results were compared to a validated RP-LC method, showing non-significant difference (p>0.05).

Development and validation of a capillary zone electrophoresis method for assessment of recombinant human granulocyte colony-stimulating factor in pharmaceutical formulations and its correlation with liquid chromatography methods and bioassay

A capillary zone electrophoresis (CZE) method was validated for the analysis of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and performed on a fused-silica capillary, with detection at 195 nm. The background electrolyte solution consisted of 50 mM sodium tetraborate solution at pH 9. The method was linear in the concentration range of 1-200 microg/mL and the limit of quantitation (LOQ) was 1 microg/mL, with acceptable validation parameters. The method was applied for the analysis of pharmaceutical formulations, and the results were correlated to the reversed-phase HPLC method (RP-HPLC), size-exclusion HPLC method (SE-HPLC) and in vitro bioassay method.

Determination of Levofloxacin in a Pharmaceutical Injectable Formulation by Using HPLC and UV Spectrophotometric Methods

Abstract The objective of this study was to develop simple and rapid methods for the determination of levofloxacin (LVF) using high performance liquid chromatography and UV spectrophotometry. LVF was separated on a reversed phase Phenomenex® C18 column (150×4.6 mm i.d., particle size 4 µm), under isocratic elution with a mixture of water:acetonitrile:phosphoric acid 0.025 M, pH adjusted to 3.0 with triethylamine (60∶20∶20, v/v/v), as the mobile phase at room temperature and at a flow rate of 1.0 mL/min. The UV detector was set to 294 nm and UV‐vis spectrophotometer at 292 nm. Both methods allowed the quantification of LVF and showed good linearity (r>0.999) in the studied range. The relative standard deviations (RSD) were 0.66 and 1.0% for HPLC and UV spectrophotometry, respectively. The accuracy determined with HPLC was 100.68 and with UV spectrophotometry was 99.61%. The methods were validated through the parameters of linearity, accuracy, precision, specificity, and robustness. The two proposed methods enabled a quantitative determination of LVF in pharmaceutical injectable formulation.

Validation of a Stability Indicating Reversed Phase LC Method for the Determination of Fluticasone Propionate in Pharmaceutical Formulations

Abstract A reversed phase liquid chromatography (RP-LC) method was validated for the determination of fluticasone propionate (FP) in nasal sprays. The LC method was carried out on a Shim-pack CLC-ODS column (150 mm × 4.6 mm I.D.), maintained at 35°C. The mobile phase consisted of acetonitrile/methanol/phosphate buffer (0.01 M, pH 4.0) (35:35:30, v/v/v), run at a flow rate of 1.0 mL/min and using photodiode array (PDA) detection at 240 nm. The chromatographic separation was obtained with retention time of 6.1 min, and was linear in the range of 0.05–150 µg/mL (r 2 = 0.9999). The specificity and stability indicating capability of the method were proven through degradation studies, which also showed that there was no interference of the excipients. The accuracy was 99.36% with bias lower than 1.12%. The limits of detection and quantitation were 0.03 and 0.05 µg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of the nasal sprays and cream pharmaceutical formulations, contributing to improve the quality control and to assure the therapeutic efficacy.

Validation of a Size-Exclusion LC Method and Assessment of rhEPO in Pharmaceutical Formulations by Liquid Chromatography and Biological Assay

Abstract A size exclusion liquid chromatography (SE-LC) method was validated for the determination of erythropoietin in pharmaceutical formulations without serum albumin. The LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm I.D.) using photodiode array (PDA) detection at 214 nm. The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M dibasic sodium phosphate, and 0.2 M sodium chloride buffer, pH 7.4, run isocratically at a flow rate of 0.5 mL/min. The chromatographic separation was obtained with retention time of 14.5 min, and was linear in the range of 5–150 µg/mL (r 2 = 0.9991). The accuracy was 101.07% with bias lower than 1.36%. The limits of detection and quantitation were 0.28 and 1 µg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of the erythropoietin in pharmaceutical dosage forms, and the content/potencies correlated to the bioassay, contributing to establish alternatives which improve the quality control, assuring the therapeutic efficacy.

Validation of an RP-LC Method for the Determination of Interferon-α2a in Pharmaceutical Formulations

Abstract A reversed phase liquid chromatography (RP-LC) method was validated for the determination of interferon-α2a in pharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm × 4.6 mm I.D.), maintained at room temperature. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. The chromatographic separation was obtained with the retention time of 32.6 min, and was linear in the concentration range of 0.5–50 MIU/mL (r 2 = 0.9999). The specificity was proven through degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.84% with bias lower than 1.87%. The limits of detection and quantitation were 0.19 and 0.5 MIU/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The proposed method was applied for the analysis of the interferon-α2a and their related proteins in parenteral dosage forms, contributing to establishing alternatives to improve the quality control assuring the therapeutic efficacy of the biological medicine.

Biological potency evaluation and physicochemical characterization of unfractionated heparins

Unfractionated heparins are used clinically as anticoagulants. The biological potency of thirteen samples of raw material and pharmaceutical formulations were assessed utilizing the 5th International Standard of heparin using the sheep plasma coagulation inhibition assay, activated partial thromboplastin time, anti-factor Xa assay, and anti-factor IIa assay, resulting in mean potencies of 101.15%, 96.15%, 98.15% and 99.37%, respectively. The samples were also evaluated by the protamine neutralization test giving results within the range of 92 - 138 IU/mg. The anti-factor IIa assay was performed showing reproducibility and significant correlation with the pharmacopoeial assays, thus demonstrating it to be a feasible alternative to the sheep plasma coagulation inhibition assay. Moreover, an analysis by nuclear magnetic resonance and capillary electrophoresis showed some peaks attributable to oversulfated chondroitin sulfate. The results show that batch-to-batch variations and the quality of samples contributed to improvements in the quality control of pharmaceutical products and to assure the safe use and clinical efficacy of this biological medicine.