Recebido em 23/11/09; aceito em 15/1/10; publicado na web em 3/5/10 A dissolution test for in vitro evaluation of tablet dosage forms containing 10 mg of rupatadine was developed and validated by RP-LC. A discriminatory dissolution method was established using apparatus paddle at a stirring rate of 50 rpm with 900 mL of deaerated 0.01 M hydrochloric acid. The proposed method was validated yielding acceptable results for the parameters evaluated, and was applied for the quality control analysis of rupatadine tablets, and to evaluate the formulation during an accelerated stability study. Moreover, quantitative analyses were also performed, to compare the applicability of the RP-LC and the LC-MS/MS methods.Keywords: rupatadine; dissolution test; RP-LC. INTRODUCTIONDissolution test has emerged in the pharmaceutical field as a very important tool to characterize drug product performance. Therefore the dissolution studies are used not only to assess batch-to-batch consistency of drug release from solid dosage forms, but they are also essential in several stages of formulation development, for screening and proper assessment of different formulations. Moreover, the in vitro dissolution studies are relevant to predict the in vivo performance of a drug release and have been used as a tool to estimate the bioavailability of the drug. [1][2][3][4][5][6] Rupatadine, chemically (8-chloro-6,11-dihydro-11-[1-[(5-methyl3-pyridinyl) methyl]-4-piperidinylidene]-5H-benzo [5,6]cyclohepta [1,2b]pyridine), is a new drug which represents a second generation of H 1 -antihistamine that inhibits both platelet activating factor (PAF) and histamine (H 1 ) effects, through its interaction with specific receptors. It is rapidly absorbed (Tmax = 0.75-1 h) after oral administration, and is subjected to extensive presystemic metabolism, with hepatic cytochrome P450 (CYP) 3A4 as the main enzyme involved in its biotransformation. Clinically is indicated for the management of diseases with allergic inflammatory conditions, such as seasonal and perennial rhinitis, and chronic urticaria. 7-9The literature describes validated liquid chromatography tandemmass spectrometry (LC-MS/MS) methods, with detection performed by positive atmospheric pressure chemical ionization (APCI) and gradient elution for LC analysis, for the simultaneous determination of rupatadine and its two active metabolites in human plasma after liquidliquid extraction and enzymatic hydrolysis, supporting pharmacokinetic studies.9-13 Moreover, sensitive isocratic LC-MS/MS methods with positive electrospray ionization (ESI) were developed and validated for the quantitation of rupatadine in human plasma without determination of the main active metabolites, 14 and with determination of the metabolite desloratadine.15 For the analysis of rupatadine in tablet dosage forms, a reversed-phase liquid chromatography (RP-LC) method was developed and validated over the concentration range of 0.5-400 µg/mL, using a C 18 analytical column and isocratic elution with UV detection at 242 nm. 16 Furthe...

Biotechnology-derived erythropoietin is a sialoglycoprotein, which stimulates erythropoiesis, and it is clinically used for the treatment of anaemia related to chronic renal failure. N-Acetylneuraminic acid content was quantified by a reversed-phase liquid chromatography method with fluorescence detection giving values higher than 108.74 ng mg À1 . An alternative in vitro TF-1 cell proliferation bioassay was studied showing a lower mean difference of the estimated potency of 2.67%, compared to the normocythaemic mice bioassay, with non-significant differences (p > 0.05). Biopharmaceutical products were also analyzed by validated reversed-phase and size-exclusion liquid chromatography methods and compared to the in vivo bioassay, showing lower mean differences of the content/potencies of 2.11 and 1.21%, respectively. Higher molecular mass forms and deamidated/sulphoxide forms showed mean bioactivities reduced to about 10%, for both. The TF-1 cell culture assay in conjunction with the determination of sialic acids represents an advance that can be correlated with the normocythaemic mice bioassay and the physicochemical methods, allowing for the establishment of alternative methods, which can be applied to monitor the stability, quality control, and thereby ensuring the therapeutic efficacy of the biological medicine.

Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were validated for the assessment of recombinant human parathyroid hormone (rhPTH 1-34). The gradient RP-LC method was carried out on a Zorbax 300 SB C(18) column (150 mm × 4.6 mm i.d.), maintained at 40 °C. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.), maintained at 25 °C. The mobile phase consisted of 0.1 M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL min(-1). Chromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and was linear over the concentration range of 1-250 μg mL(-1) (r(2) = 0.9997) and 2-300 μg mL(-1) (r(2) = 0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.49% and 100.22%, with bias lower than 1.12% and 0.81% respectively. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p < 0.05). Chromatographic methods were applied for the content/potency assessment of rhPTH and related proteins in biopharmaceutical injectable dosage forms, and the results were correlated with those of in vitro and in vivo bioassays. It is concluded that the employment of the methods in conjunction allows a great improvement in monitoring stability, contributing to evaluate alternatives which improve the quality control and thereby assure the therapeutic efficacy of the biotechnology-derived medicine.

A size-exclusion LC method was validated for the determination of interferon-a2a (rhlFN-alpha2a) in pharmaceutical formulations without interference from human serum albumin. Chromatographic separation was performed on a BioSep-SEC-S 2000 column (300 x 7.8 mm id). The mobile phase consisted of 0.001 M monobasic potassium phosphate, 0.008 M sodium phosphate dibasic; 0.2 M sodium chloride buffer, pH 7.4, run at a gradient flow rate and using photodiode array detection at 214 nm, was used. Chromatographic separation was achieved with a retention time of 17.2 min, and the analysis was linear over the concentration range of 1.98 to 198 microg/mL (r2 = 0.9996). The accuracy was 101.39%, with bias lower than 1.67%. The LOD and LOQ were 0.87 and 1.98 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for precision and robustness. The method was applied to the assessment of rhlFN-alpha2a and related proteins in biopharmaceutical dosage forms, and the content/potencies were correlated to those given by a validated RP-LC method and an in vitro bioassay. It was concluded that use of the methods in conjunction allows a great improvement in monitoring stability and QC, thereby ensuring the therapeutic efficacy of the biotechnology-derived medicine.

A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11), based on the ICH guidelines. The method was carried out on a Jupiter C4 column (250 mm × 4.6 mm i.d.), maintained at 25 C. The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and the mobile phase B was acetonitrile with 0.1% TFA, run at a flow rate of 1 mL/min, and using a photodiode array (PDA) detection at 214 nm. Separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1-200 μg/mL (r 2 = 0.9995). Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p > 0.05). The method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay.