Maximiliano da Silva Sangoi

Development and validation of an UV spectrophotometric method for the determination of aliskiren in tablets

For determination of aliskiren in commercial samples, an analytical UV spectrophotometric method was developed and validate according to ICH guideline. The method was linear in the range between 40 and 100 μg mL-1 (r2 = 0.9997, n = 7) and exhibited suitable specificity, accuracy, precision, and robustness. It is simple, it has low cost, and it has low use polluting reagents. Therefore, the proposed method was successfully applied for the assay and dissolution studies of aliskiren in tablet dosage forms, and the results were compared to a validated RP-LC method, showing non-significant difference (P > 0.05).

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability-indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused-silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5-100 and 60-1200 μg/mL for HCTZ and ALI, respectively (r(2) >0.9997). The stability-indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.

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.

Fesoterodine stress degradation behavior by liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry

In the present study, a rapid validated stability-indicating LC method was established and comprehensive stress testing of fesoterodine was carried out according to ICH guidelines. Fesoterodine was subjected to stress conditions of acid and basic hydrolysis, oxidation, photolysis and thermal decomposition. The degradation products formed under stress conditions were investigated by LC-UV and LC-ESI-MS. Successful separation of the drug from its degradation products was achieved on a monolithic C(18) column (100 mm × 4.6mm i.d.) maintained at 45°C using acetonitrile-methanol-0.03 mol L(-1) ammonium acetate (pH 3.8) (30:15:55, v/v/v) as the mobile phase. The flow rate was 2.4 mL min(-1) and the detection wavelength was 208 nm. Validation parameters such as specificity, linearity, precision, accuracy, and robustness were evaluated. Chromatographic separation was obtained within 2.5 min and it was suitable for high-throughput analysis. Fragmentation patterns of degradation products formed under different stress conditions were studied and characterized through LC-ESI-MS fragmentation. Based on the results, a drug degradation pathway was proposed, and the validated LC method was successfully applied to the quantitative analysis of fesoterodine in tablet dosage forms, helping to improve quality control and to assure therapeutic efficacy.

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).

RAPID SIMULTANEOUS DETERMINATION OF ALISKIREN AND HYDROCHLOROTHIAZIDE FROM THEIR PHARMACEUTICAL FORMULATIONS BY MONOLITHIC SILICA HPLC COLUMN EMPLOYING EXPERIMENTAL DESIGNS

A simple, rapid, precise, and stability-indicating HPLC method was developed and validated for simultaneous determination of hydrochlorothiazide (HCTZ) and aliskiren (ALI) in pharmaceutical formulations. The HPLC method was carried out on a monolithic C18 column (100 mm × 4.6 mm id), maintained at 45°C. The mobile-phase consisted of acetonitrile-sodium phosphate (pH 4.0; 30 mM) (33:67, v/v), run at a flow rate of 2.4 mL/min, using photodiode array detection at 208 nm. Validation parameters such as the specificity, linearity, precision, accuracy, and robustness were evaluated according to the ICH guidelines. The method was linear in the range of 5–200 µg/mL (r2 > 0.9992) for both drugs. The specificity and stability indicating capability of the method were demonstrated through degradation studies, which also showed that there was no interference from the excipients. Plackett-Burman experimental design and a 23 full factorial design were employed to estimate the robustness and intermediate precision, respectively. The chromatographic separation was obtained within 2 min and it was suitable for high-throughput analysis. The proposed HPLC method was successfully applied for the simultaneous quantitative analysis of HCTZ and ALI in tablet dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.

Determination of phenobarbital in human plasma by a specific liquid chromatography method: application to a bioequivalence study

A liquid chromatography method was developed and validated for the determination of phenobarbital in human plasma using phenytoin as internal standard. The drugs were extracted from plasma by liquid-liquid extraction and separated isocratically on a C12 analytical column, maintained at 35 oC, with water:acetonitrile:methanol (58.8:15.2:26, v/v/v) as mobile phase, run at a flow rate of 1.2 mL/min with detection at 205 nm. The method was linear in the range of 0.1-4 μg/mL (r2=0.9999) and demonstrated acceptable results for the precision, accuracy and stability studies. The method was successfully applied for the bioequivalence study of two tablet formulations (test and reference) of phenobarbital 100 mg after single oral dose administration to healthy human volunteers.

Determination of cetirizine in tablets and compounded capsules: comparative study between CE and HPLC

A capillary electrophoresis (CE) method was developed and validated for determination of cetirizine dihydrochloride in tablets and compounded capsules. The electrophoretic separation was performed in an uncoated fused-silica capillary (40 cm x 50 μm i.d.) using 20 mmol L-1 sodium tetraborate buffer (pH 9.3) as background electrolyte, a hydrodinamic sample injection at 50 mBar for 5 s, 20 KV applied voltage at 25 °C, and detection at 232 nm. The proposed method was compared with the high performance liquid chromatographic (HPLC) method previously validated for this drug, and statistical analysis showed no significant difference between the techniques.

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.