Vítor Todeschini

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.

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.

Delapril and manidipine measurements by liquid chromatography-tandem mass spectrometry in a pharmaceutical formulation.

A simple, specific, fast and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of delapril (DEL) and manidipine (MAN) from their combination formulation was developed and validated using fesoterodine as the internal standard (IS). The LC-MS/MS method was carried out on a Luna C8 column (50 × 3.0 mm i.d., 3 μm) with a mobile phase consistingof methanol and 10 mmol L−1 ammonium acetate (90:0, v/v), run at a flow rate of 0.25 mL min−1. The mass spectrometry method was performed employing positive electrospray ionization operating in multiple reaction monitoring mode, monitoring the transitions of m/z 453.1 → 234.1 for DEL, m/z 611.1 → 167.0 for MAN and m/z 412.2 → 223.0 for IS. The total analysis time was 3 min and the method was linear in the concentration range of 6–1080 ng mL−1 and 2–360 ng mL−1 for DEL and MAN, respectively. Parameters investigated for the method validation, such as the specificity, linearity, precision, accuracy and robustness, gave results within the acceptable range. Moreover, the proposed method was successfully applied for the simultaneous determination of DEL and MAN and the results were compared to validated liquid chromatography and capillary electrophoresis methods showing non-significant differences (P = 0.9).

A comparative study on the analytical performance of a charged aerosol detector and an ultraviolet detector for the RP-LC analysis of dabigatran etexilate in capsules

The reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of dabigatran etexilate (DAB) in capsules using an ultraviolet detector (UV) and a charged aerosol detector (CAD) connected in series. The RP-LC method was carried out on a GL Sciences Inc. Inertsil ODS-2 column (250 mm × 4.6 mm I.D.). The mobile phase consisted of acetonitrile and ammonium acetate buffer (pH 5.5; 10 mM) (65 : 35, v/v) (flow rate 1.0 mL min−1). CAD detection was performed using a nitrogen pressure of 35 psi and a range of 100 pA and the UV detection at 225 nm. The chromatographic separation was obtained with a retention time of 6.7 min. Validation parameters such as specificity, linearity, precision, accuracy and robustness were evaluated. The method was linear over the concentration range of 1–70 μg mL−1 in both detectors. The specificity and stability-indicating capability of the method were proven through the degradation studies, which also show that there was no interference from the excipients. The method was precise and accurate, with the Plackett–Burman experimental design used to evaluate robustness, delivering results within the acceptable range. The method was successfully applied for DAB analysis and the results of the CAD and UV detectors were compared, showing a non-significant difference (P > 0.05).

SIMULTANEOUS DETERMINATION OF DELAPRIL AND MANIDIPINE IN A PHARMACEUTICAL FORMULATION BY A STABILITY-INDICATING RP-LC METHOD

Simple, precise, and stability-indicating reversed-phase liquid chromatography (RP-LC) method was developed and validated for simultaneous determination of delapril (DEL) and manidipine (MAN) in pharmaceutical dosage form, using the Plackett-Burman experimental design for robustness evaluation. The LC method was carried out on a C8 column (250 mm × 4.6 mm i.d., 5 µm), maintained at 35°C. The mobile-phase consisted of acetonitrile and a solution of triethylamine 0.3% pH 3.0 (adjusted with phosphoric acid) (55:45; v/v), run at a flow rate of 1.2 mL · min−1 and using photodiode array (PDA) detection at 220 nm. The chromatographic separation was obtained within 7 min and was linear in the range of 3–120 µg · mL−1 (r2 = 0.9997) for DEL and 1–40 µg · mL−1 (r2 = 0.9998) for MAN. Validation parameters such as the specificity, linearity, precision, accuracy, and robustness were evaluated, giving results within the acceptable range. Stress studies were carried out and no interference of the degradation products was detected. The proposed method was successfully applied for the simultaneous quantitative analysis of DEL and MAN in the tablet dosage form, contributing to improve the quality control and to assure the therapeutic efficacy.

Delapril and Manidipine Main Degradation Products: LC-UV and LC-ESI-MS Evaluations, Decay Kinetic, and in vitro Cytotoxicity Studies

A simple, stability-indicating, and compatible method by liquid chromatography with ultraviolet (LC-UV) and mass spectrometry (LC-MS) detections was developed for the evaluation of delapril (DEL) and manidipine (MAN) degradation products formed under alkaline and photolytic forced conditions, respectively. The chromatographic separation of all compounds was obtained within 7 min and carried out on a C18 column with mobile-phase composed by ammonium acetate and acetonitrile for both techniques. Thereby, the main degradation products detected were studied by LC coupled to electrospray ionization mass spectrometry (LC-ESI-MS). Based on retention times and molecular weight confirmation, a comprehensive degradation pathway for both drugs and the identity of its major products formed could be suggested, without complicated isolation or purification processes. The degradation kinetics of the drugs was also evaluated and could be best described as first-order process for DEL (R2 = 0.9991) and zero-order process for MAN (R2 = 0.9867). Furthermore, no evidence of cytotoxicity in human mononuclear cells was observed for DEL and MAN degraded samples. The proposed LC-UV method was successfully validated according ICH guidelines and thus helping to improve the quality control of pharmaceuticals.

Pinaverium Bromide: Development and Validation of Spectrophotometric Methods for Assay and Dissolution Studies

This study presents the development and validation of UV spectrophotometric methods for the determination of pinaverium bromide (PB) in tablet assay and dissolution studies. The methods were satisfactorily validated according to International Conference on Harmonization guidelines. The response was linear (r2 > 0.99) in the concentration ranges of 2-14 μg/mL at 213 nm and 10-70 μg/mL at 243 nm. The LOD and LOQ were 0.39 and 1.31 μg/mL, respectively, at 213 nm. For the 243 nm method, the LOD and LOQ were 2.93 and 9.77 μg/mL, respectively. Precision was evaluated by RSD, and the obtained results were lower than 2%. Adequate accuracy was also obtained. The methods proved to be robust using a full factorial design evaluation. For PB dissolution studies, the best conditions were achieved using a United States Pharmacopeia Dissolution Apparatus 2 (paddle) at 50 rpm and with 900 mL 0.1 M hydrochloric acid as the dissolution medium, presenting satisfactory results during the validation tests. In addition, the kinetic parameters of drug release were investigated using model-dependent methods, and the dissolution profiles were best described by the first-order model. Therefore, the proposed methods were successfully applied for the assay and dissolution analysis of PB in commercial tablets.

Chemometric-Assisted Spectrophotometric Method for the Simultaneous Quantitative Determination of Ezetimibe and Simvastatin in Their Combined Dosage Forms

The multivariate method, partial least-squares (PLS), was used as a calibration procedure for the simultaneous UV spectrophotometric determination of ezetimibe and simvastatin in their pharmaceutical forms. The method was developed and satisfactorily validated according to International Conference on Harmonization guidelines with respect to specificity, linearity, precision, accuracy, and robustness. In this study, the PLS algorithms are based on the absorption spectra of 25 different mixtures of drugs obtained by a multilevel factorial design. The method was linear in the concentration range of 2-8 μg/mL for ezetimibe and 4-16 μg/mL for simvastatin (r2 > 0.99; n = 7) at wavelengths of 238 and 247 nm, respectively. The LOD and LOQ were 0.28 and 0.93 μg/mL for ezetimibe and 0.16 and 0.53 μg/mL for simvastatin, respectively. Precision and accuracy data, evaluated by RSD, were lower than 2%. The method, which proved to be robust, was performed with a 2n full-factorial design. The validated method is simple and low cost, has a low use of polluting reagents, and is environmental friendly. Therefore, the proposed method was successfully applied for the simultaneous quantitative analysis of ezetimibe and simvastatin in commercial formulations.

Stability-indicating comparative methods using mekc and lc for determination of olmesartan medoxomil

A stability-indicating method using MEKC was validated for the analysis of olmesartan medoxomil in tablets. Successful separation was achieved using a fused silica capillary (40 cm x 50 µm i.d.); background electrolyte consisted of a combination of 10 mmol L-1 borate buffer and 5 mmol L-1 anionic detergent sodium dodecyl sulfate (95:5; v/v) pH 6.5; hydrodynamic mode at 50 mBar for 5 s; 25 kV separation voltage at 25 oC; and column temperature 25 oC with detection at 257 nm. The proposed method, validated following ICH guidelines, was applied to the determination of this antihypertensive with good results compared with an LC method.