Patricia M. Castellano

Chemometric determination of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate in synthetic mixtures and pharmaceutical formulations

Different chemometric methods such as classical least squares (CLS), principal components regression (PCR) and partial least squares with one dependent variable (PLS-1) applied on UV spectral data (0 D) and on their first derivatives (1 D) were evaluated for the simultaneous quantification of samples containing mixtures of amiloride hydrochloride, atenolol, hydrochlorothiazide and timolol maleate. Their performances were compared by means of ANOVA tests, which evidenced that 0 D-PCR, 0D-PLS-1, 1D-PCR, 1D-PLS-1, were reproducible and gave statistically similar results, while 0 D-CLS and 1D-CLS displayed higher variances than the former and failed to comply with the Levenes variance homogeneity test at different stages of the method comparison and validation process. The four statistically equivalent procedures were successfully applied to the analysis of synthetic samples with two to four analytes and to commercial tablet preparations containing amiloride hydrochloride and hydrochlorothiazide alone or in association with atenolol or timolol maleate.

A spectrophotometric-partial least squares (PLS-1) method for the simultaneous determination of furosemide and amiloride hydrochloride in pharmaceutical formulations.

A numerical method, based on the use of spectrophotometric data coupled to PLS-1 multivariate calibration, is reported for the simultaneous determination of furosemide and amiloride hydrochloride in synthetic samples and commercial tablets. The method was applied in the concentration ranges of 8.0-13.0 mg l(-1) for furosemide and 1.0-1.6 mg l(-1) for amiloride hydrochloride. Its accuracy and precision were determined, and it was validated by the analysis of synthetic mixtures of both drugs. The method was successfully applied to the quantitation of furosemide and amiloride hydrochloride in three different pharmaceutical formulations, providing results in agreement with those obtained by HPLC. It allowed the rapid, accurate and precise simultaneous estimation of the concentration of both analytes of interest in spite of their important spectral overlap, high concentration relationship and the presence of small amounts of different, unmodelled, absorbing excipients.

Simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in synthetic samples and pharmaceutical formulations by multivariate analysis of spectrophotometric data

The use of multivariate spectrophotometric calibration for the simultaneous analysis of synthetic samples and commercial tablet preparations containing hydrochlorothiazide (HCT) and amiloride hydrochloride (AMH) is reported. Partial least squares (PLS-1) analysis of electronic absorption spectral data allowed the rapid and accurate resolution of mixtures in which the analyte ratios were approximately 10:1, without the need of a previous separation step and without interference from other sample constituents. The method, validated by the analysis of synthetic mixtures of both drugs, where accuracy over the linear working range as well as inter- and intra-assay precision were determined, was used in the concentration ranges of 21.7-30.4 mg l(-1) for HCT and 1.8-3.0 mg l(-1) for AMH. The proposed method was successfully applied to the evaluation of the stability of the stock solutions of the analytes in MeOH-H(2)O and to the elaboration of drug dissolution profiles of commercial tablets, results being concordant with those furnished by the USP technique. The method was also employed for the determination of drug content in two different pharmaceutical formulations, providing results that were in excellent agreement with those obtained by HPLC.

Development and validation of an HPLC method for the determination of process-related impurities in pridinol mesylate, employing experimental designs.

A simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound pridinol mesylate, has been developed and validated. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C(18) column with a mobile phase containing 50mM potassium phosphate buffer (pH 6.4), MeOH and 2-propanol (20:69:11, v/v/v), delivered at a flow rate of 1.0mLmin(-1). UV detection was performed at 245nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r(2)>0.99) over the range 0.05-2.5% (5-250% with regards to the 1% specification limit for both process-related impurities); it is also precise, regarding repeatability (RSD</=1.5% for all of the analytes) and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1-102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.

PCA-CR analysis of dissolution profiles. A chemometric approach to probe the polymorphic form of the active pharmaceutical ingredient in a drug product

A simple chemometric approach to differentiate among the three crystalline polymorphs of the model drug Furosemide (FUR) in a pharmaceutical dosage form is presented. The proposed method is based on the principal component analysis with confidence regions (PCA-CR) comparison of the dissolution profiles of the test pharmaceutical formulation, and formulations containing the different polymorphs, employed as the corresponding references. For the elaboration of the references, FUR polymorphs I, II and III were prepared, characterized and compounded with the excipients found in the test commercial formulation. The dissolutions were carried out in a discriminating HCl-KCl dissolution medium (pH 2.2), and the corresponding profiles were constructed from the absorbances (274 nm) of the dissolution samples. PCA-CR was able to differentiate among the three crystalline polymorphs of FUR and to confirm the presence of polymorph I in the test sample, with 99% statistical confidence. The PCA-CR results were compared with those obtained by a bootstrap-mediated implementation of Moore and Flanners difference factor (f(2)). The same conclusion was reached employing an f(2)-based comparison, despite its inability to differentiate between polymorphs II and III. Therefore, PCA-CR may be considered a complementary and useful tool for probing the polymorphic form present in a pharmaceutical formulation.

Validated stability-indicating HPLC method for the determination of pridinol mesylate. Kinetics study of its degradation in acid medium.

The stability of pridinol mesylate (PRI) was investigated under different stress conditions, including hydrolytic, oxidative, photolytic and thermal, as recommended by the ICH guidelines. Relevant degradation was found to take place under acidic (0.1N HCl) and photolytic (visible and long-wavelength UV-light) conditions, both yielding the product resulting from water elimination (ELI), while submission to an oxidizing environment gave the N-oxidation derivative (NOX). The standards of these degradation products were synthesized and characterized by IR, (1)H and (13)C NMR spectroscopy. A simple, sensitive and specific HPLC method was developed for the quantification of PRI, ELI and NOX in bulk drug, and the conditions were optimized by means of a statistical design strategy. The separation employs a C(18) column and a 51:9:40 (v/v/v) mixture of MeOH, 2-propanol and potassium phosphate solution (50mM, pH 6.0), as mobile phase, delivered at 1.0 ml min(-1); the analytes were detected and quantified at 220 nm. The method was validated, demonstrating to be accurate and precise (repeatability and intermediate precision levels) within the corresponding linear ranges of PRI (0.1-1.5 mg ml(-1); r=0.9983, n=18) and both impurities (0.1-1.3% relative to PRI, r=0.9996 and 0.9995 for ELI and NOX, respectively, n=18). Robustness against small modifications of pH and percentage of the aqueous mobile phase was ascertained and the limits of quantification of the analytes were also determined (0.4 and 0.5 microg ml(-1); 0.04% and 0.05% relative to PRI for ELI and NOX, respectively). Peak purity indices (>0.9997), obtained with the aid of diode-array detection, and satisfactory resolution (R(s)>2.0) between PRI and its impurities established the specificity of the determination, all these results proving the stability-indicating capability of the method. The kinetics of the degradation of PRI in acid medium was also studied, determining that this is a first-order process with regards to drug concentration, with an activation energy of 25.5 Kcal mol(-1) and a t(1/2)=10,830 h, in 0.1N HCl at 38 degrees C.

DEVELOPMENT AND VALIDATION OF AN HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF AMLODIPINE, HYDROCHLOROTHIAZIDE, AND VALSARTAN IN TABLETS OF THEIR NOVEL TRIPLE COMBINATION AND BINARY PHARMACEUTICAL ASSOCIATIONS

The novel triple combination between Amlodipine (AML), Hydrochlorothiazide (HCT), and Valsartan (VAL) provides a new option for treating hypertension. The development and validation of an HPLC method for their simultaneous determination in pharmaceutical combinations, employing experimental design strategies, is reported. The drugs were separated on a C18 column at 30°C, using a 38:62 (v/v) mixture of 30 mM phosphate buffer (pH 5.5) and MeOH as mobile phase, delivered at 1.0 mL min−1. Detection was performed at 234 nm. Despite the wide difference in analytes’ concentrations, the method showed good linearity (r2 > 0.995) in the ranges 7.0–13.0 µg mL−1, 17.6–32.8 µg mL−1, and 226.2–420.2 µg mL−1 for AML, HCT, and VAL, respectively, being specific (peak purity >0.999), accurate (bias of analyte recoveries <2.0%), and precise (inter- and intra-day variations <2%). It was also robust to small changes in flow rate (±0.05 mL min−1), pH (±0.1 unit) and proportion of MeOH (±3%) in the mobile phase. The method was applied to the assay of AML, HCT, and VAL in tablets of their novel association and formulations containing the HCT-VAL and AML-VAL binary combinations.

EXPERIMENTALLY DESIGNED, VALIDATED HPLC SIMULTANEOUS DETERMINATION OF PRIDINOL AND DICLOFENAC IN THEIR COMBINED PHARMACEUTICAL FORMULATIONS, WHICH ALLOWS LIMITING DICLOFENAC RELATED COMPOUND A

The development and validation of an HPLC method for the determination of pridinol and diclofenac in their combined formulations and the simultaneous limit testing of diclofenac related compound A is described. The separation was performed on a C18 column. Experimental design and response surface strategies were employed for optimizing detection wavelength (225 nm) and mobile phase composition [MeOH:2-propanol:phosphate buffer (50 mM, pH 5.5), 48:9:43 (v/v/v), 1 mL min−1], and for validation purposes. The method was successfully applied to the quality control of commercial brands of tablets and capsules. Found impurity levels were below 0.1% (LOQ = 0.02%). Stressed samples were also evaluated.

STRESS TESTING OF VALSARTAN. DEVELOPMENT AND VALIDATION OF A HIGH PERFORMANCE LIQUID CHROMATOGRAPHY STABILITY-INDICATING ASSAY

Stress testing studies were carried out on Valsartan (VAL) under hydrolytic (acidic, basic, and neutral), photolytic, oxidative, and thermal (in the solid state) conditions. Relevant degradation was observed when the drug was exposed to photolytic conditions, where two degradants (DP-1 and DP-2) were produced and when submitted to acid hydrolysis, which yielded one degradation product (DP-3). A high performance liquid chromatography method for the simultaneous determination of VAL and its degradation products was developed, optimized employing experimental design strategies, and validated, employing fully characterized standards of the degradation products. The results proved the stability-indicating property of the method.

Development of a Dissolution Test for Fenbendazole-Praziquantel Capsules Using UV-PLS Method

A dissolution test for capsules containing 50 mg of praziquantel and 500 mg of fenbendazole was developed and validated. The optimal conditions were an USP apparatus 2 with paddles rotating at 75 rpm, 900 mL dissolution medium (a mixture of 300 mL of ethanol and 600 mL 0.5 mol L HCl), at 37.0 ± 0.5 °C. Both analytes achieved with sink conditions. A published highperformance liquid chromatography (HPLC) method was used to monitoring dissolution test during the optimization. Additionally, a chemometrics method based on UV-VIS spectrophotometry and partial least-squares (PLS) was developed and validated for the simultaneous determination of both analytes in the dissolution media. The coefficients of determination were 0.9986 and 0.9959 for fenbendazole and praziquantel, respectively, and the elliptical joint confidence region (EJRC) test concluded that constant and proportional biases were absent. The optimized model was applied to build dissolution profile and its results did not show statistical differences with HPLC method.