Rafał Pietraś

Normal-phase TLC separation of some antiarrhythmics. Densitometric determination of mexiletine hydrochloride in capsules

A normal-phase (NP) TLC method has been established for separation of the five antiarrhythmics - disopyramide, flecainide, mexile-tine, tocainide, and verapamil. The analysis was performed in horizontal chambers on aluminum oxide 60 F254 and silica gel 60 F254 TLC plates. The best mobile phases for separation of the compounds were tetrahydrofuran-hexane-25% ammonia, 5 + 4.8 + 0.2 (v/v), on the alumina plates and chloroform-tetrahydrofuranethanol-25% ammonia, 8.1 + 1.9 + 2 + 0.1 (v/v), on the silica plates. The substances were identified by use of different reagents and under UV irradiation at λ= 254 nm. Quantification of mexiletine hydrochloride in Mexicord capsules was performed densitometrically at λ= 210 nm. A good correlation coefficient (r = 0.9974) was obtained for the calibration plot constructed in the concentration range 20–45 μg per band. The active substance was extracted from the formulation with methanol (recovery 97.01 ± 2.39%, mean ± SD). The RSD expressing the precision of the proposed method was 5.23%. The procedure was simple and rapid and the results were reliable.

Simultaneous densitometric determination of quinapril and hydrochlorothiazide in the combination tablets

A new, simple, and accurate TLC method, using normal- and reversed-phase techniques and densitometric detection, has been developed for measurement of quinapril and hydrochlorothiazide in combination tablets. UV detection at λ = 210 nm was used to quantify the analytes. The drugs were chromatographed on silica gel 60 F254 HPTLC plates and on octadecilsilane (RP-18) TLC plates, in horizontal chambers, with ethyl acetate—acetone—acetic acid, 8 + 2 + 0.5 (v/v) and methanol—0.07 m phosphate buffer, pH 2.5, 6 + 4 (v/v), respectively, as mobile phases. The active substances were extracted from tablets with methanol (96% < mean recovery < 104%). Calibration curves were constructed in the range 0.4 to 2.4 µg µL−1 for quinapril and 0.25 to 1.5 µg µL−1 for hydrochlorothiazide, with good correlation (r ≥ 0.998). The precision (RSD < 4.4%) and accuracy (2.91 < RE < 3.92) were satisfactory for TLC−densitometric determination of quinapril in combination with hydrochlorothiazide in commercial tablets.

VALIDATED STABILITY-INDICATING HPTLC METHOD FOR THE DETERMINATION OF PROPAFENONE HYDROCHLORIDE IN TABLETS AND THE GC-MS IDENTIFICATION OF ITS DEGRADATION PRODUCTS

A novel stability-indicating TLC method for determination of propafenone hydrochloride (PRO) in bulk drug and POLFENON tablets. Normal-phase high performance thin layer chromatographic separation was achieved by use of silica gel 60 F254 plates and mixture of chloroform–methanol–acetic acid 99.5% (7.9:2:0.1, v/v/v) as the mobile phase. Linear calibration function was obtained (r 2 = 0.9987) with respect to peak area in the concentration range of 0.1–3.2 µg/spot. Densitometric analysis of PRO and its forced-degradation products was carried out in the absorbance mode at 316 nm. Limits of detection and quantification were 0.02 and 0.08 µg/spot, respectively. PRO was subjected to stress conditions for obtainment of hydrolytic, oxidative, photolytic, and thermal degradation products. The GC-MS technique was applied to identification of the degradants. The elaborated method was validated using ICH guidelines. The linearity, accuracy (99.24%), precision (intraday RSD 1.61%), and specificity were satisfactory. The validated stability indicating method enables reproducible and selective analysis of propafenone hydrochloride in pharmaceutical dosage form.

Stress degradation study of two oral antidiabetics, gliclazide and glipizide, and chemical analysis by LC and LC/MS methods

AbstractKinetic study of degradation of two oral antidiabetics, gliclazide and glipizide, was performed using new HPLC method which was validated in terms of selectivity, sensitivity, linearity, precision and accuracy. The stress degradation was performed in 0.2 M HCl, 0.2 M NaOH as well as in acetate and phosphate buffers over the pH range 3.8–8.3 at 30 and 70°C.In strong acidic and alkaline media gliclazide was almost fully degraded while glipizide showed much higher stability. Generally, degradation processes of gliclazide and glipizide were observed as the first order reactions while the rates of decomposition for both drugs were smallest at pH 8.3. The samples of gliclazide and glipizide stressed in strong acid and alkali at 70°C were additionally analyzed using an LC/MS method and some products of decomposition were detected and identified.It was concluded that glipizide was more resistant to very high or very low pH and would have higher stability compared to gliclazide. Such comparisons have not been performed so far for these valuable drugs. Additional LC/MS study showed that during decomposition of sulfonylureas, different degradation pathways were possible.

New HPLC method for in vitro dissolution study of antihypertensive mixture amlodipine and perindopril using an experimental design

AbstractA new HPLC method was developed for the determination of amlodipine and perindopril in their binary mixture as a part of a routine control of combined formulations. For the first time an HPLC method was used for an in vitro dissolution study of tablets containing the above drugs. The presented method was validated to meet official requirements and this validation included specificity, stability, linearity, precision and accuracy. Chromatography was carried out using a LiChrospher RP-18 column, a mixture containing acetonitrile and phosphate buffer of pH 3.0 (50:50, v/v) as mobile phase and UV detection at 225 nm. The dissolution test was performed using 900 mL of phosphate buffer at pH 5.5 containing 1% cetylpyridini chloride (CPC) at 37°C and 75 rpm, using the paddle method. Robustness procedure was done according to the plan defined by the Plackett-Burman design. The effects of acetonitrile content, pH of the buffer and flow rate of the mobile phase, column temperature, pH and CPC content in the dissolution medium as well as rotation speed of the paddle were considered. After that, both graphical and statistical methods were used for identification of significant and non-significant effects.

RP-TLC separation of antiarrhythmic drugs. Densitometric analysis of flecainide in tablets

The chromatographic behavior of some antiarrhythmic compounds has been studied on commercially available TLC plates coated with octylsilane and octadecylsilane silica gel (RP-C8 and RP-C18, respectively) with organic-aqueous mobile phases containing citrate or acetate buffers at different pH. The best separations of individual and mixed drug standards were achieved on both RP-C8 and RP-C18 with 3:7 (v/v) tetrahydrofuran-citrate buffer pH 4.45 as mobile phase. To determine the usefulness of these chromatographic systems for analysis of tablet samples, flecainide acetate was identified and quantified by U.V. densitometry at two wavelengths, 225 and 310 nm. Linear relationships were obtained between peak height or peak area and amount in the range 6.0 to 12.0 μg per spot, the correlation coefficient, r, was ~0.990. The method was successfully applied to the analysis of flecainide in a pharmaceutical preparation, with satisfactory precision (RSD 1.14–5.93%) and accuracy (96.19–103.59%).

Chemometric comparison of thin-layer chromatography, gradient high-performance liquid chromatography, and computational methods for lipophilicity assessment of model compounds

Thin-layer chromatography (TLC) on two adsorbents (RP18 and CN) and with six modifiers (acetonitrile, acetone, dioxane, propan 2-ol, methanol, and tetrahydrofurane), followed by classical RM value extrapolation (previous results), was chemometrically compared with new one-run gradient high-performance liquid chromatography (HPLC) (C18, C18e, CN, and DIOL columns, acetonitrile, and methanol as modifiers) and, additionally, with seven computational algorithms (ALOGPs, AClogP, ALOGP, MLOGP, KOWWIN, XLOGP2, and XLOGP3) as a lipophilicity assessment tool on 35 model compounds with known lipophilicity. The statistical significance of intercepts and slopes of Collander equation (log P — retention dependence) and their values were compared. Whole results data set was subjected to scaled principal component analysis, which allowed exploring two main trends in these data. It can be concluded that one-run gradient HPLC does not outperform TLC in lipophilicity determination. Very good correlations were obtained between real log P and computational approaches; however, this is not a surprise for such simple molecules.

Dissolution profiles of perindopril and indapamide in their fixed-dose formulations by a new HPLC method and different mathematical approaches

Abstract A new HPLC method was introduced and validated for simultaneous determination of perindopril and indapamide. Validation procedure included specificity, sensitivity, robustness, stability, linearity, precision and accuracy. The method was used for the dissolution test of perindopril and indapamide in three fixed-dose formulations. The dissolution procedure was optimized using different media, different pH of the buffer, surfactants, paddle speed and temperature. Similarity of dissolution profiles was estimated using different model-independent and model-dependent methods and, additionally, by principal component analysis (PCA). Also, some kinetic models were checked for dissolved amounts of drugs as a function of time.

Influence of the regression technique on the extrapolated retention values of single-point adsorption compounds on silica gel

The retention (RM) values of nine one-point adsorption model compounds: diphenylamine, indol, 2-naphtol, 1-naphtol, 1-naphtylamine, 4-toluidine, carbazole, 4-chloraniline, and thymol were investigated on silica gel using six modifiers: acetone, dioxane, hexane, isopropanol, methylethylketone, ethyl acetate, and tetrahydrofurane (in hexane). These compounds showed small but visible curvilinearity of dependence of RMvs. modifier concentration. This curvilinearity is very similar among the investigated compounds, so relative differences of extrapolated RM are almost the same (strictly intercorrelated) regardless of the regression technique used. We have compared several robust and weighted regression methods and investigated their impact on extrapolated values. It can be concluded that one should primarily consider weighted regression with 1/x weights during retention extrapolation. It seems to be a better alternative than classical regression (better extrapolation) and also better than polynomial approaches (better stability).