Anna Berecka

Revisiting thin-layer chromatography as a lipophilicity determination tool—A comparative study on several techniques with a model solute set

The lipophilicity of a compound is a fundamental property related to pharmaceutical and biomedical activity. As many approaches are mixed together in every-day published studies, the subject needs some standardization. The paper presents a comparative study on several approaches of TLC lipophilicity determination: a single TLC run, extrapolation of a retention, principal component analysis of a retention matrix, PARAFAC on a three-way array and a PLS regression. All techniques were applied to 35 model solutes with simple molecules, using nine concentrations of six modifiers: acetonitrile, acetone, dioxane, propan-2-ol, methanol and tetrahydrofurane. The elaborated comparative analysis formed several general recommendations. Methanol and dioxane were the best modifiers, while acetonitrile gave the worst and inacceptable correlation of retention with lipophilicity. Surprisingly, good correlations were obtained for the single TLC runs and this method is underestimated in the literature. The advanced chemometric processing proposed recently, such as PCA, PARAFAC and PLS did not show a visible advantage comparing to classical methods. A need to use a robust regression and robust correlation measures, due to presence of significant outliers, was also noticed and studied.

Reversed‐Phase Thin‐Layer Chromatography of Three New Oral Antidiabetics and Densitometric Determination of Pioglitazone

Abstract The thin‐layer chromatographic behavior of new oral antidiabetic drugs, pioglitazone, rosiglitazone, and repaglinide has been investigated. For reversed‐phase (RP) chromatography, chemically bonded cyanopropyl plates with mobile phases comprising 1,4‐dioxane with phosphate buffers were used. The influence of the pH on the separation of the drugs was also examined. Then, a simple, rapid, and stability‐indicating high performance thin‐layer chromatographic method has been developed and validated for the quantitative determination of pioglitazone in tablets. Analysis was performed with 1,4‐dioxane–phosphate buffer of pH 4.4 (5:5) as the mobile phase. Detection and quantification were performed by classical densitometry at the wavelength of maximum absorption of pioglitazone, 266 nm. A calibration plot was constructed in the range of 0.4–2.4 µg/10 µL and was linear with a good correlation coefficient (r = 0.9957). Precision was validated by replicate analyses of standard solutions, and accuracy by analysis of fortified samples. The precision of the proposed chromatographic method expressed as mean relative standard deviation (RSD) was 4.99% and 2.57%, respectively, for the lowest and the highest calibration levels. Recovery from the fortified samples ranged from 98.09% to 103.28%. The mean (±SD) recovery from tablets was 99.79% ± 1.57%.

Quantitative analysis of repaglinide in tablets by reversed-phase thin-layer chromatography with densitometric UV detection

A simple, rapid, and stability-indicating thin-layer chromatograph-ic method has been developed for quantitative determination of repaglinide in tablets. Analysis was performed on RP-8 TLC plates with acetonitrile—pH 6.0 phosphate buffer, 60 + 40 (% v/v), as mobile phase. Detection and quantification were performed by classical densitometry at the wavelength of maximum absorption of repaglinide, 225 nm. A calibration plot constructed in the range 0.6–3.6 µg/10 µL was linear with a good correlation coefficient (r = 0.998 ± 0.001, mean ± SD, n = 5). Limits of quantitation and detection of repaglinide were 0.27 µg/10 µL and 0.08 µg/10 µL, respectively. Instrumental precision established at three concentrations of the drug ranged from 3.92 to 0.97% for the lowest and highest concentrations of repaglinide, respectively. The mean intra-day and inter-day variability, including three concentrations of re-paglinide, were 1.93 and 2.25% (n = 9), respectively. Recovery from model mixtures, at three levels of addition, ranged from 103.06 to 102.49% for the lowest and highest levels, respectively. Total mean ± SD recovery was 102.71 ± 2.04% (n = 15). The mean ± SD recovery from commercially available tablets was 101.85 ± 1.83% (n = 10). The effect of pH, temperature, and UV light on degradation of repaglinide was also investigated. The analytical method presented was found to be simple, reliable, and convenient for routine pharmaceutical analysis. Its analytical performance fulfilled acceptance criteria established for TLC methods in the official literature.

Normal- and Reversed-Phase Thin-Layer Chromatography of Seven Oral Antidiabetic Agents

The chromatographic behavior of seven oral antidiabetic drugs -chlorpropamide, tolbutamide, glibenclamide, metformin, pioglitazone, rosiglitazone, and repaglinide - has been investigated. Normal-phase chromatography was performed on silica gel and alumina layers with mixtures of chloroform, diethyl ether, and ethyl acetate as mobile phases. For more effective resolution aqueous ammonia or acetic acid was added to the mobile phases. Silica gel enabled better separation than alumina. Reversed-phase chromatography was performed on octadecyl-bonded silica gel (RP-18) with mixtures of acetonitrile or 2-propanol with phosphate buffer as mobile phases. The effect of pH on the separation of the drugs was also examined. For separation of these drugs reversed-phase chromatography was more effective than use of normal-phase mode.

Rapid HPTLC Determination of Rosiglitazone in Pharmaceutical Formulations

Abstract A new, simple, rapid, and stability‐indicating high‐performance thin layer chromatographic (HPTLC) method has been developed and validated for the determination of rosiglitazone in tablets. Analysis was performed on silica gel 60F254 plates in horizontal chambers with chloroform–ethyl acetate–25% ammonium hydroxide (5:5:0.1, v/v) as mobile phase. Detection and quantification were performed by classical densitometry at 240 and 254 nm. The active substance was extracted from tablets with ethanol. Calibration plots were constructed in the range 0.2–1.0 µg/10 µL and were correlated with good correlation coefficients (r 240 = 0.9993; r 254 = 0.9994). Precision was validated by replicate analyses of standard solutions, and accuracy by analysis of fortified samples. The precision of the proposed chromatographic method, expressed as mean RSD was 3.58 and 2.76% for 240 nm, and 8.23 and 6.56% for 254 nm, for the lowest and the highest calibration levels, respectively. The mean recoveries from the fortified samples ranged from 89.48% to 99.38% for 240 nm, and from 89.05% to 100.89% for 254 nm. The mean recoveries from tablets were 101.95% and 103.2% for assays at 240 and 254 nm, respectively.

Quantitative analysis of gliclazide and glipizide in tablets by a new validated and stability-indicating RPTLC method

A simple, rapid, and stability-indicating RPTLC method has been established and validated for quantitative analysis of two oral antidiabetic agents, the sulfonylureas gliclazide and glipizide, in tablets. Analysis was performed on RP-18 plates with 60% (v/v) acetonitrile in pH 2.3 phosphate buffer as mobile phase. Compact spots were obtained for gliclazide (RF 0.38 ± 0.02, mean ± SD) and glipizide (RF 0.51 ± 0.03). Detection and quantification were performed by classical densitometry at 215 nm, the wavelength of maximum absorption of gliclazide and glipizide. Calibration plots were constructed in the range 0.8–1.8 µg per 10 µL (per spot) for both drugs and were linear with good correlation coefficients (r = 0.998 ± 0.001, mean ± SD, for gliclazide and r = 0.993 ± 0.002 for glipizide). The limits of detection and quantification were 50 and 200 ng per spot, respectively, for gliclazide and 60 and 300 ng for glipizide. The method was validated for robustness, precision, accuracy, stability, and specificity. The drugs were subjected to acidic and alkaline hydrolysis, oxidation, photo-degradation, and heating and were found to be susceptible to some stress conditions. However, the degradation products were well resolved from the pure drugs with significantly different RF values.

Retention behavior of new oral antidiabetic drugs in reversed-phase chromatography

The thin-layer chromatographic behavior of three of the newest oral antidiabetic agents, pioglitazone, rosiglitazone, and repaglinide, has been studied in reversed-phase systems. Chromatography was performed on RP-8 as adsorbent with buffer–organic modifier binary mobile phases of widely different composition. Phosphate buffers of pH 2.4, 4.4, 6.0 and 7.9 were used with three organic mobile-phase modifiers, acetonitrile, 2-propanol, and methanol; the concentration of organic modifier was varied between 20 and 80% (v/v). Plates were developed in horizontal chambers, visualized by UV illumination at λ = 254 nm, and scanned with a densitometer. The effect of the mobile phase on retention was studied. The selectivity of the chromatographic systems is discussed. The linearity of relationships between RM and modifier volume fraction, molar fraction, and logarithm of the molar fraction was calculated.

Standardized Reversed-Phase Thin-Layer Chromatographic Study of the Lipophilicity of Five Anti-Diabetic Thiazolidinediones

Five anti-diabetic thiazolidinediones have been chromatographed on C18 TLC plates with binary mobile phases containing water and the organic modifiers acetone, 1,4-dioxane, or methanol. Linear relationships were obtained between the RM values of the compounds and the concentration of organic modifier in the mobile phase. These RM values enabled calculation of RM0 values by extrapolation. Calibration equations were then obtained for nine standards of known lipophilicity in the range 0.83-6.04. From these equations the partition coefficients log PEXP were calculated for the drugs. Lipophilicity values were also calculated by use of computational methods. Finally, interesting relationships were obtained between experimental and theoretical log P values and pharmacological data from the literature.

DENSITOMETRIC AND VIDEODENSITOMETRIC DETERMINATION OF NADOLOL AND PINDOLOL IN TABLETS BY QUANTITATIVE HPTLC

ABSTRACT New, simple, precise, and rapid high performance thin layer chromatography (HPTLC) methods have been developed for the determination of nadolol (N) and pindolol (P) in tablets. The stationary phase was silica gel 60F254 and the mobile phase was ethyl acetate-methanol-glacial acetic acid (49+49+2, v/v). Detection and quantification were done densitometrically at 270 nm and by videodensitometric scanning at 254 nm. In a densitometric procedure, the linearity range was 0.2–1.2 µg/10 µL for N and P. In a videodensitometric assay, the linearity ranges were 2.0–12.0 and 0.2–1.2 µg/10 µL for N and P, respectively. Precision was validated by replicate analyses of standard solutions, and accuracy by analysis of fortified samples. In densitometric procedure, the precision (RSD) obtained for the standard solutions ranged from 1.14 to 2.80 and from 0.74 to 1.85% for N and P, respectively. For the videodensitometric assay, the RSD values ranged from 0.68 to 2.36 and from 0.79 to 3.20% for N and P, respectively. The results obtained by both techniques were compared.

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.