Lipophilicity is often estimated by thin-layer chromatography (TLC) as a fundamental property related to biological and pharmaceutical activity. In a recent study, we contributed some standardization to the subject by performing a comparative analysis of the different approaches in the field. This part presents an analogous study on silica, the use of which in normal-phase systems seems to be a new trend of recent times. We have compared several approaches of TLC lipophilicity determination: a single TLC run, Soczewiński-Wachtmeister equation coefficients, principal component analysis (PCA) of the retention matrix, and PARAFAC on a three-way array. All techniques were applied to 35 model solutes with simple molecules, using nine concentrations of six modifiers: acetone, dioxane, ethyl acetate, methylethylketone, propan-2-ol, and tetrahydrofuran. Comparative analysis points to several general recommendations. Ethyl acetate seems to give the best correlations with lipophilicity, and the correlations of single retention values, extrapolated values (intercepts), or C0 are satisfactory (around 0.8) to use in normal-phase method in lipophilicity estimation. However, the correlation is quite worse and the normal-phase method should be treated as a method supplementary to the reversed-phase method. Neither PCA nor PARAFAC carried out on the retention matrix improved the correlations.
The paper discusses the usefulness of self modelling multivariate curve resolution (nonnegative matrix factorization) as a chemometric tool for the analysis of inhomogeneous spots captured by densitometer in multivariate way. The discussion is based on two examples: a spot of decomposed aspirin with comparative spot of pure salicylic acid, and spots of overlapped ciprofibrate and clofibric acid. It is concluded that this approach works well in the case of thin-layer chromatography (TLC) and the algorithm finds reliable spectral and concentration profiles, even with high overlap and spectral similarity. Nonlinearity does not affect this algorithm in visible manner. The computation can be performed with free software and can be recommended as good method to analyze inhomogeneity and to obtain additional proof of spot contamination with estimates of the spectral profiles.
New simple TLC methods with densitometry and videoscanning have been developed for the quantitation of bezafibrate in Bezamidin tablets and ciprofibrate in Lipanor capsules. Analysis was performed on HPTLC Diol F
plates with hexane-tetrahydrofuran, 8 + 2, as mobile phase. Detection was performed by densitometry at
= 227 nm and videoscanning at
= 254 nm. Calibration plots were constructed in the range 5–30 µg per spot for both drugs. The calibration data were tested using several regression models and the optimum models were selected (quadratic for videoscanning and nonlinear
was always >0.995). The active substances were extracted from tablets with methanol. The linearity of the method was tested by spotting different amounts of extracted solution (15–30 mg). The recovery function was always sufficiently linear, with an insignificant intercept and slope very close to unity. Accuracy was tested by quantitating three fortified samples (50, 100, and 150%); this resulted in homogeneous results without significant differences. Recovery measured by use of densitometry was 100.3% (
7.84%) for bezafibrate and 98.01% (
6.12%) for ciprofibrate. Videodensitometry resulted in recovery of 96.16% (
9.8%) and 97.8% (
11.2%), respectively. The
-test for two means showed there was no significant difference between the precision and accuracy of the methods.
values and mobile phase composition have been determined for the six antihyperlipidemic agents-bezafibrate, ciprofibrate, clofibrate, clofibric acid, fenofibrate, and gemfibrozil. The drugs were separated on silica gel, CN, and Diol plates using mobile phases containing
-hexane as weakly polar diluent and five polar modifiers: acetone, dioxane, ethyl methyl ketone, ethyl acetate, and tetrahydrofuran. The optimum mobile phases were also investigated on alumina, NH
, and polyamide phases for comparison. The linearity of relationships between
and modifier volume fraction, molar fraction, and logarithm of molar fraction was calculated. Plates were developed in horizontal chambers, visualized under UV irradiation at
= 254 nm, and scanned with a densitometer in the multi-wavelength scan mode. Most results fitted the
> 0.9; for approximately half
> 0.99. Separation of all the drugs was achieved on Diol plates with mobile phases containing 20–30% of each modifier in
-hexane, and with hexane-acetone, 9 + 1, on CN plates. Five drugs were separated using the same mobile phases on silica gel. The best separation was obtained on Diol plates with tetrahydrofuran-hexane, 2 + 8, as mobile phase.
Two new simple and accurate HPTLC methods for analysis of ziprasidone in capsules have been developed and validated. The first method was normal phase chromatography on silica gel F
HPTLC plates with hexane-dioxane-propylamine 1:9:0.4 (
) as mobile phase. The second method was reversed phase chromatography on RP8 F
HPTLC plates with tetrahydrofuran-pH 9.0 phosphate buffer 5:5 (
) as mobile phase. The silica gel plates were developed to a distance of 9 cm and the RP8 plates to a distance of 4.5 cm. Both analyses were performed in horizontal chambers and the plates were scanned by videodensitometry at 254 nm. The calibration plots were linear in the ranges 0.2–1.2 μg and 0.1–1.1 μg ziprasidone (per spot) for the NP-HPTLC and RP-HPTLC methods, respectively. The precision and accuracy of the methods were fully compared between themselves and also with the control method — classical densitometry at 243 nm. No significant differences were observed. The NP-HPTLC method was also used for study of the stability of ziprasidone in solutions. The methods can be used for routine quality-control analysis.
Authors:Łukasz Komsta, Urszula Dunin and Robert Skibiński
During our previous studies, we discovered that reversed-phase thin-layer chromatography (RP-TLC) plates can behave in typical normal-phase manner, which is not widely known nor used in the literature. Therefore, we decided to investigate this behavior more comprehensively on RP-8 plates. We used 35 model compounds, and performed chemometric screening mixture design approach for 20 popular solvents. This gave us the possibility to estimate separate retention effect for each solvent being the measure of average solvent strength. The results were compared to the previous study done on silica gel. It can be concluded that RP-8 plates can be used in typical normal-phase systems and their behavior does not differ substantially from silica gel plates.
Authors:Robert Skibiński, Łukasz Komsta and Ilona Kosztyła
Two new simple and accurate methods for determination of quetiapine in tablets-normal phase (NP) and reversed-phase (RP) high performance thin layer chromatography (HPTLC), each with densitometric and videodensitometric detection, were developed and validated. NP-HPTLC was developed with HPTLC silica F
plates and hexane-dioxane-propylamine 1:9:0.4 (
) as mobile phase. RPHPTLC was carried out using HPTLC RP8 F
plates with tetrahydrofuran-phosphate buffer, pH 9.0, 5:5 (
) as mobile phase. The silica gel plates were developed to a distance of 9 cm and RP8 plates to a distance of 4.5 cm. Both analyses were performed in horizontal chambers and scanned with a densitometer at 243 nm and a video-densitometer at 254 nm. Calibration plots were linear in the range 0.2–1.2 μg quetiapine per spot for NP-HPTLC and in the range 0.1–1.1 μg for RP-HPTLC. The precision and accuracy of the four methods were fully compared and no significant differences were observed. The methods can be used in routine pharmaceutical analysis.
Authors:Łukasz Komsta, Wojciech Markowski and Genowefa Misztal
The retention factor
is used in several criteria generally known as chromatographic response functions (CRF). In TLC and HPTLC most of these are based on differences between the retention factors of two substances, which are summed or multiplied. There are also other functions, for example the multispot response function (
) which enables the quality of a separation to be estimated. Although good CRF criteria should have well-defined distribution and range, current criteria based on the differences do not satisfy this requirement. Only
has a clearly defined range (0 to 1), but its distribution is unstable. In this paper two new independent coefficients:
(retention uniformity) and
(retention distance) are proposed; these always have values within the range 0 to 1 and stable density, irrespective of the number of compounds separated. Their reliable mathematical properties have been tested in wide range by Monte-Carlo simulations. An example is given of their use in the separation of fibrate-type antihyperlipidemic drugs by normal and reversed-phase TLC (114 systems).