Authors:Lionel Paillat, Christine Périchet, Sophie Lavoine, Uwe Meierhenrich, and Xavier Fernandez
A simple, sensitive, selective, precise, and robust high-performance thin-layer chromatography (HPTLC) method was developed and validated for the quantification of vanillin β-d-glucoside, p-hydroxybenzoic acid (APHB), vanillic acid, p-hydroxybenzaldehyde (PHB), and vanillin in vanilla fruits, beans, and extracts. The analysis was performed on HPTLC glass plate precoated with silica gel 60F254 as stationary phase. Vertical development was carried in an automated vertical developing chamber (ADC2), saturated, and preconditioned 5 min with the developing solvent n-hexane-chloroform-methanol-acetic acid (5:36:4:0.5, vol). Athin-layer chromatography scanner was used for spectrodensitometric scanning and analysis in absorbance mode at 254 nm, 280 nm, and 313 nm. The calibration plots showed good linear relationships in the concentration ranges 24–120 ng per spot for vanillin β-d-glucoside, 20–100 ng per spot for acids, and 8–40 ng per spot for aldehydes. The method was validated for precision, accuracy, and robustness. Detection and quantification limits were determined. Statistical analysis of the data revealed that the method is reproducible and selective for the determination of vanillin β-d-glucoside, APHB, vanillic acid, PHB, and vanillin in vanilla fruits, beans, and extracts.
Sunscreens like 4-tert-butyl, 4-methoxydibenzoylmethane (BMDM) exhibit poor photostability, when exposed to sunlight. Conventionally, photostability of BMDM and other sunscreens can be determined using high-performance liquid chromatography (HPLC) or spectroscopic methods. We have developed a rapid, easy screening, and potentially high-throughput high-performance thin-layer chromatographic (HPTLC) method for quantifying the photostability of BMDM in the presence of known stabilizers/other chemical ingredients. The method is based on spotting the silica gel coated HPTLC plates (in duplicate) with the sunscreen molecules of interest along with a known photostabilizer. The plate is then exposed to solar simulated sunlight for a specific time. Ascending chromatography was performed in a thin-layer chromatographic (TLC) chamber using n-hexane-ethyl acetate 9:1 (v/v) as the mobile phase (∼10 mL). Densitometric analysis was done to determine the amount of sunscreen that remained after solar exposure. This method also involves separation of sunscreens and their photodegraded products on the HPTLC plate. The results obtained with this method were compared well with those obtained with the conventional method.
Planar chromatography (TLC and HPTLC) is affected by many factors. Although one major advantage of the technique, its enormous flexibility, is based on this, the same flexibility easily can become a major obstacle to obtaining reproducible qualitative and quantitative results. Although literature on theoretical aspects and fundamental concepts of TLC is abundant, standardized practical approaches are rather rare.This publication, in the 100th issue of JPC, is an attempt to spark a discussion about an internationally harmonized and standardized approach to modern HPTLC. On the basis of theoretical considerations and practical examples proposals are made which could help increase the reliability and comparability of qualitative and quantitative results obtained by HPTLC. To ensure the survival of planar chromatography as a trustworthy method a pragmatic solution might be an acceptable compromise both for experts and for new-comers to the field.
Authors:Milena Lambri, Michaël Jourdes, Yves Glories, and Cédric Saucier
An HPTLC method has been developed to give qualitative and quantitative information on red wine pigments. The samples are prepared by solid-phase extraction (SPE) on C
cartridges. Analytical determination is done by chromatography on C
silica gel plates with isocratic elution with methanol-water-trifluoracetic acid, 55 + 45 + 1 (
). This method leads to the clear separation of the three classes of
anthocyanins with good statistical repeatability and reproducibility for wines of different vintages. It also enables evaluation of the amount of polymeric pigments in red wines.
Authors:Beate Fuchs, Jürgen Schiller, Rosmarie Süß, Ariane Nimptsch, Martin Schürenberg, and Detlev Suckau
Lipids are important natural products and essential in nutrition, cosmetic formulations, pharmaceuticals, etc. Lipids and, particularly, phospholipids are of substantial medical interest (some are molecules with messenger function) and of diagnostic potential (for instance, the lipoproteins in human blood). Among the different soft-ionization mass spectrometric methods that enable detection of the intact lipid molecules, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has several advantages, for instance, simple performance, high sensitivity, and robustness against contaminants. Additionally, MALDI-TOF MS analyzes a solid sample. This enables (in contrast with isotropic solutions) acquisition of spatially-resolved mass spectra (‘mass spectrometric imaging’). However, separation of complex mixtures into the individual lipid classes is normally required to enable detection of all the components. It will be shown with the example of a lipid extract from hens’ egg yolk that MALDI-TOF MS can be easily combined with TLC, enabling detection of as little as picomole amounts of lipids directly on the HPTLC plate. This results in sensitivities higher than those from established staining procedures. Additionally, because of the substantial spatial resolution, lipids separated by normal-phase TLC may not only be differentiated according to differences of their headgroups but also according to differences of their fatty acyl composition. Finally, MS-MS experiments, providing further insights into the structures of the relevant lipids, can be also performed directly on the HPTLC plate. Although the HPTLC-MALDI coupling can be easily established, there are different points to which special attention should be paid. Aspects of matrix application, data acquisition (including the stability of lipids and reproducibility), and data evaluation will be emphasized in this paper
Authors:Bhavini Patel, Bhanubhai Suhagia, Chaganbhai Patel, and Hiral Panchal
A quantitative high-performance thin-layer chromatography (HPTLC) method for determination of darunavir ethanolate (DRV) in tablets has been established and validated. DRV from the formulations was separated and identified on silica gel 60 F254 HPTLC plates with toluene-ethyl acetate-methanol 7.0:2.0:1.0 (ν/ν) as mobile phase. The plates were developed to a distance of 8 cm. Quantification was performed at λ = 267 nm. Well-resolved bands were obtained for DRV. The method was validated for specificity, precision, robustness, and accuracy. The calibration plot for DRV standard was linear in the range 250–1750 ng per band with r = 0.9994, slope = 0.4253, and intercept = 44.81. The limits of detection and quantification were 15.28 and 45.84 ng per band, respectively. The method is selective, sensitive, and specific, with potential application in pharmaceutical analysis.
Clarithromycin used for the treatment of respiratory tract infection. Anew alternative simple, rapid, sensitive, and selective high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of clarithromycin in plasma. HPTLC analysis was performed on pre-coated silica gel 60F254 plates. Separation was carried out using ethyl acetate-methanol-15% ammonium acetate (pH 10.6) as solvent system. Densitometry measurements were performed in absorbance mode at 506 nm after derivatising with xanthydrol solution. The RF of the drug was found to be 0.62. The method was linear over the range of 0.1–3.0 μg mL−1 (r2 = 0.9974). The mean extraction recoveries were over 85%. The intra-day and inter-day precision (%CV) of the assay was in the range of 0.79–4.55%. The accuracy was found to be above 95%. The method was successfully applied to a pharmacokinetic study of clarithromycin 250 mg tablet in healthy human male volunteers.
Aqueous micellar bile salt, sodium deoxycholate (NaDC) solution as additive in acetonitrile (ACN) in the ratio (1:5 ν/ν) was identified as the most favorable mobile phase for on-plate identification and resolution of three series of common sugars (disaccharide, pentose and hexose) on commercially available high performance silica gel plates. Effect of mobile phase composition and the presence of heavy metal cations as impurities in the analyte sample have been investigated to optimize experimental conditions for the separation. The lowest possible amount of all sugar on high-performance thin-layer chromatography (HPTLC) plate has been determined at nanogram level. This method was successfully applied for identification and separation of sugars in pharmaceutical formulations [cough syrup (Honitus), multivitamin syrup (Becozinc)] and biological matrix (human blood).