High-performance Thin-layer Chromatographic Determination Research Articles

Sustainable and smart HPTLC determination of silodosin and solifenacin using a constructed two illumination source chamber with a smartphone camera as a detector: Comparative study with conventional densitometric scanner

The tremendous revolution in the built-in digital camera of smartphones has opened new horizons for its applicability as a portable, easily accessible, and user-friendly detector in different analytical techniques. In this article, we proposed and developed a new and inexpensive detector for simultaneous analysis of silodosin and solifenacin. The innovation makes use of a smartphone camera coupled with high-performance thin layer chromatographic (HPTLC) approach. Firstly, chromatographic separation was attained using HPTLC plates with an ecofriendly mobile phase composed of ethyl acetate, ethanol & 25% w/w ammonia (3.0: 7.0: 0.3, by volume). The developed plates were then visualized using Dragendorff's reagent and then photographed via smartphone's rear-facing camera fixed on a fabricated two-illumination source chamber. The intensities of drug spots were quantified using an open-source image analysis software, ImageJ, over concentration ranges of 2.0–30.0 μg/band. Moreover, the study was extended to compare the obtained results with a benchtop densitometric method at 215.0 nm where the densitometric one gave linear response ranging from 0.1 to 7.0 μg/band for silodosin and 0.1–6.0 μg/band for solifenacin. The fast, simple, reliable and green merits of the proposed HPTLC/smartphone method promted it as an excellent platform for assaying marketed combined tablets and assuring their content uniformity. Moreover, the high sensitivity of the densitometric method was exploited, for the first time, for determining the residual content of the cited drugs on manufacturing equipment surfaces during cleaning validation. Finally, the environmental impact of the methods was appraised via three state-of-the-art greenness metrics.

HPTLC determination of ergosterol in wheat and structure elucidation by NMR: Toward confirming method selectivity

Ergosterol (ERG) is considered as an excellent fungal biomarker, therefore it was utilized for the determination of the degree of fungi infection in wheat grains. A novel, fast, cost-effective and selective high performance thin layer chromatography (HPTLC) with reflectance/absorbance densitometry method was developed and validated for ERG determination. Separation was attained on pre-coated silica gel HPTLC plate with ethyl acetate: petroleum ether (6: 4 v/v) as a mobile phase. This system gave compact bands at retardation factor (Rf) of 0.45 ± 0.002 and quantification was performed at 282 nm over concentration range of 40–600 ng/band. The lower detectable limit was 10.79 ng per band which is three times more sensitive than the previously reported TLC method. The developed method could be utilized efficiently for the detection of ERG in pure powders and in fungi infected wheat samples. Resolution of ERG from other compounds in wheat grain extract allowed utilizing preparative TLC for separating and purifying ERG in a sufficient quantity for structure elucidation by nuclear magnetic resonance spectroscopy (NMR) for the first time. H1 and C13 NMR spectra indicated the purity of ERG extracted from the scrapped silica of preparative TLC plate at the Rf of the standard ERG verifying the method selectivity.

High-performance thin-layer chromatographic method development and determination of bio-enhancer from Piper trichostachyon: an ethnomedicinal plant

High-performance thin-layer chromatographic method development and determination of bio-enhancer from Piper trichostachyon: an ethnomedicinal plant

Vortex-assisted dispersive solid phase microextraction using Fe3O4/FeOOH magnetic nanocomposites for high-performance thin-layer chromatographic determination of zolmitriptan in rabbit plasma samples

In this work, a fast, versatile, and convenient dispersive solid-phase micro-extraction (DSPME) method is combined with a spectro-densitometric technique for the analysis of zolmitriptan (ZOLM) in biological fluids. Fe3O4/FeOOH magnetic nanocomposites (MNCs) were prepared by a co-precipitation method in aqueous solutions and utilized subsequently as a sorbent in DSPME. By coupling DSPME with high-performance thin-layer chromatography (HPTLC) with fluorescence detection, the preconcentration and determination of (ZOLM) in presence of metoclopramide (MET) and paracetamol (PARA), which are prescribed as an adjuvant therapy with ZOLM, was accomplished. Adsorption capability was assessed using both Langmuir and Freundlich adsorption isotherm models. The adsorption data was fitted to Langmuir adsorption isotherm model as reflected by high determination coefficient (R2 = 0.9944). Moreover, adsorption kinetics was assessed by pseudo-first and pseudo-second order kinetic models. The data was fitted to pseudo-second order kinetics, which proves that ZOLM interaction with the adsorbent is a chemisorption process. Surface complexation with MNCs was suggested to explain the pH dependence of ZOLM sorption. The key parameters of extraction and desorption steps (including pH, extraction time, sample volume, magnetic adsorbent amount, and desorption circumstances) were evaluated. Optimized conditions for solid phase microextraction of ZOLM were pH 2.9, 5.0 mg Fe3O4/FeOOH MNCs, 15 min vortex-assisted extraction time and 3 × 200 μL of methanol: 33% ammonia; 4:1 as eluent. The analysis was achieved using ACN: dichloromethane: 33% ammonia (22.5: 6.0: 1.5, v/v/v) as a mobile phase and the fluorescence detection was carried out at 223 nm. The proposed DSPME method was successfully applied for trace quantification of ZOLM in rabbits’ plasma (n = 6) after oral administration with a linearity range of 50.0 – 400.0 ng mL−1 (R2 = 0.9931), a detection limit of 12.0 ng mL−1 and extraction recovery of 97.27–99.89% with an RSD < 2% (n = 9). Moreover, the selectivity of the proposed approach for analysis of ZOLM in the presence of MET and PARA is demonstrated.

DEVELOPMENT OF QUALITY CONTROL PARAMETERS FOR STANDARDIZATION OF A NOVELMUCILAGE OBTAINED FROM OKRA (ABELMOSCHUS ESCULENTUS (L.) MOENCH) FRUIT

Objective: The main objective was to focus on qualitative and quantitative analysis of isolated okra mucilage by High-performance Thin Layer Chromatography to set up the quality control parameters for the isolated mucilage.&#x0D; Methods: High-performance thin-layer chromatography (HPTLC) were applied for the identification of components present in methanolic and ethanolic fruit extract of okra at 254 nm and 356 nm. Quantitative analysis of amino acid ascorbic acid and total polyphenol content was determined.&#x0D; Results: The results showed that the yield percentage for methanolic and ethanolic fruit extract of the okra fruit mucilage was found to be 13.5 and12.5% respectively. HPTLC determination of methanolic and ethanolic okra fruit extract showed the presence of 8 components with Rf values in the range of 0.14 to 0.62 and 0.14 to 0.54 respectively when detected at wavelengths 254 nm and at 356 nm. The total amino acid in okra fruit methanolic extract was found to be 11.45%w/w. The ascorbic acid content in methanolic okra fruit extract and ethanolic okra fruit extract was found to be 0.24 %w/w and 0.18% w/w respectively. The total phenolic contents (tannic acid equivalents, mg/g) in the methanolic and ethanolic okra fruit extracts were calculated to be 4.6 % w/wand 5.3% w/w respectively.&#x0D; Conclusion: The data revealed dual benefits like it can act as a potential novel functional ingredient with health-promoting application due to the presence of ascorbic acid and total phenolics contents and at the same time the data provided guidelines for quality control parameters for the isolated okra fruit

Rapid HPTLC determination of patulin in fruit-based baby food in Turkey

Mycotoxin contamination is an ongoing problem for all kinds of food commodity, and it is necessary to investigate the extent of this problem in baby food products. It is seen that factors such as developing technology, urbanization, and intense work tempo change dietary habits in every age group. The aim of this study was to provide a current overview on patulin (PAT) contamination of fruit-based baby food in Turkey. Safety levels of products were evaluated based on the provisional maximum tolerable daily intake (PMTDI) value predefined by the regulatory agencies. A total of 58 baby foods from Turkey market were investigated in this study. A high-performance thin-layer chromatography (HPTLC) method was developed by using acidic QUECHERS (quick-easy-cheap-effective-rugged-safe) extraction process. PAT was detected in 12 of 58 with a concentration between 0.13 and 2.98 mg/L; the mean value and the median value were found 1.71 mg/L and 1.92 mg/L, respectively. Based on PMTDI values recommended as 0.4 μg/kg bm/day, it was found that 11 of the 12 PAT-containing products were above the legal limit. The presence of PAT-containing baby food above the permitted values will be the subject of acute or chronic intoxications in the clinic, forensics in the consumer courts, and financial obligations of the questioned producers during the importation of the product at customs.

High-performance thin-layer chromatographic determination of trigonelline content in various extracts and different varieties of some commercial coffees available in the Saudi Arabian market

The proposed research study was undertaken to establish a simple, rapid and sensitive high-performance thin-layer chromatography (HPTLC) method for the quantification of trigonelline (TGN) in methanolic, aqueous and hydroalcoholic extracts of different brands of coffee samples with different colours due to roasting time. The proposed HPTLC methodology was carried out using glass coated silica gel 60 F254 plates. The mobile phase used was quaternary mixture of ethyl acetate:methanol:water:formic acid 5.4:2.3:1.5:0.8 (%, v/v) which furnished a compact spot of TGN at Rf 0.24 ± 0.01. The developed plate was scanned and quantified densitometrically at 267 nm. Linear regression analysis presented a good relationship between peak area and amount of TGN in the range of 100–700 ng/band. The method was validated as per the International Conference on Harmonization (ICH) recommendations. The maximum amount of TGN was recorded in roasted Arabic coffee among different brands studied. The amounts of TGN in methanolic, aqueous and hydroalcoholic extract of roasted Arabic coffee were obtained as 0.74, 0.80 and 0.79% w/w, respectively. The proposed analytical methodology will be useful to enumerate the therapeutic dose of TGN in different herbal products.

Simultaneous high-performance thin-layer chromatographic determination of ondansetron and pantoprazole in their pure forms and spiked human plasma

The therapeutic combination of ondansetron (OND) and pantoprazole (PAN) is widely prescribed for the treatment of many gastrointestinal tract (GIT) disorders. For this reason, a new, simple, very sensitive, accurate and precise HPTLC method was proposed for the determination of OND and PAN in pure forms, normal saline infusion solution, and spiked human plasma. Merck HPTLC aluminum plates pre-coated with silica gel 60 F254 were used as the stationary phase, and the mobile phase consisted of chloroform-methanolethyl acetate (6:2:2 v/v). Using this system resulted in compact , symmetric peaks with the best resolution between peaks. The RF values were 0.50 ± 0.12 for OND and 0.73 ± 0.09 for PAN. The densitometric scanner was set at 302 nm using a deuterium lamp as the source of radiation. The detection and quantification limits were 7.50 and 22.50 ng per spot for OND and 15.81 and 47.42 ng per spot for PAN. The proposed method was successively applied for the analysis of the studied drugs in their pharmaceutical dosage forms, normal saline solution, and spiked human plasma. It gives excellent percent of recovery. Excellent agreement between the proposed method and the reported methods was achieved when the results were matched with respect to accuracy and precision.

Stability-indicating high-performance thin-layer chromatographic determination of ondansetron in pure form and pharmaceutical formulations

A new, simple, precise, accurate, stability-indicating, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the determination of ondansetron hydrochloride in pure form and pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of chloroform–methanol–ethyl acetate (7:2:1, v/v). This system was found to give a compact spot of ondansetron (RF value of 0.67 ± 0.011); ondansetron was subjected to acid and alkali hydrolysis, oxidation, and photodegradation. The wavelength of TLC scanner was set at 302 nm for both detection and quantitation. The calibration curves were linear over the range of 25–500 ng spot−1. The limit of detection was 4.9 ng spot−1, and the limit of quantitation was 14.7 ng spot−1. The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines, and the results were acceptable. The propo...

Simultaneous HPTLC Determination of Rhein and Aloe-emodin in Senna Alata Leaves from Thailand and their Commercial Products

A high performance thin layer chromatographic (HPTLC) method was developed and validated for simultaneous determination of rhein and aloe-emodin, major anthraquinone constituents, in S. alata leaves. The separation was performed on a silica gel 60 F₂₅₄ HPTLC plate using ethyl acetate: methanol: water (100:17:10, v/v/v) as the development system. Validation of the analytical method for rhein and aloe-emodin promoted acceptable parameters. Good linearity in the range of 40-480 ng/band was obtained while intra-day and inter-day precisions were shown to be precise with relative standard deviations of less than 5%. The average percentage recoveries of rhein and aloe-emodin were 98.8% and 98.9%, suggesting acceptable accuracy. The content of rhein and aloe-emodin in S. alata leaves collected from 5 different provinces in Thailand analyzed by the validated HPTLC method were in the ranges of 0.098 ± 0.017 - 0.30±0.02%, w/w, and 0.081 ± 0.0006 - 0.34 ? 0.0009 %, w/w, respectively. Five commercial products of S. alata tea available in the market were purchased and analyzed for rhein and aloe-emodin contents. The contents of rhein and aloe-emodin in the tea samples were in the ranges of 0.085 ± 0.004 - 0:23 ± 0.04%, w/w, and 0.096 ±0.006 - 0.30 ± 0.01 %, w/w, respectively. The HPTLC method is rapid, reliable, sensitive and economical for routine analysis of rhein and aloe-emodin contents in-S. alata leaf raw materials and its commercial products.

High-performance thin-layer chromatographic determination of three therapeutic phenolic components in Leucas species

This study was designed to develop a simple, sensitive, selective, and precise high-performance thin-layer chromatography (HPTLC) fingerprint and quantitative estimation method for the analysis of three phenolic compounds from nine Leucas species. The developed HPTLC method was validated according to the International Conference on Harmonization guidelines. The method permits reliable quantification of one important phenolic acid (gallic acid) and two hydroxycinnamic acids (caffeic and ferulic acids) in 50% hydroethanolic extracts on silica gel with toluene—ethyl acetate—formic acid 8:2:1 (v/v) as the mobile phase. The system showed good resolution and separation of caffeic, ferulic, and gallic acids (at Rf values 0.48, 0.60, and 0.30, respectively) from the other constituents of the extract. Densitometric scanning was done at 300 nm in absorbance mode. All the results obtained by the developed method were statistically compared for validation parameters, for accuracy and good precision. Caffeic, ferulic,...

HPTLC Determination of Three Gliptins in Binary Mixtures with Metformin.

A single, simple, selective and validated high performance thin layer chromatographic (HPTLC) method was developed for the determination of either linagliptin (LGP), saxagliptin (SGP) or vildagliptin (VGP) in their binary mixtures with metformin (MET) in pharmaceutical preparations using environmentally preferable green mobile phase system. Separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using methanol-0.5% w/v aqueous ammonium sulfate (8 : 2, v/v) as mobile phase. Densitometric measurement of the spots was performed at 225 nm for LGP/MET mixture and at 208 nm for both SGP/MET and VGP/MET mixtures. The linear regression analysis data were used for the regression line in the range of 0.05-0.5 µg/band for LGP and SGP and 0.2-2 and 5-40 µg/band for VGP and MET, respectively. The method was validated and showed good performances in terms of linearity, limits of detection and quantitation, precision, accuracy, selectivity and specificity. The calculated percentage relative error values and percentage relative standard deviation for intra- and interday precision studies did not exceed 2%. The developed method was satisfactorily applied for the analysis of pharmaceutical preparations and proved to be specific and accurate for the quality control of the cited drugs in their dosage forms.

Erratum to: Simultaneous High-Performance Thin-Layer Chromatographic Determination of Indole Acetic Acid, Indole Butyric Acid, and Absisic Acid in in vitro Seedling of Watermelon Exposed to Heavy Metals

Erratum to: Simultaneous High-Performance Thin-Layer Chromatographic Determination of Indole Acetic Acid, Indole Butyric Acid, and Absisic Acid in in vitro Seedling of Watermelon Exposed to Heavy Metals

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.) using high performance thin layer chromatography.

Evaluation of quantitative variation of secondary metabolites in Bergenia ciliata (Haw.) using high performance thin layer chromatography.

High-performance thin layer chromatography determination of astaxanthin in Euphausia superba oil

The Antarctic krill (Euphausia superba) is a kind of marine zooplankton belonging among Crustacea [1]. It is the principal prey of many marine predators by reason of its gigantic population [2]. The study of Antarctic krill has become a hot research field in the past few decades. It is not only the key organism in the southern ocean but also the biggest fishery resource in the world. With its high content of astaxanthin as a valuable natural product [2, 3], it urgently awaits development and utilization. The ketocarotenoid astaxanthin (3,3 -dihydroxy, -carotene-4,4 -dione) and astaxanthin esters are the principal components of Antarctic krill pigment [4], and astaxanthin is one of most important carotenoids by virtue of its biologic functions as a vitamin A precursor and as a much more efficient antioxidant than -carotene and vitamin E [5]. It is also a natural coloring (in fish muscles, bird feathers, yolks, etc.), supports the immune system, has an anti-tumor effect, and protects from ultraviolet radiation [6–8]. Overall, astaxanthin can play an important role in healthcare and cosmetic manufacture, with enormous commercial and industrial prospects. As a general rule, high-performance liquid chromatography (HPLC) is preferred in astaxanthin assay due to its wide use all around the world [9], whereas the HPTLC technique with high sensitivity and resolution can also function excellently in the separation and purification of bioactive compounds from natural materials. In short, we aim to establish and validate an HPTLC method that is adapted to astaxanthin determination in Antarctic krill oil and that can be applied to other materials that contain astaxanthin. The astaxanthin in Antarctic krill is mostly in the form of astaxanthin esters for its molecular stabilization [10]. Some are diesters and others are monoesters, attributed to the two hydroxyls that can combine with different fatty acids at both ends of the astaxanthin molecule [11]. While the concentration of free astaxanthin was amall, all of it and other impurities are fat soluble and can be dissolved in low-polarity reagents; thus, n-hexane–acetone (7:2, v/v) was chosen to be the mobile phase after repeated attempts, combined with the high-performance silica gel 60 F254, which can accomplish the HPTLC separation within 10 min. The Rf value of the free astaxanthin was 0.32. It was necessary to apply the saponification procedure to release the astaxanthin combined with fatty acids because the concentration of free astaxanthin was from 5 1.0% to 85 5.0%, so that the qualification and quantification of astaxanthin can be carried out more successfully, as well as the further purification, since there was no standard for astaxanthin esters. KOH was essential in saponification, but it can result in significant degradation of astaxanthin, especially when the reaction temperature was high. There was almost no degradation of astaxanthin at 4 C [12]; therefore it was selected as the saponification temperature, and the maximum concentration of free astaxanthin can reach 89.7466%. The linear regression equation of astaxanthin after saponification is Y = 1309.2935 + 47.9693X (where Y is the response and X is the mass of astaxanthin). The correlation coefficient was found to be 0.9990, the RSD (relative standard deviation) was 2.56%, and the astaxanthin in the saponification sample was 0.7045 mg·mL–1; more data are shown in Table 1. The three main components in the original sample were separated without interference from other impurities, and the spectral evaluation of the samples showed the same maximum absorption ( max 476 nm) as the standard; therefore, the specificity of this method is verified. The RSD of intraday precision was 1.68% and the value of the interday precision was 5.07%. Both values were determined when the plates were stored in the dark. The accuracy was clarified as percent recovery, and the average recovery of astaxanthin was 98.53%.

Development and validation of a stability-indicating HPTLC determination of zafirlukast in bulk drug and pharmaceutical dosage form

A simple, selective, precise, and sensitive stability-indicating highperformance thin-layer chromatographic (HPTLC) method has been developed and validated for the analysis of zafirlukast both in bulk drug and pharmaceutical dosage form. The separation was performed on a pre-coated silica gel 60 F254 plates using toluene-methanol-acetone (7:2:1 v/v) as mobile phase. Densitometric analysis was performed in reflectance-absorbance mode at 240 nm. The linear regression analysis data for the calibration plot showed good linear relationship with r2 = 0.999 in the range of 50–400 ng spot−1. The mean value of slope and intercept were 15.35 ± 0.341 and 337.81 ± 1.52, respectively. The minimum amount of zafirlukast that could be authentically detected and quantified was 16.43 and 49.80 ng spot−1, respectively. The drug was subjected to acidic, basic, oxidative, photolytic, and thermal conditions. The drug undergoes degradation under acidic, basic, H2O2, photolytic, and oxidative conditions. The degradation products...

Simultaneous high-performance thin-layer chromatographic determination of indole acetic acid, indole butyric acid, and Absisic acid inin vitroseedling of watermelon exposed to heavy metals

A new method based on the use of high-performance thin-layer chromatography (HPTLC) was developed for the simultaneous determination of the plant growth hormones, indole acetic acid, indole butyric acid, and absisic acid in in vitro seedling of watermelon. The measurements were performed at the associate maximum absorption wavelength of hormones. Calibration plots were constructed in the range of 50–600 ng per spot for indole acetic acid, 50–600 ng per spot for indole butyric acid, and 50–600 ng per spot for absisic acid. The limits of quantification (LOQs) determined experimentally were as follows: 10, 10, and 6 ng per spot, respectively, for indole acetic acid, indole butyric acid, and absisic acid. The method was successfully applied to watermelon tissues exposed to Cu2+, Co2+, and Pb2+ for determination of its indole acetic acid, indole butyric acid, and absisic acid concentrations.

Simultaneous HPTLC determination of strychnine and brucine in strychnos nux-vomica seed.

Objective:A simple, sensitive, and specific thin layer chromatography (TLC) densitometry method has been developed for the simultaneous quantification of strychnine and brucine in the seeds of Strychnos nux-vomica.Materials and Methods:The method involved simultaneous estimation of strychnine and brucine after resolving it by high performance TLC (HPTLC) on silica gel plate with chloroform–methanol–formic acid (8.5:1.5:0.4 v/v/v) as the mobile phase.Results:The method was validated as per the ICH guidelines for precision (interday, intraday, intersystem), robustness, accuracy, limit of detection, and limit of quantitation. The relationship between the concentration of standard solutions and the peak response was linear within the concentration range of 50–1000 ng/spot for strychnine and 100–1000 ng/spot for brucine. The method precision was found to be 0.58–2.47 (% relative standard deviation [RSD]) and 0.36–2.22 (% RSD) for strychnine and brucine, respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.75% for strychnine and 100.52% for brucine, respectively.Conclusions:The HPTLC method for the simultaneous quantification of strychnine and brucine was found to be simple, precise, specific, sensitive, and accurate and can be used for routine analysis and quality control of raw material of S. nux-vomica and several unani and ayurvedic formulations containing this as an ingredient.

Stability-Indicating HPTLC Determination of Ambroxol Hydrochloride in Bulk Drug and Pharmaceutical Dosage Form

A simple, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of ambroxol hydrochloride both as a bulk drug and in formulations was developed and validated. The method employed HPTLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of methanol-triethylamine (4:6 v/v). The system was found to give a compact spot for ambroxol hydrochloride (R(f) value of 0.53 +/- 0.02). Densitometric analysis of ambroxol hydrochloride was carried out in the absorbance mode at 254 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r(2) = 0.9966 +/- 0.0013 with respect to peak area in the concentration range 100-1000 ng/spot. The mean value +/- standard deviation of slope and intercept were 164.85 +/- 0.72 and 1168.3 +/- 8.26 with respect to peak area. The method was validated for precision, recovery, and robustness. The limits of detection and quantitation were 10 and 30 ng/spot, respectively. Ambroxol hydrochloride was subjected to oxidation and thermal degradation. The drug undergoes degradation under oxidation and heat conditions. This indicates that the drug is susceptible to oxidation and heat. Statistical analysis proves that the method is repeatable, selective, and accurate for the estimation of said drug. Stability indicating of new chemical entities is an important part for the drug development of ambroxol hydrochloride and for its estimation in plasma and other biological fluids; the novel Statistical analysis proves that the method is repeatable and selective for the analysis of ambroxol hydrochloride as bulk drug and in pharmaceutical formulations. The proposed developed HPTLC method can be applied for identification and quantitative determination of ambroxol hydrochloride in bulk drug and dosage forms. This work is to determine the purity of the drug available from the various sources by detecting the related impurities.

Quantitative high-performance thin-layer chromatography determination of common liposome components and critical parameters influencing the analysis results

The aim of this study was to investigate the performance of a newly devised high-performance thin-layer chromatography (HPTLC) method in quantifying common liposome membrane components, including the five phospholipids (PLs), phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, as well as cholesterol, cholesteryl hemisuccinate, and linoleic acid. Besides strictly keeping to a standardized procedure, three parameters were particularly critical for proper quantification. First, a relative humidity of higher than 60% caused migration distances to increase and reduced the resolution of the PLs on a silica-gel 60 HPTLC plate. Second, PLs underwent oxidative combustion during storage for 2 or 24 hours on an HPTLC plate, with peak losses of up to 25–44%. These losses could be prevented by storage under nitrogen and, to some extent, by the addition of the antioxidant, DL-α-tocopherol. Third, even with automated sample application, the accuracy and consistency of the application volume proved to be an important cause of error and needs routine verification. Considering these parameters, the method was found to accurately and precisely determine the composition of three different liposome preparations. The recovery was 97.2–101.8%, compared to secondary methods, and consistent over different days and with different operators (mean RSD of the recovery: 2.03 ± 1.16%, n = 9). The working range was determined to be 100–300 ng in the case of the PLs (individual limit of determination between 40 and 80 ng) and 20–60 ng in the case of cholesterol (limit of determination: 16 ng).