The quantitative detection of estrogens and antithyroid drugs by thin-layer and high performance thin-layer chromatography in animal tissue (author's transl)]

Methods of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) were developed for the determination of estrogens and antithyroid drugs in extracts of animal tissues. TLC proved to be suitable for quantities in the range of 200-2000 ng, with a detection limit of 50-200 ng, HPTLC in the range of 10-200 ng, showing calibration curves of good linearity even in extracts. By HPTLC better detection limits, better separation and faster ascending could be achieved than by TLC.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

Simultaneous estimation of pantoprazole and domperidone in pure powder and a pharmaceutical formulation by high-perfomance liquid chromatography and high-performance thin-layer chromatography methods

This paper describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous estimation of pantoprazole (PANT) and domperidone (DOM) in pure powder and capsule formulations. The HPLC separation was achieved on a Phenomenex C18 column (250 mm id, 4.6 mm, 5 pm) using 0.01 M, 6.5 pH ammonium acetate buffer-methanol-acetonitrile (30 + 40 + 30, v/v/v, pH 7.20) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol (60 + 40, v/v) as the mobile phase. Quantification was achieved with ultraviolet (UV) detection at 287 nm over the concentration range 400-4000 and 300-3000 ng/mL with mean recovery of 99.35+/-0.80 and 99.08+/-0.57% for PANT and DOM, respectively (HPLC method). Quantification was achieved with UV detection at 287 nm over the concentration range 80-240 and 60-180 ng/spot with mean recovery of 98.40+/-0.67 and 98.75+/-0.71% for PANT and DOM, respectively (HPTLC method). These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of PANT and DOM in pure powder and capsule formulations.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

Estimation of trans-resveratrol in herbal extracts and dosage forms by high-performance thin-layer chromatography

A simple, sensitive and precise high-performance thin-layer chromatographic (HPTLC) method of analysis of trans-resveratrol in Polygonum cuspidatum root extracts and in dosage forms was developed and validated. The separation was carried out on a TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase, eluted with chloroform-ethylacetate-formic acid (2.5 : 1 : 0.1) as mobile phase. Densitometric analysis of trans-resveratrol was carried out in the absorbance mode at 313 nm. This system was found to give compact spot for trans-resveratrol (Rf value of 0.40+/-0.03). A good linear regression relationship between peak areas and the concentrations was obtained over the range of 0.5-3.0 microg/spot with correlation coefficient 0.9989. The limit of detection and quantification was found to be 9 and 27 ng/spot. The method was validated for precision and recovery. The spike recoveries were within 99.85 to 100.70%. The RSD values of the precision in the range 0.37-1.84%. The proposed developed HPTLC method can be applied for identification and quantitative determination of trans-resveratrol in herbal extracts and dosage forms.     

Fingerprint profiling of acid hydrolyzates of polysaccharides extracted from the fruiting bodies and spores of Lingzhi by high-performance thin-layer chromatography

Modern extraction and planar chromatographic instrumentation were employed for the fingerprint profiling of carbohydrates from an important and popular medicinal mushroom commonly known as Lingzhi. For the first time, the feasibility of employing the high-performance thin-layer chromatography (HPTLC) peak profiles (fingerprints) of carbohydrates for the screening of various Lingzhi species/products was demonstrated. An analytical procedure was developed such that upon acid hydrolysis of the polysaccharides extracted from various Lingzhi samples, fingerprint profiles that reveal the relative amounts of the degradation products, such as mono- and oligosaccharides, can be obtained using HPTLC plates (Si 50000) for separation and 4-aminobenzoic acid as the post-chromatographic derivatization reagent for detection. Also, using automated multiple development (AMD), the acid hydrolyzates from Lingzhi, consisting of simple and more complex sugars, can be separated simultaneously with high degree of automation. An important finding was that unique fingerprint patterns were observed in the monosaccharide profiles between two highly valued Lingzhi species, Ganoderma applanatum and Ganoderma lucidum, under total or partial acid hydrolysis conditions. Additionally, the HPTLC fingerprint profiles of carbohydrates were obtained from the extracts of the spores and fruiting bodies of Lingzhi and compared.     

Analysis of flavonol aglycones and terpenelactones in Ginkgo biloba extract: A comparison of high-performance thin-layer chromatography and column high-performance liquid chromatography

Advancements in automated high-performance thin-layer chromatography (HPTLC) have made it feasible to assess its use for the quantitative analysis of marker compounds in botanical preparations. We report here the findings of method comparisons for the terpenelactones and flavonol aglycones by column high-performance liquid chromatography (HPLC) with evaporative light scattering and UV detection, and HPTLC with a scanning densitometer. For the HPTLC assay of terpenelactones, total bilobalide, ginkgolide A, and ginkgolide B consistently achieved <70% of the total determined using HPLC, regardless of variations to postchromatographic derivatization time and temperature. Accuracy testing showed the possibility of a matrix interference. In contrast, a good relationship (95%) was determined between HPTLC and HPLC for determination of total flavonol glycosides (calculated from combined quercetin, kaempferol, and isorhamnetin) from an acid-hydrolyzed Ginkgo biloba L. (GBE) sample. The HPTLC flavonol aglycone method also performed well in terms of accuracy (overall average of 96% recovery for the 3 aglycones) and consecutive plate repeatability (overall percent relative standard deviation of 4.4). It is demonstrated that HPTLC can be a time-saving complement to HPLC for routine analysis of the flavonol glycosides in GBE.     

High-performance thin-layer chromatography method for monitoring norfloxacin residues on pharmaceutical equipment surfaces

This paper presents a high-performance thin-layer chromatography (HPTLC) method with direct fluorescence measurement for the determination of norfloxacin. The method was validated for the monitoring of norfloxacin residues on stainless steel surfaces at the allowed limit of 10 mg of norfloxacin per square meter. However, it can be adapted for lower amounts of residues owing to the low detection limit of norfloxacin (about 5 ng) and can also be used for other surface materials. Test solutions were analyzed by the new HPTLC method and the known HPLC method for comparison. Accuracy and precision of the new HPTLC method, with a subsequent quantification by densitometer or video system, are comparable with those of the HPLC method.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

Standardization of Crude Extract of Neem Seed Kernels (Azadirachta indica A. Juss) and Commercial Neem Based Formulations Using HPTLC and Extended Length Packed-Columns SFC Method

Two chromatographic techniques are described for the separation and quantitative determination of azadirachtin A and B, salannin, and nimbin present in the crude extract of neem seed kernels and commercial neem based formulations. The high performance thin-layer chromatography (HPTLC) separation of markers was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using ethylacetate-benzene (7.0: 3.0, v/v) as mobile phase. The other technique was based on extended length packed column supercritical fluid chromatographic (PC-SFC) separation of the markers using Cyano column (250 mm × 4.6 mm I.D, S-5.0 μ) and Kromasil 100 NH₂ column (250 mm × 4.6 mm I.D, 5.0 μ) connected in series. The detection was carried out using photodiode array detector at 338 K using methanol-modified carbon dioxide (10%) as the mobile phase at flow rate of 2.0 mL min⁻¹. The current study assesses the effect of extending column length during PC-SFC experiment to obtain maximum resolution between a number of unknown components and known markers present in neem seed extracts. Both the chromatographic methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of quantitative determination of markers by means of HPTLC and PC-SFC method.     

High-performance thin-layer chromatographic determination of six major ginsenosides in Panax ginseng

A densitometric determination of six major ginsenosides in Panax ginseng, separated by high-performance thin-layer chromatography (HPTLC), was optimized. Simple extraction and clean-up methods of the target constituents and the development of standardized conditions of chromatoplates with a quaternary-solvents system allowed an efficient saponins recovery from the plant material and their selective separation. After exposure of the chromatograms to thionyl chloride vapors and further heating, stable reaction products of ginsenosides, which showed absorption maxima at lambda=275 nm as well as a fluorescence (lambda(excitation)=366 nm, lambda(emission)=400 nm), allowed the application of a sensitive and reproducible method for their simultaneous determination. The method was validated by spiking the ginseng extracts with pure standards.     

Quantitative analysis of phenobarbital in dosage form by thin-layer chromatography combined with densitometry

In this paper, a high-performance thin-layer chromatography (HPTLC) method combined with densitometry has been described. Chromatography was performed on silica gel Si 60F254 plates using dichloromethane-ethyl acetate-formic acid (9.5 +/- 0.5 +/- 0.1, v/v) mobile phase. This method has been successfully applied for the determination of phenobarbital in pharmaceuticals. Obtained results were comparable with traditionally used column high-performance liquid chromatography (HPLC) methods. For the proposed procedure, linearity (r > 0.999), sensitivity (limit of detection 0.4 microg/spot), recovery (97.8-102.1%), and repeatability were found to be satisfactory. The HPTLC-densitometry method has many advantages, such as simplicity, reasonable sensitivity, rapidity, and low cost, and it can be successfully used in routine quality control of multidrug preparations containing barbiturates.     

Sensitive determination of buspirone in serum by solid-phase extraction and two-dimensional high-performance liquid chromatography

A selective and sensitive determination of buspirone in serum by high-performance liquid chromatography is described. The procedure is based on separation on a C18 column. A solid-phase extraction procedure is used for sample clean-up. The retention on the first column is based on the hydrophobic interaction of buspirone with the stationary phase, and the retention on the second column is based on ionic interactions due to the presence of sodium lauryl sulphate in the mobile phase as well as hydrophobic interaction. This allows for good separation of buspirone from impurities and consequently allows lower detection limits than previously reported for liquid chromatographic methods. Detection by ultraviolet absorbance gives a detection limit of 0.2 ng/ml.     

Thin-layer chromatography and multivariate data analysis of willow bark extracts

In most cases the pharmacological activity of plant extracts is not assigned to single components and often not all active ingredients are known. Approaches other than those considering single compounds only to analyze plant material have proven helpful for a better characterization of extracts in their entirety. In this study extracts of willow bark are analyzed by high-performance thin-layer chromatography (HPTLC) and two different pharmacological tests [the 2,2'-azobis (2-amidinopropane) dihydrochloride reaction and the xanthine/xanthine oxidase reaction] with the help of multivariate data analysis. Described are two models using the results of the chromatographic study of 22 various extracts of willow bark and their pharmacological properties. The chromatographic data are obtained by a special TLC scanner that enables measurement of HPTLC tracks simultaneously in the range of lambda = 200-400 nm. Additionally, the developed models are used to predict the activity of another three extracts of willow bark demonstrating the quality of the model.     

Simultaneous determination of atorvastatin calcium and ramipril in capsule dosage forms by high-performance liquid chromatography and high-performance thin layer chromatography

Two simple and accurate methods to determine atorvastatin calcium and ramipril in capsule dosage forms were developed and validated using HPLC and HPTLC. The HPLC separation was achieved on a Phenomenex Luna C18 column (250 x 4.6 mm id, 5 microm) in the isocratic mode using 0.1% phosphoric acid-acetonitrile (38 + 62, v/v), pH 3.5 +/- 0.05, mobile phase at a flow rate of 1 ml/min. The retention times were 6.42 and 2.86 min for atorvastatin calcium and ramipril, respectively. Quantification was achieved with a photodiode array detector set at 210 nm over the concentration range of 0.5-5 microg/mL for each, with mean recoveries (at three concentration levels) of 100.06 +/- 0.49% and 99.95 +/- 0.63% RSD for atorvastatin calcium and ramipril, respectively. The HPTLC separation was achieved on silica gel 60 F254 HPTLC plates using methanol-benzene-glacial acetic acid (19.6 + 80.0 + 0.4, v/v/v) as the mobile phase. The Rf values were 0.40 and 0.20 for atorvastatin calcium and ramipril, respectively. Quantification was achieved with UV densitometry at 210 nm over the concentration range of 50-500 ng/spot for each, with mean recoveries (at three concentration levels) of 99.98 +/- 0.75% and 99.87 +/- 0.83% RSD for atorvastatin calcium and ramipril, respectively. Both methods were validated according to International Conference on Harmonization guidelines and found to be simple, specific, accurate, precise, and robust. The mean assay percentages for atorvastatin calcium and ramipril were 99.90 and 99.55% for HPLC and 99.91 and 99.47% for HPTLC, respectively. The methods were successfully applied for the determination of atorvastatin calcium and ramipril in capsule dosage forms without any interference from common excipients.     

Rapid determination of abamectin in lettuce and cucumber by high-performance liquid chromatography.

A rapid, sensitive and reliable method is presented for the determination of trace amounts of abamectin in lettuce and cucumber. Abamectin consists of greater than or equal to 80% avermectin B1a and less than or equal to 20% avermectin Bb. Vegetables were extracted with ethyl acetate and the extract was purified by solid-phase extraction using Sep-Pak silica cartridges. The purified extracts were analysed by high-performance liquid chromatography with a 5 microm Zorbax ODS column and UV detection under isocratic conditions. The method yields recoveries for avermectin B1a and B1b of 76-109% in the 0.054-0.54 ng/kg range. The limit of detection of the method is 40 microgram/kg each of avermectin B1a and B1b in vegetables.     

Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review

Fluoroquinolones are one of the most promising and intensively studied drugs of contemporary anti-infective chemotherapy. New fluoroquinolone antibacterials with improved pharmacokinetic properties and a broad spectrum of activity have been developed, opening new windows of opportunity for clinical use. To our knowledge, no comprehensive and critical review of the analytical methods for the determination of these agents, which correspond to the third- and fourth-generation quinolones, has yet been published. This work summarizes for the first time most of the liquid chromatographic methods reported in the literature for the separation and quantification of the new fluoroquinolones in biological matrices and pharmaceutical formulations. A systematic and detailed survey of physicochemical properties, sample preparation procedures, and chromatographic and detection conditions is presented herein. In the course of this review several liquid chromatographic methods are discussed: reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IEX-HPLC), hydrophilic interaction liquid chromatography (HILIC), high-performance thin-layer chromatography (HPTLC) and other chiral chromatographic methods. Their advantages, applicability and limitations are also examined.     

Quantitative Determination of Aucubin in Seven Plantago Species Using HPLC,HPTLC, and LC-ESI-MS Methods

A reversed-phase high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of aucubin in Plantago lanceolata. The analyses were carried out on Zorbax SB-C₁₈ column with an aqueous phosphoric acid and acetonitrile gradient. The correlation coefficient of calibration curve showed good linearity (r > 0.9995), with average recoveries between 96.7 and 104.5%. The developed method was applied for quantification in P. atrata, P. bellardii, P. coronopus, P. holosteum, P. reniformis, and P. schwarzenbergiana. The aucubin content in plant extracts was compared by HPLC, HPTLC, and LC-ESI-MS techniques and no significant differences between the conducted methods were observed.     

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.     

Development of a tandem thin-layer chromatography-high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products

A solid-phase extraction clean-up and and a liquid chromatographic method with ultraviolet detection were developed for the analysis of 51 corticosteroids in cosmetic samples in order to screen commercial samples for the presence of undeclared synthetic corticosteroids. A thin-layer chromatographic analysis was carried out on silica gel plates, using different eluants and detection reagents. When such a preliminary chromatographic separation gave some indications about the presence of steroid compounds, the methanol extracts from real samples were applied to a solid-phase extraction C₁₈ cartridge, and the analytes eluted with ethyl ether. The high-performance liquid chromatographic separation was then carried out for the identification and determination of the analytes using a Purospher RP-18 column, an isocratic or a gradient elution with a mixture acetonitrile-water and a photodiode-array detector. The accuracy of the method was determined by spiking experiments on home-made cosmetic samples. The analytical recoveries were satisfactory.