Thin-layer Chromatography Screening Methods Research Articles

Development of quantitative HPTLC methods for dolutegravir, lamivudine, and tenofovir disproxil fumarate in a combination pharmaceutical product using a model process published earlier for transfer of minilab TLC screening methods to HPTLC-densitometry

High-performance thin-layer chromatography (HPLTC)–densitometry methods are described for the analysis of the anti(retro)virals dolutegravir (D), lamivudine (L), and tenofovir disoproxil fumarate (TDF) in a pharmaceutical tablet product. To the best of our knowledge, no previous quantitative planar chromatography method has been reported in the literature for this combination formulation. The method for L was transferred from a thin-layer chromatography (TLC) screening method published in the Global Pharma Health Fund (GPHF) Minilab Manual designed for identification of counterfeit and substandard drug products using a model process published earlier. D and TDF are not included in the list of drugs for which TLC screening methods are published for the Minilab, but HPTLC–densitometry procedures were developed for them using the transfer process guidelines. L was analyzed simultaneously with TDF on Merck Premium Purity silica gel 60 F plates using the mobile phase ethyl acetate–methanol–acetone–concentrated ammonium hydroxide (30:7:3:1) and densitometric scanning at 254 nm. D was analyzed on a second plate by scanning at 366 nm after chromatography with the chloroform–methanol–formic acid (32:8:2) mobile phase. Data for all three drugs are shown to meet the requirements of the model transfer process for calibration curve r values, assay of tablets relative to their label values, peak purity/peak identity tests, and validation by standard addition analysis of samples spiked at 50%, 100%, and 150% of the label value of active ingredients. A TLC screening method for TDF in the combination product was developed and published online with open access.

Advances in the thin layer chromatographic analysis of counterfeit pharmaceutical products: 2008–2019

Publications reporting thin layer chromatography (TLC) screening and high performance TLC (HPTLC)-densitometry quantification analyses of counterfeit pharmaceutical products are reviewed for the 2008–2019 period. Screening using TLC methods published in the Global Pharma Health Fund (GPHF) Minilab Manual and U.S. Food and Drug Administration (FDA) Compendium, as well as in other sources, are covered. Also included are publications on TLC analysis hyphenated with Raman and mass spectrometry; analyses of counterfeit traditional herbal medicines; earlier published reviews; transfer of screening methods for counterfeit pharmaceutical products in the Minilab Manual and FDA Compendium to HPTLC-densitometry using a model process; development of HPTLC-densitometry methods for pharmaceutical products not included in the Minilab Manual or FDA Compendium using the model process followed by development of corresponding Supplemental FDA Compendium TLC screening methods; and modified Minilab methods with simplified detection based on heating of silica gel F layers to produce fluorescence quenching zones (thermochemical activation) rather than detection using spray, dip, or vapor phase derivatization reagents. Some thoughts on future prospects for the field are also offered.

Development of quantitative HPTLC-densitometry methods following a model process for transfer of TLC screening methods for pharmaceutical products containing moxifloxacin HCl, ofloxacin, amoxicillin trihydrate, acetylsalicylic acid + acetaminophen + caffeine, nimesulide, irbesartan, and pantoprazole

Transfer of thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab Manual and U.S. Food and Drug Administration (FDA) Compendium protocols for detecting falsified or substandard drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods suitable for support of regulatory authorities was performed following a previously published model process for moxfloxacin HCl, ofloxacin, and amoxicillin trihydrate. In addition, the guidelines of the process were followed to develop and validate HPTLC-densitometry methods for pharmaceutical products containing acetylsalicylic acid + acetaminophen + caffeine, nimesulide, irbesartan, and pantoprazole that are not included in the Minilab Manual or FDA Compendium. The new methods involved silica gel 60 F254 plates; semiautomated bandwise sample and standard solution application; ascending mobile phase development of plates in a glass chamber; and automated slit scanning densitometry for detection, identification, and quantification. Data for standard and sample solution preparation, calibration curves, assay of pharmaceutical products compared to label declarations, standard addition validation, and peak purity and peak identity are provided. Supplemental FDA Compendium screening methods corresponding to the HPTLC-densitometry methods for caffeine, nimesulide, irbesartan, pantoprazole, and acetylsalicylic acid co-formulated with acetaminophen and caffeine were developed and published online with open access on the website www.layloff.net.

Development of quantitative HPTLC–Densitometry methods for the analysis of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide using a model process developed earlier for the transfer of TLC screening methods

A model process, previously developed in a series of studies, allows for the transfer of thin-layer chromatography (TLC) methods for qualitative screening of counterfeit drug products published in the Global Pharma Health Fund (GPHF) Minilab manual and US Food and Drug Administration (FDA) Compendium of Unofficial Methods for Screening of Pharmaceuticals by TLC to quantitative high-performance TLC (HPTLC)–densitometry methods. This article describes HPTLC–densitometry methods developed and validated according to this model process for pharmaceutical products of amiodarone HCl, carvedilol, doxylamine succinate, magnesium salicylate, metoprolol succinate, nebivolol HCl, and salicylamide, for which qualitative screening methods have not been published in the Minilab manual or FDA Compendium. These methods use relatively inexpensive and nontoxic “green solvents” for sample and standard solution and mobile phase preparation, Merck Premium Purity silica gel 60 F254 plates, automated standard and sample solution bandwise application, and automated densitometry for the assessment of peak purity and identity and quantification. Corresponding to the quantitative HPTLC–densitometry methods, qualitative TLC screening methods for these drug products were developed and posted online with open access as supplements to the FDA Compendium.

Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel

ABSTRACTTransfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods was performed following a previously published model process. The developed and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, relatively nontoxic, readily available solvents and other reagents; silica gel 60 F254 plates; automated bandwise sample and standard solution application; ascending mobile phase development of plates in a chamber; and automated slit scanning densitometry for detection, identification, and quantification. Validation data for methods developed in an early version of the transfer model process that did not include standard addition validation are reported for pharmaceutical products containing amitriptyline HCl, amodiaquine, diphenhydramine HCl, and mebendazole.

Development of quantitative HPTLC–densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of cefixime, cefuroxime axetil, cephalexin⋅H2O, ciprofloxacin HCl, levofloxacin, and metronidazole

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund Minilab kit protocols for detecting fake or markedly substandard drugs in pharmaceutical products in the field to quantitative high-performance TLC (HPTLC)–densitometry methods was carried out using a model process published earlier. The developed and validated methods for tablets or capsules containing cefixime, cefuroxime axetil, cephalexin·H2O, ciprofloxacin HCl, levofloxacin, and metronidazole involved use of EMD Millipore Premium Purity silica gel 60 F254 plates, automated sample and standard solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification.

Transfer of silica gel TLC screening methods for clarithromycin, azithromycin, and amodiaquine + artesunate to HPTLC–densitometry with detection by reagentless thermochemical activation of fluorescence quenching

ABSTRACTA previously published model process was used for the transfer of silica gel thin-layer chromatography (TLC) screening methods for clarithromycin, azithromycin, and amodiaquine + artesunate pharmaceutical formulations published in the Global Pharma Health Fund E.V. Minilab manual for the identification of counterfeit drugs to high-performance TLC–densitometry quantitative methods that can be used in support of regulatory compliance actions. In these new methods, detection of clarithromycin, azithromycin, and artesunate was achieved by thermochemical activation involving simple reagent-free heating of the layer to produce derivatives of the drugs that quench fluorescence under 254 nm ultraviolet light. Additional drugs with TLC screening methods published in the Minilab manual and/or Compendium of Unofficial Methods for Rapid Screening of Pharmaceuticals by Thin Layer Chromatography that do not naturally quench fluorescence were studied and also found to be detectable by thermochemical activation on silica gel layers. The conditions of the thermochemical activation were studied, and in situ spectra of drug zones before and after heating were obtained. Heat activation of fluorescence quenching seems to be a widely applicable detection method that is safer and more convenient than the use of chemical spray, dip, or vapor phase reagents.

Use of a model process for transfer of minilab TLC screening methods for quinine sulfate, mefloquine, and dihydroartemisinin—piperaquine phosphate tablets to quantitative HPTLC—densitometry methods

Transfer of four thin-layer chromatography (TLC) Global Pharma Health Fund Minilab mobile kit protocols for detecting fake pharmaceutical products to quantitative high-performance TLC (HPTLC)—densitometry methods was carried out using a model process published earlier. The developed and validated methods were for the drugs quinine sulfate, mefloquine, dihydroartemisinin, and piperaquine phosphate. EMD Millipore Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 for detection, identification, and quantification were used. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software, and accuracy was estimated by the standard addition approach. HPTLC gives better efficiency, selectivity, and resolution than TLC, and the new methods overcome the deficiencies in technology related to manual application and visual zone comparison that do not allow the Minilab TLC procedures to support regulatory compliance actions. These new methods should be fully validated according to International Conference on Harmonization guidelines or by interlaboratory studies if required by their applications. In addition, a previously reported transferred simultaneous HPTLC–densitometry method for lumefantrine and artemether was used to analyze a new combination tablet to demonstrate its applicability.

Transfer of TLC screening methods designed for use in developing countries to quantitative HPTLC-densitometry methods for diazepam and amodiaquine tablets

Transfer of two rapid thin-layer chromatography (TLC) screening methods used to detect markedly substandard and fake pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods is demonstrated using a model procedure that was published earlier. These TLC methods for diazepam and amodiaquine are contained in a Compendium of methods by Kenyon and Layloff and a Minilab method manual from Global Pharma Health Fund E.V., respectively, for use in countries with limited resources. Merck HPTLC Premium Purity silica gel 60 F254 glass plates, automated standard and sample solution application with a CAMAG Linomat 4, and automated densitometry with a CAMAG Scanner 3 were used for detection, identification, and quantification. Sample peak identity and purity validation were carried out by spectral comparison checks available in the winCATS software. Accuracy was estimated by the standard addition approach with overspotting standard and sample solutions. HPTLC gives better efficiency, sel...

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.

Determination of pah in soil samples by high-performance thin-layer chromatography (HPTLC)

A thin-layer Chromatographic screening-method is presented for the determination of polycyclic aromatic hydrocarbons (PAH) in soil samples. This screening method is intended for a quick overview of the composition of a contaminant. The developed method of separation is well suited for the semi-quantitative determination of PAH in soil samples, and can be used to identify samples that require further analysis by means of GC or HPLC. The separation of 8 PAH groups with a maximum of 2 PAH (discernible by selective excitation of fluorescence) each is possible. The results of the analysis based on PAH group separation can be regarded as semi-quantitative. The extraction of PAH is effected by means of a solvent mixture consisting of n-hexane-acetone (1:1, v/v). The extraction is aided by ultrasonic treatment. The extract is purified by application to an activated silica gel column (solid-phase extraction). The qualitative analysis can be carried out either by visual observation or by fluorimetric scanning (TLC-Scanner). The characteristic fluorescent colours facilitate a reliable visual identification of PAH. The applicability of the method is shown and a comparison to other analytical methods is carried out. Some of the remarkable features of this method are its user-friendly handling, the low consumption of solvents and the applicability without the necessity for extra equipment.

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti

The periplasmic cyclic beta-1,2-glucans produced by bacteria within the Rhizobiaceae family provide functions during hypo-osmotic adaptation and plant infection. In Rhizobium meliloti, these molecules are highly modified with phosphoglycerol and succinyl substituents, and it is possible that the anionic character of these glucans is important for their functions. In the present study, we have used a thin-layer chromatographic screening method to identify a novel R. meliloti mutant specifically blocked in its ability to transfer phosphoglycerol substituents to the cyclic beta-1,2-glucan backbone. Further analysis revealed that the cyclic glucans produced by this mutant contained elevated levels of succinyl substituents. As a result, the overall anionic charge on the cyclic beta-1,2-glucans was found to be similar to that of wild-type cells. Despite this difference in cyclic beta-1,2-glucan structure, the mutant was shown to effectively nodulate alfalfa and to grow as well as wild-type cells in hypo-osmotic media.

A two-dimensional high-performance thin-layer chromatographic screening method for sulphonamides in animal tissues.

A procedure for the identification of sulphonamides in edible animal tissues by two-dimensional high-performance thin-layer chromatography is described. Sixteen sulphonamides can be detected at an absolute level of 10 ng. The absolute detection limit of the sulphonamide standards is 1 ng. After extraction of the sulphonamides with chloroform/acetone, the acidified extract is concentrated and purified using a cation-exchange solid phase extraction column. The column is treated with ammonia vapour and the sulphonamides are then eluted with methanol. Blank samples of edible animal tissues spiked with sulphonamides (frequently used in Belgium) result in very good separation.

Thin-layer chromatographic screening method for the tranquillizers azaperone, propiopromazine and carazolol in pig tissues

A procedure is described for the detection of azaperone, propiopromazine and carazolol in pig muscle, liver and kidney tissue. The method comprises extraction from an alkaline tissue homogenate with diethyl ether, followed by cleaning up and concentration of the extract on a silica gel solid-phase extraction column. Two-dimensional thin-layer chromatography on a silica plate was used for the detection of the tranquillizers. Detection levels were 25 μg kg −1 for propiopromazine, 50 μg kg −1 for azaperone (or its metabolite azaperol) and 125 μg kg −1 for carazolol. In pigs treated with the usual doses the presence of propiopromazine and azaperol could be established in kidney tissue 8 h after administration, whilst in injection sites all three tranquillizers could be detected.

Rapid thin-layer chromatographic screening method for the detection of five sulfonamides in swine tissues: collaborative study.

A rapid thin-layer chromatographic screening method for five sulfonamides in swine tissues was studied collaboratively in nine Dutch laboratories. All collaborators were sent fifteen swine muscle and fifteen swine kidney samples. These were obtained from treated swine or from swine tissues spiked with sulfonamides at levels ranging from 0 to 0.4 mg/kg. The study indicates that the method is suitable for the screening of animal tissues for these sulfonamides. False scores occasionally occur at the lowest levels.

An improved thin-layer chromatographic method for urinary oligosaccharide screening

A modified thin-layer Chromatographic screening method is presented for the rapid diagnosis of glycoprotein storage disorders based upon excess oligosaccharide excretion. The system has been used successfully in the diagnosis of mannosidosis, GM 1 gangliosidosis, mucolipidoses types I and III, aspartylglycosaminuria and fucosidosis.

Detection of Twelve Mycotoxins in Mixed Animal Feedstuffs, Using a Novel Membrane Cleanup Procedure

A multimycotoxin thin layer chromatographic screening method is described which is applicable to most animal feedstuffs. Interference from nonspecific lipid, pigment, and other components of simple and mixed feeds is reduced to a minimum by using a membrane cleanup step. Aflatoxins B1, B2, G1, and G2, citrinin, diacetoxyscirpenol, ochratoxin A, patulin, penitrem A, sterigmatocystin, T-2 toxin, and zearalenone may be reliably detected. The sensitivity of the method is generally low for mixed feeds but even so aflatoxin B1 can be detected at a level of 3 ppb and ochratoxin A at 80 ppb. While the basic method is less sensitive for sterigmatocystin (330 ppb), patulin (600 ppb), zearalenone (1000 ppb), and the trichothecenes (1000-4000 ppb), it may be adapted so as to reduce the above detection limits when the presence of these toxins is suspected. Lower levels may be detected in extracts of simple feeds.

An improved method for rapid, large-scale thin-layer chromatographic urine screening for drugs of abuse

This report describes a rapid, reliable, thin-layer chromatographic screening method, suitable for large scale operations, for the detection of drug abuse. By utilizing microdot sample application on 100 mμ thin-layer silica gel plates, sample size has been diminished with an increase in sensitivity. Drugs are detected using a separate series of chromophoric spray reagents for the acid and basic drugs, including a new reagent N,2,6-trichlorobenzoquinoneimine for the visualization of cyclic imide type compounds (barbiturates). Confirmation of drug identification to eliminate false positive results, is achieved, using the technique of microcrystallography.