Thin-layer Chromatographic Screening Research Articles

Crispene A, B, C and D, Four New Clerodane Type Furanoid Diterpenes from Tinospora crispa (L.).

Background:Tinospora crispa (L.) is used to alleviate the symptoms of diabetes mellitus in folk medicine. It is also used for hypertension and to treat malaria, remedy for diarrhea, and as vermifuge.Materials and Methods:Stems of T. crispa were collected, sun dried for several days followed by oven dried for 24 h at a considerably low temperature and then ground into coarse powder. The powdered stems were soaked in methanol at room temperature for 14 days with occasional shaking. The extract was collected by filtration, and the solvent was evaporated under reduced pressure in a rotary evaporator to obtain a solid residue which was then subjected to fractionation using the modified Kupchan partitioning method into n-hexane, CCl4, CHCl3 and aqueous soluble fractions. The n-hexane soluble fraction was chromatographed over sephadex (LH-20) and the column was eluted with n-hexane: CH2Cl2:MeOH (2:5:1) followed by CH2Cl2:MeOH (9:1) and MeOH (100%) in order to increase the polarities. The column fractions were then concentrated and subjected to thin layer chromatography screening and the fractions with a satisfactory resolution of compounds were rechromatographed over silica gel to isolate the pure compounds.Results:Four new furanoid diterpenes of clerodane types, Crispene A, B, C, and D (1–4), including one known furanoid diterpene glucoside, borapetoside E (5), were isolated from the stems of T. crispa. The structures of these compounds were elucidated by means of extensive spectroscopic analysis and by comparison of their spectral data with closely related compounds.Conclusion:We have reported four new furanoid diterpenes of clerodane types, including one known furanoid diterpene glucoside. This is the first report of any clerodane diterpene having olefinic bond between C-6 and C-7.SUMMARY Crispene A, B, C, and D, four new furanoid diterpenes of clerodane types from Tinospora crispaCrispene C, an unusual furanoid diterpene with olifinic bond between C-6 and C-7First report of Crispene D as a free aglycone, though it was earlier reported as an enzymatic hydrolysis product. Abbreviation used: TLC: Thin layer chromatography, NMR: Nuclear magnetic resonance, COSY: Correlation spectroscopy, NOE: Nuclear overhauser effect, HPLC: High-performance liquid chromatography, ESI-MS: Electrospray ionization mass spectroscopy

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...

Uniaxially aligned electrospun cellulose acetate nanofibers for thin layer chromatographic screening of hydroquinone and retinoic acid adulterated in cosmetics

Uniaxially aligned cellulose acetate (CA) nanofibers were successfully fabricated by electrospinning and applied to use as stationary phase for thin layer chromatography. The control of alignment was achieved by using a drum collector rotating at a high speed of 6000rpm. Spin time of 6h was used to produce the fiber thickness of about 10μm which was adequate for good separation. Without any chemical modification after the electrospinning process, CA nanofibers could be readily devised for screening hydroquinone (HQ) and retinoic acid (RA) adulterated in cosmetics using the mobile phase consisting of 65:35:2.5 methanol/water/acetic acid. It was found that the separation run on the aligned nanofibers over a distance of 5cm took less than 15min which was two to three times faster than that on the non-aligned ones. On the aligned nanofibers, the masses of HQ and RA which could be visualized were 10 and 25ng, respectively, which were two times lower than those on the non-aligned CA fibers and five times lower than those on conventional silica plates due to the appearance of darker and sharper of spots on the aligned nanofibers. Furthermore, the proposed method efficiently resolved HQ from RA and ingredients commonly found in cosmetic creams. Due to the satisfactory analytical performance, facile and inexpensive production process, uniaxially aligned electrospun CA nanofibers are promising alternative media for planar chromatography.

High-performance thin-layer chromatography screening of multi class antibiotics in animal food by bioluminescent bioautography and electrospray ionization mass spectrometry

The world-wide usage and partly abuse of veterinary antibiotics resulted in a pressing need to control residues in animal-derived foods. Large-scale screening for residues of antibiotics is typically performed by microbial agar diffusion tests. This work employing high-performance thin-layer chromatography (HPTLC) combined with bioautography and electrospray ionization mass spectrometry introduces a rapid and efficient method for a multi-class screening of antibiotic residues. The viability of the bioluminescent bacterium Aliivibrio fischeri to the studied antibiotics (16 species of 5 groups) was optimized on amino plates, enabling detection sensitivity down to the strictest maximum residue limits. The HPTLC method was developed not to separate the individual antibiotics, but for cleanup of sample extracts. The studied antibiotics either remained at the start zones (tetracyclines, aminoglycosides, fluoroquinolones, and macrolides) or migrated into the front (amphenicols), while interfering co-extracted matrix compounds were dispersed at hRf 20–80. Only after a few hours, the multi-sample plate image clearly revealed the presence or absence of antibiotic residues. Moreover, molecular information as to the suspected findings was rapidly achieved by HPTLC-mass spectrometry. Showing remarkable sensitivity and matrix-tolerance, the established method was successfully applied to milk and kidney samples.

The need for vigilance: False-negative screening for adenylosuccinate lyase deficiency caused by deribosylation of urinary biomarkers

ObjectivesAdenylosuccinate lyase deficiency (dADSL) is a rare inherited metabolic disorder. Biochemical diagnosis of the disease is based on the determination of enormously elevated urinary levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) and succinyladenosine (SAdo). We report a case of false negative screening for dADSL caused by deribosylation of the urinary biomarkers SAICA-riboside and SAdo. Design and methodsA thin-layer chromatography (TLC) method with Pauly reagent detection of SAICA-riboside was used as a screening method. High-performance liquid chromatography with diode-array detection (HPLC-DAD) and LC–MS/MS methods were used for the identification and quantitative determination of SAICA-riboside, SAdo, succinylaminoimidazole carboxamide (SAICA) and succinyladenine (SA). ResultsFollowing a negative TLC screening in a known case of dADSL, we analyzed urine using HPLC-DAD. The concentration of SAICA-riboside was 2.7mmol/mol creatinine (below the TLC detection limit), and we detected the two abnormal metabolites identified by LC–MS/MS as SAICA and SA. We showed that SAICA and SA were produced by deribosylation of SAICA-riboside and SAdo in the patient's urine. Studies performed by monitoring the production of SAICA and SA after the addition of SAICA-riboside and SAdo to the patient's urine and to urine samples from patients with urinary tract infections suggested that deribosylation is facilitated by bacterial enzymes. ConclusionsScreening methods for the diagnosis of dADSL may be falsely negative due to bacteria-mediated deribosylation of SAICA-riboside and SAdo. HPLC-DAD or LC–MS/MS analyses allowing for simultaneous detection of SAICA-riboside, SAdo and their deribosylation products SAICA and SA should be preferentially used for the diagnosis of dADSL in urine.

TLC screening of thraustochytrid strains for squalene production

Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as “+” (high), 29 as “±” (medium), and 109 as “−” (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L−1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with “+” squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with “±” and “−” squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene.

TLC and TLC-MS screening of ursolic, oleanolic and betulinic acids in plant extracts

Separation of three triterpenic acids (ursolic, oleanolic and betulinic acid) was achieved on different thin-layer chromatography (TLC) (silica gel 60) and high-performance thin-layer chromatography (HPTLC) sorbents (silica gel 60, C2 RP and C18 RP) using several developing solvents, based on the non-polar diluent n-hexane, and ester (methyl acetate, ethyl acetate, ethyl propionate) as selector. Anisaldehyde and molybdophosphoric acid detection reagents were used. Finally, a simple method on a C18 RP HPTLC plate was developed using n-hexane-ethyl acetate (5:1 v/v) as a developing solvent in a horizontal developing chamber. The method was used for the screening of ursolic, oleanolic and betulinic acids in different vegetable extracts. Other plant triterpenoids (lupeol, α-amyrin, β-amyrin, cycloartenol, lupenone, friedelin, lupeol acetate, cycloartenol acetate) and phytosterols (β-sitosterol, stigmasterol) did not interfere. TLC-MS was used as a tool for the additional confirmation of the presence of ursoli...

TLC and HPLC screening ofp-coumaric acid,trans-resveratrol, and pterostilbene in bacterial cultures, food supplements, and wine

A thin-layer chromatographic (TLC) method for fast screening of trans-resveratrol, pterostilbene, and p-coumaric acid in samples of recombinant bacterial cultures, food supplements, and wine was developed. The separation was performed on high-performance thin-layer chromatography (HPTLC) silica gel 60 plates using n-hexane-ethyl acetate-formic acid (20:19:1, v/v) as developing solvent in tank configuration of horizontal developing chamber, in which better resolution between trans-resveratrol and p-coumaric acid than in sandwich configuration of the same chamber or in automatic developing chamber (ADC) was obtained. Compounds were detected before and after post-chromatographic derivatization (three detection reagents) by image analyzing system (at 366 nm or white light) and by densitometer (absorption-reflectance and fluorescence mode). The lowest densitometric limits of detection (LOD) 2 ng for trans-resveratrol (303 nm), 5 ng for pterostilbene (303 nm), and 15 ng for p-coumaric acid (286 nm) were found b...

Determination of Cefacetrile and Cefuroxime Residues in Milk by Thin-Layer Chromatography

A simple thin-layer chromatography screening method was developed for the determination of two cephalosporins (cefacetrile and cefuroxime) in milk. Only two developments of TLC plates with concentrating zones are required: pre-development with hexane, as a clean-up procedure to remove lipids from milk samples, and a proper development with methanol-toluene-ethyl acetate-98% formic acid (5:20:65:10) phase. The video camera was used to detect spots on chromatograms. The recoveries of both antibiotics in milk were calculated over five days from the preparation of the samples. The best results, obtained on the second day of the experiment, were 97.66% for cefacetrile and 86.13% for cefuroxime.

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C 18 RP) thin-layer chromatography (TLC) and C 18 RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C 18 RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C 18 RP-TLC but not on the C 18 RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C 18 RP be performed before HPLC analysis.

A functional-based approach to the retention in thin layer chromatographic screening systems

A QSRR approach, based on functional group counts, useful for prediction of the retention of any small organic solute with small molecule in seven TLC screening systems is presented. The equations are based on 154 possible substituents as the only molecular descriptors involved. The model optimization was performed by uninformative variable elimination-partial least squares (UVE-PLS), reducing the variables involved. The final equations, with 7–32 variables, explain 32–60% of overall explained variance and their predictive ability ( Q 2 ) includes in range 25–54% (LOO crossvalidation) and 12–43% (external validation). Their simplicity allows to computation without any complicated software.

Quick prediction of the retention of solutes in 13 thin layer chromatographic screening systems on silica gel by classification and regression trees

The use of classification and regression trees (CART) was studied in a quantitative structure-retention relationship (QSRR) context to predict the retention in 13 thin layer chromatographic screening systems on a silica gel, where large datasets of interlaboratory determined retention are available. The response (dependent variable) was the rate mobility (RM) factor, while a set of atomic contributions and functional substituent counts was used as an explanatory dataset. The trees were investigated against optimal complexity (number of the leaves) by external validation and internal crossvalidation. Their predictive performance is slightly lower than full atomic contribution model, but the main advantage is the simplicity. The retention prediction with the proposed trees can be done without computer or even pocket calculator.

Antimicrobial activities of Garcinia kola seeds extracts on dental caries-causing microorganisms

Garcinia kola Heckel seeds (Family Guttiferae) also known as African wonder nut or bitter cola find use in folkloric medicine in Southern Nigeria for the treatment of tooth ache and prevention of dental caries [1]. We have examined the effect of the crude ethanolic extract and chromatographic fractions of this Nigeria edible seed against clinical isolates of dental caries-causing microorganisms-Streptococcus mutans and Streptococcus viridans. The cup plate method using blood agar as well as broth dilution methods was employed in the antimicrobial assay. Of the six pooled vacuum liquid chromatographic (VLC) fractions examined, fraction N (hexane: ethylacetate 70: 30) exhibited the highest activity with MIC's of 1.50mg ml-1 and 0.33mg ml-1 against S.mutans and S.viridans respectively. Corresponding MIC's for gentamicin reference standard was found to be 0.04µg ml-1 and 0.13µg ml-1. Thin-layer chromatographic screening showed that the major constituents of Fraction N are flavonoid related compounds which also showed free radical scavenging activity when sprayed with 2,2 diphenyl-1-picrylhydrazil (DPPH) radical.

Prediction of the retention in thin layer chromatography screening systems by atomic contributions

A novel atomic-contribution system for predicting of R M values is presented and validated on 13 thin layer chromatography screening systems on silica gel, where the large experimental datasets (198–761 R M values) are available. The R M is predicted with error less than 0.5 in majority of solutes (besides several outliers), which corresponds to difference in R F equal to 0.28 in the worst case. The system was validated by dividing the data into training and validation datasets, proving its accuracy. The main reason of larger errors in outliers are: large conjugated heterocycles, quarternary ammonium cations, large amount of polar atoms or very simple but unique molecules. The calculations are very easy and can be performed on free software or even manually. The presented method can be used in the retention prediction of new solutes in existing chromatographic screening systems.

Field Performance of a Thin-Layer Chromatography Assay for Detection of Nevirapine in Umbilical Cord Blood

Purpose: Although cord blood surveillance can measure the effectiveness of nevirapine (NVP)-based programs for the prevention of mother-to-child HIV transmission (PMTCT), it requires the ability to detect nevirapine in plasma. At present, the only validated method is high-performance liquid chromatography (HPLC), a technique poorly suited for most resource-constrained settings. Method: We evaluated the field performance for a simple and inexpensive thin-layer chromatography (TLC) assay for NVP detection. We developed a conditional probability model to compare 2 testing algorithms: HPLC alone, and TLC screening followed by HPLC confirmation of negative results. Results: When compared to HPLC, sensitivity of TLC was 0.67 (95% confidence interval [CI] 0.49–0.84) and specificity was 0.84 (95% CI 0.69–0.95). In this sample — where overall NVP coverage was 49% — positive predictive value was 0.80 and negative predictive value was 0.72. At baseline with population NVP coverage of 33%, cost per specimen was lower in the TLC-HPLC testing algorithm ($40 vs. $50), and the proportion of false results was acceptable (11%). As population NVP coverage increased, cost-efficiency improved and error rate dropped substantially. Conclusion: TLC is reasonably sensitive and specific for NVP detection. A 2-step testing algorithm incorporating TLC and HPLC provides cost-efficiency at little expense to test performance.

Proficiency Testing as a Tool to Assess the Performance of Visual TLC Quantitation Estimates

The use of rapid and inexpensive nonlaboratory-based screening tests for drug quality assessments is recommended as a component of a drug quality assurance program in poor resource settings. We have established routine Minilab test procedures to screen product quality and a proficiency testing program to determine the competency of the inspectors and reliability of results. Samples for the proficiency testing were prepared by pulverizing a standard reference tablet of the appropriate drug and making serial dilutions with starch to obtain concentrations of 0, 40, and 100%. The samples, which were labeled only with the drug name and an identifying letter, were given to inspectors for quality screening using Minilab procedures. In round 1 of the proficiency test, only 3 of 28 substandard samples were correctly identified. Round 2 of the proficiency test, which was administered after a performance qualification test for the analytical method, showed much improvement: 19 of 27 substandard drugs were correctly identified, while 5 out of 9 inspectors made the correct inference on the quality of 45 samples. However, in both rounds, 2 inspectors failed to identify substandard samples, indicating that their technical competencies need to be improved for the reliability of the results. Although the thin-layer chromatography screening methods provide a rapid means for drug quality assessment, they need to be put in the hands of competent users. The inclusion of a proficiency test in the screening program provides a measure of determining competency of the personnel and reliability of the results.

Field Performance of a Thin-Layer Chromatography Assay for Detection of Nevirapine in Umbilical Cord Blood

Purpose: Although cord blood surveillance can measure the effectiveness of nevirapine (NVP)-based programs for the prevention of mother-to-child HIV transmission (PMTCT), it requires the ability to detect nevirapine in plasma. At present, the only validated method is high-performance liquid chromatography (HPLC), a technique poorly suited for most resource-constrained settings. Method: We evaluated the field performance for a simple and inexpensive thin-layer chromatography (TLC) assay for NVP detection. We developed a conditional probability model to compare 2 testing algorithms: HPLC alone, and TLC screening followed by HPLC confirmation of negative results. Results: When compared to HPLC, sensitivity of TLC was 0.67 (95% confidence interval [CI] 0.49–0.84) and specificity was 0.84 (95% CI 0.69–0.95). In this sample — where overall NVP coverage was 49% — positive predictive value was 0.80 and negative predictive value was 0.72. At baseline with population NVP coverage of 33%, cost per specimen was lower in the TLC-HPLC testing algorithm ($40 vs. $50), and the proportion of false results was acceptable (11%). As population NVP coverage increased, cost-efficiency improved and error rate dropped substantially. Conclusion: TLC is reasonably sensitive and specific for NVP detection. A 2-step testing algorithm incorporating TLC and HPLC provides cost-efficiency at little expense to test performance.

Investigation of phenolic acids in yacon (Smallanthus sonchifolius) leaves and tubers

Thin-layer chromatographic (TLC) screening of crude extracts of dried leaves and tubers of yacon (Smallanthus sonchifolius, Asteraceae) and products of acid hydrolysis of tubers on the silica gel HPTLC plates using the developing solvents ethyl acetate–formic acid–water (85:10:15, v/v/v) and n-hexane–ethyl acetate–formic acid (20:19:1, v/v/v) proved the presence of chlorogenic, caffeic and ferulic acid. These phenolic acids were isolated from the crude extract of yacon leaves by preparative TLC, and identified after elution by HPLC/MS, as well as by direct injection of the crude extract into the HPLC/MS system. Acid hydrolysis of tubers released the increased amount of phenolic acids (e.g. caffeic acid and ferulic acid), flavonoid quercetin and an unidentified flavonoid, which was detected by TLC analysis.Ferulic acid, isomers of dicaffeoylquinic acid and still an unidentified derivative of chlorogenic acid (Mr=562) as constituents of yacon leaves and ferulic acid as constituent of yacon tubers are reported here for the first time. These acids gave significant contribution to the radical scavenging activity detected directly on the TLC plate sprayed with 1,1-diphenyl-2-picrylhydrazyl (DPPH).

Mycotoxins in South African traditionally brewed beers

Traditionally brewed alcoholic beverages are regularly consumed by most ethnic black South Africans. Maize and barley, both of which are used for producing locally brewed alcoholic beer, are frequently contaminated by mycotoxin-producing moulds. The study was undertaken to investigate whether these toxins are present in raw grains and the traditional beers imbibed by the local black African population. It was established that the raw ingredients (sorghum, sorghum malt grains, maize grits), commercially produced traditional beers (Utshwala and Utshwala special) and home-brewed beers (Umqombotha, Isiqatha, Imfulamfula) were contaminated by bacteria and fungi (both yeasts and moulds). The contaminating moulds were isolated and identified. The contaminated samples were analysed for aflatoxins B1, B2, G1 and G2, zearalenone, citrinin, deoxynivalenol, and ochratoxin A using a multi-mycotoxin thin-layer chromatography screening method and the toxins were quantified by high-performance liquid chromatography. Grain samples were infected by Aspergillus flavus, A. alliaceus, A. clavatus, Penicillium spp., Rhizopus spp. and Mucor spp. Sorghum malt grain samples contained the toxin zearalenone. No mycotoxin-producing fungi were present in the fermented beers but two of six commercial beer samples contained aflatoxins (200 and 400 μ gl-1) and 45% (13 of 29) of the home-brewed beers had zearalenone (range 2.6–426 μgl-1) and/or ochratoxin A (3-2340 μgl-1).

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.