Identification and quantification of glucose degradation products in heat-sterilized glucose solutions for parenteral use by thin-layer chromatography

During heat sterilization of glucose solutions, a variety of glucose degradation products (GDPs) may be formed. GDPs can cause cytotoxic effects after parenteral administration of these solutions. The aim of the current study therefore was to develop a simple and quick high-performance thin-layer chromatography (HPTLC) method by which the major GDPs can be identified and (summarily) quantified in glucose solutions for parenteral administration. All GDPs were derivatized with o-phenylenediamine (OPD). The resulting GDP derivatives (quinoxalines) were applied to an HPTLC plate. After 20 minutes of chamber saturation with the solvent, the HPTLC plate was developed in a mixture of 1,4-dioxane-toluene-glacial acetic acid (49:49:2, v/v/v), treated with thymol-sulfuric acid spray reagent, and heated at 130°C for 10 minutes. Finally, the GDPs were quantified by using a TLC scanner. For validation, the identities of the quinoxaline derivatives were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glyoxal (GO)/methylglyoxal (MGO) and 3-deoxyglucosone (3-DG)/3-deoxygalactosone (3-DGal) could be identified and quantified in pairs, glucosone (2-KDG), 5-hydroxymethylfurfural (5-HMF), and 3,4-dideoxyglucosone-3-ene (3,4-DGE) each individually. For 2-KDG, the linearity of the method was demonstrated in the range of 1–50 μg/mL, for 5-HMF and 3,4-DGE 1–75 μg/mL, for GO/MGO 2–150 μg/mL, and for 3-DG/3-DGal 10–150 μg/mL. All GDPs achieved a limit of detection (LOD) of 2 μg/mL or less and a limit of quantification (LOQ) of 10 μg/mL or less. R2 was 0.982 for 3.4-DGE, 0.997 for 5-HMF, and 0.999 for 2-KDG, 3-DG/3-DGal, and GO/MGO. The intraday precision was between 0.4 and 14.2% and the accuracy, reported as % recovery, between 86.4 and 112.7%. The proposed HPTLC method appears to be an inexpensive, fast, and sufficiently sensitive approach for routine quantitative analysis of GDPs in heat-sterilized glucose solutions.

Modified method for qualitative thyroestatic substances determination in muscle tissue and urine of food producing animals and the application of Planar Thin Layer Chromatography 2D and 4x4D horizontal development.

Substances with thyreostatic action, also known as anti-hormones [AHs], are extracted by methanol in the presence of an internal standard. Percolation of the methanol extract through a mercurated adsorption mini-column allows a selective and reproducible extraction of the AHs by creating a specific complex with Hg ions. After elution of the Ahs with an acidic sodium chloride solution, the pH of the eluate is adjusted with buffer to 8,0 and allowed to react with NBDCl, a reagent which reacts only with thiols and amines. After reaction, the AH-NBD complexes formed are extracted in acid medium using TBME and after it has been dried down and reconstituted with TBME to the required volume, the ether fraction is spotted on to a HPTLC plate for horizontal development using sophisticated instrumentation. A cysteine water solution reacts with the AH-NBD complexes which are not fluorescent and they are converted into cystein-NBD complexes which are strongly fluorescent at 366 nm. This method, based on Verbeke R, de Brabander HF, 1984 method, has been modified in some steps concerning the mini-column preparation, the elution, the buffer replacement and chromatography step. The limit of detection no the plate, has been determined to be lower than lng by direct UV observation at 366nm, while the lower limit of detection achieved in the sample was 25 μg/kg.

Fingerprint Study of Curcuma xanthorrhiza Rhizome by High Performance Thin Layer Chromatography (HPTLC)

The rhizome of Curcuma xanthorrhiza Roxb is intensively used in Indonesia as traditional medicine. It is widely used for hepatoprotective and anti inflammatory activities. To ensure the quality of its extract, we have studied the fingerprint or phytochemical analysis. This research was aimed to produce a chromatogram profile of the rhizome by HPTLC. The HPTLC fingerprint chromatogram of C. xanthorrhiza rhizome was performed using HPTLC plate of silica gel 60 F254 as the stationary phase and chloroform-methanol (97:3) as the mobile phase. Spot detection was carried out by TLC photo documentary system at 254 and 366 nm and TLC scanner at 427 nm. The developed method was validated according to ICH guidelines by determination of specificity and precision. We found that the specifity and precission of the method were met the acceptance criteria. In conclusion, the developed method is valid and could be used for quality control and standardization of herbal medicine containing C. xanthorrhiza rhizome.

Fingerprint Study of Curcuma xanthorrhiza Rhizome by High Performance Thin Layer Chromatography (HPTLC)

The rhizome of Curcuma xanthorrhiza Roxb is intensively used in Indonesia as traditional medicine. It is widely used for hepatoprotective and anti inflammatory activities. To ensure the quality of its extract, we have studied the fingerprint or phytochemical analysis. This research was aimed to produce a chromatogram profile of the rhizome by HPTLC. The HPTLC fingerprint chromatogram of C. xanthorrhiza rhizome was performed using HPTLC plate of silica gel 60 F254 as the stationary phase and chloroform-methanol (97:3) as the mobile phase. Spot detection was carried out by TLC photo documentary system at 254 and 366 nm and TLC scanner at 427 nm. The developed method was validated according to ICH guidelines by determination of specificity and precision. We found that the specifity and precission of the method were met the acceptance criteria. In conclusion, the developed method is valid and could be used for quality control and standardization of herbal medicine containing C. xanthorrhiza rhizome.

QUANTIFICATION OF TWO MARKER COMPOUNDS IN GMELINA ARBOREA ROXB. STEM BARK USING VALIDATED THIN LAYER CHROMATOGRAPHIC-DENSITOMETRIC METHOD

Gmelina arborea Roxb. (Verbenaceae) is known as Gambhari in Ayurvedic System of medicine and being used as immuno-modulator since ancient time. Plant sterols such as β-sitosterol and Lupeol are reported to have such activity. A simple, accurate, precise, rapid and reproducible TLC densitometric method was developed for simultaneous estimation of β-sitosterol and Lupeol in plant stem bark. Compounds were separated on silica gel 60 F254 HPTLC plate using Toluene: Ethyl acetate: Formic acid (8.8:1.3:0.1, v/v/v) using Anisaldehyde-Sulphuric acid as the derivatizing reagent. TLC Analysis was performed at 520 nm. Rf value of β-sitosterol and Lupeol were found to be 0.34±0.02 and 0.46±0.01 respectively. Linearity range was 150-350 ng/spot and 300-700 ng/spot with correlation coefficient 0.998 and 0.999 for β -sitosterol and Lupeol, respectively. Method was validated as per ICH guideline. The accuracy was found to be 100.55-102.86% and 99.40-101.56% for β-sitosterol and Lupeol, respectively by recovery studies. The amount of β-sitosterol and Lupeol was found to be 4.4 mg % and 3.6 mg %. This method can be employed for routine quality control analysis of β-sitosterol and Lupeol in stem bark of Gmelina arborea Roxb.

Extraction of Artemisinin, an Active Antimalarial Phytopharmaceutical from Dried Leaves of Artemisia annua L., Using Microwaves and a Validated HPTLC-Visible Method for Its Quantitative Determination

A simple, rapid, precise, and accurate high-performance thin-layer chromatographic method coupled with visible densitometric detection of artemisinin is developed and validated. Samples of the dried Artemisia annua leaves were extracted via microwaves using different solvents. This method shows the advantage of shorter extraction time of artemisinin from leaves under the influence of electromagnetic radiations. Results obtained from microwave-assisted extraction (MAE) were compared with hot soxhlet extraction. Chromatographic separation of artemisinin from plant extract was performed over silica gel 60 F254 HPTLC plate using n-hexane : ethyl acetate as mobile phase in the ratio of 75 : 25, v/v. The plate was developed at room temperature 25 ± 2.0°C. Artemisinin separation over thin-layer plate was visualized after postchromatographic derivatization with anisaldehyde-sulphuric acid reagent. HPTLC plate was scanned in a CAMAG’s TLC scanner 3 at 540 nm. Artemisinin responses were found to be linear over a range of 400–2800 ng spot−1 with a correlation coefficient 0.99754. Limits of detection and quantification were 40 and 80 ng spot−1, respectively. The HPTLC method was validated in terms of system suitability, precision, accuracy, sensitivity (LOD and LOQ), and robustness. Additionally, calculation of plate efficiency and flow constant were included as components of validation. Extracts prepared from different parts of the plant (leaves, branches, main stem, and roots) were analyzed for artemisinin content, in which, artemisinin content was found higher in the leaf extract with respect to branches and main stem extracts; however, no artemisinin was detected in root extract. The developed HPTLC-visible method of artemisinin determination will be very useful for pharmaceutical industries, which are involved in monitoring of artemisinin content during different growth stages (in vitro and in vivo) of A. annua for qualitative and quantitative assessment of final produce prior to commercial-scale processing for assessment of cost-benefit ratio.

Determination of Organophosphorus and Carbamate Insecticides in Fresh Fruits and Vegetables by High-Performance Thin-Layer Chromatography-Multienzyme Inhibition Assay

HPTLC-enzyme inhibition assay was applied to different fruit and vegetable samples after individual spiking with organophosphate and carbamate pesticides at their maximum residue limits documented by the European Commission. Samples were extracted according to the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, including cleanup by primary secondary amine sorbent. Additional cleanup was performed on the HPTLC plate by a prechromatographic step to separate most coextracted matrix compounds from 20 different pesticides under study. With both rabbit liver esterase and cutinase from Fusarium solani pisi as enzyme sources, mean recoveries from apples, cucumbers, grapes, nectarines, plums, tomatoes, and lemons were in the ranges 86–109, 95–129, 96–114, and 90–111% for chlorpyrifos, paraoxon, parathion, and pirimicarb, respectively, with a mean RSD of 8.5% for all samples.

EVALUATION OF RE-USED HPTLC PLATE FOR QUALITATIVE AND QUANTITATIVE ANALYSIS

The eficiency chromatography of HPTLC plate is better than conventional TLC plate but it more expensive. Due to the higher price of HPTLC plate compare to conventional TLC plate, the application of ";re-used"; HPTLC plate has been tested. After being used for analyzing samples, the plates were developed again with the mobile phase, then dried for 60 min before using for the next measurement. The qualitative chromatography profile showed that ";re-used"; HPTLC plate has less contaminant than new HPTLC plate and the RSD of the precision quantitative testing of the plates reused three times showed a value > 2%. Keywords: reused, HPTLC, TLC, quantitative, qualitative