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JPC - Journal of Planar Chromatography - Modern TLC
Authors: Yuping Wang, Dongyuan Wang, Jie Wang, Zhili Xiong, Hongxia Zhang, Gaohong She, Jian Li, and Shengtao Xiao

A new AMD instrument made in our laboratory is described in this paper. The main advantages of the instrument are very low cost both of construction and in use. Compared with ascending development in conventional instruments, a laboratory-made horizontal sandwich chamber is used for development, and, with the help of a series of special accessories, no obvious mobile phase remains in the distributor after each step; this saves a large amount of solvent. All the components of the instrument are easy to obtain, so the average worker in a chemical laboratory could construct all the instrument except the control unit. An application of the instrument is described; the results obtained were satisfactory.

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Conditions have been established for separation of eight ginsenosides by HPTLC with automated multiple development. The ginsenosides were separated on silica gel 60F 254 with methanol-chloroform mobile phases. Two gradient programs and two different values of the increment in the development distance were compared. Factors affecting extraction of the ginsenosides from leaves were investigated. The results show that AMD is suitable for screening of ginsenosides in extracts from ginseng.

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Glycosylceramides (GlyCers) are precursors of ceramides (Cers) that are major components of the outer layer of human skin, the stratum corneum. A Cer deficiency is associated with skin diseases such as psoriasis and atopic dermatitis and can be treated with Cer-containing semisolid formulations. Plants may serve as alternative sources for expensive semisynthetic Cer production. Since the GlyCer contents of plants vary widely, there is a need to develop a rapid, simple, selective, and precise method for GlyCer quantification in plants. In the present study, an effective and validated automated multiple development‒high-performance thin-layer chromatography (AMD‒HPTLC) method has been developed for GlyCer quantification in 9 different plant materials. An 18-step gradient elution program (n-hexane, chloroform, ethyl acetate, methanol) led to a clear separation of bands from complex matrices and allowed densitometric analysis for quantification purposes. Apple pomace and wheat germs yielded 26.8 and 39.5 mg of GlyCer per 100 g plant material, respectively, while the yields of coffee grounds were below the limit of quantification. The GlyCer contents of the seeds of six Fabaceae species, namely, Albizia grandibracteata, Albizia gummifera, Albizia lebbeck, Albizia schimperiana, Acacia etbaica, and Robinia pseudoacacia, ranged from 9.4 to 23.1 mg per 100 g plant material. GlyCers were separated by preparative thin-layer chromatography (TLC) and identified by offline high-performance liquid chromatography–mass spectrometry (HPLC–MS). Intact GlyCers were detected in the Fabaceae species for the first time. A simple AMD–HPTLC screening and quantification technique for GlyCers was developed, which may serve as a tool in searching plant GlyCers for a possible “phyto”-Cer production.

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A first attempt at the separation of fructo-oligosaccharides (FOS) and inulin mixtures is presented. Preliminary results obtained by automated multiple development (AMD) of diol layers with an acetonitrile-acetone-water polarity gradient are reported. The method is very promising and highly suitable for simple, direct analysis of complex mixtures of fructo-oligosaccharides (FOS) and inulin in samples of natural origin.

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For the purpose of qualitative and quantitative analysis of flavonoids from eight species of Indocalamus, Indocalamus latifolius (Keng) McClure, I. herklotsii McClure, I. decorus Q. H. Dai, I. barbatus McClure, I. sinicus (Hance) Nakai, I. longiauritus Hand.—Mazz, I. guangdongensis H. R. Zhao et Y. L. Yang, I. pedalis (Keng) Keng f., a simple, efficient, accurate and low-cost method was constructed. Six flavonoids, vitexin, isovitexin, orientin, isoorientin, quercetin and tricin, were selected as marker flavonoids. The multistage development was performed in Automated Multiple Development (AMD2) and solvents in different ratios were used as mobile phase. The developed plates were scanned by TLC Scanner3. The components separated well and R F values of the six marker flavonoids were 0.22, 0.31, 0.38, 0.45, 0.57, and 0.88, respectively. The method was validated for precision, accuracy, selectivity and repeatability, which were expressed as coefficient of variation (CV [%]). The concentrations and kinds of flavonoids were significantly different in these six species of Indocalamus. The concentration of total flavonoids in the dry leaves was in the range of 0.11–0.40%, and the decreasing order of concentration was I. barbatus, I. sinicus, I. longiauritus, I. guangdongensis, I. decorus, I. pedalis, I. latifolius, I. herklotsii; the concentration of total flavonoids in I. barbatus was the maximum among these eight species of Indocalamus, and they would be more useful in extracting natural bamboo flavonoids.

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The intestinal microflora Bifidobacteria has been adapted to utilize, as a source of energy, complex carbohydrates that escape hydrolysis by human digestive enzymes, for example fructo-oligosaccharides (FOS). The ability of intestinal microflora to utilize carbohydrates in complex mixtures is of particular interest, even though few data have been reported. This paper presents the results obtained using an innovative analytical approach based on instrumental HPTLC-AMD (automated multiple development) to investigate the metabolic behavior of Bifidobacterium adolescentis MB 239 as a case study. Raffinose, FOS (sucrose, 1-kestose, nystose, fructosyl-nystose), lactose, and their monomeric moieties glucose, galactose, and fructose were simultaneously present as carbon sources in the solution to be fermented by the bacterium. The method proposed has enabled quantitative monitoring of sugar concentrations during the entire time-course of the processes. The complex sugar mixtures were separated by use of acetonitrile-acetone-water mobile phases on diol layers derivatized with 4-aminobenzoic acid.

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A new stability-indicating HPTLC assay has been established for leuprolide acetate. Silica gel 60F 254 was used as stationary phase. Samples and reference standards were applied to the same precleaned and activated HPTLC plates and chromatography was performed in an automated multiple development chamber (AMD2) with five-step isocratic incremental multiple development with 1 min drying time between each step and layer conditioning with the mobile phase before each development. The mobile phase, ethyl acetate-methanol-25% aqueous ammonia, 60 + 30 + 10, resulted in dense, compact zones for the analyte and related substances. Leuprolide acetate was subjected to acidic, basic, and oxidative degradation. The peaks of the degradation products were well resolved from the main peak with significantly different migration distances. Densitometric evaluation was performed at λ = 280 nm. The calibration function of the analyte was linear in the range 107–422 ng and the correlation coefficient was 0.9914. The limits of detection and quantitation were 25 and 107 ng, respectively. The recovery and relative standard deviation obtained from between-days analysis were 97.06–102.0% and 1.34–2.90%, respectively. The method was shown to be suitable for use as a stability-indicating analytical procedure for assay of leuprolide acetate. The method enables high throughput and is easy to perform.

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Sunscreen products are meant to protect people from damaging UVA and UVB radiation. However, in some formulations the UV filters they contain can react and form many photodegradation products. Their potential toxicity has not yet been investigated. In this study effect-specific analysis has been used to evaluate the bioactivity of photodegradation products in sunscreens. HPTLC-bioluminescence coupling with the luminescent bacterium Vibrio fischeri was used. Problems in method development were because of the sensitivity of the bacteria and the wettability of HPTLC plates. A separation system using HPTLC LiChrospher plates and automated multiple development (AMD) with tert -butyl methyl ether- n -hexane was chosen. Detection was by UV in addition to Vibrio fischeri . First, biodetection was performed on pure standard solutions of the UV filters. UV filters with molecular weight >400 had no bioactivity; these included all newer UV filters (not in use before 1998). Five commercially available sunscreens with different UV filter combinations were then analyzed. They were irradiated on microscope slides with artificial light and natural sunlight and on the skin with natural sunlight. For extraction, a mixture of ethanol and acetone was used. The bioactivity which can be indicative of (cyto)toxic effects of the photodegradation products was higher than that of the corresponding UV filter. In comparison of HPLC-DAD and LC-MS with detection with Vibrio fischeri , a high signal in chemical-physical detection did not always correspond to high bioactivity, and vice versa. It was shown that biodetection with Vibrio fischeri was a suitable method for examination of photodegradation products in sunscreens, making this bioassay a useful addition to conventional analytical methods.

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