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5 figures.-- Supporting information available., The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface., Financial support for this project was provided by DGA-FEDER (E25_20R, N&SB), AES PI21/00036, CSIC 202180E076, ISCIII PI19/01007, DGA E25-20R, and ERC Advanced Grant CADENCE (grant No. ERC-2016-ADG-742684).

In the current work, computational image scanning densitometry technique was used instead of conventional and traditional instruments like IC, HPLC, GC and UV/visible spectroscopy for quantification of micro samples of amino acids (lysine hydrochloride, β-alanine, asparagine monohydrate, glutamic acid). The general reaction between locating reagent and amino acid is: This technique has accuracy at the parts per billion level and precision as indicated from percent recovery from sample analysis, which is quite good. Due to certain limits associated with conventional techniques, this technique may be presented as a progressive and important event in the history of evaluation at micro level. Keywords: amino acids, densitometry, micro level.

6 figures, 4 tables., This work is a review on HPTLC contribution to lipid analysis in complex matrices, in the period from 2010 to now, lapse of time where hyphenation with other techniques, especially Mass Spectrometry, has experienced an important growth. Scanning densitometry (SD)-UV/FL of separated lipids, as the core of detection and centerpiece for hyphenation, and its coupling with MS using soft ionization techniques (ESI, APCI, MALDI, DESI and others), are the central axis of this work. The occasional intercalation in this coupling of an on-plate biological assay for effect-direct analysis (EDA) of lipids, as well as the combination of SD with radio-densitometry (RD) using isotopically labeled-lipids are also covered topics. The described techniques make possible to develop strategies for obtaining qualitative and different levels of quantitative information, including untargeted lipids species. HPTLC of lipids has been used for: comparative purposes; fingerprinting; semi-quantitative determination, identification of species; or quantitative determination of a given individual lipid. Goals in molecular biology and biochemistry-related samples have been: preparative isolations; control of purity; verification of metabolic products; on-plate biological assays; profiles in cells; analysis of products from the cellular metabolism; measurement of enzymatic activities; monitoring lipid transport across membranes or at biological interfaces; or monitoring lipid dynamics., One of us (J.G.) thanks to Spanish Plan Nacional I + D+i (CTQ2016-76846R project). This work was supported by DGA-ESF [project 25_20R].

6 figures, 2 tables.-- Supplementary information available.-- © 2021. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/, Identification of 19 molecular species of globotriaosylceramides (Gb3) in extracts from a Fabry's plasma patient and a healthy control was performed by High-Performance Thin-Layer Chromatography (HPTLC)-densitometry and online coupling to Mass Spectrometry (MS). Separation was carried out on LiChrospher plates using Automated Multiple Development (AMD). Densitometry was performed on twin plates by combining detection in the visible at 550 nm, through previous on-plate orcinol derivatization, and by Ultraviolet 190 nm, using a non-impregnated plate. The latter was directly coupled to an ion-trap mass spectrometer through an automated elution-based interface. Gb3 molecular species, which were identified by HPTLC- Electrospray Mass Spectrometry (+)-MS and confirmed by MS/MS or HPTLC-Atmospheric Pressure Chemical Ionization Mass Spectrometry (+)-MS, are: five isoforms of saturated Gb3; seven isoforms of methylated Gb3; and seven species with two additional double bonds. Twelve of these species were previously reported as biomarkers of Fabry's lysosomal disorder using a Liquid Chromatography-MS-based method, and the other seven are structurally similar, closely related to them. Saturated Gb3 isoforms migrated on LiChrospher plate in one of the separated peaks corresponding to the migration zone of ceramide trihexosides standard. Instead, methylated and unsaturated Gb3 species co-migrated with sphingomyelin species. Ion intensity ESI-MS profiles show that saturated Gb3 species in Fabry's plasma were in higher concentration than in control sample. Before applying the Thin-Layer Chromatography (TLC)-MS interface on HPTLC separated peaks, its positioning precision was first studied using ceramide tri-hexosides as model compound. This provided information on Gb3 peak broadening and splitting during its migration., This work was supported by DGA-FEDER (E25_17R, N&SB). One of us (J.G.) thanks to Spanish Plan Nacional I+D+i (CTQ2016-76846-R project).

Hypericum perforatum L. is the most commonly used herb for treating depression. Due to the popularity of this botanical, there is a potential for economically driven adulteration of St. John's wort (SJW) products. The goal of this study was to investigate SJW ingredients suspected to be adulterated based on simple preliminary HPTLC tests. Commercial samples were analyzed by HPTLC following the United States Pharmacopeia (USP) monograph methodology, with additional visualization under white light. A number of these samples presented odd methanolic solution colors and unconventional HPTLC fingerprints, suggesting the presence of other species and/or extraneous polar additives. To achieve identification and separation of the polar additives, a new reversed-phase HPTLC method was developed. The adulterants were identified as synthetic dyes in the amounts of 0.51 to 1.36% by weight. Identities of the dyes were confirmed by scanning densitometry and HPTLC-MS. A modified USP method with additional detection mode permitted the identification of eight SJW samples adulterated with dyes and six others with flavonoid fingerprints different from those specified by USP from a total of 37 samples of dry extracts, finished products, and bulk raw herb. A decision flowchart is proposed to guide the detection of adulteration of SJW in a systematic fashion.

Autoantibody detection is a useful and obligatory tool for clinicians and researchers in diagnosing autoimmune diseases. Line blotting is a simple, sensitive, and flexible technique for fast semiquantitative detection of multiple antibodies. Line blotting enables the detection of antibodies on membrane strips coated with thin parallel lines of several purified, biochemically characterized antigens, which are fixed onto a synthetic support. Each strip can contain more than ten antigens, thus allowing simultaneous screening for multiple antibodies. Here, we describe the principle of line blotting and how the buffer composition can interfere with the results of autoantibody detection.

Background: The proposed HPTLC method combines features of the existing methods for (1) the detection of sibutramine and (2) for the detection of phosphodiesterase type 5 inhibitors and analogs. Objective: The method permits effective screening for the presence of nine adulterants in finished products, including tablets, capsules, and “instant coffee” powders. Methods: All products were prepared for analysis using the same simple procedure: ultrasound-assisted extraction in methanol for 30 min followed by centrifugation or filtration. Results: The retardation factor (RF) values of individual zones afford preliminary identification of potential adulterants. Scanning densitometry enables comparison of recorded UV spectra with those of known standard compounds and provides further structural information. Conclusions: The method was successfully applied to 12 commercial products. Of those, nine products tested positive for at least one undeclared component.

Background Anthocyanins are abundant secondary metabolites responsible for most blue to blue-black, and red to purple colors of various plant organs. In wheat grains, anthocyanins are accumulated in the pericarp and/or aleurone layer. Anthocyanin pigmented wheat grains can be processed into functional foods with potential health benefits due to the antioxidant properties of the anthocyanins. The grain anthocyanin content can be increased by pyramidizing the different genes responsible for the accumulation of anthocyanins in the different grain layers. Our objective was to develop a high-performance thin-layer chromatography (HPTLC) method that allows the determination of both the anthocyanin profile and the total pigment concentration. Thereby, selection of breeding lines with significantly higher grain anthocyanin content from purple pericarp × blue aleurone wheat crosses should become more efficient than selection based on only visual scoring of grain color and the unspecific determination of anthocyanin concentration by UV/Vis spectroscopy. Results A wide variability in the grain anthocyanin content was observed in breeding lines and check varieties. The highest concentration of anthocyanins was observed in deep purple (i.e. combination of the purple pericarp and blue aleurone genetics) grained breeding lines, followed by blue aleurone and purple pericarp genotypes. Determination of the total anthocyanin content was included into the chromatographic analysis, rendering an additional photometric analysis unnecessary. Ten target zones were identified in anthocyanin pigmented wheat grains; four of these zones were typically for blue aleurone types, five for purple pericarp types, and one (i.e. kuromanin glucoside) was characteristic for both. Chemometrics applied to the anthocyanin profile recorded by scanning densitometry revealed that peak heights and peak areas are highly correlated and that seven out of the ten target zones were responsible for about 90% of the total variation in the germplasm. Multivariate analysis of these seven target zones allowed not only a separation of the genetic material into purple, blue and deep purple grained genotypes, but also the identification of genotypes with a specific anthocyanin pattern. Thereby, the original classification by visual scoring was overruled in about one-third of the breeding lines. Conclusions The presented HPTLC method with à côté calibration allowed the profiling of the pigments and quantification of wheat grain anthocyanin content in a single analysis, replacing UV/Vis spectroscopy with subsequent HPLC analysis. Moreover, no sample preparation apart from extraction and filtration is required, and more than 15 samples can be evaluated in one analysis run, corresponding to several dozens of samples per day. Hence, the method fulfills the requirements for screening methods in early generations of a plant breeding program such as high-throughput, small sample size, high repeatability, fast determination, and reasonable costs per sample. Combined with multivariate statistical analysis, the anthocyanin pattern allowed the validation of the genetic background in the offspring of purple × blue wheat crosses and, therefore, the efficient selection of genotypes exhibiting both the cyanidin and delphinidin aglycon.

A high-performance thin-layer chromatographic method combined with a sample preparation procedure has been developed for simultaneous determination of sunset yellow and azorubine synthetic dyes in powders for jelly desserts containing a variety of other ingredients. Some different extraction solvents and sample preparation steps (solid phase extraction procedures on RP-18 and polyamide cartridges) were tested for the quantitative isolation and purification of dyes. The best recovery results were achieved by using methanol for extraction procedure followed by solid phase extraction on the polyamide cartridges for the purification step. For the quantitative evaluation of the chromatographic spots, visible slit-scanning densitometry and digital processing of images of chromatographic plates (using ImageDecipher-TLC software with pure color-green and blue and integrate color- grey channels selection) were compared. The best statistical parameters for the linear functions of the calibration were obtained using...

Introduction: We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). Methods: Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. Results: MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. Conclusions: A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs. Muscle Nerve, 2012

Total polyphenols, tannins, β-sitosterol, and bitterness values were determined in native and cultivated populations of Teucrium L. species from Croatia (T. arduini L., T. botrys L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.). Teucrium species have long been present in folk medicine for diverse medicinal uses, but little is known about polyphenols, phytosterols, and bitter principles. Spectrophotometrically determined contents of total polyphenols (TP) and tannins (T) significantly varied among investigated Teucrium species and were somewhat higher in native populations. The highest TP and T contents were measured in native specimens of T. montanum (TP: 13.68%; T: 3.48%). Scanning densitometry was used for measurements of β-sitosterol levels in plant samples ranged from 0.056% (T. montanum) to 0.129% (T. botrys), and it was not significantly different between native and cultivated specimens of the same plant species. Bitterness values were similar for native and cultivated samples of the same plant species; the highest was measured for T. montanum (15659). The present study suggests that Teucrium species growing in Croatia have potential for cultivation and might be a valuable source of natural bioactive compounds.

A double-development TLC method has been developed for simultaneous qualitative and quantitative analysis of hydrophilic and lipophilic constituents of Salvia miltiorrhiza (Danshen). The optimized mobile phases dichloromethane-ethyl acetate-formic acid 22:24:10 (ν/ν) and petroleum ether-ethyl acetate-cyclohexane 25:11:14 (ν/ν) were used for the double development on nano-silica gel 60F254 plates. Their characteristic TLC profiles were observed under UV light at 254 and 365 nm and the bands were then revealed by reaction with 5% H2SO4 in EtOH. Quantification of twelve compounds was achieved by densitometry at 260 or 290 nm, with reference at 400 nm. Linearity was quite good (R2 > 0.99) within the ranges tested. This method could be used for quality control of Danshen.

A sensitive and precise quantitative densitometric HPTLC method has been established for analysis of palmitoyl hexapeptide both as the bulk drug and in formulations,. Silica gel 60 F254 plates were used with toluene-ethanol 9:1 as mobile phase. This system gave compact spots for palmitoyl hexapeptide (RF 0.33 ± 0.02). Densitometric quantification was performed in absorbance mode at 221 nm and well resolved bands were obtained. Regression analysis of the calibration data revealed a good linear relationship (mean value of r2 = 0.99910 ± 0.003) between response and amount of palmitoyl hexapeptide in the range 10–30 ng per band. The mean slope and intercept were 114.5 ± 0.002 and 311.1 ± 1.32, respectively. The method was validated for precision, recovery, robustness, ruggedness, and specificity. The limits of detection and quantification were 3 and 9 ng per band, respectively. The method is selective and specific with potential application in cosmetics and pharmaceutical analysis.

The purpose of this paper is to report a new procedure for analysis of analytes in complex mixtures by combination of different modes of multidimensional planar chromatography (MDPC) on monolayer or multiphase plates. The procedure described for separation of complex mixtures of compounds is inexpensive and can be applied to routine analysis of analytes in samples of natural origin, e.g., in water or plant extracts, after preliminary clean-up and concentration, e.g., by solid-phase extraction (SPE). Application of multidimensional planar chromatography (MDPC) and modern fiber optical TLC densitometric scanners with DAD is especially useful for correct identification of components of difficult, complicated mixtures, e.g., pesticides in plant extracts (clofentezine in Herba Thymi).

Keywords: equine haemoglobins; α-chain variants; isoelectric focusing; quantitation; scanning densitometry

Summary Neutral lipids were measured by scanning densitometry after postchromatographic derivatization. Neutral lipids are often derivatized with phosphomolybdic acid reagent, which reacts to form highly visible, blue zones. Uniform application of this reagent to HPTLC plates is critical if quantification of neutral lipid bands is desired. In this paper we compare the quantitative and qualitative results observed by use of three methods of application of derivatization reagent — manual spraying, manual dipping, and use of the Derivapress. Results showed that dipping is the best method of reagent application for achieving quantitative data, and spraying is the best method for qualitative purposes. The Derivapress proved to be a simple and economical device for application of phosphomolybdic acid reagent to HPTLC plates. It enabled qualitative and quantitative analysis of neutral lipids.

It is often desirable, when conducting Western blot analyses, to accurately quantify the relative expression of multiple target proteins in a single sample. A common problem occurs: however, when the target proteins vary beyond the linear range of the detection system; thus precluding accurate densitometric analysis for all samples. For example, analysis of teleost immunoglobulin structure under non-reducing but denaturing conditions, yields multiple, differentially polymerized forms (redox forms) within a single sample, which can exceed single log differences in concentration, as visualized by chemiluminescent and X-ray film development. To resolve this difficulty an efficient technique has been developed that uses dilutions of a single sample, allowing accurate quantification of target proteins within their potentially unique and varied linear range of detection. Upon consideration of the respective dilution factor that yields an appropriate estimate, the multiple targets can be quantified. When the results from this technique are compared to other systems possessing more expansive linear ranges, the results obtained are comparable to within 1%. Thus, laboratories without access to more sensitive and costly densitometric instrumentation can still employ standard densitometric analysis to accurately quantify multiple targets in a single sample.

Natural colorings are frequently used in foods. In terms of food sanitation, the establishment of accurate and rapid analytical methods for natural colorings is required. Recently, the analytical methods of carotenoid colorings, quinoid colorings, flavonoid coloring, and anthocyanin coloring have been reported using reversed phase TLC with scanning densitometry. This paper reviews practical analytical methods of the above natural colorings in foods.

A quantitative densitometric high-performance thin-layer chromatographic method for determination of nebivolol hydrochloride in pharmaceutical preparations has been established and validated. Nebivolol hydrochloride from the formulations was separated and identified on silica gel 60 F 254 HPTLC plates with toluene-ethyl acetate-methanol-formic acid, 8 + 6 + 4 + 1 ( v / v ), as mobile phase. The plates were developed to a distance of 8 cm. Densitometric quantification was performed at λ = 285 nm by reflectance scanning. Well resolved bands were obtained for nebivolol hydrochloride. The method was validated for precision, recovery, robustness, specificity, and ruggedness. The calibration plot for nebivolol hydrochloride standard was linear with r = 0.9991, slope = 3.291, and intercept = 864.50. The limits of detection and quantification were 18.65 and 62.18 ng per band, respectively. The method is selective and specific with potential application in pharmaceutical analysis.

This chapter introduces the role of high-performance thin-layer chromatography (HPTLC) in the field of food analysis. HPTLC is well suited to food analysis as shown by many interesting examples analyzed with validated and standardized methods leading to results comparable to those obtained by other chromatographic techniques. Advances in automation and hyphenation made this inexpensive technique attractive for many issues in food analysis. For screening of food for bioactive compounds, residues, or contaminants, the direct detection with a bioassay (effect-directed analysis) widens the analyte range when compared to target analysis. Taking into consideration some of HPTLC's advantages like flexibility, matrix robustness, or cost- and time-effectiveness, it is surprising that quantitative HPTLC is not used more often.

We performed identification tests of aristolochic acid in Xixin, Mutong, Muxiang, and Fangji by reversed-phase TLC/scanning densitometry using aristolochic acid I as a marker compound. The developing solvents used were: I) acetonitrile/methanol/water mixture solution (3 : 1 : 1) and II) 2-butanone/methanol/sodium sulfate(1→20) mixture solution (2 : 1 : 1). The Rf value of aristolochic acid I was 0.54 using the I system and 0.57 using the II system. The maximum absorption wavelengths of aristolochic acid I observed by scanning densitometry were 254 and 325 nm.

Thin-layer chromatography in combination with fiber optical (diode array) scanning densitometry has been used for identification of fenitrothion in apples and fresh apple juice. The technique enabled parallel recording of chromatograms and in-situ UV-visible spectrometry in the range λ = 191–612 nm.

We performed identification tests of the main components (ginsenoside-Rg1, gentiopicroside, puerarin, geniposide, schizandrin, and [6]-gingerol) of the following crude drugs from the Japanese Pharmacopoeia, 14th edition (JP14): ginseng, red ginseng, gentian, Japanese gentian, pueraria root, gardenia fruit, schisandra fruit, and ginger). The identification was carried out with reversed-phase thin-layer chromatography using water, acetonitrile, methanol, 2-butanone, and n-hexane as developing solvents. A single spot could be separated from other components (Rf value: 0.44–0.59). In addition, spectral information could be simultaneously obtained by scanning densitometry. Thus, the main components of crude drugs in the JP14 could be identified by this method simply, rapidly, and accurately.

A rapid and reliable HPTLC method for analysis of aristolochic acids (AA) has been developed on the basis of a TLC procedure previously published by the German Drug Codex (DAC). The new method enables visual detection of the acids with certainty at very low levels (400 pg absolute of aristolochic acid A) in plant material and can therefore be used for screening Chinese drugs to ensure their safety on the basis of the absence of AA. Qualitatively Stephania tetrandra can be distinguished from various Aristolochia spp., Asarum spp. , and other drugs with similar Chinese names, on the basis of fingerprints.At least five different aristolochic acids can be detected in Aristolochia spp. The method is very specific and sensitive and is suitable for establishing and documenting adulteration of Stephania raw material with less than 1% Aristolochia fangji . By use of scanning densitometry quantitative determination of AA can be performed at low concentrations (1 ppm). Validation data (ICH) including linearity, limi...

A simple and rapid analytical method of oil-soluble coal tar dyes in cosmetics was established using reversed-phase TLC/scanning densitometry. Eleven kinds of oil-soluble coal tar dyes were able to be separated completely on reversed-phase TLC plates by the complementary use of 2 solvent systems. The solvent systems were A; n-hexane/2-butanone solution (5 : 1, v/v), solvent system B; acetonitrile/methanol solution (5 : 1, v/v). Then we measured visible absorption spectra of spots developed on the reversed-phase TLC plates by scanning densitometer to identify these coal tar dyes. The proposed method was successfully applied to the identification of oil-soluble coal tar dyes in commercial cosmetics.

BACKGROUND Gangliosides are membrane-bound glycolipid molecules particularly prominent in neural tissue. Changes in ganglioside expression during embryologic development result from a shift in biosynthesis from the fetal b pathway to the adult a pathway. Tumor gangliosides may play a role in the clinical behavior of certain subtypes of neuroblastoma. Because neuroblastoma, which presents in infancy, has a different biologic and clinical phenotype than that which presents in older children, the authors determined whether differences in ganglioside biosynthesis exist between these two neuroblastoma subgroups. METHODS Sixty-eight tumor specimens (25 diagnosed by screening and 43 diagnosed clinically) were obtained from the Quebec Neuroblastoma Screening Project. Gangliosides were isolated and purified by solvent partitioning, separated by high performance thin-layer chromatography, and quantitated by scanning densitometry. The sum of a and b pathway gangliosides were determined for each tumor. RESULTS Gangliosides of the b (fetal) pathway predominated in both screened and clinically diagnosed tumors of patients younger than 1 year of age. Twenty-three of 25 screened patients (92%) and 21 of 23 patients with clinically diagnosed tumors at younger than 1 year of age (91%) had tumor b pathway ganglioside content greater than 60%. In contrast, tumors of only 8 of 20 patients 1 year or older (40%) had b pathway ganglioside predominance. Predominance of b pathway tumor gangliosides correlated with improved outcome. Event free survival was significantly higher among patients with b pathway ganglioside tumor content greater than 60% versus those with b pathway ganglioside tumor content less than 60% (118.1 ± 3.9 months vs. 69.2 ± 8.6 months, P < 0.01). CONCLUSIONS Fetal patterns of ganglioside biosynthesis predominate in neuroblastoma tumors from patients younger than 1 year of age and adult patterns of ganglioside biosynthesis predominate in tumors from older children, supporting the view that neuroblastoma consists of distinct but overlapping disorders, and that gangliosides may play a biologic role in the clinical differences among these patients. Cancer 2001;91:785–93. © 2001 American Cancer Society.

A quantitative densitometric high-performance thin-layer chromatographic method for analysis of sertraline hydrochloride, estimated as sertraline, in pharmaceutical preparations has been established and validated. Sertraline was separated and identified on silica gel 60 F 254 HPTLC plateswith chloroform-ethyl acetate-triethylamine 5:3:0.2 ( v/v ) as mobile phase. The plates were developed to a distance of 7 cm. Densitometric quantification was performed at 279 nm by reflectance scanning. Well resolved bands were obtained for sertraline. The method was validated for precision, recovery, specificity, and ruggedness. The calibration plot for sertraline was linear with r = 0.9988. The method is selective and specific with potential application in pharmaceutical analysis.