High-performance Thin-layer Chromatography Mass Research Articles

Application of high-performance thin layer chromatography mass spectrometry (HPTLC MS) in foodomics authenticity

BackgroundChemometric analysis is also a powerful tool that can be used in the preliminary stages of analytical methods optimization, but is also efficient in data processing. These new approaches may be the key to the analysis of proteins, carbohydrate, sphignolipids, polyphenols in different food components. Although there are challenges in identifying and annotating those compounds due to the limited availability of analytical standards and structural diversity. Scope and approachChromatograms are a useful tool for learning more about the chemical make-up of the food being tested. This information can occasionally be implicit or presented in a subtle way. Therefore, to extract it, chemometric tools and data mining techniques are needed. A chromatogram's fingerprint allows for the possibility of performing both identity and quality testing on food. This viewpoint aims to offer a current assessment of chromatographic fingerprinting technique in the area of food authentication. Additionally, this methodology's limitations, absence from official analytical methods, and future directions are discussed. Novel techniques have been employed in the past few decades, ranging from high-pressure thin liquid chromatography (HPTLC) to mass spectrometry (MS) and other spectroscopic methods. In the last decade research developments, and the application of analytical methods in qualitative and quantitative studies of food components and their adulterants is reviewed in the present work. High-pressure Thin liquid chromatography (HPTLC) is hyphenated with high resolution mass spectrometry is particularly addressed due to its applicability in the targeted/untargeted metabolomic analysis of all food components. Key findings and conclusionsIn the current study, the HPTLC-MS technique was used to examine food components and food adulteration. The HPTLC-MS hyphenated techniques were found to be an extremely helpful method for the detection of food adulterants as well as the various food components, such as protein, carbohydrates, lipids, essential oils, and flavonoids, which were found to be effective as medicinally.

Validated simultaneous high-performance thin-layer chromatography‒mass spectrometry method for analysis of citalopram prochlorperazine, midazolam, and chlorodiazepoxide in urine for forensic analysis

Drug breakthroughs and development have been a great blessing to mankind. Problem arises when these are used in non-medical context such as use for recreational purposes, using drugs outside the label directions, and use with malicious intent as found in homicidal cases. Young generations get trapped into vicious cycle of drug addiction that has deleterious effects on their mind and body. Drugs including psychomotor drugs, opioids, narcotics, inhalants, stimulants, and central nervous system depressants are easily accessible. Forensic science plays a crucial role in investigation pertaining to drug abuse and overdose. Complex biological and non-biological matrices are submitted to forensic science laboratories for analysis and interpretation of the effects of drugs/toxicants/poisons on human body and environment. The aim of this study was to develop a method for the determination of various prescription drugs in the urine using high-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) technique. New combination of solvent systems was tried for the simultaneous detection of frequently used prescription drugs in urine. The method was developed and validated using HPTLC‒MS for the quantitative estimation of benzodiazepines, anti-depressants, and tranquilizers in urine using a common solvent system for all the drugs. Mobile phase cyclohexane‒toluene‒diethylamine (7:1.5:1.5, V/V) with one drop strong ammonia solution was used as mobile phase. Method validation was carried out according to the International Council of Harmonization guidelines. The present HPTLC‒MS method being simple, sensitive, precise, and accurate can be used for the detection and quantitative estimation of benzodiazepines, anti-depressants, and tranquilizers in biological specimens.

HPTLC–MS method for the determination of benzodiazepines in urine samples

Misuse and abuse of prescription drugs has become a global health problem in the last few years as the illicit drug cartels have grown exponentially. The non-medical use of prescribed medications, such as benzodiazepines, synthetic opioids, stimulants and tranquilizers, has increased rapidly due to their easy availability. Nowadays benzodiazepines are not only misused/abused, but also play a significant part in facilitating various crimes such as robbery, date rapes, sexual harassment, which has made its forensic examination important and critical. Considering the gravity of these crimes, an attempt has been made to develop and validate simultaneous quantification method for seven benzodiazepines (clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam) using high-performance thin-layer chromatography–mass spectrometry (HPTLC–MS) from urine. Quantitative analysis of clonazepam, lorazepam, alprazolam, diazepam, flurazepam, oxazepam and nitrazepam was carried out using HPTLC–MS. MS operating in selective ion-monitoring mode was attempted for the accurate identification of the drugs under study. Small-volume liquid extraction technique using diethyl ether–chloroform (80:20, v/v) was used for the extraction of urine. Pre-coated HPTLC (silica gel G 60 F254) plates were developed using the mobile-phase chloroform–glacial acetic acid (9:1, v/v). The screening of HPTLC plates was carried out by ultraviolet light, and the m/z ratio of drugs was obtained by extracting spots from plates using a TLC–MS interface. Method validation was carried out according to the International Council for Harmonisation guidelines. The current HPTLC–MS method is simple, sensitive, precise and accurate, and it can be used for the detection and quantitative estimation of benzodiazepines in urine.

New HPTLC methods for analysis of major bioactive compounds in mate (Ilex paraguariensis) tea

New HPTLC (high-performance thin-layer chromatography) methods to evaluate major bioactive compounds in mate (Ilex paraguariensis) tea were developed. Identification of compounds was based on HPTLC-MS (high-performance thin-layer chromatography–mass spectrometry), parallel to other techniques. Quantitative evaluation of the main methylxanthines and phenolic compounds in this species was accomplished by two HPTLC methods which can be performed consecutively on one single plate. A solvent system for the separation of the identified saponins is also described for qualitative evaluations. The methods for quantification were validated in terms of: robustness; LOD and LOQ (limits of detection and quantification); repeatability; intra-day and inter-day variation; ruggedness; and accuracy. The methods have proven to be suitable for evaluation of mate tea aqueous and methanolic extracts, with the advantages of: high-throughput; reduced need of plates and application procedures; and high sensitivity – low LODs and LOQs were achieved. Aqueous extracts of nineteen mate teas of different type, geographical origin and age were evaluated in terms of methylxanthines, phenolic compounds and pH. By means of principal component analysis, distinctive characteristics between mate teas could be observed.

A novel digitally optimized rapid quantification of carcinogenic aryl azo amines from various food matrices by HPTLC-MS

A rapid, precise and reproducible high-performance thin-layer chromatography-mass spectroscopy (HPTLC-MS) method was developed and validated for identification and quantification of toxic aryl azo amines from chili oils, pickles and related food matrices using Merck thin-layer chromatography (TLC) silica gel F-254 plates, as a stationary phase adsorbent by CAMAG HPTLC system. Extraction of azo food colors was done using method specified as per ISO standard. The use of tert-butyl-methyl ether as final extraction solvent resulted in minimal sample clean up and high efficiency. Calibration was performed to combat matrix effect. Good linearity levels were observed for the concentrations of aniline, 2, 4-xylidine and 4 aminoazobenzene of 2–14 ppm per band. The limits of detection and quantification found for aniline, 2,4-xylidine and 4-aminoazobenzene were found to be 0.0015, 200, and 400 ppm and limit of quantification (LOQ) values were found to be 0.004, 600, and 1200 ppm, respectively, with a correlation coefficient of 99.94 %. This study thus highlights an economically viable, commercially sustainable yet highly scientific technique of HPTLC-MS methodology with structural elucidation of banned azo amines in food and related products for their identification duly detectable at trace levels in a digitized form, which can enable material integrity-related prediction capacity of suspected food matrices.

High performance thin-layer chromatography–mass spectrometry methods on diol stationary phase for the analyses of flavan-3-ols and proanthocyanidins in invasive Japanese knotweed

We developed the first four HPTLC methods for the separation of proanthocyanidins according to degree of polymerization on HPTLC diol F254S plates. Acetonitrile, ethyl acetate, ethyl acetate–formic acid (9:0.1, v/v) and toluene–acetone–formic acid (3:6:1, v/v) were used as developing solvents and 4-dimethylaminocinnamaldehyde (DMACA) as the detection reagent. Each of these methods enables separation of standards of procyanidin dimers from procyanidin trimer (procyanidin C1) and separation of B-type dimers (procyanidins B1, B2, B3) from A-type dimers (procyanidins A1, A2). Based on these HPTLC methods we developed four new HPTLC-MS/MS methods for analyses of proanthocyanidins on HPTLC diol F254S plates and we identified B-type proanthocyanidins from monomers up to decamers in crude extracts of invasive Japanese knotweed (Fallopia japonica Houtt., Polygonum cuspidatum Sieb. & Zucc.) rhizomes. Monomers, monomer gallates, dimers, dimer gallates, dimer digallates, trimers, trimer gallates, tetramers, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes using developing solvents acetonitrile and toluene–acetone–formic acid (3:6:1, v/v). Ethyl acetate enabled separation from monomers up to hexamer gallates and ethyl acetate–formic acid (9:0.1, v/v) from monomers up to hexamers. During the five hours of stability testing of (–)-epicatechin and procyanidin B2 standards on HPTLC diol plates developed with all solvents we observed enhanced absorption at 280 nm. This was a totally unexpected phenomenon. This new discovery confirmed what we reported in our previous study on HPTLC silica gel. Enhanced absorption is influenced by the developing solvent (more than 30%), the stationary phase (up to 24%) and the light (up to 15%).

Analysis of nor-ephedrine by high performance thin layer chromatography-mass spectroscopy

Analysis of nor-ephedrine by high performance thin layer chromatography-mass spectroscopy

High-performance thin-layer chromatography and high-performance thin-layer chromatography–mass spectrometry methods for the analysis of phenolic acids

High-performance thin-layer chromatography (HPTLC) and HPTLC–mass spectrometry (MS)/(MSn) methods for analyses of phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) were developed. Separation was performed on HPTLC silica gel plates with and without fluorescent indicator (F254) in a saturated twin-trough chamber using n-hexane–ethyl acetate–formic acid (12:8:2, v/v) as the developing solvent. The developed HPTLC method is also suitable for the preliminary screening of some flavonoids (flavone, apigenin, luteolin, chrysin, quercetin, myricetin, kaempferide, kaempferol, hesperetin, naringenin, pinocembrin), although some interferences of phenolic acids with flavonoids were observed. The effect of pre-development on the HPTLC analysis of phenolic acids on the detection by densitometry and mass spectrometry was also explored. Pre-devel...

HPTLC-MS as a Neoteric Hyphenated Technique for Separation and Forensic Identification of Drugs

Drugs are traditionally been identified on basis of chromatographic-spectroscopic hyphenated techniques in instrumental analysis. Gas chromatography (GC) and Liquid chromatography (LC) hyphenated with mass spectroscopy (MS) i.e. GC-MS and LC-MS give reliable and confirmatory results in drugs identification. In the present work the novel hyphenated technique High Performance Thin Layer Chromatography-Mass Spectroscopy (HPTLC-MS) has been used. This technique provides efficient, quick and simple method for identification and separation of Narcotic drugs and psychotropic substances. The drugs under study are Papaverine, Methadone, Cocaine, Ketamine, Caffeine, Codeine, Diazepam, Thebaine, Heroin, Methamphetamine, Carbamazepine, Morphine, Narcotine and Ephedrine. The present study comprising of sixteen drugs has been carried out on CAMAG HPTLC instrument with automatic sampling. Thin layer chromatography (TLC) plates were developed in various solvent systems, scanned under TLC scanner and the results in terms of Retention Factor (Rf value) and UV spectrum (λmax) are presented in the manuscript. Using hyphenated technique of HPTLC-MS (MS 2020 SHIMADZU) spots of these drugs from TLC plate was lifted with CAMAG TLC-MS interface and confirmed by the mass spectrum of the individual drugs by their m/z values thus delivering fast and accurate confirmatory result on the TLC plate.

High performance thin-layer chromatography–mass spectrometry enables reliable analysis of physalins in different plant parts of Physalis alkekengi L.

We developed first HPTLC and HPTLC–MS/MS methods which enable characterization of structurally similar and complex biologically active compounds – physalins – from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate–toluene–formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive – formic acid – was efficiently solved by two successive plate pre-developments with methanol–formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC–MS/MS method, but also required a slightly modified developing solvent ethyl acetate–toluene–formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC–MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.

High performance thin-layer chromatography–mass spectrometry of Japanese knotweed flavan-3-ols and proanthocyanidins on silica gel plates

On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol–formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6′-O-malonyl)-hexoside).

Determination of ranolazine in tablet formulations by high-performance thin-layer chromatography—mass spectrometry using reflectance scanning densitometry

A new quantitative densitometric high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the analysis of ranolazine (RZ) both in bulk and formulations. RZ was separated and identified on silica gel 60 F254 HPTLC plates with butanol—acetic acid—water (6:2:2 v/v) as the mobile phase. Densitometric quantification was performed at λ = 270 nm by reflectance scanning which facilitated well-resolved band for the main drug (RF 0.56 ± 0.02). Response to RZ was a linear function of concentration in the range of 100–400 ng, with a correlation coefficient, slope, and intercept of 0.99912 ± 0.00017, 8.684 ± 0.582, and 492.147 ± 2.67, respectively. The minimum amount of RZ that could be authentically detected and quantified was 14.90 and 49.67 ng band−1, respectively. Additionally, the peak identities as well as the purities were confirmed by mass spectrometry. The electrospray ionization (ESI+) mass spectra showed the [M + H]+ ion for RZ detected at m/z 428.3 being acquired directly ...

Correct assignment of lipophilic dye mixtures? A case study for high-performance thin-layer chromatography–mass spectrometry and performance data for the TLC–MS Interface

The TLC–MS Interface, the successor of the ChromeXtract, has been available for elution head-based coupling of high-performance thin-layer chromatography with mass spectrometry (HPTLC–MS) since 2009, and is meanwhile widespread in use, mainly for compound confirmation. Until now, quantitative performance data has not been reported in detail and thus were investigated in this study. The performance data of HPTLC–electrospray ionization (ESI)-MS via the TLC–MS Interface showed good mean precisions (%RSD, n=5) for 6 dyes in a commercially available dye mixture investigated at two different concentrations (7.3% for the 1:8 dilution, and 10.1% for the 1:16 diluted) in a selected worst case scenario. The respective mean precisions of absorbance measurements were ≤1.3%. For calibrations by HPTLC–ESI-MS, the mean determination coefficient was 0.9975 for the 6 dyes (versus 0.9997 for absorbance measurement). HPTLC–MS analysis revealed the incorrect assignment of components in two commercially available dye mixtures. Using an additional software (MassWorks) that delivered a 100 times increased mass accuracy, the proposal of molecular formulae was shown to be obtainable under certain conditions with a low resolution single quadrupole mass spectrometer and in the case of helpful information such as the double bond equivalents. This enabled the identification of the incorrectly assigned unknown dyes and clearly demonstrated the benefit of using HPTLC–MS for zone confirmation.

HPTLC-MS analysis of melamine in milk: standardization and validation

Melamine containing large amount of nitrogen may be deliberately added to milk in order to increase the protein content for economic adulteration in food stuffs. High performance thin layer chromatography-mass spectrometry (HPTLC-MS)-based method of quantification of melamine has been developed using Silica gel 60 F254s HPTLC plates, with optimized mobile phase iso-propanol/dichloromethane/water, 5:2.5:3, v/v/v in a twin trough chamber saturated (5 min) at pH 6.8. Validation was carried out by testing its specificity, linearity (1–50 μg), accuracy (95.908–98.369%), inter- and intraday precision (1.578 and 1.670%), respectively, limits of detection (2.688 ng) and quantification (8.146 ng). HPTLC in combination with MS has been found suitable for routine analysis of melamine added as adulterant to market-purchased milk samples.

BACKGROUND MASS SIGNALS IN TLC/HPTLC–ESI-MS AND PRACTICAL ADVICES FOR USE OF THE TLC-MS INTERFACE

Among the different thin-layer and high-performance thin-layer chromatography mass spectrometry (TLC/HPTLC-MS) approaches, the elution head-based TLC-MS Interface is increasingly used due to its good detectability, reliability, and versatility in its application. The influence of different solvents, either used for prewashing or chromatography, on the mass signal background was studied. It was shown that the use of acidic solvents can cause intense background signals (acid sodium clusters) and ion suppression in the analyte mass spectra. Using a mixture of methanol–water 3:1 (v/v) for plate prewashing, background mass signals could be reduced to a minimum for both ionization modes. The TLC/HPTLC-MS background mass signals obtained from different plates with the green manganese activated zinc silicate fluorescence indicator F254, and without, did not show substantial differences. As sodium is ubiquitary in solvent, tubing, glassware, adsorbent, and so forth, the sodium adduct was often formed and observed, whereby the extent of formation was different depending on the plate type. Recommendations for plate handling for use in TLC/HPTLC-MS are provided, as well as practical advices with regard to integration of the inline filter, various sorts of pressure increase, and leakage using the TLC-MS Interface.