Normal-phase High-performance Thin-layer Chromatography Research Articles

Simultaneous determination of lesinurad and febuxostat in commercial fixed-dose combinations using a greener normal-phase HPTLC method

Abstract So far, no documented method for simultaneously analyzing lesinurad (LND) and febuxostat (FBX) has been reported for either traditional high-performance thin-layer chromatography (HPTLC) or a green HPTLC technique. In order to determine LND and FBX simultaneously in commercially available fixed-dose combo tablets, this study devised a normal-phase HPTLC method that is fast, sensitive, and green. The green eluents for the simultaneous analysis of LND and FBX were a mixture of ethyl acetate:ethanol:water at 70:20:10 (v/v/v) ratio. The new approach’s greenness was predicted utilizing four distinct greenness tools: the National Environmental Method Index, Analytical Eco-Scale, ChlorTox, and Analytical GREENness approaches, and the results revealed a significantly greener profile. The current method operated on a linear scale between 30 and 1,000 ng·band−1. It was confirmed that the current approach is sensitive, accurate, precise, robust, and green. The LND and FBX contents of commercially available tablet products A and B were found to be within the range of 100 ± 2%, indicating that the existing methodology for simultaneously determining LND and FBX in pharmaceutical combination products is applicable. The results of the current methodology indicated that LND and FBX could be consistently measured in pharmaceutical combination products simultaneously using the current approach.

Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

Investigation of the estrogenic potential of 15 rosé, white and red wines via effect-directed ten-dimensional hyphenation

Wine is consumed for thousands of years all over the world, however, its estrogenic potential is still underexplored. A non-target effect-directed screening was developed to reveal estrogen-like and antiestrogen-like compounds in 15 rosé, white and red wine samples of different origin and grape variety. Normal-phase high-performance thin-layer chromatography multi-imaging detection (NP-HPTLC−UV/Vis/FLD) was combined with the planar yeast estrogen screen (pYES) bioassay or the duplex planar yeast antagonist estrogen screen (pYAES) bioassay on the same adsorbent surface. Up to nine estrogen-like compound zones were detected and further characterized via heart-cut elution from the planar bioautogram to orthogonal reversed phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection (DAD) and high-resolution tandem mass spectrometry (HRMS/MS). Among the tentatively assigned estrogen-like substances, the HRMS/MS signals pointed to hexylresorcinol and diethyl esters from organic acids for the first time. This highlights the method suitability for non-target complex mixture screening and rapid dereplication. The 10D hyphenation NP-HPTLC−UV/Vis/FLD–pYAES−heart cut–RP-HPLC–DAD–HRMS/MS proved to be an efficient and powerful tool for detecting estrogens as well as antiestrogens in the matrix-rich wine samples. High-throughput capability and substantial reduction in the required resources for analysis were demonstrated by this straightforward hyphenation, if compared to bioassay-guided fractionation. The 10D information (via orthogonal chromatographic, versatile spectrometric and duplex endocrine activity data) obtained during a single chromatographic run for many samples in parallel was advantageous for the tentative molecule assignment.

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone−1 for flutamide and 20.1 ± 5.1 ng zone−1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

Strain-specific quantification of probiotic Bacillus subtilis in feed by imaging high-performance thin-layer chromatography

The European Union has banned the use of antibiotic growth promoters in animal production, which has led to increased use of probiotic microorganisms. These feed additives result in higher costs for farmers, which is why the demand for a quality control system to quantify probiotics in feeds has increased in recent years. Imaging high-performance thin-layer chromatography (HPTLC) was proven to be a robust method for determining the probiotic Bacillus subtilis DSM 29784 strain based on the production of selective bacterial metabolites and thus characteristic metabolite pattern. However, to quantify the specific probiotic strain in the feed, identification of a strain-specific metabolite not produced by genetically very similar bacteria is necessary. Compared to five bacteria with high genetic similarity, a strain-specific metabolite was formed in the probiotic bacteria by a two-step cultivation procedure. Among others, antimicrobial properties were found for this metabolite, which indicated probiotic activity. The hyphenation of normal-phase HPTLC with reversed-phase high-performance liquid chromatography diode array detection and high-resolution mass spectrometry allowed the preliminary assignment of this strain-specific metabolite to the molecular formula C35H44N6O2 (580.3527 Da). This metabolite, produced each time via an upstream cultivation process to generate the standard levels, was used for the quantification of probiotic active cells in the feed. Data on selectivity, linearity, detection limit, recovery, and precision have shown the good performance of the method.

Determination of Colchicine in Pharmaceutical Formulations, Traditional Extracts, and Ultrasonication-Based Extracts of Colchicum autumnale Pleniflorum (L.) Using Regular and Greener HPTLC Approaches: A Comparative Evaluation of Validation Parameters.

In the literature, there is a scarcity of greener analytical approaches for colchicine (CLH) analysis. As a result, efforts were made in this study to develop and validate a greener reversed-phase high-performance thin-layer chromatography (HPTLC) technique for CLH analysis in traditional extracts (TE) and ultrasonication-based extracts (UBE) of commercial Unani formulations, commercial allopathic formulations, and Colchicum autumnale Pleniflorum (L.) obtained from Egypt and India. This new technique was compared to the regular normal-phase HPTLC method. The greenness profile of both methods was estimated using the Analytical GREENness (AGREE) approach. In the 100–600 and 25–1200 ng/band ranges, regular and greener HPTLC procedures were linear for CLH analysis, respectively. For CLH analysis, the greener HPTLC method was more sensitive, accurate, precise, and robust than the regular HPTLC method. For CLH analysis in TE and UBE of commercial Unani formulations, commercial allopathic formulations, and C. autumnale obtained from Egypt and India, the greener HPTLC method was superior in terms of CLH content compared to the regular HPTLC method. In addition, the UBE procedure was superior to the TE procedure for both methods. The AGREE scores for regular and greener reversed-phase HPTLC methods were found to be 0.46 and 0.75, respectively. The AGREE results showed excellent greener profile of the greener HPTLC method over the regular HPTLC technique. Based on several validation criteria and pharmaceutical assay findings, the greener HPTLC method is regarded as superior to the regular HPTLC approach.

Multiplex planar bioassay detecting estrogens, antiestrogens, false-positives and synergists as sharp zones on normal phase

BackgroundPhytoestrogens are found in many plants used in traditional medicines. Increasingly, plant extracts (botanicals) are also being added to foods or marketed as dietary supplements. Especially such powder formulations are susceptible to adulteration and falsification, given the global processing chain. To detect estrogen-like compounds in such multicomponent mixtures, non-target screening for hormonally active or endocrine disrupting compounds in plant products is becoming more important. Unfortunately, the current planar yeast estrogen screen (pYES) is prone to zone diffusion on the normal-phase high-performance thin-layer chromatography (NP-HPTLC) plate due to long incubation times in the aqueous bioassay. PurposeThe present study aimed to reduce zone diffusion on NP plates, which provides the basis for extending pYES to a multiplex bioassay, offering 4 different biological activity principles, followed by targeted identification of active zones. Study design and methodsThe reduction of substance diffusion via a polyisobutyl methacrylate polymer coating was studied. After successful zone fixation (fix), a multiplex bioassay was developed, in which a 17β-estradiol-strip was applied along each sample track to detect synergists and antagonists (A), and for verification (V), a 4-methyl umbelliferone-strip to exclude false-positives. After multiplex bioassay screening of 68 botanicals, the zones with hormonal activities were heart-cut eluted to reversed-phase high-performance liquid chromatography−diode array detection−high-resolution tandem mass spectrometry (RP-HPLC–DAD–HESI-HRMS/MS). ResultsThe separated substances were successfully fixed by the chromatogram coating. The zone sharpness (achieved after the bioassay) made it possible to add two strips, the 17β-estradiol-strip for antagonistic and synergistic, and the 4-methyl umbelliferone-strip for false-positive effect detection, resulting in a multiplex bioassay. Using the 12D hyphenation NP-HPTLCfix–UV/Vis/FLD–pYAVES–FLD heart-cut RP-HPLC–DAD–HESI-HRMS/MS, it was possible to obtain information on estrogens, antiestrogens, false-positives, and synergists, and (tentatively) assign 17 hormonally active compounds, of which only 7 have been known to affect the human estrogen receptor, while another 4 had structural similarity to common phytoestrogens and antiestrogens. ConclusionsThe streamlined 12D hyphenation including a multiplex bioassay has been shown to differentiate hormonal effects, leading to new insights and better understanding. It can generally be used to identify unknown hormonally active compounds in complex samples.

Simultaneous Estimation of Rhein and Aloe-Emodin in Traditional and Ultrasound-Based Extracts of Rheum palmatum L. (Rhubarb) Using Sustainable Reverse-Phase and Conventional Normal-Phase HPTLC Methods

The greenness indices of literature analytical procedures for the simultaneous measurement of rhein and aloe-emodin have not been determined. As a consequence, the first goal of this study was to design and validate a sensitive and sustainable reverse-phase high-performance thin-layer chromatography (HPTLC) method for the simultaneous estimation of rhein and aloe-emodin in a traditional extract (TE) and ultrasound-based extract (UBE) of commercial Rhubarb and Rhubarb plant extracts in comparison to the conventional normal-phase HPTLC method. The second goal was to determine the greenness indices for both methods using the AGREE approach. For the sustainable reverse-phase HPTLC approach, the method was linear in the 50–1000 ng/spot range for rhein and 25–1000 ng/spot range for aloe-emodin. However, for the conventional normal-phase HPTLC approach, the method was linear in the 50–600 ng/spot range for rhein and 100–600 ng/spot range for aloe-emodin. The limit of detection (LOD) for rhein and aloe-emodin was 16.81 ng/spot and 8.49 ng/spot, respectively, using the sustainable analytical method. However, the LOD for rhein and aloe-emodin was 18.53 ng/spot and 39.42 ng/spot, respectively, using the conventional analytical method. For the simultaneous determination of rhein and aloe-emodin, the sustainable analytical method was more sensitive, accurate, precise, and robust than the conventional analytical method. The amount of rhein and aloe-emodin was higher in the UBE of commercial Rhubarb and Rhubarb plant extract over their TE. For the simultaneous quantification of rhein and aloe-emodin in the TE and UBE of marketed Rhubarb and Rhubarb plant extract, the sustainable analytical method was superior to the conventional analytical method. The AGREE index for the sustainable reverse-phase and conventional normal-phase HPTLC methods was determined to be 0.78 and 0.49, respectively, indicating an excellent greenness profile of the sustainable reverse-phase HPTLC method over the conventional normal-phase HPTLC approach. The sustainable analytical method was found to be superior to the conventional analytical method based on these results.

Determination of Gefitinib Using Routine and Greener Stability-Indicating HPTLC Methods: A Comparative Evaluation of Validation Parameters

“High-performance thin-layer chromatography (HPTLC)” methods for gefitinib (GFT) estimation are scarce in the literature. In addition, greener analytical techniques for GFT estimation are also lacking in the literature. Accordingly, an attempt was undertaken to invent and validate a sensitive and greener normal-phase HPTLC method for GFT analysis in commercial tablets in comparison to the routine normal-phase HPTLC method. The greenness index for both methods was assessed using “Analytical GREENness (AGREE)” methodology. GFT detection was carried out using both methods at 332.0 nm. In the 30–700 ng/band and 20–1400 ng/band ranges, the routine and greener HPTLC assays were linear for GFT estimation. The greener HPTLC method was highly sensitive, more accurate, more precise, and more robust than the routine HPTLC assay for GFT estimation. Both methods were able to detect GFT in the presence of its degradation products, suggesting the stability-indicating property of both methods. The assay of GFT in commercial tablets was 92.45% and 99.74% using the routine and greener HPTLC assays, respectively. The AGREE index for routine and greener analytical assays was predicted to be 0.44 and 0.77, respectively, indicating the excellent greenness index of the greener HPTLC assay over the routine HPTLC assay. The greener HPTLC assay is considered superior to the routine HPTLC assay based on these results.

Determination of Thymol in Commercial Formulation, Essential Oils, Traditional, and Ultrasound-Based Extracts of Thymus vulgaris and Origanum vulgare Using a Greener HPTLC Approach.

In the literature, greener analytical approaches for determining thymol in its commercial formulations, plant-based phytopharmaceuticals, and biological fluids are scarce. As a result, the goal of this study is to develop and validate a normal-phase “high-performance thin-layer chromatography (HPTLC)” method for determining thymol in commercial formulations, essential oils, traditional extracts (TE), and ultrasound-based extracts (UBE) of Thymus vulgaris and Origanum vulgare obtained from various geographical regions. The greener mobile phase for thymol analysis was a binary combination of cyclohexane and ethyl acetate (85:15, v/v). The derivatized densitometric analysis of thymol was carried out under visible mode at 530 nm utilizing anisaldehyde-sulfuric acid as a derivatizing/visualizing agent. In the 10–2000 ng/band range, the greener normal-phase HPTLC method was linear. Furthermore, for thymol analysis, the proposed analytical approach was simple, quick, inexpensive, accurate, precise, robust, sensitive, and greener. The thymol contents in commercial formulation were computed as 7.61% w/w. In general, the thymol contents were maximum in essential oils of T. vulgaris and O. vulgare compared to the other sample matrices studied. The thymol contents of TE of T. vulgaris and O. vulgare of different geographical regions were significantly low compared to their UBE extract. Using 12 distinct components of green analytical chemistry, the overall “analytical GREEnness (AGREE)” scale for the proposed analytical approach was computed 0.79, showing the good greener nature of the proposed analytical approach. Overall, the greener normal-phase HPTLC technique was found to be reliable for determining thymol in commercial formulations and plant-based phytopharmaceuticals.

Simultaneous Determination of Caffeine and Paracetamol in Commercial Formulations Using Greener Normal-Phase and Reversed-Phase HPTLC Methods: A Contrast of Validation Parameters.

There has been no assessment of the greenness of the described analytical techniques for the simultaneous determination (SMD) of caffeine and paracetamol. As a result, in comparison to the greener normal-phase high-performance thin-layer chromatography (HPTLC) technique, this research was conducted to develop a rapid, sensitive, and greener reversed-phase HPTLC approach for the SMD of caffeine and paracetamol in commercial formulations. The greenness of both techniques was calculated using the AGREE method. For the SMD of caffeine and paracetamol, the greener normal-phase and reversed-phase HPTLC methods were linear in the 50–500 ng/band and 25–800 ng/band ranges, respectively. For the SMD of caffeine and paracetamol, the greener reversed-phase HPTLC approach was more sensitive, accurate, precise, and robust than the greener normal-phase HPTLC technique. For the SMD of caffeine paracetamol in commercial PANEXT and SAFEXT tablets, the greener reversed-phase HPTLC technique was superior to the greener normal-phase HPTLC approach. The AGREE scores for the greener normal-phase and reversed-phase HPTLC approaches were estimated as 0.81 and 0.83, respectively, indicated excellent greenness profiles for both analytical approaches. The greener reversed-phase HPTLC approach is judged superior to the greener normal-phase HPTLC approach based on numerous validation parameters and pharmaceutical assays.

Is Our Natural Food Our Homeostasis? Array of a Thousand Effect-Directed Profiles of 68 Herbs and Spices.

The beneficial effects of plant-rich diets and traditional medicines are increasingly recognized in the treatment of civilization diseases due to the abundance and diversity of bioactive substances therein. However, the important active portion of natural food or plant-based medicine is presently not under control. Hence, a paradigm shift from quality control based on marker compounds to effect-directed profiling is postulated. We investigated 68 powdered plant extracts (botanicals) which are added to food products in food industry. Among them are many plants that are used as traditional medicines, herbs and spices. A generic strategy was developed to evaluate the bioactivity profile of each botanical as completely as possible and to straightforwardly assign the most potent bioactive compounds. It is an 8-dimensional hyphenation of normal-phase high-performance thin-layer chromatography with multi-imaging by ultraviolet, visible and fluorescence light detection as well as effect-directed assay and heart-cut of the bioactive zone to orthogonal reversed-phase high-performance liquid chromato-graphy−photodiode array detection−heated electrospray ionization mass spectrometry. In the non-target, effect-directed screening via 16 different on-surface assays, we tentatively assigned more than 60 important bioactive compounds in the studied botanicals. These were antibacterials, estrogens, antiestrogens, androgens, and antiandrogens, as well as acetylcholinesterase, butyrylcholinesterase, α-amylase, α-glucosidase, β-glucosidase, β-glucuronidase, and tyrosinase inhibitors, which were on-surface heart-cut eluted from the bioautogram or enzyme inhibition autogram to the next dimension for further targeted characterization. This biological-physicochemical hyphenation is able to detect and control active mechanisms of traditional medicines or botanicals as well as the essentials of plant-based food. The array of 1,292 profiles (68 samples × 19 detections) showed the versatile bioactivity potential of natural food. It reveals how efficiently and powerful our natural food contributes to our homeostasis.

A rapid, sensitive, and greener stability-indicating normal-phase HPTLC method with univariate calibration for the estimation of chlorhexidine acetate in its commercial formulations

Till date, there is no report on high-performance thin-layer chromatography (HPTLC) analysis of chlorhexidine (CHD) or its salts like CHD acetate (CHD-A), CHD gluconate, and CHD hydrochloride. Therefore, a rapid, sensitive, and greener normal-phase HPTLC method has been reported for the analysis of CHD-A in its four different commercial dosage forms. The quaternary combination of ethyl acetate: ethanol: water: formic acid (75:10:10:5, v v v v−1) was optimized as the green solvent system/mobile phase for CHD-A analysis. The CHD-A detection was carried out at 264 nm. The greener normal-phase HPTLC assay was linear in the range of 10–2000 ng band−1. In addition, the proposed method was accurate, precise, sensitive, and selective for CHD-A analysis. The greener normal-phase HPTLC method was able to detect CHD-A in the presence of its degradation products, suggesting the stability-indicating property of this method. The content of CHD-A in commercial products A, B, C, and D was detected as 0.18, 2.02, 1.46, and 0.19% w w−1, respectively. The greenness scale for the greener normal-phase HPTLC assay was estimated as 0.88 utilizing “analytical GREEnness (AGREE)” calculator, suggested the greener nature of the normal-phase HPTLC assay. Overall, these data suggested that the greener normal-phase HPTLC method can be successfully used for the determination of CHD-A in its commercial products.

Rapid, Highly-Sensitive and Ecologically Greener Reversed-Phase/Normal-Phase HPTLC Technique with Univariate Calibration for the Determination of Trans-Resveratrol

The rapid, highly-sensitive and ecologically greener reversed-phase (RP)/normal-phase (NP) high-performance thin-layer chromatography (HPTLC) densitometric technique has been developed and validated for the determination of trans-resveratrol (TRV). The reversed-phase HPTLC-based analysis of TRV was performed using ethanol–water (65:35, v v−1) combination as the greener mobile phase, while, the normal-phase HPTLC-based estimation of TRV was performed using chloroform–methanol (85:15, v v−1) combination as the routine mobile phase. The TRV detection was carried out at 302 nm for RP/NP densitometric assay. The linearity was recorded as 10–1200 and 30–400 ng band−1 for RP and NP HPTLC techniques, respectively. The RP densitometric assay was observed as highly-sensitive, accurate, precise and robust for TRV detection in comparison with the NP densitometric assay. The contents of TRV in commercial formulation were recorded as 101.21% utilizing the RP densitometric assay, while, the contents of TRV in commercial formulation were found to be 91.64% utilizing the NP densitometric assay. The greener profile of RP/NP technique was obtained using the analytical GREEnness (AGREE) approach. The AGREE scales for RP and NP densitometric assays were estimated 0.75 and 0.48, respectively. The recorded AGREE scale for the RP densitometric assay indicated that this technique was highly green/the ecologically greener compared to the NP densitometric assay. After successful optimization of analytical conditions, validation parameters, AGREE scale and chromatography performance, the RP densitometric assay with univariate calibration was found to be better than the NP densitometric assay for the analysis of TRV.

On-surface autosampling for liquid chromatography−mass spectrometry

An on-surface multi-purpose autosampler was built for liquid chromatography−mass spectrometry (LC−MS) based on the autoTLC–MS interface, taking advantage of open-source hard- and software developments as well as 3D printing. Termed autoTLC−LC−MS system, it is introduced for orthogonal hyphenation of normal phase high-performance thin-layer chromatography with reversed phase high-performance LC (HPLC) and high-resolution MS (HRMS). For verification of its functionality, a multi-class antibiotic mixture was applied as a calibration band pattern on an adsorbent layer and detected by the Bacillus subtilis bioassay. This effect-image was uploaded as a template in the updated TLC–MS_manager software. The clicked-on antibiotic zones were sequentially eluted without intervention from the planar counterpart (without bioassay) via a monolithic HPLC column into the HRMS system. For elution of antibiotics of 7 structural classes at 5 different calibration levels, the new on-surface autosampler achieved intra-day precisions of 2.1–14.1%, while inter-day precisions ranged 2.5–16.1% (all n = 3). The new hyphenation offers potential for planar sample clean-up prior to HPLC, concentration of liquid samples, increase of peak capacity and proof of peak purity or isomers. The integrated autoTLC−LC−MS system enabled high sample throughput, efficiency and reproducibility for the first time through fully automated TLC-LC-MS sequence operation. Its contact-closure signal functionality, versatile 3D printed planar sample holder and open-source software made it readily adjustable for new analytical tasks. Undoubtedly, any planar material can be investigated for leachables, such as textiles, foils, papers and other packagings, as well as planar biological samples for ingredients.

Non-target bioanalytical eight-dimensional hyphenation including bioassay, heart-cut trapping, online desalting, orthogonal separations and mass spectrometry

It is still a challenge to discover and identify individual bioactive compounds directly in multicomponent mixtures. Current workflows are too tedious for routine use. Hence, the hyphenation of separation and detection techniques is a powerful tool to maximize the information obtained by a single sample run. A robust eight-dimensional (8D) hyphenation was developed. Orthogonal separations, biological assay detection, analyte trapping, desalting, and physico-chemical detections were arranged in the following order, i.e. 1) normal phase high-performance thin-layer chromatography (NP-HPTLC) separation, 2) Vis detection, 3) UV detection, 4) fluorescence detection (FLD), 5) bioassay for effect-directed analysis (EDA), 6) heart-cut trapping/desalting/elution to reversed phase high-performance liquid chromatography (RP-HPLC) separation, 7) photodiode array (PDA) and 8) mass spectrometry (MS) detection. For the first time, the hyphenation exploited online analyte trapping to desalt the eluted bioactive zone from the plate containing highly salted bioassay media. Subsequent valve switching guided the trapped analyte(s) to the main column, followed by multiple detection. As proof-of-principle, cinnamon samples were analyzed by NP-HPTLC−UV/Vis/FLD−EDA−RP-HPLC−PDA−MS, whereby a bioactive zone was separated into two distinct peaks detected by PDA and MS to be 2-methoxy cinnamaldehyde and cinnamaldehyde. The developed 8D hyphenation is applicable for routine, allowing the non-target high-throughput screening of complex samples for individual bioactive compounds.

Quantitative analysis of cabozantinib in pharmaceutical dosage forms using green RP-HPTLC and green NP-HPTLC methods: A comparative evaluation

In this study, a novel antitumor drug cabozantinib (CZN) was estimated by a validated green reversed-phase high-performance thin-layer chromatography (RP-HPTLC) and normal-phase high-performance thin-layer chromatography (NP-HPTLC) techniques in the marketed tablets and capsules. The RP-HPTLC densitometry analysis of CZN was carried out using “RP-18 silica gel 60 F254S HPTLC plates”. Green acetone/water (85:15, v v−1) combination was applied as the mobile phase for a RP-HPTLC-densitometry. However, green NP-HPTLC analysis of CZN was conducted using “NP-18 silica gel 60 F254S HPTLC plates”. Green ethyl acetate/ethanol (97.5:2.5, v v−1) combination was applied for a green NP-HPLTC-densitometry. The detection of CZN was performed at λmax = 247 nm for both of the techniques. The RP-HPTLC technique showed high sensitivity for the quantification of CZN in comparison to NP-HPTLC-densitometry. For a green RP-HPTLC-densitometry, the CZN content of marketed tablets and capsules were found to be 101.17% and 98.77%, respectively. However, for a green NP-HPTLC-densitometry, the CZN content of the marketed tablets and capsules were found to be 96.12% and 94.25%, respectively. Accordingly, the green RP-HPTLC technique was identified better than a green NP-HPTLC technique. Hence, the RP-HPTLC-densitometry can be successfully used for the quantification of CZN in pharmaceutical dosage forms containing CZN as one of the constituents.

Combining Normal/Reversed-Phase HPTLC with Univariate Calibration for the Piperine Quantification with Traditional and Ultrasound-Assisted Extracts of Various Food Spices of Piper nigrum L. under Green Analytical Chemistry Viewpoint.

Due to unavailability of sustainable analytical techniques for the quantitation of piperine (PPN) in food and pharmaceutical samples, there was a need to develop a rapid and sensitive sustainable analytical technique for the quantitation of PPN. Therefore, the current research presents a fast and highly sensitive normal/reversed-phase high-performance thin-layer chromatography (HPTLC) technique with classical univariate calibration for the quantitation of PPN in various food spices of black pepper with traditional (TE) and ultrasound-assisted extracts (UBE) of various food spices of Piper nigrum L. under green analytical chemistry viewpoint. The amount of PPN in TE of four different spices of black pepper—namely BPMH, BPLU, BPSH, and BPPA—was found to be 309.53, 304.97, 282.82, and 232.73 mg g−1, respectively using a sustainable normal-phase HPTLC technique. However, the amount of PPN in UBE of BPMH, BPLU, BPSH, and BPPA was recorded as 318.52, 314.60, 292.41, and 241.82 mg g−1, respectively using a sustainable normal phase HPTLC technique. The greenness of normal/reversed-phase HPTLC technique was predicted using AGREE metric approach. The eco-scale was found to be 0.90, suggested excellent greenness of normal/reversed-phase technique. UBE of PPN was also found to be superior over TE of PPN. Overall, the results of this research suggested that the proposed normal/reversed-phase densitometry technique could be effectively used for the quantitation of PPN in food and pharmaceutical samples.

A rapid and sensitive stability-indicating green RP-HPTLC method for the quantitation of flibanserin compared to green NP-HPTLC method: Validation studies and greenness assessment

A rapid and sensitive stability-indicating green reversed-phase high-performance thin-layer chromatography (SI-G-RP-HPTLC) method was established and validated for the detection of flibanserin (FBN) compared to the stability-indicating green normal-phase high-performance thin-layer chromatography (SI-G-NP-HPTLC) method. The G-RP-HPTLC-densitometry analysis of FBN was conducted using acetone/water (80:20, v/v) solvent system as the mobile phase, while, the G-NP-HPTLC-densitometry analysis of FBN was conducted using ethyl acetate/methanol (95:5, v/v) solvent system. The quantitation of FBN was carried out at λmax = 204 nm for both of the techniques. The SI-G-RP-HPTLC and SI-G-NP-HPTLC methods were found to be linear in the range of 100–1600 and 200–1600 ng/band, respectively. The system suitability parameters for both of the methods were found to be acceptable for the quantitation of FBN. The SI-G-RP-HPTLC technique was found to be more rapid, accurate, precise, and sensitive for the determination of FBN compared to the SI-G-NP-HPTLC technique. The SI-G-RP-HPTLC technique was observed as more selective and stability-indicating for the determination of FBN in the presence of its degradation products. The FBN contents of commercial film-coated tablets were found to be 98.76% using the SI-G-RP-HPTLC technique, while, the FBN contents of commercial film-coated tablets were recorded as 96.28% using the SI-G-NP-HPTLC technique. The greenness of both of the green methods was assessed using AGREE software which utilized 12 principles of green analytical chemistry. The eco-scales of SI-G-RP-HPTLC and SI-G-NP-HPTLC methods were predicted as 0.86 and 0.80, respectively. The obtained eco-scale values for both of the methods suggested excellent greenness of both of the methods. Based on these observations and validation studies, the SI-G-RP-HPTLC methodology was considered as superior over the SI-G-NP-HPTLC technique for pharmaceutical analysis. Accordingly, the SI-G-RP-HPTLC technique could be utilized for the quantitation and stability evaluation of FBN in commercial products containing FBN.

HPTLC METHOD FOR ESTIMATION AND QUANTIFICATION OF β-SITOSTEROL FROM IN-VIVO AND IN-VITRO SAMPLES OF MERREMIA AEGYPTIA AND MERREMIA DISSECTA

Objective: A normal-phase high-performance thin-layer chromatography (HPTLC) method has been developed and validated for estimation and quantitation of beta-sitosterol from the methanolic fraction of different plant parts of two medicinally important plants viz. Merremia aegyptia and Merremia dissecta. These plants have been reported to possess antimicrobial, antioxidant, and anti-inflammatory activities.
 Methods: Chromatographic separation of beta-sitosterol from the methanolic extracts of plant parts of M. aegyptia and M. dissecta was performed on TLC aluminum plates pre-coated with silica gel 60F254 using a suitable mobile phase. The densitometric scanning was done after derivatization at ????-580 nm for ????-sitosterol.
 Result: Only M. dissecta leaf sample was reported to contain ????-sitosterol (4.6 ng/μl), whereas other samples such as seed, stem, and callus extracts of M. aegyptia and M. dissecta did not showed its presence.
 Conclusion: The developed HPTLC method is simple, rapid, and precise and can be used for routine analysis and quantification of ????-sitosterol and other useful plant bioactives that are phytopharmaceutically important.