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•Novel green HPTLC method for simultaneous quantitation of sofosbuvir and ledipasvir.•Forced degradation stability study with HPTLC-ESI-MS detection of the products.•Structure ...elucidation of each fragment in the mass spectra and fragmentation pattern.•Eco-Scale greenness assessment with a 93 score (excellent green)•Method validation and application on synthetic mixtures and pharmaceutical tablets.
Recently, the analytical community has shown increasing interest in developing more environment-friendly practices by eliminating or reducing the use of hazardous chemicals and solvents which are extremely dangerous to human health and the environment. Thus in this study, a simple, rapid, inexpensive and eco-friendly HPTLC method with densitometric detection was designed, optimized and validated for the simultaneous quantitation of the antiviral drugs sofosbuvir (SOF) and ledipasvir (LED) in pure powder, synthetic mixtures, combined pharmaceutical formulation and in the presence of their stressed degradation products with high sensitivity and no interference. The separation was carried out on silica gel F254 plates, using green mobile phase ethyl acetate: water: ethanol (94:5:1v/v/v) for development followed by densitometric detection of SOF and LED at 250 and 320 nm, respectively. Both drugs had second order polynomial calibration curves within ranges 100–5000 ng/spot and 100–3000 ng/spot for SOF and LED, respectively, with correlation coefficients >0.9998 and the limits of detection were 30 and 29 ng/spot and the limits of quantification were 90 and 88 ng/spot for SOF and LED, respectively. The method was validated in terms of linearity, accuracy, precision, selectivity, and robustness. The drugs were subjected to acidic and basic hydrolysis, oxidation and photolytic conditions as per ICH guidelines, followed by detection of the degradation products and structure elucidation of their fragments with the aid of the HPTLC-ESI- MS technique. The greenness of the proposed method was assessed using the analytical Eco-Scale as an assessment tool with a 93-point score. So, it is considered excellent green.
Introduction
Depending on their terpenoid and phenolic constituents plant resins can be classified as diterpenoid, triterpenoid or phenolic resins; thereby the profile of diterpenes and triterpenes ...is considered as genus‐ or even species‐specific.
Objectives
We aimed to develop a simple, rapid, inexpensive, sensitive and specific method for the identification of resin‐specific triterpenoid and phenolic compounds in plant resins using (HP)TLC (high‐performance) thin‐layer chromatography combined with APCI‐MS (atmospheric pressure chemical ionisation mass spectrometry) and post‐chromatographic detection reactions.
Methods
Twenty resin samples from different plant species were analysed. Different extraction procedures, post‐chromatographic detection reagents as well as various sorbents and solvents for planar chromatography were tested. To evaluate the potential of the optimised (HP)TLC‐APCI‐MS methods, parameter such as limit of detection (LOD) was determined for selected marker compounds.
Results
Our protocol enabled qualitative analyses of chemotaxonomic molecular markers in natural resins such as dammar, mastic, olibanum and benzoin. For the first time, the application of thionyl chloride‐stannic chloride reagent for a specific post‐chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic colour sequences. For triterpene acids, triterpene alcohols and phenolic compounds, detection limits of 2–20 ng/TLC zone and a system precision with a relative standard deviation (RSD) in the range of 3.9%–7.0% were achieved by (HP)TLC‐APCI‐MS. The applicability of the method for the analysis of resin‐based varnishes was successfully tested on a mastic‐based varnish. Thus, the method we propose is a helpful tool for the discrimination of resins and resin‐based varnishes with respect to their botanical origin.
In this study, we developed a simple, rapid, cost‐effective, sensitive and specific method for the identification of chemotaxonomic molecular markers in plant resins such as dammar, mastic, olibanum and benzoin using (HP)TLC combined with APCI‐MS and post‐chromatographic detection reactions. For the first time, the application of thionyl chloride‐stannic chloride reagent for a specific post‐chromatographic detection of triterpenes is reported, sometimes even allowing discrimination between isomers based on their characteristic color sequences.
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•β-lactam antibiotics are highly unstable, which may affect their antibacterial activity.•Stability studies using HPTC/MS and HPTLC/UV for following up β-Lactam antibiotics is ...demonstrated.•Ambient storing conditions are critical factors in maintenance of quality of different combinations of β-lactam antibiotics.•Decrease in the intact contents of unstable antibiotic may lead to bacterial resistance.•Degradation rate of Magna-biotic®, Unasyn® and Sulbacef® was fitted to first order degradation kinetics.
Stability testing of an active substance or final product is very crucial to ensure drug efficacy during shelf life. Simple and specific monitoring of β-lactam antibiotic combinations (with β-lactamase inhibitors) under ambient storage conditions was performed to better understand the kinetics of their degradation in powder for injection. The stored samples were analysed using HPTLC/MS and HPTLC/UV. The method employed HPTLC aluminum pre-coated plates with silica gel 60 F254 as the stationary phase. The used mobile phase systems consisted of ethyl acetate: acetonitrile: glacial acetic acid: water (5.5:3.0:2.0:1.0, v/v/v/v), for Magna-biotic® and ethyl acetate: acetonitrile: glacial acetic acid: water (5.0:3.0:2.0:1.0, v/v/v/v) for Unasyn® and Sulbacef®. The investigated mixtures were subjected to conditions resembling those found in a storage facility during different time intervals. The degradation behavior of powders for injection of the investigated mixtures, was found to be fitted to first-order kinetics, which is measured by observing the drug's starting concentration drop over time. The obtained results ensure the method's ability to assess the degradation kinetics of the tested combinations in the presence of their degradation products. The current study examines the drug's stability against high storage temperatures. MS detection was employed to elucidate the chemical structures of degradants and to confirm the suggested degradation pathway of the investigated mixtures in powder of injection. As a result, it is recommended that suitable protection measures against high temperatures must be followed during storage and handling of the investigated powder for injection of β-lactam antibiotics mixtures in order to maintain their biological efficiency.
•DESI/MS/HRToFMS was used to analyze and image plant extracts after RP-HPTLC.•Extracts of 3 Silene species were profiled for ecdysteroids using two solvent systems.•DESI/MS was used to image HPTLC ...plates after both 1 and 2-dimensional development.•A range of phytoecdysteroids were detected and identified and imaged in the extracts.•The additional resolution provided by 2-dimensional TLC increased MS data quality.
Reversed-phase high performance thin-layer chromatography (RP-HPTLC) on C18 bonded silica gel was combined with desorption electrospray ionization (DESI) and high resolution time of flight mass spectrometry (HRToFMS) to detect, characterize and image (MSI) phytoecdysteroids (plant-derived insect moulting hormones) in ethanolic extracts of members of the Silene plant family. As seen previously for silica gel, DESI provided a simple and convenient method for recovering polar polyhydoxysteroids from RP-HPTLC plates for the purposes of both the MS and MSI of extracts obtained from three species of the Silene family (Silene otites, S. nutans and S. viridiflora). Using RP-HPTLC/DESI/MSI/HRToFMS a number of ecdysteroids, including 20-hydroxyecdysone, polypodine-B, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone were identified in these extracts. Differences were noted in the mass spectra obtained depending upon both the stationary phase on which they were separated, and the temperatures used in the heated transfer line used for introduction into the ion source. Ecdysteroids detected after chromatography on C18 bonded silica showed increased fragmentation due to water loss compared to those imaged from silica. In addition, the benefits of the additional resolution provided by 2-dimensional TLC for increasing spectral quality compared to a 1-dimensional separation are demonstrated.
•Qualitative and quantitative comparison of wild-growing and cultivated elderberries.•Qualitative and quantitative comparison of dried and fresh elderberry extracts.•First effect directed analysis ...using five different (bio)assays.•Coupled to A. fischeri, B. subtilis, pYES, acetylcholinesterase and tyrosinase assays.•HPTLC–MS investigation of unknown anthocyanin zones and bioactive zones.
A healthy diet is an important factor in a healthy lifestyle that is becoming increasingly important in today's society. The fruits of European elder (Sambucus nigra L.) are a rich source of bioactive compounds like anthocyanins. In this study, dried and fresh fruits of four cultivated and six wild growing plants were investigated for their anthocyanin pattern and content as well as their bioactive compounds. After separation on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate, 2-butanone, formic acid and water, the plates were quantitatively evaluated by densitometry and also subjected to various (bio)assays to investigate the samples for compounds acting as radical-scavengers, antimicrobials, estrogens, and acetylcholinesterase or tyrosinase inhibitors. The mean contents for the two most abundant anthocyanins in European elderberries, confirmed by HPTLC–ESI-MS, ranged from 159 to 647mg/100g in fresh and from 166 to 2764mg/100g in dried fruits for cyanidin-3-sambubioside, and from 112 to 521mg/100g in fresh and 95 to 226mg/100g in dried fruits for cyanidin-3-glucoside. Additionally, the anthocyanin content was higher in berries of cultivars than of wild growing plants. The anthocyanins’ radical scavenging activity and antimicrobial effect against Aliivibrio fischeri were confirmed. Further, a radical scavenging compound affecting A. fischeri and acting as acetylcholinesterase inhibitor was tentatively assigned by its protonated molecule at m/z 456 as either ursolic or oleanolic acid by HPTLC–ESI-MS. HPTLC hyphenated with bioassays and mass spectrometry was selected as method of choice for fingerprinting, pattern recognition, and bioprofiling of elderberry samples as well as quantitation and confirmation of bioactive compounds therein.
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•Integrated standardization of propolis by chromatographic, spectroscopic, biological and palynological analyses was executed.•A dual visualization approach with four image analysis ...software adopting different algorithms were comparatively evaluated.•Multivariate analysis was used to discriminate between propolis types and to find health-relevant phytoconstituents.•HPTLC-ESI-MS were utilized for precise identification of the discriminatory phytoconstituents.•Pollen analysis was implemented to determine the botanical origin of Egyptian propolis for the first time.
An integrated method for standardization of propolis using chromatographic, spectroscopic, palynological and biological analyses was implemented in this study. Digitally-enhanced HPTLC images using dual visualization approach with four image analysis software packages adopting different digitalization algorithms were comparatively evaluated i.e.; Sorbfil TLC View®, ImageJ®, JustTLC® and Gel Analyzer®. ImageJ® and Gel Analyzer® showed superior figures of merits in case of flavonoids and phenolic acids quantitation, respectively. Unsupervised and supervised multivariable pattern recognition models were constructed for comprehensive discrimination between the three propolis types (blue, orange and green) collected from various geographical locations worldwide. HPTLC-ESI-MS was utilized for precise identification of the discriminatory phytoconstituents. Fingerprint-efficacy relationship analysis was conducted via an Orthogonal Projection to Latent Structure multivariate model to unravel the bio-efficient markers in terms of α-glucosidase and α-amylase inhibition as main targets. 3,4-Dimethoxycinnamic acid, caffeic acid, isoferulic acid, rosmarinic acid, and quercetin were found to be the main health-relevant markers. A complementary fast, simple and readily available UV spectroscopic method using aluminium chloride as bathochromic shift reagent was used as an independent method for prediction of aforementioned biomarkers using a validated Partial Least Squares Regression model. In addition, palynological analysis was implemented to determine the botanical origin of Egyptian propolis for the first time. Twenty-eight pollen types assigned to 13 families were identified, with Asteraceae being the highest representative one. The investigated samples lacked dominant pollen types, which reflected the multifloral origin of Egyptian propolis. Identification of plant sources of propolis, which directly affect its chemical composition and subsequently its biological efficacy represents an integral part of its standardization.
•First validated HPTLC method for quantitation of 11 anthocyanes.•First demonstration of HPTLC–MS analysis of unknown anthocyane zones.•First effect directed analysis with regard to Vibrio fischeri ...bioactivity response.•First analysis of anthocyanes in pomace and supplemented feed.•Reversed and normal phase separations of anthocyanes.
An efficient HPTLC method was developed, which required minimal sample preparation for quantitation of the main anthocyanes in pomace, animal feed as well as various foods. The best separation of 11 anthocyanes was achieved on HPTLC plates silica gel 60 F254 with a mixture of ethyl acetate–2-butanone–formic acid–water for anthocyanins and ethyl acetate–toluene–formic acid–water for anthocyanidins. Due to the high flexibility of the HPTLC method, both anthocyane groups could be developed in a combined 2-step method. The second development was only necessary if anthocyanidins were detected in the samples. This normal phase separation was found superior to the best separation achieved on RP-18 phases with a mixture of water–n-propanol–formic acid. Absorbance measurement was performed using the multi-wavelength scan at 505 (or 510), 520, 530 and 555nm. The correlation coefficients of the calibrations ranged between 0.9993 and 0.9999 for the 11 anthocyanes. LOQs were all ≤90ng/zone, most even ≤30ng/zone and for pn-3-glc and pg-3-glc even ≤7ng/zone. With regard to the analysis of mv-3-glc in grape seed/marc meal and supplemented animal feed samples, the mean repeatabilities were 1.4% (laboratory 1) and 1.8% (laboratory 2). The intermediate precisions within a laboratory over several months were ≤6.7%. The ruggedness of the method was ≤5.5%. The method was transferred to other sample types. Juice and wine samples, which were from the same plant source, showed a comparable anthocyanin pattern, whereas the pattern was characteristically different between plant sources. Unknown anthocyanin sample components were analyzed via HPTLC–ESI-MS by eluting the zones of interest with the TLC–MS Interface, which was helpful for further characterization of unknowns. An interesting tool was demonstrated by effect-directed analysis with regard to radical scavenging properties and general bioactivity based on detection with Vibrio fischeri bacteria.
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•Coffee beans roasted differently and coffees prepared differently were studied for effects.•Storage conditions influenced the bioactive potential of the coffee brews.•Antioxidative ...components were identified using HPTLC-EDA-MS.•Chlorogenic acid, caffeine, caffeoyl-β-d-glucopyranoside and melanoidines were highly active.•Caffeine and chlorogenic acid as important quality parameters were exemplarily quantified.
Coffee is an inherent part of our daily nutrition and seems to have protective effects against diseases, whereby it is often not fully understood, which ingredients are responsible for the observed effect. Hence, a non-targeted bioactivity profiling was developed to investigate 27 hand-filtered coffee brews of differently roasted coffee beans and 14 differently prepared and stored coffee brews. After separation, multi-imaging, and densitometry, six planar effect-directed assays were performed to reveal individual antioxidative, antibacterial, anti-cholinesterase, anti-diabetic, and estrogenic effects. Individual compounds were mainly responsible for the observed effects, e.g. 5-O-caffeoylquinic acid regarding antioxidative potential and α-glucosidase inhibition, while coffee brews made by a fully automated coffee machine showed the highest antioxidative potential. Unlike preparation and storage conditions, applied roasting conditions and origin of coffee samples played a less important role. Therefore, the way we daily consume our coffee has an impact on the magnitude of potential health effects.
•Fast and cost-effective analysis of steviol glycosides.•Minimized sample preparation.•First time HPTLC–ESI-MS of steviol glycosides.•First time benchmarking of methods for steviol glycosides.•First ...HPTLC method for analysis of steviol glycosides in food.
A high-performance TLC (HPTLC) method was newly developed and validated for analysis of 7 steviol glycosides in 6 different types of food and Stevia formulations. After a minimized one-step sample preparation, 21 samples were developed in parallel, allowing an effective food screening. Depending on the sample application volume, the method was suited to analyze food sample concentrations in the mg/kg range. LOQs of stevioside in natural yoghurt matrix spiked at 0.02, 0.13 and 0.2% were determined by the calibration curve method to be 12ng/band (peak height). ANOVA was successfully passed to prove data homogeneity in the working range (30–600ng/band). The accuracy (recovery tolerance limit, 92–120%), repeatability (3.1–5.4%) and intermediate precision (4.0–8.4%) were determined for stevioside in milk-based matrix including sample preparation and recovery rates at 3 different concentration levels. For the first time, the recording of HPTLC–ESI-MS spectra via the TLC-MS Interface was demonstrated for rebaudioside A. HPTLC contents for rebaudioside A were compared with results of two (U)HPLC methods. The running costs and analysis time of the three different methods were discussed in detail with regard to screening of food products.
HPTLC silica gel, amino and C18 plates in combination with different developing solvents were explored for the first HPTLC chemical profiles of flavonoids and sugars in crude extracts from leaves and ...fruits of caigua (Cyclanthera pedata Scrabs) harvested in Slovenia and Italy. New HPTLC and HPTLC-MS methods for analyses of flavonoids are based on HPTLC silica gel (preconditioned with water) or C18 plates in combination with developing solvents ethyl acetate-water-formic acid (17:3:2, v/v) or 5% formic acid in methanol-water (7:3, v/v), respectively. Detection was performed before (for flavonoids) and/or after post-chromatographic derivatization with Natural product (NP) reagent for flavonoids and diphenylamine-aniline-phosphoric acid (DAP) reagent for sugars. HPTLC-MS/(MS
n
) analyses on silica gel and C18 stationary phases enabled tentative identification of several compounds in crude extracts from leaves and fruits. HPTLC-DPPH
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assay combined with image analyses and HPTLC-MS/(MS
n
) analyses, applied for direct screening of antioxidant activity of separated chromatographic zones (compounds) on HPTLC silica gel plates, confirmed activity in several zones of all crude extracts, in which some active compounds were tentatively identified. Apigenin 6-C-glucoside (isovitexin) and luteolin 8-C-glucoside (orientin) were found in all crude extracts, but the last was the most active free radical scavenger. Both compounds were also identified in dietary supplement product produced from caigua fruits. Crude extracts from leaves showed much higher antioxidant activity than those from fruits. The highest antioxidant activity was determined for crude extract from leaves harvested in Slovenia, while among crude extract from fruits the highest activity was determined for the extract from fruits harvested in Italy.
