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

Chromatography Chronicles: Unveiling the Power of Reversed-phase High-performance Thin Layer Chromatography in Pharmaceutical Analysis

Pharmaceutical analysis is critical in ensuring the quality and safety of drug substances and formulations. High-performance thin-layer Chromatography (HPTLC) has emerged as a powerful analytical technique in the pharmaceutical industry due to its numerous advantages, including high separation efficiency, cost-effectiveness, and ease of sample preparation. One of its variants, Reversed-Phase High-Performance Thin-Layer Chromatography (RP-HPTLC), has gained immense popularity for analyzing nonpolar and slightly polar compounds, including drugs and their metabolites. This review paper draws attention to history and the recent developments in RP-HPTLC for pharmaceutical analysis. It highlights the advantages and limitations of RP-HPTLC, discussing its applications in drug analysis, impurity determination, stability-indicating assays, and more. In this study, recent advances in RP-HPTLC instrumentation and techniques were reviewed, including hyphenated methods, such as Reversed-Phase High-Performance Thin-Layer Chromatography coupled with Mass Spectrometry and Reversed-Phase High-Performance Thin-Layer Chromatography coupled with Nuclear Magnetic Resonance. Through this comprehensive analysis, the authors aim to underscore the potential of RP-HPTLC as a reliable and efficient analytical technique in the pharmaceutical industry and shed light on future trends in this field.

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).

A greener RP-HPTLC-densitometry method for the quantification of apremilast in nanoformulations and commercial tablets: Greenness assessment by analytical eco-scale, ChlorTox, and AGREE methods

The greener “high-performance thin-layer chromatography (HPTLC)” methods for apremilast (APM) analysis in pharmaceutical products and biological fluids are not currently available in the literature. Accordingly, this study involves the development and validation of a rapid, simple, economical, and greener reversed-phase HPTLC methodology for the estimation of APM in prepared nanoparticles (NPs), nanoemulsion, and marketed tablets. The estimation of APM was conducted using “RP-18 silica gel 60 F254S HPTLC plates” as the stationary phase. The combination of ethanol/water (65:35, v/v) was used as the greener mobile phase for APM analysis. The greenness of the method was predicted using three different approaches, namely Analytical Eco-Score (AES), ChlorTox, and the Analytical GREENness (AGREE) approaches. The λmax = 238 nm was used for the APM detection. By contrasting its single band at Rf = 0.61 ± 0.01 with those of pure APM, the HPTLC peaks for APM in an prepared NP formulation, nanoemulsion, and marketed tablets were identified. In the concentration range of 100–700 ng/band, the proposed analytical methodology was linear. The values of AES, ChlorTox, and AGREE were determined to be 93, 0.66 g, and 0.89, respectively, demonstrated an outstanding greener profile for the existing method. The amount of APM in the tablet, NP formulation, and nanoemulsion was found to be 98.40, 101.60, and 99.37 %, respectively. According to the findings of validation tests and pharmaceutical analysis, the suggested analytical technique could be successfully applied for the routine examination of APM in marketed tablets and laboratory generated nanoformulations.

Chemical and cytotoxicity profiles of 11 pink pepper (Schinus spp.) samples via non-targeted hyphenated high-performance thin-layer chromatography

IntroductionPink pepper is a worldwide used spice that corresponds to the berries of two species, Schinus terebinthifolia Raddi or S. molle L. (Anacardiaceae). Toxic and allergic reactions by ingestion or contact with these plants were reported, and classical in vitro studies have highlighted the cytotoxic properties of apolar extracts from the fruits.ObjectivesPerform a non-targeted screening of 11 pink pepper samples for the detection and identification of individual cytotoxic substances.MethodsAfter reversed-phase high-performance thin-layer chromatography (RP-HPTLC) separation of the extracts and multi-imaging (UV/Vis/FLD), cytotoxic compounds were detected by bioluminescence reduction from luciferase reporter cells (HEK 293 T-CMV-ELuc) applied directly on the adsorbent surface, followed by elution of detected cytotoxic substance into atmospheric-pressure chemical ionization high-resolution mass spectrometry (APCI-HRMS).ResultsSeparations for mid-polar and non-polar fruit extracts demonstrated the selectivity of the method to different substance classes. One cytotoxic substance zone was tentatively assigned as moronic acid, a pentacyclic triterpenoid acid.ConclusionThe developed non-targeted hyphenated RP-HPTLC–UV/Vis/FLD–bioluminescent cytotoxicity bioassay–FIA–APCI-HRMS method was successfully demonstrated for cytotoxicity screening (bioprofiling) and respective cytotoxin assignment.

Normal- and reversed-phase high-performance thin-layer chromatography methods for the simultaneous determination of remogliflozin etabonate and metformin hydrochloride antidiabetic drugs in bulk and tablet formulation

Normal- and reversed-phase high-performance thin-layer chromatography methods for the simultaneous determination of remogliflozin etabonate and metformin hydrochloride antidiabetic drugs in bulk and tablet formulation

Simultaneous Detection of Chlorzoxazone and Paracetamol Using a Greener Reverse-Phase HPTLC-UV Method

In the literature, greener/eco-friendly analytical techniques for simultaneous estimation of chlorzoxazone (CZN) and paracetamol (PCT) are scarce. As a consequence, greener reverse-phase high-performance thin-layer chromatography with ultraviolet (HPTLC-UV) detection was developed and validated for simultaneous estimation of CZN and PCT in commercial capsules and tablets. The greenness of the proposed HPTLC-UV technique was assessed quantitatively by utilizing the “Analytical GREENness (AGREE)” methodology. For simultaneous estimation of CZN and PCT, the greener HPTLC-UV technique was linear in the 40–1600 ng band−1 and 30–1600 ng band−1 ranges, respectively. Furthermore, the suggested HPTLC-UV methodology proved sensitive, accurate, precise, and robust for simultaneous detection of CZN and PCT. The assay of CZN in marketed capsules and tablets was found to be 99.01 ± 1.53 and 100.87 ± 1.61%, respectively, using the suggested HPTLC-UV method. The assay of PCT in commercial capsules and tablets was found to be 98.31 ± 1.38 and 101.21 ± 1.67%, respectively. The AGREE index for the greener HPTLC-UV technique was found to be 0.79, suggesting an excellent greenness profile for the proposed HPTLC-UV technique. These results and data suggested the suitability of the greener HPTLC-UV methodology for simultaneous estimation of CZN and PCT in commercial formulations.

Ecofriendly Validated RP-HPTLC Method for Simultaneous Determination of the Bioactive Sesquiterpene Coumarins Feselol and Samarcandin in Five Ferula Species Using Green Solvents

An environmentally friendly unreported rapid and simple reverse-phase high-performance thin-layer chromatography (RP-HPTLC) has been designed for the simultaneous determination of bioactive sesquiterpene coumarins feselol and samarcandin in the methanol extract of five Ferula species. The method was developed using glass plates coated with RP-18 silica gel 60 F254S and a green solvent system of ethanol–water mixture (8:2 v/v) as mobile phase. After development, the plates were quantified densitometrically at 254 for feselol and samarcandin. Feselol and samarcandin peaks from methanol extract of five Ferula species were identified by comparing their single band at Rf = 0.43 ± 0.02 and Rf = 0.60 ± 0.01, respectively. Valid linear relationships between the peak areas and concentrations of feselol and samarcandin in the range of 1000–7000 ng/band respectively were obtained. The method was subjected to the validation criteria of the international conference on harmonization (ICH) for precision, accuracy, and robustness. The new method provides an analytical tool to enumerate the therapeutic doses of feselol and samarcandin in herbal formulations and/or crude drugs. The obtained results indicated that F. drudeana was the richest species in the more active samarcandin, with 0.573% w/w, while F. duranii had the largest quantity of the less active feselol, 0.813% w/w. F. drudeana was superior to the other species in the sum of the two active compounds, 1.4552% w/w, and was consequently expected to be the most active aphrodisiac among the five studied species.

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.

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.

High performance Reversed-Phase Thin-Layer Chromatography-Desorption electrospray ionisation - time of flight high resolution mass spectrometric detection and imaging (HPTLC/DESI/ToFMS) of phytoecdysteroids

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.

Simultaneous Estimation of Telmisartan, Chlorthalidone, Amlodipine Besylate and Atorvastatin by RP-HPLC Method for Synchronous Assay of Multiple FDC Products Using Analytical FMCEA-Based AQbD Approach.

Numerous reversed-phase high-pressure liquid chromatography (RP-HPLC) and high-performance thin-layer chromatography (HPTLC) techniques have been published for the estimation of fixed-dose combinations (FDCs) of telmisartan (TEL). No published literature has been reported to date which described the synchronous estimation of FDCs of TEL using a single chromatography condition. Hence, the RP-HPLC method has been developed and validated for synchronous analysis of FDCs of TEL using an enhanced analytical quality by design (AQbD) approach to save time, cost and solvent for analysis. The implementation of AQbD was initiated with the identification of failure modes (FMs) using the Ishikawa diagram, and their critical effect analysis was carried out by risk priority number ranking and filtering method. The identified critical FMs were optimized by design of experiments-based response surface modeling using the Box-Behnken design. The method operable design region was navigated and control strategy was framed to mitigate the risk of critical FM. The RP-HPLC method was developed using Shim-Pack octadecyl silane C18 column and acetonitrile: 1.0%v/v triethylamine (pH6.5 adjusted using perchloric acid; 42:58, %v/v). The developed method was found to be validated as per the International Council For Harmonization Q2 (R1) guideline. The method was applied for the synchronous assay of seven different FDCs of TEL and assay results were found in good compliance with the respective labeled claim.

A Green High-Performance Thin-Layer Chromatography Method for the Determination of Caffeine in Commercial Energy Drinks and Formulations.

The literature on green analytical approaches for caffeine estimation is limited. As a consequence, this study aimed to establish a reverse-phase high-performance thin-layer chromatography (HPTLC) technique for caffeine estimation in a variety of commercial energy drinks (ED) and pharmaceutical formulations that is rapid, sensitive, and green. The combination of ethanol-water (55:45 v v−1) was used as a mobile phase. The detection of caffeine was carried out at 275 nm. The green reverse-phase HPTLC method was linear in the concentration range of 50–800 ng band−1. Furthermore, the developed method for caffeine estimation was simple, quick, economical, accurate, precise, robust, sensitive, and green. The amount of caffeine in different marketed ED (ED1–ED10) was recorded in the range of 21.02–37.52 mg 100 mL−1 using the developed HPTLC method. However, the amount of caffeine in different commercial formulations (F1–F3) was estimated as 10.63–20.30 mg 100 mL−1 using the same method. The “analytical GREEnness (AGREE)” scale for the developed analytical method was predicted to be 0.80, utilizing 12 distinct components of green analytical chemistry, indicating the HPTLC approach’s excellent greener profile. Overall, the developed method for estimating caffeine in marketed ED and dosage forms was found to be reliable.

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.

A Greener HPTLC Approach for the Determination of β-Carotene in Traditional and Ultrasound-Based Extracts of Different Fractions of Daucus carota (L.), Ipomea batatas (L.), and Commercial Formulation

Various analytical approaches for determining β-carotene in vegetable crops and commercial dosage forms have been documented. However, neither the qualitative nor quantitative environmental safety and greener aspects of the literature analytical methodologies of β-carotene analysis have been assessed. As a result, the goal of this research is to develop and validate a reversed-phase “high-performance thin-layer chromatography (HPTLC)” approach for determining β-carotene in traditional (TE) and ultrasound-assisted (UBE) extracts of different fractions of Daucus carota (L.), Ipomea batatas (L.), and commercial formulation. The greener mobile phase for β-carotene analysis was a ternary mixture of ethanol, cyclohexane, and ammonia (95:2.5:2.5, v v v−1). The detection of β-carotene was done at a wavelength of 459 nm. In the 25–1000 ng band−1 range, the greener reversed-phase HPTLC approach was linear. Other validation factors for β-carotene analysis, including as accuracy, precision, robustness, and sensitivity, were likewise dependable. The contents of β-carotene were found to be maximum in hexane: acetone (50:50%) fractions of TE and UBE of D. carota and I. batatas compared to their acetone and hexane fractions. The amount of β-carotene in hexane: acetone (50:50%) portions of TE of D. carota, I. batatas and commercial formulation A was estimated to be 10.32, 3.73, and 6.73 percent w w−1, respectively. However, the amount of β-carotene in hexane: acetone (50:50%) portions of UBE of D. carota, I. batatas and commercial formulation A was estimated to be 11.03, 4.43, and 6.89 percent w w−1, respectively. The greenness scale for the proposed HPTLC strategy was calculated as 0.81 using the “analytical GREEnness (AGREE)” method, indicating that the proposed HPTLC methodology has good greenness. The UBE approach for extracting β-carotene outperformed the TE procedure. These results indicated that the greener reversed-phase HPTLC approach can be utilized for the determination of β-carotene in different vegetable crops, plant-based phytopharmaceuticals, and commercial products. In addition, this approach is also safe and sustainable due to the utilization of a greener mobile phase compared to the toxic mobile phases utilized in literature analytical approaches of β-carotene estimation.

Separation of some vitamins in reversed-phase thin-layer chromatography and pressurized planar electrochromatography with eluent containing surfactant

The separation of some water- and fat-soluble vitamins via micellar systems of reversed-phase high-performance thin-layer chromatography (HPTLC) and pressurized planar electrochromatography (PPEC) was subjected to research. Hence, the influence of the mobile phase composition (surfactant and acetonitrile concentration, eluent buffer pH) on the migration distances and zone separation of some vitamins (thiamine, riboflavin, niacin, pyridoxine, cyanocobalamin, folic acid, ergocalciferol and α-tocopherol) was investigated. Our results indicated that the applied technique has an impact on the solute order. Comparing the system capacity of HPLC and PPEC (measured as height of the theoretical plate) for the mobile phase systems with and without surfactant shows differences, especially for fat-soluble vitamin. The variances and reproducibilities (% RDS) values of the vitamin are less in PPEC than in TLC. Moreover, the migration distances of water-soluble vitamins are longer than fat-soluble ones. Overall, eluent consisting of 50% acetonitrile, 18.75 mM SDS, the buffer of pH 6.99 via the PPEC technique was most appropriate for determining the investigated vitamins in the artificial mixture and the two commercially available vitamin combinations.

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.

Highly Sensitive and Ecologically Sustainable Reversed-Phase HPTLC Method for the Determination of Hydroquinone in Commercial Whitening Creams

Hydroquinone (HDQ) is a natural depigmenting agent, which is commonly used in skin-toning preparations. The safety and greenness of analytical methods of HDQ quantification were not considered in previous literature. Therefore, a highly sensitive and ecologically greener reversed-phase high-performance thin-layer chromatography (RP-HPTLC)-based assay was established for HDQ estimation in four different commercial whitening creams (CWCs). The binary ethanol–water (60:40, v·v−1) mixture was utilized as the green solvent system. The estimation of HDQ was carried out at 291 nm. The present RP-HPTLC-based assay was linear in the 20–2400 ng band−1 range. The present analytical method was highly sensitive based on the detection and quantification data. The other validation parameters, such as accuracy, precision, and robustness, were also suitable for the determination of HDQ. Maximum HDQ quantities were obtained in CWC A (1.23% w·w−1) followed by CWC C (0.81% w·w−1), CWC D (0.43% w·w−1), and CWC B (0.37% w·w−1). The analytical GREEnness (AGREE) score for the present analytical method was estimated as 0.91, indicating the excellent greener characteristics of the present RP-HPTLC assay. These results suggest that the present analytical method is highly sensitive and ecologically sustainable for the quantitation of HDQ in its commercial formulations.

A Sustainable Reversed-Phase HPTLC Method for the Quantitative Estimation of Hesperidin in Traditional and Ultrasound-Assisted Extracts of Different Varieties of Citrus Fruit Peels and Commercial Tablets

Hesperidin (HSP) is a bioactive flavanone glycoside, present abundantly in the variety of citrus fruits. The environmental safety and sustainability of the reported analytical assays of HSP analysis have not been considered in the literature. Hence, a sensitive and sustainable “reversed-phase high-performance thin-layer chromatography (RP-HPTLC)” method has been developed and validated for HSP analysis in traditional (TE) and ultrasound-based (UBE) extracts of four different varieties of citrus fruit peels and its commercial tablet dosage forms. The binary combination of green solvents such as ethanol-water (50:50, v v−1) was used as the mobile phase. The detection of HSP was performed at 287 nm. The sustainable RP-HPTLC method was linear in 20–2000 ng band−1 range. The studied validation parameters, including accuracy, precision, robustness, sensitivity were acceptable for HSP analysis. The content of HSP in TE of four different varieties of citrus fruits including grapefruit peels (Citrus paradisi), mosambi peels (Citrus limetta), lemon peels (Citrus lemon), and orange peels (Citrus sinensis) was detected as 8.26, 6.94, 5.90, and 6.81% w w−1, respectively. The content of HSP in TE of commercial formulations A and B was detected as 5.31 and 5.55% w w−1, respectively. However, the content of HSP in UBE of grapefruit peels, mosambi peels, lemon peels, and orange peels was detected as 11.41, 8.86, 7.98, and 8.64% w w−1, respectively. The content of HSP in UBE of commercial formulations A and B was detected as 6.72 and 6.92% w w−1, respectively. The greenness score of the sustainable RP-HPTLC method was predicted as 0.83 using analytical GREEnness (AGREE) metric approach, indicated the excellent greenness profile of the RP-HPTLC method. UBE procedure for HSP was superior over its TE procedure. These observations and results suggested that the present RP-HPTLC method can be successfully used for the quantitative estimation of HSP in the variety of citrus fruit peels and its commercial formulations. In addition, this method is simple, rapid, precise, accurate, and economical compared to the reported analytical methods of HSP analysis. It is also safe and sustainable method due to the use of ethanol-water solvents systems, as both the solvents are green solvents compared to the solvents used in reported analytical methods of HSP analysis.

Rapid, Sensitive, and Sustainable Reversed-Phase HPTLC Method in Comparison to the Normal-Phase HPTLC for the Determination of Pterostilbene in Capsule Dosage Form

The greenness evaluation of literature analytical methods for pterostilbene (PT) analysis was not performed. Accordingly, the rapid, sensitive, and green/sustainable reversed-phase high-performance thin-layer chromatography (RP-HPTLC) method was developed and compared to the normal-phase (NP)-HPTLC (NP-HPTLC) for the estimation of PT with a classical univariate calibration. The RP quantification of PT was performed using green solvent systems; however, the NP analysis of PT was performed using routine solvent systems. The PT was detected at 302 nm for both of the methods. The greenness scores for the current analytical assays were evaluated by the analytical GREEnness (AGREE) metric approach. The classical univariate calibration for RP and NP methods indicated the linearity range as 10–1600 and 30–400 ng band−1, respectively. The RP method was more reliable for PT analysis compared to the NP method. The PT contents in commercial capsule dosage form were found to be 100.84% using the RP method; however, the PT contents in commercial capsule dosage form were determined as 92.59% using the NP method. The AGREE scores for RP and NP methods were 0.78 and 0.46, respectively. The sustainable RP-HPTLC assay was able to detect PT in the presence of its degradation products, and hence it can be considered as a selective and stability-indicating method. Accordingly, the RP-HPTLC method with univariate calibration has been considered as a superior method over the NP-HPTLC method for PT analysis.

Eco-friendly stability-indicating RP-HPTLC method for sildenafil analysis, characterization and biological evaluation of its oxidized stress degradation product

A feasible and cost effective reverse-phase high-performance thin layer chromatography (RP-HPTLC) based method was developed for the quantification of sildenafil (SLD) using eco-friendly EtOH:H2O (9.5:0.5 v/v) as mobile phase. SLD was subjected to stress conditions according to the International Conference on Harmonization (ICH) guidelines. The drug undergoes significant structural changes under oxidative stress condition to the N-oxide derivative. The oxidation product Sildenafil N-oxide (SDL N-oxide) designated in the European Pharmacopeia (EP) as impurity B was characterized utilizing 1D- and 2D-NMR as well as High Resolution Electrospray Ionization Mass Spectroscopy. The aphrodisiac potency of SDL N-oxide in comparison with SLD was evaluated in vivo using rats as experimental animal model. The evaluation based on the following parameters: mount, intromission and ejaculation latencies (ML, IL and EL, respectively), mounting and intromission frequencies (MF and IF, respectively), and postejaculatory interval (PEI). SLD N-oxide expressed similar aphrodisiac effect to SLD but with less potency. Molecular docking of SDL N-oxide along with the parent drug SLD, indicated a strong binding affinity and similar binding pattern within the active site of phosphodiesterase 5 (PDE5). However, the docking score of SLD N-oxide was slightly lower as compared to SLD in agreement with the biological study findings.