Reliable High-performance Thin-layer Chromatography Research Articles

HPTLC quantification of garcinol in three endemic Garcinia species of Assam, estimation of secondary metabolites and evaluation of in vitro antioxidant activity

AbstractGarcinol, the main polyisoprenylated benzophenone present in Garcinia species. A simple, rapid, reliable high-performance thin-layer chromatography (HPTLC) technique has been developed for simultaneous detection and quantification of garcinol in three endemic Garcinia sp. of Assam, India. While G. mangostana and G. indica have garnered extensive recognition, these lesser-known species have not received proportionate level of scientific exploration, therefore has served as the subject of investigation. Chromatographic separation was achieved using pre-coated silica gel 60 F254 plates. The optimized mobile phase (Toluene-Ethyl acetate-Formic acid, 5:4:1V/V) at 276 nm produced well-defined and sharp peaks of garcinol, maintaining a constant RF value of 0.75. The regression equation demonstrated a linear relationship with a correlation coefficient of 0.9955. The method was validated in accordance with International Conference of Harmonization guidelines (ICH), encompassing assessments for precision, accuracy, repeatability and robustness, all of which yielded low percentage of relative standard deviation values, attesting to its excellent performance. The content of garcinol found in the extracts were in the range of 0.58–0.98% w/w. In secondary metabolite analysis, significant amount of phenols (26.06 ± 1.65–57.51 ± 2.62 gallic acid equivalent mg g−1), flavonoids (11.27 ± 1.06–21.09 ± 1.34 quercetin equivalent mg g−1) and ascorbic acid (3.56 ± 0.12–3.65 ± 0.11 mg/10 g) contents were detected in the extracts. Further, the extracts exhibited good anti-radical activity, which may be attributed to the presence of important secondary metabolites. Therefore, this research establishes the three endemic Garcinia sp. as promising source of garcinol. Additionally, the developed method can be applied for routine quality control analysis of herbal formulations containing garcinol.

High-Performance Thin-Layer Chromatography hyphenated with image processing and chemometrics as a tool for forensic discrimination of Cannabis sativa L. chemotypes

Cannabis sativa L. is the most widely cultivated, trafficked, and abused illicit drug. The reliable distinction between drug type and fiber type C. sativa remains a topic of interest for forensic chemists. For the first time, we applied a combination of simple, cost-effective, and reliable High-Performance Thin-Layer Chromatography (HPTLC) with image analysis as well as sophisticated multivariate tools for differentiating C. sativa drug, fiber and intermediate chemotypes. After extraction, major cannabinoids from 43 seized C. sativa were separated using the HPTLC method. Peak areas of Δ9-THC, CBN, and CBD were calculated by simple image processing, and Xfactor = [THC + CBN]/CBD was determined as criteria used to discriminate three C. sativa chemotypes. The obtained results were compared with Gas Chromatography with Flame Ionization Detection (GC-FID) as the reference method for the determination of Xfactor.Principal Component Analysis and Hierarchic Cluster Analysis were applied to classify three C. sativa chemotypes according to their chemical pattern. The proposed approach clearly distinguishes 26 drugs, 13 fiber, and 4 intermediate chemotypes in seized C. sativa, aligning with GC-FID analysis. The results showed that the HPTLC technique in combination with multivariate methods is an accurate and reliable tool for high-throughput and forensic screening of three C. sativa chemotypes. Compounds such as Δ9-THC and CBD were marked as the most important cannabinoids responsible for the classification of the seized C. sativa chemotypes.

Validation of a high-performance thin-layer chromatography method for the quantitative determination of trehalulose

A novel, simple and reliable high-performance thin-layer chromatography (HPTLC) method was developed and validated for the quantification of trehalulose in stingless bee honey. The chromatographic separation was performed using silica gel 60 F254 HPTLC plates and 1-butanol‒2-propanol‒aqueous boric acid solution (5 mg/mL) (30:50:10, V/V) as the mobile phase. The retardation factor (RF) for trehalulose was found to be 0.045. The method showed linearity over the concentration range of 100–800 ng per band with a coefficient of correlation (R) of 0.9996. The limit of detection and limit of quantification for trehalulose were found to be 20.04 ng per band and 60.72 ng per band, respectively and the mean per cent recovery of trehalulose was 101.8%. The method has been validated for its specificity, linearity, sensitivity, precision, accuracy, repeatability and robustness following the International Council for Harmonisation Q2 (R1), and it has been successfully applied in the determination of trehalulose in stingless bee honey.

Simultaneous determination of lupeol and β-sitosterol by high-performance thin-layer chromatographic method in Crataeva nurvala Buch-Ham. stem bark

Crataeva nurvala Buch-Ham. (Capparidaceae) is an important medicinal plant, the bark of which is extensively used in Ayurveda as a remedy for kidney stones, urinary tract infections, prostate-related disorders, and benign prostatic hypertrophy. In this study, a sensitive and reliable high-performance thin-layer chromatography (HPTLC) method has been developed for the simultaneous quantification of two bioactive markers, lupeol and β-sitosterol, in the stem bark of C. nurvala using precoated silica gel G 60 F254 HPTLC plates as the stationary phase and toluene–methanol (9.6:0.4, v/v) as the mobile phase. Detection and quantification were carried out by densitometric scanning at 545 nm after derivatization using 1% anisaldehyde–sulfuric acid reagent. The method was validated for linearity, inter-day and intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The linear regression analysis data for the calibration plots showed a good linear relationship (r2 = 0.987–0.990) in the concentration range of 2 to 6 μg for lupeol and β-sitosterol, both with respect to areas. The proposed method provides good resolution of lupeol and β-sitosterol from other constituents present in the stem bark of C. nurvala. It was found to be simple, accurate, precise, and reproducible and would potentially be a useful tool for routine analysis of C. nurvala.

Quantitative and chemical fingerprint analysis for quality control of Zingiber zerumbet based on HPTLC combined with chemometric methods

A simple, reliable high-performance thin-layer chromatography (HPTLC) method was developed for chemical fingerprinting of Zingiber zerumbet and quantitative estimation of zerumbone. Thirty-six batches of Z. zerumbet were collected from five eco-regions of eastern India. Zerumbone content varied from 52.4 to 214.6 mg/g (dry weight) in methanolic extract of Z. zerumbet rhizomes. Zerumbone content was in the following order: moist deciduous forests of the lower Gangetic plains > Brahmaputra valley evergreen forest > Odisha semi-evergreen forests > Sundarbans freshwater swamp forests > moist deciduous forest of the eastern highlands. Relative Standard Deviation (RSD) of the relative peak areas (RPA) and relative retention times (RRT) of eight characteristic peaks in repeatability and stability test were <3%, and the fingerprinting method was confirmed to be suitable for Z. zerumbet rhizomes. Chemometric approaches like hierarchical cluster analysis (HCA) and principal component analysis (PCA) were employed to classify Z. zerumbet samples based upon their eco-region. Consistent results were achieved showing Z. zerumbet samples could be effectively grouped according to their eco-region. The PCA loading plots identified three probable chemical markers, which might be useful in discriminating the samples. This combinative approach could be used for quality assessment of Z. zerumbet and for the formulations containing zerumbone.

A validated high-performance thin-layer chromatography method for quantification of bavachin, bakuchiol, and psoralen from Psoralea corylifolia seeds

A sensitive and reliable high-performance thin-layer chromatography method has been developed for the simultaneous estimation of bavachin, bakuchiol, and psoralen in Psoralea corylifolia seeds. The methanolic extract of the seeds was applied on a silica gel G 60 F254 plate. The plate was developed using toluene–ether, 1:1, saturated with 10% acetic acid (v/v) as the mobile phase and detection and quantification were performed by densitometric scanning at 254 nm. The correlation coefficients were found to be 0.999, 0.999, and 0.999 for bavachin, bakuchiol, and psoralen, respectively, in the developed method. The accuracy of the method was confirmed by conducting recovery studies at different levels using the standard addition method. The average recovery of bavachin, bakuchiol, and psoralen was found to be close to 98% confirming the accurateness of the method. The proposed method offers a new, sensitive, specific, and precise gauge for quantification of bavachin, bakuchiol, and psoralen representing all the categories of bioactive constituents in P. corylifolia.

DEVELOPMENT AND VALIDATION OF A HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY FOR THE DETERMINATION OF TERBUTALINE SULFATE, BROMHEXINE HYDROCHLORIDE, AND ETOPHYLLINE IN PHARMACEUTICAL DOSAGE FORM

Objective: The study aimed to development and validation of simple, precise, and reliable high-performance thin-layer chromatography (HPTLC) for the determination of terbutaline sulfate (TBS), bromhexine hydrochloride (BRH), and etophylline (ETP) in pharmaceutical dosage form.&#x0D; Methods: A simple, precise, rapid, and accurate HPTLC method was developed for the estimation of TBS, BRH, and ETP in pharmaceutical dosage form. Pre-coated silica gel G60 F254 aluminum sheet (10 cm2×10 cm2 and thickness 0.2 mm) was used as stationary phase while mobile phase consisting of benzene: methanol:glacial acetic acid 8:0.5:1.5 v/v/v detection at 275 nm. The present method had validated according to ICH guidelines.&#x0D; Results: Migration distance found 80 mm at 275 nm. The retention factor found to be 0.24, 0.57, and 0.68, respectively. The detector response was linear in the concentration range of 60–210 ng/band, 2400–8400 ng/band, and 96–336 ng/band, respectively. The linear regression equation being Y=32.20x−562.9, Y=11.79x−1711, and Y=1.756x−5636, respectively. The limit of detection for TBS 0.677 μg, for BRH 8.123 μg, and for ETP 57.915 μg and limit of quantification to be 2.053, 24.617, and 175.5 μg, respectively, were found. The developed method validated by ICH guideline, i.e., accuracy, precision, robustness, specificity, and system suitability.&#x0D; Conclusion: In this study, we had developed a simple, fast, and reliable HPTLC method for the determination of TBS, BRH, and ETP in pharmaceutical dosage form.

A simple method to discriminate Guangchenpi and Chenpi by high-performance thin-layer chromatography and high-performance liquid chromatography based on analysis of dimethyl anthranilate

Citri Reticulatae Pericarpium (CRP), the dried pericarp of Citrus reticulata Blanco, can be divided into “Guangchenpi” (GCP, the dried pericarps derived from Citrus reticulata ‘Chachi’) and “Chenpi” (CP, the dried pericarps derived from other cultivars of Citrus reticulata Blanco). To discriminate between GCP and CP, a simple and reliable high-performance thin-layer chromatography (HPTLC) method was firstly developed to analyze the volatile compound dimethyl anthranilate, and a high-performance liquid chromatography (HPLC) method was established to simultaneously quantify dimethyl anthranilate and three predominant flavonoids (hesperidin, nobiletin and tangeretin) in CRP samples. Both the HPTLC analysis and HPLC-orthogonal partial least squares discrimination analysis (OPLS-DA) indicated that GCP can be effectively distinguished from CP based on analysis of dimethyl anthranilate. Our results indicated that dimethyl anthranilate can be used as a marker compound for discrimination of GCP and CP. This work provided a convenient approach which might be applied for quality evaluation of CRP.

Quantification of herbal drug hypoxoside from the roots of South African Hypoxis hemerocallidea using cost-effective high-performance thin-layer chromatography–densitometry validated method

Hypoxis (Hypoxidaceae) consists of about 90 species of plants reported worldwide, of which 76 occur in Africa. As many as 41 species are indigenous to countries belonging to the Southern African Development Community (SADC), including South Africa. Of all the Hypoxis species, Hypoxis hemerocallidea has versatile application in traditional health care system of over 85% of South Africans and is regarded as one of the most ethnomedicinally important and most marketed species in South Africa. H. hemerocallidea corm’s water or alcoholic extract is widely used as traditional medicine for the treatment of benign hypertrophy and urinary tract infections as well as for boosting the immune system of people living with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS) among others. However, the use of other parts of Hypoxis plant as medicine is vital for conservation purposes. The roots attached on the corm of H. hemerocallidea contain hypoxoside, but the roots are usually ripped off during the preparation of Hypoxis-containing traditional medicines and other herbal products. A developed and validated, affordable but reliable high-performance thin-layer chromatography (HPTLC) densitometry for the rapid and repeatable visualization and quantitative determination of hypoxoside from the roots of H. hemerocallidea was performed. After thin-layer chromatography analysis, the hypoxoside resolved and was visualized at RF of 0.30 in CHCl3–MeOH–H20 (70:30:2 v/v). The method was linear with R2 of 0.9876 over a calibration range of 0.20 × 10−4–1.80 × 10−3 mg mL−1. The limits of detection (LOD) and quantification (LOQ) were 5.08 × 10−4 and 1.65 × 10−3 mg mL−1, respectively, while the percentage recovery and the method repeatability (%RSD) were 84.10 and 4.98, respectively. The roots of H. hemerocallidea were found to contain 4.101 × 10−4 mg mL−1 of hypoxoside.

Development and validation of a high-performance thin-layer chromatography—densitometry method for the quantitative estimation of morphine in the classical Ayurvedic formulation Kamini Vidrawan Ras

Kamini Vidrawan Ras is a classical Ayurvedic medicine, referred to in Bhaisjya Ratnavali, a book recognized by the Drugs and Cosmetics Act of India. Its usefulness has been stated in erectile dysfunction, impotence, and premature ejaculation. Opium is the major ingredient of formulation which is highly addictive as it contains narcotic alkaloids (morphine, codeine, and thebaine). Opium is a natural product; hence, the morphine content varies from 4–21%. The aim of this study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography— densitometry method for the quantitative estimation of morphine in the tablets of the said Ayurvedic medicines. Aluminum-backed silica gel F254 (20 cm × 10 cm) was used as the stationary phase and ethyl acetate, methanol, and ammonia (85:10:5, v/v) as the mobile phase. The RF value for morphine was 0.36, and the quantitative evaluation of the bands over plates was performed in the reflectance—absorbance mode at 280 nm. The regression anal...

Development of Validated High-performance Thin-layer Chromatography Method for Simultaneous Determination of Quercetin and Kaempferol in Thespesia populnea.

Introduction:Thespesia populnea L. (Family: Malvaceae) is a well-known medicinal plant distributed in tropical regions of the world and cultivated in South Gujarat and indicated to be useful in cutaneous affections, psoriasis, ringworm, and eczema. Bark and fruits are indicated in the diseases of skin, urethritis, and gonorrhea. The juice of fruits is employed in treating certain hepatic diseases. The plant is reported to contain flavonoids, quercetin, kaempferol, gossypetin, Kaempferol-3-monoglucoside, β-sitosterol, kaempferol-7-glucoside, and gossypol. T. populnea is a common component of many herbal and Ayurvedic formulation such as Kamilari and Liv-52.Objective:The present study aimed at developing validated and reliable high-performance thin layer chromatography (HPTLC) method for the analysis of quercetin and kaempferol simultaneously in T. populnea.Method:The method employed thin-layer chromatography aluminum sheets precoated with silica gel as the stationary phase and toluene: ethyl acetate: formic acid (6:4:0.3 v/v/v) as the mobile phase, which gave compact bands of quercetin and kaempferol.Result:Linear regression data for the calibration curves of standard quercetin and kaempferol showed a good linear relationship over a concentration range of 100-600 ng/spot and 500-3000 ng/spot with respect to the area and correlation coefficient (R2) was 0.9955 and 0.9967. The method was evaluated regarding accuracy, precision, selectivity, and robustness. Limits of detection and quantitation were recorded as 32.06 and 85.33 ng/spot and 74.055 and 243.72 ng/spot for quercetin and kaempferol, respectively.Conclusion:We concluded that this method employing HPTLC in the quantitative determination of quercetin and kaempferol is efficient, simple, accurate, and validated.

Development and validation of a high-performance thin-layer chromatographic-densitometric method for the quantification of swertiamarin in traditional bitters and formulations

Summary Plant bitters, traditionally used against loss of appetite and fever, owe their therapeutic efficacy to the content of iridoids such as swertiamarin. The aim of the study was to develop a simple, precise, rapid, and reliable high-performance thin-layer chromatography (HPTLC) method for the estimation of swertiamarin in traditional bitters and formulations. Aluminum-backed silica gel 60 F 254 plates (20 × 10 cm) were used as stationary phase and ethyl acetate– methanol–water (80:15:5, v/v) as the mobile phase. Densitometric quantification was carried out at 246 nm. Regression analysis of the calibration plot showed good linear relationship between peak-area vs. swertiamarin concentration. Linearity was found to be in the range of 200–1200 ng per band. The method was validated as per the International Conference on Harmonization (ICH) guidelines. The limit of detection (LOD) and quantification (LOQ) for swertiamarin were found to be ~38 and ~115 ng per band, respectively. The developed method was found to be linear (r 2 = 0.9981), precise (relative standard deviation [RSD] ≤1.42% and ≤1.83% for intra-day and inter-day precision), accurate (mean recovery ranged from 97.69 ± 1.46 to 100.03 ± 0.61%), specific, and robust. The results showed good correlation with data obtained from HPLC analysis used as the reference method. The method was employed with a high degree of precision and accuracy for the estimation of swertiamarin in Enicostemma littorale, Swertia chirata, and an in-house formulation.

Method development for simultaneous detection of ferulic acid and vanillin using high-performance thin layer chromatography

Abstract Background A simple, accurate, and reliable high-performance thin-layer chromatography (HPTLC) method was developed for separation and detection of ferulic acid and vanillin. Methods Separation of ferulic acid and vanillin was carried out on 20 × 10 cm thin layer chromatography (TLC) plates using mobile phase containing toluene/1, 4-dioxan/acetic acid in the ratio 9:2.5:0.4 (v/v). The FA and vanillin were scanned at 320 and 312 nm, respectively. Method was validated for linearity, accuracy, precision, robustness, limit of detection, limit of quantification, and specificity. Results Retention factor (Rf) obtained for ferulic acid and vanillin was 0.48 and 0.56, respectively. The correlation coefficients, 0.9975 and 0.9991 with an average recovery of 98.77% and 98.45% obtained for ferulic acid and vanillin respectively by this method were satisfactory. Conclusion The optimized method was found to be efficient, precise, accurate, specific, and economic. Therefore, the method would be useful for both qualitative and quantitative routine analysis in pharmaceutical, food industry, and research laboratories.

Ionic Liquids as Mobile Phase Additives for Feasible Assay of Naphazoline in Pharmaceutical Formulation by HPTLC-UV-Densitometric Method

A specific and reliable high-performance thin layer chromatography method with densitometry detection has been developed for the determination of naphazoline nitrate in nasal drops. The best separation of the basic analyte, without spot tailing, was achieved by using a mobile phase composed of acetonitrile-water (60:40, v/v), adding 1.5 % (v/v) imidazolium-class ionic liquid and covering the plates with a stationary phase based on RP-18 with F254S (10 × 20 cm). The presented results confirm that imidazolium tetrafluoroborate ionic liquids are efficient suppressors of free silanols, which are considered to be responsible for troublesome and irreproducible chromatographic determinations of basic compounds. The developed chromatographic system was found to be convenient in use and to provide a repeatable assay of naphazoline nitrate in nasal drops, which could not be obtained with the use of standard silanol suppressing mobile phase additives such as triethylamine or dimethyloctylamine.