{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,8]],"date-time":"2026-01-08T20:59:19Z","timestamp":1767905959956,"version":"3.49.0"},"reference-count":50,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,2,9]],"date-time":"2022-02-09T00:00:00Z","timestamp":1644364800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006261","name":"Taif University","doi-asserted-by":"publisher","award":["TURSP-2020\/124"],"award-info":[{"award-number":["TURSP-2020\/124"]}],"id":[{"id":"10.13039\/501100006261","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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 \u201chigh-performance thin-layer chromatography (HPTLC)\u201d 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\u20132000 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 \u201canalytical GREEnness (AGREE)\u201d 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.<\/jats:p>","DOI":"10.3390\/molecules27041164","type":"journal-article","created":{"date-parts":[[2022,2,9]],"date-time":"2022-02-09T21:22:15Z","timestamp":1644441735000},"page":"1164","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Determination of Thymol in Commercial Formulation, Essential Oils, Traditional, and Ultrasound-Based Extracts of Thymus vulgaris and Origanum vulgare Using a Greener HPTLC Approach"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6728-8614","authenticated-orcid":false,"given":"Ahmed I.","family":"Foudah","sequence":"first","affiliation":[{"name":"Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6109-0885","authenticated-orcid":false,"given":"Faiyaz","family":"Shakeel","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammed H.","family":"Alqarni","sequence":"additional","affiliation":[{"name":"Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4313-3896","authenticated-orcid":false,"given":"Abuzer","family":"Ali","sequence":"additional","affiliation":[{"name":"Department of Pharmacognosy, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0922-9819","authenticated-orcid":false,"given":"Sultan","family":"Alshehri","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9179-4373","authenticated-orcid":false,"given":"Mohammed M.","family":"Ghoneim","sequence":"additional","affiliation":[{"name":"Department of Pharmacy Practice, College of Pharmacy, AlMaarefa University, P.O. Box 71666, Ad Diriyah 13713, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7632-3426","authenticated-orcid":false,"given":"Prawez","family":"Alam","sequence":"additional","affiliation":[{"name":"Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173, Al-Kharj 11942, Saudi Arabia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"233","DOI":"10.4103\/0250-474X.91570","article-title":"Seasonal variation in essential oil content and composition of Thyme, Thymus serpyllum L. cultivated in uttarakhand hills","volume":"73","author":"Verma","year":"2011","journal-title":"Indian J. Pharm. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1186\/s13568-017-0344-y","article-title":"Thymol and carvacrol induce autolysis, stress, growth inhibition and reduce the biofilm formation by Streptococcus mutans","volume":"7","author":"Khan","year":"2017","journal-title":"AMB Express."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1688","DOI":"10.1002\/ptr.6109","article-title":"Thymol, thyme, and other plant sources: Health and potential uses","volume":"32","author":"Salehi","year":"2018","journal-title":"Phytother. Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1186\/s13568-019-0893-3","article-title":"Chemical diversity in leaf and stem essential oils of Origanum vulgare L. and their effects on microbicidal activities","volume":"9","author":"Khan","year":"2019","journal-title":"AMB Express."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Grulova, D., Caputo, L., Elshafie, H.S., Baranova, B., De Martino, L., Sedlak, V., Gogalova, Z., Poracova, J., Camele, I., and De Feo, V. (2020). Thymol chemotype Origanum vulgare L. essential oil as a potential selective bio-based herbicide on monocot plant species. Molecules, 25.","DOI":"10.3390\/molecules25030595"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"12016","DOI":"10.3390\/molecules200712016","article-title":"Studies on chemical composition, antimicrobial and antioxidant activities of five Thymus vulgaris L. essential oils","volume":"20","author":"Mancini","year":"2015","journal-title":"Molecules"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"380","DOI":"10.3389\/fphar.2017.00380","article-title":"Pharmacological properties and molecular mechanisms of thymol: Prospects for its therapeutic potential and pharmaceutical development","volume":"8","author":"Meeran","year":"2017","journal-title":"Front. Pharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/s00210-013-0917-5","article-title":"Characterization of anticonvulsant and antiepileptogenic potential of thymol in various experimental models","volume":"387","author":"Sancheti","year":"2014","journal-title":"Arch. Pharmacol."},{"key":"ref_9","first-page":"28","article-title":"Spectrophotometric assay of thymol in various samples by coupling with diazotized p-nitroaniline","volume":"18","author":"Bashir","year":"2007","journal-title":"Raf. J. Sci."},{"key":"ref_10","first-page":"99","article-title":"Determination of thymol in pure and pharmaceutical preparation by diazotization-coupling method with 2,4-dichloroaniline as the coupling agent","volume":"2","author":"Dhahir","year":"2011","journal-title":"J. Kufa Chem. Sci."},{"key":"ref_11","first-page":"27","article-title":"Spectrophotometric determination of thymol in pharmaceutical preparation via oxidative coupling reaction with 2,4-dinitrophenylhydrazine in the presence of potassium periodate","volume":"55","author":"Fadhil","year":"2014","journal-title":"Iraqi J. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"129","DOI":"10.13057\/nusbiosci\/n130117","article-title":"Thymol quantitative analysis in medicinal formulation types through employing of nano-technology and antimicrobial activity in some pathogenic bacterial isolates","volume":"13","author":"Barrak","year":"2021","journal-title":"Nusant. Biosci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1002\/jps.3030470215","article-title":"Colorimetric determination of thymol in thyme oil","volume":"47","author":"Fibranz","year":"1958","journal-title":"J. Am. Pharm. Assoc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"332","DOI":"10.21123\/bsj.2015.12.2.332-339","article-title":"Flow injection spectrophotometric determination of thymol using 4-aminoantipyrene and copper (II) nitrate","volume":"12","year":"2015","journal-title":"Baghdad Sci. J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"18","DOI":"10.22401\/JNUS.18.2.03","article-title":"Flow injection determination of thymol in pharmaceutical samples via oxidative coupling reaction with 2,4-dinitrophenylhydrazine","volume":"18","author":"Hadi","year":"2015","journal-title":"J. Al-Nahrain Univ."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1007\/s12161-016-0562-y","article-title":"Voltametric determination of thymol in Oregano using CeO2 modified electrode in Brij\u00ae 35 micellar medium","volume":"10","author":"Ziyatdinova","year":"2017","journal-title":"Food Anal. Methods"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"8583","DOI":"10.1109\/JSEN.2019.2922045","article-title":"Voltammetric detection of thymol (THY) using polyacrylamide embedded graphite molecular imprinted polymer (PAM@G-MIP) electrode","volume":"19","author":"Naskar","year":"2019","journal-title":"IEEE Sens. J."},{"key":"ref_18","first-page":"1049","article-title":"Determination of thymol using HPLC-ED with glassy carbon paste electrode","volume":"52","author":"Zima","year":"2007","journal-title":"Chem. Anal."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"154","DOI":"10.4103\/0973-1296.59961","article-title":"A validated high performance liquid chromatography method for the analysis of thymol and carvacrol in Thymus vulgaris L. volatile oil","volume":"6","author":"Hajimehdipoor","year":"2010","journal-title":"Phcog. Mag."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1016\/j.jchromb.2016.04.011","article-title":"Development and validation of a selective HPLC-UV method thymol determination in skin permeation experiments","volume":"1022","author":"Angelo","year":"2016","journal-title":"J. Chromatogr. B"},{"key":"ref_21","first-page":"8","article-title":"Quantification of thymol content in different extracts of Zataria multiflora by HPLC method","volume":"2","author":"Aghamohammadi","year":"2016","journal-title":"Pharm. Biol. Res."},{"key":"ref_22","first-page":"00330","article-title":"Validation of a high performance liquid chromatography method to quantify thymol in nanocapsules of bioactive essential from Lippia sidoides","volume":"10","author":"Louchard","year":"2017","journal-title":"Int. J. Complement. Alt. Med."},{"key":"ref_23","first-page":"1","article-title":"HPLC method for determination the content of thymol and carvacrol in thyme tincture","volume":"50","author":"Dedic","year":"2018","journal-title":"Bull. Chem. Technol. Bos. Herz."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"43","DOI":"10.3934\/medsci.2020004","article-title":"Simultaneous analytical determination of methyl salicylate and thymol in selected Malaysian traditional medicines","volume":"7","author":"Shahariz","year":"2020","journal-title":"AIMS Med. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"578","DOI":"10.2174\/1573412915666190220102628","article-title":"Ultrasound assisted dispersive solid phase microextraction of thymol and carvacrol in pharmaceutical products using graphene oxide as an adsorbent prior to analysis by high performance liquid chromatography","volume":"16","author":"Ghobadloo","year":"2020","journal-title":"Curr. Pharm. Anal."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"320","DOI":"10.1556\/JPC.23.2010.5.2","article-title":"HPTLC analysis of thymol in extracts of Satureja hortensis L. obtained by different techniques","volume":"23","author":"Soran","year":"2010","journal-title":"J. Planar Chromatogr."},{"key":"ref_27","first-page":"478","article-title":"Validated HPTLC analysis method for quantification of thymol content in Trachyspermum ammi and polyherbal Unani formulation arq zerra","volume":"4","author":"Haque","year":"2012","journal-title":"Int. J. Pharm. Pharm. Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3827","DOI":"10.14233\/ajchem.2013.13806","article-title":"HPTLC method development and validation of secondary metabolite-thymol from Sufof-E-Mohazzil: A Unani formulation","volume":"25","author":"Patil","year":"2013","journal-title":"Asian J. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"102916","DOI":"10.1016\/j.arabjc.2020.102916","article-title":"Novel HPTLC-densitometric method for concurrent quantification of linalool and thymol in essential oils","volume":"14","author":"Alqarni","year":"2021","journal-title":"Arabian J. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1071","DOI":"10.1021\/ac60189a011","article-title":"Determination of thymol isomers by gas liquid chromatography using lanolin","volume":"34","author":"Porcaro","year":"1962","journal-title":"Anal. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/0003-2697(75)90559-X","article-title":"Gas chromatographic determination of thymol","volume":"69","author":"Noall","year":"1975","journal-title":"Anal. Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4055","DOI":"10.5958\/0974-360X.2020.00717.9","article-title":"Determination of thymol in thyme extract and its pharmaceutical forms using gas chromatography method","volume":"13","author":"Raghad","year":"2020","journal-title":"Res. J. Pharm. Technol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2174667","DOI":"10.1155\/2021\/2174667","article-title":"Quantitative determination of p-cymene, thymol, neryl acetate, and \u03b2-caryophyllene in different growth periods and and parts of Eupatorium fortunei Turcz. by GC-MS\/MS","volume":"2021","author":"Nan","year":"2021","journal-title":"J. Anal. Methods Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1556\/AAlim.2007.0032","article-title":"Determination of eucalyptol, camphor, menthol and thymol in thyme honey by GC-FID","volume":"37","author":"Tsigouri","year":"2008","journal-title":"Acta Aliment."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/S0378-4347(01)00518-7","article-title":"Determination of thymol in human plasma by automated headspace solid-phase microextraction-gas chromatographic analysis","volume":"767","author":"Kohlert","year":"2002","journal-title":"J. Chromatogr. B"},{"key":"ref_36","first-page":"837","article-title":"Determination of thymol and carvacrol in plasma and milk of dairy cows using solid-phase microextraction","volume":"24","author":"Fiori","year":"2013","journal-title":"J. Braz. Chem. Soc."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"4045","DOI":"10.1039\/c3nj01059g","article-title":"Sensitive determination of thymol based on CeO2 nanoparticle-decorated graphene hybrid film","volume":"37","author":"Zhao","year":"2013","journal-title":"New J. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1016\/j.talanta.2005.12.030","article-title":"Liquid chromatographic determination of phenol, thymol and carvacrol in honey using fluorimetric detection","volume":"69","author":"Vinas","year":"2006","journal-title":"Talanta"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Chang, X., Sun, P., Ma, Y., Han, D., Zhao, Y., Bai, Y., Zhang, D., and Yang, L. (2020). A new method for determination of thymol and carvacrol in Thymi herba by ultraperformance convergence chromatography (UPC2). Molecules, 25.","DOI":"10.3390\/molecules25030502"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Alqarni, M.H., Alam, P., Foudah, A.I., Muharram, M.M., and Shakeel, F. (2021). Combining normal\/reversed-phase HPTLC with univariate calibration for piperine quantification with traditional and ultrasound-assisted extracts of various food spices of Piper nigrum L. under green analytical chemistry viewpoint. Molecules, 26.","DOI":"10.3390\/molecules26030732"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Alam, P., Iqbal, M., Ezzeldin, E., Khalil, N.Y., Foudah, A.I., Alqarni, M.H., and Shakeel, F. (2020). Simple and accurate HPTLC-densitometry method for quantification of delafloxacin (a novel fluoroquinolone antibiotic) in plasma samples: Application to pharmacokinetic study in rats. Antibiotics, 9.","DOI":"10.3390\/antibiotics9030134"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/j.microc.2019.04.074","article-title":"Green RP-HPLC method for simultaneous determination of moxifloxacin combinations: Investigation of the greenness for the proposed method","volume":"148","author":"Ibrahim","year":"2019","journal-title":"Microchem. J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1195","DOI":"10.1002\/jssc.201900997","article-title":"Digitally enhanced thin layer chromatography for simultaneous determination of norfloxacin tinidazole with the aid of Taguchi orthogonal array and desirability function approach: Greenness assessment by analytical eco-scale","volume":"43","year":"2020","journal-title":"J. Sep. Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"105582","DOI":"10.1016\/j.microc.2020.105582","article-title":"Determination of the abused intravenously administered madness drops (tropicamide) by liquid chromatography in rat plasma; an application to pharmacokinetic study and greenness profile assessment","volume":"159","author":"Abdelrahman","year":"2020","journal-title":"Microchem. J."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1181","DOI":"10.1093\/jaoacint\/qsaa006","article-title":"A green HPLC method for determination of nine sulfonamides in milk and beef, and its greenness assessment with analytical eco-scale and greenness profile","volume":"103","author":"Duan","year":"2020","journal-title":"J. AOAC Int."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"10076","DOI":"10.1021\/acs.analchem.0c01887","article-title":"AGREE-Analytical GREEnness metric approach and software","volume":"92","author":"Wojnowski","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"10343","DOI":"10.1021\/acs.analchem.9b01872","article-title":"What color is your method? Adaptation of the RGB additive color model to analytical method evaluation","volume":"91","author":"Nowak","year":"2019","journal-title":"Anal. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/s40508-016-0051-z","article-title":"Tools and techniques for solvent selection: Green solvent selection guide","volume":"4","author":"Byrne","year":"2016","journal-title":"Sustain. Chem. Proc."},{"key":"ref_49","unstructured":"(2005). Validation of Analytical Procedures\u2013Text and Methodology, International Conference on Harmonization (ICH)."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"105960","DOI":"10.1016\/j.microc.2021.105960","article-title":"A rapid and sensitive stability-indicating RP-HPTLC method for the quantitation of flibanserin compared to green NP-HPTLC method: Validation studies and greenness assessment","volume":"164","author":"Foudah","year":"2021","journal-title":"Microchem. J."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/4\/1164\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:17:15Z","timestamp":1760134635000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/27\/4\/1164"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,9]]},"references-count":50,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["molecules27041164"],"URL":"https:\/\/doi.org\/10.3390\/molecules27041164","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,9]]}}}