High-performance Thin-layer Chromatography Method Development, Validation, and Simultaneous Quantification of Four Compounds Identified in Standardized Extracts ofOrthosiphon stamineus.
BACKGROUND
Orthosiphon stamineusis a medicinal herb widely grown in Southeast Asia and tropical countries. It has been used traditionally as a diuretic, abdominal pain, kidney and bladder inflammation, gout, and hypertension.


OBJECTIVE
This study aims to develop and validate the high-performance thin layer chromatography (HPTLC) method for quantification of rosmarinic acid (RA), 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF), sinensitin (SIN) and eupatorin (EUP) found in ethanol, 50% ethanol and water extract ofO. stamineusleaves.


METHODS
HPTLC method was conducted using an HPTLC system with a developed mobile phase system of toluene: ethyl acetate: formic acid (3:7:0.1) performed on precoated silica gel 60 F254 TLC plates. The method was validated based on linearity, accuracy, precision, limit of detection, limit of quantification (LOQ), and specificity, respectively. The detection of spots was observed at ultraviolet 254 nm and 366 nm.


RESULTS
The linearity of RA, TMF, SIN, and EUP were obtained between 10 and 100 ng/spot with high correlation coefficient value (R2) of more than 0.986. The limit of detection was found to be 122.47 ± 3.95 (RA), 43.38 ± 0.79 (SIN), 17.26 ± 1.16 (TMF), and 46.80 ± 1.33 ng/spot (EUP), respectively. Whereas the LOQ was found to be 376.44 ± 6.70 (RA), 131.45 ± 2.39 (SIN), 52.30 ± 2.01 (TMF), and 141.82 ± 1.58 ng/spot (EUP), respectively.


CONCLUSIONS
The proposed method showed good linearity, precision, accuracy, and high sensitivity. Hence, it may be applied in a routine quantification of RA, SIN, TMF, and EUP found in ethanol, 50% of ethanol and water extract ofO. stamineusleaves.


CONCLUSIONS
HPTLC method provides rapid estimation of the marker compound for routine quality control analysis.The established HPTLC method is rapid for qualitative and quantitative fingerprinting ofOrthosiphon stamineusextract used for commercial product.Four identified markers (RA, SIN, EUP and TMF) found in three a different type ofO. stamineusextracts specifically ethanol, 50% ethanol and water extract were successfully quantified using HPTLC method.Abbreviations Used: HPTLC: High-performance thin layer chromatography; RA: Rosmarinic acid; TMF: 3'-hydroxy-5,6,7,4'-tetramethoxyflavone; SIN: Sinensitin; EUP: Eupatorin; E: Ethanol; EW: 50% ethanol; W: Water; BK: Batu Kurau; KB: Kepala Batas; S: Sik; CJ: Changkat Jering; SB: Sungai Buloh.
Department of Pharmacology, EMAN Research and Testing Laboratories, School of Pharmaceutical Sciences, Universiti Sains, 11800 Minden, Pulau Pinang, Malaysia.
Suzana
S
Hashim
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains, 11800 Minden, Pulau Pinang, Malaysia.
Hooi Kheng
HK
Beh
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains, 11800 Minden, Pulau Pinang, Malaysia.
Mohamad Shahrul Ridzuan
MS
Hamil
Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Universiti Sains, 11800 Minden, Pulau Pinang, Malaysia.
Zhari
Z
Ismail
Department of Pharmacology, EMAN Research and Testing Laboratories, School of Pharmaceutical Sciences, Universiti Sains, 11800 Minden, Pulau Pinang, Malaysia.
Amin Malik Shah Abdul
AM
Majid
 rosmarinic acid; Eupatorin; Orthosiphon stamineus; high-performance thin-layer chromatography; sinensitin; validation
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