A Simple Isocratic HPLC Method for the Simultaneous Determination of Sinensetin, Eupatorin, and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone in Orthosiphon stamineus Extracts

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Journal of Acupuncture and Meridian Studies

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J Acupunct Meridian Stud 2012;5(4):176e182

- RESEARCH ART ICLE -

A Simple Isocratic HPLC Method for theSimultaneous Determination of Sinensetin,Eupatorin, and 30-hydroxy-5,6,7,40-tetramethoxyflavone in Orthosiphon stamineusExtracts

Mun Fei Yam 1,2,*, Elsnoussi Ali Hussin Mohamed 1, Lee Fung Ang 1, Li Pei 2,Yusrida Darwis 1, Roziahanim Mahmud 1, Mohd. Zaini Asmawi 1, Rusliza Basir 3,Mariam Ahmad 1

1 School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia2 Fujian University of Traditional Chinese Medicine, Fujian, China3 Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia

Available online Jun 15, 2012

Received: Aug 28, 2011Revised: Nov 27, 2011Accepted: Nov 30, 2011

KEYWORDS30-hydroxy-5,6,7,40-

*

Coht

tetramethoxyflavone;eupatorin;flavonoids;O stamineus;HPLC;sinensetin

Corresponding author. School of PhE-mail: [email protected]

pyright ª 2012, International Pharmtp://dx.doi.org/10.1016/j.jams.201

AbstractOrthosiphon stamineus extracts contain three flavonoids (30-hydroxy-5,6,7,40-tetra-methoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reportedhigh performance liquid chromatography- ultraviolet (HPLC-UV) methods for the deter-mination of these flavonoids have several disadvantages, including unsatisfactory sepa-ration times and not being well validated according to International Conference onHarmonisation of Technical Requirements for Registration of Pharmaceuticals forHuman Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC methodwith isocratic elution of acetonitrile: isopropyl alcohol: 20 mM phosphate buffer (NaH2-

PO4) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1 ml/minute, a column temperature of25�C, and ultraviolet (UV) detection at 340 nm was developed. The method was vali-dated and applied for quantification of different types of O stamineus extracts and frac-tions. The method allowed simultaneous determination of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052e250 mg/ml. The limits of detection and quantification, respectively, were 0.0076

armaceutical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.

acopuncture Institute2.05.005

Isocratic HPLC method in O stamineus extracts 177

and 0.061 mg/ml for 30-hydroxy-5,6,7,40-tetramethoxyflavone, 0.0153 and 0.122 mg/mlfor sinensetin and 0.0305 and 0.122 mg/ml for eupatorin. The percent relative standarddeviation (% RSD) values for intraday were 0.048e0.368, 0.025e0.135, and 0.05e0.476for 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin, respectively,and those for intraday precision were 0.333e1.688, 0.722e1.055, and 0.548e1.819,respectively. The accuracy for intraday were 91.25%e103.38%, 94.32%e109.56%, and92.85%e109.70% for 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupator-in, respectively, and those for interday accuracy were 97.49%e103.92%, 103.58%e104.57%, and 103.9%e105.33%, respectively. The method was found to be simple, accu-rate and precise and is recommended for routine quality control analysis of O stamineusextract containing the three flavonoids as the principle components in the extract.

1. Introduction

Orthosiphon stamineus Benth (Lamiaceae) is a shrubavailable in various parts of Malaysia and Indonesia. InMalaysia, it grows along forest edges and roadsides and onwasteland and is easily propagated through nodded stemcuttings. O stamineus is used as a remedy for catarrh of thebladder, and as a medicine for various disorders such asnephritis, nephrolithiasis, hydronephrosis, vesical calculi,arteriosclerosis, rheumatism, inflammation, gout, and dia-betes [1,2]. Previous phytochemical investigation of theplant revealed the presence of terpenoid and polyphenoliccompounds which contributed to the therapeutic effects ofO stamineus [3,4]. Lipophilic flavonoids isolated fromO stamineus showed radical-scavenging activity towardsthe diphenylpicryl-hydrazyl radical and inhibition of15-lipoxygenase from soybeans [4,5].

Orthosiphon stamineus has been found to be a richsource of flavonoids, namely 30-hydroxy-5,6,7,40-tetrame-thoxyflavone, sinensetin, and eupatorin. Currently very fewmethods for the quantification of these three flavonoidshave been reported [5,6]. Moreover, the published methodsare not well validated according to the InternationalConference on Harmonisation (ICH) standard guidelines(requirement include specificity, accuracy, linearity,precision, detection range, detection limit, quantificationlimit and system robustness) for drug quantification andsome reported methods required long separation retentiontimes. Therefore, the aim of the present study was todevelop a reversed phase-high performance liquid chro-matography (RP-HPLC) method for 30-hydroxy-5,6,7,40-tet-ramethoxyflavone, sinensetin, and eupatorin quantificationaccording to ICH standard protocol. Hence, this studyprovided a well-developed and validated method forquantification of 30-hydroxy-5,6,7,40-tetramethoxyflavone,sinensetin, and eupatorin from a plant extract.

2. Materials and methods

2.1. Chemicals and reagents

30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, andeupatorin were obtained from Indofine Chemical Company(Hillsborough, New Jersey, USA). Sodium dihydrogen phos-phate (NaH2PO4) was purchased from Sigma (St. Louis,

Missouri, USA). HPLC-grade acetonitrile and isopropylalcohol were purchased from Merck (Darmstadt, Germany).

2.2. HPLC instrumentation

HPLC analysis was performed using a Shimadzu-LC system(Shimadzu Corporation, Kyoto, Japan) equipped witha CBM-20A controller, LC-20AT pump, DGU-20A5 degasser,SIL-20A auto-sampler, SPD-20AV detector, and CTO-10ASvpcolumn oven.

2.3. Chromatographic conditions

Chromatographic separations were achieved using anAgilent Eclipse Plus C18 (250� 4.6 mm i.d.; 5 mm) (Agi-lent Technologies, California, USA). A Zorbax guardfittings kit packed with replaceable Eclipse Plus C18Guard column (12.5� 4.6 mm i.d.; 5 mm) (Agilent Tech-nologies, California, USA) was used to protect theanalytical column. A reverse-phase HPLC assay wascarried out using an isocratic system with a flow rate of1 ml/min, a column temperature of 25�C, a mobile phaseof acetonitrile:isopropyl alcohol: 20 mM phosphatebuffer (NaH2PO4) (30:15:55 v/v), with pH adjusted to 3.5using 85% phosphoric acid. The ultraviolet (UV) detectionwas set at 340 nm. The injection volume was 20 ml ofsolution. Total run time was less than 20 min for eachinjection. Data was acquired and processed with LC-Solution software (Shimadzu Corporation, Kyoto,Japan). The peaks were detected at 340 nm and identi-fied using reference standards of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin.

2.4. Preparation of stock and work solutions

Stock solutions of sinensetin, eupatorin, and 30-hydroxy-5,6,7,40-tetramethoxyflavone were prepared separately inmethanol at a the concentration of 1 mg/ml. Stock wasstored at 4�C and protected from light. The standardsolutions were prepared by diluting the stock solutionswith mobile phase to obtain a series of concentrationsranging from 0.0365 ng/ml to 500 mg/ml for sinensetin,eupatorin, and 30-hydroxy-5,6,7,40-tetramethoxyflavone,respectively.

178 M.F. Yam et al.

3. Validation of the HPLC method

3.1. System suitability

The system suitability parameters were verified with sixreplicate analyses of three QC standard solutions (0.36, 60,and 100 mg/ml for 30-hydroxy-5,6,7,40-tetramethoxy-flavone, sinensetin, and eupatorin, respectively). The meanvalues of the six injections were used to evaluate theo-retical plate (N), tailing factor (Tf), and resolution factor(Rs) for the 30-hydroxy-5,6,7,40-tetramethoxyflavone,sinensetin and eupatorin (criteria N> 2000, TfZ 0.8e2.0,Rs� 1.5). The percent relative standard deviation (% RSD)values were used for evaluation of precision for 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin(criteria � 2.0%).

3.2. Linearity and range

The linear range of the calibration curve was determined bytriplicate injection at the lower limit (1.9 ng/ml) ofdetection to the upper limit of detection (250 mg/m). Thelinearity range was found to be between 3.8 ng/ml to125 mg/ml. The peak heights of the chromatograms wereplotted against the concentrations of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin to obtain

Table 1 Chromatographic properties using different mobilesinensetin, and eupatorin.

Property Compound Mobile pha

Acetonitrilacetonitril

30:10:60

Retention time (tR) 30-hydroxy-5,6,7,40-tetramethoxyflavone

7.463

Sinensetin 10.863Eupatorin 12.924

Tailing factor (T ) 30-hydroxy-5,6,7,40-tetramethoxyflavone

1.151

Sinensetin 1.083Eupatorin 1.027

Capacity factor (k0) 30-hydroxy-5,6,7,40-tetramethoxyflavone

2.11

Sinensetin 3.526Eupatorin 4.385

Number oftheoretical plate (N )

30-hydroxy-5,6,7,40-tetramethoxyflavone

8441

Sinensetin 10223Eupatorin 11099

Resolution (Rs) 30-hydroxy-5,6,7,40-tetramethoxyflavone

d

Sinensetin 9.010Eupatorin 4.478

Separation factor (a) 30-hydroxy-5,6,7,40-tetramethoxyflavone

d

Sinensetin 1.671Eupatorin 1.244

the calibration curve. The y-intercept, slope, and correla-tion coefficient (r2) were determined by linear regressiondata analysis, which were then used to calculate the ana-lyte concentration in each sample. Linearity and limit oflinearity (LOL), defined as the concentration at which thecalibration curve departs from linearity, were evaluated byquantity of quality control (QC) samples with concentrationlower and greater than the range of calibration curves.

3.3. Limit of quantification and detection

The limit of quantification (LOQ) and limit of detection(LOD) of each standard was determined from the calibra-tion curve by using the signal-to-noise (S/N) method. TheLOD is the smallest concentration of the analyte that givesa measurable signal/response where the S/N in ratio is 3 orgreater. The LOQ is the smallest concentration of theanalyte, which gives a response that can be accuratelyquantified and was used to determine the peak signal-to-peak noise (S/N) around the analyte retention, where theS/N ratio is 10 or greater.

3.4. Precision and accuracy

The interday and intraday precision and accuracy of themethod were determined by assaying three QC standard

phase to analyze 30-hydroxy-5,6,7,40-tetramethoxyflavone,

se

e:isopropyl alcohol: phosphate buffer (pH 3.5);e:methanol: phosphate buffer (pH 3.5)a

25:20:55 30:15:55 25:20:55a

4.683 5.555 15.014

5.636 7.067 26.9176.08 8.314 28.5121.256 1.196 1.017

1.201 1.141 0.9831.156 1.096 0.9480.951 1.267 5.418

1.348 1.884 10.2161.712 2.393 10.885344 6207 1151

6243 7366 133076913 8191 13753d d d

3.521 4.945 15.2832.914 3.578 1.673d d d

1.418 1.487 1.8851.269 1.270 1.065

Figure 1 Effect of different combination of mobile phase on flavonoids retention time. ACNZ Acetonitrile; EUPZ eupatorin;MeOHZmethanol; IPAZ isopropyl alcohol; PBZ 20 mM phosphate buffer; SENZ sinensetin; TMFZ 30-hydroxy-5,6,7,40-tetramethoxyflavone.

Isocratic HPLC method in O stamineus extracts 179

solutions and LOQ standard solution in replicates (nZ 6).Precision was reported as percentage of relative standarddeviation (% RSD) and accuracy was reported as % nominalconcentration.

3.5. Robustness of the method

Robustness of the method is a measure of its capacity toremain unaffected by small but deliberate variations inmethod parameters. The method parameters under testwere pH (� 0.5), temperature (þ5, þ10�C) and differentbrands of a similar column (Thermo, hypersil Gold C18(Thermo Scientific, Massachusetts, USA) and Agilent ZorbaxC18 Columns (Agilent Technologies, California, USA)). The

Table 2 System suitability study for the determination of 30-hydr

Compound ug/ml Retentiontime (tR) (minute)

30-hydroxy-5,6,7,40-tetramethoxyflavone

0.36 Mean 5.573%RSD 0.0104

60 Mean 5.572%RSD 0.021

100 Mean 5.569%RSD 0

Sinensetin 0.36 Mean 7.090%RSD 0.008

60 Mean 7.087%RSD 0.062

100 Mean 7.086%RSD 1.54E-14

Eupatorin 0.36 Mean 8.36%RSD 0

60 Mean 8.363%RSD 0.014

100 Mean 8.36%RSD 0

Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate brateZ 1 ml/minute.

analysis was conducted using the optimum mobile phase indifferent brands of columns.

3.6. Application of the HPLC method

The different extracts of O stamineus were prepared inmethanol at 10 mg/ml as a stock solution. The stock solu-tion was then diluted in mobile phase to 1 mg/ml as samplesolution. The total amount of 30hydroxy-5,6,7,40-tetrame-thoxyflavone, sinensetin, and eupatorin in extracts werequantified using the developed HPLC method (nZ 3). Thecontent of these three compounds was expressed asa percentage of the dried extract.

oxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin.

Peakheight

Tailingfactor (T )

Theoreticalplate

Resolution (Rs)

3559.333 1.195 6289.437 d

1.209 0.174 0.174 d

425259.000 1.203 6178.214 d

0.088 0.802 0.115 d

734833.300 1.204 6135.053 d

0.031 0.048 0.072 d

3164.333 1.126 7513.112 2.9840.156 0.578 0.381 0.432401314.300 1.142 7327.153 2.1550.062 0 0.062 1.522642918 1.147 7260.259 2.1310.014 0 0.084 0.1412869.667 1.096 8317.501 3.660.1408 0.570 0.244 0.079380001 1.095 8502.064 3.6290.020 0.053 6.8 0.016654744 1.094 8066.285 3.6140.0190 0 0.037 0.016

ufferZ 30:15:55 (pH 3.5); column temperatureZ 25�C; flow

Table 3 Linear regression analysis parameters for the determination of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin,and eupatorin.

Compounds Linearity range (mg/ml) Linear regression parameters

Slope y-intercept r2

30-hydroxy-5,6,7,40-tetramethoxyflavone 0.03052e250 7678.2514 e287.3824 0.9999Sinensetin 0.03052e250 6894.8863 e329.1236 1Eupatorin 0.03052e250 6027.8718 1061.1482 1

Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate bufferZ 30:15:55 (pH 3.5); column temperatureZ 25�C; flowrateZ 1 ml/minute.

180 M.F. Yam et al.

4. Results and discussion

4.1. Method development and optimization

30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, andeupatorin are flavonoids which are almost insoluble inwater, but soluble in organic solvents such as acetonitrileand methanol. Reversed-phase (RP) HPLC was used becauseit is suitable for the analysis for both non-polar and polaranalytes. During the development of RP-HPLC, a polarmobile phase consisting of a mixture of acetonitrile-isopropyl alcohol-phosphate buffer (pH 3.5) were used.The optimized ratio of acetonitrile:isopropyl alcohol:20 mM phosphate buffer (pH 3.5) was 30:15:55 v/v. A flowrate of 1 ml/min of the optimized mobile phase resulted ina 30hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, andeupatorin retention time of 10 minutes and less, a reducedtime for the whole analytical process. This optimized HPLCcondition also resulted in good symmetrical peaks withtailing factor < 1.2 (Table 1 and Fig. 1).

Table 4 Precision (% RSD) and accuracy for the determination oeupatorin.

Compound/concentration (mg/ml)

Intraday

Mean� SD Precision(% RSD)

30-hydroxy-5,6,7,40-tetramethoxyflavone

0.36 0.35� 0.0005 0.36860 66.34� 0.025 0.048100 108.93� 0.06 0.098

Sinensetin0.36 0.35� 0.005 0.13560 6.34� 0.017 0.025100 108.93� 0.057 0.052

Eupatorin0.36 0.33� 0.002 0.47660 65.68� 0.22 0.225100 109.70� 0.05 0.05

Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate brateZ 1 ml/minute.

4.2. System suitability

Six replicate injections of the system suitability solution gave%RSD values and peak height within 2%, indicating low vari-ation of themeasured values (Table 2). The tailing factors (T)for 30hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, andeupatorin demonstrated symmetry of all peaks (T< 2). Thisis shown in Table 2. The resolutions between 30hydroxy-5,6,7,40-tetramethoxyflavone and sinensetin and betweensinensetin and eupatorin were 2.131e2.984 and 3.614e3.65,respectively, indicating a high degree of peak separation(R> 2). This is shown in Table 2. The efficiency of thecolumn, as expressed by the number of theoretical plates,was more than 6000. This indicated that the developed HPLCcondition is suitable for sample analysis.

4.3. Method validation

4.3.1. LinearityThe slopes, y-intercepts, and correlation coefficients (r2)obtained from regression analysis are shown in Table 3. The

f for 30hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and

Interday

Accuracy Mean� SD Precision(% RSD)

Accuracy

91.25 0.34� 0.006 1.688 97.49103.46 62,35� 0.208 0.333 103.92103.38 103,91� 0.455 0.438 103.91

94.32 0.36� 0.004 1.055 103.58109.56 62.74� 0.527 0.840 104.57108.93 104.46� 0.755 0.722 104.46

92.85 0.364� 0.007 1.811 103.90109.46 63.2� 0.346 0.548 105.33109.70 104.64� 0.981 0.938 104.64

ufferZ 30:15:55 (pH 3.5); column temperatureZ 25�C; flow

Table 5 Chromatographic properties in robustness study of developed method.

Property Compound Column

AgilentZorbax C18a

ThermoHypersilGold C18a

Agilent Eclipse Plus C18

pHb Temperature (�C)c

3 3.5 4 25 30 35

Retention time (tR) 30-hydroxy-5,6,7,40-tetramethoxyflavone

5.555 5.356 5.676 5.555 5.637 5.555 5.435 5.328

Sinensetin 7.067 6.407 7.245 7.067 7.188 7.067 6.909 6.770Eupatorin 8.314 7.009 8.563 8.314 8.479 8.314 8.018 8.558

Tailing factor (T ) 30-hydroxy-5,6,7,40-tetramethoxyflavone

1.196 1.204 1.191 1.196 1.190 1.196 1.211 1.225

Sinensetin 1.141 1.175 1.136 1.141 1.133 1.141 1.147 1.158Eupatorin 1.096 1.132 1.083 1.096 1.085 1.096 1.102 1.117

Capacity factor (k0) 30-hydroxy-5,6,7,40-tetramethoxyflavone

1.267 1.232 1.365 1.267 1.349 1.267 2.792 1.470

Sinensetin 1.884 1.670 2.019 1.884 1.995 1.884 3.822 2.138Eupatorin 2.393 1.920 2.568 2.393 2.533 2.393 4.596 2.967

Number oftheoretical plate (N )

30-hydroxy-5,6,7,40-tetramethoxyflavone

6207 6134 6298 6207 6248 6207 6266 6370

Sinensetin 7366 6969 7439 7366 7395 7366 7457 7597Eupatorin 8191 7405 8300 8191 8248 8191 8225 8372

Resolution (Rs) 30-hydroxy-5,6,7,40-tetramethoxyflavone

d d d d d d d d

Sinensetin 4.945 3.619 5.043 4.945 5.004 4.945 2.366 3.079Eupatorin 3.578 1.903 3.702 3.578 3.649 3.578 3.291 2.388

Separation factor (a) 30-hydroxy-5,6,7,40-tetramethoxyflavone

d d d d d d d d

Sinensetin 1.487 1.355 1.479 1.487 1.479 1.487 1.187 1.232Eupatorin 1.270 1.150 1.272 1.270 1.270 1.270 1.212 1.145

a Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate bufferZ 30:15:55 (pHZ 3.5); column temperatureZ25�C; flowrateZ 1 ml/minute.b Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate bufferZ 30:15:55 (pHZ 3.5); column temperatureZ 25�C; flow

rateZ 1 ml/minute.c Mobile phase: acetonitrile:isopropyl alcohol: 20 mM phosphate bufferZ 30:15:55 (pHZ 3.5); flow rateZ 1 ml/minute.

Table 6 Content (mg/mg) of 30hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin in various extracts andfractions.

Extract/Fraction Method of extraction/fractionation

Content of extract and fraction (mg/mg)

30hydroxy-5,6,7,40-tetramethoxyflavone

Sinensetin Eupatorin

100% ethanol extract Maceration 5.3627� 0.0128 19.2758� 0.0160 19.9621� 0.003775% ethanol extract Maceration 1.7321� 0.0167 5.9480� 0.0026 6.1967� 0.000350% ethanol extract Maceration 1.0150� 0.0007 3.7642� 0.0188 3.0315� 0.025225% ethanol extract Maceration 0.3846� 0.0074 1.1474� 0.0135 0.9784� 0.0136Methanol extract Maceration 0.4554� 0.0164 1.1217� 0.0262 0.9345� 0.0248Chloroform fraction frommethanol extract

Liquid fractionation 7.4251� 0.0413 29.9070� 0.0172 46.6413� 0.0326

Ethyl acetate fractionfrom methanol extract

Liquid fractionation 1.6018� 0.0416 1.4176� 0.0029 1.4809� 0.0051

n-Butanol fraction frommethanol extract

Liquid fractionation 0.1752� 0.0012 0.0794� 0.0002 d

Water fraction frommethanol extract

Liquid fractionation d d d

Chloroform extract Soxhlet 5.7693� 0.0038 14.4699� 0.0078 22.5564� 0.0198

Isocratic HPLC method in O stamineus extracts 181

182 M.F. Yam et al.

calibration curves were linear in the tested concentrationranges. The regression equations were yZ 7678.2514xe287.3824 (r2Z 0.9999), yZ 6894.8863xe329.1236 (r2Z 1),and yZ 6027.8718xþ 1061.1482 (r2Z 1) for 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, and eupatorin,respectively (Table 3).

4.3.2. LOD and LOQThe LOD was 0.0076 mg/ml for 30-hydroxy-5,6,7,40-tetrame-thoxyflavone, 0.0153 mg/ml for sinensetin, and 0.0305 mg/ml for eupatorin. The LOQ was 0.061 for 30-hydroxy-5,6,7,40-tetramethoxyflavone, 0.122 mg/ml for sinensetin and0.122 mg/ml for eupatorin.

4.4. Precision and accuracy

The intraday and interday precision and accuracyfor determination of 30-hydroxy-5,6,7,40-tetramethoxy-flavone, sinensetin, and eupatorin are shown in Table 4.The %RSD values for intra-day were 0.048e0.368,0.025e0.135, and 0.05e0.476 for 30-hydroxy-5,6,7,40-tet-ramethoxyflavone, sinensetin, and eupatorin, respec-tively, and those for intraday precision were 0.333e1.688,0.722e1.055, and 0.548e1.819, respectively. The lowvalues of %RSD (< 2%) reflect the high precision of themethod. The accuracy for intra-day were 91.25%e103.38%, 94.32%e109.56%, and 92.85%e109.70% for30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin, andeupatorin, respectively and those for inter-day accuracywere 97.49%e103.92%, 103.58%e104.57%, and 103.9%e105.33%, respectively. All percentage recoveries werewithin 97%e105%, indicating the good accuracy of themethod.

4.5. Robustness

The method was unaffected by small, deliberate variationsin chromatographic parameters. The parameters testedwere pH of mobile phase, temperature and different C18columns. The robustness results (Table 5) showed that theretention time and other parameters were not significantlyaffected under testing condition.

4.6. Application of the HPLC method

The method was used to quantify the amount of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinensetin and eupatorin invarious types of O stamineus extracts and fractions The

amount of 30-hydroxy-5,6,7,40-tetramethoxyflavone, sinen-setin, and eupatorin content in the extracts of fractions(mg/mg, w/w) were 0.1752e7.4251, 0.0794e29.9070, and0.9345e46.6413 mg/mg (w/w), respectively (Table 6).

5. Conclusion

In this work, a new method for the simultaneous determi-nation of total 30-hydroxy-5,6,7,40-tetramethoxyflavone,sinensetin, and eupatorin in a plant extract was developed.The experimental conditions, including mobile phasecomposition, column temperature and flow rate wereoptimised to produce high-resolution and reproduciblepeaks. The method was validated in compliance with theICH guidelines [7]. It is suitable for the simultaneousdetermination of O stamineus extracts with excellentprecision, accuracy and linearity. The method is isocraticwith UV-detection RP-HPLC, and the sample preparationand assay procedure are simple and rapid.

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4. Yam MF, Asmawi MZ, Basir R. An investigation of the anti-inflammatory and analgesic effects of Orthosiphon stamineusleaf extract. J Med Food. 2008;11:362e368.

5. Akowuah GA, Zhari I, Norhayati I, Sadikun A, Khamsah SM.Sinensetin, eupatorin, 3-hydroxy-5, 6, 7, 4-tetramethoxyflavoneand rosmarinic acid contents and antioxidative effect ofOrthosiphon stamineus from Malaysia. Food Chemistry. 2007;87:559e566.

6. Akowuah GA, Ismail Z, Norhayati I, Sadikun A. The effects ofdifferent extraction solvents of varying polarities on poly-phenols of Orthosiphon stamineus and evaluation of the freeradical-scavenging activity. Food Chemistry. 2005;3:311e317.

7. ICH. International Conference on Harmonisation of TechnicalRequirements for Registration of Pharmaceuticals for HumanUse, guideline for industry Q2B: Validation of analytical proce-dures: methodology. Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm073384.pdf.