Advanced
HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines
HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines
Journal of Pharmacopuncture. 2014. Oct, 17(4): 36-49
Copyright ©2014, KOREAN PHARMACOPUNCTURE INSTITUTE
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : September 12, 2014
  • Accepted : October 16, 2014
  • Published : October 30, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Lee Fung Ang
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
Mun Fei Yam
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
Yvonne Tan Tze Fung
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
Peh Kok Kiang
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia
Yusrida Darwin
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia

Abstract
Objectives:
Quercetin and curcuminoids are important bioactive compounds found in many herbs. Previously reported high performance liquid chromatography ultraviolet (HPLC-UV) methods for the detection of quercetin and curcuminoids have several disadvantages, including unsatisfactory separation times and lack of validation according the standard guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use.
Methods:
A rapid, specific, reversed phase, HPLC-UV method with an isocratic elution of acetonitrile and 2% v/v acetic acid (40% : 60% v/v) (pH 2.6) at a flow rate of 1.3 mL/minutes, a column temperature of 35°C, and ultraviolet (UV) detection at 370 nm was developed. The method was validated and applied to the quantification of different types of market available Chinese medicine extracts, pills and tablets.
Results:
The method allowed simultaneous determination of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the concentration ranges of 0.00488 ─ 200 μg/mL, 0.625 ─ 320 μg/mL, 0.07813 ─ 320 μg/mL and 0.03906 ─ 320 μg/mL, respectively. The limits of detection and quantification, respectively, were 0.00488 and 0.03906 μg/mL for quercetin, 0.62500 and 2.50000 μg/mL for bisdemethoxycurcumin, 0.07813 and 0.31250 μg/mL for demethoxycurcumin, and 0.03906 and 0.07813 μg/mL for curcumin. The percent relative intra day standard deviation (% RSD) values were 0.432 ─ 0.806 μg/mL, 0.576 ─ 0.723 μg/mL, 0.635 ─ 0.752 μg/mL and 0.655 ─ 0.732 μg/mL for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and those for intra day precision were 0.323 ─ 0.968 μg/mL, 0.805 ─ 0.854 μg/mL, 0.078 ─ 0.844 μg/mL and 0.275 ─ 0.829 μg/mL, respectively. The intra day accuracies were 99.589% ─ 100.821%, 98.588% ─ 101.084%, 9.289% ─ 100.88%, and 98.292% ─ 101.022% for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and the inter day accuracy were 99.665% ─ 103.06%, 97.669% ─ 103.513%, 99.569% ─ 103.617%, and 97.929% ─ 103.606%, respectively.
Conclusion:
The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of commercial Chinese medicine products containing the flour flavonoids as their principle components in the extracts.
Keywords
1. Introduction
Quercetin is a category in the class of flavonoids, and a sub class of flavonol. Flavonoids are plant polyphenolics found as pigments in fruits, vegetables, seeds, nuts, flowers, barks and leaves. It is also found in medicinal botanicals, such as Ginkgo biloba, Hypericum perforatum (St. John’s Wort), and Sambucum canadensis (El-der) [ 1 ]. The International Union of Pure and Applied Chemistry’s (IUPAC’s) name for quercetin is 3, 3’, 4’, 5, 7-pentahydroxyflavone (or its synonym 3, 3’, 4’, 5, 7-pentahydroxy-2-phenylchromen-4-one). Fig.1 shows the chemical structure of quercetin. The hydroxyl (-OH) groups attached at positions 3, 5, 7, 3’, and 4’ and the catechol B-ring produce the antioxidant properties of quercetin [ 2 , 3 ]. The antioxidant and the free radical scavenging properties of quercetin have been reported to contribute to anti carcinogenic and anti inflammatory effects, and haves been extensively studied by researchers around the world [ 2 ].
Extensive amounts of in vitro and in vivo animal research on quercetin’s pharmacological activities have been carried out, suggesting that quercetin might be used as a new therapeutic approach to decrease blood pressure [ 4 ], to inhibit fibronectin production by keloid derived fibroblasts [ 5 ], to inhibit neointimal hyperplasia in the abdominal aorta of rats [ 6 ], to treat gout [ 7 ], to inhibit asthmatic syndrome [ 8 ] and to promote dermal wound healing [ 9 ].
Curcumin, commercially available in a mixture of curcumins (curcuminoids), contains ─ 77% pure curcumin, ─ 17% demethoxycurcumin and ─ 3% bisdemethoxycurcumin [ 10 ] (Fig 1 ). Curcuminoids are derived from Curcuma longa Linn, one of the most popular medicinal herbs, and are a polyphenolic. These compounds are yellow pigments and have been, commonly used as a dietary spices, natural coloring agents in foods, household medicines and insect repellents in South and Southeast Asia for thousands of years [ 11 ]. Curcumin and its synthetic derivatives (curcuminoids) show an array of pharmacological properties, such as antibacterial [ 12 - 14 ], antioxidant [ 13 , 15 - 16 ], anti inflammatory [ 17 , 18 ], anti tumor [ 19 , 20 ] and anti proliferation [ 18 , 21 ] properties. Curcumin/curcuminoids also possess potency as medicines for the treatment of diseases, including Alzheimer’s disease [ 22 , 23 ], cancer [ 24 , 25 , 26 ], diabetes, gastric ulcers [ 27 ], malaria [ 28 , 29 ] and for the treatment of wounds [ 30 - 32 ].
A variety of methods for quantitatively detecting curcumin and quercetin contents have been reported. Among these, spectrophotometric methods are the most commonly used [ 33 - 36 ]. Thin layer chromatography (TLC) or column chromatography was usually used for separation of curcuminoids [ 37 - 39 ]. High performance liquid chromatography (HPLC) [ 40 - 45 ] and, high performance thin layer chromatography (HPTLC) [ 39 , 46 , 47 ] are the commonly used methods for quantitatively detecting the quercetin and curcuminoids contents. Some advanced methods have been developed for the analysis of curcuminoids contents, namely, ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS) [ 48 ], ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS) [ 49 ], high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) [ 50 ] and electrochemical-HPLC [ 51 ].
For the above techniques, spectrophotometric methods are not available to quantify the individual curcuminoids due to the curcumin derivative’s being also absorbed at the same wavelength. Furthermore, LC-MS and/or qTOF are complicated and need expensive instrumentation. Even though HPTLC and TLC are widely used to study the fingerprints of plants, these methods are not suitable for analyzing compounds in combinations of herbs products like Chinese medicinal materials (because such products normally contain more than one herb). For simultaneous determination of quercetin and curcuminoids, HPLC method is the recommended technique because it uses separation, identification and quantification of the analytes from plant extracts, foods, pharmaceutical products, and body fluids.
In the present study, a simple isocratic reversed phase HPLC method was developed according to international conference harmonisation (ICH) guidelines [ 52 ] for the simultaneous quantitative detection of quercetin and curcuminoids. The method was also validated by using market available traditional Chinese medicine materials such as granules, pills and tablets.
2. Materials and Methods
Curcumin (mixture of curcumin, demethoxycurcumin, and bisdemethoxycurcumin) was obtained from Acros Organics, USA. Quercetin anhydrous was obtained from Sigma, USA. The HPLC grade acetonitrile and methanol were purchased from J.T. Baker, USA. Analytical grade acetic acid was obtained from QRëC, Malaysia. Nylon membrane filters 0.45 μm were purchased from Whatman, England.
HPLC analysis was performed using a Shimadzu-LC system (Shimadzu, Japan) equipped with an CBM-20A controller, LC-20AT pump, DGU-20A5 prominence degasser, SIL-20A auto sampler, SPD-20AV detector and CTO-10ASvp column oven.
Chromatographic separations were achieved using a Thermo Hypersil Gold column (250 mm × 4.6 mm I.D.: 5 μm). A security guard column (Zorbax Eclipse Plus) packed with a replaceable C-18 cartridge (12.5 mm × 4.6 mm ID.: 5 mm) was used to protect the analytical column. A reverse phase HPLC assay was carried out using an isocratic elution with a flow rate of 1.3 mL/minutes, a column temperature of 35°C, a mobile phase of acetonitrile and 2% v/v acetic acid (pH 2.60) (40% : 60% v/v) and a detection wavelength of 370 nm. The injection volume was 20 μL of each solutions. The total run time was 18.5 minutes for each injection. Data were acquired and processed with LC-Solution Software. Solvents and distilled water were prior filtered through a 0.45-μm nylon membrane by using a set of glass bottles with the aid of a vacuum pump (Fisherbrand FB 70155, Fisher Scientific, UK).
Twenty mg of a mixture of curcumin (containing mainly curcumin, demethoxycurcumin and bisdemethoxycurcumin) and 20 mg of quercetin were accurately weighed using a microbalance (Sartorius, MC5, Germany) and dissolved in 20 mL of HPLC grade methanol in a 20 mL volumetric flask. The mixtures were diluted to 320 μg/mL with HPLC grade methanol; and were then serially doubling diluted to 1.22 ng/mL. These solutions were used as calibration standards for the quantitative determinations of the limit of detection (LOD), the limit of quantification (LOQ) and yhe limit of linearity (LOL), and for the linear range analysis. Three quality control (QC) samples at concentrations of 3.75 μg/mL, 100 μg/mL and 160 μg/mL, respectively, were prepared from the stock solution. All solutions were stored in tightened screw cap bottles to avoid evaporation and were protected from light, and were kept in a refrigerator (4°C) for not more than two weeks.
PPT Slide
Lager Image
Chemical structures of quercetin, and the curcuminoids: curcumin, demethoxycurcumin and bisdemethoxycurcumin.
Standard solutions with concentrations in the range from of 1.22 ng/mL to 320 μg/mL were injected in duplicate into the HPLC unit. The LOD and LOQ of quercetin (QUE), bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC) and curcumin (CUR) were determined in a at the lower concentration range based on the signal to-noise ratio. According to The United Sates Pharmacopeia (USP), the LOD and the LOQ are in terms of 2 or 3 times, and 10 times the noise level respectively. The LOL was determined by plotting a calibration curve (mean value of the peak areas against the concentrations) beginnings with the LOQ concentration and proceeding to the data point that deviated from the regression line. The coefficient of determination (R 2 ≥ 0.999) was used as a guideline to evaluate the model fit of a regression equation.
Linear ranges for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin included concentrations of 1.25, 5, 20, 40, 80, 140 and 200 μg/mL. Separate calibration curves were constructed for quercetin, bisdemethoxycurcumin demethoxycurcumin and curcumin by plotting the peak areas against the concentrations, and the methods were evaluated by determining the coefficient of determination (R 2 ). Unknown assay samples were quantified by referencing them to these calibration curves.
QC samples (3.75, 100 and 160 μg/mL) were used to validate intra day and inter day accuracies and precisions. Intra day precisions and accuracies were determined by using a replicate analysis (n = 6) of the QC samples on the same day under the same analytical conditions. Inter day precisions and accuracies were tested by using a replicate analysis (n = 3) of the same QC samples on six consecutive days. The precision is calculated from the mean of the accuracy and the relative standard deviation (RSD). Accuracy is a measure of how close the experimental value to the true value, and is expressed as a percent. The experimental value was calculated from the calibration curve by using the linear regression equation, y = mx + c. The constant m is the slope of the curve. The constant c is the y intercept and can be determined by extrapolating the straight line to the y axis.
Four variation parameters of robustness were studied: change in organic composition by ± 2.0% (Table 4a ) , change in acetic acid concentration by ± 1.0% v/v (effect of buffer pH) (Table 4b ), change in the flow rate of ± 0.1 mL/min (Table 4c ) and change in the column temperature of ± 5.0°C (Table 4d ). The retention time, peak area, resolution, tailing factor, theoretical plate number and capacity factor values obtained from the variation parameters were compared to those obtained for the normal method conditions. The differences were analyzed by using SPSS version 20, and a one way analysis of variance (ANOVA), followed by Tukey’s test. P-values < 0.05 were considered significant.
The system suitability parameters were assessed by using six replicate analysis of the QC sample at 160 μg/mL. The acceptance criteria were in accordance with the guidelines of the Centre for Drug Evaluation and Research [ 53 ].
The method developed in this study was used to quantitatively determination the quercetin and the curcuminoid contents of extracts, pills and tablets made from Chinese medicinal plants.
3. Results
The LOD and the LOQ were determined based on the signal to noise (S/N) ratio, with the S/N > 3 and the S/N > 10 for the LOD and the LOQ, respectively. The LODs of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin were 0.00488, 0.62500, 0.07813 and 0.03906 μg/mL, respectively. The LOQs of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin were 0.03906, 2.5000, 0.31250 and 0.07813 μg/mL, respectively (Table 1 ) The linearity for detecting quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin was tested against a mixture of calibration standards with concentration ranging from 1.22 ng/mL to 320 μg/mL. The LOL of each compound was determined from a separate calibration curve. Quercetin was linear up to 200 μg/mL, while bisdemethoxycurcumin, demethoxycurcumin and curcumin were linear up to 320 μg/mL.
LOD, LOQ, LOL and linear regression analysis parameters for QUE, BDMC, DMC and CUR
CompoundsLOD (μg/mL)LOQ (μg/mL)LOL (μg/mL)Regression analysis (1.25 — 200 μg/mL)
slopey-interceptCoefficient of determination(R2)
QUE0.004880.0390620070055.859131521.414330.99993
BDMC0.625002.500003201807.72930— 440.281800.99984
DMC0.078130.3125032010011.5579540.135010.99985
CUR0.039060.0781332034176.440883645.088900.99993
LOD, limit of detection; LOQ, limit of quantification, LOL, limit of linearity; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
PPT Slide
Lager Image
Chromatograms of quercetin and curcuminoids. QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
Linear calibration curves in the range from 1.25 to 200 μg/ mL were constructed for each compound by plotting the peak area against the concentration. The retention times and the peak areas are tabulated in (Table 2 ) The values of R 2 , the y-intercept and the slope for each compound’s calibration plot are shown in (Table 1 ) A regression analysis of the data showed a linear relationship for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, with excellent R 2 values of 0.99993, 0.99984, 0.99985 and 0.99993 μg/mL, respectively.
Retention times and responses data for calibration standards of QUE, BDMC, DMC, and CUR
Concentration(μg/mL)Retention time (n = 5)Peak area (n = 5)
Mean (min)RSD (%)Mean (min)RSD (%)
QUE
1.253.9700.117949370.676
53.9720.0663679650.739
203.9720.04114382400.624
403.9730.05527816850.508
803.9720.02955829290.437
1403.9720.04897356180.866
2003.9720.053140739380.368
BDMC
1.2513.8230.30818591.611
513.8400.09588431.181
2013.8420.093370861.089
4013.8430.087715601.044
8013.8460.1171436591.073
14013.8460.1342494621.835
20013.8490.0603634570.850
DMC
1.2515.2140.227147050.273
515.2290.096526920.540
2015.2300.0742046020.665
4015.2320.0733984460.436
8015.2370.0997981530.867
14015.2360.12013842201.416
20015.2420.03920155830.158
CUR
1.2516.7080.199466450.856
516.7180.0771825150.901
2016.7190.0617019820.700
4016.7200.06413585910.299
8016.7250.09627377510.423
14016.7250.10847493550.897
20016.7340.06768669710.313
RSD, relative standard deviation; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
The peaks of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin were well separated at different retention times with resolutions of 32.195, 2.887 and 2.830 for quercetin-bisdemethoxycurcumin, bisdemethoxycurcumin-demethoxycurcumin and demethoxycurcumin-curcumin, respectively. No interferences or excipient peaks co eluted with the analytes were observed, indicating the method is selective and specific in relation to the medium and excipients used in this study (Fig 2 ), (Table 2 ).
Precision and accuracy data for the intraday and the inter-day variations for the three QC samples are summarized in (Table 3 ). The RSD values for the intraday and the inter day precisions were < 1%. For the accuracy test, the intraday and the inter day accuracies ranges from 98.292% to 103.617%, confirming the accuracy of the method.
Precisions and accuracies for intraday and interday repetitions for the quantitative detection of QUE, BDMC, DMC and CUR
Concentration(μg/mL)Intra day*Inter day
PeakResponsePrecision(RSD, %)Accuracy (%)PeakResponsePrecision(RSD, %)Accuracy (%)
QUE
3.752631510.43299.5892633500.32399.665
10070645990.717100.82172214700.646103.060
160112216110.806100.010112182870.968100.070
BDMC
3.7562430.57698.58861810.85497.669
1001822930.723101.0841866830.878103.513
1602868510.65499.327462880400.80599.738
DMC
3.75377000.635100.310376870.466100.276
10010100040.752100.88010374100.078103.617
16015904980.65199.28915949890.84499.569
CUR
3.751296180.65598.2921291520.29797.929
10034562180.732101.02235445350.275103.606
16054486750.71199.57654540120.82999.673
*Intra day repetitions for each concentration were analyzed on the same day. †Inter day repetitions for each concentration, were analyzse on six consecutive days. RSD, relative standard deviation; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
(a). Robustness – change in organic composition
System suitabilityCompoundChange in the normal organic composition of acetonitrile: 2% acetic acid
(A) Normal condition(B) 38% : 62% v/v(C) 42% : 58% v/v
Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)
Retention time, tR(minutes)QUE3.9930.6904.2510.1553.7610.040
BDMC13.9510.34217.6450.37411.2800.084
DMC15.3400.29119.5430.28512.3300.084
CUR16.8290.24521.6170.29613.4640.082
Peak areaQUE68530440.43368369340.44168674450.117
BDMC1674170.6471615040.8011464840.578
DMC9408360.4049031910.7819653070.071
CUR33025930.23632061340.55533673090.114
Resolution, RQUE------
BDMC32.4980.37936.4490.47129.1200.063
DMC2.9080.2083.2722.3692.7360.181
CUR2.8500.2373.2431.6482.6660.124
Tailing factor, TfQUE1.3710.2541.3470.1151.3920.074
BDMC1.5330.3641.2832.3141.0800.200
DMC1.1600.4841.0830.1511.4310.082
CUR1.0940.0941.0760.2841.1140.037
Theoretical plate, NQUE8752.1331.4638857.7910.3128520.1710.238
BDMC15931.8891.14716311.0110.05816303.1300.103
DMC14298.2871.76116569.4741.02914210.3210.233
CUR16008.0491.20216543.7540.53515157.5080.340
Capacity factor, k’QUE0.6800.3440.7770.9060.6010.327
BDMC4.8780.2023.8000.2093.8000.209
DMC5.4630.2327.2141.5924.2470.206
CUR6.0970.2538.0380.4814.7290.209
(b). Robustness – change in acetic acid concentration
System suitabilityCompoundChange in the acetic acid concentration (% v/v )
(A) Normal condition(B) 1.0% (pH 2.73)(C) 3.0% (pH 2.48)
Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)
Retention time, tR(minutes)QUE3.9720.1754.0540.0643.8930.071
BDMC13.8680.31014.5490.08613.1770.167
DMC15.2550.26516.0170.08514.5420.153
CUR16.7430.21317.5900.08416.0280.141
Peak areaQUE70394830.56269669500.52569528330.630
BDMC1804750.5411768850.5751524390.895
DMC10007160.7369871280.5519562660.670
CUR34333790.75434287620.53334287620.558
Resolution, RQUE------
BDMC32.3270.17233.2540.24431.9500.268
DMC2.9000.3702.9740.3033.0330.527
CUR2.8400.4292.9040.3392.9660.608
Tailing factor, TfQUE1.3660.0771.3640.2151.3700.110
BDMC1.4931.3771.4630.3311.0600.139
DMC1.1601.0751.1370.1031.3250.823
CUR1.0850.1481.0920.0501.0830.108
Theoretical plate, NQUE8711.9930.2678877.5460.4608548.9480.269
BDMC15740.5570.39716067.8080.68916308.1460.664
DMC14041.1810.70114691.5800.67514241.0821.031
CUR15793.0190.47216098.2390.70115531.3420.811
Capacity factor, k’QUE0.6561.7830.6801.4840.6100.803
BDMC4.7981.2025.0360.6584.4490.511
DMC5.3330.9885.6370.6985.0140.478
CUR6.0161.4166.2950.6285.6290.443
(c). Robustness – change in flow rate
System suitabilityCompoundChange in flow rate
(A) Normal condition(B) 1.2 mL/minutes(C) 1.4 mL/minutes
Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)
Retention time, tR(minutes)QUE3.9720.1754.2910.1053.6960.130
BDMC13.8680.31014.9530.32112.9090.333
DMC15.2550.26516.4420.28414.2350.279
CUR16.7430.21318.0380.26215.6680.298
Peak areaQUE70394830.56276062720.66265305710.497
BDMC1804750.5411942160.7531671111.593
DMC10007160.73610780760.7149287071.345
CUR34333790.75437001340.69031853251.198
Resolution, RQUE------
BDMC32.3270.17232.7790.19932.0470.928
DMC2.9000.3702.9210.6082.9362.014
CUR2.8400.4292.8640.7232.8681.647
Tailing factor, TfQUE1.3660.0771.3600.1831.3710.287
BDMC1.4931.3771.4901.8911.5391.614
DMC1.1601.0751.1571.4471.1812.364
CUR1.0850.1481.0810.1011.0870.207
Theoretical plate, NQUE8711.9930.2679148.3470.4298249.4300.420
BDMC15740.5570.39716035.1031.34215696.0462.851
DMC14041.1810.70114374.9441.03613420.2200.844
CUR15793.0190.47216216.0131.85415379.1652.364
Capacity factor, k’QUE0.6561.7830.6610.8320.6270.762
BDMC4.7981.2024.7800.9424.7503.067
DMC5.3510.6615.3550.4975.3502.846
CUR5.9660.6325.9850.5005.8620.427
(d). Robustness – change in column temperature
System suitabilityCompoundChange in column temperature
(A) Normal condition(B) 30°C(C) 40°C
Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)Mean (n = 6)RSD (%)
Retention time, tR(minutes)QUE3.9560.0314.0630.0743.8610.162
BDMC13.6730.07014.6470.17212.8100.268
DMC15.0370.06415.9800.15314.1670.236
CUR16.5020.06417.4230.14315.6570.196
Peak areaQUE76284830.25276205250.25476333410.259
BDMC1964930.2612028700.2531723970.136
DMC10910990.30011245670.28110584040.205
CUR37385440.24438363060.28536439100.196
Resolution, RQUE------
BDMC31.9461.43732.5600.23331.4710.267
DMC2.8721.3592.6980.3343.1550.481
CUR2.8290.5752.7180.3053.1060.559
 Tailing factor, TfQUE1.3430.0561.3290.2671.3510.124
BDMC1.5510.4211.2330.5251.0890.077
DMC1.1860.2411.0980.2681.4911.051
CUR1.0970.0821.0940.1071.0980.129
Theoretical plate, NQUE8734.2370.3008837.8100.4488619.4730.295
BDMC15779.1750.59515065.1040.60016276.5451.203
DMC13866.2061.17515742.7650.47515286.7040.311
CUR15846.7060.79115917.9870.39415793.3490.183
Capacity factor, k’QUE0.6981.5980.7171.9450.6160.692
BDMC4.8550.1155.1900.9894.3660.214
DMC5.4490.3495.7520.9664.9621.315
CUR6.0580.2016.3620.9425.5811.015
The normal conditions of HPLC are a mobile phase of acetonitrile: 2% acetic acid (pH 2.60) = 40% : 60 % v/v, flow rate 1.3 mL/min at UV wavelength of 370 nm and column temperature at 35°C. RSD, relative standard deviation; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
System suitability parameters, calculation formula and recommendations
ParameterFormulaRecommendation
PrecisionRSD = S/x̄*100RSD ≤ 1% for n ≥ 5
Resolution, RR = (tR2 – tR1)/(1/2)(tw1 –tw2)> 2
Tailing factor, TfTf = Wx/2f≤ 2
Theoretical plates, NN = 16(tR/tw)2Column efficiency ≥ 2000
Capacity factor, kk’ = (tR – t0)/t0> 2
S, standard deviation; x̄ , mean of the data; tR, retention time of analyte 1; tw, peak width measured to the baseline of the extrapolated straight sides to baseline; Wx, width of the peak determined at either 5% (0.05) or 10% (0.10) from the baseline of the peak height; f, distance between peak maximum and peak front at Wx; t0, elution time of the void volume or non retained components.
System suitability testing
ParameterQUEBDMCDMCCUR
MeanRSD (%)MeanRSD (%)MeanRSD (%)MeanRSD (%)
Retention time, tR3.9700.02113.8400.02715.2300.02516.7230.021
Peak area112216110.8062868510.65415904980.65154486750.711
Resolution, R--32.1950.3212.8870.3642.8300.370
Tailing factor, Tf1.3690.1081.5010.2611.1650.1441.0810.051
Theoretical plate, N8803.7850.35915552.3980.86513763.1450.64615568.2520.910
Capacity factor, k’0.6840.8464.8700.4155.4600.4066.0930.391
RSD, relative standard deviation; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin. N, number of theoretical plates; k’, capacity factor; Mean of six replicate injections of quality control (QC) standard of 160 μg/mL.
Robustness is a measure of the method’s capability to remain unaffected by small, but deliberate, variations in the method parameters [ 52 ]. The robustness parameters tested were the mobile phase’s composition, the concentration of acetic acid (pH effect), the flow rate and the column temperature. The results are tabulated in Table 4( a - d ). The retention times for all four compounds due to variations in the parameters were significantly different compared to those for the normal parameters. The peak area for curcumin was not significantly different after changing the acetic acid concentration from 2% to 3%, but was significantly different after changing the concentration from 2% to 1%. Quercetin, bisdemethoxycurcumin and demethoxycurcumin were shown to have significant differences in their peak area when the concentration of acetic acid was changed. Changes in the acetonitrile’s composition and temperature were shown not to cause significant differences in quecetin’s peak areas, however significant differences were seen in curcumin, bisdemethoxycurcumin and demethoxycurcumin peak areas. Increasing or decreasing the flow rate by 0.1 mL/min from normal conditions significantly raised or reduced the values of the peak areas of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin. Although changes in experimental conditions changed the retention time, the peak area and the values of the system’s suitability parameters, the four analyzed peaks were still well resolved from each other and from additional small peaks and showed good resolution in the tested parameters (Fig 3 ).
The system suitability criteria were in accordance with the Centre for Drug Evaluation and Research (CDER) guidelines [ 53 ] and are summarized in (Table 5 ) The mean values of the six replicate injections of 160 μg/mL QC standards were used to evaluate the retention time, the peak area, the resolutions for the analyte peaks, the tailing factor, the number of theoretical plates and the capacity factor. The results for the system suitability parameters are shown in (Table 6 ) The RSD values for the tested parameters were < 1%, indicating the precision of the method. The tested parameters passed the criteria under the CDER guidelines except for the capacity factor value for quercetin (< 2) [ 53 ]. This is because the retention time of quercetin was quite fast and just 1 minute behind the solvent peak. However, the quercetin peak was well resolved from the solvent peak and from the front additional small peak.
PPT Slide
Lager Image
Combined chromatograms of quercetin (QUE), bisdemethoxycurcumin (BMDC), demethoxycurcumin (DMC), curcumin (CUR) analyzed at different conditions: (a) acetonitrile: 2% acetic acid at a flow rate of 1.3 mL/minutes, 35°C (b) acetonitrile: different acetic acid concentrations (40% : 60% v/v) at a flow rate of 1.3 mL/min, 35°C (c) acetonitrile: 2% acetic acid (40% : 60% v/v) at different flow rates, 35°C (d) acetonitrile: 2% acetic acid (40% : 60% v/v) at a flow rate of 1.3 mL/min at different temperatures.
The proposed method was applied to quantitatively detect the quercetin and curcuminoids in Chinese medicines such as plant granule extracts, tablets and pills. The results of 19 samples are summarized in (Table 7 ). In the tested samples, BDMC had the highest concentration compared to the other two curcuminoids tested (DMC and CUR), and was found in the formulations of granule extracts, tablets and pills (such as samples 12, 13, 15, 16, 18 and 19) (Table 7 ). The preference of BDMC over CUR in the medicine might be due to its strong biological properties, which its use as a cure for diseases or as a supplement for certain purposes. Quercetin was found in most of the tested samples, indicating that this compound is common and useful for treatment. (Fig 4 ) shows the chromatograms for the quercetin and the curcuminoids found in the tested samples.
Concentration of QUE, CUR, DMS and BDMC in Chinese medicines
NoChinese medicineTypeConcentration (mean ± S.D*) (μg/100 mg)
QUEBDMCDMCCUR
1Gao liang jiang (高良姜)Single plant granule extract0.7532N.D134.87390.5270
2Jin qian cao (金钱草)Single plant granule extract4.0618N.DN.D0.8263
3Yu jin (郁金)Single plant granule extract0.319569.106027.228627.1020
4E su (莪术)Single plant granule extract0.598379.592242.69828.6812
5Jiang huang (姜黄)Single plant granule extract3.6523N.D933.8122796.0621
6Yu xing cao (鱼腥草)Single plant granule extract1.7930N.DN.D1.3424
7Ting li zi (葶苈子)Single plant granule extract1.3604N.DN.DN.D
8Tu si zi (菟丝子)Single plant granule extract3.9300N.DN.DN.D
9Di yu (地榆)Single plant granule extract0.8962N.DN.DN.D
10Kui hua (愧花)Single plant granule extract311.0307N.DN.DN.D
11Sang ju yin (桑菊饮)Formulation granule extract0.7402N.D0.35580.2537
12Chai hu su gan san(柴胡疏肝散)Formulation granule extract0.2029126.884348.34081.6417
13Xiao yao san (逍遥散)Formulation granule extract0.499197.92032.55340.4301
14Long dan xie gan tang(龙胆泄肝汤)Formulation granule extract11.14825.21111.28170.1236
15Sang ju gan mao pian(桑菊感冒片)Tablet17.3489173.61552.8579N.D
16Dan zhi xiao yao pian(丹栀逍遥片)Tablet7.8101135.18921.08830.2624
17Long dan xie gan pian(龙胆泄肝片)TabletN.D5.53526.74280.2378
18Bu zhong yi qi (补中益气)Tablet0.9052623.13385.94850.5964
19Xiao yao wan (逍遥丸)Pill12.01579.795111.74711.1516
*n = 3; N.D, not detected; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
4. Discussion
The HPLC method was developed by optimization of the mobile phase conditions so that quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin peaks could be simultaneously detected by using the same solvent system and an isocratic method. The flow rate, acetic acid concentration and column temperature were varied to determine the chromatographic conditions giving the best separation and the shortest analysis time. UV visible sperctrophotometry in the wavelength from 200 to 500 nm was used for the detection of quercetin and curcuminoids; 370 nm was chosen as appropriate wavelength for the analysis of quercetin and curcumin derivatives.
The retention times for quercetin (3.97 minutes), bisdemethoxycurcumin (13.84 minutes), demethoxycurcumin (15.23 minutes) and curcumin (16.72 minutes) were reasonable because the method is simple and general. The chromatograph peaks for mixtures of curcumin were identified based on their percentages in the mixtures. Most of the commercially available curcumin/turmeric products contain mixtures of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Among these, curcumin (46% ─ 72%) is the major compound, followed by demethoxycurcumin (11% ─ 28%) and bisdemethoxycurcumin (3% ─ 14%). All four analyte peaks were well separated from each other and from small additional peaks.
The linear ranges of quercetin (0.039 ─ 200 μg/mL), bisdemethoxycurcumin (2.500 ─ 320 μg/mL), demethoxycurcumin (0.313 ─ 320 μg/mL) and curcumin (0.078 ─ 320 μg/mL) are suitable for the analysis of most the pharmaceutical products, containing the compounds and for the analysis of crude herbs. The low LOD and LOQ values indicate that the method provides adequate sensitivity. The R2 values > 0.999 for the regression model for the calibration curves confirm the good linearity of the method.
The accuracies ranged from 98.292% ─ 103.617%, and the precisions were less than 1% which indicate that the proposed method is well validated and suitable for quantitatively detecting curcuminoids and quercetin simultaneously in pharmaceutical products, herb materials and various turmeric and quercetin containing products.
System suitability testing is important to ensure the performance of the system before and during the analysis. As defined in the United States Pharmacopeia/National Formulary (USP/NF) [ 54 ] system suitability parameters were established as a direct result of the ruggedness and the robustness of the experiments. The system suitability testing proved that the proposed method will allow the separation of all four anaytes and will produce satisfactory peak shapes.
PPT Slide
Lager Image
Chromatograms for Chinese medicinal plant extracts (a) containing quercetin and (b) containing curcuminoids. QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.
5. Conclusion
A simple isocratic RP-HPLC method with UV detection has been developed for simultaneous detection of quercetin, curcumin, demethoxycurcumin and bisdemethoxycurcumin. The analytes were well separated and detected within 19 minutes. This method was validated for specificity, linearity, precision, accuracy and robustness as per ICH guidelines. The data showed good selectivity and sensitivity, a wide linear range, precision and accuracy. The method was sensitive to HPLC conditions; that is, changes in the mobile phase’s composition, the pH, the column temperature and the flow rate affected the retention time and response, but did not affected the separation of the compounds. In addition, each parameter showed good repeatability of the retention time and response. In conclusion, the proposed method is simple, easy and cost effective, no specific solvent is involved and it utilizes common HPLC instruments with UV detectors. Hence, this UV-HPLC method is suitable for routine analysis of quercetin and curcuminoid formulations or products.
Conflicts of interest. The authors declare that there are no conflict of interest.
References
Kelly GS 1998 Quercetin Altern Med Rev 3 140 - 143
Harwood M , Danielewska-Nikiel B , Borzelleca JF , Flamm GW , Williams GM , Lines TC 2007 A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/ carcinogenic properties Food Chem Toxicol 45 (11) 2179 - 2205    DOI : 10.1016/j.fct.2007.05.015
Materska M 2008 Quercetin and its derivatives: chemical structure and bioactivity - a review Pol J Food Nutr Sci 58 (4) 407 - 413
Larson AJ , Symons JD , Jalili T 2012 Therapeutic potential of quercetin to decrease blood pressure: review of efficacy and mechanisms Am Soc Nutrition 3 39 - 46    DOI : 10.3945/an.111.001271
Phan TT , Lim IJ , Sun L , Chan SY , Bay BH , Tan EK , et al 2003 Quercetin inhibits fibronectin production by keloid- derived fibroblasts. implication for the treatment of excessive scars J Dermatol Sci et al 33 (3) 192 - 194    DOI : 10.1016/j.jdermsci.2003.08.008
Huang BF , Wang W , Fu YC , Zhou XH , Wang X 2009 The effect of quercetin on neointima formation in a rat artery ballon injury model Pathol Res Pract 205 (8) 515 - 523    DOI : 10.1016/j.prp.2009.01.007
Zhu JX , Wang Y , Kong LD , Yang C , Zhang X 2004 Effects of Biota orientalis extract and its flavonoid constituents, quercetin and rutin on serum uric acid levels in oxonate- induced mice and xanthine dehydrogenase and xanthine oxidase activities in mouse liver J Ethnopharmacol 93 (1) 133 - 140    DOI : 10.1016/S0378-8741(04)00162-X
Park HJ , Lee CM , Jung ID , Lee JS , Jeong YI , Chang JH , et al 2009 Quercetin regulates Th1/Th2 balance in a murine model of asthma Int Immunopharmacol et al 9 (3) 261 - 267    DOI : 10.1016/j.intimp.2008.10.021
Gomathi K , Gopinath D , Ahmed MR , Jayakumar R 2003 Quercetin incorporated collagen matrices for dermal wound healing processes in rat Biomaterials 24 (16) 2767 - 2772    DOI : 10.1016/S0142-9612(03)00059-0
Sandur SK , Pandey MK , Sung B , Ahn KS , Murakami A , Sethi G , et al 2007 Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism Carcinogenesis et al 28 (8) 1765 - 1773
Pothitirat W , Gritsanapan W 2005 Quantitative analysis of curcumin, demethoxycurcumin and bisdemethoxycurcumin in the crude curcuminoid extract from curcuma longa in Thailand by TLC-densitometry Warasan Phesatchasat 32 (1-2) 23 - 30
Bhawana RK , Buttar HS , Jain VK , Jain N 2011 Curcumin nanoparticles: preparation, characterization and antimicrobial study J Agric Food Chem 59 (5) 2056 - 2061    DOI : 10.1021/jf104402t
Parvathy KS , Negi PS , Srinivas P 2009 Antioxidant, antimutagenic and antibacterial activities of curcumin-β-diglucoside Food Chem 115 (1) 265 - 271
Wang Y , Lu Z , Wu H , Lv F 2009 Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens Int J Food Microbiol 136 (1) 71 - 74    DOI : 10.1016/j.ijfoodmicro.2009.09.001
Barzegar A 2012 The role of electron-transfer and H-atom donation on the superb antioxidant activity and free radical reaction of curcumin Food Chem 135 (3) 1369 - 1376
Grinberg LN , Shalev O , Tønnesen HH , Rachmilewitz EA 1996 Studies on curcumin and curcuminoids: XXVI. Antioxidant effects of curcumin on the red blood cell membrane Int J Pharm 132 (1-2) 251 - 257    DOI : 10.1016/0378-5173(95)04377-2
Khan MA , El-Khatib R , Rainsford KD , Whitehouse MW 2012 Synthesis and anti-inflammatory properties of some aromatic and heterocyclic aromatic curcuminoids Bioorg Chem 40 30 - 38
Ravindran J , Subbaraju GV , Ramani MV , Sung B , Aggarwal BB 2010 Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro Biochemical Pharmacology 79 (11) 1658 - 1666    DOI : 10.1016/j.bcp.2010.01.033
Anto RJ , Kuttan G , Babu KVD , Rajasekharan KN , Kuttan R 1996 Anti-tumour and free radical scavenging activity of synthetic curcuminoids Int J Pharm 131 (1) 1 - 7    DOI : 10.1016/0378-5173(95)04254-7
Ruby AJ , Kuttan G , Babu D , Rajasekharan KN , Kuttan R 1995 Anti-tumour and antioxidant activity of natural curcuminoids Cancer Letter 94 (1) 79 - 83    DOI : 10.1016/0304-3835(95)03827-J
Simon A , Allais DP , Duroux JL , Basly JP , Durand-Fontanier S , Delage C 1998 Inhibitory effect of curcuminoids on MCF-7 cell proliferation and structure-activity relationship Cancer Letters 129 (1) 111 - 116    DOI : 10.1016/S0304-3835(98)00092-5
Ahmed T , Gilani AH 2009 Inhibitory effect of curcuminoids on acetylcholinesterase activity and attenuation of scopolamine- induced amnesia may explain medicinal use of turmeric in Alzheimer’s disease Pharmacol Biochem Behav 91 (4) 554 - 559
Villaflores OB , Chen YJ , Chen CP , Yeh JM , Wu TY 2012 Curcuminoids and resveratrol as anti-Alzheimer agents Taiwan J Obstet Gynecol 51 (4) 515 - 525    DOI : 10.1016/j.tjog.2012.09.005
Kunnumakkara AB , Anand P , Aggarwal BB 2008 Curcumin inhibits proliferation, invasion, angiogenisis and metastasis of different cancers through interaction with multiple cell signaling proteins Cancer Letters 269 (2) 199 - 225    DOI : 10.1016/j.canlet.2008.03.009
Shoji M , Nakagawa K , Watanabe A , Tsuduki T , Yamada T , Kuwahara S , et al 2014 Comparison of the effects of curcumin and curcumin glucuronide in human hepatocellular carcinoma HepG2 cells Food Chem et al 151 126 - 132    DOI : 10.1016/j.foodchem.2013.11.021
Kim T , Davis J , Zhang AJ , He X , Mathews ST 2009 Curcumin activates AMPK and suppresses gluconeogenic gene expression in hepatoma cells Biochem Biophys Res Commun 388 (2) 377 - 382    DOI : 10.1016/j.bbrc.2009.08.018
Mahattanadul S , Nakamura T , Panichayupakaranant P , Phdoongsombut N , Tungsinmunkong K , Bouking P 2009 Comparative antiulcer effect of bisdemethoxycurcumin and curcumin in a gastric ulcer model system Phytomedicine 16 (4) 342 - 351    DOI : 10.1016/j.phymed.2008.12.005
Jain K , Sood S , Gowthamarajan K 2013 Modulation of cerebral malaria by curcumin as an adjunctive therapy Braz J Infect Dis 17 (5) 579 - 591    DOI : 10.1016/j.bjid.2013.03.004
Nayak A , Tiyaboonchai W , Patankar S , Madhusudhan B , Souto EB 2010 Curcuminoids-loaded lipid nanoparticles: novel approach towards malaria treatment Colloids Surf B Biointerfaces 81 (1) 263 - 273    DOI : 10.1016/j.colsurfb.2010.07.020
Jagetia GC , Rajanikant GK 2004 Role of curcumin, a naturally occurring phenolic compound of turmeric in accelerating the repair of excision wound, in mice wholebody exposed to various doses of γ-radiation J Surg Res 120 (1) 127 - 138
Li X , Nan K , Li L , Zhang Z , Chen H 2012 In vivo evaluation of curcumin nanoformulation loaded methoxy poly(ethyleneglycol)- graft-chitosan composite film for wound healing application Carbohydr Polym 88 (1) 84 - 90
Panchatcharam M , Miriyala S , Gayathri VS , Suguna L 2006 Curcumin improves wound healing by modulating collagen and decreasing reactive oxygen species Mol Cell Biochem 290 (1-2) 87 - 96    DOI : 10.1007/s11010-006-9170-2
Aneja G , Dave U , Vadodaria K 2012 Simultaneous estimation of piperine, quercetin, and curcumin in a mixture using u.v-visible spectrophotometer and method validation International Journal of Therapeutic Applications 8 14 - 17
Askal HF , Saleh GA , Backheet EY 1992 A selective spectrophotometric method for determination of quercetin in the presence of other flavonoids Talanta 39 (3) 259 - 263    DOI : 10.1016/0039-9140(92)80030-H
Kuntić V , Pejić N , Mićić S , Vukojević V , Vujić Z , Malešev D 2005 Determination of quercetin in pharmaceutical formations via its reaction with potassium titanyloxalate. Determination of the stability constants of the quercetin titanyloxalato complex J Serb Chem Soc 70 (5) 753 - 763
Sharma K , Agrawal SS , Gupta M 2012 Development and validation of UV spectrophotometric method for the estimation of curucmin in bulk drug and pharmaceutical dosage forms IJDDR 4 (2) 375 - 380
Kulkarni SJ , Maske KN , Budre MP , Mahajan RP 2012 Extraction and purification of curcuminoids from Turmeric (curcuma longa L.) Int J Pharm Technol 1 (2) 81 - 84
Revathy S , Elumalai S , Benny M , Antony B: , purification and 2011 ) by column chromatography Journal of Experimental Sciences 2 (7) 21 - 25
Sheikh S , Asghar S , Ahmad S 2013 Development of HPTLC method and its validation for the estimation of curcuminoids from polyherbal mouth ulcer gel formulation IOSR J Pharm Biol Sci 3 (1) 29 - 34    DOI : 10.9790/3013-31102934
Careri M , Corradini C , Elviri L , Nicoletti I , Zagnoni I 2003 Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and winemaking byproducts J Agric Food Chem 51 (18) 5226 - 5231
Ishii K , Furuta T , Kasuya Y 2003 High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection J Chromatogr B 794 (1) 49 - 56    DOI : 10.1016/S1570-0232(03)00398-2
Jayaprakasha GK , Rao LJM , Sakariah KK 2002 Improved HPLC method for the determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin J Agric Food Chem 50 (13) 3668 - 3672    DOI : 10.1021/jf025506a
Rajalakshmi PV , Senthil KK 2009 Direct HPLC analysis of quercetin in exudates of abutilon indicum (Linn). malvaceae J Pharm Sci Technol 1 (2) 80 - 83
Wichitnithad W , Jongaroonngamsang N , Pummangura S , Rojsitthisak , P 2009 A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts Phytochem Anal 20 (4) 314 - 319    DOI : 10.1002/pca.1129
Zhang J , Jinnai S , Ikeda R , Wada M , Hayashida S , Nakashima K 2009 A simple HPLC-fluorescence method for quantitation of curcuminoids and its application to turmeric products Analyt Sci 25 (3) 385 - 388    DOI : 10.2116/analsci.25.385
Ashraf K , Mujeeg M , Ahmad A , Amir M , Mallick MN , Sharma D 2012 Validated HPTLC analysis method for quantification of variability in content of curcumin in Curcuma long L (turmeric) collected from different geographical region of India Asian Pac J Trop Biomed S584 - S588
Paramasivam M , Poi R , Banerjee H , Bandyopadhyay A 2009 High-performance thin layer chromatographic method for quantitative determination of curcuminoids in Curcuma longa germplasm Food Chem 113 (2) 640 - 644    DOI : 10.1016/j.foodchem.2008.07.051
Verma MK , Najar IA , Tikoo MK , Singh G , Gupta DK , Anand R , et al 2013 Development of a validated UPLCqTOF- MS method for the determination of curcuminoids and their pharmacokinetic study in mice DARU Journal of Pharmaceutical Sciences et al 21 11 -
Avula B , Wang YH , Khan IA 2012 Quantitative determination of curcuminoids from the roots of Curcuma longa, Curcuma species and dietary supplements using an UPLC-UV-MS method J Chromatograph Separat Techniq 3 (1) 1000120 -
Li W , Xiao H , Wang L , Liang X 2009 Analysis of minor curcuminoids in Curcuma longa L. by high performance liquid chromatography-tandem mass spectrometry Se Pu 27 (3) 264 - 269
Long Y , Zhang W , Wang F , Chen Z 2014 Simultaneous determination of three curcuminoids in Curcuma long L. by high performance liquid chromatography coupled with electrochemical detection J Pharm Anal 4 (5) 325 - 330
1996 Validation of analytical procedures: text and methodology Q2 (R1). ICH Harmonised Tripartite Guideline [internet]. ICH Switzerland Available from: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1__Guideline.pdf.
USA: Center for Drug Evaluation and Research (CDER). 1994 Reviewer guidance-validation of chromatographic methods [internet]. FDA U.S Available from: http://www.fda.gov/downloads/Drugs/Guidances/UCM134409.pdf
2008 General chapters <621> Chromatography Glossary of Symbols [internet]. USP Pharmacists’ Pharmacopeia USA Available from: http://www.usp.org/sites/default/files/usp_pdf/EN/products/usp2008p2supplement3.pdf