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) \[[@B001]\]. 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 \[[@B002], [@B003]\]. 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 \[[@B002]\].

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 \[[@B004]\], to inhibit fibronectin production by keloid derived fibroblasts \[[@B005]\], to inhibit neointimal hyperplasia in the abdominal aorta of rats \[[@B006]\], to treat gout \[[@B007]\], to inhibit asthmatic syndrome \[[@B008]\] and to promote dermal wound healing \[[@B009]\].

Curcumin, commercially available in a mixture of curcumins (curcuminoids), contains ─ 77% pure curcumin, ─ 17% demethoxycurcumin and ─ 3% bisdemethoxycurcumin \[[@B010]\] (Fig [1](#F001){ref-type="fig"}). 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 \[[@B011]\]. Curcumin and its synthetic derivatives (curcuminoids) show an array of pharmacological properties, such as antibacterial \[[@B012] - [@B014]\], antioxidant \[[@B013], [@B015] - [@B016]\], anti inflammatory \[[@B017], [@B018]\], anti tumor \[[@B019], [@B020]\] and anti proliferation \[[@B018], [@B021]\] properties. Curcumin/curcuminoids also possess potency as medicines for the treatment of diseases, including Alzheimer's disease \[[@B022], [@B023]\], cancer \[[@B024], [@B025], [@B026]\], diabetes, gastric ulcers \[[@B027]\], malaria \[[@B028], [@B029]\] and for the treatment of wounds \[[@B030] - [@B032]\].

A variety of methods for quantitatively detecting curcumin and quercetin contents have been reported. Among these, spectrophotometric methods are the most commonly used \[[@B033] - [@B036]\]. Thin layer chromatography (TLC) or column chromatography was usually used for separation of curcuminoids \[[@B037] - [@B039]\]. High performance liquid chromatography (HPLC) \[[@B040] - [@B045]\] and, high performance thin layer chromatography (HPTLC) \[[@B039], [@B046], [@B047]\] 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) \[[@B048]\], ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS) \[[@B049]\], high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) \[[@B050]\] and electrochemical-HPLC \[[@B051]\].

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 \[[@B052]\] 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.

![Chemical structures of quercetin, and the curcuminoids: curcumin, demethoxycurcumin and bisdemethoxycurcumin.](2093-6966-v17-n04-036-g001){#F001}

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](#T04a){ref-type="table"}) , change in acetic acid concentration by ± 1.0% v/v (effect of buffer pH) (Table [4b](#T04b){ref-type="table"}), change in the flow rate of ± 0.1 mL/min (Table [4c](#T04c){ref-type="table"}) and change in the column temperature of ± 5.0°C (Table [4d](#T04d){ref-type="table"}). 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 \[[@B053]\].

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](#T001){ref-type="table"}) 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

  Compounds   LOD (μg/mL)   LOQ (μg/mL)   LOL (μg/mL)   Regression analysis (1.25 --- 200 μg/mL)                   
  ----------- ------------- ------------- ------------- ------------------------------------------ --------------- ---------
  QUE         0.00488       0.03906       200           70055.85913                                1521.41433      0.99993
  BDMC        0.62500       2.50000       320           1807.72930                                 --- 440.28180   0.99984
  DMC         0.07813       0.31250       320           10011.55795                                40.13501        0.99985
  CUR         0.03906       0.07813       320           34176.44088                                3645.08890      0.99993

LOD, limit of detection; LOQ, limit of quantification, LOL, limit of linearity; QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.

![Chromatograms of quercetin and curcuminoids. QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.](2093-6966-v17-n04-036-g002){#F002}

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](#T002){ref-type="table"}) The values of R^2^, the y-intercept and the slope for each compound's calibration plot are shown in (Table [1](#T001){ref-type="table"}) 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)              
  ---------------------- ------------------------ ------------------- ---------- -------
  QUE                                                                            
  1.25                   3.970                    0.117               94937      0.676
  5                      3.972                    0.066               367965     0.739
  20                     3.972                    0.041               1438240    0.624
  40                     3.973                    0.055               2781685    0.508
  80                     3.972                    0.029               5582929    0.437
  140                    3.972                    0.048               9735618    0.866
  200                    3.972                    0.053               14073938   0.368
  BDMC                                                                           
  1.25                   13.823                   0.308               1859       1.611
  5                      13.840                   0.095               8843       1.181
  20                     13.842                   0.093               37086      1.089
  40                     13.843                   0.087               71560      1.044
  80                     13.846                   0.117               143659     1.073
  140                    13.846                   0.134               249462     1.835
  200                    13.849                   0.060               363457     0.850
  DMC                                                                            
  1.25                   15.214                   0.227               14705      0.273
  5                      15.229                   0.096               52692      0.540
  20                     15.230                   0.074               204602     0.665
  40                     15.232                   0.073               398446     0.436
  80                     15.237                   0.099               798153     0.867
  140                    15.236                   0.120               1384220    1.416
  200                    15.242                   0.039               2015583    0.158
  CUR                                                                            
  1.25                   16.708                   0.199               46645      0.856
  5                      16.718                   0.077               182515     0.901
  20                     16.719                   0.061               701982     0.700
  40                     16.720                   0.064               1358591    0.299
  80                     16.725                   0.096               2737751    0.423
  140                    16.725                   0.108               4749355    0.897
  200                    16.734                   0.067               6866971    0.313

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](#F002){ref-type="fig"}), (Table [2](#T002){ref-type="table"}).

Precision and accuracy data for the intraday and the inter-day variations for the three QC samples are summarized in (Table [3](#T003){ref-type="table"}). 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\   Intra day^\*^   Inter day^†^                                 
  (μg/mL)                                                                       
  ---------------- --------------- -------------- ---------- ---------- ------- ---------
  QUE                                                                           

  3.75             263151          0.432          99.589     263350     0.323   99.665

  100              7064599         0.717          100.821    7221470    0.646   103.060

  160              11221611        0.806          100.010    11218287   0.968   100.070

  BDMC                                                                          

  3.75             6243            0.576          98.588     6181       0.854   97.669

  100              182293          0.723          101.084    186683     0.878   103.513

  160              286851          0.654          99.32746   288040     0.805   99.738

  DMC                                                                           

  3.75             37700           0.635          100.310    37687      0.466   100.276

  100              1010004         0.752          100.880    1037410    0.078   103.617

  160              1590498         0.651          99.289     1594989    0.844   99.569

  CUR                                                                           

  3.75             129618          0.655          98.292     129152     0.297   97.929

  100              3456218         0.732          101.022    3544535    0.275   103.606

  160              5448675         0.711          99.576     5454012    0.829   99.673
  ---------------------------------------------------------------------------------------

\*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 suitability              Compound    Change in the normal organic composition of acetonitrile: 2% acetic acid                                                 
  ------------------------------- ----------- -------------------------------------------------------------------------- ----------- ---------- ----------- ---------- -------
  Retention time, t~R~(minutes)   QUE         3.993                                                                      0.690       4.251      0.155       3.761      0.040
  BDMC                            13.951      0.342                                                                      17.645      0.374      11.280      0.084      
  DMC                             15.340      0.291                                                                      19.543      0.285      12.330      0.084      
  CUR                             16.829      0.245                                                                      21.617      0.296      13.464      0.082      
  Peak area                       QUE         6853044                                                                    0.433       6836934    0.441       6867445    0.117
  BDMC                            167417      0.647                                                                      161504      0.801      146484      0.578      
  DMC                             940836      0.404                                                                      903191      0.781      965307      0.071      
  CUR                             3302593     0.236                                                                      3206134     0.555      3367309     0.114      
  Resolution, R                   QUE         \-                                                                         \-          \-         \-          \-         \-
  BDMC                            32.498      0.379                                                                      36.449      0.471      29.120      0.063      
  DMC                             2.908       0.208                                                                      3.272       2.369      2.736       0.181      
  CUR                             2.850       0.237                                                                      3.243       1.648      2.666       0.124      
  Tailing factor, T~f~            QUE         1.371                                                                      0.254       1.347      0.115       1.392      0.074
  BDMC                            1.533       0.364                                                                      1.283       2.314      1.080       0.200      
  DMC                             1.160       0.484                                                                      1.083       0.151      1.431       0.082      
  CUR                             1.094       0.094                                                                      1.076       0.284      1.114       0.037      
  Theoretical plate, N            QUE         8752.133                                                                   1.463       8857.791   0.312       8520.171   0.238
  BDMC                            15931.889   1.147                                                                      16311.011   0.058      16303.130   0.103      
  DMC                             14298.287   1.761                                                                      16569.474   1.029      14210.321   0.233      
  CUR                             16008.049   1.202                                                                      16543.754   0.535      15157.508   0.340      
  Capacity factor, k'             QUE         0.680                                                                      0.344       0.777      0.906       0.601      0.327
  BDMC                            4.878       0.202                                                                      3.800       0.209      3.800       0.209      
  DMC                             5.463       0.232                                                                      7.214       1.592      4.247       0.206      
  CUR                             6.097       0.253                                                                      8.038       0.481      4.729       0.209      

###### (b). Robustness -- change in acetic acid concentration

  System suitability              Compound    Change in the acetic acid concentration (% v/v )                                                 
  ------------------------------- ----------- -------------------------------------------------- ----------- ---------- ----------- ---------- -------
  Retention time, t~R~(minutes)   QUE         3.972                                              0.175       4.054      0.064       3.893      0.071
  BDMC                            13.868      0.310                                              14.549      0.086      13.177      0.167      
  DMC                             15.255      0.265                                              16.017      0.085      14.542      0.153      
  CUR                             16.743      0.213                                              17.590      0.084      16.028      0.141      
  Peak area                       QUE         7039483                                            0.562       6966950    0.525       6952833    0.630
  BDMC                            180475      0.541                                              176885      0.575      152439      0.895      
  DMC                             1000716     0.736                                              987128      0.551      956266      0.670      
  CUR                             3433379     0.754                                              3428762     0.533      3428762     0.558      
  Resolution, R                   QUE         \-                                                 \-          \-         \-          \-         \-
  BDMC                            32.327      0.172                                              33.254      0.244      31.950      0.268      
  DMC                             2.900       0.370                                              2.974       0.303      3.033       0.527      
  CUR                             2.840       0.429                                              2.904       0.339      2.966       0.608      
  Tailing factor, T~f~            QUE         1.366                                              0.077       1.364      0.215       1.370      0.110
  BDMC                            1.493       1.377                                              1.463       0.331      1.060       0.139      
  DMC                             1.160       1.075                                              1.137       0.103      1.325       0.823      
  CUR                             1.085       0.148                                              1.092       0.050      1.083       0.108      
  Theoretical plate, N            QUE         8711.993                                           0.267       8877.546   0.460       8548.948   0.269
  BDMC                            15740.557   0.397                                              16067.808   0.689      16308.146   0.664      
  DMC                             14041.181   0.701                                              14691.580   0.675      14241.082   1.031      
  CUR                             15793.019   0.472                                              16098.239   0.701      15531.342   0.811      
  Capacity factor, k'             QUE         0.656                                              1.783       0.680      1.484       0.610      0.803
  BDMC                            4.798       1.202                                              5.036       0.658      4.449       0.511      
  DMC                             5.333       0.988                                              5.637       0.698      5.014       0.478      
  CUR                             6.016       1.416                                              6.295       0.628      5.629       0.443      

###### (c). Robustness -- change in flow rate

  ---------------------------------------------------------------------------------------------------------------
  System suitability      Compound    Change in flow rate                                                 
  ----------------------- ----------- --------------------- ----------- ---------- ----------- ---------- -------
  Retention time, t~R~\   QUE         3.972                 0.175       4.291      0.105       3.696      0.130
  (minutes)                                                                                               

  BDMC                    13.868      0.310                 14.953      0.321      12.909      0.333      

  DMC                     15.255      0.265                 16.442      0.284      14.235      0.279      

  CUR                     16.743      0.213                 18.038      0.262      15.668      0.298      

  Peak area               QUE         7039483               0.562       7606272    0.662       6530571    0.497

  BDMC                    180475      0.541                 194216      0.753      167111      1.593      

  DMC                     1000716     0.736                 1078076     0.714      928707      1.345      

  CUR                     3433379     0.754                 3700134     0.690      3185325     1.198      

  Resolution, R           QUE         \-                    \-          \-         \-          \-         \-

  BDMC                    32.327      0.172                 32.779      0.199      32.047      0.928      

  DMC                     2.900       0.370                 2.921       0.608      2.936       2.014      

  CUR                     2.840       0.429                 2.864       0.723      2.868       1.647      

  Tailing factor, T~f~    QUE         1.366                 0.077       1.360      0.183       1.371      0.287

  BDMC                    1.493       1.377                 1.490       1.891      1.539       1.614      

  DMC                     1.160       1.075                 1.157       1.447      1.181       2.364      

  CUR                     1.085       0.148                 1.081       0.101      1.087       0.207      

  Theoretical plate, N    QUE         8711.993              0.267       9148.347   0.429       8249.430   0.420

  BDMC                    15740.557   0.397                 16035.103   1.342      15696.046   2.851      

  DMC                     14041.181   0.701                 14374.944   1.036      13420.220   0.844      

  CUR                     15793.019   0.472                 16216.013   1.854      15379.165   2.364      

  Capacity factor, k'     QUE         0.656                 1.783       0.661      0.832       0.627      0.762

  BDMC                    4.798       1.202                 4.780       0.942      4.750       3.067      

  DMC                     5.351       0.661                 5.355       0.497      5.350       2.846      

  CUR                     5.966       0.632                 5.985       0.500      5.862       0.427      
  ---------------------------------------------------------------------------------------------------------------

###### (d). Robustness -- change in column temperature

  System suitability              Compound    Change in column temperature                                                 
  ------------------------------- ----------- ------------------------------ ----------- ---------- ----------- ---------- -------
  Retention time, t~R~(minutes)   QUE         3.956                          0.031       4.063      0.074       3.861      0.162
  BDMC                            13.673      0.070                          14.647      0.172      12.810      0.268      
  DMC                             15.037      0.064                          15.980      0.153      14.167      0.236      
  CUR                             16.502      0.064                          17.423      0.143      15.657      0.196      
  Peak area                       QUE         7628483                        0.252       7620525    0.254       7633341    0.259
  BDMC                            196493      0.261                          202870      0.253      172397      0.136      
  DMC                             1091099     0.300                          1124567     0.281      1058404     0.205      
  CUR                             3738544     0.244                          3836306     0.285      3643910     0.196      
  Resolution, R                   QUE         \-                             \-          \-         \-          \-         \-
  BDMC                            31.946      1.437                          32.560      0.233      31.471      0.267      
  DMC                             2.872       1.359                          2.698       0.334      3.155       0.481      
  CUR                             2.829       0.575                          2.718       0.305      3.106       0.559      
  Tailing factor, T~f~            QUE         1.343                          0.056       1.329      0.267       1.351      0.124
  BDMC                            1.551       0.421                          1.233       0.525      1.089       0.077      
  DMC                             1.186       0.241                          1.098       0.268      1.491       1.051      
  CUR                             1.097       0.082                          1.094       0.107      1.098       0.129      
  Theoretical plate, N            QUE         8734.237                       0.300       8837.810   0.448       8619.473   0.295
  BDMC                            15779.175   0.595                          15065.104   0.600      16276.545   1.203      
  DMC                             13866.206   1.175                          15742.765   0.475      15286.704   0.311      
  CUR                             15846.706   0.791                          15917.987   0.394      15793.349   0.183      
  Capacity factor, k'             QUE         0.698                          1.598       0.717      1.945       0.616      0.692
  BDMC                            4.855       0.115                          5.190       0.989      4.366       0.214      
  DMC                             5.449       0.349                          5.752       0.966      4.962       1.315      
  CUR                             6.058       0.201                          6.362       0.942      5.581       1.015      

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

  Parameter               Formula                                   Recommendation
  ----------------------- ----------------------------------------- --------------------------
  Precision               RSD = S/x̄^\*^100                          RSD ≤ 1% for n ≥ 5
  Resolution, R           R = (t~R2~-- t~R1~)/(1/2)(t~w1~--t~w2~)   \> 2
  Tailing factor, T~f~    Tf = Wx/2f                                ≤ 2
  Theoretical plates, N   N = 16(t~R~/t~w~)^2^                      Column efficiency ≥ 2000
  Capacity factor, k      k' = (t~R~-- t~0~)/t~0~                   \> 2

S, standard deviation; x̄ , mean of the data; t~R~, retention time of analyte 1; tw, peak width measured to the baseline of the extrapolated straight sides to baseline; W~x~, 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 W~x~; t~0~, elution time of the void volume or non retained components.

###### System suitability testing

  Parameter              QUE        BDMC    DMC         CUR                                     
  ---------------------- ---------- ------- ----------- ------- ----------- ------- ----------- -------
  Retention time, t~R~   3.970      0.021   13.840      0.027   15.230      0.025   16.723      0.021
  Peak area              11221611   0.806   286851      0.654   1590498     0.651   5448675     0.711
  Resolution, R          \-         \-      32.195      0.321   2.887       0.364   2.830       0.370
  Tailing factor, T~f~   1.369      0.108   1.501       0.261   1.165       0.144   1.081       0.051
  Theoretical plate, N   8803.785   0.359   15552.398   0.865   13763.145   0.646   15568.252   0.910
  Capacity factor, k'    0.684      0.846   4.870       0.415   5.460       0.406   6.093       0.391

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 \[[@B052]\]. 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](#T04a){ref-type="table"} - [d](#T04d){ref-type="table"}). 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](#F003){ref-type="fig"})

The system suitability criteria were in accordance with the Centre for Drug Evaluation and Research (CDER) guidelines \[[@B054]\] and are summarized in (Table [5](#T005){ref-type="table"}) 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](#T006){ref-type="table"}) 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) \[[@B053]\]. 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.

![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.](2093-6966-v17-n04-036-g003){#F003}

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](#T007){ref-type="table"}). 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](#T007){ref-type="table"}). 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](#F004){ref-type="fig"}) shows the chromatograms for the quercetin and the curcuminoids found in the tested samples.

###### Concentration of QUE, CUR, DMS and BDMC in Chinese medicines

  No   Chinese medicine                    Type                           Concentration (mean ± S.D\*) (μg/100 mg)                         
  ---- ----------------------------------- ------------------------------ ------------------------------------------ ---------- ---------- ----------
  1    Gao liang jiang (高良姜)            Single plant granule extract   0.7532                                     N.D        134.8739   0.5270
  2    Jin qian cao (金钱草)               Single plant granule extract   4.0618                                     N.D        N.D        0.8263
  3    Yu jin (郁金)                       Single plant granule extract   0.3195                                     69.1060    27.2286    27.1020
  4    E su (莪术)                         Single plant granule extract   0.5983                                     79.5922    42.6982    8.6812
  5    Jiang huang (姜黄)                  Single plant granule extract   3.6523                                     N.D        933.8122   796.0621
  6    Yu xing cao (鱼腥草)                Single plant granule extract   1.7930                                     N.D        N.D        1.3424
  7    Ting li zi (葶苈子)                 Single plant granule extract   1.3604                                     N.D        N.D        N.D
  8    Tu si zi (菟丝子)                   Single plant granule extract   3.9300                                     N.D        N.D        N.D
  9    Di yu (地榆)                        Single plant granule extract   0.8962                                     N.D        N.D        N.D
  10   Kui hua (愧花)                      Single plant granule extract   311.0307                                   N.D        N.D        N.D
  11   Sang ju yin (桑菊饮)                Formulation granule extract    0.7402                                     N.D        0.3558     0.2537
  12   Chai hu su gan san(柴胡疏肝散)      Formulation granule extract    0.2029                                     126.8843   48.3408    1.6417
  13   Xiao yao san (逍遥散)               Formulation granule extract    0.4991                                     97.9203    2.5534     0.4301
  14   Long dan xie gan tang(龙胆泄肝汤)   Formulation granule extract    11.1482                                    5.2111     1.2817     0.1236
  15   Sang ju gan mao pian(桑菊感冒片)    Tablet                         17.3489                                    173.6155   2.8579     N.D
  16   Dan zhi xiao yao pian(丹栀逍遥片)   Tablet                         7.8101                                     135.1892   1.0883     0.2624
  17   Long dan xie gan pian(龙胆泄肝片)   Tablet                         N.D                                        5.5352     6.7428     0.2378
  18   Bu zhong yi qi (补中益气)           Tablet                         0.9052                                     623.1338   5.9485     0.5964
  19   Xiao yao wan (逍遥丸)               Pill                           12.015                                     79.7951    11.7471    1.1516

\*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) \[[@B054]\] 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.

![Chromatograms for Chinese medicinal plant extracts (a) containing quercetin and (b) containing curcuminoids. QUE, quercetin; BDMC, bisdemethoxycurcumin; DMC, demethoxycurcumin; CUR, curcumin.](2093-6966-v17-n04-036-g004){#F004}

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.

**Conflict of interest** The authors declare that there are no conflict of interest.
