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High Performance Liquid Chromatographic Method with Fluorescence Detection for the Estimation of Thymoquinone in Nigella sativa Extracts and Marketed Formulations

Research Article ºÚÁÏÍø
Muzaffar Iqbal1,2*, Prawez Alam3 and Md Tarique Anwer4
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, KSA, 11451
2Bioavailability Research Laboratory, College of Pharmacy, King Saud University, Riyadh, KSA, 11451
3College of Pharmacy, Salman Bin Abdulaziz University Al Kharj, KSA -11942
4College of Pharmacy, Jazan University, Jazan, KSA
*Corresponding author: Muzaffar Iqbal
Department of Pharmaceutical Chemistry College of Pharmacy
King Saud University, Riyadh, KSA
Fax: +966-1-467-6220
E-mail: muziqbal@gmail.com; muziqbal@ksu.edu.sa
 
Received Janary 09, 2013; Published February 20, 2013
 
Citation: Iqbal M, Alam P, Anwer MT (2013) High Performance Liquid Chromatographic Method with Fluorescence Detection for the Estimation of Thymoquinone in Nigella sativa Extracts and Marketed Formulations. 2:655 doi:10.4172/scientificreports.655
 
Copyright: © 2013 Iqbal M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
Abstract
 
A simple, economic, robust, reproducible, selective, and precise high-performance liquid chromatography (HPLC) method developed and validated for the estimation of thymoquinone in two different extract and marketed formulations. The mobile phase composed of 20-mM KH2PO4 buffer (pH adjusted to 2.7 ± 0.05 using 50% OPA) and acetonitrile at a ratio of 60:40 eluted at a flow rate of 1 mL/min. Thymoquinone was monitored using florescence detector set at 274 excitation and 340 emission wavelength, having column oven temperature of 40°C and sample cooler temperature of 8°C ± 0.2°C. The linear regression analysis data for the calibration curve shows good linear relationship with correlation coefficient of 0.998 in the concentration range of 0.07-12 μg/mL. The limit of detection and limit of quantification were 0.023 and 0.07 μg/mL, respectively. The developed method was validated for accuracy, precision, reproducibility, and robustness as per ICH guidelines. The proposed method with high degree of precision and accuracy is employed for the estimation of thymoquinone in methanolic and petroleum extract of Nigella sativa as well as in formulation. Statistical analysis proved that the method is precise, reproducible, selective, and accurate for the estimation of thymoquinone for quality control purpose.
 
Keywords
 
Thymoquinone; Nigella sativa; HPLC; Chromatography
 
Introduction
 
Nigella sativa is an herbaceous plant belongs to a botanical family Ranunculaceae, grows in the Middle East, Central Europe and Western Asia. It is commonly known as black Seed or Black Caraway Seed and traditionally used for the prevention and cure of many ailments for over 2000 years [1-3]. Its seed oil had been used in Arab traditional herbal medicine for the treatment of arthritis, lung diseases and hypercholesterolemia [4]. Recent studies also showed that the Nigella sativa oil may be considered as a potential adjuvant therapy in rheumatoid arthritis [5]. Commercial use of these seeds has been extended too many products including shampoos, oils and soaps [6]. In experimental study, Nigella sativa has shown antineoplastic and antioxidant affects both in vitro and in vivo animal model. The antitumor activity of Nigella sativa seed was tested on the human hepatoma HepG2 cell line by Thabrew et al. [7]. Aqueous and alcohol extracts of Nigella sativa were found to be effective in vitro in inactivating MCF-7 breast cancer cells [8], where as methanolic, and chloroform extracts of Nigella sativa effectively killed HeLa (human epithelial cervical cancer) cells by inducing apoptosis [9]. Khan and Sultana reported the chemo-preventive effect of Nigella sativa against ferric nitrilotriacetate (Fe-NTA)-induced renal oxidative stress, hyper-proliferative response and renal carcinogenesis [10]. Active constituents of black seed identified by HPLC, include thymoquinone, dithymoquinone, thymohydroquinone and thymol [11]. But studies had shown that the biological activity of Nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly (30- 48%) thymoquinone (THQ) [12-14].
 
Standardization of herbal formulations in terms of quality of raw materials, manufacturing practices and composition is important to ensure quality and optimum levels of active principles for their biopotency. Since THQ is principal bioactive component of black seed, simple robust method is required for quantification of this active constituent which has been used for quality control and standardization plant and its formulation as a marker compound. In literature, several analytical methods for the determination of THQ have been reported. Methods include HPTLC method in Nigella sativa extracts and formulations [15] polographic [16] and HPLC with UV detection [ 11,17] in black seed oil and UPLC with UV detection in bulk drug and formulations [18]. Here we developed a simple sensitive, highly precise robust HPLC method with florescence detection for the determination of THQ in plant extracts as well as marketed formulations [19].
 
Experimental
 
Chemicals and reagent
 
THQ (purity >99%), was obtained from Sigma Aldrich USA. Its chemical structure is presented in figure 1. Acetonitrile and methanol of HPLC grade were obtained from Winlab laboratory, UK, whereas orthophasphoric acid and potassium dihydrogen phosphate were obtained from BDH and Avonchem Laboratory England. All reagents were of analytical grade unless stated otherwise. All aqueous solutions and buffers were prepared using water that was purified using Milli-QR Gradient A10R (Millipore, Moscheim Cedex, France) having pore size 0.22 μm.
 
Figure 1: Chemical structure of THQ.
 
Plant material and marketed formulations
 
Crude black seed was purchased from a local market and authenticated by a Taxonomist at the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. Baraka capsule, black seed rub and Nigella sativa oil were also purchased from a local market. The powdered seeds of Nigelia sativa (5 g) were extracted by percolation at room temperature with methanol till exhaustion. Another 5 gm were separately extracted similarly with petroleum ether. The solvent was evaporated to dryness under reduced pressure by use of a rotary vacuum evaporator and the residue was separately dissolved in methanol in 50 mL volumetric flask. Accurately 1 ml of the Nigella sativa oil was separately dissolved in 10 mL methanol in volumetric flasks. Accurately weighed 5 gram of black-seed rub also dissolved in 50 ml methanol and filtered it and used for analysis. Accurately weighed 1.622 g of the Baraka capsules was separately dissolved in 25 mL methanol in volumetric flasks.
 
Instrumentation and chromatographic conditions
 
The analysis was performed on Waters Alliance HPLC systems 2695 separation module connected to Waters 2475 Multi Lambda florescence detector. The LC system consisted of a water binary pump, model 1525 (Milford, MA, USA), with an autosampler model 717. Waters quaternary solvent delivery system was used to operate the isocratic flow. Data acquisition was carried out using Empower software. The chromatographic separations were performed using a symmetry LC-18 stainless steel column (150 mm × 3.9 mm, 5 μm). An isocratic mobile phase consisted of 20-mM KH2PO4 buffer (pH adjusted to 2.7 ± 0.05 using 50% orthophosphoric acid) and acetonitrile at a ratio of 60:40 eluted at a flow rate of 1 mL/min. Prepared buffer (20-mM KH2PO4) was filtered through a Millipore membrane filter (0.2 μm) from Nihon, Millipore (Yonezawa, Japan) and degassed in an ultrasonic bath for 5 min. The injection volume was 25 μL and effluent was monitored using florescence detection set at 274 excitation and 340 emission wavelength, having column oven temperature of 40°C and sample cooler temperature of 8°C ± 0.2°C. Calibration curves were constructed by calculating the area vs. concentration of standard analyte.
 
Preparation of stock, calibration and quality control sample
 
10 mg of THQ was dissolved in 10 ml of methanol to produce the stock solution of 1 mg/ml respectively. Serial dilution of THQ was made with the help of MS Exel 2007 in water: methanol (50:50) solution to obtain the calibration range of 0.07–12 μg/ml. Serial dilution of THQ stock solution also made to obtained four different concentration levels of 0.07, 0.24, 3, and 10 μg/mL which was used for quality control sample.
 
Linearity and standard curve
 
Calibration curves of THQ were constructed by using the analyte peak area versus nominal concentrations of the analytes. The linearity of the method was determined by analysis of standard plots associated with a six-point standard calibration curve of 0.07-12 μg/ml. The data were subjected to statistical analysis using a linear-regression model. Least squares linear regression analysis of the data gave slope, intercept and correlation coefficient data. Curves were best fitted using a least square linear regression model y = mx + b, weighted by 1/x2, in which y is the peak area ratio, m is slope of the calibration curve, b is the y-axis intercept of the calibration curve, and x is the analyte concentration. Back calculations were made from these curves to determine the concentration of THQ in each calibration standard, and the resulting calculated parameters were used to determine concentrations of analyte in quality control or unknown samples in plant extracts and formulations. The correlation coefficient R2>0.98 was desirable for all the calibration curves.
 
Limit of detection and limit of quantification
 
Limit of detection (LOD) and limit of quantitation (LOQ) were determined by standard deviation (S y/x) method. For the determination of LOD and LOQ, blank sample was injected in triplicate to the chromatograph, and then peak area of this blank was recorded. The LOD and LOQ were determined using the slope of the calibration curve and Sy/x of the blank sample by following formulae: LOD = 3.3×S y/x /S and LOQ = 10×Sy/x /S; where Sy/x is the standard deviation of the blank response and S is the slope of the calibration curve.
 
Accuracy as recovery
 
Recovery was determined by standard addition method. The pre analyzed samples of THQ (10 μg/mL) were spiked with the extra 0, 50, 100, and 150% of the standard THQ, and the mixtures were reanalyzed by the proposed method. The percent (%) recovery of samples, percentage relative standard deviation (% RSD), and standard error were calculated at each concentration level.
 
Precision and accuracy
 
The within-run and between-run accuracy and precision of the assays were determined by assaying calculated four quality control samples in triplicate over a period of three days. The concentrations represented the entire range of the calibration curves. The lowest level was the expected limit of quantitation (LOQ) for each analyte. The second level was within 3 times of LOQ. Calibration curves were prepared and analyzed daily and linear models were used to determine concentrations in the quality control samples. The nine measured concentrations per concentration level (triplicates from three runs) were subjected to estimate the between-run precision. Percent accuracy was determined (using the data from the precision assessment) as the closeness of spiked samples to the nominal value of in-house standards.
 
Reproducibility
 
Reproducibility of the method was checked by obtaining precision on a different instrument, which was analyzed by another person in a different laboratory. Both intra-day and inter-day precision was calculated at four different concentration levels 0.07, 0.24, 3, and 10 μg/ mL in triplicates.
 
Robustness
 
Robustness was carried out to evaluate the influence of small but deliberate variation in the chromatographic conditions for the determination of THQ. Robustness of the method was determined by changing the flow rate (1.1 and 0.9 mL/min) and ratio (58:42 and 62:38) of mobile phase.
 
Quantification of THQ in extracts and formulations
 
Different concentrations (25, 50, 100 and 200 μl) of methanolic (5 gm/50 ml), petroleum extracts (5 gm/50 ml) and Baraka capsule (1.62 gm/25 ml) were diluted with methanol to make up the volume 1 ml. Similarly Nigella sativa oil (1 mg/ml) was first diluted with methanol to get concentration of 100 μg/ml. Then 10, 20, 40 and 80 μl of it was further diluted with methanol to make up the volume 1 ml. Vortex all above samples vigorously and 25 μl of it injected and chromatograms were obtained in the same conditions as that of standard THQ. The peak area corresponding to the Rt of standard THQ was recorded, and content of the same was calculated from the regression equation obtained from calibration curve.
 
Results
 
Optimization of chromatographic condition
 
The LC method carried out in this study aimed at developing a reverse phase sensitive chromatographic system capable of eluting and resolving THQ and from a standard solution Nigella sativa extracts and marketed formulations. The preliminary investigations were directed toward the effect of several variables on the system suitability of the method. The parameters assessed included the type and percentage of organic modifiers, the flow rate, the type and the concentration of the buffer and the pH of the mobile phase.
 
Initially experiment was started on HPLC connected with photo diode array detector at different detection wavelength. But sensitivity of analyte was very low as no detection happened at ng/ml level. Then analyte was scanned on spectroflourimeter, and the compound was identified as florescence sensitive at excitation 274 and emission 340 wavelengths. The robustness test showed that to create a chromatogram with a minimal variation in separation, one should strictly control the fraction of organic modifier and the pH of the mobile phase. Buffers (disodium monohydrogen phosphate, Potassium dihydrogen orthophosphate) at different pH with varying concentrations of organic phases (methanol, acetonitrile) were tried. Optimum resolution and appropriate retention times of analyte was obtained with potassium dihydrogen orthophosphate buffer and acetonitrile as an organic modifier. So the experiment started by utilizing isocratic flow condition by 20 mM potassium dihydrogen orthophosphate and acetonitrile at different concentration ratio and different pH range between (2.5 to 6.5) by orthophosphoric acid. Adjusting the pH to 2.9 of 20-mM KH2PO4 buffer and its flow with acetonitrile at a ratio of 60:40 associated with better resolution of analyte.
 
 
Calibration curve and linearity
 
The calibration curve area versus concentration (μg/mL) was found linear in the range of 0.07-10 μg/mL. Statistical calculations were done at 5% level of significance. One-way analysis of variance (ANOVA) test was performed to compare the results. The linear regression data for the calibration curve showed a good linear relationship over the concentration ranges of 0.07-12 μg/mL with respect to peak area as shown in table 1. The correlation coefficient value (R2) was found to be 0.998, which was highly significant (Table 1) (p<0.05). The linear regression equation was y = 788235x + 34591. No significant differences were observed in the slope of standard curves (p ≥ 0.05).
 
Table 1: Linear regression data for the calibration curve (n=3).
 
Limit of detection and limit of quantification
 
The analysis was carried out at decreasing concentrations to determine the minimal concentration with a signal-to-noise ratio of 3:1. Under the experimental conditions, the LOD value was 0.023 μg/ mL whereas LOQ values were 0.07 μg/mL respectively. In addition, the analyte peak of the LOQ samples was identifiable, discrete, and reproducible with accuracy within ≤ 20% and a precision of ≤ 20% ( Figures 2 and 3). Table 1 shows the results of statistical analysis of the experimental data, such as the slope, the intercept, and the correlation coefficient obtained by the linear square treatment of the result along with the standard deviation of the slope (Sb) and intercept (Sa).
 
Figure 2: Representative chromatogram of blank sample showing on interference at retention time of THQ.
 
Figure 3: Representative chromatogram of THQ at LOQ level.
 
Recovery
 
The accuracy of proposed method was calculated by recovery analysis, which afforded recovery of 99.21-99.82% after spiking the additional standard drug solution to the previously analyzed test solution. The % RSD values were found in the range of 1.42-1.97. These results indicated the accuracy of the proposed method.
 
Precision
 
The intra-day and inter-day precision values were also studied using different concentration of standard THQ in the range concentration range of calibration curve. Calibration curve and quality control samples were prepared and three replicate measurements were conducted in a period of three days.
 
The intra-day coefficient of variation values (precision) was in the range of 2.28 to 6.92 where as inter-day coefficient of variation values (precision) was in the range of 1.21 to 7.55 (Table 2). These results indicate that the method has good precision and are within the acceptance limit of ≤ 15% and ≤ 15% for precision and accuracy respectively.
 
Table 2: Precision of the proposed method.
 
Reproducibility
 
Reproducibility of the method was checked by obtaining precision of the method in another laboratory using different instruments and analyzed by another person but in the same condition. Both intra-day and inter-day precision was examined in labs. There were no significant differences observed in the % RSD values of intra-day and inter-day precision, which indicates the reproducibility of the method.
 
Robustness
 
The robustness of a method describes its ability to withstand small changes in its operating conditions. A method which is unaffected by change in experimental parameters, within the control limits defined in the method protocol, is said to rugged. The small changes made included the flow rate, and composition of mobile phase (Tables 3 and 4). It was found that the percentage recoveries of THQ were good under most conditions, and remained unaffected by small changes of experimental conditions.
 
Table 3: Robustness of the method by changing the mobile phase ratio.
 
Table 4: Robustness of the method by changing the flow rate.
 
Application of the method for the quantification of THQ in various extracts and formulations
 
The peaks of THQ from sample solution were identified by comparing their retention times obtained from the peaks with those of standard. It was observed that there is no any interaction peak of other constituents or excipients detected at retention time of THQ which indicated the suitability of this method for the routine analysis of THQ in marketed formulations (Figure 4). Samples were injected at different level of dilution and calculated concentration showed good precision. Amount of THQ obtained in different extracts and formulation are presented in table 5.
 
Figure 4: Representative chromatogram of THQ obtained after injection of 25μl concentration level of 1.4 gm/25 ml of Baraka Capsule.
 
Table 5: Measured concentration of THQ in different extracts and formulations (μg/ml).
 
Conclusion
 
In conclusion, a reliable HPLC florescence method with high sensitivity, robust, reproducible and short elution time was developed for the determination of THQ in extracts and various formulations. This method was validated for its selectivity, accuracy as recovery, precision and robustness and was successfully applied for the estimation of THQ in various extracts and formulations.
 
Acknowledgement
 
Authors are grateful to the Research Center of the College of Pharmacy and the Deanship of the Scientific Research of the King Saud University for their funding and support.
 
 
References