Abstract
A new high-performance liquid chromatographic (HPLC) method for determination of triclosan (TCS) and flurbiprofen (FBP) was successfully developed and validated at a single wavelength. The method involves extraction of the targeted drugs from nanogels and simulated saliva by using methanol as the extractant. The Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm) was used for the chromatographic separations. The effects of various parameters were extensively evaluated and optimized. The optimal HPLC conditions were acetonitrile and 0.001 M citric acid (90:10, v/v) with a pH of 3.24 as the mobile phase, at a 0.3 mL/min flow rate under isocratic elution mode. Excellent sensitivity and specificity were achieved by ultraviolet (UV) detection at 242 nm. The method also demonstrated excellent linearity within the test range of 10–100 μg/mL with the correlation coefficient (R2) of 0.9998 for both the analytes. The practical applicability of the method was demonstrated by recovering TCS and FBP from nanogels and simulated saliva. The recovery of the analytes from the nanogels and the spiked simulated saliva samples was in the range of 97–98% and 96–99%, respectively, and their respective relative standard deviation (RSD) was less than 0.9% in both cases. System suitability parameters were found to be within acceptable limits. The method is simple, specific, and precise, and to the best of our knowledge, it is the first reported validated quantitative HPLC method for the concurrent determination of TCS and FBP in a pharmaceutical dental product. The method can be useful in the routine quality control analysis of dental formulations with TCS and FBP contents or products with a similar composition.
Introduction
The recent innovations in dental drug delivery systems have provided an avenue for simultaneous delivery of multiple drugs from a unit dosage form system to the intended mouth cavity area. The combination of two or more active agents in a unit delivery system is important for achieving the desired broad therapeutic effects for the purpose of countering certain dental illnesses, such as periodontal disease, and especially in conditions where the therapeutic effect of a single active agent is inadequate.
Triclosan (5-chloro-2-(2,4-dichlorophenoxy) phenol) (Figure 1a) is a non-ionic antimicrobial agent which has a broad spectrum antibacterial effect, as well as proven efficacy against various plaque-forming bacteria [1, 2]. It has been used extensively in oral health care products like oral rinses and dentifrices [3, 4] due to its efficacy in preventing and decreasing bacterial plaque [5–7]. Researchers have investigated the efficacy of TCS in periodontal disease treatment and found the drug to be a suitable candidate for its treatment. A study which involved 60 adult human subjects with recurrent periodontal disease was conducted by treating the subjects with dentifrice containing 0.3% TCS. The results demonstrated that this dentifrice with the TCS content reduced bone loss and the frequency of deep periodontal pockets [7]. Similar results were obtained when nanoparticles (NPs) loaded with 9.09% of TCS were used to treat beagle dogs with induced periodontal disease [2].
Structures of triclosan (a) and flurbiprofen (b)
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00286
Besides the pathogenic bacteria that causes periodontal disease, inflammation also plays a significant role in the pathogenesis of the disease [8–12]; hence, the need for anti-inflammatory drugs, such as FBP, to counter the inflammation and pain [13, 14]. Flurbiprofen, (RS)-2-(2-fluorobiphenyl-4-yl) propanoic acid (Figure 1b), is a member belonging to the phenylalkanoic acid class of non-steroidal anti-inflammatory drugs (NSAIDs) that have been used in adjuvant therapy for treating periodontitis in both animals and humans. In two separate studies, a significant reduction in bone loss was observed when adult human subjects with moderate to severe periodontitis were treated topically with toothpaste containing 1% w/w of FBP or systemically with 50 mg of FBP, both twice daily. This suggested the influence of FBP on bone metabolism [15, 16]. FBP blocks the cyclooxygenase enzyme which causes inflammation and pain [15]. The drug is used for not only relieving pain in periodontitis, but also for slowing the disease process [16].
Looking at the potential benefits of the described drugs, it is worthwhile to attempt exploring the combination of the duo in the treatment of localized oral cavity disease (such as periodontitis). A team of researchers investigated the therapeutic efficacy of 0.3% each of TCS and FBP gel on periodontitis cases. The gel was applied intracrevicularly once daily for a week by 16 volunteers that participated in the study. The results demonstrated significant reductions in the clinical parameters which indicated that local delivery of 0.3% of TCS and FBP gel can be used in periodontal therapy [17]. This study and other similar investigations have presented new analytical challenges and demanded a reliable quantification method for these drug combinations in pharmaceutical formulations. There are several reported analytical methods in the literature for the estimation of TCS or FBP either alone [5, 18–23] or in combination with another drug [24–29] from various media samples. However, there is no published data on the concurrent quantification of the two drugs (TCS and FBP) from any dental formulation or simulated saliva. Separate determination of the two drugs from a pharmaceutical product is costly and time-consuming and may not be suitable in a pharmaceutical firm setting; hence, a simple, rapid, economical, and accurate analytical method is needed to address the problem. Therefore, in this work, we present the development, validation, and application of a new HPLC method for the concurrent determination of TCS and FBP from a nanogel formulation and simulated saliva. Simulated saliva is an acceptable medium that has been widely used for the quantification of drugs from localized drug delivery systems that are meant to be used within the oral cavity [30].
Experimental
Chemicals and Reagents
High purity TCS was purchased from Bio Basic Canada Inc. (Markham Ontario, Canada). FBP was purchased from FDC limited (Mumbai, India). Poly-ε-caprolactone (PCL) with ̴ 14,000 molecular weight, Chitosan (medium molecular weight), and Kolliphor® P188 were purchased from Sigma-Aldrich, USA. HPLC grade acetonitrile was purchased from Fisher Scientific (Leicestershire, England). HPLC grade methanol was from Merck (Darmstadt, Germany). AR grade acetone was from QReC® Asia, (Selangor, Malaysia). Citric acid anhydrous, sodium phosphate dibasic, and sodium hydroxide pellets were purchased from R&M (Essex, England). Trisodium citrate dehydrate was from Citrique Belge (Belgium). Potassium phosphate monobasic and sodium chloride were from Bendosen laboratory chemicals (Malaysia). Water was produced in-house by the Favorit W4L water system (Genristo Ltd., England).
The mobile phase was filtered through nylon filters (0.45 μm) containing a titan membrane disc (Sun Sri, USA) and sonicated with Power sonic 405 (Seoul, Korea) before use. All sample solutions were filtered through a 0.45 μm polytetrafluoroethylene (PTFE) syringe membrane filter (Acrodisc, Pall Corporation, USA) prior to their injection into the HPLC system.
Apparatus and HPLC Conditions
The HPLC system was a Shimadzu (Japan) equipped with an ultraviolet–visible (UV–vis) detector, solvent delivery unit (LC-20AD), degasser, column oven, auto sampler, and communication bus module. Data acquisition and processing were carried out using LabSolutions software installed in a desktop computer system. An Agilent ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm) (USA) was used for the chromatographic separations. The oven temperature for the column was set and maintained at 30 °C. The HPLC mobile phase used was acetonitrile and 0.001 M citric acid (90:10, v/v) with a pH of 3.24, at a flow rate of 0.3 mL/min. Ten-microliter injection volumes of calibration standard solutions/test samples were analyzed without further dilution, under an isocratic elution mode. For each injection, a run time of 20 min was allowed in order to ensure full detection of both drugs and their clearance from the system before the next injection. Following the preliminary UV–vis spectrophotometric analysis by spectrophotometer (Perkin Elmer, Lambda 25; Singapore), FBP was found to have a maximum absorbance at 205 and 247 nm (Figure 2a) while TCS was found to have a maximum absorbance at 207, 236, and 282 nm (Figure 2b). The chosen optimum wavelength for the HPLC system which provided the best sensitivity for both analytes was 242 nm.
The spectrograms of FBP (a) at maximum absorbance of 205 and 247 nm and TCS (b) at maximum absorbance of 207, 236 and 282 nm, respectively
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00286
Preparation of Standard Solutions and Standard Calibration Curves
The stock solution was prepared by accurately weighing 10 mg each of TCS and FBP, using a Sartorius microbalance (Goettingen, Germany), and dissolving them in 100 mL methanol to produce a 100 μg/mL solution. From this stock solution, different diluted concentrations were prepared ranging from 10 to 90 μg/mL, using methanol as a diluting solvent. Each standard solution (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg/mL) was injected in 5 replicates into the HPLC system. The peak areas were recorded and plotted against the corresponding concentrations to yield the standard calibration curves of TCS and FBP. Least square linear regression with error estimation was used to determine the y-intercept, slope, and correlation coefficients of the calibration graphs.
Preparation of the Simulated Saliva
Simulated saliva was prepared based on the previously reported method [30, 31]. Briefly, sodium chloride (8 g), sodium phosphate dibasic (2.38 g), and potassium phosphate monobasic (0.19 g) were accurately weighed and then transferred into a 1 L volumetric flask containing 500 mL of distilled water. The flask was agitated for 20 min using a STIK® incubator shaker (Shanghai, China), and the volume was made to the mark with distilled water. The resultant solution had a pH of 6.8.
Preparation of Spiked Simulated Saliva Samples
Accurately weighed quantities (200 mg) of TCS and FBP were treated with 100 mL of simulated saliva under sonication for 30 min at 45 °C and magnetic stirring for 2 h at 500 rpm using a WiseStir® thermostatic magnetic stirrer (Wertheim, Germany). This formed the stock sample solution of a 2 mg/mL concentration. From this stock sample solution, 0.3, 1, and 1.8 mg/mL concentrations were prepared. Then, 1 mL each of 0.3, 1, and 1.8 mg/mL solution was transferred into 20 mL volumetric flasks containing 15 mL of methanol. The mixtures were then agitated for 20 min and sonicated for 30 min at 45 °C in order to extract the TCS and FBP. The volume was made up to the mark with methanol to obtain working test solutions having final concentrations of 15, 50, and 90 μg/mL, respectively. The resultant solutions were filtered and then analyzed. An unspiked sample was also prepared and analyzed as per the described procedure. Similarly, more samples of the same concentrations as stated above were prepared as per the above described procedure by using acetonitrile and 0.1 M sodium hydroxide as extracting solvents.
Preparation of the Nanogel Formulation
A nanogel was prepared using a three-stage procedure. Firstly, TCS-loaded NPs were prepared by solvent displacement, according to our modified reported method [32]. Briefly, 50 mg of PCL and 10 mg of TCS were dissolved in acetone by sonication for 30 min at 45 °C, and then magnetic stirring was carried out for 2 h at 500 rpm. This polymer-drug solution was slowly injected into a 0.2% Kolliphor® P188 aqueous solution (about 50 mL) under continuous magnetic stirring at 1000 rpm. The acetone was completely evaporated by mild heating at 45 °C and prolonged magnetic stirring for 24 h. The resultant TCS-loaded NPs suspension was concentrated and dried using a rotary evaporator (EYELA N-1000, Japan); and then, it was kept in a refrigerator at 4 °C until further use.
Secondly, 10 mg of FBP was mixed with chitosan powder (400 mg) by geometric dilution in a glass mortar. This mixture was then slowly added into 20 mL of a 0.3 M citric acid solution in a beaker under magnetic stirring, which was allowed to continue for 24 h to make a uniform FBP loaded chitosan hydrogel solution. Then, the hydrogel solution was cooled in a refrigerator to 4 °C.
The last stage was the formation of the nanogel which was carried out by slowly adding the previously prepared TCS-loaded NPs into the FBP-loaded hydrogel solution under magnetic stirring for 30 min. Similarly, a blank nanogel was prepared without any TCS and FBP content.
Recovery of Triclosan and Flurbiprofen
An amount of the nanogel was digested in acetone and treated in the simulated saliva and methanol with the aid of sonication and magnetic stirring; finally, it was diluted with methanol to correspond with a supernatant (originated after centrifugation of the treated nanogel) with a final concentration of 12 μg/mL each of TCS and FBP. The supernatant was filtered through a 0.45 μm PTFE syringe filter and 10 μL was injected directly into the HPLC system without further dilution. Similarly, an unloaded nanogel formulation sample was prepared and analyzed as per the above described procedure.
Method Validation
The proposed HPLC method was validated as per standard guidelines [33, 34], and it involved an assessment of the validation characteristics, such as specificity, linearity, range, accuracy, precision, system suitability, limit of detection (LOD), and limit of quantification (LOQ).
In order to demonstrate specificity, chromatograms of TCS and FBP of standard solutions were compared with chromatograms of the sample solutions with and without the analytes.
The linearity of the calibration graph was demonstrated by preparing ten different concentrations of working standard solutions over a range of 10–100 μg/mL. The calibration curves were developed by plotting the chromatograms' peak areas against the corresponding concentrations. The relevant plot parameters, such as the correlation coefficient, residual sum of squares, slope of the regression line, and y-intercept, were calculated. The spiked samples were prepared at concentration levels that fell within the range that covered the expected analytes' contents.
Accuracy was established by the recovery of the TCS and FBP from the nanogel formulation and simulated saliva samples with known analyte contents (15, 50, and 90 μg/mL). The amount of each drug was calculated from previously made calibration curves, and the percentage recovery and percentage relative error (% RE) were considered as indicators of accuracy.
Intra-day and inter-day precision was determined by repeated injections (n = 6) of the spiked simulated saliva sample solutions within the same day and on six different days, respectively. The percentage recoveries and percentage relative standard deviations (% RSD) were determined as a measure of precision.
In order to ensure that the complete system met the required expectations under the experimental conditions of the tests, system suitability testing was carried out by injecting 10 replicates of the standard and test sample solutions with known amounts of the analytes. The desirable system suitability parameters, such as tailing factor, United States Pharmacopeia (USP) number of theoretical plates, USP resolution, injection precision, and repeatability, were calculated and recorded.
The LOD and LOQ were demonstrated based on the signal-to-noise ratio. The lowest amount of analytes in the samples which yielded peaks three times greater than the height of the corresponding blank sample's noise was determined as the LOD. On the other hand, the amount of analytes in the samples which yielded peaks ten times greater than the height of the corresponding blank sample's noise was determined as the LOQ.
Results and Discussion
Selection of Extraction Solvent
TCS and FBP recovery was carried out using three different extraction solvents, i.e., methanol, acetonitrile, and 0.1 M sodium hydroxide. The drugs were extracted according to the procedure described under the preparation of the spiked simulated saliva samples, and the same experimental conditions were maintained when using all the solvents for the recovery. In each round of tests, six replicates were analyzed. The percentage recovery of TCS was 99.4%, 92%, and 43%, while that of FBP was 98.9%, 90%, and 19% for methanol, acetonitrile, and 0.1 M sodium hydroxide, respectively. Hence, methanol gave the best percentage recovery for both TCS and FBP when compared with the other extraction solvents. Therefore, methanol was selected as the extraction solvent for this study.
Selection of Mobile Phase and Chromatogram Conditions
Various mobile phase solvents at different combination ratios were tried while developing the method. The organic solvents that were tested for suitability were acetonitrile and methanol while the aqueous solvents were ultra-pure water, citric acid, and citrate buffer. The optimal HPLC conditions were achieved by keeping one parameter constant while varying the other parameters. The determining parameters used for selection were a separation of analyte peaks from the excipients, impurities, or other agents; reproducibility of retention times (tR); and peak's sharpness, resolution, and symmetry. Unfit tailing occurred in one of the analyte's peaks (TCS) with no symmetry when a higher ratio of water was used, i.e., acetonitrile and water (75:25, v/v). When acetonitrile and citrate buffer (pH 3–5) at different ratios were used as the effluent to separate the TCS and FBP, no improvement in the peaks' parameters was observed. Similar results were obtained when acetonitrile was substituted with methanol.
However, after several experimental trials, which involved varying the ratio of the acetonitrile–methanol to water, acetonitrile–methanol to citric acid (pH 3–5), and acetonitrile–methanol to citrate buffer, TCS and FBP were successfully fractionated with the solvent combination of acetonitrile–citric acid (pH 3.24), ratio 90:10, v/v under isocratic condition. The effect of the flow rate from 0.2 to 1 mL/min was also studied.
The best peak parameters for TCS and FBP were obtained at the pH of 3.24. A low pH is required for the analysis of acidic pharmaceuticals in order to prevent the dissociation of acidic active agents in the samples [22, 27]. The optimum flow rate was 0.3 mL/min. This maintained the HPLC pump pressure at around 5.3 (±0.3) MPa. Figure 3c depicts the HPLC chromatogram under optimal conditions. The tR for FBP and TCS were around 10.1 min and 12.5 min, respectively.
Chromatograms of loaded (a) and unloaded (b) nanogel formulation; standard solution (c); spiked (d) and unspiked (e) simulated saliva solution; methanol (f) and mobile phase (g) of the proposed method. 1,2Peaks of simulated saliva constituents. 3Peak of poly-ε-caprolactone
Citation: Acta Chromatographica Acta Chromatographica 30, 4; 10.1556/1326.2017.00286
To assure the accuracy and precision of the collected HPLC data, system suitability tests were carried out and the proposed method's parameters (Table 1) for both the analytes were found to be within acceptable limits, i.e., tailing factor ≤2, USP number of theoretical plates >2000, USP resolution >2, and injection precision and repeatability's RSD ≤1% for n ≥ 5 [34].
The proposed method validation results
Parameter | Triclosan | Flurbiprofen | Acceptable limits [34, 35] |
---|---|---|---|
System suitability (n = 10) | |||
t R (±SD) | 12.47 (0.03) | 10.12 (0.01) | – |
Tailing factor (±SD) | 0.88 (0.04) | 1.30 (0.08) | ≤2 |
Number of theoretical plates (±SD) | 4337.60 (168.04) | 8650.10 (432.69) | >2000 |
Resolution (±SD) | 3.93 (0.10) | 2.50 (0.15) | >2 |
Injection repeatabilitya (% RSD) | 0.84 | 0.84 | ≤1% |
Linear regression | |||
Regression equation | y = (66,733.6)x + (−24,379.3) | y = (152,214.3)x + (−62,608.7) | – |
Slope | 66,733.6 | 152,214.3 | – |
y-Intercept | −24,379.3 | −62,608.7 | – |
R 2 | 0.9998 | 0.9998 | >0.999 |
Ranges (μg/mL) | 10–100 | 10–100 | – |
Accuracy | |||
Average recovery (%) | 97.25b, 99.17c | 101.04b, 97.98c | 100 ± 2% |
Average RE (%) | −2.75d, −0.83e | 1.04d, −2.02e | ±2% |
Average RSD (%) | 0.06f, 0.30g | 0.19f, 0.76g | – |
Precision | |||
Intra-day precisionh (% RSD) | 0.20 | 0.61 | ≤2% |
Inter-day precisioni (% RSD) | 0.33 | 0.38 | ≤2% |
LOD (μg/mL) | 0.02 | 0.01 | – |
LOQ (μg/mL) | 0.06 | 0.03 | – |
t R, retention time (in minutes); SD, standard deviation; RSD, relative standard deviation; RE, relative error; LOD, limit of detection; LOQ, limit of quantification.
RSD values for injection repeatability of the method (n = 10).
Percentage recovery values of analyte from nanogel formulation (n = 6).
Percentage recovery values of analyte from spiked samples at three concentration levels (n = 6).
Percentage relative error values of analyte from nanogel formulation (n = 6).
Percentage relative error values of analyte from spiked samples at three concentration levels (n = 6).
RSD values for recoveries of analyte from nanogel formulation (n = 6).
RSD values for recoveries of analyte from spiked samples at three concentration levels (n = 6).
RSD values for precision of six samples of the analytes analysis within the same day (n = 6).
RSD values for precision of analytes samples analysis for six consecutive days (n = 6).
Calibration Curve, Linearity, and Range
Both calibration curves for TCS and FBP exhibited good linearity within the analyzed concentration range. The regression coefficient (R) and correlation coefficient (R2) requirements of R ≥ 0.999 and R2 ≥ 0.995, respectively, were considered acceptable [26, 34], and were achieved for the two analytes. Statistical data for the calibration curves of the two drugs are summarized in Table 1.
Accuracy and Precision
Accuracy and precision were demonstrated by spiking simulated saliva with 15, 50, and 90 μg/mL concentrations of TCS and FBP which were then analyzed 6 times. Three different concentration levels were chosen for both drugs to be within the working linearity range. The selected levels represent lower, middle, and higher concentrations. The accuracy results are presented in Table 1.
The intra-day variation study of TCS and FBP was conducted by analyzing the spiked simulated saliva sample solutions prepared as described earlier, using the present analytical procedure on the same day. The inter-day variation study was carried out as stated under the intra-day study but for six consecutive days. The percentage recovery and % RSD values were calculated by repeating the analysis in six replicates, and the respective results are presented in Table 1. The precision requirement for the RSD ≤1% for n ≥ 5 [34] was fulfilled for both the TCS and FBP measurements. The RSD ≤0.86% was achieved throughout the proposed method, and all of the data showed good agreement amongst the individual results under test. Repeatability was demonstrated by ten injections of the experimental samples.
Practical Applicability and Recovery
The practical applicability of the present HPLC method was demonstrated in a real sample of nanogel formulation under optimal conditions. The real sample analyzed contained a concentration of 12 μg/mL each of TCS and FBP. The analysis results are summarized in Table 1. The real sample's percentage recovery values for TCS and FBP were 97.25% and 101.04%, respectively. From Figure 3a, the chromatogram of the real sample obtained by the proposed method indicates that the FBP and TCS peaks were well-resolved at 10.1 min and 12.5 min, respectively, without any interference from various excipients of the nanogel formulation.
Specificity
The specificity of the method was adequate as there was no interference from the nanogel or simulated saliva constituents in any peak region for both drugs. The tR for standard sample chromatograms (Figure 3c) and that of the nanogel formulation (Figure 3a) and spiked simulated saliva sample (Figure 3d) were the same, which indicates the specificity of the proposed method. Figure 3 depicts a comparison between the chromatograms of the drugs' loaded and unloaded nanogel formulation, standard solution, spiked and unspiked simulated saliva sample solutions, extraction and dilution solvent (methanol), and mobile phase for the proposed method.
Limit of Detection and Limit of Quantification
The LOD and LOQ for the proposed method were determined. The determination was carried out based on the signal-to-noise ratio, and the results are reported in Table 1. The LOD was the injected quantity which yielded a peak with a height three times that of the height of the baseline's noise level, whereas the LOQ was the injected quantity which yielded a peak with a height ten times that of the height of the baseline's noise level.
Conclusions
A new, rapid, simple, specific, and precise HPLC method for the determination of TCS and FBP was successfully developed and validated. The determination was carried out from a nanogel formulation and simulated saliva. The developed method met the validation characteristic requirements for accuracy, precision, repeatability, specificity, linearity, range, and system suitability. TCS and FBP were extracted from the nanogel formulation and simulated saliva and quantified without any interference from the excipient constituents or impurities. The developed method was found to be better than the reported methods in terms of convenience, simplicity, and economy, as none of them demonstrated a concurrent determination of the analytes. The developed method is suitable for concurrent determination of TCS and FBP in dental formulations and can be utilized for routine quality control evaluations.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was funded by Grant 304/PFARMASI/6315018 from short term grant of Universiti Sains Malaysia (USM). Mr. Nafiu Aminu and Mr. Nasir Hayat Khan wish to gratefully acknowledge the support provided by Universiti Sains Malaysia through the USM fellowships.
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