Journal of Chromatographic Science Advance Access published July 12, 2014 Journal of Chromatographic Science 2014;1– 8 doi:10.1093/chromsci/bmu071
Article
Validated Chromatographic Methods for Simultaneous Determination of Tolfenamic Acid and Its Major Impurities Nada S. Abdelwahab1, Nouruddin W. Ali1, Marco M. Zaki1* and M. Abdelkawy2 1
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmed Hegazy St. Beni-Suef 62514, Egypt, and 2Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St, Cairo 11562, Egypt *Author to whom correspondence should be addressed. Email:
[email protected] Received 12 August 2013; revised 29 May 2014
Two accurate, selective and precise chromatographic methods, namely thin-layer chromatography (TLC)-densitometric method and reversed phase high-performance liquid chromatography (RP-HPLC) method, were developed and validated for the simultaneous determination of tolfenamic acid (TOL) and its two major impurities, 2-chlorobenzoic acid (CBA) and 3-chloro-2-methylaniline (CMA), which are also reported to be its related substances. The developed TLC-densitometric method depended on separation and quantitation of the studied drugs on silica gel 60F254 TLC plates. Hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v) was used as a developing system and the separated bands were UV-scanned at 240 nm. Linear relationships were obtained in the range of 10–100 mg band21 for the drug and in the range of 0.1–1 mg band21 for the studied impurities. The developed RP-HPLC depended on chromatographic separation of the studied drugs on a C18 column using 0.05 M KH2PO4 buffer (pH 3):acetonitrile (45:55, v/v) as a mobile phase delivered at constant flow rate of 1 mL min21 with UV detection at 230 nm. Calibration curves for TOL and the two impurities were constructed over the concentration ranges of 10–100 mg mL21 for TOL and 0.01–0.1 mg mL21 for both CBA and CMA. Factors affecting the developed methods have been studied and optimized. Further, methods validation has been carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for determination of the studied drug in its bulk powder and in pharmaceutical formulation. The methods showed no significant difference when compared with the reported RP-HPLC one. The developed methods have advantages of being more sensitive and specific than the published methods.
Introduction Tolfenamic acid (TOL) is 2-[(3-chloro-2-methylphenyl)amino] benzoic acid (1) (Figure 1). It is an anthranilic acid derivative, which is related to mefenamic acid, and is a nonsteroidal antiinflammatory drug (NSAID) used in the treatment of acute attacks of migraine. It is also given for the relief of mild-to-moderate pain in disorders such as dysmenorrhea, rheumatoid arthritis or osteoarthritis (2). Two major impurities for TOL have been reported in the British Pharmacopoeia (BP) (3), namely 2-chlorobenzoic acid (CBA) and 3-chloro-2-methylaniline (CMA) (Figure 1) and they are also reported to be its related substances. After an intensive literature review, different analytical methods have been found for the determination of TOL acid alone or with some other NSAIDs including titrimetric (3), spectrophotometric (4), spectrofluorimetric (5) and reversed phase
high-performance liquid chromatography (RP-HPLC) methods (6– 14). Details of conditions used in the reported HPLC methods are summarized in Table I. The published methods aimed only for determination of TOL without caring about the possibility of presence of drug impurities in TOL samples. It is very important for manufacturers of drug products to monitor the level of anticipated process-related substances and drug impurities before commercial release to prove the consistency of the manufacturing process. Hence this research paper presented for the first time rapid, efficient and validated HPLC and thin-layer chromatography (TLC)-densitometric methods for separation and quantification of TOL and its process-related impurities. Each of the developed methods has been successfully applied for resolving the three components in a single run using a single detection wavelength. Furthermore, they are successfully able to determine the studied impurities with high sensitivity (up to 0.1% of TOL) which agreed with the impurity profile criteria reported by International Conference on Harmonization (ICH) (15) and BP (3).
Experimental Materials Pure samples TOL was kindly supplied by The Egyptian Co. for Pharmaceutical & Chemical Industries (EPSI), Bayad El-Arab, Beni Suef, Egypt. Its purity was reported to be 99.5% according to the company certificates. 2-chlorobenzoic acid (CBA) and 3-chloro-2-methylaniline (CMA) were kindly supplied by Sigma-Aldrich, Cairo, Egypt. Their purity was reported to be 99.50% according to the company analysis certificates. Pharmaceutical formulation Fastgrainw (B.N. 186080511), labeled to contain 200 mg of TOL, is manufactured by EPSI. Chemicals and solvents All chemicals and solvents used throughout this work were of analytical grade and were used without further purification. Chloroform, glacial acetic acid, hexane, acetone and potassium dihydrogen phosphate were obtained from El NASR Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt. Acetonitrile HPLC grade was obtained from SDS, Peypin, France. Deionized water was purchased from SEDICO Pharmaceuticals Co., Cairo, Egypt.
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Then, the prepared buffer solution was filtered through the previously mentioned HPLC filter. Stock standard solutions (1,000 mg mL21) Stock standard solutions of TOL, CBA and CMA were prepared by weighing accurately and separately 0.1 g of pure powder of each into three separate 100 mL volumetric flasks. Then, 75 mL methanol was added, shaking well and then the volume was completed to the mark with methanol. First working standard solutions (100 mg mL21) Working standard solutions of TOL, CBA and CMA were prepared by accurately transferring 10 mL each of TOL, CBA and CMA from their respective stock standard solutions (1,000 mg mL21) into three separate 100-mL volumetric flasks, then completing to the mark with methanol (for the TLC-densitometric method) or 0.05 M KH2PO4 buffer ( pH 3):acetonitrile (45:55, v/v) (for the HPLC method) to get 100 mg mL21 working solutions of each.
Figure 1. Chemical structures of the drug and the two impurities.
Instruments TLC-densitometric method The method was carried out using a sample applicator for TLC linomat V with a 100-mL syringe (CAMAG; Muttenz, Switzerland). The TLC Scanner 3 densitometer (CAMAG) was used for scanning controlled by winCATS software (V 3.15, CAMAG). The slit dimensions ¼ 3.000.45 mm, scanning speed ¼ 20 mm s21 and the data resolution ¼ 100 mm step21 all these requirements were taken into consideration. Finally, a UV lamp with a short wavelength of 254 nm (VL-6.LC; Marne-la-Vallee Cedex 1, France) was used for scanning until optimization of the proposed method for detection the sample spots which had been applied on TLC plates (20 cm 10 cm) coated with silica gel 60 F254 (Merck, Germany) with 0.2 mm thickness. HPLC method Shimadzu 2010C integrated HPLC was equipped with a quaternary gradient pump, a 2010C UV-VIS detector, a 2010C column oven and a 2010C programmable auto sampler controlled by CLASS-VP software. The Eclipse XDB C18 column 25 cm (4.6 mm 5 mm) was used as a stationary phase. For filtration of formulation solution, a Nylon-66 membrane syringe filter (Npore; Ghaziabad, India) was used.
Prepared solutions Buffer solution First, 0.05 M KH2PO4 buffer was prepared by dissolving 6.8 g of KH2PO4 in deionized water and the volume was completed to 1 L and then pH was adjusted to 3 with an aqueous H3PO4 buffer. 2 Abdelwahab et al.
Second working standard solutions (1 mg mL21) of CBA and CMA Second working standard solutions of CBA and CMA were prepared by accurately transferring 1 mL each of CBA and CMA from their respective first working standard solutions (100 mg mL21) into two separate 100-mL volumetric flasks, then completing to the mark with 0.05 M KH2PO4 buffer ( pH 3):acetonitrile (45:55, v/v) (for HPLC method) to get 1 mg mL21 working solutions of each. Laboratory prepared mixtures Different laboratory prepared mixtures containing different ratios of TOL, CBA and CMA were prepared by accurately transferring different volumes of each drug from its respective working standard solutions (100 mg mL21) to 10-mL volumetric flasks and then completing the volume using the suitable solvent.
Procedures Method validation
Linearity and range TLC-densitometric method: Accurately measured aliquots equivalent to 10 – 100 mg band21 of TOL were separately transferred from its stock standard solution (1,000 mg mL21) and aliquots equivalent to 0.1 – 1 mg band21 each of CBA and CMA were separately transferred from their working standard solutions (100 mg mL21) and applied in triplicates on TLC aluminum plates (20 10 cm), prewashed with methanol and preactivated at 1008C for 15 min. Samples were applied as bands using the CAMAG TLC sampler. The band length was 4 mm and the bands were applied 15 mm from the bottom edge of the plate. Ascending development was performed in a chromatographic tank previously saturated for an hour with hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v). The migration distance was 80 mm from the lower edge and the developed plates were then air dried. TOL, CBA and CMA bands were scanned at 240 nm, and the calibration curves were constructed by plotting the mean integrated peak area versus
Table I Illustration of Several Published HPLC Methods Technique
Analytes
Chromatographic conditions
HPLC (6)
TOL in pharmaceutical preparations and biological fluids
HPLC (7)
Flurbiprofen, carprofen, naproxen, vedaprofen, 5-hydroxyflunixin, niflumic acid, mefenamic acid, meclofenamic acid and TOL in bovine milk
HPLC with fluorescence detection (8)
Three nonsteroidal anti-inflammatory drugs and salicylic acid in pharmaceuticals and human serum
HPLC with fluorescence detection (9)
Neutral and acidic pharmaceuticals in wastewater
HPLC (10)
Anthranilic acid derivatives in pharmaceuticals and human urine
HPLC (11)
TOL and cyclodextrin complexes
HPLC (12)
TOL in human plasma
HPLC (13)
Anthranilic acid derivatives
HPLC (14)
Anthranilic acid derivatives and its application to forensic chemistry
Isocratic elution C18 column Mobile phase: 0.05 M acetate buffer, (pH 4.6): methanol (18:82, v/v) l ¼ 282 nm Isocratic elution Column: C18 Mobile phase: acetonitrile:water (85:15, v/v) l ¼ 227 nm “Dual-mode” gradient elution ACEw column C18 Mobile phase (acetonitrile –0.1% aqueous orthophosphoric acid) Isocratic elution Column C18 Mobile phase: methanol:phosphate buffer solution (pH 3.6) (80:20, v/v) Isocratic elution Wakosil ODS 5C 18 column Mobile phase: acetate buffer solution (pH 5):acetonitrile (11:9, v/v) l ¼ 280 nm Isocratic elution C18 column Mobile phase:methanol: 0.07 M phosphate buffer solution (pH 3.2) (90:10, v/v) l ¼ 286 nm Isocratic elution C18 column Mobile phase: acetonitrile:10 mM phosphoric acid (60:40, v/v) l ¼ 280 nm Gradient elution Lichrosorb-RP 18 column Mobile phase: 0.065 M ammonium acetate:methanol l ¼ 282 nm Isocratic elution C18 column Mobile phase:acetonitrile:H2O (80:20, v/v) l ¼ 282 nm
the corresponding concentrations from which the regression equations were computed.
been performed to confirm the accuracy of the developed method.
HPLC method: Accurately measured aliquots equivalent to 10 – 100 mg mL21 of TOL were separately transferred from its stock standard solution (1,000 mg mL21) and aliquots equivalent to 0.01 – 0.1 mg mL21 each of CBA and CMA were separately transferred from their respective second working standard solutions (1 mg mL21) into three series of 10-mL volumetric flasks, then the volume was completed to the mark with the mobile phase. Triplicate 20 mL injections were made for each concentration maintaining the flow rate at 1 mL min21 and the effluent was UV- scanned at 230 nm. The chromatographic separation was performed on a C18 column using 0.05 M KH2PO4 buffer ( pH 3):acetonitrile (45:55, v/v) as a mobile phase. The peak areas were recorded and the calibration curves relating the obtained integrated peak areas to the corresponding concentrations were constructed.
Precision: Repeatability was evaluated by assaying three concentrations of TOL (20, 60 and 90 mg band21) for each method, CMA and CBA (0.2, 0.5 and 0.8 mg band21) for TLC and (0.02, 0.04 and 0.06 mg mL21) for HPLC three times intra-daily. Intermediate precision was evaluated by assaying the three chosen concentrations of TOL, CMA and CBA in triplicates on three successive days using the procedure stated under chromatographic conditions. The mean recoveries and SD values were then calculated.
Accuracy TLC-densitometric method and HPLC methods: The accuracy of the proposed methods was assessed by analyzing samples with different concentrations of pure TOL, CMA and CBA within their linearity ranges that were estimated by applying the procedure. The concentrations of TOL, CMA and CBA were calculated each from its corresponding regression equation and then the mean recoveries and SD values for each component were calculated. Moreover, a standard addition technique has
Specificity TLC-densitometric method: The specificity of the suggested TLC-densitometric method was evaluated by its application for analysis of synthetic mixtures containing TOL, CMA and CBA in different concentrations, 40 mL of each mixture was applied in triplicates to TLC plates and the method under the chromatographic conditions were then followed. Concentrations of the studied components were calculated from the previously computed regression equations. HPLC methods: The specificity was ascertained by application of the developed method to laboratory prepared mixtures containing different ratios of TOL, CMA and CBA following the procedure under linearity. Further, specificity was confirmed by calculating system suitability testing parameters such as Simultaneous Determination of Tolfenamic Acid and Its Major Impurities 3
capacity factor, resolution and selectivity factor for the separated chromatographic peaks.
Sensitivity TLC-densitometric method and HPLC methods: Sensitivity of the method was established with respect to limit of detection (LOD) and limit of quantification (LOQ) for TOL impurities. A series of concentrations of drug solution and its impurities were injected; LOD and LOQ were established by slope method as mentioned below. LOQ =
3:3 Standard deviation of the response Slope of the calibration curve
LOQ =
10 Standard deviation of the response Slope of the calibration curve
accurately weighed and transferred to a 100-mL volumetric flask, 75 mL methanol was added and the prepared solution was ultrasonicated for about 30 min. The solution was then cooled well; the volume was completed with methanol to get 1,000 mg mL21 stock solution and then filtered. For TLC, concentration equivalent to 40 mg band21 was transferred from the sample solution (1,000 mg mL21) and applied in triplicates on the TLC plates. For HPLC, concentration equivalent to 40 mg mL21 was prepared and then 20 mL injections were made in triplicates. Construction of calibration curves for each method was then followed and the concentrations of TOL were calculated from the corresponding regression equation. Results Validation of the proposed method was performed according to ICH guidelines (15).
Robustness TLC-densitometric method: It is the capacity of the method to remain unchanged with small changes in method parameters, e.g., changing in the developing system acetic acid +0.01%, change in scanning wavelength +1 nm and saturation time +5 min. The effects of these changes on Rf values and peaks areas were observed. HPLC methods: It was tested by testing the effect of small deliberate changes in the chromatographic conditions on Rt values and peak areas of the studied component changes, such as changing acetonitrile % in the mobile phase +1, changing in buffer pH +0.1 pH, changing mobile phase flow rate (+0.05 mL min21) and changing the scanning wavelength (+ 1 nm).
System suitability testing parameters TLC-densitometric method and HPLC methods: An overall system suitability testing was done to determine if the operating system was performed properly. Parameters such as resolution (Rs ), peak asymmetry and selectivity factors (a) were calculated.
Application to pharmaceutical formulation Twenty Fastgrainw tablets were powdered and mixed well. An amount of the powdered tablets equivalent to 0.1 g of TOL was
Linearity and range The linearity of the proposed methods was evaluated and it was evident in the range of 10 – 100 mg band21 for TOL and 0.1 – 10 mg band21 for both CBA and CMA in the TLC-densitometric method and in the range of 10 – 100 mg mL21 for TOL and 0.01– 0.1 mg mL21 for both CBA and CMA for the RP-HPLC method. Regression and analytical parameters are shown in Table II. Accuracy The accuracy of the proposed methods was calculated from the corresponding regression equations. Good percentage recoveries were obtained and are shown in Table II. Accuracy was further assessed by applying the standard addition technique on Fastgrainw tablets, where good recoveries were obtained, revealing the good accuracy of the proposed methods and proving that excipients did not interfere (Table III). Precision The proposed method provides acceptable intra- and interday variation, indicating the good precision of the method and revealing that it is suitable for the quality control of the suggested components. Good % standard deviation values were obtained.
Table II Regression and Analytical Parameters of the Proposed Methods for Determination of TOL, 2-Chlorobenzoic Acid and 3-Chloro-2-Methylaniline Parameters
TLC
HPLC
TOL Range Slope Intercept Correlation coefficient Accuracy Mean + RSD% Precision (RSD %) Repeatability Intermediate precision LODa LOQb a
10 –100 mg band 0.0333 0.3942 0.9999
4 Abdelwahab et al.
21
0.10 –1.00 mg band 0.4169 0.0075 0.9999
CBA 21
0.10 –1.00 mg band 0.5350 0.0057 0.9999
TOL 21
10.00 –100.00 mg mL 0.2073 20.7033 0.9999
CMA 21
0.01– 0.1 mg mL 59.8480 0.0430 0.9999
CBA 21
0.01 –0.1 mg mL21 79.3160 0.1714 0.9999
100.04 + 0.7383
100.06 + 0.6387
99.94 + 0.7538
99.77 + 1.064
100.50 + 0.7330
99.35 + 1.214
0.355 0.471 2.65 8.01
0.174 0.353 0.03 0.09
0.753 0.658 0.027 0.08
0.293 0.486 2.8 8.4
0.899 0.959 0.002 0.006
0.415 0.818 0.003 0.009
LOD ¼ (SD of the response/slope) 3.3. LOQ ¼ (SD of the response/slope) 10.
b
CMA
Table II confirms the repeatability and good intermediate precision of the developed methods. Specificity The selectivity of the proposed methods was evident from the TLC and HPLC chromatograms shown in Figures 2 and 3, respectively. In addition, the specificity of the methods was proved from the good recovery percentages obtained when they were applied for determination of TOL, CBA and CMA laboratory prepared mixtures (Table IV). Moreover, the good results were obtained where these methods were applied for analysis of Fastgrainw tablets (Table III), confirming that there was no interference from tablet excipients. Sensitivity Low values of LOD and LOQ shown in Table II proved high sensitivity after the developed methods and revealed that the developed methods met BP specifications for CBA and CMA detection limits. Table III Results of Analysis of TOL in Fastgrainw Tablets and Results of Statistical Analysis with the Reported HPLC One Items
TOL TLC method w
Robustness The methods were found to be robust and deliberate small changes in the studied factors did not lead to significant changes in Rt (or Rf ) values, area or symmetry of the peaks. System suitability testing parameters System suitability testing parameters for TLC-densitometric and RP-HPLC methods were based on the concept that the equipment, electronics, analytical operations and samples constitute an integrated system that could be evaluated as a whole. Acceptable values for all the studied parameters were obtained and are shown in Tables V and VI.
Discussion Organic impurity can arise during the manufacturing process and storage of the drug substances. The criteria for their acceptance up to certain limits are based on pharmaceutical studies or known safety data (17, 18). The use of chromatography to determine drug-related impurities is becoming established within industrial pharmaceutical analysis laboratories. As per BP (3) specifications, each of TOL impurities must not exceed 0.1% of the TOL sample. Hence, in this work, we develop sensitive and robust RP-HPLC and TLC-densitometric methods to detect and quantify TOL and its impurities.
HPLC method
Reported methodd (12)
100.28 + 0.817 5 99.92 + 1.151 0.0982
100.32 + 0.580 5
Method developments and optimization
—
TLC-densitometric method The developed TLC-densitometric method depends on the difference in retardation factor (Rf ) values of CBA (Rf ¼ 0.35), TOL (Rf ¼ 0.50) and CMA (Rf ¼ 0.88) (Figure 2). According to the literature review, the majority of the published TLC-densitometric methods developed for separation of other drugs related to the same chemical class of TOL such as mefenamic acid were built
a
Fastgrain tablets B.N. (186080511) % Founded + RSD 99.80 + 0.771 N 5 b 100.14 + 0.891 Standard addition c 1.214 Student t-test (2.365)
(continued)
Figure 2. TLC chromatogram of a mixture of (A) CBA, (B) TOL, (C) CMA using hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v) as a mobile phase.
Simultaneous Determination of Tolfenamic Acid and Its Major Impurities 5
on the basis of the use of normal stationary TLC plates (19). Therefore, our work mainly depended on the use of normal stationary phase. Parameters affecting the method performance were tested in order to obtain maximum chromatographic separation, such as the used developing system, scanning wavelength, band and
Figure 3. HPLC chromatogram of mixture of TOL, CBA and CMA using 0.05 M KH2PO4 buffer ( pH 3):acetonitrile (45:55, v/v) as a mobile phase.
slide dimensions. Different developing systems with different ratios were tried such as hexane:acetone (7:3, v/v), hexane:chloroform:acetone (8.5:1.5:1.5,v/v/v), hexane:chloroform:acetone (6.5:3.5:2.5, v/v/v/v) and hexane:chloroform:acetone (7.5: 2.5: 2, v/v/v). Using the first developing system, CBA was highly retained on the stationary phase and appeared on the base line. On the other hand, using the second developing CMA eluted rapidly with the developing system and appeared near the front line. On using the third system, CBA and TOL were found to have the same Rf value. Upon using the last one, the resolution among CBA and TOL was slightly enhanced. To improve the chromatographic separation, some modifications have been performed on the last developing system. Satisfactory separation of the three components was observed upon using hexane:chloroform:acetone:acetic acid (75:25:20:0.1, v/v/v/v). Further, different scanning wavelengths (207, 240 and 360 nm) were tested depending on the UV spectrums of the three components (Supplementary data, Figure S1), but the best sensitivity was obtained on the UV spectrum at 240 nm. Different band dimensions were tested in order to obtain sharp and symmetrical peaks. The optimum bandwidth chosen was 4 mm and the interspace between bands was 8.9 mm. The slit dimensions of a scanning light beam should ensure complete coverage of the band dimensions on the scanned track without interference from the adjacent bands. Different slit dimensions were tried and 3 mm 0.45 mm proved to be the slit dimensions of choice, which provided the highest sensitivity. RP-HPLC method A validated isocratic RP-HPLC method with UV detection has been developed for the simultaneous quantitation of TOL, CBA and CMA. It depends on the chromatographic separation of the three components using a C18 column and a mixture of 0.05 M KH2PO4 buffer ( pH 3):acetonitrile (45:55, v/v) as a mobile phase with UV detection at 230 nm. All of the experimental conditions affecting the method performance were investigated. According to the literature review, all the reported methods depended on using a C18 column as stationary phase for the separation of TOL and all of them used acetonitrile as an organic solvent, hence we started our trials on a C18 column with developing system consisting of acetonitrile and phosphate buffer. Bad resolution between the drug and the studied impurities were observed. Improving the chromatographic separation was started using 0.05 M KH2PO4 buffer:acetonitrile (60:40, v/v) as a mobile phase, but bad resolution among TOL, CBA and CMA was obtained. Then different ratios of the
Table IV Determination of TOL, 2-Chlorobenzoic Acid and 3-Chloro-2-Methylaniline in Laboratory Prepared Mixtures by the Proposed Methods TLC
HPLC
MIX TOL:CMA:CBA
TOL % founda
CMA % founda
CBA % founda
MIX TOL:CMA:CBA
TOL % founda
CMA % founda
CBA % founda
100:0.1:0.1 100: 1: 1 10:1.01:1.0 50:0.8:0.8
99.80 98.8 99.20 100.40
101.30 99.70 99.50 99.10
101.6 100.4 101.10 100.20
Mean + SD
99.55 + 0.700
99.90 + 0.966
100.83 + 0.645
10:0.01:0.01 10:0.02:0.02 10:0.1:0.1 100:0.02:0.02 100:0.08:0.08 Mean + SD
99.50 99.80 101.2 98.7 100.40 99.92 + 0.942
101.30 98.70 100.60 99.50 98.60 99.74 + 1.185
101.60 100.40 99.80 101.10 99.20 100.42 + 0.966
a
Average of three determinations.
6 Abdelwahab et al.
Conclusion
Table V System suitability testing parameters of the developed TLC-densitometric method Parameters
CBA
TOL
CMA
Symmetry factor Resolution (Rs) Retention factor (k0 ) Selectivity (a)
1.03 1.6 2.5 1.6
1.00
1.08 3.5 7.8 1.95
4
Table VI System Suitability Testing Parameters of the HPLC Method Parameters
Obtained value (CBA)
Obtained value (CMA)
Obtained value (TOL)
Reference value (16)
Resolution Selectivity (a) Tailing factor (T) Retention factor (K0 ) Column efficiency (n)
3.2 2 1.1 1.14 900.00
1.0 2.29 1,111.11
7.78 3.18 1.38 7.29 1,495.11
0.028
0.023
0.017
R . 1.5 .1 ,1.5 –2 or ,2 1 –10 acceptable Increase with efficiency of the separation The smaller the value the higher the column efficiency
HETPa
This work provides accurate, precise and sensitive chromatographic methods for determination of the studied mixtures without preliminary separation steps. The suggested TLC-densitometric method provides good resolution between the studied components within short analysis time and less money consuming. The HPLC method is more sensitive, robust, accurate and specific one but needs higher cost instruments and chemicals. Moreover, the suggested methods have advantages of being more selective than the reported RP-HPLC one (12). The developed methods are successfully applied for quantification of TOL, CBA and CMA and so that they can be considered as the first developed stability, indicating methods for determination of TOL and its reported related impurities which is very important for safety assessment of TOL preparations.
Supplementary data Supplementary data are available at Journal of Chromatographic Science online.
a
HETP, height equivalent to theoretical plate (cm plate21).
References organic modifier were tested (20 – 70%) and it was found that upon using acetonitrile more than 55%, CBA and CMA eluted together as one peak with a small retention time (Rt) value. On the other hand, decreasing the acetonitrile ratio in the mobile phase (,50%) did not affect the resolution, but increase the analysis time. Furthermore, phosphate buffer with different pH values was tested ( pH 2 – 6) where the optimum separation with symmetric untailed peaks was obtained when using 0.05 M KH2PO4 buffer, pH 3. The effect of the mobile phase flow rate (1, 1.5 and 2 mL min21) on the separation was also tested, where the use of flow rate of 1 mL min21 gave the optimum chromatographic separation within reasonable analysis time. Scanning wavelength (210, 230 and 254 nm) was tested in order to improve the sensitivity of the developed method, where scanning at 230 nm gave the best signal-to-noise ratio. Finally, a satisfactory separation was obtained by using the mixture of 0.05 M KH2PO4 buffer ( pH 3):acetonitrile in the ratio of 45:55 (v/v) as a mobile phase, maintaining the flow rate at 1 mL min21 with UV detection at 230 nm, where CBA was separated after 1.5 min, CMA was separated after 2.3 min and TOL after 5.8 min, and no interference was found among the separated peaks. A typical chromatogram is shown in Figure 3. After methods development and optimization, they were successfully applied for determination of TOL in Fastgrainw tablets where good results were obtained. In addition, the standard addition technique has been done and no interference from excipients was observed, confirming the accuracy of the developed methods (Table III). Finally, the statistical comparison of the results obtained by the proposed methods and the reported HPLC (12) one has been carried out. The values of the calculated t and F were found to be less than the tabulated ones, which revealed that there was no significant difference with respect to both accuracy and precision between the two proposed methods and the reported one.
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Simultaneous Determination of Tolfenamic Acid and Its Major Impurities 7
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