untitled European Journal of Chemistry 3 (4) (2012) 447‐454 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2012 EURJCHEM DOI:10.5155/eurjchem.3.4.447‐454.681 European Journal of Chemistry Journal homepage: www.eurjchem.com Development and validation of spectrophotometric methods for simultaneous determination of sitagliptin and simvastatin in binary mixture Sherif Abdel‐Naby Abdel‐Gawad a and Zeinab Abdelaziz Elsherif b,* a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt b National Organization for Drug Control and Research, Giza, 11126, Egypt *Corresponding author at: National Organization for Drug Control and Research, Giza, 11126, Egypt. Tel.: +20.3.5857481; Fax: +20.3.5855587. E‐mail address: zelsherif@gmail.com (Z.A. Elsherif). ARTICLE INFORMATION ABSTRACT Received: 20 September 2012 Received in revised form: 24 October 2012 Accepted: 24 October 2012 Online: 31 December 2012 KEYWORDS Simple, selective and precise spectrophotometric methods were adopted for simultaneous determination of sitagliptin (SIT) and simvastatin (SIM) in new co‐formulated pharmaceutical dosage form. In the first method, SIT was determined by measuring its zero order absorbance at 266.4 nm in the range of 40‐360 µg/mL in the presence of up to 70% of SIM. While, the two cited drugs were determined simultaneously using third derivative method by measuring the sum of peak amplitudes (peak & valley) at 275.3‐280.3 nm and 240.5‐244.7 nm in the ranges of 40‐360 µg/mL and 2‐18 µg/mL for SIT and SIM, respectively. In the second method, the first derivative of ratio spectra method was applied by measuring the peak height at 255.9 and 275.2 nm using 18 µg/mL SIM as devisor over a concentration range of 40‐360 µg/mL of SIT and at 228.3, 240.5 and 248 nm using 100 µg/mL of SIT as divisor over a concentration range 2‐18 µg/mL SIM. In the third method the ratio subtraction spectrophotometric method was used, where SIM can be determined by dividing the spectra of the mixtures by the spectrum of SIT (40 µg/mL) followed by subtracting the constant absorbance value of the plateau, then finally multiply the produced spectrum by the spectrum of the devisor. Laboratory prepared mixtures were successfully tried for the three compositions of tablets (10, 20 and 40 mg of SIM) with 100 mg of SIT. The developed methods were validated as per International Conference of Harmonization guidelines. Sitagliptin Validation Simvastatin Ratio subtraction Spectrophotometric analysis Derivative spectrophotometry 1. Introduction Sitagliptin (SIT), (R)‐4‐oxo‐4‐[3‐(trifluoromethyl)‐5, dihydro[1,2,4]triazolo[4,3‐a]pyrazin‐7(8H)‐yl]‐1‐(2,4,5‐ trifluorophenyl) butan‐2‐amine, is an oral dipeptidyl peptidase‐ 4 (DPP‐4) inhibitor, which improves glycaemic control by inhibiting DPP‐4 inactivation of the incretin hormones glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) (Figure 1). This increases active incretin and insulin levels and decreases glucagon levels and post‐glucose‐load glucose excursion [1,2]. Simvastatin (SIM), butanoic acid, 2,2‐dimethyl‐,1,2,3,7,8,8a‐ hexahydro‐3,7‐dimethyl‐8‐[2(tetrahydro‐4‐hydroxy‐6‐oxo‐2H‐ pyran‐2‐yl)‐ethyl]1‐naphthalenyl ester, is a lipid‐lowering agent that is derived synthetically from fermentation products of Aspergillus terreus (Figure 1). After oral ingestion simvastatin, an inactive lactone, is hydrolyzed to corresponding ortho‐hydroxy acid leading to the inhibition of 3‐hydroxy 3‐ methyl glutaryl‐coenzyme A (HMG‐Co A) reductase, respon‐ sible for catalysing the conversion of HMG‐Co A to mevalonate, which is an early and rate limiting step in cholesterol biosynthesis [3,4]. Recently, U.S. Food and Drug Administration (FDA) [5] has approved a fixed‐dose combination tablet consisting of sitagliptin and simvastatin. This is the first product to combine a type 2 diabetes drug with a cholesterol lowering drug in one tablet. Many techniques like UV‐visible spectrophotometry [6,7], HPLC [8‐13] and flourimetry [14] have been reported for the determination of SIT alone or in presence of the combination with other drugs. On the other hand, SIM could be determined either alone or in presence of its metabolites or in combination with other drugs using different techniques like UV‐visible spectrophotometry [15‐17], HPLC [18‐24] and LC/MS/MS [25‐ 28]. (a) (b) Figure 1. Chemical structure of (a) Sitagliptin (C16H15F6N5O) and (b) Simvastatin (C25H38O5). 448 Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 For the new co‐formulated dosage form, a few methods were published for the determination of both drugs when present in combination. These methods comprise the use of simultaneous equation spectrophotometric method [29], RP‐ HPLC [30,31] and in human plasma by LC‐MS/MS and its application to a human pharmacokinetic [32]. The goal of the present work is to develop validated, simple, accurate, precise, economic spectrophotometric methods due to its wide availability in most quality control laboratories for the simultaneous quantification of the combined diabetes with cholesterol lowering drug tablet. 2. Experimental 2.1. Instrumentation A double beam UV‐vis spectrophotometer (SHIMADZU, Japan) model UV‐1601 PC with matched quartz cell (1 cm path length) connected to IBM compatible computer and HP 680 inkjet printer (Hewlett Packard, USA). The bundled UVPC personal spectroscopy software version 3.7 was used; at a spectral bandwidth 2 nm and scanning speed of 2800 nm/min. 2.2. Chemicals and materials Pure simvastatin and sitagliptin phosphate monohydrate were kindly supplied by Merck Sharp & Dohme International, USA. The marketed formulation studied was Juvisync™ tablets manufactured by Merck Sharp Dohme International, USA BNO G011008 each tablet contains: 128.5 mg sitagliptin phosphate monohydrate equivalent to 100 mg sitagliptin free base and 20 mg simvastatin. Distilled water from "Aquatron" Automotive water Still A 4000 (Bibby Sterillin Ltd., Staffordshire, UK). Methanol from E. Merck, Darmstadt, Germany. Methanol spectroscopy grade is purchased from El‐NASR Pharmaceutical Chemicals Co., Abu‐Zaabal, Cairo, Egypt. 2.3. Standard Solutions Sitagliptin phosphate monohydrate standard solution (1 mg/mL) was prepared by dissolving 100 mg of the pure drug in 30 mL of 70% methanol into 100 mL measuring flask with continuous shaking for about 10 minutes. The volume was completed to the mark with the corresponding solvent. Simvastatin standard solution (0.1 mg/mL) was prepared by dissolving 10 mg of the pure base in the same solvent by the same manner to get the desired final concentration. 2.4. Procedures 2.4.1. Linearity Aliquots of standard solutions of SIM (0.1 mg/mL) and SIT (1 mg/mL) equivalent to 20‐180 µg and 0.4‐3.6 mg in 70% methanol were accurately and separately transferred into a series of 10 mL volumetric flasks and the volume of each was completed to the mark with the same solvent. For determination of SIT by the zero order method, the absorbencies of SIT were measured at 266.4 nm. For simultaneous determination of both drugs by the third derivative method, the peak amplitudes (peak & valley) were measured at 275.3‐280.3 nm and 240.5‐244.7 nm for SIT and SIM, respectively. For simultaneous determination of both drugs by the first derivative of ratio spectra method, the peak heights were measured at 255.9 and 275.2 nm using 18 µg/mL SIM as devisor to determine SIT in a concentration range of 40‐360 µg/mL and at 228.3, 240.5 and 248.0 nm (peak amplitude) using 100 µg/mL SIT as divisor to determine SIM in a concentration range 2‐18 µg/mL SIM. For determination of SIM by the ratio subtraction method, SIM can be determined by dividing the spectra of the mixtures of both drugs by the spectrum of SIT (40 µg/mL) followed by subtracting the constant absorbance value of the plateau and finally multiply the produced spectrum by the spectrum of the devisor. 2.4.2. Accuracy Accuracy was assured by carrying out the previously mentioned procedures under linearity for the determination of different concentrations of pure SIT and SIM. The concentrations were calculated from the corresponding regression equations. 2.4.3. Precision 2.4.3.1. Intraday precision (Repeatability) Three concentrations of each drug were analyzed three times intraday using the previously mentioned procedures. The percentage recoveries of each drug and its relative standard deviation were calculated using the suggested methods. 2.4.3.2. Intermediate precision Three concentrations of each drug were analyzed on three successive days using the procedure stated under linearity. The percentage recoveries of each drug and its relative standard deviation were calculated using the suggested method. 2.4.4. Analysis of marketed formulation Ten tablets (Juvisync™ tablets) were weighed and finely powdered. An amount of powdered equivalent to 64.25 mg SIT phosphate monohydrate and 10 mg SIM was transferred into a 100 mL round bottom flask; 30 mL 70% methanol were added and stirred for 30 minutes then filtered through 0.5 μm whatman paper into 100 mL measuring flask. The residue was washed with 2 x 20 mL 70% methanol, and then the volume was completed to the mark with the same solvent and mixed well. One ml of the resulted solution was transferred to 10 mL measuring flask then the volume was completed to the mark using the same solvent and mixed well. The general procedure was followed as mentioned before and the concentration of drug was calculated from the corresponding regression equation. 3. Results and discussion A fixed‐dose of sitagliptin and simvastatin in their new co‐ formulated pharmaceutical dosage form [5] is the first product to combine a type 2 diabetes drug with a cholesterol lowering drug in one tablet. The aim of this work is to develop simple and accurate methods for the simultaneous determination of the new combination of SIT and SIM in tablets. Molecular absorption spectroscopy has been extensively used for the determination of drugs in pharmaceutical preparation with a view to the development of analytical methods. The use of this technique for pharmaceutical analyses has the inherent constraint that most active drugs absorb in the UV region and exhibit strongly overlapped spectra that impede their simultaneous determination. The zero‐order absorption spectra (D0) of a mixture of SIT and SIM at the ratio of their presence in tablets showed overlapping (Figure 2) which allows the analysis of SIT in presence of SIM at 266.4 nm, but prevents the analysis of SIM. As SIT is soluble in water and slightly soluble in methanol while, SIM is insoluble in water and freely soluble in methanol trails were made to dissolve the mixture of the two drugs in methanol:water mixture. Different ratios of methanol and Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 449 water were tried and 70% were chosen; which fulfills complete solubility. A calibration curve is constructed relating the absorbance of zero order spectra of SIT at 266.4 nm to the corresponding concentrations where SIM shows no absorbance, the regression equation is computed. ASIT = 0.0033C + 0.0237 r2 = 0.9997 (1) where C is the concentration of SIT in µg/mL, ASIT is the peak amplitude of the zero order spectrum of SIT at 266.4 nm, and r is the correlation coefficient. Figure 2. Zero order absorption spectra of 10 µg/mL simvastatin (—) and 100 µg/mL sitagliptin (……). 3.1. Derivative spectrophotometric method (3D) For further improvement of the selectivity to resolve the overlap present between SIT and SIM in the mixture, a simple third derivative method (3D) [33‐38] is applied. The method is based on measuring the sum of peak amplitudes (peak & valley) at 275.3‐280.3 nm and 240.5‐244.7 nm (Figure 3) for SIT and SIM, respectively. Figure 3. Third derivative spectra of 10 µg/mL simvastatin (—) and 100 µg/mL sitagliptin (——). The main instrumental parameters that affect the shape of the derivative spectra such as the speed, the wavelength increment over which the derivative is obtained (Δλ) and degree of smoothing were optimized to give a well resolved peak. Linearity relationship was obtained between the peak amplitudes and the concentration over the range of 40‐360 µg/mL and 2‐18 µg/mL for SIT and SIM, respectively (Figure 4 and 5), from which the linear regression equation were computed and found to be: 3DSIM = 0.1156 ‐ 0.0217 r2 = 0.9994 for SIM (2) 3DSIT = 0.0039 + 0.0279 r2 = 0.9995 for SIT (3) where 3D is the sum of peak amplitudes (peak & valley) of the spectra, C is the corresponding concentration and r is the correlation coefficient. Figure 4. Third derivative spectra of simvastatin in the range of 2‐18 µg/mL. Figure 5. Third derivative spectra of sitagliptin in the range of 40‐360 µg/mL. 3.2. Ratio‐spectra derivative spectrophotometric method (DD1) As can be seen in Figure 2, the absorption spectra of SIT and SIM; the maximum wavelengths of the two compounds are close to each other and their spectra overlap at 200‐260 nm; which can’t permits the determination of the cited drugs. Therefore, the simultaneous determination of SIT and SIM is impossible by classical spectrophotometry and it is necessary to use another method to solve this problem. Salinas et al. [39] designed a spectrophotometric method, which is based on the derivation of the ratio‐spectra for resolving binary mixtures. The main advantage of the ratio‐ spectra derivative spectrophotometry is the chance of doing easy measurements in correspondence of peaks so it permits the use of the wavelength of highest value of analytical signals. Moreover, the presence of a lot of maxima and minima is another advantage by the fact that these wavelengths give an opportunity for the determination of active compounds in the presence of other compounds and excipients which possibly interfere the assay. In this method the absorption spectrum of the mixture (absorbance at each wavelength) is divided by the absorption spectrum of a standard solution of one of the components, and the first derivative of the ratio spectrum is 450 Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 obtained. The concentration of the other component is then determined from a calibration graph. The main parameters that affect the shape of the ratio spectra which are wavelength, scanning speed, the concentration of the standard solution used as a divisor, the wavelength increment over which the derivative is obtained and the smoothing function are carefully tested. Accordingly, the first derivative of the ratio spectra presented in Figure 6 and 7 for sitagliptin and simvastatin in the different concentration may provide a good proof for this understanding. Different concentrations of divisor were also tried for SIT and SIM which give the best regarding average recovery percent when they were used for the prediction of SIT and SIM concentrations in bulk powder as well as in laboratory prepared mixtures. Figure 6. DD1‐Spectra of sitagliptin in the range of 40‐360 µg/mL using 18 µg/mL simvastatin as divisor. Figure 7. DD1‐Spectra of simvastatin in the range of 2‐18 µg/mL using 100 µg/mL sitagliptin as divisor. The method was applied by measuring the peak height at 255.9 and 275.2 nm using 18 µg/mL SIM as devisor over a concentration range of 40‐360 µg/mL of SIT (Figure 6) and at 228.3, 240.5 and 248.0 nm using 100 µg/mL of SIT as divisor over a concentration range of 2‐18 µg/mL SIM (Figure 7). The linear regression equations are found to be: PSIT = 0.0086C + 0.053 r2 = 0.9995 (4) PSIM= 0.0120C ‐ 0.0841 r2 = 0.9994 (5) where, C is the concentration in µg/mL, P is the peak amplitude of the first derivative of the ratio spectrum curve and r is the correlation coefficient. 3.3. Ratio subtraction method The ratio subtraction technique [40] depends on that, if you have a mixture of two drugs X and Y of overlapping spectra, you can determine X by dividing the spectrum of the mixture by known concentration of Y as a divisor (Yʹ). The division will give a new curve that represents (X/Y) + Constant. If we subtract this constant, then multiply the new curve obtained after subtraction by Yʹ (the divisor), therefore we can obtain the curve of X again. This can be summarized as the following: = = + Constant (6) Constant – Constant = (7) . (8) This constant can be determined directly from the curve by the straight line which is parallel to λ axis in this region. Practically, the ratio subtraction method starts by scanning zero‐ order spectra of the prepared standard solutions of SIM in 70% methanol (Figure 2), then the linearity is checked between absorbance at the selected wavelength at 237.5 nm and the corresponding concentration of SIM. The method depends on that, when a mixture of SIM (X) and SIT (Y); where the spectrum of (Y) is more extended (Figure 2), the determination of (X) could be done by scanning the zero order absorption spectra of the laboratory‐prepared mixtures (SIM and SIT), dividing them by carefully chosen concentration (40 µg/mL) of standard SIT (Y = divisor) producing a new ratio spectra that represent (X/Y) + constant as shown in Figure 8 then subtraction of the absorbance values of these constants (Y/ Yʹ) in plateau as shown in Figure 9, followed by multiplication of the obtained spectra by (Yʹ) the divisor as shown in Figure 10. Figure 8. Absorption spectra of laboratory prepared mixtures of sitagliptin and simvastatin in the ratio of 100:10 (—), 60:12 (.…) and 40:16 (— —). Finally, the original spectra of SIM (X) could be obtained which are used for direct determination of SIM at 237.5 nm and calculation of the concentration from the corresponding regression equation. A linear correlation is obtained between the absorbance and the corresponding concentration of SIM at 237.5 nm. The regression equation is: PSIM= 0.059C+0.0217 r2 =0.9999 (9) where C is the concentration of SIM in µg/mL, PSIM is the peak amplitude of the zero order spectrum of SIM at 237.5 nm, and r is the correlation coefficient. On the other hand for determination of SIT alone a calibration curve is constructed relating the absorbance of zero order spectra of SIT at 266.4 nm to the corresponding concentrations where SIM shows no absorbance. Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 451 Table 1. Determination of simvastatin and sitagliptin phosphate monohydrate in laboratory prepared mixtures by the proposed methods. Claimed Ratio (µg/mL) Simvastatin Ratio Subtraction D3‐Method DD1‐Method 237.5 nm 240.5‐244.7 nm 228.3 nm 240.5 nm 248 nm Found* Recovery Found* Recovery Found* Recovery Found* Recovery Found* Recovery 10:128.5 9.96 99.60 10.02 100.20 10.08 100.80 9.95 99.50 10.04 100.40 12:77.1 12.04 100.33 11.98 99.83 12.01 100.08 11.94 99.50 12.07 100.58 16:51.4 15.98 99.88 15.86 99.13 16.05 100.31 15.97 99.81 15.99 99.94 Mean ± S.D. 99.94±0.368 99.72±0.543 100.40±0.368 99.60±0.120 100.31±0.330 * Average of three determinations. Table 2. Determination of simvastatin and sitagliptin phosphate monohydrate in laboratory prepared mixtures by the proposed methods. Claimed Ratio (µg/mL) Sitagliptin D3‐Method DD1‐Method D0‐Method 275.3‐280.3 nm 255.9 nm 275.2 nm 266.4 nm Found* Recovery Found* Recovery Found* Recovery Found* Recovery 10:128.5 128.40 99.92 128.60 100.08 128.60 100.08 128.29 99.84 12:77.1 77.05 99.94 77.15 100.06 77.04 99.92 77.12 100.03 16:51.4 51.51 100.21 51.35 99.90 51.26 99.79 51.50 100.19 Mean ± S.D. 100.02±0.162 100.01±0.099 99.93±0.145 100.02±0.175 * Average of three determinations. Table 3. Determination of simvastatin in pharmaceutical formulation and application of standard addition technique. Pharmaceutical Formulation Simvastatin Taken (µg/mL) Found * %±S.D. Standard Addition Technique Juvisync™ tablets containing 20 mg Simvastatin and 128.5 mg Sitagliptin phosphate monohydrate equivalent to 100 mg Sitagliptin base BNOG011008. 10.00 101.32±0.924 Pure Added (µg/mL) Pure Found * (µg/mL) Recovery (%) Ratio Subtr. D3 DD1 Ratio Subtr. D3 DD1 228.3 nm 240.5 nm 248 nm 228.3 nm 240.5 nm 248 nm 2 1.99 2.01 2.02 1.99 1.98 99.50 100.50 101.00 99.50 99.00 4 4.03 3.99 3.96 3.99 3.98 100.75 99.75 99.00 99.75 99.50 6 6.02 5.98 5.97 5.97 5.97 100.33 99.50 99.67 99.50 99.50 Mean 100.19 99.92 99.89 99.58 99.33 S.D. 0.636 0.520 1.018 0.144 0.289 R.S.D. 0.635 0.520 1.019 0.145 0.291 * Average of three determinations. Figure 9. Absorption spectra of laboratory prepared mixtures of sitagliptin and simvastatin in the ratio of 100:10 (— ), 60:12 (….) and 40:16 (——) after division on the spectrum of 40 µg/mL sitagliptin and subtraction of the constant value. The selectivity of the proposed procedures is assessed by the analysis of laboratory prepared mixtures containing different ratios of the two drugs, where satisfactory results are obtained over the calibration ranges as shown in Tables 1 and 2. The proposed procedures are also applied for the determination of SIT and SIM in Juvisync™ tablets. The validity of the proposed procedures is further assessed by applying the standard addition technique (Table 3 and 4). Results obtained by the proposed procedures for the determination of pure samples of SIT and SIM are statistically compared to those obtained by the reference method [16] (D2‐method for determination of simvastatin at 243.5 nm). The results showed no significant differences between the proposed methods and the reported one as presented in Table 5 and 6; the observed good agreement between proposed method and the reference method, The high percentage recoveries (99.33‐100.96) and low %R.S.D. (0.291‐1.337) values confirm the suitability of the proposed method for the routine determination of these components in the new combined formulation. Figure 10. Absorption spectra of laboratory prepared mixtures of sitagliptin and simvastatin in the ratio of 100:10 (—), 60:12 (…..) and 40:16 (——) after division on the spectrum of 40 µg/mL sitagliptin and subtraction of the constant value then multiplication in the spectrum of 40 µg/mL sitagliptin. 3.4. Method validation Validation was done according to ICH recommendations [41]. Linearity of the methods was evaluated by analyzing different concentrations of SIT and SIM ranging between 40‐ 360 µg/mL and 2‐18 µg/mL, respectively (Table 7 and 8). Each concentration was made in triplicate. The assay was performed according to the experimental conditions. The percentage recovery was calculated for marketed formulation by standard addition of pure drugs at four known concentrations an excellent recovery were obtained at each level. 452 Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 Table 4. Determination of sitagliptin in pharmaceutical formulation and application of standard addition technique. Pharmaceutical Formulation Sitagliptin Taken (µg/mL) Found* %±S.D. Standard Addition Technique Juvisync™ tablets containing 20 mg Simvastatin and 128.5 mg Sitagliptin phosphate monohydrate equivalent to 100 mg Sitagliptin base BNOG011008. 64.25 99.18±0.896 Pure Added (µg/mL) Pure Found * (µg/mL) Recovery (%) D0 D3 DD1 D0 D3 DD1 255.9 nm 275.2 nm 255.9 nm 275.2 nm 40 40.65 40.73 39.75 40.51 101.63 101.83 99.38 101.28 80 79.21 81.24 78.98 79.96 99.01 101.55 98.73 99.95 120 121.01 119.40 121.11 119.84 100.84 99.50 100.93 99.87 Mean 100.49 100.96 99.68 100.37 S.D. 1.344 1.272 1.130 0.792 R.S.D. 1.337 1.260 1.134 0.789 * Average of three determinations. Table 5. Statistical comparison between the proposed methods for the determination of sitagliptin and a reference method. Parameter Sitagliptin Reference Method * D3 DD1 D0 275.3‐280.3 nm 255.9 nm 275.2 nm 266.4 nm Mean±S.D. 100.25±0.526 100.39±0.650 100.42±0.547 99.96±0.381 100.12±0.489 R.S.D. 0.525 0.647 0.546 0.381 0.488 n 4 4 4 4 4 Variance 0.277 0.423 0.299 0.145 0.239 F‐value ** (9.55) 1.159 1.770 1.251 1.648 ‐ Student's t‐test ** (1.943) 0.362 0.663 0.813 0.516 ‐ * Reference colorimetric method for the determination of sitagliptin by condensation of its primary amino group with acetyl acetone and formaldehyde then measuring the produced color at 430 nm. ** Values in parenthesis are the theoretical values of t and F at p = 0.05. Table 6. Statistical comparison between the proposed methods for the determination of simvastatin and a reference method. Parameter Simvastatin Reference Method * D3‐Method DD1‐Method 240.5‐244.7 nm 228.3 nm 240.5 nm 248 nm Mean±S.D. 100.00±0.797 100.24±0.723 100.17±0.466 100.39±0.366 100.27±0.808 R.S.D. 0.797 0.721 0.465 0.365 0.806 n 4 4 4 4 3 Variance 0.635 0.523 0.217 0.134 0.653 F‐value ** (9.55) 1.028 1.249 3.009 4.873 ‐ Student's t‐test ** (1.943) 0.443 0.052 0.210 0.270 ‐ * D2‐method for determination of simvastatin at 243.5 nm. ** Values in parenthesis are the theoretical values of t and F at p = 0.05. Table 7. Assay validation results of the proposed methods. Parameter Simvastatin D3‐Method DD1‐Method 240.5‐244.7 nm 228.3 nm 240.5 nm 248 nm Accuracy 100.00±0.797 100.24±0.723 100.17±0.466 100.39±0.366 Specificity and Selectivity 99.72±0.543 100.40±0.368 99.60±0.120 100.31±0.330 Precision Repeatability * 98.95±0.0.981 100.96±0.961 101.34±0.694 99.32±0.0.812 Intermediate Precision * 101.35±0.894 99.17±1.013 101.54±0.0.789 100.84±0.0.741 Robustness 99.68±0.541 100.95±0.514 101.07±0.941 101.24±0.514 Linearity, Slope 0.1156 0.0946 0.1404 0.0866 Linearity, Intercept ‐0.0217 0.0412 0.0447 0.0357 Linearity, Correlation coefficient (r) 0.9997 0.9997 0.9998 0.9997 Range (µg/mL) 2‐18 2‐18 2‐18 2‐18 LOD ** (µg/mL) 1 1 1 1 LOQ ** (µg/mL) 2 2 2 2 * Intra‐day and inter‐day relative standard deviation of the average of three concentrations of the studied drug. ** LOD and LOQ are obtained experimentally. Table 8. Assay validation results of the proposed methods. Parameter Sitagliptin D3‐Method DD1‐Method D0‐Method 275.3‐280.3 nm 255.9 nm 275.2 nm 275.3‐280.3 nm Accuracy 100.25±0.526 100.39±0.650 100.42±0.547 99.96±0.381 Specificity and Selectivity 100.02±0.162 100.01±0.099 99.93±0.145 100.02±0.175 Precision, Repeatability * 99.51±0.1.123 99.42±0. 594 101.91±0.426 102.01±1.094 Intermediate Precision * 100.94±1.184 99.91±0.0.694 99.12±0.0.912 10159±1.278 Robustness 98.99±0.621 99.34±0.895 102.37±1.024 99.63±0.845 Linearity, Slope 0.0039 0.0086 0.012 0.0033 Linearity, Intercept 0.0279 0.053 0.0841 0.0237 Linearity, Correlation coefficient (r) 0.9997 0.9997 0.9998 0.9997 Range (µg/mL) 40‐360 40‐360 40‐360 40‐360 LOD ** (µg/mL) 20 20 20 20 LOQ ** (µg/mL) 40 40 40 40 * Intra‐day and inter‐day relative standard deviation of the average of three concentrations of the studied drug. ** LOD and LOQ are obtained experimentally. Abdel‐Gawad and Elsherif / European Journal of Chemistry 3 (4) (2012) 447‐454 453 Table 9. Results of accuracy for the simultaneous determination of simvastatin and sitagliptin by the proposed methods. Claimed Simvastatin (µg/mL) D3‐Method DD1‐Method 240.5‐244.7 nm 228.3 nm 240.5 nm 248 nm Found* Recovery Found* Recovery Found* Recovery Found* Recovery 4 4.03 100.75 4.04 101.00 3.98 99.50 4.03 100.75 8 8.05 100.63 7.96 99.50 8.03 100.38 7.99 99.88 12 11.91 99.25 11.97 99.75 12.03 100.25 12.05 100.42 16 15.90 99.38 16.11 100.69 16.09 100.56 16.08 100.50 Mean ± S.D. 100.00±0.797 100.24±0.723 100.17±0.466 100.39±0.366 * Average of three determinations. Table 10. Results of accuracy for the simultaneous determination of simvastatin and sitagliptin by the proposed methods. Claimed Sitagliptin (µg/mL) D3‐Method DD1‐Method D0‐Method 275.3‐280.3 nm 255.9 nm 275.2 nm 266.4 nm Found* Recovery Found* Recovery Found* Recovery Found* Recovery 80 79.85 99.81 80.96 101.20 80.79 80.79 79.69 99.61 160 161.51 100.94 160.84 100.53 161.12 161.12 159.64 99.78 240 240.91 100.38 239.15 99.65 240.68 240.68 239.87 99.95 320 319.58 99.87 320.58 100.18 319.15 319.15 321.56 100.49 Mean ± S.D. 100.25±0.526 100.39±0.650 100.42±.547 99.96±0.381 * Average of three determinations. The respective % Recovery and %R.S.D.s for the two drugs are shown in Table 3 and 4 where, the relative standard deviation is <1.5 in the assay of raw materials and tablets by the three proposed method. Accuracy: The accuracy of the results was checked by applying the proposed methods for determination of different samples of SIT and SIM. The concentrations were obtained from the corresponding regression equations. From which the percentage recoveries were calculated with mean percentage recovery shown in Table 9 and 10. Accuracy of the methods was further assured by the use of the standard addition technique, it was performed by addition of known amounts of pure SIT (40, 80 and 120 µg/mL) and SIM (2, 4 and 6 µg/mL) to known concentrations of the pharmaceutical preparation (64.25 for SIT and 10 µg/mL for SIM) the resulting mixtures were assayed, and the results obtained were compared with the expected results (Table 3 and 4). The good recoveries of standard addition technique suggested good accuracy of the proposed methods. Selectivity: The selectivity of the methods was achieved by the analysis of different laboratory prepared mixtures of SIT and SIM within the linearity range. Satisfactory results (Table 3 and 4); which prove that the proposed methods, in addition to its selectivity to the cited drug no interference from the presence of formulation matrix. Robustness: Robustness which is the prove that the method is not affected by small deliberated change was tested i.e. by trying to apply the proposed methods using small variation of ratio of the solvent mixture used and the degree of the smoothing of the derivative curves and no effects was observed. Repeatability: Three concentrations of SIT and SIM were analyzed three times intra‐daily using the proposed methods. The percentage recoveries and relative standard deviation were calculated (Table 7 and 8). Intermediate precision: The previous procedures were repeated inter‐daily on three different days for the analysis of the chosen concentrations. The percentage recoveries and relative standard deviation were calculated (Table 7 and 8). 3.5. Application of the method in tablets The proposed methods were applied for the determination of SIT and SIM in their combined pharmaceutical formulation. Laboratory prepared mixtures were successfully tried for the three compositions of tablets (10, 20 and 40 mg of SIM) with 100 mg of SIT, the results are shown in Table 1. The high percentage recoveries (99.60‐100.31%) and low %CV (0.099‐ 0.543) values confirm the suitability of the proposed methods for the routine determination of these components in new combined formulation. Moreover, the proposed methods is proved to be much more sensitive than the published HPLC method [30] especially for SIM which is the lower concentration (linearity rang 2‐18 µg/mL; while the reported method is 20‐200 µg/mL). 4. Conclusion The proposed methods are simple and do not require sophisticated technique or instrument. The methods also, offer a practical potential for the simultaneous determination of the cited drugs, without prior separation, especially with its advantages of acceptable sensitivity and high selectivity. 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