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Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments
Accepted Manuscript Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments Omar Abdel-Aziz, Miriam F. Ayad, Mariam M. Tadros PII: DOI: Reference:
S1386-1425(14)01890-3 http://dx.doi.org/10.1016/j.saa.2014.12.102 SAA 13152
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
9 October 2014 8 December 2014 28 December 2014
Please cite this article as: O. Abdel-Aziz, M.F. Ayad, M.M. Tadros, Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa. 2014.12.102
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Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments
Omar Abdel-Aziz, Miriam F. Ayad, Mariam M. Tadros*
Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt.
The authors certify that this article is original and unpublished and is not being considered for publication elsewhere.
________________________________________________________________________ * Corresponding author Tel: +201223345260; fax: +20224051107 E-mail address: [email protected]
1
Abstract: Simple, selective and reproducible spectrofluorimetric and spectrophotometric methods have been developed for the determination of vildagliptin and saxagliptin in bulk and their pharmaceutical dosage forms. The first proposed spectrofluorimetric method is based on the dansylation reaction of the amino group of vildagliptin with dansyl chloride to form a highly fluorescent product. The formed product was measured spectrofluorimetrically at 455 nm after excitation at 345 nm. Beer’s law was obeyed in a concentration range of 100-600 µgml-1. The second proposed spectrophotometric method is based on the charge transfer complex of saxagliptin with tetrachloro-1,4-benzoquinone (p-chloranil). The formed charge transfer complex was measured spectrophotometrically at 530 nm. Beer’s law was obeyed in a concentration range of 100-850 µgml-1. The third proposed spectrophotometric method is based on the condensation reaction of the primary amino group of saxagliptin with formaldehyde and acetyl acetone to form a yellow colored product known as hantzsch reaction, measured at 342.5 nm. Beer’s law was obeyed in a concentration range of 50-300 µgml-1. All the variables were studied to optimize the reactions' conditions using factorial design. The developed methods were validated and proved to be specific and accurate for quality control of vildagliptin and saxagliptin in their pharmaceutical dosage forms. Keywords: Vildagliptin; Saxagliptin; Experimental design; Dansylation; Charge transfer reaction; Hantzsch reaction.
Introduction: Vildagliptin (VLG), S-1-[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2carbonitrile
(Fig.1a)
and
Saxagliptin
(SXG),
(1S,
3S,
5S)
‐2‐
[(2S)
‐2‐amino‐2‐(3‐hydroxy‐1‐adamantyl) acetyl] ‐2‐azabicyclo [3.1.0] hexane‐3‐carbonitrile (Fig.1b) are novel oral hypoglycemic drugs of the dipeptidyl peptidase 4-inhibitor class (DPP‐4) [1-2]. DPP-4 inhibitors represent a new therapeutic approach for treatment of type-II diabetes; that functions to stimulate glucose-dependent insulin release and reduce glucagon’s levels. This is done through inhibition of the inactivation of incretins, particularly glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), thereby improving glycemic control [3-5]. Literature review showed many methods for determination of VLG; based on the charge transfer complexes of
VLG with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 2
(DDQ), 7,7,8,8-tetracyanoquinodimethane (TCNQ) and tetrachloro-1,4-benzoquinone (Pchloranil) [6]. Also; literature survey reveals that many chromatographic methods have been developed [7-13]. In addition; SXG has been estimated by LC‐MS/MS [14], HPLC methods [15-19] and by spectrophotometric method, in which SXG was estimated at 208 nm in methanol [20]. Another spectrophotometric method based on charge transfer reaction using DDQ and TCNQ was reported [21]. The aim of the first method is to present new spectrofluorimetric method based on the dansylation reaction for determination of VLG in bulk and in pharmaceutical dosage form. Furthermore, the established method should be rapid to be applied for routine quality control analysis of VLG in pharmaceutical dosage form. Spectrofluorimetry has long been applied in the field of pharmaceutical analysis of many drugs [22-24]. A necessary condition for a compound to fluoresce is that it absorbs light in the UV or visible region of the spectrum or reacts with a reagent to give a fluorescent product. Accordingly, A certain group in the compound as the primary amine for example react with the reagent to obtain a highly fluorescent product [22-24]. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols. Several pharmaceutical compounds have been determined through this approach [25–31]. The aim of the second and third methods is to present new spectrophotometric methods based on a charge transfer reaction and Hantzsch reaction for the determination of SXG in bulk and pharmaceutical dosage forms. Spectrophotometry continues to be very popular, because of its simplicity and versatility. Charge transfer reactions have been widely used for determination of electron donating compounds through interaction with π-acceptors [32-42]. Among the electron acceptors mostly used in literature is tetrachloro-1,4-benzoquinone (p-chloranil) [40-42].
3
Hantzsch reaction of primary amines with formaldehyde and acetyl acetone forms a yellow colored product that can be measured spectrophotometrically. The quantification of many drugs in its bulk and pharmaceutical dosage forms were carried out using Hantzsch reaction [43-49]. Hantzsch reaction is a known condensation reaction that was reported in the literatures as a useful pathway for pyrrole and pyridine synthesis, In the same manner, acetylacetone together with formaldehyde react with aliphatic amines by Hantzsch reaction forming a yellow product that can be measured spectrophotometrically [43-49]. The proposed method for determination of SXG (primary amine compound) was based on Hantzsch condensation reaction using acetylacetone as β‐diketone and formaldehyde as an aldehyde to form a colored condensation product. Optimization of the reaction conditions of all the proposed methods were optimized by experimental design using Minitab ® program with the advantage of finding a classic tool for estimating the mutual significance of multiple factors and fulfill most of the important optimality criteria [50-52]. 2. Experimental 2.1. Instrumentation BIO-TEK
spectrofluorimeter,
Italy,
SFM
25
software
was
used
for
spectrofluorimetric measurements while the spectrophotometric determinations were done using a Double-beam Shimadzu (Japan) 1601 PC UV-Visible spectrophotometer connected to a computer fitted with UVPC personal spectroscopy software version 3.7 (Shimadzu). Jenway digital pH meter was used to adjust and determine the hydrogen ion concentration (pH) of the buffer solutions.
2.2. Samples: 4
Pharmaceutical grade VLG, certified to contain 99.70 % and Galvus® tablets nominally containing 50 mg VLG per tablet (batch no. V6498) were kindly supplied from Novartis Europharm limited company (London, U.K.). Pharmaceutical grade SXG certified to contain 99.85 % and Onglyza® tablets (batch number 0J57932) nominally containing 5 mg of SXG per tablet were kindly supplied by Bristol-Myers Squibb/AstraZeneca EEIG (United Kingdom). 2.3. Reagents: 1-Dimethyl aminonaphthalene-5-sulphonyl chloride (dansyl chloride), purchased from Sigma (St. Louis, USA). A stock solution containing 0.1% of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 0.001% solution (solution a) [25]. Carbonate: Bicarbonate buffer (pH 9): Prepared by dissolving 2.1 g of sodium carbonate in 200 ml distilled water and 1.7 g of sodium bicarbonate in 200 ml distilled water and then mixing 10 ml of the carbonate solution with 115 ml of bicarbonate solution and completed to 500 ml [26]. Higher pH values (1112) had been avoided to prevent hydroxylation of dansyl chloride [25]. p-chloranil was supplied from Sigma Aldrich Chemie GmbH (Steinheim, Germany) and freshly prepared 0.5% solution was prepared in DMF (solution b) [40]. Acetyl acetone: 8.4 % v/v solution was freshly prepared by mixing 8.4 ml of acetyl acetone with 40 ml of acetate buffer (pH 5) and diluted to 100 ml with distilled water [49]. Formaldehyde (34 ‐ 40 %): Working formaldehyde solution was prepared by mixing 20 ml of formaldehyde with 40 ml of acetate buffer (pH 5) and diluted to 100 ml with distilled water. Solution (c) was prepared using 20 ml of acetyl acetone and 10 ml of formaldehyde with the commonly used ratio 2:1 as reported [49]. Acetate buffer (pH 5): Prepared by dissolving 13.6 g of sodium acetate and 6 ml of glacial acetic acid in
5
sufficient water to produce 1000 ml [49]. Methanol, acetone, acetonitrile and dimethylformamide of analytical spectrophotometric grade were used. 2.4. Stock standard solutions: Stock standard solutions of VLG (2 mgml-1) were prepared by dissolving 200 mg of VLG in acetonitrile in a 100 ml volumetric flask and completing to volume with acetonitrile. Stock standard solutions of SXG (2 mgml-1) were prepared by dissolving 200 mg of SXG in dimethylformamide in a 100 ml volumetric flask and completing to volume with dimethylformamide for the charge transfer experiment. Stock standard solutions of SXG (1 mgml-1) were prepared by dissolving 100 mg of SXG in methanol in a 100 ml volumetric flask and completing to volume with methanol for the Hantzsch experiment. 2.5. General procedures and calibration graphs 2.5.1. Spectrofluorimetric method using dansyl chloride: Aliquots of VLG containing (1–6 mg) were transferred into a set of 10 ml volumetric flasks, treated with acetonitrile to keep the volume at 3 ml for all the flasks, 0.5 ml of the carbonate bicarbonate buffer was added and mixed well followed by 1 ml of 0.001% of dansyl chloride reagent. The reaction mixture was left for 50 min, and then completed to the mark with acetone. The fluorescence intensity of the reaction product was measured at 455 nm after excitation at 345 nm. Blank experiment was carried out simultaneously. The corrected fluorescence intensity was plotted versus the final drug concentration (µg ml−1) to get the calibration graph and the corresponding regression equation was derived.
2.5.2. Spectrophotometric method using p-chloranil: Aliquots of SXG containing (1–8.5 mg) were transferred into a set of 10 ml volumetric flasks, treated with 2 ml p-chloranil solution (b), allowed to stand for 40 min
6
at room temperature and diluted to volume with dimethylformamide. The absorbance was measured at 530 nm against reagent blank, plotted against its corresponding concentration and then the regression parameters were computed. 2.5.3. Spectrophotometric method using formaldehyde and acetyl acetone: Aliquots of SXG containing (0.5–3 mg) were transferred into a set of test tubes, completed to 4 ml with the acetate buffer (pH = 5) treated with 3 ml of solution (c), allowed to stand for 10 minutes on boiling water bath (100°C), cooled, quantitatively transferred to 10 ml volumetric flasks and diluted to volume with distilled water. The absorbance was measured at 342.5 nm against reagent blank, plotted against its corresponding concentration and then the regression parameters were computed. 2.5.4. Assay of VLG and SXG in tablets and standard addition technique Twenty tablets of VLG were weighed. An accurately weighed amount of the finely powdered tablets equivalent to 200 mg of VLG was made up to 100 ml with acetonitrile, the solution was filtered. Twenty tablets of SXG were weighed and the coats were removed by carefully rubbing with a clean tissue wetted with methanol. An accurately weighed amount of the finely powdered tablets equivalent to 200 mg of SXG was made up to 100 ml with dimethylformamide for the second method and an accurately weighed amount of the finely powdered tablets equivalent to 100 mg of SXG was made up to 100 ml with methanol for hantzsch method. The solutions were filtered, procedures were continued as mentioned under 2.5., and then the concentrations were calculated using the corresponding equations. The standard addition technique was applied by adding different known concentrations of the pure drugs to different known concentrations of drugs in dosage form and the procedures mentioned above were adopted. The concentrations were calculated using the corresponding regression equations. 3. Results and discussion:
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The secondary amine of VLG reacts with dansyl chloride as in figure [2], while the primary amine of SXG reacts with p-chloranil and undergoes hantzsch reaction as in figure [3]. 3.1. Formation of the dansylation product: The secondary amine of VLG reacts with dansyl chloride to obtain a highly fluorescent product according to the proposed reaction in figure [2]. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols. Several pharmaceutical compounds have been determined through this approach [25–31]. λmax of measurements is shown in Table [I] and the excitation emission spectrum of the dansylation product of VLG with dansyl chloride is shown in figure [4]. 3.2. Formation of the charge transfer complex: The charge transfer reagent applied in this work is p-chloranil. Primary, secondary and tertiary amines, both aliphatic and aromatic were shown to react with p-chloranil to produce a blue to purple colour. It was reported that p-chloranil could react with amines to form either mono substituted aminoquinones or radical ion pairs. It appears from literature survey that the reaction of p-chloranil with different drugs may differ according to the structure of the drug, solvent used and temperature [32-42]. λmax of measurements is shown in Table [I] and the absorption spectrum of the reaction product of SXG with p-chloranil is shown in figure [5]. 3.3. Formation of Hantzsch product: Hantzsch reaction [43] is a condensation reaction that allows the formation of dihydropyridine derivatives by condensation of an aldehyde with two equivalents of a βketoester in the presence of ammonia. In the same manner, acetylacetone (β-ketoester) together with formaldehyde (aldehyde) reacts with aliphatic amines by Hantzsch reaction forming a yellow colored product that can be measured spectrophotometrically. The 8
quantification of certain sulpha drugs [44] (sulfacetamide sodium, sulfadiazine, sulfadimidine, and sulfathiazole), kanamycin [45], lisinopril [46], gabapentin [47], cefrozil [47], tranexamic acid [48], and sitagliptin phosphate [49] in its bulk and pharmaceutical dosage forms were carried out using Hantzsch reaction according to the proposed mechanism in figure [3]. λmax of the absorption spectra [table 1] of the reaction product of SXG with acetyl acetone and formaldehyde is shown in figure [6]. 3.4. Factorial design experiment and methodology: Different parameters affecting the reactions such as amount of the reagents, reaction time, heating time and stability of the formed product have been investigated by experimental design using Minitab ® program (figures 7-15). In the first step of the investigation, experimental design was used to determine variables which have statistically important influence on the reaction behavior. In the screening design (tables II-III), two levels were used so that the factors were considered as discrete variables. The effect of each factor was tested using a Student (t) test with a corresponding p-value. The factors whose p-values were less than 0.05 were considered as “statistically significant” [50-52]. A graphical display of the ordered standardized effect (the absolute value of the estimated effect divided by its standard error estimate) of each factor was given in a Pareto chart (Figures 7, 8 and 9). A factor was considered as “statistically significant” if its standardized effect exceeded a threshold. A line in the Pareto chart indicated the threshold for a test at level 0.05. In the three proposed methods, the only factor that had been considered as statistically insignificant was the stability of the formed reaction product (Figures 7, 8 and 9). In the second step of the optimization (table IV), response surface methodology (RSM) was applied, where a collection of mathematical and statistical techniques for analyzing the effect of several independent variables on dependent variables and provides its graphical representation. The effect of 9
two variables can be represented as a surface in three-dimensional space and the influence of two variables on the response can be clearly seen in the investigated region. Furthermore, the response surface methodology enables the prediction of the response between and slightly outside the investigated area, as well as visualization and rapid selection of optimal conditions (Figures 10-15). In case of the dansylation experiment, Reaction time and amount of reagent were optimum at the maximum level while the amount of buffer was optimum at the minimum level (Figures 10 and 13). While in the charge transfer experiment and hantzsch experiment, both reaction time and amount of the reagent were optimum at the maximum level (Figures 11, 12, 14 and 15). Maximum level (+1) and minimum level (-1) had been discussed in (table II). 3.4. Effect of solvent: Although many solvents have been used to carry out the dansylation of VLG, charge transfer complex of SXG and hantzsch reaction of SXG, using the appropriate solvent in the described work was critical. The highest fluorescence of the dansylation reaction was attained when dissolving the VLG in acetonitrile and completion after the reaction with acetone [25]. Higher sensitivity of SXG reactions was attained in dimethylformamide for pchloranil method [40] and dissolving in methanol and completion with water for Hantzsch method [49]. 3.5. Methods Validation 3.5.1. Linearity Linearity was studied for both VLG and SXG. The regression equations were also computed. The linearity of the calibration curves were validated by the high value of correlation coefficients. The analytical data of the calibration curves including standard
10
deviation for the slope and intercept (Sb, Sa), confidence limits of the slope, the intercept and the standard error of estimation are summarized in (Table I). 3.5.2. Accuracy Accuracy of the results was calculated by % recovery of 5 different samples for each experiment. The results obtained including the mean of the recovery and standard deviations are displayed in (Table I). The procedure mentioned under 2.5.1., 2.5.2. and 2.5.3. were repeated using VLG concentrations (150-550 µgml-1), SXG concentrations (150-750 µgml-1) and SXG concentrations (75-275 µgml-1), respectively. 3.5.3. Precision 3.5.3.1 Repeatability: Three concentrations of VLG (250, 400 and 550 µg.mL−1) using the procedure mentioned under (2.5.1.), SXG (250, 400 and 550 µg.mL−1) using the procedure mentioned under (2.5.2.) and SXG (160, 200 and 240 µg.mL−1) using the procedure mentioned under (2.5.3.) were analyzed three times, within the same day. The %RSD was calculated and found to be less than 1% in the three concentrations, as shown in (Table I). 3.5.3.2 Intermediate precision: The above mentioned concentrations were analyzed on three successive days and the %RSD was calculated as shown in (Table I). 3.5.4. Results of determination of VLG and SXG in pharmaceutical dosage form The methods had been successfully applied to the pharmaceutical dosage forms and to check the validity of the proposed method, the standard addition technique was applied and the results are shown in (tables V-VI). 3.5.5. Limit of detection (LOD) & limit of quantification (LOQ)
11
The limit of detection (LOD) and limit of quantification (LOQ) were calculated as LOD = 3.3 (ơ/S) and LOQ = 10 (ơ/S), where ‘ơ’ represents standard deviations of the intercept and ‘S’ is the slope of the calibration line, as shown in (Table I). 3.5.6. Statistical analysis Statistical analysis of the results obtained by the proposed methods and the reference methods [9 and 20] for VLG and SXG was carried out by “SPSS statistical package version 11”. The significant difference between the reference method and the described method was tested by one way ANOVA (F-test) at P=0.05 as shown in (Tables VII-VIII). The test ascertained that; there was no significant difference among the methods. For SXG, Hantzsch method was more sensitive than the charge transfer method according to LOD and LOQ values (table I), consequently it can be used for the quality control laboratories for the determination of SXG in its pharmaceutical dosage form. 4. Conclusion: The proposed spectrofluorimetric and spectrophotometric methods have the advantages of simplicity, precision, accuracy and convenience for the quantitation of VLG and SXG. Hence, the proposed methods can be used for the quality control of the cited drugs and can be extended for their routine analysis in dosage forms as an economic alternative to the time consuming HPLC reported methods. 5. References [1] B. Sekaran and P. Rani, Int J Pharm Pharm Sci. 2 (2010) 138. [2] D.J. Augeri, J.A. Robl, D.A. Betebenner, et al. J Med Chem. 48(2005) 5025. [3] R. Nirogi, V. Kandikere, K. Mudigonda, et al, Biomed Chromatogr. 22 (2008) 214. [4] S. Dhillon, Drugs. 70 (2010) 489. [5] J. Green and M. Feinglos, Vasc Health Risk Manag. 4 (2008) 743. 12
[6] R. El-bagary, E. Elkady, B. Ayoub, Int J Biomed Sci. 7 (2011) 55. [7] A. Pharne, B. Santhakumari, A. Ghemud, et al, Int J Pharm Pharm Sci. 4 (2012) 119. [8] A. Malakar, B. Bokshi, D. Nasrin, Int J Pharm life Sci. 1 (2012) 1. [9] M. Mohammad, E. Elkady, M. Fouad, European Journal of Chemistry. 3 (2012) 152. [10] R. El-bagary, E. Elkady, B. Ayoub, Int. J. Biomed.Sci. 7 (2011) 201. [11] A. Barden, B. Salamon, E. Schapoval, J Chrom sci . 50 (2012) 426. [12] B Thangabalan, P Kumar, J. Pharm. Res. 7 (2013) 113. [13] D varma, A Rao, S Dinda, et al, IRJP. 4 (2013)122. [14] A. Fura, A. Khanna, V. Vyas, et al. Drug Metab Dispos. 37 (2009) 1164. [15] R. Inturi, I. Tagaram. IJPRD. 3 (2011) 45. [16] X. Xua, R. Demersb, H. Gua. J Chromatogr. 889 (2012) 77. [17] C. Hess, F. Musshoff, B. Madea. Analytical and bioanalytical chemistry. 400 (2011) 33. [18] S. Inturi, R. Inturi, I. Tagaram. Int. j. sci. pharm. edu. res. 1 (2011) 27. [19] M. Mohammad, E. Elkady, M. Fouad. European Journal of Chemistry. 3 (2012) 152. [20] R. kalaichelvi, E. Jayachandran. Int. J. Pharm. Pharm. Sci. 3 (2011) 180. [21] R. I. El-bagary, E. F. Elkady, B. Ayoub, Int. J. Biomed.Sci. 8 (2012) 204.
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15
N
N
O H2N
N HO
N HO
H
N H H
O
A
B
Fig.1 Chemical structures of vildagliptin (a) and saxagliptin (b).
OH O N NH
Cl
S O
N O
dansyl chloride
N
Vildagliptin
Dansylation reaction in the presence of carbonate bicarbonate buffer, pH = 9
OH O N N
S O
N O N
Highly fluorescent dansylation product
Fig.2 Dansylation reaction of vildagliptin with dansyl chloride.
H H
The lone pair of the nitrogen will attack the partial positive carbon
H OH
H OH
N N NH2
H2C
O
N
H2 C
O
formaldehyde
HN
OH
O
N
Followed by elimination of one molecule of water H
H OH N
CH2
N O
N
Acetyl acetone will attack the partially positive carbon of imine O
O
acetylacetone H
H
H OH N
N N
Hantzsch product after the hydrogen bond cyclic intermediate
H OH N
HN
CH2
O
N
H
CH2
O
O O
O
O
Fig.3 The proposed mechanism of hantzsch reaction of saxagliptin with acetyl acetone and formaldehyde.
Fig.4 A typical excitation emission spectrum of the dansylation product after the reaction of vildagliptin (400 µg mL-1) with dansyl chloride (0.001%).
Fig.5 Absorption spectra of the colored product of saxagliptin (400 μgml-1) and pchloranil (0.5%) in dimethylformamide.
Fig.6 Absorption spectrum of the colored product of hantzsch reaction of saxagliptin (200 μgml-1) with acetyl acetone and formaldehyde.
Fig.7 Pareto chart of the effects in the factorial design of dansylation experiment
Fig.8 Pareto chart of the effects in the factorial design of the charge transfer experiment
Fig.9 Pareto chart of the effects in the factorial design of the Hantzsch experiment
Fig.10 Surface plot of the effects in the factorial design of dansylation experiment
Fig.11 Surface plot of the effects in the factorial design of the charge transfer experiment
Fig.12 Surface plot of the effects in the factorial design of the Hantzsch experiment
Fig.13 Contour plot of the effects in the factorial design of dansylation experiment
Fig.14 Contour plot of the effects in the factorial design of the charge transfer experiment
Fig.15 Contour plot of the effects in the factorial design of the Hantzsch experiment
Table (I): Results obtained by the proposed methods for determination of vildagliptin and saxagliptin
Item λmax of measurements Obedience of Beer’s law Regression equation Regression coefficient (r) Accuracy LOD µg.ml-1 LOQ µg.ml-1 Intraday %RSD Interday %RSD *Sb *Sa Confidence limit of the slope Confidence limit of the intercept Standard error of the estimation
Dansylation reaction λexc =345 nm, λemi =455 nm 100-600 µg.ml-1 F431 = 0.3062 Cµg/ml- 0.8867 0.9994 99.76 ± 0.95 15.05 50.17 0.14-0.22 0.21-0.34 0.0037 1.75 0.3062 ± 0.54 0.8867 ± 0.0033 1.54
*Sb: Standard error of intercept, Sa: Standard error of slope.
Charge transfer reaction 530 nm 100-850 µg.ml-1 A = 0.0013 Cµg/ml+ 0.0006 0.9995 100.48 ± 1.41 32.32 97.94 0.11-0.25 0.20-0.31 2.1 x 10-5 1.4x 10-2 0.0013± 1.82 x 10-5 0.0006 ± 1.26 x 10-8 0.013
Hantzsch reaction 342.5 nm 50-300 µg.ml-1 A = 0.0031 Cµg/ml + 0.0011 0.9997 100.44 ± 2.01 9.84 29.81 0.18-0.27 0.23-0.29 4.54 x 10-5 1.08 x 10-2 0.0031± 1.19 x 10-5 0.0011 ± 1.41 x 10-7 0.0095
Table (II): Investigated variables and their domains used in the design of experiments
Investigated variables and their domains Investigated levels Variables
Low level (-1)
High level (+1)
Dansylation experiment Reaction time
10 minutes
50 minutes
Amount of reagent
0.2 ml of solution (a)
1 ml of solution (a)
Amount of buffer
0.5 ml of carbonate
2 ml of carbonate
bicarbonate buffer pH = 9
bicarbonate buffer pH = 9
10 minutes
60 minutes
Reaction time
10 minutes
40 minutes
Amount of reagent
0.5 ml of (Solution b)
2 ml of (Solution b)
Stability of the formed complex
10 minutes
60 minutes
Heating time
10 minutes
30 minutes
Amount of reagent
0.75 ml of solution (c)
3 ml of solution (c)
Stability of the formed product Charge transfer experiment
Hantzsch experiment
Stability of the Hantzsch reaction 10 minutes
60 minutes
Table (III): Screening of variables using factorial design in dansylation experiment (A1-D1), charge transfer experiment (A2-C2) and hantzsch experiment (A3-C3) Experiments A1 B1 C1 D1 Relative Flourescence 1 -1 -1 1 1 15.3 2 1 -1 -1 1 28.2 3 -1 -1 -1 -1 12.6 4 1 1 -1 -1 90.4 5 -1 1 -1 1 38.9 6 -1 1 -1 -1 41.1 7 1 1 -1 1 90.2 8 -1 1 1 -1 31.1 9 -1 -1 -1 1 14.8 10 -1 -1 1 -1 10.1 11 1 -1 1 1 20.4 12 1 1 1 -1 42.1 13 1 -1 -1 -1 25.1 14 1 -1 1 -1 20.6 15 -1 1 1 1 30.7 16 1 1 1 1 40.6 A1: Reaction time, B1: Amount of reagent, C1: Amount of buffer, D1: Stability of the formed dansylation product
A2 -1 -1 1 1 -1 1 -1 1
B2 -1 -1 -1 1 1 1 1 -1
C2 1 -1 1 -1 1 1 -1 -1
Absorbance 0.204 0.213 0.297 0.522 0.261 0.524 0.233 0.303
A2: Reaction time, B2: Amount of reagent, C2: Stability of the formed complex
A3 1 1 -1 1 -1 -1 -1 1
B3 1 -1 1 -1 -1 1 -1 1
C3 -1 1 -1 -1 -1 1 1 1
Absorbance 0.217 0.099 0.611 0.121 0.312 0.615 0.313 0.211
A3: Heating time, B3: Amount of reagent, C3: Stability of the formed Hantzsch product
Table (IV): Optimization of variables using factorial design in dansylation experiment (A1-C1), charge transfer experiment (A2-B2) and hantzsch experiment
Experiments
A1
B1
C1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.00000 0.00000 1.00000 0.00000 0.00000 0.00000 -1.00000 -1.00000 0.00000 1.00000 0.00000 0.00000 -1.68179 0.00000 0.00000 0.00000 -1.00000 -1.00000 1.00000 1.68179
1.00000 0.00000 -1.00000 0.00000 0.00000 0.00000 1.00000 1.00000 0.00000 -1.00000 0.00000 1.68179 0.00000 0.00000 -1.68179 0.00000 -1.00000 -1.00000 1.00000 0.00000
-1.00000 0.00000 1.00000 1.68179 0.00000 0.00000 1.00000 -1.00000 0.00000 -1.00000 0.00000 0.00000 0.00000 -1.68179 0.00000 0.00000 1.00000 -1.00000 1.00000 0.00000
Relative Flourescence 90.1 23.7 40.9 26.3 22.9 24.1 30.7 40.2 23.5 25.2 23.1 81.4 12.7 62.3 14.6 23.9 15.2 13.4 40.7 75.3
A2
B2
1.41421 0.00000 -1.00000 1.00000 0.00000 0.00000 0.00000 0.00000 1.00000 -1.00000 0.00000 0.00000 0.00000 0.00000 0.00000 -1.41421 -1.41421 0.00000 0.00000 0.00000 0.00000 1.41421 -1.00000 -1.00000 1.00000 1.00000
A: Reaction time, B: Amount of reagent, C: Amount of buffer
Absorbance 0.342 0.206 0.291 0.288 0.201 0.292 0.289 0.125 0.097 0.287 0.463 0.187 0.527
A3
B3
0.00000 0.00000 -1.41421 0.00000 -1.00000 1.00000 -1.00000 -1.00000 0.00000 0.00000 0.00000 1.41421 0.00000 0.00000 0.00000 -1.41421 0.00000 0.00000 0.00000 0.00000 1.00000 -1.00000 1.00000 1.00000 1.41421 0.00000
Absorbance 0.272 0.010 0.616 0.266 0.109 0.137 0.294 0.105 0.268 0.278 0.074 0.138 0.085
Table (V): Pharmaceutical dosage form and standard addition results obtained by dansylation method for determination of vildagliptin
Standard addition technique Pharmaceutical dosage form
Galvus® tablets B.N.: V6498
Claimed
50 mg
% found ± S.D.
100.10 ± 0.80
Mean ± S.D.
*Mean of three determinations.
Amount taken dosage form (µgml-1)
Amount added (µgml-1)
*R % added
100 150 200 250 300
500 400 300 200 100
102.29 97.89 99.83 99.94 100.26 100.04 ± 1.56
Table (VI): Pharmaceutical dosage form and standard addition results obtained by the charge transfer method and hantzsch method for determination of saxagliptin
Pharmaceutical dosage form
Claimed
Onglyza® tablets B.N.: 0J57932
5 mg
*Mean of three determinations.
Standard addition technique (Charge transfer experiment) Amount taken Amount added *R % dosage form (µgml-1) added (µgml-1) 100 450 101.23 150 350 98.70 200 250 100.14 100.69 ± 1.43 250 150 102.05 300 50 100.22 100.47 Mean ± S.D. ± 1.26
% found ± S.D. (Charge transfer experiment)
% found ± S.D. (Hantzsch experiment)
99.49 ± 1.61
Standard addition technique (Hantzsch experiment) Amount Amount taken added *R % added dosage form -1 (µgml ) (µgml-1) 50 250 101.81 75 200 98.06 100 150 101.51 125 100 101.94 150 50 99.34 100.53 ± Mean ± S.D. 1.74
Table (VII): Statistical comparison between the results of the proposed dansylation method and the reference method for the determination of vildagliptin Statistical Term Mean S.D.± S.E. ± %RSD n V t (*2.306)
Reference Method**
Proposed spectrofluorimetric method
100.01 0.99 0.44 0.99 5 0.98
99.76 0.95 0.42 0.95 5 0.90 0.41
* Figures in parentheses are the theoretical t and F values at (p=0.05). No significant difference between groups of vildagliptin by using one way ANOVA with F equals 0.17 and p equals 0.69. **Reference method: aliquots of standard solutions containing 5‐200 µg/ml VLG were measured using HPLC with UV detection at 208 nm [9].
Table (VIII): Statistical comparison between the results of the proposed charge transfer method and the reference method for the determination of saxagliptin
Statistical Term Mean S.D.± S.E. ± %RSD n V t (*2.306)
Reference Method**
Charge transfer method
Hantzsch method
100.20 1.10 0.49 1.10 5 1.21
100.48 1.41 0.63 1.40 5 1.99 0.40
100.44 2.01 0.90 2.00 5 4.04 0.23
* Figures in parentheses are the theoretical t and F values at (p=0.05). No significant difference between groups of saxagliptin by using one way ANOVA with F equals 0.05 and p equals 0.95. ** Reference method: aliquots of standard solutions in methanol containing 5‐40 µg/ml SXG were measured using methanol as a blank [20].
1. The first method is based on dansylation of vildagliptin. 2. The spectrophotometric method is based on the charge transfer of saxagliptin 3. The other spectrophotometric method is based on hantzsch reaction of saxagliptin 4. The variables were studied using experimental factorial design 5. The developed methods were validated for quality control of vildagliptin and saxagliptin
1
S1386-1425(14)01890-3 http://dx.doi.org/10.1016/j.saa.2014.12.102 SAA 13152
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
9 October 2014 8 December 2014 28 December 2014
Please cite this article as: O. Abdel-Aziz, M.F. Ayad, M.M. Tadros, Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa. 2014.12.102
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Compatible validated spectrofluorimetric and spectrophotometric methods for determination of vildagliptin and saxagliptin by factorial design experiments
Omar Abdel-Aziz, Miriam F. Ayad, Mariam M. Tadros*
Analytical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt.
The authors certify that this article is original and unpublished and is not being considered for publication elsewhere.
________________________________________________________________________ * Corresponding author Tel: +201223345260; fax: +20224051107 E-mail address: [email protected]
1
Abstract: Simple, selective and reproducible spectrofluorimetric and spectrophotometric methods have been developed for the determination of vildagliptin and saxagliptin in bulk and their pharmaceutical dosage forms. The first proposed spectrofluorimetric method is based on the dansylation reaction of the amino group of vildagliptin with dansyl chloride to form a highly fluorescent product. The formed product was measured spectrofluorimetrically at 455 nm after excitation at 345 nm. Beer’s law was obeyed in a concentration range of 100-600 µgml-1. The second proposed spectrophotometric method is based on the charge transfer complex of saxagliptin with tetrachloro-1,4-benzoquinone (p-chloranil). The formed charge transfer complex was measured spectrophotometrically at 530 nm. Beer’s law was obeyed in a concentration range of 100-850 µgml-1. The third proposed spectrophotometric method is based on the condensation reaction of the primary amino group of saxagliptin with formaldehyde and acetyl acetone to form a yellow colored product known as hantzsch reaction, measured at 342.5 nm. Beer’s law was obeyed in a concentration range of 50-300 µgml-1. All the variables were studied to optimize the reactions' conditions using factorial design. The developed methods were validated and proved to be specific and accurate for quality control of vildagliptin and saxagliptin in their pharmaceutical dosage forms. Keywords: Vildagliptin; Saxagliptin; Experimental design; Dansylation; Charge transfer reaction; Hantzsch reaction.
Introduction: Vildagliptin (VLG), S-1-[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2carbonitrile
(Fig.1a)
and
Saxagliptin
(SXG),
(1S,
3S,
5S)
‐2‐
[(2S)
‐2‐amino‐2‐(3‐hydroxy‐1‐adamantyl) acetyl] ‐2‐azabicyclo [3.1.0] hexane‐3‐carbonitrile (Fig.1b) are novel oral hypoglycemic drugs of the dipeptidyl peptidase 4-inhibitor class (DPP‐4) [1-2]. DPP-4 inhibitors represent a new therapeutic approach for treatment of type-II diabetes; that functions to stimulate glucose-dependent insulin release and reduce glucagon’s levels. This is done through inhibition of the inactivation of incretins, particularly glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), thereby improving glycemic control [3-5]. Literature review showed many methods for determination of VLG; based on the charge transfer complexes of
VLG with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone 2
(DDQ), 7,7,8,8-tetracyanoquinodimethane (TCNQ) and tetrachloro-1,4-benzoquinone (Pchloranil) [6]. Also; literature survey reveals that many chromatographic methods have been developed [7-13]. In addition; SXG has been estimated by LC‐MS/MS [14], HPLC methods [15-19] and by spectrophotometric method, in which SXG was estimated at 208 nm in methanol [20]. Another spectrophotometric method based on charge transfer reaction using DDQ and TCNQ was reported [21]. The aim of the first method is to present new spectrofluorimetric method based on the dansylation reaction for determination of VLG in bulk and in pharmaceutical dosage form. Furthermore, the established method should be rapid to be applied for routine quality control analysis of VLG in pharmaceutical dosage form. Spectrofluorimetry has long been applied in the field of pharmaceutical analysis of many drugs [22-24]. A necessary condition for a compound to fluoresce is that it absorbs light in the UV or visible region of the spectrum or reacts with a reagent to give a fluorescent product. Accordingly, A certain group in the compound as the primary amine for example react with the reagent to obtain a highly fluorescent product [22-24]. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols. Several pharmaceutical compounds have been determined through this approach [25–31]. The aim of the second and third methods is to present new spectrophotometric methods based on a charge transfer reaction and Hantzsch reaction for the determination of SXG in bulk and pharmaceutical dosage forms. Spectrophotometry continues to be very popular, because of its simplicity and versatility. Charge transfer reactions have been widely used for determination of electron donating compounds through interaction with π-acceptors [32-42]. Among the electron acceptors mostly used in literature is tetrachloro-1,4-benzoquinone (p-chloranil) [40-42].
3
Hantzsch reaction of primary amines with formaldehyde and acetyl acetone forms a yellow colored product that can be measured spectrophotometrically. The quantification of many drugs in its bulk and pharmaceutical dosage forms were carried out using Hantzsch reaction [43-49]. Hantzsch reaction is a known condensation reaction that was reported in the literatures as a useful pathway for pyrrole and pyridine synthesis, In the same manner, acetylacetone together with formaldehyde react with aliphatic amines by Hantzsch reaction forming a yellow product that can be measured spectrophotometrically [43-49]. The proposed method for determination of SXG (primary amine compound) was based on Hantzsch condensation reaction using acetylacetone as β‐diketone and formaldehyde as an aldehyde to form a colored condensation product. Optimization of the reaction conditions of all the proposed methods were optimized by experimental design using Minitab ® program with the advantage of finding a classic tool for estimating the mutual significance of multiple factors and fulfill most of the important optimality criteria [50-52]. 2. Experimental 2.1. Instrumentation BIO-TEK
spectrofluorimeter,
Italy,
SFM
25
software
was
used
for
spectrofluorimetric measurements while the spectrophotometric determinations were done using a Double-beam Shimadzu (Japan) 1601 PC UV-Visible spectrophotometer connected to a computer fitted with UVPC personal spectroscopy software version 3.7 (Shimadzu). Jenway digital pH meter was used to adjust and determine the hydrogen ion concentration (pH) of the buffer solutions.
2.2. Samples: 4
Pharmaceutical grade VLG, certified to contain 99.70 % and Galvus® tablets nominally containing 50 mg VLG per tablet (batch no. V6498) were kindly supplied from Novartis Europharm limited company (London, U.K.). Pharmaceutical grade SXG certified to contain 99.85 % and Onglyza® tablets (batch number 0J57932) nominally containing 5 mg of SXG per tablet were kindly supplied by Bristol-Myers Squibb/AstraZeneca EEIG (United Kingdom). 2.3. Reagents: 1-Dimethyl aminonaphthalene-5-sulphonyl chloride (dansyl chloride), purchased from Sigma (St. Louis, USA). A stock solution containing 0.1% of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 0.001% solution (solution a) [25]. Carbonate: Bicarbonate buffer (pH 9): Prepared by dissolving 2.1 g of sodium carbonate in 200 ml distilled water and 1.7 g of sodium bicarbonate in 200 ml distilled water and then mixing 10 ml of the carbonate solution with 115 ml of bicarbonate solution and completed to 500 ml [26]. Higher pH values (1112) had been avoided to prevent hydroxylation of dansyl chloride [25]. p-chloranil was supplied from Sigma Aldrich Chemie GmbH (Steinheim, Germany) and freshly prepared 0.5% solution was prepared in DMF (solution b) [40]. Acetyl acetone: 8.4 % v/v solution was freshly prepared by mixing 8.4 ml of acetyl acetone with 40 ml of acetate buffer (pH 5) and diluted to 100 ml with distilled water [49]. Formaldehyde (34 ‐ 40 %): Working formaldehyde solution was prepared by mixing 20 ml of formaldehyde with 40 ml of acetate buffer (pH 5) and diluted to 100 ml with distilled water. Solution (c) was prepared using 20 ml of acetyl acetone and 10 ml of formaldehyde with the commonly used ratio 2:1 as reported [49]. Acetate buffer (pH 5): Prepared by dissolving 13.6 g of sodium acetate and 6 ml of glacial acetic acid in
5
sufficient water to produce 1000 ml [49]. Methanol, acetone, acetonitrile and dimethylformamide of analytical spectrophotometric grade were used. 2.4. Stock standard solutions: Stock standard solutions of VLG (2 mgml-1) were prepared by dissolving 200 mg of VLG in acetonitrile in a 100 ml volumetric flask and completing to volume with acetonitrile. Stock standard solutions of SXG (2 mgml-1) were prepared by dissolving 200 mg of SXG in dimethylformamide in a 100 ml volumetric flask and completing to volume with dimethylformamide for the charge transfer experiment. Stock standard solutions of SXG (1 mgml-1) were prepared by dissolving 100 mg of SXG in methanol in a 100 ml volumetric flask and completing to volume with methanol for the Hantzsch experiment. 2.5. General procedures and calibration graphs 2.5.1. Spectrofluorimetric method using dansyl chloride: Aliquots of VLG containing (1–6 mg) were transferred into a set of 10 ml volumetric flasks, treated with acetonitrile to keep the volume at 3 ml for all the flasks, 0.5 ml of the carbonate bicarbonate buffer was added and mixed well followed by 1 ml of 0.001% of dansyl chloride reagent. The reaction mixture was left for 50 min, and then completed to the mark with acetone. The fluorescence intensity of the reaction product was measured at 455 nm after excitation at 345 nm. Blank experiment was carried out simultaneously. The corrected fluorescence intensity was plotted versus the final drug concentration (µg ml−1) to get the calibration graph and the corresponding regression equation was derived.
2.5.2. Spectrophotometric method using p-chloranil: Aliquots of SXG containing (1–8.5 mg) were transferred into a set of 10 ml volumetric flasks, treated with 2 ml p-chloranil solution (b), allowed to stand for 40 min
6
at room temperature and diluted to volume with dimethylformamide. The absorbance was measured at 530 nm against reagent blank, plotted against its corresponding concentration and then the regression parameters were computed. 2.5.3. Spectrophotometric method using formaldehyde and acetyl acetone: Aliquots of SXG containing (0.5–3 mg) were transferred into a set of test tubes, completed to 4 ml with the acetate buffer (pH = 5) treated with 3 ml of solution (c), allowed to stand for 10 minutes on boiling water bath (100°C), cooled, quantitatively transferred to 10 ml volumetric flasks and diluted to volume with distilled water. The absorbance was measured at 342.5 nm against reagent blank, plotted against its corresponding concentration and then the regression parameters were computed. 2.5.4. Assay of VLG and SXG in tablets and standard addition technique Twenty tablets of VLG were weighed. An accurately weighed amount of the finely powdered tablets equivalent to 200 mg of VLG was made up to 100 ml with acetonitrile, the solution was filtered. Twenty tablets of SXG were weighed and the coats were removed by carefully rubbing with a clean tissue wetted with methanol. An accurately weighed amount of the finely powdered tablets equivalent to 200 mg of SXG was made up to 100 ml with dimethylformamide for the second method and an accurately weighed amount of the finely powdered tablets equivalent to 100 mg of SXG was made up to 100 ml with methanol for hantzsch method. The solutions were filtered, procedures were continued as mentioned under 2.5., and then the concentrations were calculated using the corresponding equations. The standard addition technique was applied by adding different known concentrations of the pure drugs to different known concentrations of drugs in dosage form and the procedures mentioned above were adopted. The concentrations were calculated using the corresponding regression equations. 3. Results and discussion:
7
The secondary amine of VLG reacts with dansyl chloride as in figure [2], while the primary amine of SXG reacts with p-chloranil and undergoes hantzsch reaction as in figure [3]. 3.1. Formation of the dansylation product: The secondary amine of VLG reacts with dansyl chloride to obtain a highly fluorescent product according to the proposed reaction in figure [2]. Dansyl chloride is a useful derivatizing agent for primary amines, secondary amines, imidazoles and phenols. Several pharmaceutical compounds have been determined through this approach [25–31]. λmax of measurements is shown in Table [I] and the excitation emission spectrum of the dansylation product of VLG with dansyl chloride is shown in figure [4]. 3.2. Formation of the charge transfer complex: The charge transfer reagent applied in this work is p-chloranil. Primary, secondary and tertiary amines, both aliphatic and aromatic were shown to react with p-chloranil to produce a blue to purple colour. It was reported that p-chloranil could react with amines to form either mono substituted aminoquinones or radical ion pairs. It appears from literature survey that the reaction of p-chloranil with different drugs may differ according to the structure of the drug, solvent used and temperature [32-42]. λmax of measurements is shown in Table [I] and the absorption spectrum of the reaction product of SXG with p-chloranil is shown in figure [5]. 3.3. Formation of Hantzsch product: Hantzsch reaction [43] is a condensation reaction that allows the formation of dihydropyridine derivatives by condensation of an aldehyde with two equivalents of a βketoester in the presence of ammonia. In the same manner, acetylacetone (β-ketoester) together with formaldehyde (aldehyde) reacts with aliphatic amines by Hantzsch reaction forming a yellow colored product that can be measured spectrophotometrically. The 8
quantification of certain sulpha drugs [44] (sulfacetamide sodium, sulfadiazine, sulfadimidine, and sulfathiazole), kanamycin [45], lisinopril [46], gabapentin [47], cefrozil [47], tranexamic acid [48], and sitagliptin phosphate [49] in its bulk and pharmaceutical dosage forms were carried out using Hantzsch reaction according to the proposed mechanism in figure [3]. λmax of the absorption spectra [table 1] of the reaction product of SXG with acetyl acetone and formaldehyde is shown in figure [6]. 3.4. Factorial design experiment and methodology: Different parameters affecting the reactions such as amount of the reagents, reaction time, heating time and stability of the formed product have been investigated by experimental design using Minitab ® program (figures 7-15). In the first step of the investigation, experimental design was used to determine variables which have statistically important influence on the reaction behavior. In the screening design (tables II-III), two levels were used so that the factors were considered as discrete variables. The effect of each factor was tested using a Student (t) test with a corresponding p-value. The factors whose p-values were less than 0.05 were considered as “statistically significant” [50-52]. A graphical display of the ordered standardized effect (the absolute value of the estimated effect divided by its standard error estimate) of each factor was given in a Pareto chart (Figures 7, 8 and 9). A factor was considered as “statistically significant” if its standardized effect exceeded a threshold. A line in the Pareto chart indicated the threshold for a test at level 0.05. In the three proposed methods, the only factor that had been considered as statistically insignificant was the stability of the formed reaction product (Figures 7, 8 and 9). In the second step of the optimization (table IV), response surface methodology (RSM) was applied, where a collection of mathematical and statistical techniques for analyzing the effect of several independent variables on dependent variables and provides its graphical representation. The effect of 9
two variables can be represented as a surface in three-dimensional space and the influence of two variables on the response can be clearly seen in the investigated region. Furthermore, the response surface methodology enables the prediction of the response between and slightly outside the investigated area, as well as visualization and rapid selection of optimal conditions (Figures 10-15). In case of the dansylation experiment, Reaction time and amount of reagent were optimum at the maximum level while the amount of buffer was optimum at the minimum level (Figures 10 and 13). While in the charge transfer experiment and hantzsch experiment, both reaction time and amount of the reagent were optimum at the maximum level (Figures 11, 12, 14 and 15). Maximum level (+1) and minimum level (-1) had been discussed in (table II). 3.4. Effect of solvent: Although many solvents have been used to carry out the dansylation of VLG, charge transfer complex of SXG and hantzsch reaction of SXG, using the appropriate solvent in the described work was critical. The highest fluorescence of the dansylation reaction was attained when dissolving the VLG in acetonitrile and completion after the reaction with acetone [25]. Higher sensitivity of SXG reactions was attained in dimethylformamide for pchloranil method [40] and dissolving in methanol and completion with water for Hantzsch method [49]. 3.5. Methods Validation 3.5.1. Linearity Linearity was studied for both VLG and SXG. The regression equations were also computed. The linearity of the calibration curves were validated by the high value of correlation coefficients. The analytical data of the calibration curves including standard
10
deviation for the slope and intercept (Sb, Sa), confidence limits of the slope, the intercept and the standard error of estimation are summarized in (Table I). 3.5.2. Accuracy Accuracy of the results was calculated by % recovery of 5 different samples for each experiment. The results obtained including the mean of the recovery and standard deviations are displayed in (Table I). The procedure mentioned under 2.5.1., 2.5.2. and 2.5.3. were repeated using VLG concentrations (150-550 µgml-1), SXG concentrations (150-750 µgml-1) and SXG concentrations (75-275 µgml-1), respectively. 3.5.3. Precision 3.5.3.1 Repeatability: Three concentrations of VLG (250, 400 and 550 µg.mL−1) using the procedure mentioned under (2.5.1.), SXG (250, 400 and 550 µg.mL−1) using the procedure mentioned under (2.5.2.) and SXG (160, 200 and 240 µg.mL−1) using the procedure mentioned under (2.5.3.) were analyzed three times, within the same day. The %RSD was calculated and found to be less than 1% in the three concentrations, as shown in (Table I). 3.5.3.2 Intermediate precision: The above mentioned concentrations were analyzed on three successive days and the %RSD was calculated as shown in (Table I). 3.5.4. Results of determination of VLG and SXG in pharmaceutical dosage form The methods had been successfully applied to the pharmaceutical dosage forms and to check the validity of the proposed method, the standard addition technique was applied and the results are shown in (tables V-VI). 3.5.5. Limit of detection (LOD) & limit of quantification (LOQ)
11
The limit of detection (LOD) and limit of quantification (LOQ) were calculated as LOD = 3.3 (ơ/S) and LOQ = 10 (ơ/S), where ‘ơ’ represents standard deviations of the intercept and ‘S’ is the slope of the calibration line, as shown in (Table I). 3.5.6. Statistical analysis Statistical analysis of the results obtained by the proposed methods and the reference methods [9 and 20] for VLG and SXG was carried out by “SPSS statistical package version 11”. The significant difference between the reference method and the described method was tested by one way ANOVA (F-test) at P=0.05 as shown in (Tables VII-VIII). The test ascertained that; there was no significant difference among the methods. For SXG, Hantzsch method was more sensitive than the charge transfer method according to LOD and LOQ values (table I), consequently it can be used for the quality control laboratories for the determination of SXG in its pharmaceutical dosage form. 4. Conclusion: The proposed spectrofluorimetric and spectrophotometric methods have the advantages of simplicity, precision, accuracy and convenience for the quantitation of VLG and SXG. Hence, the proposed methods can be used for the quality control of the cited drugs and can be extended for their routine analysis in dosage forms as an economic alternative to the time consuming HPLC reported methods. 5. References [1] B. Sekaran and P. Rani, Int J Pharm Pharm Sci. 2 (2010) 138. [2] D.J. Augeri, J.A. Robl, D.A. Betebenner, et al. J Med Chem. 48(2005) 5025. [3] R. Nirogi, V. Kandikere, K. Mudigonda, et al, Biomed Chromatogr. 22 (2008) 214. [4] S. Dhillon, Drugs. 70 (2010) 489. [5] J. Green and M. Feinglos, Vasc Health Risk Manag. 4 (2008) 743. 12
[6] R. El-bagary, E. Elkady, B. Ayoub, Int J Biomed Sci. 7 (2011) 55. [7] A. Pharne, B. Santhakumari, A. Ghemud, et al, Int J Pharm Pharm Sci. 4 (2012) 119. [8] A. Malakar, B. Bokshi, D. Nasrin, Int J Pharm life Sci. 1 (2012) 1. [9] M. Mohammad, E. Elkady, M. Fouad, European Journal of Chemistry. 3 (2012) 152. [10] R. El-bagary, E. Elkady, B. Ayoub, Int. J. Biomed.Sci. 7 (2011) 201. [11] A. Barden, B. Salamon, E. Schapoval, J Chrom sci . 50 (2012) 426. [12] B Thangabalan, P Kumar, J. Pharm. Res. 7 (2013) 113. [13] D varma, A Rao, S Dinda, et al, IRJP. 4 (2013)122. [14] A. Fura, A. Khanna, V. Vyas, et al. Drug Metab Dispos. 37 (2009) 1164. [15] R. Inturi, I. Tagaram. IJPRD. 3 (2011) 45. [16] X. Xua, R. Demersb, H. Gua. J Chromatogr. 889 (2012) 77. [17] C. Hess, F. Musshoff, B. Madea. Analytical and bioanalytical chemistry. 400 (2011) 33. [18] S. Inturi, R. Inturi, I. Tagaram. Int. j. sci. pharm. edu. res. 1 (2011) 27. [19] M. Mohammad, E. Elkady, M. Fouad. European Journal of Chemistry. 3 (2012) 152. [20] R. kalaichelvi, E. Jayachandran. Int. J. Pharm. Pharm. Sci. 3 (2011) 180. [21] R. I. El-bagary, E. F. Elkady, B. Ayoub, Int. J. Biomed.Sci. 8 (2012) 204.
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[31] K. Bagdonaite; G. Viklund; K. Skog; etal. J Biochem Biophys Methods. 69 (2006) 215. [32] E. Nour, S. Alqaradawi, A. Mostafa, et al. J mol Struct.980 (2010) 218. [33] A. Fakhroo, H. Bazzi, A. Mostafa, et al. Spectrochim Acta A Mol. Biomol. Spectrosc.75 (2010) 134. [34] L. Shahada, A. Mostafa, E. Nour, et al. J mol Struct.933 (2009) 1. [35] M. Zayed, S. Khalil and H. El-qudaby. Spectrochim Acta A Mol. Biomol. Spectrosc. 62 (2005) 461. [36] A. Mostafa and H. Bazzi. J mol Struct. 983 (2010)153.
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[37] G. Mohamed, F. El-Dien and N. Mohamed. Spectrochim Acta A Mol. Biomol. Spectrosc. 68 (2007) 1244. [38] F. El-Dien, G. Mohamed and E. Farag, Spectrochim Acta A Mol. Biomol. Spectrosc. 64 (2006) 210. [39]M. Abdel-Hamid and M. Abuirjeie. Talanta. 35 (1988) 242. [40] R. Elbagary, E. Elkady, B. Ayoub. Int. J. Biomed.Sci. 7 (2011) 55. [41] M. Refat, S. El-Korashy, I. El-Deen, et al. J mol Struct. 980 (2010) 124. [42] F. Siddiqui, M. Arayne, N. Sultana, et al. Eur J Med Chem. 45 (2010) 2761. [43] A. Hantzsch, Chemische Berichte, 14 (1881) 1637. [44] A. Amin, M. Zareh, Microchimica Acta, 124 (1996) 227. [45] A. Ahmad, N. Hoda, M. Ahmad, J. Anal. Chem, 61 (2006) 870. [46] F. El-Yazbi, H. Abdine, R. Shaalan, J. Pharmaceut. Biomed., 19 (1999) 819. [47] M. Ayad, M. El-Henawee, H. Abdellatef, Alex. J. Pharmaceut. Sci, 9 (2005) 157. [48] K. El-Aroud, A. Abushoffa, H. Abdellatef, Chem. Pharmaceut. Bull., 55 (2007) 364. [49] C. Sekaran, A. Rani, I. J. Pharm. Pharm. Sci, 2 (2010) 4. [50] S. Furlanetto, S. Orlandini, N. Porta, J pharm biomed anal. 28 (2002) 1161. [51] M. Odeniyi, K. Jaiyeoba. Farmacia. 57 (2009) 157. [52] W. Sangoi, S. Sangoi, P. Oliveira, J chromatog sci. 49 (2011) 170.
15
N
N
O H2N
N HO
N HO
H
N H H
O
A
B
Fig.1 Chemical structures of vildagliptin (a) and saxagliptin (b).
OH O N NH
Cl
S O
N O
dansyl chloride
N
Vildagliptin
Dansylation reaction in the presence of carbonate bicarbonate buffer, pH = 9
OH O N N
S O
N O N
Highly fluorescent dansylation product
Fig.2 Dansylation reaction of vildagliptin with dansyl chloride.
H H
The lone pair of the nitrogen will attack the partial positive carbon
H OH
H OH
N N NH2
H2C
O
N
H2 C
O
formaldehyde
HN
OH
O
N
Followed by elimination of one molecule of water H
H OH N
CH2
N O
N
Acetyl acetone will attack the partially positive carbon of imine O
O
acetylacetone H
H
H OH N
N N
Hantzsch product after the hydrogen bond cyclic intermediate
H OH N
HN
CH2
O
N
H
CH2
O
O O
O
O
Fig.3 The proposed mechanism of hantzsch reaction of saxagliptin with acetyl acetone and formaldehyde.
Fig.4 A typical excitation emission spectrum of the dansylation product after the reaction of vildagliptin (400 µg mL-1) with dansyl chloride (0.001%).
Fig.5 Absorption spectra of the colored product of saxagliptin (400 μgml-1) and pchloranil (0.5%) in dimethylformamide.
Fig.6 Absorption spectrum of the colored product of hantzsch reaction of saxagliptin (200 μgml-1) with acetyl acetone and formaldehyde.
Fig.7 Pareto chart of the effects in the factorial design of dansylation experiment
Fig.8 Pareto chart of the effects in the factorial design of the charge transfer experiment
Fig.9 Pareto chart of the effects in the factorial design of the Hantzsch experiment
Fig.10 Surface plot of the effects in the factorial design of dansylation experiment
Fig.11 Surface plot of the effects in the factorial design of the charge transfer experiment
Fig.12 Surface plot of the effects in the factorial design of the Hantzsch experiment
Fig.13 Contour plot of the effects in the factorial design of dansylation experiment
Fig.14 Contour plot of the effects in the factorial design of the charge transfer experiment
Fig.15 Contour plot of the effects in the factorial design of the Hantzsch experiment
Table (I): Results obtained by the proposed methods for determination of vildagliptin and saxagliptin
Item λmax of measurements Obedience of Beer’s law Regression equation Regression coefficient (r) Accuracy LOD µg.ml-1 LOQ µg.ml-1 Intraday %RSD Interday %RSD *Sb *Sa Confidence limit of the slope Confidence limit of the intercept Standard error of the estimation
Dansylation reaction λexc =345 nm, λemi =455 nm 100-600 µg.ml-1 F431 = 0.3062 Cµg/ml- 0.8867 0.9994 99.76 ± 0.95 15.05 50.17 0.14-0.22 0.21-0.34 0.0037 1.75 0.3062 ± 0.54 0.8867 ± 0.0033 1.54
*Sb: Standard error of intercept, Sa: Standard error of slope.
Charge transfer reaction 530 nm 100-850 µg.ml-1 A = 0.0013 Cµg/ml+ 0.0006 0.9995 100.48 ± 1.41 32.32 97.94 0.11-0.25 0.20-0.31 2.1 x 10-5 1.4x 10-2 0.0013± 1.82 x 10-5 0.0006 ± 1.26 x 10-8 0.013
Hantzsch reaction 342.5 nm 50-300 µg.ml-1 A = 0.0031 Cµg/ml + 0.0011 0.9997 100.44 ± 2.01 9.84 29.81 0.18-0.27 0.23-0.29 4.54 x 10-5 1.08 x 10-2 0.0031± 1.19 x 10-5 0.0011 ± 1.41 x 10-7 0.0095
Table (II): Investigated variables and their domains used in the design of experiments
Investigated variables and their domains Investigated levels Variables
Low level (-1)
High level (+1)
Dansylation experiment Reaction time
10 minutes
50 minutes
Amount of reagent
0.2 ml of solution (a)
1 ml of solution (a)
Amount of buffer
0.5 ml of carbonate
2 ml of carbonate
bicarbonate buffer pH = 9
bicarbonate buffer pH = 9
10 minutes
60 minutes
Reaction time
10 minutes
40 minutes
Amount of reagent
0.5 ml of (Solution b)
2 ml of (Solution b)
Stability of the formed complex
10 minutes
60 minutes
Heating time
10 minutes
30 minutes
Amount of reagent
0.75 ml of solution (c)
3 ml of solution (c)
Stability of the formed product Charge transfer experiment
Hantzsch experiment
Stability of the Hantzsch reaction 10 minutes
60 minutes
Table (III): Screening of variables using factorial design in dansylation experiment (A1-D1), charge transfer experiment (A2-C2) and hantzsch experiment (A3-C3) Experiments A1 B1 C1 D1 Relative Flourescence 1 -1 -1 1 1 15.3 2 1 -1 -1 1 28.2 3 -1 -1 -1 -1 12.6 4 1 1 -1 -1 90.4 5 -1 1 -1 1 38.9 6 -1 1 -1 -1 41.1 7 1 1 -1 1 90.2 8 -1 1 1 -1 31.1 9 -1 -1 -1 1 14.8 10 -1 -1 1 -1 10.1 11 1 -1 1 1 20.4 12 1 1 1 -1 42.1 13 1 -1 -1 -1 25.1 14 1 -1 1 -1 20.6 15 -1 1 1 1 30.7 16 1 1 1 1 40.6 A1: Reaction time, B1: Amount of reagent, C1: Amount of buffer, D1: Stability of the formed dansylation product
A2 -1 -1 1 1 -1 1 -1 1
B2 -1 -1 -1 1 1 1 1 -1
C2 1 -1 1 -1 1 1 -1 -1
Absorbance 0.204 0.213 0.297 0.522 0.261 0.524 0.233 0.303
A2: Reaction time, B2: Amount of reagent, C2: Stability of the formed complex
A3 1 1 -1 1 -1 -1 -1 1
B3 1 -1 1 -1 -1 1 -1 1
C3 -1 1 -1 -1 -1 1 1 1
Absorbance 0.217 0.099 0.611 0.121 0.312 0.615 0.313 0.211
A3: Heating time, B3: Amount of reagent, C3: Stability of the formed Hantzsch product
Table (IV): Optimization of variables using factorial design in dansylation experiment (A1-C1), charge transfer experiment (A2-B2) and hantzsch experiment
Experiments
A1
B1
C1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1.00000 0.00000 1.00000 0.00000 0.00000 0.00000 -1.00000 -1.00000 0.00000 1.00000 0.00000 0.00000 -1.68179 0.00000 0.00000 0.00000 -1.00000 -1.00000 1.00000 1.68179
1.00000 0.00000 -1.00000 0.00000 0.00000 0.00000 1.00000 1.00000 0.00000 -1.00000 0.00000 1.68179 0.00000 0.00000 -1.68179 0.00000 -1.00000 -1.00000 1.00000 0.00000
-1.00000 0.00000 1.00000 1.68179 0.00000 0.00000 1.00000 -1.00000 0.00000 -1.00000 0.00000 0.00000 0.00000 -1.68179 0.00000 0.00000 1.00000 -1.00000 1.00000 0.00000
Relative Flourescence 90.1 23.7 40.9 26.3 22.9 24.1 30.7 40.2 23.5 25.2 23.1 81.4 12.7 62.3 14.6 23.9 15.2 13.4 40.7 75.3
A2
B2
1.41421 0.00000 -1.00000 1.00000 0.00000 0.00000 0.00000 0.00000 1.00000 -1.00000 0.00000 0.00000 0.00000 0.00000 0.00000 -1.41421 -1.41421 0.00000 0.00000 0.00000 0.00000 1.41421 -1.00000 -1.00000 1.00000 1.00000
A: Reaction time, B: Amount of reagent, C: Amount of buffer
Absorbance 0.342 0.206 0.291 0.288 0.201 0.292 0.289 0.125 0.097 0.287 0.463 0.187 0.527
A3
B3
0.00000 0.00000 -1.41421 0.00000 -1.00000 1.00000 -1.00000 -1.00000 0.00000 0.00000 0.00000 1.41421 0.00000 0.00000 0.00000 -1.41421 0.00000 0.00000 0.00000 0.00000 1.00000 -1.00000 1.00000 1.00000 1.41421 0.00000
Absorbance 0.272 0.010 0.616 0.266 0.109 0.137 0.294 0.105 0.268 0.278 0.074 0.138 0.085
Table (V): Pharmaceutical dosage form and standard addition results obtained by dansylation method for determination of vildagliptin
Standard addition technique Pharmaceutical dosage form
Galvus® tablets B.N.: V6498
Claimed
50 mg
% found ± S.D.
100.10 ± 0.80
Mean ± S.D.
*Mean of three determinations.
Amount taken dosage form (µgml-1)
Amount added (µgml-1)
*R % added
100 150 200 250 300
500 400 300 200 100
102.29 97.89 99.83 99.94 100.26 100.04 ± 1.56
Table (VI): Pharmaceutical dosage form and standard addition results obtained by the charge transfer method and hantzsch method for determination of saxagliptin
Pharmaceutical dosage form
Claimed
Onglyza® tablets B.N.: 0J57932
5 mg
*Mean of three determinations.
Standard addition technique (Charge transfer experiment) Amount taken Amount added *R % dosage form (µgml-1) added (µgml-1) 100 450 101.23 150 350 98.70 200 250 100.14 100.69 ± 1.43 250 150 102.05 300 50 100.22 100.47 Mean ± S.D. ± 1.26
% found ± S.D. (Charge transfer experiment)
% found ± S.D. (Hantzsch experiment)
99.49 ± 1.61
Standard addition technique (Hantzsch experiment) Amount Amount taken added *R % added dosage form -1 (µgml ) (µgml-1) 50 250 101.81 75 200 98.06 100 150 101.51 125 100 101.94 150 50 99.34 100.53 ± Mean ± S.D. 1.74
Table (VII): Statistical comparison between the results of the proposed dansylation method and the reference method for the determination of vildagliptin Statistical Term Mean S.D.± S.E. ± %RSD n V t (*2.306)
Reference Method**
Proposed spectrofluorimetric method
100.01 0.99 0.44 0.99 5 0.98
99.76 0.95 0.42 0.95 5 0.90 0.41
* Figures in parentheses are the theoretical t and F values at (p=0.05). No significant difference between groups of vildagliptin by using one way ANOVA with F equals 0.17 and p equals 0.69. **Reference method: aliquots of standard solutions containing 5‐200 µg/ml VLG were measured using HPLC with UV detection at 208 nm [9].
Table (VIII): Statistical comparison between the results of the proposed charge transfer method and the reference method for the determination of saxagliptin
Statistical Term Mean S.D.± S.E. ± %RSD n V t (*2.306)
Reference Method**
Charge transfer method
Hantzsch method
100.20 1.10 0.49 1.10 5 1.21
100.48 1.41 0.63 1.40 5 1.99 0.40
100.44 2.01 0.90 2.00 5 4.04 0.23
* Figures in parentheses are the theoretical t and F values at (p=0.05). No significant difference between groups of saxagliptin by using one way ANOVA with F equals 0.05 and p equals 0.95. ** Reference method: aliquots of standard solutions in methanol containing 5‐40 µg/ml SXG were measured using methanol as a blank [20].
1. The first method is based on dansylation of vildagliptin. 2. The spectrophotometric method is based on the charge transfer of saxagliptin 3. The other spectrophotometric method is based on hantzsch reaction of saxagliptin 4. The variables were studied using experimental factorial design 5. The developed methods were validated for quality control of vildagliptin and saxagliptin
1