A NOVEL METHOD FOR ESTIMATION OF DIPEPTIDYL PEPTIDASE-4 INHIBITOR IN BULK AND PHARMACEUTICAL DOSAGE FORMS BY VISIBLE SPECTROPHOTOMETRY

A NOVEL METHOD FOR ESTIMATION OF DIPEPTIDYL PEPTIDASE-4 INHIBITOR IN BULK AND PHARMACEUTICAL DOSAGE FORMS BY VISIBLE SPECTROPHOTOMETRY

Vinutha Kommineni^*1, N. Prasanna Lakshmi ¹, B. Ramya ¹ and Chandra Bala Sekharan ² and O. Ahmed ³

Department of Pharmaceutical Analysis ¹, Sri Venkateswara College of Pharmacy, Hyderabad - 500081, Telangana, India.

Department of Biochemistry ², International Medical and Technological University, Dar Es Salaam, Tanzania.

Department of Pharmaceutical Chemistry ³, Deccan school of Pharmacy, Hyderabad - 500001, Telangana, India.

ABSTRACT: A new, simple and low cost spectrophotometric method for the determination of teneligliptin, a new DPP IV inhibitor drug in bulk and pharmaceutical dosage form using sodium nitroprusside was developed. The absorbance maximum (λ_max) of the resulted colored product was at 445 nm. As different solvents were tried methanol was found to be the most suitable solvent. The Beer’s law has been obeyed in the concentration range of 4 - 20 µg/ml with the detection limits of 0.09µg/ml and limit of Quantification of 0.28µg/ml. The correlation coefficient was found to be (r = 0.9999). The reaction ratio between teneligliptin and sodium nitroprusside was studied and found to be 1:1. The method was validated and results obtained for the assay of two different brands of teneligliptin tablets were compared. The repeatability and reproducibility of the developed method were evaluated and the obtained results quoted.

New spectrophotometric method, Teneligliptin, Sodium nitroprusside, Validation

INTRODUCTION: Teneligliptin, a novel DPP-4 Class 3 inhibitor is a long acting oral hypoglycemic agent. It is characterized by 5 consecutive rings namely - thiazole, pyyrolidine, piperazine, pyrazole and benzene ¹. DPP-4 inhibitors increase the levels of active GLP-1 and GIP by inhibiting DPP-4 enzymatic activity.  These inhibitors improve hyperglycemia in a glucose-dependent manner by increasing serum insulin levels and decreasing serum glucagon levels ^{2, 3, 4}.

Literature survey revealed many methods with different analytical techniques for individual or simultaneous determination of teneligliptin. The technique includes HPTLC ⁵, UPLC ⁶, HPLC ^{7 - 10}, LC-MS-MS ¹¹, UV ^{13, 14} and Visible ^{15, 16} spectrophotometric methods. The techniques other than spectrophotometry require lengthy procedures, highly sophisticated analytical instruments and some amount of the organic solvents contributing towards high cost of the analysis.

Spectrophotometry is the most common, convenient and favourite tool in analytical laboratories due to its inherent sensitivity and low costing. So, we aimed to report the original approach that allows a simple, rapid and improved spectrophotometric determination of teneligliptin avoiding lengthy procedure and minimizing the use of hazardous chemicals. Many spectrophotometric methods adapted SNP as an analytical reagent in their applications, including determination of   bowel-specific aminosalicylate drug ¹⁷, selective angiotensin AT1 receptor blocker ¹⁸, piperazine antihistamine ¹⁹, antiprotozoal ²⁰ aminoacid lysine derivative ²¹, aminoglycoside ²².

This paper deals with the use of Sodium nitroprusside as a chromogen for the spectro-photometric assay of Teneligliptin.

In the present work, simple and sensitive visible spectrophotometric method was developed and validated for the analysis of teneligliptin with broad linearity, good precision and accuracy. These methods could be applied for the quantitative determination of the teneligliptin in their tablet formulations.

MATERIALS AND METHODS:

Instrumentation:

• A double Beam UV Spectrophotometer: Lab India-3000 with UV Win software and 1cm quartz cell.
• Weighing Balance:

Preparation of Reagents and Solutions:

Standard Stock Solution of Teneligliptin: Teneligliptin working standard was procured from Hetero Pharma Labs Pvt. Ltd., Hyderabad, India.  Standard stock solution of Teneligliptin was prepared by dissolving accurately weighed 10 mg of drug in 20 ml of 0.1 M NaOH in 50 ml volumetric flask and diluted upto the mark with distilled water.

Standard Solution of Sodium Nitroprusside: 0.4% w/v of sodium nitroprusside was prepared by dissolving 0.400g in distilled water in 100 ml volumetric flask and diluted upto the mark with water.

Standard Solution of Hydroxyl Amine HCl: 0.4% w/v of Hydroxyl amine HCl was prepared by dissolving 0.400 g in distilled water in 100ml volumetric flask and diluted upto the mark with water.

Standard Solution of Sodium Carbonate: 0.4% w/v of Sodium Carbonate was prepared by dissolving 0.400 g in distilled water in 100 ml volumetric flask and diluted upto the mark with water.

Preparation of Sample Solution:

General Procedure: Aliquots (0.2 - 1.0 ml) of Teneligliptin standard solution were transferred in to 10 ml volumetric flask. To each flask 1ml of Sodium nitroprusside, 0.9 ml of Hydroxyl amine HCl, and 1 ml of Sodium Carbonate solutions were added. The volume was then made upto the mark with water and absorbance of each solution was measured at 445 nm.

Preparation of Sample Solution: The tablet formulation of Teneligliptin labelled to contain 20 mg was purchased. Twenty tablets were accurately weighed and finely powdered in a mortar. A portion of tablet powder equivalent to 20 mg was weighed and transferred into 50 ml volumetric flask. 20 ml of 0.1 M NaOH was added and the mixture was sonicated for 15 min. The mixture was filtered through Whatman No. 1 filter paper. The solution was made up to the mark with distilled water and contents were analysed by the proposed method.

RESULTS AND DISCUSSION:

Method Development: The reaction of Sodium nitroprusside with amino group has been reported, these reactions demonstrate that Sodium nitroprusside is a valuable reagent in the development of simple spectrophotometric methods for the quantification of many pharmaceutical amines ^{17 - 22}.  Tenegliptin acts as a donor due to the presence of cyclic secondary nitrogen. The mechanism is based on the electron transfer from donor Tenegliptin to acceptor Sodium nitroprusside. Sodium nitroprussside in the presence of hydroxylamine and alkali exists as aquoferrocyanide (Fe(CN)₅.H₂O)^3-. The electron transfer results in the formation of yellow colored complex characterized by high degree of covalency and electron delocalization.

The reaction was studied under various conditions of Sodium nitroprusside, Hydroxyl amine HCl, Sodium carbonate concentration and reaction time was studied to determine the optimum conditions for the method.

FIG. 1: PROPOSED REACTION PATHWAY BETWEEN SODIUM NITROPRUSSIDE AND TENELIGLIPTINE

The effect of the volume of 0.4% w/v Hydroxyl amine HCl on the absorbance of the yellow colored complex was studied in the range of 0.2 - 2.0 ml. The absorbance increases with the increase in the volume of Hydroxyl amine HCl up to 0.8 ml. Further addition of Hydroxyl amine HCl showed decrease in the absorbance. Therefore, 0.8 ml of 0.4% w/v Hydroxyl amine HCl was chosen as an optimum value Fig. 2.

The effect of volume of 0.4% w/v Sodium carbonate on the absorbance of yellow colored complex was studied in the range 0.2 - 2.0 ml. The absorbance increases with increase in the volume of Sodium carbonate and becomes constant at 1ml. Further addition of Sodium carbonate does not show any change in the absorbance. Hence 1ml of 0.4% w/v Sodium carbonate was selected as an optimum value Fig. 3.

FIG. 2: EFFECT OF VOLUME OF HYDROXYL AMINE HCl

FIG. 3: EFFECT OF VOLUME OF SODIUM CARBONATE

FIG. 4: EFFECT OF VOLUME OF SODIUM NITROPRUSSIDE                               

FIG. 5: EFFECT OF TIME

The effect of volume of 0.4% w/v Sodium nitroprusside on the absorbance of yellow colored complex was studied in the range 0.2 - 2.0 ml. The absorbance increases with increase in the volume of Sodium nitroprusside and becomes constant at 1ml. Further addition of Sodium nitroprusside does not show any change in the absorbance. Hence 1ml of 0.4% w/v Sodium nitroprusside was selected as an optimum value Fig. 4.

The effect of time on the formation of the yellow colored complex was also optimized. At room temperature, the color intensity of the product increased by the elapse if time, maximum absorbance was obtained at 2 minutes and remained constant for 20 minutes. The results indicated that the yellow colored product obtained was stable for 10 hrs Fig. 5.

In order to choose proper solvent for dilution, different solvents (methanol, dichloro methane, chloroform, isopropanol and Acetonitrile) were tested. The highest absorbance values were obtained when methanol was used as diluting solvent. In methanol medium, the absorbance values were found to be stable for 10 hrs. Hence methanol was chosen as dilution solvent.

Association Constant and the Free Energy Changes of the Complexes: The association constant of complex was determined by employing the Benesi - Hildebrand method ²³, besides the association constant was calculated by using the following equation:

[Ao]/Aλ=1/ε+ (1/Kc.ε).1/[Do]

Where;

[Do]  = Concentration of the drug,

[Ao] = Concentration of the reagent,

Aλ = Absorbance of the complex at 445 nm,

ε = Molar absorptivity of the complex at 445 nm,

Kc = Association constant of the complex.

The ΔG° (the standard free energy of complexation)   and   the   association   constant Kc are related by the following equation ²⁴

ΔG° = −2.303 RT log Kc

Where;

ΔG° = Free energy change of the complex,

R = Gas constant (1.987 cal mol⁻¹ degree⁻¹),

T = Temperature in Kelvin,

K = Association constant (L mol⁻¹) of the drug-reagent complex.

The results are summarized in the Table 1. The negative values of the standard free energy indicated that the complexes are stable and started to form spontaneously.

TABLE 1: THERMODYNAMIC STUDIES, LINEARITY AND SENSITIVITY OF THE PROPOSED METHODS

Stiochiometry of Ion Pair Complex: Jobs method of continuous variation was employed to determine the composition of the ion pair complex ²⁵. For this, solutions of identical concentrations (1.1 × 10^-3 M) of the drug and reagent were prepared and mixed in varying volume ratios such that total volume of each solution was constant. The absorbance of the resulting complex was measured at 445 nm. The absorbance values were plotted against mole fraction of drugs. The stiochiometry ratio observed for drug dye ion pair complex was 1:1 as evident from the results in Fig. 6.

FIG. 6:   MOLE FRACTION OF DRUG                                            

FIG. 7: λ_max OF COMPLEX

Method Validation:

Specificity: Specificity of proposed spectro-photometric method was evaluated by analyzing standard solution of Teneligliptine in the presence of common excipients of pharmaceutical preparations such as lactose, sucrose, starch, mannitol and magnesium stearate. The results indicated that there is no interference from excipients Fig. 7.

Sensitivity: According to the ICH guidelines, the sensitivity parameters like molar absorptivity, Sandell’s sensitivity, Limit of Detection and Limit of Quantification were calculated and summarized in Table 1.

Linearity: The relation between the absorbance and final concentration of Teneligliptine was found to be linear over the concentration range of 4 - 20 μg/ml. Results are shown in Fig. 8.

FIG. 8: LINEARITY GRAPH OF TENELIGLIPTINE

Precision: The repeatability (intra-day precision) of the proposed method was determined by replicate analysis (n = 5) of standard solutions at three concentration levels (2 μg/ml, 6 μg/ml and 10 μg/ml). The intermediate precision (inter-day precision) was conducted by repeating the analysis over a period of three consecutive days. The precision of the methods   was expressed as standard deviation (SD) and percentage relative standard deviation (% RSD). The results are summarized in Table 2. The SD and % RSD obtained by both methods are found to be in the acceptable range. Therefore, it can be considered to be satisfactory.

Accuracy: The accuracy of the proposed method was established by performing intra-day and inter-day assays by determining at different levels of drug concentrations [lower concentration (50%), intermediate concentration (100%) and higher concentration (150%)] within 1 day and 3 consecutive days, respectively. The accuracy of the methods is expressed as percentage recoveries and percentage error. The results obtained by both the methods are found to be in the acceptable range. Therefore, we can say it can be considered as satisfactory. In addition, accuracy and validity of the proposed methods were determined by standard addition technique. The pre analyzed samples were spiked with additional 50, 100 and 150% were once again analyzed by the proposed methods. The accuracy of the methods was evaluated by percentage recovery of the Teneligliptine.

The average recovery and percentage standard deviation values Table 3 of the methods lying in the acceptable range show that the methods are accurate.

TABLE 2: ACCURACY AND PRECISION OF THE PROPOSED METHODS

TABLE 3: RESULTS OF STANDARD ADDITION TECHNIQUE OF PROPOSED METHOD

Robustness: The robustness of the proposed methods was checked for each operational parameter and investigated. The operational parameters were:

Volume of Sodium Carbonate: 1 ± 0.1 mL

Volume of 0.4% SNP: 1 ± 0.1 mL

Volume of 0.4% HA HCl: 0.9 ± 0.1 mL

The robustness of the methods was assessed by analyzing the Teneligliptine at two different concentration levels (4 and 20 μg/mL). The percent recovery and % RSD of the method Table 4 was found to be satisfactory, indicating that the method is robust.

TABLE 4: ROBUSTNESS OF PROPOSED METHOD

Application of the proposed methods to analysis of Teneligliptine in tablet formulations: It is obvious from the above mentioned results that the proposed methods gave satisfactory results with Teneligliptine in bulk. Therefore, Teneligliptine tablet formulations were subjected to the analysis of their Teneligliptine contents by the proposed methods. The percent recovery and % RSD Table 5 clearly showed no interference of any excipients of formulation, thus proving accuracy and precision in the quantification of Teneligliptine.

TABLE 5: RESULTS OF ANALYSIS IN TABLET FORMULATION

Comparison of the Proposed M with the Reported Spectrophotometric Methods: Three UV spectrophotometric methods ^{5, 13, 14} and two visible spectrophotometric methods ^{15, 16} were reported for the assay of Teneligliptine. The comparison between the proposed method and reported spectrophotometric methods is summarized in Table 7. Most of the reported spectrophotometric methods were not fully validated according to the ICH guidelines ²⁶.

TABLE 7: COMPARISON BETWEEN PROPOSED AND REPORTED SPECTROPHOTOMETRIC METHODS

NR-Not Reported

CONCLUSION: A sensitive visible spectrophoto-metric method for the determination of Teneligliptine have been developed and validated. The present methods demonstrate that alkaline sodium nitroprusside can be used for the quantitative determination of Teneligliptine in bulk and in tablet dosage forms.

The reagent used in the developed method is cheap and readily available. From the values of molar absorptivity, Sandell's sensitivity, LOD and LOQ, it was observed that the new method is more sensitive than the reported methods.

Tedious procedures, extractions and sophisticated instrumentation associated with chromatographic methods ^{6 - 10} are not involved in the proposed method. The proposed method is more selective, sensitive and accurate than the spectrophotometric methods reported ^{5, 13 - 16}. The proposed method is fully validated and found to be sensitive, accurate, reproducible, selective, robust and rugged. These advantages support the application of the proposed method in routine quality control analysis of Teneligliptine.

ACKNOWLEDGEMENT: I express my sincere thanks to Dr. M. Bhagavan Raju, principal for his support and encouragement throughout my research work. I am also thankful to Sri Venkateshwara College of Pharmacy for providing chemicals and instruments and Hetero Labs Pvt. limited Hyderabad for providing drug samples for research.

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Kommineni V, Lakshmi NP, Ramya B and Sekharan CB and Ahmed O: A novel method for estimation of dipeptidyl peptidase-4 inhibitor in bulk and pharmaceutical dosage forms by visible spectrophotometry. Int J Pharm Sci Res 2018; 9(6): 2351-58. doi: 10.13040/IJPSR.0975-8232.9(6).2351-58.

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