Quality by Design Based Analytical Method Development and Validation of Sitagliptin Phosphate by RP-HPLC

 Syed Aslam Hamid*, Pallavi Patharkar, Umar Attar, Shaikh Md Moiz ,

Mohd Shoeb Nawaz

Dr.Vedprakash Patil Pharmacy College Aurangabad, Maharashtra.

*Correspondence: aslamsayyd854@gmail.com ; Tel.: (+91 9923067418)

DOI: https://doi.org/10.71431/IJRPAS.2025.4701  

INTRODUCTION

Sitagliptin, also known as MK-0431, is a new type of drug that works by inhibiting an enzyme called DPP-4, beta-amino acid-derived inhibitor of dipeptidyl peptidase 4 (DDP-4) with hypoglycemic activity. It is used to treat high blood sugar levels and has the advantage of being easily absorbed when taken orally. This drug is used to treat type 2 diabetes, either on its own or in combination with other medications like metformin or thiazolidinediones.DDP-4 enzyme degrades two gastrointestinal hormones, GLP-1 and GIP, which are released after eating a meal, by preventing GLP-1and GIP inactivation. They stimulate the pancreas to release more insulin and less glucagon, which in turn helps to regulate blood sugar levels and bring them back to a healthy range. This medication can cause mild side effects like headaches, leg swelling, and respiratory infections. However, in rare cases, it may lead to more serious issues like severe allergic reactions, low blood sugar, kidney damage, pancreas inflammation, and joint pain.^[1-3]

Chemical formula for sitagliptin phosphate is (3R)-3-amino-1-[3-(trifluoromethyl)-6,8-dihydro-5H-[1,2,4] triazolo [4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one;phosphoric acid, shown in fig. no. 01, molecular weight 505.31 g/mol. State of matter is white to off- white, crystalline, non-hygroscopic powder, oral dosage form [Tablet]. Melting point of sitagliptin phosphate is 214 ^oC. The drug has solubility in methanol, water, is barely soluble in ethanol, acetone, and acetonitrile, and insoluble in isopropanol and isopropyl acetate. Patented by Merck & Co and it is sold under brand name Januvia. Sitagliptin was granted US FDA approval on October 16, 2006.^[4-5]

Literature review shows that various analytical methods have been developed to estimation of sitagliptin in combination with other drugs like metformin, simvastatin and gliclazide in pharmaceutical formulations. Additionally, some methods have been reported for detecting sitagliptin in biological samples like blood or plasma. Furthermore, analytical method development by UV spectroscopy is also reported.^[6-9] QbD based method development of sitagliptin phosphate in combination with metformin is reported.^[6]

RP-HPLC analytical method development of sitagliptin phosphate by QbD approach have not been reported before. Hence RP-HPLC by in a QbD is performed. The aim of present analytical research is to develop and validate rapid and sensitive RP- HPLC method by QbD for determination of Sitagliptin Phosphate. 

Figure 01: Structure of Sitagliptin Phosphate

MATERIAL AND METHODS

Chemicals and reagents

Sitagliptin phosphate was kindly supplied by Lupin Pharmaceuticals Ltd, Pune (India), as gift sample. Methanol of HPLC grade (Merck, Mumbai, India), HPLC grade water (Mylochem, India).

Instrumentation

Chromatographic measurements were made on a RP-HPLC Shimaduz (Japan), P series model, using a Shimaduz chromatographic software (LAB solution). Absorbance spectra were recorded using UV- double beam spectroscopy (Shimaduz, Japan), mobile phase degassed using Branson sonicator (USA). Experimental design, data analysis were performed using Design- Expert 13 software.

Chromatographic condition

The separation was carried on C-18 column (150×3.6mm, 5um particle size) with mobile phase of 70:30 v/v) degassed in a sonicator for 10 min and filtered through 0.2µ membrane filter before use. Peak was obtained at 2.48 min with flow rate of 1ml/min. Prior to the injection of drug solution, column was equilibrated with mobile phase flowing through the system. Detection was done using PDA at 267nm. A good separation and peak symmetry for the drug were obtained with the above chromatographic condition. The HPLC method for Sitagliptin Phosphate was optimized for various parameters, mobile phase and flow rate two variables and three levels using 3² factorial design.

Preparation of reference standard solution

Transfer 10 mg of Sitagliptin Phosphate working standard into a 100ml volumetric flask, dissolve in Methanol and Water mixture in the ratio of 70:30 v/v. Take 1ml of the above solution and dilute up to 10 ml in volumetric flask with above solvent mixtrue. The resulting solution contains 10µg/mL of Sitagliptin Phosphate as working standard solutions.

Wavelength selection for analysis:

Appropriate dilutions of Sitagliptin Phosphate were prepared and sample were scanned using UV spectrometer in the range of 200 to 400 nm. An absorbance maximum was obtained at 267nm.

Method development of sitagliptin phosphate by RP- HPLC:

Initial trial and error for obtaining satisfactory method were carried out. Trial no. 01, 02, 03 mobile phase were (50:50 ml/min), (90:10 ml/min), (70:30 ml/min) respectively. Chromatographic separation was carried out at flow rate 1ml/min, C-18 column.

RP-HPLC ANALYTICAL METHOD OPTIMIZATION BY QbD

Selection of Quality Target Product Profile

The Quality Target Product Profile (QTPP) is crucial for determining the factors that impact the quality attributes of the drug. In this case, the QTPP identified the key performance indicators for the HPLC method as retention time, theoretical plate count, USP tailing and peak asymmetry, which are essential for ensuring the accuracy and precision of the analysis.^[10]

Critical Quality Attribute (CQA):

From the software generated result the critical factors which affect the tailing factor and retention time was determined. Factors such as flow rate and mobile phase concentration were found to be critical.

Method design:

The screening was done by 3² factorial design using Design Expert 13 software.^[11-12] The two factors selected are flow rate and ratio of mobile phase and water and three levels selected are low, medium and high. The total 09 runs were obtained, the response for the design was tailing factor and retention time. The results of the 09 runs were then put in the design to get the response. Experimental design its variables and levels are shown in table no.01.

Table 01 : Experimental design (3²) variables and levels

Table 02 : Factorial design runs for sitagliptin phosphate

Analytical method validation:

The optimized chromatographic method was validated as per the International Conference on Harmonization (ICH) Q2 (R1) guidelines. For linearity, range, precision, robustness and LOD and LOQ, standard solution of 10µg/ml of Sitagliptin Phosphate was prepared by diluting and mixing drug with methanol and water (70:30 v/v).

Linearity:

As per ICH guidelines the linearity of analytical procedure is its ability (within in a given range) to obtain test results which are directly proportional to the concentration of analyte in sample. Standard calibration curves were prepared with five different concentrations by making serial volume to volume dilution of stock solution with methanol and water (70- 30v/v) over the range of 10, 20, 30, 40 and 50µg/ml. Three replicate injections of each concentration were made to determine the linearity of Sitagliptin Phosphate over the concentration range. Linear concentration curves of peak area verses drug concentration were plotted sing linear least squares regression and evaluated for linearity.^[13]

LOD and LOQ:

The LOD and LOQ of Sitagliptin Phosphate by the proposed method were determined wing calibration standards. LOD and LOQ values were calculated as 3.3σ/S and 10 σ /S, where S is the slope of the calibration curve and a is the standard deviation of Y-intercept.^[13]

Robustness:

The robustness of the method was evaluated by analyzing the system suitability parameters after flow rate (±0.1ml/min), and wavelength (±2nm). A working standard of 10 μg/mL was used during the experiment and followed by calculation of the mean percent recovery and % RSD.

Precision:

The reproducibility of proposed method was determined by performing API assay at different time intervals (3hour interval) on same day (Intra-day precision) and on three different days (Inter-day precision) at concentration Sitagliptin Phosphate at the concentration 20, 30 and 40 µg/ml.

Accuracy:

Accuracy is the nearness of a measured value to the true or accepted value. Accuracy indicates the deviation between the mean value found and the true value. It is determined by applying the method to samples to which known amounts of analyte have been added. These should be analysed against standard and blank solutions so ensure that no interference exists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay. It may often be expressed as the recovery by the assay of known, added amounts of analyte.

Assay of the sitagliptin phosphate tablets:

Twenty tablets were weighed and crushed to make to a fine powder. A portion of the powdered tablets equivalent to 100 mg was accurately weighed into 100 ml grade volumetric flask and 30 mL methanol was added. The volumetric flask was sonicated for 20 min to ensure complete dissolution of the sitagliptin phosphate then the solution was made up to the volume with methanol. Solution was filtered through a 0.45 µm nylon filter and completed as previously mentioned under preparation of stock and standard solution for RP-HPLC.^[4]

RESULTS and DISCUSSION

The separation was carried on C-18 column (150×3.6mm, 5um particle size) with mobile phase of 70:30 v/v) degassed in a sonicator for 10 min and filtered through 0.2µ membrane filter before use. Peak was obtained at 2.48 min with flow rate of 1ml/min. Prior to the injection of drug solution, column was equilibrated with mobile phase flowing through the system. Detection was done using PDA at 267nm. Using 3² factorial desing.

Trial no.1: Peak shape is sharp but max intensity is not increasing as increase in concentration. Peak shape is proper and tailing is observed is within the range i.e.0.74. The retention time is 2.84 min. Area is found to be 157467. Mobile phase concentration affects the intensity of the peak, chromatogram shown in fig no. 02.

Trial no.02: Peak shape is sharp with tailing factor 1.797 and having multiple peaks. Peak shape is proper and tailing is observed is within the range i.e.1.797. The retention time is 1.705 min. Area is found to be 588143. Change in mobile phase composition to (90:10) affects the tailing factor, chromatogram shown in fig no. 02.

Trial no.03 Peak shape is sharp and proper with tailing factor 1.385. Hence method is suitable. Peak shape is proper and tailing is observed is within the range i.e.1.385. The retention time is 2.478 min. Area is found to be 441390. Mobile phase composition of (70:30) is suitable of experiment, chromatogram shown in fig no. 02.

Figure. 02: Chromatograms of trial no. 01, 02, 03.

RP-HPLC ANALYTICAL METHOD OPTIMIZATION BY QBD:

Method design:

The results of the 09 runs as per the 3² factorial design were then put in the design to get the best response. The best response was found to be run no.3 with flow rate of 1ml/min and mobile phase ratio of 70:30 (methanol: water) as shown in table no.03

Optimized condition: For both responses retention time and tailing factor. Mobile phase composition methanol: water (70:30 ml/min), flow rate 1ml/min, wavelength 267, injection volume 20µl. The optimized chromatogram is shown in fig no. 5.2 with retention time of 2.478 and tailing factor of 1.385. RP-HPLC optimized condition chromatogram obtained by 3² factorial design.

Table no. 0.3 – Factorial design runs for sitagliptin phosphate

Response 1: Retention Time

Response surface plot was studied to visualize effect of factor so as to develop design space for robust method 3D graph are given below in Fig.no.03. From the 3D graph it can be observed that as the ratio of methanol is decreased the retention time get increases and if flow rate decreased the retention time increases. Hence from the 3D surface plot it can be concluded that the optimized HPLC chromatographic condition for analysis of sitagliptin phosphate is mobile phase in the ratio of methanol:water (70:30) and flow rate is 1ml/min.

Figure 03- Response surface (3D) plot showing the effect of Flow rate (A) and mobile phase (B) on retention time.

Response 2: Tailing Factor

Response surface plot was studied to visualize effect of factor so as to develop design space for robust method 3D graph are given below in Fig.no.04. From the 3D graph it can be observed that as the ratio of methanol is increased the USP tailing get increases and if flow rate decreased the USP tailing increases. Hence from the 3D surface plot it can be concluded that the optimized HPLC chromatographic condition for analysis of sitagliptin phosphate is mobile phase in the ratio of methanol:water (70:30) and flow rate is 1ml/min.

Figure 04- Response surface (3D) plot showing the effect of Flow rate (A) and mobile phase (B) on USP tailing factor.

Analytical method validation:

Linearity:

The linearity of the developed method was assured by a linearity plot for concentrations ranging from 10-180 μg/ml. The calibration curve (fig.no.05) was constructed using linear regression analysis by plotting the peak area against the concentration. The correlation coefficient was found to be R2 = 0.9991 which reveals the excellent relationship between peak area and the concentration. %RSD was found to be 5.61% which is within the specification that should be less than 10%.

Figure 05 - Calibration curve of Sitagliptin Phosphate

LOD and LOQ:

The LOD and LOQ values were observed to be 0.851 and 2.580 μg/ml, respectively.

Robustness:

After making minute changes in each component, like the flow rate and wavelength. No considerable changes were observed in the peak area. The insensitivity against premeditated minor modification in the method parameters shows the robustness of the QbD developed method. Change in wavelength i.e, ±2nm and change in flow rate i.e ± 0.1 ml/min.

Precision:

The precision is the degree of repeatability of an analytical method under normal operational conditions. It was studied with respect to both repeatability (intraday) and intermediate (intraday) precision studies. The intra and inter-day variability or precision data are summarized in (Table no.0.4, 05) and % RSD found for intra and inter-day precision was within the specification. Considerable changes were not observed in the peak area.^[14]

Table 04- Intra-day precision parameters.

Table 05- Inter-day precision parameters.

Accuracy:

Accuracy of the analytical method was performed at 80, 100, and 120 levels of the standard concentration of 30 μg/mL which showed recovery between 98.90 and 102.17 %. Data shown in (Table No. 06) indicate that the developed method has a high level of accuracy with a mean % RSD of 1.62739%.^[15]

Table 06–Method accuracy.

Assay of the sitagliptin phosphate tablets:

The average area of three injections was found to be 296493, and the assay percentage was 98.83, shown in (table no. 5.9) and all the three chromatograms are shown in fig. no. 5.7. The retention time was found to be between 2.97 to 2.98 and tailing factor and peak area was found to be between 1.6 to 1.831 and 2899894 to 2998262 respectively.

Summary and Conclusion:

In present study the analytical method was developed by QbD method and validated as per ICH guidelines for determination of Sitagliptin phosphate. In this project, as per our objective RP-HPLC method was developed by implementing 3² factorial design with mobile phase methanol and water (70:30 %v/v). The flow rate optimized to 1.0 mL /min and the maximum wavelength was observed at 267 nm. The retention time for sitagliptin phosphate was found to be 2.478 min and tailing factor was found to be 1.385. The study was done by using 3² factorial design. In this study interaction of 2 factors were selected as dependent variables i.e mobile phase composition and flow rate and independent variables i.e retention time and tailing factor were evaluated through the runs in design. Effect of such critical process parameter on critical quality attribute of the method was studied.

Optimum conditions were obtained. Replicates of run having optimized condition were taken to confirm the predicted response with actual response. The Quality by Design approach has been successfully used to develop RP-HPLC method for Sitagliptin phosphate. All key aspect of QbD were tried to be implemented in study. Systematic approach was utilized to develop an efficient and robust method which includes beginning with determination of analytical analytical target profile characteristic and design of experiment. Two factors that were determined to significantly affect the peaks were then analysed to determine their interactions and linear effects with the least possible runs by using 3² factorial design. The statistical analysis was also performed by determining analysis of variance (ANOVA) and its was revealed that the experiment model applied was significant. The run having flow rate 1ml/min and mobile phase (70:30) was selected replicates of run having optimized condition were taken to authorise the predicted response with authentic response. The proposed RP- HPLC method using QbD approach provides simple accurate, and reproducible quality analysis for determination of Sitagliptin phosphate.

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