RP-HPLC Method Development and Validation for Simultaneous Estimation of Dapagliflozin and Sacubitril-Valsartan Sodium Complex Salt in Synthetic Mixture

MATERIALS AND METHODS:

5.1 Instruments:

Table 5.1: List of  Instrumnets

5.2 Chemicals & Reagents

Table 5.2 :  List of Chemical and Reagents

5.3 Identification of API:

The identity of the drug substances was confirmed through comparison of their infrared (IR) spectra with         corresponding reference         standards, complemented       by             evaluation       of physicochemical parameters such as melting point and solubility.

5.4.2 Determination of Melting Point

The prepared sample was introduced into a capillary tube and placed in a digital melting point apparatus. The temperature at which the sample transitioned from solid to liquid was recorded as its melting point.

5.4.3 IR Spectra of Drug:

•           Identification of DAP, SAC and VAL was carried out using infrared spectroscopy by comparing their spectra with those of standard reference materials.

•           For sample preparation, the drug substances were finely powdered to ensure particle size was sufficiently small to allow effective transmission of IR radiation. The powdered samples were then dispersed in a suitable medium (e.g., Nujol) and placed between KBr plates for analysis. Each sample was examined individually.

•           The characteristic absorption peaks obtained were recorded and interpreted from the IR spectra.

5.5 HPLC Method Development:

The development of a reliable and sensitive RP-HPLC method involved systematic optimization of key chromatographic parameters. The following factors were investigated:

5.5.1    Selection of Column:

The chromatographic behavior of DAP, SAC and VAL was evaluated using a Cosmosil C18 column (250 mm × 4.6 mm, 5 µm), selected based on their polarity and compatibility with reversed-phase conditions.

5.5.2    Selection of Diluents:

DAP, SAC and VAL analytes exhibited good solubility in methanol; therefore, methanol was chosen as the diluent for preparation of all standard and sample solutions.

5.5.3    Selection of Wavelength:

The optimal detection wavelength was determined by scanning the drug solutions over a range of 200–400 nm using a UV spectrophotometer. The wavelength providing maximum absorbance was identified as 228 nm and selected for analysis.

5.5.4    Selection of Mobile Phase:

The composition of the mobile phase was optimized by considering factors such as solvent type, buffer selection, pH, and their respective ratios. The physicochemical properties of the analytes, including solubility and molecular characteristics, were also taken into account. Buffers were incorporated to maintain a stable pH environment during analysis.

It is generally observed that acidic compounds exhibit improved retention under lower pH conditions, whereas basic compounds show enhanced retention at higher pH values. Various mobile phase compositions were evaluated through iterative trials, and system suitability parameters such as theoretical plate count, tailing factor, peak symmetry, and resolution were monitored to achieve optimal chromatographic performance.

5.5.5    Selection of pH:

The pH of the mobile phase plays a critical role in analyte separation. For most HPLC applications, an effective pH range lies between 2 and 8, within which optimal separation can typically be achieved.

5.6 Preparation of Solutions:

5.6.1    DAP Stock solution (50 µm/ml): Accurately weighed 50 mg of DAP working standard was transferred into a 100 mL volumetric flask. Approximately 50 mL of methanol was added, and the solution was sonicated for 5 minutes to ensure complete dissolution. The volume was then adjusted to the mark with methanol. A further dilution was prepared by transferring 5 mL of this solution into a 50 mL volumetric flask and diluting to volume with methanol.

5.6.2    SAC stock solution (970 µm/ml): About 970 mg of sacubitril was accurately weighed and transferred into a 100 mL volumetric flask. Methanol was added followed by sonication for 5 minutes. The final volume was adjusted with methanol. A further dilution was prepared by transferring 10 mL of this solution into a 100 mL volumetric flask and diluting to volume with methanol.

5.6.3    VAL stock solution (1030 µm/ml): About 1030 mg of Valsartan was accurately weighed and transferred into a 100 mL volumetric flask. Methanol was added followed by sonication for 5 minutes. The final volume was adjusted with methanol. A further dilution was prepared by transferring 10 mL of this solution into a 100 mL volumetric flask and diluting to volume with methanol.

5.6.3    Mixed standard solution (5 µm/ml DAP + 97 µm/ml SAC+ 103 µm/ml VAL): Aliquots of 1 mL each from the respective stock solutions of DAP, SAC and VAL were transferred into a 10 mL volumetric flask. The volume was made up to the mark with methanol, followed by sonication for 5 minutes to ensure uniform mixing.

5.6.4    Preparation of Synthetic mixture:SAMPLE PREPARATION:

Synthetic mixture was prepared for 10 tablets.[39-40]

Table No. 5.3 : Preparation of Synthetic mixture

Sample Stock: Synthetic mixture 360 mg (equivalent to 5 mg DAP + 97 mg SAC + 103 MG VAL) was weighed on butter paper. Powder was then transferred to 100 mL volumetric flask. To this 30 mL methanol was added and active contents were extracted in sonication for 15 minutes. The flask was allowed to cool down on room temperature and then the volume was made with methanol. The solution was then filtered through 0.45 µ Whatman PVDF syringe filter.

Sample (05 µm/ml DAP + 97 µm/ml SAC + 103 µm/ml VAL  ): An aliquot of 1 mL of the filtered solution was transferred into a 10 mL volumetric flask and diluted to volume with the selected diluent to obtain the final sample solution.

5.7 Trials of Method Development:

Table 5.4 : List of MP various trials for HPLC

Table 5.5 Optimization of RP-HPLC chromatographic condition

5.8  System Suitability Criteria(n=5) :

•           System suitability was assessed by performing five consecutive injections of a standard solution containing DAP (05 µg/mL), SAC (97 µg/mL) and VAL (103 µg/mL) under optimized chromatographic conditions.

•           The chromatographic system was considered acceptable when the resolution between adjacent peaks exceeded 2.

•           The efficiency of the column was evaluated through theoretical plate count, which was required to be greater than 2000, while peak symmetry was assessed using the asymmetry factor, expected to be below 1.5.

•           The capacity factor (k′) was maintained within the range of 1 to 10 to ensure adequate retention.

•           The selectivity or separation factor (α) was required to be greater than 1, indicating effective differentiation between analyte peaks.

5.9  Method Validation:

5.9.1    The developed RP-HPLC method was validated in accordance with ICH Q2 (R2) guidelines by evaluating parameters including linearity, specificity, accuracy, precision, detection limit (DL), quantification limit (QL), and robustness.

5.9.2    Linearity (n=5):

Calibration standards were prepared by transferring appropriate aliquots from the standard stock solution (SSS) into a series of 10 mL volumetric flasks and diluting to volume with methanol. Concentration ranges of 5–15 µg/mL for DAP and 100–300 µg/mL for SAC and VAL were obtained. Calibration curves were constructed by plotting peak area against corresponding concentrations for both analytes.

5.9.3    Specificity (n=6):

Specificity was evaluated by analyzing diluent, placebo (excipients), and standard solutions to assess potential interference at the retention times of the analytes. Standard solutions containing DAP (05 µg/mL), sacubitril (97 µg/mL) and valsartan (103 µg/mL) were analyzed. Additionally, placebo samples spiked with known amounts of analytes were examined to determine any interference from excipients or related impurities.

5.9.4    Accuracy (n=3):

•           Accuracy of the method was verified through recovery studies using the standard addition technique at three concentration levels: 50%, 100%, and 150%.

•           A placebo mixture containing all excipients (excluding active drugs) was prepared and treated similarly to the formulation. An amount equivalent to one tablet weight was transferred into a 100 mL volumetric flask, dissolved in methanol with sonication, diluted to volume, and filtered to obtain placebo stock solution.

50% Level:

•           Appropriate quantities of DAP (5 mg) and sacubitril–valsartan (100 mg) were added to the placebo solution, dissolved, and diluted to obtain intermediate concentrations. A further dilution yielded final concentrations of 5 µg/mL and 100 µg/mL, respectively. The solution was filtered and analyzed in triplicate.

100% Level:

•           Standard additions equivalent to 10 mg DAP and 25 mg sacubitril–valsartan were prepared and processed similarly to obtain final concentrations of 10 µg/mL and 200 µg/mL, respectively. Analysis was performed in triplicate.

150% Level:

•           Standard additions of 15 mg DAP and 30 mg sacubitril–valsartan were used to prepare final concentrations of 15 µg/mL and 300 µg/mL, respectively. Each sample was analyzed in triplicate.

5.9.5 Precision:

I.         Repeatability (n=6):

•           A working solution containing DAP (10 µg/mL) and sacubitril–valsartan (200 µg/mL) was prepared from the stock solution. Six replicate injections were performed, and the peak areas were recorded. Precision was expressed as % relative standard deviation (%RSD).

II.        Intraday Precision (n=3):

•           Samples at 50%, 100%, and 150% levels (5, 10, and 15 µg/mL for DAP; 100, 200, and 300 µg/mL for sacubitril–valsartan) were analyzed three times within the same day. The %RSD was calculated to evaluate repeatability within a single day.

III.      Interday Precision (n=3):

•           The same concentration levels were analyzed on different days to assess variability over time. %RSD values were calculated to determine method consistency across days.

5.9.6 DL and QL:

•           The DL and QL for both analytes were estimated using statistical calculations based on the standard deviation of the response and slope of the calibration curve.

5.9.7 Robustness :

•           Robustness was assessed by introducing small, deliberate variations in chromatographic conditions such as flow rate, detection wavelength, column temperature, and mobile phase composition. The effect of these variations on system performance was evaluated to determine method reliability.

5.9.8 Assay of synthetic mixture (n=6):

•           For assay determination, accurately weighed quantities of DAP (10 mg) and SVN (200 mg), along with appropriate excipients, were transferred into a 100 mL volumetric flask. The mixture was dissolved in diluent with sonication and diluted to volume.

•           The solution was filtered through a 0.45 µm membrane filter, and an aliquot of 1 mL was further diluted to 10 mL with mobile phase to obtain final concentrations of 10 µg/mL for DAP and 200 µg/mL for sacubitril–valsartan. This solution was used for assay analysis.

SUMMARY AND CONCLUSION

•           A reverse-phase high-performance liquid chromatography (RP-HPLC) method was successfully developed for the simultaneous determination of DAP and sacubitril– valsartan.

•           The proposed analytical procedure enabled effective separation of both analytes and was subsequently validated in accordance with ICH Q2 (R2) guidelines.

Table 7.1 Summary of Validation Parameters of RP-HPLC method