Method Development and Validation of Desidustat in Solid Dosage Form by RP-HPLC Method

Method Development 

Different trial chromatographic conditions were evaluated to obtain a sharp, symmetrical peak with acceptable system suitability parameters. The final conditions were selected based on retention time, peak shape, resolution, and reproducibility. The method was then optimized for routine assay of Desidustat in tablet dosage form.  

Method Validation 

The method was validated according to ICH Q2(R1) guidelines for the following parameters: 

•      Specificity.  

•      Linearity.  

•      Range.  

•      Accuracy.  

•      Precision.  

•      Limit of detection.  

•      Limit of quantitation.  

•      Robustness.  

•      System suitability. 

LINEARITY AND RANGE: 

Accuracy levels details:  

Refer Following table for each sample:  

Precision (Repeatability) Sample details are as follows:  

Intermediate Precision Sample details are as follows:  

Sample Preparation 

Tablet samples were prepared according to the procedure described in the thesis. The sample solution was filtered and analyzed under the optimized chromatographic conditions. Placebo and blank solutions were also prepared for specificity assessment.  

 RESULTS System Suitability 

The system suitability parameters met the acceptance criteria. The method provided a satisfactory retention time, good peak symmetry, and acceptable chromatographic performance for routine analysis. The thesis acceptance limits for system suitability included theoretical plates greater than 2000, capacity factor less than 1, resolution greater than 1.5, tailing factor less than 2, and %RSD less than 2.  

Specificity 

Blank and placebo chromatograms showed no interference at the retention time of Desidustat, demonstrating the specificity of the method. The analyte peak was well resolved and free from co-eluting peaks.  

Linearity 

The detector response was linear in the concentration range of 10–50 µg/mL. The calibration curve showed a correlation coefficient greater than 0.999, indicating excellent linearity.  

Accuracy 

Accuracy was evaluated at 50%, 100%, and 150% levels. The percentage recovery values were within acceptable limits, confirming that the method is accurate for tablet analysis.  

Precision 

Repeatability, intra-day precision, and inter-day precision studies showed %RSD values below 2.0%, indicating that the method is precise and reproducible.  

Robustness 

The method remained unaffected by small deliberate variations in flow rate, mobile phase composition, and temperature, demonstrating robustness.  

Assay 

The assay of the marketed tablet formulation showed satisfactory recovery and confirmed the suitability of the proposed method for routine quality control analysis.  

Solubility study Desidustat  

Fig.  UV spectrum of Solution 1 as blank

Fig. Typical chromatogram of Trial 1

Fig. Typical chromatogram of Trial 2  Trial

3:

Fig. Typical chromatogram of Trial 3  Trial

4:

Fig. Typical chromatogram of Trial 4  Conclusion: From the observations of trials first to four, it was concluded that chromatographic conditions in trial four gives better peak, good retention time and tailing factor therefore chromatographic conditions in trial four was used for method validation   Table. Optimized Chromatographic Conditions  

8.4. System suitability test  

Fig. Typical chromatogram Standard solution 1 of system suitability solution

Analysis of Marketed Test samples (Assay)  

a) Rytstat100mg Tablet: Weight of 20 tablets = 6.0480gm  

Average weight of tablet = 6.0480 /20 =0.3024gm = 302.4mg 

Table. Assay results of Rytstat 100Tablet

Fig. Typical chromatogram Tablet sample_1  

1) % Assay found should be in the range of 90-110%.  

Data interpretation:   

From the above results, it can be concluded that the assay result is within the limit for selected marketed test sample and sample can be used for validation.  

8.6. VALIDATION OF RP-HPLC METHOD  1) FILTRATION STUDY:  

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on tablet test sample.  

Table. Results of Filter study

Fig.No. Typical chromatogram of unfiltered sample

Fig. Typical chromatogram of sample filtered through 0.45µ PVDF filter

Fig. Typical chromatogram of sample filtered through 0.45µ Nylon filter Acceptance criteria:% Absolute difference of filtered samples NMT 2.0 w.r.t. Unfiltered sample.

Table. Results of Solution stability

Fig. Typical chromatogram of Standard solution Initial

Fig. Typical chromatogram of Test solution After 24 Hrs

Table. Results of Specificity

 Chromatograms:   

Fig. Typical chromatogram of Placebo solution Acceptance criteria:

Blank: There should be no Interference at R.T. of Desidustat  

Placebo: There should be no Interference at R.T. of Desidustat  

Data interpretation: Blank and placebo were not having interference at R.T. of Desidustat. Hence developed chromatographic method passed the criteria for specificity. 

Table. Linearity Data for Desidustat

Fig. Calibration curve of Desidustat

Table. Data of linearity of  Desidustat:

Fig. Typical chromatogram of Linearity 50%

Fig. No. 47 Typical chromatogram of Linearity 100%

Fig.  Typical chromatogram of Linearity 150% Conclusion:

From the calibration curve it was concluded that the Desidustat shows linear response in the range of 5.0015.00μg/ml. The Regression value was found well within the limit.  

 Limit of Detection (LOD) and Limit of Quantitation (LOQ):  

σ = 14985.08 (Residual standard deviation of a regression line)  

S = 717915.12(Slope)  

Detection limit (LOD):  

LOD = 3.3 σ / S  

LOD = 3.3 x 14985.08/ 717915.12  

LOD = 0.07µg/mL  

Quantitation limit (LOQ):  

LOQ = 10 σ / S  

LOQ = 10 x 14985.08/ 717915.12  

LOQ = 0.21µg/mL  

 ACCURACY (RECOVERY):  

Table. Result and statistical data of Accuracy of Desidustat

Overall Recovery: 100.30 %

% RSD for Overall Recovery: 0.513  

Fig.  Typical chromatogram of sample 1 of Accuracy 50%

Fig. Typical chromatogram of sample 1 of Accuracy 100%

Fig. Typical chromatogram of sample 1 of Accuracy 150% Acceptance criteria:

% Recovery for each level and overall recovery: 98.0 to 102.0%  

% RSD for each level and overall recovery: NMT 2.0  

Data interpretation: Recovery of analytical procedure was found well within acceptance criteria at all 3 levels. % Recovery not get hampered by changed in analyte concentration.  

 PRECISION   

Precision of an analytical method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample. Precision of an analytical method is usually expressed as standard deviation or relative standard deviation. Precision was performed on Test sample.   

Table No.Result of Intra- day and Inter- Day Precision for Desidustat test sample   

Chromatograms:  

Fig. Typical chromatogram of Repeatability precision (Sample 1)

Fig. Typical chromatogram of Inter-day precision (Sample 1)

Acceptance criteria:   

% Assay: % Assay value for each sample (Individual sample) and mean assay value for precision (6 samples), mean assay value intermediate precision (6 samples), and mean assay value for precision plus intermediate precision sample (12 samples): 90-110%  

% RSD: % RSD for precision study samples (6 samples), Intermediate precision study samples (6 samples) and precision plus intermediate precision sample (12 samples): NMT 2.0  Data interpretation: 

% Assay and % RSD was found well within acceptance limit and hence method is precise (Reproducible).  

 ROBUSTNESS:  

The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.  

Table. Result of Robustness study:

Chromatograms:   

Fig. Typical chromatogram of Standard +2 NM

Fig. Typical chromatogram of Standard -2 NM  C.

Change in Flow rate by + 10% (1.1 mL/min)

Fig. Typical chromatogram of Standard +10% F.R.

D.        Change in Flow rate by - 10% (0.9 mL/min)

Fig. Typical chromatogram of Standard -10% F.R.

E.         Change in Column Oven temperature by +2°C:

Fig. Typical chromatogram of Standard +2°C C.O.T.

F.         Change in Column Oven temperature by -2°C:

Fig. Typical chromatogram of Standard -2ºC C.O.T.