INTRODUCTION:^1,2,3

Vadadustat is used to treat anemia caused by chronic kidney disease (CKD) in patients who have been on dialysis for at least 3 months. This medicine works by increasing erythropoietin (protein) to help the body make more red blood cells. It is also used to lessen or avoid the need for blood transfusion.

Category:

Vadadustat is a hypoxia-inducible factor prolyl hydroxylase (HIF PH) inhibitor.

It's a class of drugs that stabilize HIF and stimulates the production of red blood cells and erythropoietin.

Structure:

Figure 1: Structure of Vadadustat

IUPAC Name:

2-[[5-(3-chlorophenyl)-3-hydroxypyridine-2-carbonyl]amino]acetic acid

Chemical formula: C₁₄H₁₁ClN₂O₄

Molecular weight: 306.70 g·mol⁻¹

MATERIALS AND METHODS:

Table: 1: Instruments used

Name	Model	Manufacturer
HPLC	Alliance	Waters
pH meter	pH700	Eutech
Weighing balance	BSA224S-CW	Sartouris
UV/VIS spectrophotometer	UV-1700	Shimadzu
Pipettes, beakers and Burettes	Class-A	Borosil
Ultra sonicator	UCA 701	Unichrome
Pump	Isocratic model	Waters

Table 2: Reagents used

Name	Grade	Manufacturer
Acetonitrile	HPLC Grade	Merck
Water (Milli Q)	HPLC Grade	In house production
Formic acid	AR Grade	Merck

Table 3: Optimized chromatographic conditions

PARAMETERS	OBSERVATION
Instrument used	Waters Alliance e-2695 HPLC
Injection volume	10µl
Mobile Phase	Acetonitrile and 0.1% Formic acid (20:80)
Column	Inertsil ODS, (150x4.6mm, 3.5µ)
Detection Wave Length	234 nm
Flow Rate	1 mL/min
Runtime	5 min
Temperature	Ambient (25° C)
Mode of separation	Isocratic mode

Mobile Phase:⁵

Mobile phase was prepared by mixing 0.1% Formic acid and Acetonitrile taken in the ratio 80:20. It was filtered through 0.45μ membrane filter to remove the impurities which may interfere in the final chromatogram.

Preparation of 0.1% Formic acid Buffer:^6,7,8

1ml of Formic acid is dissolved in 1litre of HPLC grade water. Filter through 0.45µ nylon filter.

RESULTS AND DISCUSSION:

System suitability:

All the system suitability parameters were within the range and satisfactory as per ICH guidelines⁵

Table 4: System suitability parameters for Vadadustat

S. No.	Parameter	Vadadustat
1	Retention time	2.855
2	Plate count	8053
3	Tailing factor	1.17
4	%RSD	0.19

Analytical Method Validation (HPLC):^4-10

The method was validated for its linearity range, accuracy, precision, and specificity. Method validation was carried out as per ICH guidelines.

Specificity:

Figure 2: Chromatogram of blank

Figure 3: Chromatogram of placebo

Figure 4: Optimized chromatogram

Discussion: Retention times of Vadadustat were 2.855 min. We did not find and interfering peaks in blank and placebo at retention times of these drugs in this method. So, this method was said to be specific.

PRECISION:

System Precision:

Table 5: System precision data of Vadadustat

S. No.	Concentration Vadadustat (µg/ml)	Area of Vadadustat
1.	150	3461273
2.	150	3449174
3.	150	3465906
4.	150	3455689
5.	150	3463622
6.	150	3466453
Mean	3460353
S.D	6727.580
%RSD	0.19

Discussion: From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for the drug. % RSD obtained as 0.19 for Vadadustat. As the limit of Precision was less than “2” the system precision was passed in this method.

Linearity:

Table 6: Results of linearity for Vadadustat

S. No.	Vadadustat
S. No.	Conc.(µg/ml)	Peak area
1	37.50	892684
2	75.00	1769102
3	112.50	2704947
4	150.00	3458539
5	187.50	4234065
6	225.00	5124103
Regression equation	y=22163.82x+53579.86
Slope	22163.82
Intercept	53579.86
R²	0.99953

Figure 5: Calibration curve for Vadadustat

Accuracy:

Table 7: Accuracy results of Vadadustat by HPLC method

% Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean %Recovery
50%	1716487	7.5	7.44	99.2	99.9
	1724096	7.5	7.47	99.6
	1743432	7.5	7.56	100.8
100%	3465234	15.0	15.02	100.1	99.6
	3430265	15.0	14.87	99.1
	3448796	15.0	14.95	99.7
150%	5172130	22.5	22.42	99.6	99.7
	5198521	22.5	22.53	100.1
	5148103	22.5	22.32	99.2

Discussion: Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean % Recovery was obtained as 99.7% for Vadadustat.

LOD and LOQ (µg/ml):

LOD and LOQ were determined by using the formula based on the standard deviation of the response and the slope. LOD and LOQ were calculated by using equations, LOD = 3.3 × σ / s and LOQ=10×σ/S., The results were presented in Table 10.

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

Table 8: Data table of LOD and LOQ

Name of drug	LOD (µg/ml)	S/N	LOQ (µg/ml)	S/N
Vadadustat	0.45	3	1.50	10

CONCLUSION:

The developed HPLC method for the estimation of selected drug is simple, rapid, accurate, precise, robust and economical. The mobile phase and solvents are simple to prepare and economical, reliable, sensitive and less time consuming.

The sample recoveries were in good agreement with their respective label claims and they suggested noninterference of formulation excipients in the estimation and can be used in laboratories for the routine analysis of selected drugs.

Since the system validation parameters of HPLC method used for estimation of selected drug in pure and have shown satisfactory, accurate and reproducible results (without any interference of excipients) as well, it is deduced that the simple and short proposed methods be most useful for analysis purpose.

The present work concluded that stability indicating assay method by RP-HPLC was simple, accurate, precise, and specific and has no interference with the placebo and degradation products. Hence these can be used for routine analysis of Vadadustat.

REFERENCES:

1. Pergola PE, Spinowitz BS, Hartman CS, Maroni BJ, Haase VH. Vadadustat, a novel oral HIF prolyl hydroxylase inhibitor, provides effective anemia treatment in nondialysis-dependent chronic kidney disease. Kidney Int. 2016; 90(5): 1115–1122.

2. Martin ER, Smith MT, Maroni BJ, Zuraw QC, deGoma EM. Clinical trial of vadadustat in patients with anemia secondary to CKD. N Engl J Med. 2021; 384(17): 1601–1612.

3. Choi D, Park S, Lee J, Kim M, Park H, et al. Pharmacokinetics and safety of vadadustat in healthy subjects. Clin Transl Sci. 2019; 12(4): 370–376.

4. Blessy M, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating methods—A critical review. J Pharm Anal. 2014; 4(3): 159–165.

5. International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use. ICH Q2(R1): Validation of Analytical Procedures: Text and Methodology. Geneva: ICH; 2005.

6. Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. 3rd ed. Hoboken, NJ: John Wiley & Sons; 2010.

7. Dong MW. Modern HPLC for Practicing Scientists. Hoboken, NJ: John Wiley and Sons; 2006.

8. Meyer VR. Practical High-Performance Liquid Chromatography. 5th ed. Chichester: Wiley; 2010.

9. USFDA. Bioanalytical Method Validation Guidance for Industry. U.S. Food and Drug Administration; 2018.

10. European Medicines Agency (EMA). Guideline on Bioanalytical Method Validation. EMA, 2011.

Received on 04.08.2025 Revised on 08.12.2025

Accepted on 04.03.2026 Published on 16.04.2026

Available online from April 18, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(2):119-122.

DOI: 10.52711/2231-5675.2026.00018

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