Development and Validation of an Eco-Friendly RP-HPLC Method for the Quantitative Estimation of Desidustat in Human Plasma

Gauri Vispute¹*, Dr. Upasana Yadav²

1.      Department of Pharmaceutical Analysis, Parul Institute of Pharmacy, Parul University, Vadodara, Gujarat, India.

2.      Department of Pharmaceutical Chemistry, Parul Institute of Pharmacy, Parul University, Vadodara, Gujarat, India.

Correspondence: gaurivispute18@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5802     

INTRODUCTION

Bioanalysis is a sub-discipline of analytical chemistry that is concerned with the quantitative analysis of xenobiotics and endogenous materials in biological matrices (1). Bioanalytical data are invaluable throughout the whole drug development process, starting with the optimization of the lead compound and continuing into late-stage clinical trials and post-marketing surveillance of the drug (1). These studies offer a basic knowledge of the pharmacokinetics of a drug through the characterization of the absorption, distribution, metabolism, and excretion of the drug (1). Moreover, bioanalysis plays a vital role in measuring toxicokinetics, bioavailability, and bioequivalence (BA/BE) and therapeutic drug monitoring to prevent toxicity in drugs with narrow therapeutic indices (2,3). All bioanalytical procedures should meet high international standards of regulatory standards to guarantee the scientific integrity of clinical data, including the ICH M10 guidelines (4).

Traditionally, renal anemia was treated with the help of iron supplementation and injectable Erythropoiesis-Stimulating Agents(9). Nevertheless, ESAs have been linked to the risks of hypertension and cardiovascular complications when used in supraphysiological doses (10) . Desidustat is a novel, second-generation, small-molecule Hypoxia-Inducible Factor Prolyl Hydroxylase inhibitor (11). Desidustat, commercially marketed as Oxemia™ and also known by the developmental codes ZYAN1 and BCP29692, is a novel therapeutic agent developed for the management of anemia. Chemically, Desidustat possesses the molecular formula C₁₆H₁₆N₂O₆ with an average molecular weight of approximately 332.31 g/mol. The melting point of the reported crystalline polymorphs of Desidustat lies within the range of 198–200 °C. Desidustat demonstrates a LogP value of approximately 0.57, suggesting moderate hydrophilic behavior with balanced lipophilicity. It acts by stabilizing the subunit of HIF-alpha, as it would be naturally adapted to hypoxia (11–13). This stabilization triggers the expression of genes involved in the production of EPO over physiological levels and enhances iron control through a decrease in the expression of hepcidin(14). The DREAM-D and DREAM-ND studies have demonstrated clinical efficacy, which is non-inferior to traditional injectable ESAs. It is important to note that Desidustat has the clinical benefit of being administered orally thrice in a day, which greatly increases patient compliance (11,15).

Figure 1 Structure of Desidustat

When evaluating the greenness of an analytical approach, many factors are taken into account, including the quantity and toxicity of reagents, generated waste, energy requirements, the number of procedural steps, miniaturization, and automation. An analytical method's environmental friendliness and safety are evaluated using the AGREE (Analytical GREEnness) approach(16). The Green Analytical Procedure Index (GAPI), assesses the environmental impact of the analytical process at various phases using five different colored pentagons. To build upon GAPI, a new instrument called the Complementary Green Analytical Method Index (ComplexGAPI) was created, which adds more fields related to the procedures carried out before the actual analytical method(17).

Although Desidustat is a therapeutically significant compound, there is a serious gap in the scientific literature since no bioanalytical procedures were documented to determine its concentration in human plasma. Although Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is a high-sensitivity technique, its regular clinical application is constrained by its high cost of operation and special personnel requirement. Reversed-Phase High-Performance Liquid Chromatography with UV detection, on the other hand, offers a relatively inexpensive, dependable, and readily available alternative to routine therapeutic drug monitoring and pharmacokinetic investigations.

MATERIALS AND METHOD

Chemicals and Reagents

Desidustat and Enarodustat reference standards were received as gift samples from Swapnroop Drugs and Pharmaceuticals, India. Blank human plasma was obtained from the Red Cross, Jalgaon. HPLC-grade methanol, acetonitrile, and orthophosphoric acid were procured from Merck Ltd., India. During the experiment, water was sourced from the Milli-Q water purifying system.

Instrumentation

An Agilent 1100 series HPLC system with a quaternary pump, autosampler, and Diode Array Detector, and controlled by ChemStation version 10.1 software, was used for analysis. Separation was done by an Agilent Zorbax Eclipse C18 column (250 mm x 4.6 mm, 5 um particle size).

Stock and Working Solutions Preparation

Standard Stock Solution: 25 mg dissolved in 50mL methanol. A major stock solution of Desidustat was made at 500 µg/mL in methanol. On the same note, an internal standard stock solution of Enarodustat was made at a concentration of 500 µg/mL in methanol.

Calibration Standards: Standard stock solution was diluted with methanol to make 6, 60, 110, 160, 210, and 360 µg/mL working standard solutions. These were further spiked into blank human plasma to produce the ultimate calibration range of 0.6, 6, 11, 16, 21, and 36 µg/mL.

Quality Control Samples: Appropriate volumes (0.12, 1.2, 5.2, and 6.2 mL) were pipetted into 10 mL volumetric flasks, and the volume was made up to 10 mL with mobile phase. These working solutions were spiked into blank human plasma to prepare QC samples with final concentrations. There were four levels of quality control in human plasma: 0.3 µg/mL, 3 µg/mL, 13 µg/mL and 15.5 µg/mL.

Internal Standard Working Solution: Stock solution of the internal standard was added to methanol to bring the initial working IS concentration to 5 µg/mL.

Plasma Sample Extraction Process

Sample cleanup was done through a protein precipitation technique [3,4]. 0.1 mL of human plasma was transferred into a clean centrifuge tube. 0.1 mL of Desidustat (API) standard solution and 0.1 mL internal standard (IS) were added to the plasma. The mixture was vortex-mixed for 1–2 minutes to ensure uniform distribution of the analyte. Protein precipitation was carried out by adding an appropriate volume of organic solvent (acetonitrile, 1.7 ml). The samples were vortexed and then centrifuged at 5000 rpm for 10 minutes to separate precipitated plasma proteins. The resulting clear supernatant was carefully collected and filtered through a 0.22 µm syringe filter.

The filtered extract was transferred into HPLC vials and injected into the HPLC system for analysis.

Chromatographic conditions

The chromatographic separation was carried out by injecting (20 µL) sample into the HPLC system connected to the Agilent Zorbax Eclipse C18 column (250 mm x 4.6mm, 5 µm particle size). It was run in isocratic mode at 1.0 mL/min and ambient column temperature. The measurement was performed at 238 nm. The mobile phase was methanol and 0.1% Orthophosphoric acid (pH 3.0).

Bioanalytical Method Validation

Method validation was performed to ensure that the developed HPLC method for quantification of Desidustat in human plasma is reliable, reproducible, and suitable for routine bioanalytical application. The method validation was carried out according to ICH M10 (2–4).

System suitability

System suitability was evaluated before analysis using six replicate injections of the system suitability sample. Retention time, theoretical plate number, tailing factor, resolution, and peak-area precision were assessed. Six replicate injections of the 36 µg/mL system suitability sample were analyzed (n = 6). The ICH parameters for system suitability were calculated. Retention time (Rt), theoretical plates, and tailing factor were considered for system suitability.

Linearity

Linearity of the developed HPLC method was evaluated to establish the relationship between concentration and detector response for Desidustat in human plasma. The calibration curve was plotted using six data points by introducing accurate volumes of a working solution into a blank plasma sample. The ultimate concentration of 0.6 to 36 µg/mL was prepared, and the peak area was noted to plot the calibration curve. The calibration curve was plotted as peak area versus concentration. Evaluated on a six-point calibration curve (0.6 to 36 µg/mL).

Precision

Bioanalytical accuracy and precision follow strict FDA and ICH M10 guidelines. Accuracy and precision must be evaluated at a minimum of 4 concentration levels. It is determined by examining five replicates of the four levels of QC on three consecutive days to determine intra-day and inter-day performance.

Accuracy and precision

The accuracy and precision of the developed bioanalytical HPLC method for Desidustat were evaluated to assess the closeness of measured values to true values and the method's repeatability. For the recovery study, the peak areas of extracted plasma samples of Desidustat at LQC, MQC, and HQC levels were determined. Recovery samples at LQC, MQC, and HQC were prepared using pre-prepared plasma quality control stocks and API standard solutions, without employing the standard addition method. Intra-day accuracy and precision were evaluated by analyzing QC samples at different concentration levels within the same day. Each QC level was analyzed in three replicates. Inter-day accuracy and precision were evaluated by analyzing QC samples on different days.

The determination of analyte recovery was conducted by comparing the analytical outcomes of extracted samples. The area of the extracted plasma samples with unextracted aqueous standards at LQC, MQC, and HQC levels.

Robustness

To evaluate the robustness of the developed HPLC method for Desidustat by introducing small, deliberate variations in chromatographic conditions and observing their effect on method performance. The analysis was performed at a fixed concentration of 3 µg/mL.

Stability

The assessment of stability was carried out as an essential At LQC and HQC levels, stability was investigated under different conditions, such as bench-top and long-term stability, by comparing the results with freshly prepared ones

Carryover and dilution integrity

Carryover was determined by injecting blank plasma following the Upper Limit of Quantification sample; dilution integrity was determined by injecting a 36 0g/mL sample with blank plasma; and robustness was measured by making small, intentional changes in flow rate (10%), wavelength (2nm), and mobile phase composition.  

Green characteristics of the method

Numerous approaches such as AGREE, GAPI, complex GAPI, and others have been developed to evaluate the environmental sustainability of analytical procedures. AGREE and GAPI use a color-coded system to assess the environmental friendliness of the method. Red indicates that the method is not eco-friendly, yellow suggests that the method may be somewhat eco-friendly, and green signifies that the method is environmentally friendly.

The AGREE score is shown in the center of the pictogram; scores below 0.50 indicate that the approach is not acceptable or environmentally friendly; scores between 0.50 and 0.70 indicate the method is suitable, and scores greater than 0.75 indicate the method developed is highly effective in terms of environmental sustainability. The AGREE pictogram displays three colors with varying intensities depending upon the inputs provided. The pale green color in AGREE indicates sustainability, safety, and reduced waste generation. Green indicates that the method is eco-friendly (16,18).

 The expanded version of GAPI, Complex GAPI was introduced in 2021. Each hexagon in a GAPI figure, whose color intensity varies with the data entered, represents an individual aspect of the process.  GAPI’s primary goal is to quantify green criteria into a pictogram by assessing variables including waste, energy, chemicals, and risks. From sample collection to final analysis, GAPI evaluates an analytical method's overall eco-friendliness. The primary benefit of Complex GAPI get a chance to access the pre-analytical process parameters, including purification techniques, reagents and solvents, instrumentation and sensor preparation (19).

RESULT AND DISCUSSION

Development of a chromatographic method

Solubility studies indicated high dissolution in acetonitrile and methanol, whereas only moderate solubility was observed in water and isopropanol. Based on these findings, methanol was selected as the solvent for preparing both stock and diluted solutions. The observed melting range of 198–202 °C closely aligned with reported values, indicating a high degree of sample purity. This consistency further supported the reliability of the material throughout the experiments. During UV–visible analysis, a distinct peak absorbance was observed at 238 nm. Accordingly, this wavelength was selected for HPLC detection, as it provided a strong signal response without interference from methanol.

Method development for the RP-HPLC analysis began with a systematic evaluation of different mobile phase compositions to achieve effective separation of Desidustat. Each trial mixture was carefully assessed, with particular emphasis on peak symmetry as a key indicator of performance. The inclusion of Enarodustat as an internal standard enabled consistent monitoring throughout the optimization process. Gradual, stepwise refinement ultimately led to the establishment of optimal chromatographic conditions.

Initial trials using a strong mobile phase composition of methanol and 0.1% orthophosphoric acid (70:30, v/v) resulted in rapid elution but poor peak resolution. Major Desidustat peak at ~4.20 min (area ~130.7 mAU·s), small late peak at ~5.46 min. Baseline remains stable; peaks are reasonably sharp. Mobile-phase change shifted RT slightly (~4.2 min) and produced an acceptable peak shape. Rejected the mobile phase because of poor resolution.

 Adjusting the composition to a more balanced ratio, with reduced organic content, led to slight improvements in separation; however, issues such as peak asymmetry and baseline disturbances persisted. A mobile phase consisting of methanol and 0.1% orthophosphoric acid (pH 3.0) in a 30:70 (v/v) ratio provided superior results, yielding well-resolved and symmetrical peaks. The 30:70 (v/v) methanol:0.1% orthophosphoric acid composition provided the most favorable chromatographic performance among the evaluated conditions, based on retention, peak symmetry, and resolution. Under optimized parameters, a clear separation of Desidustat and the internal standard was achieved within 8 minutes. This performance was facilitated by a Zorbax Eclipse C18 column, whose stationary-phase properties enabled effective interaction with and resolution of these moderately water-soluble compounds in plasma samples.

The mobile phase was optimized to a composition of methanol and 0.1% orthophosphoric acid (pH 3.0) in a 30:70 (v/v) ratio and was operated in isocratic mode at a flow rate of 1.0 mL/min. The injection volume was set at 20 µL. Detection was carried out at 238 nm, corresponding to the λmax of Desidustat as determined from preliminary UV–visible spectrophotometric analysis. The total run time was 8 minutes, with Desidustat and Enarodustat eluting at approximately 3.9–4.0 minutes and 6.3–6.4 minutes, respectively. Optimized chromatographic conditions are shown in Table 1.

Table 1 Optimized Chromatographic Conditions for HPLC Method Development

Figure 2: UV spectra of Desidustat

Figure 3: Chromatogram of Desidustat and IS from plasma matrix

Bioanalytical Method Validation

A key step in the development of drugs and biological products is the quantitative assessment of drug concentration in biological matrices (such as serum, plasma, blood, urine, and saliva). In the pharmaceutical sector, bioanalysis is crucial since it aids in the quantitative assessment of both high molecular weight substances like proteins and low molecular weight medications. Critical judgments supporting the safety and efficacy of a medicinal therapeutic ingredient or product depend on the results of several animal toxicity research, pharmacokinetic studies, and preclinical PK investigations, including bioavailability and bioequivalency studies. As a result, validation guarantees that the specified bioanalytical method yields sensitive and repeatable data.

Validation studies confirmed that the method complied with ICH M10 guidelines, demonstrating its reliability for routine clinical analysis. Consistent performance was observed across varying conditions throughout all evaluation phases. Given the critical importance of accuracy in testing, each step was conducted in strict adherence to established protocols. Alignment with peer-reviewed procedures ensured clarity and reproducibility of results, while even minor deviations were carefully monitored during repeated trials.

System suitability

System suitability ensures the instrument and column are performing together to generate accurate, reproducible data before and during sample analysis. Table 2 shows the system suitability parameters of desidustat and IS in human plasma.

Table 2: System suitability parameters of desidustat and IS in human plasma

Linearity

The method exhibited a strong linear relationship between concentration and peak area over the range of 0.6 to 36 µg/mL. The regression equation was determined to be y = 69.51x – 7.897 with an exceptional correlation coefficient (r²) of 0.9999. The plotted data is of peak area versus concentration. Table 3 provides linearity data of desidustat, and Figure 4 shows the linearity curve of desidustat

Figure 4: Overlay of linearity

Figure 5: Linearity curve of desidustat in human plasma

Table 3: Regression and Linearity Parameters

Accuracy and Precision

The extent to which an analytical method's mean test findings closely resemble the analyte's actual value (concentration) is known as its accuracy. When an analytical method is applied repeatedly to several aliquots of a single homogeneous volume of biological matrix, the precision of the method describes the closeness of individual analyte measurements. At least five determinations per concentration should be used to gauge precision. At least five determinations per concentration should be used to gauge precision.

It is advised to use at least three concentrations within the anticipated concentration range. With the exception of the LLOQ, where it should not exceed 20% of the coefficient of variation (CV), the precision calculated at each concentration level should not exceed 15% of the CV. Precision is further separated into two categories: between-run, inter-batch precision or repeatability, which measures precision over time and may involve various analysts, tools, reagents, and labs, and within-run, intra-batch precision or repeatability, which evaluates precision during a single analytical run.

It is advised to use at least three concentrations within the anticipated concentration range. With the exception of the LLOQ, where it should not exceed 20% of the coefficient of variation (CV), the precision calculated at each concentration level should not exceed 15% of the CV. Precision is further separated into two categories: between-run, inter-batch precision or repeatability, which measures precision over time and may involve various analysts, tools, reagents, and labs, and within-run, intra-batch precision or repeatability, which evaluates precision during a single analytical run.

Replicate examination of samples with known analyte concentrations determines accuracy.

At each of the four QC concentrations - LLOQ, LQC, MQC, and HQC - the method delivered results close to the true values, showing strong reliability. Performance remained consistent across repeated measurements, indicating low variability under controlled conditions.

Intra-day plus inter-day accuracy showed results between 96.61% and 102.86%. These fall comfortably inside the accepted range of 85–115%. Although slight variations occurred, none breached minimum standards. Performance stayed consistent across testing periods. The data met the criteria without exception. Table 4 shows Intra-day Accuracy and Precision of Desidustat in Human Plasma

Measured variation stayed tightly within 1.14% across tests, while at the lowest detectable level it narrowed further, reaching just 0.32%. This reflects strong consistency when repeated under similar conditions. Intra-day accuracy and precision were within acceptable limits as per ICH M10 guidelines

Table 4: Intra-day Accuracy and Precision of Desidustat in Human Plasma

Table 5: Inter-day accuracy and precision of desidustat in human plasma

Accuracy

The recovery was evaluated at low, medium, and high QC levels to assess the consistency and efficiency of the extraction procedure. The % coefficient of variation (%CV) for recovery at each QC level was required to be not more than 15%. Accuracy expressed as % Recovery.

 Table 6: Accuracy results

Robustness

To evaluate the robustness of the developed HPLC method for Desidustat by introducing small, deliberate variations in chromatographic conditions and observing their effect on method performance. Even when the flow rate shifted slightly - by 10% - the approach held steady. Detection wavelength altered by 2 nm, yet results stayed consistent. Mobile phase makeup varied, but performance did not waver. In every case, %RSD stayed under 2%. Stability emerged clearly across all tested conditions.

Table 7: Robustness Parameter

Carryover

Following a high-concentration injection, no notable carryover appeared in blank plasma, where the signal reached just below the LLOQ level. Response levels remained minimal despite prior exposure to concentrated samples. The absence of lingering effects suggests limited residue transfer during analysis. Signal detection stayed close to baseline under these conditions.

Dilution Integrity

For samples exceeding the calibration range (36 µg/mL), the method maintained an accuracy of 95.16% and a %RSD of 0.15 after dilution with blank plasma. This confirms the method's validity for analyzing high-concentration clinical samples.

Green characteristics of the method

 AGREE score generates a circular clock-like pictogram with a central score and color code like green, light green, yellow, and red with 12 distinct assessments. The optimized HPLC method shows an AGREE score of 0.8 (Figure 6a), which reflects higher compliance with green analytical chemistry principles. Organic modifiers like acetonitrile lower the score due to safety and waste principles.

A complex GAPI shows a standardized visual pictogram evaluating environmental sustainability. It evaluates the standard 15 steps of an analytical method from the original GAPI tool. Extra hexagonal specifically added in the Complex GAPI framework to incorporate pre-laboratory operations, sample collection, preservation, and transport(20). In this study, the Complex GAPI score was found to be 77 (Figure 6b) using parameters such as mobile phase, sample collection, storage, toxic solvents, and energy consumption.

a: AGREE score                                                                        b: Complex GAPI

Figure 6: Greenness values of proposed analytical method

CONCLUSION

An RP-HPLC method was developed and validated for the quantification of Desidustat in human plasma. The method uses conventional HPLC-UV instrumentation and therefore provides a potentially accessible alternative for laboratories without LC-MS/MS capability. Chromatographic analysis was performed using an Agilent Zorbax Eclipse C18 column, with a mobile phase consisting of methanol and 0.1% orthophosphoric acid (30:70, v/v) delivered at a constant flow rate of 1.0 mL/min. Sample preparation was simplified through protein precipitation using acetonitrile, avoiding complex extraction procedures. Reliable separation was achieved through careful control of flow rate and mobile phase composition.

The method demonstrated excellent linearity over the studied range. Accuracy was within 96.61–102.86%, complying with ICH M10 guidelines, while precision remained robust with %RSD values ≤1.14%. Recovery was consistent, ranging from 85% to 97% across experiments.

Stability studies confirmed that the analyte remained unaffected under various storage conditions, indicating resilience within the plasma matrix. Owing to its sensitivity and reproducibility, the method is well-suited for monitoring drug concentrations over time, particularly in patients with renal impairment.

The method is assessed for its greenness using the AGREE approach and Complex GAPI, and proved the greenness of the method. A high-performance liquid chromatography (HPLC) method's greenness assessment has the advantage of systematically identifying and minimizing environmental pollution, reducing hazardous chemical waste, lowering energy consumption, and safeguarding operator safety.

REFERENCES