Method Development and Method Validation of Daprodustat - Using Reverse phase High Performance Liquid Chromatography Method

The separation of non-volatile species or compounds that are thermally sensitive was made easier by the use of HPLC, a type of liquid chromatography, for the qualification and analysis of mixtures of chemical and synthetic chemicals.¹ A wide range of materials, including proteins, nucleic acids, amino acids, hydrocarbons, sugars, terpenoids, pesticides, steroids, antibiotics, metals, organic species, and a number of inorganic chemicals, are separated using this adaptable approach.² Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) technique was used to separate molecules according to how hydrophobic they were.³ In RP-HPLC, an organic solvent and water mixture makes up the mobile phase, whereas the stationary phase has hydrophobic characteristics.⁴ Greater hydrophobicity molecules have a tendency to bind to the phase that is stationary, from which they are eluted by increasing the concentration of organic solvent⁵ Being an inhibitor of hypoxia – related factor prolyl hydroxylase (HIF PH), daprodustat was prescribed to treat anemia caused by chronic renal disease in people receiving dialysis for a period of minimum four months.⁶ It worked by raising a naturally occurring chemical in the body that stimulates the creation of red blood cells.⁷ When compared to other treatments, oral administration of Daprodustat had certain benefits and drawbacks.⁸

Experimental Methodologies

MATERIALS & METHODS

The primary HPLC system was the Alliance model manufactured by waters, renowned for its precision and reliability in separating and analyzing compounds within a liquid sample.^[9] Supporting equipment included a pH meter by Eutech for monitoring the acidity or alkalinity of solutions, UV/VIS spectrophotometer, specifically the UV-1700, for qualifying the absorbance of substances at various wavelengths. A Unichrome UCA 701 ultrasonic bath facilitated sample preparation through rapid and thorough mixing and degassing, enhancing experimental reproducibility. Lastly, an Isocratic model pump was utilized for maintaining constant solvent flow rates during chromatographic separation processes.¹⁰

Reagents & Chemicals

The essential chemicals used in HPLC methodology included HPLC grade acetonitrile(ACN) and water (Milli Q), Hexane Sulfonic acid, and Ortho Phosphoric acid, all crucial for chromatographic separations. These chemicals were sourced from reputable manufacturers, ensuring high-quality standards and analytical reliability.

Drug profile ¹¹

Figure 1: Molecular Structure of Daprodustat.

IUPAC Name: 2-[(1, 3-dicyclohexyl-2, 4, 6-trioxo-1, 3-diazinane-5-carbonyl) amino] acetic acid.

Molecular Equation: - C₁₉ H₂₇ N₃ O₆

Mol. wt.: 393.440 gram/mole.

Method Development

Selection of wavelength (λmax)

The drug estimation process relied on the utilization of the isobestic wavelength, a critical parameter in determining drug concentration. The wavelength at which the molar absorption factor is constant for compounds that can merge is known as the isobestic point. Hence, this specific wavelength was employed to ensure the precise estimation of the drug. The maximum absorption wavelength of the drug solution in a blend of Acetonitrile and Hexane Sulphonic Acid (HSA) at pH 2.5/OPA (50:50) served as the reference blank.The absorption curve, in particular, clearly showed an isobestic spot at 235 nm. As a result, 235-nm became the standard wavelength for the HPLC chromatographic method's sensor.Molecular structure can be determined from the Fig:1

Selection of chromatographic method

During the chromatographic condition’s selection phase, numerous trials were conducted, and the most effective trial was singled out for method optimization. The specific conditions were meticulously recorded and summarized in Table-1. In the mobile phase, Acetonitrile was combined with Hexane Sulphonic Acid (HSA) at pH 2.5/OPA (50:50). For separation purposes, the Waters X-Terra RP-18 column (150mm×3.5µm) was deemed optimal. The detection wavelength was set to 235 nm, with an injection volume of 10 µl, a flow rate of 1ml/min, and a total run time of 4 minutes.¹² The peak corresponding to Daprodustat manifested at 2.627 minutes, exhibiting a peak area of 2942254 and a tailing factor of 1.01. This particular trial underwent optimization to ensure precision and accuracy in the analysis.¹³

Method selection criteria

Utilizing a trial and error approach, the mobile phase consisted of Acetonitrile (ACN) combined with Hexane Sulphonic Acid (HSA) at pH 2.5/OPA (50:50). The chromatographic analysis was performed using a Waters X-Terra RP-18 column (150 mm × 3.5 µm). Detection was performed at a wavelength of 235 nm, using an injection volume of 10 µl and a flow rate of 1ml/min. Four minutes was set as the optimal run time. Within this trial, the Daprodustat peak emerged at 2.627 minutes, displaying a peak area of 2942254 and a tailing factor of 1.01. The Daprodustat peak was visually identified in Figure 5.

Setting up the mobile phase

The movable phase composition involved a meticulous blending of HSA at pH 2.5/OPA and ACN, adhering to a precise ratio of 30:70.¹⁴ Subsequently, it undergone filtration using a 0.45μ membrane filter to effectively eliminate any impurities that could potentially compromise the integrity of the final chromatogram.¹⁵

Preparing the diluent

In the experimental setup, the obtained mobile phase was used as diluent for the standard and sample solutions.

Establishing a standard solution

A precise six mg of the Daprodustat working standard was first weighed and then transferred to a 10 ml volumetric flask which is dry and was cleaned.¹⁶ After adding the diluent, the mixture was subjected to ultrasound until total dissolution was attained. The stock solution was then made by adjusting the volume to the appropriate level using the same solvent.¹⁷ The prepared stock solution was then pipetted out into another volumetric flask with a capacity of 10 ml in a volume of 1 ml. Dilution was then performed up to the mark using diluent, yielding a dosage of 60 ppm of Daprodustat.

Preparation of sample solution

A Daprodustat sample weighing 91 mg was accurately measured and placed into a 10 mL dry volumetric flask for cleaning.To fully dissolve the material, diluent was added, and then centrifugation and sonication were used for 30 minutes.¹⁸To make the stock solution, the volume was precisely measured using the same solvent and filtered using an injection filter with a 0.45 µ opening.¹⁹ The 10 ml volumetric flask was filled with 1 ml of this solution, which was then diluted with diluent to produce a 60 ppm dose of daprodustat.²⁰

Procedure

The chromatographic system was stocked with 10 µL of the standard and sample, and the regions of the sample and standard peaks were measured.The location of the Daprodustat peak was established. The Percentage Assay was then computed using the relevant formulas.The ideal conditions were given in the Table:1

Problem solving

Percentage Assay =

WS = Working Standard

The analytical procedure underwent validation for several key parameters including whether the system is suitable, linear, accurate, precise, repeatable, and robust is an important consideration.

RESULTS

System Suitability

In accordance with the standards established by ICH, all system suitability criteria were determined to be satisfactory, meaning they fell within the specified range. Suitability parameters were mentioned in Table:2

Table 1: Ideal Conditions for Chromatography

Table 2: System suitable specifications for Daprodustat.

Figure 2: Chromatogram of Standard.

How well the analytical method measured the target analyte in the absence of blank and known contaminants was used to determine the method's specificity. For this purpose, chromatograms of several types were recorded and examined, including blank Fig:3 , standard Fig:2 , and sample chromatograms Fig:4. The specificity of the drug's reaction was confirmed Fig:5 when it was noted that the blank's chromatogram did not respond at the drug retention times.

Figure 3: Blank graph chromatogram.

Figure 4:Placebo graph chromatogram.

Linearity

Below are the concentration levels at which Daprodustat was shown to have a linear response. It was confirmed that it fulfilled the specified requirements for approval. Verify the Table 3 for the linearity results

Table 3: Results of Linearity for Daprodustat

Range

Analytical precision, accuracy, and linearity were demonstrated within the required levels of the analyte, which were actually defined as the interval containing both the upper and lower levels. The outcome should be a correlation coefficient of at least 0.999.

System Accuracy

Table 4: System Accuracy results of Daprodustat

Precision

By evaluating a homogeneous sample from a single batch six times to assure consistent results, the recurrence of an analytical method under normal circumstances was tested for, which is known as precision.Additionally, six injections of solutions containing 60 ppm of Daprodustat were used to confirm the accuracy of the device Refer to the Table 5 for details

Table 5: Precision for Daprodustat

Robustness

We purposefully changed the mobile phase composition, flow rate and temperature variation as part of the Robustness investigation to see how these affected the approach values of robustness were given in Table 6.

Table 6: Robustness