RP-HPLC Method Development and Validation of Ferrous Ascorbate and Folic Acid by Using Analytical Quality by Design Approach in Bulk and Formulation Dosage Form

The combination of ferrous ascorbate and folic acid is widely used in antianemic therapies, necessitating robust analytical methods for quality control. While spectrophotometric and conventional RP-HPLC methods exist for individual or combined analysis, these approaches often lack systematic optimization of critical method parameters. Analytical Quality by Design (AQbD) addresses this gap through science-based risk evaluation and experimental design (DoE), aligning with ICH Q14 guidelines to enhance method robustness and regulatory flexibility (1). Traditional methods for these compounds face challenges due to their divergent physicochemical properties - ferrous ascorbate's iron complex structure (λ_max 265 nm) versus folic acid's conjugated pteridine ring system (λ_max 280-344 nm) ^[8]. Previous attempts at simultaneous RP-HPLC analysis encountered co-elution issues and mobile phase incompatibility, forcing separate analytical procedures. The AQbD framework enables systematic optimization of chromatographic parameters (column chemistry, pH, organic modifier ratio) through quality target profiles and risk assessment matrices (2). This study introduces an AQbD-driven RP-HLC method that simultaneously quantifies both analytes in pharmaceutical formulations. By employing central composite design to optimize critical method variables, we overcome the resolution challenges documented in earlier isocratic elution attempts. The approach integrates method operable design space concepts with validation per ICH Q2(R1), addressing the need for stability-indicating methods in complex matrices containing ascorbate degradation products (3).

MATERIALS:

Analytical Samples: Pure ferrous ascorbate and folic acid were procured as active pharmaceutical ingredients (APIs) for method development. Marketed formulations of the combination (tablet dosage form) were sourced for validation studies.

Chemicals Potassium dihydrogen phosphate (KH?PO?), acetonitrile (ACN), methanol, sodium hydroxide (NaOH), hydrochloric acid (HCl), and orthophosphoric acid (OPA) were utilized for the preparation of the mobile phase.

Instrumentation A photo-diode array (PDA) detector, a Fourier transform infrared (FT-IR) spectrometer, a UV/Vis spectrophotometer, and a reversed-phase high-performance liquid chromatography (RP-HPLC) equipment were all incorporated in the device.

Software used included Design-Expert® (vX) for experimental design, response surface methodology (RSM), and statistical analysis (4).

METHOD:

Authentication of Pure Drug Samples

FT-IR Spectroscopy was employed to confirm the molecular identity of ferrous ascorbate and folic acid. For ferrous ascorbate, the FT-IR spectrum revealed characteristic peaks corresponding to C=O stretching (1700 cm?¹) and Fe-O vibrations (600–800 cm?¹). Folic acid had a number of different absorption bands, one of which was the stretching of C=O (1680 cm?¹) and NH bending vibrations (1550 cm?¹). These spectral data aligned with literature-reported peaks, confirming sample authenticity (5).

Fig.1. IR Spectra of ferrous ascorabte.

Fig.2. FT-IR Spectrum of Folic Acid.

RP-HPLC Method Development

Mobile Phase Optimization focused on balancing analyte solubility, retention, and resolution. A potassium dihydrogen phosphate buffer (pH 3.0–4.5) was selected for its stability and compatibility with ferrous ascorbate, while acetonitrile (ACN) replaced methanol due to superior peak symmetry and lower viscosity. The detection wavelength (225 nm) was chosen based on UV overlay spectra to maximize absorbance for both analytes. Standard Stock Solutions were produced by dissolving ten milligrams of ferrous ascorbate in distilled water (100 µg/mL) and 10 mg of folic acid in 0.1 N NaOH (100 µg/mL), followed by sonication and dilution (6).

Column Selection favored a C18 (150 mm × 4.6 mm, 5 µm) for its ability to retain polar ferrous ascorbate and resolve folic acid’s conjugated structure. Flow Rate Optimization tested 0.8–1.2 mL/min: 1.0 mL/min provided optimal resolution (R >2.0) and peak symmetry (tailing factor <2.0) while minimizing run time.

Design of Experiments (DoE) evaluated three trials:

1. Trial 1: Phosphate buffer (6.5 g/L) with methanol (35:65 v/v, pH 4.5) yielded poor resolution (R ≈1.6) despite adequate peak symmetry.

Fig.3.Chromatogram Of Trial 1

2. Trial 2: Ammonium acetate buffer (385 mg/1000 mL) with ACN (50:50 v/v, pH 5.2) improved peak sharpness but required further pH adjustment.

Fig.4. Chromatogram of Trial 2

3. Trial 3: Adjusted phosphate buffer (11.16 g/L KH?PO? + 5.5 g/L K?HPO?, pH 6.4) with methanol (60:40 v/v) achieved superior resolution (R ≈2.5) and theoretical plates (NTP >3000).

Fig.5. Chromatogram of Trial 2

Statistical Modeling via Design-Expert® identified critical interactions: flow rate significantly influenced retention times, while pH and organic modifier ratios affected peak symmetry and resolution. Response surface plots guided the selection of Trial 3 as the optimized method.

Table.4. Trials Data

1)RT-1:

Fig.7.predicted vs actual of RT-1

Fig.9.Interaction of Flow Rate and Rt-1.

2) RT-2:

Fig.11.predicted vs actual of RT-2.

Fig.13. Interaction of Flow Rate And RT-2.

3) NTP-1:

Fig.15.Predicted Vs Actual Of NTP-1

Fig.17.Interaction Of NTP-1

4) NTP-2:

Fig.19.Predicted Vs Actual Of NTP-2.

Fig.21.Interaction Of NTP-2

Method Validation:

Accuracy was assessed via recovery studies at 80%, 100%, and 120% target concentrations, yielding 98–102% recovery for both analytes.

Precision (% RSD ≤2%) was confirmed through intra-day (repeatability) and inter-day (reproducibility) testing.

Specificity ensured no co-elution with excipients or degradation products, validated via resolution >2.0.

System Suitability parameters included tailing factor <2.0 and NTP >3000.

Linearity was established over 50–150 µg/mL (ferrous ascorbate) and 10–300 µg/mL (folic acid) with regression coefficients (r²) ≥0.999.

Robustness tested variations in pH (±0.2), flow rate (±0.1 mL/min), and column temperature (±2°C), demonstrating <2% RSD.

Degradation Studies exposed samples to acid hydrolysis (0.1N HCl) and UV light, with peak purity assessed via PDA detection (7).

Software and Data Analysis

Design-Expert® facilitated Plackett-Burman screening to identify critical parameters (pH, organic modifier) and Central Composite Design (CCD) for optimization. ANOVA and RSM models quantified variable interactions, while regression equations (e.g., y = mx + c) validated linearity. Response surfaces visualized the impact of flow rate and mobile phase composition on retention times and theoretical plates (8).

RESULTS:

4.1 System Suitability

System suitability parameters were evaluated using five replicate injections of standard solutions. For ferrous ascorbate, retention time (tR) averaged 2.7 ± 0.1 min with a tailing factor (T) of 1.2–1.8 and theoretical plates (NTP) exceeding 3000 (Table 15). Folic acid exhibited tR of 6.8 ± 0.2 min, T <2.0, and NTP >2500. Resolution (R) between analytes was consistently >2.0, satisfying ICH Q2(R1) requirements. Blank chromatograms (Fig. 28) confirmed no interference at the target wavelengths12 (10).

Fig.22.Chromatogram of Std.5

Table.5. Result of system suitability of Ferrous Ascorbate and folic Acid.

4.2 Precision

System Precision: Five replicate injections of standard solutions (ferrous ascorbate: 50 µg/mL; folic acid: 100 µg/mL) yielded % RSD of 0.5–1.5% for retention times and 1.0–2.0% for peak areas (Table 22). Method Precision involved analyzing six sample replicates (80–120% labeled claim), with % RSD ≤2.0 for both intra-day and inter-day studies (Table 29). These results align with the precision reported in literature (e.g., % RSD ≤2.0 in UKEssays study) (2,5).

Fig.24.Chromatogram of Std.5 (Sysytem Precision)

Table.6. Precision of Ferrous ascorbate and Folic Acid

4.3 Accuracy

Recovery studies at 50%, 100%, and 150% spiked levels demonstrated 98–102% recovery for both analytes. For ferrous ascorbate, mean recovery was 100.2% (n=6), while folic acid showed 99.8% (n=6). Chromatograms of spiked samples confirmed peak purity and absence of co-elution with excipients, corroborating specificity studies(1,5).

Fig.25.Chromatofram Of Std. Soln 4 For Accuracy.

Table 7. Result of Accuracy for Ferrous Ascorbate.

Fig.26.Chromatofram of 100% Spike Inj.1 For Accuracy.

Table.8. Result of Accuracy for Folic Acid.

4.4 Linearity

Calibration curves for ferrous ascorbate (50–375 µg/mL) and folic acid (100–750 µg/mL) exhibited r² ≥0.999. Linear regression equations were:

• Ferrous ascorbate: Y=23397x+28729Y = 23397x + 28729Y=23397x+28729
• Folic acid: Y=188287x+150052Y = 188287x + 150052Y=188287x+150052

l) Chromatographic outcomes of Solution L6 inj.1 for lineartity:

Fig.27.Chromatogram of Solution L6 Inj.1 For Linearlity.

Fig.28.linearity figure for Ferrous Ascorbate.

Table.9. Result of linearity for Ferrous Ascorbate.

m) Chromatographic outcomes of Solution L6 inj.2 for lineartity:

Fig.29.Chromatogram of Solution L6 Inj.2 For Linearlity.

Fig.30.linearity figure for folic acid.

Table.10. Result of linerity for Folic acid.

4.5 Robustness

Flow rate variations (0.9–1.1 mL/min) had minimal impact: retention times shifted by <2%, and system suitability parameters remained within limits. Chromatograms at altered flow rates confirmed stable resolution and peak symmetry, validating method robustness per ICH guidelines26.

e) Chromatographic outcomes of Std.Solⁿ 4 for Robustness of flow rate(0.9mL/min):

Fig.31.Chromatogram of Std.Solⁿ 4 For Robustness of Flow Rate (0.9 Ml /Min)

Table.11. Robustness result of ferrous ascorbate and folic acid for 0.9 mL/min

j) Chromatographic outcomes of Std.Solⁿ 3for Robustness of flowrate(1.1mL/min):

Fig.32.Chromatogram Of Std.Solⁿ 3 For Robustness Of Flow Rate (1.1 Ml /Min) .

Table.12. Robustness result of Ferrous ascorbate and Folic acid for 1.1 mL/min