AQbD based green UPLC method to determine mycophenolate mofetil impurities and Identification of degradation products by QToF LCMS

Abstract

We report an ideal method for quantifying impurities in mycophenolate mofetil drug substances and their oral suspension preparations. We developed a systematic and eco-friendly analytical approach utilizing quality by design (QbD) and green chemistry principles. Initially, the critical method parameters (CMPs) were screened using a D-optimal design. The robust final method conditions were optimized using a systematic central composite design (CCD). Through graphical and numerical optimization, the protocol conditions were augmented. The pH of mobile phase buffer (25 mM KH₂PO₄) (MP-A), initial gradient composition (% MP-A), flow rate (mL min^-1), and column oven temperatures (°C) are 4.05, 87, 0.4, and 30, respectively. The best possible separation between the critical pairs was achieved while using the Waters Acquity UPLC BEH C₁₈ (100 × 2.1) mm, 1.7 µm analytical column. A mixture of water and acetonitrile in the ratio of 30:70 (v/v) was used as mobile phase-B for the gradient elution. The analytical method was validated in agreement with ICH and USP guidelines. The specificity results revealed that no peaks interfered with the impurities and MPM. The mean recovery of the impurities ranged between 96.2 and 102.7%, and the linearity results r > 0.999 across the range of LOQ - 150%. The precision results (%RSD) ranged between 0.8 and 4.5%. The degradation products formed during the base-induced degradation were identified as isomers of mycophenolic acid and sorbitol esters using Q-ToF LC-MS and their molecular and fragment ion peaks. The developed method eco-friendliness and greenness were assessed using analytical greenness (AGREE), green analytical procedure index (GAPI), and analytical eco score, and found it is green.

Figure 1

Interaction effect plots from the screening D-optimal split-plot design. R1: Number of impurity peaks separated with a minimum resolution of 1.5; R2: Rs between Imp-H and MPM; R3: Rs between MPM and Imp-D; R4: Rs between Imp-D and Imp-F; and R5: Rt of the late eluting peak.

Figure 2

2D-Normal plots and 3D-Response surface plots for the responses (R1–R5). R1: Rs between Imp-E & A; R2: Rs between Imp-D & MPM; R3: Rs between MPM & Imp-F; R4: Rs between Imp-F & B; R5: Rs between Imp-B & C.

Figure 3

The overlay plots from the graphical optimization.

Figure 4

Interference study chromatograms from the final method. Chromatographic conditions: MP-A: pH 4.05 KH₂PO₄ (25 mM); MP-B: Water: ACN = 30:70 (v/v); Initial gradient composition MP-A:MP-B (87:13); Column: Acquity UPLC BEH C18 (100 mm × 2.1 mm), 1.7 µm; Flow rate: 0.4 mL min⁻¹; Column temperature: 30 °C; Injection volume: 5 µL; and Wavelength detection: 215 nm.

Figure 5

An overlaid chromatogram from the stress study experiments. Chromatographic conditions: MP-A: pH 4.05 KH₂PO₄ (25 mM); MP-B: Water: ACN = 30:70 (v/v); Initial gradient composition MP-A:MP-B (87:13); Column: Acquity UPLC BEH C18 (100 mm × 2.1 mm), 1.7 µm; Flow rate: 0.4 mL min⁻¹; Column temperature: 30 °C; Injection volume: 5 µL; and Wavelength detection: 215 nm.

Figure 6

Positive mode low energy mass spectrums (a) Unknown at RRT 0.84; (b) Unknown at RRT 0.85; (c) Unknown at RRT 0.87; (d) Mycophenolic acid sorbitol ester.

Figure 7

Fragmentation pattern of sorbitol ester of mycophenolic acid and its isomers.

Figure 8

Method greenness assessment tools pictograms.