Accurate Mass Identification of an Interfering Water Adduct and Strategies in Development and Validation of an LC-MS/MS Method for Quantification of MPI8, a Potent SARS-CoV-2 Main Protease Inhibitor, in Rat Plasma in Pharmacokinetic Studies

Abstract

MPI8, a peptidyl aldehyde, is a potent antiviral agent against coronavirus. Due to unique tri-peptide bonds and the formyl functional group, the bioassay of MPI8 in plasma was challenged by a strong interference from water MPI8. Using QTOF LC-MS/MS, we identified MPI8•H2O as the major interference form that co-existed with MPI8 in aqueous and biological media. To avoid the resolution of MPI8 and MPI8•H2O observed on reverse phase columns, we found that a Kinetex hydrophilic interaction liquid chromatography (HILIC) column provided co-elution of both MPI8 and MPI8•H2O with a good single chromatographic peak and column retention of MPI8 which is suitable for quantification. Thus, a sensitive, specific, and reproducible LC-MS/MS method for the quantification of MPI8 in rat plasma was developed and validated using a triple QUAD LC-MS/MS. The chromatographic separation was achieved on a Kinetex HILIC column with a flow rate of 0.4 mL/min under gradient elution. The calibration curves were linear (r2 > 0.99) over MPI8 concentrations from 0.5−500 ng/mL. The accuracy and precision are within acceptable guidance levels. The mean matrix effect and recovery were 139% and 73%, respectively. No significant degradation of MPI8 occurred under the experimental conditions. The method was successfully applied to a pharmacokinetic study of MPI8 after administration of MPI8 sulfonate in rats.

Keywords: LC-MS/MS; MPI8; MPI8•H2O adduct; SARS-CoV-2; method development and validation; pharmacokinetics.

Figure 1

Chemical structures of MPI8, its prodrug MPI8 sulfonate, and antipyrine (AP, internal standard).

Figure 2

MRM chromatograms for MPI8 (transitions m/z 601.4→545.3 and 601.4→157.2): Not-fully-separated multiple peaks on a Kinetex F5 column (A) and an Acquity HSS-T3 column (B); distinctly separated double peak chromatograms of MPI8 on a Synergi Fusion-RP column (C); single peak with good peak shape on a Kinetex HILIC column (D). The time program of the gradient: (A,B,D) Phase B was initially kept at 5% for 0.5 min, increased from 5% to 90% in the next 2.5 min, then decreased to 5% in 1.0 min, and kept stably at 5% for 1 min; (C) Phase B was initially at 20% for 0.5 min, increased from 20% to 90% in 4.5 min, kept at 90% for 2 min, decreased to initial concentration (20%) in 1 min and equilibrated for 2 min.

Figure 3

HR MS and MS/MS spectra of MPI8 and MPI8•H₂O using an extracted sample from MPI8 spiked plasma at retention time of 4.5 min after column elution on an X500B QTOF mass spectrometer; (A1,A2) and (B1,B2) in positive mode, (C1,C2) and (D1,D2) in negative mode; the proposed fragmentation of product ions are shown on MS/MS spectra: (A1) protonated MPI8 [MPI8 + H]⁺ MS (m/z 601.3594), (A2) protonated MPI8 [MPI8 + H]⁺ MS/MS; (B1) [MPI8•H₂O + Na]⁺ MS (m/z 641.3502) (B2) [MPI8•H₂O + Na]⁺ MS/MS; (C1) deprotonated MPI8 [MPI8 −H]⁻ MS (m/z 599.3433), (C2) deprotonated MPI8 [MPI8 − H]⁻ MS/MS; (D1) [MPI8•H₂O + Cl]⁻ MS (m/z 653.3325), (D2) [MPI8•H₂O + Cl]⁻ MS/MS.

Figure 4

HR-MS extracted ion chromatograms of MPI8 and MPI8•H₂O eluted on a Synergi Fusion-RP column; the peak at 5.2 min was MPI8; the peak at 4.5 min was MPI8•H₂O, and the MPI8 signal was due to the in-source fragmentation: (A) MPI8 ([MPI8 + H]⁺) m/z 601.3596 in pink and MPI8•H₂O ([MPI8•H₂O + Na]⁺) m/z 641.3521 in blue in positive mode (B) MPI8 ([MPI8 −H]⁻) m/z 599.3450 in pink and MPI8•H₂O ([MPI8•H₂O + Cl]⁻) m/z 653.3323 in blue in negative mode.

Figure 5

The peak height and retention time of MPI8 (100 ng/mL) on a Kinetex HILIC column in isocratic elution at different concentrations of the mobile phase component of acetonitrile, from 5% to 90%. When the acetonitrile composition in the mobile phase was less than 10%, MPI8 was completely retained on the column, and the retention time mark was 5 min, which was the total running time of the test.

Figure 6

Representative MRM chromatograms: (A1) MPI8 in double blank rat plasma, (A2) IS in double blank plasma; (B1) MPI8 (0.5 ng/mL) spiked in blank rat plasma, (B2) IS (5 ng/mL) spiked in blank rat plasma; (C1) MPI8 in a rat plasma sample at 30 min after IV administration of MPI8 sulfonate at a single dose of 5 mg/kg, (C2) IS (5ng/mL) in a rat plasma sample at 30 min after IV administration of MPI8 sulfonate at a single dose of 5 mg/kg. There was no interference at the retention times of the analyte and IS, and no carryover for both IS (≤5% of average response) and MPI8 (≤20% of LLOQ).

Figure 7

Mean plasma concentration vs. time profiles of MPI8 in rat plasma after i.v. (n = 3) and oral (n = 3) administration (normal scale plots in normal graph and semi-log plots in inset graph). Twelve time-point plasma samples from rats dosed intravenously and 13 time-point plasma samples from rats dosed orally were collected. Three replicates for each time point were analyzed and reported as mean ± SD for each time-point.