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  1. Discovery of an Unknown and Unstable Trimebutine Metabolite and its Significant Impact of Reproducibility and Accuracy of N-Desmethyltrimebutine Quantification by LC-MS/MS Catherine Dicaire1, Maxime Ranger2, Patrick Colin2, Jean-Nicholas Mess1, Milton Furtado1, and Fabio Garofolo1* 1Algorithme Pharma Inc., Laval (Montréal), QC, Canada, 2gIcare Pharma, Montréal, QC, Canada 343 amu Table 2: Post-Extraction Increase in Concentrations for N-Desmethyltrimebutine in Subject Samples Figure 4: Product Ion Scans (Top pane: Glucuronide, Bottom pane: N-Desmethyltrimebutine) Because the reference material for this newly found metabolite was not readily available and because of its extreme instability, it became very difficult to find an adequate procedure to prevent the observed conversion. Accordingly, it was decided to completely hydrolyze the glucuronide to N-desmethyltrimebutine and to quantify the total concentration of N-desmethyltrimebutine. Acidification of the human plasma during sample preparation enabled the hydrolysis of the glucuronide and the reproducibility of the reaction was monitored through ISR evaluations. As shown in Table 5, the ISR showed a rugged hydrolysis procedure. Furthermore, the plasma stabilities evaluations were performed using incurred samples (Table 6) rendering this method a successful fit-for-purpose assay. OVERVIEW METHODS • Purpose • Determination of bioanalytical impact on N-desmethyltrimebutine quantitation due to extreme instability of an unknown phase II metabolite. • Method • Incurred sample reanalysis (ISR) evaluation was assessed. • Precursor Ion and MRM Scans were performed on freshly extracted samples. • Stabilities on the incurred samples were evaluated. • Results • The results of the ISR showed a positive bias for N-desmethyltrimebutine concentrations. • An increase was observed when incurred samples were re-injected over time. • Precursor Ion Scan on freshly extracted incurred samples enable the detection of an unknown conjugated phase II metabolite. • This metabolite proved to be unstable in the reconstitution solution, as well as in human plasma. • A hydrolysis procedure was developed to quantitate total N-desmethyltrimebutine. • SAMPLE PREPARATION • Protein precipitation with acetonitrille (ACN) 1:4 using stabled isotope labeled (SIL) internal standard (IS). The supernatant is further diluted with ACN:H2O 60:40% v/v to match the mobile phase composition. • CHROMATOGRAPHY • HPLC Agilent Technologies Series 1100 pumps and autosampler • Analytical Column: Waters XBridge C18 (30X2.1)mm, 3.5µ • Reverse phase isocratic elution at high pH • Flow rate: 0.45 mL/min • Runtime: 3.00 minutes • DETECTION • Turbo Ion Spray ESI(+) • AB SCIEX LC-MS/MS API3000 One explanation for such an increase in concentration over-time is the conversion of an unknown metabolite in the extracted samples. In order to verify this theory, precursor ion scans of m/z 343 were performed on freshly extracted incurred samples vs. incurred samples with a long post-extracted stability (Figure 2). Table 5: Incurred Sample Reproducibility Results for N-Desmethyltrimebutine (with hydrolysis) In order to stabilize the glucuronide in the reconstitution solution, different organic composition as well as different pH were tried. The results are presented in Table 3 and showed that the best conditions were in 100% ACN, although the metabolite still showed conversion over-time. Figure 2: Precursor Ion Scan of m/z 343 Freshlyextracted incurred sample: Same sample ~72 hours post-extract: Table 3: Reconstitution solution stability for N-Desmethyltrimebutine N-glucuronide RESULTS The ISR results showed a positive trend for N-desmethyltrimebutine (Table 1) but not for trimebutine. Accordingly, this trend could not be attributed to any manipulation errors and further investigation was needed. INTRODUCTION Trimebutine is a non-competitive spamolytic agent used in the treatment of irritable bowel syndrome. It is extensively metabolized through ester hydrolysis or N-demethylation, where N-desmethyltrimebutine is the main active metabolite observed in human plasma. Furthermore, glucuronide conjugation of the hydrolysis product is also reported in the literature, although direct conjugation of trimebutine or its demethylated products is not mentioned. This case study demonstrates the presence of a never before identified trimebutine N-glucuronide metabolite. This conjugated metabolite proved to be highly unstable in human plasma and post-extraction. The reproducible quantification of total N-desmethyltrimebutine was achieved through acidic hydrolysis of the conjugated metabolite. Table 6: Long-Term (11 Days at -80ºC), Short-Term (11.6 hours at 4ºC) and Freeze-Thaw (4 cycles) Combined Stabilities in human Plasma for N-Desmethyltrimebutine Table 1: Incurred Sample Reproducibility Results for N-Desmethyltrimebutine These scans enable the detection of a mass at m/z 550 in freshly extracted samples, which decreased over-time in post-extracted incurred samples. This peak corresponds to N-desmethyltrimebutine plus a glucuronide moiety (374 amu + 176 amu). The fragmentation pattern of the newly discovered metabolite shows similar product ions as N-desmethyltrimebutine, thus suggesting that the glucuronide functionality is attached to the amine group (Figures 3 and 4). Furthermore, some incurred samples were tested for short-term and long-term stabilities in human plasma and the results demonstrated that the metabolite is not stable at 4ºC for 8 hours or at -20ºC and converted to N-desmethyltrimebutine (Table 4). Table 4: Long-Term and Short-Term Conversion of N-desmethyltrimebutine N-Glucuronide in Human Plasma CONCLUSION Figure 1: Trimebutine and N-Desmethyltrimebutine Structures • A failure in ISR evaluation enabled the identification of a never-before-identified N-glucuronide metabolite for trimebutine impacting the quantitation of N-Desmethyltrimebutine due to its extreme instability. • This discovery may raise concerns on previously performed metabolism studies for trimebutine since the analytically detected levels of the main active metabolite (N desmethyltrimebutine) may be significantly impacted by the decomposition of its unstable conjugated metabolite. Upon multiple re-injections of subject samples batches, a correlation was suspected between the time post-extraction and the observed increase in N-desmethyltrimebutine concentrations. To confirm this behavior, freshly extracted subject samples, along with a calibration curve and QCs, were injected repeatedly over a 27 hours period. The results showed that the concentration in QC samples remained constant whereas the concentrations in incurred samples increased by as much as 40% (Table 2). Figure 3: Proposed Structure for N-Glucuronide Metabolite * CORRESPONDING AUTHOR