* C ORRESPONDING AUTHOR

1. Use of High Resolution Mass Spectrometry (HRMS) to Solve Severe Issues Due to Isotopic Distribution in Regulated Bioanalysis Richard Lavallée, Nicolaos Soilis, Jean-Nicholas Mess and Fabio Garofolo* Algorithme Pharma Inc., Laval (Montreal), QC, Canada Intensity (cps) Linear (weighted 1/x2) r2 = 0.9945 Table 4: Precision and accuracy of aripiprazole in presence of dehydro-aripiprazoleusing a TripleTOFTM5600 high resolution MS Aripiprazole Dehydro-aripiprazole Aripiprazole Dehydro-aripiprazole 37Cl isotope Figure 4: Representative chromatograms of extracted blank and LLOQ (0.500 ng/mL) of aripiprazole using a TripleTOFTM5600 high resolution MS Figure 2: Representative chromatogram of aripiprazole (blue), aripiprzole-D8 (red) and dehydro-aripiprzole (green) Table 1: Isotopic contribution at 1:1 using a API5000TM triple quadrupole mass spectrometer METHODS OVERVIEW • SOLUTION PREPARATION • For isotopic contribution determination, all drugs and stable labeled internal standards solutions were prepared separately at a concentration of 100ng/mL. • SAMPLE EXTRACTION • Aripiprazole, dehydroaripiprzole and aripiprazole-D8 were extracted by LLE from human plasma samples. • Analytical range: 0.500 to 150.00 ng/mL • CHROMATOGRAPHY • Agilent Technologies Series 1100 • Ascentis Express C18, (30 X 2.10) mm, 2.7µm • 10mM HCOONH4 pH 4.0 : MeOH 45:55% v/v • 1.50 minutes run time • DETECTION • AB SCIEX API5000TM operated in MRM mode • TripleTOFTM5600 operated in full scan TOFMS mode at 30K resolution • Electrospray positive ionization for both systems • Purpose • To demonstrate the advantages of using HRMS over triple quadrupole mass spectrometers to solve bioanalytical issues related to isotopic distribution in a regulated environment. • Method • Isotopic contribution of drugs on their stable labeled internal standards was evaluated in solution prepared at equimolar concentrations. • Aripiprazole precision and accuracy was evaluated in plasma extracts spiked with and without dehydro-aripiprazole. • Results • HRMS was successful in reducing the isotopic contribution of drugs to their stable labeled internal standards. • Quantification of aripiprazole on a triple quadrupole mass spectrometer showed inaccuracy for samples containing dehydro-aripiprazole. • Accuracy of these samples was maintained on the Q-TOF due to its high resolving power. Blank Table 2: Isotopic contribution at 1:1 using a TripleTOFTM5600 high resolution MS Table 3: Precision and accuracy of aripiprazole in presence of dehydro-aripiprazoleusing a API5000TM triple quadrupole LLOQ Figure 5: Calibration curve of aripiprazole using a TripleTOFTM5600 high resolution MS On the HRMS system, the resolution of 30K was still too low to allow full resolution of aripiprazole from dehydro-aripiprazole 37Cl isotope (Figure 3). Nevertheless, using a narrow extraction window of 10mDa, the isotopic contribution was kept below 5%. The same samples previously injected on the API5000TM were re-analyzed by HRMS on the TripleTOFTM5600. The data presented in Table 4 demonstrate that HRMS was able to solve a bioanalytical issue where the triple quadrupole failed. As per quantification data, the system was proven to be sensitive and linear (Figures 4 and 5) RESULTS: ISOTOPIC CONTRIBUTION OF DRUG AND INTERNAL STANDARD INTRODUCTION Interference due to isotopic distribution is a common issue bioanalytical scientists must deal with during LC-MS/MS method development. As an example, clopidogrel contributes to its internal standard clopidogrel-D3 at 7% (theoretical value, unit resolution MS). Therefore, the ratio of drug and IS must be carefully adjusted to avoid any bias in quantification. Furthermore, some dehydrogenated metabolites may interfere with the parent drug quantification due to isotopic distribution. This is the case for dehydro-aripiprazole which contributes to aripiprazole at 68.5% (theoretical value, unit resolution MS). In most cases, specific action must be taken to circumvent these issues, which is time consuming. In this study, HRMS will be evaluated as a powerful tool to easily solve bioanalytical issues caused by isotopic distribution. Three different drugs and their corresponding stable labeled internal standards were used to evaluate if HRMS can solve typical interference from isotopic distribution observed on triple quadrupole mass spectrometer. The isotopic contribution from clopidogrel to clopidogrel-D3 was determined to be 3.57% when using a triple quadrupole. This can be explained since unit resolution cannot distinguish between clopidogrel-13C37Cl isotope (m/z 325.0673) and clopidogrel-D3 (m/z 325.0857). However, when analyzed under HRMS on the TripleTOFTM5600 (30K resolution, 10mDa XIC), this contribution dropped by a factor of 5.5 times (0.65%). Similarly, the isotopic contribution from lamotrigine to lamotrigine-13CD3 was determined to be 13.21% on a triple quadrupole while it decreased by 13 times (1.00%) in HRMS (10mDa XIC). Narrowing the mass extraction window to 5 mDa further decreased even further the contribution to 0.31%. However, for riluzole and riluzole-13C15N2, where isotopic contribution was already low on a triple quadrupole (0.41%), HRMS did not succeed to lower this value (0.50% at 10 mDa XIC). Data are from isotopic distribution experiments are presented in Table 1 and Table 2. RESULTS: ISOTOPIC CONTRIBUTION OF DRUG AND METABOLITE Using LC-MS/MS on a triple quadrupole, chromatographic separation is usually needed when metabolites can isotopically contribute to the parent drug. This is the case for aripiprazole and dehydro-aripiprazole (Figure 1) where dehydro-aripiprazole theoretically contributes to aripiprazole at 68.5% (unit resolution MS). A calibration curve of aripiprazole and quality control (QC) samples (fortified with and without dehydro-aripiprazole at equimolar concentration) were extracted and injected on an API5000TM. The chromatographic conditions were adjusted in order to have co-elution of aripiprazole and dehydro-aripiprazole (Figure 2). The data was acceptable in term of linearity (linear weighted 1/x2 regression) and showed precision and accuracy for the QC samples without dehydro-aripiprazole; however, the low and high QC spiked with dehydroaripiprazole had nominal values of 148.3% and 145.8%, respectively, due to isotopic the contribution of dehydro-aripiprzole (Table 3). Figure 3: Overlay of HRMS scans of aripiprzole (blue) and dehydro-aripiprazole (red) Figure 1: Aripiprazole and Dehydro Aripiprazole CONCLUSION Aripiprazole C23H27Cl2N3O2 m/z 448.1559 Dehydro Aripiprazole C23H25Cl2N3O2 m/z 446.1402 In conclusion, we have shown that HRMS can simply and efficiently solve typical bioanalytical issues caused by isotopic contribution. Moreover, the HRMS was able to deliver acceptable quantification performance in regards to sensitivity, linearity and precision and accuracy. * CORRESPONDING AUTHOR