* C ORRESPONDING AUTHOR

1. An effective approach to reduce the impact of Raloxifene glucuronide In-Source/Interface Conversion on Raloxifene quantification by LC-MS/MS Eugénie-Raphaëlle Bérubé, Sylvain Latour, Milton Furtado and Fabio Garofolo* Algorithme Pharma Inc., Laval (Montreal), Quebec, CANADA Raloxifene MRM: 474.2/112.2 No in-source/interface conversion of glucuronide to raloxifene Raloxifene glucuronide MRM: 650.2/112.2 Raloxifene Raloxifene-4’-Glucuronide Raloxifene-6-Glucuronide Raloxifene-6,4’-Diglucuronide Raloxifene MRM: 474.2/112.2 Raloxifene glucuronide MRM: 650.2/112.2 Raloxifene MRM: 474.2/112.2 Raloxifene glucuronide MRM: 650.2/112.2 Figure 4: Raloxifene and its glucuronides metabolites chromatographic elution profile under HILIC condition when reconstituted with 100% ACN. OVERVIEW As a consequence, the precision of raloxifene quantification is affected throughout the batches. In Table 1, the impact can be observed on the % deviation at multiple QC levels. Furthermore, the presence of such interferences induces high % CV values, which can lead to a rejected batch. To overcome this issue, a more laborious extraction is needed to selectively extract the parent compound. Another option is to use a gradient for the elution of the glucuronides; however, the time required for column re-equilibration will lower the throughput of the assay. METHOD Figure 2. Raloxifene and its glucuronides metabolites chromatographic elution profile under HILIC condition when reconstituted with mobile phase. Two sets of calibration curves of raloxifene in plasma (10.00 to 1000.00 pg/mL) and QC samples containing raloxifene 4-glucuronide, raloxifene-6-glucuronide and raloxifene 4,6-diglucuronide (Figure 1) were extracted using a protein precipitation with MeOH and evaporated to dryness. One set was reconstituted with mobile phase while the other was reconstituted with 100% ACN. • Purpose • Overcome in-source/interface CID conversion of raloxifene glucuronide metabolites to the parent drug by selecting a reconstitution solution in which the glucuronides are not soluble. • Method • Calibration curves of raloxifene in plasma and QCs containing raloxifene glucuronides were extracted by precipitation of proteins using MeOH. • The supernatant was evaporated to dryness and then reconstituted with mobile phase or with 100% ACN. • The impact of the in-source CID conversion of raloxifene glucuronides on raloxifene quantification was evaluated and compared for both reconstitution solutions. • Results • The calibration curve and QC’s reconstituted with the mobile phase are impacted by the late elution of the raloxifene glucuronide, when no impact is noticed for samples reconstituted with ACN. Raloxifene MRM: 474.2/112.2 In source/interface conversion of glucuronide to raloxifene Raloxifene Raloxifene glucuronide MRM: 650.2/112.2 Table 1: Raloxifene calibration curve and precision table when reconstituted with mobile phase. Raloxifene 4’-glucuronide Raloxifene 6’-glucuronide Raloxifene 4’-glucuronide Accordingly, the high amount of glucuronides metabolites present in subject samples can affect raloxifene quantification of subsequent injected samples if co-elution with raloxifene occurs. The chromatograms presented inFigure 3, demonstrate the impact of theses metabolites on a Low QC chromatogram (upper pane), compared to the expected chromatogram of the same QC level, when no interferences is observed. Table 2: Raloxifene precision table when reconstituted with 100% ACN . Figure 3: Raloxifene QC5 sample chromatograms when reconstituted with the mobile phase. Figure 1: Structures of Raloxifene and glucuronides INTRODUCTION a) QC sample affected by in-source CID of glucuronide from previous injection. CHROMATOGRAPHY: -HPLC Agilent Technologies 1100 Series -Waters Atlantis HILIC (30 x 2.10)mm, 3 μm -Isocratic mobile phase of 20mM ammonium formate pH 3.0 and ACN. Raloxifene, an oral selective estrogens selector modulator, undergoes extensive first-pass metabolism by glucuronidation. The presence of these conjugates in blood may impact the bioanalytical assay and result in more laborious extraction procedure or chromatographic conditions. Additionally, using HILIC chromatography, which allows for more sensitive detection of raloxifene, can become difficult to manage for the glucuronides as they elute later. Since glucuronides are well retained in HILIC mode, the possible back conversion to the parent drug by in-source collision induced dissociation (CID) may affect the subsequent injection. In the present work, we described how the use of the appropriate reconstitution solution can eliminate the presence of glucuronides in the post-extracted sample and overcome their impact on raloxifene quantification due to their in-source conversion to the parent compound. DETECTION: -Positive mode ESI(+)/MS/MS on AB SCIEX API5000. The evaluation of raloxifene glucuronide in-source conversion impact on raloxifene was evaluated by monitoring their respective SRM over 10 minutes in isocratic mode. Elution of Raloxifene 6’-glucuronide On the other hand, using 100% ACN as reconstitution solution did not allow to solubilize the glucuronides, since they are poorly soluble in this media. As an effect, they are not present or present in a much lower concentration in the post extracted samples. Therefore, no impact from the late eluted glucuronide in-source/interface conversion is observed; in Figure 4, the presence of glucuronide or diglucuronide is much lower than that observed in Figure 2. Their presence is so insignificant, that no in-source conversion is observed at the SRM transition of raloxifene. As a result, the run time of the assay can be shortened to 2 minutes, improving the throughput of the method. Consequently as demonstrated in Table 2, the results obtained when using ACN as reconstitution solution establishes better precision. b) QC sample not affected by in-source CID of glucuronide from previous injection. RESULTS The HILIC chromatography gave good retention and sensitivity for raloxifene with a 2 minute run time. However, when the QC samples containing the glucuronides were reconstituted with the mobile phase, the in-source CID interfered with the chromatography of raloxifene. As demonstrated in Figure 2, the glucuronide compounds elutes until 10 minutes and the in-source/interface conversion can impact the quantitation of raloxifene, producing a bias. CONCLUSION In conclusion, an easy approach such as investigating the solubility properties of the glucuronides can be preferable to laborious extraction or longer chromatography when dealing with the presence of these metabolites. * CORRESPONDING AUTHOR