1. * CORRESPONDING AUTHOR Glucagon Bioanalysis by LC-MS: “Unprecedented Level of Sensitivity (10pg/mL) for a Novel Formulation” Jean-Nicholas Mess 1, Louis-Philippe Morin 1, Mauro Aiello 2, Xavier Misonne 2, Gary Impey 2, Johnny Cardenas 2, Josée Michon 1 and Fabio Garofolo 1* 1 Algorithme Pharma Inc., Laval (Montréal), QC, Canada 2 AB SCIEX, Concord, ON, Canada INTRODUCTION CONCLUSION Ligand Binding Assay (LBA) is currently the most common approach for Large Molecule quantification. However, LBA may suffer from cross reactivity, lack of specificity and selectivity for very low quantitation analysis. In the past several years, LC-MS based assays showed to be as effective and also complementary to LBA for Large Molecule quantification. Currently, we are working on a study for a novel formulation of glucagon, which should be easier to administer than the currently available formulations, resulting in a higher standard of Type 1 diabetes patient care. As previously published by our group, a bioanalytical method for glucagon in human plasma (lower limit of quantification (LLOQ): 100 pg/mL) was validated under FDA guidelines on an QTRAP®5500 mass spectrometer. However, this LLOQ is not adequate enough to cover the pharmacokinetic profile of this novel glucagon formulation. Therefore, the method was re-developed on two different instruments (QTRAP®6500 and TripleTOF TM 5600) to reach higher level of selectivity and sensitivity. The present work describes the challenges and solutions encountered using novel mass spectrometers to reach unprecedented level of sensitivity for the analysis of low plasma concentration of glucagon. METHODS OVERVIEW PurposePurpose –The challenging development of an LC-MS/MS method for the quantification of glucagon at the low pg/mL level to support bioanalysis of a new formulation. MethodMethod –Samples were extracted by SPE and analysed by LC-MS on an AB SCIEX QTRAP®5500, QTRAP®6500 and TripleTOF TM 5600 operated in ESI+ –The triple quadrupoles were used in MRM (unit resolution) while the Q-ToF was used in TOF MS (30K Resolution) or MRM HS (15K resolution) ResultsResults –On the Q-ToF, the use of a targeted approach (MRM HS ) led to optimal sensitivity over generic TOFMS mode, but did not allow to decrease the validated LOQ (100 pg/mL) achieved on the API5000 TM. – On the QTRAP®6500, the LLOQ of 10 pg/ml was achieved with good precision and accuracy. SAMPLE EXTRACTION Range of 10 – 10 000 pg/mL in human plasma 225 µL of sample extracted by solid phase extraction using Oasis µElution MAX 96 well plates.CHROMATOGRAPHY Agilent Technology Series 1100 pumps and autosampler Zorbax 300 SB-C18, 50x2.1mm, 3.5µm Gradient elution of 0.1% HCOOH and ACN 4.0 minutes run timeDETECTION Triple Quadrupoles: AB SCIEX QTRAP®5500 and QTRAP®6500 MRM mode ESI(+) (unit resolution) The [M+5H] 5+ (m/z 697.5 → 694.1) was monitored Q-ToF AB SCIEX TripleTOF TM 5600 TOF MS mode (ESI+) 100 – 1000 m/z, 100 ms accumulation Mass extraction window: 20 mDa MRM HS mode (ESI+) Q1 set at 697.5 ([M+5H] 5+ ) Sum of 3 most abundant isotopes of [M+5H-NH 3 ] 5+ product ion Mass extraction window: 20 mDa Figure 2A Figure 2B The TripleTOF TM 5600 was tested in different acquisition modes to determine optimal conditions. First, the full scan TOFMS mode (30K resolution) was evaluated. This mode is generic and is the easiest to use. The 3 most abundant isotopomers of the +5 charged state of glucagon were summed using a mass extraction window (MEW) of 20mDa (Figure 2A). Moreover, this acquisition mode also allows to sum different charged states in order to increase sensitivity and assay ruggedness. The 3 most abundant isotopomers of each of the +3 to +5 charged states were summed using a MEW of 20 mDa (Figure 2B). This led to a 2 fold increase in sensitivity. Table 2: Glucagon Precision and Accuracy by MRM on QTRAP®6500 Figure 3: Representative Chromatograms of a Glucagon on QTRAP®6500 RESULTS ACKNOWLEDGEMENTS The authors would like to acknowledge Suma Ramagiri from AB Sciex for her support and valuable advice during the completion of the TripleTOF TM 5600 experiments. Figure 1: Structure of Glucagon S/N = 254 S/N = 29 S/N = 62 A) TOF MS: Sum of the 3 Most Intense Isotopomers of the +5 Charged States : MEW 20 mDa B) TOF MS: Sum of the 3 Most Intense Isotopomers of Each of the +3 to +5 Charged States : MEW20 mDa B) TOF MS: Sum of the 3 Most Intense Isotopomers of Each of the +3 to +5 Charged States : MEW 20 mDa C) MRM HS : Sum of the 3 Most Intense Isotopomers of Product Ion [M+5H-NH] 5+ : MEW20 mDa C) MRM HS : Sum of the 3 Most Intense Isotopomers of Product Ion [M+5H-NH 3 ] 5+ : MEW 20 mDa Figure 2: TripleTOF TM 5600 Acquisition Mode Evaluation on a Mid-QC of Glucagon (2000 pg/mL) Table 1: Glucagon Precision and Accuracy by MRM HS on TripleTOF TM 5600 Figure 2C Finally, MRM HS mode (15K resolution), which can be considered as a targeted approach and resemble standard MRM experiment performed on a triple quadrupole, was also evaluated and the 3 most abundant isotopomers of the +5 charge state product ion (neutral loss of NH 3 ) were summed (Figure 2C). Over 4-fold gain in sensitivity was obtained in MRM HS compared to TOF MS mode which allowed to reach an LLOQ of 100.0 pg/mL. Table 1 Overall, the data showed that it is possible to have the same sensitivity and performance on a TripleTOF TM 5600 as on an QTRAP®5500 but not to reach the targeted LLOQ of 10 pg/mL. Precision and Accuracy for Glucagon obtained on the TripleTOF TM 5600 is shown in Table 1. Figure 3 Figure 4 Table 2 The validated assay was simply transferred from the QTRAP®5500 to QTRAP®6500 to evaluate its performance. The QTRAP®6500 was able to reach an LLOQ of 10 pg/ml which is 10 times lower than our validated LLOQ on QTRAP®5500 (Figure 3). The calibration curve was linear (weighted 1/x 2 ) over three orders of magnitude with a coefficient of correlation of 0.9990 (Figure 4). Precision and Accuracy for Glucagon obtained on the QTRAP®6500 for a range of 10.0 to 10 000.0 pg/mL is shown in Table 2. Figure 4: Calibration Curve for Glucagon Regression type: Linear 1/x 2 Coefficient Correlation: r = 0.9990 Peak Area Ratio A) Extracted Blank B) Extracted LLOQ (10.0 pg/mL) C) Extracted ULOQ (10000.0 pg/mL) This research demonstrated that: The TripleTOF TM 5600, when specifically used for targeted quantification (MRM HS ) can match, but not exceed the sensitivity of the QTRAP®5500 triple quadrupole. The QTRAP®6500 significantly improved the sensitivity of our assay in order to reach an LLOQ of 10.0 pg/mL The outcome of this research showed that it is possible to develop an LC-MS/MS method more sensitive than the standard LBA for the challenging quantification of Glucagon at low pg/mL level.