1. Intensity (cps) Time (min) Intensity (cps) * CORRESPONDING AUTHOR Challenge in Trying to Reach Femtogram per Milliliter (fg/mL) Sensitivity in Plasma for the Quantification of a Cyclic Peptide: Desmopressin Louis-Philippe Morin 1, France Landry 1, Jean-Nicholas Mess 1, Kelli Jonakin 2, Mauro Aiello 2, Xavier Misonne 2, Gary Impey 2, Johnny Cardenas 2 and Fabio Garofolo 1* Louis-Philippe Morin 1, France Landry 1, Jean-Nicholas Mess 1, Kelli Jonakin 2, Mauro Aiello 2, Xavier Misonne 2, Gary Impey 2, Johnny Cardenas 2 and Fabio Garofolo 1 * 1 Algorithme Pharma Inc., Laval (Montréal), QC, Canada 2 AB SCIEX, Concord, ON, Canada INTRODUCTION CONCLUSION Desmopressin is a synthetic cyclic peptide that is closely related to the human hormone arginine vasopressin. It is an antidiuretic used for the treatment of diabetes insipidus and nocturnal enuresis. It acts by increasing water reabsorption in the renal collecting ducts of the kidneys and therefore reduces urine production. LC-MS/MS assays for the quantification of desmopressin generally involve laborious extraction and chromatographic procedures in order to achieve low pg/mL quantification limits. Moreover, large plasma sample volumes (1mL) are often required for the analysis, which is not always compatible with pre-clinical studies in animals or raises safety concerns for human studies. In this research, the IonDrive™ technology is used to reach low pg/mL sensitivity and to attempt fg/mL quantification. METHODS: SENSITIVITY COMPARISON OVERVIEW PurposePurpose –Use of IonDrive™ technology to attempt desmopressin lower limit of quantification (LLOQ) at fg/mL level. MethodMethod –Desmopressin was extracted from plasma by SPE –Sensitivity comparison was performed on a QTRAP®5500 and QTRAP®6500. ResultsResults –An LLOQ of 2 pg/mL was reached for the quantification of desmopressin in human plasma on the QTRAP®5500. –The QTRAP®6500 was found 4 times more sensitive for the detection of desmopressin. SAMPLE Desmopressin was spiked post extraction in crashed plasma matrixCHROMATOGRAPHY Agilent Technologies Series 1100 pumps and autosampler (QTRAP®5500) and Shimadzu LC20AD (QTRAP®6500) Zorbax 300SB-C18, 50x2.1mm, 3.5µm Gradient elution of 0.1% HCOOH and ACNDETECTION AB SCIEX QTRAP®5500 and QTRAP®6500 MRM mode ESI(+) The [M+2H] 2+ (m/z 535.4 → 328.2) was monitored for desmopressin Figure 5: LOD and LLOQ on the QTRAP® 6500 RESULTS: DESMOPRESSIN BIOASSAY In order to evaluate if a novel generation of triple quadrupole mass spectrometer would allow to reach fg/mL level of sensitivity for the quantification of demopressin, standards were prepared in crashed plasma matrix which was found to reduce adsorptive losses. Post-extraction spiked approach was used to limit any extraction variability and therefore to allow for an unbiased comparison between the QTRAP®5500 and the QTRAP®6500. For sake of comparison, the samples were first injected on a QTRAP®5500 under generic LC ‑ MS/MS parameters. The lower limit of detection (LOD) was found to be 80 fg on- column, which had a signal-to-noise ratio (S/N) of 4.5 with a coefficient of variation (%CV) of 13.1% (n=4). This was found too variable for quantification and therefore, the lower limit of quantification was set at 160 fg on-column (S/N of 8.4 and %CV of 2.5, n=4). According to the above results, the use of the IonDrive™ technology combined with an in-house-developed specific clean & selective extraction/enrichment can allow the quantification of desmopressin in plasma at fg/mL level. An LLOQ of 500 fg/mL could be obtained on the QTRAP®6500 with as low as 250µL of plasma sample. Figure 1: Structure of Desmopressin A method for the quantification of the cyclic peptide Desmopressin was developed in our laboratory using as low as 250 µL of plasma samples. This method utilises a specific and selective extraction on Oasis µELution plate which allows to concentrate the analyte up to 3.3 fold. Figure 2 Figure 3 Table 1 Using this extraction procedure and fast reversed phase chromatography, an LLOQ of 2.00 pg/mL was achieved within 3 minutes run time on a QTRAP®5500 (Figure 2). The assay was linear (weighted 1/x 2 ) over a range of 2.00 to 250.00 pg/mL (Figure 3). Precision and accuracy meet acceptance criteria (Table 1) Figure 4: LOD and LLOQ on the QTRAP® 5500 METHODS:DESMOPRESSIN BIOASSAY SAMPLE EXTRACTION Range of 2.00 – 250.00 pg/mL in plasma 250 µL of sample extracted by solid phase extraction using Oasis µElution SPE. Samples were eluted in a final volume of 75µL and 60µL were injected.CHROMATOGRAPHY Agilent Technologies Series 1100 pumps and autosampler XBridge BEH300 C18, 50x2.1mm, 3.5µm Gradient elution in 2.4 minutes run timeDETECTION AB SCIEX QTRAP®5500 MRM mode ESI(+) The [M+2H] 2+ was monitored for desmopressin (m/z 535.4 → 328.2) and for the ISTD vasopressin (m/z 542.9 → 328.2). Figure 2: Representative Chromatograms of Extracted Desmopressin Samples Injected on the QTRAP®5500 A) Extracted Blank B) Extracted LLOQ (2.00 pg/mL) C) Extracted ULOQ (250.00 pg/mL) Intensity (cps) Regression type: Linear 1/x 2 Coefficient Correlation: r = 0.9953 Concentration (pg/mL) Peak Area Ratio CalibrantNominal pg/mL Back Calculated pg/mL Deviation % P12.001.92-4.2 P24.004.215.1 P35.005.295.8 P412.5011.92-4.6 P537.5035.58-5.1 P662.5069.1910.7 P7100.00102.312.3 P8175.00164.98-5.7 P9212.50199.95-5.9 P10250.00253.871.5 Figure 3: Calibration Curve for Desmopressin Concentration (pg/mL) LOQ QC 2.00 Low QC 6.00 Mid QC 50.00 High QC 187.50 1.916.8653.80179.56 2.105.3449.53184.50 2.075.4750.93186.38 1.745.7853.03181.11 1.986.2952.51170.19 2.21---51.47175.70 Mean2.005.9551.88179.57 S.D.0.160.631.555.93 N6566 % C.V.8.210.53.03.3 % Nominal100.099.2103.895.8 Table 1: Within Run Precision and Accuracy for Desmopressin ACKNOWLEDMENTS Algorithme Pharma would like to thank AB Sciex for their availability and support throughout the QTRAP®6500 performance evaluation. RESULTS: SENSITIVITY COMPARISON 80 fg on-column S/N = 4.5 %CV 13.1 160 fg on-column S/N = 8.4 %CV 2.5 Intensity (cps) Time (min) Table 2. The exact same samples were analyzed on a QTRAP®6500 to evaluate the IonDrive™ technology. The lower limit of detection (LOD) was determined as 20 fg on-column which had a S/N of 4.6 with a coefficient of variation (%CV) of 11.8% (n=4). The lower limit of quantification was set at 40 fg on- column (S/N of 8.1 and %CV of 6.0, n=4). Data are summarized in Table 2. Therefore, in the case of desmopressin, the QTRAP® 6500 was deemed 4 times more sensitive than the previous generation of tandem mass spectrometer. Table 2: Sensitivity Comparison Between QTRAP®5500 and QTRAP®6500 Desmopressin load on-column QTRAP® 5500QTRAP® 6500 S/N%CVS/N%CV fg n = 4 205.311.8 408.16.0 804.513.1 1608.42.5 20 fg on-column S/N = 4.6 %CV 11.8 40 fg on-column S/N = 8.4 %CV 6.0