For Research Use Only. Not for Use In Diagnostic Procedures. Drug Discovery and Development SCIEX Sub-Picogram Level Quantitation of Desmopressin in Small Volumes of Human Plasma Using a Trap-Elute Microflow Using the QTRAP® 6500+ System with OptiFiow™ Turbo V Source and M5MicroLC System Rahul Baghla, Khatereh Motamedchaboki, Remco van Soest and Lei Xiong SCIEX, Redwood City, California, USA Desmopressin is a synthetic analog of vasopressin, a natural pituitary hormone with antidiuretic properties. The deamination of vasopressin in the N-terminal 1 position and the replacement of 8-l-arginine with 8-d-arginine result in the formation of desmopressin. It has a longer duration of antidiuretic activity than that of the natural hormone and is essentially devoid of other associated pharmacological effects such as vasoconstriction and contraction of smooth muscles in the uterus or in the intestine1. Therapeutically, desmopressin reduces urine production, restricts water elimination from the kidneys by binding to the Vasopressin V2 receptors (V2R) in renal-collecting ducts, thereby facilitating increased water reabsorption. The longer halflife of desmopressin over vasopressin offers additional therapeutic advantages, and typical doses of desmopressin to treat diabetes insipidus and bedwetting range between 0.200 to 1.20 mg per day, resulting in very low plasma concentrations. The sub picogram/mL quantitation of desmopressin in human plasma using an analytical flow HPLC methodology was published in a previously described method2, in which 1 mL human plasma was used for desmopressin quantification at 0.5 pg/mL. In this current work, microflow LC combined with the OptiFlow Turbo V Source was used to quantitate the desmopressin at the same level 0.5 pg/mL in human plasma with 3.3 times less consumption of plasma samples and injecting 3.3x less volume to ensure enough sample for 5 reinjections. A similar LLOQ was observed while injecting 7x less sample on column compared to previous analytical flow assay2. Key Features of the SCIEX Microflow LC-MS/MS Solution • M5 MicroLC system provides: - Microfluidic flow control for accurate flow rates down to 1 pL/min - Trap-elute option for fast and large volume sample loading - Flexibility to couple with any microflow LC column • OptiFlow™ Turbo V Source on the QTRAP® 6500+ LC-MS/MS system provides: - Easy setup with no probe or electrode position optimization - Robust performance and long electrode lifetime

For Research Use Only. Not for Use In Diagnostic Procedures. Methods Sample Preparation: The sample preparation method is modified from the previously published technical note2 to obtain cleaner extracts. Desmopressin spiked human plasma samples in the range from 0.5 to 250 pg/mL, with 25 pg/mL of internal standard were extracted using weak cation exchange cartridges (WCX, Waters). Cartridges were conditioned with 1 mL of methanol followed by 1 mL of 100 mM ammonium acetate in water. 0.3 mL of spiked plasma mixed with 0.3 mL of 5% acetic acid in water was loaded on the pre-conditioned cartridge....

For Research Use Only. Not for Use In Diagnostic Procedures. Source/Gas Value Parameter Table 3. MS Conditions for Microflow Analysis. Name Desmopressin_11 Desmopressin_2 Desmopressin_d52 Source/Gas Parameter Curtain gas: Ion source gas 1: Ion source gas 2: LC-MS Conditions for Analytical Flow Analysis: To identify the sensitivity difference between analytical flow and microflow analysis, each sample was analyzed using a QTRAP 6500+ system coupled with an ExionLC™ AD HPLC system. Table 4 describes the liquid chromatography conditions for analytical flow analysis. The MRM parameters are identical...

Figure 4. Extracted Ion Chromatograms of Desmopressin in Extracted Human Plasma using Microflow LC. Data is shown for the A) double blank; B) blank; C) 0.5 pg/mL; D) 1.0 pg/mL; e) 2.5 pg/mL f) 5.0 pg/mL. To determine the sensitivity difference between the microflow and analytical flow analysis, the same set of samples were analyzed with both microflow and analytical flow LC-MS systems with the same injection volume. Figure 6. Comparing Analytical Flow Results to the Microflow Results. Extracted ion chromatograms of desmopressin and desmopressin d-5 in extracted human plasma are shown. Analytical...

References 1. Gudlawar SK., Pilli NR., Siddiraju S., & Dwivedi J. (2017). Highly sensitive assay for the determination of therapeutic peptide desmopressin in human plasma by UPLC-MS/MS. Journal of Pharmaceutical Analysis, 7(3), 196-202. http://doi.org/10.1016/i.ipha.2013.11.002 2. Baghla R., Guttikar S., et al. A Sub-picogram quantification method for desmopressin in plasma using the SCIEX Triple Quad™ 6500 System, SCIEX Technical Note AB Sciex is doing business as SCIEX. © 2018 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property...