(T1230-04-24) SPE-LC/MS Bioanalytical Quantification of the Biotherapeutic Peptide, Semaglutide from Plasma

Purpose: The work described here uses a selective sample preparation strategy and LC-MS compatible sample storage plates with high performance surfaces to mitigate semaglutide loss due to non-specific binding. In addition to this, we will show that optimized UPLC separation and column chemistry, along with a highly sensitive tandem quadrupole mass spectrometry method can produce significantly lower limits of quantification.

Methods: LC-MS/MS analysis was performed using a Xevo™ TQ-XS Tandem Quadrupole MS (ESI+) and chromatographic separation using an ACQUITY I-Class PLUS UPLC System and ACQUITY UPLC Peptide CSH C18 100 Å, 1.7 um 2.1 mm x 50 mm (p/n: 186005296), at a column temperature of 65°C. A flow rate of 0.3 mL/min using 0.1% formic acid in water (MP A) and 0.1% formic acid in acetonitrile (MP B) was employed. Starting LC conditions 65% mobile phase A and 35% mobile phase B were held for 0.25 minutes, followed by an increase to 75% MP over 2.75 minutes. MP B was then increased to 90% to flush the column and returned to starting gradient conditions at 3.4 minutes. Total analysis time was 5 minutes. The injection volumes of the extracted samples were 10 µLs. Sample Preparation & Extraction: Semaglutide was added to commercially available rat plasma to prepare calibration curve and quality control (QC) samples. Samples concentrations were between 0.120–1,000 ng/mL. Standard curve and QC plasma samples were prepared in triplicate prepared in protein low bind 1.5 mL polypropylene tubes. Sample extraction used a 1:1 protein precipitation step with methanol followed by reversed-phase SPE in the 96-well µElution format. A 3-step (load, wash, and elute) SPE protocol was employed, whereas the entirety (600 µL) of the diluted 2:1 PPT supernatant samples were loaded onto the extraction plate, followed by a 200 µL wash with 5% methanol. A 2x25 µL elution of the purified sample was performed using a 75/25 acetonitrile/water solution containing 5% formic acid, collected in a QuanRecovery 96-well collection plate. Subsequent LC-MS analysis of the SPE extracted sample was performed.

Results: Optimization of chromatography was essential to the success of this assay. During method development of semaglutide, with evaluation focused on an ACQUITY UPLC Peptide BEH C18 (300 Å) 1.7 µm Column and the CSH ACQUITY UPLC Peptide CSH C18 Column (130 Å) 1.7 µm Column, both well-documented for excellent peptide separation performance. The CSH column packed with C18 particles containing a low-level positive surface charge provided superior chromatography, with a better MS response (2X > peak intensity) ) and reduced peak tailing of semaglutide. An additional challenge working with semaglutide was encountered during sample preparation and SPE purification due to a high degree of semaglutide non-specific binding (NSB) to consumables and difficulty maintaining peptide solubility throughout this process. As part of the sample preparation and extraction protocol development, consumables used during sample preparation and LC-MS analysis were evaluated, with key focus on polypropylene consumables designed specifically to mitigate NSB due to peptide hydrophobic interaction. In this evaluation, standard polypropylene and QuanRecovery 96-well sample plates were compared. QuanRecovery are designed specifically to minimize peptide-surface interaction mitigating hydrophobic NSB loss. Using QR plates a 3X increase in peptide area counts was seen over standard polypropylene plates for a 1 ng/mL semaglutide solution. Semaglutide plasma extraction employing a PPT sample pretreatment step prior to SPE purification proved to be critical in improving recovery and specificity of semaglutide extraction from plasma. Traditional sample pretreatments of acidic and/or basic aqueous solutions, failed to adequately disrupt semaglutide-plasma protein binding, yielding < 20% recovery from plasma. Employing a protein precipitation (1:1) with 5% formic acid methanol pretreatment resulted in 80–100% recovery semaglutide without precipitating the peptide itself. Following PPT sample pretreatment, the semaglutide PPT supernatant was diluted 1:2 with water prior to loading onto the Oasis HLB µElution SPE plates. A 75% acetonitrile containing 5% formic acid elution solution was employed. This ensured optimum semaglutide SPE elution while maintaining adequate solubility of semaglutide. In addition use of the 96-well µElution SPE format eliminated need for evaporation/reconstitution where potential losses of semaglutide can occur.

Conclusion: The combination of a selective reversed-phase µElution SPE cleanup, mitigation of NSB with proper diluent and elution solutions and use of low bind consumables, such as QuanRecovery MaxPeak HPS 96-well plates, and chromatographic separation using the Peptide CSH C18 UPLC Column to minimize peak tailing, enabled sensitive, accurate, and robust semaglutide quantification from plasma.

Acknowledgements: Research use only.


Semaglutide LC column evaluation, comparing the ACQUITY UPLC Peptide BEH C18 (300 Å) 1.7 µm Column and the ACQUITY UPLC Peptide CSH C18 Column (130 Å) 1.7 µm Column (0.4 mL/min @ 65°C), demonstrating increased MS response and reduced peak tailing with the Peptide CSH C18 Column.


Mitigation of hydrophobic NSB to collection vessels using QuanRecovery™ with MaxPeak HPS 96-well Sample Collection plate, demonstrating a 3X increase in MS peak response vs standard polypropylene plates.


LC-MS bioanalytical quantification performance of semaglutide following PPT-reversed phase SPE extraction from plasma