(M0930-01-02) A Low Level, Carryover Free and Wide Range, LC-MS/MS Method for Quantitation of Semaglutide from Human Plasma

Purpose: Semaglutide is a peptide used as an antidiabetic medication for the treatment of type-2 diabetes. It is also used as an anti-obesity medication for weight loss. Recently, studies have shown that semaglutide also works on the brain, suggesting its potential utility for various diseases, including Parkinson's disease and Alzheimer's disease. The non-specific adsorption of semaglutide on column, HPLC flow path and high background noise at low level in complex matrix like human plasma makes quantitation of semaglutide difficult at low level. To overcome these challenges, we developed an MRM based LC-MS/MS method for quantifying semaglutide. This method is well suited for pharmacokinetic studies of semaglutide because it offers low limit of quantification, no observable carryover, and has a wide dynamic range.

Methods: Semaglutide reference standard and human plasma were used to prepare calibration standards and quality control (QC) samples. Precursor ion selection and MRM optimization at different collision energies and voltages was done using automated software tools. Two product ions were optimized for each MRM, with the most abundant product ion preferred for quantification.  The LC method was developed using an inert UHPLC column (Shim-pack Claris) to elute semaglutide, method conditions are shown in table format. Using the developed LC method and optimized MRMs, LLOQ of 0.2 ng/mL and upper limit of quantification (ULOQ) of 600 ng/mL was selected for the study. For quantification, a wide linearity batch ranging from 0.2 to 600 ng/mL was processed in human plasma. For QC check lower limit quality control (LLQC), lower quality control (LQC), medium quality control (MQC) and higher quality control (HQC) samples were processed in replicates and were quantified against the linearity.

Results: Validation parameters such as specificity, linearity, accuracy, precision and carryover were studied as per ICH M10 Guidelines. System precision was evaluated by calculating variation of the peak area and retention time (RT) of six replicates of 300 ng/mL processed semaglutide standard. The % RSD was found to be less than 5 for peak area, whereas the difference in RTs for 6 replicate injections was found to be within +0.1 min. Specificity of the method was determined by comparing the response of blank sample (reagent and matrix) against reporting level. Response in reagent/matrix blank sample was well within (less than 20%) the reporting limit and met the acceptance criteria. For linearity study, processed calibration standards were used. All calibration standards were found within 85 to 115 % accuracy as shown in table format.  QC samples at 4 different levels - LLQC, LQC, MQC and HQC were processed in replicates and quantified for accuracy and precision study. The observed results were within acceptance criteria of % RSD ± 15 %. Carryover was assessed by analyzing blank sample after injecting highest calibration standard, the area response at the retention time of semaglutide for the blank sample analyzed after highest calibration standard was found to be less than 20.0 % of the area response of the LLOQ standard.

Conclusion: A highly sensitive and precise method for quantifying GLP-1 peptide in human plasma was developed using the Shimadzu LC-MS/MS system.  By combining the use of internal and external rinsing of the autosampler with inert coating technology in the analytical column, potential sources of carryover have been mitigated to the extent that no carryover is observed that exceeds baseline noise. This method effectively addresses common challenges, including achieving low-level LLOQ, wide dynamic range, and carryover-free detection. The results met the accuracy and precision standards of ICH M10 guidelines, confirming the reliability of the method.