Methods

Samples and reagents: Liraglutide, LC-MS grade acetonitrile and methanol, and ammonium acetate were purchased from Sigma Aldrich. Insulin lispro was purchased from USP. Rat plasma (Sprague Dawley, K2 EDTA) was purchased from BioIVT. 

Sample preparation: Liraglutide and insulin lispro (internal standard, IS) were spiked into the extracted rat plasma. Stock solutions of liraglutide and the IS were prepared with acetonitrile/5mM ammonium acetate (70%/30%, v/v) and a serial dilution was performed to prepare working solutions.

Rat plasma was extracted using protein precipitation with acetonitrile/methanol (70%/30%, v/v). The resulting supernatant was diluted and used as the biological matrix for this assay.

Chromatography: Analytes were separated using a Halo ESC18 column (2.1 mm × 50 mm, 2.7 µm, 160 Å) on an ExionLC system. Total method time was 8.5 min at a flowrate of 0.5 mL/min. Mobile phase A was composed of 0.1% formic acid in water while mobile phase B was composed of 0.1% formic acid in acetonitrile. Mobile phase gradient is summarized in Table 1. Operating column temperature was 40 °C. Injection volume was 10 µL.

Mass spectrometry: MS analysis was performed on a SCIEX 7500 system. The optimized MRM parameters are summarized in Table 2.

Monitored MRM transitions for liraglutide and the IS are summarized in Table 3 below. For all the MRM transitions, an entrance potential (EP) of 10 V was used.

Data processing: MRM data were processed with SCIEX OS software 2.0 using the MQ4 integration algorithm. Linear regression with 1/x weighting was used for quantification of liraglutide.

Quantification results 

In this workflow, a sensitive LC-MRM method was developed for the quantification of GLP-1 analogue, liraglutide, in rat plasma. All method parameters were carefully optimized to ensure the best sensitivity. Quantification was performed in positive ion MRM mode. The 4+ multiply charged precursor ion for liraglutide, m/z 939.06, displayed the highest signal. Two fragment ions of the precursor ion of m/z 939.06 were monitored including m/z 1064.3 and m/z 1128.6. The y-ion fragment at m/z 1064.3 produced the most sensitive signal and was used for quantification.

Calibration curves were analyzed in triplicates. The linear range was determined to be between 0.5 ng/mL and 1000 ng/mL. A linear dynamic range (LDR) of 3.3 orders of magnitude was achieved (Figure 3)

The LLOQ was determined based on the requirements that the %CV of the average of the concentration is below 20% and accuracy is between 80% and 120%. For the concentrations above the LLOQ, the %CV of the mean of the calculated concentration was required to be below 15% while accuracy was required within the range from 85% to 115%.

The LLOQ for liraglutide was determined to be 0.5 ng/mL as shown in Figure 3. No significant interference was observed in the matrix blank.

Exceptional accuracy and precision were achieved for liraglutide across the concentration range of 0.5 ng/mL to 1000 ng/mL (Table 4). Overall, the %CV was less than 8% for all measured concentrations, demonstrating high reproducibility.

A highly robust and sensitive method for the quantification of liraglutide in a complex biological matrix was demonstrated.

Conclusion
 

• An ultra-sensitive MRM based GLP-1 analogue quantification workflow using SCIEX 7500 system was developed
  
  
• Liraglutide in rat plasma was quantified at 0.5 ng/mL in rat plasma with outstanding reproducibility, accuracy, and linearity with a LDR of 3.3 orders of magnitude
  
  
• Low-levels of quantification suggest applications in pharmacokinetics and pharmacodynamics studies of peptide therapeutics
  
  
• A cumulative gain in sensitivity was observed for GLP-1 analogue assays in complex matrices as a result of the combined improvements in front-end technology including the OptiFlow Pro ion source, E Lens probe, and D Jet ion guide

References
 

1. Enabling new levels of quantification. SCIEX technical note, RUO-MKT-02-11886-A.