11 Sensitivity and Linearity of IP and LC-MS/MS Method
A calibration line of SXN101959 was generated from 0.5 ng/mL to 1000 ng/mL in both dog and human plasma. Similar sensitivity was achieved for both plasmas (using both MAb and PAb beads) and in both cases, a linear response was achieved. Figure 2 demonstrates an example calibration line of 0.5 ng/mL to 1000 ng/mL.
2. ELISA
Vs.LC-MS/MS
The comparison of the LC-MS/MS and ELISA values demonstrated good agreement and correlation for the two approaches (R>0.91 for both peptides). Figure 4 shows the correlation of the LC-MS/MS and ELISA values for the 39 rat plasma samples.
Figure 4. Comparison of LC-MS/MS and ELISA values for 39 rat plasma samples.
Figure 2. Calibration line of SXN101959 in extracted human plasma. Conclusion Rat PK Sample Analysis
An administration of SXN101959 to rats had previously been performed and samples analysed using an ELISA [4]. Some of these samples were analysed using an LC-MS/MS, approach with MAb beads. The concentrations of SXN101959 were compared in two ways:
The developed IP and LC-MS/MS methodology showed good sensitivity and selectivity for SXN101959 in dog and human plasma. The analysis of 39 rat plasma samples by both ELISA and LC-MS/MS demonstrated good agreement in the assigned values. This demonstrates that IP based LC-MS/MS methods can be sensitive for high molecular weight proteins, and can also generate complimentary data to established ELISA based approaches.
1. LC-MS/MS Inter-Peptide Analysis
The concentration of SXN101959 assigned to 39 unknown rat plasma samples using peptides from the light and heavy chain demonstrated high agreement (Figure 3). The assigned values were also very close – with only 2 of the 39 values being greater than ± 20% different.
References 1. Lacy, DB et al, 1998, Nature Structural Biol, 5, 898-902
2. Somm et al. (2012) A botulinum toxin-derived targeted secretion inhibitor down regulates the GH/IGF1 axis. Journal of Clinical Investigation. Aug 1. pii: 63232. doi: 10.1172/JCI63232.
3. Harper et al. (2012) The targeted secretion inhibitor (TSI) SXN101959 produces dose-dependent inhibition of pulsatile growth hormone secretion in male rats after intravenous administration. Endocr Rev 2012 33: Sun-669
4. Martinez et al. (2012) PK model approach to understanding the efficacy of SXN101959 in rats. Endocr Rev 33, Sat-741
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Figure 3. Light Vs. heavy chain peptide concentrations for SXN101959 in 39 rat samples.
Interested in publishing a Technical Article?
Contact Gwyneth on +44 (0)1727 855574 or email:
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INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2013
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