J RYKL¹, KB KRUEGER<sup>2

1</sup>Shimadzu Schweiz GmbH, Römerstr. 3, 4153 Reinach, Schweiz, ²Uniklinikum Freiburg, Hugstetter Str. 55, 79106 Freiburg, Deutschland

Peptide hormones are water- soluble amino acid based hormones and act on the surface of target cells via second messengers. These hormones have an effect on the endocrine system of humans.
The concentration of these hormones in plasma can be measured using a mass spectrometry based assay.
Gonadoliberin, also known as luteinizing hormon-releasing hormone (LHRH), is a peptide consisting of 10 amino acids, which is synthesized in the hypothalamus. Reproduction in male and female mammals is critically dependent on the secretion of gonadoliberin. It is released in pulses and triggers the production of gonadotropins, which stimulate the growth and release of eggs by the ovary.
We used this peptide as a model for the establishment of a mass spectrometry based assay for the quantitation of peptides in body fluids.
Using the free of charge peptide quantitaton software Skyline and the LCMS 8060 triple quadrupole we developed and validated the assay regarding the following points :
1) Matrix effects from the blood plasma
2) Avoiding false positive results
3) Easy and reliable sample preparation
4) Finding the best fragment ions of the peptide for quantitation
5) Correct setting of mass spectrometry parameters like dwell time and collision energy
6) The correct choise of HPLC column and HPLC conditions
7) Reproducibility and accuracy of the mass spectrometry signal
8) Limit of quantitation and linearity of assay.
After the optimizaton of the parameters we were able to detect low picogram amounts of this peptide hormone in the human blood plasma with very good accuracy and linearity.
The quantitation of other clinical relevant peptide hormones is also possible using our established workflow.