Simultaneous quantification of the 22-kDa isoforms of human growth hormone 1 and 2 in human plasma by multiplexed immunocapture and LC-MS/MS.

• We present an LC-MS/MS method for the simultaneous quantification of human growth hormone 1 and 2 using multiplexed immunocapture. • The method was validated according to international guidelines. • The described method uses two monoclonal antibody reagents to extract GH1 and GH2 from plasma, and LC-MS/MS to quantify two specific signature peptides which are released from the isoforms upon tryptic digestion. An LC-MS/MS method is presented for the simultaneous quantification of two structurally closely related protein biomarker isoforms, the 22-kDa isoforms of human growth hormone 1 and human growth hormone 2, in human plasma. It is based on multiplexed immunocapture using two monoclonal antibodies immobilized on magnetic beads, tryptic digestion and quantification of two specific signature peptides plus an additional peptide for estimation of total growth hormone related concentrations. A full validation according to international guidelines was performed across the clinically relevant concentration ranges of 0.5 to 50 ng/mL for growth hormone 1, and 2 to 50 ng/mL for growth hormone 2 and demonstrated satisfactory method performance in terms of accuracy, precision, stability and absence of interference. The method's applicability for routine analysis and its ability to effectively distinguish between GH1 and GH2 was demonstrated by the analysis of plasma samples from pregnant individuals to study the changes in growth hormone levels during pregnancy. [ABSTRACT FROM AUTHOR]