Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC–MS/MS

Abstract

An LC–MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50–10,000 ng/mL and 13C 2 15N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine. Interday bias (% RE) and precision (% CV) were −4.2 and 12.3% at the LLOQ, and less than ±0.9 and 8.3% for higher concentrations, respectively. Glycine was stable in artificial CSF for at least 5 h at room temperature, 55 days at −70 °C (−60 to −80 °C range), and through three freeze–thaw cycles.