This application note describes a method using high resolution accurate mass (HRAM) LC/MS for the quantitation of cystine and related metabolites in white blood cells. Excellent assay performance was achieved for cystine quantitation in white blood cells (WBCs) using the ultra-high resolving power and mass accuracy of an Agilent 6530 Accurate-Mass Q-TOF LC/MS System. Further, related metabolites were successfully identified and quantitatively profiled. The HRAM LC/MS data acquired in this study can be retrospectively analyzed to search for more metabolites and biomarkers without sample re-injection.
This document presents a high resolution accurate mass (HRAM) LC/MS approach for quantitating cystine and profiling related metabolites in complex biological samples, specifically white blood cells (WBCs). The method utilizes the ultra-high resolving power and mass accuracy of the Agilent 6530 Accurate-Mass Q-TOF LC/MS System connected to an Agilent 1290 Infinity LC System.
The study compares the performance of this HRAM method against a previously validated multiple reaction monitoring (MRM) method using triple quadrupole mass spectrometry. Results indicate that the HRAM method offers excellent sensitivity, linearity, dynamic range, precision, accuracy, and reproducibility, comparable to the MRM method. Additionally, the accurate mass data allows for the retrospective identification of 44 related metabolites without requiring sample re-injection.
| Mass resolving power | 10K |
|---|---|
| Ionization mode | Positive |
| Drying gas temperature | 300 °C |
| Sheath gas temperature | 400 °C |
| Capillary voltage | 3,750 V |
| Fragmentor voltage | 200V |
| Mass range | 100-1,000 m/z |
| Acquisition rate | 2 Hz |
The LOQ of cystine in WBCs is 0.02 uM, or 40 fmol on-column.
The cystine calibration curve shows excellent linearity in the dynamic range of 0.02 - 4 uM.
MassHunter Workstation Software (B.03.01) is used for data acquisition, and MassHunter Quantitative Analysis Software (version B.04.00) is used for quantitation.