Development of measurement of leukocyte cystine in granulocytes by liquid chromatography-tandem MS method

Cystinosis is a rare hereditary disorder that causes cystine to accumulate in lysosomes due to impaired cystine transport. Glomerular damage is delayed with cysteamine treatment. Cystine concentration measurement in leukocytes is the gold standard in the diagnosis of cystinosis and cysteamine treatment follow-up. In this thesis, it is aimed to develop an ESI tandem MS method with high sensitivity and accuracy for the measurement of cystine concentration in the leukocyte. Samples were taken into ACD tubes and granulocyte isolation was performed. N-ethylmaleimide was used to avoid oxidation of cysteine to cysteine and interference with SH groups. Cystine was derivatized with 3N butyl-HCl, then di-butyl ester derivative ESI was measured by tandem MS. The method was found to be linear in the range of 0 - 41.38 mol / L. Within run and between days precision (% CV) was <6% and the quantitation limit (LoQ) was 0.095 µmol / L. Recovery was found as 92.2-107.4%. No significant carry-over was observed. Hemolysis was observed to cause significant interference (bias 20%). It was found that cystine remained stable for 30 days in samples stored at -80 0C and should not be freeze-thawed. As a result, a new ESI-tandem MS method has been developed for the measurement of cystine in granulocytes in this study. Cysteine oxidation and disulfide exchange reactions of cystine were inhibited by NEM. Analytical evaluation has shown that the method is sufficient. Considering the evidence obtained, this method is suitable for measuring intra-leukocyte cystine concentration in cystinosis and cysteamine monitoring. Clinicians can use the test reliably for routine measurements.