Development an lc ms ms method for the determination of leukocyte cystine level

Cystinosis is an autosomal recessive disorder characterized by the accumulation of cystine in lysosomes, causing irreversible damage to many organs, especially to the kidneys. In order to diagnose cystinosis and to monitor the treatment with cysteamine, leukocyte cystine concentrations must be measured. Cystine binding protein assay, ion exchange chromatography, HPLC and LC MS MS are among the methods used to determine the leukocyte cystine levels. Although there are several methods for determining leukocyte cystine concentration, no studies have been conducted to determine the effect of blood collection tubes and sample preparation on leukocyte cystine levels. The principal aim of this thesis is to develop a new LC MS MS method for the determination of leukocyte intracellular cystine concentration, validate the method and examine the possible influence of the type of blood collection tubes and pretreatments on the measurement. The secondary aim is to test the use of homocystine molecule as an internal standard, which is very rarely found at the intracellular level and resembles cystine. In the developed method, 50/50% acetonitrile/water solvent system containing 0.05% formic acid were used as the mobile phase. The flow program is isocratic, the flow rate is 0.2 ml / min and the total measurement time is 3 minutes. Influence of the protein precipitation agent on the measurement was investigated in terms of sensitivity and matrix effect. As protein precipitation agents, 12% sulphosalicylic acid (SSA), 12% trichloroacetic acid (TCA) and acetonitrile (ACN) were tested and the most sensitive results were obtained with 12% TCA The effect of blood collection tubes on cystine measurement was assessed in terms of recovery, matrix effect, and protein content. For this purpose, tubes containing most commonly used anticoagulants Li-Heparin, K2-EDTA, Na-Citrate were used for comparison. In the scope of method validation parameters, selectivity, linearity, determination and quantitation limits, transport effect, reproducibility, recovery, matrix effect, reference interval validation and lysate stability studies were carried out. The method developed was found to be linear in the range of 0.078-100 M for leukocyte lysate cystine. The limit of detection was found as 0.0192 M and the limit of quantification was found as 0.0582 M. In both methods using D6-cystine as the internal standard and the method using homocystine as the internal standard, % CV values obtained from the intra-day and inter-day reproducibility studies are below 15% which is suitable for validation criteria. While recovery rate was found 94-106 % for the D6-cystine method, these values are between 94%-106% in the homocystine method. The results obtained from the D6-cystine and homocystine method are compared with each other, and the R2 value of the obtained graph is 0.9838. Lysate stability was studied for 4 months at room temperature at + 4 ° C and -20 ° C. For all three temperatures, the lysate was found to be stable for 4 months.