B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were <10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine