Accurate 24‐h Urine Cystine Quantification for Patients on Cystine‐Binding Thiol Drugs
Topics: Nephrology Cystinuria
Mikel CC, Goldfarb DS, Ponte A et al.
10.1007/s00240-022-01364-9
Summary
A method for urine cystine quantification was validated for patients with cystinuria receiving a cystine-binding thiol drug (CBTD)1
Background
Cystinuria is a genetic disorder that leads to recurrent kidney and bladder stones, which affect quality of life and kidney function.2-4
Cystinuria treatment aims to reduce stone formation by increasing the solubility of cystine in urine.5 For large stones, or when conservative management is not effective, a cystine-binding thiol drug (CBTD) may be required.6
The level of urinary cysteine should be monitored to adjust the dose of CBTD appropriately.5 However, distinguishing free cystine from cysteine-thiol drug complexes is challenging.7 A valid, reliable, and practical method for quantifying urinary cystine is needed.1
Aim
This study aimed to validate a high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for quantifying urinary cystine for patients with cystinuria receiving a CBTD.1
Approach
Calibration, linearity, and range of detection in urine samples
The HPLC–MS/MS method had been previously used for determining cystine levels in whole kidney cells.1,8 For this study, the method was applied to urine samples and a calibration curve was plotted for the range 0.03-1.00 mg/mL.1 The limit of detection (LOD) and limit of quantification (LOQ) were calculated from this curve.1
Accuracy and precision
Six replicates of target concentrations were used as calibrators.1
Accuracy was determined by mean accuracy percentage.1 Repeatability was assessed with the calculated standard deviation (SD) and coefficient of variation (CV).1
Intermediate precision was defined by the relative percent difference (RPD) of three sets of calibrators.1
Potential interferences and matrix effects
To assess the potential of the HPLC–MS/MS method for interference from exogenous compounds, nine mixtures were prepared containing common over-the-counter (OTC), prescription, and illicit drugs.1
To assess the potential for interference from endogenous compounds, urine samples from patients without cystinuria were assessed to determine a false positive rate.1
Matrix effects were assessed by adding cystine solutions (2 mg/mL) in equal volume to blank urine or each of the nine interference mixtures.1
Quantification of cystine level in patient urine samples
Twenty-four hour urine samples were gathered from 24 patients with cystinuria who were receiving CBTD.1
Findings
The HPLC–MS/MS method was validated for its accuracy, repeatability, precision, linearity, LOD, and LOQ1
Cystine was separated from cysteine-tiopronin drug complexes in under three minutes by HPLC.1 The calibration curve showed a linear response (R2=0.998). LOD was 0.002 mg/mL and LOQ was 0.005 mg/mL.1
Accuracy, repeatability, and precision were determined with six replicates of calibrators, giving the following results1:
- Accuracy (mean accuracy percentage): 97.7-102.3%
- Repeatability (coefficient of variation): 1.2-3.3%
- Precision (RPD): 1.2-9.3%
No cystine false positives were seen for exogenous compounds, such as other drugs, or endogenous compounds1
For one interference mixture, a significant matrix effect was seen.1 This mixture contained acetaminophen, caffeine, cotinine, ibuprofen, naproxen, nicotine, phentermine, and pseudoephedrine.1
The HPLC–MS/MS method quantified cystine levels from patient urine samples1
Mean cystine concentrations were1:
- Aliquot A: 111.10 g/L (Range: 51.31-179.46 g/L)
- Aliquot B (cystine solubilized with sodium carbonate): 242.21 g/L (Range: 61.14-741.80 g/L)
Key takeaway
Using a HPLC–MS/MS method, urinary cysteine can be quantified reliably for patients with cystinuria receiving a CBTD.1
Footnotes
CBTD, cystine-binding thiol drug; CV, coefficient of variation: HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; OTC, over-the-counter; MS/MS, tandem–mass spectrometry; RPD, relative percent difference; SD, standard deviation.
- Mikel CC et al. Urolithiasis. 2022;50(6):721-727.
- Prot-Bertoye C et al. Clin J Am Soc Nephrol. 2015;10:842-851.
- Woodard LE et al. BMC Nephrol. 2019;20:227.
- Parr JM et al. BJU Int. 2015;116(suppl 3):31-35.
- Eisner BH et al. J Endourol. 2020;34:1103-1110.
- Pearle MS. J Urol. 2014;192:316-324.
- Goldfarb DS et al. Kidney Int. 2006;69:1041-1047.
- Jamalpoor A et al. Biomed Chromatogr. 2018;32:e4238.
MA-DS-24-0043 | November 2024