Analysis of Cystine in Urine

Authors: Mégane Moreau, Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Urine, Cystine, LDTD-MS/MS
From: Phytronix Technologies, Québec, Canada
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Introduction

The rapid quantification of cystine in urine can be useful for the detection of cystinuria, an autosomal recessive disorder (MariaLuisa Cabello-Tomás, 1999). Newborn screening for this disease can be performed by thin layer chromatography (Auray-Blais et al., 2021). Cystinuria is characterized by the formation of cystine kidney stones. Overexcretion of poorly soluble cystines causes accumulation of stones in the kidneys due to precipitation (Chillarón et al. 2010). Previously, the screening test for cystine in urine was qualitative . However, rapid quantification of cystine in urine can be helpful in confirming the diagnosis. Additionally, a high concentration of cystine in the urine can lead to kidney stones (obstruction) or kidney failure (Wear et al., 2005).

Our goal for this application note is to develop a diagnostic tool to rapidly quantify cystine in urine, which will allow the rapid diagnosis of certain autosomal disorders, such as cystinuria.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing an efficient sample preparation, which will increase the speed of the analysis process and confirm diagnoses more quickly.

Sample Preparation Method

Stock solutions of Cystine (2000 µg/mL) and Cystine-d6 (100 µg/mL) were prepared in sodium hydroxide solution (50 mM). Then, artificial urine samples were spiked to generate a calibration curve and QCs.

 

Derivatization and Extraction procedure

 

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:

3-second ramp to 55% power

 

MS/MS

MS model: QTrap® System 5500, Sciex

Curtain: 20

CAD: 8

IonSpray Voltage: 5500

Temperature, GS1 and GS2 set to zero.

Scan Time: 100 msec

Total run time: 0.15 minutes per sample

Analysis Method: Positive MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Transition CE (V)
Cystine 353.0 → 130.0
353.0 → 208.0
20
Cystine-d6 359.0 → 131.0
359.0 → 211.0
20

 

Results and Discussion

 

Validation Test

Calibration curves ranging from 1 to 100 mg/mL were prepared in synthetic urine. Two sets of QCs were prepared. One set in synthetic urine (QC-L, QC-M and QC-H) and a second set in standard urine (UQC-L and UQC-M). Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

 

Table 2 shows the inter-day correlation coefficients for Cystine. Values greater than 0.995 are obtained. Figure 3 shows a typical calibration curve result for Cystine.

Cystine calibration curve graph
Figure 3 – Cystine calibration curve

 

Table 2 – Inter-day calibration curve correlation coefficients

Cystine R
Curve 1 Y = 0.07186 x – 0.00555 0.99921
Curve 2 Y = 0.07124 x – 0.01314 0.99812
Curve 3 Y = 0.06890 x – 0.04453 0.99882
Curve 4 Y = 0.06882 x – 0.04509 0.99919

 

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

For the intra and inter-run precision and accuracy experiment, each QC was analyzed in sextuplicate, on four different runs. Table 3 and 4 show the intra and inter-run precision and accuracy results for Cystine. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 3 – Intra-Run Precision and Accuracy of Cystine

Cystine QC-L1 QC-M1 QC-H1 UQC-L2 UQC-M2
Conc (mg/mL) 3 50 70 11.6 61.6
N (μg/mL) 6 6 6 6 6
Mean (μg/mL) 2.9 50.4 73.8 11.4 61.1
%CV 5.4 3.2 4.4 6.6 6.3
%Nom 98.3 100.8 105.5 98.1 99.2

 

Table 4 – Inter-Run Precision and Accuracy of Cystine

Cystine QC-L1 QC-M1 QC-H1 UQC-L2 UQC-M2
Conc (mg/mL) 3 50 70 11.6 61.6
N (μg/mL) 24 24 24 24 24
Mean (μg/mL) 3.0 50.0 69.3 11.6 61.9
%CV 7.1 4.0 8.8 7.8 6.7
%Nom 99.3 100.0 98.9 100.4 100.4
Note 1:. Cystine spiked in synthetic urine sample
Note 2: Cystine spiked in urine sample

 

 

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers protected from light. After a week, sample extracts are spotted on a LazWell™ plate, dried and analyzed. The precision and accuracy results of QCs in synthetic urine samples are reported in Table 5. Results of QCs in urine samples are reported in Table 6. All the results are within the acceptable criteria range for one week at 4°C.

 

Dry Stability of Samples Spotted in LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 4 hours before analysis. The precision and accuracy results of QCs in synthetic urine samples are reported in Table 5. Results of QCs in urine samples are reported in Table 6. All the results are within the acceptable criteria range for 4 hours at room temperature.

Table 5 – Wet and Dry Stability of Cystine in Synthetic Urine Sample

Parameters Dry stability
(4 hours / RT)
Wet stability
(1 week / 4°C)
QC QC-L QC-M QC-H QC-L QC-M QC-H
Conc. (mg/mL) 3 50 70 3 50 70
N 6 6 6 6 6 6
Mean (mg/mL) 3.1 52.1 73.9 3 52.6 72.6
%CV 6.2 7.0 8.6 3.3 3.3 8.4
%Nom 103.8 104.1 105.6 100.37 105.2 103.7

 

Table 6 -Wet and Dry Stability of Cystine in Urine Sample

Parameters Dry stability
(4 hours / RT)
Wet stability
(1 week / 4°C)
QC UQC-L UQC-M UQC-L UQC-M
Conc. (mg/mL) 11.6 61.6 11.6 61.6
N 6 6 6 6
Mean (mg/mL) 11.4 58.7 11.4 62.5
%CV 4.7 7.1 2.6 5.2
%Nom 95.5 95.3 98.4 101.5

 

Cross validation study

Urine samples from real patients were tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage difference between the values ​​is evaluated. A difference of less than 15% is obtained. The results are reported in Table 7.

Table 7 – Cross validation between LC-MS/MS and LDTD-MS/MS

Cystine U1 U2 U3 U4 U5 U6 U7 U8 U9 U10 U11 U12
LC  (mg/mL) 11.3 20.9 17.2 25.9 16.2 27.1 19.1 29.3 3.0 12.3 11.2 19.6
LDTD (mg/mL) 13.1 23.4 18.4 28.1 17.0 28 19.4 32 2.5 12.1 11.3 20.9
%Diff (%) 7.5 5.6 3.5 4.1 2.4 1.6 0.8 4.4 -8.6 -0.8 0.3 3.2

 

Conclusion

The Luxon Ion Source® combined with the Sciex QTrap® 5500 mass spectrometer system enables the rapid analysis of cystine in urine. This method of analysis can thus be applied at the clinical level in order to diagnose certain autosomal recessive disorders such as cystinuria.