measurement of urine cystine using liquid chromatography
TRANSCRIPT
Measurement of Urine Cystine using Liquid
Chromatography Tandem Mass Spectrometry
Joanne E Wear, Brian G KeevilDepartment of Clinical
BiochemistryWythenshawe Hospital
Introduction• Cystine • Cystine transport• Cystinuria• Methods for the measurement of
cystine
Introduction cont.
• Measurement by HPLC-MS/MS– Sample preparation– HPLC– MS/MS – Assay characteristics
• Conclusion
Cystine• Cystine is a disulphide-linked homodimer of
cysteineCOOH-CHNH2-CH2-SH
COOH-CHNH2-CH2-S-S-CH2-CHNH2-COOH • Essential component of glutathione • Excreted via the kidneys• Reabsorbed at renal tubules using a sodium
independent heteromeric amino acid transporter which consists of a heavy subunit rBAT and a light subunit b0,+AT
N
N
C
S S
b0,+ transport system
Amino acid transport• The b0,+ transporter expressed on the cell
membrane in the epithelium of the renal tubules and brush border of the small intestine
• The dibasic amino acids lysine, arginine and ornithine are also transported in exchange for neutral amino acids
• Sequential mechanism – ternary complex with substrate bound at either side of membrane
Cystinuria• Autosomal recessive disease• Excessive excretion of cystine in the
urine• recurrent urolithiasis• Progressive renal failure• Incidence 1:7000
– Heterozygotes 1:20-200– Homozygotes 1:15000 (US)
Cystinuria cont.• Caused by defect in b0,+ system • Classification based on genotyping• Mutation in either SLC3A1 (rBAT) or
SLC7A9 (b0,+AT)
Cystinuria Subtype
Mutation
A Mutation in both copies of SLC3A1 B Mutation in both copies of SLC7A9
AB Mutation in one copy each of SLC3A1 and SLC7A9
Cystinuria cont.
• Cystinuria diagnosed when urine cystine >100 mg/day*
• Dibasic amino acids measured as 2nd line test
• Treatment – large fluid intake, urine alkalinisation (pH 7.5-8), thiol agents e.g. captopril
*Morton AR, Iliescu EA, Wilson JWL (2002). Nephrology :1. Investigation and treatment of recurrent kidney stones. CMAJ 166 (2) p213-218.
Methods for measurement of cystine
• Qualitative cyanide-nitroprusside method
• Thin layer chromatography• Reverse phase HPLC (cysteine)• GC with flame photometric detection• LC-MS - qualitative• Proton NMR
Sample preparation
• 10 µL centrifuged sample, standard or QC
• 30 µL d4 cystine internal standard (50 mg/L)
• 500 µL distilled water
HPLC
• Waters 2795 Alliance HT LC system• Phenomenex SecurityGuard 4 x 3
mm SCX column + Waters Atlantis C18 column 50 x 2.1 mm, 5 µm
• Flow rate 0.3 mL/minute• Elution time 0.89 minutes• Injection-to -injection time ~3.5
minutes
•Step-wise gradientHPLC cont.
A = 2 mmol/L Amm Ac, 0.1% (v/v) formic acidB = 2 mmol/L Amm Ac, 0.1% (v/v) formic acid in MeOHC = 100 mmol/L Amm Ac, 0.1% (v/v) formic acid
Mobile phase (% v/v) Time (minutes) A B C
0 75 5 20 0.2 5 5 90 0.4 75 5 20
Mass Spectrometry• Waters Quattro Micro Tandem
mass spectrometer with Z spray source
•ES+ mode, column temperature 50°C, desolvation gas flow 630 L/hour
Mass spectra for cystineParent ion
Daughter ion
Mass spectra for d4 cystine
Parent ion
Daughter ion
Ion suppresssiond4 cystine
Cystine
LLOQ
• Analyte response at least 5 times that of blank*
• Analyte peak response identifiable, discrete, reproducible with precision of 20%, accuracy of 80-120%
• LLOQ 10 mg/L, with CV of 2%, bias 115%.
*Guidelines for industry: Bioanalytical Method Validation. U.S. Department of Health and Human Services,Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). May 2001
Chromatogram
Internal Standard(d4 cystine)
Cystine
Linearity
0
200
400
600
800
1000
0 200 400 600 800 1000
Concentration (mg/L)
LC
MS
/MS
re
spo
nse
• r2 = 0.9998, y = 1.0236x - 1.374
Precision and accuracy
• Determined using a minimum of 5 determinations per concentration level.
• Mean within 15% of theoretical value, CV <15%
Within batch precision and accuracy
Between batch precision and accuracy
Recovery and stability
• Mean recovery 103% (91-118%, n=9)• Bias after 5 days at room
temperature <7.5% (n=8)• Bias after 5 freeze-thaw cycles <10%
(n=8)• Post preparative stability measured -
bias <5% after 24 hours (n=9)
Stability of a single extract over 17 hours
0.6
0.7
0.8
0.9
1
0 50 100 150 200 250 300
Injection number
Pe
ak
are
a r
ati
o
Reference Interval• Normal values below 79 mg/day
(below 95th percentile)
Conclusion
• Simple, robust method for the measurement of cystine
• No derivitisation• Quick sample preparation• High throughput