Measurement of Urine Cystine using Liquid Chromatography ...
Transcript of Measurement of Urine Cystine using Liquid Chromatography ...
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Measurement of Urine Cystine using Liquid
Chromatography Tandem Mass Spectrometry
Joanne E Wear, Brian G KeevilDepartment of Clinical
BiochemistryWythenshawe Hospital
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Introduction• Cystine • Cystine transport• Cystinuria• Methods for the measurement of
cystine
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Introduction cont.
• Measurement by HPLC-MS/MS– Sample preparation– HPLC– MS/MS – Assay characteristics
• Conclusion
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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
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N
N
C
S S
b0,+ transport system
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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
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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)
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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
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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.
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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
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Sample preparation
• 10 µL centrifuged sample, standard or QC
• 30 µL d4 cystine internal standard (50 mg/L)
• 500 µL distilled water
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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
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•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
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Mass Spectrometry• Waters Quattro Micro Tandem
mass spectrometer with Z spray source
•ES+ mode, column temperature 50°C, desolvation gas flow 630 L/hour
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Mass spectra for cystineParent ion
Daughter ion
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Mass spectra for d4 cystine
Parent ion
Daughter ion
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Ion suppresssiond4 cystine
Cystine
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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
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Chromatogram
Internal Standard(d4 cystine)
Cystine
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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
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Precision and accuracy
• Determined using a minimum of 5 determinations per concentration level.
• Mean within 15% of theoretical value, CV <15%
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Within batch precision and accuracy
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Between batch precision and accuracy
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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)
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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
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Reference Interval• Normal values below 79 mg/day
(below 95th percentile)
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Conclusion
• Simple, robust method for the measurement of cystine
• No derivitisation• Quick sample preparation• High throughput