Measurement of Urine Cystine using Liquid Chromatography Tandem Mass Spectrometry

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Measurement of Urine Cystine using Liquid
Chromatography Tandem Mass Spectrometry

• Joanne E Wear, Brian G Keevil
• Department of Clinical Biochemistry
• Wythenshawe 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
  cysteine
• COOH-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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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 17000
• Heterozygotes 120-200
• Homozygotes 115000 (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)

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Cystinuria cont.

• Cystinuria diagnosed when urine cystine gt100
  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 ?L centrifuged sample, standard or QC
• 30 ?L d4 cystine internal standard (50 mg/L)
• 500 ?L 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 ?m
• Flow rate 0.3 mL/minute
• Elution time 0.89 minutes
• Injection-to -injection time 3.5 minutes

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HPLC cont.

• Step-wise gradient

A 2 mmol/L Amm Ac, 0.1 (v/v) formic acid B 2
mmol/L Amm Ac, 0.1 (v/v) formic acid in MeOH C
100 mmol/L Amm Ac, 0.1 (v/v) formic acid
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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 cystine
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Mass spectra for d4 cystine
Parent ion
Daughter ion
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Ion suppresssion
d4 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

• 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 lt15

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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, n9)
• Bias after 5 days at room temperature lt7.5 (n8)
• Bias after 5 freeze-thaw cycles lt10 (n8)
• Post preparative stability measured - bias lt5
  after 24 hours (n9)

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Stability of a single extract over 17 hours
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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