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