5. CDT
Women tend to have higher than men.
Pregnancy was found to be correlated with increased CDT
levels.
γGT
Nor is it a specific marker of chronic heavy alcohol use,
because other liver diseases or certain drugs also increase
serum levels of γGT.
15. First trimester of pregnancy
Measure of alcohol exposure
(PEth in blood)
FASD low
risk
FASD high
risk
Measure of
alcohol exposure
(FAEE in
meconium)
Purpose of Study
Birth
Early diagnosis of FASD
Early diagnosis
of FASD
PEth: HighPEth: Low
19. Sample Preparation
Anders Helander, Molecular Species of the Alcohol Biomarker Phosphatidylethanol in
human Blood Measured by LC-MS, Clinical Chemistry, 55:7, 1395-1405 (2009)
1. 100 ul Blood + 600ul Isopropanol + 50ul Pprop (IS, 1.35 umol/L)
2. Mix for 10min.
3. Add Hexane (2 * 450 ul)
4. Mix for 10min.
5. Centrifuge for 10min (2000 g, 4℃)
6. Supernatants transfer to new glass tube
7. Evaporate under N2 gas at 30℃ using heating block
8. Dissolved in 50 ul hexane + 50 ul acetonitrile + 75 ul isopropanol
9. Transfer to 0.3 ml glass auto-sampler vial.
22. Blood sample
Blood collect into EDTA tubes
Store at 4℃ for < 3days
PEth is stable for at least 3 weeks in refrigerated
blood sample
23. LC-ESI-MS
Negative ion mode
MS condition
Drying gas flow : 10.0 ψ
Nebulizer gas : 20 ψ
Drying gas temperature : 350 ℃
Capillary voltage : 3000 V
Column : C4 (50 mm Ⅹ 3 mm, 5um)
24. LC system
Gradient mode
Sol A : 20% 2mmol/L ammonium acetate
80% acetonitrile
Sol B : 100% isopropanol
LC-ESI-MS
Sol A
Sol B
2 4 6 8(min)
100(%)
50
0
25. LC-ESI-MS
• Flow rate : 200ul/min
• Injection volume : 10ul
• Reconditioning the column with
90% A and 10% B for 10min