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Simultaneous Analysis of Alcohol Metabolites and Barbiturates in Urine by LC-MS/MS

LC_CF0773
PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct Ion 1Product Ion 2
1.EtG-d50.76100225.984.7-
2.EtG0.80500220.974.985
3.EtS-d51.70100129.797.7-
4.EtS1.78500124.896.879.7
5.Phenobarbital-d52.54100236.042.0-
6.Phenobarbital2.55500231.2188.042.0
7.Butalbital-d52.5710022842.0-
PeakstR (min)Conc.
(ng/mL)
Precursor IonProduct Ion 1Product Ion 2
8.Butalbital2.58500223180.042.0
9.Amobarbital-d52.7410023042.0-
10.Pentobarbital-d52.7410023042.0-
11.Amobarbital2.75500225182.042.0
12.Pentobarbital2.76500225182.042.0
13.Secobarbital-d52.9310024242.0-
14.Secobarbital2.93500237194.042.0
ColumnRaptor EtG/EtS (cat.# 9325A12)
Dimensions:100 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Guard Column:UltraShield UHPLC PreColumn filter, 0.2 µm (cat.# 25809)
Temp.:30 °C
Standard/SampleEthyl-ß-D-glucuronide (cat.# 34101)
Ethyl-ß-D-glucuronide-d5 (cat.# 34102)
Ethyl sulfate sodium salt (cat.# 34103)
Ethyl sulfate-d5 sodium salt (cat.# 34104)
Phenobarbital (cat.# 34037)
Pentobarbital (cat.# 34036)
Butalbital (cat.# 34032)
Secobarbital (cat.# 34038)
Diluent:Water, 0.1% formic acid
Inj. Vol.:10 µL
Mobile Phase
A:Water, 0.1% formic acid
B:Acetonitrile, 0.1% formic acid
Time (min)Flow (mL/min)%A%B
0.000.5955
1.200.58020
1.210.56535
3.000.55545
4.000.5955
5.000.595stop
DetectorMS/MS
Ion Source:Electrospray
Ion Mode:ESI-
Sample PreparationHuman urine (alcohol free) was fortified with EtG, EtS, phenobarbital, butalbital, amobarbital, pentobarbital, and secobarbital in order to prepare the calibration standards and QC samples.

50 µL of urine was diluted with 950 µL of the working internal standard (100 ng/mL EtG-d5, EtS-d5, and barbiturates-d5 in 0.1% formic acid in water). Samples were vortexed at 3500 rpm for ten seconds to mix, centrifuged at 3000 rpm for five minutes at 10 °C, and injected for LC-MS/MS analysis.