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Simultaneous Quantitation of 43 Drugs in
Human Urine with a “Dilute-and-Shoot”
LC-MS/MS Method
Xiang He and Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Application Note
576
Key Words
TSQ Quantum Access MAX, forensic toxicology, drugs of abuse, pain
management drugs, urine, quantitation
Goal
The goal of this work was to develop a simple “dilute-and-shoot” liquid
chromatography-tandem mass spectrometry (LC-MS/MS) method for the
simultaneous quantitation of 43 drugs of abuse, including pain management
drugs, in human urine for forensic toxicology purposes. The drugs to be
analyzed included opioids, amphetamines, benzodiazepines, cocaine,
buprenorphine, methadone, and some of their metabolites. An additional
objective was to use ultra-high-pressure liquid chromatography (UHPLC)
to improve throughput and sensitivity of the method.
Introduction
LC-MS/MS has become more accepted as the tool for
quantitative analysis of drugs in forensic toxicology
laboratories. This technique enables simultaneous
detection of multiple analytes of interests and is
compatible with a simple “dilute-and-shoot” sample
preparation method for urine samples.
Methods
Sample Preparation
Nine individual human urine and pure water samples
were spiked with 20 and 200 ng/mL of the 43 drugs of
abuse, pain management drugs, and with internal
standards (IS). The samples were then mixed with
β
-glucuronidase and incubated at 60 °C for hydrolysis.
Methanol was added to the mixture and the supernatant
was diluted with water. The final dilution factor was 20.
The mixture was centrifuged at 17,000
g
for 5 minutes.
Fifty microliter injections of the supernatant were analyzed
by LC-MS/MS.
Blank human urine was used as the matrix for calibration
samples. The concentrations of the calibrators were 1, 2,
5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL.
Concentration of the internal standards in all samples was
250 ng/mL.
LC-MS/MS Conditions
LC-MS/MS analysis was performed on a Thermo Scientific
™
Accela
™
1250 pump and Accela Open autosampler
coupled to a Thermo Scientific TSQ Quantum Access
MAX
™
triple stage quadrupole mass spectrometer. The
analytical column was a Thermo Scientific Accucore
™
PFP
column (50 × 2.1 mm, 2.6 µm particle size) maintained at
room temperature. Details of the LC gradient and mobile
phases (MP) are as follows:
Time Flow rate Gradient
MPA MPB MPC
(min)
(mL/min)
(%)
(%)
(%)
0.00
0.75
Step
95
5
0
0.50
0.75
Ramp
60
40
0
2.60
0.75
Ramp
5
95
0
4.50
1.00
Step
0
100
0
5.50
1.00
Step
0
0
100
5.75
1.00
Step
95
5
0
MPA: 10 mM NH
4
Ac and 0.1% formic acid in water
MPB: 10 mM NH
4
Ac and 0.1% formic acid in methanol
MPC: acetonitrile/isopropanol/acetone 9:9:2 (v/v/v)
The mass spectrometer was operated with a heated
electrospray ionization (HESI-II) source in positive
ionization mode. The MS conditions were as follows:
Spray voltage (V)
4000
Vaporizer temperature (°C)
300
Sheath gas pressure (arbitrary units)
50
Auxiliary gas pressure (arbitrary units)
15
Capillary temperature (°C)
300