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Simultaneous Quantitation of 19 Drugs in
Human Plasma and Serum by LC-MS/MS
Xiang He and Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Application Note
571
Key Words
TSQ Vantage, drug monitoring research, clinical research, CSS,
plasma, serum
Goal
To develop a simple, fast, and sensitive LC-MS/MS method for the
simultaneous quantitation of 19 drugs in human plasma and serum.
Introduction
Liquid chromatography combined with tandem mass
spectrometry (LC-MS/MS) has become an accepted tool
for quantitative analysis of drugs in clinical research
laboratories. LC-MS/MS enables simultaneous, sensitive
detection and quantitation of multiple analytes of interest.
In this study, 19 drugs of various types, including
antipsychotics, antiepileptics/anticonvulsants, antianginals,
and antidepressants, were monitored and simultaneously
quantitated using LC-MS/MS.
Experimental
Sample Preparation
Nineteen drugs (Table 1) and 15 isotopically labeled
internal standards of the drugs were used in this research.
Table 1. Drug analytes
To assess signal recovery and determine the best dilution
factor, 9 randomly chosen individual human-donor plasma
samples were spiked with the 19 drugs at 40 ng/mL and
15 isotopically labeled internal standards at 100 ng/mL.
These samples were mixed (1:3, v/v) with a 1:1 methanol/
acetonitrile mixture. The samples were vigorously vortexed
and stored at -30 °C for 30 min. The samples were then
centrifuged at 17,000
g
for 5 min. Supernatant (20 µL)
was drawn off and diluted 10-fold, 20-fold, and 50-fold
with 10% methanol in water to final dilution factors of
40x, 80x, and 200x.
Calibration and linearity standards were prepared by
spiking a matrix of charcoal-stripped human serum (CSS)
with the 15 internal standards at 100 ng/mL and the 19
drug analytes at 4, 10, 20, 40, 100, 200, and 400 ng/mL.
The samples were processed as above and diluted to a
final dilution factor of 200x.
For accuracy and precision testing, CSS samples were
spiked with the 15 isotopically labeled internal standards
at 100 ng/mL and the 19 drugs at both 40 ng/mL and
200 ng/mL. The samples were processed as above and
diluted to a final dilution factor of 200x.
Also for accuracy and precision testing, 9 individual
human-donor plasma samples were spiked with the 15
isotopically labeled internal standards at 100 ng/mL and
the 19 drugs at 40 ng/mL. The samples were processed as
above and diluted to a final dilution factor of 200x.
Analytes
Amitriptyline
Dothiepin
Nortriptyline
Bromazepam
Doxepin
Oxazepam
Clobazam
Flunitrazepam
Perhexilline
Clomipramine
Imipramine
Temazepam
Clonazepam
Lamotrigine
Trimipramine
Clozapine
Levetiracetam
Diazepam
Nitrazepam