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Quantitative LC-MS Screening for Illicit Drugs
Using Ultrahigh Resolution Mass Analysis and
Accurate Mass Confirmation
Kevin J. McHale and Mark Sanders, Thermo Fisher Scientific, Somerset, NJ
Forensic toxicology use only.
Introduction
There is increasing demand to rapidly identify and quan-
tify illicit drugs in human samples for forensic purposes.
By using ultrahigh resolution, accurate mass spectrometry
detection coupled to liquid chromatography separation,
a large number of both expected and unexpected com-
pounds can be easily screened and quantified without prior
knowledge.
Goal
To evaluate a simple LC-MS method using a benchtop
ultrahigh resolution mass spectrometer to quantitatively
screen for 46 illicit drugs of abuse in urine with little
sample preparation.
Experimental
Sample Preparation
Blank human urine diluted with 25% acetonitrile was
spiked with varying concentrations of 46 drugs of abuse
and their corresponding isotopically-labeled standards at
50 ng/mL.
HPLC
HPLC analyses were performed using a Thermo Scientific
Accela liquid chromatography system. Gradient elution
with a Thermo Scientific Hypersil GOLD PFP column (100
x 2.1 mm; 3 µm) was used at a flow rate of 350 µL/min.
The injection volume was 10 µL.
Mass Spectrometry
MS detection was carried out on a Thermo Scientific
Exactive benchtop LC-MS system with a heated electro-
spray ionization (HESI) source in positive ion mode at
a mass resolution of 50,000 FWHM via external mass
calibration.
Results and Discussion
LC-MS quantification of 46 drugs of abuse was accom-
plished via the calculated area ratios of the compound to
its heavy-labeled internal standard. Table 1 gives a listing
of the targeted drugs of abuse, their limits of quantitation
(LOQ), and their measured mass errors at the LOQ in
urine.
Example extracted ion chromatograms for metham-
phetamine and benzoylecgonine in urine are shown in
Figure 1. At a mass resolution of 50,000 FWHM, at least
10 data points were obtained across the LC peaks. Apply-
ing extracted ion chromatograms of ±5 ppm, along with
the isotopically-labeled internal standards for confirma-
tion, all drugs of abuse were easily identified.
.
Application
Note: 499
Key Words
• Exactive LC-MS
• Accela UHPLC
• Drug Screening
• Drug Quantitation
• Forensic
Toxicology
Methamphetamine
Benzoylecgonine
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time (min)
0
50000
0
10000
20000
AA: 68775
AA: 245518
163.10
163.15
m/z
0
50
100
0
50
100
163.1326
C
8
H
19
O
3
C
8
H
19
O
3
C
10
H
15
O
2
163.1229
163.1328
a
b
Figure 2 shows the nicotine mass spectrum in urine
(a) with an isobaric interference at
m/z
163.1326 and
a simulated spectrum (b) at 20,000 FWHM. Although
nicotine and its isobaric interference have a large relative
mass difference of ~60 ppm, the absolute mass difference
is only 0.0097 u. As seen in the simulated spectrum (b) at
20,000 FWHM, the nicotine ion is not resolved from the
interference. Only ultrahigh mass resolution of 50,000
FWHM provides the necessary selectivity to resolve these
compounds and therefore allows for confident identifica-
tion and quantification.
Conclusion
The Exactive benchtop LC-MS system provides easy
confirmatory and quantitative analysis of 46 illicit drugs
at LOQs of 0.5 – 5 ng/mL in urine for forensic toxicol-
ogy. Owing to the sensitivity of the Exactive system, urine
samples require only dilution with solvent to achieve this
level of performance.
Figure 2: Nicotine mass spectrum in urine (a) and simulated spectrum (b) at
20,000 FWHM
Figure 1: Example extracted ion chromatograms (± 5 ppm) at LOQs in urine
