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Analysis of Multiple Illicit Drugs, Methadone,
and their Metabolites in Oral Fluid Using
a Linear Ion Trap Mass Spectrometer
Min He, Gargi Choudhary, Diane Cho, Karen Salomon and Julian Phillips, Thermo Fisher Scientific, San Jose, CA, USA
Key Words
• LXQ
• Surveyor Plus
•
Drugs of Abuse
Forensics
• Hypersil GOLD
Columns
• MS
3
Quantification
Application
Note: 366
Introduction
Traditionally, the analysis of urine samples has been the
major approach for the detecting of drugs of abuse.
1
However, a common risk for this type of analysis is
adulteration or manipulation of the sample at the point
of collection. As an alternative, the analysis of oral fluid
provides an easy method of sample collection and has the
advantage of providing a relatively clean matrix. Because
of the reduced sample volume, this technique requires a
high sensitivity and robust analytical method to make an
attractive alternative to conventional methods.
In this report, a rapid and rugged LC-MS/MS method
using the Thermo Scientific LXQ is described for analyz-
ing a mixture of twenty drugs and their metabolites using
intelligent automated mass spectrometry (INTAMS).
The detection limits for the mixture of drugs and dynamic
range are superior to results reported previously.
2
In
addition, this method provides for the simultaneous identi-
fication and quantification of drugs and their metabolites.
Experimental Conditions
Sample Preparation:
Ten milliliters of oral fluid collected from a volunteer
were protein precipitated using 30 mL acetonitrile. The
sample was vortexed and then centrifuged at 5,000 rpm
for 10 minutes. The supernatant was evaporated to
dryness under nitrogen and reconstituted in 5 mL water.
Table 1 provides a list of 20 drugs along with the parent
and product ion masses. For quantification experiments,
known amounts of a stock solution of the 20 drug mixture
were spiked into the treated oral fluid to prepare the stan-
dards in concentrations ranging from 50 fg/µL to 1 ng/µL.
HPLC:
LC System: Thermo Scientific Surveyor Plus
Column: Thermo Scientific Hypersil GOLD
™
(20
×
2.1 mm, 1.9 µm particle size)
Mobile phase:
(A) water with 0.1% formic acid and 10 mM
ammonium acetate
(B) acetonitrile with 0.1% formic acid
Flow rate: 400 µL/min
Injection volume: 10 µL
Gradient:
t (min)
A% B%
0.00
95
5
0.10
95
5
1.00
85
15
4.20
50
50
4.21
95
5
7.00
95
5
Mass Spectrometer:
The LXQ linear ion trap mass spectrometer was operated
in positive atmospheric pressure chemical ionization (APCI)
mode. The corona discharge needle voltage was 4.5 kV and
the vaporizer temperature was 400°C. The capillary tem-
perature was 220°C and the sheath gas flow was 25 units.
All scan events were acquired with one micro scan. No
internal standard was used. The set up of the acquisition
method using INTAMS is shown in Figure 1.
Results and Discussions
INTAMS data acquisition software was used for the
simultaneous identification of 20 drugs in oral fluid.
The extracted ion chromatogram is shown in Figure 2.
INTAMS software enables the maximum number of scans
to be acquired under a given chromatographic peak by
obtaining MS/MS spectra on only the masses identified
within a specified time window which helps facilitate a
faster duty cycle.
Compound
Parent ion
m/z
Product ions
m/z
EEE
a
214.3
196.2
Normorphine
272.3
201.0
AEM
b
182.3
150.1, 122.1
Morphine
286.3
229.1, 211.2
Norcodeine
286.3
243.3, 225.3, 215.0
Codeine
300.3
175.0, 225.3
6-Acetylmorphine
328.3
268.3, 193.2
m-Hydroxybenzoylecgonine
306.2
168.2
Benzoylnorecgonine
276.2
154.1
Benzoylecgonine
290.3
168.2
Acetylcodeine
342.3
282.3, 225.2
Heroin
370.3
310.2, 328.2, 268.3
Cocaine
304.3
182.1
Norcocaine
290.2
168.1, 136.2
Cocaethylene
318.3
196.2
Norcocaethylene
304.2
182.1, 136.1
Methadol
312.3
223.1, 249.2, 171.2
EDDP
c
278.0
249.2
Propoxyphene
340.1
266.1
Methadone
310.9
266.2
Table 1: List of 20 drugs and metabolites with their respective parent and
product ion masses. EEE: ecgonine ethyl ester; AEM: anhydroecgonine
methyl ester; EDDP: 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium
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