Page 337 - CRFTApplication Compendium_090613

Application
Note: 439
Key Words
• Accela™ UHPLC
System
• MSQ Plus MS
Detector
• Drugs of Abuse
• Hypersil GOLD™
PFP Columns
• Sensitivity
UHPLC/MS: An Efficient Tool for
Determination of Illicit Drugs
Guifeng Jiang, Thermo Fisher Scientific, San Jose, CA, USA
Goal
Optimize a UHPLC/MS method with respect to stationary
phase, mobile phase, and detector settings to achieve
picogram level quantitation of fourteen drugs and metabolites
employing a 12 minutes separation.
Introduction
Gas chromatography-mass spectrometry (GC-MS) is
commonly employed for the separation and identification
of drugs and metabolites in forensic toxicology, using
electron impact (EI) or chemical ionization (CI).1 This
methodology has become a “gold standard” in terms of
admissibility and defensibility in court because of its good
sensitivity, excellent selectivity and a high degree of
standardization.2 However, laborious and time consuming
derivatization procedures and sample clean ups are
mandatory in most cases.
LC/MS methods eliminate the need to derivatize and
often simplify sample preparation. However, long run times
and low separation efficiency limit the utility of conventional
HPLC. Ultra high performance liquid chromatography
(UHPLC) performs separations 5 to 10 times faster than
conventional HPLC by employing sub-2 µm diameter
particles. The 1-2 second peak widths and relatively high
separation efficiency of UHPLC are more competitive with
capillary GC, making UHPLC-MS an attractive alternative
method for illicit drug analysis.
This application note illustrates the separation and
detection of a mixture of 14 illicit drugs/metabolites by ultra
high performance liquid chromatography-mass spectrometry
(UHPLC-MS). The drugs/metabolites are separated on a
Hypersil GOLD PFP, 1.9 µm, 100 x 2.1 mm column
and detected by a fast scanning single quadrupole mass
spectrometer.
Experimental Conditions
1. Drug Standard Preparation
Pseudoephedrine, ephedrine, amphetamine, methampheta-
mine, 3,4-methylenedioxy-N-methamphetamine (3,4-MDMA),
oxycodone, hydrocodone, clonazepam, noscapine,
cocaine, caffeine, tetrahydrocannabinol (THC), cannabinol
and cannabidiol standards (1 mg/mL in methanol) were
purchased from Alltech-Applied Science (State College,
PA, USA). The above fourteen compounds were mixed
with the optimized molar ratio in the range of 1 to 100
and diluted to 0.1 ppm with methanol to make the drug
mixture standards.
2. Chromatographic Conditions
Chromatographic analyses were performed using the
Accela UHPLC system (Thermo Scientific, San Jose, CA).
The chromatographic conditions were as follows:
LC Column: Hypersil GOLD, 1.9 µm, 20 x 2.1 mm
Hypersil GOLD, 1.9 µm, 50 x 2.1 mm
Hypersil GOLD, 1.9 µm, 100 x 2.1 mm
Hypersil GOLD, aQ (polar endcapped C18), 1.9 µm,
100 x 2.1 mm
Hypersil GOLD PFP (perfluorinated phenyl), 1.9 µm,
100 x 2.1 mm
Hypersil GOLD PFP (perfluorinated phenyl), 1.9 µm,
50 x 2.1 mm
Column Temperature: 45 °C
Injection: 1 µL Partial Loop Injection, 25 µL Loop Size
Syringe Speed: 8 µL/sec
Flush Speed: 100 µL/sec
Flush Volume: 400 µL
Wash Volume: 100 µL
Flush/Wash Source: Bottle with methanol
Gradients: Method I
Column: Hypersil GOLD PFP 1.9 µm, 100 x 2.1 mm
A: Water (0.06% acetic acid)
B: Acetonitrile (0.06% acetic acid)
C: Methanol (0.06% acetic acid)
Flow Rate: 1000 µL/min
Time (min) Eluent A% Eluent B% Eluent C%
0.00 95.0 1.0 4.0
0.10 88.0 2.4 9.6
5.00 85.0 3.0 12.0
13.00 5.0 19.0 76.0
13.90 5.0 19.0 76.0
14.00 95.0 1.0 4.0
15.00 95.0 1.0 4.0
For the other gradient methods used, see Appendix A
for details.
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