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Quantitation of 47 Forensic Compounds in Urine by HPLC-MS
Overview
Purpose:
To demonstrate analytical workflow for robust, fast and cost efficient analysis
of large panel of drugs in urine samples.
Methods:
Hydrolyzed and diluted urine samples were analyzed with 8 minute LC-MS
method on a dual channel LC system coupled to triple quadrupole mass spectrometer.
Results:
We demonstrated simple and cost efficient method producing data which
meet forensic toxicology lab requirements for sensitivity and robustness.
Introduction
Forensic toxicologists face an ever-expanding list of compounds for analysis. The need
to reliably quantitate large-panel assays with ion ratio confirmation is continually
increasing. Large panel assays are required in order to speed sample analysis time,
lower analytical costs and obtain results quicker while keeping good data quality.
Methods
Sample Preparation
•Take a 200-µL aliquot of urine sample.
•Add 100 µL
β
-glucuronidase and incubate 2 hours at 60 C.
•Cool samples and add 100 µL of Internal Standard spiking solution in methanol.
•Vortex and then centrifuge.
•Dilute sample 15 fold with water to minimize matrix effects.
•Inject 10 µL onto analytical column.
Note: Deuterated analogs of each analyte were used as internal standards.
Liquid Chromatography
•Column: Thermo Scientific ™Accucore
TM
PFP 2.6 µm , 5 0x 2.1 mm
•Mobile phase:
•A: 10 mM ammonium formate, 0.1% formic acid in water (Fisher
Scientific™Optima™ LC/MS)
•B: 10 mM ammonium formate, 0.1% formic acid in methanol water
((Fisher Scientific™ Optima™ LC/MS)
•Ambient temperature
•LC Gradient (Table 1)
Table 1. Liquid chromatography program for the method, including solvent
composition, flow rate, and timing
Results
Mass Spectrometry
A Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer with a
HESI ionization probe in polarity switch mode was used as the detector. Two SRM
transitions were collected for each analyte, and one SRM transition was collected for
each internal standard. The SRM transitions collection time scheme is presented in
Figure 1.
Data Analysis
All data acquisition and quantification for this method was performed using Thermo
Scientific ™ TraceFinder™ software version 3.2.
Step
Start
(min)
Time
(s)
Flow
(mL/min)
%A
%B
1
0.00
30
0.5
100.0
0
2
0.5
30
0.5
80
20
3
1.0
180
0.5
30
70
4
3
120
0.5
0
100
5
5
6
1.0
0
100
6
5.01
114
1
-
100.0
7
7.0
6
1
100
0
8
7.01
54
1
100
0
Method Performance E
Calibration standards an
Concentration of the high
individual analytes stock
The following method per
•
LOQ:
precision within
•
Linearity range
: all cali
•
Precision:
5 replicates
•
Matrix effects
: Internal
calculated against interna
Figure 1.
SRM transition
Deuterated internal stand
analyzed with the same a
Figure 1. Calibration cu
standards showing qu
Morphine
nicotine
Zonisamide-neg
Zonisamide
Oxymorphone
Hydromorphone
7-aminoclonazepam
Meprobamate
phenobarbital
Benzoylecgonine
gabapentin
codeine
Pregabalin
6 MAM
noroxycodone
butalbital
Oxycodone
7-aminoclonazepam
2-1000 ng/mL
Morphine
50-1000 ng/mL
7-aminoclonazepam
2-1000 ng/mL