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Quantitative Forensic Analysis of Opiates,
Opioids, and Their Metabolites in Human
Urine Without Hydrolysis
Sarah Fair Wandland, Kerry Hassell, Joseph Herman, Thermo Fisher Scientific, Franklin, MA
Application Note 601
Key Words
Opiates, opioids, metabolites, sample preparation liquid chromatography
(SPLC), mass spectrometry (MS), forensic toxicology
Goal
To develop a quantitative forensic method for analysis of opiates, opioids,
and their metabolites in human urine without the time-consuming step of
hydrolysis.
Introduction
Analysis of opiate and opioid metabolites in urine is most
often done with a hydrolysis step that make total sample
preparation time up to 24 hours. The method described
here eliminates the hydrolysis step by analyzing the
conjugated metabolites intact using a Thermo Scientific
™
Prelude SPLC
™
system for sample preparation and a
Thermo Scientific
™
TSQ Endura
™
triple quadrupole
mass spectrometer for analysis.
Experimental
Sample Preparation
Urine samples, which were free of opiates, were diluted
two-fold with water and methanol (95:5) containing
internal standards. There were a total of 10 deuterated
internal standards in solution at a concentration of
50 ng/mL. After the addition of the internal standards,
50 µL of each sample were injected onto the analytical
column at a temperature of 27 °C.
Calibration standards containing all 19 compounds at
concentrations ranging from 5 to 500 ng/mL were prepared
in urine. Quality control (QC) samples were also prepared
in urine at three levels: 12, 225, and 400 ng/mL.
SPLC Method Parameters
Instrumentation
Prelude SPLC system (Figure 1)
Analytical column
Thermo Scientific
™
Accucore
™
aQ column
(100 x 2.1 mm, 2.6 μm particle size),
catalog # 17326-102130
Mobile phase A
0.1% formic acid in water
(Fisher Chemical brand)
Mobile phase B
0.1% formic acid in methanol
(Fisher Chemical brand)
Gradient
Refer to Table 1
Table 1. Gradient details
Step Start
(min)
Time
(s)
Flow
(mL/min) Grad.
%A %B
1 0.00
20
0.40
Step 100.0 0.0
2 0.33
5
0.40
Step 92.0 8.0
3 0.42
50
0.40
Step 92.0 8.0
4 1.25
5
0.40
Step 75.0 25.0
5 1.33 130
0.40 Ramp 65.0 35.0
6 3.50
45
0.40
Step
0.0 100.0
7 4.25 100
0.40
Step 100.0 0.0
Figure 1. Prelude SPLC system with TSQ Endura triple quadrupole
mass spectrometer