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Verification of an LC-MS/MS Forensic Method for 19 Opioids, Opiates, and Their Metabolites in Human Urine Without Hydrolysis
Overview
Purpose:
This is the verification of a forensic method containing a panel of 19 opiates
without the labor-intensive step of hydrolysis. These compounds were analyzed using
a Thermo Scientific™ Prelude SPLC™ system for chromatographic separation coupled
with a Thermo Scientific™ TSQ Endura™ triple quadrupole mass spectrometer.
Methods:
All compounds were spiked into human urine, diluted, and injected onto the
Prelude SPLC system equipped with a Thermo Scientific™ Accucore™
aQ analytical
column. After elution from the analytical column, the compounds were simultaneously
analyzed using the TSQ Endura MS with a heated electrospray ionization (HESI-II)
probe in positive mode. The total method time was approximately four minutes,
allowing for rapid quantification of all analytes and 10 internal standards.
Results:
All 19 opiates, opioids, and their metabolites were verified successfully and
passed all acceptance criteria. These compounds were analyzed without hydrolysis,
drastically reducing the total sample preparation time.
Introduction
It is currently common practice in many forensic laboratories to analyze opiates and
opioids using hydrolysis during sample preparation. This process can take up to
24 hours to complete so that all conjugated metabolites are converted back to their
parent molecules. We introduce a method that alleviates these time constraints,
allowing for an efficient, sensitive analysis of the intact metabolites. Such metabolites
(for example, morphine glucuronides) have a reputation for being difficult to
chromatograph and detect at low levels. The Prelude SPLC system equipped with an
Accucore aQ column offers great retention of all analytes and resolution of isobars.
The highly sensitive TSQ Endura MS reproducibly detects the low end of the
calibration range for even the least responsive analytes and provides accurate
quantification of these somewhat problematic compounds.
Methods
Sample Preparation
The analytes of interest were spiked into human urine at various concentrations to
make calibrators and controls. Each sample was divided into two parts; one set was
hydrolyzed prior to analysis and the second set was analyzed directly. Hydrolysis was
performed by adding 1 M ammonium acetate buffer containing
b
-glucuronidase to
samples and incubating overnight at 60
°
C. The nonhydrolyzed samples had the same
volume of ammonium acetate buffer added but without any
b
-glucuronidase present
and no incubation. Isotopically labeled internal standards were then added to all the
samples. The samples were vortexed and centrifuged. The supernatant was removed
from the pellet and transferred into clean vials for LC-MS/MS analysis.
Liquid Chromatography
The LC method is shown in Table 1. A Prelude SPLC system, seen in Figure 1, was
used in LX mode, equipped with a 2.1 x 100 mm, 2.6 µm particle size Accucore aQ
analytical column. The mobile phases consisted of 0.1% formic acid in (A) water and
(B) methanol. Using less than 2 mL of solvent, per injection, the LC system was able to
successfully resolve all isobaric compounds within this method. The total method time
of 4.25 min, when multiplexed, allows for results every 2 min for all 19 compounds.
Step
Start
(min)
Time
(s)
Flow
(mL/min)
Grad
%A
%B
1
0.00
20
0.40
Step
100.0
-
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
-
100.0
7
4.25
100
0.40
Step
100.0
-
TABLE 1. Liquid chromatography program for the method, including solvent
composition, flow rate, and timing
FIGURE 1. Prelude S
Mass Spectrometry
A TSQ Endura triple q
positive mode was use
Data Analysis
All data acquisition an
Scientific™ Aria™ MX
software version 3.1.
Results
Calibration standards co
5
–
500 ng/mL were prep
prepared in urine at thre
tested by using five repli
quantitating them using
Carryover was calculate
quantitation (LLOQ) by t
limit of quantitation (UL
signal. Additionally, auto
QC samples that were r
to a freshly prepared cal
The assay precision ha
calibration standard lev
calibrations standard lev
passed acceptance crite
chromatograms at the lo
in Figure 2. Additionally,
glucuronides in Figure 3
for oxymorphone-3
b
-glu
R
2
> 0.9900.
Lastly, all mass spectro
the Thermo Scientific™
transitions and paramet
spectrometers required
analysis.