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Quantitation of Six Synthetic Opioids
in Urine Using a Triple Stage Quadrupole
LC-MS System
Kristine Van Natta, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Introduction
Synthetic opioids have analgesic, antitussive and anti-
addictive effects. However, they are also abused for their
psychoactive effects and are often diverted from lawful
prescriptions to unlawful recreational use. Simple, robust
and precise analytical methods are needed to quantify
these compounds in biological matrices for forensic
purposes.
Goal
To develop a specific and robust dilute and shoot quan-
titative method for the analysis of six synthetic opioids
and their primary metabolites in urine. These compounds
include: methadone, EDDP, merperidine, normeperidine,
propoxyphene and norpropoxyphene.
Methods
Sample Preparation
Urine was mixed with methanol containing deuterated
analog internal standards. The supernatant was diluted
with water prior to liquid chromatography-mass
spectrometry (LC-MS) analysis.
HPLC Conditions
Chromatographic analysis was performed using Thermo
Scientific Accela 600 HPLC pumps and a Thermo
Scientific Hypersil GOLD aQ column (50 x 4.6 mm,
1.9 µm particle size). The total run time was 5 minutes.
MS Conditions
MS analysis was carried out on a Thermo Scientific
TSQ Quantum Ultra triple stage quadrupole mass
spectrometer equipped with a heated electrospray
ionization (HESI-II) probe. Two selected reaction
monitoring (SRM) transitions were monitored for each
compound to provide ion ratio confirmations (IRC).
Application
Note: 545
Key Words
• TSQ Quantum
Ultra
• Transcend
LX-2 system
• Forensic
Toxicology
Validation
Standard curves were prepared by fortifying pooled blank
human urine with analytes. Quality control (QC) samples
were prepared in a similar manner at concentrations
corresponding to the low (LQC), a middle (MQC) and
high (HQC) end of the calibration range. Intra- and
Inter- run variability and robustness were determined
by analyzing five replicates of each QC level with a
calibration curve on three different days. Matrix effects
were investigated by spiking seven different lots of human
urine with analytes at 50 ng/mL and calculating peak area
recovery.
Results and Discussion
The method is linear from 20 to 5,000 ng/mL with
R
2
values > 0.99 for all six compounds. Figure 1 shows
the representative calibration curves. All IRCs passed
within 20% of the standards average. All calibrators back
calculate to within 15% of nominal, 20% for the limit of
quantitation (LOQ). All quality controls quantitated to
within 15% of nominal for the middle and high controls
and within 20% for the low control. Inter-assay %CV was
less than 10% for all QC levels. Table 1 shows quality
control statistics for the validation runs.
No matrix effects were observed during validation.
All samples showed recoveries within 20% of nominal.
Internal standard variation was less than 5% between the
different lots. Table 2 shows matrix effects testing results.
Figure 2 shows a reconstructed SRM chromatogram
at LOQ.