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Quantitation of Six Opiates in Urine Using
a Triple Stage Quadrupole LC-MS System
Kristine Van Natta, James Byrd, Marta Kozak; Thermo Fisher Scientific, San Jose, CA
Introduction
The natural opiates morphine and codeine are widely
prescribed drugs for their analgesic, antitussive and
antidiarrheal effects. However, they are also widely
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
quantitative method for the analysis of primary natural
opiates and their metabolites in urine. These compounds
include: morphine, codeine, oxymorphone, oxycodone,
hydromorphone and hydrocodone.
Methods
Sample Preparation
Urine was spiked with deuterated analog internal
standards and hydrolyzed with ß-glucuronidase.
Methanol was added to the hydrolysis mixture and the
resulting mixture was centrifuged. Supernatant was further
diluted and subject to 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.
Application
Note: 546
Key Words
• TSQ Quantum
Ultra
• Transcend
LX-2 system
• Forensic
Toxicology
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).
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-run
variability and robustness were determined by analyzing
six replicates of each QC level with a calibration curve.
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 10 to 6,000 ng/mL with R
2
values > 0.99 for all six compounds. Figure 1 shows cali-
bration curves for the six compounds. All calibrators back
calculate to within 15% of nominal (20% for LOQ). All
quality controls quantitated to within 15% of nominal for
the middle and high controls and within 20% for the low
control. %CV was less than 10% for all QC levels, except
for codeine LQC which was 17.2%. 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. Table
2 shows matrix effects testing results.