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Quantitation of Six Opioids in Urine with
Super-Dilution and Microflow LC-MS/MS
Xiang He and Marta Kozak, Thermo Fisher Scientific, San Jose, CA
Application Note 570
Key Words
TSQ Vantage, Microflow, LC-MS/MS, Forensic Toxicology
Goal
To quantitate six opioids in urine with 500-fold urine dilution and microflow
LC-MS/MS for forensic toxicology use, using the Thermo Scientific Dionex
UltiMate 3000 RSLCnano LC system and the Thermo Scientific TSQ
Vantage mass spectrometer.
Introduction
Morphine, codeine, hydromorphone, hydrocodone,
oxymorphone and oxycodone are some of the most
abused opioids in the United States. Liquid chromatography-
tandem mass spectrometry (LC-MS/MS) has been widely
used for their quantitation in forensic toxicology. The
analytical methods typically use normal LC flow rates
(~0.5 mL/min) and sample preparation usually involves
solid phase extraction (SPE) for sensitive detection.
Microflow LC uses significantly lower flow rates (15 to
50 μL/min). With the same sample amount and identical
LC peak width, the reduction in LC flow rate results in a
much-improved detection limit for concentration-
dependent detection techniques such as electrospray
ionization (ESI) mass spectrometry. Because of this
sensitivity increase, we can achieve a similar analytical
performance for sensitive measurements of urine opioids
for forensic toxicology purposes with a simple “dilute-
and-shoot” approach.
Our goal was to use a super-dilution approach to improve
the dilute-and-shoot detection of opioids in urine by
minimizing matrix effects, and to compensate the
sensitivity decrease from super-dilution by using
microflow LC. We anticipated savings in solvent
consumption and the cost of waste disposal, better
environmental conservation, and improved longevity of
the LC-MS/MS system.
Methods
Sample Preparation
Urine samples were spiked with internal standards (IS) and
then mixed with
β
-glucuronidase and incubated at 60 °C for
hydrolysis. Methanol was added to the mixture and the
supernatant was diluted. The tested dilution factors were
100, 250 and 500. The mixture was centrifuged at 17,000
g
for 5 minutes, and 20 µL of supernatant was injected for
microflow LC-MS/MS analysis.
LC-MS/MS Conditions
LC-MS/MS analysis was performed on a TSQ Vantage
™
triple stage quadrupole mass spectrometer coupled to an
UltiMate
™
3000 RSLCnano LC system equipped with a
microflow flow rate selector. The microflow LC plumbing
was set up in “pre-concentration on a trapping column”
mode (Figure 1). The temperature of the columns was
maintained at 35 °C. The trapping column was a Thermo
Scientific Hypersil GOLD PFP drop-in guard cartridge
(10 × 1 mm, 5 μm particle size) in the guard holder, and
the analytical column was a Hypersil GOLD™ PFP column
(100 × 0.32 mm, 5 μm particle size). LC connections were
made with Thermo Scientific Dionex nanoViper fingertight
fittings. The LC gradients for sample loading and analytical
elution are shown in Figure 2. The mass spectrometer was
operated with a heated electrospray ionization (HESI-II)
source in positive ionization mode. Data was acquired in
selected-reaction monitoring (SRM) mode. Detailed source
parameters and SRM settings are shown in Figure 3. For
each analyte, two SRM transitions were monitored. One
of them was used as the quantifier and the other as
qualifier. The signal ratio between the qualifier and the
quantifier was used to evaluate the validity of the results,
and any ratio outside 20% (relative to the ratio) was
considered an invalid data point.