Page 200 - CRFTApplication Compendium_090613

2 Development of a Dilute-and-Shoot LC-MS/MS Method with 21 Opiates in Urine
Introduction
Opiates are primarily central nervous system depressants and narcotic analgesics,
which can be abused and often lead to addiction. Pain management physicians need
to verify that patients are taking their prescribed medications as directed to monitor
patients for compliance. The number of new pain management forensic laboratories in
operation are increasing worldwide to keep up with sample demand.
Traditionally, urine opiates forensic testing has been by competitive immunoassays.
While this method is fast and relatively inexpensive, it often lacks specificity, since the
antibodies are directed against drug groups or classes, not at specific drug
compounds. Other more specific methods, such as GC/MS, were used to identify or
quantitate the concentration of a specific opiate. But GC/MS requires extensive sample
preparation (derivatization) and suffers from long analytical run times.
LC-MS/MS is emerging as a new “gold standard” for opiates testing in current pain
management forensic laboratories. A typical workflow includes acid or enzyme
hydrolysis of opiates glucuronides into free opiates prior to LC-MS/MS analysis. This
strategy might simplify the numbers of drugs to be analyzed, but also suffers from
disadvantages such as long incubation times (2 hours+) and poor analyte recovery.
Detection of opiate metabolites can also provide important evidence that the parent
drug was possibly being abused. This work demonstrates a simple dilute-and-shoot
quantitative method of 21 opiates, metabolites and common glucuronides by liquid
chromatography tandem mass spectrometry (LC-MS/MS), without the limitation of
time-consuming hydrolysis. This method provides broad drug coverage, fast turn-
around time and compares favorably to hydrolysis analysis.
Methods
Urine specimens were diluted 4-fold with high purity water and 25 µl injected directly
into The LC-MS/MS system (with addition of Internal Standard [IS}). A Thermo
Scientific™ Transcend™ TLX-2 HPLC coupled with Thermo Scientific™ TSQ Quantum
Ultra™ mass spectrometer was used for LC-MS/MS analysis in Selected Reaction
Monitoring (SRM) mode. Two Thermo Scientific™ Syncronis™ C18, 3x100 mm 5 µm
columns were used to achieve multiplexing capability. The LC run time is 8 minutes
with MS data collection window of 3.5 minutes for all 21 Opiates, metabolites and
select glucuronides.
All calibration standards, synthetic urine and deuterated internal standards used for
quantitation were purchased from Cerilliant (Roundrock, TX). Surine was used as urine
matrix to make calibration standards. Pilot study urine samples were obtained from
commercial reference lab.
HPLC Method Parameters
LC: TLX-2 HPLC
Analytical Column: Syncronis C18, 3x100 mm, 5 µm column
Solvent A 0.1% formic acid in water
Solvent B 0.1% formic acid in methanol
Mass Spectrometer Parameters
MS: Thermo Scientific™ TSQ Quantum Ultra™ triple stage quadrupole
Polarity: Positive mode
MS Ionization Source: Heated electrospray (HESI)
Spray Voltage: 4000 V
Sheath Gas Pressure (N2): 60 arbitrary units
Auxiliary Gas Pressure (N2): 15 arbitrary units
Vaporizer Temperature: 600 C
Capillary Temperature: 350 C
Collision Gas Pressure: 1.5 mTorr
Q1 Peak Width: 0.7 Da
Q3 Peak Width: 0.7 Da
Results and Di
A multiplex quantitative meth
established. The 21 drugs incl
normeperidine, O-desmethyltr
oxycodone, oxymorphone, mo
norhydrocodone, Morphine-3-
codeine-6-beta-D-glucuronide,
hydromorphone-3-beta-D-gluc
parent mass and fragmentatio
only be achieved by complete
We have optimized the LC se
mobile phases and pH conditi
16 opiates and 5 glucuronides
3x100 mm, 5 µm columns.
Data Acquisition and Proces
Thermo Scientific™ Xcalibur
Quality Control
Each analyte and internal stan
to pass pre-set target ratios (q
ion ratios criteria is as follows:
>50%
20-50%
10-20%
<10%
Target ratio(
FIGURE 1. Chromatogram l