AI-64310-CRFT

Quantitation of Opiates to Low ng/mL

Levels in Urine for Forensic Use Using

an Affordable, High-Resolution,

Accurate-Mass Mass Spectrometer

Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, San Jose, CA

Application Note

615

Key Words

Q Exactive Focus, opiates, morphine, codeine, hydromorphone,

hydrocodone, oxymorphone, oxycodone, TraceFinder, forensic toxicology,

drugs of abuse, PRM, parallel reaction monitoring

Goal

To evaluate the performance of th

e Thermo Scientific ™ Q Exactive ™ Focus hybrid quadrupole-Orbitrap ™ mass spectrometer a

s a quantitative platform

for HPLC-MS analysis of opiates in human urine to low ng/mL levels for

forensic toxicology.

Introduction

Forensic toxicologists need an economical instrument

capable of both screening a large number of compounds

and quantifying smaller panels to industry-established

limits. Here we present a method for quantitation of six

opiates—morphine, codeine, hydromorphone,

hydrocodone, oxymorphone, and oxycodone—in human

urine down to low ng/mL levels. This work was

performed on a

Q Exactive Focus hybrid quadrupole- Orbitrap mass spectrometer.

Methods

Sample Preparation

Samples were processed by enzymatic hydrolysis followed

by urine dilution. Briefly, an aliquot of urine was spiked

with stable-isotope-labeled internal standards and

incubated with ß-glucuronidase enzyme. The resulting

mixture was centrifuged and further diluted before an

aliquot was analyzed by gradient HPLC and a Q Exactive

Focus MS. Calibrators and controls were prepared by

spiking compounds into blank synthetic urine in the range

of 1 to 5000 ng/mL.

Liquid Chromatography

Gradient elution was performed using a

Thermo Scientific ™ Dionex ™ UltiMate ™ 3000 RSLC

system with OAS autosampler (Figure 1). Mobile phases

consisted of 10 mM ammonium acetate with 0.1% formic

acid in water and methanol (Fisher Chemical brand) for

solvents A and B, respectively. The column used was a

Thermo Scientific ™ Accucore ™ PFP, 2.6 µm particle size, 50 x 2.1 mm fused core (p/n 17426-052130). The

gradient was run from 0 to 70% mobile phase B over

3.3 minutes followed by a column wash at 100% B and

re-equilibration to starting conditions. The total run time

was 5.3 minutes.

Mass Spectrometry

Compounds were detected on a

Q Exactive Focus MS

equipped with a

Thermo Scientific ™ Ion Max ™ source

and

a heated electrospray ionization (HESI-II) sprayer. Data

was acquired in parallel reaction monitoring (PRM)

mode. In this mode, a single precursor ion is selected in

the quadrupole with an isolation width of 3.0

m/z

and

fragmented in the HCD cell. The resulting MS/MS

product ions are detected in the Orbitrap detector at a

resolution of 35,000.

Method Evaluation

The method precision and accuracy were evaluated by

running a calibration curve and quintuplicate replicates

of quality controls on three different days. Additionally,

internal-standard response was assessed in 58 donor

samples obtained from a collaborator laboratory and

compared to a sample prepared in water to determine

matrix effects.

Figure 1. Q Exactive Focus MS with UltiMate 3000 RSLC HPLC

pump and UltiMate 3000 OAS autosampler.