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Rapid Analysis of Opiates from

Low Volume Whole Blood Samples by

LC-MS/MS Utilizing TurboFlow Methods

Peter Ashton, Alex Allan, Bob Ardrey, Triple A Forensics Ltd., Oldham, UK

Shane McDonnell, Sarah Robinson, Thermo Fisher Scientific, Hemel Hempstead, UK

Introduction

The opiate morphine, and its derivatives, are medicines often

used for pain-relief, cough-relief and as anti-diarrhoeals. For

example, codeine and dihydrocodeine (morphine derivatives)

are available in over-the-counter preparations in combination

with paracetamol (acetaminophen) and are slowly meta-

bolized to morphine and dihydromorphine respectively.

However, the semi-synthetic opiate diacetylmorphine (heroin)

is subject to wide abuse and has become such a major

social problem that it is responsible for almost half of

the drug-related deaths in the UK.

1

Heroin is deacetylated very rapidly (half-life ca. 3 mins in

plasma) to its major active metabolite 6-monoacetylmorphine

(6-MAM), which readily penetrates the blood-brain barrier

to produce the desired euphoric effects.

2

6-MAM also has

a short plasma half-life of about 38 minutes (producing

morphine), and thus, its detection in blood is very important

to the forensic toxicologist in establishing the recent use of

heroin.

3

As a product of heroin metabolism, via 6-MAM,

or from its own administration, morphine also undergoes

further metabolism. The conjugation step produces inactive

morphine-3-glucuronide (M3G) and the potently active

morphine-6-glucuronide (M6G) along with other minor

ones, including diglucuronides.

The forensic toxicologist is often asked to interpret

results and possibly account for time of death in opiate

(especially heroin) abuse cases. This task can be made easier

if it is possible to identify and quantify the components

such as 6-MAM, morphine, codeine, dihydrocodeine and

the glucuronides in whole blood rather than urine. The

volume of a human whole blood sample, however, may

often only be available in the low microlitre range, thus

presenting sample preparation and analysis sensitivity issues.

The analysis of free- and protein-bound opiate analytes

in human whole blood by LC-MS/MS is routinely done

after rigorous sample cleanup via solid phase extraction or

liquid-liquid extraction in order to minimize ion suppression

in the ionization source of the mass spectrometer. These

clean up steps can be lengthy, laborious and expensive.

Here we present a method to quantitatively analyze opiate

compounds present in whole blood utilizing a simple, fast,

low-volume extraction procedure followed by a Thermo

Scientific TurboFlow method, an online extraction and

chromatography coupled with selected reaction monitoring

tandem mass spectrometry.

Goal

To replace laborious off line sample preparation with

TurboFlow

methodology and tandem mass spectrometry

for the analysis of opiates in acetonitrile extracts from low

volume whole blood samples.

Experimental

Sample Preparation

Horse blood was spiked with a mixture of opiates (codeine,

morphine, 6-MAM, M3G, M6G and d6-codeine) at

concentrations ranging from 1 ng/mL to 500 ng/mL.

150 µL spiked whole blood was mixed with 200 µL

acetonitrile and vortexed. The resulting sample was then

centrifuged for 10 min at 300 rpm. The supernatant was

placed into a 96-well microtitre plate and 10 µL of the

supernatant was used for the analysis.

TurboFlow Methodology

Thermo Scientific Transcend TLX-1 system

Column:

Thermo Scientific TurboFlow Cyclone MAX 0.5 x 50 mm

Mobile phase A:

0.1% formic acid

Mobile phase B:

0.1% formic acid in acetonitrile

Mobile phase C:

10 mM ammonium bicarbonate pH 9

Mobile phase D:

10 mM ammonium acetate pH 6

Analytical LC

Column:

Thermo Scientific Hypersil GOLD aQ 50 x 2.1 mm, 1.9 µm

Mobile phase A:

0.1% formic acid

Mobile phase B:

0.1% formic acid in acetonitrile

The eluent gradients for both pumps are shown in Table 1.

Key Words

• Transcend TLX-1

• TurboFlow

Technology

• TSQ Quantum

Ultra

• Whole Blood

• Opiates

Application

Note: 461

Table 1: Thermo Scientific Aria operating software gradient programs for the Transcend

TLX-1 system with TurboFlow method and analytical LC method.

Flow rate is reported as mL/min.

lacitylanA

dohteM wolFobruT

Step Start

Sec Flow Grad %A %B %C %D Tee Loop Flow Grad %A %B

1 00:00 30 1.50 Step

-

-

100

-

==== out

0.30 Step 100

0

2 00:30 60 0.20 Step 100

-

-

-

T

in

0.10 Step 100

0

3 01:30 60 1.50 Step

-

-

-

100 ==== in

0.30 Ramp 5

95

4 02:30 120 1.50 Step 99

1

-

-

==== in

0.30 Step

5

95

5 04:30 60 1.50 Step

-

-

100

-

==== out

0.30 Step 100

0

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