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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