Automated DPX process - Gerstel

analyst to plan, optimize, and set up the
whole process.
From theory to practice
In order to test the MPS-DPX-CIS-
GC/MS system in practice, the scientists
analyzed blood and urine samples
that had been spiked with different drugs
and pharmaceuticals. Compounds determined
included amphetamines, benzodiazepines,
cocaine and methadone as well
as tetrahydrocannabinol (THC) and metabolites.
For details, please see the graphic
representations on this page. The analysis
was performed using deuterated internal
standards: For blood samples, d5-PCP
(0.2 ppm) was used. For the determination
of benzodiazepines, d5-Nordiazepam (0.2
ppm) and d5-OH-Alprazolam (0.2 ppm)
were used. For the opiates, equivalent deuterated
compounds were used (each at a
level of 0.1 ppm).
16
TIC
GERSTEL Solutions Worldwide Innovation
EIC
Drug Average RSD
Amphetamine 13.9 %
Methamphetamine 14.4 %
Meperidine 5.8 %
PCP 2.2 %
Methadone 3.3 %
Methaqualone 3.6 %
Amitriptyline 3.1 %
Cocaine 3.8 %
Cis Doxepin 2.8 %
Imipramine 3.3 %
Trans Doxepin 3.2 %
Pentazocine 5.3 %
Codeine 4.2 %
Desipramine 6.4 %
Required manual sample
preparation steps
Preparing blood samples: 0.5 mL of acetonitrile
was added to a 0.25 mL sample of
whole blood followed by mixing to precipitate
proteins in the sample. The mixture
was centrifuged and the supernatant transferred
to a clean labelled test tube containing
0.1 mL of 0.1 M HCl.
Preparing urine samples for the determination
of benzodiazepines: In order to
determine the total level of free, bound and
metabolized residues of drugs and pharmaceuticals
in urine, hydrolysis must be performed
of the respective conjugates, such
as for example glucuronides of benzodiazepines,
which are metabolites of the drugs
that are formed to facilitate excretion of the
substances from the body.
The hydrolysis reaction is started by
add ing 10 µL of a solution of the enzyme
b-glucuronidase and 50 µL of a 0.1 M sodi-
Drugs of abuse in blood: Total ion chromatogram of a DPX extract
of 250 µL whole blood spiked at 0.5 ppm with drugs of abuse using
d5-PCP as internal standard. Chromatograms from the 5th and
20th injections are shown alongside each other to demonstrate
the ruggedness of the analysis. The insert shows the extracted
ion chromatogram (EIC) from the 5th injection. The sample was
protein precipitated with 0.5 mL acetonitrile, and the supernatant
was transferred to clean tubes. After adding 0.1 mL of 0.1 M HCl,
automated DPX was performed. 1) Meperidine, 2) d5-PCP (ion
205), 3) PCP, 4) Methadone, 5) Methaqualone, 6) Amitriptyline, 7)
Cocaine, 8) cis-Doxepin, 9) Imipramine, 10) trans-Doxepin, 11)
Desipramine, 12) Pentazocine, 13) Codeine (Septum bleed from
vial cap after repeat injections from the same vial).
um phosphate buffer with pH 4 to a 0.2 mL
sample of urine. The mixture is kept at 55 °C
for two hours and is then allowed to cool to
room temperature. Acetonitrile (0.25 mL)
is then added in order to preci pitate the
enzyme. Following centrifugation, the supernatant
is transferred to a clean, labelled
test tube and 200 µL of 0.1M HCl is added.
The prepared samples are then placed in the
MPS sample tray.
Automated Disposable Pipette Extraction
(DPX) is subsequently performed on
the prepared samples using the MultiPurpose
Sampler (MPS) equipped with 1 mL
CX tips from DPX Labs (www.dpxlabs.
com). As the name suggests, CX tips contain
a novel and unique cation exchange material
with additional slightly apolar characteristics.
The DPX process is completely
automated: 250 µL of a 30 % solution
of acetonitrile in water is dispensed onto
the DPX sorbent inside the tip for condi-
Analysis conditions
CIS: 1 min solvent purge (150 mL/
min); splitless; temperature
program: 80 °C (1 min) –
12 °C/min – 300 °C (3 min)
Column: 30 m HP5 (Agilent Technologies);
di = 0.25 mm; df = 0.25 µm
Pneumatics: He, constant flow, 1.5 mL/min
GC oven: 100 °C (0.5 min) – 20 °C/min
– 300 °C (12.5 min)
MSD: Selectable Full Scan
or SIM mode
GERSTEL Solutions Worldwide – March 2009