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Thermo Scientific Poster Note
•
PN64227-MSACL-EN 0814S
mobile phases to be completed in a fraction of the time and effort.
Methods
Sample Preparation:
“Neat” specimens were prepared in HPLC
-grade solvents -
acetonitrile, methanol, water - using standards purchased from
Cerilliant
(Round Rock, TX).
Blood serum specimens and corresponding calibrators and quality
controls (QCs) were subjected to protein precipitation by mixing 1:2
with acetonitrile containing internal standard (IS) - 25-OH-VitD3-d6 or
d3-methylmalonic acid. After centrifugation, 50 uL of supernatants
from the 25-OH-Vitamin D (VitD) batches were injected directly into
the UHPLC system. From the methylmalonic acid (MMA) batches,
100 uL of supernatants were evaporated to dryness by heated
nitrogen flow. The residues were derivatized by 100 uL of 10% acetyl
chloride in butanol for 15 minutes. After evaporation to dryness and
reconstitution with 100 uL of 50% methanol in water, 10 uL injections
of each sample preparation were made into the UHPLC system.
Urine specimens and corresponding calibrators and QCs to be
analyzed for buprenorphine & norbuprenorphine (Bup/Norbup) were
hydrolyzed by incubating a mixture of 150 uL of
β
-glucuronidase
solution (10,000 U/mL, pH 5) with 200 uL of specimen, and 50 uL of
IS solution containing - buprenorphine-d3 & norbuprenorphine-d4 for
1.5 hours at 60°C. Each preparation was then mixed with 200 uL of
cold methanol and refrigerated for 10 minutes before centrifugation.
20 uL injections of supernatants from each preparation were made
into the UHPLC system.
Urine specimens and corresponding calibrators and QCs to be
analyzed for ethyl-glucuronide & ethyl-sulfate (EtG/EtS) were diluted
1:10 with water and then spiked with 50 uL of IS solution containing
EtG-d3 & EtS-d3 before making 20 uL injections into the UHPLC
system.
Note:
β
-glucuronidase powder was purchased from Sigma-Aldrich. All
other reagents and consumables were from Thermo Fisher Scientific.
Liquid Chromatography:
The UHPLC system was a Thermo
Scientific™ Transcend™ II LX4 equipped with binary
-solvent pumps
and a dual-arm autosampler configuration. The columns and mobile
phase conditions for each method are described with the results.
Mass Spectrometry
The Thermo Scientific™ TSQ Endura™ triple
-quadrupole mass
spectrometer was used with APCI when multiplexing VitDs with MMA
batches or HESI when multiplexing Bup/Norbup with EtG/EtS
batches. Ion source and MS/MS conditions are described with the
results.
System Control & Data Analysis
Thermo Scientific™
TraceFinder
™ with Aria™ MX software was used
to control the Transcend II LX4 and Endura MS/MS systems, submit
FIGURE
Start
FIGURE
Desired
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