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The Utilization of Novel Platform in a LC-MS/MS Work ow for the Analysis of Vitamin D, Testosterone, Immunosuppressants, Chemotherapeutics and Cortisol
TABLE 1. Method Range, L
TABLE 2. Intraday Accurac
Overview
Purpose:
To demonstrate the validity of the Prelude Sample Preparation Liquid
Chromatography (SPLC) system, a new LC/MS/MS platform that reduces solvent
consumption, requires less maintenance, and is easier to use then traditional systems.
Methods:
Prelude SPLC
TM
, Turbulent Flow Chromatography, LC/MS/MS, Multiplexing
Results:
Methods for 25-hydroxy-vitamin D2 and D3, testosterone, the immunosuppressant
drugs Sirolimus, Tacrolimus, Everolimus, and Cyclosporine A, the chemotherapeutic drugs
Busulfan, Docetaxel, Methotrexate and Imatinib, and cortisol were validated using a Prelude
SPLC
TM
LC/MS/MS platform.
Introduction
A new LC system was specifically designed to reduce instrument maintenance, down time,
and operating costs for high-throughput, LC/MS/MS applications which require sample clean-
up prior to HPLC analysis. The Prelude SPLC System utilizes syringe pumps designed to
deliver the volume of mobile phase required for each sample analysis with a single push of
the piston. This pump design greatly reduces the wear and tear on pump seals and check
valves, because the pistons in dual piston reciprocating pumps can move several hundred if
not thousands of times per sample run. The majority of maintenance required on traditional
HPLC pumps results from the wear of the seals and check valves; therefore, syringe pumps
are more robust than traditional HPLC pumps. The Prelude SPLC Systemʼs also have
extremely low dead volumes making rapid changes in mobile phases possible. The time
required for many of the steps in a method to occur are reduced resulting in shorter run times
and lower solvent costs for equivalent methods.
In order to prove the utility of the Prelude SPLC platform, several LC/MS methods that are
currently used by clinical researchers were validated. The successful validation of such a wide
range of analytes using the new platform demonstrates that the Prelude SPLC offers a viable
alternative to existing LC/MS systems. Reduced system void volumes resulted in methods that
had run times 20-30% shorter then their equivalent methods run on a conventional HPLC and
produce a corresponding reduction in mobile phase consumption.
Methods
All samples were vortexed, mixed with internal standard solution and centrifuged.
Supernatant was removed and transferred into sampling containers for LC-MS/MS analysis.
On-line sample clean-up using a 0.5x50 mm ThermoScientific HTLC-C18 XL TurboFlow
column was followed by chromatographic separations of 25-OH-D
2
, 25-OH-D
3
,
immunosuppressants, chemotherapeutics, cortisol and testosterone using a 50x2.1mm, 2.6
µm particle size ThermoScientific Accucore PFP analytical column. The detector was a TSQ
Vantage triple quadrupole mass spectrometer with HESI-II ionization probe in positive mode.
Mobile phases were (A) 10 mM ammonium formate in water, (B) 10 mM ammonium formate in
Methanol, and (C) 45/45/10 acetonitrile/isopropanol/acetone. All run times were 4 minutes or
less and when mutiplexed the effective analysis time was reduced to 2 minutes per sample.
The Immunosuppresants were run in spiked human whole blood with cell lysis and protein
precipitation occurring at the same time as the addition of the internal standard. Testosterone
analysis was performed in spiked testosterone depleted human plasma. Chemotherapeutics
were run in spiked human plasma. Cortisol was run using synthetic urine but was validated
against human urine samples containing known levels of Cortisol
Results
Accuracy and precision experiments were performed for system verification from three
separate preparations on calibrators and controls on three different days . The interday and
intraday accuracy and precision results were obtained for 25-OH-D
2
and 25-OH-D
3
at a
concentration range of range of 2-100 ng/mL. The range for testosterone was 0.02-10 ng/mL.
Immunosuppressants and chemotherapeutics were analyzed in ranges from 1-2000 ng/mL.
The method range for cortisol was 3.62 - 362 ng/mL (0.1-10 nM). The method precision had
RSD values were less than 15.0% for all compounds tested. Additionally, accuracy was ±15%
of the theoretical value for all the methods. The correlation coefficient values for all the
compounds ranged from 0.991 to 0.999, showing linearity throughout all concentrations and
analytes. All the analytes passed carryover, benchtop stability, autosampler stability, and
Compound Name
(
L
Cyclosporin A
2.
Sirolimus
1.7
Everolimus
1.9
Tacrolimus
1.
Testosterone
0.1
Cortisol
Busulfan
0.
Docetaxel
0.
Imatinib
1
Methotrexate
0.1
25-hydroxy Vit D2
0.
25-hydroxy Vit D3
1.
TABLE 3. Interday Accurac
Compound Name
(
L
Cyclosporin A
Sirolimus
Everolimus
Tacrolimus
Testosterone
Cortisol
Busulfan
Docetaxel
Imatinib
Methotrexate
25-hydroxy Vit D2
25-hydroxy Vit D3
Compound Name Metho
Cyclosporin A
Sirolimus
Everolimus
Tacrolimus
Testosterone
Cortisol
Busulfan
Docetaxel
Imibitib
Methotrexate
25-hydroxy Vit D2
25-hydroxy Vit D3