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Quantitation of Seven Designer Cathinones in Urine Using Q Exactive Mass Spectrometer
Overview
Purpose:
To evaluate various scan modes available through high-resolution, accurate-
mass analysis to determine suitability for
in vitro
plasma protein binding assay
analysis.
Methods:
An
in vitro
plasma protein binding assay was analyzed using various scan
modes available to a high-resolution, accurate-mass analysis LC-MS system and the
results compared to data obtained using a triple quadrupole mass spectrometer.
Results:
The lower limit of detection was found to be between 5 nM and 50 nM in full
scan mode. The 5 nM was detected for a majority of the samples analyzed using full
scan mode. The signal response was determined to be linear across 3 orders of
magnitude for most test compound calibration curves. The results for the calculated
amount of the free fraction remaining (% Free) for the binding assay demonstrated a
good correlation between the results for the high-resolution, accurate-mass analysis
and the results collected using LC-MS/MS analysis. Sample analysis performed using
SIM mode provided a lower limit of detection of 5 nM for all compounds in the assay
calibration curve demonstrating an improvement in sensitivity for several compounds
in the more targeted scan mode.
Introduction
High-resolution mass spectrometers are becoming increasingly more powerful and
capable of sophisticated scanning experiments that offer new solutions to complex
challenges. Additionally, assays that fall into a well defined and routine workspace,
such as
in vitro
screening assay in early drug discovery, will also benefit from the ease
of use and high performance of high-resolution mass spectrometric analysis but do not
require all available scan capabilities needed for more complex applications. In this
evaluation several different full scan and SIM analyses were used to analyze a protein
plasma binding assay with an Thermo Scientific™ Orbitrap™ mass analyzer and the
results compared to previous analysis performed using traditional LC-MS/MS on a
triple quadrupole mass spectrometer.
Methods
Sample Preparation
A set of 24 of commercially available drug compounds was selected based on reported
binding properties and molecular weight and incubated in an
in vitro
plasma protein
binding assay in triplicate at a concentration of 10 µM. Samples were incubated for 6.5
hours in a dialysis block followed by protein precipitation. Protein precipitation was
performed by first adding 150 mL of acetonitrile containing internal standard compound
(Alprenolol) to a 96-well 340-mL V-bottomed storage plate followed by addition of 50
mL of each of the assay samples. Calibration curves were also generated for each
compound. A working stock solution of 50 mM in DMSO was first made for each
compound. A five-point standard curve at concentrations of 5, 50, 500, 1000 and 2000
nM was prepared for each compound by serial dilution from the working stock solution
into a blank mixed matrix using an eight channel pipette
1
.
Liquid Chromatography
Gradient elution was accomplished using water (A) + 0.1% Formic Acid (v/v) and
Acetonitrile (B) + 0.1% Formic Acid (v/v). The gradient was held at 98% aqueous for
0.25 minutes, ramped to 98% B over 0.35 minutes, and held at 98% B for 0.2 minutes
before returning to the starting conditions at 2% B for a 0.4 minute equilibration time.
Chromatographic separation was performed using a C18, 2.1 x 30 mm, 3µm column
with 5uL injections made for each sample. All injections were completed using a
Thermo Scientific™ Accela™ Open system with DLW (Dynamic Load and Wash) and
with Thermo Scientific™ Accela™ 1250 pumps at a flow rate of 900 µL/min.
Mass Spectrometry
Samples were analyzed using both a Thermo Scientific™ Exactive™ Plus mass
spectrometer in Full Scan mode (
m/z
220 – 900) and a Thermo Scientific™ Q
Exactive™ mass spectrometer in both Full Scan (
m/z
220 – 900) and SIM mode with
each using a resolution setting of 35,000 (FWHM) at
m/z 200
and a spectral speed of
7 Hz. Generic ion source conditions were used for all sample collection including
vaporizer temperature (350 °C), capillary temperature (300 °C), sheath gas of 45
arbitrary units, and an auxiliary gas of 10 arbitrary units. The instrument was
calibrated in positive ion mode before sample acquisition using Thermo Scientific™
Pierce™ LTQ Velos™ ESI Positive Ion Calibration Solution.
Results
Scan Mode Signal Res
Each compound analyze
a concentration curve to
compounds were serially
ranging from 5 nM to 200
analysis. The calibration
regression and 1/x
2
weig
of more than 20% of the
The majority of the comp
the required sensitivity a
dilution and correlate wel
quadrupole mass spectr
mode is displayed below
FIGURE 1. Calibration
analysis, (B) Q Exactiv
Exactive Plus Full Sca
Data Analysis
Data was acquired using
software. Chromatograp
performed and reported
Gubbs Inc., GMSU Gubb
measurements in the buf
area measurement in the
of unbound compound (
compound replicate was
obtained using a triple q
the % Free values for ea
analyzed.
0
2
4
6
8
0
200
40
Peak Area Ratio
R^2 = 0.99326
0
1
2
3
4
5
0
200
400
Area Ratio
R^2 = 0.99775
0
2
4
6
8
0
200
400
Area Ratio
R^2 = 0.99987
0
2
4
6
0
200
400
Area Ratio
R^2 = 0.99967
(D)
(C)
(B)
(A)