0.4 pg/mL - XenoBiotic Laboratories

An Extremely Sensitive LC-MS/MS Method for Quantitation of Fluticasone Propionate (0.4 pg/mL) in Human Plasma
Xinping Fang, Dawei Zhou, and Jinn Wu
XenoBiotic Laboratories, Inc., 107 Morgan Lane, Plainsboro, NJ 08536 USA,
Overview
An extremely sensitive and specific liquid
chromatographic-tandem mass spectrometric (LC-MS/MS)
method capable of quantifying fluticasone propionate in
human plasma is described.
In this method,
method 0.5
0 5 mL of plasma was used.
used The drug
was extracted from plasma using a reverse phase C18 solid
phase extraction procedure. The analytical separation was
performed on a reverse phase C18 column with Acquity
UPLC. Detection was achieved using a Waters Xevo TQ-S
tandem mass spectrometer employing electrospray ionization
in the positive ion mode along with multiple reaction
monitoring (MRM). The lowest limit of quantitation is 0.400
pg/mL .
The method has been successfully validated and
applied to sample analysis in bioequivalent studies.
Introduction
Fluticasone propionate is an inhaled bronchodilator used to
relieve the nasal symptoms of seasonal and year-round
allergies. It helps to reduce the inflammation that leads to
symptoms including congestion, sneezing, itchy, and runny
nose. Two year ago, we published a validated method for
determination of fluticasone propionate with the lowest limit
of quantitation (LLOQ) of 1 pg/mL in human plasma. With
this method, several thousand clinical samples have been
analyzed. Recent clinical trials involve a lower dose level to
improve the efficacy and safety. Since the circulating levels of
fluticasone propionate are very low, it was necessary to further
increase assay sensitivity to provide an adequate
pharmacokinetic assessment. Here we report a more
ruggedness analytical method capable of quantifying
fluticasone propionate from 0.5 mL of human plasma at the
level of 0.4 pg/mL. Currently, this is the most sensitive
bioanalytical method for the determination of fluticasone
propionate concentrations human plasma samples.
Results and Discussion
Sample Preparation
The fluticasone propionate and the internal standard
(fluticasone propionate-D3) were mixed with the buffer
solution and transferred onto the preconditioned Strata C18-E
96-well plate (Phenomenex). The plate was preconditioned
with 0.8 mL of methanol and 0.8 mL of water. The loaded
plate
l were washed
h d with
i h 1.6
1 6 mL
L off water, andd 0.8
0 8 mL
L off 20%
methanol in water. Both analyte and internal standard were
then eluted using 90% methanol in water.
+MS2 (501.10) CE (20): 1.927 to 1.994 min from
Sample 4 (100 ng/mL MSMS) of test032613.wiff (Turbo
Spray), subtracted (1.692 to 1....
3.2e4
275.4
Max. 3.2e4 cps.
313.2
MRM of 2 Channels ES+
501 > 313 (Fluticasone Propionate)
6.00e3
(A)
2.0e4
%
Mobile Phase B)
Gradient
Flow rate:
Injection Volume:
1.4e4
205.5
369.0
341.2
361.1
251.2
8000.0
481.2
463.2
501.4
333.2
109.1
100
160
220
280
340
400
460
Method Recovery
0
540
m/z, Da
0.20
0.80
1.40
LLOQ
13012011M094
2.00
2.60
(B)
Daughters of 501ES+
2.80e7
%
Mass Spectrometry
MS System:
Condition:
MRM Transition:
Fluticasone Propionate:
Fluticasone Propionate-D3
0
204.7
332.9
358.8 389.0
406.9
109.0
154.9
0
100
160
220
280
340
m/z, Da.
481.1
400
460
540
504.2
MRM of 2 Channels ES+
501> 313 (FluticasonePropionate)
2.00e4
100
Kinex 2.6 µ C18 column
313.2
313.2
1.40
2.00
2.60
3.40
Time
•
The objective of method development for quantitation
of fluticasone propionate in human plasma was to achieve the
best sensitivity in human plasma with 0.5 mL of plasma volume.
In our previous article, we have validated a method capable
quantifying 1 pg/mL of fluticasone propionate from 0.6 mL of
human plasma
plasma. In the sample preparation procedure of this
study, SPE preconditioning, washing solution, and eluting
solution were optimized with different volumes, different
percentage, and different type of organic solution. A good
extraction recovery (~79%), and low matrix effect (~ -9%) were
obtained. Therefore, there is a little room to be improved in
sample extraction part.
Ascentis Express 2.7 µC18 column
501.2
0.80
Figure 3. LC-MS/MS (MRM) ion chromatograms of blank plasma (A), and
lowest calibration standard with 0.4 pg/mL (B)
Figure 1. Fluticasone propionate positve full scan mass spectrum
collected at previous mass spectrometer (A), and the one collected at
Xevo TQS mass spectrometer (B)
Waters Xevo TQ-S System
LC/(+)ESI-MS/MS (MRM)
0.20
501.0
274.9
Gemini‐ NX 3 µ C18 column
BEH 1.7 µ C18 column
%
QC
Conc. (pg/mL)
LLOQ
0.400
Low
1.20
Medium
80.0
High 160
0.400 to 200 pg/mL
0.997
Accuracy
RE%
2.75
0.83
‐1.13
‐3.13
Compared with Nominal Value (%)
> 78.50
Time
FP
313.0
293.0
(B)
3.40
MRM of 2 Channels ES+
501 > 313 (Fluticasone Propionate)
6.00e3
1.45
100
12660 MS1M013 89 (1.487) Cm (84:89-(61:67+109:118))
100
Acquity UPLC, I Class
BEH C18, 50 x 2.0 mm, 1.7 
Water with 0.04% ammonium
hydroxide
10% IPA in methanol
0.5 mL/min
10 µL
Validation data summary of fluticasone propionate in human plasma
Inter‐Batch (n=18)
LC-MS/MS Conditions
Liquid Chromatography:
HPLC System:
Analytical Column:
Mobile Phase A)
Table I.
Calibration Range
Coefficient of Determination (R 2; Mean)
Accuracy & Precision
FP
0.0
The eluted samples were evaporated to dryness in 96-Well
Nitrogen Evaporator (EvapArray ) at about 35 °C. The
residue was redissolved in 125 µL of 50% methaol in
water.
Blank
13012011M002
100
293.1
(A)
2.6e4
Freeze/Thaw
Bench‐Top
Long‐Term Storage Stability
Condition
3 Cycles, ‐20 ºC
21 hrs, Room Temperature
3 month, ~ ‐20 ºC
Precision
CV%
12.9
9.34
2.27
1.99
Accuracy
RE%
‐3.70~2.50
3.20
‐15.0~‐2.50
•
In order to further improve the method sensitivity, we focused on
how to improve the LC chromatography and mass spectrometer
sensitivity. In this method development, we have screened different
column with different manufactures (Figure 2)
2). Acquity UPLC BEH
column gives lower noise background, higher peak response (better
column recovery), and narrower peak width. Another key factor is the use
of the extremely sensitive Xevo@ TQ-S mass spectrometer. The use of the
StepWave ion guide in Xevo@ TQ-S resulted in a 5-fold increase in
sensitivity, compared to previous generations of mass spectrometer
(Figure 1). The ion pattern is very similar but the absolute response of
TQ-S system is much higher. Figure 3 shows the fluticasone propionate
ion chromatograms of calibrator with zero and 0.4 pg/mL (LLOQ)
concentration.
•
Excellent linearity was obtained with the average Coefficient of
determination of 0.997. The inter-day precision (CV%) and accuracy
(RE%) for all QC plasma samples, including LLOQ were <13% and <3%,
respectively (Table I). Three freeze/thaw cycles, ambient temperature
storage QC samples for up to 21 h prior to analysis, and 3 month ~-20 °C
long-term storage QC samples appeared to have little effect on the
quantitation.
Structures
0
0 10
0.10
0 70
0.70
1 30
1.30
Time
1 90
1.90
A
2 50
2.50
B
O O
S
H
F
O
H
Conclusion
O O
HO
HO
Figure 2. The LC performance comparison of four brand column.
Acquity UPLC BEH column gives highest peak response
and lowest background (giving a better S/N ratio)
O
D 3C
O
S
H
F
F
H
O
F
F
Figure 4. Chemical structure of fluticasone propionate (A), and
fluticasone propionate-d3 (B)
F
This is a most sensitive bioanalytical method for
quantitation of fluticasone propionate in human plasma in current
clinical sample analysis. The method is validated to demonstrate its
ultra sensitivity, highly selectivity and robustness.

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