Microsoft word - fp 2011 presentation(10-11-2011).docx
Development of a fg/mL Level LC-MS/MS Method
for Fluticasone Propionate in Human Plasma
Mohammed Abrar, John Allanson, Philip Dalton, Ian Smith and Helen Young
Unilabs York Bioanalytical Solutions, Cedar House, Northminster Business Park, Upper Poppleton, York YO26 6QR, UK
MATERIALS AND METHODS
There were no significant interferent peaks in the regions of the
Lower Limit of Quantitation
The objective of this work is to investigate a sensitive LC-
MRM chromatograms at the retention time of FP and
MS/MS method for the determination of fluticasone
Thpe previous LLOQ for fluticasone propionate achieved
3-FP, in six different individual human plasma samples.
propionate in human plasma, exploiting the latest
at York Bioanalytical Solutions was 3.0 pg/mL.
technologies, enabling a lower limit of quantitation (LLOQ)
A comparison of the chromatography obtained during
The method was shown to be specific for the determination of
previous work with that currently obtained at 0.5 pg/mL FP
FP and free from matrix-related modification of ionisation
is given below (see figures 2,3 & 4)
effects when spiked with FP at the level of 1.5 pg/mL (mean
Fluticas one 3 pg/m L
bias and CV, 14.2% and 2.7% respectively).
Fluticasone propionate (FP) is a potent, synthetic corticosteroid
Sample processing was automated using the Hamilton robotic
used to treat asthma and allergic rhinitis.
platform enabling fast and consistent batch preparation. Total
The modifications to the analytical procedures to achieve this
sample processing time for two plates was 40 minutes.
A 3 pg/mL LLOQ assay for FP was developed and validated at
T im e (m inu te s )
York Bioanalytical Solutions in 20091 and has been applied for
–We pre-treat the plasma with zinc
the analysis of over 30,000 clinical samples for FP.
sulphate/ammonium hydroxide mixture to eliminate protein
At the time the method was developed the LLOQ of 3 pg/mL
Figure 2 – Original assay showing extracted human plasma sample spiked with
binding. The introduction of this step increased SPE recovery
Fluticasone Propionate at 3.0 pg/mL retention time 3.4 mins.
was the best that could be achieved with the available
from 30% to >90%. The SPE extraction was also optimised for
technology. The limitation of this method was that later time
both volumes and solvent strengths used during wash and
points for lower dose regimes were below the LLOQ of 3
- automate the procedure using robotics, provide more
Figure 1 - Hamilton Star Robotic System
consistent results and reduce the risk of outside contamination
– Fused core technology columns provide
excellent chromatographic resolution whilst being able to
withstand high back pressures, relatively high flow rates and
Figure 3 - Current work showing extracted human plasma sample spiked with
Fluticasone Propionate at 0.50 pg/mL retention time 2.9 mins.
– Fluticasone gives 2 to 3 fold higher
absolute response in ESI mode compared to APcI in pure
standards but in extracted samples the signal to noise in APcI
is 5 times greater than in ESI. This is due to the higher
background noise observed in ESI in the presence of matrix,
hence we chose to use APcI mode to achieve the lower level of
Structure of Fluticasone Propionate
Figure 4 - Current work showing extracted human plasma blank and no peak is observed at retention time of 2.9 mins.
The objective of this work was therefore to develop a sensitive
Chromatography changed from narrow bore reverse phase
LC-MS/MS method for the determination of FP in human
column to a fused core column. Run time decreased from 6.0
plasma, with a lower limit of quantitation of 0.5 pg/mL, using
Linearity was demonstrable from the 0.5 to 40 pg/mL using
the advances in technology since 2008 and the vast
experience gained at YBS working with FP.
a linear regression of peak area ratio with a 1/x weighting.
Key characteristics of the method:
Figure 4 - Waters Xevo TQ-S LC-MS/MS System
By making judicious changes to the analytical procedure
and the application of new technologies it is possible to
LLOQ for fluticasone propionate by a factor of 6. This
Original method was on an SCIEX API 5000 MS utilising Turbo
method will enable the clinical profile of fluticasone
IonSpray transferred to a Water TQ-S MS utilising APCI and
propionate to be followed for a longer period of time and at
lower doses than can be achieved at present.
Precision and Bias
The intra assay precision and bias was within acceptable limits,
precision ranged between 3.1% and 14.4%, bias ranged
1. Poster Presentation at the 10th Annual Land ‘O’Lakes Bioanalytical Conference, WI, USA, 2009 entitled ‘A Sensitive LC-MS/MS Method for the Quantitation of
Fluticasone Propionate in Human Plasma’ by J Allanson et al.
Selectivity and Matrix Effects
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