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

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 Sample Preparation
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. Matrix effects
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). INTRODUCTION
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 Sample preparation –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 Chromatography
Chromatography – 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. Mass Spectrometry – 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 Linearity
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. Mass Spectrometry
Key characteristics of the method:
Figure 4 - Waters Xevo TQ-S LC-MS/MS System CONCLUSION
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, References
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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