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Addressing the Issues of Matrix Resolution and Measurement in Bioanalytical Assays

Presented by Diego Rodriguez Cabaleiro, Waters Europe, Guyancourt, France
Authors: Geneen Baynham [1], Paul D Rainville [1], Marian Twohig [1], Robert S Plumb [1] and Ian Wilson [2]

[1] Waters Corporation, Milford, MA 01757
[2] AstraZeneca, Macclesfield, UK.

In quantitative bioanalysis the analytical technique of choice is LC/MS/MS due to the high sensitivity and selectivity that it affords.  Quantitative bioanalytical methods development is complicated by matrix interferences which can alter the response of the analyte. 

Matrix effects, resulting from co-eluting matrix components that compete for charge in the ionisation process, manifest themselves as suppression or occasionally enhancement of the analyte signal.  Matrix effects are caused by numerous factors all of which can cause significant errors in the accuracy and precision of a bioanalytical assay, Phospholipids, Subject differences, Impurities, Degradation Products, Co-eluting metabolites

Monitoring the presence and impact of matrix effects can complicate and prolong the development of a robust, sensitive assay. 

In this poster we evaluate the use sub 2um particle LC on the matrix effects observed.  We will also demonstrate how the background matrix interference can be monitored in a single run analytical using a mass spectrometer equipped with a novel fast switching quadrupole MS.  We will also demonstrate how the matrix effects can be determined and quantified by an integrated matrix calculator tool.  We will show how the use of these tools can significantly shorten the time taken to develop a sensitive, robust bioanalytical LC/MS/MS assay. 

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LC/MS/MS Quantification of Fluticasone Furoate in Human Plasma at the fg/mL Level

Presented by Diego Rodriguez Cabaleiro, Waters Europe, Guyancourt, France
Authors: Jennifer Keller [1]; Joanne Mather [2]; Nancy Zheng [1]; Zong-Ping Zhang [1]

[1} PPDI, Middleton,, WI;
[2] Waters Corporation, Milford, MA, USA

Novel Aspect: 

New generation of LC/MS/MS methodology allowing previously unobtainable levels of sensitivity for the low exposure compound Fluticasone furoate.

Introduction 

Fluticasone furoate is used in the management of asthma and chronic obstructive pulmonary disease; fluticasone is a trifluorinated corticosteroid which has potent anti-inflammatory activity. As this compound undergoes extensive first-pass metabolism, there is negligible systemic exposure. In order to accurately define the pharmacokinetics of this compound it is necessary to be able to detect the compounds down to the sub- pg/mL level. Historically LC/MS/MS instrumentation has not provided sufficient sensitivity to quantify the plasma concentrations of this compound at the fg/mL level. In this paper we demonstrate the quantification of fluticasone at the sub pg/mL level using LC/MS/MS.

Methods 

The fluticasone furoate was extracted from 1 mL of human plasma using a LLE method. The final volume of the extract was 100µL. The chromatography was operated in reversed-phase UPLC with gradient and optimized to provide separation of the analytes of from the endogenous components in the sample over 5.5 minutes. Basic mobile phase (0.1% NH4OH) gave the greatest sensitivity for the analyte. The LC/MS analysis of the fluticasone was performed on an ACQUITY UPLC system employing sub 2µm particle technology coupled to a Xevo TQ-S tandem quadrupole mass spectrometer operated in MRM mode. A stable label isotope was used as the internal standard for the analyte in this study.

Preliminary Data 

As Fluticasone furoate is dosed by the inhaled route the majority of the drug is directed to the lungs. The remainder is eliminated in the liver to via hydrolysis of the S-fluoromethyl carbothioate function to form the inactive 17?-carboxylic acid metabolite. These factors result in circulating levels in the 0.5pg/mL – 1pg/mL level. The high protein binding, greater than 99%, also present a challenge when developing a high sensitivity assay. The various extraction procedures were evaluated such as liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase/liquid-liquid extraction (SLE+) and protein precipitation in tandem with SPE such as HLB by varying the following parameters: extraction buffers, extraction solvents, and PH. The combination of Zinc Sulphate dilution to LLE gave a reproducible high extraction efficiency sample prep approach with extraction recovery more than 95% and significantly lowered background noise. The sub 2um separation resolved the active compound from endogenous interferences and metabolites. The assay cycle time was 5.5 mins with a peak width of 2 secs at base. The novel ion guide optics in the MS resulted in sufficient sensitivity to detect and quantify the compound at very low levels. The limit of quantification fluticasone was determined to be 0.2 pg/mL. The peak width of the fluticasone analyte was calculated to be approximately 3 seconds at the base, this allows for a high resolution separation of the analyte from the endogenous material in the sample. The calibration was shown to be linear over the range of 0.2 - 100pg/mL. The signal to noise of the 0.2pg/mL standard was determined to be 7:1. This level of sensitivity allows for the accurate determination of the pharmacokinetics of the Fluticasone furoate in plasma.