Quantitative determination of BAF312, a S1P-R modulator, in human urine by LC-MS/MS: Prevention and recovery of lost analyte due to container surface adsorption

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC-MS/MS method for the determination of a drug candidate. BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the non-specific loss' of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption call be quickly identified by using freshly spiked urine calibration standaids and pre-pooled QC samples during a LC-MS/MS feasibility run. The resulting low recovery of all analyte in urine samples can be prevented through the use of additives. such as the non-ionic surfactant Tween-80, CHAPS and others. to the container pilot to urine sample collection. If the urine samples have not been transferred from the bulk container, the 'non-specific binding' of all analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective beta-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of beta-cyclodextrin to untreated urine samples does not recover the 'lost' analyte due to non-specific binding or container surface adsorption in the case of BAF312, a dynamic range of 0 0200-20 0 ng/ml in human urine was validated with all overall accuracy and precision for QC sample results ranging from -3.2 to 5 1% (bias) and 3 9 to 10 2% (CV), respectively. Pre- and post-addition of 0 5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid-liquid extraction in combination with an isocratic LC separation was employed The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at <=-60 degrees C and for at least 3 months when stored at <=-60 degrees C The current work Could serve as a simple example in trouble shooting non-specific binding or container Surface adsorption in quantitative analysis Of urine samples (C) 2010 Published by Elsevier B.V.