P3: Hair and whole blood matrix effect in identification of a model analyte and determination of its limit of detection

Abstract

Any kind of biological material matrix may represent a serious problem during real samples analyses, especially using LCMS method. It causes both signal intensity loss and/or signal to noise ratio (S/N) decrease. Unfortunately, this phenomena touches selected ions sometime in a different way. It means that even deuterated standards in some cases cannot protect from precision disturbance or even substance misidentification. The study aim was to elaborate a control method of a hair or whole blood matrix influence on a limit of detection (LOD) in identification and quantitation aspects. Waters Alliance 2695 Separations Module with Quattro Micro Micromass were applied in the research. Analyses were performed using LiChroCART 55x4 Purospher RP-18 column from Merck as well as by direct injection without separation in flow injection analysis (FIA). The mobile phase consisted of 0.1 % (v/v) formic acid in acetonitrile and water. Flow rate was set to 0.5 ml/ min. Hair samples free of drugs were used in testing. Hair were powdered in ball-mill and 50 mg weighed samples were macerated in 1 ml of methanol during 72 hours in 37°C. Results were compared to extract of 0,5 ml whole blood sample (also free of drugs) obtained by ethyl acetate at pH 9. Short segments of mass range were set (25 amu) to improve sensitivity in scan mode. SIR and MRM modes were applied in LOD testing. Morphine was engaged as a model drug for matrix influence investigation. Ions map for m/z 50 to m/z 450 was created both for blood and hair matrix. That allowed to compare possible interferences from those matrices. Signal intensity and S/N were used for calculations. Plots of S/N versus analyte concentration appeared to better show LOD than signals intensity. Some ions (not the weakest) or MRM transitions disappeared from chromatograms of morphine causing lack of all parameters for appropriate identification although analyte was present in higher than LOD concentration. Standard infusion during real sample analysis a few seconds after analyte retention time helped to solve problem of analyte presence in a sample. Matrix effect very often cannot be avoided but always should be controlled.