A method for the direct injection and analysis of small volume human blood spots and plasma extracts containing high concentrations of organic solvents using revered-phase 2D UPLC/MS

Abstract

The emergence of micro sampling techniques holds great potential to improve pharmacokinetic data quality, reduce animal usage, and save costs in safety assessment studies. The analysis of these samples presents new challenges for bioanalytical scientists, both in terms of sample processing and analytical sensitivity. The use of two dimensional LC/MS with, at-column-dilution for the direct analysis of highly organic extracts prepared from biological fluids such as dried blood spots and plasma is demonstrated. This technique negated the need to dry down and reconstitute, or dilute samples with water/aqueous buffer solutions, prior to injection onto a reversed-phase LC system. A mixture of model drugs, including bromhexine, triprolidine, enrofloxacin, and procaine were used to test the feasibility of the method. Finally an LC/MS assay for the probe pharmaceutical rosuvastatin was developed from dried blood spots and protein-precipitated plasma. The assays showed acceptable recovery, accuracy and precision according to US FDA guidelines. The resulting analytical method showed an increase in assay sensitivity of up to forty fold as compared to conventional methods by maximizing the amount loaded onto the system and the MS response for the probe pharmaceutical rosuvastatin from small volume samples.

A New Phase of Drug Discovery? The Rise of Pk/Pd Means Extremely Challenging Work at The Coalface

“…Assays may need therefore to be superior to eventual clinical assays in terms of selectivity and sensitivity. Technologies such as 2D LC-MS may become routine [10];…”

The Use of Dried Blood Spots for the Quantification of Antihypertensive Drugs

“…The only problem with simvastatin is recovery, which did not exceed 68%. Rosuvastatin was assayed by 2-dimensional analytical-scale chromatography with at-column dilution enabling the injection of large sample volumes of organic extracts of DBS [ 73 ]. Analysis of rosuvastatin was performed in MRM mode with simple mixing extraction by means of 100 μ L of MeOH in the concentration range 5–80 ng/mL.…”

“…In all cases, accuracy and precision were less than 15% within the tested range, and therefore accurate pipetting during preparation of DBS may not be necessary. To minimize the influence of spot homogeneity on measurement, the same location of a spot (center) [ 29 , 48 – 51 , 53 , 54 , 61 – 63 , 69 , 73 ] or the whole spot [ 52 , 66 ] has been punched. No difference between punching the central area and punching an off­center site of DBS has been observed for pregabalin [ 61 ] and guanfacine [ 62 ].…”

“…Bosentan DBS and plasma concentrations have also been compared in a Bland–Altman plot, and a limited systematic difference between the measurements was found [ 71 ]. For rosuvastatin, quantification methods for either plasma or DBS have been developed, but comparison experiments for plasma and DBS samples have not been carried out [ 73 ]. For nifedipine, only photostability has been compared between DBS and liquid aqueous and biological matrices [ 60 ].…”