Screening ionisation and chromatography conditions for quantitative LC/MS methods.

To develop an optimal quantitative LC/MS method with high sensitivity, high selectivity and robustness in a limited time period can be very challenging, especially for methods in which many analytes are to be quantified. In this study the relevant options are reviewed and a simple screening strategy of mass spectrometric and chromatographic conditions is presented. The strategy is divided into two stages, mass spectrometric ionisation screening and reversed phase LC column screening. The objective of the first stage is to find out how sensitivity is affected by ionisation technique, ionisation polarity and buffer. The compounds are dissolved in different buffers covering a broad pH range. Thereafter they are injected using flow injection analysis without LC column, evaluating both electrospray and atmospheric pressure chemical ionisation (APCI). In the second stage the buffers yielding the best sensitivity and selectivity in the ionisation screening stage are used as mobile phase buffers to LC column screening with different stationary phases applying a shallow gradient. The aim is to find the combinations of column(s) and buffer(s) that give symmetric peaks, adequate retention and selectivity. Finally the retention is adjusted using isocratic or gradient elution. The strategy provides a simple and practical experimental design that allows fast screening a large range of ionisation and chromatographic conditions especially for multiple compounds. The examples included in this study demonstrate that optimal buffer, ionisation technique, ionisation polarity and column cannot be predicted from compound properties such as structure and pK(a).

Determination of ximelagatran, melagatran and two intermediary metabolites in plasma by mixed-mode solid phase extraction and LC-MS/MS.

An analytical method was developed for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and the two intermediate metabolites, OH-melagatran and ethyl-melagatran in human plasma. Extraction of plasma was carried out on a mixed mode bonded sorbent material (C8/SO(3)(-)). All four analytes, including their isotope-labelled internal standards, were eluted at high ionic strength with a mixture of 50% methanol and 50% buffer (0.25 M ammonium acetate and 0.05 M formic acid, pH 5.3) with an extraction recovery above 80%. The extracts were demonstrated to be clean in terms of a low concentration of albumin and lysoPC. The sample extraction was fully automated and performed in 96-well plates using a Tecan Genesis pipetting robot. Analysis of the extracts were performed with liquid chromatography followed by positive electrospray ionization mass spectrometry. The low organic content and the low pH of the extracts allowed for, after dilution 1:3 with buffer, direct injection onto the LC-column. The four analytes were separated on a C18 analytical LC-column using gradient elution with the acetonitrile concentration varying from 10 to 30% (v/v) and the ammonium acetate and acetic acid concentration kept constant at 10 and 5 mmol/L, respectively, at a flow rate of 0.75 mL/min. Linearity was achieved over the calibrated range 0.010-4.0 micromol/L with accuracy and relative standard deviation in the range 96.9-101.2% and 6.6-17.1%, respectively at LLOQ, and in the range 94.7-102.6% and 2.7-6.8%, respectively at concentrations above 3 x LLOQ. The method replaces a manual method, and displays the advantages of having a fully automated sample clean-up, no evaporation/reconstitution step, high recovery, and complete LC-separation of all four analytes.