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).
Clinical Pharmacology & DMPK, AstraZeneca R&D Mölndal, Pepparedleden 3, S-431 83 Mölndal, Sweden.
Henrik
H
Sillén
Niklas
N
Magnell
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*