LC-MS has conquered the bioanalytical laboratory

The introduction and rapid advancement of atmospheric pressure ionization sources for mass spectrometers in the eighties and nineties has revolutionized the bioanalytical laboratory in the pharmaceutical industry. Electro-spray ionization (ESI), atmospheric pressure chemical ionization (APCI) and more recently, atmospheric pressure photo-ionization (APPI) allow robust and easy-to-operate coupling of Liquid Chromatographic separations to Mass Spectrometric detection. More than 90% of the assays for pre-clinical and clinical studies are nowadays developed on LC-MS systems. Decisive benefits, responsible for the massive breakthrough of LC-MS, are high sensitivity and selectivity, especially in MS/MS mode (precursor-ion selection followed by selected fragment detection) and speed of analysis. Speed being a result of the high selectivity, because the LC separation time can be reduced significantly: LC-MS run times of less than 2 minutes are not exceptional. For quantitative analysis, triple-quadrupoles have proven to be the most suitable mass detectors, in terms of precision and linearity. Positive-ion ESI seems to become the dominating ionization method because most drugs have basic functionalities and ESI has a higher success-rate as compared to APCI in bioanalytical method development in the popular 0.2 - 1ml/min flow rate region.

The importance of sample prep for LC-MS

In spite of the unrivaled selectivity of MS detection, good sample clean-up remains a prerequisite for reliable LC-MS performance. This is mainly due to a phenomenon named matrix ion suppression (or ionization suppression): matrix components of biological samples interfere with the ionization process of the analytes, causing significant loss of sensitivity and unreliable quantitation. In addition, most matrix components such as proteins are non-volatile and will rapidly contaminate the MS inlet. The chromatographic answer to ionization suppression is increasing the resolution between analyte and matrix components i.e. longer retention times. In other words: if you want to maximize LC-MS throughput you need to maximize sample clean-up. At Spark we are continuously working on improved on-line sample clean-up, targeted on reduction of ionization suppression. Our most recent advancement is temperature-assisted on-cartridge protein precipitation for (significantly!) enhanced deproteinization of biological samples. Obviously, enhanced deproteinization will also have a beneficial effect on LC-column life time. Another favorable aspect of on-line SPE is enrichment of the analyte. Enrichment factors up to 10,000 have been reported for environmental water analysis by loading 1 Liter water sample on the SPE cartridge. For bioanalysis a sample volume of several ml plasma is no problem and > tenfold enrichment is therefore not exceptional. As a result, better sensitivity is obtained or a less sensitive MS system is required for adequate assay LOD’s.

On-line sample prep compatibility with high throughput LC-MS

Once sample clean-up is adequate to allow for short LC-MS run times, the challenge for on-line SPE is to keep pace with the speed of the LC-MS run to make sure that it will not become the speed limiting component of the SPE-LC-MS system. Rapid sampling and fast, high-pressure SPE permit SPE cycle times in the order of 1 minute. The use of a dual cartridge clamping system allows parallel processing of SPE and LC-MS. As a result, in most cases the contribution of the SPE procedure to the total SPE-LC-MS assay cycle time is zero!


	Symbiosis system