Application Notes: General

By William Goodman, PerkinElmer, Inc.

The analysis of polar compounds in support of clinical and pre-clinical pharmacokinetic studies requires an analytical methodology capable of achieving ultra-low detection and quantification limits. The high sensitivity afforded by coupling HPLC with tandem mass spectrometry (MS–MS) has made it the technique of choice in this environment, but it is subject to the following limitations when reversed phase liquid chromatography (RPLC) is used

Multidimensional liquid chromatography (MDLC) techniques are essential for the separation of highly complex proteomic samples. Advantages of off-line MDLC techniques over on-line approaches include high flexibility in choice of column dimensions and mobile-phase compositions, and the ability to reanalyse sample fractions. Here we present a fully automated off-line two-dimensional chromatographic approach for the analysis of proteomic samples using an UltiMate 3000 system optimized for proteomics MDLC.

Combining an ultra fast LC system (e.g., Agilent 1200RRLC, Waters UPLC) with an accurate mass TOF mass spectrometer creates a powerful system for information-rich high-throughput analyses. However, for de novo formula generation and confirmation the residual mass accuracy tolerance of 3–5 ppm can still leave significant ambiguity in the proposed formula. Consequently, skilled manual inspection or further measurements deploying additional analytical techniques (NMR or MS–MS) are frequently required to arrive at a confident formula assignment.

Using ACQUITY UPLC technology with triple quadrupole MS detection enhances the selectivity, sensitivity and throughput in quantitative bioanalytical studies. Detection limits for these methods are being driven lower and lower as drugs become more potent.

Calvados is an apple-brandy of Normandy (France). Mashed apples are fermented to obtain cider, which is then distilled to give the young spirit.

The process of investigating a suspicious fire includes many different types of analyses. An essential step in confirming the presence of a liquid accelerant is gas chromatography (GC); detection with a mass spectrometer (MS) provides an accurate confirmation of both the presence and identity of an accelerant. In arson analysis, the sample preparation for GC–MS analysis is typically performed by headspace or solvent extraction.

PLOT columns are often used in GC analyses when it is necessary or desirable to retain one class of solutes in favour of other solutes that have little or limited interactions with the surface of the stationary phase. With a PLOT column, chromatographers can even cause lower boiling point compounds to elute well after higher boiling point compounds, thus providing better qualitative and quantitative separations for the solutes of interest.

Semiquantitative elemental analysis by ICP-MS is a powerful tool for quick screening of unknown samples for a wide range of elements.

One problem frequently encountered in LC–MS is the appearance of mass peaks, which appear totally unrelated to the samples run - "ghost" mass peaks. It is impossible to differentiate whether these signals come from an unknown component in the sample co-eluting with a known peak, or from an impurity in the mobile phase or from some residual contamination "bleeding" from the column.

Preliminary studies of biodiesel samples by a high speed LC–MS system using electrospray ionization and a patented cone-wash feature demonstrate that LC–MS reduces the analysis time to 20 minutes and reveals information about higher molecular weight compounds in biodiesel while still detecting many low molecular weight chemicals, including FAMEs, at high sensitivity.

The use of 30 mm UPLC columns coupled with Oasis SPE in µElution format was investigated to increase the speed of quantitative bioanalytical methods while maintaining sensitivity and resolution of closely related analytes.

The analysis of crude oil by means of different atmospheric pressure ionization (API) techniques is described. Crude oil is analysed without any separation prior to API-Fourier transform mass spectrometry. The use of a quadrupole/hexapole device to selectively enhance a certain mass range is demonstrated. Automated generation of molecular formulas from accurate mass measurements enables rapid compound identification.

Solid-phase extraction (SPE) has revolutionized sample preparation. Variations on the technique offer enhanced recovery, greater speciation and reduced solvent and sample consumption over other techniques. Micro-extraction packed sorbent (MEPS) is the miniaturization of conventional SPE from millilitre to microlitre bed volumes that allows SPE to be used with very small samples. The manipulation of the small volumes is achieved with a precision gas tight syringe. With a typical void volume of 7 μL, the volume of solvent eluted from MEPS is compatible with GC and LC inlets making it ideal for integration into an automated sampling system for on-line SPE.

Mixed mode chromatography combines aspects of ion exchange chromatography and conventional reversed-phase (RP) chromatography. The combination of both hydrophobic and ion-exchange properties allows for independent control of retention for ionizable and neutral molecules.

UltraPerformance LC (UPLC) has been widely accepted by chromatographers because of improvements over HPLC in the sensitivity, resolution and speed of separations. As scientists begin to use this technology for impurity and metabolite profiling, they will need to transfer the methods to preparative LC to isolate and purify their compounds for further research. Therefore, it is necessary to systematically transfer UPLC assays not only to HPLC, but, more importantly, to preparative chromatography. In this application, we provide information on how to scale a UPLC impurity/degradant separation to a preparative LC separation.

The exploration of myxobacterial metabolite profiles by LC–MS screening for the presence of new natural products is described. Extracts from fermentations of Myxococcus strains are analysed by UPLC-coupled ESI-TOF mass spectrometry and the obtained data are processed using principal component analysis (PCA). The generation of molecular formulae from accurate mass measurements facilitates rapid compound identification.

Brian De Borba and Jeffrey S. Rohrer, Dionex Corporation

Xiaodong Liu and Christopher Pohl, Dionex Corporation

Eric S. Grumbach, Diane M. Diehl and Jeffrey R. Mazzeo, Waters Corporation