HPLC, UHPLC

Addressing the Issues of Very Sharp LC Peaks Width in Quantitative LC/MS/MS

Mar 09 2010 Comments 0

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The advent of the electrospray interface for mass spectrometry in the late 1980’s [1] had a dramatic impact on the field of analytical chemistry. The sensitivity and specificity that mass spectrometry confered on analytical detection could now be combined with the sample compatibility and selectivity of liquid chromatography to produce, perhaps, the most powerful and exploited hyphenated analytical technology, LC/MS(MS) [2].

The application of LC/MS(MS) has moved from the early adopters for structural elucidation to impurity measurement, metabolite profiling [2] and bioanalysis [3]. In the pharmaceutical industry, natural products analysis, proteomics [4] and metabonomics [5]. In academia water quality testing, environmental monitoring and food safety testing to name but a few. Indeed the doping control of the sports that we watch on TV relies heavily on LC/MS as well as GC/MS [6].

The hyphenation of mass spectrometry with LC and the subsequent increase in demand for this mode of sample analysis required better utilisation of expensive MS detector technology. Many scientists quickly realized
that the increased specificity of MS and MS/MS technology reduced the need for complete analyte chromatographic resolution and analysis times could be significantly shortened from the typical 25-30 minutes to
just 3-5 minutes. Indeed many scientists in the bioanalytical field tried to remove chromatography altogether from the analysis process - relying solely on the selectivity and specificity of the mass spectrometer for the
analysis [7]. Whilst this approach had some success, factors such as ion suppression, specificity from metabolites and absolute sensitivity for low systemic exposure compounds still required some form of separation prior to mass analysis. The advent of a commercial sub 2 μm chromatographic system in the early 2000’s gave both high throughput, with analysis times in the 1-2 minute range, and high resolving power (efficiency), typically 3-5 times greater than that of an equivalent length conventional column. Many bioanalytical scientists were quick to see the potential of this form of chromatography [8], as with the increase in chromatographic efficiency came sharper peaks and hence more sensitivity, many researchers quoting figures in the 3-8 fold range, with analysis times cut by a similar figure.


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