Identifying and quantifying analytes in highly complex samples, such as extracts from food, animal feed, pesticides, mycotoxins, and veterinary drugs, is a major analytical challenge for mass spectrometry. Accurate screening of these samples is critical in regulated environments such as food and animal-feed analysis.

Triple-quadrupole mass spectrometers have been used for identifying and quantifying analytes in complex samples, though that approach comes with a number of limitations, including the inability to re-interrogate the data postacquisition, as well as the inability to interrogate unknowns and the limited number of compounds per analysis. Time-of-flight (TOF) instruments have also been used for this application, but the technology's low resolution (15,000) has led to inaccurate mass measurements caused by unresolved background matrix interferences.

A growing record of successful experiments has shown that the ability to perform high-resolution liquid chromatography–tandem mass spectrometry (LC–MSⁿ) analyses is the key to measuring accurate mass, determining elemental compositions, and performing highly specific quantification on highly complex samples.

While the advantages of high resolution have been demonstrated, there remains significant confusion in the scientific community about what constitutes a high-resolution approach to MS. This article will attempt to clarify the terms, technology, and methods used in high-resolution MS and then present a brief case study that shows the application of a high-resolution LC–MSⁿ approach in greater depth.

Defining "High Resolution"

The application of high-resolution technology in MS is fundamentally the same as in photography: a high-resolution photograph shows great detail; a low-resolution photograph is blurry. However, the terms high (mass) resolution and accurate mass are often (incorrectly) used interchangeably. High resolution is necessary to separate peaks from one another and to ensure that ions of only one kind contribute to a particular measurement. The measurement may be an accurate mass determination or a highly specific quantification.

High resolution is particularly important for experiments involving complex mixtures, such as samples generated from a matrix (for example, biological, environmental), because these will often contain a significant number of background ions. In such cases, high resolving power will make the difference between detecting analytes at low concentration or not detecting these analytes due to the masking effect of isobaric matrix interferences.

Because two terms, resolution and resolving power, are used to characterize mass spectrometers, it is useful to look at their definitions.

The International Union of Pure and Applied Chemistry (IUPAC) defines resolving power in MS as
"The ability to distinguish between ions differing in the quotient mass/charge by a small increment. It may be characterized by giving the peak width — measured in mass units, expressed as a function of mass — for at least two points on the peak, specifically at 50% and at 5% of the maximum peak height."

The IUPAC defines resolution in MS (m/Δm) as
"10% valley definition: Let two peaks of equal height in a mass spectrum at masses m and m – Δm be separated by a valley, which at its lowest point is just 10% of the height of either peak. For similar peaks at a mass exceeding m, let the height of the valley at its lowest point be more (by any amount) than 10% of either peak height."
"Peak-width definition: For a single peak made up of singly charged ions at mass m in a mass spectrum, the resolution may be expressed as m/Δm, where Δm is the width of the peak at a height that is a specified fraction of the maximum peak height." It is recommended that one of three values — 50%, 5% or 0.5% — always be used. For an isolated symmetrical peak recorded with a system that is linear in the range between 5% and 10% of the peak, the 5% peak-width definition is technically equivalent to the 10% valley definition. A common standard is the definition of resolution based upon Δm being full width of the peak at half its maximum height, sometimes abbreviated FWHM. Preferably, this abbreviation should be defined the first time it is used.