Advances in LC–MS for Food Analysis

Within the wide range of hyphenated techniques, liquid chromatography–mass spectrometry (LC–MS) has recently emerged to a central role in different fields, including food analysis. In this review, the most recent LC–MS approaches are discussed, as well as the technical requirements for linking an LC system to a mass spectrometer. The advantages of on-line two-dimensional liquid chromatography (2DLC) in the "comprehensive" mode are also illustrated and selected applications for the analysis of common foodstuffs, such as triacylglycerols, carotenoids and polyphenols, are described. Finally, future trends for LC–MS in the food analysis are reported.

Food products are complex mixtures containing both organic and inorganic constituents. The analysis of food products is generally directed towards the assessment of food safety and authenticity, the control of a technological process, the determination of nutritional values and the detection of molecules with a possible beneficial or toxic effect on human health.

Consequently, one of the most stringent demands of food chemistry is directed towards the continuous improvement and development of powerful analytical techniques (1) to analyse the major and minor components of food samples.

Liquid chromatography–mass spectrometry (LC–MS) is an increasingly valuable tool in food analysis and has been widely applied to the analysis of many food products (2). Recent achievements in instrumentation and data processing have allowed LC–MS to play a central role in food-related analysis. However, when dealing with the extreme complexity of many real-world samples, one-dimensional chromatography may not provide sufficient analytical results. As a consequence, considerable research has recently been devoted to the development of multidimensional LC techniques (MDLC), with enhanced resolving power (3).

The purpose of this review is to acquaint the reader with some of the existing recent applications of LC–MS-based techniques in food analysis. Topics covered will include MS analysis of LC-amenable food compounds, namely triacylglycerols, carotenoids and polyphenols. Technical sections will briefly introduce both theoretical and practical concerns of this hyphenated technique, also in the multidimensional "comprehensive" mode (LC×LC–MS).

Liquid Chromatography–Mass Spectrometry (LC–MS)

The potential benefits arising from the hyphenation of LC to MS become clear if the limitations of the two independent techniques are considered, and to what extent such a combination may alleviate them. Peak overlapping sometimes precludes unambiguous identification, even if disposing of reference standard material. Even the most widely used ultraviolet (UV) detector can rarely provide unambiguous data on the separated analytes, regardless of the degree of chromatographic separation obtained; the situation is further complicated if quantitative determination is also desired.

On the other hand, mass spectral data are, in many instances, specific enough to support positive identification and in discriminating nonisobaric compounds, providing the analyst with structural information, in addition to the molecular weight.

MS systems can also be used with non-UV absorbing analytes and can be operated in the full scan mode, viz total ion current (TIC), or, more specifically, in tandem MS (MS–MS) experiments or in the selected ion monitoring (SIM) mode. SIM operation is preferred for the development of selective and sensitive quantitative assays, while tandem MS data, generated by using soft ionization techniques, provide structural information which can help in the identification of unknown analytes.

Mass spectrometry allows for quantitative determination to be performed accurately, precisely and with high sensitivity (at the picogram level), using isotopicallylabeled compounds as internal standards (ISs). Whenever the quantification, or even the detection of a target trace component, in SIM mode, is hampered by the presence of high background ions with the same m/z values, constant neutral loss or precursor ion scanning techniques help in distinguishing the ions of interest from unspecific matrix components. The so-called selected reaction monitoring (SRM) mode enhances selectivity and lowers detection limits, therefore reducing sample consumption, analysis times and the need for clean-up procedures (4).

LC–MS plays a central role in both basic and applied research because of significant advances in interface technology and ionization techniques and it also has a broad range of applicability and high sensitivity for the analysis of high-polar and highmolecular mass compounds.

In addition, the replacement of the older sector machines with ion trapping instruments (IT), quadrupoles (Q), timeof-flight (ToF) systems and a variety of hybrid instruments characterized by high resolution, enhanced sensitivity, as well as increased mass accuracy over a wide dynamic range. Among these are the ion mobility time-of-flight (IM-ToF), quadrupole ToF (Q-ToF), ion trap-ToF (IT-ToF) and linear ion trap-Fourier transform ion cyclotron resonance (FT-ICR).

Ultimate generation singlequadrupoles allow for high speed scanning (up to 15,000 amu/sec) and ultrafast polarity switching; the small size and the possibility to perform tandem MS make them ideal for benchtop LC–MS. On the other hand, ToF instruments present a number of advantages: high speed (up to 20,000 Hz), high resolution (using a reflectron), virtually no limit on mass range, femtogram-level sensitivity, sub-ppm mass accuracy, improved in-spectrum dynamic range without loss in sensitivity, high mass resolution and feasibility to use as a second stage in tandem MS experiments, in combination with either an IT-ToF or a Q-ToF.

From the quantitative standpoint, the linear dynamic range depends on the type of source employed; electrospray ionization (ESI) is characterized by a dynamic range over 2–3 orders of magnitude, and currently represents the most common choice for routine LC–MS analysis. However, atmospheric-pressure chemical ionization (APCI) and atmospheric pressure photo-ionization (APPI) techniques offer greater sensitivity and a wider dynamic range (4–5 orders of magnitude), though their use for large bio-molecules is precluded (5). Liquid chromatography nano-electrospray ionization (LC–nano-ESI) operation has become feasible in recent years (6), boosting the sensitivity of LC–MS techniques. The newly developed interfaces are suitable for linkage with capillary-type LC columns, operated in the µL-to-nL flow range; current configurations using goldcoated capillaries or automated chips allow analyte detection down to the femtomole level.