HPLC-MS analysis

HPLC mass spectrometry as an analytical tool

High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an ideal tool for the identification, quantitation, and mass analysis of components, often used to determine the chemical composition and purity of chemicals. HPLC-MS has high sensitivity and is optimal for performing precise and reproducible quantitative analyses. The method is ideal for detecting heat-labile compounds, such as proteins and vitamins in food products. Food contaminants are also often quantified using HPLC-MS.

The operating principle

In HPLC-MS, the two techniques (HPLC and MS) are connected by an interface that transfers the separated components from the liquid chromatograph column into the mass spectrometer ion source. An interface is needed since HPLC operates at high pressure and the MS system has a high vacuum.

HPLC includes a mobile phase and a stationary phase, both of which can be modified to suit the sample matrix and the desired properties to be determined. Usually, the mobile phase is adjusted to the sample, and the stationary phase is adjusted to work well with the mobile phase. The degree of compound separation is based on the compound’s affinity for the mobile phase.

HPLC methods can be divided into two main categories based on the properties of the stationary and mobile phases. A combination of a polar stationary phase and a non-polar mobile phase is called "normal-phase chromatography" and the opposite of that, the combination of a non-polar stationery and a polar mobile phase is called "reverse-phase chromatography".

Normal-phase HPLC

In normal-phase HPLC, the column is usually filled with silica particles. The silica is polar and will bind to polar molecules in the mobile phase. This means that the least polar compounds elute first and the most polar compounds last. Normal-phase HPLC is suitable for highly hydrophobic or hydrophilic compounds, compounds that are not soluble in water, and compounds that may decompose in water. The ideal use of normal-phase HPLC is the separation of isomers.

Reverse-phase HPLC

In reverse-phase HPLC, the stationary phase usually consists of C8 or C18 silica (silica derivatized with alkyl chains). Unlike in normal-phase HPLC, in reverse-phase HPLC, the most polar compounds elute first and the least polar compounds last. The stationary phase can be composed of different solids to meet different needs.

Combining HPLC with mass spectrometry

Once compounds have been separated using HPLC, they are identified and their contents are determined by mass spectrometry (MS), which creates a mass spectrum that is unique for every compound. In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios. 

It is also possible to combine HPLC with detectors other than the mass spectrometer. In HPLC-DAD analysis, for example, a diode-array detector is used instead. The choice of the most appropriate detector depends on the analyte and the goal of the analysis. In general, HPLC-MS is more suitable for identifying unknown components, while the DAD-detector works well in quantifying known components. For certain analytes with fluorescent properties, the HPLC-FLD method (with a fluorescence detector) can yield excellent results.

Samples and sample preparation

HPLC-MS is suitable for samples in liquid form. For solid sample matrices, sample preparation is necessary. Compounds that are being determined must be extracted into a solvent and filtered before analysis.