Figure 4.1: Scheme of a HPLC system. Specific types of columns and solvents are used to separate and detect various classes of phytochemicals (LaboratyInfo)
In an HPLC system a small volume of sample extract, e.g. 10 µl, is added to the solvent flow and brought onto the column by the injector (mostly an autosampler). The various compounds present in the extract will then elute from the column at different time points, hence the name chromatography, based on their differential affinity for the stationary phase (the column material) and the mobile phase (the solvents). Under optimal chromatographic conditions (column type and temperature, solvent flow rate and composition, injection volume, etcetera) the compound(s) of interest are eluting as symmetric baseline-separated peaks. Compounds eluting from the column can be detected using a suitable detector, depending on the compound to be analysed. The most frequently used detector in HPLC is still the light absorbance detector, which can be a single or a dual wavelength detector, or preferably a photodiode array detector (also abbreviated as PDA or DAD) (Figure 4.2). The single and dual wavelength detector can detect the light absorbance of compounds at only one or two wavelength(s), respectively, while a DAD can detect the compounds at a large range of wavelengths simultaneously (e.g. 220-700nm). Single and dual wavelengths detectors are set at the wavelength at which the compound of interest absorbs maximal or most specific, in order to obtain highest detection sensitivity. Checking for exact co-elution (exact retention time overlap) with the authentic standard allows identification and integration (quantification) of the peak area corresponding to the target compound. While single and dual wavelengths detectors are relatively cheap, they are prone to erroneous compound identification and quantification: due to co-elution by other components an overestimation of the level of target compound can easily occur, especially in cases when analysing crude and thus complex food or plant extracts.
Figure 4.2: Principle of absorbance spectrum detection by a photo diode array (PDA) detector, frequently used in HPLC analyses (Crawford Scientific)
Since most phenolic compounds (including flavonoids) and carotenoids show a characteristic absorbance spectrum in the UV and visible light range (mostly between 240 and 700 nm), they can best identified and quantified using a PDA detector. Such PDA detector not only can be set at a specific optimal wavelength (like the single wavelength detector) but also enables absorbance spectrum comparison for the eluting compounds with standards (Figure 4.3). A PDA detector is a bit more expensive, but clearly much more versatile and accurate in quantification than single or dual wavelength detectors.
Figure 4.3: HPLC-PDA analysis of carotenoids in tomato fruits. Lower panel indicates the 450 nm absorbance (AU) of the various compounds eluting from the column at different time points (minutes) after injection of the tomato extract. The reference compound astaxanthin was used as internal standard (IS). Upper panel shows the online-recorded absorbance spectra (from 240 to 740 nm) of the eluted compounds: both absorbance spectra and retention times are compared with standards to confirm the identity of compounds. Source: Bioscience lab, Wageningen Plant Research, Wageningen, The Netherlands.
Tocopherols (vitamin E) are mostly less abundantly present in plant foods, absorb UV-light less efficiently and do not show a typical absorbance spectrum like carotenoids or flavonoids; they are therefore better and more sensitively detected by using a fluorescence detector (e.g. Moco et al. 2007). Figure 4.4 shows the principle of a fluorescence detector used in HPLC analyses.
Figure 4.4: principle of a fluorescence detector, used in HPLC analysis for specific compounds showing fluorescence, like tocopherols (vitamin E) (Chromatography Online)
Reduced vitamin C (ascorbic acid) is usually also analyzed by HPLC with UV detection; its optimal detection wavelength is around 260 nm. PDA detection, enabling matching its corresponding absorbance spectrum, is again preferred over single or dual wavelengths detection. Nevertheless, the absorbance spectrum of vitamin C is rather a-specific as compared to for instance carotenoids and flavonoids. Vitamin C analysis is therefore prone to overestimation, due to co-elution (peak overlap) with other food components also absorbing at this wavelength. This is especially true for food extracts with a relative low vitamin C concentration (less than 1 mg per 100 g). If total vitamin C (reduced plus oxidized form) is to be determined, the extract can be pre-incubated with a strongly reducing agent like dithiothreitol (DTT) in order to get all vitamin C in its reduced form. Contents of either forms can be determined by comparing vitamin C in the extract both with and without this reduction step.
Folates can be analysed by using a folate-dependent microbial assay or by HLPC. For the microbiological assay, the polyglutamate tail of the natural folates should be removed firstly, as the microorganism used in the assay does not response to folate forms with chains larger than diglutamate. Removal of the glutamate chain is achieved by incubating with a gamma-glutamyl hydrolyse enzyme. However, this enzyme is poorly commercially available and therefore often purified in-house from natural sources such as rat plasma. As alternative of the microbiological assay for determining total folate, HPLC at acidic conditions can be used to determine both folic acid and the natural, reduced folates; UV detection at 290 nm for folic acid and fluorescence detection at 300nm excitation with 360nm emission for the natural folates (Lawrance 2014).
Phytate (or phytic acid, or inositol-P6 or InsP6) is predominantly present in unprocessed food, but can be degraded during processing (Sandberg and Scheers 2016). Therefore, not only phytate but a broad range of inositol-phosphates may be consumed, which may differ in their affinity to minerals and thus in their potential for reducing mineral bioavailability (Harland and Narula 1999). Phytate neither absorbs UV or visible light, nor show fluorescence, which characteristic prevents its direct detection using HPLC analysis. Spectrophotometric assays exist based on colouring the amount of organic phosphorus. These colouring methods for phytate assume that InsP6 comprises all or nearly all of the organic phosphate of the food stuff to be analysed. However, since plant may contain a diverse range of other organic phosphates, including InsP1-5, sugar phosphates and nucleotides, these methods are prone to overestimate the real phytate contents. Therefore, a method of phytate quantification based on a colourimetric assay of phosphate needs to be validated well for each food stuff separately. Since recently, commercial colourimetric kits are available that use an enzyme (phytase) that specifically release phosphate from food phytate (McKie and McCleary 2016); this assay is thus . HPLC separation coupled to a post-column chemical colouring reaction can also be applied (e.g. Phillippy et al. 2004; Sandberg and Scheers 2016). This HPLC method enables the detection of both phytate and the other inositol-phosphates.
Alternative, or next to, detection based on UV-Vis light absorbance or fluorescence, many analytical methods nowadays apply mass spectrometry (MS) (Wikipedia) as detection system. In a mass spectrometer compounds eluting from the chromatographic system are converted into charged structures (ions or radicals), which via a series of electronic filters are sorted and detected based on their mass-to-charge ratio. MS is generally more sensitive and specific than other detection methods. The specific masses and fragmentation pattern of detected molecules can be used to identify compounds and to quantify targets. MS can be coupled to various separation techniques like HPLC (HPLC-MS or UPLC-MS; in short LCMS), gas chromatography (GC-MS) and capillary electrophoresis (CE-MS). In LCMS a so-called electrospray ionization (ESI) source is most often used to generate molecular ions that are either positively or negatively charged. Especially phenolic compounds (including flavonoids) can be well detected using LCMS with ESI, under exactly the same LC conditions as their classical HPLC-PDA analysis (e.g. Moco et al. 2006). Carotenoids can also be well detected using LCMS, although mostly a different ionization source, so-called atmospheric pressure chemical ionization (APCI), is applied to generate ions of these apolar compounds (e.g. Schweiggert et al. 2005). A precondition in LCMS is that the chromatographic eluent used to separate the compounds should be compatible with the ionisation conditions in the MS. For instance, non-volatile acidifying agents in the eluent, like buffered phosphate that is frequently used in chromatographic separation of highly polar nutrients like ascorbic acid, is incompatible with MS. For the analysis of minerals, the state-of-the-art technique is nowadays inductively coupled plasma-MS (ICP-MS), which allows sensitive and simultaneous detection of a large range of elements (e.g. Hansen et al. 2013). Similar to other detection methods, MS-based approaches make use of authentic standards to verify the identification of the chromatographic peak and to construct calibration curves for quantification.
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