Liquid Chromatography: Mass Spectrometry of Isoflavones

Abstract

High-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is particularly suited to the separation and analysis of a wide range of biological compounds. When investigating such compounds in complex matrices (e.g., blood, tissue, urine, foods), specificity of the detection method becomes an important issue. This can be overcome by extensive purification procedures before HPLC or CE analysis, but at the cost of a lot of effort and often unknown losses during the extraction and purification procedures. Such losses can be overcome by the addition of isotopically labeled internal standards, subject to their availability. The coupling of HPLC or CE with mass spectrometry provides a very specific method of detection. Compounds are transferred as ions to the gas phase by two types of spraying technologies, electrospray ionization (ESI) and heated nebulizer-atmospheric pressure chemical ionization. Although the mass-to-charge ratios of the molecular ions can be used to provisionally identify a compound, full confirmation of structure requires collision-induced dissociation and analysis of the resulting fragment ions. Combination of specific parent ion-daughter ion pairs allows for the quantitative measurement of isoflavones in the 1-5 pmol range with coefficients of variation for duplicate samples in the range of 5-9%. CE places greater demands on the mass spectrometer than does HPLC, because it generates narrow peak widths. A mass spectrometer with a scanning quadrupole analyzer does not enable full exploitation of the power of CE; in this context, an instrument with an ESI interface and a time-of-flight analyzer is ideal.

Differentiation of isomeric flavone/isoflavone aglycones by MS2 ion trap mass spectrometry and a double neutral loss of CO

“…These two masses are 2 u lower than those of the ions formed by the retro-Diels-Alder (RDA) mechanism 1,3 A À displayed in Table 1 and described by Fabre et al 9 They were completely absent from the spectra of hydroxylated isoflavones, or present only at very low abundance (<2% for 1-iso). It is worth noting that Fabre et al 9 could not detect the ion m/z 149 in the 1-iso IT-MS 2 spectra, while Barnes et al 6 used this difference to distinguish 1-fla from 1-iso.…”

“…In the past, the fragmentation behaviour of flavonoids has been studied in detail by electron ionization (EI) and gas chromatography (GC)/MS. Flavonoids have also been characterized by LC/MS/MS and different soft ionisation and mass separation techniques 6–11. In recent years, API‐LC/MS/MS has become the method of choice for analysis of isoflavones 6,7…”

“…[6][7][8][9][10][11] In recent years, API-LC/MS/MS has become the method of choice for analysis of isoflavones. 6,7 Isoflavone structures differ from the isomeric flavones only by the position of the B ring at the phenylchroman skeleton (Table 1). This positional isomerism is ideal to study the influence of a very defined structural difference on fragmentation in API-CID-MS n .…”

“…To our knowledge, no systematic study has been reported before on the fragmentation behaviour of isomeric flavone and isoflavones by API-MS n . Barnes et al, 6 Hughes et al 12 and Fabre et al 9 have shown that the isomers genistein and apigenin can be differentiated by the fragments m/z 133 and 149 in the API-MS/MS spectra recorded in the negative ion mode. The aim of this study was to investigate whether isomeric flavones/isoflavones can be differentiated by their API-IT-MS n spectra.…”

Phytochemical analysis of isoflavonoids using liquid chromatography coupled with tandem mass spectrometry

“…Presently reverse phase-HPLC (RP-HPLC) is widely used for the analysis of most of the herbal extracts. Normal phase HPLC is also used for the analysis of polar isoflavonoids whose retention in reverse phase is low (Barnes et al 1994(Barnes et al , 1999(Barnes et al , 2006(Barnes et al , 2002Coward et al 1993;Eldridge 1982;Franke et al 1995;Fukutake et al 1996;Griffith and Collison 2001;Liggins et al 1998;Wang and Murphy 1994). The longer the alkyl chain attached to the adsorbent, the higher will be the retention of the components.…”