Analysis of phospholipid molecular species by liquid chromatography — atmospheric pressure chemical ionisation mass spectrometry of diacylglycerol nicotinates

Abstract

A method using liquid chromatography — atmospheric pressure chemical ionisation mass spectrometry was evaluated for determining the molecular species composition of phospholipids (phosphatidylcholines from soybean, egg yolk and bovine liver) after conversion to diacylglycerol nicotinate derivatives. The structures could be deduced from pseudo-molecular ions ([MH-123]⁺) and three pairs of monoacyl containing fragment ions. All molecular species in mixed peaks were readily identified and many minor components, earlier not encountered in the samples under investigation, were identified. Acyl chain regioisomers were readily distinguished by the ratio of the [MH–RCHCO]⁺ ions. Molecular species differing only in the position of the double bonds in one polyunsaturated acyl chain were separated on the basis of retention times. A half quantitative estimation of the molecular species composition of complex samples was achieved by a combination of UV detection and, for mixed peaks, the areas of [MH-123]⁺ ions.

Introduction

Phospholipids occur in cell membranes as mixtures of molecular species, the complexities of which depend on the size of degree of unsaturation of the acyl chains according to the biological source. A complete qualitative and quantitative analysis of a complex mixture of molecular species presents a considerable challenge. Different analytical approaches have been taken.

In one approach, intact phospholipids were separated into molecular species by reversed-phase high-performance liquid chromatography (RP-HPLC) (Kuksis et al., 1991a, Bell, 1997, Olsson and Salem, 1997). The identities of molecular species have been verified by using a combined liquid chromatography-mass spectrometry (LC-MS) technique (Kim and Salem, 1993). Thermospray LC-MS has been used (Kim and Salem, 1986, Kim and Salem, 1987) but improved quantification and sensitivity was possible with electrospray ionisation (ESI) (Kim et al., 1994, Fang and Barcelona, 1998, Brouwers et al., 1999) and this technique is now usually favoured. Fast atom bombardment (FAB) or liquid secondary ion (LSI) MS has also been utilised (Chen et al., 1992, Singleton et al., 1999). ESI (Kerwin et al., 1994, Han and Gross, 1995, Smith et al., 1995, Karlsson et al., 1996) and FAB (Jensen et al., 1986, Chen and Claeys, 1996, Chen, 1997, Tavana et al., 1998) tandem mass spectrometry have been used extensively to characterise molecular species of phospholipids without the need for prior HPLC separation.

Another approach is to convert the complex lipids to diacylglycerols (using phospholipase C), derivatise the free hydroxyls and examine by gas chromatography (GC) or RP-HPLC. Trimethylsilyl (TMS) and tert-butyldimethylsilyl (t-BDMS) ethers are suitable for GC analysis, and excellent resolution and quantitation are possible on polar columns (Myher and Kuksis, 1982, Myher and Kuksis, 1989). Structures can be verified by GC-electron impact (EI) MS on non-polar columns (Kuksis et al., 1984, Kuksis et al., 1991b). The same derivatives have been analysed by HPLC with short wavelength UV detection (Kuksis et al., 1991a) but with UV-absorbing derivatives such as benzoates, dinitrobenzoates, and pentafluorobenzoates (PFB) a wider range of mobile phase solvents can be used and quantitative analysis is possible (Kuksis et al., 1991a, Bell, 1997). Greater sensitivity can be achieved by using anthroyl and naphthyl derivatives, for example, and fluorescence detection (Bell, 1997).

Verification of the structures of molecular species can be attained by LC-MS. This approach is particularly useful for determining the composition of mixed peaks in complex samples but there have been relatively few studies. Kuksis and colleagues (Pind et al., 1984, Kuksis et al., 1987, Kuksis et al., 1989, Kuksis et al., 1991b) have employed the direct liquid inlet interface to obtain positive and negative CI mass spectra of the t-BDMS ethers and PFB derivatives of the diacylglycerols derived from a variety of natural phospholipids. Quantitation using molecular or pseudomolecular ions compared favourably with GC of the TMS ethers (Pind et al., 1984, Kuksis et al., 1991b). The molecular species of red cell membrane phospholipids (Kuypers et al., 1991) and glycosyl–inositolphospholipid anchors (Butikofer et al., 1992) were determined by LC coupled to UV detection followed by thermospray MS of the diradylglycerol benzoate derivatives. The mass spectra were informative, but quantitation of mixed peaks was only possible for those compounds that could be calibrated using synthetic standards.

The nicotinate derivatives of individual standard diacylglycerols have been examined by GC-EIMS (Zollner et al., 1994, Zollner and Lorbeer, 1995, Zollner and Schmid, 1996). A useful feature of these derivatives was that not only could the size and degree of unsaturation of the acyl chains be determined but the positions of the double bonds and other functional groups could be located. Another feature of the mass spectra was that acyl chain regioisomers could be distinguished using the relative intensities of two ions corresponding to the loss of each acyl residue (Zollner, 1997). An LC-MS method, using a particle-beam interface, was developed to obtain EIMS of the nicotinate derivatives of diacylglycerol mixtures, thereby obtaining maximum structural information on natural phospholipids (Dobson et al., 1998). In the present study, a method for identifying and quantifying phospholipid molecular species as nicotinate derivatives by RP-HPLC in conjunction with UV detection and APCI-MS was examined.