Table 1.
MRM transitions used for LC-MS analysis of lysophospholipids.
Fig 1.
Selected LC/MS chromatograms of sn-2 acyl LPC species incubated at 37°C in Tris-HCl buffer, pH 7.4.
The aqueous solution of each sn-2 acyl LPC (2 mg/ml) was incubated at 37°C, and aliquots were taken out at the indicated time. The samples were diluted with equal volume of methanol, and analyzed by LC/MS/MS in the MRM mode, as described in Methods.
Fig 2.
Identification of the regioisomers of LPC from the product ion spectra.
The product ion spectra were obtained for the early eluting (peak 1) and late eluting (peak 2) isomers of each LPC as described in Methods. The intensity of the molecular ion as percentage of that of phosphocholine ion (m/z 184) differentiates the two regioisomers, with the sn-1 acyl isomer showing much higher percent of molecular ion [17]. The relative intensities of the molecular ion, expressed as percent of phosphoryl choline ion (m/z 184.1) for the various LPCs are as follows: 16:0 LPC (4.7% for peak 1 and 65.5% for peak 2); 18:1 LPC (3.7% for peak 1 and 44.8% for peak 2); .20:4 LPC (10.2% for peak 1 and 60.5% for peak 2); 22:6 LPC (15.3% for peak 1, and,172% for peak 2). In addition, the choline fragment ion (m/z 104.1) is completely absent from the sn-2 acyl isomers, but was prominently present in all the sn-1 acyl isomers.
Fig 3.
Stability of aqueous dispersions of sn-2 acyl LPCs at various temperatures.
The sn-2 acyl LPCs were dispersed in Tris-HCl buffer, pH 7.4 at a concentration of 2 mg/ml and incubated under nitrogen in the dark at the indicated temperature for 24 h. Aliquots were taken out at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h, brought to room temperature, diluted with equal volume of methanol and subjected to LC/MS/MS analysis as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.
Fig 4.
Stability of sn-2 acyl LPCs in organic solvent.
The sn-2 acyl LPCs were dissolved in chloroform: methanol (2:1 v/v) at a concentration of 1 mg/ml, and stored at the indicated temperature for various periods. Aliquots were taken out at the indicated time points, brought to room temperature (22°C), and subjected to LC/MS, as described in Methods. The percentage of the sn-2 acyl isomer remaining was calculated from the area counts of the two isomers. The values shown are mean ± SD of 3 separate experiments.
Fig 5.
Effect of serum albumin on the isomerization of sn-2 acyl LPC at 37°C.
Freshly prepared sn-2 acyl LPCs were complexed with BSA in PBS, pH 7.4, and incubated at 37°C for the indicated periods. The isomer composition was then determined by LC/MS/MS as described in Methods. Chromatograms of only selected time periods are shown. The time course of the isomerization of each LPC is shown in Fig 6.
Fig 6.
Time course of isomerization of albumin-bound sn-2 acyl LPCs at 37°C.
Freshly prepared sn-2 acyl LPCs were complexed with 0.1% BSA in PBS buffer at pH 7.4, and incubated at 37°C for the indicated periods of time. The percent of the sn-2 acyl isomer remaining after each time point was determined by LC/MS/MS. The values shown are mean ± SD of 3 separate analyses. Note that the error bars too small to be visible at some points.