Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan
Received date: January 28, 2013; Accepted date: April 25, 2013; Published date: April 26, 2013
Citation: Shintani H (2013) HPLC Analysis of Phospholipids and their Hydroperoxides with Chemiluminescence Detection. Pharmaceut Anal Acta 4:225. doi: 10.4172/2153-2435.1000225
Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Biomembranes contain phosphatidylcholine, phosphatidylethanolamine, phos.phatidylserine, and phosphatidylinositol as major phospholipids. Here we describe the separation of these phospholipids by HPLC, and the simultaneous detection of their primary oxidation products, hydroperoxides, by means of hydroperoxide-specific chemiluminescence detection [1-9].
• Prepare tissue homogenate. For example, homogenize minced rat liver (0.5 g) in ChelexR 100-treated Tris-HCI (5 mM, pH 7.4, 5 ml) containing sucrose (0.25 M) by means of a TeflonR homogenizer.
• Discard solids after centrifugation at 3000 rpm for 10 min.
• Extract oxidized and unoxidized phospholipids with 2 vol. chloroform.methanol (2:1, v/v) containing 2,6-di-tert-butyl-4- methylphenol (100 μM; to prevent oxidation).
• Centrifuge at 12,000 rpm for 3 min.
• Analyse aliquots (20 μL) of the organic phase by HPLC equipped with UV and chemiluminescence detection. Column: 250 mm × 4.6 mm i.d., 5 μm, aminopropylsilyl (Supelco) with a 20 mm × 4.6 mm i.d., 5 μm, silica gel guard column (Supelco): Mobile phase: 6:3:1 (v/v) methanol.tert-butyl alcohol-aqueous monobasic sodium phosphate (40 mM) at a flow rate of 1.0 ml/ min. Chemiluminescence reagent: aqueous borate buffer (100 mM; 38.14 g sodium tetraborate decahydrate/L) is prepared, and the pH is adjusted to 10 with sodium hydroxide. lsoluminol (177.2 mg, final concentration 1 mM) is dissolved in methanol (500 mL) and borate buffer (500 ml), and microperoxidase (5 mg) is added. The flow rate for the chemiluminescence reagent is 1.5 ml/min. The UV detector is operated at 210 nm.
• Analyse aliquots (10 μL) of methanolic phosphatidylcholine hydroperoxide (PC-OOH: standard, 10 μM).
Figure 1 depicts the separation and detection of 10 pmol each of PC-OOH, phosphatidylethanolamine hydroperoxide (PE-OOH), phosphatidylserine hydroperoxide (PS-OOH), and phosphatidylinositol hydroperoxide (PI-OOH) .
The concentration (μM) of hydroperoxides in the homogenate can be calculated by use of the equation:
[hydroperoxide] = [(100/20 × 3 × 0.46) × peak area for hydroperoxide] (peak area for standard)
because the organic and aqueous phases are present in the ratio 46:54 (v!v), respectively.
• Levels of phospholipids can be estimated from the absorption at 210 nm.
• PC-OOH and PE-OOH were the major oxidation products of rat liver homogenate .
• PC-OOH and PE-OOH can be baseline separated. However the separation of PS-OOH and PI-OOH was not satisfactory and in this status determination cannot be attained. In that meaning further HPLC analytical condition must be studied by changing constituent of mobile phase, selection of column, choice of gradient elution and/or use of solid phase extraction and so on.