Comprehensive approach to the quantitative analysis of mitochondrial phospholipids by HPLC-MS

Abstract

A normal-phase HPLC-MS method was established to analyze mitochondrial phospholipids quantitatively as well as qualitatively. An efficient extraction procedure and chromatographic conditions were developed using twelve standardized phospholipids and lysophospholipids. The chromatographic conditions provided physical separation of phospholipids by class, and efficient ionization allowed detection of low abundance phospholipids such as phosphatidylglycerol and monolysocardiolipin. The chromatographic separation of each class of phospholipid permitted qualitative identification of molecular species without interference from other classes. This is advantageous for mitochondrial lipidomics because the composition of mitochondrial phospholipids varies depending on tissue source, pathological condition, and nutrition. Using the method, seven classes of phospholipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, cardiolipin, and monolysocardiolipin) were detected in rat heart and skeletal muscle mitochondria and all but phosphatidylserine were quantified. The concentration was calculated using standard curves with an internal standard generated for each class of phospholipid. The method was validated for intraday and interday variation and showed excellent reproducibility and accuracy. This new method, with each step documented, provides a powerful tool for accurate quantitation of phospholipids, a basic structural component of mitochondrial membranes.

Figure 1

Mass spectra of phospholipids from skeletal muscle mitochondria. Mass spectra ranges are derived from the ion chromatogram eluting at the same retention time window as standardized phospholipids.

Figure 2

Representative ion chromatogram of skeletal muscle mitochondrial phospholipids. Mass ranges for each chromatographic peak are PE, 737-800, PME, 704-708; PG, 744-775; PI, 860-915; CL,1420-1520; MLCL, 1185-1189; PC, 740-885.

Figure 3

Representative standard curves of six classes of phospholipids with PME as internal standard. Standardized phospholipids with six different concentrations were extracted and analyzed by HPLC-MS. Each data point represents the relative areas of chromatographic peaks of phospholipid compared to that of PME at a given concentration.

Figure 4

The comparison of mass responses of two species in CL and three species in PC over a range of concentrations.

Figure 5

Recovery of standardized phospholipids extracted from BSA and from pre-packed silica gel SPE column (n=6). Standardized phospholipids were mixed with 0.2% BSA and extracted with ACN:MeOH (dark grey), ACN:IPA (light grey), method of Folch (black) or method of Christiansen (white). The extracts were subjected to the silica gel column and recovery was determined by comparing the amounts of standardized phospholipids recovered from extraction and silica gel column to the same amount of control standardized phospholipids using PME as an external standard.

Figure 6

Relative recovery of mitochondrial phospholipids (n=6). Mitochondria were extracted by the method of Folch (black) or by the method of Christiansen (white) and the amount of phospholipids extracted were compared using PME as an external standard.

Figure 7

Standardized phospholipids were added to mitochondria (circle) or BSA (square), extracted, and analyzed by HPLC-MS. The amount of each class of phospholipid was calculated using the standard curve and plotted against the amount added to mitochondria (10 μg for PE and PC, 30 μg for PG, PI, CL, and MLCL).

Figure 8

Mass spectra of CL and MLCL extracted from rat heart and mouse heart mitochondria. The mass spectra show increased amounts of CL and MLCL species containing docosahexaenoic acid (22:6) and oleic acid (18:1) in mouse heart. The structure of the peaks was assigned based on their molecular weights.