Enrichment of Phospholipids from Biological Matrices with Zirconium Oxide-Modified Silica Sorbents Xiaoning Lu, Jennifer E. Claus, and David S. Bell Supelco, Div. of Sigma-Aldrich Bellefonte, PA 16823 USA T413042 sigma-aldrich.com/analytical
Introduction There has been increasing interest in detection and structural characterization of phospholipids in biological matrices such as plasma and tissues. Phospholipids are the building blocks of cellular membranes and play important roles in various cellular responses. Studies have also shown the alteration of phospholipids in blood has been associated with various diseases such as heart attack and vascular diseases and, therefore, are believed potential molecular markers for diseases. Phospholipids are commonly extracted from biological matrices by liquid-liquid extraction (LLE) methods. However, the LLE methods tend to be time consuming and labor-intensive. The present work describes a simple and rapid solid phase extraction (SPE) procedure for extraction and enrichment of phospholipids from biological matrices using zirconium oxide-modified silica particles. In the procedure, biological samples such as plasma and serum were subjected to protein precipitation with organic solvents followed by filtration or centrifugation to remove the protein precipitates. The resulting organic extracts were then loaded onto the zirconium oxide-modified silica particles, either packed in 96-well plates or a single cartridge. 2
Introduction (contd.) After wash, the phospholipids are eluted from the particles by 5% ammonium hydroxide in the organic solvents. The entire procedure generally takes less than 15 min and multiple samples (e.g. 96 samples) can be processed in parallel with 96-well plates packed with the sorbents. The applicability of the method has been demonstrated by the extraction of seven classes of phospholipids with varied polar head groups including phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and sphingomyelin from plasma samples. 3
Phospholipid Structure O R1O O P OR2 O - OR3 Phospholipid Phospholipid R1 R2 R3 Phosphatidic Acid (PA) Fatty Acyl Fatty Acyl H Phosphatidylcholine (PC) Fatty Acyl Fatty Acyl -CH 2 -CH 2 -N +(CH 3 ) 3 Phosphatidylethanolamine (PE) Fatty Acyl Fatty Acyl -CH 2 -CH 2 -NH 2 Phosphatidylglycerol (PG) Fatty Acyl Fatty Acyl -CH 2 -CH(OH)-CH 2 (OH) OH OH Phosphatidylinositol (PI) Fatty Acyl Fatty Acyl HO OH OH Phosphatidylserine (PS) Fatty Acyl Fatty Acyl Lysophosphatidylcholine (LPC) Fatty Acyl H -CH 2 -CH(COOH)-NH 2 -CH 2 -CH 2 -N +(CH 3 ) 3 4
Experimental 2.1. Phospholipid Standards and MRM Transitions # PL short name Full name Molecular Formula MW (mono) ESI ions (+) MRM(+) 1 PA 16:0/16:0 2 PC 16:0/16:0 3 PE 18:1/18:1 1,2-Dipalmitoyl-sn-glycero-3- phosphate acid C35H69O8P 648.472 666.7 ([M+NH4]+) 666.7/551.7 (NL115) 1,2-Dipalmitoyl-rac-glycero-3- phosphocholine C40H81NO8P 734.57 734.6 (M+) 734.6/184.1 1,2-Dioleoyl-sn-glycero-3-744.7/603.7 phosphoethanolamine C41H78NO8P 743.546 744.6 [M+H]+ (NL141) 4 PG 16:0/18:1 5 PI 38:4 Not sure 6 PS 16:0/16:0 1-Palmitoyl-2-oleoyl-sn-glycero-3- phospho-rac-(1-glycerol) C40H77O10P 748.525 C47H83O13P (18:2/20:2) C47H83O13P (18:0/20:4)886.557 1,2-Dipalmitoyl-sn-glycero-3- phospho-l-serine C38H74NO10P 735.505 766.7, [M+NH4]+ 749.7, [M+H]+ 904.8, [M+NH4]+ 736.6, [M+H]+ 758.6, [M+Na]+ 749.7/577.7 (NL172) 904.8/627.8 (NL277) 736.7/551.7 (NL185) 7 SM 16:0 N-Palmitoyl-D-sphingomyelin C39H80N2O6P 703.575 703.6 (M+) 703.7/184.1 5
Experimental (contd.) 2.2. LC-MS Analysis LC-MS conditions: instrument: Agilent 1200 HPLC with 3200 QTRAP (AB/Sciex ) HPLC column: Ascentis C18, 15 cm x 2.1 mm (Sigma Cat. No. 581365-U) mobile phase (isocratic): 20% water/30% methylene chloride/50% methanol, with 10 mm ammonium formate or 5 mm ammonium methylmalonic acid (MMA) flow rate: 0.2 ml/min temp.: 35 C det.: MS, ESI(+), MRM mode column pressure: 1277 psi (88 bar) injection: 2 µl 6
Experimental (contd.) 2.3. Phospholipid Enrichment Procedure The phospholipid enrichment using HybridSPE 96-well plates was performed either in the on-line or off-line modes. In the on-line mode, the biological sample, e.g. plasma and serum, are protein crashed with organic solvents inside the wells of the plates. Whereas in the off-line mode, the protein crashing step is carried out outside the plates. In either mode, a solvent-toplasma ratio of 9:1 was employed for optimum protein crashing results and maximum extraction of phospholipids from the plasma. After sample loading, the samples are pulled through the plate via vacuum typically at 10 in Hg for 3 min. Extended vacuum may be needed for more viscous samples. A washing was then performed to remove the loosely or non-specific bindings to the sorbents. Finally, the phospholipids were eluted off the sorbents with 5% ammonium hydroxide (NH 4 OH) in organic solvents. The collected samples were directly injected for LC-MS analysis. Alternatively, the samples were dried with nitrogen and reconstituted into the LC-MS mobile phase. 7
Flow Chart of the Phospholipid Enrichment Procedure On-line Flow Chart + 100 µl Plasma 900 µl Solvent Off-line Flow Chart 100 µl Plasma 900 µl Solvent +Centrifugation (e.g. 5000 g x 3 min.) Sample loading Mixing (e.g. by vortex) Vacuum (10 in Hg for 3 min.) Vacuum (10 in Hg for 3 min.) Flow thru Washing (optional) Flow thru Washing (optional) Elution: NH 4 OH in solvent Wash solvent Recovered Phospholipids Elution: NH 4 OH in solvent Wash solvent Recovered Phospholipids 8
Results and Discussion 3.1. LC-MS of the Phospholipids SM16:0/16:0 PC16:0/16:0 703.7/184.1 734.8/184.2 PS16:0/16:0 736.7/551.7 PE18:1/18:1 744.7/603.7 PI18:0/20:4 904.8/627.8 PG16:0/18:1 749.7/577.7 PA16:0/16:0 666.7/551.7 MP with 5 mm (NH 4 ) 2 MMA MP with 10 mm NH 4 FA 2 4 6 8 10 12 14 16 18 Time, min 9
Results and Discussion (contd.) 3.2. Phospholipid Enrichment Mechanism Lewis Base Hydroxide Phosphate Fluoride Citrate Sulfate Acetate Formate Chloride rel. Strength on Zirconia Strongest Weakest 10
Results and Discussion (contd.) 3.3. Phospholipid Enrichment 96-well plate 5 µm PTFE (Teflon) Frit Zirconia coated silica particles, 50 mg 0.2 µm filter membrane 11
Results and Discussion (contd.) 3.4. Recovery of Phospholipid Enrichment Recovery of neat phospholipid standards and phospholipids spiked in rabbit plasma. MRM Neat Flow-through Neat Elute Spike Flow-through Spike Elute PA 16:0/16:0 666.7/551.7 0% 1% 0% 0% PC 16:0/16:0 734.6/184.1 0% 96% 0% 154% PE 18:1/18:1 744.7/603.7 0% 94% 0% 106% PG 16:0/18:1 749.7/577.7 0% 34% 0% 41% PI 38:4 904.8/627.8 0% 0% 0% 0% PS 16:0/16:0 736.7/551.7 0% 19% 0% 26% SM 16:0 703.7/184.1 0% 94% 0% 793% Experimental: Neat phospholipid standards of 200 ng/ml each were prepared in water/methanol/dichloromethane (10/45/45). The rabbit plasma were protein crashed with methanol/dichloromethane (50/50) at ratio of 1:9, and then centrifuged at 6000 g for 3 min. The resulting supernatant were collected and spiked with phospholipid standards at 200 ng/ml. In either case, 400 μl of the neat or spiked phospholipid samples were loaded onto the HybridSPE plate and pulled through under vacuum at 10 in Hg for 3 min. The flowthrough were collected for LC-MS analysis. The bound phospholipid on the sorbent were eluted with 400 μl of 5% ammonium hydroxide in methanol/dichloromethane (1:1), and 12 collected for LC-MS analysis.
Results All the tested phospholipids were completely captured by the plate, as indicated by the fact that no phospholipids were detected in the flow-through of the neat or spiked phospholipid samples. PA and PI strongly bind to the phase and could not be eluted off the plate by the explored elution solvent, as reflected by the zero recovery of neat or spiked phospholipids. PC, PE, and SM were readily eluted and completely recovered from the plate. Relatively lower recovery for PG (30%-40%) and PS (20-30%) were observed, indicating the elution is not complete. The high recoveries of spiked PC (154%) and SM (793%) from rabbit plasma were due to the presence of endogenous PC and SM in the rabbit plasma. 13
Conclusion A generic phospholipid enrichment method has been devised for 7 classes of phospholipids except PA and PI, which could not be eluted off the zirconia sorbents. The method utilizes the selective affinity of zirconia toward phosphate groups of the different phospholipids. The method can be performed with high throughput, with the aid of a 96-well plate. In addition, an LC-MS method has been established for separation and detection of phospholipids on a C18 reversed-phase column with high separation efficiency (e.g. nice peak shapes). Further work will be necessary to elaborate conditions for elution of PA and PI off the zirconia sorbents. Ascentis and HybridSPE are registered trademarks of Sigma-Aldrich Co. LLC. 14