1232
Anal. Chem. 1981, 53,
1232-1235
Determination of Phospholipids by a Combined Liquid Chromatograph-Automated Phosphorus Analyzer Jukka K. Kaltaranta" Technical Research Centre of Finland, Food Research Laboratory, 02 150 Espoo 15, Finland
Samuel
P. Bessman
DepaHment of Pharmacology and Nutrition, University of Southern California School of Medicine, Los Angeles, California 90033
An automatic phosphorus analyzer was evaluated for phospholipld quantltations and as a detector In the HPLC analysis of phospholipids. The relative standard deviation of the analyses wlth 10 nmol loads of Individual phosphollplds averaged 3.2 % (range 1.1-4.7 % ). Considerlng the reproducibility and the linearity of the method, the range from 1 to 100 nmol is suggested as a practical worklng range. A two-step elution system was developed to separate individual phospholipids from natural samples on a sllica column. Neutral lipids present in the total lipid extracts do not interfere wlth the phosphollpid determlnation if the phosphorus analyzer is used for detection.
High-performance liquid chromatography (HPLC) has limited applications in lipid chemistry concerning phospholipids. Since phospholipids have no specific absorption maxima, the commonly used UV-VIS detection does not offer an effective quantitation device. Due to the unsaturated centers of the fatty acid moieties and the presence of the polar end groups, direct UV detection at the 200-nm region can be applied for phospholipids ( 1 , 2 ) . However, this complex absorption behavior makes the quantitation very difficult because, depending on the average unsaturation degree, the molar extinction coefficients of individual phospholipids vary for each lipid source. Similar difficulties are found if a flame ionization detection (FID) is applied for the lipid quantitation since the molar responses of various lipid components are different (3, 4 ) . Several derivatization methods have been developed for the detection and quantitation of lipids (5). Ethanolamine and serine containing phospholipids can easily be derivatized whereas suitable derivatives of choline containing lipids are not easily synthesized. The quantitation problem can be solved by collecting the eluate in fractions for various subsequent chemical determinations. Fluorometric methods (6) and phosphorus microassays based on the method of Fiske and Subbarow (7) can be applied although they are laborious and time-consuming for routine use. Phosphorus-containing compounds can also be quantitated with an automatic phosphorus analyzer (8). Most applications with this instrument have been made on the analysis of phosphorylated metabolic intermediates of carbohydrates and nucleotides although studies on the phospholipid precursors have also been reported (9-1 I). In this study we evaluated the automatic phosphorus analyzer for the quantitative determination of individual phospholipids. The analyzer was also combined with a HPLC system to provide a direct quantitative detection of phospholipids in the eluate. Reference compounds as well as natural lipid extracts were chromatographed on a silica column to demonstrate the separation.
MATERIALS AND METHODS Reference Compounds and Reagents. Reference phospholipids were purchased from Sigma Chemical Co. (St. Louis, MO) and they included L-a-phosphatidylcholine (PC), L-a-lysophosphatidylethanolamine (LPE), L-a-phosphatidylserine (PS), and sphingomyelin (SPH) from bovine brain, and L-a-phosphatidylethanolamine (PE) from bovine liver. L-O-LYSOphosphatidylcholine (LPC) and L-a-phosphatidic acid (PA) were of egg yolk origin, and L-a-phosphatidylinositol (PI) was from soybean. A brain lipid extract (type V, Sigma Chemical Co.) was used t o demonstrate the analysis of a partially purified lipid sample. Organic solvents were of HPLC grade from Fisher Scientific Co. (Fair Lawn, NJ) and were used as such after degassing. Other chemicals were of ACS or equal grade. Deionized water was redistilled before use. Lipid Extraction. The total lipids of a rainbow trout roe product were extracted according to the method of Bligh and Dyer (12). The total lipid extract was used for the phospholipid analysis without further purification. Chromatographic Instrumentation. A Milton Roy Instrument minipump (LDC Division, Milton Roy Co., Riviera Beach, FL) was applied to provide the solvent flow from a reservoir through a rotary injection valve (Altex Scientific, Inc., Berkeley, CA) to the HPLC column. A Spherisorb S5W guard column (4.6 mm X 40 mm, Applied Science Division, Milton Roy Co., State College, PA) was connected between the injection valve and the analytical pPorasil column (3.9 mm X 300 mm, Waters Associates, Inc., Milford, MA). All the connections were made by using either Teflon or stainless steel tubing. Phosphorus Analyzer. The automatic phosphorus analyzer (Alsab Scientific Products, Inc., Los Angeles, CA) consisted of a dry ashing unit, a peristaltic pump, a debubbler, and a colorimeter connected to an integrating recorder. The eluate from the column was directed into a rotating table containing 40 silica cups. At 30-9intervals the analyzer oxidized the aliquots with nitric acid, dried, and ashed them, changing organic phosphorus to inorganic phosphorus. After the phosphorus was ashed, molybdenum blue color reagents were automatically added to cooled cups and following a 2-min incubation the solution was withdrawn by a peristaltic pump and pumped through the 25-fiL continuous flow cell of the colorimeter. The color measurement was made at 820 nm. Evaluation of the Phosphorus Analyzer. The analytical conditions of the phosphorus determination including the ashing temperature, composition of the oxidizing reagent, and a possible interference of the organic solvents were investigated. The reference compounds were individually studied for the reproducibility of the analysis by adding the compounds in appropriate solvents directly into the cups with an adjustable micropipet (Pipetman, Rainin Instruments Co., Inc., Woburn, MA). The linearity of the analysis method was studied under simulated chromatographic conditions for each reference compound at 12 load levels ranging from 0.4 to 200 nmol. For each peak an equal quantity of a compound was measured into a band of five successive CUPS to form a peak corresponding to 0.4-200 nmol of the total phospholipid. The total sample volume in each cup was adjusted to 0.2 mL with methanol to simulate a flow rate of 0.4 mL/min. The recorded peaks of five separate analyses at each of the 12 load
0003-2700/81/0353-1232$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
Table I. Relative Standard Deviations of Phospholipid Determinationsa relative standard deviation (%, n = 5) phospholipid load, nmol PC LPC PE LPE PS PA SPH 1 10 100
slope r value a
5.2 4.2 3.2
3.9 2.0 1.2
12.1 1.1
1.015 0.999
1.020 1.000
1.058 0.999
8.6
8.8 4.7 4.2 0.964 1.000
6.1
5.1 2.9 4.4
1.078 0.999
1.023 0.999
5.0 2.9
PI
7.8 4.7 1.8 1.038 0.997
1233
KH,PO,
9.0 3.4 8.8
3.9 1.9 0.9 1.031 1.000
1.061 1.000
For abbreviations see Materials and Methods.
levels were measured, and the average values were used to describe the linearity. HPLC Analysis of Lipids. The silica column was equilibrated with a mixture of acetonitrile, methanol, and water, 8015:6.5 (by volume). The flow rate was limited to 0.4 mL/min by the digestibility of the phosphorus analyzer. The elution of the reference mixtures and the lipid extracts was started with the abovementioned solvent system. After 15 min the solvent ratios were changed to 50:45:6.5 (by volume) and the elution was continued until the LPC peak appeared on the chromatogram. All the reference mixtures and lipid extracts were filtered through an Acrodisc CR filter (Gelman Sciences, Ann Arbor, MI) before the analysis. RESULTS Phosphorus Determination. The application of the automatic phosphorus analyzer for the quantitation of phosphorylated metabolic intermediates of carbohydrates and nucleotides has been well established (13, 14). These conditions were also found appropriate for the determination of phospholipids with an exception of the ammonium tetraborate concentration in the oxidizing reagent. Borate is an essential component for smooth oxidation and color development in the ashed samples. In this study the regular borate concentration, 10 mM, was tripled to maintain reproducible results. When separating phosphorylated intermediates by the means of ion-exchange chromatography, a borate solution is used for elution and the eluate provides the additional borate needed for ashing (14). In the phospholipid analysis the total borate has to be provided as a component of the oxidizing reagent. The addition of organic solvents, chloroform, methylene chloride, methanol, 2-propanol, benzene, or acetonitrile into the reaction mixture did not interfere with the phosphorus determination. The solvents were quickly evaporated before the conventional oxidation and ashing. Reproducibility and Linearity of Phospholipid Analysis. Reproducibility of the phospholipid analysis was studied separately for each reference compound on 12 load levels from 0.4 through 200 nmol. For a comparison, similar determinations were made with inorganic phosphate, KH2P04, which requires no ashing before the color development. The relative standard deviations of the peak areas of five successive analyses at the three load levels, 1, 10, and 100 nmol, are presented in Table I. By use of a linear regression analysis, the slope of the regression line as well as the correlation coefficients ( r value) as determined by the 12 points were calculated, and these values are included in Table I. HPLC Analysis of Phospholipids. An isocratic elution system with a mixture of acetonitrile, methanol, and water has earlier been used on a silica column to separate phosphatidylcholine from sphingomyelin (1) and to determine phosphatidylcholine in chocolate (15). Isocratic systems were also investigated in this study to separate the most abundant phospholipid components of animal tissues. A good separation of the reference compounds was obtained when acetonitrilemethanol-water, 5045:6.5, was used as the solvent (Figure 1). Some overlapping of phosphatidylserine with phosphatidylethanolamine as well as between phosphatidylethanolamine
PC
0.05 A U
Figure 1. Separation of a reference phospholipid mixture on a silica column using an isocratic solvent system acetonitrile-methanol-water, 50:45:6.5, and the flow rate 0.4 mL/min. PI = external phosphate standard, 20 nmol. For other abbreviations see Materials and Methods.
I
0.1 A U
PI
il
P:
0
I1
I
30
60
9 0 rnin TIME
Figure 2. Separation of a reference phospholipid mixture on a silica column. A two-step elution was started with acetonitrile-methanolwater, 80:15:6.5, and continued after 15 rnin with the same solvents applying the ratios 50:45:6.5. The flow rate was 0.4 mL/min. PI = external phosphate standard, 20 nmol. For other abbreviations see Materials and Methods. and its lyso form was found when analyzing natural samples with large variations in the concentrations of individual lipids. Therefore a two-step method applying these solvents in the ratios 80:15:6.5 and 50:45:6.5 was developed. In this system a separation of the less polar phospholipids is obtained with an increase of about 20% in the total analysis time (Figure 2). The stepwise system was now applied to the analysis of partially purified phospholipid fraction of bovine brain (Figure 3) as well as to the analysis of the total lipid extract of rainbow
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
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0
O.5AUFS
TIME [min]
60
30
*0,25AUFS*-
0.25AUFS
-4
t
Flgure 3. Separation of a partially purified phospholipid fraction of bovine brain on a silica column. The conditions were as in Figure 2. UNK = unidentified phosphorus compound.
Pi
-
I
0
I
30 0.5 AUFS
1
90
00 >
