J. A. Singleton

High-performance liquid chromatography analysis of peanut phospholipids. II. Effect of postharvest stress on phospholipid composition

Peanuts are harvested in late September, and sometimes the harvest season can extend through most of October. When weather patterns delay harvest, the result may cause an immature crop, curing problems, rain damage, and freeze damage. All of the above stress situations can affect oil quality and flavor of the peanuts by altering phospholipid composition. Such changes are related to refining problems as well as flavor problems. A new high-performance liquid chromatography (HPLC) method was used for the analysis of phospholipids from postharvest stressed peanuts. The concentrations of phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were higher in immature seed when compared to mature seed. A slight increase in concentration was observed for phosphatidylglycerol (PG), and a decrease in phosphatidylinositol occurred in immature peanuts. All phospholipids increased in concentration except PG when peanuts were cured at a high temperature (40°C). When peanut seeds were frozen at −16°C (before curing), a significant increase in concentration was observed for PA and PG, whereas the concentrations of PC and PE decreased to very low levels when compared to the control. Where concentration permitted, molecular species were separated on a reverse-phase column by HPLC.

Lipoxygenase isozymes of peanut.

Lipoxygenase was isolated and partially purified from peanut seed by ammonium sulfate precipitation, gel filtration, and ion exchange column chromatography. Three isozymes of lipoxygenase were identified. Two had pH optima of 6.2, and the other an optimum of 8.3. Molecular weight of each isozyme was 7.3×104, as determined by gel filtration. The alkaline optimum isozyme was not inhibited by NaCN and was inhibited by CaCl2 except at very low concentrations. The acid optimum isozymes were inhibited by NaCN and were stimulated by CaCl2 concentrations up to ca. 0.7 mM.

Characterization of peanut oil tricacyglycerols by HPLC, GLC and EIMS

Twenty-three peanut oil triacylglycerols have been characterized by liquid chromatography, gas chromatography and electron impact mass spectrometry. High resolution was achieved using two 8-cm × 6.2-mm reverse phase columns in series, and 20 of the triacylglycerols were separated in an analysis time of less than 45 min. Triacylglycerols were identified by analyzing each liquid chromatography fraction for carbon number, fatty acid composition and mass fragmentation pattern. The combined application of these methods permitted the identification of triacylglycerols representing combinations of all of the fatty acids present in peanut oil.

High-performance liquid chromatography analysis of peanut phospholipids. I. Injection system for simultaneous concentration and separation of phospholipids

This paper discusses the details of a high-performance liquid chromatography method for the simultaneous concentration and separation of phospholipids or other trace compounds by direct oil injection using two different solvent systems. The system equilibrates and concentrates phospholipids on a silica column using hexane. At the same time, an analytical column is equilibrating and separating phospholipids using two binary solvent mixtures. This system eliminates a preconcentration step previously accomplished by solid-phase extraction, open-column chromatography, and other previously used methodology. Other advantages include: a 40% reduction in analysis time, elimination of a second transfer of labile compounds, decreased solvent use, and a simpler array of solvents to separate phospholipids. The method described has broader applications, such as trace organic compounds in water supplies, and trace metals with appropriate modifications for the particular analysis.

Isolation of isomeric hydroperoxides from the peanut lipoxygenase- linoleic acid reaction

Hydroperoxides were isolated from the peanut lipoxygenase-linoleic acid reaction mixture and were separated as their methyl esters by high performance liquid chromatography. Mass spectrometry and infra-red analysis indicated the isolated hydroperoxides to be 13-hydroperoxy-cis-9,trans- 11-octadecadienoic acid; 13-hydroperoxy-trans- 9,trans- 11-octadeca-dienoic acid; and 9-hydroperoxy-trans-l0,trans- 12- octadecadienoic acid. The percentages of the hydro-peroxides in the reaction mixture were 72.8%, 3.6%, and 23.6% under the conditions used. 1 Paper No. 4973 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.

Calcium activation of peanut lipoxygenase

Peanut lipoxygenase isozyme 1 (pH optimum, 8.3) was strongly activated by 0.5–1.0 mM Ca++, and the rate of activation was maximum when the ratio of substrate to Ca++ was ca. 2∶1. Peanut lipoxygenase isozymes 2 and 3 (pH optima, 6.2) were activated by calcium but did not have an optimum level of activity. Calcium differentially activated peanut lipoxygenase causing the rate of pentane production to increase much more rapidly than the rate of oxygen consumed by the enzyme reaction. At pH 6.2, in the absence of calcium, the percentages of the hydroperoxide isomers produced by peanut lipoxygenase were 74.9% 13-hydroperoxycis-9,trans-11-octadecadienoic acid (13 LOOHcis-trans), 2.6% 13-hydroperoxytrans-9,trans-11-octadecadienoic acid (13 LOOHtrans-trans) and 22.5% 9-hydroperoxy 10, 12-octadecadienoic acid (9 LOOH). The presence of 1 mM Ca++ at pH 6.2 did not significantly affect the percentage distribution of the hydroperoxides produced. However, at pH 8.3, the percentage distribution of hydroperoxides produced was 45.2% 13 LOOHcis-trans, 10.9% 13 LOOHtrans-trans and 43.9% 9 LOOH in the absence of Ca++ and 57.0% 13 LOOHcis-trans, 8.0% 13 LOOHtrans-trans and 35.0% 9 LOOH in the presence of 1 mM Ca++.

Aerobic pentane production by soybean lipoxygenase isozymes

The effects of oxygen on production of pentane and compounds absorbing at 234 nm and 285 nm by soybean lipoxygenase isozymes I and II were examined in a model system. Aerobic conditions increased pentane production. Differences in dienone formation (A285) and diene conjugation (A234) indicate the reaction sequences of the 2 isozymes are not the same.

A preconcentration and subsequent gas liquid chromatographic analysis method for trace volatiles

A glass column containing a porous polymer was used to concen-trate headspace volatiles from enzymatically mediated reactions and inserted directly into the injection port of a gas liquid chromatog-raphy (GLC) for elution and separation of adsorbed volatiles. The polymer column was placed in an entrainment system attached to a water aspirator at 30 psi to collect volatiles produced by the en-zymatic reaction. A useful chromatogram was obtained from 1 g of raw material by this method. Volatiles collected in this manner could be stored on the polymer matrix at ambient temperatures without deleterious effects for subsequent GLC analysis. Multiple columns of the same or different trapping material could also be used in the entrainment system.

Computation of conversion factors to determine the phospholipid content in peanut oils

Peanut oil was separated into the various lipid classes by column chromatography. The polar lipid fraction which contained phospho-lipids was separated into individual major components by 2-dimen-sional thin layer chromatography. Conversion factors for calculating the concentration of total phospholipid in peanut oil from percent elemental phosphorus were determined by estimation of molecular weights for the respective components. A conversion factor of 23.6, 24.8, 26.6, 22.2, and 24.4 was found for phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidic acid, and total phospholipid, respectively. These factors also were used to convert µg of phosphorus into µg of phospholipid.

Enrichment of phospholipids from neutral lipids in peanut oil by high-performance liquid chromatography

Phospholipids from crude peanut oil were enriched on a 2-cm silica column and subsequently separated from neutral lipids within the chromatographic system without prior concentration. Hexane effectively removed the bulk neutral lipids, leaving the adsorbed phospholipids on the silica precolumn. Individual phospholipids were separated from the remaining neutral lipids and from each other by using two mixed solvents and a gradient program. This method separates the phospholipids in approximately 27 min after the desired enrichment level has been reached.