LC–MS Identification of Wax Esters in Cloudy Canola Oil - - Spectroscopy
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LC–MS Identification of Wax Esters in Cloudy Canola Oil


Special Issues
pp. s16-s21

Canola largely contains unsaturated fatty acids, thus rendering it clear in most cases after extraction and refining. However, canola can become cloudy even at room temperature. The cloudiness is thought to be largely because of the presence of wax esters transferred to the oil from the plant tissues during extraction and increased saturated fatty acid content. Methods have previously used saponification to measure the fatty alcohol and fatty acid components of the wax esters. Others have used solid-phase extraction with gas chromatograph y–mass spectrometry. This study shows that high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC–QTOF-MS) can directly analyze the wax esters with a simple filter sample preparation step. Using HPLC–QTOF-MS also allows for direct analysis of other fatty components in the sample such as triacylglycerols.

Canola oil is predominately composed of unsaturated fatty acids, thus rendering it clear in most cases after extraction and refining. However, there are instances in which canola can become cloudy even at room temperature. Cloudy canola oil is not typical for the consumer and, therefore, has a negative connotation. Dewaxing is a process in which solids in an oil can be removed, but this added processing step can be costly. Typically, canola oil does not require dewaxing, which, in addition to the low amount of saturated fatty acids, makes it a desirable option for salad oils. Industrially, a cold test is used to see if the oil will become hazy upon storage (1). The oil is heated to 130 C and then immersed in an ice water bath maintained at 0 C for 5.5 h. The sample is examined for any evidence of crystals or cloudiness. The sample must be completely clear to pass the test. A lack of clarity indicates the potential for the oil to become hazy over time.

The cloudiness sometimes observed in canola and other oils is thought to be mainly because of the presence of wax esters (2–5) transferred to the oil from the plant tissues and increased levels of saturated fatty acids (2). The increased wax esters can arise in different growing seasons because of varying environmental conditions. Wax esters are made up of a fatty acid and fatty alcohol. These components tend to be long carbon chains 20–28 carbons in length. They can be saturated or unsaturated, although saturated chains are reported more often (2–4). Waxes can act as an organogelator in liquid oils. An organogelator must have a balance of insolubility and solubility in the solution (6). Liquid oils and waxes can have such a relationship. The gelling of waxes with liquid oils has been studied. Sunflower wax esters have been shown to be able to create an organogel in soybean oil at a concentration of 0.5% (7).

Methods have previously used saponification to measure the fatty alcohol and fatty acid components of the wax esters (2). Other studies have used solid-phase extraction with silica gel and gas chromatography–mass spectrometry (GC–MS) (8) as well as silver nitrate–impregnated silica gel with liquid chromatography (LC) (9) to characterize the wax ester fractions of olive and sunflower oils. Studies that have not used a mass spectrometer for detection characterize the composition of the waxes by their number of carbons and degree of unsaturation (8,9). This type of analysis is also common for analyzing triacylglycerols. However, there can be different isomers of each wax. Therefore, mass spectrometry is needed to fully elucidate the composition of each type of wax ester. This study confirms that high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC–QTOF-MS) can directly detect the wax esters with a simple sample filtration preparation step. HPLC–QTOF-MS also allows for direct analysis of other fatty components in the sample such as triacylglycerols.

Experimental

Sample Preparation

Canola oil with visible cloudiness at room temperature was filtered through a Buchner funnel fitted with black filter paper for visual confirmation of collection of white material. Material on the filter paper was scraped off and collected for analysis. Approximately 10 mg of the collected material was diluted in 25 mL of 80:20 methylene chloride–acetonitrile.

Standards were purchased from NuChek Prep and included lignoceryl lignocerate, behenyl behenate, behenyl oleate, arachidyl oleate, stearyl oleate, and stearyl linoleate. Approximately 1 mg of standard was diluted in 25 mL of 80:20 methylene chloride–acetonitrile.

LC–QTOF-MS Method Parameters

The collected material was analyzed on an Agilent 1290 HPLC system coupled to an Agilent 6520 QTOF-MS system with an electrospray ionization (ESI) source. Mobile-phase A consisted of 80:20 water–2-propanol and mobile-phase B was 80:10:10 butanol–water–2-propanol, each with 25 M ammonium formate added. The mobile phase was ramped from 30% B to 100% B over a period of 24 min at 0.25 mL/min. A 100 mm 3.0 mm, 2.7-m d p Agilent Poroshell 120 EC-C18 column was used and held at 50 C. The ESI was used in positive mode with the nebulizer set at 30 psig. The drying gas was heated to 325 C and introduced at a flow rate of 4 L/min. Full-scan data were acquired by scanning from m/z 200 to m/z 1500. Targeted MS-MS was done on the waxes using a mass-dependent formula with a slope of three and y-intercept of 10 for the collision energy.


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