A New Method for Determination of Campesterol, Stigmasterol and Β-Sitosterol in Edible Oils by Supercritical Fluid Chromatography
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Journal of Food and Nutrition Research (ISSN 1336-8672) Vol. 58, 2019, No. 4, pp. 363–369 A new method for determination of campesterol, stigmasterol and β-sitosterol in edible oils by supercritical fluid chromatography NINGLI QI – YIJUN LIU – LIANGKUN LIAO – XIAO GONG – CHUNLIANG YANG Summary This study aimed to develop a rapid method for separating three major phytosterols (stigmasterol, campesterol and β-sitosterol) in edible oils by supercritical fluid chromatography using the ultra-performance convergence chromato- graphy system. The samples were prepared by alkaline saponification followed by n-hexane extraction and then sepa- rated on a high strength silica C18 column with selectivity for bases using acetonitrile-methanol (50 : 50) as a co-solvent with a gradient elution (98 : 2 to 80 : 20) and UV-spectrophotometric detection at 210 nm. The optimized method yielded a linear calibration curve ranging from 0.07 ng·ml-1 to 200 ng·ml-1 for stigmasterol, from 0.12 ng·ml-1 to 200 ng·ml-1 for campesterol and from 0.06 ng·ml-1 to 200 ng·ml-1 for β-sitosterol. The limit of detection and limit of quantification of the phytosterols were 20–42 ng·ml-1 and 75–117 ng·ml-1, respectively. The recovery rates ranged from 96.4 % to 101.2 % with relative standard deviations of 1.7–3.8 %, depending on the sterol type and the specific sample. The developed method is simple and sensitive, as demonstrated by its successful use for phytosterol characterization in edible oils. Keywords campesterol; stigmasterol; β-sitosterol; edible oils; supercritical fluid chromatography Vegetable oils are used for cooking and fry- where campesterol, stigmasterol and β-sitosterol ing as well as in food formulations. The vegetable were found to be dominant and most frequent- oil consumption in China has markedly increased ly studied [2]. Phytosterols have attracted great with a remarkable income growth in the last two attention due to their nutritional properties and decades. The annual consumption of vegetable oil biological effects, for example their anti-hypocho- exceeds 30 million tons in China [1], making it one lesterolemic, anti-inflammatory, anti-oxidative and of the largest importing countries in the world. anti-tumour activities. They are now widely used The nutritional value and antioxidant characteris- in pharmaceuticals, nutritional supplements and tics of the edible plant oils have gained immense cosmetics [3–5]. attention in terms of technology research and new The official methods for separation and quan- product development. titative analysis of phytosterols are conventionally Phytosterols are a group of compounds simi- based on gas chromatography (GC). However, it lar to cholesterol, which naturally occur in plants usually requires chemical derivatization for fa- and differ from cholesterol in carbon side chains vourable peak shape, better sensitivity and resolu- and/or presence or absence of a double bond. tion, and a higher stability for labile unsaturated They are found in many edible plants, nuts, sterols with thermal instability [6]. In general, seeds, vegetables and edible oils. So far, 200–300 liquid chromatography (LC) has better operat- different types of phytosterols have been success- ing conditions, including milder temperatures and fully separated and identified in botanical sources, pressure conditions for column separation, non- Ningli Qi, Chunliang Yang, Center for Food Quality Supervision and Testing of Ministry of Agriculture and Rural Affairs (Zhanjiang), Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Renmingdadaonan No. 48, 524001 Zhanjiang, China. Yijun Liu, Liangkun Liao, Xiao Gong, Key Laboratory of Tropical Crop Products Processing of Ministry of Agriculture and Rural Affairs, Agricultural Products Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Renmingdadaonan No. 48, 524001 Zhanjiang, China. Correspondence author: Xiao Gong, e-mail: [email protected]; Chunliang Yang, e-mail: [email protected] © 2019 National Agricultural and Food Centre (Slovakia) 363 Qi, N. et al. J. Food Nutr. Res., Vol. 58, 2019, pp. 363–369 destructive analysis as well as several available de- (99.6 %), stigmasterol (98.1 %) and β-sitosterol tectors and has been widely used for sterol sepa- (98.6 %) were obtained from ANPEL Labora- ration [5, 7, 8]. ROCCO and FANALI [9] developed tory Technologies (Shanghai, China). HPLC-grade a nano-LC method for the determination of stig- methanol and acetonitrile were obtained from masterol, campesterol and β-sitosterol in extra- Merck Chemicals (Shanghai, China). Carbon virgin olive oil, after sample treatment, with good dioxide (99.9 %) was obtained from Zhanjiang sensitivity, precision and speed, the analysis taking Oxygen Plant (Zhanjiang, China). High-puri- 20 min. ZARROUK et al. [10] for the first time re- ty deionized water was prepared by a Milli-Q ported on LC with positive ion, atmospheric ultrapure purification system (Millipore, Billerica, pressure chemical ionization and ion trap mass Massachusetts, USA). All other chemicals and spectrometry, which was successfully applied to reagents were of analytical grade. direct determination of sterols and lipids with no sample preparation and derivatization required. Standard solutions and calibration Seven plant sterols and stanols could be separated The working standard solutions of individual within 8.5 min using an ultra-high-performance sterols were prepared by dissolving the respective LC method, with a low limit of detection (LOD) reference compounds separately in methanol. A linear value of 400–600 mg∙ml-1 was obtained [11]. regression equation was prepared from five increas- Supercritical fluid chromatography (SFC) inte- ing concentrations by diluting the stock solution in grates the advantages of both ultraperformance methanol. A linear relationship between peak area LC and supercritical fluid separation techniques. and concentrations (0.06–200.00 mg·ml-1) was ob- It exhibits good performance in terms of thermal tained, and the linear regression equation for each stability and volatility in GC analysis, significantly standard was used for phytosterol quantification in reduces the analysis time of LC separation and the selected oil samples. can also reduce organic solvent usage. As SFC is effective, less time-consuming and cost-effective in Sample preparation terms of separation and determination, it is exten- The procedure for phytosterol extraction re- sively applied in foodomics, biopharmaceuticals, ported by BEDNER et al. [14] was slightly modified drug epidemiology and environmental monitoring and applied to the sample preparation. Briefly, [12, 13]. 1.0 g edible oil sample was weighed, transferred To date, no studies on SFC use for separation into a 50 ml saponification bottle and mixed of phytosterols from food, including edible oils with 2.0 mol·l-1 ethanolic potassium hydroxide in China, have been reported. The present study (10 ml). The mixture was immediately vortexed aimed to develop a simple and rapid method for and placed in a boiling water bath in darkness simultaneous separation of three major phyto- for 30 min. After saponification, the sample was sterols, namely, campesterol, stigmasterol and cooled down in an ice-water bath, 10 ml of de- β-sitosterol, using an ultra-performance conver- ionized water was added and the resulting solu- gence chromatography (UPC2) system coupled to tion was transferred into a separatory funnel. The diode array detector (DAD). Further, it was effi- unsaponifiable fraction was twice extracted with ciently validated and used in phytosterol analysis 10 ml of n-hexane and washed with distilled water and characterization of edible oils that are widely until neutral pH. The extracts were combined and consumed in South China. dried under a constant nitrogen gas flow supplied using an N-EVAP 112 nitrogen evaporator (Or- ganomation Associates, Worcester, Massachu- MATERIALS AND METHODS setts, USA), while the samples were maintained at room temperature. The residues were dissolved in Raw materials HPLC-grade methanol (2.0 ml) and then filtered Nine brands of edible oils, including palm through a syringe nylon membrane filter (pore size oil, tea tree oil, linseed oil, blend oil, camellia 0.22 μm). oil, colza oil, soybean oil, olive oil and sun flower oil, were obtained from local retail outlets in Instrument and separation conditions Zhanjiang, China. They were all produced in 2016 Phytosterols were quantitatively and qualita- and represented approximately 90.0 % of the ve- tively analysed using ACQUITY UPC2 system getable oil consumption in South China. (Waters, Milford, Massachusetts, USA) consist- ing of a Diode Array Detector (DAD) detec- Chemicals and reagents tor, an autosampler, an automated back pressure The standard compounds of campesterol regulator (ABPR), a column oven, a binary sol- 364 Determination of phytosterols using supercritical fluid chromatography vent delivery pump and a convergence chro- RESULTS AND DISCUssION matography manager. The separation was car- ried out on an ACQUITY UPC2 high strength Optimization of chromatographic conditions silica C18 with selectivity for bases (HSS C18 SB) Chromatographic conditions were optimized column (100 mm × 3.0 mm, particle size 1.7 μm; for good separation according to the following Waters). The elution process started at 98 % of steps. First, the column screening experiment A (supercritical CO2) and 2 % of B (acetonitrile- using four kinds of packing particles