Composition of total and esterified 4α-monomethylsterols and triterpene alcohols in virgin olive oil

Esterified 4α-monomethylsterols and triterpene alcohols were isolated from virgin olive oil by combined column chromatography-thin layer chromatography and their composition was assessed gas chromatographically using both packed and capillary columns. The compositions of the esterified 4α-monomethylsterol and triterpene alcohol fractions were compared with those of the total fractions which were isolated after saponification. Significant differences were observed between the distribution patterns of the total and esterified triterpene alcohol fractions, especially in the percentages of 24-methylenecycloartanol, butyrospermol and cycloartenol.     

The detection of the presence of hazelnut oil in olive oil by free and esterified sterols

Genuine olive and hazelnut oils from diverse geographical origins, as single varieties and blends, were mixed at different percentages and analysed by the method based on the quantification of free and esterified sterols. Two formulas based on three sterols (Campesterol, Δ⁷-stigmastenol and Δ⁷-avenasterol) together with empirical decision rules were able to detect the presence of hazelnut oil in olive oil when the percentage of the former was more than 6–8%, although this figure was much lower in the most of the adulterations. Results of univariate and multivariate statistical procedures based on the analysis of 116 samples are presented in support of the method efficiency.     

酶法快速测定植物油中游离甾醇

目的 基于甾醇在胆固醇氧化酶的作用下与显色剂产生显色反应,建立了固相萃取结合酶法测定植物油中游离甾醇含量的快速检测方法。方法 采用固相萃取（Solid Phase Extraction, SPE）结合酶法40 min内实现对植物油中的游离甾醇含量测定,并应用于植物油中游离甾醇含量的测定。结果 方法学验证的结果显示,植物甾醇含量在浓度范围0.3~7.5 mg/mL之间线性良好,检出限为0.018 mg/g,日内精密度为7.94%,日间精密度为6.88%,回收率在84.75%~99.50% 之间,实际样品的检测结果显示,酶法与气相色谱-串联质谱法（gas chromatography-tandem mass spectrometry, GC-MS）的相对误差在±20%以内,且酶法的检测效率比GC-MS法提高3倍。常见的几种植物油中,游离甾醇含量较高的是玉米油和菜籽油,分别为1.53~3.66 mg/g和1.98~3.02mg/g。结论 本研究建立的SPE结合酶法检测植物油中游离甾醇的快速检测方法稳定性、重现性好、灵敏可靠,可满足植物油中游离甾醇的现场批量快速检测。     

橄榄油中甾醇组成及总量测定方法的探讨

介绍了甾醇组成及总量测定在橄榄油评价中的意义,比较了3种甾醇测定方法优缺点,详细探讨了COI/T.20/Doc.no.10《气相色谱法测定甾醇组成及甾醇含量》测定方法中影响测定结果的操作要点等,以求通过对方法探讨和分析,提高COI/T.20/Doc.no.10方法的可操作性.通过对实例样品的分析可以看出,橄榄油理事会制定的COI/T.20/Doc.no.10方法是一种值得推荐的可行的分析方法.     

Analysis of free and esterified sterols in edible oils by online reversed phase liquid chromatography-gas chromatography (RPLC-GC) using the through oven transfer adsorption desorption (TOTAD) interface

An online reversed phase liquid chromatography-gas chromatography (RPLC-GC) method is proposed to quantify free, total and esterified sterols of edible oils. To determine free sterols the diluted oils are injected into the liquid chromatograph, where free sterols are separated from triglycerides and the sterol fraction is automatically transferred to the gas chromatograph to be analysed. To determine total sterols the samples were saponified with potassium hydroxide in ethanolic solution and the unsaponifiable fraction was extracted with diethyl ether. The extract was then analysed by RPLC-GC, avoiding the laborious thin layer chromatography step used in the Official European Union (EU) Method. The relative standard deviations (RSDs) from the absolute peak area varied from 7.6% to 15.8%. Limits of detection (LODs) were less than 8.5 mg/kg. No variability in retention time was observed. The method was applied to the determination of total sterols in edible oil samples and the results were compared with those obtained with the Official EU Method. Good agreement was found between both methods, except in the case of campesterol.     

Comparative study of different extraction techniques for the analysis of virgin olive oil aroma

Headspace solid-phase microextraction (HS-SPME), simultaneous distillation/extraction (SDE) and closed-loop stripping analysis (CLSA) coupled to gas chromatography/mass spectrometry (GC/MS) were used to study virgin olive oil, with the goal of detecting large numbers of characteristic volatile and semi-volatile compounds. More than one hundred compounds were detected in the olive oil extracts, and their percent amounts obtained by each technique were calculated. Qualitative and quantitative differences of virgin olive oil volatile profiles were observed applying the three extraction techniques. SPME showed a higher affinity for alcohols and ketones, while CLSA achieved the highest percentages of esters and hydrocarbons. Finally, the highest extraction of total terpenoid compounds occurred with SDE and CLSA, where CLSA allowed extracting the highest percentages of the most of them. SDE extraction caused the thermal degradation of the oil sample, which resulted in a high percentage of aldehydic compounds.     

Influence of free fatty acids, sterols and phospholipids on volatile compounds in olive oil headspace determined by solid phase microextraction-gas chromatography

In order to study the influence of some polar compounds, naturally present in virgin olive oils (VOOs), on the volatiles release, the volatiles-free olive oil matrix was enriched with free fatty acids (FFA) (concentration range from 0.35 to 1.40 g/100 g), sterols (1.0–2.5 g/kg) and phospholipids (1.6–6.0 g/kg). Sixteen standards of VOO aroma compounds were dissolved in thus obtained olive oil matrices. Volatile compounds were analysed by headspace solid phase microextraction with DVB/CAR/PDMS fibre and gas chromatography with flame ionization detection. Evaluation of the data by statistical analysis revealed that FFA, sterols and phospholipids, in concentration ranges considered in this study, generally did not significantly influence the determination of VOO volatiles. The most important changes occurred in the release of the three alcohols (Z-2-penten-1-ol, 1-hexanol and E-3-hexen-1-ol) and 3-methylbutyl acetate which slightly decreased upon the increase of FFA concentration.     

Minor compounds in the phenolic fraction of virgin olive oils

The phenolic fraction of 34 virgin olive oils was analysed by gas chromatography–mass spectrometry (GC–MS) with the aim to identify new compounds at low level. Twenty-seven compounds previously described in olive oils were identified; several new minor compounds with phenolic structure were detected in the samples: amongst them, 4-hydroxyphenylacetaldehyde, trans-isoeugenol (trans-2-methoxy-4-(1-propenyl)-phenol), 1,4-dihydroxy-2,6-dimethoxybenzene, 3,4-dihydroxybenzyl alcohol and 3,4-dihydroxyphenylacetic acid were identified by their mass spectra and confirmed using standards. In 34 virgin olive oils (cv. Nocellara del Belice) the mean concentrations for these five substances were always below 0.2 mg kg⁻¹ and only in two samples the level of 3,4-dihydroxyphenylacetic acid reached 1.47 and 1.97 mg kg⁻¹, respectively. These compounds could be used to characterise olive oils; low concentrations of 3,4-dihydroxyphenylacetic acid may show the initial autoxidation processes of olive oils.     

Composition of total sterols (4-desmethyl-sterols) in extravirgin olive oils obtained with different extraction technologies and their influence on the oil oxidative stability

The composition and antioxidant activity of total sterols in extravirgin olive oils obtained with different extraction technologies from olives harvested at two ripening stages, were studied. The antioxidant activity was evaluated with an oxidative stability instrument (OSI), by using a model system (made of a mixture of treated/untreated commercial refined peanut oil) enriched with the total sterol fractions of the extravirgin olive oils. No correlation was found between the OSI time and the extraction technologies, the ripening stages or the actual amount of sterols added. No significant differences were observed in the percent composition of sterols of extravirgin olive oils produced with different technologies during the same harvesting period. The latter, however, had a significant effect on the percent of β-sitosterol and 5-avenasterol in extravirgin olive oils produced with the same technology.     

Sterolic composition and triacylglycerols of Oueslati virgin olive oil: comparison among different geographic areas

The main triglycerides (TG) identified in the Oueslati virgin olive oil were 1,2,3‐trioleylglycerol (OOO), 2,3‐dioleyl‐1‐palmitoylglycerol (POO) and 2,3‐dioleyl‐1‐linoleylglycerol (LOO) representing more than 80% of the total area of peaks in the chromatogram. Other minor triacylglycerols were 2,3‐dioleyl‐1‐stearoylglycerol (SOO), 2‐oleyl‐3‐palmitoyl‐1‐stearoylglycerol (SOP), 1‐linolenoyl‐2‐oleyl‐3‐palmitoylglycerol (LnOP), 1, 2‐dilinoleyl‐3‐palmitoylglycerol (LLP), 1, 3‐dioleyl‐2‐linolenoylglycerol (OLnO), 1‐linolenoyl‐2‐linoleyl‐3‐oleylglycerol (LnLO) and 1,2,3‐trilinoleylglycerol (LLL). The sterol profile of Tunisian virgin olive oils produced from Oueslati cultivar was established by gas chromatography using a flame ionisation detector (GC‐MS). More than 10 compounds were identified and characterised. As expected for virgin olive oil, the main sterols found in all Oueslati olive oils were β‐sitosterol, Δ5‐avenasterol, campesterol and stigmasterol. Cholesterol, 24‐methylenecholesterol, clerosterol, campestanol, sitostanol, Δ7‐stigmastenol, Δ5, 24‐stigmastadienol and Δ7‐avenasterol were also found in all samples, but in lower amounts. Most of these compounds are significantly affected by the geographical origin of the oil. Besides the sterol components, two triterpene dialcohols, erythrodiol and uvaol, were also detected.     

Separation and determination of sterols in olive oil by HPLC-MS

This study presents the first liquid chromatographic method for the identification and quantification of seven phytosterols in olive oil. Sterols were identified and quantified by liquid chromatography with mass spectrometry detection in positive APCI (atmospheric pressure chemical ionisation) mode. The samples were saponified by refluxing with 2 N ethanolic KOH, and the non-saponificable fraction was extracted with diethyl ether. This fraction was subjected to thin layer chromatography (TLC) on silica gel plates and then the band of sterols was isolated and extracted with methanol. Sterols were quantified by LC-MS, on a Waters Atlantis 5 μm dC₁₈2,1 × 150 mm column with a gradient of acetonitrile/water (0.01% acetic acid) at a flow of 0.5 mL min⁻¹; column temperature 30 °C. The method presents values between 123 and 677 ng mL⁻¹ for detection limits, with relative standard deviations between 4.0% and 5.4% at a concentration of 5 mg L⁻¹ for each sterol. Sterol contents were determined in a virgin olive oil, a refined olive oil, an olive-pomace oil and a crude olive-pomace oil.     

The influence of ripening and crop year on quality indices,polyphenols, terpenic acids,squalene, fatty acid profile,and sterols in virgin olive oil (Koroneiki cv.) produced by organic versus non‐organic cultivation method

The chemical composition of virgin olive oil is determined by numerous factors. The aim of this study was to evaluate the influence of the olive ripening stage, and crop year on the quality indices of Koroneiki cv. virgin olive oil of organic vs non‐organic cultivation. Drupes of organic and of non‐organic cultivation were sampled at four successive ripening periods in crop years 2000 and 2004. Quality indices, total and simple polyphenols, terpenic acids, squalene, fatty acid profile, and sterols were measured. Statistical analysis demonstrated that peroxide value differed according to cultivation method and according to crop year. Organic Koroneiki cv. olive oils exhibited higher total phenols content. Total phenols differed mainly according to crop year and maturation phase, but also according to cultivation method. Total unsaturated and saturated fatty acids differed according to cultivation method, crop year and maturation. Oleic and palmitoleic acids varied according to cultivation method and according to maturation process. Cholesterol, campesterol and stigmasterol differed according to maturity, while β‐sitosterol differed according to crop year. Overall, olive oil from organic cultivation was of superior quality compared to non‐organic, while composition of olive oils was greatly variable during maturation, whereas it was also affected by the crop year.     

Multivariate analysis of NMR fingerprint of the unsaponifiable fraction of virgin olive oils for authentication purposes

A new approach to the geographical characterisation of virgin olive oils (VOOs) based on the ¹H NMR fingerprint of the unsaponifiable matter is presented. The ¹H NMR spectra of the unsaponifiable fraction of virgin olive oils from Spain, Italy, Greece, Tunisia, Turkey, and Syria were analysed by several pattern recognition techniques (LDA, PLS-DA, SIMCA, and CART). PLS-DA (PLS-1 approach) obtained the best classification results for all classes. Moreover, ¹H NMR spectra of the bulk oil, and its corresponding unsaponifiable fraction, as well as the subfractions of the unsaponifiable fraction (alcohol, sterol, hydrocarbon, and tocopherol fractions) were studied in the search for the markers that multivariate techniques revealed to be related to the geographical origin of olive oils. Additionally, a preliminary study regarding ¹H NMR data of the bulk oil and the corresponding unsaponifiable fraction of VOOs suggested that these spectral data contained complementary information for the geographical characterisation of VOOs.     

NMR spectroscopy: a powerful tool for the analysis of polyphenols in extra virgin olive oil

Extra virgin olive oil (EVOO), a key component of the Mediterranean diet, has aroused interest in recent years due to its health properties. Nuclear magnetic resonance (NMR) spectroscopy is an appropriate tool for the accurate quantification of minor compounds in complex food matrices, such as polyphenols in olive oil. Flavonoids, lignans, secoiridoids and phenolic acids and alcohols in EVOO have been identified and quantified by NMR. This review provides an overview of the major developments in the structural elucidation of polyphenol compounds in EVOO. © 2019 Society of Chemical Industry     

Coupling MOS sensors and gas chromatography to interpret the sensor responses to complex food aroma: Application to virgin olive oil

This paper describes a novel approach to determine the individual contribution of volatile compounds to the overall sensor responses of metal oxide semiconductor (MOS) sensors when they are applied to a complex mixture of compounds such as food aroma. A methodology entailing the use of a sensor array or electronic nose (EN) as non-destructive detector in-parallel with the flame ionization detector of a gas chromatograph (GC) is described and illustrated in the context of virgin olive oil aroma analysis. Aspects related to the experimental set up and data processing are examined. The capability of gas chromatography for obtaining qualitative and quantitative information of the volatile compounds present in virgin olive oil was used to find relationships between these compounds and the sensor responses by means of a coupled system GC–EN. The sensor responses that resulted from this coupling (sensorgrams) served to prove the sensor sensitivity to alcohols, aldehydes, and those compounds that are responsible to sensory defects in virgin olive oil (e.g. aldehydes and acids).     

High-power gradient diffusion NMR spectroscopy for the rapid assessment of extra-virgin olive oil adulteration

A high gradient diffusion NMR spectroscopy was applied to measure diffusion coefficients (D) of a number of extra-virgin olive, seed, and nut oils in order to ascertain the suitability of this rapid and direct method for discrimination of adulterated olive oils. Minimum adulteration levels that could be detected by changes in D were 10% for sunflower (SuO) and soybean oil (SoO), and 30% for hazelnut (HO) and peanut oil (PO). Qualitative and quantitative prediction of adulteration was achieved by discriminant analysis (DA). The highest prediction accuracy (98–100%) was observed only when two DA models were concomitantly used for sample classification. The first DA model provided recognition of high adulterated EVOO with more than 20% of SuO or SoO, and 30% with PO, whilst the second model could differentiate EVOO adulterated with 10% of SuO or SoO, and more than 30% of HO. The validation test performed with an independent set of randomly adulterated EVOO samples gave 100% classification success. The high accuracy levels together with minimal requirements of sample preparation, and short analyses time, prove the high-power gradient diffusion NMR spectroscopy as an ideal method for rapid screening of adulteration in valuable olive oils.     

Looking through the qualities of a fluorimetric assay for the total phenol content estimation in virgin olive oil,olive fruit or leaf polar extract

As an alternate to the Folin-Ciocalteu assay (F-C) we propose a fluorimetric estimation of the total phenol content in virgin olive oil (VOO), olive fruit and leaf polar extracts. Phenol content was determined at excitation/emission wavelengths set at 280/320 nm. Standard operational procedures (slit widths, temperature, pH) and method validation were carried out according to Eurachem guidelines. The qualities of the proposed assay are better than those of the F-C one, as the procedure is more sensitive (LOD and LOQ values 10-fold lower), three times faster, needs no reagents and most importantly, is not destructive for the sample that can be further used in HPLC or other assays. Data for VOO extracts correlated well with the colorimetric ones (r = 0.69, n = 65). HPLC coupled with diode array and fluorescence detectors supported the above findings. Good correlations were also found between the respective data for olive fruit and leaf extracts (r = 0.96, n = 18).     

液液萃取-UPLC快速测定初榨橄榄油中叶绿素铜钠的含量

建立了液液萃取-超高效液相色谱（UPLC）快速测定初榨橄榄油中叶绿素铜钠含量的方法。初榨橄榄油样品经80%的乙醇溶液提取后，在UPLC条件为采用SVEA C18色谱柱（50 mm×1.0 mm, 1.7 μm），柱温30 ℃，乙腈-水（体积比95∶ 5）为流动相，等度洗脱，流速0.3 mL/min，进样量2.0 μL，检测波长405 nm下，可实现初榨橄榄油中叶绿素铜钠的有效分离，整个分析过程仅需6 min。通过方法学考察，该方法的检出限及定量限分别为0.20 mg/L和0.50 mg/L，叶绿素铜钠加标回收率均在84.00%~97.90%之间，方法的重复性、精密度和稳定性较好。所建立的方法可用于初榨橄榄油中叶绿素铜钠含量的快速测定。     

Optimisation,characterisation and quantification of phenolic compounds in olive cake

The phenolic compounds in extracts from pressed olive cake were investigated. Free phenolic compounds were extracted from olive cake using methanol. To liberate bound phenolic compounds, the olive cake was subjected to basic and acidic hydrolysis followed by methanol extraction. The individual phenolic compounds and antioxidant activity of the extracts were determined. The highest total phenolic content and antioxidant activity were obtained using methanol extraction for 12 h at 70 °C. The RP-HPLC profiles for full-fat and defatted olive cake showed that protocatechuic acid, hydroxybenzoic acid, sinapic acid, p-coumaric acid, rutin and hesperidin were the predominant free phenolic compounds. Meanwhile, syringic acid, sinapic acid, caffeic acid and protocatechuic acid were the predominant bound phenolic acids. A positive correlation was observed between total phenolic content and antioxidant activity. The results indicated that most of the phenolic compounds in olive products were present in their free forms (75–90% of total phenolic content), while bound phenolic compounds were only a small proportion (10–25%) of total phenolic content.     

Direct determination of phenolic compounds and phospholipids in virgin olive oil by micellar liquid chromatography

A simple HPLC method is reported for fast separation and determination of phenolic compounds (tyrosol, caffeic acid, p-coumaric acid and oleuropeina) and phospholipids (phosphatidylethanolamine and phosphatidylcholine) in virgin olive oil samples. The samples were diluted with 2-propanol and injected into the column directly without previous extraction. Samples with an olive oil content of up to 65% were injected without any problems. The analytes were separated on a C-18 column by a micellar mobile phase containing 0.07 M SDS and 2.5% 2-propanol at pH 3, and were detected at 210 nm. Linear calibration curves [r² > 0.997] were obtained with detection limits ranging from 0.052 to 0.16 μg/g and 1 to 8.6% repeatability for the phenolic compounds. Several virgin olive oil samples were analysed and the recovery values were around 110%.