固相萃取-气相色谱法测定橄榄油中4种脂肪酸乙酯

doi:10.3724/SP.J.1123.2022.09011

摘要/Abstract

摘要：

橄榄油中脂肪酸乙酯(FAEEs)的含量是衡量其产品品质的重要指标之一,GB/T 23347-2021《橄榄油、油橄榄果渣油》中要求特级初榨橄榄油中FAEEs应≤35 mg/kg。目前橄榄油中FAEEs的国际标准检测方法是硅胶柱层析-气相色谱法(GC),该方法存在操作复杂、耗时长、试剂消耗量大等缺点。本研究建立了硅胶固相萃取(Si SPE)-气相色谱分析橄榄油中棕榈酸乙酯、亚油酸乙酯、油酸乙酯和硬脂酸乙酯4种FAEEs的检测方法。考察了载气类型,选定He气作为载气;筛选了内标化合物,确定使用十七碳烯酸乙酯(顺-10)作为内标物质;优化了SPE条件,对比了不同品牌Si SPE柱对回收率的影响,确定了称取0.05 g橄榄油,使用正己烷提取,1 g/6 mL Si SPE柱净化的前处理方法。该方法处理一个样品大约仅需2 h前处理时间和23 mL试剂。对优化后的方法进行方法学验证,结果表明,4种FAEEs在0.1~5.0 mg/L的范围内线性关系良好(决定系数(R²)>0.999),方法检出限为0.78~1.11 mg/kg,定量限为2.35~3.33 mg/kg,在低、中、高3个加标水平(4、8、20 mg/kg)下的回收率为93.8%~104.0%,相对标准偏差为2.2%~7.6%。用建立的方法对15个橄榄油样品进行测试,发现3个特级初榨橄榄油样品的FAEEs总量超过了35 mg/kg,表明市场上可能存在以次充好的现象。与国际标准检测方法相比,该方法前处理过程简便,操作时间短,试剂用量少,检测成本低,精密度高,准确性好,可以为橄榄油检测标准的完善提供有效的理论和数据参考。

关键词: 固相萃取, 气相色谱, 脂肪酸乙酯, 棕榈酸乙酯, 亚油酸乙酯, 油酸乙酯, 硬脂酸乙酯, 橄榄油

Abstract:

The fatty acid ethyl ester (FAEE) content of olive oil is an important indicator of its quality. At present, the international standard method used to detect FAEEs in olive oil is silica gel (Si) column chromatography-gas chromatography (GC); however, this technique presents a number of disadvantages, including complex operation, long analysis times, and high reagent consumption. In this study, a method based on Si solid phase extraction (SPE)-GC was established to determine four FAEEs in olive oil, namely, ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate. First, the effects of the carrier gas were investigated, and He gas was ultimately selected as the carrier gas. Next, several internal standards were screened, and ethyl heptadecenoate (cis-10) was determined as the optimal internal standard. The SPE conditions were also optimized, and the effects of different brands of Si SPE columns on the recoveries of analytes were compared. Finally, a pretreatment method in which 0.05 g of olive oil was extracted with n-hexane and purified through a Si SPE column (1 g/6 mL) was developed. A sample could be processed within approximately 2 h using a total reagent volume of about 23 mL. Validation of the optimized method revealed that the four FAEEs have good linearities within the range of 0.1-5.0 mg/L (coefficients of determination (R²)>0.999). The limits of detection (LODs) of the method were within 0.78-1.11 mg/kg, and its limits of quantification (LOQs) were in the range of 2.35-3.33 mg/kg. The recoveries ranged from 93.8% to 104.0% at all spiked levels tested (4, 8, and 20 mg/kg), and the relative standard deviations were 2.2%-7.6%. Fifteen olive oil samples were tested using the established method, and the total FAEEs of three extra-virgin olive oil samples were found to exceed 35 mg/kg. Compared with the international standard method, the proposed method has the advantages of simpler pretreatment process, shorter operation time, lower reagent consumption and detection cost, high precision, and good accuracy. The findings provide an effective theoretical and practical reference for improving olive oil detection standards.

Key words: solid phase extraction (SPE), gas chromatography (GC), fatty acid ethyl esters (FAEEs), ethyl palmitate, ethyl linoleate, ethyl oleate, ethyl stearate, olive oil

中图分类号:

O658

陆慧媛, 王利娟, 张炯恺, 张驰中, 李天娟, 季瑞雪, 沈伟健. 固相萃取-气相色谱法测定橄榄油中4种脂肪酸乙酯[J]. 色谱, 2023, 41(4): 359-365.

LU Huiyuan, WANG Lijuan, ZHANG Jiongkai, ZHANG Chizhong, LI Tianjuan, JI Ruixue, SHEN Weijian. Determination of four fatty acid ethyl esters in olive oil by solid phase extraction-gas chromatography[J]. Chinese Journal of Chromatography, 2023, 41(4): 359-365.

图/表 10

图1 不同载气条件下FAEEs标准溶液的色谱图

Fig. 1 Chromatograms of fatty acid ethyl ester (FAEE) standard solution under different carrier gases C16∶0E: ethyl palmitate; C17∶1E: ethyl heptadecenoate (cis-10); C18∶0E: ethyl stearate; C18∶1E: ethyl oleate; C18∶2E: ethyl linoleate.

图2 以(a) C17∶1E或(b) C17∶0E作内标的FAEEs标准溶液以及(c)无内标的样品溶液的色谱图

Fig. 2 Chromatograms of the FAEE standard solution with (a) C17∶1E or (b) C17∶0E as the internal standard and (c) sample solution without the internal standard C17∶0E: ethyl heptadecanoate.

图3 采用不同洗脱方案时FAEEs的洗脱结果

Fig. 3 Elution results of the FAEEs under different elution plans 0.05-0.2 g: sample amounts; 1 g/6 mL, 2 g/6 mL and 3 g/10 mL: Si SPE column specification.

图4 采用不同品牌的Si SPE柱净化时FAEEs的洗脱曲线图

Fig. 4 Elution curves of the FAEEs during purification with different brands of Si SPE columns

图5 不同品牌的硅胶固相萃取柱净化后FAEEs的回收率(n=3)

Fig. 5 Recoveries of FAEEs after purification with different brands of Si SPE columns (n=3)

图6 两种前处理方法所得的样品色谱图

Fig. 6 Chromatograms of samples processed using two pretreatment methods a. this method; b. COI/T.20/Doc. No 28/Rev.2 method. C17∶0M: methyl heptadecanoate.

图7 两种前处理条件下样品的加标回收率(4 mg/kg)

Fig. 7 Recoveries of standards spiked in sample processed using two pretreatment methods (4 mg/kg)

表1 FAEEs的回归方程、决定系数(R2)、检出限和定量限

Table 1 Regression equations, coefficients of determination (R2), LODs, and LOQs of the FAEEs

Compound	Regression equation	R²	LOD/ (mg/kg)	LOQ/ (mg/kg)
C_16∶0E	y=0.5051x-0.0104	0.9999	0.78	2.35
C_18∶2E	y=0.3806x-0.0099	0.9999	0.85	2.56
C_18∶1E	y=0.4944x-0.0074	0.9999	1.11	3.33
C_18∶0E	y=0.4825x-0.0085	0.9999	0.82	2.45

表2 FAEEs在橄榄油中3个水平下的平均回收率及相对标准偏差(n=6)

Table 2 Average recoveries (AR) and RSDs of FAEEs in olive oil at three spiked levels (n=6)

Compound	Background/ (mg/kg)	Added/ (mg/kg)	AR/ %	RSD/ %
C_16∶0E	3.25	4	95.3	4.3
		8	99.8	6.0
		20	104.0	2.2
C_18∶2E	1.68	4	100.0	6.5
		8	102.8	2.3
		20	100.2	4.3
C_18∶1E	3.78	4	99.3	6.1
		8	93.8	3.6
		20	102.7	2.8
C_18∶0E	3.79	4	96.7	3.3
		8	100.3	7.6
		20	100.1	4.2

表3 实际橄榄油样品中FAEEs的测试结果

Table 3 Detection results of FAEEs in actual olive oil samples

No.	Grade	Origin	Contents/(mg/kg)
No.	Grade	Origin	C_16∶0E	C_18∶2E	C_18∶1E	C_18∶0E	SUM
1	refined+extra virgin olive oil	Italy	N. D	16.09	7.47	N. D	23.56
2	extra virgin olive oil	Spain	2.48	3.41	3.16	N. D	9.05
3	extra virgin olive oil	Spain	3.09	13.83	10.74	N. D	27.66
4	extra virgin olive oil	Spain	3.25	1.68	3.78	3.79	12.5
5	extra virgin olive oil	Spain	3.31	7.76	7.86	N. D	18.93
6	extra virgin olive oil	Spain	3.87	7.89	20.57	N. D	32.33
7	extra virgin olive oil	Greece	8.08	7.13	41.54	3.17	59.92
8	extra virgin olive oil	Italy	N. D	5.79	13.37	N. D	19.16
9	extra virgin olive oil	Spain	4.43	8.99	21.27	N. D	34.69
10	Refined+extra virgin olive oil	Spain	4.02	12.32	46.14	N. D	62.48
11	extra virgin olive oil	Spain	14.78	39.2	195.01	8.56	257.55
12	extra virgin olive oil	Portugal	3.36	6.15	9.98	N. D	19.49
13	extra virgin olive oil	Spain	6.18	5.25	18.33	3.12	32.88
14	extra virgin olive oil	Italy	4.95	20.48	28.52	N. D	53.96
15	refined+extra virgin olive oil	Italy	2.87	16.79	14.66	N. D	34.32

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