g-C3 N4 富集结合毛细管电泳与电感耦合等离子体质谱联用分析西瓜中硒形态

doi:10.3724/SP.J.1123.2020.06015

摘要/Abstract

摘要：

硒是人体所必需的微量元素之一，对人身体健康非常重要。该工作称取2.0 g三聚氰胺于反应釜中，在600℃下密闭加热2 h，冷却至室温，取0.5 g上述产物置于烧杯中在室温下超声10 h，静置30 min，取上层乳白色溶液于60℃下烘干，即得类石墨烯氮化碳（g-C₃ N₄ ）。所制备的g-C₃ N₄ 经傅里叶变换红外光谱（FT-IR）、X射线粉末衍射（XRD）、场发射环境扫描电子显微镜（SEM）进行表征，证明g-C₃ N₄ 已经成功合成。建立了毛细管电泳-电感耦合等离子体质谱（CE-ICP-MS）联用技术测定西瓜中硒形态的新方法。用胃蛋白酶作提取剂及超声波辅助提取西瓜中的硒形态，经g-C₃ N₄ 富集、长100 cm内径100 μm毛细管分离，电泳电压为22 kV，缓冲液为8 mmol/L NaH₂ PO₄ -12 mmol/L H₃ BO₃ -0.2 mmol/L十六烷基三甲基溴化铵（CTAB）（pH 9.2），于11 min内完全分离6种硒形态。所建立的方法检测速度快、灵敏度高，采用CH₄ 作动态反应气消除干扰。硒脲（SeUr）、硒代胱氨酸（SeCys₂ ）、硒代蛋氨酸（SeMet）、亚硒酸钠（Se（Ⅳ））、硒酸钠（Se（Ⅵ））、硒代乙硫氨酸（SeEt）的线性范围（按Se计）分别为0.017~20 μg/L、0.091~50 μg/L、0.032~40 μg/L、0.023~60 μg/L、0.015~75 μg/L、0.015~30 μg/L。各硒形态的线性相关系数均大于0.9995，SeUr、SeCys₂ 、SeMet、Se（Ⅳ）、Se（Ⅵ）、SeEt的检出限分别为6.2、30、11、8.2、48、5.5 ng/L，回收率为96.0%~106%，RSD < 3%。将本方法应用于西瓜中硒形态分析，结果令人满意。

关键词: 毛细管电泳, 电感耦合等离子体质谱, 硒, 形态分析, 西瓜

Abstract:

Selenium is one of the essential trace elements in the human body, and it plays a critical role in human health. In this work, 2.0 g melamine was placed in an alumina crucible, which was heated in a box-type resistance furnace for 2 h at 600 ℃, at the heating rate of 3 ℃/min, and then cooled to room temperature. After cooling, yellow graphite phase carbon nitride (g-C₃ N₄ ) nanosheets were obtained. Subsequently, 500 mg of the nanosheets was dispersed in 50 mL water with ultrasonication for 10 h in order to remove the residual un-exfoliated g-C₃ N₄ nanoparticles and large-sized nanosheets. The obtained suspension was centrifuged at about 10000 r/min, followed by drying at 60 ℃ to produce g-C₃ N₄ . The prepared g-C₃ N₄ was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), and field emission-environmental scanning electron microscopy (SEM) analyses. Given that the selenium content in actual samples is very low, high sensitivity, and accuracy are imperative for selenium detection. The combination of capillary electrophoresis (CE) with inductively coupled plasma-mass spectrometry (ICP-MS) can greatly improve the sensitivity, accuracy, and speed of the analysis. A novel method based on CE-ICP-MS was established for the determination of selenourea (SeUr), L-selenocystine (SeCys₂ ), DL-selenomethionine (SeMet), selenite (Se(Ⅳ)), selenate (Se(Ⅵ)), and selenoethionine (SeEt) in watermelon. The selenium species in watermelon were extracted by ultrasonication with pepsin as an extractant and g-C₃ N₄ enrichment. The enrichment factor of g-C₃ N₄ ranged from 12 to 29. Six selenium species were completely separated within 11 min in a 100-cm-long capillary with 100 μm internal diameter, at an applied voltage of 22 kV, using a buffer solution of 8 mmol/L NaH₂ PO₄ -12 mmol/L H₃ BO₃ -0.2 mmol/L cetyl trimethyl ammonium bromide (CTAB; pH 9.2). The interference in the selenium detection was eliminated using a dynamic reaction cell with CH₄ . The linear correlation coefficients of all the selenium species were greater than 0.9995. Under the optimal conditions, the limits of detection (3 σ, σ for standard deviation, as Se) for SeUr, SeCys₂ , SeMet, Se(Ⅳ), Se(Ⅵ), and SeEt were 6.2, 30, 11, 8.2, 48, and 5.5 ng/L, respectively. The linear range (as Se) for SeUr, SeCys₂ , SeMet, Se(Ⅳ), Se(Ⅵ), and SeEt were 0.017-20 μg/L, 0.091-50 μg/L, 0.032-40 μg/L, 0.023-60 μg/L, 0.015-75 μg/L, and 0.015-30 μg/L, respectively. The recoveries ranged from 96.0% to 106%, and the relative standard deviations (RSDs; n =5) were less than 3%. The developed method is simple, rapid, and sensitive, and it is also suitable for the detection of selenium species in other food and environmental samples.

Key words: capillary electrophoresis (CE), inductively coupled plasma-mass spectrometry (ICP-MS), selenium, speciation analysis, watermelon

冯金素, 曹玉嫔, 莫桂春, 唐莉福, 邓必阳. g-C₃ N₄ 富集结合毛细管电泳与电感耦合等离子体质谱联用分析西瓜中硒形态[J]. 色谱, 2020, 38(10): 1224-1231.

FENG Jinsu, CAO Yupin, MO Guichun, TANG Lifu, DENG Biyang. Selenium speciation in watermelon by g-C₃ N₄ enrichment combined with capillary electrophoresis-inductively coupled plasma-mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(10): 1224-1231.

图/表 10

表1 毛细管电泳及电感耦合等离子体质谱工作条件

Table 1 Operating conditions for CE and ICP-MS

CE conditions		ICP-MS conditions
CTAB: cetyl trimethyl ammonium bromide.
Electrophoresis buffer	8 mmol/L NaH₂ PO₄ -12 mmol/L	RF power	1100 W
	H₃ BO₃ -0.2 mmol/L CTAB (pH=9.2)	plasma gas flow	15 L/min
Capillary	100 cm×100 μm i. d.	auxiliary gas flow	1.2 L/min
Separation voltage	22 kV	nebulizer gas flow	0.92 L/min
Injection way	0.04 MPa×12 s	pulse voltage	800 V
		CH₄ flow rate	1.1 mL/min
		monitored species	⁸⁰ Se

图1 缓冲液浓度对硒形态保留时间的影响

Fig. 1 Effect of buffer solution concentration on retention time of selenium species a. 4 mmol/L NaH2 PO4 -6 mmol/L H3 BO3 -0.2 mmol/L CTAB; b. 6 mmol/L NaH2 PO4 -9 mmol/L H3 BO3 -0.2 mmol/L CTAB; c. 8 mmol/L NaH2 PO4 -12 mmol/L H3 BO3 -0.2 mmol/L CTAB; d. 10 mmol/L NaH2 PO4 -15 mmol/L H3 BO3 -0.2 mmol/L CTAB; e. 12 mmol/L NaH2 PO4 -18 mmol/L H3 BO3 -0.2 mmol/L CTAB. CSeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), SeEt =30 μg/L. For other conditions, see Table 1 .

图2 缓冲液pH对6种硒形态保留时间的影响

Fig. 2 Effect of the pH of buffer solution on retention time of selenium species CSeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), SeEt =30 μg/L. For other conditions, see Table 1 .

图3 6种硒形态的CE-ICP-MS图

Fig. 3 Electropherogram of six selenium species using CE-ICP-MS CSeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), SeEt =30 μg/L. For other conditions, see Table 1 .

图4 类石墨烯氮化碳对硒形态的吸附率

Fig. 4 Adsorption percentages of selenium species using g-C3 N4 For detection conditions, see Table 1 .

图5 NaOH溶液浓度对硒形态洗脱的影响

Fig. 5 Effect of NaOH concentration on the desorption of selenium species Mass concentration: SeUr 20 μg/L, SeCys2 50 μg/L, SeMet 40 μg/L, Se(Ⅳ) 60 μg/L, Se(Ⅵ) 75 μg/L, SeEt 30 μg/L. For detection conditions, see Table 1 .

图6 超声时间对硒形态吸附和洗脱的影响

Fig. 6 Effect of ultrasonic time on adsorption and elution of selenium species CSeUr, SeCys2 , SeMet, Se(Ⅳ), Se(Ⅵ), SeEt =20 μg/L. For detection conditions, see Table 1 .

表2 用CE-ICP-MS检测硒形态的分析性能(n =5)

Table 2 Analytical performance of selenium species using CE-ICP-MS (n =5)

Selenium species	Linear range/(μg/L)	EF	LOD¹⁾ /(ng/L)	RSD/%	LOD²⁾ /(μg/L)
EF: enrichment factor; LOD: 1) this method; 2) reported method.
SeUr	0.017-20	17	6.2	2.2
SeCys₂	0.091-50	21	30	3.3	0.9^[18] , 0.3^[23] , 0.42^[24]
SeMet	0.032-40	18	11	2.4	5.6^[18] , 0.2^[23] , 0.56^[24]
Se(Ⅳ)	0.023-60	18	8.2	1.9	0.4^[18] , 0.15^[23] , 0.36^[24]
Se(Ⅵ)	0.15-75	29	48	3.5	0.4^[18] , 0.15^[23]
SeEt	0.015-30	12	5.5	2.7	1.2^[18] , 0.35^[23]

图7 (A) 西瓜汁和(B)西瓜渣中硒形态的CE-ICP-MS图

Fig. 7 Electropherograms of selenium species in (A) watermelon juice and (B) watermelon slag using CE-ICP-MS a. ordinary watermelon; b. selenium-rich watermelon; c. selenium-rich watermelon+10 μg/L SeCys2 . For detection conditions, see Table 1 .

表3 富硒西瓜样品中硒形态测定结果及回收率(n =5)

Table 3 Analytical results and recoveries for selenium species in selenium-enriched watermelon samples (n =5)

Selenium species	Original/ (ng/g)	Added/ (ng/g)	Found/ (ng/g)	Recovery/ %	RSD/ %
WJ: watermelon juice. WS: watermelon slag.
SeCys₂ ^WJ	9.10	50.0	60.5	103	2.10
SeMet^WJ	30.0	100	129	99.0	1.60
Se(Ⅳ)^WJ	22.0	100	120	98.0	2.90
Se(Ⅵ)^WJ	7.50	50.0	60.5	106	1.80
SeEt^WJ	6.90	50.0	55.2	96.6	2.40
SeCys₂ ^WS	19.0	50.0	67.0	96.0	2.20
SeMet^WS	5.30	50.0	54.6	98.6	1.90
SeEt^WS	2.71	50.0	53.2	101	1.40

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g-C₃ N₄ 富集结合毛细管电泳与电感耦合等离子体质谱联用分析西瓜中硒形态

Selenium speciation in watermelon by g-C₃ N₄ enrichment combined with capillary electrophoresis-inductively coupled plasma-mass spectrometry

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