在线固相萃取-二维液相色谱法同时测定儿童配方奶粉中维生素A、D、E

黄燕; 林灵超; 蒋婷婷; 周雅雯; 周小靖

doi:10.13386/j.issn1002-0306.2022060239

在线固相萃取-二维液相色谱法同时测定儿童配方奶粉中维生素A、D、E

doi: 10.13386/j.issn1002-0306.2022060239

浙江福立分析仪器股份有限公司，浙江台州 317500

基金项目: 浙江省重点研发计划项目（2021C01128）。

详细信息

作者简介:
黄燕（1985−），女，硕士，工程师，研究方向：多维液相色谱系统的开发及应用，E-mail：huangy@cnfuli.com.cn

通讯作者:
周小靖（1957−），男，博士，高级工程师，研究方向：色谱分离分析系统的开发及应用，E-mail：zhouxj@cnfuli.com.cn

中图分类号: TS207.3
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出版历程
- 收稿日期: 2022-06-27
- 刊出日期: 2023-05-01

Simultaneous Determination of Vitamins A, D and E in Children's Formula Milk Powder by Combining Online Solid Phase Extraction and Two-Dimensional Liquid Chromatography

Zhejiang Fuli Analytical Instruments Co., Ltd., Taizhou 317500, China

摘要

摘要: 建立在线固相萃取（SPE）-二维液相色谱法同时测定儿童配方奶粉中脂溶性维生素A、D₂、D₃和α-、β-、γ-、δ-维生素E含量的方法。样品经皂化后直接进样，以聚合物为填料的PLRP-S色谱柱为在线固相萃取柱净化皂化液，采用SPE泵反向洗脱目标组分，并通过引入在线稀释将目标组分聚焦在第一维色谱柱柱头。以NuovaSil^TM PFP（4.6 mm×150 mm，3 μm）为第一维色谱柱，甲醇-0.1%甲酸水溶液为流动相进行梯度洗脱，分离维生素A和E的4 种异构体。以ChromCore PAH（4.6 mm×150 mm，3 μm）为第二维色谱柱，甲醇-乙腈（90:10，v/v)为流动相，分离维生素D₂和D₃。结果表明，维生素A在5.0~500.0 ng/mL、维生素D在1.0～100.0 ng/mL、维生素E在50.0～5000.0 ng/mL线性范围内线性良好，相关系数均大于0.9999。维生素A、D₂、D₃和α-、β-、γ-、δ-维生素E的定量限分别为5.11、1.22、1.25、83.82、57.27、56.61、54.82 µg/100 g。加标回收率为91.20％~104.47％，相对标准偏差为0.94%~3.34%。结果表明本文建立的在线固相萃取-二维液相色谱系统，前处理简单、自动化程度高、分析效率高、准确度高、重复性好，适用于儿童配方奶粉中维生素A、D、E的含量测定。
- 在线固相萃取 /
- 二维液相色谱 /
- 在线稀释 /
- 维生素A、D、E /
- 儿童配方奶粉
Abstract: A method was developed for the simultaneous determination of fat-soluble vitamins A, D₂, D₃ and α-, β-, γ-, δ-vitamin E in children's formula milk powder by combining online solid phase extraction (SPE) and two-dimensional liquid chromatography. The saponified samples were directly injected into the constructed system and purified by using the polymeric-based PLRP-S column as the SPE column. Then the target analytes were eluted in back-flush mode from the SPE column by SPE pump and refocused on the head of the first-dimensional column by using online dilution. Vitamins A and four E homologues were separated on a NuovaSil^TM PFP column (4.6 mm×150 mm, 3 μm) as the first-dimensional column using a mobile phase consisting of methanol and 0.1% formic acid in water by gradient elution, and vitamins D₂ and D₃ were separated on a ChromCore PAH column (4.6 mm×150 mm, 3 μm) as the second-dimensional column using a mobile phase consisting of methanol and acetonitrile (90:10, v/v). The results showed good linearity in the range of 5.0~500.0 ng/mL for vitamin A, 1.0~100.0 ng/mL for vitamin D and 50.0~5000.0 ng/mL for vitamin E, and the correlation coefficients were all better than 0.9999. The limits of quantification for vitamins A, D₂, D₃ and α-, β-, γ-, δ-vitamins E were 5.11, 1.22, 1.25, 83.82, 57.27, 56.61, 54.82 µg/100 g, respectively. The spiked recoveries were 91.20%~104.47%, and the relative standard deviations were 0.94%~3.34%. The results show that the method established in this paper has the advantages of simple pretreatment, high automation, high analytical efficiency, high accuracy and good reproducibility, and is suitable for the determination of vitamins A, D and E in children's formula milk powder.
- online solid phase extraction /
- two-dimensional liquid chromatography /
- online dilution /
- vitamins A, D and E /
- children's formula milk powder

HTML全文

图 1 在线SPE-二维液相色谱系统流程图

Figure 1. The scheme of online SPE two-dimensional liquid chromatography

下载: 全尺寸图片幻灯片

图 2 混合标准溶液的第一维色谱图

注：λ=325 nm, 294 nm；V_A：500 ng/mL，V_E：5000 ng/mL。

Figure 2. One-dimensional chromatogram of mixed standard solution

下载: 全尺寸图片幻灯片

图 3 混合标准溶液的第二维色谱图

注：λ=264 nm；V_D：20 ng/mL。

Figure 3. Two-dimensional chromatogram of mixed standard solution

下载: 全尺寸图片幻灯片

图 4 儿童配方奶粉样品的第一维色谱图

注：λ=325 nm, 294 nm。

Figure 4. One-dimensional chromatogram of children's formula milk powder

下载: 全尺寸图片幻灯片

图 5 儿童配方奶粉样品的第二维色谱图

注：λ=264 nm。

Figure 5. Two-dimensional chromatogram of children's formula milk powder

下载: 全尺寸图片幻灯片

表 1 三泵梯度洗脱程序

Table 1. Gradient programs of 3 pumps

SPE泵				第一维泵			第二维泵
时间（min）	A （%）	B （%）	C （%）	时间（min）	A （%）	B （%）	时间（min）	A （%）	B （%）
0	80	0	20	0	20	80	0	90	10
5	80	0	20	11	20	80	45	90	10
5.1	0	90	10	20	87	13
20	0	90	10	28	95	5
20.1	0	100	0	35	95	5
35	0	100	0	35.1	20	80
35.1	80	0	20	45	20	80
45	80	0	20

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表 2 阀切换过程及说明

Table 2. Switching instructions for two-position six-way valve

步骤	阀1切换位置	阀2切换位置	说明
1	1-6	1-6	SPE泵上样，目标物被保留，样品基质和强碱液被淋洗到废液中。
2	1-2	1-6	SPE泵反向冲洗转移目标物至第一维分析柱，同时第一维泵采用高水相进行在线稀释，使目标物在第一维色谱柱柱头聚焦。
3	1-6	1-6	第一维泵分离分析V_A和4种V_E，并完成V_D的净化，SPE泵对SPE柱进行清洗和平衡。
4	1-6	1-2	将V_D馏分切割至捕集柱，阀切换的起始时间为V_D色谱峰起点出峰时间减去0.1 min，阀切换的结束时间为V_D色谱峰终点出峰时间加上0.1 min。
5	1-6	1-6	第二维泵分离分析V_D₂ 和V_D₃。

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表 3 V_A、V_D和V_E的线性回归方程、线性范围、相关系数、检出限和定量限

Table 3. Regression equations, linear ranges, correlation coefficient, LODs and LOQs of V_A, V_D and V_E

组分名	回归方程	线性范围（ng/mL）	线性相关系数	方法检出限（µg/100 g）	方法定量限（µg/100 g）
V_A	y=0.0007x+0.6315	5.0～500.0	0.99998	1.53	5.11
V_D₂	y=0.0009x+0.0099	1.0～100.0	0.99996	0.37	1.22
V_D₃	y=0.0008x+0.2035	1.0～100.0	0.99993	0.38	1.25
α-V_E	y=0.0229x+55.838	50.0～5000.0	0.99993	25.15	83.82
β-V_E	y=0.0139x+33.115	50.0～5000.0	0.99993	17.18	57.27
γ-V_E	y=0.0141x+18.368	50.0～5000.0	0.99995	16.98	56.61
δ-V_E	y=0.0152x+29.373	50.0～5000.0	0.99991	16.45	54.82

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表 4 V_A、V_D和V_E加标回收率与精密度（n=6）

Table 4. Spiked recoveries and RSD of V_A, V_D and V_E (n=6)

组分名	本底值（μg/100 g）	加标量（μg/100 g）	平均值（μg/100 g）	平均加标回收率（%）	RSD （%）
V_A	363.48	10	372.96	94.80	1.32
		400	752.27	97.20	1.61
		800	1199.2	104.47	1.13
V_D₂	ND	2	1.84	92.10	3.34
		10	9.39	93.85	2.41
		20	19.18	95.89	2.36
V_D₃	7.47	2	9.29	91.20	1.65
		10	17.01	95.36	2.25
		20	26.79	96.59	1.52
α-V_E	5250	100	5346.32	96.32	0.94
		4000	9239.33	99.73	1.47
		8000	13579.36	104.12	1.29
β-V_E	240	100	335.54	95.54	2.67
		4000	4146.18	97.65	2.15
		8000	8154.44	98.93	1.15
γ-V_E	2620	100	2717.43	97.43	1.02
		4000	6568.64	98.72	1.21
		8000	10678.71	100.73	1.42
δ-V_E	510	100	607.25	97.25	1.18
		4000	4426.02	97.90	2.09
		8000	8499.06	99.86	1.53
注：ND表示未检出，表5同。

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表 5 实际样品中V_A、V_D和V_E的测定结果（n=3）

Table 5. Determination results of V_A, V_D and V_E in samples (n=3)

样品名称		V_A（µg/100 g）	V_D₃（µg/100 g）	α-V_E（mg/100 g）	β-V_E（mg/100 g）	γ-V_E（mg/100 g）	δ-V_E（mg/100 g）
圣元优强儿童配方奶粉	本方法	363.48±2.43	7.47±0.26	5.25±0.08	0.24±0.01	2.62±0.03	0.51±0.03
	国标	355.59±3.57	7.33±0.42	5.07±0.12	0.23±0.02	2.53±0.05	0.48±0.04
	相对偏差（%）	2.19	1.89	3.49	6.45	3.50	6.06
伊利金领冠儿童配方奶粉	本方法	405.67±1.38	6.54±0.07	1.63±0.02	ND	0.78±0.01	0.16±0.01
	国标	412.55±3.06	6.69±0.09	1.71±0.03	ND	0.83±0.02	0.17±0.01
	相对偏差（%）	1.68	2.27	4.79		6.21	7.81
合生元阿尔法星学龄前儿童配方奶粉	本方法	289.23±0.34	4.78±0.02	1.37±0.01	ND	0.53±0.02	0.11±0.01
	国标	283.37±2.16	4.57±0.03	1.32±0.02	ND	0.49±0.02	0.10±0.01
	相对偏差（%）	2.05	4.49	3.72		7.84	6.57
爱他美学龄前儿童成长奶粉	本方法	283.48±1.27	4.82±0.03	2.83±0.05	ND	1.02±0.03	0.32±0.02
	国标	275.50±1.89	4.65±0.05	2.72±0.05	ND	0.99±0.03	0.31±0.02
	相对偏差（%）	2.86	3.59	3.96		3.49	4.47
美赞臣安儿健儿童配方奶粉	本方法	515.42±2.17	6.26±0.12	4.27±0.13	0.13±0.01	1.85±0.06	0.14±0.01
	国标	498.49±3.76	6.13±0.13	4.18±0.13	0.12±0.02	1.76±0.07	0.13±0.01
	相对偏差（%）	3.34	2.10	2.13	7.17	5.16	5.13
卡洛塔妮儿童配方羊奶粉	本方法	558.63±1.36	8.05±0.21	4.82±0.11	ND	1.65±0.03	0.12±0.01
	国标	547.77±3.82	7.68±0.23	4.68±0.12	ND	1.56±0.03	0.11±0.01
	相对偏差（%）	1.96	4.70	2.95		5.93	6.90

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