超高效液相色谱-四极杆-飞行时间质谱法测定防脱发化妆品中19种非法添加化学成分

doi:10.3724/SP.J.1123.2021.08019

摘要/Abstract

摘要：

建立了超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)法,结合高分辨质谱数据库,用于快速筛查及定量分析防脱发化妆品中19种非法添加化学成分。实验比较了提取溶剂的影响,优化了色谱条件和质谱条件。采用ACQUITY UPLC BEH C18色谱柱(100 mm×2.1 mm, 1.7 μm)进行色谱分离,以乙腈和2 mmol/L甲酸铵水溶液(含0.05%甲酸)为流动相进行梯度洗脱,运行时间为20 min。在电喷雾电离源、正离子扫描模式下,采用MS^E模式采集19种化合物的质谱信息。根据标准品的色谱保留时间、母离子和碎片离子精确质量数及丰度比,在UNIFI软件中构建了非法添加化学成分的筛查数据库。结果表明,19种化合物线性关系良好,检出限为0.025~0.05 μg/g,定量限为0.075~0.15 μg/g。以溶液型防脱育发剂和膏霜型防脱洗发水两种常见防脱发化妆品为代表性基质进行加标回收试验,在不同的添加水平下,19种化合物的平均回收率为68.6%~118.8%,相对标准偏差(RSD)为0.3%~10.3%。通过对比保留时间以及母离子和碎片离子的精确质量数,该方法可用于77批样品的筛查。最终两批育发剂产品中均确证检出米诺地尔和非那雄胺两种非法添加化学成分,并对米诺地尔的裂解途径进行了推测;采用基质匹配外标法定量,结果表明其中米诺地尔含量高达60 mg/g,非那雄胺含量高达0.31 mg/g,说明防脱发类化妆品中存在多种化学组分同时添加的情况,且添加的药物含量非常高,具有很高的安全风险。该方法操作简便、灵敏度高、重现性好,能够同时用于防脱发化妆品中多种类型非法添加化学药物的筛查和定量分析,为防脱发化妆品的安全监管提供了强有力的技术手段。

关键词: 超高效液相色谱, 高分辨质谱, 非法添加, 防脱发化妆品, 筛查

Abstract:

Cosmetic products for hair loss prevention are often mixed with prohibited substances such as hormones, antibiotics, and forbidden pharmacologically active substances. Although drugs increase the efficacy of cosmetic products, they cause skin irritation and allergic reactions, upon long-term exposure. Given the increasing number of hair loss prevention cosmetics on the market, the need to guarantee product safety calls for efficient and reliable methods to identify illegal ingredients in these products. Chromatography combined with high-resolution mass spectrometry offers the advantages of high resolution and high throughout, thus being a powerful technique for simultaneously detecting illegal ingredients in cosmetics. In this study, an ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) method for detecting 19 illegal chemical components was established. Combined with the scientific database, a screening platform for hair loss prevention cosmetics was constructed. The effect of extraction solvent was investigated. The chromatographic and mass spectrometry conditions were optimized. Under the optimal conditions, separation was achieved within 20 min on an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm). Acetonitrile and 2 mmol/L ammonium formate solution containing 0.05% formic acid were used as mobile phases for gradient elution. The 19 compounds were detected by positive ion electrospray ionization (ESI) in the MS^E mode. The chromatographic retention time, precursor ions, product ions of the target analytes, and abundance ratio were collected to construct a screening database with UNIFI software. The 19 compounds were well separated, with good linearity. The limits of detection (LODs) and limits of quantification (LOQs) were 0.025-0.05 μg/g and 0.075-0.15 μg/g, respectively. Hair lotion and shampoo, which are commonly marketed as hair loss prevention cosmetics, were selected as the respective matrices for the recovery experiment. The average recoveries of the 19 compounds ranged from 68.6% to 118.6%, and the relative standard deviations (RSDs) were 0.3%-10.3%. Then, 77 batches of cosmetic samples were detected and screened under the same conditions. The TOF-MS information, including the retention time, ion addition mode, mass-to-charge ratio of the parent ions and fragment ions, as well as the abundance ratio, were compared between the cosmetic samples and the standard MS information with UNIFI software. Finally, two batches of samples that were illegally adulterated with minoxidil and finasteride were identified. The ESI fragmentation pathway of the product ions from minoxidil was also proposed. The matrix matching external standard method was used to determine the amounts of minoxidil and finasteride in the two batches of hair lotion, and they were as high as 60 mg/g and 0.31 mg/g, respectively. This result revealed that multiple chemical components were simultaneously added to hair loss prevention cosmetics. Furthermore, the amount of the illegally added drug was very high, indicating high safety risk for consumers using such cosmetics. The present method has the advantages of simple operation, high sensitivity, and good reproducibility. It can be used for rapid screening and simultaneous quantitative analysis of various illegal chemicals in hair loss prevention cosmetics.

Key words: ultra-performance liquid chromatography (UPLC), high resolution mass spectrometry (HRMS), illegally added, hair loss prevention cosmetics, screening

中图分类号:

O658

董亚蕾, 牛水蛟, 乔亚森, 黄传峰, 王海燕, 孙磊. 超高效液相色谱-四极杆-飞行时间质谱法测定防脱发化妆品中19种非法添加化学成分[J]. 色谱, 2022, 40(4): 343-353.

DONG Yalei, NIU Shuijiao, QIAO Yasen, HUANG Chuanfeng, WANG Haiyan, SUN Lei. Determination of 19 illegally added chemical ingredients in hair loss prevention cosmetics by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry[J]. Chinese Journal of Chromatography, 2022, 40(4): 343-353.

图/表 12

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No.	Compound	Chinese name	Chemical formula	Precursor ion	t_R/ min	Observed (m/z)	Fragment 1 (m/z)	Fragment 2 (m/z)
1	bifonazole (B)	联苯苄唑	C₂₂H₁₈N₂	[M+H]⁺	7.15	311.1542	165.0699	243.1168
2	clotrimazole (C)	克霉唑	C₂₂H₁₇ClN₂	[M-C₃H₃N₂]⁺	6.98	277.0777	165.0699	241.1012
3	dutasteride (D)	度他雄胺	C₂₇H₃₀F₆N₂O₂	[M+H]⁺	11.59	529.2293	461.2033	95.0837
4	econazole (ECZ)	益康唑	C₁₈H₁₅Cl₃N₂O	[M+H]⁺	8.59	381.0322	125.0153	89.0386
5	epristeride (EPS)	依立雄胺	C₂₅H₃₇NO₃	[M+H]⁺	10.66	400.2851	344.2227	72.0425
6	finasteride (FNS)	非那雄胺	C₂₃H₃₆N₂O₂	[M+H]⁺	7.22	373.2852	305.2603	317.2237
7	fluconazole (FCZ)	氟康唑	C₁₃H₁₂F₂N₆O	[M+H]⁺	2.8	307.1111	220.0681	238.0786
8	griseofulvin (G)	灰黄霉素	C₁₇H₁₇ClO₆	[M+H]⁺	6.03	353.0781	285.0524	165.0546
9	hydrocortisone (H)	氢化可的松	C₂₁H₃₀O₅	[M+H]⁺	3.77	363.2161	109.0648	119.0491
10	ketoconazole (K)	酮康唑	C₂₆H₂₈Cl₂N₄O₄	[M+H]⁺	5.54	531.1566	489.1455	495.1794
11	methyltestosterone (MT)	甲睾酮	C₂₀H₃₀O₂	[M+H]⁺	7.23	303.2313	285.2213	109.0648
12	miconazole (MCZ)	咪康唑	C₁₈H₁₄Cl₄N₂O	[M+H]⁺	9.71	416.9934	158.9763	122.9996
13	minoxidil (MXD)	米诺地尔	C₉H₁₅N₅O	[M+H]⁺	2.63	210.1355	164.0933	193.1327
14	naftifine (N)	萘替芬	C₂₁H₂₁N	[M+H]⁺	5.68	288.1743	141.0699	117.0699
15	progesterone (P)	孕酮	C₂₁H₃₀O₂	[M+H]⁺	9.95	315.2326	109.0648	97.0648
16	spironolactone (S)	螺内酯	C₂₄H₃₂O₄S	[M-CH₃OS]⁺	7.91	341.2112	107.0853	181.1108
17	testosterone (T)	睾酮	C₁₉H₂₈O₂	[M+H]⁺	6.45	289.2164	97.0648	109.0648
18	triamcinolone acetonide (TA)	曲安奈德	C₂₄H₃₁FO₆	[M+H]⁺	5.09	435.2174	415.2115	397.2010
19	triamcinolone acetonide	醋酸曲安奈德	C₂₅H₃₁FO₈	[M+H]⁺	8.28	477.2284	457.2221	321.1485
	acetate (TAA)

No.	Compound	Chinese name	Chemical formula	Precursor ion	t_R/ min	Observed (m/z)	Fragment 1 (m/z)	Fragment 2 (m/z)
1	bifonazole (B)	联苯苄唑	C₂₂H₁₈N₂	[M+H]⁺	7.15	311.1542	165.0699	243.1168
2	clotrimazole (C)	克霉唑	C₂₂H₁₇ClN₂	[M-C₃H₃N₂]⁺	6.98	277.0777	165.0699	241.1012
3	dutasteride (D)	度他雄胺	C₂₇H₃₀F₆N₂O₂	[M+H]⁺	11.59	529.2293	461.2033	95.0837
4	econazole (ECZ)	益康唑	C₁₈H₁₅Cl₃N₂O	[M+H]⁺	8.59	381.0322	125.0153	89.0386
5	epristeride (EPS)	依立雄胺	C₂₅H₃₇NO₃	[M+H]⁺	10.66	400.2851	344.2227	72.0425
6	finasteride (FNS)	非那雄胺	C₂₃H₃₆N₂O₂	[M+H]⁺	7.22	373.2852	305.2603	317.2237
7	fluconazole (FCZ)	氟康唑	C₁₃H₁₂F₂N₆O	[M+H]⁺	2.8	307.1111	220.0681	238.0786
8	griseofulvin (G)	灰黄霉素	C₁₇H₁₇ClO₆	[M+H]⁺	6.03	353.0781	285.0524	165.0546
9	hydrocortisone (H)	氢化可的松	C₂₁H₃₀O₅	[M+H]⁺	3.77	363.2161	109.0648	119.0491
10	ketoconazole (K)	酮康唑	C₂₆H₂₈Cl₂N₄O₄	[M+H]⁺	5.54	531.1566	489.1455	495.1794
11	methyltestosterone (MT)	甲睾酮	C₂₀H₃₀O₂	[M+H]⁺	7.23	303.2313	285.2213	109.0648
12	miconazole (MCZ)	咪康唑	C₁₈H₁₄Cl₄N₂O	[M+H]⁺	9.71	416.9934	158.9763	122.9996
13	minoxidil (MXD)	米诺地尔	C₉H₁₅N₅O	[M+H]⁺	2.63	210.1355	164.0933	193.1327
14	naftifine (N)	萘替芬	C₂₁H₂₁N	[M+H]⁺	5.68	288.1743	141.0699	117.0699
15	progesterone (P)	孕酮	C₂₁H₃₀O₂	[M+H]⁺	9.95	315.2326	109.0648	97.0648
16	spironolactone (S)	螺内酯	C₂₄H₃₂O₄S	[M-CH₃OS]⁺	7.91	341.2112	107.0853	181.1108
17	testosterone (T)	睾酮	C₁₉H₂₈O₂	[M+H]⁺	6.45	289.2164	97.0648	109.0648
18	triamcinolone acetonide (TA)	曲安奈德	C₂₄H₃₁FO₆	[M+H]⁺	5.09	435.2174	415.2115	397.2010
19	triamcinolone acetonide	醋酸曲安奈德	C₂₅H₃₁FO₈	[M+H]⁺	8.28	477.2284	457.2221	321.1485
	acetate (TAA)

No.	Compound	Linear range/(μg/L)	R²	LOD/(μg/g)	LOQ/(μg/g)	RSD/%
1	bifonazole	5-50	0.9950	0.050	0.15	3.9
2	clotrimazole	2-50	0.9950	0.025	0.075	1.0
3	dutasteride	2-50	0.9995	0.025	0.075	3.2
4	econazole	2-50	0.9995	0.025	0.075	1.3
5	epristeride	2-50	1.000	0.025	0.075	3.6
6	finasteride	2-50	0.9991	0.025	0.075	1.1
7	fluconazole	2-100	0.9999	0.035	0.10	4.5
8	griseofulvin	2-100	0.9997	0.025	0.075	0.5
9	hydrocortisone	2-50	0.9999	0.025	0.075	3.5
10	ketoconazole	2-100	0.9991	0.025	0.075	2.3
11	methyltestosterone	2-50	0.9999	0.025	0.075	1.3
12	miconazole	2-100	1.000	0.025	0.075	2.2
13	minoxidil	2-100	0.9994	0.035	0.10	8.2
14	naftifine	2-100	0.9997	0.025	0.075	1.8
15	progesterone	2-100	0.9998	0.025	0.075	4.1
16	spironolactone	2-100	0.9991	0.035	0.10	5.9
17	testosterone	2-100	1.000	0.025	0.075	1.6
18	triamcinolone acetonide	2-100	0.9986	0.025	0.075	4.1
19	triamcinolone acetonide acetate	2-100	0.9974	0.025	0.075	4.6

No.	Compound	Linear range/(μg/L)	R²	LOD/(μg/g)	LOQ/(μg/g)	RSD/%
1	bifonazole	5-50	0.9950	0.050	0.15	3.9
2	clotrimazole	2-50	0.9950	0.025	0.075	1.0
3	dutasteride	2-50	0.9995	0.025	0.075	3.2
4	econazole	2-50	0.9995	0.025	0.075	1.3
5	epristeride	2-50	1.000	0.025	0.075	3.6
6	finasteride	2-50	0.9991	0.025	0.075	1.1
7	fluconazole	2-100	0.9999	0.035	0.10	4.5
8	griseofulvin	2-100	0.9997	0.025	0.075	0.5
9	hydrocortisone	2-50	0.9999	0.025	0.075	3.5
10	ketoconazole	2-100	0.9991	0.025	0.075	2.3
11	methyltestosterone	2-50	0.9999	0.025	0.075	1.3
12	miconazole	2-100	1.000	0.025	0.075	2.2
13	minoxidil	2-100	0.9994	0.035	0.10	8.2
14	naftifine	2-100	0.9997	0.025	0.075	1.8
15	progesterone	2-100	0.9998	0.025	0.075	4.1
16	spironolactone	2-100	0.9991	0.035	0.10	5.9
17	testosterone	2-100	1.000	0.025	0.075	1.6
18	triamcinolone acetonide	2-100	0.9986	0.025	0.075	4.1
19	triamcinolone acetonide acetate	2-100	0.9974	0.025	0.075	4.6

No.	Compound	Spiked/(μg/L)	Hair lotion matrix		Shampoo matrix
No.	Compound	Spiked/(μg/L)	Recovery/%	RSD/%	Recovery/%	RSD/%
1	bifonazole	13.6	76.7	8.1	83.2	6.2
		27.2	86.2	3.6	77.4	2.1
2	clotrimazole	5.6	109.8	3.3	74.0	3.2
		11.2	110.7	0.9	87.4	5.7
3	dutasteride	10.1	71.9	4.8	82.5	4.2
		20.2	77.5	4.1	113.0	2.0
4	econazole	15.1	111.7	2.5	106.8	2.4
		30.3	107.6	0.7	76.9	9.8
5	epristeride	8.0	95.8	7.3	117.4	7.3
		16.0	117.5	3.4	81.5	7.3
6	finasteride	10.1	95.0	3.3	68.6	2.6
		20.2	93.8	0.3	99.8	3.2
7	fluconazole	6.8	101.2	5.5	71.9	2.2
		13.6	97.3	1.8	77.7	5.1
8	griseofulvin	6.2	88.6	2.5	94.4	0.9
		12.4	82.7	1.0	88.5	2.2
9	hydrocortisone	3.9	82.1	2.2	96.3	3.8
		7.7	118.1	5.2	114.1	8.5
10	ketoconazole	9.2	88.4	4.2	84.0	3.0
		18.4	83.8	0.6	94.4	4.6
11	methyltestosterone	9.8	116.9	6.1	84.4	3.4
		19.6	113.0	1.7	104.8	7.9
No.	Compound	Spiked/(μg/L)	Hair lotion matrix		Shampoo matrix
No.	Compound	Spiked/(μg/L)	Recovery/%	RSD/%	Recovery/%	RSD/%
12	miconazole	7.9	118.8	3.6	105.5	6.2
		15.8	113.8	0.6	89.1	5.0
13	minoxidil	4.9	113.7	5.0	78.9	10.3
		9.8	105.8	0.7	93.2	3.7
14	naftifine	4.3	103.7	2.4	74.1	3.2
		8.6	97.6	0.7	108.9	4.1
15	progesterone	9.7	116.3	6.3	95.5	7.2
		19.5	104.8	1.7	89.2	1.1
16	spironolactone	10.5	74.4	4.9	73.9	6.1
		21.1	97.7	1.0	80.8	9.6
17	testosterone	10.5	112.6	6.2	85.1	4.1
		21.1	104.1	1.2	114.7	6.0
18	triamcinolone acetonide	10.8	88.8	9.0	83.1	5.7
		21.5	100.4	3.0	100.0	6.5
19	triamcinolone acetonide acetate	4.9	79.2	9.3	77.9	0.3
		9.8	109.1	2.7	115.9	2.7