超高效液相色谱-串联质谱法测定鸡肉中4种蛋白酶抑制剂

doi:10.3724/SP.J.1123.2019.04047

摘要/Abstract

摘要：

建立了鸡肉中4种蛋白酶抑制剂（沙奎那韦、利托那韦、奈非那韦、茚地那韦）的超高效液相色谱-串联质谱（UPLC-MS/MS）检测方法。样品经30%（v/v）乙腈水溶液（含1%（v/v）三氯乙酸）振荡提取、混合型阳离子交换MCX柱净化，采用Luna^® C8色谱柱（150 mm×2 mm，3 μm），以0.2%（v/v）甲酸水溶液（含5 mmol/L乙酸铵）和乙腈为流动相进行梯度洗脱，采用电喷雾电离（ESI⁺）源和多反应监测（MRM）模式检测。结果表明，在0.1~20.0 μg/L范围内，4种目标化合物呈良好的线性关系，相关系数（r²）大于0.99，方法的定量限（S/N=10）为0.20~0.90 μg/kg；鸡肉组织中4种目标化合物在1.0、2.0和10.0 μg/kg 3个水平下的平均加标回收率为69.0%~106.0%，日内和日间相对标准偏差（RSD）为2.2%~13.8%（n=6）和3.6%~14.6%（n=3）。该法简单、高效、灵敏、准确，可用于鸡肉中沙奎那韦、利托那韦、奈非那韦、茚地那韦残留量的测定。

关键词: 超高效液相色谱-串联质谱, 固相萃取, 沙奎那韦, 利托那韦, 奈非那韦, 茚地那韦, 鸡肉

Abstract:

A method was developed for the determination of four protease inhibitors (saquinavir, ritonavir, nelfinavir and indinavir) in chicken using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted by shaking with 30% (v/v) acetonitrile aqueous solution (containing 1% (v/v) trichloroacetic acid), and purified by using mixed-mode cationic-exchanger (MCX) cartridges. The samples were separated on a Luna^® C8 column (150 mm×2 mm, 3 μm) using 0.2% (v/v) formic acid aqueous solution (containing 5 mmol/L ammonium acetate) and acetonitrile as the mobile phases with gradient elution. The determination was carried out by using an electrospray ion source in the positive and multiple-reaction monitoring (MRM) modes. The calibration curves showed good linearities in the range of 0.1-20.0 μg/L, and the correlation coefficients (r²) were greater than 0.99. The limits of quantification (LOQs, S/N=10) of the four protease inhibitors varied from 0.20 μg/kg to 0.90 μg/kg. At the spiked levels of 1.0, 2.0, and 10.0 μg/kg, the average recoveries of the four protease inhibitors were ranging from 69.0% to 106.0%. The intra-day and inter-day relative standard deviations (RSDs) were 2.2%-13.8% (n=6) and 3.6%-14.6% (n=3), respectively. The method is simple, efficient, sensitive and accurate, and it can be used to detect residues of saquinavir, ritonavir, nelfinavir and indinavir in chicken.

Key words: ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), solid phase extraction (SPE), saquinavir, ritonavir, nelfinavir, indinavir, chicken

吕佳乐, 刘正才, 姚闽娜, 林元地. 超高效液相色谱-串联质谱法测定鸡肉中4种蛋白酶抑制剂[J]. 色谱, 2020, 38(2): 212-217.

Lü Jiale, LIU Zhengcai, YAO Minna, LIN Yuandi. Determination of four protease inhibitors in chicken by ultra performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(2): 212-217.

图/表 7

表1 4种蛋白酶抑制剂的主要质谱参数

Table 1 Main MS parameters of the four protease inhibitors

No.	Compound	Retention time/min	Parent ion (m/z)	Daughter ions (m/z)	Collision energies/V	Declustering potential/V
* Quantitative ion.
1	saquinavir	4.27	671.4	570.5^*, 416.2, 367.2	45, 51, 43	140
2	ritonavir	5.24	721.5	426.2, 296.2^*, 268.2	30, 30, 36	120
3	nelfinavir	4.29	568.6	467.2, 330.2^*, 239.2	36, 39, 39	130
4	indinavir	3.87	614.6	465.3, 421.1^*, 364.2	32, 43, 40	130

图1 4种蛋白酶抑制剂的二级离子碎片质谱图及其主要裂解方式

Fig. 1 Secondary ion fragment mass spectra of the four protease inhibitors and their main fragmentation pathways

图2 不同提取溶剂对4种蛋白酶抑制剂回收率的影响(n=3)

Fig. 2 Influence of different extraction solvents on the recoveries of the four protease inhibitors (n=3)

图3 4种目标化合物在固相萃取柱上的洗脱曲线

Fig. 3 Elution curves of the four target compounds on SPE column

表2 4种蛋白酶抑制剂的线性范围、线性方程、相关系数、检出限和定量限

Table 2 Linear ranges, linear equations, correlation coefficients (r2), limits of detection (LODs) and limits of quantification (LOQs) of the four protease inhibitors

Compound	Linear range/(μg/L)	Linear equation	r²	LOD/(μg/kg)	LOQ/(μg/kg)
y: peak area; x: mass concentration, μ g/L.
Saquinavir	0.2-20.0	y=4.81×10³x+6.71×10²	0.9998	0.30	0.90
Ritonavir	0.1-20.0	y=2.53×10⁴x-1.01×10⁴	0.9987	0.08	0.30
Nelfinavir	0.1-20.0	y=2.48×10⁴x+6.89×10²	0.9998	0.06	0.20
Indinavir	0.2-20.0	y=7.45×10³x+3.75×10²	0.9999	0.20	0.50

图4 (a) 空白鸡肉样品和(b)加标鸡肉样品(1.0 μ g/kg)中4种目标化合物的MRM色谱图

Fig. 4 MRM chromatograms of the four protease inhibitors in (a) the blank chicken and (b) spiked chicken samples (1.0 μ g/kg)

表3 鸡肉中4种蛋白酶抑制剂的加标回收率和日内、日间精密度

Table 3 Spiked recoveries and intra-day and inter-day precisions of the four protease inhibitors in chicken samples

Compound	Spiked/(μg/kg)	Found/(μg/kg)	Recovery/%	RSDs/%
Compound	Spiked/(μg/kg)	Found/(μg/kg)	Recovery/%	Intra-day (n=6)	Inter-day (n=3)
Saquinavir	1.0	0.92	92.0	13.8	13.2
	2.0	2.12	106.0	8.2	6.6
	10.0	10.23	102.3	7.6	7.5
Ritonavir	1.0	0.81	81.0	5.6	14.6
	2.0	1.70	85.0	5.2	4.4
	10.0	8.83	88.3	3.4	6.1
Nelfinavir	1.0	0.69	69.0	11.4	10.2
	2.0	1.55	77.5	3.9	3.6
	10.0	7.78	77.8	2.2	3.8
Indinavir	1.0	0.88	88.0	6.2	5.3
	2.0	1.77	88.5	6.6	6.2
	10.0	8.57	85.7	4.8	4.6

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