超高效液相色谱-Orbitrap高分辨质谱用于减肥和壮阳类保健食品中32种非法添加药物的快速筛查和确证

doi:10.3724/SP.J.1123.2021.12009

摘要/Abstract

摘要：

建立了基于超高效液相色谱-Orbitrap高分辨质谱的快速筛查及确证减肥和壮阳类保健食品中32种非法添加药物的分析方法,并总结了数据库建立和应用的相关要点。研究对象聚焦于非法添加药物的衍生物,在对比正负离子模式下各化合物响应强度的基础上建立了高分辨质谱信息库,对提取溶剂、色谱柱温度等实验条件进行了详细探究,尽可能给出了较宽的标准曲线线性范围。使用Hypersil gold vanquish色谱柱(100 mm×2.1 mm, 1.9 μm),梯度洗脱,流量0.3 mL/min,正、负离子全扫描/数据依赖的二级扫描模式,在17 min内完成32种目标化合物的数据采集,通过TraceFinder软件进行快速定性筛查和定量。结果显示在17 min内32种化合物能得到较好分离;2种基质加标溶液中32种化合物的一级质谱离子精确质量数的实测值与理论值均在5×10^-6误差之内,二级碎片离子质量数的实测值与理论值均在1×10^-5误差之内;方法学验证结果表明,所有化合物均显示出优异的线性关系,相关系数(r²)均大于0.99;固体基质中除达泊西汀、羟基硫代豪莫西地那非、硫代豪莫西地那非、硫代西地那非、去甲基硫代西地那非的回收率较低外,其余27种化合物的回收率为50.5%~84.5%,相对标准偏差(RSD)为1.2%~13%,液体基质中32种化合物的回收率范围为60.4%~109.3%, RSD为0.77%~8.2%;在48份减肥及壮阳类保健食品中检出1份阳性样品,检出率2.08%。该方法操作简单,结果定性准确,可用于减肥及壮阳类保健食品中32种非法添加药物的快速筛查及确证。

关键词: 超高效液相色谱, 高分辨质谱, 非法添加药物, 保健食品, 快速筛查

Abstract:

A novel method based on ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) was developed for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods. In addition, the key points of the database establishment and application are summarized. This research focused on the derivatives of illegally added drugs. An HRMS database was established by comparing the response intensity of each compound in the positive and negative modes. The experimental conditions such as the type of extraction solvent and chromatographic column temperature were explored in detail. The analytes were separated on a Hypersil gold vanquish column (100 mm×2.1 mm, 1.9 μm) by gradient elution with acetonitrile/water (containing 0.1%(v/v) formic acid) as the mobile phase at a flow rate of 0.3 mL/min. Positive and negative ion full scanning/data-dependent secondary scanning mode was used to collect the 32 target compounds within 17 min, and TraceFinder software was used to screen the fragment ions. All the 32 compounds could be well separated within 17 min. The measured and theoretical values of the exact mass of the 32 compounds in the two matrix-spiked solutions were within an error of 5×10^-6, and the MS² fragment ions were within an error of 1×10^-5. All the compounds showed an excellent linear relationship, with correlation coefficients (r²) above 0.99. Except dapoxetine, hydroxythiohomo sildenafil, thiohomo sildenafil, thiosildenafil, desmethyl thiosildenafil, the recoveries ranged from 50.5% to 84.5% in the solid matrix, with the relative standard deviations (RSDs) ranging from 1.2% to 13%. The recoveries were 60.4% to 109.3% in the liquid matrix, with the RSDs ranging from 0.77% to 8.2%. The matrix effect (ME) values of the 32 compounds ranged from 0.61 to 0.95 in the solid matrix and from 0.73 to 1.09 in the liquid matrix. Thiohomo sildenafil, desmethyl thiosildenafil, and chlorpretadalafil exhibited strong matrix inhibitory effects in the solid matrix. Therefore, solid and liquid negative matrix extracts were used to prepare a series of mixed standard solutions in order to reduce the ME values. The limits of detection (LODs) were 0.02 mg/kg for the 32 drugs in the liquid sample and 0.02 mg/kg for 29 compounds in the solid sample; the LODs for chlorothalidone, udenafil, and desmethyl thiosildenafil in the solid sample were 0.04 mg/kg. When the retention time in the self-built database matches the sample collection method, it should be used as one of the screening conditions. As for the selection of the matching mode, if the identify mode is selected, the retention time is a necessary condition for compound confirmation. When the retention time does not meet the requirements, subsequent screening of the fragment ions and isotope abundance ratios will not be performed. If the confirm mode is selected, the retention time is the optional condition for compound confirmation. When the retention time does not meet these requirements, subsequent matching of other conditions such as fragment ions and isotope information is required. Isotope information is very important in HRMS and is an effective supplement to the first-order extracted mass. Therefore, its use is recommended, but the isotope abundance ratio will be even lower when the target content is very low in the complex matrix, which may affect isotope matching. In addition, if the fragment ions are not detected in the screening results of the TraceFinder software but can be extracted in the data browser, their intensity threshold in the screening conditions can be further reduced to find the corresponding fragment ions. One positive sample was detected among 48 healthy food samples, with a detection rate of 2.08%. This method has the advantages of simple operation and high accuracy. It can be used for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods.

Key words: ultra-high performance liquid chromatography (UHPLC), high-resolution mass spectrometry (HRMS), illegally added drugs, health foods, rapid screening

中图分类号:

O658

徐红斌, 张申平, 杜茹芸, 周静, 翁史昱. 超高效液相色谱-Orbitrap高分辨质谱用于减肥和壮阳类保健食品中32种非法添加药物的快速筛查和确证[J]. 色谱, 2022, 40(6): 531-540.

XU Hongbin, ZHANG Shenping, DU Ruyun, ZHOU Jing, WENG Shiyu. Ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry for rapid screening and identification of 32 illegally added drugs in slimming and anti-impotence health foods[J]. Chinese Journal of Chromatography, 2022, 40(6): 531-540.

图/表 6

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No.	Compound	Formula	Measured mass (m/z)	Fragments (m/z)	t_R/min	Adduct
1	avanafil (阿伐那非)	C₂₃H₂₆ClN₇O₃	484.18585	375.12159, 233.10332	6.00	[M+H]⁺
2	acetilacid (那非乙酰酸)	C₁₈H₂₀N₄O₄	357.15515	329.12390, 300.08511	6.85	[M+H]⁺
3	dapoxetine (达泊西汀)	C₂₁H₂₃NO	306.18439	261.12695, 183.08032	6.67	[M+H]⁺
4	xanthoanthrafil (苯酰胺那非)	C₁₉H₂₃N₃O₆	390.16553	151.07516, 135.04373	6.95	[M+H]⁺
5	piperiacetidenafil (苯噻啶红地那非)	C₂₄H₃₁N₅O₃	438.24939	297.12422, 341.16089	5.93	[M+H]⁺
6	benzocaine (苯佐卡因)	C₉H₁₁NO₂	166.08601	138.05492, 120.04429	6.31	[M+H]⁺
7	bendroflumethiazide (苄氟噻嗪)	C₁₅H₁₄F₃N₃O₄S₂	420.03149	289.04599, 327.96851	7.13	[M-H]^-
8	yohimbine (育亨宾)	C₂₁H₂₆N₂O₃	355.20090	212.12802, 144.08075	5.43	[M+H]⁺
9	hydroxyacetildenafil (羟基红地那非)	C₂₅H₃₄N₆O₄	483.27103	297.13419, 127.08654	5.73	[M+H]⁺
10	hydroxythiohomo sildenafil (羟基硫代豪莫西地那非)	C₂₃H₃₂N₆O₄S₂	521.19958	299.09589, 327.12759	6.74	[M+H]⁺
11	hydroxyvardenafil (羟基伐地那非)	C₂₃H₃₂N₆O₅S	505.22235	344.14746, 169.09691	5.73	[M+H]⁺
12	thiohomo sildenafil (硫代豪莫西地那非)	C₂₃H₃₂N₆O₃S₂	505.20401	299.09607, 113.10741	6.92	[M+H]⁺
13	thiosildenafil (硫代西地那非)	C₂₂H₃₀N₆O₃S₂	491.18918	341.14389, 407.12027	6.82	[M+H]⁺
14	N-desmethylsibutramine hydrochloride	C₁₆H₂₄ClN·HCl	266.16666	125.01524, 139.03091	6.72	[M+H]⁺
	(盐酸N-单去甲基西布曲明)
15	N,N-didesmethylsibutramine hydrochloride	C₁₅H₂₂ClN·HCl	252.15092	125.01521, 139.03085	6.69	[M+H]⁺
	(盐酸N,N-双去甲基西布曲明)
16	chlorthalidone (氯噻酮)	C₁₄H₁₁CLN₂O₄S	337.00583	146.02345, 189.97272	5.70	[M-H]^-
17	lorcaserin (氯卡色林)	C₁₁H₁₄ClN	196.08885	179.06218, 167.06212	5.52	[M+H]⁺
18	hydrochlorothiazide (氢氯噻嗪)	C₇H₈ClN₃O₄S₂	295.95804	268.94708, 204.98419	3.06	[M-H]^-
19	torasemide (托拉塞米)	C₁₆H₂₀N₄O₃S	347.11835	262.06564, 195.07954	6.04	[M-H]^-
20	gendenafil (庆地那非)	C₁₉H₂₂N₄O₃	355.17612	327.14514, 285.13467	7.44	[M+H]⁺
21	emodin (大黄素)	C₁₅H₁₀O₅	269.04623	225.05583, 241.05090	8.11	[M-H]^-
22	indapamide (吲达帕胺)	C₁₆H₁₆O₃N₃ClS	366.06686	132.08073, 117.05728	6.75	[M+H]⁺
23	desmethyl sildenafil (去甲基西地那非)	C₂₁H₂₈N₆O₄S	461.19626	283.11868, 255.12376	5.97	[M+H]⁺
24	desmethyl thiosildenafil (去甲基硫代西地那非)	C₂₁H₂₈N₆O₃S₂	477.17288	299.09586, 315.09033	6.79	[M+H]⁺
25	nortadalafil (去甲他达拉非)	C₂₁H₁₇N₃O₄	376.12857	262.08588, 254.09195	6.69	[M+H]⁺
No.	Compound	Formula	Measured mass (m/z)	Fragments (m/z)	t_R/min	Adduct
26	N-desethyl acetildenafil (去乙基红地那非)	C₂₃H₃₀N₆O₃	439.24466	297.13419, 313.12927	5.78	[M+H]⁺
27	N-desethyl vardenafil (去乙基伐地那非)	C₂₁H₂₈N₆O₄S	461.19577	344.14749, 299.11346	5.97	[M+H]⁺
28	rimonabant (利莫那班)	C₂₂H₂₁Cl₃N₄O	463.08517	380.99548, 299.01343	9.08	[M+H]⁺
29	lidocaine (利多卡因)	C₁₄H₂₂N₂O	235.18016	86.09662, 58.06575	4.33	[M+H]⁺
30	chlorpretadalafil (他达拉非甲基氯化物)	C₂₂H₁₉ClN₂O₅	337.00583	146.02345, 189.97272	7.91	[M+H]⁺
31	dimethylsildenafil (二甲基西地那非)	C₂₃H₃₂N₆O₄S	489.22803	283.11920, 344.14856	6.14	[M+H]⁺
32	udenafil (乌地那非)	C₂₅H₃₆N₆O₄S	517.25854	283.11874, 299.11380	6.22	[M+H]⁺

No.	Compound	Formula	Measured mass (m/z)	Fragments (m/z)	t_R/min	Adduct
1	avanafil (阿伐那非)	C₂₃H₂₆ClN₇O₃	484.18585	375.12159, 233.10332	6.00	[M+H]⁺
2	acetilacid (那非乙酰酸)	C₁₈H₂₀N₄O₄	357.15515	329.12390, 300.08511	6.85	[M+H]⁺
3	dapoxetine (达泊西汀)	C₂₁H₂₃NO	306.18439	261.12695, 183.08032	6.67	[M+H]⁺
4	xanthoanthrafil (苯酰胺那非)	C₁₉H₂₃N₃O₆	390.16553	151.07516, 135.04373	6.95	[M+H]⁺
5	piperiacetidenafil (苯噻啶红地那非)	C₂₄H₃₁N₅O₃	438.24939	297.12422, 341.16089	5.93	[M+H]⁺
6	benzocaine (苯佐卡因)	C₉H₁₁NO₂	166.08601	138.05492, 120.04429	6.31	[M+H]⁺
7	bendroflumethiazide (苄氟噻嗪)	C₁₅H₁₄F₃N₃O₄S₂	420.03149	289.04599, 327.96851	7.13	[M-H]^-
8	yohimbine (育亨宾)	C₂₁H₂₆N₂O₃	355.20090	212.12802, 144.08075	5.43	[M+H]⁺
9	hydroxyacetildenafil (羟基红地那非)	C₂₅H₃₄N₆O₄	483.27103	297.13419, 127.08654	5.73	[M+H]⁺
10	hydroxythiohomo sildenafil (羟基硫代豪莫西地那非)	C₂₃H₃₂N₆O₄S₂	521.19958	299.09589, 327.12759	6.74	[M+H]⁺
11	hydroxyvardenafil (羟基伐地那非)	C₂₃H₃₂N₆O₅S	505.22235	344.14746, 169.09691	5.73	[M+H]⁺
12	thiohomo sildenafil (硫代豪莫西地那非)	C₂₃H₃₂N₆O₃S₂	505.20401	299.09607, 113.10741	6.92	[M+H]⁺
13	thiosildenafil (硫代西地那非)	C₂₂H₃₀N₆O₃S₂	491.18918	341.14389, 407.12027	6.82	[M+H]⁺
14	N-desmethylsibutramine hydrochloride	C₁₆H₂₄ClN·HCl	266.16666	125.01524, 139.03091	6.72	[M+H]⁺
	(盐酸N-单去甲基西布曲明)
15	N,N-didesmethylsibutramine hydrochloride	C₁₅H₂₂ClN·HCl	252.15092	125.01521, 139.03085	6.69	[M+H]⁺
	(盐酸N,N-双去甲基西布曲明)
16	chlorthalidone (氯噻酮)	C₁₄H₁₁CLN₂O₄S	337.00583	146.02345, 189.97272	5.70	[M-H]^-
17	lorcaserin (氯卡色林)	C₁₁H₁₄ClN	196.08885	179.06218, 167.06212	5.52	[M+H]⁺
18	hydrochlorothiazide (氢氯噻嗪)	C₇H₈ClN₃O₄S₂	295.95804	268.94708, 204.98419	3.06	[M-H]^-
19	torasemide (托拉塞米)	C₁₆H₂₀N₄O₃S	347.11835	262.06564, 195.07954	6.04	[M-H]^-
20	gendenafil (庆地那非)	C₁₉H₂₂N₄O₃	355.17612	327.14514, 285.13467	7.44	[M+H]⁺
21	emodin (大黄素)	C₁₅H₁₀O₅	269.04623	225.05583, 241.05090	8.11	[M-H]^-
22	indapamide (吲达帕胺)	C₁₆H₁₆O₃N₃ClS	366.06686	132.08073, 117.05728	6.75	[M+H]⁺
23	desmethyl sildenafil (去甲基西地那非)	C₂₁H₂₈N₆O₄S	461.19626	283.11868, 255.12376	5.97	[M+H]⁺
24	desmethyl thiosildenafil (去甲基硫代西地那非)	C₂₁H₂₈N₆O₃S₂	477.17288	299.09586, 315.09033	6.79	[M+H]⁺
25	nortadalafil (去甲他达拉非)	C₂₁H₁₇N₃O₄	376.12857	262.08588, 254.09195	6.69	[M+H]⁺
No.	Compound	Formula	Measured mass (m/z)	Fragments (m/z)	t_R/min	Adduct
26	N-desethyl acetildenafil (去乙基红地那非)	C₂₃H₃₀N₆O₃	439.24466	297.13419, 313.12927	5.78	[M+H]⁺
27	N-desethyl vardenafil (去乙基伐地那非)	C₂₁H₂₈N₆O₄S	461.19577	344.14749, 299.11346	5.97	[M+H]⁺
28	rimonabant (利莫那班)	C₂₂H₂₁Cl₃N₄O	463.08517	380.99548, 299.01343	9.08	[M+H]⁺
29	lidocaine (利多卡因)	C₁₄H₂₂N₂O	235.18016	86.09662, 58.06575	4.33	[M+H]⁺
30	chlorpretadalafil (他达拉非甲基氯化物)	C₂₂H₁₉ClN₂O₅	337.00583	146.02345, 189.97272	7.91	[M+H]⁺
31	dimethylsildenafil (二甲基西地那非)	C₂₃H₃₂N₆O₄S	489.22803	283.11920, 344.14856	6.14	[M+H]⁺
32	udenafil (乌地那非)	C₂₅H₃₆N₆O₄S	517.25854	283.11874, 299.11380	6.22	[M+H]⁺

No.	Compound	Solid sample				Liquid sample
No.	Compound	Linear equation	Linear range^*	r²	LOD/ (mg/kg)	Linear equation	Linear range^*	r²	LOD/ (mg/kg)
1	avanafil	y=2.41×10⁶x+4.55×10⁷	5-500	0.9924	0.02	y=2.13×10⁶x+3.66×10⁷	5-500	0.9941	0.02
2	acetilacid	y=2.20×10⁶x+4.16×10⁶	5-200	0.9985	0.02	y=2.03×10⁶x+2.14×10⁷	5-500	0.9970	0.02
3	dapoxetine	y=5.47×10⁶x+4.90×10⁷	5-500	0.9972	0.02	y=5.00×10⁶x+7.34×10⁷	5-500	0.9960	0.02
4	xanthoanthrafil	y=5.76×10⁵x+7.49×10⁶	5-500	0.9917	0.02	y=5.22×10⁵x+4.97×10⁶	5-500	0.9977	0.02
5	piperiacetidenafil	y=3.00×10⁶x+8.73×10⁶	5-200	0.9988	0.02	y=2.81×10⁶x+6.18×10⁷	5-500	0.9922	0.02
6	benzocaine	y=4.12×10⁶x-9.63×10⁶	5-200	0.9914	0.02	y=3.74×10⁶x+3.60×10⁷	5-500	0.9970	0.02
7	bendroflumethiazide	y=1.15×10⁶x+1.90×10⁶	5-500	0.9943	0.02	y=1.01×10⁶x+1.52×10⁷	5-500	0.9951	0.02
8	yohimbine	y=2.81×10⁶x+1.01×10⁶	5-200	0.9952	0.02	y=6.53×10⁶x+3.99×10⁶	5-200	0.9982	0.02
9	hydroxyacetildenafil	y=1.29×10⁶x+1.55×10⁵	5-200	0.9983	0.02	y=9.64×10⁵x+7.38×10⁵	5-200	0.9952	0.02
10	hydroxythiohomo sildenafil	y=1.38×10⁶x+2.81×10⁶	5-200	0.9962	0.02	y=7.16×10⁵x+5.72×10⁶	5-500	0.9978	0.02
11	hydroxyvardenafil	y=3.93×10⁵x+4.73×10⁵	5-200	0.9976	0.02	y=9.49×10⁵x+3.17×10⁵	5-200	0.9932	0.02
12	thiohomo sildenafil	y=2.27×10⁶x+4.87×10⁶	5-200	0.9938	0.02	y=1.35×10⁶x+1.45×10⁷	5-500	0.9942	0.02
13	thiosildenafil	y=2.25×10⁶x-3.53×10⁶	5-200	0.9995	0.02	y=1.03×10⁶x+1.31×10⁷	5-500	0.9943	0.02
14	N-desmethylsibutramine	y=2.82×10⁶x+1.10×10⁶	5-200	0.9975	0.02	y=3.38×10⁶x+1.48×10⁷	5-200	0.9945	0.02
	hydrochloride
15	N,N-didesmethylsibutramine	y=1.27×10⁶x+1.05×10⁶	5-200	0.9982	0.02	y=1.43×10⁶x+5.65×10⁶	5-200	0.9904	0.02
	hydrochloride
16	chlorthalidone	y=2.24×10⁵x+2.05×10⁵	5-200	0.9966	0.04	y=3.20×10⁵x+2.39×10⁶	5-500	0.9983	0.02
17	lorcaserin	y=2.98×10⁶x+3.97×10⁶	5-200	0.9945	0.02	y=3.03×10⁶x+1.11×10⁷	5-200	0.9946	0.02
18	hydrochlorothiazide	y=3.00×10⁵x-1.73×10⁵	5-200	0.9997	0.02	y=2.98×10⁵x+1.04×10⁶	5-200	0.9943	0.02
19	torasemide	y=9.78×10⁵x+2.87×10⁶	5-500	0.9983	0.02	y=8.06×10⁵x+1.05×10⁶	5-500	0.9964	0.02
20	gendenafil	y=3.63×10⁶x+2.27×10⁷	5-500	0.9985	0.02	y=3.29×10⁶x+4.60×10⁷	5-500	0.9949	0.02
21	emodin	y=2.60×10⁶x+2.51×10⁶	5-200	0.9916	0.02	y=2.96×10⁶x+1.20×10⁶	5-200	0.9926	0.02
22	indapamide	y=5.13×10⁵x+6.57×10⁶	5-500	0.9921	0.02	y=4.69×10⁵x+3.35×10⁶	5-500	0.9986	0.02
23	desmethyl sildenafil	y=1.12×10⁶x+1.76×10⁶	5-200	0.9940	0.02	y=8.79×10⁵x+5.67×10⁶	5-200	0.9904	0.02
24	desmethyl thiosildenafil	y=1.02×10⁶x+2.03×10⁶	5-500	0.9985	0.04	y=7.90×10⁵x+7.91×10⁶	5-500	0.9979	0.02
25	nortadalafil	y=4.84×10⁶x-1.01×10⁶	5-500	1	0.02	y=4.35×10⁵x+3.97×10⁶	5-500	0.9974	0.02
26	N-desethyl acetildenafil	y=7.81×10⁵x-4.59×10⁵	5-200	0.9983	0.02	y=7.06×10⁵x+7.71×10⁵	5-200	0.9962	0.02
27	N-desethyl vardenafil	y=6.65×10⁶x+1.03×10⁶	5-200	0.9967	0.02	y=8.87×10⁵x+5.66×10⁶	5-200	0.9904	0.02
28	rimonabant	y=2.46×10⁶x+2.68×10⁶	5-200	0.9953	0.02	y=2.74×10⁶x+2.04×10⁶	5-200	0.9981	0.02
29	lidocaine	y=5.97×10⁶x+1.78×10⁷	5-200	0.9954	0.02	y=5.42×10⁶x+2.67×10⁷	5-200	0.9929	0.02
30	chlorpretadalafil	y=1.81×10⁵x+5.23×10⁴	5-500	0.9998	0.02	y=1.67×10⁵x+8.56×10⁵	5-500	0.9990	0.02
31	dimethylsildenafil	y=1.84×10⁶x+8.15×10⁶	5-200	0.9946	0.02	y=1.16×10⁶x+2.08×10⁷	5-500	0.9917	0.02
32	udenafil	y=4.67×10⁶x+1.09×10⁶	5-200	0.9982	0.04	y=1.51×10⁶x+4.89×10⁷	5-200	0.9940	0.02

No.	Compound	Solid sample				Liquid sample
No.	Compound	Linear equation	Linear range^*	r²	LOD/ (mg/kg)	Linear equation	Linear range^*	r²	LOD/ (mg/kg)
1	avanafil	y=2.41×10⁶x+4.55×10⁷	5-500	0.9924	0.02	y=2.13×10⁶x+3.66×10⁷	5-500	0.9941	0.02
2	acetilacid	y=2.20×10⁶x+4.16×10⁶	5-200	0.9985	0.02	y=2.03×10⁶x+2.14×10⁷	5-500	0.9970	0.02
3	dapoxetine	y=5.47×10⁶x+4.90×10⁷	5-500	0.9972	0.02	y=5.00×10⁶x+7.34×10⁷	5-500	0.9960	0.02
4	xanthoanthrafil	y=5.76×10⁵x+7.49×10⁶	5-500	0.9917	0.02	y=5.22×10⁵x+4.97×10⁶	5-500	0.9977	0.02
5	piperiacetidenafil	y=3.00×10⁶x+8.73×10⁶	5-200	0.9988	0.02	y=2.81×10⁶x+6.18×10⁷	5-500	0.9922	0.02
6	benzocaine	y=4.12×10⁶x-9.63×10⁶	5-200	0.9914	0.02	y=3.74×10⁶x+3.60×10⁷	5-500	0.9970	0.02
7	bendroflumethiazide	y=1.15×10⁶x+1.90×10⁶	5-500	0.9943	0.02	y=1.01×10⁶x+1.52×10⁷	5-500	0.9951	0.02
8	yohimbine	y=2.81×10⁶x+1.01×10⁶	5-200	0.9952	0.02	y=6.53×10⁶x+3.99×10⁶	5-200	0.9982	0.02
9	hydroxyacetildenafil	y=1.29×10⁶x+1.55×10⁵	5-200	0.9983	0.02	y=9.64×10⁵x+7.38×10⁵	5-200	0.9952	0.02
10	hydroxythiohomo sildenafil	y=1.38×10⁶x+2.81×10⁶	5-200	0.9962	0.02	y=7.16×10⁵x+5.72×10⁶	5-500	0.9978	0.02
11	hydroxyvardenafil	y=3.93×10⁵x+4.73×10⁵	5-200	0.9976	0.02	y=9.49×10⁵x+3.17×10⁵	5-200	0.9932	0.02
12	thiohomo sildenafil	y=2.27×10⁶x+4.87×10⁶	5-200	0.9938	0.02	y=1.35×10⁶x+1.45×10⁷	5-500	0.9942	0.02
13	thiosildenafil	y=2.25×10⁶x-3.53×10⁶	5-200	0.9995	0.02	y=1.03×10⁶x+1.31×10⁷	5-500	0.9943	0.02
14	N-desmethylsibutramine	y=2.82×10⁶x+1.10×10⁶	5-200	0.9975	0.02	y=3.38×10⁶x+1.48×10⁷	5-200	0.9945	0.02
	hydrochloride
15	N,N-didesmethylsibutramine	y=1.27×10⁶x+1.05×10⁶	5-200	0.9982	0.02	y=1.43×10⁶x+5.65×10⁶	5-200	0.9904	0.02
	hydrochloride
16	chlorthalidone	y=2.24×10⁵x+2.05×10⁵	5-200	0.9966	0.04	y=3.20×10⁵x+2.39×10⁶	5-500	0.9983	0.02
17	lorcaserin	y=2.98×10⁶x+3.97×10⁶	5-200	0.9945	0.02	y=3.03×10⁶x+1.11×10⁷	5-200	0.9946	0.02
18	hydrochlorothiazide	y=3.00×10⁵x-1.73×10⁵	5-200	0.9997	0.02	y=2.98×10⁵x+1.04×10⁶	5-200	0.9943	0.02
19	torasemide	y=9.78×10⁵x+2.87×10⁶	5-500	0.9983	0.02	y=8.06×10⁵x+1.05×10⁶	5-500	0.9964	0.02
20	gendenafil	y=3.63×10⁶x+2.27×10⁷	5-500	0.9985	0.02	y=3.29×10⁶x+4.60×10⁷	5-500	0.9949	0.02
21	emodin	y=2.60×10⁶x+2.51×10⁶	5-200	0.9916	0.02	y=2.96×10⁶x+1.20×10⁶	5-200	0.9926	0.02
22	indapamide	y=5.13×10⁵x+6.57×10⁶	5-500	0.9921	0.02	y=4.69×10⁵x+3.35×10⁶	5-500	0.9986	0.02
23	desmethyl sildenafil	y=1.12×10⁶x+1.76×10⁶	5-200	0.9940	0.02	y=8.79×10⁵x+5.67×10⁶	5-200	0.9904	0.02
24	desmethyl thiosildenafil	y=1.02×10⁶x+2.03×10⁶	5-500	0.9985	0.04	y=7.90×10⁵x+7.91×10⁶	5-500	0.9979	0.02
25	nortadalafil	y=4.84×10⁶x-1.01×10⁶	5-500	1	0.02	y=4.35×10⁵x+3.97×10⁶	5-500	0.9974	0.02
26	N-desethyl acetildenafil	y=7.81×10⁵x-4.59×10⁵	5-200	0.9983	0.02	y=7.06×10⁵x+7.71×10⁵	5-200	0.9962	0.02
27	N-desethyl vardenafil	y=6.65×10⁶x+1.03×10⁶	5-200	0.9967	0.02	y=8.87×10⁵x+5.66×10⁶	5-200	0.9904	0.02
28	rimonabant	y=2.46×10⁶x+2.68×10⁶	5-200	0.9953	0.02	y=2.74×10⁶x+2.04×10⁶	5-200	0.9981	0.02
29	lidocaine	y=5.97×10⁶x+1.78×10⁷	5-200	0.9954	0.02	y=5.42×10⁶x+2.67×10⁷	5-200	0.9929	0.02
30	chlorpretadalafil	y=1.81×10⁵x+5.23×10⁴	5-500	0.9998	0.02	y=1.67×10⁵x+8.56×10⁵	5-500	0.9990	0.02
31	dimethylsildenafil	y=1.84×10⁶x+8.15×10⁶	5-200	0.9946	0.02	y=1.16×10⁶x+2.08×10⁷	5-500	0.9917	0.02
32	udenafil	y=4.67×10⁶x+1.09×10⁶	5-200	0.9982	0.04	y=1.51×10⁶x+4.89×10⁷	5-200	0.9940	0.02

No.	Compound	Recoveries (RSDs) in solid sample/%			Recoveries (RSDs) in liquid sample/%
No.	Compound	0.2 mg/kg	0.6 mg/kg	1.0 mg/kg	0.2 mg/kg	0.6 mg/kg	1.0 mg/kg
1	avanafil	74.2(2.2)	53.9(5.2)	74.7(10)	72.5(2.5)	85.7(3.4)	86.5(5.2)
2	acetilacid	78.6(1.8)	82.7(2.8)	78.3(5.1)	70.2(3.8)	88.3(2.0)	92.4(2.1)
3	dapoxetine	50.5(1.5)	36.9(3.1)	49.9(4.1)	71.8(2.1)	96.1(2.0)	96.9(3.1)
4	xanthoanthrafil	69.9(4.8)	54.5(3.7)	75.3(4.2)	77.6(3.4)	98.7(2.0)	94.4(1.2)
5	piperiacetidenafil	72.7(2.0)	76.9(2.6)	74.6(7.8)	88.2(4.3)	92.1(5.0)	94.9(6.0)
6	benzocaine	84.3(12)	73.4(5.1)	70.0(3.0)	78.5(6.4)	91.2(8.2)	109.3(5.2)
7	bendroflumethiazide	77.5(4.0)	81.2(3.2)	73.1(3.7)	75.0(5.3)	84.3(2.5)	86.3(3.6)
8	yohimbine	74.3(2.4)	77.0(3.8)	78.7(6.0)	66.6(2.5)	86.7(2.7)	94.1(2.1)
9	hydroxyacetildenafil	71.6(2.9)	76.4(3.4)	70.4(7.4)	62.1(3.4)	77.1(1.5)	83.4(2.8)
10	hydroxythiohomo sildenafil	69.7(3.2)	41.0(3.7)	59.2(9.3)	77.2(4.3)	91.3(2.1)	85.9(1.6)
11	hydroxyvardenafil	67.6(3.4)	70.9(3.4)	67.8(8.5)	71.2(2.9)	82.2(1.5)	87.7(2.9)
12	thiohomo sildenafil	49.8(3.2)	23.4(3.1)	36.7(9.2)	64.9(5.1)	86.7(2.6)	88.5(2.2)
13	thiosildenafil	64.3(5.0)	36.5(2.6)	53.2(10)	74.3(4.4)	93.4(1.6)	94.0(1.7)
14	N-desmethylsibutramine hydrochloride	69.6(2.2)	74.2(2.7)	70.7(3.0)	67.7(2.8)	88.1(2.4)	98.1(1.2)
15	N,N-didesmethylsibutramine hydrochloride	66.2(3.3)	81.2(2.8)	70.1(5.0)	60.4(1.1)	83.1(2.5)	90.5(1.0)
16	chlorthalidone	84.1(5.8)	59.6(5.9)	65.7(5.1)	80.5(3.2)	102.0(3.0)	99.3(2.5)
17	lorcaserin	72.6(2.1)	75.8(3.6)	74.5(5.4)	76.0(1.6)	88.5(1.6)	94.4(2.1)
18	hydrochlorothiazide	74.1(7.4)	83.7(2.0)	73.3(4.3)	78.6(2.0)	88.2(1.3)	89.6(0.99)
19	torasemide	78.6(5.5)	84.5(3.8)	77.1(5.0)	73.3(4.5)	93.3(3.8)	92.1(2.3)
20	gendenafil	80.3(1.2)	54.3(3.9)	73.5(5.5)	85.3(3.7)	107.3(1.2)	100.0(2.3)
21	emodin	64.5(2.4)	73.0(3.1)	66.7(2.4)	81.6(4.5)	86.8(3.0)	90.2(2.5)
22	indapamide	78.7(5.1)	80.4(1.4)	77.5(8.1)	78.8(3.0)	91.3(1.9)	91.9(2.1)
23	desmethyl sildenafil	68.5(4.5)	74.6(3.4)	70.5(8.4)	74.6(2.5)	87.2(0.77)	90.3(2.8)
24	desmethyl thiosildenafil	45.8(6.2)	24.9(3.2)	38.3(11)	71.4(3.2)	95.9(2.9)	93.2(2.3)
25	nortadalafil	69.2(13)	71.9(5.5)	71.9(8.6)	68.4(7.9)	95.0(4.7)	91.4(3.0)
26	N-desethyl acetildenafil	76.4(2.6)	78.8(1.7)	74.2(8.2)	76.3(3.1)	88.0(1.2)	91.2(1.4)
27	N-desethyl vardenafil	68.5(4.5)	74.6(3.4)	70.5(10)	74.6(2.5)	87.2(0.78)	90.2(2.8)
28	rimonabant	71.7(3.6)	72.3(1.8)	69.3(7.1)	91.7(1.6)	96.1(2.1)	99.9(3.7)
29	lidocaine	75.4(1.6)	74.7(2.9)	75.7(7.9)	80.0(1.4)	89.4(2.9)	95.5(2.4)
30	chlorpretadalafil	53.7(6.3)	72.9(5.2)	73.5(10)	69.5(7.5)	79.5(2.3)	89.8(5.7)
31	dimethylsildenafil	72.2(3.7)	54.6(2.6)	72.2(6.2)	79.8(5.2)	104.1(3.4)	97.5(2.1)
32	udenafil	70.7(3.6)	51.3(3.4)	70.6(8.5)	67.2(2.8)	85.7(1.8)	88.7(2.5)