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

doi:10.3724/SP.J.1123.2021.12009

Abstract

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

CLC Number:

O658

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.

Figures/Tables 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)