Rapid determination of multiple prohibited and restricted veterinary drug residues in fish and shrimp by ultra high performance liquid chromatography-tandem mass spectrometry

doi:10.3724/SP.J.1123.2019.09017

Abstract

Abstract:

A method was established for the rapid determination of 28 prohibited and restricted veterinary drugs of five classes (sulfonamides, quinolones, chloramphenicols, nitrofurans metabolites and triphenylmethane) in fish and shrimp by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled with simultaneous extraction and purification. After optimizing the sample pretreatment methods and chromatographic conditions, the samples were hydrolyzed with hydrochloric acid. The five kinds of residual drugs were simultaneously extracted with acetonitrile after nitrofuran metabolites derivatized with 2-nitrobenzaldehyde. The extraction solutions were dehydrated with sodium chloride and salted out. The acetonitrile layer was purified with n-hexane and concentrated to near dryness. The residues were dissolved in 1.0 mL methanol. Then, the solutions were purified by dispersive solid phase extraction with C18, N-(n-propyl)ethylenediamine (PSA) and amino bonded silica gel (NH2) sorbents. The residues were separated on a ZORBAX C18 chromatographic column (50 mm×2.1 mm, 1.8 μm). The MS data were acquired in positive and negative multiple reaction monitoring (MRM) modes, and quantitated by the isotope internal standard method. With this approach, the linear relationships of the 28 veterinary drugs were good. The linear correlation coefficients were greater than 0.99. The limits of detection and limits of quantification were in the range of 0.1-0.8 μg/kg and 0.3-2.7 μg/kg, respectively. The average recoveries at three spiked levels (1, 5, and 20 fold of the LOQs) were 70.0%-120% with relative standard deviations of 0.9%-10.0%. The method is simple, rapid, accurate and sensitive, and it is suitable for the rapid determination of various prohibited and restricted drugs in fish and shrimp.

Key words: ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), dispersive solid phase extraction, simultaneous extraction and purification, prohibited and restricted veterinary drugs, fish, shrimp

CHEN Xinglian, LIN Tao, LIU Xingyong, MEI Wenquan, WANG Li, GENG Huichun, CHENG Long, WANG Luxiang. Rapid determination of multiple prohibited and restricted veterinary drug residues in fish and shrimp by ultra high performance liquid chromatography-tandem mass spectrometry[J]. Chinese Journal of Chromatography, 2020, 38(5): 538-546.

Figures/Tables 6

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Compound	Chinese name	Retention time/min	Precursor ion (m/z)	Product ions (m/z)	DP/V	CEs/V	IS
DP: declustering potential; CE: collision energy; * quantitative ion.
Sulfadiazine (SD)	磺胺嘧啶	1.9	251	156^*/185	55	23/27	SDX-D₃
Sulfathiazole (ST)	磺胺噻唑	2.1	285	156^*/108	70	19/30	SDX-D₃
Norfloxacin (NOR)	诺氟沙星	2.2	320	276^*/233	80	26/35	NOR-D₅
Fleroxacin (FLE)	弗罗沙星	2.2	370	326^*/269	80	26/37	NOR-D₅
Ofloxacin (OFL)	氧氟沙星	2.2	362	318^*/261	60	25/35	NOR-D₅
Ciprofloxacin (CIP)	环丙沙星	2.3	332	288^*/245	80	25/33	CIP-D₈
5-Morpholine-methyl-3-amino-	5-甲基吗啉-3氨基-	2.3	335	291^*/262	66	18/24	D₅-AMOZ
2-oxazolidinone (AMOZ)	唑烷基酮
Sulfamerazine (SMR)	磺胺甲基嘧啶	2.3	265	156^*/172	50	25/24	SDX-D₃
Pefloxacin (PEF)	培氟沙星	2.3	334	316^*/290	118	27/24	NOR-D₅
Lomefloxacin (LOM)	洛美沙星	2.4	352	308^*/265	60	27/31	CIP-D₈
Thiamphenicol (TAP)	甲砜霉素	2.4	354	185^*/290	-75	-28/-16	CAP-D₅
Enrofloxacin (ENR)	恩诺沙星	2.5	360	316^*/245	80	28/36	ENR-D₅
Sulfamethazine (SMT)	磺胺二甲基嘧啶	2.6	279	156^*/204	55	22/20	SDX-D₃
Sulfamethizol (SMTZ)	磺胺甲噻二唑	2.6	271	108^*/92	60	33/36	SDX-D₃
Florfenicol (FF)	氟甲砜霉素	2.7	356	336^*/185	-70	-14/-27	CAP-D₅
Sulfamonomethoxine (SMM)	磺胺间甲氧嘧啶	3.0	281	156^*/215	65	25/25	SDX-D₃
Sulfachloropyridazine (SCP)	磺胺氯哒嗪	3.1	285	156^*/108	50	23/35	SDX-D₃
Chloramphenicol (CAP)	氯霉素	3.1	321	152^*/257	-70	-24/-17	CAP-D₅
Sulfadoxine(SDX)	磺胺邻二甲氧嘧啶	3.3	311	156^*/108	70	31/35	SDX-D₃
Semicarbazide (SEM)	氨基脲	3.3	209	192^*/166	51	15/17	¹³C¹⁵N₂-SEM
Sulfamethoxazole (SMZ)	磺胺甲基异唑	3.4	254	156^*/147	50	23/22	SDX-D₃
1-Aminohydantoin (AHD)	1-氨基-2-内酰脲	3.4	249	134^*/104	60	18/33	¹³C₃-AHD
Sulfisoxazole (SIZ)	磺胺二甲异唑	3.5	268	156^*/113	45	20/23	SDM-D₆
3-Amino-2-oxazolidinone (AOZ)	3-氨基-唑烷基酮	3.8	236	134^*/104	61	18/33	D₄-AOZ
Sulfaquinoxalin (SQX)	磺胺喹啉	3.9	301	156^*/108	55	24/37	SDM-D₆
Sulfadimethoxine (SDM)	磺胺间二甲氧嘧啶	3.9	311	156^*/108	70	31/35	SDM-D₆
Malachite green (MG)	孔雀石绿	4.9	329	313^*/208	101	53/46	MG-D₅
Leucomalachite green (LMG)	隐色孔雀石绿	6.8	331	239^*/315	76	45/32	LMG-D₆
Norfloxacin-D₅(NOR-D₅)	诺氟沙星-D₅	2.2	325	307	80	26
Ciprofloxacin-D₈ (CIP-D₈)	环丙沙星-D₈	2.3	340	322	90	30
D₅-5-Morpholine-methyl-3-amino-	5-甲基吗啉-3氨基-	2.3	340	296	70	18
2-oxazolidinone (D₅-AMOZ)	唑烷基酮-D₅
Enrofloxacin-D₅(ENR-D₅)	恩诺沙星-D₅	2.4	365	321	90	30
Sulfadoxine-D₃(SDX-D₃)	磺胺邻二甲氧嘧啶-D₃	3.2	314	156	70	31
¹³C¹⁵N₂-Semicarbazide (¹³C¹⁵N₂-SEM)	¹³C¹⁵N₂-氨基脲	3.3	212	168	57	15
Chloramp henicol-D₅ (CAP-D₅)	氯霉素-D₅	3.3	326	157	-75	-22
¹³C₃-1-Aminohydantoin (¹³C₃-AHD)	¹³C₃-1-氨基-2-内酰脲	3.4	252	134	61	18
D₄-3-Amino-2-oxazolidinone (D₄-AOZ)	D₄-3-氨基-唑烷基酮	3.8	240	134	68	20
Sulfadimethoxine-D₆(SDM-D₆)	磺胺间二甲氧嘧啶-D₆	3.9	317	156	70	31
Malachite green-D₅ (MG-D₅)	孔雀石绿-D₅	4.9	334	318	66	54
Leucomalachite green-D₆ (LMG-D₆)	隐色孔雀石绿-D₆	6.7	337	321	66	32

Compound	Chinese name	Retention time/min	Precursor ion (m/z)	Product ions (m/z)	DP/V	CEs/V	IS
DP: declustering potential; CE: collision energy; * quantitative ion.
Sulfadiazine (SD)	磺胺嘧啶	1.9	251	156^*/185	55	23/27	SDX-D₃
Sulfathiazole (ST)	磺胺噻唑	2.1	285	156^*/108	70	19/30	SDX-D₃
Norfloxacin (NOR)	诺氟沙星	2.2	320	276^*/233	80	26/35	NOR-D₅
Fleroxacin (FLE)	弗罗沙星	2.2	370	326^*/269	80	26/37	NOR-D₅
Ofloxacin (OFL)	氧氟沙星	2.2	362	318^*/261	60	25/35	NOR-D₅
Ciprofloxacin (CIP)	环丙沙星	2.3	332	288^*/245	80	25/33	CIP-D₈
5-Morpholine-methyl-3-amino-	5-甲基吗啉-3氨基-	2.3	335	291^*/262	66	18/24	D₅-AMOZ
2-oxazolidinone (AMOZ)	唑烷基酮
Sulfamerazine (SMR)	磺胺甲基嘧啶	2.3	265	156^*/172	50	25/24	SDX-D₃
Pefloxacin (PEF)	培氟沙星	2.3	334	316^*/290	118	27/24	NOR-D₅
Lomefloxacin (LOM)	洛美沙星	2.4	352	308^*/265	60	27/31	CIP-D₈
Thiamphenicol (TAP)	甲砜霉素	2.4	354	185^*/290	-75	-28/-16	CAP-D₅
Enrofloxacin (ENR)	恩诺沙星	2.5	360	316^*/245	80	28/36	ENR-D₅
Sulfamethazine (SMT)	磺胺二甲基嘧啶	2.6	279	156^*/204	55	22/20	SDX-D₃
Sulfamethizol (SMTZ)	磺胺甲噻二唑	2.6	271	108^*/92	60	33/36	SDX-D₃
Florfenicol (FF)	氟甲砜霉素	2.7	356	336^*/185	-70	-14/-27	CAP-D₅
Sulfamonomethoxine (SMM)	磺胺间甲氧嘧啶	3.0	281	156^*/215	65	25/25	SDX-D₃
Sulfachloropyridazine (SCP)	磺胺氯哒嗪	3.1	285	156^*/108	50	23/35	SDX-D₃
Chloramphenicol (CAP)	氯霉素	3.1	321	152^*/257	-70	-24/-17	CAP-D₅
Sulfadoxine(SDX)	磺胺邻二甲氧嘧啶	3.3	311	156^*/108	70	31/35	SDX-D₃
Semicarbazide (SEM)	氨基脲	3.3	209	192^*/166	51	15/17	¹³C¹⁵N₂-SEM
Sulfamethoxazole (SMZ)	磺胺甲基异唑	3.4	254	156^*/147	50	23/22	SDX-D₃
1-Aminohydantoin (AHD)	1-氨基-2-内酰脲	3.4	249	134^*/104	60	18/33	¹³C₃-AHD
Sulfisoxazole (SIZ)	磺胺二甲异唑	3.5	268	156^*/113	45	20/23	SDM-D₆
3-Amino-2-oxazolidinone (AOZ)	3-氨基-唑烷基酮	3.8	236	134^*/104	61	18/33	D₄-AOZ
Sulfaquinoxalin (SQX)	磺胺喹啉	3.9	301	156^*/108	55	24/37	SDM-D₆
Sulfadimethoxine (SDM)	磺胺间二甲氧嘧啶	3.9	311	156^*/108	70	31/35	SDM-D₆
Malachite green (MG)	孔雀石绿	4.9	329	313^*/208	101	53/46	MG-D₅
Leucomalachite green (LMG)	隐色孔雀石绿	6.8	331	239^*/315	76	45/32	LMG-D₆
Norfloxacin-D₅(NOR-D₅)	诺氟沙星-D₅	2.2	325	307	80	26
Ciprofloxacin-D₈ (CIP-D₈)	环丙沙星-D₈	2.3	340	322	90	30
D₅-5-Morpholine-methyl-3-amino-	5-甲基吗啉-3氨基-	2.3	340	296	70	18
2-oxazolidinone (D₅-AMOZ)	唑烷基酮-D₅
Enrofloxacin-D₅(ENR-D₅)	恩诺沙星-D₅	2.4	365	321	90	30
Sulfadoxine-D₃(SDX-D₃)	磺胺邻二甲氧嘧啶-D₃	3.2	314	156	70	31
¹³C¹⁵N₂-Semicarbazide (¹³C¹⁵N₂-SEM)	¹³C¹⁵N₂-氨基脲	3.3	212	168	57	15
Chloramp henicol-D₅ (CAP-D₅)	氯霉素-D₅	3.3	326	157	-75	-22
¹³C₃-1-Aminohydantoin (¹³C₃-AHD)	¹³C₃-1-氨基-2-内酰脲	3.4	252	134	61	18
D₄-3-Amino-2-oxazolidinone (D₄-AOZ)	D₄-3-氨基-唑烷基酮	3.8	240	134	68	20
Sulfadimethoxine-D₆(SDM-D₆)	磺胺间二甲氧嘧啶-D₆	3.9	317	156	70	31
Malachite green-D₅ (MG-D₅)	孔雀石绿-D₅	4.9	334	318	66	54
Leucomalachite green-D₆ (LMG-D₆)	隐色孔雀石绿-D₆	6.7	337	321	66	32

Level	Factors
Level	Extraction reagent (A)	V(dipotassium phosphate solution) (B)/mL	Extraction times (C)
1	ethyl acetate	0	1
2	acetonitrile	13	2
3	ethyl acetate-acetonitrile	26	3
	(1:1, v/v)

Level	Factors
Level	Extraction reagent (A)	V(dipotassium phosphate solution) (B)/mL	Extraction times (C)
1	ethyl acetate	0	1
2	acetonitrile	13	2
3	ethyl acetate-acetonitrile	26	3
	(1:1, v/v)

Compound	Linear range/(μg/L)	Linear equation	r	LOD/(μg/kg)	LOQ/(μg/kg)
y: peak area ratio of analyte to the internal standard; x: mass concentration of analyte, μg/L.
SD	1.0-300	y=0.0268x+0.0193	0.9990	0.7	2.3
ST	1.0-300	y=0.0282x+0.0520	0.9994	0.7	2.3
NOR	1.0-300	y=0.0139x+0.0108	0.9990	0.7	2.3
FLE	1.0-300	y=0.0929x+0.0735	0.9993	0.6	2.0
OFL	1.0-300	y=0.1100x+0.1280	0.9989	0.5	1.7
CIP	1.0-300	y=0.0206x+0.0365	0.9958	0.5	1.7
AMOZ	0.5-80	y=0.0377x-0.0050	0.9991	0.2	0.7
SMR	1.0-300	y=0.0309x+0.0013	0.9985	0.6	2.0
PEF	1.0-300	y=0.1230x-0.0263	0.9991	0.8	2.7
LOM	1.0-300	y=0.0500x+0.1230	0.9992	0.7	2.3
TAP	0.2-80	y=0.1830x+0.0012	0.9994	0.2	0.7
ENR	1.0-300	y=0.0678x+0.0761	0.9990	0.6	2.0
SMT	1.0-300	y=0.0251x+0.0258	0.9992	0.6	2.0
SMTZ	1.0-300	y=0.0141x+0.0416	0.9993	0.8	2.7
FF	0.2-80	y=0.4480x+0.0001	0.9992	0.2	0.7
SMM	1.0-300	y=0.0030x+0.0489	0.9989	0.7	2.3
SCP	1.0-300	y=0.0260x+0.0348	0.9989	0.7	2.3
CAP	0.2-40	y=0.0468x+0.0011	0.9991	0.1	0.3
SDX	1.0-300	y=0.0712x+0.1080	0.9984	0.5	1.7
SEM	0.5-80	y=0.0429x-0.0025	0.9991	0.2	0.7
SMZ	1.0-300	y=0.0301x+0.0753	0.9987	0.6	2.0
AHD	0.5-80	y=0.0336x-0.0046	0.9989	0.2	0.7
SIZ	1.0-300	y=0.0678x+0.0146	0.9994	0.8	2.7
AOZ	0.5-80	y=0.0350x-0.0042	0.9993	0.2	0.7
SQX	1.0-300	y=0.0821x+0.1111	0.9994	0.7	2.3
SDM	1.0-300	y=0.1530x+0.2700	0.9984	0.6	2.0
MG	0.5-100	y=0.0552x-0.0217	0.9998	0.1	0.3
LMG	0.5-100	y=1.3900x-5.5600	0.9994	0.2	0.7