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Analytical and Bioanalytical Chemistry

, Volume 410, Issue 22, pp 5373–5389 | Cite as

Target screening of 105 veterinary drug residues in milk using UHPLC/ESI Q-Orbitrap multiplexing data independent acquisition

  • Jian Wang
  • Daniel Leung
  • Willis Chow
  • James Chang
  • Jon W. Wong
Paper in Forefront
Part of the following topical collections:
  1. Food Safety Analysis

Abstract

This paper presents a multi-class target screening method for the detection of 105 veterinary drug residues from 11 classes in milk using ultra-high performance liquid chromatography electrospray ionization quadrupole Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap). The method is based on a non-target approach of full mass scan and multiplexing data-independent acquisition (Full MS/mDIA). The veterinary drugs include endectocides, fluoroquinolones, ionophores, macrolides, nitroimidazole, NSAIDs, β-lactams, penicillins, phenicols, sulfonamides, and tetracyclines. Veterinary drug residues were extracted from milk using a salting-out and solid-phase extraction (SOSPE) procedure, which entailed the precipitation of milk proteins by an extraction buffer (oxalic acid and EDTA, pH 3) and acetonitrile, a salting-out acetonitrile/water phase separation using ammonium sulfate, and solid-phase extraction for clean-up using polymeric reversed-phase sorbent cartridges. The Q-Orbitrap Full MS/dd-MS2 (data-dependent acquisition) was used to acquire product-ion spectra of individual veterinary drugs to build a compound database and a mass spectral library, whereas its Full MS/mDIA was utilized to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 μg/kg. The in-spectrum mass correction or solvent background lock-mass correction was used to minimize mass error when building the compound database from experimental dd-MS2 accurate mass data. Retention time alignment and response threshold adjustment were used to eliminate or reduce false negatives and/or false positive rates. The validated method was capable of screening 58% and 96% of 105 veterinary drugs at 1.0 and 10.0 μg/kg, respectively, without manually evaluating every compound during data processing, which will reduce the workload in routine practice.

Keywords

UHPLC/ESI Q-Orbitrap Veterinary drug residues Compound database Target screening Milk Multiplexing data-independent acquisition 

Introduction

Veterinary drugs have been widely used in veterinary medicine and animal production to treat and prevent animal diseases and to enhance growth rate and feed efficiency. Incorrect administration of the drug or improper withdrawal time after treatment could lead to the presence of drug residues in foods of animal origin. The residues may provoke allergic reactions in some hypersensitive individuals or encourage the spread of drug-resistant pathogenic bacterial strains [1, 2, 3]. Furthermore, veterinary drugs present in milk can have negative implications on microbial processes (for example cheese and yogurt production [4, 5]). Therefore, the residue levels of veterinary drugs should not exceed the maximum residue limits set by regulatory agencies in milk to ensure the safety of food supply. This justifies the need for analytical methods that are capable of quantifying and screening an increasingly large number of veterinary drug residues that are potentially used for food production in routine monitoring programs [6].

Veterinary drug residues in food can be determined through biological screening methods such as microbial inhibition tests, immunochemical methods etc., and quantitative and confirmatory methods such as liquid chromatography coupled to mass spectrometry (LC-MS). Historically, veterinary drug residues, which were typically in a group of less than 20 compounds, were analyzed by a single-class or related families using LC/MS/MS. A single-class method was relatively easy to optimize for both extraction and instrument parameters because of the similar physical and chemical properties of veterinary drugs.

In the last decade, the use and development of high resolution mass spectrometric instrumentation such as LC-Orbitrap and LC-TOF (Time of Flight) have increased rapidly. Their applications for quantitation and target screening of chemical residues in food are advantageous because of their high mass-resolving power and accurate mass measurement, multi-function data acquisition, and powerful data processing capacity. The Orbitrap and TOF mass spectrometers (i.e., high-end models) offer high mass-resolution (>20,000 FWHM), accurate mass measurement (<5 ppm), excellent Full MS scan sensitivity, and complete mass spectrometric information. The Full MS scan data allow for the screening of targeted analytes, quantitation of selected compounds, and retrospective analysis (knowns or unknowns) even when appropriate standards are not available at the time of analysis. When operated in Full MS scan mode with generic instrument parameter settings, these instruments have been increasingly used in target approach based on accurate masses to quantify or screen veterinary residues in food. Examples of quantitative and/or screening methods based on Full MS scan that have been reported for multi-veterinary drugs at various concentration levels include screening of 150 veterinary drugs in milk using TOF [7]; 100 veterinary drugs in egg, milk, fish, and animal tissues using TOF [8, 9]; 54 veterinary drugs in honey and 59 veterinary drugs in milk using Q-TOF [10]; 100 veterinary drugs in milk using Q-Orbitrap [6]; 105 veterinary drugs in milk using Q-Orbitrap [11]; 90 veterinary drugs in royal jelly using Q-TOF [12]. Those applications focused on a generic sample preparation and a full set of calibration standards for quantifying or screening of as many as possible veterinary drugs in food.

Recently, target screening based on non-target approach of data acquisition and retrospective data processing has become one type of promising application for the purpose of screening using high resolution mass spectrometers such as TOF or Orbitrap [13, 14, 15, 16]. Data were acquired using a combination of Full MS scan, all in fragmentation (AIF) and data-independent acquisition (DIA), etc. The screening of targeted chemical contaminants, for example veterinary drugs and pesticides, was accomplished using accurate masses of the targeted precursor ion (i.e., pseudomolecular ion) along with retention time or characteristic fragment ion by specific software for automated data mining and exploitation. These types of analyses are often referred to as screening methods or semi-automated mass spectrometry-based detections. The screening concept offers laboratories an effective means to extend their analytical scope to chemical contaminants, which potentially have a low probability of being present in the samples. The residues that occur more frequently would continue to be monitored using validated quantitative multi-residue methods [17].

This study was designed to develop a systematic and detailed protocol for the development of a compound database (CDB) of the retention time and the exact or accurate mass of a precursor and its fragments and a mass spectral (MS) library of a product ion spectrum, and their applications for target screening of 105 veterinary drugs in milk based on multiplexing data independent acquisition using a Q-Orbitrap. Full MS/dd-MS2 (data-dependent acquisition) was utilized to acquire product-ion spectra of veterinary drugs for individual standards to obtain exact masses of fragments for a CDB and a MS library. The CDB was built based on theoretical and experimental mass data. Accurate mass, retention time, and response threshold were three key parameters that were used to build a functional and working CDB, and they were optimized or adjusted to reduce false negatives and/or false positives. Full MS/mDIA (multiplexing data-independent acquisition) was used to acquire sample data from milk for target screening of veterinary drugs fortified at 1.0 or 10.0 μg/kg. The method was validated according to SANTE/11945/2015 [17], which provided guidance for target screening validation. This qualitative method significantly reduced the workload for data processing in routine practice. The entire procedure, including sample extraction and data processing, allowed for high-throughput testing of routine samples, which could benefit veterinary drug residue monitoring programs.

Material and methods

Chemicals and reagents

Ten batches of whole milk (4 or 8L/batch) were collected from 10 different local farms. All milk samples, which were tested free of veterinary drug residues using the previously developed UHPLC/ESI Q-Orbitrap method [11], were kept at –20 °C. Pierce LTQ ESI positive ion calibration solution (10 mL) was purchased from ThermoFisher Scientific (Rockford, IL, USA). The calibration solution, which includes n-butylamine (m/z 74), caffeine (m/z 195 and its fragment m/z 138), Ultramark 1621 (m/z 1022, 1122, 1222, 1322, 1422, 1522, 1622, 1722, 1822), and MRFA (m/z 524), was used to tune and calibrate the Q-Orbitrap. Ammonium acetate (reagent grade), ammonium sulfate (reagent plus, >99.0%), formic acid (LC-MS grade, ~98%), ammonium formate (for mass spectrometry, >99.0%), oxalic acid (reagent plus, >99.0%), ammonium hydroxide (28%–30%), and LC-MS acetonitrile (Chromasolv, 2.5 L) were purchased from Sigma-Aldrich Corp. (Oakville, Canada). Acetic acid (glacial acetic acid, reagent grade, 99.7%), acetonitrile (distilled in glass), methanol (distilled in glass), and EDTA disodium salt were obtained from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Water (18.2 MΩ⋅cm) used for reagent and sample preparation was obtained from a Barnstead Nanopure system (Thermo Scientific, Marietta, OH, USA). Veterinary drug standards were obtained from Sigma-Aldrich Corp. or Toronto Research Chemicals (ON, Canada). A LC vial was a 0.45 μm PVDF Syringeless Filter Device Mini-UniPrep with polypropylene housing (GE Healthcare UK Limited, Little Chalfont, UK). OASIS HLB Plus 225 mg cartridges were purchased from Waters Corporation (Milford, MA, USA).

Preparation of standard solutions

Individual veterinary drug standard stock solutions were generally prepared at a concentration of 1000 or 2000 μg/mL in methanol, acetonitrile, or water. Intermediate veterinary drug working solutions were prepared at 5.0 μg/mL in methanol from stock solutions. Stock and intermediate solutions were stored at –20 °C. Two-level veterinary drug standard mix working solutions for sample fortification were prepared by transferring 0.4 and 4.0 mL of 5.0 μg/mL into two separate 100 mL volumetric flasks for their respective concentration levels, and then making up to volume with acetonitrile. The resulting concentrations were 0.020 and 0.200 μg/mL, which were used for sample fortification at 1.0 or 10.0 μg/kg. All working solutions were stored at 4 °C.

UHPLC/ESI Q-Orbitrap parameters

UHPLC/ESI Q-Orbitrap system consisted of an Accela 1250 LC pump and an Accela open autosampler coupled with a Q-Exactive mass spectrometer (ThermoFisher Scientific, Germany). The system was controlled by Xcalibur 3.1 with Tune 2.8 SP1 Build 2806. The UHPLC/ESI Q-Orbitrap instrument parameter settings were the same as those we used for the target screening of 448 pesticide residues in fruits and vegetables [15].
  1. (a)

    Ultra-high pressure liquid chromatography

     
UHPLC mobile phases A and B consisted of 4 mM ammonium formate and 0.10% formic acid in water and methanol, respectively. The UHPLC column utilized was a Hypersil Gold, 100 mm × 2.1 mm, 1.9 μm column (Thermo Scientific, Marietta, OH, USA). The UHPLC guard column was an Accucore aQ 10 × 2.1 mm, 2.6 μm Defender cartridge (Thermo Scientific, Marietta, OH, USA). The UHPLC gradient profile and flow rate are shown in Table 1. Column oven temperature was set at 45 °C and auto-sampler temperature was set at 5 °C. Injection volume was 5 μL and the total run-time was 14 min.
  1. (b)

    Q-Orbitrap parameters

     
Table 1

UHPLC gradient profile

Steps

Time

Mobile phase A (%)

Mobile phase B (%)

Flow rate (μL/min)

0

0.00

88.0

12.0

300

1

8.00

5.0

95.0

300

2

9.00

0.0

100.0

300

3

11.00

0.0

100.0

300

4

11.10

88.0

12.0

300

5

14.00

88.0

12.0

300

The Q-Exactive ion source was equipped with a heated electrospray ionization (HESI) probe and the Q-Orbitrap was tuned and calibrated using positive LTQ calibration solution once per week.

The workflow of target screening is shown in Fig. 1. To obtain the product-ion spectra that were used to build an in-house compound database and a mass spectral library, the Q-Exactive was operated in Full MS/dd-MS2. During the Full MS scan, the Q-Exactive mass-resolution was set at 70,000 FWHM; AGC target: 1.0E6; maximum IT: 250 ms; and scan range m/z 80 to 1100. If the targeted mass of a compound from the inclusion list was detected within ±5 ppm mass tolerance, the precursor ion was isolated by the quadrupole and sent to the HCD (higher energy collisional dissociation) cell for fragmentation via the C-trap. The inclusion list consisted of the masses of precursor ions in forms of [M+H]+, [M+NH4]+, and [M+Na]+ for veterinary drugs. The precursor ion was fragmented with a stepped normalized collision energy (NCE) to generate product-ion spectra. For dd-MS2, the Q-Exactive mass-resolution was set at 35,000 FWHM; AGC target: 2E5; maximum IT: 120 ms; isolation window: m/z 1.0; NCE/stepped NCE: 20, 40 and 60; underfill ratio: 10%; intensity threshold: 1.7E5; apex trigger: 2 to 4 s; and dynamic exclusion: 10.0 s. Each veterinary drug standard was injected twice into the system at 50 μg/L (ppb) or higher concentration.
Fig. 1

Workflow for building Compound database and mass spectral library using UHPLC/ESI Full MS/dd-MS2 and sample analysis by UHPLC/ESI Full MS/mDIA, and data process for target screening

For target screening of veterinary drugs in milk samples, data acquisition was achieved through Full MS (m/z 100–1000) and mDIA (m/z 100–900) (Fig. 2). For Full MS, the Q-Exactive mass-resolution was set at 70,000 FWHM; AGC target: 3.0E6; maximum IT: 200 ms; scan range: m/z 100 to 1000. For mDIA with segments of mass ranges covering from m/z 100 to 900, the Q-Exactive mass-resolution was set at 17,500 FWHM; AGC target: 1.0E6; maximum IT: auto; loop count: 10; MSX (multiplexing) count: 4; isolation window: m/z 52.0; and NCE/stepped NCE 20, 40, 60. Thus, for loop counts from 1 to 8, a collection of ions isolated by the quadruple in every m/z 50 mass increment from m/z 100 to 500 were sent to HCD cell for fragmentation, and then to Orbitrap via the C-trap in eight steps. For loop count 9 with MSX (multiplexing) count 4, a collection of ions isolated by the quadruple for mass ranges in segments of m/z 500–550, 600–650, 700–750, and 800–850 were sent to HCD cell sequentially for fragmentation, and then all fragments stored in HCD were sent to Orbitrap via the C-trap in one step. The same procedure was used for loop count 10 with MSX count 4 for mass ranges in segments of m/z 550–600, 650–700, 750–800, and 850-900. The inclusion list and MSX ID for mDIA are shown in Table 2 and Fig. 2A. The masses in the list were centered with each isolation window of m/z 52. Other Q-Exactive generic parameters were: sheath gas flow rate set at 60; Aux gas flow rate: 30; Sweep gas flow rate: 2; Spray voltage (KV): 3.50; Capillary temperature (°C): 350; S-lens level: 55.0 and Heater temperature (°C): 350 as reported elsewhere [15, 18].
Fig. 2

(A) Q-Orbitrap multiplexing data independent acquisition. IN: isolation window number. IL: inclusion list. IW: isolation window; (B) target screening for oleandomycin in milk at 10.0 μg/kg by compound database; (C) target screening for oleandomycin in milk at 10.0 μg/kg by mass spectral library

Table 2

Method editor - Inclusion List

 

Mss (m/z)

Polarity

MSX ID

1

125.00000

Positive

1

2

175.00000

Positive

2

3

225.00000

Positive

3

4

275.00000

Positive

4

5

325.00000

Positive

5

6

375.00000

Positive

6

7

425.00000

Positive

7

8

475.00000

Positive

8

9

525.00000

Positive

9

10

575.00000

Positive

10

11

625.00000

Positive

9

12

675.00000

Positive

10

13

725.00000

Positive

9

14

775.00000

Positive

10

15

825.00000

Positive

9

16

875.00000

Positive

10

Sample extraction and clean-up

The sample preparation was based on a two-step process of salting-out acetonitrile/water extraction (Step 1) and solid-phase extraction clean-up (Step 2) known as salting-out and solid-phase extraction (SOSPE) reported elsewhere [11].
  1. (a)

    Step 1 Extraction

     
A milk sample (5 g) was weighed into 50 mL centrifuge tubes (VWR International, Edmonton, Alberta, Canada). Two hundred fifty μL per two-level veterinary drug standard working solution for sample fortification was added into each centrifuge tube to provide 1.0 or 10.0 μg/kg equivalent residue in sample. After 15 min, 5 mL of extraction buffer and 10 mL of acetonitrile were added to the sample. The extraction buffer contained 0.86% oxalic acid and 0.74% EDTA disodium salt, and its pH was adjusted to 3.0 using ammonium hydroxide. The sample mixture was capped and shaken for 30 s by hand and then centrifuged at 3000 rpm (~2100 × g) for 5 min using a centrifuge. The supernatant was transferred into another 50 mL centrifuge tube, to which 1 g of ammonium sulfate was added. The sample mixture was shaken by hand to mix for 2 min, and then was left to stand for 2 min. A phase separation was observed from the mixture.
  1. (b)

    Step 2 SPE clean-up

     

Polymeric reversed-phase sorbent OASIS HLB Plus cartridges were used for SPE clean-up. OASIS HLB Plus 225 mg cartridges were attached to 25 mL syringe barrels and set up for solid-phase extraction using a Visiprep 24-port SPE Vacuum Manifold (Sigma-Aldrich Corp). The cartridges were preconditioned sequentially with 10 mL of methanol, 10 mL of water, and 2 mL of extraction buffer. The sample extracts from Step 1 were separated into three layers after centrifugation (3000 rpm or ~2100 × g, 3 min). The top acetonitrile layer (~10 mL) was transferred to a 16 × 125 mm disposable test tube and retained for later to be loaded onto OASIS HLB cartridges. The lower aqueous layer was transferred onto the preconditioned Oasis HLB cartridges under vacuum at –2 to –3 inHg with a flow rate of ~1 mL/min. The very thin middle white layer (~1 or 2 mm) was discarded and should be not transferred onto the cartridges. Oasis HLB cartridges were rinsed further with 2 mL of extraction buffer and allowed to run dry. The previously retained acetonitrile layer, which served as eluting solvent as well, was loaded onto the OASIS HLB cartridges through syringe barrels. The flow (~1 mL/min) of the eluting solvent was maintained under vacuum at –2 to –3 inch Hg and the eluent was collected into a 16 × 125 mm disposable glass test tube. The OASIS HLB cartridges were run dry under vacuum. Then an additional 5 mL of methanol was dispensed onto the cartridges to elute further. The eluent was collected into the same test tube as above (acetonitrile layer). The OASIS HLB cartridges were run dry under vacuum. The eluent (~15 mL) was capped and inverted to thoroughly mix. Three mL of eluent was transferred into individual 5 mL PYREX brand centrifuge tubes, which was pre-calibrated with 1 mL volume accuracy (VWR International, Edmonton, Alberta, Canada). The sample extracts were evaporated to 0.1–0.2 mL, which took approximately 20 min, using an N-EVAP nitrogen evaporator (Organomation Associates Inc., Berlin, MA, USA) at 50 °C under a stream of nitrogen. Then the extracts were reconstituted by making up to 0.5 mL with acetonitrile and then to 1.0 mL with 0.1 M ammonium acetate. The extracts were vortexed for 30 s. Five hundred μL of sample extracts was transferred into a 0.45 μm PVDF Syringeless Filter Device Mini-UniPrep vial and pressed to filter. Sample extracts were ready to be injected to UHPLC/ESI Q-Orbitrap MS for analysis.

Experimental design and method validation

The method was validated at 1.0 and 10.0 μg/kg using a total of 10 different raw milk blank samples. For each matrix, samples were spiked at 1.0 or 10.0 μg/kg, in duplicate. The experiment was repeated on three different days. The positive screen of a veterinary drug should meet the criterion that it can be detected in at least 95% of the 20 samples (i.e., an acceptable false-negative rate of ≤5%) in the batch [17].

Data processing

The Target Screening function of TraceFinder 3.3 (ThermoFisher Scientific, USA) was used for data processing based on the compound database developed in-house. For target screening parameter settings, response threshold was set individually for each veterinary drug (Table 3, column 5); mass accuracy: 5 ppm for both precursor ion and fragment; retention time window: 60 s; minimum number of fragments: 1; and MS order: MS2.
Table 3

UHPLC/ESI Q-Orbitrap compound database of veterinary drug for target screening

Compound name

Class

Total number

Chemical formula

Response thresholda

Adduct

Retention time (min)

Massb

Exact

Accurate

Accurate

Accurate

Accurate

Precursor

Fragment 1

Fragment 2

Fragment 3

Fragment 4

1c

2

3

4

5

6

7

8

9

10

11

12

Abamectin B1a

Endectocides

7

C48H72O14

45669

[M+Na]+

10.01

895.48143

455.24118

751.40208

183.06277

449.25116

Doramectin

Endectocides

 

C50H74O14

28604

[M+Na]+

10.28

921.49708

183.06294

353.20852

777.41936

449.25095

Emamectin B1a

Endectocides

 

C49H75NO13

188440

[M+H]+

9.13

886.53112

158.11764

82.06581

126.09159

302.19602

Eprinomectin B1a

Endectocides

 

C50H75NO14

21719

[M+Na]+

9.88

936.50798

352.17300

490.27760

208.09455

368.16782

Ivermectin

Endectocides

 

C48H74O14

36832

[M+Na]+

10.39

897.49708

183.06296

329.20878

753.41828

457.25590

Moxidectin

Endectocides

 

C37H53NO8

110311

[M+Na]+

10.23

662.36634

361.21410

257.11459

467.20533

632.35540

Selamectin

Endectocides

 

C43H63NO11

14286

[M+H]+

10.55

770.44739

333.24268

276.12336

608.35853

626.36911

Cinoxacin

Fluoroquinolones

17

C12H10N2O5

1206754

[M+H]+

4.62

263.06625

217.06089

189.02952

207.04015

235.07135

Ciprofloxacin

Fluoroquinolones

 

C17H18FN3O3

759427

[M+H]+

3.76

332.14050

231.05653

288.15061

245.10859

 

Danofloxacin

Fluoroquinolones

 

C19H20FN3O3

840566

[M+H]+

3.80

358.15615

82.06582

255.05647

314.16631

340.14451

Difloxacin

Fluoroquinolones

 

C21H19F2N3O3

645499

[M+H]+

4.14

400.14672

299.09911

356.15689

285.08335

 

Enoxacin

Fluoroquinolones

 

C15H17FN4O3

713503

[M+H]+

3.51

321.13575

250.06232

232.05174

204.05705

303.12400

Enrofloxacin

Fluoroquinolones

 

C19H22FN3O3

796469

[M+H]+

3.85

360.17180

316.18210

245.10864

286.09874

203.06171

Flumequine

Fluoroquinolones

 

C14H12FNO3

1120466

[M+H]+

6.17

262.08740

220.04050

238.05104

244.07663

202.03018

Lomefloxacin

Fluoroquinolones

 

C17H19F2N3O3

747621

[M+H]+

3.95

352.14672

265.11476

308.15691

237.08351

251.09913

Marbofloxacin

Fluoroquinolones

 

C17H19FN4O4

673901

[M+H]+

3.25

363.14631

72.08155

320.10422

205.04102

233.07227

Nalidixic Acid

Fluoroquinolones

 

C12H12N2O3

901948

[M+H]+

5.94

233.09207

205.06084

187.05026

159.05536

215.08107

Norfloxacin

Fluoroquinolones

 

C16H18FN3O3

702412

[M+H]+

3.64

320.14050

233.10856

276.15075

231.05651

300.13427

Ofloxacin

Fluoroquinolones

 

C18H20FN3O4

778102

[M+H]+

3.51

362.15106

261.10344

318.16131

221.07221

205.04095

Orbifloxacin

Fluoroquinolones

 

C19H20F3N3O3

773924

[M+H]+

3.96

396.15295

295.10532

352.16297

267.03754

254.06615

Oxolinic Acid

Fluoroquinolones

 

C13H11NO5

925124

[M+H]+

5.10

262.07100

244.06026

234.03972

216.06571

160.03929

Pipemidic Acid

Fluoroquinolones

 

C14H17N5O3

547814

[M+H]+

2.94

304.14042

217.10852

233.06700

189.07725

276.10928

Sarafloxacin

Fluoroquinolones

 

C20H17F2N3O3

519324

[M+H]+

4.08

386.13107

299.09908

342.14130

366.12483

270.09641

Sparfloxacin

Fluoroquinolones

 

C19H22F2N4O3

927417

[M+H]+

4.57

393.17327

292.12568

251.08661

349.18362

264.05798

Lasalocid

Ionophores

5

C34H54O8

480536

[M+NH4]+

9.88

608.41569

237.18500

255.19546

281.24745

337.27380

Monensin

Ionophores

 

C36H62O11

383189

[M+NH4]+

10.01

688.46304

461.32600

421.29464

617.40440

635.41464

Narasin

Ionophores

 

C43H72O11

288345

[M+NH4]+

10.50

782.54129

747.50369

729.49278

373.23704

391.24790

Nigericin

Ionophores

 

C40H68O11

527723

[M+NH4]+

10.43

742.50999

657.43615

125.09842

675.44666

461.32628

Salinomycin

Ionophores

 

C42H70O11

400063

M+NH4

10.28

768.52564

733.48808

715.47794

391.24743

373.23714

Erythromycin

Macrolides

8

C37H67NO13

200515

[M+H]+

6.88

734.46852

83.04991

158.11777

576.37463

558.36436

Neospiramycin I

Macrolides

 

C36H62N2O11

273123

[M+2H]++

4.83

350.22496

174.11263

160.13332

192.12318

88.07637

Oleandomycin

Macrolides

 

C35H61NO12

272208

[M+H]+

6.29

688.42665

158.11759

116.07098

544.34785

98.09696

Roxithromycin

Macrolides

 

C41H76N2O15

450550

[M+2H]++

7.48

419.26956

83.04981

158.11762

98.09690

116.07094

Spiramycin I

Macrolides

 

C43H74N2O14

169401

[M+2H]++

5.17

422.26428

174.11274

101.06035

142.12289

145.08610

Tilmicosin

Macrolides

 

C46H80N2O13

623752

[M+2H]++

5.98

435.29030

174.11267

143.07043

126.12802

116.07104

Tylosin A

Macrolides

 

C46H77NO17

42168

[M+H]+

6.67

916.52643

174.11254

88.07634

772.44722

318.19078

Tylosin B

Macrolides

 

C39H65NO14

10000

[M+H]+

6.34

772.44778

174.11269

88.07642

132.10218

598.35881

2-methyl-4(5)-nitroimidazole

Nitroimidazoles

13

C4H5N3O2

292182

[M+H]+

1.60

128.04545

82.05308

111.04295

56.03763

98.04786

Dimetridazole

Nitroimidazoles

 

C5H7N3O2

85119

[M+H]+

2.75

142.06110

96.06870

56.05026

112.06346

95.06091

Etanidazole

Nitroimidazoles

 

C7H10N4O4

115995

[M+H]+

1.13

215.07748

102.05548

126.06643

169.08459

138.06627

HMMNI

Nitroimidazoles

 

C5H7N3O3

19969

[M+H]+

2.01

158.05602

95.04969

105.04518

141.00044

140.04548

Ipronidazole

Nitroimidazoles

 

C7H11N3O2

307181

[M+H]+

4.66

170.09240

124.09977

109.07649

84.08144

140.09446

Ipronidazole-OH

Nitroimidazoles

 

C7H11N3O3

206493

[M+H]+

3.93

186.08732

168.07673

121.07630

82.06576

128.04561

Metronidazole

Nitroimidazoles

 

C6H9N3O3

228665

[M+H]+

2.26

172.07167

128.04566

82.05321

111.04314

 

Metronidazole-OH

Nitroimidazoles

 

C6H9N3O4

69831

[M+H]+

1.57

188.06658

126.02997

123.05553

144.04036

68.05021

Nimorazole

Nitroimidazoles

 

C9H14N4O3

140783

[M+H]+

1.30

227.11387

114.09173

100.07622

140.04549

70.06587

Ornidazole

Nitroimidazoles

 

C7H10N3O3Cl

186176

[M+H]+

3.79

220.04835

128.04563

82.05318

93.01076

111.04313

Ronidazole

Nitroimidazoles

 

C6H8N4O4

116527

[M+H]+

2.21

201.06183

140.04540

55.04245

110.04779

94.05312

Ternidazole

Nitroimidazoles

 

C7H11N3O3

232802

[M+H]+

2.96

186.08732

128.04561

82.05316

111.04307

98.04799

Tinidazole

Nitroimidazoles

 

C8H13N3O4S

485564

[M+H]+

2.85

248.06995

121.03208

128.04567

110.08429

202.07713

5-hydroxyflunixin

NSAIDS

3

C14H11F3N2O3

166046

[M+H]+

7.59

313.07945

295.06892

280.04543

226.07376

 

Flunixin

NSAIDS

 

C14H11F3N2O2

1303904

[M+H]+

7.90

297.08454

279.07393

264.05047

259.06789

210.07885

Phenylbutazone

NSAIDS

 

C19H20N2O2

676190

[M+H]+

7.99

309.15975

120.04485

92.05019

188.10734

211.08683

Ampicillin

Penicillins

6

C16H19N3O4S

42693

[M+H]+

3.81

350.11690

106.06569

160.04284

192.04794

174.05516

Cloxacillin

Penicillins

 

C19H18ClN3O5S

17021

[M+H]+

6.79

436.07285

160.04279

114.03763

178.00553

277.03748

Dicloxacillin

Penicillins

 

C19H17Cl2N3O5S

10000

[M+H]+

7.10

470.03387

160.04264

114.03755

310.99819

211.96640

Oxacillin

Penicillins

 

C19H19N3O5S

12478

[M+H]+

6.58

402.11182

160.04275

114.03763

144.04449

243.07651

Penicillin G

Penicillins

 

C16H18N2O4S

15269

[M+H]+

6.05

335.10600

160.04280

114.03765

176.07075

189.06936

Penicillin V

Penicillins

 

C16H18N2O5S

10277

[M+H]+

6.52

351.10092

229.06425

257.05901

333.09008

211.05356

Florfenicol

Phenicols

2

C12H14Cl2FNO4S

91417

[M+NH4]+

3.94

375.03429

241.00517

339.99659

206.03647

170.05978

Thiamphenicol

Phenicols

 

C12H15Cl2NO5S

30725

[M+Na]+

3.15

377.99402

    

Dapsone

Sulfonamides

26

C12H12N2O2S

877058

[M+H]+

3.23

249.06922

108.04485

92.05008

156.01137

110.06046

Sulfabenzamide

Sulfonamides

 

C13H12N2O3S

216102

[M+H]+

4.35

277.06414

156.01134

108.04483

92.05006

120.05589

Sulfacetamide

Sulfonamides

 

C8H10N2O3S

64257

[M+H]+

1.79

215.04849

142.00426

106.04870

159.03089

156.01161

Sulfachloropyridazine

Sulfonamides

 

C10H9ClN4O2S

172526

[M+H]+

3.73

285.02075

156.01141

108.04489

92.05011

120.05600

Sulfadiazine

Sulfonamides

 

C10H10N4O2S

386598

[M+H]+

2.20

251.05972

156.01135

108.04487

92.05009

120.05604

Sulfadimethoxine

Sulfonamides

 

C12H14N4O4S

654854

[M+H]+

4.92

311.08085

156.07688

108.04490

92.05012

245.10340

Sulfadoxine

Sulfonamides

 

C12H14N4O4S

865642

[M+H]+

4.11

311.08085

108.04492

156.01146

92.05014

140.04560

Sulfaethoxypyridazine

Sulfonamides

 

C12H14N4O3S

588523

[M+H]+

4.54

295.08594

108.04486

156.01137

92.05007

140.08186

Sulfaguanidine

Sulfonamides

 

C7H10N4O2S

219453

[M+H]+

1.11

215.05972

142.00407

156.01134

108.04483

92.05006

Sulfamerazine

Sulfonamides

 

C11H12N4O2S

524732

[M+H]+

2.93

265.07537

108.04487

156.01139

92.05008

190.02814

Sulfameter

Sulfonamides

 

C11H12N4O3S

512801

[M+H]+

3.24

281.07029

108.04489

156.01140

126.06646

215.09280

Sulfamethazine

Sulfonamides

 

C12H14N4O2S

651560

[M+H]+

3.48

279.09102

124.08708

204.04367

156.01119

149.02325

Sulfamethizole

Sulfonamides

 

C9H10N4O2S2

179325

[M+H]+

3.32

271.03179

156.01137

108.04485

92.05008

110.06047

Sulfamethoxazole

Sulfonamides

 

C10H11N3O3S

435152

[M+H]+

3.84

254.05939

108.04488

92.05011

156.01140

188.08195

Sulfamethoxypyridazine

Sulfonamides

 

C11H12N4O3S

608731

[M+H]+

3.55

281.07029

108.04487

156.01139

92.05007

126.06644

Sulfamonomethoxine

Sulfonamides

 

C11H12N4O3S

337562

[M+H]+

3.91

281.07029

108.04490

156.01143

126.06648

215.09283

Sulfamoxole

Sulfonamides

 

C11H13N3O3S

552607

[M+H]+

3.30

268.07504

108.04487

156.01140

113.07134

92.05008

Sulfanilamide

Sulfonamides

 

C6H8N2O2S

10000

[M+H]+

1.27

173.03792

93.05784

108.04471

156.01118

128.14344

Sulfanitran

Sulfonamides

 

C14H13N3O5S

30297

[M+H]+

6.08

336.06487

134.06023

156.01142

198.02211

294.05429

Sulfaphenazole

Sulfonamides

 

C15H14N4O2S

523226

[M+H]+

4.63

315.09102

158.07143

92.05010

108.04492

222.03348

Sulfapyridine

Sulfonamides

 

C11H11N3O2S

645554

[M+H]+

2.73

250.06447

108.04486

156.01138

92.05009

184.08705

Sulfaquinoxaline

Sulfonamides

 

C14H12N4O2S

270661

[M+H]+

5.13

301.07537

156.01135

108.04485

92.05010

146.07132

Sulfathiazole

Sulfonamides

 

C9H9N3O2S2

231112

[M+H]+

2.52

256.02089

156.01141

108.04489

92.05010

110.06051

Sulfisomidine

Sulfonamides

 

C12H14N4O2S

398006

[M+H]+

2.35

279.09102

124.08721

108.04486

204.04390

156.01135

Sulfisoxazole

Sulfonamides

 

C11H13N3O3S

318659

[M+H]+

4.08

268.07504

108.04487

156.01140

113.07135

236.07152

Trimethoprim

Sulfonamides

 

C14H18N4O3

1216776

[M+H]+

3.25

291.14517

123.06686

230.11639

261.09829

275.11391

4-epitetracycline

Tetracyclines

5

C22H24N2O8

39702

[M+H]+

3.21

445.16054

410.12328

98.06062

428.13381

392.11267

Chlortetracycline

Tetracyclines

 

C22H23ClN2O8

24711

[M+H]+

4.88

479.12157

462.09501

303.04155

154.04992

444.08462

Doxycycline

Tetracyclines

 

C22H24N2O8

45025

[M+H]+

5.60

445.16054

428.13362

321.07557

267.06506

339.08632

Oxytetracycline

Tetracyclines

 

C22H24N2O9

21747

[M+H]+

3.82

461.15546

426.11848

201.05486

444.12921

337.07094

Tetracycline

Tetracyclines

 

C22H24N2O8

50674

[M+H]+

3.76

445.16054

410.12311

154.04985

98.06058

427.14979

Cefamandole

β-Lactams

13

C18H18N6O5S2

10000

[M+H]+

4.29

463.08529

158.02704

185.03808

141.04804

112.02189

Cefapirin

β-Lactams

 

C17H17N3O6S2

26489

[M+H]+

2.44

424.06315

152.01693

292.05799

124.02215

181.04356

Cefazolin

β-Lactams

 

C14H14N8O4S3

10000

[M+H]+

3.52

455.03729

156.01137

295.06072

323.05573

252.04382

Cefoperazone

β-Lactams

 

C25H27N9O8S2

21710

[M+H]+

3.98

646.14968

143.08159

290.11344

148.03930

360.06463

Cefotaxime

β-Lactams

 

C16H17N5O7S2

21851

[M+H]+

3.51

456.06422

125.00445

167.02742

156.02258

324.05799

Cefoxitin

β-Lactams

 

C16H17N3O7S2

10000

[M+NH4]+

3.86

445.08462

215.04842

339.04627

197.03782

359.02797

Cefquinome

β-Lactams

 

C23H24N6O5S2

21308

[M+H]+

2.90

529.13224

134.09652

167.02740

324.05814

140.01650

Ceftiofur

β-Lactams

 

C19H17N5O7S3

28136

[M+H]+

5.12

524.03629

125.00445

95.01332

241.03893

285.01087

Cefuroxime

β-Lactams

 

C16H16N4O8S

10000

[M+NH4]+

3.38

442.10271

336.06445

179.04523

211.01742

364.06130

Cephacetrile

β-Lactams

 

C13H13N3O6S

10000

[M+NH4]+

2.48

357.08633

252.04365

206.03828

280.03851

234.03330

Cephalothin

β-Lactams

 

C16H16N2O6S2

10000

[M+Na]+

4.97

419.03420

315.02327

359.01297

204.00908

247.98110

Cephradine

β-Lactams

 

C16H19N3O4S

42693

[M+H]+

3.74

350.11690

158.02711

108.08126

176.07068

136.07580

Desacetyl cephapirin

β-Lactams

 

C15H15N3O5S2

27336

[M+H]+

1.12

382.05259

152.01659

112.02200

193.04324

292.05732

Total

 

105

         

Percentage (%)

           

aNumbers that are underlined are response thresholds set to default of 10000 due to low sensitivity of a veterinary drug in UHLPC/ESI Q-Orbitrap

bThe masses of fragments are corrected based on the mass accuracy of a precursor or 214.08963

cColumn number

Results and discussion

Target screening workflow

The workflow of semi-automated qualitative analysis for target screening is shown in Fig. 1. Full MS/dd-MS2 was used to acquire product ion spectra of individual veterinary drugs. The mass-resolution for Full MS was set at 70,000 FWHM, the resolution for dd-MS2 was at 35,000 FWHM, and the isolation window was at 1.0 m/z. The product-ion spectra provided the accurate masses of fragments that were used to build both the compound database (CDB) and the mass spectral (MS) library. The retention times were obtained from the extracted chromatograms of Full MS using the exact masses of individual veterinary drugs. Both CDB and MS library were tested for their applicability for target screening. The development and application of a CDB based on exact (or accurate mass) and retention time are the main focus in the current study since the mass accuracy and retention time tolerance are well accepted criteria for identification and are associated with the identity of a veterinary drug for target screening. The CDB, which included compound name, elemental composition, exact or accurate mass, retention time, and response threshold, was first organized in Microsoft Excel and then imported into TraceFinder. MS library was created using Thermo Library Manager 2.0 by importing individual product ion spectra of veterinary drugs into the library.

For sample data acquisition, Full MS/mDIA (multiplexing data independent acquisition), which acquired spectra of precursor ions as well as their fragments from per defined mass range in multiple steps or events, was utilized. The Q-Orbitrap first performed one Full MS at 70,000 FWHM, followed by mDIA at 17,500 FWHM in 10 steps or loops (Fig. 2A, B, and Table 2). For loop counts from 1 to 8 or MSX ID 1 to 8, a collection of ions isolated by the quadruple in every m/z 50 mass increment from m/z 100 to 500 were sent to the HCD cell for fragmentation, and then fragments were sent to the Orbitrap via the C-trap in eight steps. However, for loop counts 9 and 10, fragments were obtained using the multiplexing function of Q-Orbitrap, i.e., MSX count 4. For example, for loop count 9, a collection of ions, which were first isolated by the quadruple for mass ranges in segments of m/z 500–550, 600–650, 700–750, and 800–850, were sent to the HCD cell sequentially (namely four events) for fragmentation. The fragments from the four events, which were stored in the HCD, were then sent to Orbitrap as one packet of ions via the C-trap in one step. The spectrum obtained in this case was also called a multiplexed spectrum. The same multiplexing is used for loop count 10 for mass ranges in segments of m/z 550–600, 650–700, 750–800, and 850–900. Multiplexing was used for the mass range of m/z 500–900 to speed up the cycle time. In this mass range, less selectivity (small isolation window) is needed, compared with the range of m/z 100–500. Therefore, the extracted chromatograms of Full MS provided the accurate mass and retention time of a precursor, and mDIA likely offered the accurate masses of the fragments generated in the HCD from a precursor for target screening.

Target screening parameters and criteria

The target screening or semi-automated qualitative analysis was performed using TraceFinder 3.3 Target Screening function. The screening parameters and criteria were based on either Retention Time (±0.5 min) and mass accuracy (≤5 ppm) of a Precursor (RTP by Full MS), or Retention Time (±0.5 min) and mass accuracy (≤5 ppm) of the precursor and a Fragment Ion (RTFI by Full MS/mDIA). A method performance acceptability criterion was set at an acceptable false-negative rate of ≤ 5%. Screening results of incurred residues by RTP approach were considered as tentative positive findings, whereas those by the RTFI were taken as confirmative positive findings.

Compound database

The accurate masses, retention times, and response thresholds were three key parameters in the compound database (CDB). The mass correction, retention time alignment, and response threshold adjustment were based on a protocol reported elsewhere [15]. Retention time alignment should be made for every batch of samples but mass correction and response threshold should be corrected and set in the beginning when building the CDB. Furthermore, response threshold can be adjusted accordingly based on routine experiences to reduce false positives if a high constant matrix background occurs.

The three parameters were first organized in a Microsoft Excel CDB template and then imported to Tracefinder 3.3 to create an executable compound database (eCDB). The exact masses of precursors were calculated theoretically from the elemental compositions while the accurate masses of their fragments were obtained from the product-ion spectra of individual standards (50 μg/L) acquired using Full MS/dd-MS2 (Table 3). The Q-Orbitrap performed a three-step normalized collision, i.e., NCE at 20, 40, and 60, to induce fragmentation. The fragments, which were generated sequentially in three NCEs and collected in the HCD, were sent altogether to the Orbitrap analyzer via C-Trap for a single scan detection. Since the stepped NCE was not optimized for each individual veterinary drug, the obtained product-ion spectra did not represent the best scenario in terms of sensitivity for identification, especially for low abundance fragments. In general, the developed CDB served the purpose for target screening of veterinary drugs at the required concentration 1.0 or 10.0 μg/kg levels as indicated in the validated results (Table 4).
Table 4

UHPLC/ESI Q-Orbitrap Full MS/mDIA target screening results of veterinary drug residues spiked in milk

Compound name

Class

Total number

Target screening (Scenario I)a

Target screening (Scenario II)a

1.0 μg/kg

10.0 μg/kg

1.0 μg/kg

10.0 μg/kg

t R (± 0.5 min)

FI (± 5 ppm)

t R and FI

LS10

t R (± 0.5 min)

FI (± 5 ppm)

t R and FI

LST10

t R (± 0.5 min)

FI (± 5 ppm)

t R and FI

LS10

t R (± 0.5 min)

FI (± 5 ppm)

t R and FI

LST10

1b

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Abamectin B1a

Endectocides

7

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

No

No

Yes

Doramectin

Endectocides

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Emamectin B1a

Endectocides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Eprinomectin B1a

Endectocides

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Ivermectin

Endectocides

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Moxidectin

Endectocides

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

No

No

No

Selamectin

Endectocides

 

No

No

No

No

Yes

No

No

Yes

No

No

No

No

Yes

No

No

Yes

Cinoxacin

Fluoroquinolones

17

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Ciprofloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Danofloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Difloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Enoxacin

Fluoroquinolones

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Enrofloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Flumequine

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Lomefloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Marbofloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Nalidixic Acid

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Norfloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Ofloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Orbifloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Oxolinic Acid

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Pipemidic Acid

Fluoroquinolones

 

Yes

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Sarafloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sparfloxacin

Fluoroquinolones

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Lasalocid

Ionophores

5

Yes

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Monensin

Ionophores

 

Yes

No

No

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

Yes

Yes

Yes

Yes

Narasin

Ionophores

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

No

No

No

No

Nigericin

Ionophores

 

Yes

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

No

No

No

No

No

Salinomycin

Ionophores

 

Yes

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Erythromycin

Macrolides

8

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Neospiramycin I

Macrolides

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Oleandomycin

Macrolides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

Yes

Yes

Yes

Yes

Roxithromycin

Macrolides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Spiramycin I

Macrolides

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Tilmicosin

Macrolides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Tylosin A

Macrolides

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Tylosin B

Macrolides

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

2-methyl-4(5)-nitroimidazole

Nitroimidazoles

13

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Dimetridazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Etanidazole

Nitroimidazoles

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

HMMNI

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Ipronidazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Ipronidazole-OH

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Metronidazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Metronidazole-OH

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Nimorazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Ornidazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Ronidazole

Nitroimidazoles

 

Yes

No

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

Yes

Yes

Yes

Yes

Ternidazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Tinidazole

Nitroimidazoles

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

5-hydroxyflunixin

NSAIDS

3

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Flunixin

NSAIDS

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Phenylbutazone

NSAIDS

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Ampicillin

Penicillins

6

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Cloxacillin

Penicillins

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

Yes

Yes

Yes

Yes

Dicloxacillin

Penicillins

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Oxacillin

Penicillins

 

Yes

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Penicillin G

Penicillins

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Penicillin V

Penicillins

 

No

No

No

Yes

Yes

No

No

Yes

No

No

No

No

Yes

No

No

Yes

Florfenicol

Phenicols

2

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Thiamphenicol

Phenicols

 

No

No

No

No

Yes

Yes

Yes

No

No

No

No

No

Yes

Yes

Yes

No

Dapsone

Sulfonamides

26

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfabenzamide

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfacetamide

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfachloropyridazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfadiazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfadimethoxine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfadoxine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfaethoxypyridazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfaguanidine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Sulfamerazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfameter

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamethazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamethizole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamethoxazole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamethoxypyridazine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamonomethoxine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfamoxole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfanilamide

Sulfonamides

 

Yes

Yes

No

Yes

Yes

Yes

Yes

Yes

No

Yes

No

Yes

Yes

Yes

Yes

Yes

Sulfanitran

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

Yes

Yes

Yes

Yes

Yes

Sulfaphenazole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfapyridine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfaquinoxaline

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfathiazole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfisomidine

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Sulfisoxazole

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Trimethoprim

Sulfonamides

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

4-epitetracycline

Tetracyclines

5

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Chlortetracycline

Tetracyclines

 

Yes

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Doxycycline

Tetracyclines

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Oxytetracycline

Tetracyclines

 

Yes

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Tetracycline

Tetracyclines

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Cefamandole

β-Lactams

13

No

No

No

No

Yes

No

No

No

No

No

No

No

No

No

No

No

Cefapirin

β-Lactams

 

Yes

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Cefazolin

β-Lactams

 

No

No

No

No

Yes

Yes

Yes

No

No

No

No

No

Yes

No

No

No

Cefoperazone

β-Lactams

 

Yes

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Cefotaxime

β-Lactams

 

Yes

No

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

Yes

Yes

Yes

Yes

Cefoxitin

β-Lactams

 

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Cefquinome

β-Lactams

 

No

No

No

No

Yes

Yes

Yes

No

No

No

No

No

Yes

No

No

No

Ceftiofur

β-Lactams

 

Yes

No

No

No

Yes

Yes

Yes

Yes

Yes

No

No

No

Yes

No

No

No

Cefuroxime

β-Lactams

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Cephacetrile

β-Lactams

 

No

No

No

No

Yes

No

No

No

No

No

No

No

No

No

No

No

Cephalothin

β-Lactams

 

No

No

No

No

Yes

No

No

No

No

No

No

No

Yes

No

No

No

Cephradine

β-Lactams

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Desacetyl cephapirin

β-Lactams

 

No

No

No

No

Yes

Yes

Yes

Yes

No

No

No

No

Yes

Yes

Yes

Yes

Total

 

105

77

64

63

71

105

95

95

94

61

55

54

58

101

88

88

90

Percentage (%)

  

73

61

60

68

100

90

90

90

58

52

51

55

96

84

84

86

atR: retention time; FI: fragment ion; LST10: library search score threshold set at 10.

bColumn number.

A veterinary drug can be ionized in the form of [M+H]+, [M+NH4]+, or [M+Na]+. Each exact mass of a precursor (protonated or adduct) was selected and entered into Xcalibur Qual Browser to determine which yielded the highest extracted ion peak and the most intense fragment spectrum. Often compound’s protonated form showed the highest abundance (Table 3, column 6). The four most abundant fragments plus the precursor were selected to build the CDB (Table 3, columns 8–12). The accurate mass of a precursor, which was eventually replaced by the exact mass, was always placed in the first position (Table 3, column 8) in the Excel template, followed by the masses of four fragments arranged according to ion abundance from high to low (Table 3, columns 9–12).
  1. (a)

    Mass correction

     
Mass correction was based on either in-spectral mass correction or solvent background lock-mass correction [15]. In brief, this was achieved using the following equations:
$$ {\displaystyle \begin{array}{c}A=B+B\times \left(\varDelta Me/1,000,000\right)\\ {}\varDelta Me=\frac{C-B}{C}\times 1,000,000\end{array}} $$
where ∆Me : mass accuracy or mass error in ppm; A: corrected mass; B: measured mass; C: exact mass or theoretical value.
When the mass error of a precursor was less than 2.5 ppm, the mass correction was made according to the mass accuracy of the precursor in the dd-MS2 product-ion spectrum and this is called in-spectral mass correction. When a precursor was not observed in the dd-MS2 product-ion spectrum or its mass error was greater than 2.5 ppm, the mass correction was made according to the mass accuracy of an ion in the background of a Full MS scan spectrum, i.e., m/z 214.08963, which is n-butyl benzenesulfonamide, and this is the solvent background lock-mass correction. The 2.5 ppm cutoff was arbitrarily chosen to reduce the probability of mass overcorrection. Columns 9–12 in Table 3 present the corrected masses for fragments and column 8 lists the exact masses of the precursors. The masses in columns 8–12 in Table 3 were used to build a compound database for target screening. There were a total of 105 veterinary drugs from 11 groups or classes (Table 3, columns 1 and 2) in the in-house developed compound database. Since eCDB was based on corrected masses (not exact masses), it can only be used for screening, not for identification.
  1. (b)

    Retention time alignment

     
Retention time (tR) alignment is critical for the software to identify an incurred compound of interest in order to match fragments in a CDB (Fig. 2B) or a MS library (Fig. 2C) so as to reduce false positive or negative rates [15]. The individual tR of the veterinary drugs were obtained from their extracted ion chromatograms using the exact masses and were input into the CDB. The tR alignment was achieved using a single stable and well-characterized compound to calculate a correction constant, which was applied to all veterinary drugs in the CDB for its current batch of samples. This correction effectively aligned the retention times to the reference and this process was termed as “tR alignment”. For example, ipronidazole, which eluted at ~4.64 min, was chosen as the reference for tR alignment. If the tR of ipronidazole was 4.64 min in the CDB while the observed tR was 4.70 min in a current batch, the time difference would be 0.06 min. Then, 0.06 min was added to the tR of each veterinary drug in the CDB.
  1. (c)

    Response threshold adjustment

     
Response threshold is a parameter that can be assigned a value for each compound in the CDB [15]. The software integrates peaks when a sample peak response surpasses the threshold. When a generic default is set as the response threshold, this could result in an increased number of false positives. For example, when the response threshold was set at 30,000, the number of false veterinary drug positives was in a range of 8 to 29 in 30 blank samples (Fig. 3A). The detected peaks of false positives resulted from small adjacent peaks, interferences, and noisy baselines, or peaks from a trace amount of veterinary drug residues present in the “blank samples” that were above the response thresholds. It is essential to set appropriate response thresholds to reduce false positives and/or false negatives.
Fig. 3

UHPLC/ESI Q-Orbitrap target screening response threshold adjustment. (A) A number of false positives from blank samples of 10 milk blank matrices prepared in triplicate. Data acquisition: Full MS/mDIA. OPA: original peak area. (B) Response thresholds of individual veterinary drugs calculated as 2% original peak areas from 20 injections at 10 μg/L or ppb in solvent

It is well known that the ionization efficiency is compound-dependent and matrix effects contribute to the responses of individual veterinary drugs in the electrospray ion source as a result of ion suppression and enhancement. For example, fluoroquinolones have much higher ionization efficiency than endectocides, penicillins, tetracyclines, and β-lactams (Fig. 3B). This is observed from the differences in peak areas of the veterinary drugs injected to the UHPLC/ESI Q-Orbitrap at the same concentration. Therefore, response thresholds should be set accordingly for each veterinary drug.

To set appropriate response thresholds for individual veterinary drugs, a mixture of standards, which was prepared at 10.0 μg/L in solvent, was injected 20 times on to the UHPL/ESI Q-Orbitrap, and data were acquired by Full MS/mDIA. Ten μg/L in solvent was equivalent to 10.0 μg/kg in sample when the final sample extracts were concentrated in 3:1. Note that 1.0 μg/L was not used since it was too low for some of the veterinary drugs to generate a quantifiable peak area or to be detected.

After data acquisition and processing, the peak areas of the individual veterinary drugs were averaged. Due to potential matrix effects (mainly ion suppression) and instrument sensitivity variation from day-to-day, the values of 2%, 10%, 20%, or 40% Original Peak Areas (OPA) at 10.0 μg/L were tested for their applicability to determine the appropriate response thresholds to be used in this study. When response thresholds were set at 2%, 10%, 20%, or 40% OPA, the number of false positives was reduced significantly compared with those from the default of 30,000 (Fig. 3A). However, in order to detect veterinary drugs at 1.0 μg/kg, 2% OPA was used to set response thresholds for target screening (Fig. 3B). To reduce false negatives, certain response thresholds were set to 10,000 (Table 3, column 5) to help compensate for the low sensitivity of a veterinary drug in UHLPC/ESI Q-Orbitrap.

Mass spectral library

Mass spectral (MS) library was built by importing individual dd-MS2 product ion spectra of veterinary drugs using Thermo Library Manger 2.0. The MS library matching was achieved using the “Reverse Search” function. Spectrum tolerance was 5 ppm. Score threshold was set at 10, 20, or 40 to explore its applicability for screening. The MS library matching served to provide additional information for confirmative screening (Fig. 2C).

Validation and results

The target screening method was validated using SANTE/11945/2015 [17] as a reference. The validation of a screening method based on targeted concentration levels (TCL) focused on detectability. The validation involved analysis of at least 20 samples spiked per TCL. Since the screening method is only intended to be used as a qualitative method, there are no requirements with regard to recovery of the analytes. In addition, there is no need for a strict criterion for the number of false positives detected.

Ten blank milk samples were spiked at 1.0 μg/kg and 10.0 μg/kg in duplicate, respectively, and the experiment was repeated on three different days. For every veterinary drug, there were a total of 20 samples per batch that were used to validate the method and to meet the criterion that a veterinary drug had to be detected in at least 95% of the samples (i.e., an acceptable false-negative rate of ≤5%).

After data acquisition, the results from samples spiked at 1.0 μg/kg and 10.0 μg/kg were processed by TraceFinder 3.3, and reviewed accordingly. Table 4 and Fig. 4 present the final validated results based on the criteria of mass accuracy ±5 ppm and tR ±0.5 min by either RTP or RTFI using 2% OPA as response thresholds. Taking Day 1 at 10.0 μg/kg as an example, 103 veterinary drugs were detected by RTP and 88 by RTFI (Fig. 4A, Data Table rows 1 and 3). The method proved to be reproducible as indicated by the repeatability of the results from 3 different days at the concentration level of 10.0 μg/kg but some variations at 1.0 μg/kg. In general, there were more veterinary drugs detected for both tentative (by RTP) and confirmative (by RTFI) screening at 10.0 μg/kg than at 1.0 μg/kg as a result of higher peak intensities at the higher concentration (Fig. 4A). At 10.0 μg/kg, almost all 105 veterinary drugs were found by RTP approach, which served the purpose of target screening.
Fig. 4

(A) UHPLC/ESI Q-Orbitrap target screening results based on compound database with tR tolerance ±0.5 min and mass accuracy ≤5 ppm; (B) target screening results based on mass spectral library with a score threshold at 10, 20, and 40, respectively. Samples were spiked at 1.0 and 10.0 μg/kg, respectively. Data acquisition: Full MS/mDIA. OPA: original peak area. RTP: retention time and precursor. FI: fragments. RTFI: retention time and fragment ion. LST: library search threshold. S I: Scenario I. S II: Scenario II.

Detectability and its reproducibility

According to both Commission Decision 2002/657/EC [19] and SANTE/11945/2015 [17], a qualitative screening method should be able to detect an analyte with a false compliant rate of <5% at the level of interest [19], or the screening method should be able to detect an analyte at the screening detection limit or reporting limit in at least 95% of the samples (i.e., an acceptable false-negative rate of 5%) [17]. The validation of detectability can be achieved using a batch of 20 samples that are analyzed in the same batch. However, it is not clear how the “reproducibility” (between-day) of detectability should be evaluated for a qualitative or screening method.

In our study, we repeated the detectability experiment on three different days (20 samples per batch). Within-day the criterion, that is, an analyte was detected in at least 95% of the samples (i.e., an acceptable false-negative rate of 5%), was followed. There were some issues with “reproducibility” from the 3-day experiments. Some veterinary drugs were detected on day-1 but not in all 3 days, especially at a low concentration level such as 1.0 μg/kg. Therefore, the final list of veterinary drugs that are included in the method can be consolidated in two ways. One represents the best detection scenario to minimize false negatives (Scenario I). Another represents the assured detection scenario of non-compliant results (Scenario II).

For Scenario I, when a veterinary drug was detected in at least 95% of the samples on one of the 3-day experiments, it was included in the method. The results in Fig. 4A (the last 4th and 3rd columns) and Table 4 (columns 4–6, 8–10) represent the best detection scenario for the method. In other words, the method was able to tentatively screen 77 veterinary drugs and to confirmatively screen 63 at 1.0 μg/kg (Fig. 4A, the last 4th column); and to tentatively screen 105 veterinary drugs and to confirmatively screen 95 at 10.0 μg/kg (Fig. 4A, the last 3rd column).

For Scenario II, only when a veterinary drug was detected in at least 95% of the samples on all 3-day experiments, it was included in the method. This was the worst case scenario in terms of the number of veterinary drugs to be detected with assured certainty for target screening. Table 4 (columns 12–14, 16–18) and Fig. 4A (the last two columns) also indicated the veterinary drugs that were detected on all three days. In this case, the method was able to tentatively screen 61 veterinary drugs and to confirmatively screen 54 at 1.0 μg/kg (Fig. 4A, the last 2nd column); and to tentatively screen 101 veterinary drugs and to confirmatively screen 88 at 10.0 μg/kg (Fig. 4A, the last column).

The number of veterinary drugs that are included in the final list are different based on the detectability between days. Since the target screening method focused on detectability, Scenario II is recommended to be adopted to consolidate the final list for the method.

As for MS library search, the “Reverse Search” was selected, and the spectrum tolerance was set at 5 ppm as a standard measure related to mass accuracy. The other parameter that was applicable for target screening was the Library Search score Threshold (LST). The LST can be selected arbitrarily between 0 and 100. In this study, we tested its value at 10, 20, and 40, and the results are shown in Fig. 4B. Apparently, the higher score threshold, the lower of numbers matched. The results from LST10 were similar to the RTP, FI, and RTFI values as observed in Fig. 4A, and specifically for each veterinary drug in Table 4. Therefore, library search with score threshold of 10 can potentially be used to provide additional information, likely visual spectra, for the certainty of target screening.

Conclusions

UHPLC/ESI Q-Orbitrap Full MS and mDIA along with a compound database can serve as a practical approach for target screening. Full MS/dd-MS2 acquired data of individual veterinary drugs to build a compound database and a MS library of 105 veterinary drugs. Accurate mass, retention time, and response threshold were three key parameters that needed to be corrected, aligned, and adjusted, respectively, when optimizing a compound database to reduce false negatives and/or false positives. Full MS/mDIA was used to acquire data for target screening of veterinary drug residues in sample. The screening parameters and criteria were based on either retention time (±0.5 min) and mass accuracy (≤5 ppm) of a precursor (RTP by Full MS), or retention time (±0.5 min) and mass accuracy (≤ 5 ppm) of the precursor and a fragment ion (RTFI by Full MS/mDIA). The screening method performance acceptability criterion was set at an acceptable false-negative rate of ≤5%, repeated on three different days. Based on the criteria and parameter settings used in this study, the RTP approach tentatively found at least 58% and 96% of the 105 veterinary drugs, whereas the RTFI confirmatively screened 51% and 84% in milk at 1.0 and 10.0 μg/kg, respectively. The RTP approach may be the preferred choice for target screening to avoid possible false negative results, whereas the RTFI approach increases the likelihood of screening a true positive of incurred residue in a sample. MS library can provide additional information or visual mass spectrum for target screen using appropriate score threshold. For future work, the exact masses of all fragments need to be determined for identification purpose in addition to target screening.

Notes

Compliance with ethical standards

Conflict of interest

There is no potential conflict of interest in current study.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jian Wang
    • 1
  • Daniel Leung
    • 1
  • Willis Chow
    • 1
  • James Chang
    • 2
  • Jon W. Wong
    • 3
  1. 1.Calgary LaboratoryCanadian Food Inspection AgencyCalgaryCanada
  2. 2.ThermoFisher ScientificSan JoseUSA
  3. 3.Center for Food Safety and Applied NutritionUS Food and Drug AdministrationCollege ParkUSA

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