Multiresidue Method for Quantification of Nitroimidazoles, Anthelmintics, Amphenicols, and Dyes in Meat, Shrimp, and Fish by Using a Modified QuEChERS-type Extraction and Isotope Dilution Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

This paper presents an analytical multiresidue method for the quantification of 7 nitroimidazoles, 19 anthelmintics, 3 amphenicols, and 4 dyes in poultry, pork, beef, horse, shrimp, and fish samples by isotope dilution ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) in a single 6 min run. Samples were prepared using a modified quick, easy, cheap, effective, rugged and safe (QuEChERS)-type extraction with ethyl acetate and Na₂SO₄-NaCl (2:1), concentration into dimethyl sulfoxide (DMSO), and defatting by liquid-liquid extraction (LLE) with n-hexane. No clean-up using solid-phase extraction (SPE) cartridges or dispersive solid-phase extraction (DSPE) was necessary. The linearity ranges (in μg kg^-1) were 0.10-0.60 for chloramphenicol, 0.50-3.0 for dyes, 1.5-9.0 for nitroimidazoles, and 5.0-30.0 for the other drugs. The limits of quantitation (LOQ) were established at the lowest calibration curve levels. Except for chloramphenicol in the horse matrix, analyte recovery and reproducibility were satisfactory (recovery (f_REC): 75.0 to 116.6%; coefficient of variation (CV): 1.2 to 29.9%). For the unapproved substances and the substances with a maximum residue level (MRL), respectively, the decision limits (CCα) were 0.14-4.10 and 5.28-1003.60 μg kg^-1, while the detection capabilities (CCβ) were 0.13-3.33 and 5.46-11.56 μg kg^-1.

Keywords:
nitroimidazoles; anthelmintics; amphenicols; dyes; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

Introduction

Nitroimidazoles, benzimidazoles, imidazothiazoles, salicylanilides, amphenicols, and dyes are drugs whose residues must be investigated in foodstuffs, such as meat, shrimp, and fish, to ensure that their residual concentrations are below the maximum residue levels (MRL) or the unapproved use levels in the case of banned substances in food-producing animals (e.g., 5-nitroimidazoles, chloramphenicol, and dyes).

Figure 1 shows the chemical structures of the investigated drug classes. 5-Nitroimidazoles are imidazoles that contain a NO₂ group on the 5-ring position and are active against anaerobic bacteria and protozoa. They are banned due to possible carcinogenicity and mutagenicity in humans and are rapidly metabolized in beef, porcine, and avian species, forming hydroxyl metabolites in the C-2 position, which should also be investigated.¹ Benzimidazoles, imidazothiazoles, and salicylanilides are used as anthelmintic agents in the control of parasitic infections in livestock animals. Amphenicols are efficient antibiotics, but chloramphenicol is a banned drug in food-producing animals due to its serious adverse effects on human health, including aplastic anemia.² Finally, triphenylmethane dyes like crystal violet and malachite green have an effect on fungus and protozoa, which would make them useful in fish and shrimp farms, mainly due to their low costs.³,⁴ However, their use in food for human consumption was banned because, once absorbed, they are quickly reduced to leuco crystal violet and leuco malachite green, which have negative side effects on human health,⁵ including carcinogenicity, teratogenicity, and mutagenicity.⁴

Figure 1
Chemical structures of investigated drug classes.

Table 1 summarizes the limits for the investigated drugs adopted in our validation studies, which are based on the Brazilian Health Regulatory Agency (ANVISA),⁶ the MMPR - EURL guidance 2022,⁷ and the European Commission Regulation 37/2010/EU.⁸ Whenever a limit was not established, it was assumed to be 5 μg kg^-1 for validation purposes.

Table 1
Limits of the investigated drugs for validation purposes

Group	Analyte	Limit / (μg kg-1)
		Poultry	Pork	Beef	Horse	Shrimp	Fish
Nitroimidazole	dimetridazolea	1.5	1.5	1.5	1.5	1.5	1.5
	dimetridazole hydroxidea	1.5	1.5	1.5	1.5	1.5	1.5
	ronidazolea	1.5	1.5	1.5	1.5	1.5	1.5
	metronidazolea	1.5	1.5	1.5	1.5	1.5	1.5
	metronidazole hydroxidea	1.5	1.5	1.5	1.5	1.5	1.5
	ipronidazolea	1.5	1.5	1.5	1.5	1.5	1.5
	ipronidazole hydroxidea	1.5	1.5	1.5	1.5	1.5	1.5
Benzimidazole	albendazoleb	100	5	100	100	5	5
	albendazole sulfoneb	100	5	100	100	5	5
	albendazole sulfoxideb	100	5	100	100	5	5
	albendazole 2-amino-sulfoneb	100	5	100	100	5	5
	fenbendazolec	50	100	100	100	5	5
	fenbendazole sulfone (= oxfendazole sulfone)c	50	100	100	100	5	5
	oxfendazolec	50	100	100	100	5	5
	oxibendazole	5	100	5	5	3	3
	febantel	50	100	100	100	5	5
	flubendazole	200	10	5	5	5	5
	aminoflubendazole	5	5	5	5	5	5
	thiabendazole	5	100	100	5	5	5
	5-hydroxy-thiabendazole	5	100	100	5	5	5
	triclabendazoled	5	5	250	5	5	5
	triclabendazole sulfoned	5	5	250	5	5	5
	mebendazole	5	5	60	5	5	5
	ketotriclabendazoled	5	5	250	5	-	-
Imidazothiazole	levamisole	10	10	10	100	3	3
Salicylanilide	closantel	5	5	1000	5	5	5
Amphenicol	chloramphenicola	0.15	0.15	0.15	0.15	-	-
	florfenicol	50	100	100	100	-	-
	thiamphenicol	50	50	5	5	-	-
Dye	malachite greena	-	-	-	-	0.50	0.50
	leuco malachite greena	-	-	-	-	0.50	0.50
	crystal violeta	-	-	-	-	0.50	0.50
	leuco crystal violeta	-	-	-	-	0.50	0.50

• ^a
  Prohibited or unauthorized pharmacologically active substances.
• ^b
  The limit refers to the sum of albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole 2-amino sulfone, expressed as albendazole.
• ^c
  The limit refers to the sum of fenbendazole, oxfendazole, and fenbendazole-sulfone, expressed as oxfendazole-sulfone.
• ^d
  The limit refers to the sum of compounds that may be oxidized to ketotriclabendazole.

In Brazil, the Ministry of Agriculture, Livestock, and Food Supply (MAPA) is responsible for monitoring the quality of foodstuffs, livestock feed, fertilizers, and vaccines, among others. As part of the National Control Plan for Residues and Contaminants (PNCRC),⁹,¹⁰ several foodstuff matrices are investigated for the presence of different classes of residues. Therefore, selective, sensitive, and high-throughput multiresidue analytical methods, which allow the quantification of a broad range of trace contaminants in the same sample and using a single chromatographic run, contribute to saving time and money and optimizing resources.

Several methods based on QuEChERS-type (quick, easy, cheap, effective, rugged and safe) extractions coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were described in the literature for the quantification of nitroimidazoles,¹,¹¹-¹⁵ anthelmintics,¹¹,¹²,¹⁶-²³ amphenicols,¹¹-¹³,¹⁵ or dyes.³-⁵,²⁴ However, to our knowledge, no previous method performed the simultaneous quantification of all four drug classes, in six food matrices (poultry, pork, beef, horse, shrimp, and fish), in a single chromatographic run.

In this work, we present a multiresidue analytical method based on a modified QuEChERS-type extraction with ethylacetate and Na₂SO₄-NaCl (2:1), concentration into dimethyl sulfoxide (DMSO), defatting by liquid-liquid extraction (LLE) with n-hexane, followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), with MS polarity switching to detect both positively and negatively charged ions in a single 6-min run. The method was optimized and validated for the simultaneous identification, confirmation, and quantification of 7 nitroimidazoles, 19 anthelmintics, 3 amphenicols, and 4 dyes in meat, shrimp, and fish samples.

Experimental

Instruments and apparatus

The chromatographic analysis was performed in a Waters UHPLC-MS/MS (Waters Technologies, São Paulo, Brazil) system, consisting of an Acquity i-class UHPLC chromatograph with autosampler, interfaced to a XeVo TQS triple quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source in both positive and negative ion-switching and multiple reaction monitoring (MRM) modes. A Waters Acquity UPLC C18 column (BEH-C18, 50 × 2.1 mm internal diameter (i.d.), 1.7 µm) (Waters Technologies, São Paulo, Brazil) was used for chromatographic separation. Data treatment was achieved using the software MassLynx^TM 1.4 Workstation and TargetLynx^TM (Waters Technologies, São Paulo, Brazil).

Reagents and chemicals

Anhydrous sodium sulfate (JTBaker, Phillipsburg, USA), sodium chloride (Lioserum, São Paulo, Brazil), ethyl acetate (Fischer Scientific, Hampton, USA), n-hexane (Honeywell, São Paulo, Brazil), dimethyl sulfoxide (Scharlab, Taboão da Serra, Brazil), formic acid (PanReac, Barcelona, Spain), ammonium formate (Sigma-Aldrich, Cotia, Brazil), and N,N-dimethylformamide (Fischer Scientific, Waltham, USA) were of analytical grade. LC MS grade methanol and acetonitrile (Supelco, Darmstadt, Germany) were used for the preparation of standard solutions and mobile phase, respectively.

Standard solutions

Individual standard stock solutions containing 200 μg mL^-1 of individual drugs and internal standards (IS) were prepared and stored at -10 °C for up to 1 year (except for solutions of dyes, which were stored for up to 6 months). Acetonitrile (ACN) was used for the dissolution of dyes, while methanol was used for the other drugs (this is valid for intermediate and working solutions). Whenever necessary, some drops of N,N-dimethylformamide were added to completely dissolve the standard material. The following analytical standards were from Dr. Ehrenstorfer GmbH (Augsburg, Germany): dimetridazole, ronidazole, metronidazole, metronidazole hydroxide, ipronidazole, albendazole 2-amino sulfone, triclabendazole, triclabendazole sulfone, chloramphenicol, florfenicol, thiamphenicol, closantel, leuco crystal violet, dimetridazole hydroxide-d₃, metronidazole-d₄, metronidazole hydroxide-d₂, ipronidazole-d₃, chloramphenicol-d₅, crystal violet-d₆, and leuco crystal violet-d₆. Dimetridazole hydroxide was from LGC Standards (Teddington, UK). Ipronidazole hydroxide, albendazole, albendazole sulfone, albendazole sulfoxide, febantel, aminoflubendazole, thiabendazole, 5-hydroxy-thiabendazole, levamisole hydrochloride, malachite green, dimetridazole-d₃, ronidazole-d₃, ipronidazole hydroxide-d₃, albendazole sulfoxide-methyl-d₃, oxfendazole-d₃, fenbendazole-d₃, febantel-d₆, flubendazole-d₃, thiabendazole-d₆, mebendazole-d₃, malachite green-d₅, and leuco malachite green-d₆ were purchased from Sigma-Aldrich (Barueri, Brazil). Fenbendazole, fenbendazole sulfone, oxfendazole, oxibendazole, flubendazole, mebendazole, leuco malachite green, and crystal violet was obtained from CPA Chem (Bogomilovo, Bulgaria). Ketotriclabendazole was from NMI (West Lindfield, Australia), whereas albendazole-d₇ was purchased from TRC (Toronto, Canada).

Intermediate solutions were prepared as follows, and stored at -10 °C for up to 1 year (except for solutions of dyes, which were stored for up to 1 month): (i) 10 μg mL^-1 of nitroimidazoles, (ii) 10 μg mL^-1 of nitroimidazoles IS, (iii) 10 μg mL^-1 of chloramphenicol, (iv) 10 μg mL^-1 of chloramphenicol IS, (v) 5 μg mL^-1 of dyes, and (vi) 5 μg mL^-1 of dyes IS. For the other drugs, no intermediate solutions were necessary.

Working solutions were prepared from the intermediate solutions (if available) or stock solutions, as follows, and stored at -10 °C for up to 6 months (except for solutions of dyes, which were stored for up to 1 day): (i) nitroimidazoles (0.15 μg mL^-1) plus chloramphenicol (0.010 μg mL^-1), (ii) nitroimidazoles IS (0.15 μg mL^-1) plus chloramphenicol IS (0.015 μg mL^-1), (iii) dyes (0.05 μg mL^-1), (iv) dyes IS (0.15 μg mL^-1), (v) remaining drugs (0.50 μg mL^-1 each), (vi) IS of the remaining drugs (0.50 μg mL^-1 each).

Precaution for dyes

Whenever dyes were investigated, the labeling of tubes and flasks was preferably made using pencils instead of pens, to avoid ink contamination, and the exposure to light was minimized, as previously described.³ Although crystal violet and leuco crystal violet in fortified fish/shrimp matrices were stable for at least 23 days when kept at -20 °C, malachite green was unstable in shrimp, and leuco malachite green was unstable in fish. Therefore, the extraction and UHPLC-MS/MS analysis were carried out on the same day.

Modified QuEChERS-type sample extraction

Muscle samples were cut into small pieces and weighed into 50-mL polypropylene centrifuge tubes (5 ± 0.05 g). The remaining muscle samples were stored in plastic bags at temperatures below -10 °C. Samples were spiked with 100 µL of IS working solutions (approximately 2 times the MRL, the minimum method performance requirement (MMPR), or the report action level (RPA)) and left to sit for 5 min. Then, a 10 mL aliquot of ethyl acetate was added and the tubes were mixed at 1500 rpm for 1 min using a 2010 Geno/Grinder tissue homogenizer (Spex Sampleprep, Metuhen, USA) and sonicated at 37 kHz for 5 min. Subsequently, the tubes received 6 g of a salting-out mixture (anhydrous Na₂SO₄:NaCl 2:1) and were again homogenized. After centrifugation at 4 °C for 10 min at 4000 rpm, the tubes were held in an ultra-freezer at a temperature below -60 °C for 10 min. The supernatants were transferred into 10-mL glass tubes containing 1 mL DMSO and evaporated in a water bath or solvent evaporating unit at 45 to 50 °C under air stream until the ethyl acetate was eliminated. Thereafter, 1 mL n-hexane was added to the tubes with DMSO, and the mixtures were vortexed, transferred into Eppendorf tubes, and centrifuged for 10 min at 4 °C and 14000 rpm. The fat-containing n-hexane upper layers were removed using vacuum and discharged, while the lower DMSO layers were filtered through 0.22 µm hydrophilic nylon syringe filters (Analítica, São Paulo, Brazil), transferred to vials, and injected onto the UHPLC-MS/MS system.

Sample preparation for method validation

The method validation was performed based on the procedures of the European Commission Decision 2002/657/EC.²⁵ Linearity range, limit of quantitation (LOQ), recovery, repeatability, within-laboratory reproducibility, selectivity, robustness, measurement uncertainty (U), decision limit for confirmation (CCα), and detection capability for screening (CCβ) were evaluated. The general procedure for fortification during validation was the following: matrix blanks (5 ± 0.05 g) were weighed into polypropylene centrifuge tubes, spiked with 100 μL of IS working solutions, fortified with working standard solutions at the required concentration levels, left to sit for 5 min, and then extracted as previously described.

Matrix-fortified calibration curves consisted of fortified matrix blanks prepared in the following concentration ranges: (i) nitroimidazoles: 1.5 to 9.0 μg kg^-1, 6 levels; (ii) chloramphenicol: 0.10 to 0.60 μg kg^-1, 6 levels; (iii) benzimidazoles, levamisole, closantel, florfenicol, thiamphenicol, and closantel: 5.0 to 30 μg kg^-1 (poultry, pork, beef, horse), 6 levels, or 2.50 to 30 μg kg^-1 (shrimp and fish), 7 levels; and (iv) dyes: 0.50 to 3.0 μg kg^-1 (only shrimp and fish), 6 levels. At least 12 fortified matrix blanks were prepared at the concentration of the first level of the calibration curves, which were initially considered to be the LOQs, to verify satisfactory recovery and reproducibility.

For the evaluation of poultry recovery and within-laboratory reproducibility, 6 replicates of fortified matrix blanks were prepared on 3 different days by 2 different analysts (a total of 18). For the other matrices, two sets of 10 fortified matrix blanks were prepared on 2 different days by 2 different analysts. The fortifications were done at the MRL, MRPL, RPA, or LOQ. For poultry, two other fortification levels were added (results not shown). Whenever the MRL exceeded the calibration curve range, additional curve calibration levels were added.

For poultry, CCα and CCβ were estimated using the ISO 11843²⁶ approach, based on the calibration curves prepared on three different days. For the other matrices, CCα and CCβ were estimated from the standard measurement uncertainty results of 20 fortified matrix blanks, according to the Commission Implementing Regulation 2021/808.²⁷

Isotope dilution UHPLC-MS/MS analysis

The UHPLC-MS/MS experimental conditions are shown in Table 2. The MS/MS conditions were optimized via infusion injection or taken from the literature. A precursor ion (parent mass) and two product ions (base and secondary fragments) were monitored for each analyte (Table 3). Analyte identity confirmation was based on retention time and area ratio between quantification and confirmation ions (ion ratio). The ion ratio of the analyte shall correspond to those of the standards, within a ± 40% relative deviation.²⁷ Analyte quantification was calculated from the equipment response (area ratio between quantification product ion and IS, multiplied by the IS concentration), except in the case of closantel, for which the peak area was used since no IS was added. At the beginning of each analysis, a reference solution (50 μL of each standard working solution and 100 μL of each IS working solution, which were evaporated and reconstituted in 1 mL DMSO) was injected into the system to check if the peaks of all investigated analytes were present and to adjust the detection windows, if necessary.

Table 2
UHPLC-MS/MS experimental conditions

Chromatographic conditions
Chromatographic column	Acquity UPLC BEH C18, 50 × 2.1 mm i.d., 1.7 µm
Mobile phase A	0.1% formic acid in 5 mmol L-1 ammonium formate
Mobile phase B	0.1% formic acid in ACN-water (95:5, v v-1)
Flow rate / (mL min-1)	0.4
Gradient program	0-2 min: 0 to 20% B
	2-3.5 min: 20 to 95% B
	3.5-4.5 min: 95% B
	4.5-5 min: 95 to 0% B
	5-6 min: 0% B
Diverter valve	0-1 min: on; 1-5 min: off; 5-6 min: on
Column temperature / °C	35
Injector temperature / °C	18
Injection volume / µL	5
MS detector
Scan mode	multiple reaction monitoring (MRM)
Ionization mode	electrospray ionization (ESI)
Polarity	switching between positive and negative modes
Capillary voltage / kV	3.0 (ESI+) and 2.4 (ESI-)
Source offset / V	80 (ESI+) and 60 (ESI-)
Source temperature / °C	150
Desolvation temperature / °C	400
Cone gas flow rate / (mL min-1)	150
Desolvation gas flow rate / (L h-1)	700
Nebulizer gas flow rate / Bar	7
Ion energy (1)	0.0 (ESI+) and 0.5 (ESI-)
Ion energy (2)	0.8 (ESI+) and 1.0 (ESI-)
Multiplier / V	560
Collision gas	argon
Collision gas flow rate / (L h-1)	0.13

Table 3
Parameters for Waters XeVo UHPLC/MS-MS

Group	Analyte	ESI	Precursor (m/z)	Base fragment (m/z)	Secondary fragment / (m/z)	Dwell time / s	Cone / V	Collision energy / eV	IS
Nitroimidazole	dimetridazole	+	142.00	96.00	81.00	0.020	25	15/25	dimetridazole-d3
	dimetridazole hydroxide	+	158.10	140.10	55.10	0.020	30	15/22	dimetridazole hydroxide-d3
	ronidazole	+	201.00	139.80	55.00	0.020	20	12/21	ronidazole-d3
	metronidazole	+	171.90	127.80	81.90	0.020	25	13/25	metronidazole-d4
	metronidazole hydroxide	+	187.90	125.90	122.90	0.020	26	16/13	metronidazole hydroxide-d2
	ipronidazole	+	169.90	123.80	108.80	0.020	34	17/24	ipronidazole-d3
	ipronidazole hydroxide	+	185.90	167.80	121.90	0.020	25	13/19	ipronidazole hydroxide-d3
Benzimidazole	albendazole	+	266.10	234.10	191.10	0.008	32	21/31	albendazol-d7
	albendazole sulfone	+	298.10	224.00	158.80	0.005	35	35/35	albendazol-d7
	albendazole sulfoxide	+	282.10	240.00	207.90	0.005	35	15/25	albendazole sulfoxide-d3
	albendazole 2-amino-sulfone	+	240.20	198.20	133.10	0.005	40	20/30	oxfendazole-d3
	fenbendazole	+	300.10	268.20	159.10	0.005	27	21/36	fenbendazole-d3
	fenbendazole sulfone	+	332.10	300.00	158.80	0.005	35	22/40	fenbendazole-d3
	oxfendazole	+	316.10	284.29	159.00	0.005	35	19/32	oxfendazole-d3
	oxibendazole	+	250.00	218.00	175.90	0.007	29	18/28	oxfendazole-d3
	febantel	+	447.10	415.20	383.20	0.015	21	13/20	febantel-d6
	flubendazole	+	314.10	282.20	122.90	0.005	23/35	35/35	flubendazole-d3
	aminoflubendazole	+	256.10	122.70	94.60	0.005	50	28/38	flubendazole-d3
	thiabendazole	+	202.00	175.00	131.00	0.005	43	26/32	tiabendazol-d6
	5-hydroxy-thiabendazole	+	218.00	191.00	147.00	0.005	42	26/32	tiabendazol-d6
	triclabendazole	-	358.90	334.10	274.10	0.015	45	27/35	flubendazole-d3
	triclabendazole sulfone	-	389.00	309.90	244.20	0.015	45	27/28	dimetridazole-d3
	mebendazole	+	296.10	264.20	104.90	0.005	34	20/33	mebendazole-d3
	ketotriclabendazole	-	329.00	184.00	182.00	0.010	45	25/25	tiabendazol-d6
Imidazothiazole	levamisole	+	205.10	178.10	123.00	0.005	35	23/35	oxfendazole-d3
Salicylanilide	closantel	+	660.70	315.10	126.80	0.050	44	33/46	-
Amphenicol	chloramphenicol	-	321.00	257.00	152.10	0.050	25	12/18	chloramfenicol-d5
	florfenicol	-	356.00	336.00	185.00	0.025	25	11/22	chloramfenicol-d5
	thiamphenicol	-	354.00	290.00	185.00	0.025	30	14/23	chloramfenicol-d5
Dye	malachite green	+	329.30	313.40	208.10	0.020	60	35/30	malachite green-d5
	leuco malachite green	+	331.30	316.40	315.40	0.020	45	20/20	leuco malachite green-d6
	crystal violet	+	372.30	356.30	240.40	0.020	100	30/40	crystal violet-d6
	leuco crystal violet	+	374.30	239.00	238.10	0.020	50	25/28	leuco crystal violet-d6
IS	dimetridazole-d3	+	145.00	99.00	-	0.020	25	15	-
	dimetridazole hydroxide-d3	+	160.90	142.80	-	0.020	30	15	-
	ronidazole-d3	+	204.00	142.80	-	0.020	20	12	-
	metronidazole-d4	+	175.80	127.80	-	0.020	25	13	-
	metronidazole hydroxide-d2	+	189.90	127.90	-	0.020	26	16	-
	ipronidazole-d3	+	172.90	126.80	-	0.020	34	17	-
	ipronidazole hydroxide-d3	+	188.90	170.80	-	0.020	25	13	-
	albendazole-d7	+	273.10	191.10	-	0.008	32	21	-
	albendazole sulfoxide-d3	+	285.10	243.00	-	0.005	35	15	-
	oxfendazole-d3	+	319.10	159.00	-	0.005	35	32	-
	fenbendazole-d3	+	302.90	268.10	-	0.005	15	20	-
	febantel-d6	+	453.20	383.10	-	0.015	30	20	-
	flubendazole-d3	+	317.10	282.10	-	0.005	35	23	-
	thiabendazole-d6	+	208.00	181.00	-	0.005	43	26	-
	mebendazole-d3	+	299.10	264.00	-	0.005	34	20	-
	chloramfenicol-d5	-	326.10	157.10	-	0.005	25	18	-
	malachite green-d5	+	334.20	318.20	-	0.020	65	32	-
	leuco malachite green-d6	+	337.30	240.30	-	0.020	35	30	-
	crystal violet-d6	+	378.40	362.20	-	0.020	65	35	-
	leuco crystal violet-d6	+	380.30	364.30	-	0.020	40	30	-

Measurement uncertainties, decision limit (CCα), and detection capability (CCβ) calculations

Expanded measurement uncertainties (U) were estimated using a “top-down” approach based on the within-laboratory reproducibility standard deviation. The calculations of CCα and CCβ were based on the Commission Implementing Regulation 2021/808.²⁷ The value of CCα for banned substances was calculated from equation 1, while equation 2 was used to calculate CCα for substances with an MRL. CCβ was calculated from equation 3.

(1) $$\mathrm{CC}\alpha =\mathrm{LOQ}+2.33{\mathrm{u}}_{\mathrm{c}}$$

(2) $$\mathrm{CC}\alpha =\mathrm{MRL}+1.64{\mathrm{u}}_{\mathrm{c}}$$

(3) $$\mathrm{CC}\beta =\mathrm{LOQ}+1.64{\mathrm{u}}_{\mathrm{c}}$$

where CCα: decision limit, CCβ: detection capability, LOQ: limit of quantitation, MRL: maximum residue level, u_c: combined measurement uncertainty (u_c = U/k), U: expanded measurement uncertainty, k: coverage factor.

Results and Discussion

Modified QuEChERS-type extraction

Previously, two other methods were validated in our laboratories.³,²⁸ The first method²⁸ was used for the quantification of nitroimidazoles, benzimidazoles, and avermectins in beef muscle and was based on the extraction of 5 g samples (2 replicates) with acetic acid-ACN (2:98, v/v) and Na₂SO₄-NaCl 2:1, clean-up of supernatants by dispersive solid-phase extraction (DSPE) with C18 MgSO₄, and evaporation until dryness. The first replicate was dissolved in 0.1% formic acid in ACN-water (95:5, v/v) (for analysis of triclabendazole, closantel, and avermectins), while the second one was dissolved in 0.1% formic acid in 5 mmol L^-1 ammonium formate (for the analysis of nitroimidazoles and benzimidazoles). The replicates were analyzed by independent UHPLC-MS/MS runs, using polarity switching to detect positively and negatively charged ions. The second method³ was developed to quantify dyes in shrimp and fish and consisted of the extraction of 2 g samples with formic acid-acetonitrile (1:99, v/v) and MgSO₄, evaporation of the supernatants, dilution in ACN-water (1:1, v/v), clean-up by DSPE with C18-MgSO₄, followed by a single UHPLC-MS/MS run (no negatively charged ions were investigated).

In this study, a multiresidue method was developed and validated to quantify nitroimidazoles, anthelmintics (benzimidazoles, imidazothiazoles, and salicylanilides), amphenicols, and dyes in meat (poultry, pork, beef, and horse), shrimp, and fish. First, the solvent was changed from acidified ACN to ethyl acetate, based on the work of Xia et al.,¹¹ and Na₂SO₄-NaCl 2:1 was used as a salting-out mixture to induce liquid-liquid partitioning. The extraction with ethyl acetate improved signal intensity and reduced noise for several analytes, mainly nitroimidazoles. Second, the clean-up was improved by adding a freezing step below -60 °C after extraction, which removes the water trapped in the frozen lower layer and facilitates transferring the ethyl acetate supernatant to new tubes containing DMSO. Third, the ethyl acetate extract was evaporated into DMSO, instead of evaporating and dissolving the residue in aqueous solutions. DMSO was chosen because it is an aprotic polar solvent able to dissolve both polar and non-polar compounds and also helps to keep analytes in solution instead of binding to proteins, as described by Whelan et al.¹⁶ and Cooper et al.¹⁷ Finally, an n-hexane LLE step was used for defatting, since the DSPE step with C18 resulted in lower recovery for three analytes: ipronidazole, ipronidazole hydroxide, and albendazole sulfoxide (it may be possible that they were trapped into the DSPE phase together with the removed fat).

Although the complexity of tissue matrices in terms of content and analyte-tissue interactions must be considered in the development and optimization of methods for the analysis of veterinary drug residues,²⁹ the optimized and validated extraction procedure has fewer steps and complexity than those currently available for the studied groups of compounds.

Chromatographic conditions

The chromatographic mobile phases were the same used in our laboratories for the analysis of nitroimidazoles and benzimidazoles, as described by Silva et al.,²⁸ and the necessary adjustments were made in the gradient elution. The peaks of some analytes showed splitting, mainly levamisole (pK_a = 6.98) at the retention times of 1.90 and 1.99 min, as well as chloramphenicol (pK_a = 5.5) at 2.95 and 3.00 min. The pH of the mobile phase A (0.1% formic acid in 5 mmol L^-1 ammonium formate) is close to 3, therefore both levamisole and chloramphenicol are fully protonated in the mobile phase, and the pH may not be responsible for peak splitting. A possible reason could be the formation of isomers or the quick elution of levamisole in acidic pH. Some authors¹⁹,²⁰ pointed out that a higher recovery of levamisole can be obtained at neutral or even basic pH values.

Isotope dilution UHPLC-MS/MS method

The selectivity of the method was ensured by spiking the samples with marked isotope IS of each analyte (or similar analytes) (Table 3), except in the case of closantel. Since DMSO was the diluent injected into the UHPLC MS/MS, the autosampler temperature was kept at 18 °C to ensure appropriate fluidity and prevent clogging of the autosampler needle or the UHPLC-MS/MS tubing. The risk of clogging was real when the autosampler temperature was set to 15 °C and the room temperature dropped overnight to around 10 °C since our laboratories generally do not require the control of low room temperatures. There was no evidence that DMSO interfered with the ionization of analytes. However, if it happened, it was minimized by spiking the samples with IS (if analytes ionized less, the same occurred with the IS) and by the high sensitivity of the MS/MS system (injection volumes of 5 μL were sufficient to produce good response intensities).

By using rapid polarity switching between positive and negative modes, it was possible to detect both positively and negatively charged ions in a single run that took only 6 min. The majority of compounds were detected in positive ionization mode [M + H]⁺, except for amphenicols¹⁵ and triclabendazole-like molecules, which were detected in negative ionization mode [M - H]^- (Table 3). Aiming to reduce the negative effect of interferents carried from the extraction into the DMSO diluent, the diverter valve was kept on from 0 to 1 min and from 5 to 6 min (Table 2), and the MRM window was restricted to the peak elution time window to maximize the ESI+ and ESI- responses of the analytes. The co-elution or the difficult separations between peaks represented no problem due to different precursor and product ions (Table 2). Figure 2 shows examples of UHPLC-MS/MS total ion chromatograms (TIC) of a fish matrix blank fortified at the LOQ with the investigated analytes (Figures S1 to S6, Supplementary Information (SI) section).

Figure 2
Examples of UHPLC-MS/MS TIC of a fish matrix blank fortified at the LOQ with nitroimidazoles, benzimidazoles, levamisole, closantel, amphenicols, and dyes. Experimental conditions: see Experimental section.

Validation results - linear ranges and limit of quantitation (LOQ)

The linear ranges (in μg kg^-1) were 0.10 to 0.60 for chloramphenicol, 0.50 to 3.0 for dyes, 1.5 to 9.0 for nitroimidazoles, and 5.0 to 30.0 for the other drugs. Determination coefficients of at least 0.9052 were obtained and complied with the acceptance criteria of R² ≥ 0.90. The first calibration curve levels were set as the LOQ, since the recovery of fortified matrix blanks at this level, as well as the coefficient of variation (CV%) of recovery results complied with the acceptance criteria.²⁷ The allowed recovery (f_REC) ranges according to concentration (c) were: (i) c ≤ 1 µg kg ¹, 50% ≤ f_REC ≤ 120%; (ii) 1 µg kg^-1 < c < 10 µg kg^-1, 70% ≤ f_REC ≤ 120%; (iii) c > 10 µg kg^-1, 80% ≤ f_REC ≤ 120%, whereas the allowed reproducibility (CV) ranges were: (i) c < 10 µg kg^-1, CV% ≤ 30%; (ii) c > 10 µg kg^-1, CV ≤ 25%²⁷ (Tables S1 and S2, SI section).

Accuracy and within-laboratory reproducibility

Analyte recovery and reproducibility were satisfactory (f_REC: 75.0 to 116.6%; CV: 1.2 to 29.9%), except for chloramphenicol in the horse matrix (acceptance criteria²⁷ were summarized above), Tables S3 and S4 (SI section).

The accuracy of results was demonstrated by satisfactory performance in proficiency testing, (|z score| ≤ 2), as follows: (i) Progetto Trieste Veterinary Drug Residues 2022 May, M1110A bovine muscle: dimetridazole, f_REC 92.25%, z-score -0.2; ronidazole, f_REC 97.93%, z-score -0.09; metronidazole, f_REC 98.45%, z-score -0.07; (ii) CFIA Proficiency Test 2021 November, T10372 bovine muscle: chloramphenicol, f_REC 93.33, 91.43, 76.00, and 120.00%, z-score -0.30, -0.39, -0.23, and -0.18; and (iii) FAPAS Food Chemistry Proficiency Test, 2020 September, T02406 eggs: dimetridazole, f_REC 109.22%, z-score 0.40; ronidazole, f_REC 109.21%, z-score 0.40.

Robustness and selectivity

The robustness of the method was assessed by using the fractional factorial design proposed by Youden and Steiner³⁰ and focused on three analytical parameters: amount of salt mixture (A: 6 g; a: 5 g), type of homogenizer (B: 2010 Geno/Grinder tissue homogenizer; b: E6003 Eberbach shaker), and ethyl acetate evaporation temperature (C: 45 °C; c: 50 °C). The capital letters (A, B, and C) denote the nominal values, while the lowercase letters (a, b, and c) stand for the alternative values. The method was considered not robust for variations in the amount of salt mixture and type of homogenizer, especially for dimetridazole hydroxide, fenbendazole, fenbendazole sulfone, oxibendazole, and albendazole. Therefore, the method shall be executed as described in the Experimental section. Considering the possible instability of dyes,²⁸ mainly malachite green, the extraction and UHPLC/MS-MS analysis were carried out on the same day, using freshly prepared working standard solutions. No significant evidence of degradation was observed.

For method selectivity evaluation, the analysis of representative blank samples did not show any interferences of signals, peaks, or ion traces at the retention times of the analytes. Furthermore, the addition of tetracyclines and macrolides to fortified matrix blanks did not interfere with the recovery of the investigated based on the t-test at 95% significance. It was assumed that matrix effects could occur based on previous studies.²⁸ Therefore, matrix-fortified calibration curves were used and all extracts were spiked with IS. Special attention was paid to the negative signal suppression of chloramphenicol when positively charged ions co-eluted with it. Usually, the chloramphenicol response was improved by adjusting the SRM windows in the Waters XeVo MS/MS method to the smallest window possible.

Measurement uncertainties, decision limit (CCα), and detection capability (CCβ)

The decision limit (CCα) is the limit at and above which it can be concluded with a probability of α (1% for banned substances and 5% for substances with an MRL) that a sample is non-compliant, while the detection capability (CCβ) is the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β (5% for banned or with an MRL substances).²⁵Table 4 shows the results for expanded measurement uncertainties (U), CCα, and CCβ. Nitroimidazoles, chloramphenicol, and dyes are prohibited or unauthorized pharmacologically active substances; therefore, CCα and CCβ shall be as low as reasonably achievable. For authorized substances, CCα shall be higher than the MRL, but as close as possible to it, and CCβ shall be lower than the MRL.²⁷ As shown in Table 4, these criteria were fulfilled.

Table 4
Expanded measurement uncertainties (U), decision limits (CCα), and detection capabilities (CCβ) determined from fortified matrix blanks with nitroimidazoles, benzimidazoles, levamisole, closantel, amphenicols, and dyes

Analyte	Concentration / (µg kg-1)	Poultry / (µg kg-1)			Pork / (µg kg-1)			Beef / (µg kg-1)			Horse / (µg kg-1)			Shrimp / (µg kg-1)			Fish / (µg kg-1)
		U	CCα	CCβ	U	CCα	CCβ	U	CCα	CCβ	U	CCα	CCβ	U	CCα	CCβ	U	CCα	CCβ
Dimetridazole	1.5	0.77	2.40	2.13	1.15	2.84	2.44	1.17	2.86	2.46	2.24	4.10	3.33	0.42	1.99	1.84	0.60	2.20	1.99
Dimetridazole hydroxide	1.5	0.53	2.12	1.93	1.92	3.74	3.08	1.06	2.74	2.37	1.69	3.47	2.88	1.30	3.01	2.57	0.62	2.22	2.01
Ronidazole	1.5	0.72	2.33	2.09	0.69	2.31	2.07	0.67	2.29	2.05	0.60	2.19	1.99	0.47	2.05	1.89	0.57	2.16	1.97
Metronidazole	1.5	0.60	2.19	1.99	0.43	2.00	1.85	0.39	1.95	1.82	0.44	2.01	1.86	0.43	2.00	1.85	0.47	2.05	1.89
Metronidazole hydroxide	1.5	0.92	2.57	2.25	0.74	2.36	2.11	0.88	2.53	2.22	1.35	3.07	2.61	0.83	2.47	2.18	0.82	2.46	2.17
Ipronidazole	1.5	0.69	2.31	2.07	0.98	2.64	2.30	0.83	2.47	2.18	0.95	2.60	2.28	1.14	2.83	2.43	0.77	2.40	2.13
Ipronidazole hydroxide	1.5	0.48	2.06	1.89	0.71	2.33	2.08	0.99	2.65	2.31	0.69	2.30	2.07	0.96	2.62	2.29	0.66	2.27	2.04
Albendazole	100/5/100/100/5/5	1.25	101.02	6.02	0.63	5.52	5.52	1.17	100.96	5.96	2.16	101.77	6.77	1.79	6.47	6.47	0.56	5.46	5.46
Albendazole sulfone	100/5/100/100/5/5	1.78	101.46	6.46	2.20	6.80	6.80	1.00	100.82	5.82	2.43	102.00	7.00	2.09	6.71	6.71	2.26	6.85	6.85
Albendazole sulfoxide	100/5/100/100/5/5	1.84	101.51	6.51	2.48	7.03	7.03	2.26	101.86	6.86	6.10	105.00	10.00	0.56	5.46	5.46	0.80	5.66	5.66
Albendazole 2-amino-sulfone	100/5/100/100/5/5	2.53	102.08	7.08	2.77	7.27	7.27	1.52	101.24	6.24	2.87	102.35	7.35	1.60	6.31	6.31	1.12	5.92	5.92
Fenbendazole	50/100/100/100/5/5	1.45	51.19	6.19	1.53	101.25	6.25	1.86	101.53	6.53	2.84	102.33	7.33	0.90	5.74	5.74	1.15	5.94	5.94
Fenbendazole sulfone	50/100/100/100/5/5	0.92	50.75	5.75	1.12	100.92	5.92	1.88	101.54	6.54	2.16	101.77	6.77	2.03	6.66	6.66	2.56	7.10	7.10
Oxfendazole	50/100/100/100/5/5	0.84	50.69	5.69	1.22	101.00	6.00	2.21	101.81	6.81	2.61	102.14	7.14	1.87	6.53	6.53	3.07	7.52	7.52
Oxibendazole	5/100/5/5/3/3	1.87	6.53	6.53	2.56	102.10	7.10	2.34	6.92	6.92	7.27	10.96	10.96	3.02	5.48	7.48	2.78	5.28	7.28
Febantel	50/100/100/100/5/5	2.38	51.95	6.95	1.62	101.33	6.33	2.97	102.44	7.44	3.42	102.81	7.81	0.81	5.66	5.66	0.68	5.56	5.56
Flubendazole	200/10/5/5/5/5	0.69	200.56	5.56	0.58	10.47	5.47	1.00	5.82	5.82	1.74	6.43	6.43	2.36	6.94	6.94	2.63	7.16	7.16
Aminoflubendazole	5/5/5/5/5/5	1.31	6.07	6.07	2.86	7.35	7.35	2.08	6.71	6.71	1.48	6.22	6.22	3.63	7.98	7.98	2.96	7.43	7.43
Thiabendazole	5/100/100/5/5/5	1.00	5.82	5.82	1.20	100.99	5.99	1.48	101.22	6.22	1.36	6.12	6.12	0.95	5.78	5.78	1.38	6.13	6.13
5-Hydroxy-thiabendazole	5/100/100/5/5/5	1.53	6.25	6.25	2.67	102.19	7.19	3.42	102.80	7.80	1.68	6.38	6.38	1.34	6.10	6.10	1.49	6.22	6.22
Triclabendazole	5/5/250/5/5/5	3.35	7.75	7.75	0.93	5.76	5.76	4.03	253.30	8.30	3.66	8.00	8.00	8.00	11.56	11.56	1.89	6.55	6.55
Triclabendazole sulfone	5/5/250/5/5/5	3.99	8.27	8.27	2.53	7.08	7.08	2.24	251.83	6.83	6.36	10.22	10.22	1.82	6.49	6.49	1.14	5.93	5.93
Mebendazole	5/5/5/60/5/5	1.03	5.85	5.85	0.76	5.62	5.62	2.52	7.07	7.07	2.66	62.18	7.18	1.81	6.48	6.48	1.28	6.05	6.05
Ketotriclabendazole	5/5/250/5	2.53	7.08	7.08	1.81	6.49	6.49	2.22	251.82	6.82	3.54	7.90	7.90	-	-	-	-	-	-
Levamisole	10/10/10/100/3/3	3.30	12.70	7.70	2.15	11.77	6.77	3.14	12.58	7.58	1.36	101.11	6.11	3.68	6.02	8.02	3.03	5.48	7.48
Closantel	5/5/1000/5/5/5	3.82	8.13	8.13	4.06	8.33	8.13	4.39	1003.60	8.13	10.57	13.67	8.13	1.80	6.48	6.48	2.92	7.39	7.39
Chloramphenicol	0.15	0.04	0.14	0.13	0.08	0.19	0.16	0.04	0.15	0.13	0.27	0.42	0.32	-	-	-	-	-	-
Florfenicol	50/100/100/100	3.15	102.58	7.58	2.97	302.44	7.44	2.73	202.24	7.24	3.41	102.79	7.79	-	-	-	-	-	-
Thiamphenicol	50/50/5/5	2.87	52.35	7.35	2.90	52.38	7.38	3.91	8.21	8.21	1.54	6.26	6.26	-	-	-	-	-	-
Malachite green	0.50	-	-	-	-	-	-	-	-	-	-	-	-	0.31	0.86	0.75	0.29	0.84	0.74
Leuco malachite green	0.50	-	-	-	-	-	-	-	-	-	-	-	-	0.08	0.59	0.56	0.09	0.60	0.57
Crystal violet	0.50	-	-	-	-	-	-	-	-	-	-	-	-	0.23	0.76	0.69	0.26	0.81	0.71
Leuco crystal violet	0.50	-	-	-	-	-	-	-	-	-	-	-	-	0.16	0.68	0.63	0.17	0.69	0.64

Method application and future perspectives

The method started to be used in the laboratory routine in 2020 after it was validated for nitroimidazoles and benzimidazoles in poultry. The other matrices were included in the validated method thereafter. In 2022 and 2023, the method scope was extended to new analytes (e.g., amphenicols, dyes). In the last four years, around 2400 samples were analyzed at the Laboratório Federal de Defesa Agropecuária de Minas Gerais (LFDA-MG) using the method as part of the MAPA PNCRC program.⁹Figure 3 shows examples of UHPLC-MS/MS TIC of albendazole for routine samples of poultry (Figure 3a), pork (Figure 3b), beef (Figure 3c), and shrimp (Figure 3d), which were spiked with the IS working solutions before extraction. The albendazole amounts in these samples were below the LOQ: the albendazole peak intensities of the quantification transition (266.1 to 191.1) at the retention time (t_R) 3.2 min are in the order of 10³, while the signal intensities in the fortified P1 (first calibration curve level, equivalent to the LOQ) are in the order of 10⁶. The second transition (266.1 to 234.1) was used for confirmation. Albendazole-d₇ signal intensity was used to estimate analyte responses and to quantify albendazole in the samples from the matrix-fortified calibration curves.

Figure 3
Examples of UHPLC-MS/MS TIC of albendazole for poultry (a), pork (b), beef (c), and shrimp (d) routine samples, compared to fortified blanks of the same matrices at the LOQ. Albendazole-d₇ was the IS. Experimental conditions: see Experimental section.

A method scope extension for nitroimidazoles in eggs has started to be validated, and good z-scores in proficiency testing were already obtained. The applicability of the method for macrocyclic lactones (e.g., avermectins) is also promising, but further studies are necessary.

Conclusions

A multiresidue UHPLC-MS/MS method, based on a modified QuEChERS-type extraction was successfully developed for the quantification of 33 drugs (nitroimidazoles, anthelmintics, amphenicols, and dyes). The residual concentrations of these drugs in foodstuffs have to be determined, either because they are banned (5-nitroimidazoles, chloramphenicol, and dyes) or have an MRL. The validation parameters were satisfactory for six different matrices (poultry, pork, beef, horse, shrimp, and fish muscle), except for chloramphenicol in the horse matrix (unsatisfactory recovery and reproducibility). Therefore, taking this exception into account, the method is suitable for the confirmation and quantification of the investigated drug residues, in compliance with the National Control Plan for Residues and Contaminants (PNCRC) of the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA). The main advantages of using the method are the easy extraction procedure and the fast and accurate quantification of all drugs in a single UHPLC-MS/MS run. This represents significant time and financial savings for routine analysis laboratories.

Supplementary Information

Supplementary data (Figures S1 to S6 present the UHPLC-MS/MS TIC of fish matrix blanks fortified at the LOQ with all the investigated analytes and their corresponding IS, Tables S1 and S2 show the LOQ, linear ranges, and determination coefficients for the matrix-fortified calibration curves, while Tables S3 and S4 show the results of analyte recovery (f_REC) and within-laboratory reproducibility (CV%) determined from fortified matrix blanks with nitroimidazoles, benzimidazoles, levamisole, closantel, amphenicols, and dyes) are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

The LFDA-MG is grateful to the other five National Agricultural Defense Laboratories (LFDA) for sharing information and resources.

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Edited by

• Editor handled this article:
  Eduardo Carasek

Publication Dates

• Publication in this collection
  25 Nov 2024
• Date of issue
  2025

History

• Received
  09 Apr 2024
• Accepted
  31 Oct 2024