Reagents and standards

3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), AMOZ-d5, (C13)₃-AHD, C13N15-N15-SEM, 2NP-AOZ, 2NP-AMOZ, 2NP-AHD and 2-nitrobenzaldehyde (2-NBA), 3-amino-5-morpholinomethyl-2-oxazolidinon (AMOZ), semicarbazide hydrochloride (SEM), AOZ-d4, 2NP-AOZ-D4, 2NP-AMOZ-D5, 2NP-(C13)₃–AHD and 2NP-C13N15-N15-SEM were obtained from Dr. Ehrenstorfer and Witega. The purity of all compounds was greater than 99%.

Methanol (MeOH) and ethylacetate (LC grade), hydrochloric acid (HCl), dimethylsulphoxide (DMSO), n-hexane and potassium hydrogen phosphate were supplied by Merck. The water was purified with a from a Milli-Q purifying system (Elga PureLab Prima).

Individual standart stock solutions of 1 mg/mL were prepared in methanol, but SEM was prepared in DMSO. Working solutions of 10 ng/mL were diluted by methanol. All standard stock soutions were stored -20 ºC, and the working solutions were stored in refrigerator.

The concentration and content of mix standard solution were used to spiked samples with AMOZ, AOZ, AHD and SEM at a 8, 16, 20 and 20 ng/mL respectively. The concentration and content of internal mix standard solutions were used AOZ-d4, AMOZ-d5, (C13)₃-AHD and C13N15-N15-SEM at a 40, 40, 100 and 100 ng/mL, respectively.

Sample preparation

Collected samples for validation were known to be negative in the screning analysis within the national program for residues control in Turkey. Only, the milk samples were centrifuged at 3500 g, +4ºC, 15 min and upper the fat layer was removed before extraction.

Two grams or 2 mL homogenised samples (milk, honey, poultry meat and fish) were weighed into 50 mL polypropylene centrifuge tubes. Standard spiking solution mix (50, 100, 150 and 200 µL), internal standard solution mix (100 µL) and 5 mL of 0.1 M HCl were added. The extraxtion tube was shaken for 2 min by vortex. 2-nitrobenzaldehyde (2-NBA) (50 mM, 300 µL) were added and the mixture shaken for 2 min by vortex. The tube were capped and incubated overnight (16 h) in the 37ºC temperature. After derivatization, the samples were cooled in room temperature and neutralized by addition 1 mL of 1 M K₂PO₄ and mixed for 2 min by vortex. Ethylacetate (5 mL) and n-hexane (3 mL) was added and mixed for 15 min by vortex and centrifuged at 4000 g for 15 min. The organic phase (6 mL) was collected into a 15 mL graduated glass tubes. The organic fraction was evaporated to dryness under a stream of nitrogen in a water bath at 42°C.

The dry residue was reconstituted with n-hexane (2 mL) and mixed for 2 min by vortex and methanol/water (5/95) (0.7 mL) was added and mixed for 2 min by vortex. 0.5 mL samples were taken from the lower phase with the help of syringe or automatic pipette and filtered using a 0.2 µm syringe filter into an autosampler vial.

Instrumentation

Chromatographic analyses were performed on a LC-MS/MS equipment consisted of a Thermo Electron TSQ Quantum Access Max, mass spectrometer controlled by the Xcalibur (2.2 SP1) software.

Chromatographic separations were achieved on Phenomenex Synergy Hydro RP (150x2.00 mm 80Å 4 µm), protected with a C18 guard column. The mobile phase was deionised water/methanol (80/20) (A) and methanol acidified with 0.1% acetic acid. The linear gradient was: 0-2 min 100% A, 2-9 min 10% A and 9-15 min 100% A and flow rate of 0.25 mL/min. Injection volume was 50 µL. The column was thermostated at 40°C. The analysis of samples were carried in the positive ESI-MS-MS ion mode.

Mass spectrometry

MS/MS parameters and precursor-product ions of each compound were tuned by direct infusion in the SRM mode and 0.25 mL/min flow rate of the mobile phase A and B) (50:50).

The concentration and content of mix standards derivatizes and internal standards used for control of the MS-detector, were 25 μg kg-1 for 2-NP-SEM-C13N15-N15 and 2-NP-(C13)₃-AHD and other standards at 10 μg kg-1.

MS/MS detector parameters are given in Table 1. The precursor-product ions of each compound and their collision energy are given in Table 2.

Table 1. MS detector parameters

Table 2. LC–MS/MS confirmation parameters for the analytes

Specificity/Selectivity

Specificity/selectivity were evaluated via analysis of blank matrix samples fortified with mixed benzimidazole and nitroimidazole standards (concentration of 1 µg kg-1 each) and with standards of nitrofuran metabolites. According to analysis no significant peaks with an S/N (signal to noise) ratios of 3 or more and chromatographic interference were being observed at the retention times of the targeted nitrofuran metabolites.

Linearity

The matrix calibration curves were at four levels: 0.4, 0.8, 1.2 and 1.6 μg kg-1 for AOZ, AMOZ, AHD and SEM, which is in accordance with the MRPL levels. For each compound we made three matrix calibration curves, using blank samples, over three days with 6 replicates at four concentration levels. No significant differences were found between the different matrix curves (r2>0.9941) shown in Table 3.

Table 3. Summary of Linearity, CC_α, and CC_β

Decision limit (CC_α) and detection capability (CC_β)

The CC_α and CC_β for banned substances were calculated with the application of the following formula;

CCα = C₁ + 2.33 x SD_wIR

where in C₁ is lowest concentration level of the validation study (MRPL) and SD_wIR is the standard deviation from within-laboratory reproducibility.

CCβ = CCα + 1.64 x SD_{wIR, CCα}

where in SD_{wIR, CCα} is standard deviation at CC_α concentration.

For each compound of CC_α and CC_β were calculated from the linearity study. The mean value CCα and CCβ were presented in Table 3.

Recovery

The method recoveries and RSDs were determined from 6 replicates at four concentration levels spiking blank samples over three days. The recovery results were observed in acceptable range of 70-110%. All the data relating to method recovery and precision were summarisedin Table 4; mean recoveries ranging and CV% values were satisfactory, required by Decision 2002/657/EC.

Table 4. Recovery of nitrofuran metabolites: between-day repeat measures

Evaluation

In order to evaluate this method, it eventually participating in the Food Analysis Performance Assessment Scheme (FAPAS), the test of “Nitrofuran Metabolites in Prawns” (FAPAS 02229, 12/05/2014, Lab No:75). AOZ total assigned value were 0.934 μg kg-1, the results of our laboratory 0.8 μg kg-1. Z-score were -0.7. The result is good and satisfactory.