Božica Solomun Kolanović

Validation of a multi-residue enzyme-linked immunosorbent assay for qualitative screening of corticosteroids in liver, urine and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was applied for the qualitative screening analysis of dexamethasone, betamethasone, flumethasone, and prednisolone in milk and urine, and dexamethasone, flumethasone and prednisolone in liver samples at levels corresponding to the European Union maximum residue limit (MRL), or at required performance levels (RPLs) for substances for which there is no established MRL. Method validation was performed according to Commission Decision 2002/657/EC criteria established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, limit of detection (LOD), limit of quantitation (LOQ), recovery, within-laboratory reproducibility, linearity and ruggedness. LODs were 0.2, 1.2 and 0.6 µg kg−1 in milk, urine and liver samples, and LOQ values were 0.3, 1.2 and 1.4 µg kg−1 in milk, urine and liver, respectively. Recoveries from spiked samples ranged from 68% to 131% for dexamethasone, from 57% to 120% for flumethasone, from 60% to 155% for betamethasone, and from 23% to 32% for prednisolone, with a coefficient of variation (CV) between 1.6% and 21.2%. The CCβ value was below the MRL/RPL for all examined matrices. Moderate variations of some critical factors in the sample pre-treatment for liver and milk samples were deliberately introduced for ruggedness evaluation and did not result in any negative effects on corticosteroid detection. The proposed method is suitable for qualitative screening analysis of corticosteroids in the above-mentioned food in conformity with the current European Union performance requirements.

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white

The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and white was measured during and after administration of a SMM/TMP combination in laying hens in doses of 8 g l(-)(1) and 12 g l(-)(1) in drinking water for 7 days. The SMM concentration reached maximal levels on day 2 of the post-treatment period for both doses (μg kg(-)(1)): 5920 and 9453 in yolk; 4831 and 6050 in white, in doses 1 and 2, respectively. Significant differences in the SMM and TMP concentrations between yolk and white in post treatment period were found. SMM dropped below the LOD (1.9 μg kg(-1)) in yolk after day 16 and 19 for doses 1 and 2. TMP reached maximal levels on day 3 after drug administration for doses 1 and 2 (μg kg(-)(1)): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured above LOD (0.3 μg kg(-)(1)) in yolk for both doses on day 37 post-treatment.

Feed additives diclazuril and nicarbazin in egg and liver samples from Croatian farms

In total 307 egg and 275 liver samples were examined for nicarbazin and 365 eggs for diclazuril over a 30-month period. Enzyme-linked immunosorbent assay methods used for quantification were validated according to European Commission Decision 2002/657/EC. Non-compliant samples were confirmed by LC-MS/MS. Mean diclazuril concentrations in egg samples were 0.31 µg kg−1, which is below the MRL. In only one egg sample, 2.26 µg kg−1 was determined by enzyme-linked immunosorbent assay, although confirmation by LC-MS/MS gave a value of 1.6 µg kg−1. Mean nicarbazin levels determined were 1.85 µg kg−1 in egg and 21.1 µg kg−1 in liver samples. Four samples, one egg and three livers, yielded elevated concentrations of nicarbazin, but only in the egg sample the LC-MS/MS method confirmed nicarbazin (106 µg kg−1) above the MRL value.

Elimination of Chloramphenicol in Rainbow Trout Receiving Medicated Feed

Elimination of Chloramphenicol in Rainbow Trout Receiving Medicated Feed Chloramphenicol muscle residue levels in rainbow trout were determined after oral administration of 84 μg kg-1d-1 of chloramphenicol for four days. Samples were taken one day before treatment and for 43 days after the treatment was over. Chloramphenicol was analysed using an in-house enzyme linked immunoassay (ELISA) validated against the criteria of the Commission Decision 2002/657/EC. Validation parameters confirmed that the method was appropriate for the detection of chloramphenicol at levels below the minimum required performance limit (MRPL) of 0.3 μg kg-1. The highest chloramphenicol levels were observed on the first day after the treatment had ended (144.3 μg kg-1). Elimination was significant over the first seven days; significant differences were detected between days 1 and 3 (p<0.001), 3 and 5 (p<0.001), and 5 and 7 (p<0.05). Chloramphenicol levels dropped below MRPL to 0.17 μg kg-1 on day 9 after the end of treatment. From day 11 to 43, chloramphenicol residues were detectable in a range from 0.091 μg kg-1 (highest) to 0.011 μg kg-1 (lowest). Our results indicate that trout muscle tissue could be compliant with health requirements for consumption 10 days after withdrawal from chloramphenicol treatment. Eliminacija kloramfenikola u kalifornijskoj pastrvi Određivani su ostaci kloramfenikola u mišićnom tkivu kalifornijske pastrve nakon oralne primjene u dozi od 84 μg kg-1d-1 tijekom 4 dana. Uzorkovanje je provedeno dan prije tretmana te tijekom 43 dana nakon tretmana. Maseni udjeli kloramfenikola određivani su primjenom in-house imunoenzimske metode (ELISA) validirane prema kriterijima Odluke Komisije 2002/657/EC. Dobiveni validacijski parametri pokazuju da je metoda prikladna za određivanje kloramfenikola na nivou manjem od vrijednosti granice najmanje zahtijevane učinkovitosti izvedbe metode (MRPL) od 0,3 μg kg-1. Najviši maseni udjeli kloramfenikola utvrđeni su prvog dana nakon završetka tretmana (144,3 μg kg-1). Statistički značajna eliminacija utvrđena je tijekom sedam dana te je značajno smanjenje određeno između prvog i trećeg (p<0,001), trećeg i petog (p<0,001) te petog i sedmog dana nakon tretmana (p<0,05). Razina kloramfenikola ispod MRPL vrijednosti utvrđena je devetog dana (0,17 μg kg-1) nakon tretmana. U vremenu od 11. do 43. dana nakon tretmana određeni su ostaci kloramfenikola od maksimalno 0,091 μg kg-1 do minimalno 0,011 μg kg-1. Prikazani rezultati pokazuju da se 10 dana nakon završetka tretmana tkivo pastrve može smatrati prikladnim za konzumaciju bez potencijalne štete za zdravlje.

Deposition and depletion of maduramicin residues in eggs after oral administration to laying hens determined by LC-MS

The coccidiostat maduramicin has been approved as a feed additive for chickens and turkeys, although it is prohibited for use in laying hens. In the present study, laying hens were divided into three groups and fed for 14 days with medicated feed containing maduramicin, at three different concentrations: 50, 100 and 500 µg kg−1. Eggs were collected during treatment and for 26 days after the end of feeding with medicated feed. Maduramicin residues were found exclusively in egg yolk, with the highest concentration in egg yolk of 459 µg kg−1 for the highest dose. The maximum concentration of maduramicin in whole egg was 16.6 µg kg−1 for the group receiving feed containing the maximum permitted level of maduramicin in feed (50 µg kg−1). The half-life of elimination of maduramicin, calculated for post-treatment days 1–10, was 6.5 days. Twelve days after drug administration, the concentration of the maduramicin in egg yolk for Group 3 (fed with 500 µg kg−1 maduramicin) still exceeded 20 µg kg−1, while the concentrations for Groups 1 and 2 were 1.2 and 2.7 µg kg−1, respectively. Graphical Abstract

Tylosin content in meat and honey samples over a two-year period in Croatia.

A total of 646 meat and 96 honey samples were examined over a 2-year period for the presence of tylosin residues. ELISA method used was validated according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. The CCβ values were 32.1 µg kg−1 in muscle and 24.4 µg kg−1 in honey. The recoveries from spiked samples ranged from 66.4–118.6%, with a coefficient of variation between 12.6% and 18.6%. All the investigated samples showed no presence of tylosin. Calculated estimated daily intakes show exposure levels lower than the acceptable daily intakes set by World Health Organization.

Essential and toxic element concentrations in monofloral honeys from southern Croatia

The concentrations of 24 elements in seven honey types (multifloral, heather, common heather, bearberry, sage, mandarin orange-blossom and honeydew) collected in southern Mediterranean regions of Croatia were determined using ICP-MS. Significant differences were found in the concentrations of Ag, As, Ba, Cu, Co, Fe, K, Mg, Mn, Mo, Na, Ni, Se, Sb, U and Th (p<0.05, all) among honeys. The highest element concentrations were determined in honeydew honeys, with the exception of multifloral (Ca, Cr, Mo, Se), common heather (Mg, Na), bearberry (Ba, Fe, Pb) and sage (Ag) honeys. Among the floral honeys, the highest concentrations were found in multifloral honey (Al, As, Be, Ca, Cr, Mn, Mo, Ni, Se, Th and U), common heather (Co, K, Mg, Na, V), sage (Ag, Cd, Cu), and bearberry (Ba, Fe, Pb, Sb, Zn). The results contribute to the evidence supporting the role of botanical origin on the elemental composition of honey.

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides, trimethoprim and dapsone in muscle, egg, milk and honey

ABSTRACT A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO3H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCβ for sulfonamides ranged between 5.6 and 8.2 µg kg−1 for eggs, between 11.1 and 69.9 µg kg−1 for milk, between 64.7 and 87.9 µg kg−1 for muscle, and between 2.7 and 5.3 µg kg−1 for honey. CCβ values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg−1 and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg−1 calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 μg/kg; detection capability, 0.56 μg/kg; limit of detection, 0.04 μg/kg; limit of quantification, 0.15 μg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 μg/kg in egg yolks and 166.5 μg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 μg/kg) and on day 37 in egg yolks (0.06 μg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Occurrence of aflatoxin M1 in raw cow, goat and sheep milk during spring and autumn in Croatia during 2016

Abstract Raw cow, goat and sheep milk samples were collected in different regions of Croatia during spring and autumn 2016. Aflatoxin M1 (AFM1) concentrations were measured in the following ranges (ng/kg): cow milk 0.93–85.4; goat milk 2.0–18.6; sheep milk 2.27–11.2. AFM1 levels exceeding the European Union maximum residue levels of 50 ng/kg were found in two cow milk samples in spring and 10 cow milk samples in autumn. The probable daily intakes (PDIs) for all three dairy species were from 0.0108 to 0.0165 ng/kg b.w./day and contributing 1.08–1.65% to the proposed provisional maximum tolerable daily intake (PMTDI; 1 ng/kg b.w./day), indicating low risk exposure for consumers. The mean positive cow milk PDI was 0.15 ng/kg b.w./day, contributing 15.2% to the PMTDI.