Metabolites In Eggs Research Articles

Organophosphate esters and their metabolites in eggs from Vietnam

ABSTRACT Data on the occurrence of organophosphate tri-esters (tri-OPEs) and their metabolites (di-OPEs) in hen’s eggs are scarce. Therefore, 200 egg samples were gathered in 2023 in Hanoi, Vietnam and analysed by UHPLC-Q-Exactive HRMS. The majority of these compounds were detected, with tris(2-ethylhexyl) phosphate (0.10–2.7 ng/g wet weight (ww)) and trihexyl phosphate (0.08–2.3 ng/g ww) being the most prevalent tri-OPEs. Significant differences in tri-OPE profiles were observed in egg samples from battery-cage and free-range farming (p < .05). Despite egg levels ranging from 0.05 to 11.2 ng/g ww, Σdi-OPE accumulation in yolk and egg white was not significantly different in (p > .05). Among di-OPEs, dibutyl phosphate was found at the highest levels in the egg white, while bis(2-ethylhexyl) phosphate had the highest levels in yolk. There was no carcinogenic human health risk associated with OPEs in eggs (HQs <1).

Natural hydrophobic deep eutectic solvent based dispersive liquid-liquid microextraction followed by liquid chromatography with tandem mass spectrometry analysis of multi-class metabolites of pesticides, phthalates, and polycyclic aromatic hydrocarbons in animal-originated foods

Demonstrated a new natural hydrophobic deep eutectic solvent (NADES) based dispersive liquid-liquid microextraction (DLLME) approach/method to extract and enrich 37 multi-class metabolites (MCM) such as pesticides, phthalates, and polycyclic aromatic hydrocarbons metabolites from a complex animal- originated food matrices. The extraction solvent (NADES) synthesized from an equimolar mixture of the terpenes, thymol and menthol. The analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing electrospray ionization, and an enzymatic deconjugation of metabolites was carried out with β-glucuronidase. The parameters were optimized using the design of experiments (DoE). Further, the method was validated, and measurement uncertainty was also calculated. The detection and quantitation limits of the method were 0.03 to 0.4 ng/mL and 0.1 to 1.32 ng/mL, respectively. Recovery and expanded uncertainty ranged from 81.3 to 112.3 % and 8.9 to 19.7 %. The pertinence of the method was proven in identifying/finding metabolites in egg and meat samples ranging from 0.31 to 7.83 ng/g and 0.51 to 9.78 ng/mL in milk samples.

Residue depletion of toltrazuril and its metabolites in egg after an authorised application in hens

Toltrazuril is effective against all coccidial parasites in most animal species. Pharmacological studies showed that toltrazuril was well absorbed in the gastrointestinal tract and rapidly metabolised to toltrazuril sulfoxide and toltrazuril sulfone (ponazuril), the latter being considered as marker residue for toltrazuril residues. The maximum residue limits (MRLs) for toltrazuril sulfone in edible tissues from poultry range from 100 μg/kg in muscle to 600 μg/kg in liver. The substance is currently not allowed for use in poultry producing eggs for human consumption. However, several veterinary medicinal products containing the active substance are authorised in the European Union for use in pullets. According to product literature, withdrawal periods for edible tissues from chickens of 12 up to 18 days as well as restrictions for use before the onset of laying must be observed. Depending on the chicken breed, the start of the laying activity can vary between the 17th and 20th week of age. In monitoring programs, certain cases of residues in eggs were detected although zero tolerance was applicable at that time. The objective of the presented study was to evaluate the residue depletion profiles of toltrazuril and its major metabolites after the treatment of 15-week-old laying hens with an authorised toltrazuril-containing veterinary medicinal product. The pullets were treated on two consecutive days with TOLTRA-K (25 mg/ml) oral solution via drinking water at a dosage of 7 mg of toltrazuril per kg of body weight per day according to the recommended posology. Individual egg samples were collected after the beginning of the laying activity and were analysed in duplicate by LC-MS/MS to determine residues of toltrazuril and its metabolites. Residues of toltrazuril sulfone in egg samples were detected for several weeks and a statistical model was established to determine the time span needed to meet the zero tolerance.

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase HPLC Coupled with a Photo Diode Array Detector

An innovative and sensitive approach using high-performance liquid chromatography-photo diode array detection (HPLC-PDAD) was developed and optimized for the simultaneous determination of abamectin (ABM), ivermectin (IVM), albendazole (ABZ) and three metabolites in eggs. The samples were extracted with acetonitrile (MeCN)/water (90:10, v/v), and the extracts containing the targets were cleaned up and concentrated by a series of liquid-liquid extraction (LLE) steps. A reversed-phase C18 column and a mobile phase consisting of 0.1% trifluoroacetic acid (TFA) aqueous solution and methanol (MeOH) were utilized to perform optimal chromatographic separation. The developed method was validated on the basis of international guidelines. The limits of detection (LODs) and quantitation (LOQs) were 2.1-10.5 µg/kg and 7.8-28.4 µg/kg, respectively. Satisfactory linear relationships were observed for the targets in their corresponding concentration ranges. The mean recoveries ranged from 85.7% to 97.21% at 4 addition levels, with intraday and interday relative standard deviations (RSDs) in the ranges of 1.68-4.77% and 1.74-5.31%, respectively. The presented protocol was demonstrated to be applicable and reliable by being applied for the detection of target residues in locally sourced egg samples.

Presence of fipronil and metabolites in eggs and feathers of ornamental hens from Italian family farms

In Italy, hens for beauty contests are bred in backyard farms, characterized by the presence of few heads and self-consumption of animal products. According to law, these small farms are not subject to animal registration on national databases and are not submitted to official inspections by official veterinarians. Fipronil belongs to the category of banned drugs for animals intended for human consumption. However, being sold the formulation for pets both online and on the market, its illicit use by farmers is conceivable. In this study, egg and feather samples were collected from 19 farms spread all over Italy, and the concentration of fipronil and its main metabolites (fipronil-sulfone, hydroxy-fipronil, fipronil-desulfinyl, and fipronil-sulfide) were analysed by HPLC-HRMS. Two farms out of 19 reached a positivity rate of 80% for fipronil; five farms reached a positivity rate of 80% for fipronil-sulfone. Analysed separately, egg albumens and yolks showed a positive correlation for the concentration of both fipronil (r 0.92) and fipronil-sulfone (r 0.74), with higher concentrations recorded in yolk due to the lipophilicity of the molecules. The risk characterization shows that the intake of these eggs is not harmful to human health: the EDI (estimated daily intake) calculated on the data we collected was below the ADI (acceptable daily intake). Fipronil-desulfinyl was detected in higher concentrations in feathers, likely as a photodegradation product of the parent molecule.

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase Hplc Coupled with a Photo Diode Array Detector

Separation and Detection of Abamectin, Ivermectin, Albendazole and Three Metabolites in Eggs Using Reversed-Phase Hplc Coupled with a Photo Diode Array Detector

Determination of fipronil and its metabolites in eggs using dispersive- solid phase extraction and gas chromatography-tandem mass spectrometry

To develop a method for determination of fipronil and its metabolites in eggs by gas chromatography-tandem mass spectrometry(GC-MS/MS)cleaned with dispersive-solid phase extraction(D-SPE). Orthogonal array design with OA16(4~4) matrices was used to optimize the efficiency of D-SPE. The targets were extracted from samples with acetonitrile, and followed by D-SPE cleanup. The extracts were analyzed by GC-MS/MS, and quantified by internal standard method with matrix correction. The calibration curves showed good linearity in the range of 1.0-250.0 μg/L. The correlation coefficients were larger than 0.99.The average recoveries spiked in eggs at the levels of 2 μg/kg, 4 μg/kg and 20 μg/kg were between 87.1% and 125.0%(n=6), and the relative standard deviations were less than 10%. The limits of determination were between 0.1 and 0.4 μg/kg, and the limits of quantitation were between 0.3 and 1.2 μg/kg. The method possesses low background, high sensitivity. It can be applied to determine the residues of fipronil and its metabolites in eggs.

Simple and efficient method for the determination of fipronil and two main metabolites in eggs by capillary liquid chromatography

Capillary liquid chromatography (CLC) with UV-diode array detection has been proposed for the first time to determine fipronil and two metabolites, fipronil-sulfide and fipronil-sulfone in hen egg samples. Under optimum conditions, analytes eluted in less than 12 min. Despite the complexity of egg samples, a simple and fast sample preparation method based on salting-out assisted liquid–liquid extraction (SALLE) was developed using acetonitrile as extraction solvent and ammonium sulfate as salting out reagent. To obtain satisfactory extraction efficiencies for the target compounds, several parameters affecting the procedure were optimized, including the nature and amount of the extraction solvent and the type and amount of salting-out reagent, among others. Validation parameters of the proposed SALLE-CLC-UV method yielded satisfactory results with repeatability and intermediate precision, expressed as relative standard deviation (RSD), below 9% and 11%, respectively and recoveries above 80%. Good linearity was obtained (R2 > 0.990) with limits of detection (LOD) below 5 μg·kg−1. The advantages of a miniaturized technique such as CLC in terms of reduced sample and solvent consumption, combined with simplicity of the SALLE procedure, make this method an attractive green alternative to traditional LC for the monitoring of these residues in egg samples.

Covalent Triazine Framework Sorbent for Solid Phase Extraction of Fipronil and its Metabolite in Eggs with Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

ABSTRACT A solid-phase extraction (SPE) method was established for fipronil and its metabolite residues (fipronil desulfinyl, fipronil sulphone and fipronil sulphide) in eggs with a covalent triazine framework (CTF) porous material as the adsorbent followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection. Multiple probes and quantum chemistry theory calculations were conducted to describe the versatile adsorption property directly and quantifiably. The conjugated structure of CTF and N-containing triazine generated π-π interactions and hydrogen bonds between the CTF and the targets, which led to high extraction efficiency and recoveries. The solid-phase extraction parameters, including amount of the adsorbent, type of eluent, amount of eluent and loading rate were investigated. Under the optimal experimental conditions, the recoveries of the analytes were between 85.5% and 103.2%, and the RSD (n = 5) was between 1.8% and 3.6%. The LODs and LOQs were 0.13–0.2 ng g−1 and 0.5–0.8 ng g−1, respectively. The sorbent can effectively reduce the interference of the matrix and meet the detection requirements of fipronil and its metabolites in eggs. These results imply that the CTF as adsorbents have great potential in the analysis of trace targets in samples with complex matrices.

Appraisal of different clean-up strategies for the determination of fipronil and its metabolites in eggs by UHPLC-MS/MS

The large-scale misuse of fipronil insecticide can be a food safety issue. Therefore, the EU has fixed a maximum residue level of 5 µg kg−1 in egg expressed as the sum of both fipronil and its sulfone metabolite. On the other hand, their determination in egg samples is not an easy task due to the high proportion of lipids and proteins. Therefore, the development of new strategies to isolate fipronil and its metabolites from egg matrix interference remains a significant challenge and is central in food analysis. In this work, three clean-up strategies (EMR-lipid, HLB PRiME, and primary secondary amine (PSA)) based on either dispersive solid-phase extraction (dSPE) or SPE in pass-through mode were evaluated in combination with the QuEChERS extraction to determine fipronil and its metabolites in chicken eggs. All extracts were analyzed with ultra high performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). Several aspects, including laboratory throughput, matrix effect, limits of quantification, cleanliness of extracts, recovery, and repeatability, were taken into account to provide a comprehensive assessment of these clean-up strategies. LOQs ranged from 0.02 to 0.03 µg kg−1 were obtained in all cases, being lower than the maximum residue levels fixed by the current legislation. The samples cleaned up by EMR‐Lipid and PSA achieved the highest average recovery, obtaining recovery values between 95.3 and 99.3%, 91.3–96.7%, 85.1–89.3% for EMR‐Lipid, PSA, and HLB PRiME, respectively. The matrix effects were only negligible for all studied analytes in the case of EMR-lipid, achieving signal suppression ranged from −8 to −4%. Thus, EMR-lipid provided the best results due to its higher effectiveness in removing co-extracted species from the extract. Finally, different egg chicken samples were tested using the selected clean-up procedure to illustrate the method applicability.

Lateral flow immunoassay for the simultaneous detection of fipronil and its metabolites in food samples

We developed a sensitive and rapid lateral flow immunochromatographic (LFI) assay for the simultaneous detection of fipronil and its metabolites in eggs and cucumbers using gold nanoparticle (GNP)-labeled monoclonal antibodies (mAbs). Anti-fipronil mAbs (1B6) were produced using two haptens and identified by heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) with half maximal inhibitory concentration (IC50) and limit of detection (LOD) values of 0.46 ± 0.07 and 0.05 ± 0.01 ng mL−1, respectively. The developed LFI strip showed high sensitivity and specificity in the detection of fipronil with cut-off and visual limit of detection (vLOD) values of 10 and 0.25 ng mL−1, respectively. Furthermore, the application of LFI in the detection of fipronil-spiked egg and cucumber samples was validated by liquid chromatography tandem mass spectrometry (LC-MS/MS). Our developed LFI assay is suitable for detection of fipronil and its metabolites in real samples.

Combination of Modified QuEChERS and Disposable Polyethylene Pipet Assisted DLLME Based on Low Density Solvent Extraction for Rapid and Sensitive Determination of Fipronil and Its Metabolites in Eggs by GC-MS

In the present study, a new method for separation and enrichment of fipronil and its metabolites in eggs was proposed by combining a modified QuEChERS procedure with a disposable polyethylene pipet assisted DLLME (DPP-DLLME). The DPP-DLLME employed low density solvent as extractant and was conducted in a cheap, disposable polyethylene pipet with an open and narrow tip upside. After phase separation, the extractant was floating on the aqueous solution and concentrated in the narrow neck of the pipet, which can be directly withdrawn by a microsyringe for instrument analysis. By coupling with GC-MS detection, a new method for analysis of fipronil and its metabolites in eggs was established. The linearity range was 0.5–200 ng/g with correlation coefficient R2 higher than 0.9967. The recoveries in spiked egg samples were in the range of 89.3–103.8% with the RSDs less than 13.6% (intra-day) and 17.5% (inter-day). The limits of quantification for the four target analytes (fipronil desulfinyl, fipronil sulfide, fipronil, fipronil sulfone) in eggs were in the range of 0.2–0.3 ng/g, which was comparable or better to the previously reported methods. Finally, the established QuEChERS-DPP-DLLME method was successfully applied to detect fipronil and its metabolites in several egg samples with different brands from local market.

Multi-Residue Analysis of Fipronil and Its Metabolites in Eggs by SinChERS-Based UHPLC-MS/MS.

A method for simultaneous detection of fipronil (F) and its metabolites fipronil desulfinyl (FD), fipronil sulfide (FS), fipronil sulfone (FSO) in chicken eggs was applied and validated. It includes single-step, cheap, effective, rugged, safe-based method (SinChERS) for sample preparation and ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) for chemical analysis. Results suggested that formic acid enhanced the recovery of 4 target residues and 1% supplementation to acetonitrile gained higher recoveries than that of 5%. SinChERS integrated extraction and clean-up steps into one, with shorter time (1.5 h) to operate and higher recoveries (97%–100%) than HLB, Envi-Carb-NH2 and quik-easy-cheap-effective-rugged-safe method (QuEChERS), and it consumed the smallest volume of extracting solvent (10 mL) as QuEChERS. Quantitative analyses using external standard method suggested the linear ranges of 4 target compounds were 1–20 μg/L with R2>0.9947. The limit of detection (S/N>3) and quantification (S/N>10) were 0.3 μg/kg and 1 μg/kg. Recoveries ranged from 89.0% to 104.4%, and the relative standard deviations (n=6) at 1, 10, and 20 μg/kg were lower than 6.03%. Thirty batches of domestic eggs (500 g each) were detected by the established SinChERS-based UHPLC-MS/MS and no target residues were detected in all samples. The method developed in this study is a rapid, sensitive, accurate and economic way for multi-residue analysis of fipronil and its metabolites in eggs.

Stress, health and the welfare of laying hens

It is essential to understand responses to stress and the impact of stress on physiological and behavioural functioning of hens, so as to assess their welfare. The current understanding of stress in laying hens is comprehensively reviewed here. Most research on stress in hens has focussed on the activity of the adrenal glands, with the most common approach being to measure corticosterone, which is the predominant glucocorticoid produced by birds in response to stress. While these measures are useful, there is a need to understand how the brain regulates stress responses in hens. A greater understanding of the sympathoadrenal system and its interaction with the hypothalamo–pituitary–adrenal axis is required. There is also a lack of knowledge about the many other peptides and regulatory systems involved in stress responses in hens. The usefulness of understanding stress in hens in terms of assessing welfare depends on appreciating that different stressors elicit different responses and that there are often differences in responses to, and impacts of, acute and chronic stress. It is also important to establish the actions and fate of stress hormones within target tissues. It is the consequences of these actions that are important to welfare. A range of other measures has been used to assess stress in hens, including a ratio of heterophils to lymphocytes and haematocrit : packed cell-volume ratio and measures of corticosterone or its metabolites in eggs, excreta, feathers and the secretions of the uropygial gland. Measures in eggs have proffered varying results while measures in feathers may be useful to assess chronic stress. There are various studies in laying hens to indicate impacts of stress on the immune system, health, metabolism, appetite, and the quality of egg production, but, generally, these are limited, variable and are influenced by the management system, environment, genetic selection, type of stressor and whether or not the birds are subjected to acute or chronic stress. Further research to understand the regulation of stress responses and the impact of stress on normal functioning of hens will provide important advances in the assessment of stress and, in turn, the assessment of welfare of laying hens.

Simultaneous determination of 58 pesticides and relevant metabolites in eggs with a multi-functional filter by ultra-high performance liquid chromatography-tandem mass spectrometry

A sensitive method for the simultaneous determination of 58 pesticides and relevant metabolites in eggs was developed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) after clean-up with a multi-functional filter (MFF) based on quick, easy, cheap, effective, rugged, and safe method (QuEChERS). The egg sample was extracted with 5 ml water and 10 ml 1% acetic acid in acetonitrile and then salt out with sodium chloride. The extracted solution was filtered directly through an MFF containing 50 mg PSA, 50 mg C18, and 150 mg magnesium sulphate before UHPLC-MS/MS analysis. The clean-up and filter procedures were integrated using the MFF to substantially improve the work efficiency. Good linearity was shown for each analyte, and all the correlation coefficients exceeded 0.99. The recoveries in the eggs at the five spiked levels were 74.4%–115.2%, and the relative standard deviations (RSDs) were less than 15.3%. The limit of detection (LOD) and the limit of quantitation (LOQ) of 58 pesticides and 8 metabolites in eggs were 0.1–1.0 μg/kg and 0.2–5.0 μg/kg, respectively. The decision limit (CCα) and detection capacity (CCβ) were 3.4–111.1 μg/kg and 6.8–122.1 μg/kg, respectively. This method has also been successfully applied in the determination of actual eggs samples. This developed method is more effective and faster in the monitoring of pesticide residue in eggs compared to the traditional analytical method.

Quick determination of fipronil and its metabolites in eggs by gas chromatography-triple quadrupole mass spectrometry

A method was established for the simultaneous determination of fipronil and its metabolites in eggs by gas chromatography-tandem mass spectrometry coupled with QuEChERS. Fipronil and its metabolites were extracted from eggs with acetonitrile, and the solution was dehydrated with 150 mg anhydrous magnesium sulfate. The extracts were purified with 50 mg N-propyl ethylenediamine (PSA) and 50 mg C18, and a capillary column pesticide Ⅱ was used. The analytes were detected in timed selected reaction monitoring (timed-SRM) mode, and were quantified using external standard method with matrix correction standard curves. A good linearity in the range of 1.0-200 μg/L with correlation coefficients (R2)>0.999 was observed. The limits of quantification (LOQs) were between 0.5 and 1.0 μg/kg. The recoveries at three spiked levels (2.0, 5.0 and 10.0 μg/kg) were in the range of 87.8% to 111.5% with RSDs between 2.0% and 9.2% (n=3). This new method satisfied the related regulations of the European Union for the determination of fipronil and its metabolites in eggs.

Determination of fipronil and its metabolites in eggs by UPLC-QqLIT-MS/MS with multistage mass spectrometry mode

A fast, sensitive, and reliable method for the determination of fipronil and its metabolites (fipronil-sulfone, fipronil sulfide, and fipronil-desulfynil) in eggs was developed by a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method and ultra-performance liquid chromatography-quadrupole-linear ion trap mass spectrometry (UPLC-QqLIT-MS/MS). The proposed multistage mass spectrometry (MS3) method provides sufficient sensitivity and excellent reproducibility. The four analytes were separated by a Waters ACQUITY UPLC® BEH C18 column and detected by AB Sciex QTRAP® 5500. The mean recoveries for the four analytes ranged from 90.7% to 105% with relative standard deviations (RSDs) of 0.7% to 4.9%. The calibration curve showed a good linearity within the concentration range of 0.05 to 20 ng/mL. The correlation coefficients (R2) were higher than 0.999. The limits of detection (LODs) obtained from the experimental results for fipronil, fipronil-sulfone, fipronil-sulfide, and fipronil-desulfinyl were 0.06, 0.02, 0.05, and 0.04 µg/kg, respectively. The MS3 method was successfully applied to detect fipronil and its metabolites in 60 egg samples purchased from Chinese supermarkets, and wholesale markets.

Determination of fipronil and its metabolites in eggs and egg products with gas chromatography-negative chemical ionization-mass spectrometry

A method was established for the determination of fipronil and its metabolites in eggs and egg products with gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS). The targets were extracted from samples with acetonitrile, and followed by a simple cleanup step known as dispersive solid-phase extraction QuEChERS. The extracts were determined by GC-NCI-MS, and quantified by external standard method with matrix correction standard curves. The recoveries were in the range of 87.0% to 99.3% at four spiked levels (0.1, 2.0, 4.0 and 20.0 μg/kg), and all RSDs were not more than 12.7% for the four analytes. The linearity of the method was good between 0.005 and 0.10 mg/L, and all LOQs were less than 0.10 μg/kg. So this method can be used to determine the residues of fipronil and its metabolites in eggs and egg products.

Rapid screening of fipronil and its metabolites in egg and egg products by solid phase extraction-liquid chromatography-quadrupole time-of-flight mass spectrometry

A method for rapid screening of fipronil and its metabolites in egg and egg products was developed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF MS). The samples were extracted by acid-acetonitrile, cleaned up by PRiME HLB SPE. The separation was performed on a Poroshell 120 EC C18 column (150 mm×3 mm, 2.7 μm) with gradient elution using water and acetonitrile as mobile phases. The target compounds were monitored under negative ionization mode with electrospray ionization (ESI) source and two databases of accurate mass and fragment ions were created. The matrix effects in four kinds of egg and egg products were considered and the quantification was carried out by internal standard method. The results demonstrated that the linear ranges were from 0.1 to 5 μg/L with good correlation coefficients (r2>0.99). The limits of detection (LODs, S/N>3) and limits of quantitation (LOQs, S/N>10) were 0.2 μg/kg and 1 μg/kg, respectively. The recoveries of fipronil and its metabolites in different matrixes spiked with 1, 2 and 5 μg/kg varied from 82.6%-98.1%, and the relative standard deviations (RSDs) were between 3.8%-9.9% (n=6). The method can effectively correct the ionization suppression. It is sensitive, accurate and suitable for the rapid screening of fipronil, fipronil sulfide, fipronil sulfone and fipronil desulfinyl in egg, egg noodle, cake and mayonnaise.

Residue depletion of amoxicillin and its major metabolites in eggs.

The depletion of amoxicillin (AMO) and its major metabolites, amoxicilloic acid (AMA) and amoxicillin-diketopiperazine-2',5'-dione (DIKETO) in the albumen, yolk and whole egg was studied after the oral dose of AMO (25 and 50mg/kg body weight) to laying hens once per day for five consecutive days. Egg samples were prepared by a simple liquid-liquid extraction procedure with acetonitrile and saturated methylene chloride and analysed using liquid chromatography-tandem mass spectrometry. The results showed that AMO, AMA and DIKETO residues were mainly distributed in the yolk, where particularly high concentrations of AMO and DIKETO were found, whereas the albumen contained high concentrations of AMA. This distribution suggested that AMO and DIKETO were depleted slowly in yolk, whereas AMA was depleted slowly in albumen. The amount of AMO residue positively correlated with the dose, and the theoretical withdrawal times, which were calculated based on the residue level falling below a safe limit, were 5.21 and 7.67days at AMO doses of 25 and 50mg/kg, respectively. Moreover, the theoretical withdrawal times for all residues in the whole egg were 8.00 and 9.11days at doses of 25 and 50mg/kg, respectively. Our findings suggested that 9days was an appropriate withdrawal time for the use of AMO in laying hens.