EU surveys insights: analytical tools, future directions, and the essential requirement for reference materials in wastewater monitoring of SARS-CoV-2, antimicrobial resistance and beyond.

BACKGROUND: Wastewater surveillance (WWS) acts as a vigilant sentinel system for communities, analysing sewage to protect public health by detecting outbreaks and monitoring trends in pathogens and contaminants. To achieve a thorough comprehension of present and upcoming practices and to identify challenges and opportunities for standardisation and improvement in WWS methodologies, two EU surveys were conducted targeting over 750 WWS laboratories across Europe and other regions. The first survey explored a diverse range of activities currently undertaken or planned by laboratories. The second survey specifically targeted methods and quality controls utilised for SARS-CoV-2 surveillance. RESULTS: The findings of the two surveys provide a comprehensive insight into the procedures and methodologies applied in WWS. In Europe, WWS primarily focuses on SARS-CoV-2 with 99% of the survey participants dedicated to this virus. However, the responses highlighted a lack of standardisation in the methodologies employed for monitoring SARS-CoV-2. The surveillance of other pathogens, including antimicrobial resistance, is currently fragmented and conducted by only a limited number of laboratories. Notably, these activities are anticipated to expand in the future. Survey replies emphasise the collective recognition of the need to enhance the accuracy of results in WWS practices, reflecting a shared commitment to advancing precision and effectiveness in WWS methodologies. CONCLUSIONS: These surveys identified a lack of standardised common procedures in WWS practices and the need for quality standards and reference materials to enhance the accuracy and reliability of WWS methods in the future. In addition, it is important to broaden surveillance efforts beyond SARS-CoV-2 to include other emerging pathogens and antimicrobial resistance to ensure a comprehensive approach to protecting public health.

Determination of four aminoglycoside antibiotics and spectinomycin in feed at cross-contamination level: Development and in-house validation of a LC-MS/MS method for enforcing EU Regulation.

Combating antimicrobial resistance is a top priority worldwide involving a concerted action by several high-level institutions and organisations in the health sector. To ensure that a meaningful progress is achieved, several campaigns and political initiatives have been launched targeting the health professionals, the industry, the farmers, and the general public. The Regulation (EU) 2019/4 on medicated feed contains provisions for the limitation and control of the contamination of non-target compound feed with 24 antimicrobials. The purpose of this work was to develop a reliable and effective method for the determination of four aminoglycoside antibiotics (apramycin, paromomycin, tobramycin and neomycin) and spectinomycin in feed at cross-contamination level, where an absolute lack of suitable methods was identified. Four candidate methods described in the literature failed to provide adequate recoveries of all analytes. Therefore, an in-depth investigation was carried out to identify the bottleneck variable. The optimised method was then in-house validated and showed performance features appropriate for the intended purpose. The selected compounds could be analysed by LC-MS/MS in five animal feeds with LOQs between 2.6 and 9.2 µg kg-1 for the AGs and between 28 and 86 µg kg-1 for spectinomycin. Using isotopically labelled internal standards, the recovery rates varied from 63 % to 103 % and the intermediate precision (RSDip) varied from 1.1 % to 14 %. This work represents a step forward in the reliable determination of antibiotics in compound feed as the developed method has shown to be precise and sensitive. It is expected that this method gains wide acceptance and can supplement the legislation with effective control tools for antibiotic residues.

Determination of urea in pet feed: assessing the suitability of different analytical techniques using proficiency test data.

The determination of urea in pet feed at contaminant levels using the spectrophotometric method described in Commission Regulation (EC) No 152/2009 has been reported by several EU laboratories to lack the required selectivity. Whilst urea is not authorised as an additive in pet feed, the control of urea in pet feed is of economic importance, because the addition of urea may unlawfully increase the apparent protein content. To investigate the capabilities of different analytical techniques, a proficiency test was organised where the participants (EU official control laboratories, laboratories from the academia and private laboratories) were free to use their method of choice for analysing three dog feed test materials, two samples of which were spiked with urea. Twenty-one laboratories submitted results using the following techniques: spectrophotometry (Implementing Regulation (EC) No 152/2009), LC-MS/MS, HPLC-UV, enzymatic-colorimetry, gravimetry and an 'in-house photometric' method. Only two laboratories that used LC-MS/MS were able to quantify urea accurately in the test material containing a mass fraction of 18.9 mg kg-1 whereas satisfactory results at the level of 258.9 mg kg-1 were obtained by one participant that used an 'in-house photometric method' and one that used the enzymatic method, in addition to the five participants using LC-MS/MS. The technique that provided the highest success rate across the three test materials was LC-MS/MS, whereas spectrophotometry, the enzymatic-based and HPLC-UV methods led to overestimated results in addition to a dispersion of results not suitable for compliance analysis. To address the determination of urea in pet feed at low levels, a better performing method than the one described in the legislation is required.

Screening methods and recent developments in the detection of anticoccidials.

This article presents a review of the current trends in the analysis of coccidiostats in various matrices, focusing principally on screening and rapid methods. Coccidiosis is an infectious disease having a high negative impact on the animal industry. Drugs are therefore necessary to prevent and/or to combat this disease. However, it is also of crucial importance that these veterinary drugs do not enter the human food chain. European legislation has therefore established the boundaries for the use of coccidiosats and has also addressed the unavoidable problem of cross-contamination of the feed, mainly caused by the use of the same production lines. Consequently there is a need for analytical methods and/or analytical strategies for the monitoring and control of the residues of anticoccidials, both in feed and in the resulting matrices for human consumption. In the frame of the European collaborative project CONffIDENCE, such attempts to establish the required analytical tools were made, which required beforehand a review of the state of the art in this domain. Aiming at this objective, in this review we consider the most interesting publications since 2000. In essence, both a rapid approach with mainly immunoassays and chromatographic methods were developed. To date, the obstacle to routine use of the first approach has been its inability to detect more than two compounds simultaneously, but recent developments in flow cytometry have made it possible to detect six coccidiostats at once. On the other hand, an increasingly popular approach for detecting multiple coccidiostats simultaneously is liquid chromatography coupled with tandem mass spectrometry. There remains a need to adapt these analytical methods to legislative requirements.

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed.

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs.

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 µg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 µg/kg, respectively.

Interlaboratory comparison of an analytical method for the determination of semduramicin in poultry feed at the authorized level using high-performance liquid chromatography coupled to postcolumn derivatization and spectrophotometric detection.

The performance characteristics of a method based on HPLC with postcolumn derivatization and spectrophotometric detection for the quantification of semduramicin in poultry feedingstuffs have been determined via a collaborative study. Semduramicin is a feed additive that is authorized for fattening chickens within the European Union at a minimum and maximum content of 20 and 25 mg/kg in feedingstuffs, respectively. The target concentration of semduramicin in the test samples ranged from 11.5 to 45.0 mg/kg. The study has been conducted with two different types of test material, namely, feedingstuff samples that have been previously ground in our laboratory and pelleted feedingstuffs. In the latter case, the laboratories participating in the study had to grind the samples prior to analysis. The obtained RSD for repeatability (RSD(r)) ranged from 2 to 10% for the ground materials, and from 2 and 7% for the pelleted materials. The RSD for reproducibility (RSDR) varied between 11 and 16% for the ground materials, and between 12 and 15% for the pelleted materials. These data indicated that grinding as an additional step in the analytical procedure did not influence the precision profile of the method. In addition, the HorRat values for all test materials were below or equal to 1.5, thus demonstrating that the obtained precision data were acceptable for the purpose of the method. Furthermore, an estimation of trueness based on statistical treatment of the results reported from the laboratories for spiked samples revealed acceptable mean recovery values of 88 +/- 4%. Based on the obtained performance profile, the method can be considered fully validated and transferable to control laboratories to be used within the framework of official control.

DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances.

Feed additives require pre-market authorisation prior to their use in the EU. For the group of coccidiostats, the EU regulations authorising these products include specifications for these substances and the major components of the feed additive formulations. Feed business operators can use only feed additives that meet these criteria. The traceability of these products is supported by the allocation of specific identification numbers that need to be printed on the feed additive label along with other information. In the present study, Direct Analysis in Real Time mass spectrometry (DART-MS) was applied to investigate the correct characterisation of feed additives that contain coccidiostats as active substances. The results of the study demonstrated that this technique allows an unequivocal identification of the active substances in the feed additive formulations when combining DART with high-resolution mass spectrometry. In addition, two feed additives containing the same coccidiostat, but different excipients could be correctly classified according to their composition. The method protocol is very simple and comprises two steps, namely the extraction of the feed additives with an organic solvent and the subsequent measurement with DART-MS. For the evaluation of the MS spectra, chemometrics was applied offering an effective method for classification. Chemometric models were established with nominal masses obtained from the analysis of the samples, thus showing that these feed additives could be correctly classified even using low-resolution mass spectrometry. Moreover, we demonstrated that molecule-specific stable isotope patterns obtained with low-resolution mass spectrometry could be used as an alternative tool for the confirmation of the active substance.

Validation of a multi-analyte HPLC method for the determination of carotenoids used as feed additives in fish and poultry feed: results of an interlaboratory study.

The performance characteristics of a multi-analyte method for the determination of all 10 carotenoids authorised as feed additives within the EU were assessed via an interlaboratory study. The analytical method is based on reversed phase high performance liquid chromatography (RP-HPLC) coupled to an optical detector set at 410 nm. The analysis is particularly challenging due to the presence of various stereoisomers of each carotenoid, and the use of these compounds via natural or synthetic formulations, requiring a special sample preparation. EU regulations specifying the conditions of use set legal limits for these substances in compound feedingstuffs ranging from 6 mg kg-1 to 138 mg kg-1, depending on the individual carotenoid and the target animal for which the feed is supplemented with this carotenoid. The purpose of the multi-analyte method validated in this paper is to facilitate the monitoring of carotenoids at relevant levels when used as feed additives in compound feedingstuffs and pre-mixtures. The interlaboratory study delivered precision data for 43 different analyte/mass fraction/matrix combinations, covering a mass fraction range of the target analytes from 2.6 mg kg-1 to 3861 mg kg-1. The relative standard deviations for repeatability (RSDr) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSDR) varied from 6.8 to 39 % with a mean value of 21 %. Given the broad scope of the method covering 10 carotenoids added to compound feedingstuffs and pre-mixtures via different formulations, this multi-analyte method is considered fit for the intended purpose.

Revision of a European Union Official Method to Enforce Legal Provisions: The Case of Diclazuril.

Background: Diclazuril is a coccidiostat currently authorized as feed additive in the European Union (EU), with a legal limit set at 1 mg/kg. For official control, an official EU method based on reversed-phase HPLC coupled with UV detection at 280 nm needs to be applied. Recently, the EU Reference Laboratory for feed additives was informed that the recovery rate for diclazuril was very low when implementing this method and performed experiments demonstrating that the indicated sorbent mass of the solid-phase extraction (SPE) was too low. Objective: Therefore, the paper presents a modified method protocol and the results of an interlaboratory study, performed on two compound feedingstuffs containing diclazuril around the legal limit. Methods: The official method was modified by using a higher SPE sorbent mass and was further subjected to validation. Results: The obtained values for the relative standard deviation for repeatability were 4.5 and 11.2%, and the corresponding values for the relative standard deviation for reproducibility were 14.3 and 18.1%; the calculated HorRat values were 0.95 and 1.14. Furthermore, acceptable mean recovery values of 98 and 111% were obtained for the two test materials, respectively. Conclusions: Based on the obtained performance profile, it was concluded that the modified official method was fit for purpose. In consequence, the official EU method will be corrected accordingly. Highlights: The highlights of this work are reflected by the following terms, namely Diclazuril, correction of EU official method, and interlaboratory study.

Determination of ionophore coccidiostats in feedingstuffs by liquid chromatography-tandem mass spectrometry Part I. Application to targeted feed.

A new and fit to purpose multi-analyte method for the determination of six coccidiostats (monensin A, salinomycin, narasin composed of its principal component narasin A and its minor component narasin I, lasalocid, semduramicin and maduramicin) in poultry and cattle compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The concentration level of the target analytes at which the validation experiments have been carried out varied between 1 and 9 mg kg(-1). The method developed involved a simple extraction of the coccidiostats from the feed samples followed by a clean-up by solid-phase extraction prior to chromatographic analysis. The analytes were quantified either by matrix-matched standards or by the standard addition technique, obtaining the following performance profile of the method for the various analyte/matrix combinations. When quantifying against matrix-matched standards, the concentration independent intermediate precision expressed in terms of relative percentage standard deviation varied between 4 and 10% and the relative percentage recovery rates ranged from 86 to 120%, depending on the target analyte and matrix. When using the standard addition technique, the corresponding values for the intermediate precision varied between 2 and 8% and the relative percentage recovery rate ranged from 73 to 115%. The limit of detection (LOD) and limit of quantification (LOQ) were different for the various analyte/matrix combinations but were in all cases below 0.014 and 0.046 mg kg(-1), respectively. Based on the obtained method performance characteristics, the method is considered suitable for the determination of ionophore coccidiostats in target feed. The main field of application of the validated method is to enforce European legislation regarding the authorisation of coccidiostats, focusing on the measurement at the authorised levels and at low level in feed during the withdrawal period at which the coccidiostats must not be added to the feed. Overall, the method proposed appears to be appropriate as a confirmatory method for the monitoring of these six ionophore coccidiostats and can therefore be considered as complementary to the official HPLC-UV methods.

Sample preparation strategy for the simultaneous determination of macrolide antibiotics in animal feedingstuffs by liquid chromatography with electrochemical detection (HPLC-ECD).

A novel and suitable clean-up method that allows, for the first time, the simultaneous determination of a rather large number of macrolide antibiotics (erythromycin, rosamicin, spiramycin, tylosin, kitasamycin and josamycin in feedingstuffs by high performance liquid chromatography with electrochemical detection (HPLC-ECD) is presented in this work. The effectiveness of the developed clean-up method allows the quantification of the target macrolides in poultry feed using standard calibration curves instead of matrix matched standards, which overcomes the general problem of finding representative blanks. Furthermore an additional back extraction included in the sample preparation procedure allows the determination of an additional macrolide (oleandomycin) with detection limits, expressed as apparent concentration in poultry feed, ranging from 0.04 to 0.22 mg kg(-1) and relative standard deviation values ranging from 3.6 to 10.1% depending on the target analyte. Moreover, this additional step has been proven to enlarge the scope of the method by the extension of its applicability, at the target level of concentration, to other animal feedingstuffs such as pig and cattle. The analysis of real feedingstuffs containing macrolides demonstrated the fitness for purpose of the whole analytical procedure as well as a good fitting between real and spiked samples. The proposed methods appeared therefore as a sound alternative in the frame of control (e.g. for post-screening purposes) and/or monitoring surveillance programmes at the target level of 1.0 mg kg(-1) established according to the reported lowest dosage of additive needed to lead a growth promoting effect.

Development and validation of a multi-analyte method for the regulatory control of carotenoids used as feed additives in fish and poultry feed.

Carotenoids are used in animal nutrition mainly as sensory additives that favourably affect the colour of fish, birds and food of animal origin. Various analytical methods exist for their quantification in compound feed, reflecting the different physico-chemical characteristics of the carotenoid and the corresponding feed additives. They may be natural products or specific formulations containing the target carotenoids produced by chemical synthesis. In this study a multi-analyte method was developed that can be applied to the determination of all 10 carotenoids currently authorised within the European Union for compound feedingstuffs. The method functions regardless of whether the carotenoids have been added to the compound feed via natural products or specific formulations. It is comprised of three steps: (1) digestion of the feed sample with an enzyme; (2) pressurised liquid extraction; and (3) quantification of the analytes by reversed-phase HPLC coupled to a photodiode array detector in the visible range. The method was single-laboratory validated for poultry and fish feed covering a mass fraction range of the target analyte from 2.5 to 300 mg kg-1. The following method performance characteristics were obtained: the recovery rate varied from 82% to 129% and precision expressed as the relative standard deviation of intermediate precision varied from 1.6% to 15%. Based on the acceptable performance obtained in the validation study, the multi-analyte method is considered fit for the intended purpose.

Optimization and validation of an analytical procedure for the determination of oxidative hair dyes in commercial cosmetic formulations.

A method has been developed and validated for the analysis of commonly used intermediates of oxidative hair dyes in commercial cosmetic formulations, including both liquid and cream forms, in dark and blonde shades. The commercial formulations are submitted to extraction by an organic solvent, and the resulting aqueous phase is analyzed by reverse-phase HPLC with a gradient elution and detection with DAD and/or ESI-MS-MS. A spectra library containing 200-400 nm spectra of the target substances and their HPLC retention times has been recorded for the identification. The quantification of the target substances is also performed after spiking of the commercial formulations, using an external calibration. The recoveries obtained are very good for all selected intermediates. The whole procedure has been found to be highly suitable for the identification and quantification of dye intermediates. Also implemented has been a database containing (a) the retention times, (b) the spectral, MS, and MS/MS characteristics of the intermediates, (c) acidity constant values of some intermediates of interest experimentally determined and compared to the available NIST values, (d) the chromatographic conditions used, (e) the behavior towards extraction of dye intermediates, and (f) matrix compounds.

Validation of an analytical procedure for the determination of oxidative hair dyes in cosmetic formulations.

A high range and variety of cosmetic formulations that contain oxidative hair dyes and matrix-forming compounds have been industrially developed over recent years and are now available on the international market. Member states of the European Union are responsible for conducting analyses of cosmetic products as deemed necessary by law and European regulation enforcement. Therefore, inspection authorities as well as the cosmetics trade and industry need validated analytical methods for the identification, characterization, and/or quality control of specific active ingredients or formulations with the aim of implementing the European Union Cosmetic Directives (76/768/ECC, 95/17/EC). In this frame, we validated a candidate reference method that enables the identification and quantification of hair dye-forming compounds. This method consists of a separation by RP-HPLC coupled with a DAD after a liquid-liquid extraction procedure for separating matrix components from the dye-forming compounds. The validation of the method includes common criteria such as the repeatability of the analysis and the establishment of figures of merit, as well as statistical evaluations and quality assurance in order to follow the recommendations of the Eurachem guide for analytical measurements.

Multi-residue method for the detection of veterinary drugs in distillers grains by liquid chromatography-Orbitrap high resolution mass spectrometry.

Distillers Grain (DG) is an important by-product of ethanol production. The ethanol production process uses only the starch portion of the plant and all the remaining nutrients, protein, fat, minerals, and vitamins remain in DGs, a valuable feed material for livestock. The use of antimicrobial drugs is helpful to limit harmful bacterial growth during the early part of the fermentation process. This can lead to the possible presence of contaminants in the by-products that are used in the food and feed industries, resulting in a major concern for the development of bacterial resistance in both humans and animals. To facilitate the detection of antimicrobial and other commonly used veterinary drugs in DGs, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed targeting a wide range of 12 chemical classes of anti-bacterial substances and drugs, such as ionophore and non-ionophore authorized coccidiostats, banned coccidiostats, macrolides, tetracyclines, nitroimidazoles, amphenicols, quinolones, sulphonamides, tranquilizers, non-steroidal anti-inflammatory drugs and benzimidazoles. Following a simple and fast extraction step with a mixture of organic solvents, the extract was directly injected into the LC coupled to an Orbitrap mass analyzer. The identification of residues is based on accurate mass measurement. The high mass resolution of 50,000 full width at half maximum (FWHM) and corresponding narrow mass windows permitted a very selective and sensitive detection of the analytes in such a complex matrix. A single-laboratory validation procedure was carried out evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance for precision and trueness, and allowed the determination of the compounds at low concentration. The proposed multi-method demonstrated that liquid chromatography coupled to an Orbitrap mass spectrometer is a promising analytical technique, suitable for official residue control of a variety of veterinary drugs in DGs supporting feed safety policies.

Analysis of antimicrobial agents in pig feed by liquid chromatography coupled to orbitrap mass spectrometry.

The emerging trend towards full scan mass spectrometry alternatives was evaluated for the identification of a wide range of anti-bacterial compounds in animal feed. The high resolving power of the orbitrap exactive provides precise mass accuracy, resulting in high selectivity which enables qualitative and quantitative analysis in complex matrices such as feed. A simple generic sample preparation procedure was applied, including extraction of the feed samples with a combination of organic solvents. The mass spectrometer was operated in full scan with polarity switching between positive and negative modes, using heated electrospray ionization (H-ESI). The detection was carried out using automatic control of the number of ions entering the mass analyzer and with the combination of the ion injection time, accurate quantitative analysis was achieved. Due to the complexity of the feed samples the resolving power proved to be the key for the discrimination between interfering masses from the matrix and the exact masses of the compounds in order to achieve mass accuracy of 5ppm. Furthermore, the use of narrow mass windows (±5ppm) improved the selectivity of the method, increasing the signal-to-noise ratio for the compounds. A thoroughly validation study was successfully performed and evaluated in pig feed. The utilization of liquid chromatography to the high resolution orbitrap exactive proved to be a powerful tool for routine analysis of undesirable anti-bacterial compounds in feed control, ensuring food safety under the EU food chain legal framework.

Separation and quantification of 15 carotenoids by reversed phase high performance liquid chromatography coupled to diode array detection with isosbestic wavelength approach.

The manuscript presents the development of a new reverse phase high performance liquid chromatography (RP-HPLC) photo diode array detection method allowing the separation and quantification of 15 carotenoids (adonirubin, adonixanthin, astaxanthin, astaxanthin dimethyl disuccinate, asteroidenone, beta-apo-8'-carotenal, beta-apo-8'-carotenoic acid ethyl ester, beta-carotene, canthaxanthin, capsanthin, citranaxanthin, echinenone, lutein, lycopene, and zeaxanthin), 10 of which are feed additives authorised within the European Union. The developed method allows for the reliable determination of the total carotenoid content in one run using the corresponding E-isomer as calibration standard while taking into account the E/Z-isomers composition. This is a key criterion for the application of the method, since for most of the analytes included in this study analytical standards are only available for the E-isomers. This goal was achieved by applying the isosbestic concept, in order to identify specific wavelengths, at which the absorption coefficients are identical for all stereoisomers concerned. The second target referred to the optimisation of the LC conditions. By means of an experimental design, an optimised RP-HPLC method was developed allowing for a sufficient chromatographic separation of all carotenoids. The selected method uses a Suplex pKb-100 HPLC column and applying a gradient with a mixture of acetonitrile, tert-butyl-methyl ether and water as mobile phases. The limits of detection and limits of quantification ranged from 0.06 mg L(-1) to 0.14 mg L(-1) and from 0.20 mg L(-1) to 0.48 mg L(-1), respectively.

Determination of semduramicin in poultry feed additive, premixture and compound feed by liquid chromatography and UV spectrophotometric detection after post-column derivatisation.

A new, simple and fit for purpose method based on liquid chromatography with UV spectrophotometric detection and post-column derivatisation (LC-UV-PCD) for the determination of semduramicin in poultry compound feed, premixtures and feed additive as well as its discrimination from other coccidiostats in poultry compound feed has been developed and single-laboratory validated. The concentration levels of the target analyte at which the validation experiments have been carried out varied between 12.8 and 51.3 mg kg(-1) in compound feed, covering the authorised levels of semduramicin according to European Union legislation. Furthermore, the method has been validated for a premixture sample containing semduramicin at 3 g kg(-1) and the feed additive containing semduramicin at 51 g kg(-1). The method developed involved a simple extraction of the coccidiostats with acetonitrile from the feed samples followed by a filtration of the supernatants. The resulting supernatants were submitted to chromatographic analysis. When analysing the feed additive and the premixture samples, the extraction solution was appropriately diluted prior to LC-UV-PCD analysis. The analytes were quantified through an external calibration curve prepared with pure semduramicin standards. The relative standard deviations for repeatability and for intermediate precision varied from 2.4 to 8.8% and from 2.6 to 8.8%, respectively, and the values for the relative recovery rate ranged from 89 to 95%. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be below 1 mg kg(-1) and 3 mg kg(-1), respectively. Moreover, the results showed a comparable performance profile, when using methyl isobutyl ketone instead of acetonitrile as extraction solvent.Based on the obtained method performance characteristics, the method is considered suitable for the determination of semduramicin in poultry compound feed at authorised level, in premixtures and in the feed additive, hence allowing the enforcement of the European Union legislation regarding the control and the monitoring in feedingstuffs.