Comparison of triple quadrupole mass spectrometry and Orbitrap high‐resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks

This paper presents a comparison of triple quadrupole tandem mass spectrometry (MS/MS) and Orbitrap high‐resolution mass spectrometry (HRMS) combined to ultrahigh performance liquid chromatography for the determination of glucocorticoids and polyether ionophores in sewage, in order to show the major benefits and drawbacks for each mass spectrometry analyser. Overall, HRMS measurements have enhanced performance in terms of confirmatory capabilities than MS/MS measurements. Moreover, similar limits of quantification, limits of detection, linear range and repeatability for glucocorticoids with both the MS/MS and HRMS methods were compared, but in the case of polyether ionophores, slightly better limits of detection and limits of quantification were obtained with the HRMS method because of the high sensitivity obtained when diagnostic ions are used for quantification instead of selected reaction monitoring transitions for these compounds. The two methods have been applied to the analysis of several influent and effluent sewage samples from sewage treatment plants located in the Tarragona region (Catalonia, Spain), showing an excellent correlation between the two methods. Copyright © 2014 John Wiley & Sons, Ltd.     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry

The confirmatory LC-MS/MS method for the determination of residues for twelve coccidiostats including ionophore antibiotics (lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin) and chemical coccidiostats (clazuril, decoquinate, diclazuril, halofuginone, nicarbazin and robenidine) in poultry liver has been developed. The sample preparation was based on extraction with acetonitrile, defatting with Alumina columns and clean-up on Oasis HLB spe. The separation of analytes was performed on PhenylHexyl column with a gradient of acetonitrile, methanol and the ammonium formate pH 4.0. For all analytes, at least 2 diagnostic fragmentation ions were monitored. The validation, performed according to the CD 2002/657/EC, proved the suitability of the method for the confirmatory analysis of the coccidiostats. For lasalocid, however, low reproducibility was observed and the proper quantification could not be performed with this method.     

LC/MS confirmation of ionophores in animal feeds.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.     

Rapid forensic selected reaction monitoring liquid chromatography/mass spectrometry determination of ionophore antibiotics found at toxic levels in animal feeds

A rapid, accurate, and selective method was developed for the forensic determination of ionophore antibiotics in animal feeds. A simple extraction procedure and liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode were used for rapid identification and confirmation of monensin and lasalocid in feed samples and for quantitation of monensin. Extracts from a homogenous portion of ground feeds were prepared using liquid-solid extraction and liquid-liquid extraction techniques. Feed extracts were further purified by a simple defatting and solvent wash step and then concentrated to dryness. Feed extract residues were reconstituted in 1 mL LC mobile phase and a 2 microL aliquot injected into the SRM LC/MS system. The latter system used a C18, 100 x 2.0 mm, LC column coupled to a PE-Sciex API 2000 tandem triple quadrupole mass spectrometer equipped with a TurbolonSpray LC/MS interface. Feed samples were extracted and analyzed for the determination of monensin and lasalocid within a couple of hours. Control feed samples fortified with monensin at concentrations from 50 ppb to 5 ppm provided a linear response and calibration curve across this range with a correlation coefficient of 0.996.     

A new approach to the analysis of nicarbazin and ionophores in eggs by HPLC/MS/MS

An HPLC/MS/MS method has been developed and validated for the quantification and confirmation of nicarbazin and ionophores (lasalocid, monensin, salinomycin, and narasin) in eggs. Nicarbazin is determined in the negative electrospray mode with a basic mobile phase that supports creation of negative ions. Consequently, our ability to maintain instrument sensitivity over time has significantly improved. The analysis of the ionophores is done in the positive electrospray mode using ammonium buffer for HPLC separation. Monitoring ammonium adduct parent ions resulted in enhanced sensitivity and better reproducibility of the ionophore analysis. The validation of this improved HPLC/MS/MS method for the detection of nicarbazin and the ionophores demonstrated excellent precision of below 10% RSD and lower LOD values (microg/kg) for nicarbazin (0.018), lasalocid (0.015), monensin (0.015), salinomycin (0.033), and narasin (0.039).     

Determination of the ionophoric coccidiostats narasin, monensin, lasalocid and salinomycin in eggs by liquid chromatography/tandem mass spectrometry

A sensitive and selective liquid chromatographic tandem mass spectrometric method (LC/MS/MS) for the simultaneous detection of the ionophoric coccidiostats narasin, monensin, lasalocid and salinomycin in whole eggs has been developed. A very simple sample preparation consisting of an extraction with an organic solvent was carried out. Sample extracts were injected into the LC/MS/MS system on a C18 column and an isocratic elution was performed. Nigericin was used as internal standard. The precursor ions produced by electrospray positive ionisation were selected for collisional dissociation with argon into product ions. Validation of the methods was performed based on Commission Decision 2002/657/EC.1 CC(alpha) was found to be 1 microg/kg for all four compounds. Monitoring of Belgian egg samples in 2004 revealed that residues of salinomycin, lasalocid and monensin could be found.     

Validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for 10 anticoccidials in eggs according to Commission Decision 2002/657/EC

A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the simultaneous detection and confirmation of halofuginone, robenidine, diclazuril, nicarbazin, monensin, narasin, lasalocid, salinomycin, maduramicin and semduramicin in whole egg has been developed and validated. The anticoccidial residues were extracted by acetonitrile, evaporated and dissolved in a sodium acetate/acetonitrile mixture. Then, the samples were injected on a C8 column in a gradient mode. Diclazuril-bis, DNC-d8 and nigericin were used as internal standards. The results of the full validation in accordance with the guidelines of the Commission Decision no 2002/657/EC are presented. This rapid and sensitive method was found suitable to confirm the anticoccidials at 1 and at 75 μg kg⁻¹ for the MRL compound lasalocid.     

Development of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein affinity chromatography

In this work we present a method for confirmatory analysis of chloramphenicol (CAP) in bovine and buffalo raw milk. CAP is extracted in acetonitrile and purified by affinity chromatography on an alpha-1-acid glycoprotein (AAG) column, then is identified and determined by ion-trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified at the minimum required performance limit (MRPL) of 0.30 ppb, by monitoring the [M-H]- ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in Decision 2002/657/EC for confirmation of prohibited veterinary drugs. The trueness and within-day and between-day repeatability data are also reported. Moreover, the loading capacity of affinity columns towards CAP was tested. This method, based on the molecular recognition between drug and AAG during the purification step to improve sample cleanup, represents a quantitative and repeatable procedure for confirmatory analysis, and fits the requirements for the routine official control of CAP residues in raw milk.     

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.     

Determination of spiramycin and neospiramycin antibiotic residues in raw milk using LC/ESI‐MS/MS and solid‐phase extraction

A simple confirmatory method for the determination of spiramycin and its metabolite neospiramycin in raw milk using LC ESI MS/MS is presented. Macrolide residues in raw milk were extracted by ACN, and sample extracts were further cleaned up and concentrated using SPE cartridges. Both spiramycin and neospiramycin were protonated in electrospray positive ion mode to form singly and/or doubly charged pseudomolecular ions. Data acquisition was achieved using multiple reaction monitoring, i.e., two transitions, for quantification and confirmation. Matrix‐matched standard calibration curves were utilized to achieve the best accuracy for the method. The method performance was evaluated according to both a conventional validation procedure and a designed experimental result. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of an overall recovery, was from 82.1 to 108.8%, and its intermediate precision was less than 20%. LC/ESI‐MS/MS method LODs (S/N ? 3:1) of spiramycin and neospiramycin were less than 1.0 μg/kg.     

Determination of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry at cross contamination levels

A confirmatory multi-residue method has been developed to allow for the detection, confirmation and quantification of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method can be used to determine halofuginone, robenidine, nicarbazin, diclazuril, decoquinate, semduramicin, lasalocid, monensin, salinomycin, narasin, maduramicin at levels relating to unavoidable carry over as stated in Regulation 2009/8/EC. Feed samples are extracted with water and acetonitrile with the addition of anhydrous magnesium sulphate and sodium chloride. The extract then undergoes a freezing out step before being diluted and injected onto the LC-MS/MS system. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation and can confirm all eleven analytes in a run time of 19 min. The sensitivity of the method allows quantification and confirmation for all coccidiostats at a 0.5% carry over level. The method was validated over three days in accordance with of European legislation; Commission Decision 2002/657/EC. Validation criteria of accuracy, precision, decision limit (CCα), and detection capability (CCβ) along with measurement uncertainty are calculated for all analytes. The method was then successfully used to analyse a number of feed samples that contained various coccidiostat substances.     

Confirmatory analysis of Trenbolone using accurate mass measurement with LC/TOF-MS

The use of accurate mass measurement as a confirmation tool is examined on a TOF-MS and compared with confirmation using a triple quadrupole mass spectrometer (QqQ-MS). Confirmation of the identity of a substance using mass-spectrometric detection has been described. However, the use of accurate mass measurement for confirmatory analysis has not been taken into account. In this study, criteria for confirmation with accurate mass are proposed and feasibility is demonstrated. Mass accuracy better than 3 ppm of the quasi-molecular ion and a fragment and their relative ratios determined with LC/TOF-MS are compared to the criteria of two transition ions and their ratio of LC/QqQ-MS. The results show that these criteria can be met for Trenbolone in samples of bovine urine and that single MS accurate mass measurement is comparable to nominal mass MS/MS for confirmation. The increase in popularity and availability of LC/TOF-MS instruments and the ease, of which exact masses can be measured, make it important to formulate criteria for this type of instrumentation. It is shown in this study that accurate mass measurement can be used for confirmatory analysis. However, more experiments need to be conducted to demonstrate the applicability of accurate mass measurement in general for residue analysis.     

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.     

Development and validation of a multi-residue liquid chromatography-tandem mass spectrometry confirmatory method for eleven coccidiostats in eggs

A confirmatory method for the determination of residues of eleven coccidiostats including ionophore antibiotics: lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin and chemical coccidiostats: decoquinate, diclazuril, halofuginone, nicarbazin and robenidine in poultry eggs was developed and validated. The sample was extracted with acetonitrile, defatted with hexane and cleaned-up on a silica SPE cartridge. The analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method performance characteristics required by Commission Decision 2002/657/EC were estimated adopting a more flexible and simple validation design. In this alternative approach the experimental study focuses on a larger dynamic range with progressively increasing validation levels. Each of them presents equal concentrations of all the analytes. On the contrary the classical Decision plan investigates a restricted concentration range necessarily different for each analyte being determined by the individual permitted limit (0.5-1.5 times the permitted limit). As a consequence each validation level involves the simultaneous fortification with complex mixtures containing different concentrations of each analyte. Adopting this alternative strategy the validation of several substances with significantly different permitted limits is considerably simplified and a deeper knowledge of the method is achieved. The results proved that the method enables the confirmation of regulated coccidiostats in eggs at the levels required in the official control of residues (CCα in the range of 2.2-174 μg kg(-1), depending on the coccidiostat). The repeatability (CV(r) in the range of 1.1-19%) and within-laboratory reproducibility (CV(Rw) in the range of 1.8-30%) are also acceptable. The procedure was successfully verified in the proficiency test and implemented in the national residue control plan.     

Direct aqueous supercritical fluid extraction coupled on-line with liquid chromatography–tandem mass spectrometry for the analysis of polyether ionophore antibiotics in water

A direct aqueous SFE system designed to extract water samples contained in vials has been coupled on-line with a reverse phase LC–MS–MS system using a single 10-port valve. An SFE trap system using C₁ stationary phase connected to a C₁₈ analytical HPLC column enabled the SFE–LC–MS–MS analysis of three polyether ionophore antibiotics in water using a step gradient. A quantitative SFE–LC–MS–MS method has been developed whereby the progress of SFE can be monitored directly on-line such that ionophore recovery profile data from a single water sample can be obtained. Using a continuous direct aqueous SFE period of 75 min, the SFE–LC–MS–MS recoveries of the ionophores were: monensin 76.2% with RSD 4.1%, lasalocid 84.6% with RSD 3.8% and narasin 91.2% with RSD 3.2%. With positive ion electrospray ionization, the SFE–LC–MS–MS system using a 4 mL water sample provided multiple reaction monitoring (MRM) limits of detection for monensin and lasalocid each equivalent to 90 ng/L whereas 30 ng/L for narasin. A two-way valve controlling carbon dioxide distribution to the SFE vessel has provided a means for the initial investigation of the recovery of ionophore sodium salts from water using static SFE.     

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 μgkg(-1)) to 18.6 (erythromycin at 25 μgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 μgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCβ) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 μgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 μgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.     

Reliability of veterinary drug residue confirmation: High resolution mass spectrometry versus tandem mass spectrometry

Confirmation of suspected residues has been a long time domain of tandem triple quadrupole mass spectrometry (QqQ). The currently most widely used confirmation strategy relies on the use of two selected reaction monitoring signals (SRM). The details of this confirmation procedure are described in detail in the Commission Decision 93/256/EC (CD). On the other hand, high resolution mass spectrometry (HRMS) is nowadays increasingly used for trace analysis. Yet its utility for confirmatory purposes has not been well explored and utilized, since established confirmation strategies like the CD do not yet include rules for modern HRMS technologies.     

Analyses of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry

Two liquid chromatography mass spectrometric techniques, i.e. ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-Tof MS) and high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS), were used for quantification, confirmation or identification of six macrolide antibiotic residues and/or their degradation products in eggs, raw milk, and/or honey. Macrolides were extracted from food samples by acetonitrile or phosphate buffer (0.1 M, pH 8.0), and sample extracts were further cleaned up using solid-phase extraction cartridges. UPLC/Q-Tof data were acquired in Tof MS full scan mode that allowed both quantification and confirmation of macrolides, and identification of their degradation products. LC/MS/MS data acquisition was achieved using multiple reaction monitoring (MRM), i.e. two transitions, to provide a high degree of sensitivity and repeatability. Matrix-matched standard calibration curves with the use of roxithromycin as an internal standard were utilized to achieve the best accuracy of the method. Both techniques demonstrated good quantitative performance in terms of accuracy and repeatability. LC/MS/MS had advantages over UPLC/Q-Tof MS in that its limits of detection were lower and repeatability was somewhat better. UPLC/Q-Tof provided ultimate and unequivocal confirmation of positive findings, and allowed degradation product identification based on accurate mass. The combination of the two techniques can be very beneficial or complementary in routine analysis of macrolide antibiotic residues and their degradation products in food matrices to ensure the safety of food supply.     

Investigations by HPLC-electrospray mass spectrometry and NMR spectroscopy into the isomerisation of salinomycin

HPLC-MS studies have indicated that certain polyether ionophore veterinary drugs are prone to degradation when stored as water-methanol solutions at ambient temperature. Salinomycin and narasin were particularly susceptible, disappearing completely within weeks to produce more polar species, which were identified as isomers of the original compounds. Lasalocid appeared to be stable under such conditions. Structural elucidation of the principal ultimate salinomycin isomerisation product was achieved by 2D NMR spectroscopy. This indicated that the isomerisation process consists of the opening of the spiro rings in the salinomycin structure with the concomitant formation of a furan moiety. The MS data indicated that the isomers retain the ability to complex alkali metal ions and may therefore retain their pharmacological activity. These discoveries may have implications both for the development of legislation covering acceptable levels of polyether ionophore residues in foodstuffs and also for analytical protocols designed to detect them.