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 chloramphenicol in aquatic products by graphene‐based SPE coupled with HPLC‐MS/MS

An effective analytical protocol using graphene‐based SPE coupled with HPLC‐MS/MS for determination of chloramphenicol (CAP) in aquatic products has been developed. In the present work, graphene was evaluated as SPE sorbents for the analytes enrichment and clean up. The target analytes were quantified by a triple‐quadrupole linear ion trap MS in multiple‐reaction monitoring mode. In addition, the proposed method was validated according to Commission Decision 2002/657/EC. The calibration curve was linear over the range of 0.5–100 ng/mL. The mean values of RSD of intra‐ and interday ranging from 1.48 to 4.29% and from 3.25 to 7.42% were obtained, respectively. In the three fortified levels, the recoveries of CAP ranging from 92.3 to 103.4% with RSDs ≤ 5.58% were obtained. The proposed method has been successfully applied to the analysis of CAP in several aquatic product samples, indicating that graphene was a potential SPE sorbent for the enrichment of trace residues in food samples.     

Determination of chloramphenicol residues in bee pollen by liquid chromatography/tandem mass spectrometry

A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.     

Quantitative determination of ochratoxin A in kidneys by liquid chromatography/mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the quantitative determination of ochratoxin A (OTA) in kidney samples was developed. Ochratoxin B (OTB) was used as internal standard. Extraction of homogenized kidney samples was done by adding a chloroform/phosphoric acid mixture. Due to restriction of the sample size, less chloroform could be used than in previously described methods. Two different columns for clean-up were compared: strong anion exchange (Bond Elut SAX) and Extrelut NT columns. The high-performance liquid chromatography (HPLC) was used with gradient elution consisting of variable mixtures of formic acid (0.3%) in acetonitrile and formic acid (0.3%) in water. The mass spectrometer was operated in the positive ESI mode using multiple reaction monitoring. For OTA the precursor ion was m/z 404 while the product ions were at m/z 239 and m/z 341. For OTB the precursor ion was m/z 370 while the product ions were at m/z 205 and at m/z 324. A calibration curve of fortified kidney samples was used to quantify OTA. Method validation was performed according to Commission Decision 2002/657/EC. Decision limit (CCalpha), detection capability (CCbeta), precision, bias, trueness, specificity and measurement uncertainty were determined. In general, the best results were obtained using SAX clean-up. CCalpha and CCbeta were 0.11 and 0.25 microg kg(-1), respectively. Within-laboratory reproducibility (% CV) was 9, 9, and 5% for OTA-fortified kidney samples of 0.5, 1, and 2.5 microg kg(-1), respectively. Trueness (%) was 75, 69, and 57% for OTA-fortified kidney samples at 0.25, 0.5, and 1 microg kg(-1), respectively. Measurement uncertainty and expanded uncertainty were 14.85 and 29.70%, respectively. All criteria as laid down in Commission Decision 2002/657/EC were fulfilled. Therefore, this LC/ESI-MS/MS method can be used to monitor kidneys for OTA in the framework of Council Directive No. 96/23/EC.     

Food safety evaluation: Detection and confirmation of chloramphenicol in milk by high performance liquid chromatography-tandem mass spectrometry

A simple and rapid procedure for extraction of chloramphenicol (CAP) in milk and analysis by high-performance liquid chromatography coupled with quadrupole mass spectrometry in tandem was developed. The method consisted of one step of liquid-liquid extraction using ethyl acetate and acidified water (10 mmol L⁻¹ formic acid) and HPLC-MS/MS detection. CAP-D5 was used as internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear, with typical r² values higher than 0.98. Absolute recovery of CAP from milk proved to be more than 95%, however CAP-D5 absolute recovery was 75%. The method was accurate and reproducible, being successfully applied to the monitoring of CAP in milk samples obtained from the Brazilian market. Decision limit (CCα) was 0.05 ng mL⁻¹ and detection capability (CCβ) was 0.09 ng mL⁻¹.     

Development, validation and data quality assurance of screening methods: a case study

Despite the growing importance of qualitative screening tests in routine laboratories involved in the EU official control, their validation is not as deeply explained in Commission Decision 2002/657/EC as the validation of quantitative confirmatory methods. At the same time, the issue of quality assurance of screening assays defining internal quality control (IQC) procedures as required by accreditation bodies is undoubtedly less developed in this analytical field. As an example the present study describes the development, the validation and the IQC implemented for a commercial enzyme linked immunosorbent assay (ELISA) able to detect 17-α-19-nortestosterone (α-NT) and 17-β-19-nortestosterone (β-NT) isomers in bullock urine. In order to select a suitable sample treatment, two SPE purification protocols were preliminary compared. The chosen method was therefore fully validated determining the mandatory parameters required by Commission Decision 2002/657/EC: specificity, detection capability and robustness. An in-depth discussion was carried out illustrating the possible validation approaches and their implications especially in the assessment of the key performance characteristic: detection capability. Finally, the control charts implemented for continuous method verification during analyses of real samples were reported.     

European analytical criteria: past, present, and future

In this paper, the past, present, and (possible) future of the European analytical criteria for residues are described. The elaboration of the revision of Commission Decision 9312561EC was a long process starting in 1996 and ending with the formation of a European Commission (EC) working group in 1998. This working group took account of developments in scientific and technical knowledge at that time and produced a draft version of the revision within 6 months. The revision, finally published in 2002 (2002/657/EC), includes new ideas on the identification of analytes and the criteria for performance assessment as well as validation procedures. Currently (2009), the evolution in analytical equipment and progress in scientific research, accompanied by recent European regulatory changes, demands an update or revision of the 2002/657/EC.     

Multiresidue Screening of Veterinary Drugs in Meat,Milk, Egg,and Fish Using Liquid Chromatography Coupled with Ion Trap Time-of-Flight Mass Spectrometry

New approaches to veterinary drug screening based on liquid chromatography-mass spectrometry (LC-MS/MS) and time-of-flight mass spectrometry (ToF/MS) are rapid and have high selectivity and sensitivity. In this study, we developed a multiresidue method for screening over 100 veterinary drug residues using ion trap (IT)-ToF/MS. The screened compounds comprised major drug classes used in veterinary practice, representing the following: amphenicols, anthelmintics, benzimidazoles, β-lactams, coccidiostats, ionophores, macrolides, non-steroidal anti-inflammatory drugs, quinolones, sulfonamides, tetracyclines, and tranquilizers. The method was developed based on chromatographic retention time, specific accurate mass, isotope distribution, and fragment data. Each compound was validated at three levels, and the mass accuracy, accuracy, and repeatability were calculated. All parameters showed acceptable values and conformed to the Commission Decision 2002/657/EC criteria. This screening method can simultaneously analyze over 100 veterinary drugs in meat, milk, eggs, and fish in a single analytical run.     

Rapid determination of chloramphenicol residues in milk powder by liquid chromatography-elektrospray ionization tandem mass spectrometry

A simple and rapid liquid chromatography tandem mass spectrometry (LC-ESI-MS-MS) confirmation method for the analysis chloramphenicol (CAP) in milk powder has been developed. Samples were extracted by using liquid-liquid extraction steps with ethyl acetate. Lipids were removed using hexsan. LC separation was achieved by using a Phenomenex Luna C-18 column and acetonitryle-water as a mobile phase. The mass spectrometer was operated in multiple reaction monitoring mode (MRM) with negative electro-spray interface (ESI-). The four transitions were monitored m/z 321-->257, 321-->194, 321-->152, 326-->157 (IS) and for quantification, the transition m/z 321-->152 was chosen. Validation of the method was done according to criteria of Decision Commission No 2002/657 EC. Validation includes the determination of specification, linearity, precision (within- and between-day), accuracy, decision limit (CC alpha) and detection capability (CC beta). Samples were fortified at CAP levels 0.30, 0.45 and 0.60 microg/kg with CAP-5d as internal standard. The precision within-day (RSD%) was lower than 12% and accuracy (RE%) ranged from -9.8 to -3.7%. The precision between-day (RSD%) was less than 15%. The limit of decision (CC alpha) and detection capability (CC beta) for milk powder 0.09 and 0.11 microg/kg. Value CC alpha and CC beta were calculated for the 321-->152 ion transition. This method has been successfully used for routine analysis.     

Method validation approach on the basis of a quadratic regression model

A method validation approach that bases on a quadratic regression model in which two types of error are incorporated is presented and applied to an experimental data set. The validation approach enables the determination of analytical performance characteristics referred to in Commission Decision 2002/657/EC (i.e., repeatability, within-laboratory reproducibility, decision limit, detection capability).     

Screening and confirmation criteria for hormone residue analysis using liquid chromatography accurate mass time-of-flight, Fourier transform ion cyclotron resonance and orbitrap mass spectrometry techniques

An emerging trend is recognised in hormone and veterinary drug residue analysis from liquid chromatography tandem mass spectrometry (LC/MS/MS) based screening and confirmation towards accurate mass alternatives such as LC coupled with time-of-flight (TOF), Fourier transform ion cyclotron resonance (FTICR) or Fourier transform orbitrap (FT Orbitrap) MS. In this study, mass resolution and accuracy are discussed for LC/MS screening and confirmation of targeted analytes and for the identification of unknowns using the anabolic steroid stanozolol and the designer beta-agonist "Clenbuterol-R" as model substances. It is shown theoretically and experimentally that mass accuracy criteria without proper mass resolution criteria yield false compliant (false negative) results, both in MS screening and MS/MS confirmation of stanozolol. On the other hand, previous medium resolution accurate mass TOFMS/MS data of the designer beta-agonist were fully confirmed by high resolution FT Orbitrap MS(n) experiments. A discussion is initiated through a proposal for additional criteria for the use of accurate mass LC/MS technologies, to be implemented in Commission Decision 2002/657/EC.     

Study of robustness based on n-way models in the spectrofluorimetric determination of tetracyclines in milk when quenching exists

An important step in the validation of an analytical procedure is the study of its robustness. In the case of spectrofluorimetric determinations, quenching introduces specific problems which are approached in this paper for the particular case of tetracyclines determination in milk. Quenching can be detected with excitation emission matrices (EEM) signals and a three-way Parallel Factor (PARAFAC) decomposition and modelled by means of a four-way PARAFAC decomposition which reproduces the physical model of this effect.The robustness of the method is evaluated by including changes in seven experimental variables: trichloroacetic acid (TCA) volume solution used in the precipitation of milk proteins, revolutions per minute, time and temperature in the centrifugation step, pH and emission-excitation slit width in the fluorimetric analyte determination and the analyst. The robustness analysis is carried out by means of a Plackett-Burman experimental design as it is suggested by European Decision 2002/657/EC (European Decision (EC) No. 2002/657/EC of 12 August 2002, implementing Council Directive 96/23/EC, concerning the performance of analytical methods and the interpretation of results, Off. J.L 221, 17/8/2002, 8). The analyte concentration will be taken as response in the Plackett-Burman experimental design instead of the signal as it is habitual in these cases. Therefore, a three-way Partial Least Squares (3-PLS) calibration models with EEM signal is needed.When an analogous study is carried out for tetracycline (TC) in the absence of chlorotetracycline (CTC) as interferent, univariate calibration is employed, being able to conclude that in the robustness analysis, different factors are significantly active when quenching exists.     

Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry

A rapid, specific and highly sensitive multi-residue method for the determination of anabolic steroid residues in bovine, pork and poultry muscle tissues was developed. The sample preparation involves enzymatic digestion followed by extraction with methanol. The crude extract was cleaned up by solid-phase extraction (SPE) combining C18 and NH2 columns. The detection was carried out by a highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using both positive and negative ionization modes. Natural and synthetic steroids covering different polarities could be extracted, concentrated and purified using one single method. Mobile phase composition and additives were optimized to achieve the highest sensitivity. The linearity was not good enough for quantitative analysis but the method was well-suited for qualitative confirmation. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.5 ng g(-1) for all the compounds in the three types of meat studied. The developed method is suitable for routine analysis in our laboratories.     

A fast and simple procedure for determination of perfluoroalkyl substances in food and feed: a method verification by an interlaboratory study

In this study, a simple, fast, and cheap sample preparation procedure for the analysis of three well-known representatives of perfluoroalkyl substances (perfluorooctane sulfonate, perfluorooctanoic acid, and perfluorooctane sulfonamide) was validated in accordance with Commission Decision 2002/657/EC. The method was based on extraction with methanol followed by a dispersive solid phase extraction cleanup step by addition of activated charcoal for fish tissue, fish feed, and milk samples. The novel analytical approach combined with liquid chromatography–tandem mass spectrometry makes it possible to achieve limits of quantification below 1 μg/kg (defined by Commission Recommendation 2010/161/EU). This method provides a high laboratory sample throughput: ten samples in 60 min. The validated procedure was successfully verified in an interlaboratory study.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Screening method for the analysis of antiviral drugs in poultry tissues using zwitterionic hydrophilic interaction liquid chromatography/tandem mass spectrometry

A screening method for the analysis of seven anti-viral drugs in poultry tissue has been developed. These include anti-influenza drugs (amantadine, rimantadine, zanamivir and oseltamivir and its carboxylate metabolite), anti-herpes drugs (acyclovir and ganciclovir) and an immunomodulator (imiquimod). Poultry tissue was extracted in acetonitrile:water:acetic acid. After sample purification, using a strong cation exchange column, the eluate was split into two fractions. The first portion was dissolved in methanol:water and the second in acetonitrile:methanol:water. Both fractions were analysed on a zwitterionic hydrophilic interaction liquid chromatography column coupled to a triple quadrupole mass spectrometer. The screening method was successfully validated according to Commission Decision 2002/657/EC in three different laboratories with CCβ concentrations of ≤10 μg kg(-1).     

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.     

Liquid–liquid microextraction methods based on ultrasound-assisted emulsification and single-drop coupled to gas chromatography–mass spectrometry for determining strobilurin and oxazole fungicides in juices and fruits

Two procedures are proposed based on ultrasound-assisted emulsification and single-drop liquid–liquid microextraction for the sensitive determination of seven strobilurin and six oxazole fungicides in fruits and juice samples. Both miniaturized techniques are coupled to gas chromatography with mass spectrometry in the selected ion monitoring mode, GC–MS(SIM). The procedures use low density organic solvents, and several factors influencing the emulsification, extraction and collection efficiency are optimized. The detection limits obtained at a signal-to-noise ratio of 3 are below the MRLs set by the European Commission. Enrichment factors are between 140–1140 for the first technique used and 80–1600 for the latter. The recoveries obtained for spiked samples are satisfactory for all compounds. The methods are validated according to the Commission Decision 2002/657/EC. Different fruit and juices are analyzed by the proposed method and none of the samples contained fungicide residues above the detection limits.     

Determination of ochratoxin A at parts-per-trillion levels in cereal products by immunoaffinity column cleanup and high-performance liquid chromatography/mass spectrometry

Ochratoxin A (OTA) is a toxic and potentially carcinogenic fungal toxin found in a variety of food commodities. A new sensitive method has been developed to quantify OTA in cereal products by reversed-phase liquid chromatography (LC) with mass spectrometric (MS) detection. Ochratoxin B was used as the internal standard. OTA was extracted from cereal products with acetonitrile-water, and the extract was diluted with a buffer; the diluted extract was cleaned up on an immunoaffinity column before LC/MS analysis. Two multiple-reaction monitoring transitions were used, one for quantification of OTA and one for confirmation of identity. The method was shown to be highly sensitive, with a low decision limit (CCalpha) of 0.012 microg/kg and a detection capability (CCbeta) of 0.021 microg/kg. Within-laboratory repeatability coefficient of variation values were 7.1, 3.7, and 3.1%, and the corresponding recoveries were 104, 106, and 103% for rice samples fortified with OTA at 0.05, 0.10, and 0.15 microg/kg, respectively. Method validation was performed according to the criteria of European Commission Decision 2002/657/EC. All criteria as presented in the Commission Decision were fulfilled. This method is the first fully validated method using immunoaffinity chromatography for cleanup and MS for detection in the analysis of cereals for OTA. The method was also successfully applied to cereal-derived products. The analytical results for determination of the OTA content of cereal products commercially available in Hong Kong are also reported.     

Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography–tandem mass spectrometry

This paper describes an analytical method for four phenolic and salicylanilide anthelmintics authorised for use within the EU (nitroxinil, oxyclozanide, rafoxanide and closantel) in bovine kidney, and the extension of this procedure to include a number of related compounds; ioxynil, niclosamide, salicylanide and 3-trifluoromethyl-4-nitrophenol (TFM). The method comprises a solvent extraction with 1% acetic acid in acetone and clean-up using a mixed-mode anion-exchange solid phase extraction column. Determination is by reversed phase LC–MS/MS. The method was validated to the latest EU requirements (Commission Decision 2002/657/EC) using both spiked and incurred tissues and was subject to second laboratory evaluation.