The pivotal role of mass spectrometry in determining the presence of chemical contaminants in food raw materials.

During recent years, a rising interest from consumers and various governmental organizations towards the quality of food has continuously been observed. Human intervention across the different stages of the food supply chain can lead to the presence of several types of chemical contaminants in food-based products. On a normal daily consumption basis, some of these chemicals are not harmful; however, for those that present a risk to consumers, legislation rules were established to specify tolerance levels or in some cases the total forbiddance of these specific contaminants. Hence, the use of appropriate analytical tools is recommended to properly identify chemical contaminants. In that context, mass spectrometry (MS)-based techniques coupled or not to chromatography offer a vast panel of features such as sensitivity, selectivity, quantification at trace levels, and/or structural elucidation. Because of the complexity of food-based matrices, sample preparation is a crucial step before final detection. In the present manuscript, we review the contribution and the potentialities of MS-based techniques to ensure the absence of chemical contaminants in food-based products.

Comprehensive analytical strategy for biomarker identification based on liquid chromatography coupled to mass spectrometry and new candidate confirmation tools.

A comprehensive analytical LC-MS(/MS) platform for low weight biomarkers molecule in biological fluids is described. Two complementary retention mechanisms were used in HPLC by optimizing the chromatographic conditions for a reversed-phase column and a hydrophilic interaction chromatography column. LC separation was coupled to mass spectrometry by using an electrospray ionization operating in positive polarity mode. This strategy enables us to correctly retain and separate hydrophobic as well as polar analytes. For that purpose artificial model study samples were generated with a mixture of 38 well characterized compounds likely to be present in biofluids. The set of compounds was used as a standard aqueous mixture or was spiked into urine at different concentration levels to investigate the capability of the LC-MS(/MS) platform to detect variations across biological samples. Unsupervised data analysis by principal component analysis was performed and followed by principal component variable grouping to find correlated variables. This tool allows us to distinguish three main groups whose variables belong to (a) background ions (found in all type of samples), (b) ions distinguishing urine samples from aqueous standard and blank samples, (c) ions related to the spiked compounds. Interpretation of these groups allows us to identify and eliminate isotopes, adducts, fragments, etc. and to generate a reduced list of m/z candidates. This list is then submitted to the prototype MZSearcher software tool which simultaneously searches several lists of potential metabolites extracted from metabolomics databases (e.g., KEGG, HMDB, etc) to propose biomarker candidates. Structural confirmation of these candidates was done off-line by fraction collection followed by nanoelectrospray infusion to provide high quality MS/MS data for spectral database queries.

Multi-screening approach to monitor and quantify 42 antibiotic residues in honey by liquid chromatography-tandem mass spectrometry.

A multi-screening approach for monitoring potential chemical contaminants in honey by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) has been developed. A total of 42 veterinary drugs (5 tetracyclines, 7 macrolides, 3 aminoglycosides, 8 beta-lactams, 2 amphenicols and 17 sulfonamides) were surveyed with the ultimate goal of unambiguously confirmed and quantified these analytes at a concentration level of 20 microg/kg. A basic sample preparation including four subsequent liquid/liquid extraction steps was necessary to adequately extract the compounds of interest from the honey. The four extracts were injected into the LC-ESI-MS/MS using a stacking injection procedure. Validation of the entire procedure was carried out according to the European Union directive 2002/657/EC at three concentration levels, i.e., 10, 20 and 30 microg/kg. Good performance data were obtained for 37 analytes, out of the 42 studied. Limit of compliance and detection limit were calculated based on an internal limit set at 20 microg/kg for all the compounds and ranged between 24-30 and 27-80 microg/kg, respectively. A limited survey on honeys of different geographical origins has demonstrated that positive honey samples were often contaminated by more than one class of drugs, thus highlighting the usefulness of such multi-screening approach to ensure and warrants the quality of honey.

Use of molecularly imprinted solid-phase extraction sorbent for the determination of four 5-nitroimidazoles and three of their metabolites from egg-based samples before tandem LC-ESIMS/MS analysis.

A nitroimidazole, molecularly imprinted polymer (MIP) was tested to extract four 5-nitroimidazoles (i.e., dimetridazole (DMZ), ipronidazole (IPZ), metronidazole (MNZ), and ronidazole (RNZ)) and three of their metabolites (i.e., DMZOH, IPZOH, and MNZOH) from egg powder samples. Various MIP templates were produced, and their selectivity was assessed on nitroimidazole standard solutions using liquid chromatography coupled with ultraviolet detection. The optimal cleanup was then used for the extraction of nitroimidazole in egg powder samples, and their quantification was achieved by isotope dilution LC-ESIMS/MS. The sample preparation entails a solubilization of the samples with water and acetonitrile followed by a MISPE cleanup step before LC-ESIMS/MS analysis. Data acquisition was achieved using selected reaction monitoring, and quantification was done with five deuterated analogues (i.e., DMZ- d(3), RNZ- d(3), IPZ- d(3), DMZOH- d(3), and IPZOH- d(3)). DMZOH- d(3) was used to quantify MNZ and MNZOH since they do not have their corresponding internal standards. The method was validated according to the European Union criteria by spiking experiments at concentration levels of 1, 2, and 3 microg/kg. At these three levels and for compounds having their own internal standards, acceptable performance data were obtained, with internal standard corrected recoveries ranging from 91 to 111%, and decision limits (CCalpha) and detection capabilities (CCbeta) were below 0.34 and 0.39 microg/kg, respectively.

LC-HRMS Metabolomics for Untargeted Diagnostic Screening in Clinical Laboratories: A Feasibility Study.

Today’s high-resolution mass spectrometers (HRMS) allow bioanalysts to perform untargeted/global determinations that can reveal unexpected compounds or concentrations in a patient’s sample. This could be performed for preliminary diagnosis attempts when usual diagnostic processes and targeted determinations fail. We have evaluated an untargeted diagnostic screening (UDS) procedure. UDS is a metabolome analysis that compares one sample (e.g., a patient) with control samples (a healthy population). Using liquid chromatography (LC)-HRMS full-scan analysis of human serum extracts and unsupervised data treatment, we have compared individual samples that were spiked with one xenobiotic or a higher level of one endogenous compound with control samples. After the use of different filters that drastically reduced the number of metabolites detected, the spiked compound was eventually revealed in each test sample and ranked. The proposed UDS procedure appears feasible and reliable to reveal unexpected xenobiotics (toxicology) or higher concentrations of endogenous metabolites. HRMS-based untargeted approaches could be useful as preliminary diagnostic screening when canonical processes do not reveal disease etiology nor establish a clear diagnosis and could reduce misdiagnosis. On the other hand, the risk of overdiagnosis of this approach should be reduced with mandatory biomedical interpretation of the patient’s UDS results and with confirmatory targeted and quantitative determinations.

Evaluation of atmospheric pressure ionization interfaces for quantitative measurement of sulfonamides in honey using isotope dilution liquid chromatography coupled with tandem mass spectrometry techniques.

A comparison was made between electrospray, atmospheric pressure chemical and atmospheric pressure photospray ionizations to evaluate the MS/MS responses of standard sulfonamides and honey spiked samples. The sample preparation entails an acidic hydrolysis followed by a liquid/liquid extraction. Full method validation was realised by LC-APPI-MS/MS. Decision limit and detection capability were calculated for each analyte (at 50 microg/kg) and ranged between 53.6 and 56.9 and 57.5 and 63.2 microg/kg, respectively. Limits of detection and of quantification ranged, respectively, at 0.4-4.5 and 1.2-15.0 microg/kg. Precursor ion scan experiments of m/z 92 were also carried out as a survey experiment, linked with an enhanced product ion scan experiment to potentially identified additional sulfonamides via a library search.

Quantitative determination of five ergot alkaloids in rye flour by liquid chromatography-electrospray ionisation tandem mass spectrometry.

A confirmatory method for detecting five ergot alkaloids, ergocristine, ergotamine, ergonovine, ergocornine and alpha-ergokryptine, in rye flour is described using high performance liquid chromatography coupled to tandem mass spectrometry detection by monitoring two transition reactions per analyte. The procedure entails a liquid-liquid extraction followed by a clean-up step using a C18 solid-phase extraction (SPE) cartridge. An analogue compound, methysergide hydrogen maleinate, was used to assess both repeatability sample preparation and potential MS response fluctuations. The method was fully validated according to the European Union (EU) criteria. Detection and quantification limits of all analytes were calculated ranging from 7 to 11 microg/kg and from 23 to 37 microg/kg, respectively. Fifteen rye flour samples were investigated with the newly developed method, and none of them were above the current Swiss limits of 200mg/kg for total ergot alkaloids.

Validation of a dried blood spot LC-MS/MS approach for cyclosporin A in cat blood: comparison with a classical sample preparation.

A rapid, selective and sensitive bioanalytical method was developed and validated for cyclosporin A (CsA) in cat blood samples using dried blood spot (DBS) coupled with high pressure liquid chromatography hyphenated to positive electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). CsA was quantified using a structural analog cyclosporin D (CsD) as internal standard in multiple reaction monitoring mode (MRM) using the transitions 1220→1203 for CsA and 1234→1217 for CsD. A 5-µL blood aliquot was spotted onto a DBS card, then after a drying step, blood spots were punched out from the cards and extracted with MeOH before injection into a LC-MS/MS system. The linear range of the method was 5-2000 ng/mL, with accuracy and precision within the FDA acceptance criteria (i.e., ±15% and ±20% for the lowest level). In the study presented herein a comparison was made between this new methodology, based on the use of DBS, and a previously developed solid phase extraction (SPE) procedure, applied to blood samples from a cat pharmacokinetic study. Good correlation (r(2)=0.97) was demonstrated between the data obtained with the two methods. The DBS methodology revealed to be cheaper, faster, less solvent-consuming and requiring less blood volume from animals than the previous SPE method. Thus, the proposed HPLC-ESI-MS/MS method, based on DBS, has demonstrated to be a suitable and advantageous approach for the analysis of CsA in cat blood.

Advantages of molecularly imprinted polymers LC-ESI-MS/MS for the selective extraction and quantification of chloramphenicol in milk-based matrixes. comparison with a classical sample preparation.

A simple and fast selective extraction of the antibiotic chloramphenicol (CAP) from milk (raw milk, skimmed milk, and milk powder) using a molecularly imprinted polymer (MIP) sorbent is described. The method entails a single centrifugation step prior to loading the supernatant onto the MIP cartridge and subsequent elution with a mixture of solvents. CAP was further analyzed by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) operating in negative ionization acquisition mode. The advantages of the MIP approach were assessed by comparing the data generated from a classical solid-phase and liquid-liquid extractions procedure, previously developed in our laboratory. A better recovery of CAP due to an enhanced selectivity and a faster turnaround time (18 samples processed within 3 h compared to 8 h with the classical approach) were evidenced when using the MIP cleanup. The analysis of CAP in raw milk was further validated according to the 2002/657/EC European Union criteria for the analysis of veterinary drug residues at the minimum required performance limit (MRPL) of 0.3 microg/kg, using CAP-d(5) as internal standard. Non-internal-standard corrected recovery values ranged between 50% and 87% over the range of concentrations considered. The decision limit (CCalpha) and detection capability (CCbeta) were calculated to be 0.06 and 0.10 microg/kg, respectively.

Mass spectral characterization of ergot alkaloids by electrospray ionization, hydrogen/deuterium exchange, and multiple stage mass spectrometry: Usefulness of precursor ion scan experiments.

Six ergot alkaloids belonging to the lysergic acid derivatives (ergonovine (EGN) and methysergide hydrogen maleinate (MHM)) and peptide-type derivatives (ergocristine (EGR), ergotamine (EGT), ergocornine (EGC) and alpha-ergokryptine (EGK)) were studied by positive electrospray tandem mass spectrometry. The fragmentation mechanisms of these compounds were studied by collision-induced dissociation (CID) using triple quadrupole and ion trap mass spectrometers, and the nature of the major product ions further confirmed by hydrogen/deuterium (H/D) exchange experiments. A common abundant product ion at m/z 223 was characteristic of the two classes of ergot alkaloids. Therefore, a precursor ion scan of m/z 223 that triggers information data acquisition (IDA) in combination with CID experiments was used to identify other potential ergot alkaloids. Using this approach, it was possible to confirm the presence of ergosine, another peptide-type ergot alkaloid, in a rye flour extract at trace levels.