Full Scan MS Research Articles

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry and isotope ion cluster

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography–electrospray ionization mass spectrometry, gas chromatography–mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS n spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.

Determination of rimonabant in human plasma and hair by liquid chromatography–mass spectrometry

Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC–MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50 μL of plasma or 10 mg of hair using diethylether. Chromatography was performed on a 150 mm × 2.1 mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/ z 463.1 for rimonabant and m/ z 555.1 for IS. The most intense product ion of rimonabant ( m/ z 380.9) and IS ( m/ z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0 ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0 pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85–115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at −20 °C. This simple and robust LC–MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.

Targeted Quantitative Analysis of Streptococcus pyogenes Virulence Factors by Multiple Reaction Monitoring

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Rapid Screening of Glutathione-Trapped Reactive Metabolites by Linear Ion Trap Mass Spectrometry with Isotope Pattern-Dependent Scanning and Postacquisition Data Mining

The present study describes a novel integrated approach for rapid analysis of reactive metabolites with a linear ion trap mass spectrometer (LTQ). In this approach, an isotope pattern-dependent scanning method was applied to the data acquisition of glutathione (GSH)-trapped reactive metabolites. Recorded full-scan MS and MS/MS data sets were further processed with neutral loss filtering, product ion filtering, and extracted ion chromatographic analysis to search for protonated molecules and MS/MS spectra of GSH adducts. To evaluate the effectiveness and reliability of the approach, GSH adducts of carbamazepine, diclofenac, 4-ethylphenol, acetaminophen, p-cresol, and omeprazole were analyzed, which were formed in human liver microsome incubations fortified with a mixture of nonlabeled GSH and stable isotope-labeled GSH at a 1:0.8 ratio. Results demonstrate that the combination of the isotope pattern-dependent scanning with the postacquisition data mining was very effective in detecting low levels of GSH adducts, regardless of their fragmentation patterns. As compared to a neutral loss scanning method performed with a triple quadrupole mass spectrometer, the LTQ-based approach had several major advantages, including the superior selectivity and sensitivity in detecting different classes of GSH adducts and the higher throughput capability of the detection and MS/MS spectral acquisition of GSH adducts in a single LC/MS run. Overall, this analytical approach provides a simple and efficient means for screening for reactive metabolites using a linear ion trap LC/MS platform.

Quantitative Proteomics Reveals Regulation of Dynamic Components within TATA-binding Protein (TBP) Transcription Complexes

Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.

LC-DAD and LC–MS–MS Analysis of Piperidine Alkaloids of Lobelia inflata L. (In Vitro and In Vivo)

An LC-DAD method was developed for determination of lobeline from in vitro and in vivo cultures of Lobelia inflata. Samples were extracted with 0.1 N HCl–acetonitrile (1:1, v/v), and purified by solid-phase extraction. Optimized conditions resulted in high recovery. LC separations were performed on an Eurosphere C8 reversed-phase column using 30:70 (v/v) acetonitrile–0.1% trifluoroacetic acid as a mobile phase. Quantitative determination of lobeline was performed by external standard method at 250 nm, in the range of 2.4–80 μg mL−1. Validation studies proved that the repeatability of the method was good and the recovery was satisfactory. In vitro organized cultures contained considerable amount of lobeline (herb: 175 μg g−1, root: 100 μg g−1). When these cultures were transplanted into the open field, the lobeline content increased significantly (herb: 323 μg g−1, root: 833 μg g−1). Plants obtained from seed propagation contained 382 μg g−1 lobeline in the herb. For direct characterization of di-substituted piperidine alkaloids in extracts of L. inflata, tandem mass spectrometric method was developed using electrospray ionization. Analysis was performed in the positive ion mode on a triple quadropole LC–MS system. LC separations were achieved on Eurosphere C8 column with a modified mobile phase (acetonitrile–30 mM ammonium formate, pH 2.80) to ensure proper molecular ionization. The identification and structural elucidation of the alkaloids were performed by comparing their changes in molecular mass (ΔM), full-scan MS–MS spectra with those of lobeline, norlobelanine and lobelanidine. These alkaloids and ten other derivatives were identified in the plant extracts. Three piperidine alkaloids were reported in L. inflata for the first time.

Rapid screening and characterization of drug metabolites using a multiple ion monitoring–dependent MS/MS acquisition method on a hybrid triple quadrupole‐linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.

Highly efficient analysis of underivatized carbohydrates using monolithic-silica-based capillary hydrophilic interaction (HILIC) HPLC

A polyacrylamide (PAAm)-modified monolithic silica capillary column of increased phase ratio, 200T-PAAm, for hydrophilic interaction liquid chromatography (HILIC) was prepared. The column showed high separation efficiency, with a theoretical plate height H = 7-20 microm at a linear velocity, u = 1-7 mm/s. From a kinetic plot analysis, it was expected that the monolithic column could provide three times faster separation than particle-packed HILIC columns under a pressure limit at 20 MPa. HILIC coupled with electrospray ionization (ESI)-mass spectrometry (HILIC-ESI-MS) using the 200T-PAAm column was employed for the analysis of underivatized carbohydrates to achieve fast and efficient separations of mixtures containing mono-, di-, and trisaccharides within 5 min. Under single MS full scan mode, 200 pg of oligosaccharides was detected by the system. The limit of detection (LOD) of the LC-ESI-MS/MS system was determined using selected reaction monitoring (SRM) to be as low as 3.2 ng/mL (attomol level) for nonreducing saccharides. The system was successfully applied to the detection of disaccharides in extracts of plant, such as corn, soybean, and Arabidopsis thaliana.

Rapid screening for cyclo‐dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. using liquid chromatography/tandem mass spectrometry

A simple and rapid qualitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for screening cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L. at sub-ppm levels. An electrospray ionization orbitrap mass spectrometer, which provides accurate full scan MS and MS/MS data, was used in this study. After simple extraction with ethanol and purification by AB-8 resin, the extracts were subjected to LC/MS/MS analysis. A high mass tolerance (10 ppm) was used in the initial screening to filter the full scan MS data. The cyclo-dopa and diketopiperazine alkaloid standards gave limits of detection (LODs) at or below 5 ng/mL. The results also indicated that the method had an acceptable precision for day-to-day use in the identification of compounds. The alkaloids could be identified based on their MS/MS data, elemental compositions, and retention behavior. This system was used to assay trace amounts of cyclo-dopa and diketopiperazine alkaloids in crude extracts of Portulaca oleracea L., leading to the identification of 5/2 confirmed/unconfirmed cyclo-dopa and 7/6 confirmed/unconfirmed diketopiperazine alkaloids, respectively. The screening method considerably reduces the time and cost involved in the identification of cyclo-dopa and diketopiperazine alkaloids in Portulaca oleracea L., as well as being a simple and convenient approach to the identification of other structural families of natural products.

Study of the Metabolites of Bencycloquidium Bromide Racemate, a Novel Anticholinergic Compound, in Rat Bile by Liquid Chromatography-Tandem Mass Spectrometry

A sensitive and specific liquid chromatographic-electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of bencycloquidium bromide (BCQB) and its metabolites in rat bile. Six healthy rats were administrated a single dose (3.0 mg kg(-1)) of BCQB by intraperitoneal (i.p.) injection. The bile were sampled from 0 h to 24 h and purified by using a C(18) solid- phase extraction (SPE) cartridge, then the purified bile samples were separated on a reversed-phase C(18) column using acetonitrile/40 mM ammonium acetate buffer (containing 0.1% formic acid) as mobile phase at gradient elution and detected by an on-line MS(n) detector. Identification and structural elucidation of the metabolites were performed by comparing the changes in molecular weight (Deltam) and full scan MS(n) spectra with those of the parent drug. Eight metabolites (such as hydroxylated and oxidized metabolites) and the parent drug were found in rat bile. Eight metabolites of BCQB were identified and hydroxylated metabolites were the major metabolites. The metabolic pathways of BCQB in vivo are proposed for the first time.

Chymotrypsin B Cached in Rat Liver Lysosomes and Involved in Apoptotic Regulation through a Mitochondrial Pathway

Lysosomes can trigger the mitochondrial apoptotic pathway by releasing proteases. Here we report that a 25-kDa protein purified from rat liver lysosomes possesses a long standing potent Bid cleavage activity at neutral pH, and the truncated Bid can in turn induce rapid mitochondrial release of cytochrome c. This protease was revealed as chymotrypsin B by biochemical and mass spectrometric analysis. Although it was long recognized as a digestive protease exclusively secreted by the exocrine pancreas, our data support that it also expresses and intracellularly resides in rat liver lysosomes. Translocation of lysosomal chymotrypsin B into cytosol was triggered by apoptotic stimuli such as tumor necrosis factor-alpha, and intracellular delivery of chymotrypsin B protein induced apoptotic cell death with a potency comparable with cathepsin B, suggestive of a lysosomal-mitochondrial pathway to apoptosis regulated by chymotrypsin B following its release. Noteworthily, either knockdown of chymotrypsin B expression by RNA interference or pretreatment with chymotrypsin B inhibitor N-p-tosyl-L-phenylalanine chloromethyl ketone significantly reduced tumor necrosis factor-alpha-induce apoptosis. These results demonstrate for the first time that chymotrypsin B is not only restricted to the pancreas but can function intracellularly as a pro-apoptotic protease.

Humoral Response Profiling Reveals Pathways to Prostate Cancer Progression

There is considerable evidence for an association between prostate cancer development and inflammation, which results in autoantibody generation against tumor proteins. This immune system-driven amplification of the autoantibody response to intracellular antigens can serve as a sensitive tool to detect low abundance serum proteomic tumor markers for prostate cancer as well as provide insight into biological processes perturbed during cancer development. Here we examine serum humoral responses in a cohort of 34 patients with either benign prostatic hyperplasia or clinically localized prostate cancer (PCa). The experimental strategy couples multidimensional liquid-phase protein fractionation of localized and metastatic prostate cancer tissue lysates to protein microarrays and subsequent mass spectrometry. A supervised learning analysis of the humoral response arrays generated a parsimonious predictor having 78% sensitivity and 75% specificity in distinguishing PCa from benign prostatic hyperplasia in a cohort of American males with elevated prostate-specific antigen. Enrichment analysis of the PCa-specific humoral signature revealed large scale immune reprogramming mediated by STAT transcription factors and the generation of autoantibodies to enzymes involved in nitrogen metabolism. Meta-analysis of independent prostate cancer gene expression data validated the presence of STAT-induced immunomodulation. Concomitant validation of elevated levels of the nitrogen metabolism pathway was obtained by direct measurement of metabolic levels of glutamate and aspartate in prostate cancer tissues. Thus, in addition to functioning as markers in prostate cancer detection, humoral response profiles can serve as powerful tools revealing pathway dysregulation that might otherwise be suppressed by the complexity of the cancer proteome.

Polymethoxylated flavones metabolites in rat plasma after the consumption of Fructus aurantii extract: Analysis by liquid chromatography/electrospray ion trap mass spectrometry

A LC with full scan MS n method was developed in order to investigate the in vivo absorption and biotransformation of polymethoxylated flavones (PMFs) by analysis of plasma samples from rats after ingestion of Fructus aurantii extract. Four parent compounds and six metabolites with intact flavonoid structures were tentatively identified. The metabolites were either glucuronides of parent compounds or glucuronides of demethylated products of parent compounds.

An integrated method for metabolite detection and identification using a linear ion trap/Orbitrap mass spectrometer and multiple data processing techniques: application to indinavir metabolite detection

A new strategy using a hybrid linear ion trap/Orbitrap mass spectrometer and multiple post-acquisition data mining techniques was evaluated and applied to the detection and characterization of in vitro metabolites of indinavir. Accurate-mass, full-scan MS and MS/MS data sets were acquired with a generic data-dependent method and processed with extracted-ion chromatography (EIC), mass-defect filter (MDF), product-ion filter (PIF), and neutral-loss filter (NLF) techniques. The high-resolution EIC process was shown to be highly effective in the detection of common metabolites with predicted molecular weights. The MDF process, which searched for metabolites based on the similarity of mass defects of metabolites to those of indinavir and its core substructures, was able to find uncommon metabolites not detected by the EIC processing. The high-resolution PIF and NLF processes selectively detected metabolites that underwent fragmentation pathways similar to those of indinavir or its known metabolites. As a result, a total of 15 metabolites including two new indinavir metabolites were detected and characterized in a rat liver S9 incubation sample. Overall, these data mining techniques, which employed distinct metabolite search mechanisms, were complementary and effective in detecting both common and uncommon metabolites. In summary, the results demonstrated that this analytical strategy enables the high-throughput acquisition of accurate-mass LC/MS data sets, comprehensive search of a variety of metabolites through the post-acquisition processes, and effective structural characterization based on elemental compositions of metabolite molecules and their product ions.

Characterization and identification of triterpenoid saponins in crude extracts from Clematis spp. by high‐performance liquid chromatography/electrospray ionization with multi‐stage tandem mass spectrometry

High-performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC/ESI-MS(n)) was applied to characterize and identify triterpenoid saponins in crude extracts from nine species of Clematis L. After separation on a Zorbax SB-C(18) column, negative ESI-MS experiments were performed. The quasi-molecular ions [M-H]-, [M+Cl]- and [M+HCOO]- were observed in the full-scan MS spectra of all compounds. The MS(n) (n = 2-4) data of the [M-H]- ions provided structural information on the sugar sequence of the oligosaccharide chains and on the aglycone of the saponins. In addition, the fragmentation mechanisms could be deduced from the fragment ions. As a result, eight saponins were unambiguously identified in C. ganpiniana by comparison with reference compounds. In addition, a new compound was tentatively identified as 3-O-ribopyranosyl --> rhamnopyranosyl --> (glucopyranosyl) --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 1), and another one was tentatively deduced to be 3-O-glucopyranosyl --> ribopyranosyl --> rhamnopyranosyl --> arabinopyranosylhederagenin 28-O-rhamnopyranosyl --> glucopyranosyl --> glucopyranosyl ester (peak 5) from the genus Clematis L. for the first time. By ESI-MS(n), non-isomeric saponins could be discriminated rapidly. It is of interest that cleavage preferentially occurrs at the ester bond at C-28 and the charge is easy to transfer onto the oligosaccharide chain when the ester bond of a monodesmosidic saponin like HNH cleaves.

Detection and Structural Characterization of Glutathione-Trapped Reactive Metabolites Using Liquid Chromatography−High-Resolution Mass Spectrometry and Mass Defect Filtering

The present study was designed to apply the mass defect filter (MDF) approach to the screening and identification of reactive metabolites using high-resolution mass spectrometry. Glutathione (GSH)-trapped reactive metabolites of acetaminophen, diclofenac, carbamazepine, clozapine, p-cresol, 4-ethylphenol, and 3-methylindole in human liver microsomes (HLM) were analyzed by HPLC coupled with Orbitrap or Fourier transform ion cyclotron resonance mass spectrometry. Through the selective removal of all ions that fall outside of the GSH adduct MDF template windows, the processed full scan MS chromatograms displayed GSH adducts as major components with no or a few interference peaks. The accurate mass LC-MS data sets were also utilized for the elimination of false positive peaks, detection of stable oxidative metabolites with other MDF templates, and determination of metabolite molecular formulas. Compared to the neutral loss scan by a triple quadrupole instrument, the MDF approach was more sensitive and selective in screening for GSH-trapped reactive metabolites in HLM and rat bile and far more effective in detecting GSH adducts that do not afford the neutral loss of 129 Da as a significant fragmentation pathway. The GSH adduct screening capability of the MDF approach, together with the utility of accurate mass MS/MS information in structural elucidation, makes high-resolution LC-MS a useful tool for analyzing reactive metabolites.

Identification of metabolites of lobeline in the rat urine by liquid chromatography–tandem mass spectrometry

This is a report about the analysis of lobeline and its metabolites in rat urine by using high-performance liquid chromatography–electrospray ionization ion trap tandem mass spectrometric method (LC/MS n ). The urine of healthy rat were sampled from 0 to 24 h after administered a single dose of lobeline (3 mg/kg) by oral gavage, then centrifuged at 10,000 rpm for 10 min to get the supernatants. The supernatants were purified by solid-phase extraction (SPE) with a C 18 cartridge. After the above purified process, the purified urine were injected into a reversed-phase C 18 column with mobile phase of methanol/water (70:30, v/v, adjusted to pH 3.5 with formic acid) and detected by an on-line MS n system. The identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass (Δ M), full-scan MS n spectra with those of the parent drug. Ten metabolites of lobeline were found in rat urine. All the metabolites were reported for the first time.

Fast screening of pesticide residues in fruit juice by solid-phase microextraction and gas chromatography–mass spectrometry

A new vanguard–rearguard analytical method for determining 54 pesticide residues in different fruit juices (natural and commercial orange, peach and pineapple juices were tested) is proposed. For that, in a first step, a fast screening (vanguard) method is applied for detecting those samples containing pesticides at concentrations above a pre-established cut-off value. In a second step, those samples are re-analyzed by a conventional pesticide residue (rearguard) method that confirms the presence of the pesticides and quantifies them. The sample process is very simple, fast and semiautomatic and therefore, it reduces significantly the average time required per sample, increases precision and minimizing human mistakes. Only 1 mL of juice sample is required for analysis. Pesticides are quickly extracted with ethyl acetate in a test tube, transferred to a mixture water:acetone 9:1 (v/v), and isolated by solid-phase microextraction (SPME). The SPME screening method only requires 10 min of SPME extraction. The SPME confirming/quantifying method requires 55 min of SPME extraction. The instrumental determination is carried out by gas chromatography–mass spectrometry (GC–MS) using a full scan acquisition mode for the screening method (less than 17 min of chromatographic run) and a tandem mass spectrometry (MS/MS) acquisition mode for the quantifying/confirming method (less than 70 min of chromatographic run). The use of full scan MS and tandem MS for the detection increase significantly the certainty of the results. Also, the combination of a solvent and SPME extractions and GC–MS/MS offers a significant selectivity and sensitivity with a proven reduction of false positive and negative cases. The use of a vanguard–rearguard strategy can reduce the 50% of the total time required for determining routinely juices in a laboratory by a traditional strategy (identification, confirmation and quantitation of the pesticides in the samples by a conventional analytical method).

Residue analysis of veterinary drugs and growth-promoting agents

Two major trends are observed in the analysis of veterinary drugs and growth-promoting agents. First is the selection of sample material for monitoring the use of registered veterinary drugs. Traditionally meat, kidney and liver were analyzed but, due to the food scandals in which meat was very often involved, the consumption of alternative products (e.g., eggs and fish) has become more popular and therefore more of interest for residue-monitoring programmes. Most food scandals started with contamination of animal feed, so feed has also gained interest for monitoring purposes. For the detection of unauthorized substances (e.g., growth-promoting agents), the matrix hair is frequently used because residues can be detected in hair even a long time after treatment. Second, the techniques used for residue analysis are moving from target-orientated methods, mainly based on liquid chromatography in combination with triple-quadrupole mass-spectrometric detection (LC–QqQ-MS), towards accurate mass full-scan MS techniques (e.g., time-of-flight (ToF)-MS and Fourier-transform(FT)-MS. Full-scan MS techniques enable retrospective analysis (i.e. without re-injecting the sample) and real multi-residue analysis (including different classes of veterinary drugs). The application of full-scan MS also has consequences for separation and extraction techniques, which have to be applicable to a broad range of compounds differing in physical and chemical properties. Experience shows that ToF-MS in combination with ultra-performance LC (UPLC) is very powerful for multi-residue analysis. The introduction of new MS techniques also has consequences for European Union (EU) criteria defined for confirmation of the identity of compounds. It is suggested that both mass-resolution and mass-accuracy data influence identity confirmation and both parameters should be implemented in the revision of European Commission Decision 2002/657/EC.

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.