Selenium speciation analysis of Misgurnus anguillicaudatus selenoprotein by HPLC–ICP–MS and HPLC–ESI–MS/MS

Analytical methods for selenium (Se) speciation were developed using high-performance liquid chromatography (HPLC) coupled to either inductively coupled plasma mass spectrometry (ICP?CMS) or electrospray ionization tandem mass spectrometry (ESI?CMS/MS). Separations of selenomethionine (Se-Met) and selenocysteine (Se-(Cys)₂) with favorable peak shape and resolution were obtained by both HPLC-ICP-MS and HPLC?CESI?CMS/MS. Both methods achieved low limits of detection, high sensitivity and favorable stability. With HPLC?CESI?CMS/MS, signal suppression was observed when complex matrix was co-eluted, but excellent structural characterization was still achieved. Thus, HPLC-ICP-MS is better for the detection of Se species, and HPLC?CESI?CMS/MS is essential for molecular identification and confirmation. A water-soluble selenoprotein from purified M. anguillicaudatus muscle tissue was analyzed by the two complementary systems (HPLC-ICP-MS and HPLC?CESI?CMS/MS) with high sensitivity and accuracy. The results demonstrated that Se-Met was the predominant selenoamino acid in the purified selenoprotein from M. anguillicaudatus muscle tissue, and the concentration of Se-Met in the selenoprotein was 6.280?mg/kg (dry mass). In addition, in HPLC-ICP-MS, an unknown Se-containing compound with similar polarity to Se-(Cys)₂ was discovered. Using complementary data from HPLC?CESI?CMS/MS, it was determined that this unknown Se-containing compound was not Se(Cys)_2.     

Finding of pesticides in fashionable fruit juices by LC–MS/MS and GC–MS/MS

Products labelled as containing extracts from two mushrooms (cordyceps plus reishi) and the juices from açaí, goji, mangosteen, noni, pomegranate, and sea buckthorn have been analysed for 174 different pesticides, using the validated QuEChERS method for sample preparation and electrospray LC–MS/MS in the positive ion mode for analysis. Pesticides were found in 10 of the 21 samples analysed. Most pesticides found were below the tolerance levels (1–6 μg/g, depending on the pesticide), but some were not. This included boscalid, dimethomorph, iprovalicarb, pyridaben, pyrimethanil, and imazalil, for which there is no tolerance reported or zero tolerance in any fruit. However, genuine açaí that was harvested in the state of Pará and lyophilised in Rio de Janeiro had no detectable pesticides, when analysed by both LC–MS/MS and GC–MS/MS, which can detect 213 more pesticides and industrial chemicals. Likewise no pesticides were found in one sample each of cordyceps plus reishi, sea buckthorn and noni.     

HPLC–PDA–ESI–MS/MS profiling and chemopreventive potential of Eucalyptus gomphocephala DC

An HPLC–PDA–ESI/MS/MS method was developed to identify the phytoconstituents of the EtOAc fraction of Eucalyptus gomphocephala DC. The antioxidant effect of the EtOAc fraction together with its sub-fractions was determined in vitro. The cytotoxicity was evaluated on different cell lines. The EtOAc fraction exhibited strong antioxidant activity, reduced the viability of all cell lines and was more active on MCF-7 and HepG-2 cell lines. Subsequently, the cytotoxicity of the sub-fractions and the isolated compounds were tested on MCF-7, HepG-2. The EtOAc fraction possessed potential antitumour promoting properties. It inhibited the stimulated NO (20%), 5-LOX (48.0%) and COX-2 (49.7%) respectively (at concentration of 20 μg/ml). This study suggests that this fraction is a source of different antioxidant and cytotoxic compounds with potential chemopreventive properties that might prevent different stages of the carcinogenesis process.     

Rapid analysis of phenolic acids in beverages by UPLC–MS/MS

A rapid method for qualitative and quantitative analysis of 17 phenolic acids (gallic acid, 3,5-dihydroxybenzoic acid, protocatechuic acid, chlorogenic acid, gentisic acid, 4-hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid, 3-hydroxybenzoic acid, 4-coumaric acid, sinapic acid, ferulic acid, 3-coumaric acid, 2-coumaric acid, salicylic acid and trans-cinnamic acid) in different beverages was developed using ultra performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS/MS). The analytes were detected in multiple reaction monitoring (MRM) mode and quantified using internal standards of deuterium-labelled 4-hydroxybenzoic (2,3,5,6-D4) and salicylic (3,4,5,6-D4) acids. Limits of detection (LODs) ranged from 0.15 to 15 pmol and the response was linear to 1000 pmol injected. Mean method precision of 4.4 RSD% (range, 2.0–9.1%) was obtained, and a mean accuracy (bias) of 1.1% (range, −14.5 to 17.5%). The applicability of this analytical approach was confirmed by the successful analysis of real samples of white wine, grapefruit juice and green tea infusion. Twelve phenolic acids were determined in the analysed beverages, in concentrations ranging from 40.8 to 9046 μg L⁻¹ and the results were compared to data from previous studies.     

Hydrolysis of β-lactoglobulin by trypsin under acidic pH and analysis of the hydrolysates with MALDI–TOF–MS/MS

Trypsin (EC 3.4.21.4) hydrolysis of food proteins are done at the optimum pH (7.8) and temperature (37 °C). Little information is available on the effect of sub-optimal conditions on hydrolysis. Bovine β-lactoglobulin (β-Lg) was hydrolysed by trypsin under acidic pH (pH 4–7) between 20 and 60 °C and the substrate concentration from 2.5% to 15% (w/v) and compared with hydrolysis at pH 7.8 and 37 °C. Aliquots were taken at different times (t = 0 up to 10 min). Samples were analysed using matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI–TOF–MS/MS) with α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxyacetophenone (DHAP) matrices. Hydrolysis patterns of β-Lg were generally similar at pH 7.8, 7, 6 and 5 while at pH 4 fewer peptides were detected except a unique fragment f(136–141). The different cleavage sites of β-Lg showed low resistance to trypsin at optimum conditions and pH 7 while being random and simultaneous. At lower pH, some cleavage sites showed increased resistance, while hydrolysis was relatively slow and ordered. Initial attack by trypsin occurred at Arg⁴⁰–Val⁴¹, Lys¹⁴¹–Ala¹⁴² and Arg¹⁴⁸–Leu¹⁴⁹ resistance was at Lys⁶⁰–Trp⁶¹, Arg¹²⁴–Thr¹²⁵ and Lys¹³⁵–Phe¹³⁶. Five domains were identified based on β-Lg resistance to trypsin in the order f(1–40) < f(41–75) < f(76–91) > f(92–138) > f(139–162). Results suggest that hydrolysis away from trypsin optimum offer better hydrolysis process control and different peptides. This strategy may be used to protect target bioactive or precursor peptides, or avoid the production of unwanted peptides.     

GC–MS analysis of essential oil of some commercial Fennel teas

Fennel teas were prepared by classical infusion or microwave decoction of unbroken and crushed fruits, three pre-packaged teabags and two instant teas. Their volatile constituents were obtained by extraction with n-hexane and analysed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC–MS), using two columns with stationary phases of different polarity. Of the constituents 85–95% were identified on the basis of their GC retention times and their mass spectra in relation to authentic compounds. No volatile constituents were detected in one sample of instant tea. Conventional teas from crushed fruits and teas prepared from the other instant tea showed the highest levels of volatile constituents. Anethole (30–90%) and/or anisaldehyde (0.7–51%) were the main constituents of all the samples. Methychavicol (0.8–4.1%), eugenol (1.5–11.3%) and fenchone (0.5–47%) were detected in most samples. Carvone (2.1–6.1%) was presenting only some teabags and camphor (2.3–2.6%) in others. The volatile constituents of only one instant tea included limonene (1.4%) and α-terpineol (0.4%).     

Application of GC–MSD and LC–MS/MS for the determination of priority pesticides in baby foods in Serbian market

Babies and small children are especially sensitive population to the exposure to environmental contaminants. Their small mass and developing systems, including brain development may show adverse health effects from even low levels of contamination on a chronic or single dose case. In this paper one extraction method and two chromatographic techniques for the determination of pesticide residues in baby food were evaluated. A liquid chromatography–tandem mass spectrometry technique combined with electrospray ionization (ESI), (LC–MS/MS) and gas chromatography–mass spectrometry detection (GC–MSD) technique were applied in the detection of 50 pesticides in baby food. So-called QuEChERS (quick, easy, cheap, effective, rugged and safe) method was used as a sample preparation procedure. The recoveries were investigated at three levels (5, 10 and 50 μg/kg) and the results obtained showed compliance with the contemporary EU requirements with a few exceptions. LOQs for most of the tested pesticides were below the EU MRLs (10 μg/kg), except deltamethrin, cypermethrin, fenvalerate, phosalone and beta-cyfluthrin (LOQs were 10 μg/kg). Both techniques were applied in the analysis of 50 samples of baby food manufactured in Serbia.     

The simultaneous determination of the ionophore antibiotics in animal tissues and eggs by tandem electrospray LC–MS–MS

A method has been developed for the simultaneous extraction and determination of the four most currently used Ionophore antibiotics (lasalocid, monensin, narasin and salinomycin) by LC–MS–MS from different animal tissues and eggs. Results show good repeatability, and mean spiked recoveries for lasalocid, monensin, narasin and salinomycin in animal livers are in the average range 93–103, 96–103, 93–102 and 97–106%, respectively, and in eggs the mean spiked recoveries are 101, 103, 98 and 102% for lasalocid, monensin, narasin and salinomycin, respectively. The detection limit is at 1 ng ml⁻¹ for all the named ionophorous compounds. A quantitation level of 50 ng g⁻¹ for lasalocid, monensin at 2.5 ng g⁻¹, and 10 ng g⁻¹ for narasin and salinomycin is achieved which represents half the action limit prescribed by the UK Regulation in compliance with the European Council Directive 96/23/EC. A high throughput of samples is achievable using this method which allows the analysis of 30–40 samples by one analyst in a day.     

Analysis of seven folates in food by LC–MS/MS to improve accuracy of total folate data

An LC–MS/MS method for analyzing seven folates in food was developed and validated. 5-Methyltetrahydrofolate, 5-formyltetrahydrofolate, 10-formylfolic acid, tetrahydrofolate and folic acid were quantified using a stable isotope dilution assay (SIDA) with deuterated analogues as internal standards. Additionally, 10-formyldihydrofolate and 5,10-methenyltetrahydrofolate were quantified using deuterated internal standards different in structure. Due to interconversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate and 10-formyldihydrofolate to 10-formylfolic acid during sample preparation, a SIDA was not considered because of a resulting double calculation of the amounts interconverting. [²H₄]-5-methyltetrahydrofolate was used as internal standard for 5,10-methenyltetrahydrofolate, due to a similar retention time, and [²H₄]-10-formylfolic acid as well as [²H₄]-5-methyltetrahydrofolate was used for 10-formyldihydrofolate, because no internal standards co-elute. To confirm that no matrix effects affect the quantitation of 5,10-methenyltetrahydrofolate and 10-formyldihydrofolate, postcolumn infusion experiments were performed. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection and limit of quantitation. The latter parameters were partly obtained by application of a dual-isotope label design including [¹³C₅]-labeled folates. The amounts of 5,10-methenyltetrahydrofolate in the purified extracts of different food samples ranged between 0.3 and 1.3 % and for 10-HCO-H₂folate between 0.05 and 8 % of the total folate amount. Correction for incomplete recovery of the latter folate during cleanup indicates even higher contents. Therefore, especially 10-formyldihydrofolate should not be neglected to obtain accurate results for folates.     

Application of an HPLC–MS/MS method for mycotoxin analysis in commercial baby foods

This article describes the validation of an analytical method for the detection of 21 mycotoxins in baby food. The analytical method is based on the simultaneous extraction of selected mycotoxins by matrix solid-phase dispersion (MSPD) followed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) using a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP®). Information Dependent Acquisition (IDA), an extra confirmation tool for samples that contain the selected mycotoxins, was used. The matrix effects were evaluated, and the corrections for the matrix effects were performed using two calibration approaches: external matrix-matched calibration and internal standard calibration. Matrix-matched calibration was ultimately used for accurate quantification, and the recoveries obtained were generally higher than 70%. The analytical method was applied to the analysis of 35 samples of commercial baby foods. No sample exceeded the maximum limit (ML) fixed by the European Union for these mycotoxins in baby food. However, this survey highlighted the occurrence of mycotoxins in cereal-based infant foods.     

Monitoring of the amphetamine-like substances in dietary supplements by LC-PDA and LC–MS/MS

Recently, amphetamine-like substances derived from the β-phenylethylamine core structure have been detected in dietary supplements. Especially, β-methylphenylethylamine (BMPEA), an amphetamine isomer, has been found in dietary supplements labeled as containing Acacia rigidula. The U. S. Food and Drug Administration determined that BMPEA is not naturally present in food and does not meet the statutory definition of a dietary ingredient. In addition, BMPEA has been classified as a psychotropic drug in South Korea and a doping substance by the World Anti-Doping Agency. The aim of this study was to determine whether dietary supplements contained amphetamine and amphetamine-like substance, including β-phenylethylamine (β-PEA) and BMPEA using LC-PDA and LC–MS/MS. In 10 of 110 samples, illegally added compounds were detected in the following ranges; β-PEA 1.4–122.0 mg/g and BMPEA 4.7–37.6 mg/g. This study will contribute to enhancement of food safety in the South Korea.     

Determination of gellan gum by capillary electrophoresis and CE–MS

Intact gellan gum (0.1% m/v) was detectable by capillary electrophoresis (CE) with UV detection. Characteristic tetrasaccharide fragments, prepared with a newly characterised gellan-degrading enzyme, provided a clearer signal that was detectable in complex food products containing other polysaccharides. Food products spiked with gellan gum could be analysed reproducibly with high accuracy and specificity by CE–ESI–MS, which is recommended as the technique of choice. Gellan gum declared as a fruit flavour drink ingredient could not be identified by CE–ESI–MS. When added to the product at the start of sample preparation, before enzyme treatment, the gum was readily detectable, demonstrating that the method was compatible with this sample type. Possible explanations for the negative results are that gellan gum was used as a trace component, with other texturing agents; that its declaration was precautionary only; or that the product contained a chemically modified form. Further work will establish whether modified gellan gums can be similarly analysed.     

LC-MS–MS characterisation of curry leaf flavonols and antioxidant activity

Curry leaf (Murraya koenegii) is a common flavouring agent in Indian foods. This study characterised the flavonol profile of curry leaf extracted with different solvents and the relative antioxidant capacity of these extracts by quantifying phenolic constituents. Flavonols were extracted using ethanol, methanol, or acetone prior to identification and quantification using liquid chromatography coupled to atmospheric pressure chemical ionisation (APCI) mass spectrometry in tandem mode (LC-MS–MS) with negative ion detection. Major curry leaf flavonols included myricetin-3-galactoside, quercetin-O-pentohexoside, quercetin-3-diglucoside, quercetin-3-O-rutinoside, quercetin-3-glucoside, quercetin-3-acetylhexoside, quercetin-O-xylo-pentoside, kaempferol-O-glucoside, and kaempferol-aglucoside. Lag-time and TBARS tests demonstrated that curry leaf phenolics prevent cupric-ion induced oxidation of LDL. The best extraction yield was obtained with 80% ethanol. Acetone extracts provided better antioxidant activity expressed as increased lag-time formation, than did ethanol or methanol extracts. Curry leaf is a rich source of flavonols that have biological activity in vitro and further studies are warranted in regards to the potential health benefits and identification of the novel flavonols whose identities remain unknown.     

Contribution of dynamic headspace GC–MS analysis of aroma compounds to authenticity testing of honey

The volatile profiles of 43 authentic honey samples of different botanical and geographical origins were obtained by means of gas chromatography–mass spectrometry. A qualitative analysis of the volatile compounds identified was performed in order to assess the marker compounds (if/when existing) for both botanical and geographical origin. The results seem to indicate the existence of certain marker compounds for the floral origins assessed (e.g. acacia, chestnut, eucalyptus, heather, lavender, lime, rape, rosemary and sunflower). Also such compounds for two geographical origins (e.g. Denmark and England) seem to exist and possible marker compounds could also be found for the honeys from The Netherlands, Spain and Portugal.     

Comparison of headspace-SPME-GC–MS and LC–MS for the detection and quantification of coumarin,vanillin, and ethyl vanillin in vanilla extract products

A headspace-solid phase micro-extraction (HS-SPME) GC–MS method has been developed for the determination of coumarin, vanillin and ethyl vanillin in vanilla products. Limits of detection ranged from 1.33 to 13.2 ng mL⁻¹. Accuracy and precision data for the method were measured and compared to those obtained using LC-ESI-MS. A survey of 24 commercially available vanilla products was completed using both techniques. No coumarin was detected in any of the samples. Examination of the GC–MS chromatograms revealed the presence of 18 other flavor related compounds in the samples. The method validation and sample analysis data using HS-SPME-GC–MS were comparable to those obtained using the LC–MS method. Because the two methods are conceptually different from one another, both methods would not be subject to the same interferences. This would allow them to be used as confirmatory methods for each other.     

Quantification of folpet and phthalimide in tea and herbal infusions by LC– high-resolution MS and GC–MS/MS

Two methods based on a modified QuEChERS sample preparation and either LC coupled to atmospheric pressure ionisation and high-resolution MS or GC coupled to electron ionisation and tripled quadrupole MS have been assessed for the quantification of folpet and phthalimide in tea and other dry herbal infusions. Both methods have been fully validated in green tea and further checked in black tea, verbena and rooibos, and they performed according to the SANTE/11813/2017 criteria at the target LOQ concentration level (50 µg/kg). These methods allow the accurate quantification of folpet in the selected matrices according to the new EU residue definition, which includes phthalimide. Phthalimide is the main metabolite and degradation product of folpet, although according to recent studies, it could be generated from different sources than folpet breakdown, such as food processing or analysis by GC.     

Quantitative analysis of 3-alkyl-2-methoxypyrazines in German Sauvignon blanc wines by MDGC–MS or MDGC–MS/MS for viticultural and enological studies

A method for trace-level analysis of 3-alkyl-2-methoxypyrazines (MPs) based on mixed-bed cation-exchange solid phase extraction followed by heart-cut multidimensional gas chromatographic analysis was applied in a trial on viticultural and enological treatments of Sauvignon blanc wines produced in Germany. The quantification was based on a stable isotope dilution assay using deuterated MPs. For method comparison, detection was either with selected ion monitoring with a quadrupole mass spectrometer (MS) or after selected reaction monitoring using a triple quadrupole MS. Comparable performance for MP detection in the lower ng L^?1 concentration range was found for both detection methods; however, in some cases, matrix problems could only be solved with MS/MS detection. It could be shown that MP levels varied considerably between the investigated vintages, with concentrations often well below 10 ng L^?1. Leaf removal as a viticultural trial was a measure to decrease MP concentrations; however, the effects observed were low in the vintages studied here. Cold maceration and stem addition to the must were found as valuable enological means for increasing the MP concentration and improving the green sensory notes typical for cold climate Sauvignon blanc wines.     

Determination of Pesticide Residues in Whole Wheat Flour Using Modified QuEChERS and LC–MS/MS

Pesticides in food are a major issue due to their intensive use in agriculture. Thus, an appropriate control of their residues in food samples should be done. In this study, a multiresidue method for the quantification of 37 pesticides in whole wheat flour was developed and validated. The modified QuEChERS (without cleanup) procedure followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) was used for analysis. The method was validated according to the European Union SANCO/12,571/ 2013 guidelines and Brazilian Manual of Analytical Quality Assurance. The following parameters were evaluated on the method validation: linearity, limit of detention, limit of quantification, matrix effect, precision, accuracy evaluating the percentage of recovery at three different spike levels, and robustness. Acceptable values were obtained. The linear range used was 1–200 μg kg^?1, resulting to r ² of >0.99. The recovery was satisfactory with 70 and 120 % and RSD of <20 % for most compounds. Assessing the samples collected in the south Brazil region, some pesticide residues were detected in whole wheat flour (carbendazim, chlorpyrifos, deltamethrin, imidacloprid, malathion, pendimethalin, pirimiphos-methyl, triamedifom, and triadimenol). The applicability of this analytical approach was confirmed by successful determination of pesticides in whole wheat flour, a complex matrix.

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Graphical Abstract ?

     

Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS

The food and agricultural products processing industries generate substantial quantities of phenolics-rich subproducts, which could be valuable natural sources of polyphenols. In oranges, the peel represents roughly 30% of the fruit mass and the highest concentrations of flavonoids in citrus fruit occur in peel. In this work we have carried out the characterisation and quantification of citrus flavonoids in methanolic extracts of bitter and sweet orange peels using CE–ESI–IT–MS. Naringin (m/z 579.2) and neohesperidin (m/z 609.2) are the major polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin (m/z 609.2) in sweet orange peels. The proposed method allowed the unmistakable identification, using MS/MS experiments, and also the quantification of naringin (5.1 ± 0.4 mg/g), neohesperidin (7.9 ± 0.8 mg/g), narirutin (26.9 ± 2.1 mg/g) and hesperidin (35.2 ± 3.6 mg/g) in bitter and sweet orange peels. CE coupled to MS detection can provides structure-selective information about the analytes. In this work we have developed a CE–ESI–IT–MS method for the analysis and quantification of main phenolic compounds in orange peels.