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.     

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.     

A rapid LC/MS/MS method for the analysis of nonvolatile antiinflammatory agents from Mentha spp

Abstract: Mints (Mentha spp.), aromatic crops grown largely for their essential oils, also are rich sources of nonvolatile antiinflammatory agents. Identification and quantitation of the constituents responsible for their antiinflammatory activity is challenging owing to the lack of suitable chromatographic methodology. In the present research, the simultaneous quantitation of antiinflammatory constituents rosmarinic acid, oleanolic acid, and ursolic acid in mints was attained by using a unique tandem HPLC column system coupled with an electrospray ionization mass detection (MRM mode). The ion mode optimization for rosmarinic acid under negative and triterpenoid acids under positive was achieved by setting 2 time segments in a single run where the polarity mode was switched from negative (0 to 10 min) to positive (10 to 40 min). For the investigated concentration ranges of antiinflammatory agents in mints, good linearities (r²≥ 0.998) were obtained for each calibration curve. Validation of precision and accuracy for this method showed that intra‐ and inter‐day repeatabilities for all analytes were less than 5.51%, and the recoveries varied from 97.8% to 99.3%. The developed LC/MS/MS assay provides a suitable quality control method for the determination of antiinflammatory constituents in Mentha spp. There is a wide range of diversity in the natural product composition for these acids across the Mentha germplasm collection evaluated. The presence of these antiinflammatory acids in post‐distilled mints shows that value‐added nutraceutical enriched products can be developed with proper processing and recovery systems in addition to the distillation and capture of the valuable volatile essential oils. Practical Application: Results from this research would benefit both commercial farmers growing mint for essential oil and those in the food industry where value‐added phytopharmaceutical enriched products can be developed with proper processing, quality control, and recovery systems during mint essential oil distillation.     

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 application of d-SPE in the QuEChERS method for the determination of PAHs in food of animal origin with GC–MS detection

This paper reports the evaluation of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for the determination of polycyclic aromatic hydrocarbons (PAHs) in food of animal origin with GC–MS detection. Although in the available literature, there is a lot of information about sample preparation method for PAHs determination in food samples, but the QuEChERS method application for PAHs determination in food of animal origin has not been reported as yet. The results showed that the best recovery ratios 72.4–110.8 % with relative standard deviation lower than 10 % for all determined compounds were received for the method with ethyl acetate as an extraction solvent, primary–secondary amine and C₁₈ sorbents and evaporation to dryness and dissolving the residues in the hexane. The limit of quantification ranged from 0.0003 to 0.0030 mg kg^?1 for pyrene and benzo[a]anthracene, respectively. This method was also used for the determination of PAHs in 15 samples of pork ham. In 8 of 15 samples selected, PAHs were identified. It was observed that in 6 cooked ham and one smoked and cooked samples, any PAHs were found. In other samples, which were smoked and roasted, some low concentration of PAHs was detected. In one sample benzo[a]pyrene (0.0015 mg kg^?1), in one sample benzo[b]fluoranthene (0.0015 mg kg^?1) and in one sample chrysene (0.0024 mg kg^?1) were detected. A number of other less harmful PAHs were also determined. There were no exceedances of maximum levels (according to Commission Regulation (EU) No 835/2011) for determined PAHs in any of the analysed samples.     

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.     

Determination of phthalate esters in non-alcoholic beverages by GC–MS and optimization of the extraction conditions

A quantificational method for 7 phthalate esters in non-alcoholic beverages was developed. Dimethyl phthalate, di-ethyl phthalate, di-propyl phthalate (DPP), di-butyl phthalate (DBP), benzyl butyl phthalate, di-(2-ethylhexyl) phthalate (DEHP), and di-octyl phthalate (DOP) were extracted from non-alcoholic beverages with the optimized solid-phase extraction method, and quantification was achieved by gas chromatography–mass spectrometry with isotope internal standard of d₄-di-(2-ethylhexyl) phthalate (DEHP-d₄). The inter-day method repeatability (RSD) was 8–13 %, whereas the intra-day method repeatability (RSD) was 9–15 %. The mean spiking recoveries were 84–105 %. A wide variety of phthalate concentrations was observed in 48 non-alcoholic beverages. DEHP was the most abundant phthalate compound followed by DBP, DPP, and DOP. DEHP was found in sport drinks (0.015–0.098 mg L^?1), tea (0.016–0.123 mg L^?1), coffee (0.028–0.159 mg L^?1), and fruit juices (0.022–0.126 mg L^?1).     

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.     

Determination of acrylamide in food using a UPLC–MS/MS method: results of the official control and dietary exposure assessment in Cyprus

ABSTRACT

The determination of acrylamide in potato products, bakery products and coffee, and the human dietary exposure is reported. The method reported is based on a single extraction step with water, followed by the clean-up of the extract using solid phase extraction columns and finally, the determination of acrylamide using UPLC–MS/MS. The MS/MS detection was carried out using an ESI interface in positive ion mode. Internal calibration was used for the quantification of acrylamide, because of the suppression/enhancement matrix effects due to the complex nature of the samples. The method performance characteristics were determined after spiking blank samples. The mean recoveries in spiked coffee samples, potato chips, breakfast cereals and crispbread ranged from 93% to 99%, with RSDs lower than 5% for both repeatability and reproducibility conditions. The estimated limits of detection and quantification of the method were 10 and 32 μg kg^?1, respectively. The method was used for monitoring acrylamide in 406 samples. Acrylamide amounts ranged from <32 to 2450 μg kg^?1. A total of 360 samples (89%) were contaminated with acrylamide, but only 14% of the samples exceeded the benchmark levels of the EU legislation. Foods with the highest mean acrylamide amounts were potato crisps (642 μg kg^?1), French fries (383 μg kg^?1) and biscuits (353 μg kg^?1). The mean and 95th percentile acrylamide exposures of adolescents in Cyprus were 0.8 and 1.8 μg kg^?1 body weight per day, respectively. The estimated levels of dietary exposure to acrylamide are not of concern with respect to neurotoxicity. However, the margins of exposure (MOEs) indicate a concern for carcinogenicity. Potato fried products (45%), fine bakery ware (21%) and potato chips (14%) contributed the most to overall acrylamide exposure.     

LC–MS/MS with atmospheric pressure chemical ionisation to study the effect of UV treatment on the formation of vitamin D3 and sterols in plants

Some plant species are known to cause calcium intoxification in grazing animals. This has been attributed to the presence of vitamin D₃-like activity. However, research into the presence of vitamin D₃ in plants has been limited. One reason for this may be limitations in the analytical methods available for unambiguous detection and quantification of vitamin D₃. This paper presents a new method for determining vitamin D₃ and its sterol precursors. The method is based on saponification and extraction followed by solid phase clean-up of the compounds from plant leaves and detection by APCI-MS. Recoveries ranged from 101% to 114% and precision from 3% to 12%. Detection limits were 2–8 ng/g fresh weight for the substances tested. In a pilot study we found that Solanum glaucohyllum Desf. and Solanum lycopersicum L. produced vitamin D₃ after UV-treatment. The preliminary results presented suggest that vitamin D₃ formation in plants is dependent on light exposure.     

HS-SPME/GC–MS and chemometrics for the classification of Balsamic Vinegars of Modena of different maturation and ageing

Head space solid-phase microextraction (HS-SPME) coupled with GC–MS analysis has been applied for the determination of the characteristic volatile profile of Balsamic Vinegar of Modena (BVM) with the aim to distinguish the less matured products (matured in wooden barrels for at least 60 days) from the aged ones (aged in wooden barrels for at least 3 years). Coupling the HS-SPME/GC–MS analysis data with multivariate statistical techniques, such as Principal Components Analysis (PCA) and Classification Trees (CT), it has been possible to classify BVMs on the basis of different maturation and ageing. A matured BVM presents an aromatic profile characterised by high contents of 3-methyl-1-butanol, 4-ethyl-phenol, and 3-methyl-1-butanol acetate, while an aged BVM is characterised by a prevalence of ethyl acetate, ethyl acetoacetate, furans, 2,3-butanediol and 2,3-butanediol acetate. This work represents a first attempt to classify Balsamic Vinegars of Modena on the basis of their maturation and ageing.     

Nicotine determination in mushrooms by LC–MS/MS with preliminary studies on the impact of drying on nicotine formation

A problem concerning significant amounts of nicotine in dried wild mushrooms (mainly Boletus edulis from China) has been reported to the European Commission. As a consequence, the European Food Safety Authority (EFSA) proposed temporary maximum residue levels (MRLs) of 0.036 mg kg^?1 for fresh wild mushrooms and 1.17 mg kg^?1 for dried wild mushrooms (2.3 mg kg^?1 for dried ceps only). The EFSA also highlighted the necessity for a monitoring and testing programme to be launched by food business operators at the start of the 2009 harvest season. In the present study, a quick and sensitive analytical method for routine analysis of nicotine in fresh and dried mushrooms was developed and validated by a single-laboratory procedure. The method, which employs an LC–MS/MS system and (±)-nicotine-d ₄ as internal standard, has a limit of quantification of 6 and 60 µg kg^?1 for fresh and dried product, respectively. Analyses of samples spiked with different levels of nicotine showed recoveries ranging from 107 to 122%, with relative standard deviations of 2.9–10.1% depending on the spiking level. The combined uncertainties, calculated at a low level for frozen (0.015 mg kg^?1) and a high level for the dried (2 mg kg^?1) matrix, were 13 and 10%, respectively. Application of the method to real samples of mushrooms purchased on the market or obtained from local producers showed nicotine levels ranging 0.01–0.04 and 0.1–4.5 mg kg^?1 in fresh/frozen and dried matrices, respectively. To establish reasons for the unexpectedly high levels of the nicotine in dried matrices, preliminary laboratory experiments involving drying mushrooms were performed under various conditions.     

Evaluation of the volatile profile of 33 Pyrus ussuriensis cultivars by HS-SPME with GC–MS

Evaluation of the volatile compounds in fruit provides useful information for plant breeding for improved fruit aroma. In this study, headspace solid-phase microextraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) was used to assess the volatile profile of 33 cultivars of the Chinese pear Pyrus ussuriensis. In all, 108 volatile compounds were identified and there were significant differences in the composition and concentration of volatiles among cultivars. On the basis of principal components analysis (PCA), the cultivars could be divided into four groups: Group 1 contained Reli, Jinxiang, Hongbalixiang, Baibalixiang and Fuwuxiang, cultivars with a high concentration of esters and a low concentration of hydrocarbons. Group 2 contained Qiuxiang, Fuanjianba, Longxiang, Guanhongxiao, Shanli24 and Wuxiangli, cultivars with high concentrations of hydrocarbons and low concentrations of esters. Group 3 contained Shatangli and Manyuanxiang, cultivars with high concentrations of aldehydes. Group 4 contained the other 25 cultivars.     

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.     

Analytical strategy for the determination of non-steroidal anti-inflammatory drugs in plasma and improved analytical strategy for the determination of authorised and non-authorised non-steroidal anti-inflammatory drugs in milk by LC–MS/MS

A sensitive and selective method for the determination of six non-steroidal anti-inflammatory drugs (NSAIDs) in bovine plasma was developed. An improved method for the determination of authorised and non-authorised residues of 10 non-steroidal anti-inflammatory drugs in milk was developed. Analytes were separated and acquired by high performance liquid chromatography coupled with an electrospray ionisation tandem mass spectrometer (ESI–MS/MS). Target compounds were acidified in plasma, and plasma and milk samples were extracted with acetonitrile and both extracts were purified on an improved solid phase extraction procedure utilising Evolute? ABN cartridges. The accuracy of the methods for milk and plasma was between 73 and 109%. The precision of the method for authorised and non-authorised NSAIDs in milk and plasma expressed as % RSD, for the within lab reproducibility was less than 16%. The % RSD for authorised NSAIDs at their associated MRL(s) in milk was less than 10% for meloxicam, flunixin and tolfenamic acid and was less than 25% for hydroxy flunixin. The methods were validated according to Commission Decision 2002 European Commission Decision. 2002. Decision(2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Off J Eur Commun, L221: 8–36.  [Google Scholar]/657/EC.     

Volatiles of grape berries evaluated at the germplasm level by headspace-SPME with GC–MS

Volatile aroma compounds are one of the important characteristics determining fruit quality. Evaluation of volatiles at the germplasm level is useful for future breeding efforts, aimed at improvement of fruit quality, via effects on fruit aroma. The composition and concentration of volatiles in berries of forty-two grape cultivars belonging to seven genotypic groups were evaluated by headspace solid-phase microextraction with GC–MS. Sixty volatiles were detected, of which C₆ compounds were the dominant volatiles in V. amurensis grape, neutral cultivars V. vinifera grapes and hybrid grapes between V. vinifera with V. thunbergii or V. amurensis. Alcohols and carbonyls were relatively low in all Vitis germplasm studied. Terpenoids were abundant in V. vinifera with muscat aroma, while esters were dominant in V. labrusca and its hybrids with V. vinifera or V. amurensis. Via principal component analysis, all the grape germplasm studied could be divided into three groups: (1) V. labrusca and its hybrids with V. vinifera or V. amurensis, (2) V. vinifera with muscat aroma and (3) the others, including V. vinifera without muscat aroma plus V. amurensis, and hybrids between V. vinifera and V. thunbergii or V. amurensis. Seasonal quantitative variations of volatiles were observed, but the qualitative volatile composition of the cultivars was consistent.     

A Comparative Study of the Ionization Modes in GC–MS Multi-residue Method for the Determination of Organochlorine Pesticides and Polychlorinated Biphenyls in Crayfish

A gas chromatography–mass spectrometry (GC–MS) multi-residue method with different ionization modes has been developed for the determination of 23 organochlorine pesticides and ten polychlorinated biphenyls (PCBs) in crayfish. The approach was based on an extraction with acetonitrile saturated by hexane (containing 0.1 % acetic acid), followed by a dispersed solid-phase extraction cleanup with primary secondary amine and octadecylsilane, and finally analyzed by GC–MS/MS operated in electron impact (EI) and positive chemical ionization (PCI), and GC–MS in negative chemical ionization (NCI), respectively. An isotope internal standard, DDT-D₈, was used for quantification. These three GC–MS procedures were validated and compared using crayfish samples fortified at three levels (5, 10 and 20 ng/g). Good linearity, accuracy, precision and sensitivity were achieved by all three ionization modes with recoveries in the range of 70 to 112 %, relative standard deviations (RSDs) below 10 % and limits of detection (LODs) in the range of 0.002 to 8 ng/g. Extremely high selectivity was obtained by both EI- and PCI-MS/MS, while PCI was more sensitive to compounds containing nitro and carbonyl groups, and EI was a better choice for dieldrin, endosulfan, and its metabolites. NCI-MS had the highest sensitivity but lower anti-interference ability and was not suitable for the determination of DDTs and PCBs. These three ionization modes can be used as complementary and alternative in routine GC–MS analysis of organochlorine pesticides and polychlorinated biphenyls in food.     

Formation of the aroma of a raw goat milk cheese during maturation analysed by SPME–GC–MS

The volatile profile of the Spanish goat raw milk cheese of the protected designation of origin (PDO) “Queso Ibores” was studied at four stages of maturation (day 1, 30, 60, and 90) by the method of solid-phase micro-extraction–gas chromatography–mass spectrometry (SPME–GC–MS) to determinate the characteristic volatile compounds of this cheese and to know the changes in the volatile profile of this cheese during maturation. According to the PDO, Ibores cheese aroma varies between sweet and mild and it has a strong taste, slightly tart. A total of 64 compounds were detected: 14 acids, 18 alcohols, 13 esters, 6 ketones and 13 compounds which could not be classified in these groups. Carboxylic acids were the most abundant volatile compounds in the headspace of Ibores cheese. Content of volatile compounds was significantly modified (P < 0.05) during ripening. The relative total amounts of acids, esters and ketones increased during the first 60 days of maturation. The most characteristic compounds of Ibores cheese aroma were butanoic, hexanoic and octanoic acids, some alcohols (2-butanol and 2-heptanol), ethyl esters of hexanoic and butanoic acids, some methyl ketones (2-butanone, 2-pentanone and 2-heptanone) and δ-decalactone.     

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.