Determination of trinexapac in wheat by liquid chromatography-electrospray ionization tandem mass spectrometry

A quantitative and confirmatory method for the analysis of trinexapac (free acid metabolite of trinexapac-ethyl) in wheat is described. Residues were extracted from wheat with acetonitrile in aqueous phosphate buffer (pH 7) overnight. The extract was directly injected into the HPLC system. Chromatographic separation was achieved on an octadecylsilica column, and detection was performed by negative ion electrospray ionization tandem mass spectrometry. The precursor ion of trinexapac [M - H](-) at m/z 223 was subjected to collisional fragmentation with argon to yield two intense diagnostic product ions at m/z 135 and 179, respectively. Accuracy and specificity for routine analysis of trinexapac were demonstrated. The validated concentration range was 10-200 microg/kg based on a 0.10 g/mL wheat sample extract. Recoveries were within the range of 71-94%, with associated relative standard deviations better than 10%. The limit of detection for trinexapac in wheat was estimated at 5 microg/kg. The method has been applied to a survey of 100 samples of wheat. In 46% of the samples analyzed, a quantifiable amount of trinexapac was detected, ranging from 10 to 110 microg/kg. It has been demonstrated that analyses of trinexapac accurately reflect the total amount of residues of the plant growth regulator, trinexapac-ethyl, in the wheat samples following field application. No residues of the parent compound, trinexapac-ethyl, in wheat were detected.     

Quantitation of carcinogenic heterocyclic aromatic amines and detection of novel heterocyclic aromatic amines in cooked meats and grill scrapings by HPLC/ESI-MS

A tandem solid-phase extraction method was used to isolate carcinogenic heterocyclic aromatic amines (HAAs) from cooked meats. The following 10 HAAs were identified by HPLC/ESI-MS/MS: 2-amino-9H-pyrido[2,3-b]indole (2-AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), and 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]); the latter HAA has not previously been reported in cooked meats. The concentrations of these HAAs ranged from <0.03 to 15 ppb in cooked meats and poultry, to 75 ppb in cooked beef extract, and to 85 ppb in grill scrapings. The product ion scan mode was used to confirm the identities of these HAAs. Six other compounds were detected that appear to contain the N-methylimidazoquinoxaline skeleton on the basis of their product ion spectra, and these compounds are probable isomers of IQx, 8-MeIQx, and DiMeIQx. A number of known HAAs and novel HAAs of unknown genotoxic potential are formed at appreciable levels in cooked meats.     

Determination of furaltadone and nifursol residues in poultry eggs by liquid chromatography-electrospray ionization tandem mass spectrometry

The use of nitrofurans as veterinary drugs has been banned from intensive animal production in the European Union (EU) since 1993. The objective of the present study was to evaluate the accumulation and depletion of furaltadone and nifursol and their side-chain metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) and 3,5-dinitrosalicylic acid hydrazide (DNSAH) in eggs after administration of therapeutic and subtherapeutic doses of the drugs to laying hens during three consecutive weeks. LC-MS/MS, with positive and negative electrospray ionization methods, was used for the determination of parent compounds and metabolites in yolk and egg white and was validated according to criteria established by Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) of the analytical methodology for metabolites were 0.1 and 0.5 μg/kg for AMOZ and 0.3 and 0.9 μg/kg for DNSAH, respectively. For the parent compounds, CCα and CCβ were 0.9 and 2.0 μg/kg for furaltadone and 1.3 and 3.1 μg/kg for nifursol, respectively. The data obtained show that the parent compounds are much less persistent than their side-chain metabolites in either yolk or egg white. Between the studied metabolites, AMOZ is the most persistent and could be detected in either yolk or egg white three weeks following withdrawal from treatment.     

Determination of 12 type A and B trichothecenes in cereals by liquid chromatography-electrospray ionization tandem mass spectrometry

A new sensitive method for the simultaneous determination of 12 trichothecenes (deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenon X, T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxyscirpenol, diacetoxyscirpenol, T-2 triol, and T-2 tetraol) by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is presented. The development of the method and investigations on the matrix influence on the MS signal are described in particular. The matrix effect was thereby minimized by using an internal standard, a special mobile phase, and specific fragmentation parameters. The sample was extracted with acetonitrile/water (84:16, v/v), and the extract was cleaned up with a MycoSep 227 column. Quantification was based on the internal standard de-epoxy-deoxynivalenol. Calibration curves were linear between 16 and 1600 ng/g, and the limits of detection ranged from 0.18 to 5.0 ng/g. The developed method was applied for the determination of trichothecenes in 120 naturally contaminated wheat and oat samples.     

Determination of five macrolide antibiotic residues in raw milk using liquid chromatography-electrospray ionization tandem mass spectrometry

A confirmatory method using liquid chromatography-electrospray ionization tandem mass spectrometry for determination of five macrolide antibiotics including spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin in raw milk is presented. Macrolides were extracted from raw milk by acetonitrile, and sample extracts were further cleaned up using solid-phase extraction cartridges. Data acquisition was achieved using multiple reaction monitoring, that is, two transitions, to provide a high degree of sensitivity and specificity. 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 a conventional validation procedure and a designed experiment were applied to study the accuracy and precision of the method. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of overall recovery, was approximately 100%, and its intermediate precision was <10%. LC-ESI/MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <0.3 microg/kg.     

Determination of oleandrin in tissues and biological fluids by liquid chromatography-electrospray tandem mass spectrometry

A rapid LC-MS/MS method, using a triple-quadrupole/linear ion trap mass spectrometer, was developed for the quantitative determination of oleandrin in serum, urine, and tissue samples. Oleandrin, the major cardiac glycoside of oleander (Nerium oleander L.), was extracted from serum and urine samples with methylene chloride and from tissues with acetonitrile. The tissue extracts were cleaned up using Florisil solid-phase extraction columns. Six replicate fortifications of serum and urine at 0.001 microg/g (1 ppb) oleandrin gave average recoveries of 97% with 5% CV (relative standard deviation) and 107% with 7% CV, respectively. Six replicate fortifications of liver at 0.005 microg/g (5 ppb) oleandrin gave average recoveries of 98% with 6% CV. This is the first report of a positive mass spectrometric identification and quantitation of oleandrin in tissue samples from oleander intoxication cases. The sensitivity and specificity of the LC-MS/MS analysis enables it to be the method of choice for toxicological investigations of oleander poisoning.     

Direct determination of paclobutrazol residues in pear samples by liquid chromatography-electrospray tandem mass spectrometry

A rapid and sensitive liquid chromatography/electrospray ionization/tandem mass spectrometry (LC-ESI-MS-MS) method has been developed for the determination of the plant growth regulator paclobutrazol in pear samples. Extraction was performed with methanol by using a high-speed blender Ultra-Turrax, and 10 microL of pear extract was directly injected in the LC-ESI-MS-MS system without any previous sample treatment. The highest sensitivity of the method was achieved under MS-MS conditions obtaining a limit of detection of 0.7 microg/kg and a quantification limit of 5 microg/kg, with a run time of only 5.5 min. Recoveries for paclobutrazol from spiked pear samples at 0.005, 0.05, and 0.5 mg/kg were around 82-102% with relative standard deviations between 2 and 7%. The method was applied to real treated and untreated samples of pears, using quality control samples as a evaluation of the method reliability. Two MS-MS transitions were selected, one for quantification (294 > 70) and the other for confirmation of the analyte (296 > 70). All the experiments were performed in compliance with good laboratory practices.     

Simultaneous determination of multiple phytohormones in plant extracts by liquid chromatography-electrospray tandem mass spectrometry

A rapid multiresidue method to quantify three different classes of plant hormones has been developed. The reduced concentrations of these metabolites in real samples with complex matrixes require sensitive techniques for their quantification in small amounts of plant tissue. The method described combines high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Deuterium-labeled standards were added prior to sample extraction to achieve an accurate quantification of abscisic acid, indole-3-acetic acid, and jasmonic acid in a single run. A simple method of extraction and purification involving only centrifugation, a partition against diethyl ether, and filtration was developed and the analytical method validated in four different plant tissues, citrus leaves, papaya roots, barley seedlings, and barley immature embryos. This method represents a clear advantage because it extensively reduces sample preparation and total time for routine analysis of phytohormones in real plant samples.     

Rapid determination of domoic acid in serum and urine by liquid chromatography-electrospray tandem mass spectrometry

A rapid, selective, and sensitive LC-MS/MS method was developed for the quantitative determination of domoic acid in serum and urine samples. Samples were prepared for analysis using an Oasis HLB SPE column. Determination was by a reversed phase HPLC using a mixture of methanol, acetonitrile, and water containing 1% acetic acid and an electrospray ionization (ESI) ion-trap mass spectrometer (Finnigan LCQ). The method was validated by analyzing five replicates each of negative control bovine serum or urine fortified with domoic acid at the 0.005 microg/g method detection limit (MDL) and at the 0.05 microg/g level. Recoveries ranged from 90 to 95% for fortifications at the MDL and from 92 to 98% for fortifications 10 times higher than the MDL. The diagnostic utility of the method was tested by analyzing samples from live animals showing clinical signs suggestive of domoic acid poisoning submitted to the veterinary toxicology laboratory.     

Analytical study of trichlorfon residues in Kaki fruit and cauliflower samples by liquid chromatography-electrospray tandem mass spectrometry

A detailed analytical study on trichlorfon residues in selected vegetables samples has been carried out, focused on the reliable quantification and confirmation of this compound, and on stability of residues under storage. As a consequence, a rapid and sensitive LC-ESI-MS/MS method has been developed for the determination of residues of this insecticide in kaki fruit (flesh and peel) and cauliflower samples. Extraction was performed with acetonitrile using a high-speed blender. After 4-fold dilution of the extract with water, 20 microL was directly injected in the LC-ESI-MS/MS system (triple quadrupole), using matrix-matched standards calibration for quantification. Under optimized MS/MS conditions, limit of detections between 0.006 and 0.013 mg/kg were reached, and a limit of quantification of 0.05 mg/kg was established, with a runtime of only 15 min. Recoveries from spiked blank samples at 0.05 and 0.5 mg/kg were in the range 83-101% with relative standard deviations lower than 10%. The method was applied to treated and untreated samples collected from field residues trials, using quality control samples analysis for the evaluation of the method. Despite the acquisition of two MS/MS transitions in selected reaction monitoring mode, the analysis of treated samples revealed the presence of a chromatographic peak close to the analyte that corresponded to a trichlorfon isobaric compound that shared the same MS/MS transitions. This unusual situation in LC-MS/MS-based procedures required the application of an efficient chromatographic separation to avoid this interference. All experiments have been made in compliance with the principles of Good Laboratory Practices (GLP) and following the European SANCO guidelines for pesticides residue analysis (PRA).     

Determination of saponins in aerial parts of barrel medic (Medicago truncatula) by liquid chromatography-electrospray ionization/mass spectrometry

Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.     

Analysis of patulin in pear- and apple-based foodstuffs by liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography electrospray ionization tandem mass spectrometry method for the determination of patulin in apple- and pear-based foodstuffs was developed. The sample preparation is based on the QuEChERS procedure involving an initial extraction step with water and acetonitrile, followed by a partitioning step after the addition of magnesium sulfate and sodium chloride. The cleanup was performed by using dispersive solid-phase extraction with a mixture of magnesium sulfate, primary secondary amine sorbent, and n-octadecylsiloxane sorbent added together to the extract. The cleaned extract was finally evaporated and reconstituted in water prior to injection. Quantitation was performed by isotope dilution using ((13)C(7))-patulin as internal standard. The method was first fully validated in three different baby food products including apple-pear juice, apple-pear puree, and infant cereals. Then the scope of application of the method was extended to pear concentrate, raw apples, apple flakes (naturally contaminated), dried apples, and yogurt. The sensitivity achieved by the method in all matrices gave limits of detection (LOD) and quantitation (LOQ) of ≤0.5 and ≤10 μg/kg, respectively, which was compliant with maximum levels settled in Commission Regulation (EC) No. 1881/2006. Method performances for all matrices also fulfilled the criteria established in the CEN/TR 16059:2010 document. Indeed, recoveries were within the 94-104% range; relative standard deviations of repeatability (RSD(r)) and intermediate reproducibility (RSD(IR)) were ≤7.5 and ≤13.0%, respectively, and trueness in an infant apple drink (FAPAS 1642) was measured at 99%.     

Determination of capsaicin and dihydrocapsaicin in capsicum fruits by liquid chromatography-electrospray/time-of-flight mass spectrometry

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with a reversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values were lower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin in fruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg(-1).     

Determination of five macrolide antibiotic residues in eggs using liquid chromatography/electrospray ionization tandem mass spectrometry

A method using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) for the determination of trace levels of five macrolide antibiotics (spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin) in eggs is presented. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring (MRM) of two or three fragment ion transitions to provide a high degree of sensitivity and specificity for both quantification and confirmation. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method. A fully nested experimental design was used to study the measurement uncertainty arising from intermediate precision and trueness or proportional bias. The overall recoveries, that is, those determined by the nested experiments, of spiramycin, tilmicosin, oleandomycin, erythromycin, and tylosin at fortified levels of 60, 100, 200, and 300 microg/kg were 96.8, 98.2, 98.3, 98.8, and 95.4%, respectively. The LC/ESI-MS/MS method detection limits (S/N > or = 3:1) of five macrolides were <1.0 microg/kg.     

Profiling and quantification of isoflavonoids in kudzu dietary supplements by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry

The kudzu vine (Pueraria sp.) is a rich source of isoflavones. Dietary supplements based on kudzu have become commercially available. In the present study, liquid chromatography coupled with negative and positive electrospray ionization tandem mass spectrometry (MS/MS) and diode array detection (DAD) has been used for the detection and characterization of isoflavonoids in kudzu dietary supplements (KDS). The MS/MS spectrum of the protonated ion of puerarin showed characteristic product ions of the C-glycoside unit itself, whereas daidzin generated an abundant Y(0)(+) aglycon ion in its product ion spectrum. A base peak due to the loss of 120 Da [M + H - 120](+) is the diagnostic ion for C-glycosides. Neutral loss scans allowed for the detection of other C- and O-glycosides in the methanolic extract of KDS, and their structures have been proposed. The concentration of isoflavonoids in the methanolic extract of commercially available KDS was quantified by using DAD-HPLC. Puerarin, rather than daidzin, was the most abundant component (8.44-30.60 mg/capsule) in commercially available KDS.     

Determination of pesticides in apple-based infant foods using liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to quantify and confirm trace levels of 13 pesticides including aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, formetanate, 3-hydroxycarbofuran, carbendazim, thiabendazole, aldicarb, propoxur, carbofuran, carbaryl, and methiocarb in apple-based infant foods such as apple sauces, apples and strawberries, apples and blueberries, and apples and plums. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring of two fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantification and confirmation. LC/ESI-MS/MS quantitative results were significantly affected by matrices, and thus, the standard addition was employed to compensate for the matrix effects to achieve the best accuracy of the method. Recoveries of 13 pesticides, spiked at 5.0, 25.0, and 45.0 microg/kg, were around 100% using the LC/ESI-MS/MS standard addition. The method detection limits (S/N > or = 3:1) of 13 pesticides were less than 0.2 microg/kg.     

Profiling of soluble proteins in wine by nano-high-performance liquid chromatography/tandem mass spectrometry

Wine proteins play an important role in a wine's quality as they affect taste, clarity, and stability. To enhance our understanding of the proteins in wine, nano-high-performance liquid chromatography (HPLC)/tandem mass spectrometry was used to profile soluble proteins in wine. Twenty proteins were identified from a Sauvignon Blanc wine including five proteins derived from the grape, 12 from yeast, two from bacteria, and one from fungi. The findings are somewhat peculiar at first glance, but reasonable explanations can account for the results. The grape proteins identified are less in number, which may be due to the availability of an incomplete database and possibly bentonite fining. The relatively large number of identified yeast proteins may be due to their complete protein database. The identified bacterial and fungal proteins could possibly be attributed to sources in the vineyard including natural infections and improper handling during harvest. The use of nano-HPLC/tandem mass spectrometry is an important tool for identifying wine proteins and understanding how they affect its characteristics.     

Quantitation of dityrosine in wheat flour and dough by liquid chromatography-tandem mass spectrometry

A method for the quantitation of dityrosine in wheat flour and dough by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) using an isotope dilution assay with the internal standard 3,3'-(13)C(2)-dityrosine in the single-reaction monitoring mode was developed. The method consisted of the release of protein-bound dityrosine by hydrolysis in 4 mol/L hydrochloric acid/8.9 mol/L propionic acid for 24 h at 110 degrees C after addition of the internal standard, cleanup by C(18) solid-phase extraction, and HPLC-MS/MS. The limit of detection of dityrosine was 80 ng/g of sample (0.22 nmol/g), and the limit of quantitation was 270 ng/g of sample (0.75 nmol/g). The method was sensitive enough to analyze wheat flour and dough and to study the effect of flour improvers on the dityrosine content. Furthermore, the effect of the mixing time was studied. The dityrosine concentration in the flour was 0.66 nmol/g. After we mixed a dough to peak consistency, the dityrosine concentration doubled and remained constant on further mixing. Overdoses of hydrogen peroxide and hexose oxidase (HOX, E.C. 1.1.3.5) resulted in a strongly increased dityrosine content, whereas no increase of the dityrosine concentration was found after the addition of ascorbic acid and potassium bromate. Calculation of the percentage of dimeric tyrosine showed that less than 0.1% of the tyrosine residues of wheat protein were cross-linked. Therefore, dityrosine residues seem to play only a very minor role in the structure of wheat gluten.     

Inhibitory effect of antioxidant-rich marinades on the formation of heterocyclic aromatic amines in pan-fried beef

The inhibitory effect of antioxidant-rich marinades containing beer and white wine (with/without alcohol) alone or mixed with herbs commonly used as meat flavoring (garlic, ginger, thyme, rosemary, and red chili pepper) on the formation of heterocyclic aromatic amines (HAs) in pan-fried beef was studied. Radical-scavenging activity was evaluated by DPPH assay, before the addition of meat to the marinade (T0) and after 4 h of meat marinating (T4). At T0, wine with herbs possessed the highest scavenging activity (73.5%), followed by wine (72.5%), dealcoholized wine with herbs (53.4%), beer and herbs (41.7%), dealcoholized wine (39.6%), and beer (25.9%). At T4, a decrease in the radical-scavenging activity of all marinades was observed, although with a similar radical-scavenging profile. All of the six marinades under the study reduced the total amount of HAs, keeping meat with good overall sensory quality. Beer marinades were more efficient than white wine marinades, and the addition of herbs provided a superior inhibitory effect, reducing around 90% of HAs. No correlation was observed between radical-scavenging activity of marinades and total or individual HAs formation. Herbs explained around 30% of inhibition of PhIP formation, whereas alcohol increased PhIP formation.