Identification of the major metabolites of prochloraz in rainbow trout by liquid chromatography and tandem mass spectrometry

The metabolic pattern of the imidazole fungicide prochloraz [N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboximide] was investigated in rainbow trout (Oncorhynchus mykiss). Following a single oral administration of [(14)C]prochloraz, levels 4.3 +/- 4.1 and 3.9 +/- 1.8% of the dose were excreted in the bile after 48 h in male and female animals, respectively. Urinary radioactivity accounted for 1.3 +/- 0.4 and 2.4 +/- 1.1% of the dose over the same period in males and females. Metabolites from both matrices were separated by reversed-phase HPLC with radioactive detection and analyzed by positive and/or negative electrospray ionization mass spectrometry. No unchanged prochloraz was detected in the analyzed excreta. The major biotransformation products in bile were the aldehyde corresponding to the cleavage of the imidazole ring, N-2-(2,4,6-trichlorophenoxy)ethylurea, and the glucuronide conjugate of 2,4,6-trichlorophenoxyethanol. In urine, the major metabolite was 2,4,6-trichlorophenoxyacetic acid. On the basis of enzymatic hydrolysis by beta-glucuronidase and LC-MS analyses, this study demonstrates that rainbow trout are able to biotransform prochloraz, mainly as glucuronide conjugates.     

Determination and confirmation of 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry

A method was developed and validated to determine 5-hydroxyflunixin in raw bovine milk using liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean recovery and percentage coefficient of variation (%CV) of 35 determinations for 5-hydroxyflunixin was 101% (5% CV). The theoretical limit of detection was 0.2 ppb with a validated lower limit of quantitation of 1 ppb and an upper limit of 150 ppb. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. A LC/MS/MS confirmatory method using the extraction steps of the determinative method was developed and validated for 5-hydroxyflunixin in milk from cattle. Briefly, the determinative and confirmatory methods were based on an initial solvent (acetone/ethyl acetate) precipitation/extraction of acidified whole milk. The solvent precipitation/extraction effectively removed incurred ((14)C) residues from milk samples. The organic extract was then purified by solid phase extraction (SPE) using a strong cation exchange cartridge (sulfonic acid). The final SPE-purified sample was analyzed using LC/MS/MS. The methods are rapid, sensitive, and selective and provide for the determination and confirmation of 5-hydroxyflunixin at the 1 and 2 ppb levels, respectively.     

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%.     

Metabolic profiling of flavonol metabolites in human urine by liquid chromatography and tandem mass spectrometry

Twenty-one flavonol metabolites have been identified by LC/ESI-MS/MS in human urine, including isomers, after the consumption of cooked onions. Metabolites identified include quercetin monoglucuronides, methyl quercetin monoglucuronides, a quercetin monoglucuronide sulfate, quercetin diglucuronides, a methyl quercetin diglucuronide, quercetin glucoside sulfates, methyl quercetin, quercetin, and kaempferol monoglucuronides. The fragmentation patterns of flavonol metabolites obtained by MS/MS were distinctive for some isomers, indicating that fragmentation patterns may be useful predictors of conjugation position. Two isomers of sulfate quercetin glucosides were also found in urine, suggesting that many of the quercetin glucosides in onion are absorbed intact and undergo metabolism to the sulfate conjugate. Additionally, the interindividual variation in urinary quercetin metabolite profiles was determined by comparing the relative level of six different quercetin metabolites excreted in the urine of healthy volunteers. The ranges of quercetin metabolites excreted were similar among volunteers, yet notable differences in the levels of metabolites among individuals were observed. This study demonstrates the potential of monitoring the range of quercetin metabolites to reveal information on interindividual biotransformation capacity in response to dietary manipulations and as a biomarker for flavonol consumption.     

Identification of puerarin and its metabolites in rats by liquid chromatography-tandem mass spectrometry

Puerarin (daidzein-8-C-glucoside) is the major bioactive isoflavone of kudzu root (the root of Pueraria lobata). Its metabolic fate, however, is not well-known. In this study, a sensitive and specific LC-ESI-MS/MS method for the determination of puerarin and its metabolites daidzein, dihydrodaidzein, and equol was developed for their analysis in biological samples. Two new metabolites of puerarin, mono- and dihydroxylated derivatives, were detected in the urine and feces of rats after oral administration. The persistence of puerarin in blood and urine as the principal metabolic form for the period of 4-72 h after oral administration suggested that puerarin is rapidly absorbed from the intestine without metabolism. Its presence in organs such as the brain suggests that this glucoside may enter tissues by specific transport pathways. Study of these metabolites may provide further understanding of the health beneficial effects of puerarin in kudzu dietary supplements.     

Determination of traces of fipronil and its metabolites in pollen by liquid chromatography with electrospray ionization-tandem mass spectrometry

Fipronil is a pesticide suspected of having harmful effects on honey bees at microgram per kilogram levels. Considering the lack of methodology, it thus appeared to be necessary to develop a method for the determination of the lowest amounts of fipronil and its metabolites in pollen. This paper describes a new analytical method with a limit of quantification (LOQ) of 0.1 microg/kg for a representative sample weight of 5 g. In the case of a field study, this tool was used for checking the possible existence of fipronil and/or metabolites in pollen samples, but none of them contained residues higher than the LOQ. This three-step rapid method uses liquid-solid solvent extraction with mechanical grinding, followed by liquid-liquid partitioning and Florisil solid-phase extraction for the two cleanup steps. The quantification is made by liquid chromatography with electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Indeed, combined with an adequate sample treatment, this technique offers good sensitivity and selectivity in such a complex matrix. The method has given good recoveries of 74-104% with relative standard deviations of 5.6-18.2%.     

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.     

Identification of fonofos metabolites in Latuca sativa, Beta vulgaris, and Triticum aestivum by packed capillary flow fast atom bombardment tandem mass spectrometry

The metabolism of fonofos, a thiophosphonate insecticide, was investigated in mature lettuce (Latuca sativa), beet (Beta vulgaris), and wheat (Triticum aestivum). Six new metabolites were identified by LC-MS and LC-MS-MS analysis using fast atom bombardment (FAB) and packed capillary LC columns with application of the on-column focusing technique. These methods provided the sensitivity required to identify unknown metabolites that were present in the mature plants at only 20-230 ppb. Structural elucidation was facilitated by use of fonofos labeled with both carbon-14 and carbon-13 in the phenyl ring. In all three plants fonofos was converted to a glucose conjugate of thiophenoxylactic acid. Oxidation of the glucose conjugate gave isomeric sulfoxides in all species examined. Thiophenoxylactic acid was found esterified to malonic acid in lettuce. In beets, S-phenylcysteine was found as its malonic acid amide. A second metabolite unique to beets was N-(malonyl)-[2[(ethoxyethylphosphinothionyl)oxy]phenyl]cysteine. This novel structure was confirmed by synthesis.     

Analysis of toxic norditerpenoid alkaloids in Delphinium species by electrospray, atmospheric pressure chemical ionization, and sequential tandem mass spectrometry

A rapid electrospray mass spectrometry method was developed for screening larkspur (Delphinium spp.) plant material for toxic norditerpenoid alkaloids. The method was calibrated using two standard alkaloids, methyllycaconitine (1) and deltaline (2), with a recovery of 92% from spiked samples and relative standard deviations of 6.0% and 8.1% for the two alkaloids, respectively. Thirty-three samples of plains larkspur, Delphinium geyeri, were analyzed. Methyllycaconitine (1) concentration was 0.27% +/- 0.08% during a 1-month period in 1997 establishing the relative risk of poisoning from the plant to be low. The method was also applied to the trace analysis (<1 ppm) of 1 in serum samples from sheep dosed different levels of the alkaloid. Electrospray ionization combined with sequential tandem mass spectrometry and HPLC coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry were used to detect and tentatively identify three new norditerpenoid alkaloids from Delphinium nuttallianum [bearline (6), 14-acetylbearline (7), 16-deacetylgeyerline (8)]. The tentative structure of the new alkaloids was predicted from the tandem mass spectra fragmentation patterns and assigning the substitution pattern for methoxy and acetyl groups at the C-14 and C-16 carbons.     

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.     

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.     

Validation of recombinant and bovine chymosin by mass spectrometry

Mass spectrometry has been used to map chymosin from a fermentative source. The copresence of the two known genetic variants A (Asp(244)) and B (Gly(244)) was ascertained in bovine chymosin. By contrast, either the A or the B genetic variant occurred in the three commercial samples of recombinant calf chymosin (RCC). Specific biomarker proteins were searched to identify the enzyme source, in both bovine chymosin and RCC samples. Analyzing the derived tryptic peptides, evidence was provided that RCC and bovine chymosin are mainly formed by (1-323), (3-323), and (40p-323) (suffix "p" denotes residues in the pro-segment region of chymosin), whereas the minor components, (4-323), (5-323), and (6-323), were only detected in bovine chymosin. Additionally, the three commercial RCC samples contained the protein species (1-323), (38p-323), (39p-323), and (40p-323) and the shorter form (3-323). Differentiation of the natural and bioengineered enzyme is based upon the detection of these unique minor components by mass spectrometry.     

Determination of flunixin in edible bovine tissues using liquid chromatography coupled with tandem mass spectrometry

An accurate, reliable, and reproducible assay was developed and validated to determine flunixin in bovine liver, kidney, muscle, and fat. The overall recovery and percent coefficient of variation (%CV) of twenty-eight determinations in each tissue for flunixin free acid were 85.9% (5.9% CV) for liver, 94.6% (9.9% CV) for kidney, 87.4% (4.7% CV) for muscle, and 87.6% (4.4% CV) for fat. The theoretical limit of detection was 0.1 microg/kg (ppb, ng/g) for liver and kidney, and 0.2 ppb for muscle and fat. The theoretical limit of quantitation was 0.3, 0.2, 0.6, and 0.4 ppb for liver, kidney, muscle, and fat, respectively. The validated lower limit of quantitation was 1 ppb for edible tissues with the upper limit of 400 ppb for liver and kidney, 100 ppb for fat, and 40 ppb for muscle. Accuracy, precision, linearity, specificity, ruggedness, and storage stability were demonstrated. Briefly, the method involves an initial acid hydrolysis, followed by pH adjustment ( approximately 9.5) and partitioning with ethyl acetate. A portion of the ethyl acetate extract was purified by solid-phase extraction using a strong cation exchange cartridge. The eluate was then evaporated to dryness, reconstituted, and analyzed using LC/MS/MS. The validated method is sensitive and specific for flunixin in edible bovine tissue.     

Mycotoxin analysis by fast atom bombardment tandem mass spectrometry

Positive fast atom bombardment tandem mass spectrometry is demonstrated to be an effective technique for determination of crude aflatoxins and sterigmatocystin-related compounds. The molecular ion was selected by the first system and bombarded to produce characteristic daughter ions that could be used to identify mycotoxins in mixtures and with the same molecular weight.     

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.     

"Cross-talk" in scheduled multiple reaction monitoring caused by in-source fragmentation in herbicide screening with liquid chromatography electrospray tandem mass spectrometry

Interferences, or "cross-talk", have been found for the liquid chromatorgraphy mass spectrometry (LC-MS) determination of chlorophenoxy acid (CPA) herbicides. The time-scheduled multiple reaction monitoring (MRM) of m/z 161.0→125.0 and 163.0→127.0 transitions were produced by 2,4-dichlorophenol and 2,4-dichlorophenoxyacetic acid. Although MRM reduces the possibility of false positives when two transitions for LC/MS-MS are used for quantification and qualitative confirmation, in the case of the structurally related CPAs here, false positives still occurred when using a mixture of standards to identify the residues. It was necessary to analyze pure individual standards to compare with the extracted retention times of the candidate CPAs in food samples.     

Separation and characterization of phenolic compounds in fennel (Foeniculum vulgare) using liquid chromatography-negative electrospray ionization tandem mass spectrometry

Liquid chromatography (LC) diode array detection (DAD) coupled to negative electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was used for the rapid and sensitive identification of water-soluble phenolic compounds in fennel waste. The plant material was first extracted and then chromatographed on Sephadex LH-20 to afford seven fractions, each of them being subjected to LC-MS analysis. Identification of the compounds was carried out by interpretation of UV, MS, and MS/MS spectra. Forty-two phenolic substances were identified, 27 of which had not previously been reported in fennel, including hydroxycinnamic acid derivatives, flavonoid glycosides, and flavonoid aglycons.     

Determination of alpha-amanitin in serum and liver by multistage linear ion trap mass spectrometry

This paper describes a rapid LC-MS/MS/MS method for the analysis of alpha-amanitin in serum and liver. Serum was initially prepared by precipitation of proteins with acetonitrile and subsequent removal of acetonitrile with methylene chloride. Liver was prepared by homogenization with aqueous acetonitrile and subsequent removal of acetonitrile using methylene chloride. For both matrices, the aqueous phase was then extracted using mixed-mode C18/cation exchange SPE cartridges and analyzed on a linear ion trap LC-MS system. Standards were prepared in extracts of control matrix. Seven replicate fortifications of serum at 0.001 mug/g (1 ng/g) of alpha-amanitin gave a mean recovery of 95% with 8.8% CV (relative standard deviation) and a calculated method detection limit of 0.26 ng/g. Seven replicates of control liver fortified at 1 ng/g gave a mean recovery of 98% with 17% CV and a calculated method detection limit of 0.50 ng/g. This is the first report of a positive mass spectrometric identification and quantitation of alpha-amanitin in serum and liver from suspect human and animal intoxications.     

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.     

Liquid chromatographic determination of cephapirin residues in milk

A liquid chromatographic (LC) method was developed for quantitative determination of cephapirin residues in milk that also resolved cephapirin from ampicillin, cloxacillin, and penicillin G. Diluted milk was passed through a C18 cartridge on which the cephapirin was adsorbed; then, interfering material was removed by washing with water and methylene chloride and cephapirin residues were eluted with methanol-acetonitrile (25 + 75). After drying, residues were dissolved in the mobile phase for injection. The LC system had an ultrasphere-ODS column with RP-18 Spheri-10 guard column and a UV detector with a 254 nm filter. The mobile phase was 85% sodium acetate (0.01M) and 15% methanol-acetonitrile (25 + 75) with a flow rate of 1 mL/min. Sensitivity was 20 ppb or less with a recovery of 61-80% in the range studied. Other beta-lactam antibiotics tested did not interfere with detection of cephapirin. Analysis of 30 samples of commercial homogenized milk obtained for a survey of antibiotics in consumer milk in Canada revealed no detectable cephapirin residues.