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.     

Multiresidue analysis of seven anticoagulant rodenticides by high-performance liquid chromatography/electrospray/mass spectrometry

Mice and rat populations are commonly controlled by two classes of rodenticide anticoagulants, coumarins and indandiones. However, poisoning of nontarget animals also often occurs. For cases such as these, a rapid, multiresidue method, which provides positive confirmation for both classes of anticoagulant rodenticides, is needed by diagnostic laboratories. A method was developed for the determination of seven anticoagulant rodenticides, coumafuryl, pindone, warfarin, diphacinone, chlorophacinone, bromadiolone, and brodifacoum, in diverse matrices, animal feed, cooked beef, and fruit-flavored beverages using high-performance liquid chromatography/electrospray/mass spectrometry. Detection was by MS/MS with electrospray ionization in negative mode. Confirmation was by retention time, m/z of molecular ion, and two parent-daughter transitions. Recoveries from selected the matrices ranged from 61 to 117%. Limits of quantitation were as low as 1.5-4.5 ng g-1. The developed method was rapid and provided the simultaneous confirmation and quantification of the seven anticoagulant rodenticides.     

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.     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

Normal phase high-performance liquid chromatography method for the determination of tocopherols and tocotrienols in cereals

The eight vitamers of vitamin E (alpha-, beta-, gamma-, and delta-tocopherols and -tocotrienols) have different antioxidant and biological activities and have different distributions in foods. Some cereals, especially oat, rye, and barley, are good sources of tocotrienols. A fast procedure for the determination of tocopherols and tocotrienols (tocols) in cereal foods was developed. It involves sample saponification and extraction followed by normal phase high-performance liquid chromatography (HPLC). The results have been compared with those found by direct extraction without saponification. The method is sensitive and selective enough to be tested on a wide variety of cereal samples. The highest tocol levels were found in soft wheat and barley ( approximately 75 mg/kg of dry weight). beta-Tocotrienol is the main vitamer found in hulled and dehulled wheats (from 33 to 43 mg/kg of dry weight), gamma-tocopherol predominates in maize (45 mg/kg of dry weight) ), and alpha-tocotrienol predominates in oat and barley (56 and 40 mg/kg of dry weight, respectively).     

Analysis of strigolactones,germination stimulants for striga and orobanche,by high-performance liquid chromatography/tandem mass spectrometry

A simple and rapid analytical method for strigolactones, germination stimulants for the root parasitic weeds witchweed (Striga spp.) and broomrape (Orobanche spp.), has been developed using high-performance liquid chromatography connected to tandem mass spectrometry (LC/MS/MS). The natural strigolactones (strigol, sorgolactone, orobanchol, and alectrol) were clearly separated and identified by LC/MS/MS. As low as 0.1 pg/microL of strigol and 0.5 pg/microL of sorgolactone could be quantified, whereas 1 pg/microL was needed for the quantification of orobanchol (S/N > 10). Using this method, it was found that red clover produces orobanchol and alectrol but not strigol. The roots of red clover seedlings were found to produce 13, 70, 58, and 65 pg of orobanchol/plant 1, 2, 3, and 4 weeks after germination, respectively.     

A routine high-performance liquid chromatography method for carotenoid determination in ultrafrozen orange juices

An isocratic reversed-phase high-performance liquid chromatography method was developed for routine analysis of the main carotenoids related to the color of orange juice, using a more selective wavelength (486 nm) in which the absorption in the red-orange region of the visible spectra is maximum. Separation was carried out using as the mobile phase the mixture methanol:acetonitrile:methylene chloride:water (50:30:15:5, v/v/v/v), to which small amounts of butylated hydroxytoluene and triethylamine were added (0.1%). Identification was made by comparison either with standards obtained by thin-layer chromatography or with spectral data previously reported. The reproducibility of the method was remarkable; coefficients of variation for the most polar xanthophylls were under 1 and 4% for retention times and areas, respectively. Its application to Valencia late ultrafrozen orange juices has shown that major carotenoids are lutein + zeaxanthin (36%), lutein 5,6-epoxide (16%), antheraxanthin (14%), and beta-cryptoxanthin (12%).     

Determination of isoxaflutole (balance) and its metabolites in water using solid phase extraction followed by high-performance liquid chromatography with ultraviolet or mass spectrometry

Balance (isoxaflutole, IXF) belongs to a new family of herbicides referred to as isoxazoles. IXF has a very short soil half-life (<24 h), degrading to a biologically active diketonitrile (DKN) metabolite that is more polar and considerably more stable. Further degradation of the DKN metabolite produces a nonbiologically active benzoic acid (BA) metabolite. Analytical methods using solid phase extraction followed by high-performance liquid chromatography-UV (HPLC-UV) or high-performance liquid chromatography-mass spectrometry (HPLC-MS) were developed for the analysis of IXF and its metabolites in distilled deionized water and ground water samples. To successfully detect and quantify the BA metabolite by HPLC-UV from ground water samples, a sequential elution scheme was necessary. Using HPLC-UV, the mean recoveries from sequential elution of the parent and its two metabolites from fortified ground water samples ranged from 68.6 to 101.4%. For HPLC-MS, solid phase extraction of ground water samples was performed using a polystyrene divinylbenzene polymer resin. The mean HPLC-MS recoveries of the three compounds from ground water samples spiked at 0.05-2 microg/L ranged from 100.9 to 110.3%. The limits of quantitation for HPLC-UV are approximately 150 ng/L for IXF, 100 ng/L for DKN, and 250 ng/L for BA. The limit of quantitation by HPLC-MS is 50 ng/L for each compound. The methods developed in this work can be applied to determine the transport and fate of Balance in the environment.     

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.     

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%.     

Development of an immunoaffinity chromatography purification and ultra performance liquid chromatography tandem mass spectrometry method for determination of 12 sulfonamides in beef and milk

A highly selective and sensitive method was developed for the simultaneous determination of 12 sulfonamides in beef and milk by immunoaffinity chromatography purification coupled to ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The MS/MS conditions, UPLC mobile phase, injection solution, sample purification process, and matrix effect were studied to optimize the operating conditions. The limits of detection (LODs) of the instrument for the studied sulfonamides ranged from 0.4 to 2.0 μg L(-1), being 1.6-8.0 μg kg(-1) for beef and 1.8-6.4 μg kg(-1) for milk. The standard solution was diluted with blank beef or milk matrix for the construction of calibration curves, which had a linear range from 10 to 200 μg kg(-1) and regression coefficients higher than 0.990 (n=10) for all the studied sulfonamides. Samples spiked at 10, 20, and 100 μg kg(-1) showed recoveries above 70% and relative standard deviations below 10%.     

Development of a new high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry method for the determination of low molecular mass organic acids in plant tissue extracts

A liquid chromatography-electrospray ionization time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of 10 low molecular mass organic acids in different plant tissue extracts. The method does not include a derivatization step. Quantification was accomplished using (13)C-labeled malic and succinic acids as internal standards. Good limits of detection (0.05-1.27 pmol) were obtained for malic, 2-oxoglutaric, succinic, quinic, shikimic, cis-aconitic, and citric acids, whereas for oxalic, ascorbic, and fumaric acids limits of detection were 255, 25, and 11 pmol, respectively. Repeatability values for the retention time and peak area were <5%, with the exception of ascorbic acid. Analyte recovery was between 92% and 110% in most cases, with the exception of oxalic (39-108%), 2-oxoglutaric (44-69%), and ascorbic (22-86%) acids, which may require specific extraction procedures and use of the corresponding (13)C-labeled organic acid as internal standards to improve accuracy. The method was applied to three types of plant materials: sugar beet leaf extracts, tomato xylem sap, and commercial orange juice.     

液相色谱串联质谱法测定有机肥料中磺胺甲噻二唑 残留量的不确定度评定

依据《有机肥料中19种兽药残留量的测定液相色谱串联质谱法》（GB/T 40462—2021）对有机肥料中磺胺甲噻二唑（SMT）的残留量进行测定,对测量结果进行不确定度评定。不确定度来源包括标准溶液配制、样品制备、测量重复性、回收率及仪器等分量。相对标准不确定度计算结果表明标准曲线拟合、标准物质纯度和标准曲线配制是影响不确定度的主要因素,应在实际试验过程中加以重点关注与控制。在95%置信水平下,取扩展因子k=2,待测肥料样品中最终结果表示为：X(SMT)=(189.82±20.12) mg/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.     

Development of analyses by high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry of bilberry (Vaccinium myrtilus) anthocyanins in human plasma and urine

Anthocyanins are potent antioxidants that may possess chronic disease preventive properties. Here, rapid, reliable, and reproducible solid-phase extraction, high-performance liquid chromatography (HPLC), and mass spectrometry techniques are described for the isolation, separation, and identification of anthocyanins in human plasma and urine. Recoveries of cyanidin-3-glucoside (C3G) were 91% from water, 71% from plasma, and 81% from urine. Intra- and interday variations for C3G extraction were 9 and 9.1% in plasma and 7.1 and 9.1% in urine and were less than 15% for all anthocyanins from a standardized bilberry extract (mirtoselect). Analysis of mirtoselect by HPLC with UV detection produced spectra with 15 peaks compatible with anthocyanin components found in mirtoselect within a total run time of 15 min. Chromatographic analysis of human urine obtained after an oral dose of mirtoselect yielded 19 anthocyanin peaks. Mass spectrometric analysis employing multiple reaction monitoring suggests the presence of unchanged anthocyanins and anthocyanidin glucuronide metabolites.     

Diagnostic determination of melamine and related compounds in kidney tissue by liquid chromatography/tandem mass spectrometry

In 2007, it was determined that melamine, ammeline, ammelide, and cyanuric acid (abbreviated as MARC for melamine and related contaminants) had been added to wheat gluten and rice protein that were subsequently incorporated into pet food. The consumption of food tainted by MARC compounds was implicated in numerous instances of renal failure in cats and dogs. A method for the analysis of MARC compounds in kidney tissue using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) has been developed. MARC analytes were extracted by homogenization of kidney tissue in 50/40/10 acetonitrile/water/diethylamine. The homogenate was centrifuged, and an aliquot of supernatant was diluted with acetonitrile, concentrated, and fortified with a stable isotope-labeled analogue of melamine. Analytes were detected using atmospheric pressure chemical ionization and multiple reaction monitoring. Quantitation of positive samples was performed using the internal standard method and five-point calibration curves ranging between 50 and 1000 ng/mL of each analyte. The method was validated by analysis of replicate kidney tissue samples fortified with the individual analytes and by analysis of kidney samples fortified with melamine cyanurate powder at two different concentrations. This method was successfully used for routine postmortem diagnosis of melamine toxicosis in animals. Melamine was also detected by this method in paraffin-embedded tissue from animals suspected to have died of melamine toxicosis.     

Identification and quantitation of sulfamethazine metabolites by liquid chromatography and gas chromatography-mass spectrometry

Methods for the identification and quantitation of carbon-14 labeled sulfamethazine [4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzenesulfonamide], N4-acetylsulfamethazine, the N4-glucose conjugate of sulfamethazine, and desaminosulfamethazine in swine tissue are described. Tissues are ground and extracted with methanol, and the 14C-labeled compounds are purified by XAD-2 column chromatography and C-18 reverse phase liquid chromatography (LC) and the 14C-labeled compounds are then methylated and identified by gas chromatography-mass spectrometry analysis. Quantitation is accomplished by measuring the amount of 14C-activity that cochromatographs (C-18 reverse phase LC) with reference compounds.     

A simple and economic method for simultaneous determination of 11 antibiotics in manure by solid-phase extraction and high-performance liquid chromatography

Purpose

A simple and highly efficient economic method for the analysis of 11 antibacterial drugs including two tetracyclines, three quinolones, four sulfonamides, chloramphenicol and tylosin, in livestock manure, was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC).

Materials and methods

The analytes were successively extracted by EDTA-McIlvaine solution and organic solvent mixture. The extracts were degreased with n-hexane and cleaned through SPE on a hydrophile-lipophile balance (HLB) cartridge. All compounds were determined on a C18 reverse phase column with gradient elution.

Results and discussion

Recoveries calculated from spiked samples of animal manures ranged from 62.65 to 99.16 % for 11 antibiotics with relative standard deviations of less than 10.0 %. Limits of detection ranged from 0.1 to 1.9 μg kg^?1, and limits of quantification ranged from 0.3 to 5.9 μg kg^?1.

Conclusions

The results show that SPE-HPLC is an inexpensive and practical method for rapid detection of multiple antibiotics in animal manure.

     

Development of liquid chromatography mass spectrometry method for analysis of organic N monomers in soil

The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC–MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC–MS method it was compared with a capillary electrophoresis–mass spectrometry (CE–MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC–MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC–MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5 μL injection ranged between 0.002 and 0.38 μmol L⁻¹, with the majority (49 out of 62) having limits of detection better than 0.05 μmol L⁻¹. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC–MS and CE–MS, with the notable exception of four ureides that were detected by LC–MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC–MS method offered advantages and disadvantages relative to CE–MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts.     

Analysis of eight capsaicinoids in peppers and pepper-containing foods by high-performance liquid chromatography and liquid chromatography-mass spectrometry

Diverse procedures have been reported for the isolation and analysis of secondary metabolites called capsaicinoids, pungent compounds in the fruit of the Capsicum (Solanaceae) plant. To further improve the usefulness of high-performance liquid chromatography (HPLC), studies were carried out on the analysis of extracts containing up to eight of the following capsaicinoids: capsaicin, dihydrocapsaicin, homocapsaicin-I, homocapsaicin-II, homodihydrocapsaicin-I, homodihydrocapsaicin-II, nonivamide, and nordihydrocapsaicin. HPLC was optimized by defining effects on retention times of (a) the composition of the mobile phase (acetonitrile/0.5% formic acid in H2O), (b) the length of the Inertsil column, and (c) the capacity values (k) of the column packing. Identification was based on retention times and mass spectra of individual peaks. Quantification was based on the UV response at 280 nm in HPLC and recoveries from spiked samples. The method (limit of detection of approximately 15-30 ng) was successfully used to quantify capsaicinoid levels of parts of the pepper fruit (pericarp, placenta, seeds, and in the top, middle, and base parts of whole peppers) in 17 species of peppers and in 23 pepper-containing foods. The results demonstrate the usefulness of the method for the analysis of capsaicinoids ranging from approximately 0.5 to 3600 microg of capsaicin equiv/g of product. The water content of 12 fresh peppers ranged from 80.8 to 92.7%. The described freeze-drying, extraction, and analysis methods should be useful for assessing the distribution of capsaicinoids in the foods and in defining the roles of these biologically active compounds in the plant, the diet, and medicine.