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.     

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.     

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.     

Analysis of bisphenol A and alkylphenols in cereals by automated on-line solid-phase extraction and liquid chromatography tandem mass spectrometry

An on-line solid-phase extraction (SPE) following a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was established for the simultaneous analysis of bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) in cereals (including rice, maize, and wheat). The target compounds were extracted by acetonitrile, purified by an automated on-line SPE cartridge, and analyzed by LC-MS/MS under the negative-ion mode. Mean recoveries fortified at three concentration levels ranged from 81.6 to 115.7%, and the coefficient of variation ranged from 4.6 to 19.9% (n = 6). The limits of quantification (LOQs) of the method were 0.5, 0.5, and 0.25 μg/kg for BPA, NP, and OP, respectively, in both rice and maize, while the LOQs in wheat were 0.5, 1.25, and 0.5 μg/kg for BPA, NP, and OP, respectively. This method was applied in the analysis of rice, maize, and wheat from a local market. As a result, NP occurred in all cereal samples at the concentration range of 9.4-1683.6 μg/kg and BPA was detected in a few samples.     

Liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of epoxidized soybean oil in food products

Epoxidized soybean oil (ESBO) is widely used as a plasticizer and stabilizer in such polymers [poly(vinyl chloride) in particular] commonly adopted for manufacturing of gaskets of the lids for glass jars and plastic films for food packaging. Human exposure to ESBO and its derivatives is likely to occur over a lifetime with a significant variation according to life stage. A reversed phase liquid chromatography interfaced with electrospray ion trap tandem mass spectrometry method for the determination of ESBO in foods was developed. A simple sample treatment procedure entailing the use of an extraction step with dichloromethane without any further cleanup was proved. Chromatographic separation was performed using two C18 columns with an aqueous acetic acid-acetone-acetonitrile mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (4 mg kg(-1)), quantitation limits, linearity (established over 2 orders of magnitude), recovery (good mean recoveries, higher than 90% for all of the signals detected), precision (RSD% < 8), and trueness. The applicability of the method to the determination of ESBO in different food matrices (in particular those rich in edible oil) was demonstrated, and the performances were compared to those reachable by the commonly well-known gas chromatography-mass spectrometry procedure.     

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.     

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.     

Liquid chromatography-electrospray tandem mass spectrometry of cis-resveratrol and trans-resveratrol: development, validation, and application of the method to red wine, grape, and winemaking byproducts

The application of liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was investigated for the analysis of trans-resveratrol in red wine, grape skin, grape pomace, and winemaking byproducts. Chromatographic elution performed under isocratic reversed-phase conditions using a C18 narrow-bore LC column allowed retention times lower than 12 min to be obtained. Qualitative analysis was performed in negative-ion (NI) full-scan and product-ion scan acquisition modes, whereas method validation in terms of linearity, detection limits, accuracy, and robustness was carried out under NI selected reaction monitoring conditions. The matrix-matched detection limit was calculated in the low parts per billion range (10 microg/L). Results of the application of the method to red wine, grape, and winemaking byproduct samples were compared with those obtained using an LC-UV/DAD technique. Determination of trans-resveratrol in the samples investigated showed that the highest concentration was found in red wine, whereas wine made from grape pomace contained the lowest content.     

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.     

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.     

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.     

A new method for the determination of carbonyl compounds in wines by headspace solid-phase microextraction coupled to gas chromatography-ion trap mass spectrometry

A new analytical method for the determination of 18 carbonyl compounds [2,3-pentadione, hexanal, (E)-2-hexen-1-al, octanal, acetoin, (E)-2-octenal, furfural, decanal, (E)-2-nonenal, benzaldehyde, 5-methylfurfural, (E,E)-2-cis-6-nonadienal, β-damascenone, phenylacetaldehyde, acetophenone, (E,E)-2,4-decadienal, benzophenone, and vanillin] in wines using automated headspace solid-phase microextraction (HS/SPME) coupled to gas chromatography-ion trap mass spectrometry (GC-ITMS) was developed. Five fibers with different polarities were tested, and a study of the influence of various factors such as time and extraction temperature, desorption time and temperature, pH, and ionic strength and content in tannins, anthocyans, sucrose, SO(2), and alcoholic degree was conducted. These factors were optimized using a synthetic wine doped with the different analytes. The proposed method affords wide ranges of linearity, good linearity (r(2) > 0.998), values of repeatability and reproducibility lower than 5.5% of RSD, and detection limits ranging from 0.62 μg/L for β-damascenone to 129.2 μg/L for acetoin. Therefore, the optimized method was applied to the quantitative analysis of the aforementioned analytes in real samples of wines.     

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.     

Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry

Red wine extracts were screened for potential wine pigments derived from anthocyanins, using a combination of nanoelectrospray tandem mass spectrometry techniques. Fourteen aglycons were considered to be of anthocyanidin origin on the basis of their MS/MS spectra. The proposed structures of the aglycons were anthocyanidin C-4 substituted with vinyl linkage between C-4 and the hydroxy group at C-5. The anthocyanidin derivatives identified in the wine extracts were vinyl, vinylmethyl, vinylformic acid, 4-vinylphenol, 4-vinylguaiacol, and vinylcatechin adducts of malvidin as well as vinylformic acid and 4-vinylphenol adducts of peonidin and petunidin. The presence of vinyl alcohol, 4-vinylcatechol, and 4-vinylsyringol adducts of malvidin was also proposed.     

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 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.     

Analysis of aldehydes in beer using solid-phase microextraction with on-fiber derivatization and gas chromatography/mass spectrometry

A new, fast, sensitive, and solventless extraction technique was developed in order to analyze beer carbonyl compounds. The method was based on solid-phase microextraction with on-fiber derivatization. A derivatization agent, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA), was absorbed onto a divinyl benzene/poly(dimethylsiloxane) 65-microm fiber and exposed to the headspace of a vial with a beer sample. Carbonyl compounds selectively reacted with PFBOA, and the oximes formed were desorbed into a gas chromatograph injection port and quantified by mass spectrometry. This method provided very high reproducibility and linearity. When it was used for the analysis of aged beers, nine aldehydes were detected: 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, pentanal, hexanal, furfural, methional, phenylacetaldehyde, and (E)-2-nonenal.     

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.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.     

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.