Determination of polycyclic aromatic hydrocarbons in soy isoflavone nutraceutical products by gas chromatography coupled to triple quadrupole tandem mass spectrometry

Thirteen polycyclic aromatic hydrocarbons have been determined in soy‐based nutraceutical products. First, an optimization of extraction procedure was performed, and a solid–liquid extraction assisted by sonication and a dilute and shoot procedure were compared, selecting the dilute and shoot approach for the extraction of target compounds, utilizing a mixture of acetone/n‐hexane (1:1 v/v) as extractant solvent. After this, a clean‐up step was needed bearing in mind the complexity of these matrices. Dispersive solid‐phase extraction, using a mixture of C₁₈ and Zr‐Sep+ (25 mg/mL each) was used. The separation was achieved by gas chromatography and detection with triple quadrupole tandem mass spectrometry. For quantification purposes, matrix‐matched calibration was used. The validation was applied at three concentration levels (20, 100 and 250 μg/kg), obtaining recoveries between 70 and 120% and precision values equal to or lower than 23%. Limits of detection and quantification were below 8 and 20 μg/kg, respectively. The method was applied in 11 samples, detecting five polycyclic aromatic hydrocarbons at concentrations ranging from 4.1 to 18.5 μg/kg.     

Re-evaluation of glyphosate determination in water by liquid chromatography coupled to electrospray tandem mass spectrometry

An analytical method based on on-line solid-phase extraction-liquid chromatography coupled to electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) for the determination of glyphosate and aminomethylphosphonic acid (AMPA) residues has been applied to the analysis of water samples within a Round Robin Study. The method had been previously validated in a variety of water samples and it fulfilled all the parameters of precision, accuracy, sensitivity and unequivocal confirmation. The results within the study that we participated were highly satisfactory in all cases with the only exception of glyphosate in groundwater samples, where surprisingly recoveries around 15% were obtained despite the use of isotope-labeled glyphosate as internal standard (I.S.). A slight modification has been introduced in the method, simply consisting of the acidification of sample with hydrochloric acid (HCl) to pH 1. Then, the sample is neutralized and immediately derivatized with 9-fluorenylmethylchloroformate (FMOC) before LC-MS/MS determination. Round Robin Study samples were reanalyzed using this approach, and the recoveries increased up to 98%. A possible explanation might be the slow kinetic interaction between glyphosate and some components of the matrix. These components might act as chelating agents, making glyphosate unavailable for derivatization and therefore for analysis. Several water samples collected at the Mediterranean area of Spain, and previously analyzed and being found to contain glyphosate, were also reanalyzed using this approach, obtaining higher concentrations (between 2 and 14 times) in most of cases.     

Fast determination of herbicides in waters by ultra-performance liquid chromatography/tandem mass spectrometry

A rapid multiresidue method for the analysis of more than 40 herbicides (such as simazine, terbuthylazine and diuron) in waters has been developed and validated by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS). Prior to chromatographic determination, the samples were extracted using a solid-phase extraction procedure. The analysis was performed on an Acquity UPLC BEH C(18) column using a gradient elution profile and a mobile phase consisting of methanol and an aqueous solution of formic acid (0.01%). Other chromatographic and MS/MS parameters were optimised in order to improve selectivity and sensitivity of the analytes. The analytes were detected using electrospray ionisation (ESI)-MS/MS in positive ion mode with multiple reaction monitoring (MRM), optimising parameters such as voltage cone, capillary voltage, source and desolvation temperature, and desolvation and cone gas flow. The optimised method provides a rapid separation (less than 10 min) of the selected herbicides in the assayed matrices, and it was validated by the analysis of spiked blank matrix samples. Good linearity was obtained and the repeatability of the method was less than 20% for the lowest calibration point. The limits of detection ranged from 0.002 to 0.02 microg/L, and the limits of quantification from 0.005 to 0.05 microg/L, which were below the values specified by the European Union. Finally, the method was successfully applied to real environmental samples from Andalusia (southern Spain). Terbuthylazine, simazine, atrazine desisopropyl and desethyl terbuthylazine were the herbicides most frequently found in water samples.     

Rapid determination of fumonisins in corn-based products by liquid chromatography/tandem mass spectrometry

A simple, fast, and robust method was developed for the determination of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) in corn-based human food and animal feed (cornmeal). The method involves a single extraction step followed by centrifugation and filtration before analysis by ultra-performance liquid chromatographylelectrospray ionization (UPLC/ESI)-MS/MS. The LC/MS/MS method developed here represents the fastest and simplest procedure (<30 min) among both conventional HPLC methods and other LC/MS methods using SPE cleanup. The potential for high throughput analysis makes the method particularly beneficial for regulatory agencies and analytical laboratories with a high sample volume. A single-laboratory validation was conducted by testing three different spiking levels (200, 500, and 1000 ng/g for FB1 and FB2; 100, 250, and 500 ng/g for FB3) for accuracy and precision. Recoveries of FB1 ranged from 93 to 98% with RSD values of 3-8%. Recoveries of FB2 ranged from 104 to 108%, with RSD values of 2-6%. Recoveries of FB3 ranged from 94 to 108%, with RSD values of 2-5%.     

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid-liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS-MS in selecting m/z 321 --> 257, 321 --> 152 (35Cl2) and m/z 323 --> 257, 323 --> 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 --> 152 versus 326 --> 157 and m/z 321 --> 257 versus 326 --> 262 against their corresponding amount ratio. Indeed, even if m/z 321 --> 152 was found to give a higher MS-MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 --> 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 microg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCalpha) and detection capability (CCbeta) of CAP in milk were calculated for m/z 321 --> 152 at 0.02 microg/kg and 0.03 microg/kg, respectively, and for m/z 321 --> 257 at 0.02 microg/kg and 0.04 microg/kg, respectively. At the lowest fortification level (i.e. 0.1 microg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 --> 257 both at 0.02 microg/kg and for m/z 321 --> 152 at 0.03 and 0.05 microg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.     

Fast liquid chromatography coupled to electrospray tandem mass spectrometry peptide sequencing for cross-species protein identification

Using a parallel microcolumn switching liquid chromatography set-up coupled to a quadrupole time-of-flight mass spectrometer, a rapid liquid chromatography/mass spectrometric (LC/MS) protein identification method is presented. Without prior sample clean-up up to 300 protein digest samples a day can be processed. Using data-directed acquisition, up to 10 fragmentation analyses for each protein sample can be acquired in the same chromatographic run that can be used for database searching. Using internal peptide sequence information, protein databases and the various nucleic acid databases can both be queried for cross-species identification of the protein sample. The method was evaluated and put into force to generate data for a tobacco cell culture protein database.     

Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry

The present study developed an improved analytical method for the simultaneous quantification of 17 kinds of Aspergillus, Fusarium and Penicillium mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode, and especially focused on the optimization of extraction, clean-up, UPLC separation and MS/MS parameters of analytes. Homogenized samples were sequentially extracted by 84% (v/v) of acetonitrile aqueous solution with the selected internal standard (zearalanone) spiking, SPE clean-up with Mycosep 226 Aflazon+ Multifunctional cartridges, filtration, concentration and secondary filtration. Using double sample injection method, the analytes were separated by UPLC BEH C18 column (100 mm x 2.1 mm I.D., 1.7 microm), and eluted with ammonium acetate/methanol and aqueous ammonia/methanol for the ESI+ and ESI- analysis, respectively. The 10 positive ions and 7 negative ions of mycotoxins were separated by gradient elution with the retention time of 6.5 and 4 min, respectively. The LOQ of selected analytes ranged from 0.01 to 0.70 microg kg(-1), which was lower than the criteria of EU, USA and other countries on the determination of the minimum limiting level of various mycotoxins in foods including baby foods and feed stuffs. Meanwhile, high correlation coefficients (r2>0.99) of 17 mycotoxins were obtained within their respective linear ranges (0.05-20 microg kg(-1) for 10 positive ions and 0.5-50 microg kg(-1) for 7 negative ions) and reasonable recoveries (70.6-119.0%) of them were also demonstrated in different spiked levels. This quantitative method has many advantages including simple pretreatment, rapid determination and high sensitivity, which could be applied to the determination and quantification of multi-component mycotoxin contaminants in complex matrixes. Meanwhile, the method successfully fulfilled the minimum limiting level requests from various countries.     

Determination of N-acetylcysteine in human plasma by liquid chromatography coupled to tandem mass spectrometry

An analytical method for the determination of total N-acetylcysteine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists of plasma digestion with dithiothreitol in order to reduce all the oxidized forms of N-acetylcysteine, and extraction with ethyl acetate followed by determination of levels by an LC–MS–MS method. The intra- and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 50 ng/ml of plasma. The concentration working range was established between 50 ng/ml and 1000 ng/ml. This method has been used in the analysis of approximately 800 human plasma samples from a clinical study with 24 volunteers; the precision of the quality controls was in the range 8.7 to 13.4% and the accuracy was in the range −5.9 to 8.5%, expressed as the RSD and the relative error, respectively.     

Quantification of tizanidine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry (MS/MS) method was developed and validated for the assay of tizanidine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by MS/MS in the selected reaction monitoring mode. The assay exhibited a linear dynamic range of 50-5000 pg/mL for tizanidine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 13%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.5 min for each sample made it possible to analyze more than 300 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.     

Simultaneous determination of zolazepam and tiletamine in dog plasma by liquid chromatography coupled to a tandem mass spectrometry

A mixture of tiletamine, a dissociative anesthetic, and zolazepam, a minor tranquilizer, has been widely used as an anesthetic or an immobilizing agent in a variety of animal species. However, interestingly, their pharmacokinetic behaviors have been published only in polar bears and pigs. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the two drugs in dog plasma. After simple protein precipitation with acetonitrile including midazolam (internal standard), the analytes were chromatographed on a reversed‐phase column with a mobile phase of 10 m m ammonium acetate aqueous solution and acetonitrile (1:4, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of zolazepam and tiletamine in plasma after a single intramuscular 10 mg dose of each in beagle dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast profiling of chemical constituents in Yiqing Capsule by ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry

An ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC-ESI-MS(n)) has been developed for structural characterization and identification of multi-constituents in Yiqing Capsule, a well-known combined herbal remedy prepared from the extract mixtures of Rhizoma Coptidis, Radix et Rhizoma Rhei and Radix Scutellariae. The UPLC analysis was performed on an Agilent ZorBax SB-C(18) column (4.6 mm×50 mm, 1.8 μm) and gradient elution of 0.1% formic acid solution and acetonitrile in 16 min. Based on their retention times and mass spectra in comparison with the data from standards or references, a total of 29 compounds including 3 phenolic acids and 4 anthraquinones from Radix et Rhizoma Rhei, 8 alkaloids from Rhizoma Coptidis and 14 flavonoids from Radix Scutellariae were unambiguously identified or tentatively characterized in the complex system. The MS data and fragmentation information of two isomers of feruloylquinic acid were first reported in Radix et Rhizoma Rhei and in Yiqing Capsules. This study is expected to be accepted as an effective and reliable pattern for comprehensive and systematic characterization of this commonly used Chinese herbal preparation.     

高效液相色谱串联质谱法同时测定水产品中24种性激素

建立了同时测定24种性激素的高效液相色谱串联质谱法,包括:睾酮、甲基睾酮、诺龙、苯丙酸诺龙、群勃龙、康力龙、勃地酮、雄烯二酮、美雄酮、炔诺酮、乙酸甲孕酮、乙酸甲羟孕酮、乙酸氯地孕酮、17α羟基孕酮、21α羟基孕酮、甲羟孕酮、左炔诺孕酮、雌酮、雌二醇、雌三醇、炔雌醇、己烷雌酚、己烯雌酚、双烯雌酚。乙酸乙酯提取2次,硅胶柱净化。采用甲醇、水作为流动相,经过CAPCELLPAK C18色谱柱分离后,采用APCI离子源,外标法定量。方法定量限为0.5～2μg/kg,加标回收率为80%～102%,相对标准偏差为6%～10%。方法实现了3类性激素的同时定量及确证分析。     

Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.     

Direct determination of beta-blockers and their metabolites in urine by liquid chromatography coupled with tandem mass spectrometry

A method is proposed for the detection and confirmation of the presence of beta-blockers and their metabolites in fivefold diluted human urine samples by ultra performance liquid chromatography coupled with electrospay ionization tandem mass spectrometry. The limits of detection for most of compounds are 5–10 ng/mL. A substantial effect of ionization suppression was observed. The determination of metabolites and glucuronides of beta-blockers without additional derivatization and extraction is described for the first time.     

Simultaneous determination of four shanzhiside methylester derivatives in rabbit plasma by liquid chromatography/tandem mass spectrometry

A simple and sensitive high‐performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous determination of shanzhiside methylester and its three derivatives in rabbit plasma. The method showed good linearity and no endogenous material interfered with the marked compounds and internal standard (IS) capatol peaks. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C₁₈ column (150 × 3.9 mm i.d., 4 µm). The mobile phase consisted of methanol and water (60:40, v/v) during a total run time of 7 min. The main mass parent ions and daughter ions pairs (m/z) for monitoring were: shanzhiside methylester, 429.0/267.4; 8‐O‐acetyl shanzhiside methylester, 470.9/411.3; loganin, 413.2/251.4; phloyoside II, 479.2/281.3; and IS 385.2/203.3. Finally, the method was applied to a pharmacokinetic study of rabbits following intravenous administration of iridoid glycosides extracted from traditional herb Lamiophlomis rotata. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantification of granisetron in human plasma by liquid chromatography coupled to electrospray tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of granisetron in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 313/138 for granisetron and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.1-20 ng/mL for granisetron in human plasma. The lower limit of quantification was 100 pg/mL with a relative standard deviation of less than 5%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Identification of leucine-enkephalin radical oxidation products by liquid chromatography tandem mass spectrometry

The oxidation of the peptide leucine-enkephalin (YGGFL) induced by the hydroxyl radical (HO*), formed under Fenton-like conditions [Cu (II)/H(2)O(2)], was studied and monitored by LC-MS. The oxidation products identified included products resultant from (a) the insertion of oxygen atoms (1-5), (b) peptide backbone cleavage (short-chain products formed by diamide pathway) and (c) radical-radical crosslinking reactions. In order to identify the modified residues, LC-MS/MS spectra were obtained. The insertion of oxygen atoms into the peptide originated hydroxide, di-hydroxide and/or hydroperoxide derivatives. In addition it was found that the aromatic amino acids are most susceptible to being hydroxylated, while the aliphatic amino acids are more prone to forming hydroperoxides. Oxidation products with double bonds were also identified. The short chain products resulted from the alpha-carbon radical of terminal amino acids (Tyr and Leu). Products resulting from cross-linking reactions between intact carbon-centered peptide radical (with and without one HO group) and a side chain radical (*C(7)H(7)O) were identified. It was found that, although all amino acids residues of the peptide undergo modifications, the N-terminal seems to be prone to oxidative modifications under these conditions.     

Quantitative and confirmative performance of liquid chromatography coupled to high-resolution mass spectrometry compared to tandem mass spectrometry

The quantitative and confirmative performance of two different mass spectrometry (MS) techniques (high-resolution MS and tandem MS) was critically compared. Evaluated was a new extraction and clean-up protocol which was developed to cover more than 100 different veterinary drugs at trace levels in a number of animal tissues and honey matrices. Both detection techniques, high-resolution mass spectrometry (HRMS) (single-stage Orbitrap instrument operated at 50 000 full width at half maximum) and tandem mass spectrometry (MS/MS) (quadrupole technology) were used to validate the method according to the EU Commission Decision 2002/657/EEC. Equal or even a slightly better quantitative performance was observed for the HRMS-based approach. Sensitivity is higher for unit mass resolution MS/MS if only a subset of the 100 compounds has to be monitored. Confirmation of suspected positive findings can be done by evaluating the intensity ratio between different MS/MS transitions, or by accurate mass based product ion traces (no precursor selection applied). MS/MS relies on compound-specific optimized transitions; hence the second, confirmatory transition generally shows relatively high ion abundance (fragmentation efficacy). This is often not the case in single-stage HRMS, since a generic (not compound-optimized) collision energy is applied. Hence, confirmation of analytes present at low levels is superior when performed by MS/MS. Slightly better precision, but poorer accuracy (fortified matrix extracts versus pure standard solution) of ion ratios were observed when comparing data obtained by HRMS versus MS/MS.     

Rapid determination of quinolones in cosmetic products by ultra high performance liquid chromatography with tandem mass spectrometry

This study developed an improved analytical method for the simultaneous quantification of 13 quinolones in cosmetics by ultra high performance liquid chromatography combined with ESI triple quadrupole MS/MS under the multiple reaction monitoring mode. The analytes were extracted and purified by using an SPE cartridge. The limits of quantification ranged from 0.03 to 3.02 μg/kg. The precision for determining the quinolones was <19.39%. The proposed method was successfully developed for the determination of quinolones in real cosmetic samples.