On‐line solid‐phase extraction coupled with liquid chromatography/electrospray ionization mass spectrometry for the determination of trace tributyltin and triphenyltin in water samples

On‐line solid‐phase extraction (SPE) for pre‐concentration and sample cleanup is one strategy to reduce matrix effects and to simultaneously improve detection sensitivity in liquid chromatography/mass spectrometry (LC/MS). This paper describes an on‐line SPE‐LC/MS method for the determination of tributyltin (TBT) and triphenyltin (TPhT) at trace levels in water samples. The direct coupling of an on‐line C₁₈ pre‐column to LC/MS was used to pre‐concentrate TBT and TPhT at trace levels from waters and to remove interfering matrix effects. Pre‐concentration was followed by separation of TBT and TPhT on a C₁₈ column using a mobile phase containing 0.1% (v/v) HCOOH/5 mM HCOONH₄ and methanol. While both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) can be interfaced with MS for the detection of TBT and TPhT, ESI‐MS was preferred for this application. The calibration curve for the targets was linear in the concentration range 0.1–30 µg L^?1. The detection limit (signal‐to‐noise (S/N) ratio = 3) was 0.02 µg L^?1 when 3.0 mL of sample was enriched on the C₁₈ pre‐column. The recoveries of TBT and TPhT in spiked waters were from 81.0 to 101.9%. The reproducibilities for the analysis of the standard mixture (10 µg L^?1) for TBT and TPhT were 13.1 and 5.0%, respectively. The developed method was an easy and fast way to analyze TBT and TPhT in water samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Improved characterization of tomato polyphenols using liquid chromatography/electrospray ionization linear ion trap quadrupole Orbitrap mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry

Tomato (Lycopersicon esculentum Mill.) is the second most important fruit crop worldwide. Tomatoes are a key component in the Mediterranean diet, which is strongly associated with a reduced risk of chronic degenerative diseases. In this work, we use a combination of mass spectrometry (MS) techniques with negative ion detection, liquid chromatography/electrospray ionization linear ion trap quadrupole‐Orbitrap‐mass spectrometry (LC/ESI‐LTQ‐Orbitrap‐MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) on a triple quadrupole, for the identification of the constituents of tomato samples. First, we tested for the presence of polyphenolic compounds through generic MS/MS experiments such as neutral loss and precursor ion scans on the triple quadrupole system. Confirmation of the compounds previously identified was accomplished by injection into the high‐resolution system (LTQ‐Orbitrap) using accurate mass measurements in MS, MS² and MS³ modes. In this way, 38 compounds were identified in tomato samples with very good mass accuracy (<2 mDa), three of them, as far as we know, not previously reported in tomato samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library search

Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the ‘screen’ and ‘confirmation’ goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI‐MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid‐liquid and solid‐phase extraction protocols were coupled to LC/ESI‐MS/MS using a 1.8‐µm particle size analytical column operated at 50°C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive‐ion ESI‐MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1–10 µg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens – based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two‐step (immunoassay and GC/MS) approach in the past. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Comparison of analytical approaches for liquid chromatography/mass spectrometry determination of the alcohol biomarker ethyl glucuronide in urine

Official guidelines originating from a European Union directive regulate requirements for analytical methods used to identify chemical compounds in biological matrices. This study compared different liquid chromatography/electropray ionization mass spectrometry (LC/ESI‐MS) and tandem mass spectrometry (LC/ESI‐MS/MS) procedures for accurate determination of the conjugated ethanol metabolite and alcohol biomarker ethyl glucuronide (EtG) in urine, and the value of combined EtG and ethyl sulfate (EtS) measurement. Analysis was carried out on 482 urines following solid‐phase extraction (SPE) sample cleanup or using direct injection of a diluted sample. SPE combined with LC/MS/MS was demonstrated to be the most selective and sensitive method and was chosen as reference method. The EtG results by different methods showed good correlation (r = 0.96–0.98). When comparing five reporting limits for EtG in the range 0.10–1.00 mg/L, the overall agreement with the reference method (frequency of true positives plus true negatives) was 82–97% for direct‐injection LC/MS/MS, 90–97% for SPE‐LC/MS, 86–98% for direct‐injection LC/MS, and 86–98% for direct‐injection LC/MS analysis of EtG and EtS. Most deviations were attributable to uncertainty in quantitation, when the value was close to a cutoff but the respective results were slightly above and below, or vice versa, the critical limit. However, for direct‐injection LC/MS/MS, despite earning 4 identification points, equally many negative results were due to a product ion ratio outside the ±20% deviation accepted by the guidelines. These results indicate that the likelihood of different analytical methods to provide reliable analytical results depends on the reporting limit applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of sub‐ppb epichlorohydrin levels in water by on‐line solid‐phase extraction liquid chromatography/tandem mass spectrometry

A new sensitive and selective method based on on‐line solid‐phase extraction (SPE) coupled to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) using a triple quadrupole mass spectrometer has been developed for the determination of epichlorohydrin (ECH) in different types of water samples. ECH is not easily determined directly by ESI‐MS as it is not readily ionized, and it has a low molecular mass and high polarity. Thus, prior derivatization of ECH was necessary, employing 3,5‐difluorobenzylamine as a derivatizing agent with Fe(III) as a catalyst. In order to achieve accurate quantification, correcting for matrix effects, losses in the derivatization process and instrumental deviations, isotope labelled ECH (ECH‐d₅) was added as an internal standard (IS) to the water samples. The method was validated based on European SANCO guidelines using drinking and other types of treated water spiked at two concentration levels (0.1 and 1.0 µg/L), the lower level having been established as the limit of quantification (LOQ) of the method. Satisfactory accuracy (recoveries between 70 and 103%), precision (RSD <20%) and linearity (from 0.05 to 50 µg/L, r >0.99) were obtained. The limit of detection (LOD) was set up at 0.03 µg/L. The method was applied to different water samples (drinking water and water samples collected from a municipal treatment water plant). In order to enhance confidence, five selected reaction monitoring (SRM) transitions were acquired, thus obtaining a simultaneous reliable quantification and identification of ECH in water, even at sub‐ppb levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of isomers of oleuropein aglycon in olive oils by rapid‐resolution liquid chromatography coupled to electrospray time‐of‐flight and ion trap tandem mass spectrometry

In this work, rapid‐resolution liquid chromatography (RRLC) coupled to electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS) and ion trap multiple mass spectrometry (IT‐MSⁿ) has been applied to separate and characterize eleven isomers of oleuropein aglycon in fourteen Spanish extra‐virgin olive oils. After the extra‐virgin olive oil sample had been dissolved in hexane and cleaned up by a diol‐bonded phase solid‐phase extraction (SPE) cartridge, the eluting extract was resolved in methanol and analyzed on an Angilent 1200 system with a 4.6 × 150 mm, 1.8 µm Zorbax Eclipse plus C18 column. Mass spectrometry was carried out on a Bruker Daltonics microTOF mass spectrometer and a Bruker Daltonics ion trap mass spectrometer. The characterization of isomers of oleuropein aglycon was based on accurate mass data and the isotope function of characteristic fragment ions in the studied compounds by TOF‐MS, and the fragment ions were further confirmed by IT‐MSⁿ. The fragmentation pathway of oleuropein aglycon was successfully elucidated and all possible transformations among isomers of oleuropein aglycon were suggested. Copyright © 2008 John Wiley & Sons, Ltd.     

Analytical study on ethephon residue determination in water by ion-pairing liquid chromatography/tandem mass spectrometry

A detailed analytical study on ethephon residue determination in water, making use of ion-pairing liquid chromatography coupled to electrospray tandem mass spectrometry (LC/MS/MS), has been carried out. Ethephon is a plant growth regulator, highly polar, which is typically present in aqueous solution in anionic form due to its acid character. Both its extraction and pre-concentration from water samples and its chromatographic retention are difficult. Several approaches for sample pretreatment have been tested including direct injection into the chromatographic system, on-line solid phase extraction (SPE) and off-line SPE, with the best results being obtained after off-line SPE, using Oasis MAX cartridges (mixed-mode strong anion-exchange). After testing several ion-pairing reagents, tetrabuthylammonium acetate (TBA) was selected. This was added to the samples before LC/MS/MS analysis to facilitate ethephon chromatographic retention. The acquisition of several specific MS/MS transitions together with the evaluation of their relative intensity ratios allowed the reliable confirmation of the analyte in samples. The optimised approach was tested in low-salinity water spiked at 0.1?µg?L^?1 level with satisfactory recovery, and a limit of detection of 0.02?µg?L^?1. To this purpose, the water sample was partially de-ionised in an initial stage, in order to remove major ions that would have interfered in analyses. The application of this methodology to more saline/complex water samples, as surface or wastewater, was problematic and a thorough optimisation of the de-ionisation conditions would be required.     

Determination of urine melamine by validated isotopic ultra‐performance liquid chromatography/tandem mass spectrometry

Little is known about melamine (MEL) analysis in children's urine. In this study, an isotopic ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and systematically validated for the analysis of MEL in urine. The method is easily performed and comprises acidification, solid‐phase extraction (SPE) and UPLC/MS/MS analysis. ¹³C₃N₃(¹⁵NH₂)₃ was used as the internal standard (IS) for calibration. Transition ions m/z 127 > 85 of MEL and m/z 133 > 89 of the IS were used for quantification and m/z 127 > 68 of MEL was used for quantitative confirmation. Recovery and precision were assessed to guarantee the applicability of the method. The limit of quantification (LOQ) was 0.01 µg/mL while the calculated method detection limit was 0.006 µg/mL. The mean recoveries ranged from 96–99%. The method was then applied to analyze urine samples from children who had potentially consumed MEL‐tainted dairy products during screening in Taiwan. Ten nephrolithiasis cases and 20 age‐ and gender‐matched controls were selected for this study. Three out of the 10 nephrolithiasis cases had elevated levels of MEL. Comparatively, twenty age‐ and gender‐matched non‐nephrolithiasis controls consuming Taiwan brand milk powder all showed MEL levels lower than the detection limit except for two children with background levels of 0.02 µg/mL. The background level in these children urine samples was established by UPLC/MS/MS analysis. Positive results of urine MEL tests might be associated with nephrolithiasis in these candidates. Measurement of urine MEL concentration can be helpful in confirming MEL‐related nephrolithiasis, but its clinical application needs further clarification. Copyright © 2009 John Wiley & Sons, Ltd.     

An analytical strategy for identification of a somatotropin‐like bioactive peptide by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry after immuno‐affinity purification from buffalo serum

The use of growth hormones, such as native and recombinant somatotropins, is forbidden in the European Union (EU), but is legal in the USA. The misuse of recombinant bovine somatotropin in Italy is suspected for enhancing milk production, thanks to its availability on the illegal market. A synthetic bioactive peptide of 27 amino acids derived from bovine somatotropin was successfully tested in France and in southern Italy for scientific purposes, to stimulate milk production, both in cows and buffaloes. This somatotropin‐like peptide (PEP‐ST), suspected for illegal use in southern Italy, was synthesized by linking the 104–113 sequence of bovine somatotropin to the 323–339 sequence of ovalbumin. Herein, a method for detection and identification of the PEP‐ST in buffalo serum is described; our strategy was based on the production of IgG anti‐PEP‐ST, used to synthesize an immuno‐affinity column for peptide purification from buffalo serum, prior to analysis by ion trap liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS). The immuno‐affinity column was successfully used to purify in a single step the bioactive PEP‐ST from buffalo serum samples spiked at 20, 50 and 200 µg/mL for confirmatory analysis. Ion trap LC/ESI‐MS/MS identification was based on detection of a multi‐charged molecular ion and its characteristic fragmentation pattern. No significant matrix interference was observed, accounting for method specificity. We consider this strategy to be a basic approach that could be improved in the perspective of the official control of illegal use of somatotropin and somatotropin‐like compounds in buffalo breeding. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of metabolites in rat plasma after intravenous administration of salvianolic acid A by liquid chromatography/time‐of‐flight mass spectrometry and liquid chromatography/ion trap mass spectrometry

Salvianolic acid A (SalA) is one of the main active constituents in Salvia miltiorrhiza (Danshen). Although the pharmacokinetics of SalA in rats after intravenous (i.v.) administration of Danshen injection has been reported, the information relevant to the metabolites of SalA in vivo is absent so far. In this study, by means of liquid chromatography with time‐of‐flight mass spectrometry (LC/TOFMS) and liquid chromatography with ion trap mass spectrometry (LC/MSⁿ) techniques, the unknown metabolites of SalA in rat plasma after i.v. administration of the purified SalA at the dose of 20 mg/kg body weight were identified. A liquid‐liquid extraction method was established to separate the metabolites from the plasma and the chromatographic separations were performed on a Xterra MS C₁₈ column (100 mm × 4.6 mm i.d., 3.5 µm) with acetonitrile/methanol/water/formic acid (20.5:19.5:64: 0.05, v/v/v/v) as the mobile phase at a constant flow rate of 0.2 mL/min. Based on the data obtained from the LC/TOFMS determination (the total ion chromatograms, MS spectra and extracted ion chromatograms), in combination with the characteristic fragment ions acquired from the LC/MSⁿ determination, five metabolites were identified as SalA‐monoglucuronide, monomethyl‐SalA‐monoglucuronide, mono‐methyl‐SalA, dimethyl‐SalA and dimethyl‐SalA‐monoglucuronide, and the possible chemical structures were deduced. The results indicated that SalA might mainly undergo two metabolic pathways in vivo in rats, which were methylation and glucuronidation. The present studies have laid a solid foundation for the metabolic mechanism of SalA in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification and structural characterization of in vivo metabolites of ketorolac using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS)

In vivo metabolites of ketorolac (KTC) have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI‐HR‐MS/MS) in combination with online hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, blood urine and feces samples were collected after oral administration of KTC to Sprague–Dawley rats. The samples were prepared using an optimized sample preparation approach involving protein precipitation and freeze liquid separation followed by solid‐phase extraction and then subjected to LC/HR‐MS/MS analysis. A total of 12 metabolites have been identified in urine samples including hydroxy and glucuronide metabolites, which are also observed in plasma samples. In feces, only O‐sulfate metabolite and unchanged KTC are observed. The structures of metabolites were elucidated using LC‐MS/MS and MSⁿ experiments combined with accurate mass measurements. Online HDX experiments have been used to support the structural characterization of drug metabolites. The main phase I metabolites of KTC are hydroxylated and decarbonylated metabolites, which undergo subsequent phase II glucuronidation pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Fast analysis of isobaric grape anthocyanins by Chip‐liquid chromatography/mass spectrometry

In a previous work, direct‐infusion electrospray ionization ion trap tandem mass spectrometry (ESI‐IT‐MS/MS) was applied to the study of anthocyanins in extracts from the skins of Clinton grapes, a non‐Vitis vinifera red grape variety qualitatively and quantitatively rich in anthocyanins. A good characterization of anthocyaninins was obtained, but it was impossible to differentiate some compounds with the same nominal mass but with different elemental composition. In this work, the capabilities of quadrupole time‐of‐flight mass spectrometry (QTOF‐MS) coupled with Chip‐liquid chromatography (LC‐Chip) were applied to the study of Clinton anthocyanins and this method provided the complete sample anthocyanin fingerprint in less than 5 min. Multi‐stage mass spectrometry (MSⁿ; n >2) was not necessary to identify isobaric compounds, nor were deuterium‐exchange experiments necessary to distinguish between compounds containing the same aglycone. The fast separation bypasses the problem of petunidin‐3‐O‐(6‐O‐acetyl)monoglucoside and delphinidin‐3,5‐O‐diglucoside quantification, present in the direct‐infusion ESI‐ITMS approach, due to overlapping with matrix interferences. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization and quantification of 10‐hydroxycamptothecine in Camptotheca acuminate and its medicinal preparation by liquid chromatography–ion trap mass spectrometry

A rapid and sensitive method for the identification and quantification of 10‐hydroxycamptothecine (HCPT) in Camptotheca acuminata Decne is described. The HCPT standard solution was directly infused into the ion trap mass spectrometers (IT/MS) for collecting the MSⁿ spectra. The electrospray ionization (ESI) mass spectral fragmentation pathway of HCPT was proposed and the ESI‐MSⁿ fragmentation behavior of HCPT was deduced in detail. The major fragment ions of HCPT were confirmed by MSⁿ in both negative ion and positive ion mode. The possible main cleavage pathway of fragment ions was studied. Quantification of HCPT was assigned in negative‐ion mode at a product ion at m/z 363 → 319 by LC‐MS. The LC‐MS method was validated for linearity, sensitivity, accuracy and precision, and then used to determine the content of the HCPT. Lastly, the LC‐MS method was successfully applied to determine HCPT in real samples of Camptotheca acuminate Decne and its medicinal preparation in the first time. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2‐isoprostanes are a family of prostaglandin F2‐like compounds that are formed by free‐radical‐catalyzed peroxidation of arachidonic acid. Several F2‐isoprostanes, but in particular 8‐epi PGF_2α, are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8‐epi PGF_2α concentrations in human plasma, whole blood, erythrocytes and urine. 8‐epi PGF_2α‐d4, a stable isotope derivative of 8‐epi PGF_2α, was used as an internal standard (IS). A 50 µL sample was focused on‐column and separated on two 3 µm particle size SUPELCOSIL? ABZ+Plus HPLC columns (15 cm × 4.6 mm and 7.5 cm × 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor‐to‐product ion transitions m/z 353.4 → 193.1 (8‐epi PGF_2α), 357.4 → 197.1 (8‐epi PGF_2α‐d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Profiling of 19-norsteroid sulfoconjugates in human urine by liquid chromatography mass spectrometry

19-Nortestosterone (nandrolone) major metabolites in human urine are excreted as sulfoconjugated and glucuroconjugated forms. A sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in negative ESI mode was developed for direct quantification of 19-norandrosterone sulfate (19-NAS) and 19-noretiocholanolone sulfate (19-NES). For both sulfoconjugates, the [M−H]⁻ ion at m/z 355 and the fragment ion at m/z 97 were used as the precursor and product ions, respectively. The purification method involved a complete and rapid separation of sulfates and glucuronides in two extracts after loading the sample on a weak anion exchange solid phase extraction support (SPE Oasis^® WAX). Then, sulfates were separated by LC (Uptisphere^® ODB, 150 mm × 3.0 mm, 5 μm) and analyzed on a linear trap and a triple quadrupole mass spectrometer. The lower limit of detection (LLOD) and lowest limit of quantification (LLOQ) were of 100 pg mL⁻¹ and 1 ng mL⁻¹, respectively. Assay validation demonstrated good performances in terms of trueness (92.0-104.9%), repeatability (0.6-7.2%) and intermediate precision (1.3-10.8%) over the range of 1-2500 ng mL⁻¹. Finally, 19-NAS and 19-NES in urine samples collected after intake of 19-norandrostenedione (nandrolone precursor) were quantified. This assay may be easily implemented to separate glucuronide and sulfate steroids from urine specimens prior to quantification by LC/MS/MS.     

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine

In equine sport, theobromine is prohibited with a threshold level of 2 µg mL^?1 in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time‐of‐flight mass spectrometry (TOFMS). Particle‐free diluted urine samples were directly injected into the LC/MS systems, avoiding the time‐consuming extraction step. 3‐Propylxanthine was used as the internal standard. The tested linear range was 0.75–15 µg mL^?1. Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra‐ and inter‐day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of cylindrospermopsin in freshwaters and fish tissue by liquid chromatography coupled to electrospray ion trap mass spectrometry

Cylindrospermopsin (CYN) is a toxic alkaloid‐like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI‐MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI‐MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in southern Italy. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterisation of oxazepam degradation products by high‐performance liquid chromatography/electrospray ionisation mass spectrometry and electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry

Oxazepam has been subjected to controlled degradation at 100°C for 3 h in 0.5 M HCl and 0.5 M NaOH. Following neutralisation of the degradation mixture and removal of salts by solid‐phase extraction (SPE), isocratic high‐performance liquid chromatography/mass spectrometry (HPLC/MS) using water/methanol (25:75 v/v) as the mobile phase was carried out using a flow diverter to collect fractions prior to their characterisation by electrospray ionisation multi‐stage mass spectrometry (ESI‐MSⁿ) and proposal of the corresponding fragmentation patterns. The elemental compositions of the degradation products and their MS fragments were evaluated using electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) which was then used to support the proposed fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.