Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe⁺), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe⁺ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled ¹³CD₃⁸²SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe⁺, the standard addition method was applied. Quality control for the accurate quantitation of TMSe⁺ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L⁻¹. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L⁻¹ for TMSe⁺, 5.6 μg Se L⁻¹ for SeMet, and 0.1 μg Se L⁻¹ for both SeGalNAc and SeGluNAc.     

Sensitive liquid chromatography–tandem mass spectrometry method for the determination of pantoprazole sodium in human urine

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed to determine pantoprazole sodium (PNT) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C₁₈ column (symmetry 3.5 μm; 75 mm × 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (90:10, v/v). The method was linear over a concentration range of 1–100 ng mL^?1. The lower limit of quantitation was 1 ng mL^?1. The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was <10.5%. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ng mL^?1 PNT) was within ±1.25% in terms of relative errors.     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Quantitative determination of taurine and related biomarkers in urine by liquid chromatography–tandem mass spectrometry

Current urinary bladder cancer diagnosis is commonly based on a biopsy obtained during cystoscopy. This invasive method causes discomfort and pain in patients. Recently, taurine and several other compounds such as L-phenylalanine and hippuric acid in urine were found to be indicators of bladder cancer. However, because of a lack of sensitive and accurate analytical techniques, it is impossible to detect these compounds in urine at low levels. In this study, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a noninvasive method was developed to separate and detect these compounds in urine. ¹⁵N₂-L-glutamine was used as the internal standard, and creatinine acted as an indicator for urine dilution. A phenyl-hexyl column was used for the separation at an isocratic condition of 0.2% formic acid in water and 0.2% formic acid in methanol. Analytes were detected in multiple-reaction monitoring with positive ionization mode. The limit of detection range is 0.18–6 nM and the limit of quantitation ranges from 0.6 to 17.6 nM. The parameters affecting separation and quantification were also investigated and optimized. Proper clinical validation of these biomarkers can be done using this reliable, fast, and simple method. Furthermore, with simple modifications, this method could be applied to other physiological fluids and other types of diseases.     

Determination of evodiamine and rutecarpine in human serum by liquid chromatography–tandem mass spectrometry

Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography–tandem mass spectrometric method (LC–MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol–water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2–1040 ng/mL and 10.2–1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18–4.00% and 2.99–5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI–MS, ESI–MS/MS, APCI–MS and APCI–MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H]⁺, m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.      

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.     

Detection of catecholamine metabolites in urine based on ultra-high-performance liquid chromatography–tandem mass spectrometry

The excretion of neurotransmitter metabolites in normal individuals is of great significance for health monitoring. A rapid quantitative method was developed with ultra-performance liquid chromatography–tandem mass spectrometry. The method was further applied to determine catecholamine metabolites vanilymandelic acid (VMA), methoxy hydroxyphenyl glycol (MHPG), dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) in the urine. The urine was collected from six healthy volunteers (20–22 years old) for 10 consecutive days. It was precolumn derivatized with dansyl chloride. Subsequently, the sample was analyzed using triple quadrupole mass spectrometry with an electrospray ion in positive and multireaction monitoring modes. The method was sensitive and repeatable with the recoveries 92.7–104.30%, limits of detection (LODs) 0.01–0.05 μg/mL, and coefficients no less than 0.9938. The excretion content of four target compounds in random urine samples was 0.20 ± 0.086 μg/mL (MHPG), 1.27 ± 1.24 μg/mL (VMA), 3.29 ± 1.36 μg/mL (HVA), and 1.13 ± 1.07 μg/mL (DOPAC). In the urine, the content of VMA, the metabolite of norepinephrine and adrenaline, was more than MHPG, and the content of HVA, the metabolite of dopamine, was more than DOPAC. This paper detected the levels of catecholamine metabolites and summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.     

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and ¹H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.     

Determination of steryl sulphates in invertebrate tissue by liquid chromatography–tandem mass spectrometry

A method for the identification and quantification of underivatised steryl sulphates in invertebrates by liquid chromatography (LC) coupled with tandem mass spectrometry (MS) involving a single cleanup step has been developed. Negative electrospray ionisation and positive and negative atmospheric-pressure chemical ionisation (APCI) spectra of steryl sulphate showed pseudomolecular ions ([M+H–H₂SO₄]⁺or [M–H]⁻). Collision-induced dissociation (CID) was efficient only in positive APCI. LC-MS in negative APCI was least susceptible to interference and possible differences in response factors. The detection limits (signal-to-noise ratio of 3) based on cholest-5-enyl-3-sulphate in positive and negative APCI modes are 3.66 and 0.73 pmol μL⁻¹, respectively. Calibration plots and response factors for cholest-5-enyl-3-sulphate relative to the internal standard, cholecalciferyl-3-sulphate, in both positive and negative polarities, were linear in the concentration range from 1.22 to 16.4 pmol μL⁻¹ with good coefficients of determination (R ²>0.98). It is suggested that the structure elucidation of steryl sulphates is best achieved in CID positive APCI mode, whereas their quantification should be carried out using negative APCI.     

Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry

A specific and sensitive multi-method based on liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization (LC–APCI–MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α/β-nortestosterone, methyltestosterone, β-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, α/β-zearalenol, α/β-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC–MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCα ranged from 0.03 to 0.14 ng/g while the detection capabilities CCβ ranged from 0.05 to 0.24 ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.     

Quantification of clomiphene metabolite isomers in human plasma by rapid-resolution liquid chromatography–electrospray ionization–tandem mass spectrometry

Since the 1960s, clomiphene citrate is used for ovulation induction. Since nonresponse to clomiphene therapy is still not well understood, interindividual variability of clomiphene metabolism has been considered to be a plausible explanation. Therefore, a comprehensive, rapid, sensitive, and specific analytical method for the quantification of (E)- and (Z)-isomers of clomiphene and their putative N-desethyl, N,N-didesethyl, 4-hydroxy, and 4-hydroxy-N-desethyl metabolites, and the N-oxides in human plasma has been newly developed, using HPLC-tandem mass spectrometry and stable isotope-labeled internal standards. All standards other than the parent drug were synthesized in our laboratory. Following protein precipitation analytes were separated on a ZORBAX Eclipse plus C18 1.8 μm column with a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile and detected on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Lower limit of quantification for metabolites ranged from 0.06 ng/mL for clomiphene-N-oxides to 0.3 ng/mL for (E)-N-desethylclomiphene. The assay was validated according to FDA guidelines. Plasma levels of clomiphene and its metabolites were measured in two women after single-dose treatment with clomiphene.     

A method for the quantification of biomarkers of exposure to acrylonitrile and 1,3-butadiene in human urine by column-switching liquid chromatography–tandem mass spectrometry

1,3-Butadiene and acrylonitrile are important industrial chemicals that have a high production volume and are ubiquitous environmental pollutants. The urinary mercapturic acids of 1,3-butadiene and acrylonitrile—N-acetyl-S-(3,4-dihydroxybutyl)cysteine (DHBMA) and MHBMA (an isomeric mixture of N-acetyl-S-((1-hydroxymethyl)-2-propenyl)cysteine and N-acetyl-S-((2-hydroxymethyl)-3-propenyl)cysteine) for the former and N-acetyl-S-2-cyanoethylcysteine (CEMA) for the latter—are specific biomarkers for the determination of individual internal exposure to these chemicals. We have developed and validated a fast, specific, and very sensitive method for the simultaneous determination of DHBMA, MHBMA, and CEMA in human urine using an automated multidimensional LC/MS/MS method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column, and subsequently determined by tandem mass spectrometry using labeled internal standards. The limits of quantification (LOQs) for DHBMA, MHBMA, and CEMA were 10 μg/L, 2 μg/L, and 1 μg/L urine, respectively, and were sufficient to quantify the background exposure of the general population. Precision within series and between series for all analytes ranged from 5.4 to 9.9%; mean accuracy was between 95 and 115%. We applied the method on spot urine samples from 210 subjects from the general population with no occupational exposure to 1,3-butadiene or acrylonitrile. A background exposure of the general population to acrylonitrile was discovered that is basically influenced by individual exposure to passive smoke as well as active smoking habits. Smokers showed a significantly higher excretion of MHBMA, whereas DHBMA levels did not differ significantly. Owing to its automation, our method is well suited for application in occupational or environmental studies. Figure Boxplots of the results from LC/ESI-MS/MS analysis of urinary excretion of CEMA reveal a strong correlation with nicotine metabolite cotinine, indicating that exposure to passive smoke as well as active smoking is the main source of exposure to acrylonitrile in the general population     

High-performance liquid chromatography–tandem mass spectrometry method for quantifying sulfonylurea herbicides in human urine: reconsidering the validation process

We have developed a method for measuring 17 sulfonylurea (SU) herbicides in human urine. Urine samples were extracted using solid phase extraction (SPE), preconcentrated, and analyzed by high-performance liquid chromatography–tandem mass spectrometry using turboionspray atmospheric pressure ionization. Carbon 13-labeled ethametsulfuron methyl was used as an internal standard. Chromatographic retention times were under 7 minutes. Total throughput was estimated as >100 samples per day. Because only one labeled internal standard was available for the analysis, we were forced to reconsider and restructure the validation process to include stringent stability tests and analyses of urine matrices of differing compositions. We describe our restructured validation process and the critical evaluation it provides for the method developed. The limits of detection (LOD) ranged from 0.05 μg/L to 0.10 μg/L with an average LOD of 0.06 μg/L. Average total relative standard deviations were 17%, 12% and 8% at 0.1 μg/L, 3.0 μg/L and 10 μg/L, respectively. Average extraction efficiencies of the SPE cartridges were 87% and 86% at 2.5 μg/L and 25 μg/L, respectively. Chemical degradation in acetonitrile and urine was monitored over 250 days. Estimated days for 10% and 50% degradation in urine and acetonitrile ranged from 0.7 days to >318 days. The influence of matrix effects on precision and accuracy was also explored. Electronic Supplementary Material Supplementary material is available for this article at For additional information, contact Anderson Olsson at     

Quantification of atrazine and its metabolites in urine by on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry

We have developed a method using on-line solid-phase extraction–high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) and isotope dilution quantification to measure atrazine and seven atrazine metabolites in urine. The metabolites measured were hydroxyatrazine, diaminochloroatrazine, desisopropylatrazine, desethylatrazine, desethylatrazine mercapturate, atrazine mercaturate and atrazine itself. Our method has good precision (relative standard deviations ranging from 4 to 20% at 5, 10 and 50 ng/mL), extraction efficiencies of 67 to 102% at 5 and 25 ng/mL, relative recoveries of 87 to 112% at 5, 25, 50 and 100 ng/mL limits of detection (LOD) ranging from 0.03 to 2.80 ng/mL. The linear range of our method spans from the analyte LOD to 100 ng/mL (40 ng/mL for atrazine and atrazine mercapturate) with R ² values of greater than 0.999 and errors about the slope of less than 3%. Our method is rapid, cost-effective and suitable for large-scale sample analyses and is easily adaptable to other biological matrices. More importantly, this method will allow us to better assess human exposure to atrazine-related chemicals. Figure A schematic representation showing the elution of the analytes from the solid-phase extraction cartridge onto the analytical column for chromatographic separation prior to MS/MS analysis     

Doping control analysis of recombinant human erythropoietin, darbepoetin alfa and methoxy polyethylene glycol-epoetin beta in equine plasma by nano-liquid chromatography–tandem mass spectrometry

Recombinant human erythropoietin (rhEPO), darbepoetin alfa (DPO) and methoxy polyethylene glycol-epoetin beta (PEG-EPO) are synthetic analogues of the endogenous hormone erythropoietin (EPO). These erythropoiesis-stimulating agents have the ability to stimulate the production of red blood cells and are commercially available for the treatment of anaemia in humans. These drugs are understood to have performance-enhancing effects on human athletes due to their stimulation of red blood cell production, thereby improving delivery of oxygen to the muscle tissues. Although their effect on horses has not been proven, these substances were thought to be similarly performance enhancing and have indeed been applied covertly to horses. As such, these protein-based drugs are prohibited by authorities in both human and equine sports. The method officially adopted by the International Olympic Committee (IOC) and World Anti Doping Agency (WADA) for the confirmation of rhEPO and/or DPO (rhEPO/DPO) in human urine is based on electrophoresis in combination with Western blotting. A shortcoming of the WADA method is the lack of definitive mass spectral data for the confirmation of a positive finding. Recently, a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the detection and confirmation of rhEPO/DPO in equine plasma was reported. However, we have not been successful in achieving the reported sensitivity. This paper presents a method for the detection and confirmation of rhEPO/DPO, as well as the newly released PEG-EPO, in equine plasma. The procedures involve immunoaffinity extraction using anti-rhEPO antibody-coated Dynabeads followed by trypsin digestion. The injected extract was further purified and concentrated using an on-line trap column in the nano-LC system. Detection and confirmation were achieved by monitoring a unique peptide segment of rhEPO/DPO/PEG-EPO using nano-liquid chromatography–tandem mass spectrometry equipped with a nanospray ionisation source operated in the selected reaction monitoring mode. rhEPO, DPO and PEG-EPO can be confirmed at 0.1, 0.2 and 1.0 ng/mL, respectively, in equine plasma.     

Comparison of supercritical fluid chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco

This study used reversed-phase liquid chromatography–tandem mass spectrometry and supercritical fluid chromatography–tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10–500 ng/mL showed excellent linearity with R² ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography–tandem mass spectrometry (86.1–95.7%) as well as supercritical fluid chromatography–tandem mass spectrometry (86.5–94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography–tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography–tandem mass spectrometry.     

Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry

A method for the simultaneous analysis of nucleosides and nucleotides in infant formula using reversed-phase liquid chromatography–tandem mass spectrometry is described. This approach is advantageous for compliance testing of infant formula over other LC-MS methods in which only nucleotides or nucleosides are measured. Following sample dissolution, protein was removed by centrifugal ultrafiltration. Chromatographic analyses were performed using a C₁₈ stationary phase and gradient elution of an ammonium acetate/bicarbonate buffer, mass spectrometric detection and quantitation by a stable isotope-labelled internal standard technique. A single laboratory validation was performed, with spike recoveries of 80.1–112.9 % and repeatability relative standard deviations of 1.9–7.2 %. Accuracy as bias was demonstrated against reference values for NIST1849a certified reference material. The method has been validated for the analysis of bovine milk-based, soy-based, caprine milk-based and hydrolysed milk protein-based infant formulae.

Determination of cyclopiazonic acid in food and feeds by liquid chromatography–tandem mass spectrometry

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol–water mixture, defatting with hexane and quantification using HPLC–MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 μg/kg while the limit of quantification was 20 μg/kg in the matrices investigated. The detector response was found to be linear over the range 25–250 μg/kg in feed and 25–500 μg/kg in wheat, peanuts and rice. The mean overall recoveries (n = 18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n = 3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.