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.

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.     

Simultaneous quantification of candesartan and irbesartan in rabbit eye tissues by liquid chromatography–tandem mass spectrometry

Diabetic retinopathy is a major cause of vision loss in adults. Novel eye-drop formulations of candesartan and irbesartan are being developed for its cure or treatment. To support a preclinical trial in rabbits, it was critical to develop and validate a new LC–MS/MS method for simultaneous quantification of candesartan and irbesartan in rabbit eye tissues (cornea, aqueous humor, vitreous body and retina/choroid). Eye tissue samples were first homogenized in H₂O-diluted rabbit plasma. The candesartan and irbesartan in the supernatants together with their respective internal standards (candesartan-d₄ and irbesartan-d₄) were extracted by solid-phase extraction. The extracted samples were injected onto a C₁₈ column for gradient separation. The MS detection was in the positive electrospray ionization mode using the multiple reaction monitoring transitions of m/z 441 → 263, 445 → 267, 429 → 207, and 433 → 211 for candesartan, candesartan-d₄, irbesartan and irbesartan-d₄, respectively. For the validated concentration ranges (2–2000 and 5–5000 ng/g for candesartan and irbesartan, respectively), the within-run and between-run accuracies (% bias) were within the range of −8.0–10.0. The percentage CV ranged from 0.6 to 7.3. There was no significant matrix interference nor matrix effect from different eye tissues and different rabbits. The validated method was successfully used in the Good Laboratory Practice (GLP) study of rabbits.     

Streamlining sample preparation and gas chromatography–tandem mass spectrometry analysis of multiple pesticide residues in tea

In this work, a new rapid method for the determination of 135 pesticide residues in green and black dry tea leaves and stalks employing gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) with a triple quadrupole was developed and validated. A substantial simplification of sample processing prior to the quantification step was achieved: after addition of water to a homogenised sample, transfer of analytes into an acetonitrile layer was aided by the addition of inorganic salts. Bulk co-extracts, contained in the crude organic extract obtained by partition, were subsequently removed by liquid–liquid extraction using hexane with the assistance of added 20% (w/w) aqueous NaCl solution. The importance of matrix hydration prior to the extraction for achieving good recoveries was demonstrated on tea samples with incurred pesticide residues. For most of the analytes, recoveries in the acceptable range of 70–120% and repeatabilities (relative standard deviations, RSDs) ≤20% were achieved for both matrices at spiking levels of 0.01, 0.1 and 1 mg kg⁻¹. Under optimised GC–MS/MS conditions, most of the analytes gave lowest calibration level ≤0.01 mg kg⁻¹, permitting the control at the maximum residue levels (MRLs) laid down in Regulation (EC) No 396/2005. The developed method was successfully applied to the determination of pesticide residues in real tea samples.     

The determination of β-agonist residues in bovine tissues using liquid chromatography–tandem mass spectrometry

We aimed to develop a rapid, simple and reproducible method based on LC–tandem mass spectrometry (LC–MS/MS) to analyze β-agonist residues (clenbuterol, zilpaterol, ractopamine and isoxsuprine) in bovine tissues. The method was validated in accordance with the European Council Decision 2002/657/EC. The samples were homogenized, and then 10 mL of an acetate buffer was added to a 5-g sample. The sample was then centrifuged at 12,000 rpm and filtered. Sodium hydroxide (2 m ) was added to adjust pH of the sample that was centrifuged again. The extract was filtered through a solid-phase extraction column. The residue was re-dissolved in 250 μL acetonitrile and then subjected to LC–MS/MS. The separation was done on a C₁₈ column. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in methanol. The mean recoveries of β-agonists were in the range of 84.3%–119.1% with relative standard deviations (%RSDs) of 0.683%–4.05%. Decision limits and detection capabilities of the analytes ranged from 0.0960 to 4.9349 μg/kg and from 0.0983 to 5.0715, respectively. This method was used to detect four β-agonists in 100 bovine muscle, 100 liver and 100 kidney tissues from a slaughterhouse. No residue was found above the maximum residue limit level.     

Determination of biocides in different environmental matrices by use of ultra-high-performance liquid chromatography–tandem mass spectrometry

A sensitive and robust method using solid-phase extraction and ultrasonic extraction for preconcentration followed by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS–MS) has been developed for determination of 19 biocides: eight azole fungicides (climbazole, clotrimazole, ketoconazole, miconazole, fluconazole, itraconazole, thiabendazole, and carbendazim), two insect repellents (N,N-diethyl-3-methylbenzamide (DEET), and icaridin (also known as picaridin)), three isothiazolinone antifouling agents (1,2-benzisothiazolinone (BIT), 2-n-octyl-4-isothiazolinone (OIT), and 4,5-dichloro-2-n-octyl-isothiazolinone (DCOIT)), four paraben preservatives (methylparaben, ethylparaben, propylparaben, and butylparaben), and two disinfectants (triclosan and triclocarban) in surface water, wastewater, sediment, sludge, and soil. Recovery of the target compounds from surface water, influent, effluent, sediment, sludge, and soil was mostly in the range 70–120?%, with corresponding method quantification limits ranging from 0.01 to 0.31?ng?L^?1, 0.07 to 7.48?ng?L^?1, 0.01 to 3.90?ng?L^?1, 0.01 to 0.45?ng?g^?1, 0.01 to 6.37?ng?g^?1, and 0.01 to 0.73?ng?g^?1, respectively. Carbendazim, climbazole, clotrimazole, methylparaben, miconazole, triclocarban, and triclosan were detected at low ng?L^?1 (or ng?g^?1) levels in surface water, sediment, and sludge-amended soil. Fifteen target compounds were found in influent samples, at concentrations ranging between 0.4 (thiabendazole) and 372?ng?L^?1 (methylparaben). Fifteen target compounds were found in effluent samples, at concentrations ranging between 0.4 (thiabendazole) and 114?ng?L^?1 (carbendazim). Ten target compounds were found in dewatered sludge samples, at concentrations ranging between 1.1 (DEET) and 887?ng?g^?1 (triclocarban).     

Determination of selected pesticides in environmental water by employing liquid-phase microextraction and liquid chromatography–tandem mass spectrometry

An optimised extraction and cleanup method for the analysis of pesticide in natural water samples is presented. Sixteen pesticides of different polarity and from the different chemical classes (organophosphates, triazines, benzimidazoles, carbamates, carbamides, neonicotinoides, methylureas, phenylureas and benzohydrazides), most frequently used in Serbia, were selected for the analysis. Liquid-phase microextraction in a single hollow fibre (HF-LPME) has been applied for sample preparation. The concentrations of pesticides were determined using HPLC-MS/MS method with electrospray ionisation. The extraction behaviour and selection of the experimental conditions was predicted based on log D and pK _a values of targeted pesticides, which were calculated applying the computer software ACD/Labs PhysChem Suite v12. The influence of the donor pH and concentration of pesticides, organic phase composition as well as the extraction time on the extraction efficiency was investigated. Optimum extraction conditions were evaluated with respect to the investigated parameters of the extraction. The extraction method was validated for 10 out of 16 studied pesticides. Linear range of the pesticides was 0.1–5 μg L⁻¹ with the correlation coefficient from 0.991 to 0.9998, and the relative standard deviation for three standard measurements was between 0.2 and 11.8%. The limits of detections ranged from 0.026 to 0.237 μg L⁻¹ and the limits of quantifications from 0.094 to 0.793 μg L⁻¹. The optimised two-phase HF-LPME method was successfully applied for determination of moderately polar as well low-polar pesticides in the environmental water samples.     

Sensitivity comparison of gas chromatography–mass spectrometry with Cold EI and standard EI

Gas chromatography–mass spectrometry (GC–MS) with Cold EI is based on interfacing GC and MS with supersonic molecular beams (SMBs) along with electron ionization of vibrationally cold sample compounds in SMB in a fly-through ion source (hence the name Cold EI). Cold EI improves all the central performance aspects of GC–MS, and in this paper, we focus on its improvement of signal-to-noise ratio (S/N) and limits of detection (LODs). We found that the harder the compound for analysis with standard EI, the greater the Cold EI gain in S/N and LOD. The lower LOD and higher S/N of Cold EI emerge from a few reasons: (a) similar ionization yield as standard EI, (b) enhanced abundance of molecular ions, (c) elimination of vacuum background noise, (d) elimination of ion source-related peak tailing and degradation, (e) ability to lower the elution temperatures via the use of high column flow rates, and (f) greater range of thermally labile and low-volatility compounds that can be analyzed. We demonstrate the superior S/N and lower LOD of Cold EI versus standard EI in a range of compounds, from the simple-to-analyze octafluoronaphthalene all the way to reserpine and an organo-metallic compound that cannot be analyzed by standard EI. These compounds include methyl stearate, cholesterol, n-C₃₂H₆₆, large polycyclic aromatic hydrocarbons, dioctyl phthalates, diundecyl phthalate, pentachlorophenol, benzidine, lambda-cyhalothrin, and methidathion. The significantly lower Cold EI LODs that can be over 1000 times better than in standard EI further result in far superior response linearity and greater measurement dynamic range.     

Quantitative analysis of phenibut in rat brain tissue extracts by liquid chromatography–tandem mass spectrometry

Phenibut (3-phenyl-4-aminobutyric acid) is a γ-aminobutyric acid mimetic drug, which is used clinically as a mood elevator and tranquilizer. In the present work, a rapid, selective and sensitive liquid chromatography–tandem mass spectrometry method for quantification of phenibut in biological matrices has been developed. The method is based on protein precipitation with acidic acetonitrile followed by isocratic chromatographic separation using acetonitrile–formic acid (0.1% in water; 8:92, v/v) mobile phase on a reversed-phase column. Detection of the analyte was performed by electrospray ionization mass spectrometry in multiple reaction monitoring mode with the precursor-to-product ion transition m/z 180.3 → m/z 117.2. The calibration curve was linear over the concentration range 50–2000 ng/mL. The lower limit of quantification for phenibut in rat brain extracts was 50 ng/mL. Acceptable precision and accuracy were obtained over the whole concentration range. The validated method was successfully applied in a pharmacological study to analyze phenibut concentration in rat brain tissue extract samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Study of the microbiodegradation of terpenoid resin-based varnishes from easel painting using pyrolysis–gas chromatography–mass spectrometry and gas chromatography–mass spectrometry

The alterations produced by microbiological attack on terpenoid resin-based varnishes from panel and canvas paintings have been evaluated using pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) and gas chromatography–mass spectrometry (GC–MS). The proposed methods include the on-line derivatisation of drying oils and diterpenoid resins using hexamethyldisilazane during pyrolysis and the application of methyl chloroformate as a derivatisation reagent for triterpenoid resins in GC–MS. Two types of specimens, consisting of model oil medium prepared from linseed oil and model spirit varnishes prepared from colophony and mastic resins dissolved in turpentine, have been used as reference materials. For a series of specimens upon which different genera of bacteria and fungi were inoculated and encouraged to grow, analyses indicated that no mechanisms that commonly occur during the attack of enzymes on drying oils and terpenoid biodegraders were observed to occur in the oil medium and varnishes studied. Thus, the degradation pathways observed in the performed trials usually occur as consequence of natural ageing. Specific trials consisting of the application of biocides to uninoculated colophony varnish resulted in the identification of processes that produce undesirable degradation of the varnish due to interactions between the biocide and the varnish components. Finally, the studied biocides—Biotin, New-Des and Nipagine—generally exhibited good inhibiting effects on the microorganisms studied, although some interesting differences were found between them regarding the application method and type of biocide.     

Liquid chromatography–tandem mass spectrometry analysis of neonicotinoid pesticides and 6-chloronicotinic acid in environmental water with direct aqueous injection

An efficient, high throughput and cost-effective direct aqueous injection approach for the analysis of neonicotinoid pesticides and a common metabolite in environmental water has been described here. The method determines eight neonicotinoid pesticides (acetamiprid, clothianidin, dinotefuran, flonicamid, imidacloprid, nitenpyram, thiacloprid, thiamethoxam) and 6-chloronicotinic acid (a common metabolite of the first generation neonicotinoids, acetamiprid, imidacloprid, nitenpyram and thiacloprid) without any sample enrichment/cleanup steps. The method detection limits are 2–8 ng/L for the neonicotinoids and 93 ng/L for 6-chloronicotinic acid. The performance of the QTRAP^®5500 mass spectrometer was compared against a 4000QTRAP^®, and a QTRAP^®6500, to provide insights for future method transfer among different generations of instrumentations. Critical mass spectrometric parameters such as collision energy were quite consistent among the three instruments evaluated. However, increased chemical background levels for some target compounds on the more sensitive instruments were observed. The application of differential ion mobility spectrometry combined with tandem mass spectrometry was demonstrated to have great potential in reducing chemical background and/or isobaric interferences inherited in sample matrices. This ISO 17025 accredited method was employed to quantitate neonicotinoids in Ontario stream water samples. Good correlation for analytical results of this direct aqueous injection approach and a previously published solid phase extraction approach warrant high confidence in data quality.     

Group-specific fragmentation of pesticides and related compounds in liquid chromatography–tandem mass spectrometry

Current strategies in the LC–MS analysis of pesticides and related compounds in environmental samples, fruits and vegetables, and biological samples mostly rely on the selection of appropriate precursor/product-ion combinations (transitions) for selected reaction monitoring (SRM), often based on automated parameter optimization and selection of the transition. Such a procedure does not require any information on the type of fragmentation reaction involved in the generation of the product ion from the selected precursor ion. However, such information does become important in untargeted screening for unknown contaminants in environmental and food samples, which are generally based on a combination of high-resolution mass spectrometry and (multistage) tandem mass spectrometry. With this in mind, the group-specific fragmentation behaviour has been studied for six classes of pesticides and herbicides, i.e., triazines, organophosphorous pesticides, phenylurea herbicides, carbamates, sulfonylurea herbicides, and chlorinated phenoxy acid herbicides. When relevant, some comparison was made between fragmentation of protonated molecules in MS–MS and of molecular ions generated by electron ionization in GC–MS.     

Sampling of benzene in environmental and exhaled air by solid-phase microextraction and analysis by gas chromatography–mass spectrometry

Benzene is classified as a Group I carcinogen by the International Agency for Research on Cancer (IARC). The risk assessment for benzene can be performed by monitoring environmental and occupational air, as well as biological monitoring through biomarkers. The present work developed and validated methods for benzene analysis by GC/MS using SPME as the sampling technique for ambient air and breath. The results of the analysis of air in parks and avenues demonstrated a significant difference, with average values of 4.05 and 18.26 μg m⁻³, respectively, for benzene. Sampling of air in the occupational environment furnished an average of 3.41 and 39.81 μg m⁻³. Moreover, the correlations between ambient air and expired air showed a significant tendency to linearity (R ² = 0.850 and R ² = 0.879). The results obtained for two groups of employees (31.91 and 72.62 μg m⁻³) presented the same trend as that from the analysis of environmental air.     

Detailed study of polystyrene solubility using pyrolysis–gas chromatography–mass spectrometry and combination with size-exclusion chromatography

Measuring polymer solubility accurately and precisely is challenging. This is especially true at unfavourable solvent compositions, when only very small amounts of polymer dissolve. In this paper, pyrolysis–gas chromatography–mass spectrometry (Py-GC-MS) is demonstrated to be much more informative and sensitive than conventional methods, such as ultraviolet spectroscopy. By using a programmed-temperature-vapourisation injector as the pyrolysis chamber, we demonstrate that Py-GC-MS can cover up to five orders of magnitude in dissolved polymer concentrations. For polystyrene, a detection limit of 1 ng mL^?1 is attained. Dissolution in poor solvents is demonstrated to be discriminating in terms of the analyte molecular weight. Py-GC-MS additionally can yield information on polymer composition (e.g. in case of copolymers). In combination with size-exclusion chromatography, Py-GC-MS allows us to estimate the molecular weight distributions of minute amounts of a dissolved polymer and variations therein as a function of time.

Determination of melamine and cyanuric acid in powdered milk using injection-port derivatization and gas chromatography–tandem mass spectrometry with furan chemical ionization

A reliable, sensitive and eco-friendly injection-port trimethylsilylated (TMS) derivatization and gas chromatography–tandem mass spectrometry (GC–MS/MS) with furan chemical ionization (furan-CI) method was developed to determine melamine and cyanuric acid in powdered milk samples. The effects of several parameters related to the TMS-derivatization process (i.e., injection-port temperature, residence time and volume of silylating agent) and of various CI agents were investigated. Addition of a solution (3 μL) of bis(trimethyl)silyltrifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) reagent to a 20-μL extract from the powdered milk sample gave an excellent yield of the tris-TMS-derivatives of melamine and cyanuric acid at an injection-port temperature of 90 °C. Furthermore, using furan as the CI agent in conjunction with tandem mass spectrometry provided the greatest sensitivity and selectivity of detection. The limits of quantitation (LOQs) for melamine and cyanuric acid were 0.5 and 1.0 ng/g in 0.5-g of powdered milk samples, respectively. The recoveries from spiked samples – after simple ultra-sonication with 5% dimethyl sulfoxide in acetonitrile coupled with n-hexane liquid–liquid extraction – ranged from 72% to 93% with relative standard deviations of lower than or equal to 18%. In three of four real powdered milk samples, melamine was detected at concentrations ranging from 36 to 1460 ng/g; and cyanuric acid was detected in two of these samples at concentrations of 17 and 180 ng/g.     

Determination of multiclass pesticides in river sediments via matrix solid-phase dispersion extraction and gas chromatography–tandem mass spectrometry

A fast and environment-friendly analytical method was implemented to determine multiclass pesticides in river sediments. Twenty-three pesticides—organochlorine pesticides, organophosphorus pesticides, and triazines—were extracted via matrix solid-phase dispersion (MSPD) and analyzed by gas chromatography–tandem mass spectrometry (GC–MS/MS). Florisil demonstrated excellent analytes uptake capability as the extractant phase, with suitable selectivity for treating complex sediment samples. Under defined extraction conditions, the MSPD–GC–MS/MS method demonstrated robustness in the n inter-day analysis of sediments from different sources, providing limit of quantifications (LOQs) between 5 and 15 ng/g, linear responses in the range between LOQs and 150 ng/g, extraction recoveries of 71%–106%, and precision, assessed as relative standard deviation below 20%. The MSPD significantly reduced samples and solvents’ consumption, providing critical environmental gains compared to traditional extraction methods like Soxhlet. Finally, the method was applied to analyze sediment samples from three different collection areas of the Subachoque River (Cundinamarca, Colombia), demonstrating a fast, efficient, confident, and profitable analytical tool for pollution control and monitoring in environmental samples. The method allowed us to determine the current use in Colombia of banned pesticides under the 2001 Stockholm Convention.     

Metabolomic analysis of amino acids and organic acids in aging mouse eyes using gas chromatography–tandem mass spectrometry

This is a metabolomics study for monitoring altered amino acid (AA) and organic acid (OA) metabolism of in eyes from aging an mouse model at 8 and 18 weeks and 18 months. Simultaneous metabolic profiling analysis of OAs and AAs was performed as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–tandem mass spectrometry. A total of 42 metabolites—24 AAs and 18 OAs—were determined and their composition values were normalized to the corresponding mean values of 8-week-old mice as the control group. Then their normalized values were plotted as star graphs, which were distorted and readily distinguishable for each age-related group. Among the 42 metabolites, 18 AAs and 11 OAs were age dependent and significantly different (p < 0.05). Principal component analysis and partial least squares discriminant analysis showed unclear separation between 8- and 18-week-old mice but clear separation between these and 18-month-old mice. In particular, the variable importance in projection scores of 4-hydroxyproline, cis-aconitic acid, glycine, isocitric acid, leucine, pipecolic acid and lysine from partial least-squares–discriminant analysis were higher than 1.3. A heatmap for the classification and visualization of 42 metabolites showed differences in metabolite changes with aging. Altered AA and OA profiles were monitored, which may explain the metabolic disturbance of AA and OA. These findings are related to mitochondrial dysfunctions related to energy metabolism and the impaired antioxidant system in the aging eye. Therefore, the present metabolomics results of the association between physiological states and altered metabolism of AA and OA will be useful for understanding the aging eye and related diseases.