Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry

A new multiresidue method has been developed and validated for the simultaneous extraction of more than two hundred pesticides, including non-polar and polar pesticides (carbamates, organochlorine, organophosphorous, pyrethroids, herbicides and insecticides) in urine at trace levels by gas and ultra high pressure liquid chromatography coupled to ion trap and triple quadrupole mass spectrometry, respectively (GC-IT-MS/MS, UHPLC-QqQ-MS/MS). Non-polar and polar pesticides were simultaneously extracted from urine samples by a simple and fast solid phase extraction (SPE) procedure using C₁₈ cartridges as sorbent, and dichloromethane as elution solvent. Recovery was in the range of 60-120%. Precision values expressed as relative standard deviation (RSD) were lower than 25%. Identification and confirmation of the compounds were performed by the use of retention time windows, comparison of spectra (GC-amenable compounds) or the estimation of the ion ratio (LC-amenable compounds). For GC-amenable pesticides, limits of detection (LODs) ranged from 0.001 to 0.436 μg L⁻¹ and limits of quantification (LOQs) from 0.003 to 1.452 μg L⁻¹. For LC-amenable pesticides, LODs ranged from 0.003 to 1.048 μg L⁻¹ and LOQs ranged from 0.011 to 3.494 μg L⁻¹. Finally, the optimized method was applied to the analysis of fourteen real samples of infants from agricultural population. Some pesticides such as methoxyfenozide, tebufenozide, piperonyl butoxide and propoxur were found at concentrations ranged from 1.61 to 24.4 μg L⁻¹, whereas methiocarb sulfoxide was detected at trace levels in two samples.     

Development of a broad toxicological screening technique for urine using ultra-performance liquid chromatography and time-of-flight mass spectrometry

Withdrawal of the support for the REMEDi HS drug profiling system has necessitated its replacement within our laboratories with an alternative broad toxicological screening technique. To this end, a novel method, based on ultra-performance liquid chromatography (UPLC) and time-of-flight (TOF) mass spectrometry, was developed for the routine analysis of urine samples. Identification was achieved by comparison of acquired data to libraries containing more than 300 common drugs and metabolites, and was based on a combination of retention time, exact mass and fragmentation patterns. Validation data for the method is presented and comprised an evaluation of the following parameters: precision; transferability of the methodology between the six collaborating laboratories; specificity; extraction recovery and stability of processed samples; matrix effects and sensitivity.This paper presents the benefits of supplementary fragmentation data with particular regard to increasing specificity and confidence of identification and its usefulness with overdosed samples. The utility of the method was assessed by the parallel analysis of 30 authentic urine samples using the REMEDi HS and UPLC-TOF. The latter provided enhanced detection, leading to the identification of twice as many drugs. Furthermore it did not miss any compounds that were identified by REMEDi HS. The UPLC-TOF findings were further verified by a combination of data from three other conventional screening techniques, i.e., GC-MS, HPLC-DAD and UPLC-MS/MS.     

Solid-phase extraction followed by liquid chromatography quadrupole time-of-flight tandem mass spectrometry for the selective determination of fungicides in wine samples

In this work, a reliable and selective procedure for the determination of thirteen fungicides in red and white wine samples is proposed. Solid-phase extraction (SPE) and liquid chromatography (LC) tandem mass spectrometry (MS/MS), based on a hybrid quadrupole time-of-flight (QTOF) system, were used as sample preparation and determination techniques, respectively. Extraction and purification of target analytes was carried out simultaneously by using a reversed-phase Oasis HLB (200mg) SPE cartridge combined with acetonitrile as elution solvent. Fungicides were determined operating the electrospray source in the positive ionization mode, with MS/MS conditions adjusted to obtain at least two intense product ions per compound, or registering two transitions per species when a single product was noticed. High selective MS/MS chromatograms were extracted using a mass window of 20 ppms for each product ion. Considering external calibration as quantification technique, the overall recoveries (accuracy) of the procedure ranged between 81% and 114% for red and white wine samples (10-20 mL), spiked at different concentrations between 5 and 100 ng mL(-1). Relative standard deviations of the above data stayed below 12% and the limits of quantification (LOQs) of the method, calculated for 10 mL of wine, varied between 0.1 ng mL(-1) for cyprodinil (CYP) and 0.7 ng mL(-1) for myclobutanil (MYC). The optimized method was applied to seventeen commercial wines produced in Spain and obtained from local supermarkets. Nine fungicides were determined, at levels above the LOQs of the method, in the above samples. The maximum concentrations and the highest occurrence frequencies corresponded to metalaxyl (MET) and iprovalicarb (IPR).     

Determination of flunitrazepam and 7-aminoflunitrazepam in human urine by on-line solid phase extraction liquid chromatography-electrospray-tandem mass spectrometry

A method using an on-line solid phase extraction (SPE) and liquid chromatography with electrospray-tandem mass spectrometry (LC-ES-MS/MS) for the determination of flunitrazepam (FM2) and 7-aminoflunitrazepam (7-aminoFM2) in urine was developed. A mixed mode Oasis HLB SPE cartridge column was utilized for on-line extraction. A reversed phase C₁₈ LC column was employed for LC separation and MS/MS was used for detection. Sample extraction, clean-up and elution were performed automatically and controlled by a six-port valve. Recoveries ranging from 94.8 to 101.3% were measured. For both 7-aminoFM2 and FM2, dual linear ranges were determined from 20 to 200 and 200-2000 ng/ml, respectively. The detection limit for each analyte based on a signal-to-noise ratio of 3 ranged from 1 to 3 ng/ml. The intra-day and inter-day precision showed coefficients of variance (CV) ranging from 4.6 to 8.5 and 2.6-9.2%, respectively. The applicability of this newly developed method was examined by analyzing several urine samples.     

Determination of fungicides in wine by mixed-mode solid phase extraction and liquid chromatography coupled to tandem mass spectrometry

A novel procedure for the determination of nine selected fungicides (metalaxyl-M, azoxystrobin, myclobutanil, flusilazole, penconazole, tebuconazole, propiconazole, diniconazole and difenoconazole) in wine samples is presented. Sample enrichment and purification is simultaneously performed using mixed-mode, anion exchange and reversed-phase, OASIS MAX solid-phase extraction (SPE) cartridges. Analytes were determined by liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure electrospray ionization (LC-ESI-MS/MS). Parameters affecting the chromatographic determination and the extraction-purification processes were thoroughly investigated. Under optimized conditions, 10 mL of wine were firstly diluted 1:1 with ultrapure water and then passed through the mixed-mode SPE cartridge at a flow of ca. 5 mLmin(-1). After a washing step with 5 mL of an aqueous NH(4)OH solution (5%, w:v), analytes were recovered with just 1 mL of methanol and injected in the LC-MS/MS system without any additional purification. The selective extraction process avoided significant changes in the ionization efficiency for red and white wine extracts in comparison with pure standards in methanol. Performance of the method was good in terms of precision (RSDs<11%) and accuracy (absolute recoveries>72%, determined against pure standards in methanol) reporting method LOQs in the range of 0.01-0.79 ngmL(-1) for target compounds, which are far below the EU maxima residue levels (MRLs) for fungicides in vinification grapes and wine. Several commercial wines from different geographic areas in Spain were analyzed. In most samples, metalaxyl-M and azoxystrobin were found at concentrations up to several ngmL(-1).     

Herbal medicine analysis by liquid chromatography/time-of-flight mass spectrometry

The fact that the effects of herbal medicines (HMs) are brought about by their chemical constituents has created a critical demand for powerful analytical tools performing the chemical analysis to assure their efficacy, safety and quality. Liquid chromatography coupled to mass spectrometry (LC–MS) is an excellent technique to analyze multi-components in complex herbal matrices. Due to its inherent characteristics of accurate mass measurements and high resolution, time-of-flight (TOF) MS is well-suited to this field, especially for qualitative applications. The purpose of this article is to provide an overview on the potential of TOF, including the hybrid quadrupole- and ion trap-TOF (QTOF and IT-TOF), hyphenated to LC for chemical analysis in HMs or HM-treated biological samples. The peculiarities of LC–(Q/IT)TOF-MS for the analysis of HMs are discussed first, including applied stationary phase, mobile-phase selection, accurate mass measurements, fragmentation and selectivity. The final section is devoted to describing the applicability of LC–(Q/IT)TOF-MS to routine analysis of multi-components, including target and non-target (unknown) compounds, in herbal samples, emphasizing both the advantages and limitations of this approach for qualitative and quantitative purposes. The potential and future trends of fast high-performance liquid chromatography (HPLC) (e.g. rapid resolution LC and ultra-performance LC) coupled to (Q)TOF-MS for chemical analysis of HMs are highlighted.     

Determination of pesticides in water samples by solid phase extraction and gas chromatography tandem mass spectrometry

A multiresidue method for the determination of more than 80 pesticides in water has been developed and validated. The proposed method is based on SPE followed by GC coupled to MS/MS. Different variables affecting SPE procedure, such as cartridges, sample volume and solvents were studied, and mass spectrometric conditions were optimised in order to increase selectivity and sensitivity. Calibration curves were linear over the range of 0.03-0.5 microg/L. Recoveries were in the range of 70-110% and repeatability was below 20% for the lowest calibration point. LODs ranged from 0.001 to 0.025 microg/L and LOQs from 0.003 to 0.076 microg/L. Finally, the method was successfully applied to the analysis of water samples from southeast of Spain.     

Simultaneous analysis of fourteen tertiary amine stimulants in human urine for doping control purposes by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry

A method for the simultaneous screening and confirmation of the presence of fourteen tertiary amine stimulants in human urine by gas chromatography-mass spectrometry (GC-MS) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) approaches were utilized for the pre-treatment of the urine samples. The study indicated that the capillary temperature played a significant role in the signal abundances of the protonated molecules of cropropamide and crotethamide under positive ion electrospray ionization (ESI) conditions. In addition, comparison studies of two different pre-treatment approaches as well as the two ionization modes were conducted. The LODs of the developed method for all the analytes were lower than the minimum required performance limit (MRPL) as set forth in the World Anti-Doping Agency (WADA) technical document for laboratories. The human urine sample obtained after oral administration of prolintane·HCl was successfully analyzed by the developed method, which demonstrated the applicability and reliability of the method for routine doping control analysis.     

Identification of isopropyl substituted β-blocking agents in human urine by gas chromatography and tandem mass spectrometry

Summary A method for the sensitive detection of isopropyl substituted β-blocking agents in human urine is presented. The sample preparation step involves enzymatic hydrolysis, solid phase extraction and derivatisation withN-methyl-N-trimethylsilyl-trifluoroacetamide. The instrumental analysis was performed using gas chromatography-mass spectrometry with an ion trap mass spectromeler. The mass spectrometer was operated in the scan as well as in the MS-MS mode.     

Multiresidue determination of UV filters in water samples by solid‐phase extraction and liquid chromatography with tandem mass spectrometry analysis

UV filters, contained in sunscreens and other cosmetic products, as well as in some plastics and industrial products, are nowadays considered contaminants of emerging concern because their widespread and increasing use has lead to their presence in the environment. Furthermore, some UV filters are suspected to have endocrine disruption activity. In the present work, we developed an analytical method based on liquid chromatography with tandem mass spectrometry for the determination of UV filters in tap and lake waters. Sixteen UV filters were extracted from water samples by solid‐phase extraction employing graphitized carbon black as adsorbent material. Handling 200 mL of water sample, satisfactory recoveries were obtained for almost all the analytes. The limits of detection and quantification of the method were comparable to those reported in other works, and ranged between 0.7–3.5 and 1.9–11.8 ng/L, respectively; however in our case the number of investigated compounds was larger. The major encountered problem in method development was to identify the background contamination sources and reduce their contribution. UV filters were not detected in tap water samples, whereas the analyses conducted on samples collected from three different lakes showed that the swimming areas are most subject to UV filter contamination.     

Comparative study of sample preparation methods; supported liquid membrane and solid phase extraction in the determination of benzimidazole anthelmintics in biological matrices by liquid chromatography-electrospray-mass spectrometry

Supported liquid membrane (SLM) and solid phase extraction (SPE) have been applied as clean-up and/or enrichment techniques for a mixture of five benzimidazole anthelmintics compounds, namely albendazole, fenbendazole, mebendazole, oxibendazole, and thiabendazole. Two biological matrices, mainly urine and milk, and ultra high purity (UHP) water were spiked with a mixture of these five compounds. Waters Oasis^® MCX and International Sorbent Technology (IST) HCX SPE sorbents were used. The liquid membrane used for clean-up and/or enrichment of these compounds was 5% tri-n-octylphosphine oxide (TOPO) dissolved in n-undecane/di-n-hexyl ether (1:1). The SLM extraction efficiencies and SPE percentage recoveries ranged between 60 and 100%. The detection limits (DLs) for different benzimidazole compounds by SPE/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L, for fenbendazole and mebendazole was 1 and 10 ng/L, respectively. Similarly, the detection limits of SLM/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L and for fenbendazole and mebendazole was 1 ng/L. The results of optimization of various parameters of the SLM method are reported.     

Liquid chromatography with time-of-flight mass spectrometry for simultaneous determination of chemotherapeutant residues in salmon

Liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS) method has been developed for simultaneous confirmation by accurate mass measurement and quantitative determination of antibiotics (enrofloxacin, oxolinic acid, flumequine, erythromycin), fungicides (malachite green MG, leucomalachite green LMG) and parasiticide (emamectin benzoate) residues in edible portion of salmon. Confirmation of chemotherapeutant residues has been based on the system of identification points (IPs) established in the Commission Decision 2002/657/EC concerning the use of mass spectrometry (MS) techniques. A validation study on matrix is presented evaluating accuracy in terms of precision (λ_ppm 0.83-1.15) and trueness (0.22-0.70 Da). Limits of detection (LODs) and limits of quantification (LOQs) were in ranges of 1-3 and 3-9 μg/kg, below the maximum residue limits (MRLs) established in current EU legislation (100-200 μg/kg) for these chemotherapeutants. Considering the EU guidelines, decision limits (CCα) and detection capabilities (CCβ) were determined (ranges of 103-218 and 107-234 μg/kg, respectively) for authorised substances. For no authorised compounds (MG and LMG), LODs were 2 and 1 μg/kg, respectively, but exceed the MRPL (minimum required performance limit) established in the legislation which corresponds to the sum of MG and LMG (2 μg/kg). Acceptable intra-day and inter-day variability, in terms of relative standard deviation (R.S.D.) of the analytical method, were obtained (2-15%). Linearity was demonstrated from the LOQs of the analytes to 600 μg/kg (r > 0.9991). The method has involved an extraction procedure based on solid-liquid extraction (SLE) with recoveries higher than 80% for most target chemotherapeutants, with exception of enrofloxacin (40%).     

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Simultaneous determination of multiveterinary drug residues in pork meat by liquid chromatography‐tandem mass spectrometry combined with solid phase extraction

An LC‐MS/MS method developed for simultaneous analysis of 54 veterinary drug residues of six families in pork meat samples, including sulfanilamide, nitroimidazoles, quinolones, macrolide antibiotics, lincosamides, and praziquantel. The pork meat sample was prepared by extraction with ACN, and clean‐up on a C₁₈ SPE cartridge. The sample was separated on a C₈ column and eluted with ACN, methanol, and formic acid. The MS/MS detector is operated in the multiple reaction monitoring mode, acquiring two specific precursor‐product ion transitions per target compound. The method showed excellent linearity (R² ≥ 0.99) and high precision (relative SD, RSD ≤ 19.8%) for all compounds. The method quantification limits of 54 veterinary drug residues were in the range of 0.3–3.0 μg/kg. Recoveries for most analytes based on matrix‐matched calibration in matrices were 20.9–121.0%. This method has been successfully applied for analysis of more than 100 pork meat samples from the local market; five of the 54 drugs were detected.     

Confirmatory and quantitative analysis of fatty acid esters of hydroxy fatty acids in serum by solid phase extraction coupled to liquid chromatography tandem mass spectrometry

A novel class of endogenous mammalian lipids endowed with antidiabetic and anti-inflammatory properties has been recently discovered. These are fatty acid esters of hydroxy fatty acids (FAHFAs) formed by condensation between a hydroxy fatty acid and a fatty acid. FAHFAs are present in human serum and tissues at low nanomolar concentrations. Therefore, high sensitivity and selectivity profiling analysis of these compounds in clinical samples is demanded. An automated qualitative and quantitative method based on on-line coupling between solid phase extraction and liquid chromatography–tandem mass spectrometry has been here developed for determination of FAHFAs in serum with the required sensitivity and selectivity. Matrix effects were evaluated by preparation of calibration models in serum and methanol. Recovery factors ranged between 73.8 and 100% in serum. The within-day variability ranged from 7.1 to 13.8%, and the between-days variability varied from 9.3 to 21.6%, which are quite acceptable values taking into account the low concentration levels at which the target analytes are found. The method has been applied to a cohort of human serum samples to estimate the concentrations profiles as a function of the glycaemic state and obesity. Statistical analysis revealed three FAHFAs with levels significantly different depending on the glycaemic state or the body mass index. This automated method could be implemented in high-throughput analysis with minimum user assistance.     

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H₂O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 μg/g to 1.04 μg/g and 1.32 μg/g to 5.29 μg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.     

Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC–QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., β-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.     

Quantitative liquid chromatography/time-of-flight mass spectrometry

Over the last decade, time-of-flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals.     

Single-step extraction and cleanup of bisphenol A in soft drinks by hemimicellar magnetic solid phase extraction prior to liquid chromatography/tandem mass spectrometry

Hemimicelles of tetradecanoate chemisorbed onto magnetic nanoparticles (MNPs) are here proposed as a sorbent for the single-step extraction and cleanup of bisphenol A (BPA) in soft drinks. The purpose of this work was to develop a simple, rapid and low-cost sample treatment suitable to assess the human exposure to BPA from this type of high consumption food. The nanoparticles were easily coated by mixing commercially available magnetite of 20–30 nm mean particle diameter with tetradecanoate at 85 °C for 30 min. The extraction/cleanup procedure involved stirring the samples (3 mL) with 200 mg of tetradecanoate-coated MNPs for 20 min, isolating the sorbent with a Nd–Fe–B magnet and eluting BPA with methanol. The extraction efficiency was not influenced by salt concentrations up to 1 M and pH values over the range 4–9. No cleanup of the extracts was needed, and the method proved matrix-independent. The extracts were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Quantitation was performed by internal standard calibration using BPA-¹³C₁₂. The limit of quantitation obtained for the method, 0.03 ng mL⁻¹, was below the usual range of concentrations reported for BPA in soft drinks (0.1–3.4 ng mL⁻¹). The proposed method was successfully applied to the determination of BPA in different samples acquired from various supermarkets in southern Spain; the concentrations found ranged from 0.066 to 1.08 ng mL⁻¹. Recoveries from samples spiked with 0.33 ng mL⁻¹ of BPA ranged from 91% to 105% with relative standard deviations from 3% to 8%.