Quantitative performance of liquid chromatography coupled to Q-Exactive high resolution mass spectrometry (HRMS) for the analysis of tetracyclines in a complex matrix

The presence of antibiotics in the environment is of increased interest and, as modern mass spectrometers become more efficient, we are increasingly aware of traces of pharmaceuticals appearing in a wide range of environmental and biological matrices. The Q-Exactive mass spectrometer is part of these innovative hybrid high-resolution mass spectrometers (HRMS) which is often associated with peptide sequencing or metabolomics but with a limited number of studies focusing on its application to the quantification of small molecules in environmental and biological matrices. It combines the high resolving power (RP) performance of the Orbitrap with the high performance selectivity of the quadrupole. Tetracyclines (TCs) are a family comprising some of the most widely used antibiotics in veterinary medicine. This study presents the quantitative performances of the Q-Exactive by illustrating a new approach to quantify TCs using liquid chromatography coupled to a HRMS in a complex matrix, i.e., swine manure. The Q-Exactive was used at high-resolution in both full scan (FS) and targeted ion fragmentation (tMS²) modes. These two modes were optimized and compared to determine the most reliable and efficient approach to quantify TCs with good accuracy. The proposed method was optimized to obtain the best selectivity and sensitivity, thus eliminating false positive and allowing the detection of trace levels of analyte. The TCs were extracted from the matrix by sonication using McIlvaine buffer followed by an off-line solid phase extraction method to concentrate and clean the extracts. Both FS and tMS² modes presented good linearity (R² > 0.991) and repeatability (RSD < 15%). Mass accuracy was acceptable with values below 2 ppm. The method detection limits (MLD) calculated from the calibration curves ranged from 2.0 to 12 ng g⁻¹ for FS mode and from 1.5 to 3.6 ng g⁻¹ for tMS² mode. Accuracy and interday/intraday relative standard deviations were below 21% for both modes studied. TCs were quantified in real samples of swine manure with concentrations ranging from 29 to 75 ng g⁻¹. This study showed the possibility of using hybrid HRMS for trace detection and quantification of TCs in a complex matrix, thus avoiding false positive while achieving good selectivity and sensitivity.     

Quantification of anthelmintic drug residues in milk and muscle tissues by liquid chromatography coupled to Orbitrap and liquid chromatography coupled to tandem mass spectrometry

A simple method for the determination of some anthelmintic drugs and phenylbutazone residues in milk and muscle was developed. Following a fast and easy extraction and evaporation procedure, the extract was injected into an ultra performance liquid chromatography system coupled to a single stage Orbitrap detector. The high mass resolution of 50,000 full width at half maximum and corresponding narrow mass windows permitted a very selective and sensitive detection of analytes without requiring fragmentation of the observed [M+H]⁺ or [M+Na]⁺ ions. This eliminated some difficulties which have plagued the analysis of compounds belonging to the group of avermectins. The analytical method was validated according to the EU commission decision for Orbitrap based, but also for more traditional tandem mass spectrometry based detection and quantification. Equal repeatability but significantly higher sensitivity for critical compounds (avermectins) was obtained for the Orbitrap based detection. A result of this study was the conclusion that analytes with poor fragmentation properties (e.g. sodium-cationized molecules) can be more easily quantified by single stage high resolution mass spectrometry than by tandem mass spectrometry.     

Quantitative profiling of phosphatidylethanol molecular species in human blood by liquid chromatography high resolution mass spectrometry

Phosphatidylethanol (PEth) is a group of aberrant phospholipids formed in cell membranes in the presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Recently published literature has demonstrated the existence of several molecular species of PEth in samples drawn from alcohol-dependent subjects. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method coupled with a lipidomic strategy was developed and validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. Chromatography was performed on a C18 column using acetonitrile, 10mM ammonium acetate, and 2-propanol as mobile phases with a 22-min gradient. HRMS experiments were performed on an LTQ-Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization source operated in negative ion mode. The theoretical masses of [M-H](-) of PEth species were calculated from the elemental chemical formula by varying the length and unsaturation grade of the fatty acid side chains; identification of PEth species in blood was achieved by searching the accurate masses of the targeted compounds in the acquired full-scan LC-HRMS chromatogram. The chemical structure of tentatively identified PEth species was elucidated through HR multiple mass experiments. The validated LC-HRMS method was selective, as warranted by HRMS at 60,000 resolution and 4 ppm accuracy. Linearity was observed in the 0.001-2.000 μM range, and limit of detection of 0.0005 μM and limit of quantitation of 0.001 μM were obtained for single PEth species. Imprecision and inaccuracy were always lower than 10% and 15%, respectively. The identification capabilities of the method were tested on blood samples collected from heavy drinkers (n=11), social drinkers (n=8), and teetotalers (n=10). The high sensitivity of the method led to the simultaneous identification of 17 different PEth molecular species in blood collected from heavy drinkers, and 2 PEth species (16:0/18:1 and 16:0/18:2) in blood collected from social drinkers.     

Rapid quantification of pharmaceuticals and pesticides in passive samplers using ultra high performance liquid chromatography coupled to high resolution mass spectrometry

The presence of both pharmaceuticals and pesticides in the aquatic environment has become a well-known environmental issue during the last decade. An increasing demand however still exists for sensitive and reliable monitoring tools for these rather polar contaminants in the marine environment. In recent years, the great potential of passive samplers or equilibrium based sampling techniques for evaluation of the fate of these contaminants has been shown in literature. Therefore, we developed a new analytical method for the quantification of a high number of pharmaceuticals and pesticides in passive sampling devices. The analytical procedure consisted of extraction using 1:1 methanol/acetonitrile followed by detection with ultra-high performance liquid chromatography coupled to high resolution and high mass accuracy Orbitrap mass spectrometry. Validation of the analytical method resulted in limits of quantification and recoveries ranging between 0.2 and 20 ng per sampler sheet and between 87.9 and 105.2%, respectively. Determination of the sampler-water partition coefficients of all compounds demonstrated that several pharmaceuticals and most pesticides exert a high affinity for the polydimethylsiloxane passive samplers. Finally, the developed analytical methods were used to measure the time-weighted average (TWA) concentrations of the targeted pollutants in passive samplers, deployed at eight stations in the Belgian coastal zone. Propranolol, carbamazepine and seven pesticides were found to be very abundant in the passive samplers. These obtained long-term and large-scale TWA concentrations will contribute in assessing the environmental and human health risk of these emerging pollutants.     

Coupling liquid chromatography to Orbitrap mass spectrometry

The Orbitrap mass analyzer has become a mainstream mass spectrometry technique. In addition to providing a brief introduction to the Orbitrap technology and its continuing development, this article reviews the most recent publications quoting the use of the Orbitrap detection for a variety of chromatographic separation techniques. Its coupling to reversed-phase liquid chromatography (LC) represents undoubtedly the most ubiquitous approach to both small molecule and proteomic analyses. Multi-dimensional LC separations have an important role to play in the proteomics applications while an ultra-high-pressure LC is more frequently encountered in the area of metabolomics and metabolite analysis. Recently, special chromatographic techniques such as hydrophilic interaction chromatography and its variations have also been also cited with the Orbitrap detection.     

Novel approaches in analysis of Fusarium mycotoxins in cereals employing ultra performance liquid chromatography coupled with high resolution mass spectrometry

Rapid, simple and cost-effective analytical methods with performance characteristics matching regulatory requirements are needed for effective control of occurrence of Fusarium toxins in cereals and cereal-based products to which they might be transferred during processing. Within this study, two alternative approaches enabling retrospective data analysis and identification of unknown signals in sample extracts have been implemented and validated for determination of 11 major Fusarium toxins. In both cases, ultra-high performance liquid chromatography (U-HPLC) coupled with high resolution mass spectrometry (HR MS) was employed. ¹³C isotopically labeled surrogates as well as matrix-matched standards were employed for quantification. As far as time of flight mass analyzer (TOF-MS) was a detection tool, the use of modified QuEChERS (quick easy cheap effective rugged and safe) sample preparation procedure, widely employed in multi-pesticides residue analysis, was shown as an optimal approach to obtain low detection limits. The second challenging alternative, enabling direct analysis of crude extract, was the use of mass analyzer based on Orbitrap technology. In addition to demonstration of full compliance of the new methods with Commission Regulation (EC) No. 401/2006, also their potential to be used for confirmatory purposes according to Commission Decision 2002/657/EC has been critically assessed.     

Analysis of multiple mycotoxins in cereals under ambient conditions using direct analysis in real time (DART) ionization coupled to high resolution mass spectrometry

Direct analysis in real time (DART) ionization coupled to an (ultra)high resolution mass spectrometer based on orbitrap technology (orbitrapMS) was used for rapid quantitative analysis of multiple mycotoxins isolated from wheat and maize by modified QuEChERS procedure. After initial evaluation of ionization efficiencies for major groups of mycotoxins achievable with DART technology, sample preparation procedure and instrument parameter settings were optimized to obtain sensitive and accurate determination of most intensively ionizing toxins (deoxynivalenol, nivalenol, zearalenon, actyldeoxynivalenol, deepoxy-deoxynivalenol, fusarenon-X, altenuene, alternariol, alternariolmethylether, diacetoxyscirpenol, sterigmatocystin). The lowest calibration levels (LCLs) estimated for the respective analytes ranged from 50 to 150 μg kg⁻¹. Quantitative analysis was performed either with the use of matrix-matched standards or by employing commercially available ¹³C-labeled internal standards (available for deoxynivalenol, nivalenol and zearalenon). Good recoveries (100-108%) and repeatabilities (RSD 5.4-6.9%) were obtained at spiking level 500 μg kg⁻¹ with isotope dilution technique. Based on matrix-matched calibration, recoveries and repeatabilities were in the range 84-118% and 7.9-12.0% (RSD), respectively. The trueness of data obtained for deoxynivalenol and zearalenon in wheat/maize by DART-orbitrapMS was demonstrated by analysis of certified reference materials (CRMs). Good agreement of these results with data generated by validated ultra-high pressure liquid chromatography-time-of-flight mass spectrometry method was documented.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

The use of high performance liquid chromatography-quadrupole time-of-flight mass spectrometry coupled to advanced data mining and chemometric tools for discrimination and classification of red wines according to their variety

In this study, the potential of high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC–QTOFMS) for metabolomic profiling of red wine samples was examined. Fifty one wines representing three varieties (Cabernet Sauvignon, Merlot, and Pinot Noir) of various geographical origins were sourced from the European and US retail market. To find compounds detected in analyzed samples, an automated compound (feature) extraction algorithm was employed for processing background subtracted single MS data. Stepwise reduction of the data dimensionality was followed by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) which were employed to explore the structure of the data and construct classification models. The validated PLS-DA model based on data recorded in positive ionization mode enabled correct classification of 96% of samples. Determination of molecular formula and tentative identification of marker compound was carried out using accurate mass measurement of full single MS spectra. Additional information was obtained by correlating the fragments obtained by MS/MS accurate mass spectra using the QTOF with collision induced dissociation (CID) of precursor ions.     

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography–high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times.     

Rapid confirmatory analysis of non-steroidal anti-inflammatory drugs in bovine milk by rapid resolution liquid chromatography tandem mass spectrometry

A rapid method has been developed to analyse carprofen (CPF), diclofenac (DCF), mefenamic acid (MFN), niflumic acid (NIFLU), naproxen (NAP), oxyphenylbutazone (OXYPHEN), phenylbutazone (PBZ) and suxibuzone (SUXI) residues in bovine milk. Milk samples are extracted with acetonitrile and sample extracts were purified on Evolute™ ABN solid phase extraction cartridges. Aliquots were analysed by rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) with a runtime of 6.5 min. The method was validated in bovine milk, according to the criteria defined in Commission Decision 2002/657/EC. CCα values of 0.46, 1.08, 0.92, 1.26, 1.29, 2.12, 0.55 and 2.86 ng mL⁻¹ were determined for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI, respectively. CCβ values of 0.79, 1.85, 1.56, 2.15, 2.19, 3.62, 0.94 and 4.87 ng mL⁻¹ were determined for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI, respectively. The measurement uncertainty of the method was estimated at 9, 28, 28, 45, 46, 45, 10 and 39% for CPF, DCF, MFN, NIFLU, NAP, OXYPHEN, PBZ and SUXI. Fortifying bovine milk samples (n = 18) in three separate assays, show the accuracy of the method to be between 82 and 108%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (5, 7.5 and 10 ng mL⁻¹) was less than 16%, respectively. The advantage of the method is that low ng mL⁻¹ levels can be detected and quantitatively confirmed rapidly in milk and that 3 batches of samples can be analysed within a single day using RRLC-MS/MS with a runtime of 6.5 min.     

Isolation and determination of paspalitrem-type tremorgenic mycotoxins using liquid chromatography with diode-array detection

A liquid chromatographic (LC) method is described for the isolation and determination of the tremorgenic mycotoxins paxilline (Penicillium paxilli NRRL 6110), paspaline, paspalinine and paspalicine (Claviceps paspali). Following a Soxhlet extraction of a mould-contaminated matrix using chloroform, the crude extract was partitioned between hexane and 80% aqueous methanol. The latter fraction, containing the desired toxin(s), was evaporated to dryness, the residue dissolved in methylene chloride and the solution analysed by liquid chromatography using a Supelcosil LC-Si column eluted with methylene chloride-diethyl ether (9 + 1, v/v). A mixture containing standards of these compounds was similarly analysed. All toxins were detected using a UV diode-array detector. The generated UV spectra and chromatographic data of the standard toxins were stored in a computer as a library and used to identify these toxins in a crude mixture. The purity of the separated peaks and the amount of toxin in the crude mixture were also determined. The toxins were isolated by selectively collecting the eluted peaks using a programmable fraction collector equipped with a peak level sensor. Further confirmation of compound identity was achieved by mass spectrometry using the direct inlet probe method. In comparison with methods used previously to isolate these toxins, the present technique is fast and allows the acquisition of complete UV spectral information and chromatographic data and the isolation of multiple toxins in a single chromatographic operation.     

Analysis of catecholamines and their metabolites in adrenal gland by liquid chromatography tandem mass spectrometry

Two simple, rapid and specific analytical methods for 13 catecholamines and their metabolites have been developed based on liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. Tyrosine, dopamine, dihydroxyphenylalanine, epinephrine, norepinephrine, 3-methoxytyramine, normetanephrine, metanephrine and isoproterenol (internal standard) were separated on a Kromasil™ Cyano analytical column by a mobile phase consisting of 60% (v/v) acetonitrile and 40% (v/v) water adjusted with formic acid to pH 3.0, and detected by positive ionization electrospray tandem mass spectrometry. While vanillymandelic acid, 3,4-dihydroxymandelic acid, homovanillic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylglycol and 5-hydroxy-2-indolecarboxylic acid (internal standard) were separated on a reversed-phase Shim-Pak VP-ODS column with the mobile phase of 60% (v/v) acetonitrile, and 40% (v/v) water adjusted with formic acid to pH 4.5 and detected in the negative ionization electrospray tandem mass spectrometry. The influence of various parameters such as column type and mobile phase composition on separation and sensitivity were investigated. The limits of detection were in the range of 0.5-20 ng mL⁻¹. The mean recoveries determined from three different concentrations of each analyte were above 85.4%. The precision of the method calculated as relative standard deviation was lower than 5.3%. Deduced from the results of real sample analysis, adrenal gland synthesizes and stores the catecholamine hormones norepinephrine and epinephrine.     

液相色谱-高分辨质谱快速筛查海水中磺胺类药物

建立了海水中15种磺胺类药物的液相色谱-静电场轨道阱高分辨质谱方法.采用C18固相萃取膜提取海水中磺胺类药物,使用Hypersil GOLD Cl8色谱柱分离,乙腈和5 mmol/L乙酸铵(含0.1％甲酸)为流动相梯度洗脱.正离子模式全扫描/数据依赖二级扫描(Full MS/dd-MS2),提取一级质谱图中准分子离子的...     

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.     

高分辨质谱技术在农药残留检测中的应用

农药残留检测是农产品中有害物质控制的重要组成部分,随着农药残留限量标准体系的发展完善,农药残留检测方法也在不断进步。近年来质谱技术发展迅速,已被广泛应用于农药残留检测领域,高分辨质谱由于具有较高的分辨率和质量精确度,在复杂基质的农药多残留高通量检测中发挥着越来越重要的作用。本文从高分辨质谱与液相色谱、气相色谱及其他分离模式联用等方面出发,简述了近5年来高分辨质谱在农药残留检测中的应用,对目前高分辨质谱在农药残留检测应用中发现的问题进行了讨论,并对其未来发展趋势进行展望。     

Multi-mycotoxin analysis in eggs using a QuEChERS-based extraction procedure and ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A reliable and rapid method has been developed for the determination of 10 mycotoxins (beauvericin, enniatin A, A1, B1, citrinin, aflatoxin B1, B2, G1, G2 and ochratoxin A) in eggs at trace levels. Ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) has been used for the analysis of these compounds in less than 7 min. Mycotoxins have been extracted from egg samples using a QuEChERS-based extraction procedure (Quick, Easy, Cheap, Effective, Rugged and Safe) without applying any further clean-up step. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification. Blank samples were fortified at 10, 25, 50 and 100 μg kg(-1), and recoveries ranged from 70% to 110%, except for ochratoxin A and aflatoxin G1 at 10 μg kg(-1), and aflatoxin G2 at 50 μg kg(-1). Relative standard deviations were lower than 25% in all the cases. Limits of detection ranged from 0.5 μg kg(-1) (for aflatoxins B1, B2 and G1) to 5 μg kg(-1) (for enniatin A, citrinin and ochratoxin A) and limits of quantification ranged from 1 μg kg(-1) (for aflatoxins B1, B2 and G1) to 10 μg kg(-1) (for enniatin A, citrinin and ochratoxin A). Seven samples were analyzed and aflatoxins B1, B2, G1, G2, and beauvericin were detected at trace levels.     

Determination of 2-isopropyl thioxanthone and 2-ethylhexyl-4-dimethylaminobenzoate in milk: comparison of gas and liquid chromatography with mass spectrometry

Liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS) have been compared for the analysis of 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB). Pressurized liquid extraction (PLE) was applied for the extraction of ITX and EHDAB from milk and milk-based beverages. Samples were homogenized with sea sand and anhydrous sodium sulfate, and were extracted with ethyl acetate at 100 °C and 10.3 × 10⁶ Pa in one cycle of 10 min at 90% flush. Both, GC-MS and LC-MS/MS were suitable to determine these photoinitiators in the PLE extracts, providing appropriate identification and quantification. The recoveries obtained ranged from 70 to 99% for ITX and from 70 to 95% for EHDAB. These recoveries were equal as those obtained by a conventional liquid–liquid partitioning with acetonitrile and tert-butyl methyl ether–hexane. The quantification limits using GC-MS, based on a signal-to-noise ratio of 10, were 0.5 μg/L for ITX and 1 μg/L for EHDAB. The repeatability of the method, as indicated by the relative standard deviations, was within the range 0.9–16.1%. The same parameters calculated using LC-MS/MS result in quantification limits of 0.1 μg/L for ITX and 0.02 μg/L for EHDAB and repeatability within the range 5.2–19.4%. These results pointed out that both techniques are appropriate to determine these compounds in food samples. The method was applied to milk and milk-based beverages from different supermarkets. The ITX and EHDAB contents ranged from 2.5 to 325 μg/L and from 8 to 126 μg/L, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of heterocyclic amines in hair by on-line in-tube solid-phase microextraction coupled with liquid chromatography−tandem mass spectrometry

Mutagenic and carcinogenic heterocyclic amines (HCAs) are formed during heating of various proteinaceous foods, but human exposure to HCAs has not yet been elucidated in detail. To assess long-term exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in hair by automated on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Using a Zorbax Eclipse XDB-C8 column, 16 HCAs were analyzed within 15 min. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL sample at a flow rate of 200 μL min⁻¹ using a Supel-Q PLOT capillary column as an extraction device. The extracted HCAs were easily desorbed from the column by passage of the mobile phase, with no carryover observed. This in-tube SPME LC–MS/MS method showed good linearity for HCAs in the range of 10–2000 pg mL⁻¹, with correlation coefficients above 0.9989 (n = 18), using stable isotope-labeled HCA internal standards. The detection limits (S/N = 3) of 14 HCAs except for MeAαC and Glu-P-1 were 0.10–0.79 pg mL⁻¹. This method was successfully utilized to analyze 14 HCAs in hair samples without any interference peaks, with quantitative limits (S/N = 10) of about 0.17–1.32 pg mg⁻¹ hair. Using this method, we evaluated the exposure to HCAs in cigarette smoke and the suitability of using hair HCAs as exposure biomarkers.     

Multi-analyte high performance liquid chromatography coupled to high resolution tandem mass spectrometry method for control of pesticide residues,mycotoxins, and pyrrolizidine alkaloids

A new reliable and highly sensitive method based on high performance liquid chromatographic (HPLC) separation and high resolution tandem mass spectrometric detection (HRMS/MS) has been developed and validated for determination of 323 pesticide residues, 55 mycotoxins, and 11 plant toxins represented by pyrrolizidine alkaloids. The method was validated for three matrices, leek, wheat, and tea differing in nature/amount of co-extracts that may cause various matrix effects. For target analytes isolation, optimized QuEChERS-based (quick, easy, cheap, effective, rugged, and safe) extraction procedure was employed. Spectral HRMS/MS library has been established providing an entire spectrum of fragment ions for each analyte, which allows unbiased identification and confirmation of target compounds. The limits of quantification (LOQs) of target analytes were below 10 μg kg⁻¹ for 82%, 81%, and 61% for matrices leek, wheat, and tea, respectively. Recoveries were in the acceptable range (70–120%) according to SANCO/12571/2013 for most of target analytes, except for highly polar ‘masked’ mycotoxin deoxynivalenol-3-glucoside with recoveries 35%, 47%, and 42% for matrices leek, wheat, and tea, respectively. The linearities of calibration curves expressed as coefficients of determination were in the range of 0.9661–1.000, and repeatabilities expressed as relative standard deviations (RSDs) at LOQs lied in the range of 0.25–13.51%. The trueness of the method was verified using several certified reference materials (CRMs) and proficiency test samples.