On-line Clean-up Research Articles

Determination of tetrodotoxin in human plasma and urine using online MCX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry

An efficient technique for quantitative analysis of tetrodotoxin (TTX) in human plasma and urine has been developed, which combines liquid chromatography-tandem mass spectrometry (LC-MS/MS) with online MCX solid phase extraction (SPE) cleanup. Sample preparation, including extraction with acetonitrile containing 0.5 % acetate acid, centrifugation, and filtration, was followed by online SPE cleanup. The whole run-time was less than 15 min, including online cleanup, chromatographic separation, and re-equilibration of the online SPE − LC-MS/MS system. The parameters of sample extraction, purification, separation, and detection were optimized. The matrix-matched internal standard calibration standard curves with linear regression coefficients larger than 0.9990 were established for quantification. The LOD and LOQ for this approach were determined to be 0.1 ng/mL and 0.3 ng/mL, respectively. The recoveries for varied concentrations of TTX in human plasma and urine were 84.9–104.2 % and 89.2–109.6 %, respectively. The matrix effects of TTX in human plasma and urine matrices were 85.5 % and 74.3 %, respectively, and both the inter- and intra-day precision values were less than 9.5 %. This analytical method was successfully employed for detecting TTX in biological samples from a poisoned patient who accidentally ingested the nassarius glans.

Liquid-liquid extraction combined with online cleanup for the simultaneous determination of PAHs by GC–MS/MS and their hydroxylated metabolites by LC-MS/MS in human fingernails

A method was developed for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and their hydroxylated metabolites (OH-PAHs) in human fingernails using liquid–liquid extraction and online purification. After surface decontamination by rinsing with acetone, the fingernails were digested with sodium hydroxide and subjected to liquid–liquid extraction with a mixture of n-hexane and methyl tertbutyl ether. The organic extract was then fractionated using a silica-based solid-phase extraction (SPE) cartridge to obtain a PAH fraction eluted with n-hexane/dichloromethane (v/v, 95:5) and an OH-PAH fraction eluted with dichloromethane/ethyl acetate (v/v, 50:50). The PAH fraction was directly injected into an online gel permeation chromatography-gas chromatography-triple quadrupole tandem mass spectrometry (GPC-GC–MS/MS) system, enabling rapid determination of 16 PAHs. A parallel online SPE liquid chromatography tandem mass spectrometry (LC-MS/MS) method was used to determine 12 OH-PAHs. Validation experiments showed that the recovery of PAH and OH-PAH were within range of 67.4%−105.1% (RSD ≤ 10.1%) and 72.8%−102.3% (RSD ≤ 10.9%), respectively, with limits of quantitation (LOQ) of 0.06–0.8 ng/g and 0.15–3.1 ng/g, respectively. Forty-two human fingernail samples from residents of Southern China were analyzed to establish background PAH and OH-PAH levels in this region. Several PAHs and OH-PAHs were detected, at concentrations of 97.5 to 3,687 ng/g for PAHs and 24.2 to 767 ng/g for OH-PAHs. The dominant homologues were two- and three-ring PAH isomers, notably naphthalene (Nap), fluorene (Flu), and phenanthrene (Phe), as well as the corresponding hydroxylated metabolites 2-OH-Nap, OH-Flu, and OH-Phe. Smoking, consuming barbecued food, and age had no significant effects on PAH exposure, but a larger sample would be required to confirm this finding. The online purification strategy presented here expedites cleanup and purification during analysis of human fingernails and should facilitate non-invasive biomonitoring of PAHs in humans, particularly when analyzing large numbers of samples.

Rapid measurement of anticoagulant rodenticides in human blood and urine using online turbulent flow chromatography coupled with liquid chromatography–tandem mass spectrometry

AbstractAnticoagulant rodenticides (ARs) are widely used to control rodents. A method based on online turbulent flow chromatography (TFC) combined with LC-MS/MS has been established for rapid quantitative determination of eight ARs in human blood and urine. This method, which does not require time-consuming pre-processing steps, renders it especially suited for use in emergency poisoning cases. Sample preparation, including extraction, centrifugation, and filtration, was followed by online clean-up using TFC. The total run-time was within 13.5 min, including online purification, chromatographic separation, and re-equilibration of the TFC system. The parameters for sample extraction, purification, separation, and detection in this study were optimized separately. The linear regression coefficients of matrix-matched calibration standard curves established for quantification were greater than 0.9976. The limit of quantification (LOQ) for the method were found to be 0.3–3.0 ng/mL in human blood and 0.06–0.6 ng/mL in urine. The recoveries of spiked target compounds at different concentrations in human blood and urine were 91.8–111.9% and 86.9–105.3%, respectively. Inter- and intra-day precision values were both less than 12.5%, and the matrix effects of human blood and urine samples for ARs were 75.3–108.6% and 102.7–130.0%, respectively. This method had successfully applied to the emergency detection of ARs in biological samples of poisoned patients.

Development, validation, and application of a multi-method for the determination of mycotoxins, plant growth regulators, tropane alkaloids, and pesticides in cereals by two-dimensional liquid chromatography\xa0tandem mass spectrometry

Mycotoxins and pesticides regularly co-occur in agricultural products worldwide. Thus, humans can be exposed to both toxic contaminants and pesticides simultaneously, and multi-methods assessing the occurrence of various food contaminants and residues in a single method are necessary. A two-dimensional high performance liquid chromatography tandem mass spectrometry method for the analysis of 40 (modified) mycotoxins, two plant growth regulators, two tropane alkaloids, and 334 pesticides in cereals was developed. After an acetonitrile/water/formic acid (79:20:1, v/v/v) multi-analyte extraction procedure, extracts were injected into the two-dimensional setup, and an online clean-up was performed. The method was validated according to Commission Decision (EC) no. 657/2002 and document N° SANTE/12682/2019. Good linearity (R2 > 0.96), recovery data between 70-120%, repeatability and reproducibility values < 20%, and expanded measurement uncertainties < 50% were obtained for a wide range of analytes, including very polar substances like deoxynivalenol-3-glucoside and methamidophos. However, results for fumonisins, zearalenone-14,16-disulfate, acid-labile pesticides, and carbamates were unsatisfying. Limits of quantification meeting maximum (residue) limits were achieved for most analytes. Matrix effects varied highly (−85 to +1574%) and were mainly observed for analytes eluting in the first dimension and early-eluting analytes in the second dimension. The application of the method demonstrated the co-occurrence of different types of cereals with 28 toxins and pesticides. Overall, 86% of the samples showed positive findings with at least one mycotoxin, plant growth regulator, or pesticide.Graphical abstract

Development and validation of LC-MS/MS method for imatinib and norimatinib monitoring by finger-prick DBS in gastrointestinal stromal tumor patients.

The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 μL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 μm, 2x50 mm) while the trapping column was a POROS R1/20 (20 μm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.

Fast LC-ESI-MS/MS analysis and influence of sampling conditions for gut metabolites in plasma and serum

In the past few years, the gut microbiome has been shown to play an important role in various disorders including in particular cardiovascular diseases. Especially the metabolite trimethylamine-N-oxide (TMAO), which is produced by gut microbial metabolism, has repeatedly been associated with an increased risk for cardiovascular events. Here we report a fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method that can analyze the five most important gut metabolites with regards to TMAO in three minutes. Fast liquid chromatography is unconventionally used in this method as an on-line cleanup step to remove the most important ion suppressors leaving the gut metabolites in a cleaned flow through fraction, also known as negative chromatography. We compared different blood matrix types to recommend best sampling practices and found citrated plasma samples demonstrated lower concentrations for all analytes and choline concentrations were significantly higher in serum samples. We demonstrated the applicability of our method by investigating the effect of a standardized liquid meal (SLM) after overnight fasting of 25 healthy individuals on the gut metabolite levels. The SLM did not significantly change the levels of gut metabolites in serum.

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

Analysis of 2-Aminobenzoic Acid-Labeled Monosaccharides and Glycoprotein-Derived Oligosaccharides by Online Cleanup Liquid Chromatography in the Reversed-Phase and Hydrophilic Interaction Liquid Chromatography Modes

A robust LC-MS/MS assay with online cleanup for measurement of serum testosterone.

Accurate measurement of low levels of testosterone is critical for diagnosis and treatment of androgen disorders. The very low concentrations of testosterone in children, females, and males with androgen suppression therapies necessitate the use of mass spectrometry-based methods. We aimed to develop a liquid chromatography with tandem mass spectrometry method with simplified sample preparation and online solid-phase extraction cleanup to achieve enhanced precision, accuracy, robustness, and cost-effectiveness. The assay was linear from 10 to 20 000pg/mL with an analytical recovery of 93-104%. The total coefficient of variation was 2.5, 1.9, and 1.7% at concentration levels of 348, 5432, and 10 848pg/mL, respectively. No significant carryover was observed from samples with concentrations up to 20 000pg/mL. No significant interference was observed from androstenedione, dehydroepiandrosterone, epi-testosterone, and estriol. Comparison with CDC Hormone Standardization program (HoSt) reference samples with defined values (n=40) showed a Deming regression slope of 0.963, intercept of 28.06pg/mL, standard error of estimate was 66.9, a correlation coefficient of 0.9996, and a mean bias of -0.6%. The method met the accuracy criteria by the CDC HoSt program. In addition, we achieved >12 000 injections on a single analytical column without significant performance deterioration due to the specific online solid-phase extraction settings.

Simultaneous determination of pesticides, mycotoxins, and metabolites as well as other contaminants in cereals by LC-LC-MS/MS

A 2D LC-MS/MS method for the simultaneous determination of 350 pesticides, 16 mycotoxins as well as the growth regulators Chlormequat and Mepiquat was developed. The method is applicable to cereals and products thereof. Attention should be paid to the simultaneous analysis of the cereal-relevant mycotoxins aflatoxin B1, B2, G1 and G2, ochratoxin A, deoxynivalenol and zearalenone. Moreover, the tropane alkaloids atropine/scopolamine could be integrated into the final method. The samples were extracted with a mixture of acetonitrile/water (80:20), diluted with acetonitrile and injected into an LC-LC-MS/MS system. There were no further manual clean-up steps. The automatic online clean-up took place during the HILIC-separation in the first dimension (YMC-Pack Diol; 2.1 × 100 mm; 5 μm, 120 Å). Here, polar matrix compounds were retained, while the majority of the analyte scope eluted in a fraction at the beginning of the analytical run. This fraction was transferred to the second dimension by a packed loop interface (Agilent Zorbax SB-C8; 4.6 × 12.5 mm; 5 μm; 80 Å). On the second column (Phenomenex Synergi Fusion RP C18; 2 × 100 mm; 2.5 μm; 100 Å), the majority of the scope was separated by a typical RP-gradient. Only some of the polar pesticides could not be transferred to the second column. They eluted directly after the transfer step from the HILIC-column to the MS/MS. The final method was sensitive enough to meet all the regulated maximum levels for pesticides in cereals according to EU Regulation 396/2005 and those for contaminants according to EU Regulation 1881/2006. Above all, the method was so robust and accurate that nearly 90% of the pesticides and all the tested mycotoxins, growth regulators and tropane alkaloids fulfilled the validation criteria of the SANTE guideline document, although the demanding criteria are only applicable to pesticides. For the verification, eight proficiency tests were passed successfully: three for the pesticide analysis, three for the mycotoxin analysis, and two for the analysis of the tropane alkaloids. In addition to the already mentioned contaminants, the six most important ergot alkaloids (e.g. ergotamine/ergotaminine) and two modified mycotoxins (deoxynivalenol-3-glucoside and zearalenone-sulfate, also known as masked mycotoxins) were detected during the routine analysis of rye and corn samples.

Rapid determination of sulfonamides in chicken using two-dimensional online cleanup mode with three columns coupled to liquid chromatography-tandem mass spectrometry

A novel method based on online cleanup mode combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. After an automated sample cleanup system with aqueous gel column, sulfonamides in chicken were detected in multiple reaction monitoring (MRM) mode. The total run time of the system, which included automated extraction, analytical chromatography and re-equilibration, was within 30 min. Different experimental processes containing extraction, purification, separation, and detection have been evaluated respectively to obtain optimized parameters. The developed method was fully validated and the efficient and superior performance of the developed method was demonstrated. The method produced linear results for all sulfonamides from 1 to 10 ng g−1 with a linearity >0.99. The intra-day precision of the method was <8.45% while the inter-day precision was <9.11%. The matrix effect was 77.5% to 105.1%. The recovery was in the range of 72.66% to 116.7% for all sulfonamides. The limit of quantitation in the chicken was 0.6 ng g−1 and the limit of detection was 0.2 ng g−1. Compared with traditional procedures, the automated sample clean-up strategy could significantly shorten the analysis time and offer higher detectability, with the advantage of sufficient sensitivity. Also, the use of gel chromatography column employed the water phase and reduced the organic reagent to achieve the level of green chemistry.

Determination of Lipophilic Marine Biotoxins in Shellfish by Online Turbulent Flow Chromatography Coupled to Liquid Chromatography–Tandem Mass Spectrometry

A novel method based on online turbulent flow chromatography (TFC) combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been established for simultaneous quantitative determination of 11 lipophilic marine toxins (OA, DTX1, DTX2, YTX, hYTX, AZA1, AZA2, AZA3, PTX2, SPX1, and GYM) in shellfish. Sample preparation, including extraction with 75% methanol aqueous solution, centrifugation, and filtration, was followed by online cleanup using TFC. The total run time was within 15.5 min, including online purification, chromatographic separation, and re-equilibration of the TFC system. Various experimental parameters including extraction, purification, separation, and detection were optimized separately in this study. The developed method was validated, and good performance characteristics were obtained. The linear regression coefficients (R2) of matrix-match calibration standard curves established for quantification were higher than 0.9945. The limits of detection and quantification of this method ranged between 0.5 and 4.0, and 1.5 and 10.0 µg/kg, respectively. The average recoveries of spiked target compounds with different concentrations ranged from 80.1 to 113.8%, the intra- and inter-day precision were lower than 12.7% and 14.9%, respectively. The matrix effects were in the range of 76.1–115.9%. The method accuracy was also confirmed with certified reference materials. The results indicate that the developed method has great potential for the routine laboratory analysis or emergency detection of lipophilic marine toxins in shellfish.

Simultaneous determination of pesticides, mycotoxins, tropane alkaloids, growth regulators, and pyrrolizidine alkaloids in oats and whole wheat grains after online clean-up via two-dimensional liquid chromatography tandem mass spectrometry

In this study, a two-dimensional liquid chromatography tandem mass spectrometry method was developed and validated for the determination of pesticide residues and contaminants in whole wheat grains and oats. The samples were extracted with a mixture of acetonitrile and water and were injected into the two-dimensional LC-MS/MS system without any further clean-up or sample preparation. Samples were analyzed with four different matrix matched calibrations. Matrix effects were evaluated by comparing analyte signals in the respective matrix matched standard with the neat solvent standards. The final method was validated according to the current Eurachem validation guide and SANTE document. The number of successfully validated analytes throughout all three validation levels in oats and wheat, respectively, were as follows: 330 and 316 out of 370 pesticides, 6 and 13 out of 18 pyrrolizidine alkaloids and 7 out of 9 regulated mycotoxins. Moreover, both plant growth regulators mepiquat and chlormequat as well as the tropane alkaloids atropine and scopolamine met the validation criteria. The majority of pesticides showed limits of detection below 1 µg kg−1, pyrrolizidine alkaloids below 0.7 µg kg−1, tropane alkaloids below 0.2 µg kg−1, growth regulators below 0.7 µg kg−1 and mycotoxins below 8 µg kg−1 in both matrices.

Development of an SPE Method Using Ionic Liquid-Based Monolithic Column for On-Line Cleanup of Human Plasma and Simultaneous Determination of Five Steroid Drugs

An 1-vinyl-3-hexyl imidazolium bromide ionic liquid-based monolithic column was prepared via in situ free radical polymerization, which was used as the sorbent of on-line solid phase extraction. The prepared material was characterized by scanning electron microscopy, nitrogen adsorption–desorption instrument and mercury intrusion porosimetry, respectively, and the results showed that the homemade monolith occupied relatively uniform porous structure and high-specific surface area of 212.53 m2 g−1. On-line solid phase extraction–high-performance liquid chromatography was performed to quantitatively analyze five steroid drugs, including betamethasone, norgestrel, halcinonide, beclomethasone dipropionate and testosterone propionate in human plasma. The determination procedure was carried out following the optimization of the mobile phase composition. Methodological validation showed that correlation coefficients of the linear regressions were in the range of 0.9989–0.9992; the values of relative standard deviation for precision were in the range of 1.39–6.15% for intra-day and 0.32–6.34% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 99.02–101.98, 93.27–102.89, 102.79–104.01, 100.04–102.61 and 100.77–102.44% for the five drugs in order, respectively; the values of relative standard deviation for repeatability calculated according to retention times and peak areas of the five drugs were in the ranges of 0.28–0.76% (n = 5) and 0.84–2.63% (n = 5), respectively. The results showed that the polymer monolithic column was feasible for an on-line solid phase extraction column, exhibiting good selectivity and high permeability.

Determination of vitamin D in oily drops using a column-switching system with an on-line clean-up by supercritical fluid chromatography

A column-switching system, composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was developed for the analysis of vitamin D in oily and fatty matrices. The SFC with the similar retention behavior as normal-phase liquid chromatography (NPLC), was applied for an on-line clean-up of oily and fatty samples, then followed by separation and detection using a reverse-phase LC-MS/MS. Three SFC columns packed with materials of different functional groups (Silica, NH2, Diol) were compared and the column with diol groups, on which the retention time of vitamin D was the longest, was finally selected for purification of the samples. 100% methanol was chosen to carry vitamin D from the clean-up column to the pre-treatment column. It was also used as the mobile phase for the separation of vitamin D on a reverse phase C18 column. Vitamin D2 and D3 were baseline separated by using this system. The linearity was calculated with a value of coefficient of determination (r2) ≥ 0.998. The linear range is from 20 ng/mL to 200 ng/mL. Two kinds of liquid vitamin D3 supplements (Baby Ddrops and Vitamin AD drops) were directly analyzed using this system without any fussy preparation procedure. The limit of detection (LOD) for vitamin D3 in the two oily samples was estimated to be 10 ng/mL. The relative standard deviations (RSD) of intra- and inter-day precision, repeatability were 1.47%, 2.43% and 1.59% for Baby Ddrops and 5.76%, 8.24% and 5.86% for Vitamin AD drops. The recoveries vary between 84.3% and 102.8% with 7.1% RSD for Baby Ddrops and 90.8–109.6% with 5.83% RSD for Vitamin AD drops, respectively. These results suggest that the method based on the SFC-RPLC/MS column-switching system is simple and suitable for analysis of vitamin D in oily and fatty samples.

Automated clean-up, separation and detection of polycyclic aromatic hydrocarbons in particulate matter extracts using a 2D-LC/2D-GC system: a method translation from two FIDs to two MS detectors

An online two-dimensional (2D) liquid chromatography/2D gas chromatography system with two mass-selective detectors has been developed on the basis of a previous system with two flame ionization detectors. The method translation involved the change of carrier gas from hydrogen to helium, column dimension and detectors. The 2D system with two mass-selective detectors was validated with use of polycyclic aromatic hydrocarbon (PAH) standards and two standard reference materials from air and diesel exhaust. Furthermore, the system was applied to a real sample, wood smoke particulates. The PAH values determined correlated well with the previous data and those from the National Institute of Standards and Technology. The system enhanced the benefits of the previous system, which were limited by the low detectability and lack of mass selectivity. This study shows an automated 2D system that is valid for PAH analysis of complex environmental samples directly from crude extracts.Graphical Schematic illustration showing on-line clean-up, separation and detection using 2D-LC/2D-GC/MS

On-line coupling of molecularly imprinted solid phase extraction with liquid chromatography for the fast determination of coumarins from complex samples

In this work, an on-line SPE-HPLC method with spectrophotometric detection was developed for the determination of coumarins in complex samples. For the on-line cleanup of samples, a molecularly imprinted polymer was packed into the column cartridge and coupled directly with HPLC (MISPE-HPLC) using a column switching system. The separation of coumarins was performed on a C18 core-shell column (100×4.6mm, 5μm) with a mobile phase consisting of 0.3% acetic acid/acetonitrile with gradient elution at a flow-rate of 1mLmin−1. The total time of the whole analytical run, including the extraction step, was 13.25min. The on-line MISPE-HPLC method was optimized and validated. The results showed good linearity (0.10–100μgmL−1) with correlation coefficients higher than 0.99. The LOD values were from 0.03 to 0.15μgmL−1. The proposed method was successfully applied for analysis of real samples (Cassia cinnamon, chamomile tea, and Tokaj specialty wines) and obtained recoveries varied from 78.7% to 112.2% with an RSD less than 9%.

On-line clean-up and LC-MS analysis of primary metabolites in cell culture supernatants

A novel automated clean-up procedure for supernatants of Chinese hamster ovary cell cultures enhances the accuracy of results obtained via LC-MS analysis of primary metabolites.

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

Automated Strong Cation-Exchange Cleanup To Remove Macromolecular Crowding Agents for Protein Hydrogen Exchange Mass Spectrometry.

Measuring amide hydrogen exchange (HX) of intrinsically disordered proteins (IDPs) in solutions containing high concentrations of macromolecular crowding agents would give new insights into the structure and dynamics of these proteins under crowded conditions. High concentrations of artificial crowders, required to simulate cellular crowding, introduce overwhelming interferences to mass spectrometry (MS) analysis. We have developed a fully automated, dual-stage online cleanup that uses strong cation-exchange (SCX) followed by reversed-phase desalting to remove Ficoll, a synthetic polymer, for HX-MS analysis of proteins under crowded conditions. We tested the efficiency of our method by measuring the HX-MS signal intensities of myoglobin peptides from crowded samples containing 300 g L-1 Ficoll and from uncrowded samples. Although there was loss of abundance relative to uncrowded myoglobin analyzed using conventional HX-MS, 97% coverage of the myoglobin sequence was still obtained. Control HX-MS experiments using unstructured peptides labeled at pD 4.0 under crowded and uncrowded conditions confirmed that Ficoll does not alter chemical exchange and that the same extent of HX is achieved in uncrowded solutions as in solutions containing 300 g L-1 of predeuterated Ficoll. We validated our method by measuring HX of CBP, the intrinsically disordered nuclear coactivator binding domain of CREB binding protein (UniProt CBP_MOUSE P45481 ), residues 2059-2117, at pD 6.5 under crowded and uncrowded conditions. Ficoll induced both protection and deprotection from HX in different regions of CBP, with the greatest deprotection occurring at the edges of helices. These results are consistent with previous observation of IDPs under the influence of synthetic polymers.

Matrix solid-phase dispersion combined to liquid chromatography–tandem mass spectrometry for the determination of paraben preservatives in mollusks

A method for the extraction and determination of seven parabens, esters of 4-hydroxybenzoic acid, widely used as preservatives in personal care products, pharmaceuticals, etc., and two chlorinated derivatives (mono- and di-chloro methyl paraben) from mollusk samples was developed by combining matrix solid-phase dispersion (MSPD) and liquid chromatography–tandem mass spectrometry. MSPD parameters, such as solvent, solid support and clean-up sorbent, were optimized. Besides, since blank problems were observed for some parabens, these were investigated and blanks were tackled by precleaning all sorbents prior to use. Under final conditions, 0.5g of freeze-dried mollusk were dispersed with 1.2g of silica and packed into a cartridge containing 3g of C18, as on-line clean-up sorbent. This cartridge was eluted with 10mL of acetonitrile, evaporated and reconstituted in methanol for analysis. In the validation stage, successful linearity (R2>0.999), recoveries (between 71 and 117% for most analytes), precision (RSD lower than 21%) and limits of detection and quantification (LOD and LOQ, lower than 0.4 and 1.4ngg−1 dry weight respectively) levels were achieved. Finally, the new methodology was applied to mussel, clam and cockle samples. Methyl paraben was above the LOQ in five of the six samples (not found in one clam sample) at concentrations up to 7ngg−1 dry weight. Ethyl paraben was found above the LOQ in mussel and cockle samples at a concentration level around 0.3ngg−1. n-Propyl paraben was only above the LOQ in one mussel sample.