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World Congress on Biosensors 2014

World Congress on Biosensors 2014
Biosensors 2014

Wednesday, 26 September 2012

Just Published: Journal of Chromatography A


A new issue of this journal has just been published. To see abstracts of the papers it contains (with links through to the full papers) click here:
Journal of Chromatography A
Selected papers from the latest issue:

Graphene-supported zinc oxide solid-phase microextraction coating with enhanced selectivity and sensitivity for the determination of sulfur volatiles in Allium species

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Suling Zhang, Zhuo Du, Gongke Li
A graphene-supported zinc oxide (ZnO) solid-phase microextraction (SPME) fiber was prepared via a sol–gel approach. Graphite oxide (GO), with rich oxygen-containing groups, was selected as the starting material to anchor ZnO on its nucleation center. After being deoxidized by hydrazine, the Zn(OH)2/GO coating was dehydrated at high temperature to give the ZnO/graphene coating. Sol–gel technology could efficiently incorporate ZnO/graphene composites into the sol–gel network and provided strong chemical bonding between sol–gel polymeric SPME coating and silica fiber surface, which enhanced the durability of the fiber and allowed more than 200 replicate extractions. Results indicated that pure ZnO coated fiber did not show adsorption selectivity toward sulfur compounds, which might because the ZnO nanoparticles were enwrapped in the sol–gel network, and the strong coordination action between Zn ion and S ion was therefore blocked. The incorporation of graphene into ZnO based sol–gel network greatly enlarged the BET surface area from 1.2m2/g to 169.4m2/g and further increased the adsorption sites. Combining the superior properties of extraordinary surface area of graphene and the strong coordination action of ZnO to sulfur compounds, the ZnO/graphene SPME fiber showed much higher adsorption affinity to 1-octanethiol (enrichment factor, EF, 1087) than other aliphatic compounds without sulfur-containing groups (EFs<200). Also, it showed higher extraction selectivity and sensitivity toward sulfur compounds than commercial polydimethylsiloxane (PDMS) and polydimethylsiloxane/divinylbenzene (PDMS/DVB) SPME fibers. Several most abundant sulfur volatiles in Chinese chive and garlic sprout were analyzed using the ZnO/graphene SPME fiber in combination with gas chromatography–mass spectrometry (GC–MS). Their limits of detection were 0.1–0.7μg/L. The relative standard deviation (RSD) using one fiber ranged from 3.6% to 9.1%. The fiber-to-fiber reproducibility for three parallel prepared fibers was 4.8–10.8%. The contents were in the range of 1.0–46.4μg/g with recoveries of 80.1–91.6% for four main sulfides in Chinese chive and 17.1–122.6μg/g with recoveries of 73.2–80.6% for three main sulfides in garlic sprout.

Highlights

► A graphene-supported zinc oxide SPME fiber via a sol–gel approach was originally developed. ► The selectivity and sensitivity of ZnO/graphene SPME coating to sulfur compounds were excellent. ► The ZnO/graphene SPME was successfully applied to determine sulfur volatiles in complex samples.

Ultra trace determination of fluorobenzoic acids in tap and reservoir water using solid-phase extraction and gas chromatography–mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Karsten Müller, Andreas Seubert
A method for the ultra trace analysis of 21 fluorobenzoic acids (FBAs) via GC–MS based on solid-phase extraction (SPE) and derivatization with BF3·MeOH is described. All fluorobenzoic acids were enriched and determined simultaneously. Solid-phase extraction on hydrophilic–lipophilic-balanced reversed-phase cartridges containing a poly(divinylbenzene-co-N-vinylpyrrolidone) polymer allowed a 250-fold enrichment of the acids if 100mL sample volume is used with extraction efficiencies between 71% and 94%. The method enables the determination of fluorobenzoic acid methyl esters (FBAMEs) down to the range of 6–44ngL−1 combined with a fast and easy sample-preparation (pH-adjusting prior to SPE and derivatization within 24h at 64°C directly in the vial). It uses low amounts of chemicals and is adaptable to larger and smaller sample volumes. Simultaneous extraction and determination of 21 fluorinated aromatic acids in reservoir samples with high salinity confirmed the applicability and reproducibility of the method.

Highlights

► New method for the derivatization of fluorobenzoic acids using BF3·MeOH. ► New SPE method using a material never used before for this application. ► Quantitative extraction of all commercially available 23 FBAs. ► Determination of all 23 fluorobenzoic acid methyl esters in the ngL−1 range. ► Capability of the method for highly saline reservoir water samples is shown.

Stir bar sorptive extraction approaches with a home-made portable electric stirrer for the analysis of polycyclic aromatic hydrocarbon compounds in environmental water

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Xiangju Mao, Bin Hu, Man He, Wenying Fan
In this study, novel off/on-site stir bar sorptive extraction (SBSE) approaches with a home-made portable electric stirrer have been developed for the analysis of polycyclic aromatic hydrocarbon compounds (PAHs). In these approaches, a miniature battery-operated electric stirrer was employed to provide agitation of sample solutions instead of the commonly used large size magnetic stirrer powered by alternating current in conventional SBSE process, which could extend the SBSE technique from the conventional off-site analysis to the on-site sampling. The applicability of the designed off/on-site SBSE sampling approaches was evaluated by polydimethylsiloxane (PDMS) coating SBSE-high performance liquid chromatography-fluorescence detection (HPLC-FLD) analysis of six target PAHs in environmental water. The home-made portable electric stirrer is simple, easy-to-operate, user friendly, low cost, easy-to-be-commercialized, and can be processed in direct immersion SBSE, headspace sorptive extraction (HSSE) and continuous flow (CF)-SBSE modes. Since the stir bar was fixed onto the portable device by magnetic force, it is very convenient to install, remove and replace the stir bar, and the coating friction loss which occurred frequently in conventional SBSE process could be avoided. The parameters affecting the extraction of six target PAHs by the home-made portable SBSE sampling device with different sampling modes were studied. Under the optimum extraction conditions, good linearity was obtained by all of three SBSE extraction modes with correlation coefficient (R) higher than 0.9971. The limits of detection (LODs, S/N=3) were 0.05–3.41ngL−1 for direct immersion SBSE, 0.03–2.23ngL−1 for HSSE and 0.09–3.75ngL−1 for CF-SBSE, respectively. The proposed portable PDMS-SBSE-HPLC-FLD method was applied for the analysis of six target PAHs in East Lake water, and the analytical results obtained by on-site SBSE sampling were in good agreement with that obtained by off-site SBSE sampling. The accuracy of the developed method was evaluated by recovery test and the recoveries for the spiked sample were found to be in the range of 87.1–122.8% for off-site CF-SBSE, 88.8–114.3% for on-site sampling, and 87.7–123.6% for off-site SBSE, respectively. The developed method is one of the most sensitive methods for PAHs determination and the home-designed SBSE system is feasible for the field sampling.

Highlights

► A portable stir bar sorptive extraction system was designed for on-site sampling. ► It can be used for off/on-site sampling with different extraction modes. ► The portable SBSE system was applied for HPLC analyzing six PAHs in lake water. ► The portable SBSE system shows a great application potential in field sampling.

Simultaneous determination of 10 β2-agonists in swine urine using liquid chromatography–tandem mass spectrometry and multi-walled carbon nanotubes as a reversed dispersive solid phase extraction sorbent

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Xiang-Dang Du, Yin-Liang Wu, Hong-Ju Yang, Ting Yang
A simple and inexpensive pretreatment procedure was developed for 10 β2-agonists (clenbuterol, ractopamine, salbutamol, bambuterol, penbuterol, tulobuterol, clorprenaline, mabuterol, cimaterol and terbutaline) in swine urine using dispersive solid phase extraction (dSPE) with multi-walled carbon nanotubes (MWCNTs). The sample was analysed after purification by ultra high performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry (UHPLC-ESI–MS/MS). The pH value of the swine urine, extraction time, type and amount of MWCNTs and type of eluent were optimised to increase the sample throughput and sensitivity. The samples were quantified using clenbuterol-D9, ractopamine-D6 and salbutamol-D3 as internal standards. The recoveries of the target compounds from swine urine samples at pH 10.0 were most efficient when using 20mg of MWCNTs with a 30–50nm outer diameter and a length of 10–30μm, while a mixture of water/methanol (90:10, v/v) with 0.5% formic acid was shown to be the most suitable solvent for desorbing the compounds from the MWCNTs. The proposed method was validated according to the European Commission Decision 2002/657/EC, which determines linearity, specificity, decision limit (CCα), detection capability (CCβ), recovery, precision and stability.

Highlights

► A simple and cheap method was developed for β2-agonists using dSPE with MWCNTs. ► The pH, extraction time, the type and amount of MWCNTs were optimised. ► The proposed method was validated according to 2002/657/EC. ► The whole method was fast, reliable, convenient and sensitive.

Investigation of protocols to extraction and quantification of folates in vegetables matrices split into liquor and fiber fraction using factorial design

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Emmanuela Prado de Paiva, Clayton Anderson de Azevedo Filho, Sabrina Gomes Ferreira, Tânia Lucia Montenegro Stamford, Jose Almiro da Paixão
The main protocols of extraction were investigated for the six folate forms in vegetable matrices, treated in two fractions, liquor and fiber. In a pilot study, it was used ammonium acetate added of 2-mercaptoetanol and ascorbic acid as extraction solution. The condition of use of protease and folate conjugase was evaluated, besides alternative treatments without enzyme use. Based on the results of this stage, it was built the factorial design 24, with three replications at the central point, using the following variables: temperature, time for reaction, molar concentration of the extraction solution and ratio sample/solution as independent variables and dependent variable, the amount of each folate form extracted as well as spectral and chromatographic parameters. In the pilot study it was verified that the enzyme use can cause an increase in the variability of the folate content, which enabled to build the factorial design without the enzyme use. The binomial time and temperature showed greatest impact on the extraction profile, besides high concentrations of ammonium acetate resulting in bifurcation of some peaks. 5-Methyltetrahydrofolate was extracted primordially in the liquor fraction, indicating that this treatment on the matrix provoked suitable extraction condition to this folate.

Highlights

► Statistic factorial design confirms the binomial impact of time and temperature. ► Low temperature and short time are indicated as the best extraction conditions. ► 5-MTHF was suitability extracted from liquor fraction of all vegetable matrices.

Planar solid phase extraction clean-up for pesticide residue analysis in tea by liquid chromatography–mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Claudia Oellig, Wolfgang Schwack
Efficient clean-up is indispensable for preventing matrix effects in multi-residue analysis of pesticides in food by liquid and gas chromatography (LC and GC) coupled to mass spectrometry (MS). High-throughput planar solid phase extraction (HTpSPE) was recently introduced as a new clean-up concept in residue analysis of pesticides in fruit and vegetables (C. Oellig, W. Schwack, 2011 [45]). Thin-layer chromatography (TLC) was used to completely separate pesticides from matrix compounds and to focus them into a sharp zone, followed by extraction of the target zone by the TLC–MS interface. As rather challenging matrices, tea samples were chosen in this study. Besides chlorophylls and polyphenols, high amount of caffeine is co-extracted resulting in strong matrix effects both in LC–MS and GC–MS. The former HTpSPE procedure was adapted to initial extracts of green and black tea resulting in colorless extracts nearly free of matrix effects and interferences, as shown for seven chemically representative pesticides (acetamiprid, penconazole, azoxystrobin, chlorpyrifos, pirimicarb, fenarimol, and mepanipyrim). LC–MS/MS calibration curves obtained in the range of 0.002–0.5mg/kg from matrix-matched standards and solvent standards were nearly identical and demonstrated the effectiveness of clean-up by HTpSPE. Mean recoveries determined by LC–MS/MS against solvent standards at spiking levels of 0.01 and 0.1mg/kg ranged between 72 and 114% with relative standard deviations (RSDs) of 0.7–4.7% (n =4), while LC–MS measurements of tea samples spiked at 1mg/kg provided recoveries of 81–104% with RSDs of 1.2–4.9% (n =6). Using LC–MS/MS, the method showed high sensitivity with signal-to-noise ratios >10 for concentrations below 0.002mg/kg. HTpSPE of one sample was done in a few minutes, while numerous samples were cleaned in parallel at minimal costs with very low sample and solvent consumption.

Highlights

► A new clean-up concept for pesticide residue analysis in tea by LC–MS is introduced. ► Planar solid phase extraction was proven to be the highly efficient clean-up method. ► Nearly matrix-free tea extracts generally avoided matrix effects in LC–MS(/MS). ► Applying pure solvent standards, method validation showed impressive results.

Investigation and optimization of particle dimensions for needle trap device as an exhaustive active sampler

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Weiqiang Zhan, Janusz Pawliszyn
Various needle trap devices (NTDs) with different designs have been developed during the past decade. A theoretical model on the fundamentals of the NTD was recently proposed, which employed the theory of frontal (gas–solid) chromatography to describe the sampling process. In the current work, different types of sorbent particles with different dimensions were packed into the needle as the adsorbent. The effects of particle dimensions, which would affect the packing density and consequently affect the capacity, the extraction efficiency and desorption efficiency of the NTD were experimentally investigated and the proposed theory was validated. The results demonstrated that NTDs packed with small particles possess higher extraction capacity and efficiency but much higher resistances to flow as well. The higher resistance did not necessarily result in poor desorption efficiency. The observed relationships among those physical parameters provide valuable guidance on how to design a NTD with high performance for future applications.

Highlights

► Frontal chromatography process is experimentally validated to describe the sampling process of a needle trap device. ► The trapping efficiency and desorption efficiency of a needle trap device in regard to particle dimensions are investigated and optimized. ► Effects of particles on the extraction of toluene, ethylbenzene and o-xylene are presented.

Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Amith D. Naik, Stefano Menegatti, Hannah R. Reese, Patrick V. Gurgel, Ruben G. Carbonell
The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

Highlights

► This paper deals with the purification of a monoclonal IgG from a plant extract. ► We used a two-step approach, with a charcoal treatment and affinity chromatography step. ► The final product shows high purity and recovery, competitive to commercial products.

Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Lieven Van Meulebroek, Julie Vanden Bussche, Kathy Steppe, Lynn Vanhaecke
Phytohormones are key signalling biomolecules and are of particular interest because of their regulating role in numerous physiological and developmental plant processes. Since the plant response to a given stimulus results amongst others from the complex interaction between phytohormones, there is a mounting interest for multiple phytohormone analysis. Therefore, with the primary aim of profiling the hormonal status of the tomato plant, a generic extraction protocol and an U-HPLC–Orbitrap-MS analysis were developed and validated for both tomato fruit and leaf tissue. To this end, eight phytohormones were considered, i.e. gibberellic acid, indol-3-acetic acid, abscisic acid, jasmonic acid, salicylic acid, zeatin, N6-benzyladenine and epibrassinolide, representing the major hormonal classes. The sample pre-treatment involved liquid extraction with a buffer of methanol, ultrapure water and formic acid (75:20:5, v/v/v), after which the extract was purified by means of an Amicon® Ultra centrifugal unit. Subsequently, analytes were chromatographically separated on a sub-2μm particles Nucleodur Gravity C18 column and detected by an Exactive™ high-resolution mass spectrometer. Validation of the analytical method demonstrated that linearity (≥0.99), precision (CV≤15%) and mean corrected recovery (between 80% and 110%) performed well for the majority of the eight targeted phytohormones. In addition, the generic nature of the extraction protocol and the full scan approach of the Orbitrap mass spectrometer allowed metabolomic profiling of the hormonal status of the tomato plant.

Highlights

► Development of a generic extraction for phytohormones from tomato plant tissue. ► Development of a full scan U-HPLC–Orbitrap-MS method for phytohormone analysis. ► Validation of the developed methods for both tomato leaf and fruit tissue. ► Demonstration of the metabolomic character of the developed methods.

Improved chemical stabilities for end-capped high performance liquid chromatography stationary phases based on poly(methyloctadecylsiloxane) thermally immobilized onto metalized silicas

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Carla G.A. da Silva, Carol H. Collins
Endcapped stationary phases were prepared after thermal immobilization of poly(methyloctadecylsiloxane) (PMODS) onto zirconized and titanized silica supports. These new stationary phases have lower densities of residual hydroxyl groups, according to infrared spectroscopy and 29Si CP-MAS NMR and as shown by the symmetrical peaks of basic compounds from the Tanaka, Engelhardt and SRM 870 test mixtures. Stability tests for the endcapped stationary phases, measured using severe alkaline conditions (70:30 (v/v) methanol:0.05mol/L K2CO3/KHCO3, pH 10, 50°C), revealed that the stabilities of these phases are greater than the stabilities of similar nonendcapped phases. The stationary phases showed good performance for the separation of basic pharmaceuticals.

Highlights

► HPLC support prepared by metallization of silica and thermal immobilization of PMODS onto the support. ► Endcapping reaction with hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS). ► Evaluation with several standardized test mixtures. ► Improvement of the chemical stability in severe alkaline conditions. ► Potential application for separation of basic pharmaceuticals.

Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Tobias K.H. Müller, Matthias Franzreb
The effect of temperature in the range from 10°C to 40°C and comparatively low ammonium sulfate (AS) concentrations of up to 0.5M on the adsorption of bovine serum albumin (BSA) on four different commercially available sepharose-based stationary phases was investigated. The determined isotherms were fitted by the Langmuir equation, and thermodynamic values were calculated by van’t Hoff analysis. The adsorption of BSA onto the chromatographic resin Butyl Sepharose 4FF showed the strongest temperature influence; however, protein unfolding effects occurred when characterizing this system by dynamic column experiments, with an unfolded BSA fraction strongly attached to the sorbent. The percentage of the unfolding fraction was determined for different operating conditions and found to increase with the concentration of the cosmotropic salt, but even stronger with increasing temperature. Temperature-induced cyclic adsorption and desorption experiments were carried out to investigate the long-term performance of Butyl Sepharose 4FF by applying purely temperature-controlled regeneration. Over a period of five cycles, the working capacity remained stable, but BSA also started to accumulate on the column due to incomplete regeneration. Finally, the possibility to fractionate different proteins with a single temperature shift was shown by the complete separation of lysozyme and BSA. The results presented indicate that temperature-induced binding and elution may offer a possibility to shift the operation conditions of HIC resins toward reduced salt concentrations, thus saving chemicals and facilitating salt removal in further downstream processing stages.

Highlights

► Temperature-dependent adsorption behavior of model proteins on HIC gels. ► Temperature as advantageous parameter regarding purification applications using HIC. ► Non-isothermal HIC resin regeneration. ► Temperature-mediated protein fractionation. ► Protein unfolding in dependence of ionic strength, temperature and pulse injections.

Size-exclusion-chromatography separation of randomly branched polymers with tetrafunctional branch points and local dispersity

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Miloš Netopilík
The SEC separation of a randomly branched polymer, in particular local dispersity due to branching, are theoretically examined. A model of the SEC separation of randomly branched polymer with tetrafunctional branch points enabling the estimation of local dispersity was developed. Measurable quantities (branching indices) were compared with real data. Local dispersity due to branching is demonstrated to depend on elution volume and degree of branching and in the area of the beginning of the elution curve it can reach non-negligible values.

Highlights

► SEC analysis of randomly branched polymer was modeled. ► Results were compared with experiment. ► Local dispersity in the frontal area of the elution curve was found.

Application of Doehlert uniform shell designs for selecting optimal amounts of internal standards in the analysis of prostaglandins and leukotrienes by liquid chromatography–tandem mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Pedro Araujo, Steve Janagap, Elisabeth Holen
A protocol for the analysis of multiple prostaglandins and leukotrienes in cell culture media by using multiple internal standards was validated. A two-factor Doehlert design was used to determine the behaviour of the relationship analyte/internal standard (namely: PGE2/PGE2-d 4, PGE3/PGE2-d 4, LTB4/LTB4-d 4 and LTB5/LTB4-d 4) and to select the optimal amounts of deuterated internal standards for quantifying simultaneously the prostaglandins and leukotrienes in cell culture media by LC–MS/MS. The selection of optimal amounts of internal standards was based on mathematical models that allow visualizing concentration regions where the response factors remain constant over a wide range of analytical concentrations. The linearity of the calibration curves for each analyte at the optimal levels suggested by the mathematical models was statistically confirmed by means of the ratio lack-of-fit to pure error. The validated protocol was successfully applied in the simultaneous quantification of pro- and anti-inflammatory eicosanoids in stimulated cod head kidney cell culture media. The two-factor Doehlert design has permitted to estimate the experimental response as a function of six variables (PGE2, PGE3, LTB4, LTB5, PGE2-d 4 and LTB4-d 4) which represents a substantial reduction of resources, time and experiments of approximately 84% (7×3 experiments) when compared with the full six-factor Doehlert design (43×3 experiments).

Highlights

► Rational design for selecting optimal amounts of multiple internal standards. ► Study of six variables with a Doehlert design intended for two variables. ► 84% reduction in resources compared to a six-factor Doehlert design.

Determination of sedatives and adrenergic blockers in blood meal using accelerated solvent extraction and Orbitrap mass spectrometry

26 September 2012, 10:35:32
Publication year: 2012
Source:Journal of Chromatography A, Volume 1260
Jeong-Heui Choi, Marc Lamshöft, Sebastian Zühlke, Ki Hun Park, Jae-Han Shim, Michael Spiteller
The detection of veterinary drugs in blood meal is needed since it is used as an environment-friendly agricultural material despite its origination from animal blood. A method using accelerated solvent extraction and liquid chromatographic linear ion trap quadrupole Orbitrap mass spectrometry was developed to determine sedatives and adrenergic blockers in blood meal. The determination method was established following optimizations of accelerated solvent extraction, dispersive solid-phase extraction and high resolution mass spectrometric detection. Linearity, sensitivity, accuracy, repeatability and reproducibility of the method were fully validated. The method was applied to commercial blood meal products.

Highlights

► Combination of ASE and LC–Orbitrap MS for sedatives and α-, β-blockers in blood meal. ► Dispersive solid-phase extraction as an efficient cleanup method. ► Good results of the sensitivity, linearity, accuracy and precision. ► Finding a metabolite of xylazine via the high resolution MS detection.

1 comment:

  1. Hi Analytical Chemistry,
    I loved reading this piece! Well written!

    Merlen Hogg
    hms

    ReplyDelete