Strong Cation Exchange Solid-phase Extraction Research Articles

Determination of Tetrodotoxin in Miso Soup byStrong Cation Exchange Solid Phase Extraction and LC-MS/MS

A method for analyzing tetrodotoxin (TTX) in miso soup samples was proposed. The samples were purified using strong cation exchange solid-phase extraction and analyzed by liquid chromatography-tandem mass spectrometry. The recovery of TTX was considerably influenced by the salt concentration in the loading solution during purification. It was observed that diluting the loading solution to reduce the salt concentration helped to maintain the recovery rate during the washing step. However, during the loading step, the benefit of dilution in improving recovery was not evident, as the enhanced retention on the solid phase caused by dilution was counteracted by losing TTX with the increased volume of the loading solution, which led to enhanced elution. This suggests that the volume of extraction solution used in the loading process is crucial in determining the recovery rate. Additionally, the use of volatile ammonium acetate as the elution solvent was explored to optimize conditions for effective recovery. Testing this method on miso soup samples with varying miso types, salt concentrations, and TTX levels resulted in consistently high recovery rates of>80%.

Pharmacokinetic study of osilodrostat and identification of mono-hydroxylated metabolite in equine plasma for the purpose of doping control.

Osilodrostat is an inhibitor of 11-beta-hydroxylase (CYP11B) and is used for the treatment of Cushing's disease but also categorized as an anabolic agent. The use of osilodrostat is prohibited in horseracing and equestrian sports. To the best of our knowledge, this is the first metabolic study of osilodrostat in equine plasma. Potential metabolites of osilodrostat were identified by differential analysis using data acquired from pre- and post-administration plasma samples after protein precipitation with liquid chromatography electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS). [Correction added on 27 January 2023, after first online publication: In the preceding sentence, "C-HRMS" was changed to "LC/ESI-HRMS" in this version.] For quantification of osilodrostat, a strong cation exchange solid-phase extraction was employed, and the extracts were analyzed using LC/ESI-triple quadrupole tandem mass spectrometry (LC/ESI-QqQ-MS/MS)to establish its elimination profile. Such extracts were further analyzed using LC/ESI-HRMS to investigate the detectability of osilodrostat and its identified mono-hydroxylated metabolite over a 2-week sampling period. Mono-hydroxylated osilodrostat was identified based on the differential analysis and mass spectrometric interpretations, and it was found to be the most abundant metabolite in plasma. Elimination profile of osilodrostat in plasma was successfully established over the 24-h post-administration period. Both osilodrostat and its mono-hydroxylated metabolite were detected up to the last sampling point at 2weeks using HRMS, and osilodrostat could be confirmed up to 8-day post-administration with its reference material using HRMS as well. For doping control, screening of both the parent drug osilodrostat and its mono-hydroxylated metabolite in equine plasma would be recommended due to their extended detection windows of up to 2weeks. Given the availability of reference material for potential confirmation in forensic samples, osilodrostat is considered the most appropriate monitoring target.

A New Broad-Spectrum Drug Screen for 127 Analytes by LC-MS/MS.

Broad-spectrum drug screening is offered by many clinical laboratories to support investigation of possible drug exposures. The traditional broad-spectrum drug screen employed at our laboratory utilizes several different analytical platforms, thus requiring relatively high volumes of sample and a cumbersome workflow. Here we describe the development and validation of a consolidated broad-spectrum drug screen assay designed to qualitatively detect 127 compounds in urine (Ur) and serum/plasma (S/P) samples. An LC-MS/MS method was developed using the Ultivo LC-MS/MS and designed to be qualitative with a 1-point calibration curve and 50% to 150% controls. Sample preparation included the addition of 122 internal standards (IS) followed by mixed-mode strong cation exchange solid-phase extraction and reverse-phase chromatographic separation on a biphenyl column. For the method described herein, ≥ 95% of analytes in urine and serum control samples had a CV of ≤20% for total imprecision. Accuracy testing included 46 external controls and demonstrated 99.9% accuracy. Method comparison studies to quantitative testing are discussed. The high level of coverage of the analytes with a stable isotope-labeled IS (SIL-IS) helped normalize for matrix effects when significant ion suppression (>25%) was present. Analyte stability in the matrix, the impact of potentially interfering compounds, and method ruggedness were demonstrated. Method limitations include limited detection of glucuronidated drugs and potential cross-contamination with samples at very high concentrations (>>100 × cutoff). The broad-spectrum drug screen method developed here qualitatively detected 127 drugs and select metabolites. This method could be used to support investigations of possible drug exposures in a clinical setting.

THE ANTIBACTERIAL PEPTIDES (AMPs) ORIGINATED FROM TRYPTIC HYDROLYSIS OF Naja sumatrana VENOM FRACTIONATED USING CATION EXCHANGE CHROMATOGRAPHY

Bioactive peptides bring revolutions in the medical field and become an attractive target for new antibiotics. The bioactive peptides can be derived from the hydrolysis of protein. This study aims to explore the antibacterial peptide from venom hydrolysate of the Spitting cobra (Naja sumatrana). The venom protein was isolated from venom using a gel filtration technique. The digestion of venom protein was performed using trypsin with and without alkylation and reduction. After that, fractionation was carried out using a strong cation exchange solid-phase extraction, followed by antibacterial activity testing. The active peptides present in the active fractions were identified using HRMS. The protein hydrolysis with pretreatment using alkylation and reduction gave almost two-fold higher than that of untreated hydrolysis. Both the venom protein and the hydrolysate showed antibacterial activity against Escherichia coli but there was no activity against Staphylococcus aureus. Antibacterial activity has been detected in numerous cation exchange fractions against Escherichia coli and Staphylococcus aureus. The four best bioactive peptides were identified with amino sequences of VYGGDSR, YTPTNK, TQFSDR, and TFQDSR. In addition, molecular docking analysis revealed that the peptides showed high-affinity scores, and the model best matched the DNA gyrase subunit B by salt bridge formation, whereas hydrogen bond interactions proved inhibition of bacterial replication as an antibacterial mechanism.

Extraction and detection of quaternary ammonium ionic liquid cations in water samples

The recent development of ionic liquids may induce environmental pollution, yet there is a lack of sensitive analytical methods to detect traces of ionic liquids, notably for quaternary ammonium ionic liquids. Here, we set up a method based on solid-phase extraction and ion chromatography combined to analyze quaternary ammonium ionic liquid cations in river water samples. Cations included tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium. We used methanesulfonic acid–acetonitrile as mobile phase in a cation-exchange column with carboxylic acid functional groups. A strong acid cation-exchange solid-phase extraction column was used for extraction and enrichment, then water–methanol was eluted to wash impurities, and finally the analytes were eluted with H3PO4–KH2PO4 buffer solution because this buffer has a low conductivity signal which avoids interferences with cation detection. We obtained a detection limit of 0.003 mg/L for river samples. Precision and accuracy met the requirements of quantitative analysis.

Sulfonic Acid-Functionalized SBA-15 as Strong Cation-Exchange Sorbent for Solid-Phase Extraction of Atropine and Scopolamine in Gluten-Free Grains and Flours.

A novel method was developed and applied to the determination of the most representative tropane alkaloids (TAs), atropine and scopolamine, in gluten-free (GF) grains and flours by HPLC-MS/MS. Accordingly a suitable sample treatment procedure based on solid-liquid extraction (SLE) and followed by strong cation-exchange solid-phase extraction (SCX-SPE) was optimized. SBA-15 mesostructured silica functionalized with sulfonic acids was evaluated as sorbent. The proposed method was fully validated in sorghum flour showing good accuracy with recoveries in the range of 93–105%, good linearity (R2 > 0.999) and adequate precision (RSD < 20%). Low method quantification limits (MQL) were obtained (1.5 and 2.4 µg/kg for atropine and scopolamine, respectively) and no matrix effect was observed thanks to the extraction and clean-up protocol applied. The method was applied to 15 types of GF samples of pseudocereals (buckwheat, quinoa and amaranth), cereals (teff, corn and blue corn, sorghum and millet) and legumes (red and green lentil, chickpea and pea). Atropine was found above the MQL in eight of them, with values between 7 and 78 µg/kg, while scopolamine was only found in teff flour, its concentration being 28 µg/kg. The method developed is an interesting tool for determining TAs in a variety of samples of GF grains and flours.

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone–releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized “hematocrit effect.”Graphical abstract

The establishment of a practical method for the determination of piperazine residues using accelerated solvent extraction and UHPLC-FLD

This article describes a reliable method for estimating piperazine residues in chicken tissues (muscle, kidney and liver) and pork via ultra-high-performance liquid chromatography (UHPLC) coupled with a fluorescence detector (FLD) using dansyl chloride (DNS-Cl) as the derivatisation reagent. After extraction by accelerated solvent extraction (ASE), the analyte was purified on a strong cation-exchange solid-phase extraction (SPE) column. Separation was achieved using an Acquity UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column with ultrapure water–acetonitrile (15:85, V/V) as the mobile phase. The results showed that when the concentration of piperazine was between the limit of quantitation (LOQ) and 200.0 μg/kg, the peak area of the derivative had a good linear relationship with the piperazine concentration, and the coefficients of determination (R2) were greater than or equal to 0.9991. When the spiked concentration of piperazine was equal to the LOQ, maximum residue limit (MRL) of 0.5, 1.0 and 2.0, the recoveries ranged from 79.64 to 99.77% and the relative standard deviations (RSDs) were 1.14–5.63%. The limits of detection (LODs) and LOQs were 0.50–1.20 and 1.80–3.50 μg/kg, respectively. The method was applied to the quantification of piperazine residues in commercial chicken tissues and pork from local supermarkets.

Chemical profiling of spermidines in goji berry by strong cation exchange solid-phase extraction (SCX-SPE) combined with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS/MS)

Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.

Isolation of mitraphylline from Uncaria tomentosa (Willd. ex Schult.) DC. barks and development of spectrophotometric method for total alkaloids determination in Cat's Claw samples.

AbstractIntroductionThe usual quality control for Uncaria tomentosa (Willd. ex Schult.) DC. barks requires highly specific analytical standards and methods based on high‐performance liquid chromatography (HPLC), which impacts the costs of the analytical process and the final products.ObjectiveTo obtain an analytical reference standard of mitraphylline by isolation from U. tomentosa barks and develop a spectrophotometric method for determination of total alkaloids in samples of U. tomentosa.MethodologyAn alkaloid‐enriched extract was obtained by acid–base partition and mitraphylline was selectively precipitated using an 80:20 v/v toluene/hexane solution. The compound was characterised by HPLC‐UV/DAD (diode‐array detector), mass spectrometry, UV‐visible, infrared (IR) and 1H‐ and 13C‐nuclear magnetic resonance (NMR) spectroscopy. Sample preparation for the spectrophotometric method consisted of an extraction with boiling methanol (3 × 10 mL, 15 min), followed by a strong cation exchange solid phase extraction (SCX‐SPE) clean‐up.ResultsMitraphylline with a purity of 98% was isolated in 0.05% m/m yield. All characterisation results were in agreement with previous published data. The spectrophotometric method showed linear range between 0.40 and 20 μg/mL; limits of detection and quantification of 0.15 and 0.49 μg/mg, respectively; dispersion of results lower than 5% for repeatability and intermediate precision; statistically proven accuracy by comparison with reference values obtained by Soxhlet and an HPLC‐UV/DAD method; and robustness in relation to sample mass extracted and extraction time.ConclusionThe methods developed to obtain mitraphylline analytical standard from U. tomentosa barks and to determine total alkaloids by spectrophotometry provided a cheaper and faster quality control alternative for U. tomentosa samples.

22 CANONICAL CORRELATION ANALYSIS OF ADHD POLYGENIC RISK AND FUNCTIONAL BRAIN CONNECTIVITY

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL−1, 0.62 μg mL−1, 4.15 μg mL−1, 0.58 μg mL−1 and 4.04 μg mL−1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Quantification of the soluble Receptor of Advanced Glycation End-Products (sRAGE) by LC-MS after enrichment by strong cation exchange (SCX) solid-phase extraction (SPE) at the protein level

The study of low abundant proteins contributes to increasing our knowledge about (patho)physiological processes and may lead to the identification and clinical application of disease markers. However, studying these proteins is challenging as high-abundant proteins complicate their analysis. Antibodies are often used to enrich proteins from biological matrices prior to their analysis, though antibody-free approaches have been described for some proteins as well. Here we report an antibody-free workflow on the basis of strong cation exchange (SCX) enrichment and liquid chromatography-mass spectrometry (LC-MS) for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). sRAGE was quantified in serum at clinically relevant low to sub ng mL−1 levels. The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and was compared to an antibody-based LC-MS sRAGE method. The SCX-based method builds upon the bipolar charge distribution of sRAGE, which has a highly basic N-terminal part and an acidic C-terminal part resulting in an overall neutral isoelectric point (pI). The highly basic N-terminal part (pIcalculated = 10.3) allowed for sRAGE to be enriched by SCX at pH 10, a pH at which most serum proteins do not bind. This study shows that ion exchange-based enrichment is a viable approach for the LC-MS analysis of several low abundant proteins following a thorough analysis of their physical-chemical properties.

Determination of four main components of gentamicin in animal tissues after solid-phase extraction by high-performance liquid chromatography/tandem mass spectrometry.

An analytical method for gentamicin in animal tissues was developed and validated. An alkaline mobile phase with an HPH C8 column was selected so that all the four gentamicin components were retained and eluted without using fluorinated ion-pairing reagents. The method is sufficiently sensitive and highly selective, using a strong cation-exchange solid-phase extraction cartridge (PCX) to clean up the samples. Different types of solid-phase extraction columns and membranes were considered to obtain a high recovery. The method was validated on spiking samples, recovery, inter- and intra-assay variation, to ensure its accuracy and precision. The LOQ (S/N ≥ 10) for gentamicin in goat meat, liver, kidney and adipose tissue was 25, 50, 30 and 30 ng/g, respectively; the LOD (S/N ≥ 3) was 5, 10, 10 and 10 ng/g, respectively. The recoveries were between 88% and 106%. The method in all animal tissues was calibrated from 10 to 1000 μg/L in the matrix-assisted standard solution. The novelty of this method is that the commonly used fluorinated ion-pairing reagent was not used in the mobile phase in our analysis, greatly reducing the contamination of the ESI source in negative mode. Moreover, the four gentamicin components were clearly separated via chromatographic separation.

Determination of gentamicin C components in fish tissues through SPE-Hypercarb-HPLC-MS/MS

In this work, an HPLC/MS/MS method for determination of gentamicin C components in fish tissues was developed based on strong cation exchange solid-phase extraction (SPE) purification coupled with Hypercarb chromatographic column in separation mode. Sample was extracted using trichloroacetic acid aqueous solution containing EDTA. Ion-pairing reagents were not needed because of the “graphite polarity retention effect” of the Hypercarb chromatographic column. HPLC-MS/MS was performed in multiple reaction monitoring (MRM) mode for simultaneous qualitative and quantitative analyses (using matrix external standard) of gentamicin C components in fish tissues. Good linearity was obtained for the target analytes within the concentration range from 0.0100 to 0.500 mg/L. The limits of quantification (LOQ) of this method were 10.0, 20.0, and 20.0 μg/kg for C1, C1a, and sum of C2 + C2a, respectively. The average recoveries of gentamicin C components were 80.0%–110% when spiked at three levels with the blank carp (Cyprinus carpio) matrix, and the relative standard deviations (RSD) were all less than 15% (n = 6). In addition, for the features of simple operation, high sensitivity and good reproducibility, the proposed method has been successfully applied for detection of gentamicin residues in fish tissues during actual breeding.

Differential Mobility Spectrometry-Mass Spectrometry (DMS-MS) in Radiation Biodosimetry: Rapid and High-Throughput Quantitation of Multiple Radiation Biomarkers in Nonhuman Primate Urine.

High-throughput methods to assess radiation exposure are a priority due to concerns that include nuclear power accidents, the spread of nuclear weapon capability, and the risk of terrorist attacks. Metabolomics, the assessment of small molecules in an easily accessible sample, is the most recent method to be applied for the identification of biomarkers of the biological radiation response with a useful dose-response profile. Profiling for biomarker identification is frequently done using an LC-MS platform which has limited throughput due to the time-consuming nature of chromatography. We present here a chromatography-free simplified method for quantitative analysis of seven metabolites in urine with radiation dose-response using urine samples provided from the Pannkuk et al. (2015) study of long-term (7-day) radiation response in nonhuman primates (NHP). The stable isotope dilution (SID) analytical method consists of sample preparation by strong cation exchange-solid phase extraction (SCX-SPE) to remove interferences and concentrate the metabolites of interest, followed by differential mobility spectrometry (DMS) ion filtration to select the ion of interest and reduce chemical background, followed by mass spectrometry (overall SID-SPE-DMS-MS). Since no chromatography is used, calibration curves were prepared rapidly, in under 2h (including SPE) for six simultaneously analyzed radiation biomarkers. The seventh, creatinine, was measured separately after 2500× dilution. Creatinine plays a dual role, measuring kidney glomerular filtration rate (GFR), and indicating kidney damage at high doses. The current quantitative method using SID-SPE-DMS-MS provides throughput which is 7.5 to 30 times higher than that of LC-MS and provides a path to pre-clinical radiation dose estimation. Graphical Abstract.

Simultaneous determination of ibuprofen and its metabolites in complex equine urine matrices by GC-EI-MS in excretion study in view of doping control

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL−1, 0.62 μg mL−1, 4.15 μg mL−1, 0.58 μg mL−1 and 4.04 μg mL−1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

Simultaneous Determination of Aminoglycoside Residues in Food Animal Muscles by Mixed-Mode Liquid Chromatography-Tandem Mass Spectrometry

A sensitive, selective, and reproducible method was developed for determining nine aminoglycoside antimicrobial residues in swine, cattle, mutton, chicken, and fish muscles by liquid chromatography-tandem mass spectrometry. The analytes were extracted with phosphate buffer solution and purified by a mixed-mode strong cation exchange solid-phase extraction cartridge. Using acetonitrile-1% formic acid solution as a mobile phase system, avoiding the use of the ion pair reagents, the target components retained well and effectively separated on the mixed-mode liquid chromatographic Obelisc R column. It was suggested that the aminoglycoside’s retention time increased with the increase of the number of amino groups in its molecule. The matrix-matched calibration curves for analytes were good linearity within the range of 5–500 μg L−1. The CCαs of analytes were between 2 and 10 μg kg−1, and CCβs were between 6 and 25 μg kg−1. In the spiked levels of 25, 50, and 100 μg kg−1, the average recoveries of analytes were in the range of 65–110% except neomycin of 60–75%, with relative standard deviation below 20%.

An Improved Method for the Quantification of Thiamine (Vitamin B1) in Cocoa-Containing Beverage Powders.

The purpose of this work was to understand low recoveries of thiamine (vitamin B1) when extracted from cocoa-containing beverage powders fortified with water-soluble vitamin B1, and to develop and validate a new procedure to improve these results. Based on the literature, previous trials have focused on two main factors: pH value prior to paper filtration and the need for solid-phase extraction (SPE) clean up. We demonstrate that by following European Standard EN 14122, recovery of thiamine in cocoa-containing beverage powders is low and dependent on the test portion (86 and 72% for 0.5 and 1.5 g test portions, respectively). Our improved method resolved this problem by keeping the pH low (around 1) prior to paper filtration, leading to a 96.3% recovery and high precision (RSDr of 3.5%). The use of strong cation-exchange SPE cartridges for cleanup prior to the thiamine oxidation reaction proved to be essential. A comparison between our improved method and EN 14122 on nine cocoa-containing beverage samples available on local market from different manufacturers showed a systematic increase in thiamine content (up to 70%) when the improved method was applied. The highest difference was observed for the sample that contained the highest amount of cocoa. However, for beverage powders that contained both cocoa and milk, no difference was observed.

Srage as biomarker for COPD, introducing novel LC-MS-based methods for the quantification of serum srage levels

Rationale: Soluble RAGE (sRAGE) is a promising biomarker for COPD and an attractive target for biomarker validation studies. Validated, reliable and clinically applicable biomarkers are needed for the early detection of COPD, identification of clinically relevant subgroups of COPD and for the selection of subjects for clinical trials. The circulating levels of sRAGE are decreased in COPD patients compared to both smoking and non-smoking controls, and are even further decreased during COPD exacerbations. Furthermore, the serum levels of sRAGE associate with neutrophilic airway inflammation, decline in FEV1, and emphysema. Methods: To date, most studies use commercially available ELISA kits for measurements of sRAGE, which have limited accuracy and reproducibility. Here, we present two novel liquid chromatography-mass spectrometry (LC-MS) methods for the quantification of sRAGE in serum. Our first approach measures the free, unbound fraction of sRAGE using an immunoaffinity enrichment-based method as sample preparation, comparable to the ELISA. Our second approach is an antibody-free method based on strong cation exchange solid-phase extraction, which likely measures the total sRAGE fraction. Both sample preparation methods are followed by LC-MS analysis in the multiple reaction monitoring mode. Results: Using the immunoaffinity enrichment-based method we are able to detect and quantify sRAGE in human serum over a large range (0.1-10 ng/mL) with high precision and accuracy (CV

Determination of Five Aminoalcohol-diterpenoid Alkaloids in the Lateral Root of Aconitum carmichaeli by HPLC-ELSD with SPE.

A convenient and accurate high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) method using solid phase extraction (SPE) was established for quantification of five aminoalcohol-diterpenoid alkaloids (ADAs), including mesaconine, aconine, hypaconine, fuziline and neoline, in the lateral roots of Aconitum carmichaeli (Fuzi) for the first time. The Fuzi extractive was purified using strong cation-exchange SPE. The chromatographic separation was performed on a Gemini C18 column (150 × 4.60 mm, 5 μm) with the mobile phase of methanol-water-diethylamine (48:52:0.01, v/v/v), adjusted to pH 10.2 with acetic acid. The detector was Alltech ELSD 2000ES (drift tube temperature: 90°C; gas flow-rate: 2.3 L/min). Five ADAs in Fuzi were well separated. The calibration curves showed good linearity (r > 0.9990) in the range of 0.0125-0.3750 mg/mL for each alkaloid. The recoveries were in the range of 95.1-105.7%, with relative standard deviations < 5.0%. This method is accurate, specific and repeatable for the determination of five ADAs in different Fuzi samples, which can be applied to the quality control of Fuzi.