Mixed-mode Anion Exchange Solid-phase Extraction Research Articles

Simultaneous determination of ethyl glucuronide, cocaine, cocaethylene, and benzoylecgonine in hair by using LC–MS/MS

Alcohol and cocaine (COC) are commonly co-used drugs that cause addiction and have harmful effects. Their abuse may threaten the health of the abuser and public safety by causing serious accidents or crimes. The recidivism rate of drug-related crimes closely correlates with alcoholism. Several incidences of alcohol consumption in combination with drug abuse have been reported. Here, liquid chromatography tandem mass spectrometric method was developed to simultaneously analyze ethyl glucuronide (EtG), a metabolite of ethanol; COC; cocaethylene (CE), an alcohol-derived metabolite of COC; and benzoylecgonine (BZE), a major metabolite of COC, to determine the concurrent use of alcohol with COC. For pre-treatment, ultracentrifugation (5 min, 50,000 g) and mixed-mode anion exchange solid-phase extraction were used to increase the recovery of target compounds and minimize the matrix effect of hair. The lower limits of quantification were: 7 pg/mg (EtG), 2 pg/mg (COC), 10 pg/mg (CE), and 1 pg/mg (BZE). The correlation coefficient (r) of the calibration curve within the quantified range of target compounds was ≥ 0.9978. The intra- and inter-day accuracies were − 6.1–9.7% and − 9.3–8.3%, and intra- and inter-day precisions were 0.5–10.3% and 0.6–14.4%, respectively. The recovery, matrix effect, process efficiency, and autosampler stability were 89.2–104.8%, 81.6–105.4%, 81.5–107.1%, and 96.6–109.7%, respectively. The novel analytical method was validated with hair samples from individuals suspected of alcohol and COC use, and the method could distinguish between independent and concurrent use. Based on the findings, the analytical approach developed in this study is anticipated to be valuable in drug and alcohol dependence tests that require the simultaneous detection of alcohol and COC abuse.

Antibody-Free Quantification of Serum Chromogranin A by Targeted Mass Spectrometry.

Chromogranin A (CgA) is a 48 kDa protein that serves as a diagnostically sensitive, but nonspecific, serum biomarker for neuroendocrine tumors. Immunoassays for CgA are not standardized and have a narrow dynamic range, which requires dilution of concentrated specimens. We developed and validated an antibody-free, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for CgA without these limitations. CgA was extracted from serum using a mixed-mode anion exchange solid-phase extraction plate, digested with trypsin, and analyzed by LC-MS/MS using well-characterized CgA calibration standards. After validation, the mass spectrometry method was compared with the CISBIO immunoassay using 200 serum specimens previously submitted for CgA analysis. Specimens with discordant results were reanalyzed by high-resolution mass spectrometry- (HRMS) -based methods to assess the contribution of truncated and post-translationally modified forms of CgA. The assay had a linear range of 50 to 50 000 ng/mL, recoveries between 89% and 115%, and intra- and interassay imprecision <10%. LC-MS/MS assay results showed a Pearson's correlation of r = 0.953 with the CISBIO immunoassay, with CgA values being a mean 2- to 4-fold higher. Concordance for CgA between the 2 assays was 80.9% (95% CI 72.8%-89.2%), showing substantial agreement. Truncation and posttranslational modification, including 2 phosphorylation sites that had not been previously observed or predicted to our knowledge, did not appear to contribute directly to discordance between the 2 assays. Quantification of CgA by LC-MS/MS provides an analytically sensitive and reproducible alternative to commercially available immunoassays.

Development and validation of a fast and reliable method for the quantification of glucagon by liquid chromatography and tandem mass spectrometry

IntroductionThe quantitation of glucagon remains a challenging immunoassay, mainly due to cross-reactivity. A sensitive, rapid and specific intact glucagon method is therefore necessary for quality routine analysis. A tandem mass spectrometry method to fulfill this objective is described in this work. MethodsGlucagon was extracted from plasma employing a mixed-mode anion exchange solid-phase extraction. Sample stability was assessed in K2-EDTA and P800 tubes at different temperatures. We compared our method to two different immunoassays. FDA and EMA guidelines were followed for validation. An external quality control program served for comparison with other laboratories. ResultsAssay imprecision was below 4%. Recoveries were within 95–103%. LoQ was 8.75 pg/mL. Total analytical CV was 2.91%. Samples were found stable at 4 °C for less than 4 h. Diasource® RIA disagreed with our method. Mercodia® ELISA provided a closer agreement, also proven by external quality control samples. ConclusionsA rapid and specific LC-MS/MS method for glucagon quantitation has been developed, validated and is suitable to routine care. The simplicity and the good performances in terms of time and specificity, could open the possibility to establish a standardized method for glucagon.

A tiered analytical approach for target, non-target and suspect screening analysis of polar transformation products of polycyclic aromatic compounds

Polycyclic aromatic compounds (PACs) possess toxicity towards humans, and their presence in the environment is unwanted. Polar transformation products (TPs) are more mobile, and can be considered emerging contaminants, as they represent a more bioavailable carrier of the same toxic properties. Acidic TPs has been proposed as an important class of polar TPs. This study presents a tiered analytical approach to investigate acidic and polar PAC TPs in environmental conditions. The tiered approach exploits target analysis for quantification of acids; suspect screening for tentative identification based on retention time and spectral matching using databases; and finally non-target analysis based on chromatography and data independent broadband MS to highlight potentially unknown analyte peaks. The approach includes a mixed-mode anion exchange solid phase extraction (MAX-SPE) to fractionate neutral and acidic compounds, and is applied to three cases: I) Photo-oxidation of six PACs generated suspected hydroxylated-, carbonylated- and carboxylated PACs but also proposed the presence of mono- and dicarboxylic acids, which have not been reported elsewhere. For a subset of four acids, conversion rates were determined. II) Recovery of spiked acids from diesel spilled harbor water was 80% by LC-MS, and diesel spill weathering was evaluated from the neutral fraction by GC-MS. III) By non-target analysis sulfonated PACs, presumable derived from photo-oxidation, were detected in run-off basins of an arctic landfarm, alongside hypothesized naturally occuring fatty acids. The tiered approach is a sensitive and versatile tool to extract information on PACs and their polar TPs from polluted environmental sites.

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography–mass spectrometry

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.

Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography–tandem mass spectrometry

This paper describes an analytical method for four phenolic and salicylanilide anthelmintics authorised for use within the EU (nitroxinil, oxyclozanide, rafoxanide and closantel) in bovine kidney, and the extension of this procedure to include a number of related compounds; ioxynil, niclosamide, salicylanide and 3-trifluoromethyl-4-nitrophenol (TFM). The method comprises a solvent extraction with 1% acetic acid in acetone and clean-up using a mixed-mode anion-exchange solid phase extraction column. Determination is by reversed phase LC–MS/MS. The method was validated to the latest EU requirements (Commission Decision 2002/657/EC) using both spiked and incurred tissues and was subject to second laboratory evaluation.

Determination of Chlorinated Acid Herbicides in Vegetation and Soil by Liquid Chromatography/Electrospray-Tandem Mass Spectrometry

The method presented uses reversed-phase liquid chromatography with negative electrospray ionization and tandem mass spectrometry to analyze 9 chlorinated acid herbicides in soil and vegetation matrixes: clopyralid, dicamba, MCPP, MCPA, 2,4-DP, 2,4-D, triclopyr, 2,4-DB, and picloram. A 20 g portion is extracted with a basic solution and an aliquot acidified and micropartitioned with 3 mL chloroform. Vegetation samples are subjected to an additional cleanup with a mixed-mode anion exchange solid-phase extraction cartridge. Two precursor product ion transitions per analyte are measured and evaluated to provide the maximum degree of confidence in results. Average recoveries for 3 different soil types tested ranged from 72 to 107% for all compounds with the exception of 2,4-DB at 56-99%. Average recoveries for the 3 different vegetation types studied were lower and ranged from 53 to 80% for all compounds.

Analysis of N‐acylhomoserine lactones after alkaline hydrolysis and anion‐exchange solid‐phase extraction by capillary zone electrophoresis‐mass spectrometry

A quantitative, specific, and sensitive method for the determination of N-acylhomoserine lactones (HSLs - a group of bacterial semiochemicals) in the form of their hydrolysis products (N-acylhomoserines, HSs) is presented. Real samples were analyzed by capillary zone electrophoresis-mass spectrometry (CZE-MS) after alkaline lactonolysis and extraction by mixed-mode anion-exchange solid-phase extraction. The presented cleanup significantly speeds up the HSL extraction procedure, strongly reduces sample consumption, and is more selective compared to the commonly used liquid/liquid extraction. Completeness of the hydrolysis reaction was examined by nuclear magnetic resonance spectroscopy. This CZE-MS method complements recently published capillary separation techniques (nano liquid chromatography-MS, partial-filling micellar electrokinetic chromatography-MS, gas chromatography-MS) and provides a possibility to differentiate quantitatively between the homoserines (as naturally occurring degradation products) besides the intact homoserine lactones. The method was found to be quantitative down to a concentration of 0.05 microg/mL (limit of quantification), while the limit of detection was determined with 0.01 microg/mL - sufficient for the analysis of culture supernatants.