Anion Exchange Solid Phase Extraction Research Articles

A novel LC-MS/MS method for the determination of favipiravir ribofuranosyl-5’-triphosphate (T-705-RTP) in human peripheral mononuclear cells

Favipiravir is a broad-spectrum antiviral that is metabolised intracellularly into the active form, favipiravir ribofuranosyl-5’-triphosphate (F-RTP). Measurement of the intracellular concentration of F-RTP in mononuclear cells is a crucial step to characterising the pharmacokinetics of F-RTP and to enable more appropriate dose selection for the treatment of COVID-19 and emerging infectious diseases. The described method was validated over the range 24 – 2280 pmol/sample. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and lysed using methanol-water (70:30, v/v) before cellular components were precipitated with acetonitrile and the supernatant further cleaned by weak anion exchange solid phase extraction. The method was found to be both precise and accurate and was successfully utilised to analyse F-RTP concentrations in patient samples collected as part of the AGILE CST-6 clinical trial.

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.

A Fast and Simple Solid Phase Extraction-Based Method for Glucosinolate Determination: An Alternative to the ISO-9167 Method.

Glucosinolates (GLSs) are a well-studied sulfur-containing compound found in Brassicaceae plants that play critical roles in plant resistance and human health. Correctly identifying and reliably quantifying the total and individual GLS content is of great importance. An improved method as an alternative to the ISO 9167-1 (ISO) method is developed in the present study. An efficient extraction and purification procedure is proposed with a commercially available dimethylaminopropyl (DEA)-based weak anion exchange solid-phase extraction (SPE) cartridge instead of using the self-prepared ion-exchange columns in the ISO method. The GLSs are identified and quantified by ultra high-performance liquid chromatography (UHPLC) high-resolution mass spectrometry (HRMS). The method demonstrates a comparable quantification of total and individual GLSs on certified rapeseeds and other Brassicaceae vegetables when compared to the ISO method. The developed SPE method is simpler and more efficient, thus allowing for applications to a large sample size with reduced analysis time, improved repeatability and accuracy, and possible automation.

Validating blood microsampling for per- and polyfluoroalkyl substances quantification in whole blood

Microsampling allows the collection of blood samples using a method which is inexpensive, simple and minimally-invasive, without the need for specially-trained medical staff. Analysis of whole blood provides a more holistic understanding of per- and polyfluoroalkyl substances (PFAS) body burden. Capillary action microsamplers (Trajan hemaPEN®) allow the controlled collection of whole blood as dried blood spots (DBS) (four 2.74 µL ± 5 %). The quantification of 75 PFAS from DBS was evaluated by comparing five common extraction techniques. Spiked blood (5 ng/mL PFAS) was extracted by protein precipitation (centrifuged; filtered), acid-base liquid-liquid extraction, trypsin protease digestion, and weak anion exchange (WAX) solid-phase extraction with analysis by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Filtered protein precipitation was the most effective extraction method, recovering 72 of the 75 PFAS within 70 to 130 % with method reporting limit (MRL) for PFOS of 0.17 ng/L and ranging between 0.05 ng/mL and 0.34 ng/mL for all other PFAS. The optimised method was applied to human blood samples to examine Inter- (n = 7) and intra-day (n = 5) PFAS blood levels in one individual. Sixteen PFAS were detected with an overall Σ16PFAS mean = 6.3 (range = 5.7–7.0) ng/mL and perfluorooctane sulfonate (branched and linear isomers, ΣPFOS) = 3.3 (2.8–3.7) ng/mL being the dominant PFAS present. To the authors knowledge, this minimally invasive self-sampling protocol is the most extensive method for PFAS in blood reported and could be a useful tool for large scale human biomonitoring studies.

Simultaneous determination of enantiomer residues of metalaxyl and its metabolite metalaxyl acid in animal muscle tissues by chiral liquid chromatography-tandem mass spectrometry

The toxicity of the two enantiomers of metalaxyl is significantly different and, for food safety concerns, stereoselective analysis of its residues is extremely important in animal muscle tissues. Herein, a simple, sensitive and reproducible method for the simultaneous stereoselective analysis of metalaxyl and its metabolite metalaxyl acid in animal-derived foods has been established based on mixed-mode anion exchange solid phase extraction and chiral liquid chromatography-tandem mass spectrometry. Under the optimized separation and sample pretreatment conditions, good linearity (r > 0.99) was obtained for four target analytes in four animal muscle tissues including bovine, chicken, swine and fish. Mean recoveries of all enantiomers in muscle tissue samples were 89.5–110.3 % with relative standard deviations below 12.4 %. The limits of quantification of the analytes were 0.3–1.0 µg/kg. The proposed method provides an effective means to monitor the enantiomer residues in real samples whether using highly active R-metalaxyl or racemic mixture drugs.

A liquid chromatography tandem mass spectrometry method for plasma glucagon

Glucagon is a peptide involved in controlling the body's blood glucose levels. The majority of analytical methods used for its quantitation are based on immunoassays that suffer from cross-reactivity with other peptides. For accurate routine analysis a liquid chromatography tandem mass spectrometry (LC-MSMS) was developed. Glucagon was extracted from plasma samples by a combination of protein precipitation using ethanol and mixed anion exchange solid phase extraction. Linearity for glucagon was above 0.99 (r2) up to a concentration range of 771 ng/L with a lower limit of quantification established at 19 ng/L. Precision of the method was below 9% (coefficient of variation). Recovery was 93%. Correlations with the existing immunoassay displayed a significant negative bias.

A Rapid Derivatization for Quantitation of Perfluorinated Carboxylic Acids from Aqueous Matrices by Gas Chromatography-Mass Spectrometry.

Ultrashort-chain perfluorinated carboxylic acids (PFCAs) are receiving more attention due to their ever-increasing presence in the environment. Methods have been established for the analysis of short- and long-chain PFCAs, while robust quantitation of ultrashort-chain species is scarce. Here, we develop a novel derivatization method using diphenyl diazomethane for quantitation of C2-C14 PFCAs in aqueous matrices. The method is highlighted by rapid completion of derivatization (<1 min) and retention and separation of ultrashort-chain (C2/C3) PFCA derivatives using H2 carrier gas (R > 1.5). A weak anion exchange solid-phase extraction procedure for analyte recovery from representative aqueous samples was developed and validated by spike and recovery from ultrapure water, synthetic ocean water, and simulated denuder extracts used for collecting gaseous PFCAs. Recoveries for PFCAs ranged from 83 to 130% for the majority of analytes and matrices. The instrument detection limits (IDLs) range from 8 to 220 fg per injection, and method detection limits (MDLs) range from 0.06 to 14.6 pg/mL for 500 mL aqueous samples, which are within an order of magnitude to conventional LC-MS/MS methods. The method was applied to the analysis of real samples of tap water, rainwater, ocean water, and annular denuder extracts. The overall method provides a cost-effective alternative to conventional LC-MS/MS methods, overcoming the typical GC-MS drawbacks of high detection limits and long sample preparation times while being able to simultaneously analyze the complete spectrum of environmentally relevant PFCAs.

An LC-MS/MS Analytical Method for Moenomycin A in Livestock Products

A simple and sensitive method for the determination of moenomycin A residues in livestock products using LC-MS/MS was developed. Moenomycin A, a residual definition of flavophospholipol, was extracted from samples with a mixture of ammonium hydroxide and methanol (1 : 9, v/v) preheated at 50℃. The crude extracted solutions were evaporated and purified by liquid-liquid partitioning between a mixture of ammonium hydroxide, methanol and water (1 : 60 : 40, v/v/v) and ethyl acetate. The alkaline layer was taken, and cleaned up using a strong anion exchange (InertSep SAX) solid phase extraction cartridge. The LC separation was performed on an Inertsil C8 column with liner gradient elution using 0.3 vol% formic acid and acetonitrile containing 0.3 vol% formic acid. Moenomycin A was detected using tandem mass spectrometry with negative ion electrospray ionization. Recovery tests were conducted using three porcine samples (muscle, fat and liver) and chicken eggs. Samples were spiked with moenomycin A at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The trueness ranged from 79 to 93% and precision ranged from 0.5 to 2.8%. The limit of quantification (S/N≥10) of the developed method is 0.01 mg/kg. The developed method would thus be very useful for regulatory monitoring of flavophospholipol in livestock products.

An Accurate Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry Method for Serum C-Peptide and Its Use in Harmonization in China.

Serum C-peptide results from various routine methods used in China are highly variable, warranting well-performing methods to serve as an accuracy base to improve the harmonization of C-peptide measurements in China. We developed an accurate isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for serum C-peptide measurement and explored its use in harmonization. After protein precipitation with ZnSO4 solution, C-peptide was extracted from serum samples by anion-exchange solid-phase extraction and quantified by ID-LC-MS/MS in positive ion mode. The precision and analytical recovery of the ID-LC-MS/MS method were assessed. Seventy-six serum samples were analyzed using the ID-LC-MS/MS method and six routine immunoassays. Ordinary linear regression (OLR) and Bland-Altman (BA) analyses were conducted to evaluate the relationship between the ID-LC-MS/MS method and routine immunoassays. Five serum pool samples assigned using the ID-LC-MS/MS method were used to recalibrate the routine assays. OLR and BA analyses were re-conducted after recalibration. The within-run, between-run, and total precision for the ID-LC-MS/MS method at four concentrations were 1.0%-2.1%, 0.6%-1.2%, and 1.3%-2.2%, respectively. The analytical recoveries for the ID-LC-MS/MS method at three concentrations were 100.3%-100.7%, 100.4%-101.0%, and 99.6%-100.7%. The developed method and the immunoassays were strongly correlated, with all R2 >0.98. The comparability among the immunoassays was substantially improved after recalibration. The performance of the ID-LC-MS/MS method was carefully validated, and this method can be used to improve the harmonization of serum C-peptide measurements in China.

Enhanced selectivity for acidic contaminants in drinking water: From suspect screening to toxicity prediction

A novel analytical workflow for suspect screening of organic acidic contaminants in drinking water is presented, featuring selective extraction by silica-based strong anion-exchange solid-phase extraction, mixed-mode liquid chromatography-high resolution accurate mass spectrometry (LC-HRMS), peak detection, feature reduction and compound identification. The novel use of an ammonium bicarbonate-based elution solvent extended strong anion-exchange solid-phase extraction applicability to LC-HRMS of strong acids. This approach performed with consistently higher recovery and repeatability (88 ± 7 % at 500 ng L-1), improved selectivity and lower matrix interference (mean = 12 %) over a generic mixed-mode weak anion exchange SPE method. In addition, a novel filter for reducing full-scan features from fulvic and humic acids was successfully introduced, reducing workload and potential for false positives. The workflow was then applied to 10 London municipal drinking water samples, revealing the presence of 22 confirmed and 37 tentatively identified substances. Several poorly investigated and potentially harmful compounds were found which included halogenated hydroxy-cyclopentene-diones and dibromomethanesulfonic acid. Some of these compounds have been reported as mutagenic in test systems and thus their presence here requires further investigation. Overall, this approach demonstrated that employing selective extraction improved detection and helped shortlist suspects and potentially toxic chemical contaminants with higher confidence.

RESEARCH ON THE INFLUENCE OF DIFFERENT TYPES OF ANION-EXCHANGE CARTRIDGES ON THE QUALITY OF [18F]NAF RADIOPHARMACEUTICAL AS PART OF PRODUCTION PROCESS DEVELOPMENT

[18F]Sodium Fluoride radiopharmaceutical is a sterile solution for intravenous administration, intendedfor skeletal visualization by positron emission tomography (PET). [18F]Sodium Fluoride for bone imaging wasintroduced in early 1960's, but with the increased availability of PET scanners in the last two decades, thisradiopharmaceutical has growing use in clinical practice for the detection of bone metastases. The productionprocess of [18F]NaF includes production of the radioisotope [18F]F- and purification and formulation of the [18F]NaFradiopharmaceutical. The radioisotope [18F]F- is produced by a cyclotron via the 18O(p,n)18F nuclear reaction,followed by recovery of [18F]F- from [18O] proton-irradiated water by adsorption and desorption from anionexchangeresins. The fluoride anions are trapped on the anion-exchange SPE (solid-phase extraction) cartridge, andall other cationic and water-soluble radionuclide impurities present in irradiated enriched water are collected in thewaste vial. Next step is desorption of the fluoride anions from the cartridge by elution with saline solution (0.9%NaCl). This study aimed to define the most appropriate type of anion-exchange SPE cartridge which could be usedfor routine production [18F]Sodium fluoride radiopharmaceutical which meets the quality requirements defined inEuropean pharmacopeia monograph. For that purpose, as part of development of in-house production method,manual productions with four different types of anion-exchange cartridges were performed. The influence of sorbentsubstrate and counter-ion of the cartridge on the final yield and the quality of the produced radiopharmaceutical wasinvestigated. The study also aimed to define the minimum volume of physiological solution required for the pHparameter to be within limits.The results have shown that the quality parameters: appearance, chemical purity, radiochemical purity andradionuclide purity were in defined acceptance criteria and did not differ when using different anion-exchangecartridges. The pH analyses have demonstrated that the type of cartridge and counter-ion influence the final pH of[18F]NaF solution. This study confirmed that the three types of anion-exchange resins (QMA-Cl-, QMA-CO32- andPS-OH-) could be used for production. In the experiments where QMA-Cl- was used, the required pH level wasobtained even without dilution. The other cartridges could be used in the [18F]NaF production process, but furtherdilution is necessary in order to obtain the pH value in acceptance criteria. On the basis of this study, the QMA-Cl- ischosen as a cartridge to be used in the further development of the in-house method for [18F]NaF radiopharmaceuticalproduction.

Determination of three oryzanols in rice by mixed-mode solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry

建立了利用高效液相色谱-串联质谱结合混合型阴离子交换固相萃取柱测定稻米中3种谷维素(环木菠萝烯醇阿魏酸酯(CA-FA)、24-亚甲基环木菠萝烯醇阿魏酸酯(24MCA-FA)和菜油甾醇阿魏酸酯(Camp-FA))含量的分析方法。实验通过优化3种谷维素的多反应监测(MRM)质谱参数,比较了在不同流动相中的分离度以及响应强度,同时考察了不同提取条件、净化条件对3种谷维素提取率和净化效果的影响,再结合外标法定量,实现了对稻米中3种谷维素的定量分析。实验结果表明,采用5 mmol/L乙酸铵水溶液为流动相A,甲醇∶乙腈=1∶1(v/v)为流动相B,梯度洗脱,3种谷维素在Agilent Eclipse XDB-C8色谱柱(150 mm×2.1 mm, 3.5 μm)上基本分离且响应强度和峰形最佳;提取条件经正交实验优化后可得:料液比为1∶20(g/mL)、提取溶剂为甲醇、浸泡时间为12 h、超声温度为40 ℃以及超声时间为20 min时,3种谷维素提取率最高;对混合型阴离子交换固相萃取柱的上样溶剂以及洗脱溶剂优化后,样品基质效应为1.6%~10.8%。在各优化条件下,3种谷维素在各自的线性范围内线性良好,相关系数(r2)均≥0.9983,检出限(LOD)为0.5~1.0 μg/L,定量限(LOQ)为2.0~3.5 μg/L。在稻米样品本底浓度2、5和10倍的加标水平下3种谷维素的平均回收率为86.1%~110.6%,相对标准偏差(RSD)为0.9%~3.2%。该方法可快速准确测定稻米中3种谷维素的含量,为后续稻米中谷维素类化合物测定及鉴定奠定基础。

Separation and characterization of sulfonates in dissolved organic matter from industrial wastewater by solid phase extraction and high-resolution mass spectrometry.

Electrospray ionization(ESI) source combined with high-resolution mass spectrometry (HRMS) has been successfully used to characterize the molecular composition of dissolved organic matter (DOM) in industrial wastewaters. However, sulfonates are commonly presenting in these wastewaters and showing strong ionization suppression for the ESI analysis, which seriously affects the characterization of other DOM components. In this paper, a novel method based on polymer anion exchange (PAX) solid phase extraction (SPE) was proposed to separate DOMs from petroleum refining and coal coking wastewaters into hydrophobic neutral, hydrophobic acids, and hydrophobic strong acids; the fractions were analyzed by negative-ion ESI Fourier transform ion cyclotron resonance mass spectrometry. The results show that PAX-SPE separated sulfonates from wastewaters and enabled the molecular characterization of oxygen-containing compounds, which are the major component of DOM in the wastewaters. Semi-quantitative analysis of sulfonates in the wastewaters was carried out and the results show that these compounds account for 11% and 2% in total organic carbon for the refinery and coal coking wastewaters, respectively. PAX-SPE with stepwise elution and followed by high-resolution mass spectrometry analysis enables a comprehensive characterization for the molecular composition of industrial wastewater DOM. The method has potential for extensive application and the results are instructive for the understanding of the molecular composition of DOM in industrial wastewaters.

Membrane Sampling Separates Naphthenic Acids from Biogenic Dissolved Organic Matter for Direct Analysis by Mass Spectrometry.

Oil sands process waters can release toxic naphthenic acids (NAs) into aquatic environments. Analytical techniques for NAs are challenged by sample complexity and interference from naturally occurring dissolved organic matter (DOM). Herein, we report the use of a poly(dimethylsiloxane) (PDMS) polymer membrane for the on-line separation of NAs from DOM and use direct infusion electrospray ionization mass spectrometry to yield meaningful qualitative and quantitative information with minimal sample cleanup. We compare the composition of membrane-permeable species from natural waters fortified with a commercial NA mixture to those derived from weak anion exchange solid-phase extraction (SPE) using high-resolution mass spectrometry. The results show that SPE retains a wide range of carboxylic acids, including biogenic DOM, while permeation through PDMS was selective for petrogenic classically defined NAs (CnH2n+zO2). A series of model compounds (log Kow ∼1–7) were used to characterize the perm-selectivity and reveal the separation is based on hydrophobicity. This convenient sample cleanup method is selective for the O2 class of NAs and can be used prior to conventional analysis or as an on-line analytical strategy when coupled directly to mass spectrometry.

A review of draft Environmental Protection Agency Method 1633: A data user's perspective

AbstractCurrently, the availability of multilaboratory‐validated analytical methods for per‐ and polyfluoroalkyl substances (PFAS) is limited. Two of the more commonly known methods are the finalized United States Environmental Protection Agency (EPA) methods for the analysis of select PFAS in drinking water samples, EPA 537.1, Determination of Selected Per‐ and Polyfluorinated Alkyl Substances in Drinking Water by Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) (EPA 600‐R‐20‐006) and EPA 533, Determination of Per‐ And Polyfluoroalkyl Substances in Drinking Water by Isotope Dilution Anion Exchange Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (EPA 815‐B‐19‐020) These two methods were developed specifically for drinking water samples, which generally do not have matrix interferences and are generally an easier matrix to analyze. EPA also finalized SW‐846 Method 8327, Per‐ and Polyfluoroalkyl Substances (PFAS) by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS), which covers the analysis of select PFAS in nonpotable water matrices. However, one of the critical deficiencies in this method is the lack of the gold standard for quantitation, isotope dilution, which has become a critical necessity for the analysis of PFAS in nondrinking water matrices to accommodate for potential matrix interferences. In addition, the United States Department of Defense (DoD) Environmental Data Quality Workgroup (EDQW) has stated that SW‐846 Method 8327 should only be used as a screening method and should not be used for the collection of definitive data. The release of Draft EPA Method 1633, Analysis of Per‐and Polyfluoroalkyl Substances (PFAS) in Aqueous, Solid, Biosolids, and Tissue Samples by LC‐MS/MS (EPA 821‐D‐21‐001), for nonpotable water matrices, is a first step toward providing the laboratory community with a standardized method that can provide accurate results for PFAS in nonpotable water matrices.

Use of liquid chromatography-tandem mass spectrometry for foscarnet quantification in human serum and cerebrospinal fluid.

Foscarnet (FCV) is used to treat cytomegalovirus, human herpesvirus, and human immunodeficiency virus infections. It is a low-molecular-weight compound containing carboxylate and phosphate groups. There are no reports on the use of liquid chromatography-tandem mass spectrometry (LC/MS/MS) to analyze FCV via bioanalysis. In the present study, we developed the ion-pair LC/MS/MS method to analyze FCV in human serum and cerebrospinal fluid (CSF). FCV was extracted from human serum and CSF by weak anion exchange (WAX) solid-phase extraction. The LC/MS/MS systems were coated with 0.1% phosphoric acid in methanol to avoid nonspecific absorption. FCV was detected using ion-pair LC/MS/MS with dibutylammonium acetate. Selected reaction monitoring transition of FCV in the negative ion mode was selected at m/z 125.1 → 62.9. FCV was selectively detected in human serum and CSF, and the liner range was 5-1000 μM (R2 = 0.99). The intraday and interday accuracy and precision were within ±15%. FCV was constantly extracted from human serum and CSF by WAX solid-phase extraction (recovery ratio = 76.0-77.9%). No matrix effect was observed. A robust LC/MS/MS method to analyze FCV was successfully developed. FCV was selectively measured using LC/MS/MS in very low extract volumes (20 μL). The method will be useful in evaluating the FCV level in human serum and CSF.

Purification and Analysis of Nucleotides and Nucleosides from Plants.

This protocol describes necessary steps to isolate and quantify nucleotides and nucleosides from plant samples. Proper sample preparation in combination with liquid chromatography coupled to mass spectrometry enables the sensitive detection and quantification of metabolites of low abundance. Utilizing a liquid-liquid extraction in combination with a weak anion-exchange solid phase extraction enables the separation of negatively charged molecules from uncharged metabolites or cations.

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.

Analytical and Clinical Performance of a Liquid Chromatography-Tandem Mass Spectrometry Method for Measuring Gastrin Subtypes G34 and G17 in Serum.

Two major forms of gastrin, gastrin-17 (G17) and gastrin-34 (G34), exist in blood. However, conventional immunoassay methods can only quantify total gastrin or G17 alone. Here, we aimed to establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify G17 and G34 simultaneously. Serum samples were prepared by anion-exchange solid-phase extraction. The analytical performance of the LC-MS/MS method was validated and the method was compared to chemiluminescence immunoassay (CLIA) and radioimmunoassay (RIA). The G17 and G34 concentrations in 245 serum samples from healthy controls, individuals with gastrinoma, and individuals with other diseases were analyzed. The total runtime of the LC-MS/MS method was 6 min. No substantial matrix effect was observed with internal standard correction. The intraassay coefficients of variation (CVs) for G17 and G34 were 4.0%-14.2% and 4.4%-10.4%, respectively, and total CVs were 5.2%-14.1% and 4.6%-12.4%, respectively. The correlation coefficient between LC-MS/MS and CLIA was 0.87, and between LC-MS/MS and RIA was 0.84. The G17+G34 concentrations for 87.5% of individuals with gastrinoma were higher than the 95th percentile of healthy controls (18.1 pg/mL), whereas the concentrations for individuals with other diseases and gastrinoma overlapped. Based on the Youden indices calculated for G17+G34, G34, and G17, the most specific biomarker was G17 (96.9% clinical specificity at 209.8 pg/mL) for gastrinoma. This method should aid in the diagnosis of diseases associated with increased gastrin concentrations.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis

BackgroundTherapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. ObjectiveThis study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. ResultsThe use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2–40 mg L−1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8–10.0%, intra-assay CV of 10.2–11.0%, and accuracy of 96.8–104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15–3.57 mg L−1 for GEN C1, 0.12–3.55 mg L−1 for GEN C1a, 0.20–5.77 mg L−1 for GEN C2, and 0.47–12.88 mg L−1 for the GEN complex, all within the linear range of the assay. ConclusionA sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.