Strong Anion-exchange Solid-phase Extraction Research Articles

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.

Determination of sulbactam in raw bovine milk by isotope dilution–ultra-high-performance liquid chromatography–tandem mass spectrometry

We developed a sensitive and selective isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of sulbactam residue in raw bovine milk. Sulbactam and internal standard, sulbactam-d5, were extracted from raw bovine milk via liquid-liquid extraction and enriched with strong anion exchange solid-phase extraction cartridges and finally analyzed by using UPLC-MS/MS with multiple reaction monitoring mode. The method was validated according to European regulations. The calibration curve showed good linearity, with a correlation coefficient of 0.9998. Decision limit and detection capability of sulbactam were determined by matrix calibration curve and were 0.0445 and 0.0517 μg/L, respectively. The recoveries of sulbactam in fortified raw bovine milk ranged from 72.1 to 91.5%, with the intra- and interday relative standard deviations ranging from 3.0 to 18.9%. Furthermore, the developed method was applied to analyzing real raw bovine milk samples collected from dairy farms in Beijing, China. Sulbactam was not determined in all samples. The proposed method could ultimately serve as a methodological foundation for the determination of sulbactam in different types of raw milk and dairy products.

Determination of free nucleotides in infant formula by solid phase extraction-liquid chromatography

A method for the determination of five free nucleotides in infant formula was established. The analytes were extracted by ethylene diamine tetraacetic acid and sodium chloride solution, and purified by strong anion exchange (SAX) solid-phase extraction. An Atlantis T3 reverse-phase column was applied to separate the five free nucleotides. A high performance liquid chromatography (HPLC) system equipped with a diode array detector was applied to detect the target compound, and a series of external standards were used for quantification. Good linearities (r≥0.999) were observed, with the range of 0.5-10 mg/L for the five free nucleotides. When the addition levels were 0.05-0.50 g/kg, the recoveries were between 91.1% and 112%, and the precisions were between 2.3%-4.7%. The limits of detection (LODs) and the limits of quantification (LOQs) for the five free nucleotides ranged from 0.0010 to 0.0015 g/kg and from 0.0030 to 0.0045 g/kg. In conclusion, the proposed method has several merits. First, the high accuracy and precision indicated that the quality control sample had a relative deviation of less than 10% to the median. Second, perfect anti-disturbance was observed for the complex goat milk powder. Thus, this method can provide technical support for supervision departments and guarantees healthy development of the dairy industry.

Highly sensitive determination of fatty acid esters of hydroxyl fatty acids by liquid chromatography-mass spectrometry.

Recently, a new class of endogenous lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), was discovered with anti-diabetic and anti-inflammatory effects in mammals. FAHFAs attracted increasing attention because of their critical physiological function. However, accurate quantitation of FAHFAs is still a challenge due to their high structure similarity and low abundance in biological samples. Herein, we developed a highly sensitive method for the determination of 16 FAHFAs (PAHSAs, OAHSAs, SAHSAs and POHSAs) in biological samples by coupling strong anion exchange solid phase extraction (SAX-SPE) with chemical labeling assisted ultra-high performance liquid chromatography/mass spectrometry (SAX-SPE-CL-UHPLC/MS). In the developed method, SAX-SPE was employed to selectively enrich and purify FAHFAs from biological samples. And then a pair of isotope labeling reagents, 2-dimethylaminoethylamine (DMED) and d4-DMED were used to label the purified samples and standard FAHFAs, respectively. The labeled samples were mixed and further subjected to UHPLC/MS analysis. Our results demonstrated that the detection sensitivities of FAHFAs increased by 7-72 folds upon DMED labeling and the limits of detections (LODs) of labeled FAHFAs ranged from 0.01 to 0.14pg. Moreover, a good separation of FAHFAs isomers was achieved on C18 column in a UHPLC system and all FAHFAs could be analyzed in 20min with sharp peak shape. The established method provided substantial sensitivity, high specificity, and broad linear dynamic range (3 orders of magnitude). Using this method, we successfully measured the contents and distribution of FAHFAs in rat white adipose, lung, kidney, thymus, liver and heart tissues. The results showed that 7 FAHFAs (13-, 12-, 9-, 5-PAHSA, 13-, 12- and 9-SAHSA) were observed in different tissues of rat. In addition, we successfully detected the above 7 FAHFAs in human serum samples; and among the 7 FAHFAs, 13-, 9-PAHSA, 13- and 12-SAHSA were found remarkably decreased in human breast cancer serum. The proposed method could be successfully applied for the detection of FAHFAs in various biological samples, which will facilitate the understanding of the physiological functions of FAHFAs.

Determination of nine phthalate ester metabolites in human urine using ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometry

A method was developed to determine trace concentrations of phthalate ester (PAE) metabolites in human urine by using ultra high performance liquid chromatography-triple quadrupole mass spectrometry. Aliquots (2 mL) of human urine samples were purified using strong anion-exchange solid-phase extraction after enzymatic deconjugation by glucuronidase for 2 h. The separation was performed on a Waters ACQUITY UPLC BEH Phenyl column (100 mm×2.1 mm, 1.7 μ m) with gradient elution by 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid aqueous solution. The concentrations of nine PAE metabolites were determined under a multiple reaction monitoring mode by negative electrospray ionization. Eight of the determined metabolites showed good linearities in the range of 0.39-200 μ g/L, and the ninth metabolite showed linearity in the range of 1.17-600 μ g/L with correlation coefficients > 0.995. The limits of detection ranged from 0.06 to 0.85 μ g/L, and the limits of quantification ranged from 0.20 to 2.80 μ g/L. The recovery rates at all three spiked levels ranged from 84.1% to 122% with relative standard deviations between 4.5% and 14.3%. The intra-and inter-day precisions were not more than 10.1% and 9.3%, respectively. The matrix effect and stability were found to be favorable for analysis. This method was utilized for the determination of 50 human urine samples. The detection rates of monocyclohexyl phthalate (MCHP) and monobenzyl phthalate (MBZP) were 0 and 44.0%, and the detection rates of the other seven PAE metabolites was 100%. This method has the advantage of convenient operation and good accuracy and stability, and is suitable for the quantitative determination of the nine PAE metabolites in human urine.

Simultaneous LC-MS/MS Analysis of Glyphosate, Glufosinate, and Their Metabolic Products in Beer, Barley Tea, and Their Ingredients

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H2O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.

Liquid chromatographic-mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria.

Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5-2500μg/mL for LA, 0.125-125μg/mL for aHBA, 7.5-375μg/mL for bHBA, 0.1-100μg/mL for pHPLA, 1-1000μg/mL for MA, 0.25-250μg/mL for MMA, 0.25-100μg/mL for EMA, and 30-1500μg/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169μg/mL, aHBA=4.70-38.4μg/mL, bHBA=7.70-38.0μg/mL, pHPLA=0.900-4.30μg/mL and aKGA=30.2-32.0) and seven in urine samples (range LA=11.2-513μg/mL, aHBA=1.50-69.5μg/mL, bHBA=8.10-111μg/mL, pHPLA=4.30-27.7μg/mL, MMA=0.300-13.3μg/mL, EMA=0.300-48.1μg/mL and aKGA=30.4-107μg/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.

Improved quantitative method for fludarabine in human plasma by liquid chromatography and tandem mass spectrometry

An improved quantitative assay was developed and validated for fludarabine in human plasma. Fludarabine and its internal standard, cladribine, were separated on a C18 analytical column after sample purification by strong anion-exchange solid-phase extraction. Quantitation was performed by electrospray triple-quadrupole mass spectrometry in positive ionization mode using multiple-reaction monitoring. This assay had excellent inter- and intra-assay precisions within 8%, and accuracies ranging from 100 to 116%. The method was linear within the concentration range of 0.2-250ng/mL using 100μL of plasma with mean R(2)=0.9999. The extraction recoveries were 85% for fludarabine and 95% for the internal standard, which represent a significant improvement over the previously published methods. We utilized this method for pharmacokinetic (PK) investigations in 215 patients. Interference peaks constantly observed in each blank plasma sample were well resolved from fludarabine using our optimized LC-MS/MS conditions, demonstrating the reliability of this improved assay. The validated method will be further applied to PK studies within our bone marrow transplant program, which will allow for optimal dose and scheduling of fludarabine in these patients.

SPE HG-AAS method for the determination of inorganic arsenic in rice—results from method validation studies and a survey on rice products

The present paper describes the development, validation and application of a method for inorganic arsenic (iAs) determination in rice samples. The separation of iAs from organoarsenic compounds was done by off-line solid-phase extraction (SPE) followed by hydride generation atomic absorption spectrometry (HG-AAS) detection. This approach was earlier developed for seafood samples (Rasmussen et al., Anal Bioanal Chem 403:2825-2834, 2012) and has in the present work been tailored for rice products and further optimised for a higher sample throughput and a lower detection limit. Water bath heating (90 °C, 60 min) of samples with dilute HNO3 and H2O2 solubilised and oxidised all iAs to arsenate (As(V)). Loading of buffered sample extracts (pH 6 ± 1) followed by selective elution of arsenate from a strong anion exchange SPE cartridge enabled the selective iAs quantification by HG-AAS, measuring total arsenic (As) in the SPE eluate. The in-house validation gave mean recoveries of 101-106% for spiked rice samples and in two reference samples. The limit of detection was 0.02 mg kg(-1), and repeatability and intra-laboratory reproducibility were less than 6 and 9%, respectively. The SPE HG-AAS method produced similar results compared to parallel high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) analysis. The SPE separation step was tested collaboratively, where the laboratories (N = 10) used either HG-AAS or ICP-MS for iAs determination in a wholemeal rice powder. The trial gave satisfactory results (HorRat value of 1.6) and did not reveal significant difference (t test, p > 0.05) between HG-AAS and ICP-MS quantification. The iAs concentration in 36 rice samples purchased on the Danish retail market varied (0.03-0.60 mg kg(-1)), with the highest concentration found in a red rice sample.

A One-Step Solid Phase Extraction Method for Bioanalysis of a Phosphorothioate Oligonucleotide and Its 3′ n-1 Metabolite from Rat Plasma by uHPLC–MS/MS

Oligonucleotide therapeutics have emerged as a promising class of drugs to treat a wide range of diseases caused by genetic abnormalities. Replacement of the phosphodiester linkage with a phosphorothioate is one of the most successful modifications made to oligonucleotides to enhance their in vivo stability. The longer elimination phase of phosphorothioates and other modified oligonucleotides requires sensitive and selective methods to quantify the parent drug and their metabolites simultaneously. Liquid chromatography tandem mass spectrometry has excellent selectivity between the parent drug and its metabolites and a wide dynamic range. However, the biological sample extraction remains a formidable challenge in developing quantitative LC-MS methods for oligonucleotides. Protein precipitation, protein digestion, liquid-liquid extraction, reversed phase solid phase extraction (SPE), strong anion exchange SPE, and combinations of them have been reported to extract oligonucleotides from biological matrices. Unfortunately, these methods either have low recoveries or present potential problems for applications with chromatography due to the large amount of matrix substances in the resulting solutions. In this study, a weak anion exchange SPE method was optimized. The recovery ranged from 60% to 80% depending on the concentration. This is the first report of a one-step SPE method with recoveries greater than 60% across the method dynamic range. This sample extraction procedure was used in combination with ultrahigh-performance liquid chromatography-tandem mass spectrometry. The lower limit of quantitation was 10ng/mL (1.3nM), and the dynamic range was 10-1,000ng/mL. The intra- and inter-day precision and accuracy were within 8.4% and 10.5%, respectively.

Development and validation of an SPE HG-AAS method for determination of inorganic arsenic in samples of marine origin

The present paper describes a novel method for the quantitative determination of inorganic arsenic (iAs) in food and feed of marine origin. The samples were subjected to microwave-assisted extraction using diluted hydrochloric acid and hydrogen peroxide, which solubilised the analytes and oxidised arsenite (As(III)) to arsenate (As(V)). Subsequently, a pH buffering of the sample extract at pH 6 enabled selective elution of As(V) from a strong anion exchange solid-phase extraction (SPE) cartridge. Hydride generation atomic absorption spectrometry (HG-AAS) was applied to quantify the concentration of iAs (sum of As(III) and As(V)) as the total arsenic (As) in the SPE eluate. The results of the in-house validation showed that mean recoveries of 101-104% were achieved for samples spiked with iAs at 0.5, 1.0 and 1.5 mg·kg(-1), respectively. The limit of detection was 0.08 mg kg(-1), and the repeatability (RSD(r)) and intra-laboratory reproducibility (RSD(IR)) were less than 8% and 13%, respectively, for samples containing 0.2 to 1.5 mg kg(-1) iAs. The trueness of the SPE HG-AAS method was verified by confirming results obtained by parallel analysis using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. It was demonstrated that the two sets of results were not significantly different (P < 0.05). The SPE HG-AAS method was applied to 20 marine food and feed samples, and concentrations of up to 0.14 mg kg(-1) of iAs were detected.

Determination of nonnitrogenous organic acids and inorganic anions in sugarcane molasses and molasses alcohol waste by ion chromatography with solid-phase extraction

A method was established for the determination of main nonnitrogenous organic acids (including acetic, lactic, succinic, maleic, tartaric, oxalic, fumaric, citric, and aconitic acids) and three inorganic anions (Cl-, SO4(2-) and PO4(3-)) in sugarcane molasses and molasses alcohol waste by ion chromatography with solid-phase extraction. The diluted sample solution was purified by strong anion exchange (SAX) solid-phase extraction (SPE) in a small packed column to remove most of sugar, pigment and other interfering matrices. The eluate obtained from the column was rinsed with diluted KOH solution and filtered through a 0.45 microm inorganic membrane, finally separated on an anion column of IonPac AS15 with the gradient elution of KOH solution and determined with a suppressed conductivity detector. The pretreatment and procedures of SPE for separating organic acids and inorganic anions from their matrix were investigated. The detection limits wee d less than 0.20 mg/L. The relative standard derivations were less than 6.7%. The organic acids and inorganic anions in three real samples (two sugarcane molasses samples and an alcohol waste sample) were determined, the recovery ranges were from 94% to 109%. The method shows good precision and linearity.

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

Optimization of solid phase extraction clean up and validation of quantitative determination of corticosteroids in urine by liquid chromatography–tandem mass spectrometry

A solid phase extraction (SPE) method for extraction and clean up of 9 synthetic corticosteroids was optimized for quantification by reversed-phase high-performance liquid chromatography/negative electrospray ionisation mass spectrometry. Clean up was accomplished using a mixed mode polymeric strong anion exchange SPE column. The final method was validated according to EU regulations for determination of residues of veterinarian drugs in products of animal origin. Initial results showed a large difference in ion suppression between samples of porcine and bovine urine. The aim of optimisation was to design a procedure that minimised this difference while using a single SPE procedure and a fast HPLC method that enabled sufficient separation of the epimers beta- and dexamethason. To include conjugated corticosteroids in the analysis, the sample was hydrolysed with Helix Pomatia β-glucuronidase/aryl sulfatase. For the final method, which included fluocinolone acetonid, triamcinolone acetonid, beclomethasone, flumethasone, dexamethasone, betamethasone, 6α-methylprednisolone, prednisone and prednisolone, a quantification based on spiked samples carried through the entire analytical procedure was used. For quantification of triamcinolone acetonid an internal standard (triamcinolon acetonid-D6) was used. Relative average recoveries from 96 to 103% were found, except for beclomethason (113%). Absolute average recoveries were 81–99%. Quantification limits (decision limits, CCα) were demonstrated to be not higher than 1 μg L −1 (3 μg L −1 for prednisone and prednisolone). The internal reproducibility, determined by triplicates from spiking at three different levels in six analytical series was 7–19% (at 2–4 μg L −1) except for prednisone and prednisolone (26–27% at 3–6 μg L −1).

Analysis of Imazaquin in Soybeans by Solid-phase Extraction and High-performance Liquid Chromatography

An analytical method for the determination imazaquin residues in soybeans was developed. The developed liquid/liquid partition and strong anion exchange solid-phase extraction procedures provide the effective cleanup, removing the greatest number of sample matrix interferences. By optimizing mobile-phase pH water/acetonitrile conditions with phosphoric acid, using a C-18 reverse-phase chromatographic column and employing ultraviolet detection, excellent peak resolution was achieved. The combined cleanup and chromatographic method steps reported herein were sensitive and reliable for determining the imazaquin residues in soybean samples. This method is characterized by recovery >88.4%, precision <6.7% CV, and sensitivity of 0.005 ppm, in agreement with directives for method validation in residue analysis. Imazaquin residues in soybeans were further confirmed by high performance liquid chromatography-mass spectrometry (LC-MS). The proposed method was successfully applied to the analysis of imazaquin residues in soybean samples grown in an experimental field after treatments of imazaquin formulation.

Quantification of cidofovir in human serum by LC–MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/ z 278.1 → 234.9 and the m/ z 288.1 → 133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within ±12.1% for quality controls. The novel LC–MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.

Determination of Imazaquin Residues in Soil by Solid-Phase Extraction and High-Performance Liquid Chromatography

A method has been developed for the quantitation of imazaquin residues in soil. The herbicide was extracted from soil with methanol-water (2 + 1, v/v) and cleaned up by strong anion-exchange solid-phase extraction cartridges. Analysis was performed by using high-performance liquid chromatography with ultraviolet detection. Average recoveries through the method ranged from 90.7 to 100.6%, with relative standard deviation equal to or lower than 6.6%. The limit of detection was estimated to be 0.0015 mg/kg, and the minimum quantitation concentration of imazaquin in soil was 0.005 mg/kg. This method was successfully applied to evaluate imazaquin residue levels in soil and its dissipation rates in a soybean field in the Xisanqi District of Beijing, People's Republic of China. The dissipation study showed that the half life of imazaquin in soil was 10.37 +/- 0.0135 days at 3 different application rates.

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.

Determination of perfluorooctanoic acid and perfluorooctanesulfonate in human tissues by liquid chromatography/single quadrupole mass spectrometry

A method is described that permits the measurement of the levels of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in human liver, kidney, adipose tissue, brain, basal ganglia, hypophysis, thyroid, gonads, pancreas, lung, skeletal muscle and blood, even in subjects not occupationally exposed to these compounds. The purification of samples involved the use of trifunctional (tC18) and strong anion-exchange (SAX) solid-phase extraction cartridges, and the analysis utilized a high-performance liquid chromatograph coupled to a single quadrupole mass spectrometer (LC/MS). The analyses were conducted on a mixed-bed reversed-phase column by gradient runs using 3 mM ammonium acetate/methanol mixtures at different proportions as the mobile phase. The detector was used in electrospray negative ion mode by recording simultaneously the ions m/z 413.0 (PFOA) and 499.0 (PFOS). Perfluorononanoic acid (PFNA), added to the samples before the purification, was used as the internal standard (ion monitored = m/z 463.6). The recovery rates of the extraction procedure ranged from 79.6 to 95.6% (CV% 1.7-7.4%) for PFOA, from 79.7 to 100.8% (CV% = 1.2-7.1) for PFOS, and from 89.1 to 102.3% (CV% = 0.9-5.2 %) for PFNA. The calibration curves were linear up to at least 400 ng of analytes per gram of tissue. The detection limits (signal-to-noise ratio = 3) were 0.1 ng/g for both PFOA and PFOS measured in all tissues except adipose tissue, where the limits were about 0.2 ng/g. The content of analytes in tissues varied from 0.3 to 3.8 ng/g (respectively: basal ganglia and lung) for PFOA, and from 1.0 to 13.6 ng/g (respectively: skeletal muscle and liver) for the linear isomer of PFOS. The method is suitable to evaluate the content of PFOA and PFOS in different tissues taken from the general population exposed to very low concentrations of these pollutants.

Gas Chromatographic–Mass Spectrometric Method for Quantitation of Trimethylsilyl Derivatives of Nerve Agent Degradation Products in Human Plasma, Using Strong Anion–Exchange Solid-Phase Extraction

For unequivocal proof of the use of nerve agents such as sarin, soman, cyclohexylsarin, VX, and Russian VX, a simple and accurate method, gas chromatography–mass spectrometry (GC–MS) after trimethylsilyl derivatization, was explored for simultaneous determination of the corresponding alkyl methylphosphonic acids (AMPAs) and of methylphosphonic acid (MPA) in human plasma. GC–MS analysis was performed after solid-phase extraction, with a strong anion-exchange cartridge, from plasma samples previously deproteinized with mercuric acetate, and then derivatization with bis(trimethylsilyl)trifluoroacetamide containing 5% trimethylchlorosilane. All five AMPA derivatives and the MPA derivative were separated to baseline within 11 minutes without interference. Linear calibration plots were obtained over concentrations ranging from 50 ng mL−1 to 5 µg mL−1. The relative standard deviation of recoveries ranged from 1.9 to 9.7% and detection limits were 22 ng mL−1 or below.