Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.     

Determination of senkirkine and senecionine in Tussilago farfara using microwave-assisted extraction and pressurized hot water extraction with liquid chromatography tandem mass spectrometry

Tussilago farfara (Kuan Donghua) is an important Chinese herbal medicine which has been shown to contain many bioactive compounds and widely used to relieve cough and resolve phlegm. However, besides therapeutic bioactive compounds, this herb has been found to contain toxic pyrrolizidine alkaloids (PAs), mainly senkirkine and traces of senecionine. In this report, conditions for microwave-assisted extraction (MAE) and pressurized hot water extraction (PHWE) were optimized for the extraction of the PAs. The results were compared against heating under reflux. It was found that the binary mixture of MeOH:H₂O (1:1) acidified using HCl to pH 2-3 was the optimal solvent for the extraction of the PAs in the plant materials. Liquid chromatography (LC) with ultra-violet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) in the positive mode was used for the determination and quantitation of senkirkine and senecionine in the botanical extract. The proposed extraction methods with LC/MS allow for the rapid detection of the major and the minor alkaloids in T. farfara in the presence of co-eluting peaks. With LC/MS, the quantitative analysis of PAs in the extract was done using internal standard calibration and the precision was found to vary from 0.6% to 5.4% on different days. The limits of detection (LODs) and limits of quantitation (LOQs) for MAE and PHWE were found to vary from 0.26 μg/g to 1.04 μg/g and 1.32 μg/g to 5.29 μg/g, respectively. The method precision of MAE and PHWE were found to vary from 3.7% to 10.4% on different days. The results showed that major and minor alkaloids extracted using MAE and PHWE were comparable to that by heating under reflux. Our data also showed that significant ion suppression was not observed in the analysis of senkirkine and senecionine in the botanical extracts with co-eluting peaks.     

Determination of organic and inorganic mercury species in water and sediment samples by HPLC on-line coupled with ICP-MS

This paper describes a preconcentration method for Hg²⁺ and MeHg⁺ in water samples using sodium diethyldithiocarbamate immobilized in polyurethane foam (PU-NaDDC) and an extraction method for several mercury species in sediment samples, including MeHg⁺, EtHg⁺ and PhHg⁺, which is simple, rapid, and uses a single organic solvent. Separation and measurement were done by high-performance liquid chromatography on-line with inductively coupled plasma mass spectrometry (HPLC/ICP-MS). Initially, the test of recovery was applied using procedures compatible with HPLC. Under the optimum extraction conditions, recoveries of 96.7, 96.3 and 97.3% were obtained for MeHg⁺, EtHg⁺, and PhHg⁺, respectively, from n = 4 spiked sediment samples. This study also demonstrates that the combination of solid-phase extraction on PU-NaDDC with HPLC separation and ICP-MS detection is an effective preconcentration procedure for simultaneous measurement of Hg²⁺ and MeHg⁺ at ultra-trace levels in water samples. The application of the proposed procedure to the determination of mercury species in drinking water sample was investigated. The proposed method clearly gave satisfactory average recoveries between 93.7 and 101.5%.     

Characterization of fulvic acids by liquid chromatography-quadrupole time-of-flight mass spectrometry

Fulvic acid standards from Suwannee River, Pony Lake, Elliot Soil, Waskish Peat, and Nordic Reservoir were characterized by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) operating in negative electrospray ionization mode. The method employed a commercially available stationary phase that resulted in a distinctive chromatographic peak for each of the fulvic acid samples that differed in width and retention time at peak maximum. The QTOF-MS, operating in TOF mode, revealed that the unique chromatographic peak shapes were the result of the relative fraction of hydrogen and oxygen contained in various fulvic acid components. Those species that contained larger amount of hydrogen displayed a larger mass defect and were retained longer on the LC column, indicating reduced polarity. This is supported by a reduction in the degree of fragmentation related to polar functional groups as the mass defect and retention time increased. Lastly, the analysis of even and odd mass (at m/z 1 greater) ion intensity ratios revealed a correlation to the percent nitrogen of the various standards.     

Application of ultra-high-pressure liquid chromatography–tandem mass spectrometry to the determination of multi-class pesticides in environmental and wastewater samples: Study of matrix effects

An ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the determination of 37 pesticides (herbicides, insecticides and fungicides) in environmental and wastewater has been developed. To efficiently combine UHPLC with MS/MS, a fast-acquisition triple quadrupole mass analyzer was used. This analyzer (minimum dwell time, 5 ms) allows acquiring up to three simultaneous transitions in the selected reaction monitoring mode for each compound assuring a reliable identification without resolution or sensitivity losses. A pre-concentration step based on solid-phase extraction using Waters Oasis HLB cartridges (0.2 g) was applied with a 100-fold pre-concentration factor along the whole analytical procedure. The method was validated based on European SANCO guidelines using surface, ground, drinking and treated water (from an urban solid residues treatment plant) spiked at two concentration levels (0.025 and 0.1 μg/L), the lowest having been established as the limit of quantification objective. The method showed excellent sensitivity, with instrumental limits of detection ranging from 0.1 to 7 pg. It was applied to environmental water samples (ground and surface water) as well as to samples of urban solid waste leachates (raw leachate and treated leachate after applying reversed osmosis) collected from a municipal treatment plant. Matrix effects have been studied in the different types of water samples analyzed, and several isotope-labelled internal standards have been evaluated as a way to compensate the signal suppression observed for most of the compounds studied, especially in wastewater samples. As a general remark, only those pesticides which response was corrected using their own isotope-labelled molecule, could be satisfactorily corrected in all type of samples, assuring in this way the accurate quantification in all matrix samples.     

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the φX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the φX174/HaeIII digest diluted from 500 μg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.     

Determination of thyreostats in urine and thyroid gland by ultra high performance liquid chromatography tandem mass spectrometry

Thyreostatic compounds could be illegally administered to animals in order to obtain a weight gain due to a higher retention of water in the edible tissue and the gastro-intestinal tract. In the European Union their use for animal production is banned since 1981. Recently a highly sensitive method exploiting the determination of thyreostats with 3-iodobenzylbromide prior to purification to determine thyreostats in urine and other matrices was reported. For the first time, the UPLC instrumentation was used to separate the 3-iodobenzyl derivatives of various thyreostats. The deuterated internal standards tapazole-d₃ and propylthiouracil-d₅ were for the first time used for the quantification of tapazole, thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil and mercaptobenzimidazole. The confirmative quantitative liquid chromatographic tandem mass spectrometric (LC–MS/MS) method was validated according to Commission Decision 2002/657/EC. The decision limit (CCα) and the detection capability (CCβ) were found to be for all compounds below the recommended value of 10 μg kg⁻¹.     

Dispersive liquid–liquid microextraction with little solvent consumption combined with gas chromatography–mass spectrometry for the pretreatment of organochlorine pesticides in aqueous samples

Dispersive liquid–liquid microextraction with little solvent consumption (DLLME-LSC), a novel dispersive liquid–liquid microextraction (DLLME) technique with few solvent requirements (13 μL of a binary mixture of disperser solvent and extraction solvent in the ratio of 6:4) and short extraction time (90 s), has been developed for extraction of organochlorine pesticides (OCPs) from water samples prior to gas chromatography/mass spectrometry analysis. In DLLME-LSC, much less volume of organic solvent is used as compared to DLLME. The new technique is less harmful to environment and yields a higher enrichment factor (1885–2648-fold in this study). Fine organic droplets were formed in the sample solution by manually shaking the test tube containing the mixture of sample solution and extraction solvent. The large surface area of the organic solvent droplets increases the rate of mass transfer from the water sample to the extractant and produces efficient extraction in a short period of time. DLLME-LSC shows good repeatability (RSD: 4.1–9.7% for reservoir water; 5.6–8.9% for river water) and high sensitivity (limits of detection: 0.8–2.5 ng/L for reservoir water; 0.4–1.3 ng/L for river water). The method can be used on various water samples (river water, tap water, sea water and reservoir water). It can be used for routine work for the investigation of OCPs.     

Simultaneous screening and confirmation of multiple classes of drug residues in fish by liquid chromatography-ion trap mass spectrometry

LC-ion trap mass spectrometry was used to screen and confirm 38 compounds from a variety of drug classes in four species of fish: trout, salmon, catfish, and tilapia. Samples were extracted with acetonitrile and hexane. The acetonitrile phase was evaporated, redissolved in water and acetonitrile, and analyzed by gradient chromatography on a phenyl column. MS² or MS³ spectra were monitored for each compound. Qualitative method performance was evaluated by the analysis over several days of replicate samples of control fish, fish fortified with a drug mixture at 1 ppm, 0.1 ppm and 0.01 ppm, and fish dosed with a representative from each drug class. Half of the 38 drugs were confirmed at 0.01 ppm, the lowest fortification level. This included all of the quinolones and fluoroquinolones, the macrolides, malachite green, and most of the imidazoles. Florfenicol amine, metronidazole, sulfonamides, tetracyclines, and most of the betalactams were confirmed at 0.1 ppm. Ivermectin and penicillin G were only detectable in the 1 ppm fortified samples. With the exception of amoxicillin, emamectin, metronidazole, and tylosin, residue presence was confirmed in all the dosed fish.     

Development and validation of an ultra-high-performance liquid chromatography coupled to time-of-flight mass spectrometry method to quantify benzoic acid and long-chain monocarboxylic acids (C12–C28) in atmospheric aerosols

An ultra-high-performance liquid chromatography/time-of-flight mass spectrometry (with negative ion electrospray ionization) methodology was developed for the simultaneous quantification of benzoic acid and 15 long-chain monocarboxylic acids (MCAs) in ambient aerosols. A fast and quantitative pressurized fluid extraction procedure was optimized using experimental design and the extracts were analyzed without any further clean-up step. Chromatographic separation was achieved on a BEH-C18 column with a mobile phase consisting of 5 mM formic acid in water/acetonitrile (90:10, v/v) and methanol. Excellent precision and accuracies in the MS mass measurements were observed. The method was validated using actual samples spiked with a solution containing either standards or ¹³C-surrogates. Matrix effects were observed for the C₁₄ MCA only. It was then applied to air particulate reference materials and atmospheric samples collected by a low-volume sampler.