Solid-phase Extraction-liquid Chromatography-tandem Mass Spectrometry Research Articles

Validated Method for Determination of Eight Banned Nitroimidazole Residues in Natural Casings by LC/MS/MS with Solid-Phase Extraction

A sensitive method based on solid-phase extraction-liquid chromatography-tandem mass spectrometry interfaced with electrospray ionization (SPE-LC-MS/MS-ESI) was developed for the simultaneous determination of 8 banned nitroimidazole (NOZ) drugs including metronidazole (MNZ), ronidazole (RNZ), dimetridazole (DMZ), tinidazole, ornidazole, secnidazole, metronidazole-OH (MNZOH, the metabolite of MNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI, the metabolite of RNZ and DMZ) in natural casings. After extraction with ethyl acetate and evaporation, the NOZs were reconstituted in ethyl acetate and purified on a strong cation-exchange SPE column, and then LC/MS/MS analysis was performed by positive ESI applying multiple reaction monitoring of 2 transition reactions for each compound. The method was validated according to the European Union requirements (Commission Decision 2002/657/EC). Specificity, linearity, decision limit (CCalpha), detection capability (CCbeta), accuracy, and precision were determined. Average recoveries of the 8 NOZs from natural animal casing fortified at 3 levels (0.1, 0.5, and 1 microg/kg) ranged from 87.3 to 116.5%. The calculated CCalpha for NOZs ranged from 0.029 to 0.049 microg/kg, and CCbeta ranged from 0.049 to 0.083 microg/kg. Repeatability was in the range of 3.35-10.1%, and within-laboratory reproducibility was <10.3%.

On-line coupling of solid-phase extraction to liquid chromatography–tandem mass spectrometry for the determination of macrolide antibiotics in environmental water

An automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) system was developed for the determination of macrolide antibiotics including erythromycin (ETM), roxithromycin (RTM), tylosin (TLS) and tilmicosin (TMC) in environmental water samples. A Capcell Pak MF Ph-1 column packed with restricted access material (RAM) was used as SPE column for the concentration of the analytes and clean-up of the sample. One milliliter water sample was injected into the conditioned SPE column and the matrix was washed out with 3 mL high purity water. By rotation of the switching valve, macrolides (MLs) were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase. The matrix effect was evaluated by the directly injection LC–MS and on-line SPE–LC–MS methods. The limits of detection (LODs) and limits of quantification (LOQs) obtained are in the range of 2–6 and 7–20 ng L −1, respectively, which means that the proposed method is suitable for trace analysis of MLs at low level concentration. The intra- and inter-day precisions are in the range of 2.9–7.2% and 3.3–8.9%, respectively. In the three fortified levels (20, 200 and 2000 ng L −1), recoveries of MLs ranging from 86.5% to 98.3% are obtained.

Development and validation of a high-throughput zwitterionic hydrophilic interaction liquid chromatography solid-phase extraction–liquid chromatography–tandem mass spectrometry method for determination of the anti-influenza drug peramivir in plasma

An assay for the analysis for the quantification of the anti-influenza drug peramivir in human plasma using high-throughput zwitterionic (ZIC) hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) in a 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The ZIC-HILIC SPE efficiently removed sources of interference present in the supernatant after protein precipitation of plasma proteins. The main advantage of the ZIC-HILIC SPE sample preparation step was that it allowed load and elution conditions to be optimised to extract only peramivir and minimize co-extraction of lipophilic phospholipids. The method was validated according to published US Food and Drugs Administration guidelines and showed excellent performance. The assay was validated over two calibration ranges (0.952–500 and 50–50,000 ng/mL) to support analysis of peramivir after intra-venous administration. The lower limit of quantification for peramivir in plasma was 1 ng/mL and the upper limit of quantification was 50,000 ng/mL. The within-day and between-day precisions expressed as RSD, were lower than 8% at all tested quality control concentrations and below 11% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range.

CB1 receptor‐mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.

Analysis of 17 polar to semi-polar pesticides in the Ebro river delta during the main growing season of rice by automated on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry

This work describes the optimization of a fully automated method based on on-line solid-phase extraction-liquid chromatography–electrospray–tandem mass spectrometry (SPE-LC–ESI-MS/MS) for the simultaneous determination of 17 medium to polar pesticides in water. The list of target analytes included organophosphates, triazines, phenylureas, anilides, chloroacetanilides, acidic herbicides and thiocarbamates. Detection limits achieved were below 5 ng/L for all compounds except metolachlor (12 ng/L), alachlor (17 ng/L), malathion (36 ng/L) and fenitrothion (50 ng/L). The method developed was used to investigate the occurrence of the target pesticides in a total of 52 water samples collected monthly (from May to August 2005) at 14 selected locations in the rice cultivation area of the Ebro river delta. The study showed high levels, in the μg/L range, of bentazone, MCPA, propanil, molinate and atrazine, in basically all the samples investigated. The remaining pesticides were present at lower levels (<0.1 μg/L) or only detected sporadically ( e.g. fenitrothion and malathion). The sampling campaign performed in July showed comparatively higher levels than the other three campaigns.

Simultaneous determination of various pharmaceutical compounds in water by solid-phase extraction–liquid chromatography–tandem mass spectrometry

A solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionisation tandem mass spectrometry (LC–ESI-MS–MS) method was developed and validated for simultaneous analysis of 11 pharmaceutical residues (propranolol, sulfamethoxazole, meberverine, thioridazine, carbamazepine, tamoxifen, mecoprop, indomethacine, diclofenac, meclofenamic acid and monensin) in environmental water samples. The collision energy chosen for the multiple reaction monitoring (MRM) experiment was optimised. A number of parameters that may affect the recovery of the pharmaceuticals, such as the type of SPE cartridges, eluents, as well as water properties including pH value, salinity and concentration of colloid and surfactant were investigated. It is shown that the Oasis HLB SPE cartridge produced the best recoveries of the target pharmaceuticals while methanol was efficient in eluting pharmaceuticals from SPE cartridges. The recovery of some target compounds was enhanced with increasing salinity, but reduced by increasing pH value, and unaffected by surfactant concentration (0–10 μg/l). The recovery of most compounds was slightly increased by the presence of colloids (0–10 mg/l), which however caused a reduction in recovery for sulfamethoxazole and meberverine. The optimised method was further verified by performing spiking experiments in river water and seawater matrices, with good recovery and reproducibility for all except two compounds. The established method was successfully applied to environmental water samples from East Sussex, UK, for the determination of the target pharmaceuticals.

High-throughput on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma

A rapid, sensitive and fully automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 μL of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 μg/L and the r 2 > 0.99 over the range investigated (10–1000 μg/L). Limits of quantification (LOQs) were estimated to be 10 μg/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD).

Combined use of biomarkers and in situ bioassays in Daphnia magna to monitor environmental hazards of pesticides in the field

The aim of this investigation was to evaluate toxicity effects of pesticides in aquatic invertebrates using in situ bioassays with the local species, Daphnia magna. Investigations were carried out in the Delta del Ebro (northeast Spain) during the main growing season of rice (May-August). Measures of energy consumption (i.e., algal grazing) and of specific biochemical responses (biomarkers) were conducted in individuals transplanted at four stations, including a clean site upstream of the affected area and the three main channels that collect and drain the water from the rice fields into the sea. Seventeen pesticides were analyzed in water by on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry. The results obtained indicated high levels of pesticides in water, with peak values of 487 microg/L for bentazone, 8 microg/L for methyl-4-chlorophenoxyacetic acid, 5 microg/L for propanil, 0.8 microg/L for molinate, and 0.7 microg/L for fenitrothion. Measured biological responses denoted severe effects on grazing rates; a strong inhibition of cholinesterases and carboxylesterases, which are specific biomarkers of organophosphorous and carbamate pesticides; and altered patterns of the antioxidant enzyme catalase and the phase II metabolizing enzyme glutathione S-transferase. Correlation analysis with pesticide residue levels converted to toxic units relative to its acute 48-h median lethal concentration of D. magna indicated significant and negative coefficients between the dominant pesticide residues and the observed biological response, thus denoting a clear cause-and-effect relationship. The results emphasize the importance of considering specific (biomarkers) as well as more generalized and ecologically related (grazing) in situ responses to identify and evaluate biological effects of environmental contaminants in the field.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma

In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Application of an innovative matrix solid-phase dispersion–solid-phase extraction–liquid chromatography–tandem mass spectrometry analytical methodology to the study of the metabolism of the estrogenic mycotoxin zearalenone in rainbow trout liver and muscular tissue

The metabolic fate of the estrogenic mycotoxin zearalenone in rainbow trout is presently unknown. In this study, the tissue concentration of zearalenone and its principal metabolites (α-zearalenol and β-zearalenol) was determined. A known amount of zearalenone was administered as a single bolus to ten fish, and the biological tissue concentration was determined at various times following administration. The analytes were extracted from liver and muscular tissue using an on-line matrix solid-phase dispersion–solid-phase extraction sample preparation protocol, and their concentration determined by HPLC–Turboionspray–tandem mass spectrometry. The results showed that zearalenone is mainly metabolized into α-zearalenol in both liver and muscular tissues. The maximum concentrations of each analyte found in liver were 76.1, 211.2 and 63.7 ng/g respectively for zearalenone, α-zearalenol and β-zearalenol, while in muscular tissue they were 10.7, 8.2 and 6.5 ng/g. These values were reached after 2 h in liver tissue and 12 h in muscular tissue. Moreover the data obtained showed that the elimination rate in liver is quite fast since 48 h after the exposure less than 7% of the maximum concentration found is still present. In muscular tissue, however, about one-third of the maximum concentration found is still present after 48 h.

Determination of three phthalate metabolites in human urine using on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry

An on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (on-line SPE–HPLC–MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE–HPLC–MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.

Determination of valdecoxib and its metabolites in human urine by automated solid-phase extraction–liquid chromatography–tandem mass spectrometry

A simple, sensitive and specific automated SPE–LC–MS–MS assay was developed and validated for determination of valdecoxib (I), its hydroxylated metabolite (II) and carboxylic acid metabolite (III) in human urine. The analytes (I, II and III) and a structural analogue internal standard (I.S.) were extracted on a C 18 solid-phase extraction cartridge using a Zymark RapidTrace™ automation system. The chromatographic separation was performed on a narrow-bore reverse phase HPLC column with a mobile phase of acetonitrile–water (50:50, v/v) containing 10 m M 4-methylmorpholine (pH 6.0). The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring with a tandem mass spectrometer. The precursor to product ion transitions of m/ z 313→118, m/ z 329→196 and m/ z 343→196 were used to measure I, II and III, respectively. The assay exhibited a linear dynamic range of 1–200 ng/ml for I and II and 2–200 ng/ml for III in human urine. The lower limit of quantitation was 1 ng/ml for I and II and 2 ng/ml for III. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 5.5 min for each sample made it possible to analyze a throughput of 70 human urine samples per run. The assay has been successfully used to analyze human urine samples to support clinical phase I and II studies.

Trace determination of priority pesticides in water by means of high-speed on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation

An integrated on-line SPE–HPLC–MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50×1 mm, 30–50 μm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API–MS/MS detection. Matrix effects on the APCI–MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE–HPLC–MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.

Determination of quaternary ammonium pesticides by liquid chromatography–electrospray tandem mass spectrometry

A method for the direct determination of paraquat, diquat, chlormequat and difenzoquat in water samples, using an on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry system was developed. No sample preparation was required and the detection limits were below the European Union maximum residue levels. The chromatographic separation was performed using an XTera MS C 8 column. The concentration of the ion pair reagent, the pH and the gradient elution were optimized to give high recoveries and good chromatographic resolution between quats. The detection was carried out using an ion trap as mass analyzer. Parameters such as the magnitude and duration of the resonant excitation voltage and the magnitude of the trapping RF voltage for full scan tandem mass spectrometry (MS–MS) experiments were studied to establish the optimal experimental conditions. Moreover, the accurate optimization of these parameters allowed MS–MS experiments of low mass ions, below m/z 200, providing unambiguous peak identification. Finally, the reproducibility of the proposed method was shown by good run-to-run and day-to-day precision values and its applicability to the determination of quats in drinking water was evaluated using spiked samples.