LC ESI-MS MS Research Articles

Mitigating Matrix Effects in LC-ESI-MS-MS Analysis of a Urinary Biomarker of Xylenes Exposure.

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) with stable isotope-labeled internal standards (SIL-ISs) is the gold standard for quantitative analysis of drugs and metabolites in complex biological samples. Significant isotopic effects associated with deuterium labeling often cause the deuterated IS to elute at a different retention time from the target analyte, diminishing its capability to compensate for matrix effects. In this study, we systematically compared the analytical performance of deuterated (2H) SIL-IS to non-deuterated (13C and 15N) SIL-ISs for quantifying urinary 2-methylhippuric acid (2MHA) and 4-methylhippuric acid (4MHA), biomarkers of xylenes exposure, with an LC-ESI-MS-MS assay. Analytical method comparison between ISs demonstrated a quantitative bias for urinary 2MHA results, with concentrations generated with 2MHA-[2H7] on average 59.2% lower than concentrations generated with 2MHA-[13C6]. Spike accuracy, measured by quantifying the analyte-spiked urine matrix and comparing the result to the known spike concentration, determined that 2MHA-[2H7] generated negatively biased urinary results of -38.4%, whereas no significant bias was observed for 2MHA-[13C6]. Post-column infusion demonstrated that ion suppression experienced by 2MHA and 2MHA-[13C6] was not equally experienced by 2MHA-[2H7], explaining the negatively biased 2MHA results. The quantitation of urinary 4MHA results between ISs exhibited no significant quantitative bias. These results underscore the importance of the careful selection of ISs for targeted quantitative analysis in complex biological samples.

Evidence of Sulfur Mustard Exposure in Human Plasma by LC-ESI-MS-MS Detection of the Albumin-Derived Alkylated HETE-CP Dipeptide and Chromatographic Investigation of Its Cis/Trans Isomerism.

Sulfur mustard (SM) is a chemical warfare agent that causes painful blisters and chemically modifies endogenous biomacromolecules by alkylation to hydroxyethylthioethyl (HETE) adducts representing valuable long-term markers for post-exposure analysis. The albumin adduct formed in human plasma in vitro (HETE bound to the side chain of cysteine 34) was isolated and cleaved by current lots of pronase primarily generating the internal modified dipeptide (HETE-cysteine-proline, HETE-CP) instead of the formerly reported HETE-CPF tripeptide. The analyte was detected by liquid chromatography-electrospray ionization tandem-mass spectrometry (LC-ESI-MS-MS). In principle, HETE-CP undergoes a dynamic on-column equilibrium of cis-trans isomerism thus requiring separation at 50°C to obtain one narrow peak. Accordingly, we developed both a novel longer lasting but more sensitive microbore (1 mm i.d., flow 30 µL/min, cycle time 60 min, LOD 50 nM) and a faster, less sensitive narrowbore (2.1 mm i.d., 200 µL/min, cycle time 16 min, LOD 100 nM, both on Atlantis T3 material at 50°C) LC-ESI-MS-MS method suitable for verification analysis. The corresponding tri- and tetrapeptide, Q(HETE)-CPF were monitored simultaneously. HETE-CP peak areas were directly proportional to SM concentrations added to plasma in vitro (0.05-100 µM). Albumin adducts formed by deuterated SM (d8-SM) served as internal standard.

Analysis of eight glycols in serum using LC-ESI-MS-MS.

A liquid chromatography coupled with electrospray tandem mass spectrometry method was developed for the analysis of ethylene glycol, diethylene glycol, triethylene glycol, 1,4-butanediol, 1,2-butanediol, 2,3-butanediol, 1,2-propanediol and 1,3-propanediol, in serum after a Schotten-Baumann derivatization by benzoyl chloride. Usual validation parameters were tested: linearity, repeatability and intermediate precision, limits of detection and quantification, carry over and ion suppression. Limits of detection were between 0.18 and 1.1 mg/L, and limits of quantification were between 0.4 and 2.3 mg/L. Separation of isomers was possible either chromatographically or by selecting specific multiple reaction monitoring transitions. This method could be a useful tool in case of suspected intoxication with antifreeze agents, solvents, dietary supplements or some medical drug compounds.

Hepatoprotective activity of LC–ESI-MS standardized Iris spuria rhizome extract on its main bioactive constituents

The study was designed to evaluate the hepatoprotective activity of Iris spuria against paracetamol induced toxicity at two different doses 100 and 200mg/kg. The extract showed significant protective activity (p>0.01) at both the doses in dose dependent manner. Administration of the plant extract restored the paracetamol induced elevated levels of serum marker and distorted hepatic tissue architecture. The lipid peroxides (LPO) and glutathione (GSH) levels were also restored towards normal in liver tissue significantly. The main chemical constituents of the extract identified by the liquid chromatography–tandem mass spectrometry (LC–ESI-MSMS) were found to be flavones and isoflavonoids. Tectoridin and iristectorigenin A were the principal compounds present in the methanolic extract of Iris spuria.

A Liquid Chromatography Tandem Mass Spectrometry Based Regulatory Compliant Method for the Determination of Tenofovir in Human Serum

A simple, rapid, and specific assay based on solid phase extraction and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS-MS) for the quantitative analysis of Tenofovir in human serum using Tenofovir D6 as internal standard (IS) have been developed. The precursor to product ion transitions of m/z 288.2/176.2 and m/z 293.9/182.3 used to measure the analyte and internal standard (Tenofovir and Tenofovir D6). The method was validated over a concentration range of 5.06-603.72 ng mL(-1). The method was validated over the parameters like selectivity, matrix effect, sensitivity, linearity, precision, accuracy, various stabilities (bench top stability, standard stock solution stability in refrigerator and at room temperature, stock dilution stability, auto sampler stability, freeze thaw stability, long term stability - 65°C±10°C & long term stability - 22°C±5°C, reagent stability, dry extract stability, wet extract stability in refrigerator and at bench top, blood stability), effect of potentially interfering drugs, dilution integrity, recovery and reinjection reproducibility. The mean % recovery of Tenofovir was 98.22% with a precision of 2.42%, The mean % recovery of Tenofovir D6 was 100.96% with a precision of 2.88%. The RSD % of intra-day and inter-day assay was≤15%.

Induction of apoptosis in human pancreatic MiaPaCa-2 cells through the loss of mitochondrial membrane potential (ΔΨm) by Gentiana kurroo root extract and LC-ESI-MS analysis of its principal constituents

The objective of the current study was to evaluate the methanolic root extract of Gentiana kurroo for antioxidant and antiproliferative activities as well as to study the effect of the extract on the induction of apoptosis in human pancreatic cancer cell line (MiaPaCa-2). The extract exerted significant antioxidant activity as verified by DPPH, hydroxyl radical, lipid peroxidation and protective oxidative DNA damage assays. The results were comparable to standard antioxidants like α-tocopherol, catechin and BHT used in such experiments. Antioxidant potential of G. kurroo may be attributed to the presence of high phenolic and flavonoid content (73±1.02 and 46±2.05mg/g extract respectively). The anti-proliferative property of Gentiana kurroo root extract was determined by sulphorhodamine B (SRB) assay against Human colon cancer cell line (HCT-116), Lung carcinoma cell line (A-549), Pancreatic cancer cell line (MiaPaCa-2), Lung cancer cell line (HOP-62) and acute monocytic leukaemia cell line (THP-1). G. kurroo root extract inhibited cancer cell growth depending upon the cell line used and in a dose dependent manner. The extract induced potent apoptotic effects in MiaPaCa-2 cells. The population of apoptotic cells increased from 11.4% in case of control to 49.6% at 100μg/ml of G. kurroo root extract. The extract also induced a remarkable decrease in mitochondrial membrane potential (ΔΨm) leading to apoptosis of cancer cells used. The main chemical constituents identified by the liquid chromatography–tandem mass spectrometry (LC–ESI-MSMS) were found to be iridoid glucosides (iridoids and secoiridoids), xanthones and flavonoids. Iridoid glucosides are the bitter principles of Gentiana species. Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4″-O-β-d-glucosyl-6′-O-(4-O-β-d-glucosylcaffeoyl)-linearoside and Swertisin were the principal compounds present in the methanol root extract of G. kurroo.

Culture condition-dependent metabolite profiling of Aspergillus fumigatus with antifungal activity

Three sections of Aspergillus (five species, 21 strains) were classified according to culture medium-dependent and time-dependent secondary metabolite profile-based chemotaxonomy. Secondary metabolites were analysed by liquid chromatography–electrospray ionisation tandem mass spectrometry (LC–ESI-MS–MS) and multivariate statistical methods. From the Aspergillus sections that were cultured on malt extract agar (MEA) and Czapek yeast extract agar (CYA) for 7, 12, and 16 d, Aspergillus sections Fumigati (A. fumigatus), Nigri (A. niger), and Flavi (A. flavus, A. oryzae, and A. sojae) clustered separately on the basis of the results of the secondary metabolite analyses at 16 d regardless of culture medium. Based on orthogonal projection to latent structures discriminant analysis by partial least squares discriminant analysis (PLS-DA), we identified the secondary metabolites that helped differentiate sections between A. fumigatus and Aspergillus section Flavi to be gliotoxin G, fumigatin oxide, fumigatin, pseurotin A or D, fumiquinazoline D, fumagillin, helvolic acid, 1,2-dihydrohelvolic acid, and 5,8-dihydroxy-9,12-octadecadienoic acid (5,8-diHODE). Among these compounds, fumagillin, helvolic acid, and 1,2-dihydrohelvolic acid of A. fumigatus showed antifungal activities against Malassezia furfur, which is lipophilic yeast that causes epidermal skin disorders.

Trace determination of β-blockers and β2-agonists in distilled and waste-waters using liquid chromatography–tandem mass spectrometry and solid-phase extraction

A highly sensitive method for simultaneous determinations of eleven β-blockers and β-agonists in distilled and waste-waters using liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS–MS) was developed, optimized and validated. The method was used for trace determinations of acebutolol, atenolol, metoprolol, propranolol, timolol, nadolol, labetalol, oxprenolol, pindolol, alprenolol and terbutaline. Oasis MCX and Clean Screen cartridges were used for solid phase extractions and an alkaline mixture of dichloromethane–propanol was used as mobile phase. Matrix effect was reduced by using methanol as a pre-eluant for removing co-extractives on the SPE cartridges and by applying the internal standard method for quantification. Using Oasis MCX-SPE cartridges, developed method gave average recoveries of 77.20–97.30% for drugs spiked at 150.00–500.00pg/ml. Intra-day precisions gave RSD of 3.367–12.489% while as inter-day precisions gave RSD of 6.425–19.768%. Detection limits of 0.11–6.74pg/ml and quantification limits of 0.14–22.88pg/ml were obtained. Signal's suppression in the range of 4.50–24.50% was recorded due to the matrix effect. Drugs spiked in wastewater at 500.00pg/ml concentrations level and stored at 4°C for 6 days, showed insignificant degradation. Developed method was successfully applied to the analysis of pharmaceutical residues in effluents wastewaters. Five β-blockers and one β-agonists were detected in Al-Ain and Abu Dhabi wastewaters at average concentrations of 3.44–19.05pg/ml. Atenolol was detected at higher average concentration ranged in 125.60–234.28pg/ml. Results obtained suggest that adopted wastewater treatment processes are not enough to degrade these compounds.

Rapid Analysis of 27 Components of Isodon serra by LC–ESI-MS–MS

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C18 column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.

Urinary N3 adenine DNA adducts in humans occupationally exposed to styrene

Urine samples from humans occupationally exposed to styrene, with mandelic acid levels ranging from 400 to 1145 mg/g creatinine and from 68 to 400 mg/g creatinine for high and low exposure group, respectively, were analysed for N3 adenine DNA adducts, namely, 3-(2-hydroxy-1-phenylethyl)adenine (N3αA) and 3-(2-hydroxy-2-phenylethyl)adenine (N3βA). A sensitive LC-ESI–MSMS method was developed with the limit of quantification of 1 pg/mL for both analytes. Peaks corresponding to N3αA and/or N3βA were found in seven of nine end-of-shift samples of the high exposure group and in six of 19 end-of-shift samples of the low exposure group. Concentration of N3αA + N3βA amounted to 2.8 ± 1.6 pg/mL (mean ± S.D.; n = 9) and 1.8 ± 1.3 pg/mL (mean ± S.D.; n = 19) in the high and low exposure group, respectively. Of other 10 samples taken the next morning after exposure, two contained low but quantifiable concentrations of N3αA and none contained N3βA. However, interfering peaks were detected also in some control urine samples. Out of 22 controls, six and two samples contained peaks co-eluting with N3αA and N3βA, respectively. Therefore, the method used was found insufficiently specific to be applicable for biological monitoring. Comparing the excretion of N3αA + N3βA to that reported previously in mice it can be estimated that at the same absorbed dose, humans excreted not more than 1/30 of the amount of adenine adducts excreted by mice. As a consequence, the damage to DNA caused by styrene 7,8-oxide (SO), a reactive metabolite of styrene, appears to be much lower in humans than in mice.

Determination of Pirfenidone in Rat Plasma by LC–MS-MS and Its Application to a Pharmacokinetic Study

A simple liquid chromatography-electrospray ionization-mass spectrometric (LC–ESI–MS-MS) method was developed for determination of pirfenidone in rat plasma. After addition of phenacetin as internal standard, protein precipitation with 10% perchloric acid was employed for the sample preparation. Chromatographic separation was achieved on a C18 column, with 40: 60 (v/v) acetonitrile-0.1% formic acid as mobile phase. The assay is linear over the range 2–2,000 ng mL−1, with a lower limit of quantitation of 2 ng mL−1. The developed method was successfully applied to pharmacokinetic studies of pirfenidone in rats following single oral administration dose of 15 mg kg−1.

Liquid chromatography–tandem mass spectrometric method for determination of salivary 17α-hydroxyprogesterone: A noninvasive tool for evaluating efficacy of hormone replacement therapy in congenital adrenal hyperplasia

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of 17α-hydroxyprogesterone (17OHP) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a highly proton-affinitive reagent, 2-hydrazinopyridine, and subjected to LC–MS–MS. Quantification was based on the selected reaction monitoring, and deuterated 17OHP was used as the internal standard. This method allowed the reproducible and accurate quantification of the salivary 17OHP using a 200-μl sample, and the limit of quantitation was 5.0 pg/ml. The developed method was applied to clinical studies. A linear relationship was found to be positive ( r 2 = 0.975) between the blood 17OHP level and the salivary 17OHP level measured using the proposed method. The result from the salivary 17OHP measurement in patients with congenital adrenal hyperplasia demonstrated that the proposed method is very useful for monitoring of the therapeutic efficacy during hormone replacement therapy.

Phototransformation of monuron induced by nitrate and nitrite ions in water: Contribution of photonitration

The nitrate and nitrite-induced phototransformation of monuron in aqueous solution has been investigated using 300–450 nm light irradiation. The degradation kinetics was estimated under various conditions of concentrations of substrate and inducer, oxygen content and pH. From the numerous photoproducts identified using LC–ESI-MS–MS and their evolution curves a degradation scheme was proposed. Besides oxidation of the N-terminus group (major pathway in the presence of NO 3 −) and a minor contribution of direct photolysis, the other photoproducts originated from Cl/OH substitution, regiospecific hydroxylation and/or nitration of the phenyl ring. The two last processes occurred competitively under the NO 3 − conditions even at “environmental” concentrations of the inducer yielding significant levels of nitro-derivatives. Their production was found to be oxygen-dependent in contrast to the NO 2 − conditions.

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.

Studies on neurosteroids XIX: Development and validation of liquid chromatography–tandem mass spectrometric method for determination of 5α-reduced pregnane-type neurosteroids in rat brain and serum

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the simultaneous determination of 5α-reduced pregnan-type neurosteroids, allopregnanolone (AP), epiallopregnanolone and 5α-dihydroprogesterone, in rat brain and serum has been developed and validated. The brain and serum steroids were extracted with methanol–acetic acid, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-positive ESI-MS–MS. The limits of quantitation (LOQ) for brain (0.25 ng/g tissue) and serum (0.25 ng/ml) assays using the derivatization–ESI-MS–MS method are 60–150-fold lower than the LOQs for their atmospheric pressure chemical ionization-MS method without derivatization. [17α,21,21,21- 2H 4]-AP was used as an internal standard. This method allowed the reproducible and accurate quantification of the brain or serum neurosteroids using a 20 mg or 20 μl sample, respectively. That is, the intra- and inter-assay coefficients of variation were below 8.2 and 6.0%, respectively, and the % accuracy values were 98.5–103.0% for all the steroids in both the brain and serum. The application of the developed method to the analysis of changes in the brain and serum neurosteroid levels by immobilization stress and ethanol administration is also presented.

Determination of salivary dehydroepiandrosterone using liquid chromatography–tandem mass spectrometry combined with charged derivatization

A sensitive liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method for the quantification of dehydroepiandrosterone (DHEA) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC–MS–MS. The derivatization with HMP was very effective for increasing the detectability of DHEA in the positive-ESI-MS. Quantification was based on the selected reaction monitoring and androsterone was used as an internal standard. This method allowed the reproducible and accurate quantification of the salivary DHEA using a 200-μl sample and the limit of quantitation for DHEA was 25 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetition was observed. The developed method was applied to clinical studies, and produced satisfactory results.

Determination of bisphenol diglycidyl ethers in topical dosage forms

A method involving extraction and LC–ESI–MS-MS detection of BADGE, BFDGE, BADGE·H 2O, BADGE·2H 2O, BADGE·HCl, BADGE·H 2O·HCl, BADGE·2HCl and BFDGE·2HCl in aqueous cream was developed and validated. Initially, empty internally lacquered aluminum container closure systems were extracted with isopropanol as an attempt to estimate the upper limit of extractable bisphenol diglycidyl ethers present in lacquer. Six of the eight potential bisphenol diglycidyl ethers were quantified. In an accelerated experiment, on aqueous cream stored in lacquered aluminum tubes at 70 °C, all derivatives except BADGE·2HCl and BFDGE·2HCl were extracted from cream samples and quantified as an attempt to estimate the upper limit of compounds leaching to the cream. Detection limits were from 0.3 ± 0.2 to 3.4 ± 0.7 μg l −1. Recoveries were determined for all compounds at three concentration levels (mean 63 ± 6%). Mean inter-day and mean intra-day precision was 7 ± 2 and 13 ± 6%, respectively. Three commercially available creams were obtained from a local community pharmacy and analysed for bisphenol diglycidyl ethers. BADGE, BADGE·H 2O, BADGE·2H 2O and BADGE·H 2O·HCl were detected and quantified. In conclusion, the developed method allows for the extraction and detection of bisphenol diglycidyl ethers originating from the epoxy phenol lacquer used in aluminum tubes. This study does not indicate that they leach into aqueous cream in significant amounts under normal storage conditions.

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC–ESI-MS–MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid–liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS–MS in selecting m/ z 321 → 257, 321 → 152 ( 35Cl 2) and m/ z 323 → 257, 323 → 152 ( 37Cl 35Cl). Two calibration curves were constructed by plotting the area ratio of m/ z 321 → 152 versus 326 → 157 and m/ z 321 → 257 versus 326 → 262 against their corresponding amount ratio. Indeed, even if m/ z 321 → 152 was found to give a higher MS–MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/ z 321 → 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 μg/kg concentration levels using d 5-CAP as internal standard. The decision limit (CCα) and detection capability (CCβ) of CAP in milk were calculated for m/ z 321 → 152 at 0.02 μg/kg and 0.03 μg/kg, respectively, and for m/ z 321 → 257 at 0.02 μg/kg and 0.04 μg/kg, respectively. At the lowest fortification level (i.e. 0.1 μg/kg), repeatability and within-laboratory reproducibility were calculated for m/ z 321 → 257 both at 0.02 μg/kg and for m/ z 321 → 152 at 0.03 and 0.05 μg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.

Liquid chromatography–electrospray quadrupole ion-trap mass spectrometry of nine pesticides in fruits

A liquid chromatographic method, with electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS), has been developed for determining acrinathrin, carbosulfan, cyproconazole, λ-cyhalothrin, kresoxim methyl, pyrifenox, pyriproxyfen, propanil, and tebufenpyrad in fruits. The ions prominent in ESI spectra were [ M + H] + and [ M + Na] +. In the mass analyzer, collision-induced dissociation fragmentation involved common pathways, for example, product ions of [ M + H] + resulted from the cleavage of the carbamic group or an oxygen bound. The utility of the method is demonstrated by the analysis of crude extracts obtained by matrix solid-phase dispersion (MSPD) using C 18 as dispersant and dichloromethane-methanol as eluent, and by solid–liquid extraction (SLE) with ethyl acetate and anhydrous sodium sulfate. Mean recoveries ranged from 51.5 to 108%, with relative standard deviations <16%, were obtained for MSPD and from 59 to 101% with relative standard deviation <17% for SLE. However, for most compounds, limits of quantification are better by SLE (0.01–0.4 mg kg −1) than by MSPD (0.05–2 mg kg −1). During the validation process, the procedure was tested for matrix effects, blanks and stability of the system. Considerably matrix effects in the ESI ionization process were detected by comparing standard calibration, and matrix calibration. Because of this, detected residues were quantified from interpolation against calibration data obtained using matrix matched standards.

Analysis by liquid chromatography–electrospray ionization tandem mass spectrometry and acute toxicity evaluation for β-blockers and lipid-regulating agents in wastewater samples

This paper describes a multiresidue method for the extraction and determination of two therapeutic groups of pharmaceuticals, lipid-regulating agents (clofibric acid, bezafibrate, gemfibrocil, fenofibrate) and β-blockers (atenolol, sotalol, metoprolol, betaxolol) in waters by solid-phase extraction followed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Recoveries obtained from spiked HPLC water, as well as, from spiked real samples (sewage treatment plants influent and effluents, river and tap water) were all above 60%, with the exception of betaxolol with a 52% recovery. The quantitative MS analysis was performed using a multiple reaction monitoring. The LC–MS–MS method gave detection limits ranging from 0.017 to 1.25 μg/l in spiked effluent. Precision of the method, calculated as relative standard deviation, ranged from 3.7 to 18.5%. Individual and combined effects on Daphnia magna were evaluated for both therapeutic groups. Individual effects in culture medium showed these compounds as not harmful and not toxic, an exception is fenofibrate that was found to be harmful, but at high, in the environment unrealistic concentrations (EC 50 of 50 mg/l). Combined effect in wastewater showed synergistic toxic effects at low concentration level (2 μg/l).