Fully automated determination of 74 pharmaceuticals in environmental and waste waters by online solid phase extraction-liquid chromatography-electrospray-tandem mass spectrometry

The present work describes the development of a fully automated method, based on on-line solid-phase extraction (SPE)-liquid chromatography-electrospray-tandem mass spectrometry (LC-MS-MS), for the determination of 74 pharmaceuticals in environmental waters (superficial water and groundwater) as well as sewage waters. On-line SPE is performed by passing 2.5 mL of the water sample through a HySphere Resin GP cartridge. For unequivocal identification and confirmation two selected reaction monitoring (SRM) transitions are monitored per compound, thus four identification points are achieved. Quantification is performed by the internal standard approach, indispensable to correct the losses during the solid phase extraction, as well as the matrix effects. The main advantages of the method developed are high sensitivity (limits of detection in the low ng L⁻¹ range), selectivity due the use of tandem mass spectrometry and reliability due the use of 51 surrogates and minimum sample manipulation. As a part of the validation procedure, the method developed has been applied to the analysis of various environmental and sewage samples from a Spanish river and a sewage treatment plant.     

Fully automated determination of N-nitrosamines in environmental waters by headspace solid-phase microextraction followed by GC-MS-MS

A fully automated method for determining nine Environmental Protection Agency N-nitrosamines in several types of environmental waters at ng/L levels is presented. The method is based on a headspace solid-phase microextraction followed by GC-MS-MS using chemical ionization. Three different fibers (carboxen/PDMS, divinylbenzene/carboxen/PDMS, and PEG) were tested. Solid-phase microextraction conditions were best when a divinylbenzene/carboxen/PDMS fiber was exposed for 60?min in the headspace of 10?mL water samples at pH 7 containing 360?g/L of NaCl, at 45°C. All compounds were analyzed by GC-MS-MS within 18?min. The method was validated using effluent from an urban wastewater treatment plant and the LODs ranged from 1 to 5?ng/L. The method was then applied to determine the N-nitrosamines in samples of different complexities, such as tap water and several influent and effluent wastewater samples from urban and industrial wastewater treatment plants and a potable water treatment plant. Although the analysis of influent industrial wastewater revealed high concentrations of some compounds (N-nitrosomorpholine and N-nitrosodimethylamine at μg/L levels), in industrial effluents and other samples, the concentrations were substantially lower (ng/L levels). The new method is suitable for the simple and reliable determination of N-nitrosamines in highly complex water samples in a completely automated procedure.     

Multiresidue fully automated online SPE-HPLC-MS/MS method for the quantification of endocrine-disrupting and pharmaceutical compounds at trace level in surface water

The present work describes the development and validation of a sensitive method for the determination of traces of diverse groups of pharmaceuticals and endocrine disruptors in surface water. Thirty-seven substances have been selected, including 10 pesticides, 6 hormonal steroids and assimilates, 12 pharmaceuticals, 5 alkylphenols, 1 chlorophenol and 3 other well-known human contaminants, 1 UV filter and 2 plasticisers. An automated online solid-phase extraction (SPE) is directly coupled to liquid chromatography–tandem mass spectrometry. Different SPE columns have been tested, and the injection volume has been optimised. The developed analytical methodology is based on the direct injection of 2.5 mL of water sample acidified at pH 1.6 on an Oasis HLB loading column (20 × 2.1 mm) with 5-µm particles. Then, the chromatographic separation is achieved on a Kinetex XB C18 (100 × 2.1 mm; 1.7 µm) column, and the quantification is realised in multiple-reaction monitoring mode. The online SPE step warrants minimal sample handling, low solvent consumption, high sample throughput, saving time and costs. This method allows the quantification of the target analytes in the lower ng/L concentration range, with limits of quantification (LQs) between 100 pg/L and 10 ng/L, 26 compounds having LQ lower than 1 ng/L. The monitoring of two selected MS/MS transitions for each compound allows the reliable confirmation of positive findings even at the LQ level. The developed and validated methodology has been applied to the analysis of various real samples from two French rivers. Twelve target compounds have been detected in the environmental samples, and the major pollutants are pharmaceuticals usually used by humans (paracetamol, carbamazepine, oxazepam, ketoprofen, trimethoprim). The pesticides atrazine and carbendazim have been ubiquitously detected in real samples too. Metronidazole, sulfamethoxazole and diuron were also frequently quantified in the water samples.     

Spectrophotometric determination of zinc at trace level in environmental samples

Summary Zinc is selectively extracted with N-hydroxy-N,N-diphenylbenzamidine and spectrophotometrically determined with 4-(2-pyridylazo)resorcinol. The molar absorptivity is 9.4×10⁴l·mol^–1 cm^–1. Relative standard deviation is ±1.5%.     

Fully automated analysis of estrogens in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive method for the determination of five estrogens, estrone, 17beta-estradiol, estriol, ethynyl estradiol, and diethylstilbestrol, was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These estrogens were separated within 8 min by HPLC using an XDB-C8 column and 0.01% ammonia/acetonitrile (60/40, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the negative ion mode were optimized for MS/MS detection of the estrogens. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r > or = 0.9996) was obtained in the concentration range from 10 to 200 pg/mL for all compounds examined. The limits of detection (S/N= 3) of the five estrogens examined ranged from 2.7 to 11.7 pg/mL. The in-tube SPME method showed 34-90-fold higher sensitivity than the direct injection method (5 microL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface water and wastewater samples were collected from the area around Asahi River, and estriol was detected at 35.7 pg/mL in the effluent of a sewage treatment plant. The recoveries of estrogens spiked into river waters were above 86%, except for estriol, and the relative standard deviations were below 0.9-8.8%.     

Analysis of natural and synthetic estrogenic endocrine disruptors in environmental waters using online preconcentration coupled with LC-APPI-MS/MS

This paper describes a fully automated online method for solid-phase extraction coupled with liquid chromatography and tandem mass spectrometry using atmospheric pressure ionization (LC-LC-APPI-MS/MS) to simultaneously detect selected dissolved natural and synthetic hormones at concentrations as low as 5 ng/L from aqueous matrices. The method shows excellent performance for the direct analysis of water and wastewater with respect to calibration curve linearity, analytic specificity, sensitivity, and carryover, as well as overall method accuracy and precision. With a runtime of 15 min, a minimum of 200 samples were analyzed using a single online solid-phase extraction (SPE) column without noticeable differences in system performance. Because of the ruggedness and simplicity of this system, generic methods can be easily developed and applied for the high-throughput analysis of a wide variety of compounds without the need to resort to laborious offline SPE sample preparation.     

Extraction,preconcentration and spectrophotometric determination of trace levels of thiosulfate in environmental waters

In the existing study, a new vortex-assisted cloud point extraction (VA-CPE) method was developed for determination of low levels of thiosulfate in environmental waters at 632 nm by spectrophotometry. The method is selectively based on charge-transfer-sensitive ion-pair complex formation of Ag(S₂O₃)₂ ^3?, which is produced by the reaction of thiosulfate with excess Ag⁺ ions with toluidine blue (3-amino-7-dimethylamino-2-methylphenazathionium chloride, TB⁺) and then its extraction into micellar phase of polyethylene glycol 4-tert-octylphenyl ether (Triton X-45) in presence of Na₂SO₄ as salting-out agent at pH 7.0. All the factors affecting complex formation and VA-CPE efficiency were optimized in detail. Under the optimized conditions, the linear calibration curves for thiosulfate were in the range of 0.2–120 and 5–180 µg L^?1 with sensitivity improvement of 81-folds and 15-folds, respectively, as a result of efficient mass transfer obtained by CPE with and without vortex, while it changed in the range of 260–3600 µg L^?1 without preconcentration at 642 nm. The limits of detection and quantification of the method for VA-CPE were found to be 0.05 and 0.22 µg L^?1, respectively. The precision (expressed as the percent relative standard deviation) was in range of 2.5–4.8% (5, 10 and 25 µg L^?1, n: 5). The method accuracy was validated by comparing the results to those of an independent 5,5′-dithiobis(2-aminobenzoic acid) (DTNB) method as well as recovery studies from spiked samples. It has been observed that the results are statistically in a good agreement with those obtained by DTNB method. Finally, the method developed was successfully applied to the preconcentration and determination of trace thiosulfate from environmental waters.     

Simultaneous ion chromatographic determination of selenium and metal ions in waters at trace level

Summary An ion-chromatographic procedure is described for the determination of selenium (VI) at μg L⁻¹ level in the presence of anions and heavy metal ions. Maximum permissible concentrations and effects from each interfering substance were investigated for the Se concentration range 12.5–1,000 μg L⁻¹. The method, optimized for the detection of SeO ₄ ²⁻ , gives results suitable for speciation analysis. Total selenium can be determined after complete conversion to selenate ion by oxidation with KMnO₄. The detection limit of selenium is 4.8 μg L⁻¹ (0.96 ng for 200 μL sample). Paper presented at the 41st Pittsburgh Conference, New York, March 5–9, 1990.     

Rapid quantification of tilidine,nortilidine, and bisnortilidine in urine by automated online SPE-LC-MS/MS

The opioid tilidine is a prodrug which is hepatically metabolized to active nortilidine and bisnortilidine. Due to the increasing abuse of tilidine by drug users and the lack of a specific immunoassay, we developed an analytical method for the quantification of tilidine, nortilidine, and bisnortilidine in urine suitable for screening. In a following step, this method was used to establish data on excretion kinetics of the substances in order to evaluate the time window of detection after a single oral dose of tilidine/naloxone and also was applied to authentic urine samples from correctional facilities. Urine samples were mixed with internal standard solution and extracted on a weak cation exchanger at pH 6 using a Symbiosis Pico system. The chromatographic separation was achieved within a 3.5-min run time on a Phenylhexyl column (50 × 2.0 mm, 5 μm) via gradient elution (methanol and 0.2% formic acid) at a flow rate of 0.50 mL/min. The ESI-MS/MS was performed on a QTrap 3,200 in positive multiple reaction monitoring mode using two mass transitions per analyte. Validating the method resulted in a lower limit of quantification of 1.0 μg/L followed by a linear calibration range to 100 μg/L for each analyte (r ² > 0.99). The analytical method allowed the detection of a single dose of a commercially available tilidine solution up to 7 days after administration. Using this highly sensitive method, 55 of 3,665 urine samples were tested positive.     

Critical evaluation of the determination of pharmaceuticals, personal care products, phenolic endocrine disrupters and faecal steroids by GC/MS and PTV-GC/MS in environmental waters

An analytical method is described for the determination of a broad range of emerging and priority pollutants, together with sewage molecular markers in environmental waters. The step-by-step study of the GC/MS analyses focuses on the effects of experimental variables using a large volume injection (LVI) technique [a programmed temperature-vaporising (PTV) inlet], the evaluation of a clean-up step using classical and newer sorbents (i.e. Al-N, Fl, NH₂, PSA, Si, CN and DIOL), and the revision of how organic matter [i.e. humic acids (HA) content] affects method performance. Reproducibility and recoveries from spiked coastal water samples at different analyte concentrations (100, 250 and 500 ng L⁻¹) as well as with different levels of spiked humic acids (2, 10 and 20 mg L⁻¹) are reported indicating a good performance of the extraction procedure with low levels of HA (<10 mg L⁻¹). The presence of HA is a critical parameter during the solid-phase extraction (SPE) procedures. Of the clean-up sorbents tested, CN and DIOL proved most efficient in cleaning-up the extracts with recoveries in the range of 66-77% and 100-114%, respectively for the selected analytes. Both GC/MS and PTV-GC/MS instrumental configurations were tested using final sewage effluents, riverine, estuarine and coastal water samples. However, limited applicability of the PTV inlet is reported for environmental applications, affording only a modest improvement in chromatographic signal-to-noise ratios.     

HPLC-MS/MS multiclass determination of steroid hormones in environmental waters after preconcentration on the carbonaceous sorbent HA-C@silica

In this study, a sensitive and multiclass method has been developed for analysis of three families of steroid hormones, i.e. progestins, oestrogens, androgens, by SPE-HPLC-ESI-MS/MS. The extraction efficiency of thermally condensed humic acids onto silica sorbent (HA-C@silica), here for the first time studied for multiclass enrichment of these sex hormones, was tested in different environmental waters (tap and river water, urban wastewater treatment plant effluent) spiked at the nanograms per litre levels (5–1000 ng L⁻¹). Quantitative adsorption was achieved using 200 mg sorbent for preconcentration of 250–1000 mL sample, at the native pH (pH = 6.5–7.7). Elution was performed by two sequential fractions (methanol followed by acetonitrile), obtaining in all the matrices investigated satisfactory recoveries (71% to 124% for river waters and 71–113% for urban wastewater treatment plant effluent) and RSDs below 15% (n = 3). The high enrichment factors (up to 4000) coupled with high-performance liquid chromatography tandem mass spectrometry quantification (MRM mode) provided low limits of detection and quantification (a few ng L⁻¹), that are suitable for environmental monitoring. Most of the analytes were detected in river water and in wastewater effluent samples (in the ng L⁻¹ concentration range), attesting their environmental diffusion. The proposed method was extended to a fourth class, Glucocorticoids, achieving good results in river samples, by the same SPE cartridge and chromatographic run.     

Screening of fluoroquinolones in environmental waters using disk-based solid-phase extraction combined to microplate fluorimetric determination and LC-MS/MS

Fluoroquinolones are in the order of the day concerning environmental contamination through anthropogenic activities, resulting in increased risk for antibiotic resistance dissemination. In this context, accessible, low-cost analytical methods are required for implementation of comprehensive surveillance and screening schemes. In this work, we propose a down-scaled disk-based solid-phase extraction system from which the eluate can be first screened by miniaturized fluorimetric reading, followed by individual determination of target fluoroquinolones (ciprofloxacin, norfloxacin, and enrofloxacin) by liquid chromatography combined to tandem mass spectrometry. The fluorimetric measurement is based on the intrinsic fluorescence of fluoroquinolones. Disk-based retention was performed after sample acidification (pH 4.0) by mixed-mode cation exchange using polystyrene divinylbenzene sulphonated sorbent. Sample loading was precisely controlled in a dedicated flow system operating at 4.0 mL min^?1. Different eluent compositions were tested, with elution performed by 1.00 mL of methanol-ammonium hydroxide (98:2, v/v), with subsequent reading of eluate in both detectors. Quantification was attained for 2–25 µg L^?1 range, with LOD values at 1 µg L^?1. The proposed approach was successfully applied to estuarine waters from the Douro River, with comparable results to a conventional SPE-LC-MS/MS procedure.     

Multielement preconcentration of trace metals from natural waters by solvent extraction with an alkylated oxine derivative

Kelex 100, a commercially available alkylated oxine derivative, is shown to be effective, in purified form, for the simultaneous extraction of trace levels of Cd(II), Co(II), Cu(II), Mn(II), Ni(II), Pb(II), and Zn(II) from natural waters into toluene. The high lipophilicity of the extractant and its chelates affords large preconcentration factors in a single batch-extraction. Back-extraction with a small volume of nitric acid provides additional enrichment for subsequent determination of total (soluble) metal by graphite-furnace atomic-absorption spectrometry (GFAAS). Calibration with standard solutions can be used, which has advantages over the method of standard additions.     

Application of dispersive liquid–liquid microextraction followed by HPLC–MS/MS for the trace determination of clotrimazole in environmental water samples

A method for the analysis of clotrimazole was developed with dispersive liquid–liquid microextraction for sample pre‐concentration and HPLC–MS/MS for analysis. A linear ion trap was used for the confirmation of clotrimazole identity in the samples. The developed method enables the analysis of clotrimazole in river water and sewage effluent from wastewater treatment plants with a LOQ of 0.7 ng/L. Environmental monitoring of clotrimazole was undertaken. Samples from river water and sewage effluents were analysed over a one‐year period. Clotrimazole was found in every tested sample with concentration range from 1 to 31 ng/L. The amount of clotrimazole in tested samples was highly dependent on sampling season. The highest results were obtained in summer and autumn.     

Determination of trace PAHs in seawater and sediment pore-water by solid-phase microextraction (SPME) coupled with GC/MS

A fast and reliable method for the determination of trace PAHs (polynuclear aromatic hydrocarbons) in seawater by solid-phase microextraction (SPME) followed by gas chromatographic (GC) analysis has been developed. The SPME operational parameters have been optimized, and the effects of salinity and dissolved organic matter (DOM) on PAHs recoveries have been investigated. SPME measures only the portion of PAHs which are water soluble, and can be used to quantify PAH partition coefficient between water and DOM phases. The detection limits of the overall method for the measurement of sixteen PAHs range from 0.1 to 3.5 ng/g, and the precisions of individual PAH measurements range from 4% to 23% RSD. The average recovery for PAHs is 88.2±20.4%. The method has been applied to the determination of PAHs in seawater and sediment porewater samples collected in Jiaozhou Bay and Laizhou Bay in Shandong Peninsula, China. The overall levels of PAHs in these samples reflect moderate pollution compared to seawater samples reported elsewhere. The PAH distribution pattern shows that the soluble PAHs in seawater and porewater samples are dominated by naphthalenes and 3 ring PAHs. This is in direct contrast to those of the sediment samples reported earlier, in which both light and heavy PAHs are present at comparable concentrations. The absence of heavy PAHs in soluble forms (<0.1-3.5 ng/L) is indicative of the strong binding of these PAHs to the dissolved or solid matters and their low seawater solubility.     

Sub PPB level determination of eperison in human plasma by GC/MS

Summary Very low concentrations (down to 0.2 ng/mL) of an antispasmodic drug (Eperison) can be extracted from human plasma and analyzed by GC/MS. For sample preparation and preconcentration, solid phase extraction (SPE) has been employed. A double focussing mass spectrometer was interfaced with a capillary GC column for the GC/MS analysis. The gas chromatograph was equipped with an on-column injector to avoid thermal decomposition.     

Ultra-trace determination of aquaculture chemotherapeutants and degradation products in environmental matrices by LC-MS/MS

Two of the most common products currently used to control parasitic sea lice in fin fish aquaculture, salmon in particular, are Slice® and AlphaMax®. Emamectin benzoate (EB) is the active ingredient in Slice® and deltamethrin is the active ingredient in AlphaMax®. Several analytical methods have been developed for the determination of the active ingredients in these products but these have been focused on specific matrices and lack the sensitivity and versatility required in environmental monitoring. Here we present a validated, versatile, and simple analytical method for the determination of EB, its desmethyl degradation product (AB), and deltamethrin in a wide range of environmental matrices (sea water, marine sediment, and tissue). Sediments and tissues were extracted by accelerated solvent extraction (ASE®) and sample cleanup was achieved by solid phase extraction (SPE) while sea water was extracted using SPE disks. Analyte identification and quantification was based on liquid chromatography tandem mass spectrometry (LC-MS/MS) instrumentation with electrospray ionization, and multiple reaction monitoring (MRM). Method detection limits for the target analytes was in the parts per trillion (pg?g^?1) level for tissue and sediment and parts per quadrillion (pg?L^?1) for water. Except for deltamethrin in sea water, method performance in terms of analyte recoveries was better than 60%, and the method precision was RSD<20%. The method was used to determine EB and AB concentrations in water, sediment and prawn tissue samples collected near salmon aquaculture sites treated with Slice®. A distinct concentration gradient was observed in the immediate vicinity (within 50 to 100?m radius) of the salmon aquaculture sites where EB was detected at low ng?g^?1 levels for tissue (EB ranged from 0.041 to 3.0?ng?g^?1) and sediment (EB ranged from 0.051 to 35?ng?g^?1) and pg?L^?1 levels (EB ranged from 3 to 209?pg?L^?1) for water samples.     

On-line preconcentration and GFAAS determination of trace metals in waters

An on-line separation preconcentration system coupled to electrothermal (graphite furnace) atomic absorption spectrometry was developed. A miniature column packed with iminodiacetic acid ethyl cellulose (IDAEC) was inserted into the loop. A peristaltic pump was used to deliver solutions. A flow of air was driven into the packed column, evacuating it between sample loading, washing and elution. The retained analyte was introduced on-line to graphite furnace using countercurrent elution with HNO₃. The system was applied for the determination of V, Co and Pb in medicinal mineral water samples, and nickel in sea water samples. The detection limits (3σ) were 0.058, 0.022, 0.067, 0.062 μg/l for Co, Pb, V, and Ni, respectively. The R.S.D. (n=5) was <5% at 0.4–1.0 μg/l concentration range.     

Fully automated potentiometric determination of ionized magnesium in blood serum

Basic observations leading to the successful application of magnesium ion-selective electrodes for the automated determination of ionized magnesium in undiluted serum are discussed. The principles of the method are described and the first numerical results are reported.