Analytical Method Development for the Simultaneous Determination of Five Human Pharmaceuticals in Water and Wastewater Samples by Gas Chromatography-Mass Spectrometry

Summary Effective analytical methods for the simultaneous determination of five pharmaceuticals from various therapeutic classes in a variety of aqueous samples have been developed and method performance data are presented. The method involves the simultaneous extraction of the selected pharmaceuticals from the aqueous phase by solid phase extraction using a hyper cross linked, polystyrene-divinylbenzene polymer based sorbent. Analytes were eluted with methanol, derivatised with N-methyl-N-trimethylsilyltrifloroacetamide and analysed by gas chromatography – electron ionisation mass spectrometry (GC-EI-MS). Recoveries of 50 to 98% were established for waters spiked with the studied compounds at the low ng L^–1 level with the highest detection sensitivities being achieved in the selected ion monitoring (SIM) mode and the quantification limit of the procedure for sample sizes of 1000 ml was approximately 5 ng L^–1 for all matrices except sewage which was only tested to 20 ng L^–1. Analysis of domestic sewage from a large treatment works demonstrate the presence of all five compounds in both influents and effluents.     

Ultrasound-assisted extraction for the analysis of phenolic compounds in strawberries

A semiautomatic method based on application of ultrasounds has been developed to leach and hydrolyse phenolic compounds, such as naringin, rutin, naringenin, ellagic acid, quercetin and kaempferol, from strawberries. Two grams of lyophilized sample was placed into a sample cell and 5 mL of acetone containing hydrochloric acid was added. The cell was immersed in a water bath and sonicated for 30 s (duty cycle 0.8 s, output amplitude 50% of the nominal amplitude of the converter, applied power 100 W and with the probe placed 2 cm from the top surface of the extraction cell) for three times: each time 5 mL extractant displaced the previous extract. When the extraction was completed, the combined extracts were evaporated for 10 min, diluted to 10 mL with water adjusted to pH 8, and transferred to a cleanup–preconcentration manifold; here the analytes were retained in two in-series minicolumns packed with HR-P sorbent and then eluted with 4 mL methanol, and injected for individual separation–quantitation into a chromatograph–photodiode array detector assembly. Optimisation of the extraction was carried out using samples spiked with 4 mg kg^–1 of each analyte. Calibration curves using the standard addition in red strawberries typically gave linear dynamic ranges of 4–40 mg L^–1 for all analytes, except for ellagic acid (40–400 mg L^–1). The r ² values exceeded 0.98 in all cases.     

Application of solid-phase extraction for determination of phenolic compounds in barrique wines

A fast, selective and sensitive chromatographic method has been developed for determination of gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, benzoic, ferulic, sinapic, cinnamic, and ellagic acids and p-hydroxybenzaldehyde, vanillin, syringaldehyde, 2-furfural, 5-methylfurfural, and 5-methoxyfurfural. The compounds from untreated wine samples were pre-concentrated and cleaned using solid-phase extraction on RP-105 polymeric sorbent. The cartridge was conditioned with methanol and water. Co-extracted ballast substances were rinsed from the sorbent with 0.1 mol L^–1 hydrochloric acid–methanol, 1:4 (v/v). Retained phenolic compounds were selectively eluted with diethyl ether. A linear mobile phase gradient containing 0.3% acetic acid and methanol was used for final baseline chromatographic separation on a Hypersil BDS C18 column. Limits of detection (LOD=3s_bl) in the range 5.2 to 181.2 g L^–1, resolution (R) better than 1.7, and repeatability of 2.7–5.1% (RSD for real samples) were achieved. The method was applied for quantification of individual phenolic compounds in barrique wines.     

Determination of carbendazim,thiabendazole, and<Emphasis Type="Italic"> o-</Emphasis>phenylphenol residues in lemons by HPLC following sample clean-up by ion-pairing

An efficient analytical method is presented involving effective sample clean-up with solid-phase extraction and HPLC-UV analysis for the simultaneous determination of carbendazim, thiabendazole, and o-phenylphenol residues in lemons. Sample preparation involves extraction with acetonitrile acidified with trifluoroacetic acid and an ethyl acetate/petroleum ether mixture. Purification of the crude extract was carried out with liquid–liquid partitioning after addition of an aqueous ammonia solution. Final clean-up was performed on polymeric reversed-phase cartridges pretreated with sodium dodecyl sulfate. Chromatographic analysis was performed on a reversed-phase HPLC column isocratically eluted with an acetonitrile/water/ammonia mixture and UV detection at 254 nm. The chromatographic method is repeatable, reproducible, and sensitive. Fungicide recoveries from lemon samples fortified at levels of 5 and 1 mg kg^–1 were 81–85% for carbendazim, 96–98% for thiabendazole, and 81–106% for o-phenylphenol with coefficients of variation of 2.5–7.4%. Detection limits for carbendazim, thiabendazole, and o-phenylphenol in lemons were 0.21, 0.27, and 0.51 mg kg^–1, respectively.     

Development of a stir-bar-sorptive extraction–liquid desorption–large-volume injection capillary gas chromatographic–mass spectrometric method for pyrethroid pesticides in water samples

Stir-bar-sorptive extraction followed by liquid desorption and large-volume injection capillary gas chromatography with mass spectrometric detection (SBSE–LD–LVI-GC–MS), had been applied for the determination of ultra-traces of eight pyrethroid pesticides (acrinathrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin, fenvalerate, and permethrin cis and trans isomers) in water samples. Instrumental calibration for selected-ion monitoring acquisition and conditions that could affect the SBSE–LD efficiency are fully discussed. By performing systematic assays on 30-mL water samples spiked at the 0.10 g L^–1 level it was established that stir-bars coated with 47 L polydimethylsiloxane, an equilibrium time of 60 min (750 rpm), 5% methanol as organic modifier, and acetonitrile as back-extraction solvent, provided the best analytical performance to monitor pyrethroid pesticides in water matrices. Good accuracy (81.8–105.0%) and remarkable reproducibility (<11.7%) were obtained, and the experimental recovery data were in good agreement with the theoretical equilibrium described by octanol–water partition coefficients (log K_O/W), with the exception of acrinathrin for which lower yields were measured. Excellent linear dynamic ranges between 25 and 400 ng L^–1 (r²>0.994), low quantification (3.0–7.5 ng L^–1) and detection (1.0–2.5 ng L^–1) limits were also achieved for the eight pyrethroid pesticides studied. The method was successfully used for analysis of tapwater and groundwater matrices spiked at the 0.10 g L^–1, revealing the suitability of the method for determination of pyrethroid pesticides in real samples. The method was shown be reliable and sensitive and a small volume of sample was required to monitor pyrethroids at ultra-trace levels, in compliance with international regulatory directives on water quality.     

Determination of Benzimidazole Fungicides by HPLC with Fluorescence Detection After Micellar Extraction

An analytical method was developed to determine the benzimidazole fungicides and their residues (benomyl, carbendazim, thiabendazole and fuberidazole) in real water samples. Analyses were performed by reverse phase (RP) HPLC with direct fluorescence detection with mobile phase methanol:water, 40:60 (v/v) with 0.6% (v/v) ammonia. The extraction of analytes from water samples was performed with the use of micellar systems. Specifically, oligoethylene glycol monoalkyl ether (Genapol X-080) and polyoxyethylene 10 lauryl ether (POLE) were used as extractants. The recoveries of fungicides obtained in spiked water samples ranged from 68% to 94% for Genapol and from 68% to 96% for POLE. The limit of detection (LOD) was lower than 6 g L^–1 for carbendazim, 7 g L^–1 benomyl, 0.15 g L^–1 for thiabendazole and 0.01 g L^–1 for fuberidazole in both surfactants.     

Determination of Celecoxib, Rofecoxib, Sodium Diclofenac and Niflumic Acid in Human Serum Samples by HPLC with DAD Detection

A reverse-phase high-performance liquid chromatographic method has been developed for the separation and simultaneous determination of two COX-2 inhibitors, celecoxib and rofecoxib, in addition to two well-known non-steroidal anti-inflammatory drugs (NSAIDs), sodium diclofenac and niflumic acid in human serum samples. Good chromatographic separation was achieved using a C18 bonded silica column applying a gradient with acetronitrile and water, from 15 to 60% acetonitrile. The mobile phase contained 0.1% trifluoroacetic acid as an organic modifier. Detection was made using a diode array detector (DAD) and the analytical parameters were established at the wavelength maximum in the UV spectrum of each drug. Linearity was studied up to 100.0 mg L⁻¹. Calibration functions, quantification and detection limits, intra- and inter-day reproducibility and accuracy were estimated for each drug. Solid phase extraction was needed to separate and concentrate the drugs from human serum samples. The method was successfully applied to determine the drugs in human serum samples at levels of 1.0 mg L⁻¹.     

Electrosynthesis of N-tert-Butyl-2-benzothiazolesulfenamide

Conditions for the production of N-tert-butyl-2-benzothiazolesulfenamide (TBBS) in an oxidative condensation of 2-mercaptobenzothiazole and tert-butylamine are determined. The electrosynthesis occurs during electrolysis of a 4.5–5.0 M NaCl solution containing a water-immiscible organic solvent extracting TBBS. The process efficiency is the highest at the following conditions: a 1 : 8 ratio between 2-mercaptobenzothiazole and tert-butylamine, a DSA, a stainless-steel cathode, a current density of 300 A m^–2, and a continuous extraction of TBBS into a 3 : 1 mixture of carbon tetrachloride and acetonitrile. Under these conditions, the TBBS yield is 98–100%, the current efficiency is 74%, the process productivity is 0.9 kg m^–2 h^–1, and the electricity consumption is 1.9 kW h kg^–1. Repeated use of the aqueous phase corrected for the source products jeopardizes neither the process characteristics nor the product quality.     

Micro-pumping flow system for spectrophotometric determination of anionic surfactants in water

A novel procedure has been developed for spectrophotometric determination of anionic surfactants in water using a solenoid micro-pump as fluid-propulsion device. The proposed method is based on substitution of methyl orange (MO) by anionic surfactants in the formation of an ion-pair with the cetyl pyridine ion (CPC⁺) at pH 5.0. The flow network comprised four solenoid micro-pumps which, under microcomputer control, enabled sample and reagent introduction, and homogenisation in the reaction zone. The system is flexible and simple to operate and control, and sensitive and precise. The analytical plot for the anionic surfactant was linear between 1.43×10^–6 and 1.43×10^–5 mol L^–1 (0.5 to 5.0 mg L^–1; R=0.997, n=5). The relative standard deviation was 0.8% (n=11) for a sample containing 5.74×10^–6 mol L^–1 (2 mg L^–1) surfactant. The limit of detection was 9.76×10^–8 mol L^–1 (0.034 mg L^–1) and the sampling throughput was 60 determinations per hour. The results obtained for washing-water samples were comparable with those obtained by use of the reference method, and no significant differences at the 95% confidence level were observed.     

Use of ICP and XAS to determine the enhancement of gold phytoextraction by <Emphasis Type="Italic">Chilopsis linearis</Emphasis> using thiocyanate as a complexing agent

Under natural conditions gold has low solubility that reduces its bioavailability, a critical factor for phytoextraction. Researchers have found that phytoextraction can be improved by using synthetic chelating agents. Preliminary studies have shown that desert willow (Chilopsis linearis), a common inhabitant of the Chihuahuan Desert, is able to extract gold from a gold-enriched medium. The objective of the present study was to determine the ability of thiocyanate to enhance the gold-uptake capacity of C. linearis. Seedlings of this plant were exposed to the following hydroponics treatment: (1) 5 mg Au L^–1 (2.5×10^–5 mol L^–1), (2) 5 mg Au L^–1+10^–5 mol L^–1 NH₄SCN, (3) 5 mg Au L^–1+5×10^–5 mol L^–1 NH₄SCN, and (4) 5 mg Au L^–1+10^–4 mol L^–1 NH₄SCN. Each treatment had its respective control. After 2 weeks we determined the effect of the treatment on plant growth and gold content by inductively coupled plasma–optical emission spectroscopy (ICP–OES). No signs of shoot-growth inhibition were observed at any NH₄SCN treatment level. The ICP–OES analysis showed that addition of 10^–4 mol L^–1 NH₄SCN increased the concentration of gold by about 595, 396, and 467% in roots, stems, and leaves, respectively. X-ray absorption spectroscopy (XAS) studies showed that the oxidation state of gold was Au(0) and that gold nanoparticles were formed inside the plants.     

Screening method for phthalate esters in water using liquid-phase microextraction based on the solidification of a floating organic microdrop combined with gas chromatography-mass spectrometry

A simple and efficient liquid-phase microextraction (LPME) technique was developed using directly suspended organic microdrop coupled with gas chromatography–mass spectrometry (GC–MS), for the extraction and the determination of phthalate esters (dimethyl phthalate, diethyl phthalate, diallyl phthalate, di-n-butyl phthalate (DnBP), benzyl butyl phthalate (BBP), dicyclohexyl phthalate and di-2-ethylhexyl phthalate (DEHP)) in water samples. Microextraction efficiency factors, such as nature and volume of the organic solvent, temperature, salt effect, stirring rate and the extraction time were investigated and optimized. Under the optimized extraction conditions (extraction solvent: 1-dodecanol; extraction temperature: 60 °C; microdrop volume: 7 μL; stirring rate: 750 rpm, without salt addition and extraction time: 25 min), figures of merit of the proposed method were evaluated. The values of the detection limit were in the range of 0.02–0.05 μg L⁻¹, while the R.S.D.% value for the analysis of 5.0 μg L⁻¹ of the analytes was below 7.7% (n = 4). A good linearity (r² ≥ 0.9940) and a broad linear range (0.05–100 μg L⁻¹) were obtained. The method exhibited enrichment factor values ranging from 307 to 412. Finally, the designed method was successfully applied for the preconcentration and determination of the studied phthalate esters in different real water samples and satisfactory results were attained.     

Spectrophotometric determination of manganese with derivatives of 1,3,3-trimethyl-2-[3-(1,3,3-trimethyl-1,3- H-indol-2-ylidene)propenyl]-3 H-indolium

A new, simple, rapid, and sensitive spectrophotometric method has been developed for the determination of manganese in sewage. The method is based on the reaction of manganese with derivatives of 1,3,3-trimethyl-2-[3-(1,3,3-trimethyl-1,3-H-indol-2-ylidene)propenyl]-3H-indolium to form a colored ion associate with a sensitive absorption maximum at 560 nm. The appropriate reaction conditions have been established: pH 8.5–10.0, 1.25–2.3×10^–3 mol L^–1 1-nitroso-2-naphthol, and 1.6–2.4×10^–4 mol L^–1 dye reagent. Beer's law is obeyed for manganese concentrations up to 4.2 mg L^–1. The limit of detection is 0.01 mg L^–1 Mn²⁺; the molar absorptivity of the ion associate was 7.5×10⁴ L mol^–1 cm^–1. The effect of various foreign ions was examined. A reaction mechanism is suggested. The developed procedure was tested for determination of manganese in sewage with satisfactory precision and accuracy.     

Determination of Compounds from Epoxy Resins in Food Simulants by HPLC-Fluorescence

Summary A fluorimetric-detection RP-HPLC method is developed for the separation and quantification of bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), its hydrolysis and chlorohydroxy derivatives, and NOGE and BADGE oligomers. This method was applied to test an epoxy resin used as a coating for food contact packaging materials with two different heat treatments in the curing process. Migration of these compounds was evaluated in three food simulants: 3% (w/v) acetic acid, 10% (v/v) ethanol, rectified olive oil and also in acetonitrile. A simple extraction procedure was also applied to obtain olive oil extracts. Quantification was performed by using external calibration, and correlation coefficients were greater than 0,996 and the limit of detection (LOD) was 0.02 mg L^–1 for all substances. HPLC-MS method was applied to confirm derivatives and oligomers identity.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, The Netherlands, February 5–7, 2003     

Rapid analysis of pyrethroids in whole urine by high-performance liquid chromatography using a monolithic column and off-line preconcentration in a restricted access material cartridge

A rapid high-performance liquid chromatography (HPLC) method using a monolithic column with UV detection at 238 nm was developed for the determination of fenpropathrin, betacyfluthrin, deltamethrin, and permethrin (cis and trans isomers) in whole urine. The method is based on the use of a monolithic chromatographic column and a restricted access material (RAM) cartridge for sample preparation. The mobile phase was water/acetonitrile (42:58 v/v), the flow rate was 3 mL min^–1, and chromatographic separation was carried out in 10 min. The separation of cis and trans isomers of permethrin was also possible under the above-mentioned conditions. Detection limits in reconstituted whole urine samples were between 0.9 g L^–1 for betacyfluthrin and 4.4 g L^–1 for fenpropathrin and trans-permethrin. Recoveries for urine samples spiked with different amounts of pyrethroids (between 19 g L^–1 and 75 g L^–1) were in the 70±6 to 90±7% range.     

Simultaneous Determination of Trimethoprim, Sulfadiazine, Florfenicol and Oxolinic Acid in Surface Water by Liquid Chromatography Tandem Mass Spectrometry

A liquid chromatographic/tandem mass spectrometric method using atmospheric pressure chemical ionisation (LC-APCI-MS-MS) was developed for simultaneous determination of trimethoprim, sulfadiazine, florfenicol and oxolinic acid in surface water. The compounds were extracted by solid phase extraction on a polymeric sorbent. Two transition products were monitored for each compound. The detection capabilities related to the transition products of lowest abundance were 1 ng L^–1 for trimethoprim, sulfadiazine and florfenicol and 2 ng L^–1 for oxolinic acid. The mean recoveries were 84%, 83%, 96% and 78% at a level of 10 ng L^–1. The relative repeatability standard deviations were less than 11% at a level of 10 ng L^–1.     

Investigation of 2-[(<Emphasis Type="Italic">E</Emphasis>)-2-(4-diethylaminophenyl)-1-ethenyl]-1,3,3-trimethyl-3H-indolium as a new highly sensitive reagent for the spectrophotometric determination of nitrophenols

A new, simple, rapid, and sensitive spectrophotometric method has been developed for the determination of nitrophenols [picric acid (PA); dinitrophenols (DNP)] in wastewater samples. The method is based on the reaction of nitrophenols with 2-[(E)-2-(4-diethylaminophenyl)-1-ethenyl]-1,3,3-trimethyl-3 H-indolium chloride reagent to form the colored ion associates, which are extracted by organic solvents. The molar absorptivity of the ion associates of PA with the investigated reagent ranges from 8.3×10⁴ to 11.3×10⁴ L mol^–1 cm^–1, depending on the extractant. Because only PA is extracted in an acidic medium with the investigated reagent, but both PA and DNP are extracted in an alkaline medium, it is possible to determine both substances in a mixture. Appropriate reaction conditions have been established. The absorbance of the colored extracts obeys Beers law in the range of 0.04–4.58 mg L^–1 PA, 1.0–18.4 mg L^–1 2,4-DNP and 1.2–14.7 mg L^–1 2,6-DNP, respectively. The limit of detections, calculated from a blank test (n=10; P=0.95), are 0.05 mg L^–1 PA, 0.9 mg L^–1 (2,4-DNP), and 1.1 mg L^–1 (2,6-DNP), respectively.     

High-Performance Liquid Chromatographic Method for Determination and Pharmacokinetic Study of Chlorogenic Acid in the Plasma of Rats After Administration of the Chinese Medicinal Preparation Luying Decoction

A simple and sensitive high-performance liquid chromatographic method has been developed for determination of chlorogenic acid in rat plasma. Chlorogenic acid was extracted from plasma samples with methanol. HPLC analysis of the extracts was performed on a C₁₈ column (250 mm × 4.6 mm i.d., 5 µm particles). The mobile phase was acetonitrile −1% formic acid (9:91, v/v). The calibration plot was linear over the range 0.0420–2.10 µg mL⁻¹ and the lower limit of quantification was 0.0420 µg mL⁻¹. The method was reproducible and reliable with intra-day precision better than 8.2%, inter-day precision better than 9.1%, accuracy within ±8.3%, and mean extraction recovery above 84.4%. The validated method was successfully applied to pharmacokinetic studies of chlorogenic acid in rat plasma after administration of Luying decoction.     

Rapid and sensitive method for the determination of acetaldehyde in fuel ethanol by high-performance liquid chromatography with UV–Vis detection

A high-performance liquid chromatography (HPLC) method for the determination of acetaldehyde in fuel ethanol was developed. Acetaldehyde was derivatized with 0.900 mL 2,4-dinitrophenylhydrazine (DNPHi) reagent and 50 L phosphoric acid 1 mol L^–1 at a controlled room temperature of 15°C for 20 min. The separation of acetaldehyde-DNPH (ADNPH) was carried out on a Shimadzu Shim-pack C₁₈ column, using methanol/LiCl_(aq) 1.0 mM (80/20, v/v) as a mobile phase under isocratic elution and UV–Vis detection at 365 nm. The standard curve of ADNPH was linear in the range 3–300 mg L^–1 per injection (20 L) and the limit of detection (LOD) for acetaldehyde was 2.03 g L^–1, with a correlation coefficient greater than 0.999 and a precision (relative standard deviation, RSD) of 5.6% (n=5). Recovery studies were performed by fortifying fuel samples with acetaldehyde at various concentrations and the results were in the range 98.7–102%, with a coefficient of variation (CV) from 0.2% to 7.2%. Several fuel samples collected from various gas stations were analyzed and the method was successfully applied to the analysis of acetaldehyde in fuel ethanol samples.     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

A rapid and sensitive GC–MS method for determination of 1,3-dichloro-2-propanol in water

A rapid analytical method for sensitive determination of 1,3-dichloro-2-propanol (1,3-DCP) in river water has been developed. 1,3-DCP is extracted from water with ethyl acetate. After filtration through sodium sulfate the ethyl acetate phase is analyzed by gas chromatography–mass spectrometry. The method uses 1,3-DCP-d₅ as internal standard. Different extraction solvents, concentrations of ammonium sulfate in the water samples, and the effect of humic acid were tested and their influence on the recovery of DCP has been evaluated. The method quantification limit was 0.1 g L^–1. For spiked water samples (0–5.2 g L^–1, n=21) a repeatability coefficient of variation of 5.4% was obtained. The average recovery rate of 1,3-DCP was 105±3% (n=21). Stability tests, which were carried out with Danube river water, led to an estimated 1,3-DCP degradation rate of 0.008±0.0008 day^–1 at 6°C.