Automated Solid-phase Extraction Research Articles

Rapid gas chromatography/mass spectrometry quinine determination in plasma after automated solid‐phase extraction

The combined use of an automatic solid-phase extraction (SPE) apparatus with Oasis MCX cartridges and gas chromatography/mass spectrometry (GC/MS) to rapidly quantify quinine in biological samples with cyproheptadine as the internal standard is described. The selected ion monitoring mode, with the quantification ions m/z 136 and 287 (qualifier ions: m/z 261, 381 and 215, 96), allows the estimation of quinine levels, respectively. Separation was completed within 12.7 min. Excellent linearity was found up to 10 000 microg/L of plasma. The limit of detection (LOD) was 12.2 microg/L and the limit of quantification (LOQ) was 40.6 microg/L. High reproducibility (intra-assay CV range 1.9-4.3%, inter-assay CV range 2.2-11.3%) and accuracy values (intra-assay range 83.2-103.7%, inter-assay range 86.8-103.7%) were obtained. Recoveries were concentration-independent (97.2% and 89.8% for 4000 and 10 000 microg/L, respectively). This sensitive, simple assay for quinine in various matrices meets the current requirements for bioanalytical assays and may be used to monitor quinine levels in patients developing severe malaria with acute renal failure during hemofiltration. The optimal quinine dose in this situation is not really established and to improve clinical care, quinine concentrations might be explored to improve efficacy and minimise potential toxicity.

Measurement of human urinary organophosphate pesticide metabolites by automated solid-phase extraction, post extraction derivatization, and gas chromatography–tandem mass spectrometry

Organophosphorus (OP) pesticides are among the most widely used pesticides in the United States. Human exposure to these pesticides may occur from their use on crops in agriculture and for pest control in residential settings. Most of the OP pesticides used in the United States are metabolized to up to three of six common urinary dialkyl phosphate metabolites. Quantification of these metabolites provides information on cumulative exposure to most OP pesticides. To accurately quantify OP pesticide metabolites in human urine, we developed a simple, highly sensitive, analytic method involving automated solid-phase extraction (SPE) of human urine, followed by post-extraction derivatization of the organophosphorus metabolites with 1-chloro-3-iodopropane, and analysis by isotope dilution gas-chromatography-tandem mass spectrometry. The styrene-divinyl benzene polymer-based SPE cartridges yielded good SPE recoveries of the metabolites because of their enhanced non-polar interactions. This method is less labor-intensive, more time-efficient, and reproducible than previously reported methods. Automation of the SPE allowed unattended extraction of urine samples, and hence, increased the sample throughput and reduced the inter- and intra-day variations. The method limits of detection were excellent for all analytes ranging from 50 pg/ml to 170 pg/ml. Relative standard deviations ranged from 2% to 12%.

Rapid Quantitation of Digoxin in Human Plasma and Urine Using Isotope Dilution Liquid Chromatography‐Tandem Mass Spectrometry

A rapid LC/MS/MS assay to quantitate digoxin in human plasma and urine has been developed and validated using a Packard liquid handling systems‐Multiprobe® II AMP8E1. Following automatic solid phase extraction on Waters Oasis HLB 30 mg 96 plates, digoxin and its internal standard (Digoxin D3) were analyzed by reverse phase chromatography (LUNA C18, 5 µm, 150×2 mm), and introduced into the mass spectrometer (Sciex ‐ API 3000) via the turboIonspray ion source operating in positive mode. The assay was validated for human plasma and human urine over a concentration range of 0.2–20 ng/mL and 1–100 ng/mL, respectively, using 0.5 mL of sample. The between day and within day coefficients of variation for all matrices were <17% at the concentrations of lower limit of quantification (LLOQ), and <13% at other quality control concentrations. The average recovery was 80.3% from plasma and 74.3% from urine. No matrix effect was observed. Freeze‐thaw stability, stability of digoxin in matrix, and stability of extracted samples were also evaluated. The mean concentration after three freeze‐thaw cycles was within±3.2% of the nominal value in plasma and within±11.4% of the nominal value in urine. After storage for 4 hours at room temperature, greater than 97.2% of digoxin remained in plasma and 96% remained in urine. Digoxin in extract was stable in the autosampler at+5°C for 77 hours in plasma and 80 hours in urine.

Determination of S‐phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography‐tandem mass spectrometric method

S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.

Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry

In order to perform comprehensive epidemiological studies where multiple metabolites of several PAHs are measured and compared in low-dose urine samples, fast and robust methods are needed to measure many analytes in the same sample. We have modified a previous method used for measuring polycyclic aromatic hydrocarbon (PAH) metabolites by automating the solid-phase extraction (SPE) and including an additional eight metabolites. We also added seven new carbon-13 labeled standards, which improves the use of isotope-dilution calibration. Our method included enzyme hydrolysis, automated SPE and derivatization with a silylating reagent followed by gas chromatography (GC), coupled with high-resolution mass spectrometry (HRMS). Using this method, we measured 23 metabolites, representing 9 parent PAHs, with detection limits in the low pg/mL range. All steps in the clean-up procedure were optimized individually, resulting in a method that gives good recoveries (69-93%), reproducibility (coefficient of variation for two quality control pools ranged between 4.6 and 17.1%, N>156), and the necessary specificity. We used the method to analyze nearly 3000 urine samples in the fifth National Health and Nutrition Examination Survey (NHANES 2001-2002).

Determination of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and alprazolam in human plasma by liquid chromatography–electrospray ionization mass spectrometry

A fast liquid chromatographic assay with mass spectrometric detection (LC/MS) has been developed and validated for the simultaneous determination of methadone (MT), its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and alprazolam, in human plasma. The extraction procedure was performed with automatic solid phase extraction, and the compounds were separated with a Sunfire column using a gradient mode. Deuterated analogues for all of the analytes of interest were used for quantitation. Limits of detection (LOD) were established between 0.5 and 1 ng/ml. Linearity was obtained over a range of 2-2,000 ng/ml with an average correlation coefficient (R(2)) of >0.99. Intra- and inter-batch coefficients of variation and relative mean errors were less than 10% for all analytes and concentrations. The recoveries were higher than 50.0% in all cases. The method proved to be suitable for evaluation of plasma obtained from patients enrolled in a MT-maintenance program who are frequently treated with alprazolam as a sedative.

Randomized, Double-Blind, Placebo-Controlled Study About the Effects of Cannabidiol (CBD) on the Pharmacokinetics of ??9-Tetrahydrocannabinol (THC) After Oral Application of THC Verses Standardized Cannabis Extract

Cannabidiol (CBD) is known to modify the effects of Delta-tetrahydrocannabinol (THC) by decreasing anxiety and antagonizing other THC-effects. As a reason, pharmacodynamic as well as pharmacokinetic mechanisms were suggested. In context of the use of cannabis-based medicine extracts for therapeutic purposes, a study was performed in a double-blind and placebo-controlled cross-over design in which each of 24 volunteers (12 male and 12 female, age 18-45 years) obtained soft-gelatin capsules with 10 mg THC (THC-set), cannabis extract containing 10 mg THC +5.4 mg CBD (CAN-set) or placebo in weekly intervals. Blood samples were taken 30 minutes before and 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 9 hours and 24 hours after the intake. The concentrations of THC, of its metabolites 11-OH-THC, THC-COOH and of CBD in the plasma samples were determined by automatic solid phase extraction, derivatization with N,O-bis(trimethylsilyl)triflouroacetamide and gas chromatography-mass spectrometry. The concentration versus time curves (maximum concentrations Cmax, corresponding time tmax and areas under the curves AUC) were evaluated by statistical methods with respect to equivalence or differences between the CAN-set and the THC-set. Furthermore, the intra-individual ratios of Cmax and AUC for 11-OH-THC/THC, THC-COOH/THC and THC-COOH/11-OH-THC were compared between the THC-set and the CAN-set. Despite the large variation of the data, evidence emerged from the total of the results that CBD partially inhibits the CYP 2C catalyzed hydroxylation of THC to 11-OH-THC. The probability for this inhibition is particularly high for oral intake because THC and CBD attain relatively high concentrations in the liver and because of the high first-pass metabolism of THC. However, the effect of CBD is small in comparison to the variability caused by other factors. Therefore, a pharmacokinetic reason for the differences determined between pure THC and cannabis extract is improbable at the doses chosen in this study. Significantly higher AUC and Cmax and shorter tmax were found for females as compared with males.

Development and validation of an automated solid phase extraction and liquid chromatographic method for the determination of piperaquine in urine

A sensitive and specific bioanalytical method for determination of piperaquine in urine by automated solid-phase extraction (SPE) and liquid chromatography (LC) has been developed and validated. Buffered urine samples (containing internal standard) were loaded onto mixed phase (cation-exchange and octylsilica) SPE columns using an ASPEC XL SPE robot. Chromatographic separation was achieved on a Chromolith Performance RP-18e (100 mm × 4.6 mm I.D.) LC column with phosphate buffer (pH 2.5; 0.1 mol/L)–acetonitrile (92:8, v/v). Piperaquine was analysed at a flow rate of 3 mL/min with UV detection at 347 nm. A linear regression model on log–log transformed data was used for quantification. Within-day precision for piperaquine was 1.3% at 5000 ng/mL and 6.6% at 50 ng/mL. Between-day precision for piperaquine was 3.7% at 5000 ng/mL and 7.2% at 50 ng/mL. Total-assay precision for piperaquine over 4 days using five replicates each day ( n = 20) was 4.0%, 5.2% and 9.8% at 5000, 500 and 50 ng/mL, respectively. The lower limit of quantification (LLOQ) was set to 3 ng/mL using 1 mL of urine, which could be lowered to 0.33 ng/mL when using 9 mL of urine and an increased injection volume.

Simultaneous and Sensitive Analysis of THC, 11-OH-THC, THC-COOH, CBD, and CBN by GC-MS in Plasma after Oral Application of Small Doses of THC and Cannabis Extract

Besides the psychoactive Delta(9)-tetrahydrocannabinol (THC), hashish and marijuana as well as cannabis-based medicine extracts contain varying amounts of cannabidiol (CBD) and of the degradation product cannabinol (CBN). The additional determination of these compounds is interesting from forensic and medical points of view because it can be used for further proof of cannabis exposure and because CBD is known to modify the effects of THC. Therefore, a method for the simultaneous quantitative determination of THC, its metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), CBD and CBN from plasma was developed. The method was based on automatic solid-phase extraction with C(18) ec columns, derivatization with N,O-bistrimethylsilyltrifluoroacetamide (BSTFA), and gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) with deuterated standards. The limits of detection were between 0.15 and 0.29 ng/mL for THC, 11-OH-THC, THC-COOH, and CBD and 1.1 ng/mL for CBN. The method was applied in a prospective pharmacokinetic study after single oral administration of 10 mg THC alone or together with 5.4 mg CBD in cannabis extract. The maximum plasma concentrations after cannabis extract administration ranged between 1.2 and 10.3 ng/mL (mean 4.05 ng/mL) for THC, 1.8 and 12.3 ng/mL (mean 4.9 ng/mL) for 11-OH-THC, 19 and 71 ng/mL (mean 35 ng/mL) for THC-COOH, and 0.2 and 2.6 ng/mL (mean 0.95 ng/mg) for CBD. The peak concentrations (mean values) of THC, 11-OH-THC, THC-COOH, and CBD were observed at 56, 82, 115, and 60 min, respectively, after intake. CBN was not detected. Caused by the strong first-pass metabolism, the concentrations of the metabolites were increased during the first hours after drug administration when compared to literature data for smoking. Therefore, the concentration ratio 11-OH-THC/THC was discussed as a criterion for distinguishing oral from inhalative cannabis consumption.

Hyphenated NMR Methods in Natural Products Research, Part 1: Direct Hyphenation

This review describes the current status of the development of techniques combining liquid chromatography and nuclear magnetic resonance and their use in the context of natural products. HPLC-NMR methods have a rapidly growing impact on natural products research by enabling structure determination of natural products directly from small amounts of extracts, i. e., prior to the investment in an often lengthy and laborious preparative-scale isolation process. This speeds up extract dereplication and helps to avoid re-isolation of already known extract constituents. Direct HPLC-NMR hyphenation techniques, defined as methods in which NMR spectra are recorded using the HPLC eluate, are described. The recently developed indirect HPLC-NMR method employing an automated solid-phase extraction interface between HPLC and NMR, which replaces the chromatographic solvent with a different solvent for NMR data acquisition, is described in the second part of this review.

Simultaneous analysis of Thebaine, 6-MAM and six abused opiates in postmortem fluids and tissues using Zymark ® automated solid-phase extraction and gas chromatography–mass spectrometry

Opiates are some of the most widely prescribed drugs in America and are often abused. Demonstrating the presence or absence of opiate compounds in postmortem fluids and/or tissues derived from fatal civil aviation accidents can have serious legal consequences and may help determine the cause of impairment and/or death. However, the consumption of poppy seed products can result in a positive opiate drug test. We have developed a simple method for the simultaneous determination of eight opiate compounds from one extraction. These compounds are hydrocodone, dihydrocodeine, codeine, oxycodone, hydromorphone, 6-monoacetylmorphine, morphine, and thebaine. The inclusion of thebaine is notable as it is an indicator of poppy seed consumption and may help explain morphine/codeine positives in cases where no opiate use was indicated. This method incorporates a Zymark ® RapidTrace™ automated solid-phase extraction system, gas chromatography/mass spectrometry, and trimethyl silane (TMS) and oxime-TMS derivatives. The limits of detection ranged from 0.78 to 12.5 ng/mL. The linear dynamic range for most analytes was 6.25–1600 ng/mL. The extraction efficiencies ranged from 70 to 103%. We applied this method to eight separate aviation fatalities where opiate compounds had previously been detected.

Evaluation of Ketamine Abuse Using Hair Analysis: Concentration Trends in a Singapore Population

This paper presents a method for the detection of ketamine in hair. Hair samples (25 mg) were washed, pulverized, and digested in hydrochloric acid (0.5 M) overnight at 45 degrees C. The samples were extracted by an automated solid-phase extraction procedure, and the extracts were subsequently analyzed using gas chromatography-mass spectrometry in selected ion monitoring mode. Linearity up to 120 ng/mg was obtained for both ketamine and norketamine with correlation coefficients of 0.9987 and 0.9985, respectively. Limit of detection was found to be at 0.4 ng/mg for both drugs while the limit of quantitation was found to be 0.6 and 0.8 ng/mg for ketamine and norketamine, respectively. The validated method was used in the analysis of 91 hair segments obtained from 54 ketamine abusers. Based upon the voluntary confession of the ketamine abusers, a correlation between the amount of ketamine detected and the frequency of abuse was observed.

Automated solid-phase extraction and high-performance liquid chromatographic determination of nitrosamines using post-column photolysis and tris(2,2′-bipyridyl) ruthenium(III) chemiluminescence

A sensitive and selective post-column detection system for nitrosamines is described. The principle upon which the detector works is that UV irradiation of aqueous solutions of nitrosamines leads to cleavage of the N NO bond. The amine generated is subsequent detected by chemiluminescence using tris(2,2′-bipyridyl) ruthenium(III), which is on-line generated by photo-oxidation of the ruthenium(II) complex in the presence of peroxydisulfate. Factors affecting the photochemical and chemiluminescent reactions were optimized to minimise their contribution to the total band-broadening. This detection system was tested for N-nitrosodimethylamine, N-nitroso-diethylamine, N-nitrosomorpholine, N-nitrosopiperidine and N-nitrosopyrrolidine, which were separated on an ODS column by isocratic reversed-phase chromatography with acetonitrile–water containing 5 mM acetate buffer at pH 4.0. A linear relationship between analyte concentration and peak area was obtained within the range 0.13–500 μg l −1 with correlation coefficients greater than 0.9995 and detection limits of between 0.03 and 0.76 μg l −1. Intra- and inter-day precision values of about 1.2% RSD ( n = 11) and 2.5% RSD ( n = 10), respectively, were obtained. The sensitivity may increase from 9 to 280 times with respect to UV detection, depending on the nitrosamine in question. An automated solid-phase extraction (SPE) system was used in conjunction with HPLC to determine nitrosamine residues in waters. Detection limits within the range 0.10–3.0 ng l −1 were achieved for only 250 ml of sample.

Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization followed by gas chromatography–mass spectrometry

A repeatable and highly sensitive analytical method using gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d 4-KT for KT and d 4-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF 3CONCH 3COCF 3, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC–MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20–2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5–1.0 and 1.5–3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2–93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.

Development and validation of a microsomal online cytochrome P450 bioreactor coupled to solid-phase extraction and reversed-phase liquid chromatography

The development and validation of an online cytochrome P450 (CYP)-based bioreactor coupled to automated solid-phase extraction (SPE) and gradient HPLC separation is described. The analytical method was checked on intra- and inter-day repeatability of the ethoxyresorufin- O-demethylation (EROD) reaction with CYP 1A1/1A2 containing β-NF induced rat liver microsomes as an enzyme source. These experiments showed that CYP activity was linearly decreased with 16% over an 11 h period. Inter-day measurements had a CV of 9.1%. Furthermore, K m and V max values of the EROD reaction, measured with the bioreactor, were 2.72 ± 0.46 μM and 7.9 ± 0.5 nmol/min/mg protein, respectively. These were in good correspondence with K m and V max values, measured with standard batch assay, which amounted 0.66 ± 0.08 μM and 6.4 ± 0.2 nmol/min/mg protein respectively. In conclusion the newly developed analytical method can be used effectively and at a microliter scale for online generation, extraction and separation of metabolites.

Correcting Common Errors in Identifying Cancer-Specific Serum Peptide Signatures

"Molecular signatures" are the qualitative and quantitative patterns of groups of biomolecules (e.g., mRNA, proteins, peptides, or metabolites) in a cell, tissue, biological fluid, or an entire organism. To apply this concept to biomarker discovery, the measurements should ideally be noninvasive and performed in a single read-out. We have therefore developed a peptidomics platform that couples magnetics-based, automated solid-phase extraction of small peptides with a high-resolution MALDI-TOF mass spectrometric readout (Villanueva, J.; Philip, J.; Entenberg, D.; Chaparro, C. A.; Tanwar, M. K.; Holland, E. C.; Tempst, P. Anal. Chem. 2004, 76, 1560-1570). Since hundreds of peptides can be detected in microliter volumes of serum, it allows to search for disease signatures, for instance in the presence of cancer. We have now evaluated, optimized, and standardized a number of clinical and analytical chemistry variables that are major sources of bias; ranging from blood collection and clotting, to serum storage and handling, automated peptide extraction, crystallization, spectral acquisition, and signal processing. In addition, proper alignment of spectra and user-friendly visualization tools are essential for meaningful, certifiable data mining. We introduce a minimal entropy algorithm, "Entropycal", that simplifies alignment and subsequent statistical analysis and increases the percentage of the highly distinguishing spectral information being retained after feature selection of the datasets. Using the improved analytical platform and tools, and a commercial statistics program, we found that sera from thyroid cancer patients can be distinguished from healthy controls based on an array of 98 discriminant peptides. With adequate technological and computational methods in place, and using rigorously standardized conditions, potential sources of patient related bias (e.g., gender, age, genetics, environmental, dietary, and other factors) may now be addressed.

GC/MS Profiling of γ-Hydroxybutyrate and Precursors in Various Animal Tissues Using Automatic Solid-Phase Extraction. Preliminary Investigations of Its Potential Interest in Postmortem Interval Determination

To quantify gamma-hydroxybutyrate (GHB) and its physiological metabolites, gamma-aminobutyric acid (GABA), 1,4-butanediol (1,4-BD), and gamma-butyrolactone (GBL) in various animal tissues (kidney, muscle, heart, liver, blood, brain cortex, thalamus, hypothalamus, hippocampus, or pons), an original gas chromatographic/mass spectrometric method with a automated solid-phase extraction by Oasis MCX cartridges on a Gilson Aspec Xli was developed. Using such apparatus allowed the limit of detection (LOD) of target compounds to be significantly lowered (LOD: 0.027, 0.025, and 5.7 microg/mL for GHB, 1,4-BD, and GABA, respectively, in 200 microL or microg of sample). After validation of each analytical step, the satisfactory performances of the apparatus in conjunction with the rapidity and ease of the extraction step make it suitable for simultaneous assay of GHB, 1,4-BD, GBL, and GABA. The method was used to test the correlation between GHB levels in tissues obtained at different times after death of male Sprague-Dawley rats and the postmortem interval. Preliminary results show a linear increase of GHB levels in relation to time of death in thoracic blood and central nervous system of animals kept at 15 and 20 degrees C.

Quantification of voriconazole in plasma by liquid chromatography-tandem mass spectrometry

A convenient liquid chromatography-tandem mass spectrometry method for the quantification of the triazole antifungal agent voriconazole in plasma samples is described. Fenbuconazole is used as an internal standard. After protein precipitation, automated solid-phase extraction is applied. Electrospray ionization in the positive mode is used and the following mass transitions are recorded: voriconazole, 350-->127; and fenbuconazol, 337-->125. The analytical run time is 4 min. The response was linear from 78 to 5000 microg/L. The total coefficient of variation (n=16) was 12.6% for a low-concentration pool (143 microg/L), 4.7% for a medium-concentration pool (419 microg/L), and 5.0% for a high-concentration pool (4304 microg/L). The method is proposed for future investigations that should be performed to test the hypothesis that therapeutic drug monitoring of voriconazole is clinically useful.

Determination of estrogenic steroids in surface water and wastewater by liquid chromatography-electrospray tandem mass spectrometry

An analytical method is presented which permits trace level determination of 17alpha-ethynylestradiol (EE2), 17beta-estradiol (E2), and estrone (E1). Using this method, the estrogenic steroids were analyzed in drinking water, surface water, and wastewater (sewage influents and effluents) at concentrations down to 0.1 ng/L. Sample volumes between 100 and 500 mL are concentrated using automated solid-phase extraction. Analysis is performed by liquid chromatography with detection by tandem mass spectrometry. Applying simple clean-up procedures and internal standard calibration, recovery losses resulting from matrix-dependent ion suppression during electrospray ionization could be compensated for all of the investigated compounds. Recoveries around 100% were obtained for all analytes after correction using the internal standards. Limits of quantification (LOQ) were between 0.1 and 0.4 ng/L for purified sewage, surface, ground, and drinking water and between 1 and 2 ng/L in the case of raw sewage. Water treatment by wastewater treatment plants (WWTPs) or by a surface water treatment plant affected the removal of all estrogenic steroids. Thus, E1, E2, and EE2 were removed in the municipal WWTPs to the extent of 93%, 93%, and 80%, respectively. In the effluents of the WWTP in Ruhleben (Berlin, Germany), E1, E2, and EE2 were detected at the low ng/L level. E2 and EE2 were, however, not present in the Berlin surface water above the LOQ (0.2 ng/L). E1 was the only compound that could be detected in surface water samples. After additional surface water treatment it was still detectable but only at trace-level concentrations with a mean value of 0.16 ng/L.

Validation of a liquid chromatographic-tandem mass spectrometric method for the determination of loperamide in human plasma

A sensitive and selective method based on liquid chromatography-tandem mass spectrometry (LC–MS/MS) has been developed for the quantitative determination of loperamide in human plasma. Automated solid-phase extraction (SPE) on disposable extraction cartridges (DEC) is used to isolate the compounds from the biological matrix and to prepare a cleaner sample before injection and analysis in the LC–MS/MS system. After conditioning, the plasma sample is loaded on the DEC filled with endcapped ethyl silica (C2 EC) and washed twice with water. The analytes are therefore eluted by dispensing methanol. The eluate is then collected and added with ammonium acetate solution in order to inject an aliquot of this final extract in the LC–MS/MS system. On-line LC–MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of loperamide. The separation is obtained on a octadecylsilica based stationary phase using a mobile phase consisting in a mixture of methanol and 5 mM ammonium acetate solution (25:75, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/ z 477 → 266 and 316 → 270 for loperamide and clonazepam, respectively. The most appropriate regression model of the response function as well as the limit of quantitation were first selected during the pre-validation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for loperamide. The method was also validated with respect to recovery, precision, trueness, accuracy and linearity.