Oasis MCX Research Articles

Detection of 36 antibiotics in coastal waters using high performance liquid chromatography-tandem mass spectrometry

Among pharmaceuticals and personal care products released into the aquatic environment, antibiotics are of particular concern, because of their ubiquity and health effects. Although scientists have recently paid more attention to the threat of antibiotics to coastal ecosystems, researchers have often focused on relatively few antibiotics, because of the absence of suitable analytical methods. We have therefore developed a method for the rapid detection of 36 antibiotic residues in coastal waters, including tetracyclines (TCs), sulfanilamides (SAs), and quinolones (QLs). The method consists of solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, using electrospray ionization (ESI) in positive mode. The SPE was performed with Oasis HLB and Oasis MCX cartridges. Chromatographic separation on a C18 column was achieved using a binary eluent containing methanol and water with 0.1% formic acid. Typical recoveries of the analytes ranged from 67.4% to 109.3% at a fortification level of 100 ng/L. The precision of the method, calculated as relative standard deviation (RSD), was below 14.6% for all the compounds. The limits of detection (LODs) varied from 0.45 pg to 7.97 pg. The method was applied to determine the target analytes in coastal waters of the Yellow Sea in Liaoning, China. Among the tested antibiotics, 31 were found in coastal waters, with their concentrations between the LOD and 212.5 ng/L. These data indicate that this method is valid for analysis of antibiotics in coastal waters. The study first reports such a large number of antibiotics along the Yellow Sea coast of Liaoning, and should facilitate future comprehensive evaluation of antibiotics in coastal ecosystems.

Analysis of β‐blockers in groundwater using large‐volume injection coupled‐column reversed‐phase liquid chromatography with fluorescence detection and liquid chromatography time‐of‐flight mass spectrometry

Atenolol, nadolol, metoprolol, bisoprolol and betaxolol were simultaneously determined in groundwater samples by large-volume injection coupled-column reversed-phase liquid chromatography with fluorescence detection (LVI-LC-LC-FD) and liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS). The LVI-LC-LC-FD method combines analyte isolation, preconcentration and determination into a single step. Significant reductions in costs for sample pre-treatment (solvent and solid phases for clean up) and method development times are also achieved. Using LC-TOF-MS, accurate mass measurements within 3 ppm error were obtained for all of the β-blockers studied. Empirical formula information can be obtained by this method, allowing the unequivocal identification of the target compounds in the samples. To increase the sensitivity, a solid-phase extraction step with Oasis MCX cartridge was carried out yielding recoveries of 79-114% (n=5) with RSD 2-7% for the LC-TOF-MS method. SPE gives a high purification of β-blockers compared with the existing methods. A 100% methanol wash was allowed for these compounds with no loss of analytes. Limit of quantification was 1-7 ng/L for LVI-LC-LC-FD and 0.25-5 ng/L for LC-TOF-MS. As a result of selective extraction and effective removal of coextractives, no matrix effect was observed in LVI-LC-LC-FD and LC-TOF-MS analyses. The methods were applied to detect and quantify β-blockers in groundwater samples of Almería (Spain).

Development and validation of a selective and sensitive LC–MS/MS method for determination of cycloserine in human plasma: Application to bioequivalence study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the determination of cycloserine in human plasma is developed using niacin as internal standard (IS). The analyte and IS were extracted from 500 μL of human plasma via solid phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on a Peerless Basic C18 (100 mm × 4.6 mm, 3 μm) column under isocratic conditions. Detection of analyte and IS was done by tandem mass spectrometry, operating in positive ion and multiple reaction monitoring (MRM) acquisition mode. The protonated precursor to product ion transitions monitored for cycloserine and niacin were at m/z 103.1 → 75.0 and 124.1 → 80.1 respectively. The method was fully validated for its selectivity, interference check, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The limit of detection (LOD) and lower limit of quantitation of the method were 0.0013 and 0.20 μg/mL respectively with a linear dynamic range of 0.20–30.00 μg/mL for cycloserine. The intra-batch and inter-batch precision (%CV) across six quality control levels was less than 8.0% for cycloserine. The method was successfully applied to a bioequivalence study of 250 mg cycloserine capsule formulation in 24 healthy Indian male subjects under fasting condition.

The UHPLC-DAD fingerprinting method for analysis of extracellular metabolites of fungi of the genus Geosmithia (Acomycota: Hypocreales)

A new simple ultra-high-performance liquid chromatography method with diode array detection (UHPLC-DAD) was developed for chemical fingerprinting analysis of extracellular metabolites in fermentation broth of Geosmithia spp. The SPE method employing Oasis MCX strong cation-exchange mixed-mode polymeric sorbent was chosen for extraction of the metabolites. The analyses were performed on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d.; particle size, 1.7 μm; Waters) using a gradient elution program with an aqueous solution of trifluoroacetic acid and acetonitrile as the mobile phase. The applicability of the method was proved by analysis of 38 strains produced by different species and isolated from different sources (hosts). The results revealed the correlation of obtained UHPLC-DAD fingerprints with taxonomical identity.

Abstract 1309: Human imatinib (IM) and desmethyl-IM blood vs. tissue levels in healthy and tumor tissue of patients treated for advanced gastrointestinal stromal tumors (GIST)

Abstract Background: Blood trough levels of 1100 ng/ml are thought to be required for effective treatment of CML with IM. In a solid tumor like GIST, there might be different access of tumors cells to the drug even depending on the harbouring organ. There are no data available comparing tissue and blood levels of IM in humans. Materials: 24 patients scheduled for tumor resection were treated with IM 400mg/d or bid for metastatic GIST and medication continued including the preop. day. Blood samples were drawn intraop. at the time of resection. Samples of tumor tissues (n=52) and healthy organ tissues the tumor grew in were taken from 28 locations: liver (n=12), omentum (n=7), stomach (n=5), small bowel (n=2), soft tissue (n=1) and lung (n=1). Methods: To quantify IM in serum and tissue specimens a LC-MS/MS assay linear for concentrations 10 – 5000 ng/ml was used. IM and internal standard D8-IM were extracted from plasma specimens and corresponding tissue of GIST-pats. with ion exchange cartridges (OASIS MCX). Extracts were injected into the HPLC system equipped with XBridge phenyl column (Waters Corp.) and the non-isocratic elution with a mobile phase consisting of methanol/ammonium formate (4mM, pH4) performed within 15 minutes at a flow rate of 0,3ml/min. Ion transitions [M + H]+ of IM (m/z = 494 –> 394), DM-IM (m/z 480–> 394) and of D8-IM (m/z 502–> 394) were monitored by MRM mode. The mass spectrometer HCT Ultra (Bruker Daltonics) was equipped with an electro spray ionization probe, operating in positive mode. Peak areas and respective concentrations of DM-IM were calculated with QuantAnalysis (Bruker Daltonics). A six-point calibration for IM was performed once a day before analytical runs and quality control specimens with 3 different concentration-ranges (20, 100 and 1000 ng/ml). The interday precisions for the low level quality control (20 ng/ml) was below 20% and below 10% for the medium (100 ng/ml) and high (1000ng/ml) – levels respectively. Tissue IM/DM-IM data are given as ng/mg tissue, and mean+/SD. Correlations were calculated using Spearman and Pearson tests. Results: IM blood levels were 997+/-265 ng/ml (range 225-2760), and DM-IM were 215 +/- 124 ng/ml (range, 29-399), correlated with r=0.74. There was a significant difference between organs: liver IM 16,8 +/6.2 (DM-IM 13.2 +/4.5), stomach IM 5.25 +/- 1.2 (DM-IM 4.12 +/- 1.1), omentum IM 1.25 (DM-IM 2.1), p<0.01 and only a weak correlation to blood IM levels (r=.45). Tumor concentrations of IM were 40+/-12 (DM-IM 18.6 +/- 14) There was only a moderate correlation (r=.486, p=.021,) between blood levels of IM and tumor tissue (DM-IM r=0.34, p 0.06). Conclusions: In humans, tissue concentrations of imatinib cannot be predicted from blood levels, neither in healthy organs, nor in GIST tumor tissue. Different tissue IM levels could explain tumor progression during therapy despite of the missing of secondary mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1309. doi:10.1158/1538-7445.AM2011-1309

Drugs of abuse and their metabolites in waste and surface waters by liquid chromatography‐tandem mass spectrometry

A solid-phase extraction and liquid chromatography-tandem mass spectrometry (SPE/LC-MS-MS) method was developed and validated for the simultaneous determination of nicotine, five drugs of abuse (morphine, cocaine, codeine, methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) and four metabolites (dihydrocodeine, 6-acetylmorphine, 11-nor-carboxy-Δ(9)-tetrahydrocannabinol, and benzoylecgonine) in water samples. A Fused-Core™ particle column was used as an alternative to sub-2-μm particles in chromatographic separations to work with low backpressures and high efficiencies in short analysis times. Drugs were extracted from waste and surface water with SPE using Oasis MCX cartridges. Electrospray (ESI) in positive and negative mode and tandem MS selected reaction monitoring mode were used for identification and quantification. Calibration by linear regression analysis with deuterated internal standards was used to compensate the matrix effects. Limits of detection were found as low as 0.5-1 ng/L (surface water) and 1-50 ng/L (wastewater). The method was applied to the analysis of different kinds of samples. Wastewater from a sewage treatment plant was collected from three sampling points (after primary, secondary, and tertiary treatments) for a week. The analysis of the samples revealed a significant presence of these drugs in samples from primary treatments, where maximum concentrations of nicotine (1105 ng/L) and benzoylecgonine (3336 ng/L) were found. Most of the compounds showed values between <LOQ and 52 ng/L after tertiary treatment. When surface waters from the Ebro River were analyzed, only benzoylecgonine showed levels between 19 and 35 ng/L.

Comparison of fluorescence reagents for simultaneous determination of hydroxylated phenylalanine and nitrated tyrosine by high‐performance liquid chromatography with fluorescence detection

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are well-known and important contributors to oxidative and nitrosative stress in several diseases. Hydroxylated phenylalanine and nitrated tyrosine products appear to be particularly susceptible targets of oxidative and nitrosative stress. We compared fluorescence reagents for their potential use in the analysis of hydroxylated phenylalanine and nitrated tyrosine products with a high-sensitivity and high-specificity HPLC-UV-FL technique. The analytes were extracted from serum via solid-phase extraction on Waters Oasis MCX cartridges. Chromatographic separation was achieved on an ODS column (Capcell Pak MG II; 150 × 2.0 mm) using a gradient mobile phase consisting of 20 mm sodium phosphate buffer (adjusted to pH 3.0) and acetonitrile. The method quantification limit for 4-nitrophenylalanine, m-tyrosine, and 3-nitrotyrosine was 0.1 μm. The relative standard deviation of the precision and accuracy was acceptable at the spiked concentration of 0.1 μm for 4-nitrophenylalanine, m-tyrosine and 3-nitrotyrosine. The method could be used for the in vitro analysis of serum samples.

Comparison of four extraction methods for the analysis of pharmaceuticals in wastewater

As one category of the most urgent emerging pollutants, pharmaceuticals have provoked much public and scientific attention due to widespread contamination in aquatic environment. In this study, two active methods by Oasis HLB and MCX and two passive methods by XAD-16 and XAD-16/7 were evaluated for determining the concentrations of 10 pharmaceuticals (carbamazepine, clofibric acid, diclofenac, gemfibrozil, ibuprofen, ketoprofen, naproxen, paracetomol, terbutaline and triclosan) in reclaimed wastewater. Recoveries of the target pharmaceuticals extracted by MCX were higher than HLB except for diclofenac and ketoprofen. For the passive methods, the addition of polar resin XAD-7 improved the recovery compared with the addition of XAD-16 only. The mean recoveries of the target analytes by XAD-16/7 ranged from 22 to 75.8%. The limit of quantification (LOQ) ranged between 25 and 280 ng/L. In addition, by comparing the accuracy and precision of XAD-16/7 method and MCX method, we further demonstrated that the XAD-16/7 method can be satisfactorily used for the analysis of pharmaceuticals in wastewater samples. We applied the method to some wastewater samples from sewage treatment plant (STP) nearby Riverside, CA to track the concentration change of pharmaceuticals in the treatment processes. The result shown that pharmaceuticals were effective reduced in STP mostly by activated sludge.

Monitoring the contents of biogenic amines in sufu by HPLC with SPE and pre-column derivatization

Sufu is a nutritious fermented food whose consumption is currently increasing in many countries. The purpose of this study was to develop an HPLC method with SPE and pre-column derivatization for monitoring the content of biogenic amine in sufu and to understand the differences in the contents of biogenic amines with different processing technologies. Biogenic amines in sufu were extracted with 6% trichloroacetic acid, derivatized with 4-chloro-3,5-dinitrobenzotrifluoride, purified with Waters Oasis MCX cartridge, separated by HPLC using gradient elution, and detected by spectrophotometric UV at 254 nm. The method linearity, calculated for each biogenic amine, has a correlation coefficient higher than 0.9915. Detection limits of the biogenic amines were ranged from 0.016 mg/kg for putrescine to 0.067 mg/kg for spermine. Spiking experiments showed that the overall recoveries ranged between 83.6% and 103.5% with relative standard deviations of 1.26–5.24%. Of the seven biogenic amines under study, at least four biogenic amines were detectable in different kinds of sufu. Tryptamine, putrescine, histamine, and tyramine were the most prevailing biogenic amines in all sufu samples. The mean values of histamine, tryptamine, phenylethylamine, tyramine, putrescine, spermidine and spermine determined in samples were found to be 18.2 mg/kg, 39.0 mg/kg, 1.1 mg/kg, 21.7 mg/kg, 34.6 mg/kg, 11.1 mg/kg, and 8.7 mg/kg, respectively. The total contents of biogenic amines in bacteria fermented sufu samples (129.0 mg/kg) were slightly lower than those in enzymatically ripened sufu samples (137.7 mg/kg). The concentrations of biogenic amines detected in sufu samples were below the maximum permissible limits.

Simultaneous determination of malachite green, gentian violet and their leuco-metabolites in shrimp and salmon by liquid chromatography–tandem mass spectrometry with accelerated solvent extraction and auto solid-phase clean-up

An automated method had been developed for the determination of Malachite green and Gentian violet as well as their leuco-metabolites in shrimp and salmon by liquid chromatography-tandem mass spectrometry with accelerated solvent extraction and auto solid-phase clean-up. The target analytes were extracted using accelerated solvent extraction (ASE) and then purified using auto solid-phase clean-up. The ASE conditions as: solvent, temperature, pressure, static time, and cell size were optimized. The optimum extraction conditions were set as the following aspects: using 22 mL ASE cell, McIlvaine buffer (pH 3)/acetonitrile/hexane (2/10/2, v/v) as the extraction solvent; pressure at 1500 psi; temperature at 60 °C; static time 5 min (static time); one cycles. The extracts were purified on OASIS MCX SPE column. Detection and quantification of Malachite green; gentian violet; leucomalachite green; leucogentian violet were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS). An averaged decision limits (CCα) and detection capability (CCβ) of the method were in the range of 0.005 μg/kg ～ 0.012 μg/kg and 0.08 μg/kg～0.13 μg/kg in shrimp and salmon. The recoveries of Malachite green; Gentian violet; Leucomalachite green; Leucogentian violet at levels of 0.1–1.0 μg/kg averaged from 82.1 to 102.9% with the relative standard derivation less than 14.6%. This method is precise, sensitive and highly efficient in extraction. What’s more, after routine applications, it’s turned out that this method is suitable for the determination of Malachite green, Gentian violet and their leuco-metabolites in shrimp and salmon.

Multi-residue analysis of drugs of abuse in wastewater and surface water by solid-phase extraction and liquid chromatography–positive electrospray ionisation tandem mass spectrometry

A new-multi residue method was developed for the environmental monitoring of 65 stimulants, opiod and morphine derivatives, benzodiazepines, antidepressants, dissociative anaesthetics, drug precursors, human urine indicators and their metabolites in wastewater and surface water. The proposed analytical methodology offers rapid analysis for a large number of compounds, with low limits of quantification and utilises only one solid-phase extraction–ultra performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry (SPE–LC–MS/MS) method, thus overcoming the drawbacks of previously published procedures. The method employed solid phase extraction with the usage of Oasis MCX sorbent and subsequent ultra performance liquid chromatography–positive electrospray ionisation tandem mass spectrometry. The usage of a 1.7 μm particle size column (1 mm × 150 mm) resulted in very low flow rates (0.04 mL min −1), and as a consequence gave good sensitivity, low mobile phase consumption and short retention times for all compounds (from 2.9 to 23.1 min). High SPE recoveries (>60%) were obtained for the majority of compounds. The mean correlation coefficients of the calibration curves were typically higher than 0.997 and showed good linearity in the range 0–1000 μg L −1. The method limits of detection ranged from 0.1 ng L −1 for compounds including cocaine, benzoylecgonine, norbenzoylecgonine and 2-oxo-3-hydroxy-LSD to 100 ng L −1 for caffeine. Method quantification limits ranged from 0.5 to 154.2 ng L −1. Intra- and inter-day repeatabilities were on average less than 10%. The method accuracy range was within −33.1 to 30.1%. The new multi-residue method was used to analyse drugs of abuse in wastewater and river water in the UK environment. Of the targeted 65 compounds, 46 analytes were detected at levels above the method quantification limit (MQL) in wastewater treatment plant (WWTP) influent, 43 in WWTP effluent and 36 compounds in river water.

Rapid wide-scope screening of drugs of abuse, prescription drugs with potential for abuse and their metabolites in influent and effluent urban wastewater by ultrahigh pressure liquid chromatography–quadrupole-time-of-flight-mass spectrometry

This work illustrates the potential of hybrid quadrupole-time-of-flight mass spectrometry (QTOF MS) coupled to ultrahigh pressure liquid chromatography (UHPLC) to investigate the presence of drugs of abuse in wastewater. After solid-phase extraction with Oasis MCX cartridges, seventy-six illicit drugs, prescription drugs with potential for abuse, and metabolites were investigated in the samples by TOF MS using electrospray interface under positive ionization mode, with MS data acquired over an m/ z range of 50–1000 Da. For 11 compounds, reference standards were available, and experimental data (e.g., retention time and fragmentation data) could be obtained, facilitating a more confident identification. The use of a QTOF instrument enabled the simultaneous application of two acquisition functions with different collision energies: a low energy (LE) function, where none or poor fragmentation took place, and a high energy (HE) function, where fragmentation in the collision cell was promoted. This approach, known as MS E, enabled the simultaneous acquisition of full-spectrum accurate mass data of both protonated molecules and fragment ions in a single injection, providing relevant information that facilitates the rapid detection and reliable identification of these emerging contaminants in the sample matrices analyzed. In addition, isomeric compounds, like the opiates, morphine and norcodeine, could be discriminated by their specific fragments observed in HE TOF MS spectra, without the need of reference standards. UHPLC–QTOF MS was proven to be a powerful and efficient technique for rapid wide-scope screening and identification of many relevant drugs in complex matrices, such as influent and effluent urban wastewater.

Simultaneous determination of 15 top-prescribed pharmaceuticals and their metabolites in influent wastewater by reversed-phase liquid chromatography coupled to tandem mass spectrometry

A fast and sensitive high performance reversed-phase liquid chromatography–tandem mass spectrometry method was developed and validated for the analysis of 15 prescription pharmaceuticals and four of their metabolites in influent wastewater. The selected pharmaceuticals belonged to various classes, such as angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, calcium antagonists, β-blockers, antidepressants, analgetics, anticonvulsants, platelet antiaggregants, and cholesterol lowering agents. They were selected from the list of top-sold prescription pharmaceuticals in Belgium. The chromatographic separation was optimized in order to achieve suitable retention times, good resolution for analytes susceptible of mass spectrometric cross-talk and high sensitivity in one single run. All compounds eluted within 9 min on a Phenomenex Kinetex C18 column, based on a newly developed technology that allows a very narrow distribution of the core–shell particles, providing high separation efficiency. Sample preparation was executed with solid-phase extraction on Oasis MCX cartridges. The method was validated by assessing specificity, selectivity, lower limit of quantification (LLOQ), linearity, accuracy, precision, extraction recovery, and matrix effects following Food and Drug Administration guidelines. The method LLOQs ranged from 0.5 to 25 ng/L. Calibration curves and LLOQs were designed to provide a good analytical performance at concentrations expected in real influent wastewater samples for each target compound. Eight deuterated analogues were used as internal standards for quantification. The method was applied to influent wastewater samples collected from 17 different wastewater treatment plants throughout Belgium. Most of the analytes were measured in the samples at concentrations above LLOQ. Seven of the compounds were for the first time reported in influent wastewater. The newly developed analytical method is currently used to assess relationships between sales figures of pharmaceuticals and their corresponding concentrations in influent wastewater.

Determination of benzimidazole residues in bovine milk by ultra-high performance liquid chromatography–tandem mass spectrometry

A method based on ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UHPLC–MS/MS) for the simultaneous determination of benzimidazole residues in bovine milk has been optimized and validated. Rapid chromatographic separation of 13 analytes in 8 min was obtained by means of UHPLC. The samples were subject to Oasis MCX solid-phase extraction cartridges for extraction and clean-up. Matrix-matched calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Mean recoveries ranged from 80% to 101% and inter-day precision was lower than 14%. Limit of detection and limit of quantification of the method ranged from 0.01 to 0.5 μg L −1 and from 0.1 to 1.0 μg L −1, respectively.

Selective Determination of Antiretroviral Agents Tenofovir, Emtricitabine, and Lamivudine in Human Plasma by a LC-MS-MS Method for a Bioequivalence Study in Healthy Indian Subjects

A selective, sensitive, rugged, and high throughput liquid chromatography tandem mass spectrometry method is developed for the determination of one nucleotide tenofovir (TFV) and two nucleosides emtricitabine (FTC) and lamivudine (3TC) reverse transcriptase inhibitors in human plasma. Plasma samples were prepared by solid-phase extraction of the analytes and acyclovir (ACV) as internal standard using Waters Oasis MCX cartridges. The chromatographic separation is achieved in a run-time of 3.0 min on an ACE 5 CN column (150 mm × 4.6 mm, 5 μm) under isocratic conditions. The mobile phase consisted of 0.5% formic acid in water and acetonitrile (55:45, v/v). The protonated precursor --> product ion transitions for TFV, FTC, 3TC, and internal standard were monitored on a triple quadrupole mass spectrometer operating in the multiple reaction monitoring (MRM) and positive ion mode. A linear dynamic range of 4.0-802 ng/mL, 15.0-3006 ng/mL, and 20.1-4023 ng/mL is established for TFV, FTC, and 3TC, respectively, using 0.2 mL plasma sample. The method is fully validated for its sensitivity, selectivity, accuracy and precision, ion suppression, matrix effect, recovery, stability, and dilution integrity. It is successfully applied to a bioequivalence study of [300(TFV) + 200(FTC) + 300(3TC)] mg tablet formulation in 43 healthy human subjects under fasting conditions.

Simultaneous determination of six alkaloids in blood and urine using a hydrophilic interaction liquid chromatography method coupled with electrospray ionization tandem mass spectrometry

A method was developed for the simultaneous determination of six toxic alkaloids (aconitine, hypaconitine, gelsemine, raceanisodamine, strychnine, brucine) in blood and urine by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray tandem mass spectrometry. Ephedrine was selected as the internal standard. Samples were extracted and cleaned up by solid-phase extraction (SPE) using Oasis MCX cartridges. Separation parameters such as organic modifier, buffer pH, and concentration of buffer salt were investigated. Gradient separation and analysis were achieved for six alkaloids on a 3-mum Atlantis HILIC column using a mobile phase consisting of 30 mM ammonium formate and acetonitrile at pH 3. Two multiple reaction monitoring (MRM) transitions for each substance were monitored to provide sufficient identification of alkaloid. The retention mechanisms were explored in the method development. Validation included assessment of linearity, limit of quantification, accuracy, and precision. Bias was less than 15.1% and precision was better than 8.3% for both blood and urine samples. A total of 54 clinical samples were examined by this validated method.

Simultaneous determination of peimine and peiminine in rat plasma by LC‐ESI‐MS employing solid‐phase extraction

A simple and reliable LC-ESI-MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid-phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C(18) column using 10 mM ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)-acetonitrile (85:15, v/v) as mobile phase. The linear range was 1-100 ng/mL for peimine and peiminine. Intra- and inter-day precisiond were less than 10%. Accuracies were within 85-115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method.

SPE and LC-MS/MS determination of 14 illicit drugs in surface waters from the Natural Park of L’Albufera (València, Spain)

A simple and robust method using solid-phase extraction (SPE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of 14 drugs of abuse and their metabolites (cocainics, amphetamine-like compounds, cannabinoids, and opiates) in surface waters has been developed. Seven SPE adsorbents (Oasis HLB, Oasis MCX, Oasis Wax, Supelselect HLB, Strata-X, Strata-XCW), amount of sorbent bed, water volume, and pH were investigated. The highest recoveries, as well as the simplest protocol, were obtained for Oasis HLB cartridges (6 mL/200 mg) using 250 mL of water. The proposed method was linear in a concentration range from 0.03-6 to 300-60,000 ng/L depending on the compound, with correlation coefficients higher than 0.998. Matrix effects have been studied in surface water samples, and several isotope-labeled internal standards have been evaluated as a way to compensate the signal suppression observed. Limits of detection (LODs) and quantification (LOQs) ranged from 0.01 to 1.54 ng/L and from 0.03 to 5.13 ng/L, respectively. Recoveries were 71-102% at the LOQ level and 77-104 at 50 ng/L. The intra-day and intermediate precisions were from 1% to 8% and from 2% to 11%, respectively. The present work reports for the first time the occurrence of drugs of abuse residues in surface water samples from the Natural Park of L'Albufera (Valencia, Spain). Codeine, cocaine, benzoylecgonine, ecgonine methylester, amphetamine, 3,4-methylendioxy methamphetamine, morphine, and methadone were quantified with median values of 11.10, 0.02, 5.59, 0.08, 0.21, 0.75 and 0.14 ng/L respectively, and 11-nor-9-carboxy-Delta9-tetrahydrocannabinol was detected in one sample at levels <LOQ.

Investigation of commercial sorbents for the analysis of opioid peptides in human plasma by on‐line SPE‐CE

In this study, we investigated the performance of several commercial sorbents (Sep-pack) C18, (t)C18, C8 and (t)C2, Oasis HLB, Isolute ENV+, Strata-X and Oasis MCX) for the determination of opioid peptides by solid-phase extraction coupled on-line to capillary electrophoresis (SPE-CE). First, standard solutions were analyzed in order to achieve the lowest LOD and the best electrophoretic separations using UV detection. The best results were obtained using C18, C8 and (t)C2 sorbents, which were examined for the analysis of spiked human plasma samples. A double-step sample clean-up pretreatment, which consisted of precipitation with acetonitrile and filtration, was needed to prevent saturation of the on-line SPE microcartridge. The filtration step was critical to obtain optimum analyte recovery and to clean up the sample matrix. A range of centrifugal filters and filtration conditions were tested and the recoveries of the sample pretreatment were evaluated by CE-ESI-MS. The LODs attained through SPE-CE-UV were approximately ten-fold better with C18 than with C8 and (t)C2. The 0.1 microg/mL LODs achieved by C18-SPE-CE-UV were further improved until we could detect 1 ng/mL concentrations of opioid peptides in plasma samples by C18-SPE-CE-ESI-MS, due to the outstanding selectivity of the MS detection.

Abstract 4697: Liquid chromatography-electrospray ionization tandem mass spectrometry analysis of 7-ethylguanine in human urine

Abstract Considerable evidence in the literature demonstrates the exposure of humans to an unknown ethylating agent. Previous studies have demonstrated the presence of 3-ethyl-Ade in urine, 7-ethylguanine (7-ethyl-Gua) in urine and human liver DNA, 7-ethyldGuo and O4-ethyl-dThd in human lung DNA, and ethylvaline in hemoglobin. Some studies also report higher levels of ethyl adducts in smokers compared to nonsmokers, and there is convincing evidence for an uncharacterized ethylating agent in cigarette smoke. To further investigate this question and evaluate the potential role of ethylating agents in smoking-induced cancer we have developed a liquid chromatography-electrospray ionization tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) method for analysis of 7-ethyl-Gua in human urine. A two-dimensional chromatography approach was used with [15N5]7-ethyl-Gua as the internal standard. Human urine was diluted with 15 mM ammonium acetate and partially purified by a solid-phase extraction cartridge (Strata-X 33 µm, 30 mg/1 mL, Phenomenex) and a mixed mode cation exchange reversed-phase extraction cartridge (Oasis MCX, 30 mg/cartridge, Waters). The fraction from this last purification step containing the analyte (30% MeOH, 5% NH4OH) was dried and redissolved in 80% acetonitrile, 0.1% formic acid and purified by HPLC on a YMC Polyamino II column (150 × 1.0 mm I.D. 5 μm). The fraction containing 7-ethyl-Gua was collected, dried, dissolved in H2O and analysed by LC-ESI-MS/MS-SRM using a Thermo Hypersil Gold PFP column (150 × 0.5 mm I.D. 3 μm). The analysis was carried out using the transition m/z 180 [M + H]+ → m/z 152 [Gua+ H]+ for 7-ethyl-Gua and m/z 185 → m/z 157 for the internal standard. Recovery was about 50%. The detection limit was approximately 10 fmol on column. The peaks attributed to 7-ethyl-Gua and the labeled internal standard had identical retention time and were observed in most of the human urine samples analyzed. The identity of the peak was confirmed by re-injecting the samples after spiking with a standard solution of 7-ethyl-Gua. Twenty-one human urine samples (1 mL) from 11 smokers and 10 nonsmokers were analyzed. 7-Ethyl-Gua was clearly detected in 8 out of 11 samples from smokers and 5 out of 10 samples from nonsmokers. These results strongly indicate that 7-ethyl-Gua is present in human urine and demonstrate that it can be detected and quantified. Further investigation is required to clarify its relationship to cigarette smoking. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4697.