96-well Plate Solid Phase Extraction Research Articles

Towards high throughput analysis using 96-well plate solid phase extraction to determine sedatives and β-blocker residues in food control monitoring

BackgroundVeterinary drugs are widely used in animal production to prevent infections and treat diseases but, this may cause a risk to consumers. Due to the high number of food samples required to monitor yearly, simple, fast, sensitive and selective analytical methods are needed in control laboratories to ensure consumers safety. Nevertheless, many analytical methodologies available in these laboratories include multiple steps and therefore are time-consuming and hinder the analysis throughput requiring significant amounts of solvents and reagents. ResultsThis work developed a 96-well plate solid phase extraction (SPE) method for the extraction of seven sedatives and a β-blocker in animal kidney by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC−MS/MS). The developed method was validated based on Implementing Regulation (EU) 2021/808 in kidney, meat and fish. The performance characteristics of the validation (1–5 μg kg−1) showed good linearity (R2 > 0.998) while decision limits (CCα) were between 1 and 1.2 μg kg−1. Trueness and precision were determined at three levels (n = 7) and the results showed values ranging from 85 to 103 % and from 1 to 9 %, respectively. The feasibility of the method was demonstrated for the residue control requirement established by EU. Method was applied to 201 samples of kidney, meat and fish. SignificanceThis study is the first to present an optimized and validated holistic method for sedatives and β-blocker using 96-well plate SPE and UHPLC−MS/MS. The method showed good performance in kidney, meat and fish samples being an universal method for any species along the same type of sample. The fast, easy, efficient, reliable and universal method showed high throughput and so reduced the analysis time by nine-fold and the required solvent amount by four times fulfilling the green chemistry principals.

Development of a High-Throughput Analytical Method for Antimicrobials in Wastewater Using an Automated Pipetting and Solid-Phase Extraction System.

Antimicrobial resistance (AMR) has emerged and spread globally. Recent studies have also reported the presence of antimicrobials in a wide variety of aquatic environments. Conducting a nationwide monitoring survey of AMR in the environment to elucidate its status and to assess its impact on ecosystems and human health is of social importance. In this study, we developed a novel high-throughput analysis (HTA) system based on a 96-well plate solid-phase extraction (SPE), using automated pipetting and an SPE pre-treatment system. The effectiveness of the system as an HTA for antimicrobials in environmental water was verified by comparing it with a conventional manual analytical system in a domestic hospital over a period of two years and four months. The results of the manual analysis and HTA using a combination of automated pipetting and SPE systems were generally consistent, and no statistically significant difference was observed (p > 0.05) between the two systems. The agreement ratios between the measured concentrations based on the conventional and HTA methods were positively correlated with a correlation coefficient of r = 0.99. These results indicate that HTA, which combines automated pipetting and an SPE pre-treatment system for rapid, high-volume analysis, can be used as an effective approach for understanding the environmental contamination of antimicrobials at multiple sites. To the best of our knowledge, this is the first report to present the accuracy and agreement between concentrations based on a manual analysis and those measured using HTA in hospital wastewater. These findings contribute to a comprehensive understanding of antimicrobials in aquatic environments and assess the ecological and human health risks associated with antimicrobials and antimicrobial-resistant bacteria to maintain the safety of aquatic environments.

A high-throughput small volume matrix based calibration using isotope dilution liquid chromatography tandem mass spectrometry analysis for 42 per and polyfluoroalkyl substances in groundwater

A novel method for the determination of per- and polyfluoroalkyl substances (PFAS) in groundwater is presented using a subsample, matrix-matched calibrators, 96-well plate solid phase extraction (SPE), and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Accuracy, precision, measurement of uncertainty (MOU), method detection limit (MDL), method quantitation limit (MQL), analytical measurement range, interferences/ion suppression, and analyte stability were determined as part of the in-house method validation. The method quantitates 42 PFAS compounds from nine different compound classes. Accuracy for the reference material (RM) and matrix spike (MS) ranged from 52.3 to 117.8 %, and precision for the MS and matrix spike duplicate (MSD) had a coefficient of variation (CV) from 2.0 % to 23.3 %. MDLs spanned from 0.07 to 1.97 ng L−1, with MQLs ranging from 0.20 to 5.90 ng L−1. Suppression studies determined that iron and manganese have effects on analytes that do not have paired isotopically labeled standards. The results from the in-house validation indicated that this Michigan Department of Health and Human Services laboratory developed test meets the necessary accuracy, precision, MDL, MQL and reporting limits requirement established by the laboratory's quality system essentials (QSEs) and select criteria from the Department of Defense (DoD) Quality Systems Manual for Environmental Laboratories and American Industrial Hygiene Association Laboratory Accreditation Program, LLC (AIHA LAP, LLC) accrediting International Standard Organization (ISO/IEC 17025:2017) check list.

Determination of phenoxyacetic herbicides, metabolites of organophosphorus and pyrethroid pesticides in human urine using solid phase extraction coupled with ultra-performance liquid chromatography-tandem mass spectrometry

Pesticides are widely used in most agricultural areas to protect food crops but adversely affect ecosystems and human beings. Pesticides have attracted great public concern due to their toxic properties and ubiquitous occurrence in the environment. China is one of the largest users and producers of pesticides globally. However, limited data are available on pesticide exposure in humans, which warrants a method for quantification of pesticides in human samples. In the present study, we validated and developed a comprehensive and sensitive method for the quantification of two phenoxyacetic herbicides, two metabolites of organophosphorus pesticides and four metabolites of pyrethroid pesticides in human urine using 96-well plate solid phase extraction (SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). For this purpose, a systematic optimization of the chromatographic separation conditions and MS/MS parameters was conducted. Six solvents were optimized for the extraction and clean-up of human urine samples. The targeted compounds in the human urine samples were well separated within 16 min in one analytical run. A 1 mL aliquot of human urine sample was mixed with 0.5 mL sodium acetate buffer (0.2 mol/L) and hydrolyzed by β-glucuronidase enzyme at 37 ℃ overnight. The eight targeted analytes were extracted and cleaned using an Oasis HLB 96-well solid phase plate and eluted with methanol. The separation of the eight target analytes was performed on a UPLC Acquity BEH C18 column (150 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% (v/v) acetic acid in acetonitrile and 0.1% (v/v) acetic acid in water. The analytes were identified using the multiple reaction monitoring (MRM) mode under negative electrospray ionization (ESI-) and quantified by isotope-labelled analogs. Para-nitrophenol (PNP), 3,5,6-tricholor-2-pyridinol (TCPY) and cis-dichlorovinyl-dimethylcyclopropane carboxylic acid (cis-DCCA) exhibited good linearities ranging from 0.2 to 100 μg/L, and 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxy benzoic acid (4F-3PBA), 2,4-dicholorphenoxyacetic acid (2,4-D), trans-dichlorovinyl-dimethylcyclopropane carboxylic acid (trans-DCCA) and 2,4,5-tricholorphenoxyacetic acid (2,4,5-T) showed linearity ranging from 0.1 to 100 μg/L with correlation coefficients all above 0.9993. Method detection limits (MDLs) and method quantification limits (MQLs) of targeted compounds were in the range of 0.02 to 0.07 μg/L and 0.08 to 0.2 μg/L, respectively. The spiked recoveries of target compounds at three levels of 0.5, 5 and 40 μg/L were 91.1% to 110.5%. The inter- and intra-day precisions of targeted analytes were 2.9% to 7.8% and 6.2% to 10%, respectively. This method was applied to the analysis of 214 human urine samples across China. The results showed that all the targeted analytes, except 2,4,5-T, were detected in human urine. The detection rates of TCPY, PNP, 3-PBA, 4F-3PBA, trans-DCCA, cis-DCCA, and 2,4-D were 98.1%, 99.1%, 94.4%, 2.80%, 99.1%, 63.1% and 94.4%, respectively. The median concentration of targeted analytes in a decreasing order were: 2.0 μg/L (TCPY), 1.8 μg/L (PNP), 0.99 μg/L (trans-DCCA), 0.81 μg/L (3-PBA), 0.44 μg/L (cis-DCCA), 0.35 μg/L (2,4-D) and below MDLs (4F-3PBA ). For the first time, we developed a method to extract and purify specific biomarkers of pesticides from human samples based on offline 96-well SPE. This method has the advantages of simple operation, high sensitivity, and high accuracy. Moreover, up to 96 human urine samples were analyzed in one batch. It is suitable for the determination of eight specific pesticides and their metabolites in large sample sizes.

Application of 96-well plate SPE method for analysis of persistent organic pollutants in low volume blood serum samples

Though many persistent organic pollutants (POPs) are closely regulated the human population is still exposed to these ubiquitous chemicals from the environment and diet. Safe management and human biomonitoring of POPs is necessary to understand the risk of exposure. Within human biomonitoring the mass of sample is often limited, therefore robust methods using smaller sample amounts are necessary. This study developed a 96-well plate solid phase extraction (SPE) method for determination of selected POPs: polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) and non-persistent novel flame retardants (NFRs) in low volume blood serum. Non-destructive clean-up coupling Oasis HLB extraction plate with Phree phospholipid removal plate was employed. Extraction efficiency was determined at low and high concentrations in certified reference materials NIST SRM 1957 and 1958, respectively. Target compounds deviated from certified values on average by 15% and 21% for SRM 1957 and SRM 1958, respectively. Observed limit of detections (LODs) ranged from 0.36 pg/mL (PCB 180) to 66.07 pg/mL (δ-HCH). The applicability for real samples is demonstrated on 48 samples from pregnant women enrolled in the pilot phase of the CELSPAC: TNG study. In total, 30 target compounds were detected in at least one sample. The method developed here provides a fast and reliable analysis of human blood serum with possibility to introduce automation for the sample preparation procedure.

Determination of eight environmental phenols in human urine samples by high-throughput solid-phase extraction-ultra-performance liquid chromatography-tandem mass spectrometry

A method combining 96-well plate solid-phase extraction with ultra-performance liquid chromatography-tandem mass spectrometry (96-well SPE LC-MS/MS) was developed for the simultaneous determination of eight environmental phenols in urine samples. The samples included seven bisphenol compounds and triclosan. The urine samples were thawed to room temperature, and the target analytes were deconjugated by β -glucuronidase/aryl-sulfatase in ammonium acetate buffer solution at 37℃ overnight. Then, the effects of three kinds of 96-well solid-phase extraction plates and different elution conditions on the purification of the urine samples and the environmental phenol recoveries were compared. The best purification effect was achieved on Oasis HLB 96-well plate (60 mg) solid phase extraction, using 30% (v/v) acetonitrile aqueous solution as the rinse solution. The target analytes were then eluted by methanol solution and evaporated to dryness using a nitrogen blower. After reconstruction with 0.5 mL methanol/water (1:1, v/v) solution, the target compounds were detected by UPLC-MS/MS. To achieve better chromatographic separation, two kinds of analytical columns (C18 and T3) and different types of mobile phases (methanol and acetonitrile as the organic phase) were also compared. The best chromatographic effect was achieved when the treated samples were separated on a C18 column (100 mm×2.1 mm, 1.7 μm) using acetonitrile/water as the mobile phase at a flow rate of 0.3 mL/min. Mass spectra were recorded by negative electrospray ionization under the multiple reaction monitoring (MRM) mode. The sample matrix effect was also evaluated. The absolute matrix effects of bisphenol A, bisphenol F, bisphenol S, bisphenol B, and bisphenol AF were in the range of 3.47% to 15.32%. Since the above mentioned matrix effect was weak, there was no need for compensation measures. On the contrary, tetrachlorobisphenol A, tetrabromobisphenol A, and triclosan showed an absolute matrix effect of 49.58% (moderate), 71.99% (strong), and 86.93% (strong), thus necessitating compensation measures. Therefore, this strategy uses a one-to-one corresponding isotope internal standard method to offset the matrix effect. Six different urine samples were used to evaluate the relative matrix effect. The relative standard deviations (RSDs) of the eight corresponding internal standard peak areas were 3.63%-9.06%, indicating that the relative matrix effect was stable. Under the optimized conditions, linearity ranges were 0.50-50 μg/L for bisphenol A and bisphenol AF; 0.05-50 μg/L for tetrachlorobisphenol A and bisphenol S; 0.01-50 μg/L for bisphenol F and tetrabromobisphenol A; 1.00-50 μg/L for bisphenol B; and 5.00-200 μg/L for triclosan. The correlation coefficients were all greater than 0.9995. At spiked levels of 2.5, 5, and 25 μg/L, the average recovery ratios of the eight target analytes were 81.01%-118.84%, while the intra-day and inter-day precisions were 0.38%-19.41% and 2.54%-17.83%, respectively. The limits of detection (LOD) were 0.002-1.09 μg/L, and the limits of quantitation (LOQ) were 0.007-3.63 μg/L. This method was successfully applied to the determination of the eight environmental phenols in 64 urine samples collected from Beijing area between 2019 and 2020. All the target environmental phenols were detected, except for bisphenol B and bisphenol AF. Bisphenol A and bisphenol S showed the highest detection rates of 100% and 96.9%, respectively. The detection rates of triclosan, tetrabromobisphenol A, tetrachlorobisphenol A, and bisphenol F were 57.8%, 46.9%, 23.4%, and 21.9%, respectively. The medium values of urinary concentration followed the order 1.44 μg/L(triclosan), 0.69 μg/L(bisphenol A), 0.086 μg/L (bisphenol S), 0.0032 μg/L (tetrabromobisphenol A), 0.00050 μg/L (tetrachlorobisphenol A), 0.00 μg/L (bisphenol F, bisphenol B, and bisphenol AF). The aforementioned results imply that the widespread environmental phenolic exposure of Beijing residents is worthy of attention. Compared with traditional solid-phase extraction methods, the method reported in this paper is time-saving, effective, and suitable for the simultaneous analysis of large quantities of samples; moreover, the small sample and organic solvent consumption make this method more environment- and operator-friendly.

QuEChERS and 96-well plate solid phase extraction for determination of vancomycin and norvancomycin in fish meat by UPLC–MS/MS

Vancomycin and norvancomycin are glycopeptide antibiotics for gram-positive bacteria infection, but indiscriminately used in aquaculture. In this study, a QuEChERS (quick, easy, cheap, effective, rugged, and safe)/96-well solid-phase extraction (SPE) plate method was used to extract vancomycin and norvancomycin in fish meat samples, and the drugs were further analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). The parameters, such as the sorbent of cation exchange resin, the proportion of acetonitrile (15%) in extractant, the mobile phase of water (0.1% formic acid)/acetonitrile, were optimized. The method was validated in terms of linearity (0.9990–0.9994), LOD (0.51 μg·kg−1), LOQ (1.73 μg·kg−1), intra-dayprecision (<5.19%), inter-day precision (<6.30%), and recovery (86.7–98.6%). Finally, the method was successfully applied to contaminated and randomly collected samples. The results indicated that the proposed method meet the daily monitoring requirements for vancomycin and norvancomycin.

Sodium 4-styrenesulfonate functionalized nanofibers mat as 96-well plate solid–phase extraction adsorbent for quantitative determination of multiple β-agonists residues in pork samples

In this work, sodium 4-styrenesulfonate functionalized polyacrylonitrile nanofibers mat (SS/PAN NFM) was firstly prepared and applied as 96-well plate solid–phase extraction adsorbent for quantitative determination of seven β-agonists residues in pork samples. The functional modification endowed the SS/PAN NFM with superior adsorption performance for target β-agonists. The adsorption process is spontaneous (ΔG < 0), the initial adsorption rate can reach 6.03–9.09 mg/g/min and the maximum adsorption capacity is calculated to be 48.3 mg/g at 298 K. Moreover, SS/PAN NFM can be reused for 12 times without degradation in adsorption capability. Combined with UPLC–MS/MS, the limits of detection can reach 0.006–0.24 μg/kg, the recoveries ranged from 87.2% to 111% and the relative standard deviations of intra-day and inter-day precisions were in the scope of 1.75%–11.6% and 5.08%–13.5%, respectively. The obtained results fully demonstrated the practicability of this method in preventing the hazard of β-agonists residues.

High-throughput 96-well solid-phase extraction for preparation of tetracycline followed by liquid chromatography and mass spectrometry analysis.

Tetracyclines abuse has frequently occurred in aquaculture against bacteria, rickettsiae, spirochetes, and mycoplasmas. In this study, a high-throughput sample preparation method was developed using 96-well plate solid-phase extraction (p-SPE) and the extract was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The experimental conditions were optimized such that the pH is 4, the eluting solvent is methanol (2 mL), and the sorbent is hydrophilic-lipophilic balance (HLB) microsphere. The whole protocol was validated, and it showed that the tetracyclines were linear with correlation coefficients ≥ 0.9990, precision and accuracy (RSD%) in 3.9-6.1%, and mean recoveries of 88.6-103.6%. To exhibit the potential of 96-well p-SPE as a routine tool for inspection and quarantine, fresh aquatic samples were tested, and among which positive samples were observed. This method was demonstrated to be promising for the purification and enrichment of tetracyclines with reduced time and labor, and indeed practically and particularly suitable for widespread tetracyclines analysis.

Longitudinal changes in persistent organic pollutants (POPs) from 2001 to 2009 in a sample of elderly Swedish men and women

BackgroundProspective cohort studies evaluating the temporal trends of background-level persistent organic pollutants (POPs) and their potential negative health effects in humans are needed. ObjectiveThe objectives of this study are to examine the five year longitudinal trend in chlorinated and brominated (Cl/Br) POP concentrations in a sample of elderly individuals and to investigate the relationship between gender, changes in body weight, plasma lipid levels and POP concentrations. MethodsIn the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study, plasma samples were collected from the same individuals over a 5 year period. Originally 992 subjects (all aged 70) were sampled between 2001 and 2004 and 814 returning subjects (all aged 75) were sampled again from 2006 to 2009. Plasma concentrations of 16 polychlorinated biphenyls (PCBs), 5 organochlorine pesticides (OCPs), octachlorinated dibenzo-p-dioxin (OCDD), and one polybrominated diphenylether (BDE 47) were determined using high-throughput 96-well plate solid phase extraction and gas chromatography-high resolution mass spectrometry (GC-HRMS). ResultsDuring the 5-year follow-up, plasma concentrations of all POPs significantly decreased (p < 0.00001). Median reductions ranged from 4% (PCB105) to 45% (PCB 99), with most reductions being in the 30–40% range. For most POPs, a larger decline was seen in men than in women. The relationship between the weight change and change in POP concentrations was generally negative, but a positive relationship between lipid levels and POP concentrations when expressed as wet-weight was observed. In general, similar changes in POP concentrations and their relationships to body weight were observed regardless of using either wet-weight (pg/mL) or lipid-normalized (ng/g lipid) concentrations. ConclusionIn this longitudinal cohort study, gender and minor, but varying changes in body weight and lipid levels greatly influenced the individual-based changes in POP concentrations. In general, our findings suggest that men and women with larger decreases in body weight and greater increases in lipid levels have the slowest decline in body burden of POPs. Based on the results from this study, either wet-weight or lipid normalized concentrations can be used to determine the percent change in POP concentrations and their relationships to physiological changes and differences.

A method for analysis of marker persistent organic pollutants in low-volume plasma and serum samples using 96-well plate solid phase extraction

The objective of this study was to develop and validate a 96-well plate solid phase extraction method for analysis of 23 lipophilic persistent organic pollutants (POPs) in low-volume plasma and serum samples which is applicable for biomonitoring and epidemiological studies. The analysis of selected markers for internal exposure: 16 polychlorinated biphenyls (PCBs), 5 organochlorine pesticides (OCPs), octachlorinated dibenzo-p-dioxin (OCDD), and polybrominated diphenylether 47 (BDE 47) was evaluated by comparing two SPE sorbents and GC-HRMS or GC–MS/MS detection. The final method extracted 23 POPs from 150 μL of serum and plasma using a 96-well extraction plate containing 60 mg Oasis HLB sorbent per well prior to GC-HRMS magnetic sector analysis. The extraction method was applied to 40 plasma samples collected for an epidemiological study. The recovery of selected POPs ranged from 31% to 63% (n = 48), and detection limits ranged from 2.2 to 45 pg/mL for PCBs, 4.2 to 167 pg/mL for OCPs, 7.8 pg/mL for OCDD and 6.1 pg/mL for BDE 47. This method showed good precision with relative standard deviations of selected POP concentrations in quality control samples (n = 48) ranging from 11% to 25%. The trueness was determined with standard reference material serum (n = 48) and the deviation from certified values ranged from 1 to 27%. Of the 23 POPs analyzed, 18 were detected in 43% to 100% of plasma samples collected for the epidemiological study. The method showed good robustness with low inter-well plate variation (11–31%) determined by twelve 96-well plate extractions, and can extract 96 samples, including quality controls and procedural blanks in 2–3 days. Comparison with GC–MS/MS analysis showed that similar concentrations (within 0.5% to 30%) of most POPs could be obtained with GC-APCI-MS/MS. Larger deviations were observed for PCB 194 (60%) and trans-nonachlor (43%). The developed method produces accurate concentrations of low-level marker POPs in plasma and serum, providing a suitable high-throughput sample preparation procedure for biomonitoring and epidemiological studies involving large sample size and limited sample volume. GC-HRMS was chosen over GC–MS/MS, however the latter showed promising results, and could be used as an alternative to GC-HRMS analysis for most POPs.

Quantitation of aldosterone in human plasma by ultra high performance liquid chromatography tandem mass spectrometry

Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC–MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis® HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters® Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0>189.1 for aldosterone and 366.0>194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r2=0.9999). Mean intra- and inter-assay imprecision and bias were all <10%. Comparison of the UPLC–MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC–MS/MS=0.789(RIA)–41.7, linear regression r2=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.

Determination of 1,25-dihydroxyvitamin D2 in rat serum using liquid chromatography with tandem mass spectrometry

Vitamin D therapy is widely used for the treatment of hyperparathyroidism associated with chronic renal failure in renal disease patients. The vitamin D prodrug, 1α-hydroxyvitamin D2 (1α(OH)D2), is used for the treatment of the end stage renal disease patients who as a result of impaired kidney function cannot convert the naturally occurring vitamin D to the active hormonal form namely 1,25-dihydroxyvitamin D2 (1,25(OH)2D2). The systemic circulating levels of this active form are in the pg/mL range and represent a significant bioanalytical challenge for therapeutic monitoring. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) is considered the gold standard for the selective and sensitive determination of small molecule therapeutics in biological matrices. However, the reported LC–MS/MS bioanalytical assays for 1,25(OH)2D2 suffer from extensive sample preparation procedures or derivatization protocols to achieve the requisite sensitivity and selectivity. In this paper, we describe an assay that employs 96-well plate solid phase extraction sample preparation combined with highly sensitive LC–MS/MS instrumentation. The utility of ultra high pressure liquid chromatography to reduce the analytical run time was also demonstrated. Employing this assay a lower limit of quantitation of 25.0pg/mL using 300μL sample aliquot of rat serum was achieved with linearity obtained over the range of 25.0–1000pg/mL. Both intra-day and inter-day coefficients of variation were <15% and accuracy across the assay range was within 100±7.24%. The application of the assay was demonstrated for the analysis of 1,25(OH)2D2 rat serum samples to support pharmacokinetic studies conducted at doses down to sub-microgram per kilogram of 1α(OH)D2.

Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography–tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04–1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.

Epicatechin, procyanidins, and phenolic microbial metabolites after cocoa intake in humans and rats

Proanthocyanidins, flavonoids exhibiting cardiovascular protection, constitute a major fraction of the flavonoid ingested in the human diet. Although they are poorly absorbed, they are metabolized by the intestinal microbiota into various phenolic acids. An analytical method, based on an optimized 96-well plate solid-phase extraction (SPE) procedure and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of 19 phenolic microbial metabolites and monomeric and dimeric flavanols in urine samples, was developed and validated. Human urine samples were obtained before and after ingestion of an acute consumption of 40 g of soluble cocoa powder and rat urines before and after the prolonged administration (2 weeks) of different diets composed of natural cocoa powder. The mean recovery of analytes using the new SPE-LC-MS/MS method ranged from 87% to 109%. Accuracy ranged from 87.5% to 113.8%, and precision met acceptance criteria (<15% relative standard deviation). Procyanidin B2 has been detected and quantified for the first time in human and rat urine after cocoa consumption. Changes in human and rat urinary levels of microbial phenolic acids and flavanols were in the range of 0.001-59.43 nmol/mg creatinine and of 0.004-181.56 nmol/mg creatinine, respectively. Major advantages of the method developed include reduction of laboratory work in the sample preparation step by the use of 96-well SPE plates and the sensitive measurement of a large number of metabolites in a very short run time, which makes it ideal for use in epidemiological studies.

Development and validation of a method for fipronil residue determination in ovine plasma using 96-well plate solid-phase extraction and gas chromatography–tandem mass spectrometry

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, undergoes a reinforcement of the regulation within the European Union (2007/52/EC directive) due to its potential effects on environment and human health. In order to assess the plasmatic concentrations of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide) in ovine, a methodology based on gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was developed and validated according to the European standard (2002/657/EC). The proposed method allows a large number of samples to be treated concurrently ( n = 80) using a reduced sample amounts (0.2 mL), and consents to reach a level of quantification of 0.1 pg μL −1. The sample preparation consisted of a single solid-phase extraction (SPE) purification on a 96-well plate filled with a styrene-divinyl-benzene phase. Linearity was demonstrated all along the investigated range of concentrations, i.e. from 0.25 to 2000 pg μL −1, with coefficient of determination ( R 2) from 0.977 to 0.994, depending on target analytes. Calculated decision limit (CCα) and detection capability (CCβ) for fipronil, sulfone and sulphide were in the range 0.05–0.16 and 0.28–0.73 pg μL −1 respectively.

Automated Solutions for Total RNA Isolation from Diverse Sample Types

In response to the increasing demands to generate larger amounts of quality data faster, specifically in the area of RNA isolation to support gene expression assays, we have adopted several automated solutions for isolating total RNA from a variety of sample types, e.g., blood, cells, and tissue. For isolation from blood, collected in PAXgene (PreAnalytiX, Hombrechtickon, CH) tubes, we use a Qiagen BioRobot Universal System (Qiagen Inc-USA, Valencia, CA). For total RNA from cells, we have adapted a 96-well plate solid-phase extraction method using Promega SV96 (Promega, Inc., Madison, WI) reagents. Finally, if starting from tissues, we use the AutoGenPrep 245 (AutoGen Inc, Holliston, MA) robot to perform a modified acid guanidine phenol chloroform isolation. The use of the AutoGenPrep 245 is preceded by tissue homogenization, usually performed on the Qiagen TissueLyser. Following total RNA isolation the RNA is quantified by QuantiT (Invitrogen Corporation, Carlsbad, CA) using a Biomek FX method that performs whole plate serial dilutions, and then creates a reaction plate with samples in duplicate. Finally, to check for genomic DNA contamination in the total RNA sample, a minus reverse transcription PCR assay is performed. In using these methods, we are able to increase our throughput without compromising data integrity.

Liquid chromatography–mass spectrometric analysis of buprenorphine and its N-dealkylated metabolite norbuprenorphine in rat brain tissue and plasma

Introduction A specific, accurate, and reproducible liquid chromatography–mass spectrometric (LC/MS) method was developed and validated that allows simultaneous measurement of the centrally acting analgesic buprenorphine and its major metabolite, norbuprenorphine, in rat brain and plasma samples. Methods A 96-well plate solid phase extraction (SPE) procedure was developed for buprenorphine and norbuprenorphine using mixed-mode cation-exchange reversed-phase sorbent. An LC method using a C8 column with isocratic mobile phase (80:20 water/acetonitrile with 20 mM ammonium acetate and 0.1% acetic acid) was developed for reproducible and selective separation. A quadrupole mass spectrometer with atmospheric electrospray ionization source under positive ion mode was used for detection. d 4-Buprenorphine and d 3-norbuprenorphine were used as internal standards. Results The calibration curves for buprenorphine and norbuprenorphine in plasma and brain tissue were linear within the range of 7 to 8333 ng/ml (plasma) and 5 to 5000 ng/g (brain). The lower limit of quantification for both buprenorphine and norbuprenorphine from brain tissue was 5 ng/g, and from plasma was 7 ng/ml. Assay accuracy and precision of back-calculated standards were within ± 15%. Discussion This method will be useful for investigation of buprenorphine's mechanism of action and clinical profile.

The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography–micro-electrospray ionization–tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent

An in vitro reactive intermediate screening assay, incorporating the use of the close analog of glutathione, glutathione ethyl ester (GSH-EE) as a conjugating agent, was developed to identify compounds that form reactive intermediates in an in vitro metabolite generating system. The biological assay consisted of substrate [s] = 10 μM, human liver microsomes, an NADPH generating system and glutathione ethyl ester. Conjugates were extracted from the biological matrix using a combination of protein precipitation and a semi-automated 96-well plate solid phase extraction (SPE) procedure. A micro-bore liquid chromatography–micro-electrospray ionization–tandem mass spectrometry (μLC–μESI–MS/MS) method detected glutathione ethyl ester conjugates using selected reaction monitoring (SRM) to simultaneously monitor for multiple MH + to [MH − 129] + transitions, where the 129 mass unit (Da) represents the neutral loss of the pyroglutamate moiety from GSH-EE. The multiple MH + to [MH − 129] + transitions (SRM mass table) were generated for potential reactive intermediates of each compound. Glutathione (GSH) and GSH-EE conjugate standards were used to evaluate MS detection sensitivity. Based on direct comparison of standard curve data, an approximate 10-fold increase in sensitivity was observed for conjugates containing GSH-EE moiety versus GSH. In vitro experiments were conducted using literature substrates acetaminophen, rosiglitazone, clozapine, diclofenac and either GSH-EE or GSH as a reactive intermediate conjugating agent. An increase in detection sensitivity was observed for each GSH-EE conjugate and in the case of acetaminophen–GSH-EE the peak area increase was approximately 80-fold. Twelve drug compounds, each having known biotransformation mechanisms, were used to further test the detection capabilities of the assay and establish a concordance to literature data. When GSH was used in the assay, conjugates were detected for 4 out of the 12 test compounds (33%). When GSH-EE was used in the assay, conjugates were detected for 10 out of the 12 test compounds (83%).

Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography–electrospray ionization mass spectrometry

There is considerable interest in quantifying morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). Available assays use gas chromatography–mass spectrometry or high-performance liquid chromatography (HPLC) with single or tandem mass spectrometry, ultraviolet, electrochemical, or fluorimetric detection. Nevertheless, few methods provide adequate sensitivity for all analytes, in a single injection, with the desired rate of sample throughput. A rapid and sensitive method for quantification of morphine, M3G and M6G from human plasma using HPLC with electrospray ionization mass spectrometry was developed using a Waters Oasis MCX 96-well plate for extracting both lipophilic morphine and its hydrophilic glucuronides, C18 separation using an isocratic mobile phase (methanol, acetonitrile and formic acid), and selected ion monitoring. Recoveries of morphine, M3G and M6G, respectively, were 81, 90 and 82% at the low (2, 25 and 2 ng/ml), 80, 77 and 75% at the medium (10, 250 and 10 ng/ml), and 74, 62 and 72% at the high (100, 1000 and 100 ng/ml) quality control samples. The limit of quantitation was 0.5 ng/ml morphine and M6G, and 5 ng/ml M3G. Analytes were validated over a linear range of 0.5–200 ng/ml morphine and M6G, and 5–2000 ng/ml M3G. This assay represents an improvement over existing methods through solid phase extraction with increased sample throughput (96-well plates), use of small samples (0.5 ml), and sub-nanogram detection.