Lab-made automated parallel-dispersive pipette extraction device for the determination of polycyclic aromatic hydrocarbons in distilled beverages (sugarcane spirits) using HPLC-DAD.

This work describes the development of a new automated parallel dispersive tip microextraction method (Au-Pa-DPX) for the determination of eleven polycyclic aromatic hydrocarbons (PAHs) in four samples of Brazilian sugarcane spirit beverages, with separation and detection done by the HPLC-DAD. The results obtained with the Au-Pa-DPX approach were also compared with those obtained via the conventional parallel manual DPX method with the same samples and optimized extraction process. Desorption solvent and cycles of desorption, cleaning and extraction were optimized using response surface methodology and univariate approaches. For the Au-Pa-DPX method, the coefficient of determination (R2) ranged from 0.9948 to 0.9997. The limits of detection and quantification were all 0.303 µg l-1 and 1.00 µg l-1, respectively. Interday and intraday precision ranged from 7.6 % to 31.7 % and 0.40 % to 15.8 %, respectively. For the manual parallel DPX method, the interday and intraday precision ranged from 8.2 % to 38.1 % and 5.40 % to 18.7 %, respectively. The relative recovery values obtained with the proposed method ranged from 53.29 to 124.94 %. The enrichment factors ranged from 15.13 to 22.35. The sum of PAH concentrations in the four samples ranged from undetected to 25.58 µg l-1. These results, when correlated to other methods, highlight the gains in regards to precision obtained with the automated apparatus. Furthermore, when compared to other methods from the literature, it is an interesting green alternative for the determination of these analytes and this sample, with high throughput (4.67 min per sample), low consumption of solvents and samples, generating less waste and reducing health risks to the analyst.

Innovative disposable pipette extraction for concurrent analysis of fourteen psychoactive substances in drug users' sweat.

Widespread consumption of drugs of abuse worldwide has caused concern: it adversely affects public health, individual safety, and social structures. Experts are particularly alarmed because new psychoactive substances have been increasingly detected in biological samples. In recent years, several studies have focused on developing methods to identify psychoactive substances in alternative biological matrices, such as sweat. This approach holds promise for monitoring substance use, especially in individuals undergoing rehabilitation. Among the commonly employed analytical procedures, extraction using disposable DPX tips stands out as a novel, miniaturized, and promising technique. This study aimed to validate and to apply a method to analyze various substances, including amphetamine, methamphetamine, MDMA, MDA, MDEA, cocaine, cocaethylene, anhydroecgonine methyl ester, dibutylone, N-ethylpentylone, 25E-NBOMe, 25CNBOMe, 2CC, 2C-E, fentanyl, and carfentanil, in sweat samples simultaneously. In this method, sweat is collected by using laboratory-developed patches, and extraction is conducted with DPX-SCX tips. Gas chromatography coupled to mass spectrometry is employed to separate, to identify, and to quantify the analytes. Validation results indicated that the quantification limit ranged from 2 to 30 ng of analyte/patch, and that the method was linear for analyte concentrations ranging from 2 to 1100 ng/patch. The validated method was applied to analyze 30 sweat samples collected from volunteers drug users and processed by using both the selected ion mode (SIM) and full scan. The method was able to detect and to quantify substances such as cocaine, cocaethylene, anhydroecgonine methyl ester, MDMA, MDA, nicotine, cotinine, caffeine, procaine, lidocaine, and ethylamphetamine simultaneously. The recovery rates ranged from 72.4 % to 97.1 %. The analytes were stable in the biological matrix. In conclusion, the validated method proved effective and allowed the target analytes to be quantified in sweat samples, highlighting that sweat is a viable matrix for analyzing drugs of abuse.

Determination of selected herbicides in sugarcane-derived foods by graphene-oxide based disposable pipette extraction followed by liquid chromatography-tandem mass spectrometry.

Sugarcane is widely cultivated in Brazil. Although there are Maximum Residue Limits of pesticides determined for this plant, there is no legislation covering alimentary products from sugarcane. In this study, Disposable Pipette Tip Extraction (DPX) technique was evaluated as a sample preparation technique for simultaneous determination of eleven herbicides followed by LC-MS/MS analysis in three sugarcane-derived food matrices: juice, candy, and syrup. First, graphene oxide anchored to silica functionalized with octadecyl silane and endcapped was synthesized, which was evaluated as a sorbent in DPX. Then, after evaluating the parameters involved in DPX extraction, the method was validated following the ICH guide. As a result, the method showed acceptable linearity (r ≥ 0.99), limits of quantification (1.0 - 5.0 ng mL-1 for juice and 5.0 - 25.0 ng g - 1 for candy and syrup, varying according to the pesticide), precision, and accuracy within the limits of the literature, and recoveries ranging from 48 - 69% (juice), 34 - 89% (candy), and 28 - 76% (syrup). Finally, the developed method was successfully applied in actual samples of the three studied matrices.

Determination of Cyclaniliprole in Fruits and Vegetables Using Disposable Pipette Extraction Cleanup and Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Despite an outstanding agent for control of Lepidoptera, the diamide insecticide cyclaniliprole (CYCP) is a suspected carcinogen. In the present study, an analytical method was developed for the determination of CYCP in six fruits and vegetables (apple, grape, peach, bell pepper, lettuce, and tomato) using ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was carried out by the acetonitrile-salting-out extraction followed by simple and fast cleanup of disposable pipette extraction tip containing styrene divinyl benzene and/or graphitized carbon black. Satisfactory linearity (r > 0.99) was obtained in the calibration range of 0.001−1 µg mL−1. Matrix effects decreased from −9.9−−17.9% to −1.0−−7.6% after the cleanup. The recoveries of CYCP at three spike levels (0.01, 0.1, and 1 mg kg−1) from different matrices were between 75.7% and 111.5%, with the intra-day (n = 5) and inter-day (n = 15) relative standard deviations lower than 12.1%. The limit of quantification was 0.01 mg kg−1. The developed method provides a good reference for routine monitoring of CYCP in these fruits and vegetables.

Bioaccumulation of organochlorine pesticides in the parasite Cosmocerca sp. (Nematoda: Cosmocercidae) and the amphibian host Amietophrynus regularis (Reuss, 1833) within Lagos metropolis, Nigeria.

BACKGROUND: Amphibian species are being threatened worldwide and chemical pollution is one of the leading causes of this decline. The use of agrochemicals such as organochlorine pesticides (OCPs) among the several health and ecological challenges it causes, the sharp amphibian population decline is most pressing.Toad specimens Amietophrynus regularis were sampled from three (3) selected areas; each comprising of natural habitat and dumpsites within Lagos metropolis. METHODS: The congeners of organochlorine pesticides were tested in the liver, intestine, and parasite (Cosmocerca sp.) of the toads and soil samples from the respective locations using gas chromatography-mass spectrometer (GC-MS). Histopathological analyses were conducted on the intestines and liver of the toads using hematoxylin and eosin (H&E) stain and then examined under the binocular dissecting microscope. RESULTS: The concentration of aldrin in the intestine of A. regularis sampled at the dumpsites was higher than the concentrations in the intestines of A. regularis in the natural habitat. The concentrations of dieldrin in the uninfected A. regularis at both dumpsite and natural habitat were higher than the concentrations in the infected A. regularis at both environments. This indicated that the parasite Cosmocerca sp. may have played a depurative role in sequestering the concentration of dieldrin in the toads irrespective of the location. The parasites exhibited marked sequestration capacity characterized by the notably high total bioaccumulation rate both in the liver and the intestine at the dumpsite. The stunted villi being the common histological alteration in the infected and uninfected toads at the dumpsite but missing in the uninfected counterparts at the natural habitat may be attributed to the differences in the background concentration of the OCP congeners. CONCLUSIONS: The parasite- Cosmocerca sp. has been shown to be a potential tool in the biomonitoring of these OCP congeners which persists in the environment. Continuous research on these congeners is a searchlight to checkmate the environment to see how compliant industries and the consumers are in terms of regulation of these chemicals.

Disposable pipette extraction: A critical review of concepts, applications, and directions.

Analytical chemistry has always been evolving towards techniques that are increasingly simple and effective and that conform to green principles. Disposable pipette extraction (DPX, also known as dispersive pipette tip solid phase extraction) is a recent technique that has become an interesting tool in sample preparation methodologies. The principle is based on a dynamic mixture between the matrix and the sorbent which allows rapid and effective extraction of analytes and provides vigorous clean-up of the samples. In the context of fitting in with green chemistry, DPX has contributed to replacing commercially available materials with natural alternative materials. The production of these materials is also simple, reduces sample/solvent volumes, consequently generates less waste and is less laborious and safer for the laboratory worker. This review is a source of information about the DPX technique, dealing with its basic concepts, procedure, optimizations, materials for the main applications published so far, which are in the food, environmental and biological (forensic) sciences.

A Fluorescence Dequenching-based Liposome Leakage Assay to Measure Membrane Permeabilization by Pore-forming Proteins.

Pore-forming toxins (PFTs) have been discovered in a wide range of organisms. Their functions are essential to the survival or virulence of many species. PFTs often interact with lipid membranes. Large unilamellar vesicles (LUV), also known as liposomes, have been commonly used as reliable membrane models for testing PFTs activity. Liposomes have great adaptability in size, lipid composition, and loading cargo. Incorporating the fluorescent dye/quencher pair, 8-Aminonaphthalene-1,3,6-Trisulfonic Acid (ANTS) and p-Xylene-Bis-Pyridinium Bromide (DPX), in liposomes is an effective approach for measuring membrane leakage. When ANTS and DPX are encapsulated in a liposome, the fluorescence of ANTS is quenched by DPX. However, disruption of liposome integrity and subsequent leakage result in measurable fluorescence emitted by ANTS. Here, we report our protocol for optimal liposome preparation for measuring liposome leakage by fluorescence dequenching.

Dispersive Solid-Phase Extraction of Multiresidue Pesticides in Food and Water Samples.

Pesticides has become an essential part of our life and have entered and bioaccumulated in water, air, soil ecosystem, and food. However, the majority of the pesticides are not biodegradable and eco-friendly, and the accumulation of them in food and the ecosystem could constitute a serious risk to human and environmental health. It is critical to understand pesticides' identities and level of residues present in environment and food. Robust analytical techniques that offer easy, fast, and reliable extraction of multiresidue pesticides in water, soil and food with matrix interference-free quantification are necessary for proper risk assessment. Although various methods have been reported for pesticides extraction in food and environment samples, dispersive solid-phase extraction (d-SPE) has become the most popular sample preparation method for pesticides analysis today. Multiresidue pesticides extraction in food and environmental sample using a novel d-SPE method, dispersive pipette extraction (DPX), is described step-by-step in this chapter.

Sub-chronic toxicity of ethanol leaf extract of Syzygium guineense on the biochemical parameters and histopathology of liver and kidney in the rats.

BACKGROUND: Syzygium guineense Wall. leaf is being used as a traditional medicine against hypertension and diabetes mellitus. Unlike its efficacy, the safety profile of this plant upon long-term administration has not been investigated yet. Therefore, this study investigated the sub-chronic toxicity of S. guineense leaves in rats. METHODS: Wistar albino rats, 10/sex/group were randomly assigned into four groups. Group I-III respectively received 250, 500, and 1000 mg/kg of body weight of 70 % ethanol extract ofS. guineense leaves for 90 consecutive days. Group IV (control) received distilled water. Throughout the experiment, clinical observations were carried out, food intake and weight of the rats also were measured. Finally, different biochemical parameters, organ weight, and histopathology of liver and kidneys were evaluated. RESULTS: Administration of 70 % ethanol extract ofS. guineense leaves decreased food intake and body weight gain of the test animals. Rats treated with 1000 mg/kg of S. guineense extract showed significantly increased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels. Serum urea levels also increased in female rats treated with 500 and 1000 mg/kg body weight of S. guineense. Moreover, the blood glucose level of rats treated with 1000 mg/kg body weight was significantly decreased compared to the control group. However, the histology of the liver and kidneys were not significantly altered by any of the doses administered. CONCLUSION: Administration ofS. guineense in rats at a dose of 1000 mg/kg body weight affected the food consumption, weight gain, and serum levels of liver and kidney enzymes suggesting that S. guineense intake at high doses may be toxic. Therefore, liberal consumption of S. guineense leaves should be taken curiously and cautiously.

Novel strategy for disposable pipette extraction (DPX): Low-cost Parallel-DPX for determination of phthalate migration from common plastic materials to saliva simulant with GC-MS.

In widespread use in commercial products as plasticizers, phthalic acid esters (phthalates) have worried researchers and society in general, given the negative impacts on living organisms, especially human health. Since they are not chemically linked to the polymeric matrix, their migration is evident for samples that come into contact with plastics that contain them, such as water, food and saliva. In this work, a new strategy is described, named parallel-disposable pipette extraction (Pa-DPX), in a fast, efficient and robust analytical method using five simultaneous extractions for the determination of migration of 6 phthalates from common plastic materials (children's toys, school supplies, dog toys and oral contact items) to saliva simulant, using gas chromatography-mass spectrometry (GC-MS). The optimized conditions were 5 extraction cycles with 1600 µL of saliva simulant and desorption with 200 µL of ethyl acetate using 5 cycles with the same aliquot. The calibration curves resulted in determination coefficients higher than 0.9915, limits of detection at 1.5 µg L-1, and the quantification limits were 5.0 µg L-1. Excellent results were obtained for repeatability (relative standard deviation ranging from 8.7% to 20.1% for 5 µg L-1) and intermediate precision, varying the day of analyses (7.9%-16.2%). The analyte recovery ranged from 75% to 114% for two different samples, in four different levels of concentration. The Pa-DPX-GC-MS method was successfully applied to determine the migration of PAE from 21 samples. At least one PAE was detected in 81% of samples, and di-n-octyl phthalate was found in higher concentration, achieving the migration of almost 30 µg per g of sample.

Residue analysis of fungicides fenpicoxamid, isofetamid, and mandestrobin in cereals using zirconium oxide disposable pipette extraction clean-up and ultrahigh-performance liquid chromatography-tandem mass spectrometry.

In the present study, a fast multiresidue method determining three novel fungicides fenpicoxamid, isofetamid, and mandestrobin in cereals was developed and validated for the first time using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Samples were extracted by QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology, and cleaned up using the disposable pipette extraction (DPX) tips containing primary secondary amine (PSA) and silica gel modified with zirconium oxide (Z-Sep) in less than 1 min. Linearity (r > 0.99) of three fungicides in the calibration range of 0.001-0.1 µg mL-1 was satisfactory. Mean recoveries (n = 15) from all matrices were between 84.8% and 100.3% as the corresponding intra-day and inter-day relative standard deviations (RSDs) were less than 10.6%. Limits of quantitation (LOQs) of all analytes in different matrices were defined at 0.01 mg kg-1. The results indicate this method can serve as a sensitive and rapid approach to monitoring contents of fenpicoxamid, isofetamid, and mandestrobin in cereals.

Disposable pipette extraction using a selective sorbent for carbendazim residues in orange juice.

In this work, a selective sorbent for carbendazim was prepared by the thermal immobilization of the poly(glycidoxypropylmethyl-co-dimethylsiloxane), PGDMS, on silica. The lab-made Si(PGDMS) sorbent was physicochemically and morphologically characterized and was used into the pipette tip for the disposable pipette extraction (DPX) of carbendazim residues of orange juice. The DPX method was optimized from a central composite design and validated according to the SANTE/11813/2017 document recommended by the European Union. The proposed method presented recoveries between 93 and 110% with RSD <16% and the limit of quantification below the MRL for carbendazim in citrus. The whole sample preparation process was carried out at less than 3 min, with good accuracy and precision without the need of any cleanup step or electrical equipment, allowing its portability for the extraction of carbendazim residues in orange juice.

A Cross-Calibration Study of GE Lunar iDXA and GE Lunar DPX Pro for Body Composition Measurements in Children and Adults.

OBJECTIVE: To cross-calibrate dual energy X-ray absorptiometry machines when replacing GE Lunar DPX-Pro with GE Lunar iDXA. METHODS: A cross-sectional study was conducted in 126 children (3-19 years) and 135 adults (20-66 years). Phantom cross calibration was carried out using aluminum phantom provided with each of the machines on both machines. Total body less head (TBLH), lumbar spine (L2-L4) and left femoral neck bone mineral density (BMD), bone mineral content (BMC), and bone area were assessed for each patient on both machines. TBLH lean and fat mass were also measured. Bland-Altman analysis, linear regressions, and independent sample t test were performed to evaluate consistency of measurements and to establish cross-calibration equations. RESULTS: iDXA measured 0.33% lower BMD and 0.64% lower BMC with iDXA phantom as compared to DPX-Pro phantom (p < 0.001). In children, TBLH-BMC, femoral BMC and area were measured 10%-14% lesser, TBLH area was higher by 1%-2% and L2-L4 area by 10%-14% by iDXA as compared to DPX-Pro. iDXA measured higher TBLH fat [15% (girls), 31% (boys)] than DPX-Pro. In adults, TBLH-BMD (1.7%-3.4%), BMC (6.0%-10.9%) and area (4.2%-7.6%) were measured lesser by iDXA than DPX-Pro. L2-L4 BMD was higher [2.7% (men), 1.8% (women)] by iDXA than DPX-Pro. Femoral BMC was 2.11% higher in men and 4.1% lower in women by iDXA as compared to DPX-Pro. In children, R2 of cross-calibration equations, ranged from 0.91 to 0.96; in adults, it ranged from 0.93 to 0.99 (p < 0.01). After the regression equations were applied, differences in BMD values between both machines were negligible. CONCLUSION: A strong agreement for bone mass and body composition was established between both machines. Cross-calibration equations need to be applied to transform DPX-Pro measurements into iDXA measurements to avoid errors in assessment. This study documents a need for use of cross-calibration equations to transform DPX-Pro body composition data into iDXA values for clinical diagnosis.

Expanding the applicability of cork as extraction phase for disposable pipette extraction in multiresidue analysis of pharmaceuticals in urine samples.

In this paper, cork is proposed as a natural and renewable material for the extraction phase in disposable pipette extraction to be applied in the multiresidue determination of pharmaceuticals in human urine by HPLC with diode array detection. The compounds carbamazepine, losartan, ketoprofen, 17­ß­estradiol, naproxen, diazepam, 17­α­ethinylestradiol, estrone, diclofenac and ibuprofen were studied. A known amount (5 mg) of cork, with a size of 200 mesh, was inserted into a pipette tip. The method optimization was carried out with univariate and multivariate approaches. The optimized conditions were sample pH adjusted to 3, urine dilution factor of 40 (158 µL of urine diluted in 6.142 mL of ultrapure water), 9 extraction cycles each performed with 700 µL of sample, and extraction time of 10 s per cycle. Desorption was performed with 85 µL of methanol applying 6 cycles of 10 s each using the same solvent aliquot. For the clean-up step, 4 cycles were carried out each with 200 µL of methanol. The limits of quantification varied from 5 to 10 µg L-1 with determination coefficients higher than 0.9919 for the calibration curves for all the analytes. Intra-day and inter-day precision and relative recovery from urine samples donated by two volunteers were assessed based on three spiked concentrations. The analyte relative recoveries ranged from 65 to 117% (n = 3) for the two samples. Intra-day precision ranged from 1.2 to 17% (n = 3) and inter-day precision varied from 11 to 21% (n = 9).

Delay knowledge and trial set count modulate use of proactive versus reactive control: A meta-analytic review.

The AX-continuous performance task (AX-CPT) and dot pattern expectancy (DPX) are the predominant cognitive paradigms used to assess the relative utilization of proactive versus reactive cognitive control. Experimental parameters vary widely between studies and systematically between different modalities (i.e., fMRI vs. EEG) with unknown consequences for the implementation of control. This meta-analytic review systematically surveyed these bodies of literature (k = 43, 73 data points) to resolve how cue-probe delay knowledge, delay length, and trial set count modulate the preferential use of proactive versus reactive control. In healthy young adults, delay knowledge and increasing trial set count each bias participants toward greater proactive control. Further, the interaction of delay knowledge and trial set count accounts for ~40% of variability in proactive/reactive control performance. As trial count varies reliably between experimental modalities, it is critical to understand how these parameters activate distinct cognitive processes and tap into different neural mechanisms for control. Subgroup analyses revealed important distinctions from our results in healthy young adults. Healthy, slightly older adults (ages 30-45 years) performed more reactively compared to healthy young adults. In addition, participants with schizophrenia showed evidence of more proactive control as trial set count increased. In light of this meta-analytic review, we conclude that delay knowledge and trial set length are important parameters to account for in the assessment of proactive versus reactive control. More broadly, this metaregression provides strong evidence that cognitive control becomes more reactive when timing demands are not known, and that both healthy persons and persons with schizophrenia shift toward proactive control with increasing repetitions of a task set.

An effective and high-throughput analytical methodology for pesticide screening in human urine by disposable pipette extraction and gas chromatography - mass spectrometry.

In this paper, the use of disposable pipette extraction (DPX) for the determination of pesticides in human urine in possible cases of poisoning is proposed for the first time. The pesticides studied were oxamyl, propoxur, carbofuran, 3­hydroxycarbofuran, carbaryl, methiocarb, terbufos, parathion methyl, malathion, chlorpyrifos and endosulfan. The pipette tip used for the extraction of these compounds was commercially acquired. It has a capacity of 5 mL and contains 20 mg of sorbent material (styrene-divinylbenzene). The optimization of the main parameters that can influence the extraction efficiency of this sample preparation technique was performed with univariate and multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions were 5 extraction cycles of 30 s and 5 desorption cycles of 15 s with 250 µL of ethyl acetate. Elution of the extract was performed in a vial containing 100 mg of anhydrous sodium sulfate. The method developed was validated, providing correlation coefficients higher than 0.9955 for all analytes, limits of detection (LOD) of 0.76 to 1.52 µg L-1, limits of quantification (LOQ) of 2.5 to 5.0 µg L-1, relative recoveries of 63 to 118%, intra-day precision of 0.7 to 15.3% and inter-day precision of 4.9 to 13.1%. An effective and rapid method for the analysis of human urine for the identification of possible cases of poisoning by pesticides was successful developed.

Simultaneous determination of neonicotinoid insecticides and insect growth regulators residues in honey using LC-MS/MS with anion exchanger-disposable pipette extraction.

In this study, we developed an anion exchanger-disposable pipette extraction (DPX) method to detect the residual concentrations of eight neonicotinoid insecticides (dinotefuran, acetamiprid, clothianidin, thiacloprid, imidachloprid, imidaclothiz, nitenpyram, and thiamethoxam) and eight insect growth regulators (IGRs; triflumuron, cyromazine, buprofezin, methoxyfenozide, tebufenozide, chromafenozide, fenoxycarb, and RH 5849) in Chinese honey samples collected from different floral sources and different geographical regions using liquid chromatography tandem mass spectrometry (LC-MS/MS). QAE Sephadex A-25 was used as the anion exchanger in the DPX column for the purification and cleanup of honey samples. Analytes were eluted with a mixture of acetonitrile and 0.1 M HCl, and the elution was subjected to LC analysis. This method was thoroughly validated for its reproducibility, linearity, trueness, and recovery. Satisfactory recovery of pesticides was obtained ranging from 72% to 111% with intraday RSDs (n = 5) of 1%-10%. High linearity (R2 ≥ 0.9987) was observed for all 16 pesticides. Limits of detection and quantification for all 16 compounds ranged from 0.3 to 3 µg/kg and from 1 to 10 µg/kg, respectively. Pesticide residues (9-113 µg/kg) were found in Chinese honey samples. The anion exchanger-DPX method was effective for removing sugars and retaining target analytes. Moreover, this method was highly reliable and sensitive for detecting neonicotinoids and IGRs in different floral sources of honey and will be applicable to matrixes with high sugar content.

Analysis of 10 ß-agonists in pork meat using automated dispersive pipette extraction and LC-MS/MS.

An analytical procedure for the analysis of 10 ß-adrenergic agonists (cimaterol, terbutaline, salbutamol, isoxsuprine, ractopamine, cimbuterol, clenbuterol, brombuterol, mabuterol and mapenterol) in pork meat was developed and validated using LC-MS/MS. An automated dispersive pipette extraction (DPX) was employed on a Hamilton Microlab® NIMBUS96® platform to extract the analytes of interest prior to LC-MS/MS analysis. The extraction time was <20 min with a total LC-MS/MS run time of 9.6 min. The method was fully validated in accordance with the international guidelines (European Commission Decision 2002/657/EC and National Standards of People's Republic of China, GB/T 22286-2008) for limit of detection, limit of quantitation, carryover, extraction efficiency, matrix effects, linearity, and within and between-run precision. The proposed method can be successfully used in the routine determination of 10 ß-adrenergic agonists in pork and as a potential solution for compliance monitoring in regulatory laboratories.

Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3).

BACKGROUND: Nuclear Receptors (NRs), including PXR and CAR, are presumed to be ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation. Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with screening tools incapable of detecting indirect activators could present liabilities. OBJECTIVE: The aim of this study was to establish assays that identify indirect activators of human PXR and CAR. METHODS: Commercially available human PXR and CAR transactivation assays were used for analyses. RESULTS: We show that transactivation assays containing full-length nuclear receptors with native promoters can identify indirect activators of human CAR and PXRwhen compared to those of commercially available assays containing only the LBD of PXR and CAR. Of these two assay systems, only human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors, phenobarbital and midostaurin were found to be direct and indirect activators of PXR while human CAR activation by phenobarbital occurs by indirect mechanisms only. CONCLUSION: Cell based transactivation assays employing the full-length receptors and native promoters identify both direct and indirect activators of either or both human PXR and CAR.