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Ethnopharmacological Relevance: The objective of traditional Chinese medicine (TCM) combination theory is to "reduce toxicity and increase efficiency", especially to solve the liver toxicity of many TCMs. Fructus Meliae Toosendan (CLZ)-Fructus Foeniculi (XHX) is a typical traditional Chinese herb pair that decreases the toxicity and increases the efficiency of the herbs. Fructus Meliae Toosendan (CLZ, cold-natured) has significant liver toxicity. However, it has been widely used in combination with Fructus Foeniculi (XHX, hot-natured) for thousands of years in TCM, in which form it shows no hepatotoxicity, indicating that the combined use of XHX and CLZ can reduce the hepatotoxicity of CLZ. Herb-herb interactions could affect herb pharmacokinetics and in vivo efficacy. The herb-herb interactions between CLZ and XHX are still unknown., Materials and Methods: This study used liquid chromatography tandem mass spectrometry (LC-MS) and gas chromatography tandem mass spectrometry (GC-MS) to establish methods for detecting toosendanin and trans-anethole, the main active substances of CLZ and XHX, respectively. Additionally, we investigated their herb-herb interactions via pharmacokinetic and pharmacodynamic studies., Results: The results indicate that the established analytical methods are suitable for detecting toosendanin and trans-anethole, and the methodology meets the requirements of biological sample testing methods. Compared with the CLZ group, the pharmacokinetic parameters C max , AUC (0-t) , AUC (0-∞) , MRT (0-t) and MRT (0-∞) of toosendanin in the CLZ-XHX group notably decreased and the values of V z/F remarkably increased. Compared with the XHX group, the pharmacokinetic parameters C max , AUC 0-t , AUC 0-∞, T max and t 1/2z of trans-anethole notably increased in the CLZ-XHX group, and the values of CL z/F and V z/F obviously decreased., Conclusion: The pharmacokinetic results indicate that XHX can significantly decrease the absorption and bioavailability and accelerate the elimination process of toosendanin in CLZ. XHX could decrease the risk of in vivo accumulation of the toxic constituent of CLZ, toosendanin, thus decreasing its toxicity. It has also been shown that CLZ can significantly increase absorption and bioavailability and attenuate the elimination process of trans-anethole in XHX, thus enhancing its efficacy. Hepatotoxicity studies indicate that CLZ has significant hepatotoxicity, and its combined use with XHX can decrease its liver-damaging properties., (© 2020 John Wiley & Sons, Ltd.)

A simple, sensitive and selective liquid chromatography-tandem mass spectrometric method was developed and validated for the quantification of α-asarone in mouse plasma with its application to pharmacokinetic studies. An electrospray ionization (ESI) with multiple reaction monitoring (MRM) mode was used to monitor the precursor-product ion transitions of 209.1 > 193.9 m/z for α-asarone and 157.8 > 114.0 m/z for allantoin. Chromatographic separation was acquired on a Sepax BR-C18 (5 μm, 120 Å 1.0 × 100 mm) column with an isocratic mobile phase consisting of methanol and 0.1% formic acid (80:20, v/v). The developed bioanalytical method was successfully validated according to the United States Food and Drug Administration (US FDA) guidelines for linearity, selectivity, accuracy, precision, recovery, matrix effect, and stability. The validated method was successfully applied to a pharmacokinetics study of α-asarone along with a combination of pharmacokinetic techniques, including small-volume serial blood sampling in mice, reducing drug doses and the number of animals used, using a simple protein precipitation method and less solvent consumption will enable its use in further bioequivalence studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Isomers β-asarone and α-asarone have recently been demonstrated to have differential pharmacological activities. Here, we report an LC-MS/MS method developed using acetonitrile to extract two isomeric phenylpropenes from rat plasma. Separation was achieved using a XDB-C 18 column (100 × 2.1 mm; i.d., 1.8 μm) with a mobile phase of methanol-0.1% formic acid (55:45, v/v) at a flow rate of 0.3 mL/min. Calibration curves ranging from 5.20 to 2080 ng/mL for β-asarone and from 3.68 to 1470 ng/mL for α-asarone were linear (r 2  ≥ 0.9938) with the lower limits of quantification being 5.20 and 3.68 ng/mL for both isomers. Intravenous administration of β-asarone (2.22 mg/kg) and α-asarone (2.36 mg/kg) in rats yielded half-lives of 13.40 ± 4.11 and 28.88 ± 7.82 min with clearance values of 0.196 ± 0.062 mL/min/kg and 0.112 ± 0.012 mL/min/kg for β-asarone and α-asarone, respectively., (Copyright © 2017 John Wiley & Sons, Ltd.)

Usnea longissima Ach. (Usnea) is used in pharmaceuticals, food and cosmetics. Evernic acid (EA), barbatic acid (BA), diffractaic acid (DA) and usnic acid (UA) are the most typical ingredients in U. longissima and exert a wide variety of biological functions. The study aimed to develop a sensitive method for simultaneous analysis of EA, BA, DA and UA in rat plasma and was applied to pharmacokinetic studies after consumption of UA and ethanol extract from U. longissima (UE). The samples were separated on a BEH C 18 column by gradient elution with 0.5% formic acid in water and in methanol. The relative molecular masses of analytes were obtained in full-scan range from 50.0 to 750.0 m/z under negative ionization mode by UPLC-Q-Exactive Orbitrap MS. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for biological specimen analysis. The pharmacokinetic results indicated that the absolute bioavailabilities of UA after oral administration of UA and UE reached 69.2 and 146.9%, respectively. Compared with UA in UE, the relative bioavailability of DA, BA and EA reached 103.7, 10.4 and 0.7% after oral administration of UE., (Copyright © 2017 John Wiley & Sons, Ltd.)

This paper describes cases of poisoning caused by new psychoactive substances such as: 25B-NBOMe (2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine) and 4-CMC (1-(4-chlorophenyl)-2-(methylamino)-1-propanone). The analytical procedure includes rapid and selective method for the extraction and determination of 4-CMC and 25B-NBOMe in blood samples using UPLC-MS/MS technique. To the best of our knowledge, this is the first report, that involves a fully validated method for quantification of new-designer drug - 4-CMC in postmortem blood samples. The biological material was also analyzed with the use of routine analytical methods: immunochemical techniques, gas chromatography with flame ionization detection and gas chromatography with electron impact mass spectrometry. The results of real samples analyses correspond to possible toxicological effects: death resulting from 25B-NBOMe - mediated hallucinations (661ng/mL of 25B-NBOMe and 0.887ng/mL of 4-CMC), fatal overdose of 25B-NBOMe and 4-CMC (66.5ng/mL of 25B-NBOMe and 2.14ng/mL of 4-CMC) and non-fatal intoxication of these drugs (38.4ng/mL of 25B NBOMe and 0.181ng/mL of 4-CMC). Additionally, O-demethylathed O, O-bis-demethylathed and glucuronidated metabolites of 25B-NBOMe in biological specimens were detected., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Peripheral leukocytes in patients with atopic dermatitis (AD) have elevated phosphodiesterase-4 activity, which is associated with production of proinflammatory mediators. OPA-15406 is a phosphodiesterase-4 inhibitor with high selectivity for phosphodiesterase-4-B., Objectives: We sought to assess effectiveness and tolerability of topical OPA-15406 in patients with AD., Methods: This was a randomized, double-blind, vehicle-controlled, phase-II study. Patients 10 to 70 years of age with mild or moderate AD received topical OPA-15406 0.3% (n = 41), OPA-15406 1% (n = 43), or vehicle (n = 37) twice daily for 8 weeks., Results: The primary end point, Investigator Global Assessment of Disease Severity score of 0 or 1 with greater than or equal to 2-grade reduction, was met at week 4 in the OPA-15406 1% group (P = .0165 vs vehicle). Mean percentage improvement from baseline Eczema Area and Severity Index score for OPA-15406 1% was notable in week 1 (31.4% vs 6.0% for vehicle; P = .0005), even larger in week 2 (39.0% vs 3.0%; P = .0001), and persisted for 8 weeks. Visual analog scale pruritus scores improved from moderate to mild within the first week in the OPA-15406 1% group (36.4% mean change; P = .0011). OPA-15406 levels in blood were negligible. Incidence of adverse events was low, with most events mild in intensity., Limitations: Further confirmatory phase-III studies are required., Conclusion: OPA-15406 ointment may provide an effective therapeutic modality for patients with mild to moderate AD., (Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.)

In the present study, an efficient and environmental friendly method (called in-syringe reversed dispersive liquid-liquid microextraction (IS-R-DLLME)) was developed to extract three important components (i.e. para-anisaldehyde, trans-anethole and its isomer estragole) simultaneously in different plant extracts (basil, fennel and tarragon), human plasma and urine samples prior their determination using high-performance liquid chromatography. The importance of choosing these plant extracts as samples is emanating from the dual roles of their bioactive compounds (trans-anethole and estragole), which can alter positively or negatively different cellular processes, and necessity to a simple and efficient method for extraction and sensitive determination of these compounds in the mentioned samples. Under the optimum conditions (including extraction solvent: 120 μL of n-octanol; dispersive solvent: 600 μL of acetone; collecting solvent: 1000 μL of acetone, sample pH 3; with no salt), limits of detection (LODs), linear dynamic ranges (LDRs) and recoveries (R) were 79-81 ng mL(-1), 0.26-6.9 μg mL(-1) and 94.1-99.9%, respectively. The obtained results showed that the IS-R-DLLME was a simple, fast and sensitive method with low level consumption of extraction solvent which provides high recovery under the optimum conditions. The present method was applied to investigate the absorption amounts of the mentioned analytes through the determination of the analytes before (in the plant extracts) and after (in the human plasma and urine samples) the consumption which can determine the toxicity levels of the analytes (on the basis of their dosages) in the extracts., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Identification of new psychoactive substances (NPS) is challenging. Developing targeted methods for their analysis can be difficult and costly due to their impermanence on the drug scene. Accurate-mass mass spectrometry (AMMS) using a quadrupole time-of-flight (QTOF) analyzer can be useful for wide-scope screening since it provides sensitive, full-spectrum MS data. Our article presents a qualitative screening workflow based on data-independent acquisition mode (all-ions MS/MS) on liquid chromatography (LC) coupled to QTOFMS for the detection and identification of NPS in biological matrices. The workflow combines and structures fundamentals of target and suspect screening data processing techniques in a structured algorithm. This allows the detection and tentative identification of NPS and their metabolites. We have applied the workflow to two actual case studies involving drug intoxications where we detected and confirmed the parent compounds ketamine, 25B-NBOMe, 25C-NBOMe, and several predicted phase I and II metabolites not previously reported in urine and serum samples. The screening workflow demonstrates the added value for the detection and identification of NPS in biological matrices.

Over the last few years, NBOMe substances have been used either as a legal alternative to lysergic acid diethylamide (LSD) or sold surreptitiously as LSD to unknown users. These NBOMe substances have been detected in blotter papers, powders, capsules and liquids. We report the deaths of two teenage male subjects that were related to 25B-NBOMe and 25I-NBOMe in Indiana during 2014. Samples were extracted via a solvent protein precipitation with acetonitrile and analyzed via ultra-performance liquid chromatography with tandem mass spectrometry. For these two cases, we describe the NBOMe instrumental analysis, toxicological results for postmortem heart blood and urine specimens and the relevant case history and pathological findings at autopsy. In the first case, 25B-NBOMe was detected in postmortem heart blood at 1.59 ng/mL; in the second case, 25I-NBOMe was detected in postmortem heart blood at 19.8 ng/mL. We also review relevant published casework from clinical toxicology and postmortem toxicology in which analytically confirmed 25B-NBOMe and 25I-NBOMe were determined to be causative agents in intoxications or deaths., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The Armed Forces are developing new explosives that are less susceptible to unintentional detonation (insensitive munitions [IMX]). 2,4-Dinitroanisole (DNAN) is a component of IMX. Toxicokinetic data for DNAN are required to support interpretation of toxicology studies and refinement of dose estimates for human risk assessment. Male Sprague-Dawley rats were dosed by gavage (5, 20, or 80 mg DNAN/kg), and blood and tissue samples were analyzed to determine the levels of DNAN and its metabolite 2,4-dinitrophenol (DNP). These data and data from the literature were used to develop preliminary physiologically based pharmacokinetic (PBPK) models. The model simulations indicated saturable metabolism of DNAN in rats at higher tested doses. The PBPK model was extrapolated to estimate the toxicokinetics of DNAN and DNP in humans, allowing the estimation of human-equivalent no-effect levels of DNAN exposure from no-observed adverse effect levels determined in laboratory animals, which may guide the selection of exposure limits for DNAN., (© The Author(s) 2015.)

The commercially available alpha-asarone injections (CA-ARE) were frequently found to cause severe anaphylactic reactions by the solubilizer contained in the formulation such as polysorbate 80 and propylene glycol. This study aimed to develop a new ARE injection using Kolliphor HS 15 as solubilizing agent (HS 15-ARE) by the dissolution method to resolve its poor solubility problem and reduce the anaphylaxis of CA-AREs caused by Polysorbate 80. The HS 15-ARE micelle showed a homogeneous round shape with the mean particle size of around 13.73 ± 0.02 nm, polydisperse index (PDI) of 0.19 ± 0.01 and solubilizing efficiency of 95.7% ± 2.4%. In vitro and in vivo studies showed that HS 15-ARE is a stable injection presenting the same pharmacokinetic profile with CA-ARE. Moreover, improved therapeutic effect was observed for HS 15-ARE in treating asthma compared to CA-ARE (p < 0.05) with no anaphylactic reactions observed. These results demonstrate that the new formulation of ARE (HS 15-ARE) has a great potential for replacing CA-AREs injections.

To study the pharmacokinetic characteristics and absolute bioavailability of α-asarone through dry powder inhalation in rats, and compare with that through oral administration and intravenous injection. A HPLC method was established for the determination of α-asarone in rat plasma to detect the changes in plasma concentrations of α-asarone through dry powder inhalation (20 mg · kg(-1)), oral administration (80 mg · kg(-1)) and intravenous injection (20 mg · kg(-1)) in rats. DAS 2.0 software was used to calculate the pharmacokinetic parameters. The absolute bioavailability of α-asarone was calculated according to AUC(0-t)) of administration routes and administration doses. According to the results, α-asarone showed good linear relations (r = 0. 999 4) at concentrations between 0.282-14.1 mg · L(-1), with the limit of detection (LOD) at 0.212 mg · L(-1). Through dry powder inhalation, oral administration and intravenous injection of α-asarone, the metabolic processes of α-asarone in rats conformed to one, two and three compartment models respectively, with the elimination half-life of (95.48 ± 48.28), (64.34 ± 27.59), (66.99 ± 29.76) min. According to the bioavailability formula, the absolute bioavailability of α-asarone through dry powder inhalation and oral administration were 78.32% and 33. 60%, respectively. This study showed that significant increase in elimination half-life and absolute bioavailability of α-asarone through dry powder inhalation, which lays a theoretical foundation for preparing α-asarone dry powder inhalers.

Aims: About one-third of patients with mild dyssynchronous heart failure suffer from atrial fibrillation (AF). Drugs that convert AF to sinus rhythm may further slowdown ventricular conduction. We aimed to investigate the electrophysiological and haemodynamic effects of vernakalant and flecainide in a canine model of chronic left bundle branch block (LBBB)., Methods and Results: Left bundle branch block was induced in 12 canines. Four months later, vernakalant or flecainide was administered using a regime, designed to achieve clinically used plasma concentrations of the drugs, n = 6 for each drug. Epicardial electrical contact mapping showed that both drugs uniformly prolonged myocardial conduction time. Vernakalant increased QRS width significantly less than flecainide (17 ± 13 vs. 34 ± 15%, respectively). Nevertheless, both drugs equally decreased LVdP/dtmax by ∼15%, LVdP/dtmin by ∼10%, and left ventricular systolic blood pressure by ∼5% (P = n.s. between drugs)., Conclusions: Vernakalant prolongs ventricular conduction less than flecainide, but both drugs had a similar, moderate negative effect on ventricular contractility and relaxation. Part of these reductions seems to be related to the increase in dyssynchrony., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

We present a case of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), an N-benzyl phenethylamines derivative, intoxication and a high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for detection and quantification of 25B-NBOMe. A 19-year-old male was found unresponsive with generalized grand mal seizure activity. On the second day of hospitalization, a friend admitted that the patient used 'some unknown drug' called 25B. Serum and urine collected were sent to the Virginia Commonwealth University Medical Center Toxicology Laboratory for analysis. An HPLC-MS/MS method for the identification and quantification of 25B-NBOMe using 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe) as the internal standard (ISTD) was developed. As this is a novel, single-case presentation, an assay validation was performed prior to testing to ensure the reliability of the analytical results. The serum and urine specimens were determined to contain 180 pg/ml and1900 pg/ml of 25B-NBOMe, respectively., (Copyright © 2013 John Wiley & Sons, Ltd.)

Recently, much attention has been given to congener analysis, which can be used to check the possibility of postoffence drinking claims in forensic toxicology. In this type of analysis, the information given by the defendant regarding the type, quantity, and time of consumption of a specific alcoholic beverage is used to calculate theoretically expected congener concentration in the blood and this is compared with the analytically determined concentrations in the blood sample. Many alcoholic drinks aromatized with essential oils of plants and fruits contain a specific congener, for example, anethole in aniseed drinks. The present study describes the GC procedure of anethole analysis in human plasma using SPE as the sample preparation method. The procedure involves the protein precipitation process, which generally degrades the protein-analyte complex, and SPE isolation of anethole from the examined materials. This analytical approach is proposed as a method of choice for the estimation of anethole concentration in human fluids after the consumption of alcoholic beverages and other foods containing the substance. The described method is characterized by a low LOD (8.33 ng/g) and a very high recovery (average recovery 98.37%) of the analyte., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

From early year 2000 different herbal products containing synthetic cannabinoids (SC) have appeared on the drug market all over the world, and new substances are frequently introduced. The prevalence of SC use in different populations is however still mainly unknown, also in Norway. This information is difficult to obtain, but studies of drivers suspected of driving under the influence of drugs (DUID), might provide important information. The aim of this study was to assess the prevalence of SC in drivers suspected of being under the influence of drugs in Norway, and investigate if SCs impair driving performance. For two periods of three and four weeks all blood samples from drivers suspected of DUID in Norway were analyzed for the presence of 12 and 18 different SCs, respectively. A new ultra performance liquid chromatography tandem mass spectrometry method was developed. A total of 726 cases were analyzed during our study period, and SCs were detected in 16 cases (2.2%) in total. The mean age of these drivers was 29.6 years. High concentrations of other psychoactive drugs were detected in all the blood samples where a SC was found. AM-2201 and JWH-018 were the most frequently detected SCs, each found in five cases. In addition RSC-4, JWH-122, JWH-081 and JWH-250 were detected. None of the drivers had reported using SCs prior to driving. Despite the limited number of SCs investigated in this 7 week study period, a considerable percent of the cases were positive. Other psychoactive drugs of abuse were always found concomitant with the SCs, and the age of these drivers indicates that experienced drug users also ingest SCs. Since other drugs were found in all the samples, the psychomotor impairment caused by the SCs is difficult to estimate. Our study shows the importance of screening analyses of biological samples from different populations to assess the prevalence of drug use, since self-reporting might be encumbered with significant under-reporting., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

β-Asarone (BAS), a phenylpropanoid from Acorus calamus Linn., has shown biological effects in the management of cognitive impairment conditions such as Alzheimer's disease. The present paper describes a selective and sensitive liquid chromatography-tandem mass spectrometric method (HPLC-MS/MS) using electrospray ionization source (ESI) for quantification of BAS in rat plasma. Briefly, the plasma samples were pre-treated using a simple solid-phase extraction method. The separation of BAS and the internal standard, caffeine, was achieved on an Agilent Zorbax XDB C(18) column (50 × 2.1 mm i.d., 5 µm) using 0.2 mL/min isocratic mobile phase flow. The detection was performed using an Applied Biosystems Hybrid Q-Trap API 2000 mass spectrometer equipped with an ESI source operated in positive mode. Also, the developed bioanalytical method was validated as per the US FDA bioanalytical guidelines over the concentration range of 9.79-4892.50 ng/mL (r(2) ≥ 0.9951) for BAS from rat plasma. The mean percentage recovery (n = 3) for the low, middle and high quality control samples was 86.92 ± 3.89, 85.30 ± 1.09 and 87.24 ± 4.03%, respectively. The applicability of the validated HPLC-MS/MS method was demonstrated by successful measurement of BAS from plasma following oral administration of Acorus calamus rhizome extracts to three female albino Wistar rats., (Copyright © 2012 John Wiley & Sons, Ltd.)

A simple and rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method has been developed and validated for the determination of 4-p-anisamidobutyric acid (ABA; or N-anysoyl-γ-aminobutiryc acid, N-anisoyl-GABA), a major active metabolite of aniracetam, in human plasma. After protein precipitation of plasma sample with methanol, ABA and the internal standard lisinopril were separated on a Venusil ASB C₁₈ column at 25 °C. The mobile phase consisted of methanol-ammonium acetate (10 mmol/L) (30:70, v/v). The detection was performed on a triple quadrupole tandem mass spectrometer with an ESI source in negative ion mode. Multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 235.8→m/z 106.6, and m/z 403.8→m/z 113.6 was used to quantify ABA and lisinopril, respectively. This is the first LC-MS/MS method for ABA with advantages of short analysis time (4.5 min per sample run) and high selectivity attributable to the MRM detection and optimized HPLC conditions. The response was linear in a concentration range of 0.0485-19.4 μg/mL in plasma. The extraction recovery of ABA was between 89.1% and 100.7%. The precision (RSD) and accuracy (RE) of the method were evaluated to be within 7.3% and from 2.5% to 6.9%. The validated method has been applied to the pharmacokinetic study after a single oral administration of aniracetam dispersible tablets to human beings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

beta-Asarone has significant pharmacological effects on the central nervous system. As a potential therapeutic agent to manage brain diseases, analysis of the pharmacokinetics of beta-asarone in brain is necessary. We used cardio-perfusion method to exclude the beta-asarone in the brain blood. The brain was divided into five regions: hippocampus, cortex, brain stem, thalamus and cerebellum, and pharmacokinetic differences were investigated. We found that concentration-time profile of beta-asarone in blood, hippocampus, cortex, brain stem and cerebellum could be adequately described by a first-order equation, consistent with a linear two-compartmental model, but a first-order equation with a linear one-compartmental model in thalamus. The half lives of beta-asarone in blood, hippocampus, cortex, brain stem, thalamus and cerebellum were 1.3801, 1.300, 1.937, 7.142, 2.832 and 8.149 h, respectively. Gender differences do not significantly influence plasma pharmacokinetics of beta-asarone.

A sensitive and specific method for the quantification of JWH-018, JWH-073, and JWH-250 and the qualitative identification of JWH-019 in whole blood was developed and validated. Samples fortified with JWH-018-d₉ and JWH-073-d₉ underwent liquid-liquid extraction and were analyzed by liquid chromatography-positive ion electrospray ionization-tandem mass spectrometry. Two transitions were monitored for all analytes except JWH-250, for which there was only one available transition. JWH-019 did not meet the stringent requirements for quantitative analysis, and thus this method is only appropriate for the qualitative identification of this compound in whole blood. The linear range was 0.1-20 μg/L for all quantitative analytes. The maximum average within and between-run imprecision was 7.9% and 10.2%, respectively, and all controls quantified within 8.2% of target concentrations. Process efficiency, a measurement that takes into effect extraction efficiency and matrix effect, was ≥ 32.0% for all quantitative analytes; similar results were obtained for the deuterated internal standards. All analytes were stable at room, refrigerated, and frozen temperatures for at least 30 days. The method was used to quantify JWH-018 and JWH-073 in a blood specimen collected from a person known to have used an herbal incense blend containing these substances.

Vernakalant is a novel antiarrhythmic agent that has demonstrated clinical efficacy for the treatment of atrial fibrillation. Vernakalant blocks, to various degrees, cardiac sodium and potassium channels with a pattern that suggests atrial selectivity. We hypothesized, therefore, that vernakalant would affect atrial more than ventricular effective refractory period (ERP) and have little or no effect on ventricular defibrillation threshold (DFT). Atrial and ventricular ERP and ventricular DFT were determined before and after treatment with vernakalant or vehicle in 23 anesthetized male mixed-breed pigs. Vernakalant was infused at a rate designed to achieve stable plasma levels similar to those in human clinical trials. Atrial and ventricular ERP were determined by endocardial extrastimuli delivered to the right atria or right ventricle. Defibrillation was achieved using external biphasic shocks delivered through adhesive defibrillation patches placed on the thorax after 10 seconds of electrically induced ventricular fibrillation. The DFT was estimated using the Dixon "up-and-down" method. Vernakalant significantly increased atrial ERP compared with vehicle controls (34 ± 8 versus 9 ± 7 msec, respectively) without significantly affecting ventricular ERP or DFT. This is consistent with atrial selective actions and supports the conclusion that vernakalant does not alter the efficacy of electrical defibrillation.

Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), and their derivatives, hydroxylated (OH) and methoxylated (MeO) PBDEs and methylsulfonylated (MeSO(2)) PCBs, were measured in sera of eight bird species collected from an e-waste recycling region in South China. Concentrations of summation operatorPCBs, ranging from 38 to 1700 ng/g lipid weight (lw), were one to two orders of magnitude higher than concentrations of summation operatorPBDEs (0.64-580 ng/g lw). The significantly positive relationship between PCB and PBDE concentrations suggested a similar pathway of exposure to these compounds. Compared with muscle in birds, serum might prefer to accumulate and/or retain less brominated/chlorinated congeners. 3-OH-BDE47 and 2'-OH-BDE68 were detected in more than 80% of the collected bird serum samples (range: not detectable (nd) to 13 and nd to 7.8 ng/g lw, respectively). The other three OH-PBDE congeners (4'-OH-BDE-17, 6-OH-BDE47, and 4'-OH-BDE-49) and two MeO-PBDE congeners (3-MeO-BDE47 and 6-MeO-BDE47) were occasionally detected in bird sera at concentrations ranging from nd to 2.5 ng/g lw. Both natural sources and metabolic transformation of PBDEs could contribute to the presence of these PBDE derivatives in the birds. The two MeSO(2)-PCB congeners (4-MeSO(2)-CB49 and 4-MeSO(2)-CB101) under investigation were detected at respective concentration ranges of nd to 12 and nd to 0.68 ng/g lw. 4-MeSO(2)-CB101 exhibited the highest concentration among the nine PCB and PBDE derivatives studied, indicating that biotransformation via the mercapturic acid pathway of PCBs might have occurred in the studied bird species.

The metabolism of the potent carcinogen estragole was investigated in humans after consumption of fennel tea by analyses of its metabolites in blood plasma and urine. Stable isotope dilution assays based on LC-MS/MS detection revealed that 1'-hydroxylation of estragole happened very fast as the concentration of conjugated 1'-hydroxyestragole in urine peaked after 1.5 h, whereas it was no longer detectable after 10 h. Besides the formation of less than 0.41% conjugated 1'-hydroxyestragole of the estragole dose administered, the further metabolite p-allylphenol was generated from estragole in a higher percentage (17%). Both metabolites were also detected in blood plasma in less than 0.75-2.5 h after consumption of fennel tea. In contrast to this, no estragole was present in these samples above its detection limit. From the results, it can be concluded that an excess of the major fennel odorant trans-anethole principally does not interfere with estragole metabolism, whereas influences on the quantitative composition of metabolites cannot be excluded. The presence of a sulfuric acid conjugate of estragole could not be confirmed, possibly due to its high reactivity and lability.

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of anethole in serum samples. Anethole is a characteristic marker for the consumption of aniseed spirits. This method enabled the detection of anethole with a limit of detection (LoD) of 3.6 ng/ml and a limit of quantification (LoQ) of 5.3 ng/ml in serum samples with a good degree of precision intraday (2.8%) and interday (4.5%). Experiments were conducted with one volunteer, in which the subject consumed the alcoholic drink ouzo on 3 different days under controlled conditions. At defined intervals, blood samples were taken from the subject. Using these blood samples, the concentration-time profiles for anethole were determined. In blood samples taken from 50 drivers who claimed to have consumed drinks containing anethole (ouzo, raki and the German aniseed liqueur "Küstennebel") before the taking of the blood sample, anethole was detected in the serum in concentrations of between 5.4 and 17.6 ng/ml in 10 cases. This is the first report describing the qualitative and quantitative determination of a beverage-characteristic aroma compound - in this case anethole - in serum samples after consumption of alcoholic beverages.

This work investigated the potential of solid lipid nanoparticles (SLNs) to improve oral bioavailablity and tissue uptake of a poorly soluble drug, alpha-Asarone. Ultrasonic homogenization method was employed to prepare alpha-Asarone-loaded SLNs (alpha-Asarone-SLNs). Particle size and distribution, pH, viscosity, drug incorporation and zeta potential of the SLNs were investigated. Pharmacokinetic study of oral administration to male rats at 10 mg/Kg suggested that the relative bioavailability of alpha-Asarone was significantly improved in alpha-Asarone-SLN group compared to alpha-Asarone solution group. Comparison of alpha-Asarone-SLN to alpha-Asarone control solution for alpha-Asarone concentrations in rat tissue showed an increased uptake of alpha-Asarone in brain and lung for the ARE-SLN group. These results indicate that alpha-Asarone-SLNs significantly enhance the absorption and tissue distribution of alpha-Asarone. SLNs offer a new approach to improve the oral bioavailability of poorly soluble drugs.

Objective: To compare the pharmacokinetics and tissue distribution of alpha-asarone in lipid emulsion and aqueous solution for injection and study the feasibility of lipid emulsion of alpha-asarone as the parenteral drug delivery system., Method: HPLC was used to determine the drug concentration in rat plasma and mice tissues after intravenous (i.v.) administration of lipid emulsion and aqueous solution of alpha-asarone at a single dose (40 mg x kg(-1)), respectively., Result: The plasma concentration-time profiles of lipid emulsion and aqueous solution of alpha-asarone after intravenous administration of them are similar and the drug concentration-time data were fitted to a two-compartment open model. The results of tissues distribution showed that distribution contents of alpha-asarone from lipid emulsion and aqueous solution in vivo are similar in lungs but lipid emulsion increased the uptake in livers and spleens, and decreased the uptake in hearts and kidneys for alpha-asarone., Conclusion: The plasma concentration-time profiles of alpha-asarone in lipid emulsion and aqueous solution are similar, but lipid emulsion significantly altered the tissue distribution of alpha-asarone, which may be beneficial to decrease its potential toxicity to heart and kidney.

In recent years, the frequent appearance of phenethylamine designer drugs on the illicit drug market has been a matter of concern for all authorities involved. New phenethylamine drugs are being introduced because these compounds are not covered by existing legislation. Therefore, the new drugs cannot be considered illicit drugs until their names are officially recognized. This paper describes a method to screen for and quantify four 2,5-methylenedioxy-derivatives of 4-thio-phenethylamine (2C-T-series) in human plasma, using capillary electrophoresis coupled with electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple liquid extraction was used for sample cleanup. The method was validated according to international guidelines.

Propenyl- and allyl-phenols, such as methylchavicol, p-anol and eugenol, have gained importance as flavoring agents and also as putative precursors in the biosynthesis of 9,9'-deoxygenated lignans, many of which have potential medicinal applications. In spite of several decades of investigation, however, the complete biosynthetic pathway to a propenyl/allylphenol had not yet been reported. We have subjected a Thai basil variety accumulating relatively large amounts of the simplest volatile allylphenol, methylchavicol, to in vivo administration of radiolabeled precursors and assays of protein preparations in vitro. Through these experiments, the biosynthesis of chavicol was shown to occur via the phenylpropanoid pathway to p-coumaryl alcohol. Various possibilities leading to deoxygenation of the latter were examined, including reduction of the side-chain double bond to form p-dihydrocoumaryl alcohol, followed by dehydration to afford chavicol, as well as formation of p-methoxycinnamyl alcohol, with further side-chain modification to afford methylchavicol. A third possibility studied was activation of the side-chain alcohol of p-coumaryl alcohol, e.g.via esterification, to form a more facile leaving group via reductive elimination. The latter was shown to be the case using p-coumaryl esters as potential substrates for a NAD(P)H-dependent reductase to afford chavicol, which is then O-methylated to afford methylchavicol.

Asarones (alpha-asarone and beta-asarone) are the active components in the traditional Chinese medicine (TCM) of Acorus tatarinowii Schott, which has been used to treat epilepsy for several thousand years. To perform the pharmacokinetics (PK) study of alpha- and beta-asarone from the TCM essential oil, a simple, rapid and sensitive method was developed for the determination of asarones from the TCM in rabbit plasma, based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) with electron ionization (EI). The extraction parameters of headspace volume, fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized. Furthermore, the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 microg/mL, R(2) > 0.99), low detection limit (<2.0 ng/mL) and good precision (RSD < 7.0%). Finally, HS-SPME followed by GC/MS was applied to fast determination of alpha- and beta-asarone in rabbit plasma at different time points after oral adminstration of the essential oil from A. tatarinowii. The experimental results suggest that the proposed method provides an alternative approach to the PK studies of volatile compounds in TCMs., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Intrinsic clearances (CLint-HLM-total) for the metabolism of NE-100, metoprolol, clarithromycin (CAM), lornoxicam and tenoxicam were predicted from in vitro data with recombinant cytochorme P450s (CYPs) using relative activity factor (RAF) and then compared with CLint-HLM observed in human liver microsomes (HLM). The predicted CLint-HLM-total correlated well with the observed CLint-HLM in HLM. When oral clearances (CLoral) of low-clearance drugs such as metoprolol, CAM, lornoxicam and tenoxicam were predicted from the in vitro data using four physiological models (well-stirred, parallel tube, distributed and dispersion models), the predicted CLoral corresponded well with the observed CLoralin vivo and were similar among the four models. For a high-clearance drug, the predicted CLoral of NE-100 in extensive CYP2D6 metabolizers (EMs) was substantially different between individual models, although the predicted CLoral in a poor metabolizer of CYP2D6 (PMs) was similar. The CLoral ratio of NE-100 between the EMs and the PMs predicted from the dispersion model, which leads to a reliable prediction for the high-clearance drug, was 48.4, but the ratio decreased depending on the increase of the NE-100 plasma concentration. The results suggest that the CLoral decrease in the EMs is caused by saturation of NE-100 metabolism mediated by CYP2D6 and is based on increases in plasma NE-100 concentrations dependent on the dose of NE-100. The study suggests that the RAF and the in vitro-in vivo scaling approaches are useful for predicting CLoral from in vitro data with recombinant CYPs without using HLM and hepatocytes.

Structure-activity relationships in rhesus monkeys for a novel mixed-onium class of ultra-short-acting nondepolarizing tetrahydroisoquinolinium neuromuscular blockers (NMBs) are described. Bis-onium chlorofumarate 20a with (1R,2S)-benzyltetrahydroisoquinolinium groups was a potent lead compound (ED(95) = 0.079 mg/kg) with an ultra-short duration of NMB effect (7.1 min) and a selectivity index (SI: defined as a ratio of the cardiovascular threshold dose to the ED(95)) similar to that of mivacurium (3). The mean threshold dose for cardiovascular effects with 20a was ca. 20 times its ED(95) value (SI = 20). A novel mixed-onium analogue of 20a was prepared by replacing the benzyltetrahydroisoquinolinium group distal to the fumarate chlorine atom with a (1S,2R)-phenyltetrahydroisoquinolinium moiety. The resulting mixed-onium chlorofumarate 24a displayed good NMB potency (ED(95) = 0.063 mg/kg), ultra-short duration of action (5.6 min) and an improved selectivity index (SI = 57). Several other mixed-onium derivatives containing octanedioate (25a; ED(95) = 0.103 mg/kg), difluorosuccinate (27c; ED(95) = 0.056 mg/kg), and fluorofumarate (28a; ED(95) = 0.137 mg/kg) linkers were also potent, ultra-short-acting NMBs with good to excellent selectivity index values (SI = 37-96). Octanedioate 25a was longer acting at higher doses compared to difluorosuccinate 27c and chlorofumarate 24a. Durations of NMB effect following a 0.4 mg/kg bolus dose (100% block) of 25a, 27c, and 24a were 16.9, 13.0, and 10.0 min, respectively. Recovery time for mixed-onium chlorofumarate 24a following a 1 h continuous infusion at 10-20 microg/kg/min (95-100% block) was ca. 5 min which is similar to that observed following a 0.2 mg/kg bolus dose of this compound and indicates a lack of cummulative effects. Preliminary studies with chlorofumarate 24a in whole human blood revealed that mixed-onium thiazolidine 29 was the major metabolite and that plasma cholinesterases do not play the primary role in duration of NMB effect. The NMB properties of 24a in rhesus monkeys led to its clinical evaluation as a possible alternative to succinylcholine.

A solid phase method for direct radioimmunoassay in plasma of the new renin inhibitor CGP 60536 has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed a good reproducibility down to plasma concentrations of 0.15 ng/ml (LOQ) with intra- and inter-assay coefficients of variation < or = 20%. The procedure, which requires only small volumes of plasma (25 microl), is simple to use and well suited for routine analysis. The method allows the investigation of the pharmacokinetics of CGP 60536 in animals and man given low oral doses of the drug.

A fully automated high-performance liquid chromatography method with fluorescence detection for the determination of the renin inhibitor CGP 60536 in animal and human plasma and urine has been developed and validated. After addition of an internal standard, the compounds were automatically extracted from 400 microl of plasma or urine with methyl alcohol-acetic acid (99:1, v/v) on 100-mg Bond-Elut CN cartridges using the Gilson ASPEC system. The on-line chromatographic separation was performed on a LiChrospher 100 RP8 5-microm particle size packed analytical column (25x0.4 cm I.D.). The mobile phase consisted of acetonitrile-0.01 M potassium dihydrogenphosphate (65:35, v/v) at a flow-rate of 0.8 ml/min. The analytes were detected using a fluorescence detector at excitation and emission wavelengths of 280 and 330 nm, respectively. The limit of quantitation was established at 4.5 ng/ml in plasma (accuracy 106% and precision 1%), and 9.0 ng/ml in urine (accuracy 101% and precision 13%). The method was applied to the investigation of the pharmacokinetics of CGP 60536.

Solagé is a combination product composed of 2% mequinol (4-hydroxyanisole) and 0.01% tretinoin (all-trans-retinoic acid) in an ethanolic solution, which is being studied for its safety and efficacy as a topical treatment for disorders of skin hyperpigmentation. The purpose of this study was to evaluate the extent of percutaneous absorption of [3H]tretinoin and to estimate the systemic exposure to mequinol from this combination product when topically applied to the backs of healthy subjects. Eight subjects received bid topical applications of nonradiolabelled 2% mequinol/0.01% tretinoin solution on a 400 cm2 area of the back for 14 days. The subjects then received a single topical application of 2% mequinol/0.01% [3H]tretinoin solution. After 12 h, the radiolabelled dose was removed and bid treatment with nonradiolabelled 2% mequinol/0.01% tretinoin solution was continued for 7 days. Plasma, urine and faecal samples were analysed for total radioactivity and plasma was analysed for both mequinol and tretinoin by GC/MS procedure. Mean percutaneous absorption of [3H]tretinoin based on the cumulative recoveries of radioactivity in the urine and faeces was about 4.5% (median 2.18%). Tretinoin concentrations in plasma did not increase above endogenous levels. This was consistent with the concentrations of radioactivity in plasma, which showed an average Cmax of 91 pg-eq/mL (median 26 ng/mL). Average Cmax and AUC(0-12 h) values for mequinol were 10 ng/mL and 33 ng h/mL, respectively. Based on the results of this study, systemic toxicity from topical application of tretinoin in this formulation is unlikely, because percutaneous absorption of tretinoin is minimal and because endogenous levels of tretinoin are not increased following bid dosing with this combination formulation. The safety of mequinol in this combination formulation is supported by the low systemic exposures of the subjects in this study compared with the systemic exposures at the highest doses in the dermal toxicity studies in mice (16.6-fold) and rats (34.6-fold).

For the highly sensitive and selective determination of NE-100, a novel sigma ligand, at levels of low picogram per milliliter of human plasma, a method with excellent reliability employing liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) combined with a column-switching technique has been developed. The method involves the use of a stable isotope labeled compound as the internal standard (I.S.), liquid-solid extraction of a plasma specimen with a C8 cartridge, automated on-line clean-up on a short trapping column, subsequent separation on a micro-bore C18 column and detection with ESI-MS-MS using m/z 356 ([M+H]+) as a precursor ion and m/z 105 as a product ion in a selected reaction monitoring mode. The detection and the quantification limits of NE-100 in plasma were 0.5 pg/ml with a signal-to-noise ratio (S/N) of 3 and 2.3 pg/ml, respectively, with an S/N of 21. The good linearity of the calibration graph was obtained in the range of 2.3 to approximately 907.0 pg/ml with excellent reliability. The developed method was applied to the determination of NE-100 in plasma obtained from the clinical trail.

Tissue distribution, excretion and metabolism studies of pentachloroanisole (PCA), an environmental metabolite of pentachlorophenol (PCP), were conducted in the beagle dog and miniature pig following single oral doses (25 mg/kg) of radiolabelled PCA. PCA was readily demethylated by both species, with a half-life of 5-8 min. The resultant PCP was the major metabolite in dogs and pigs. In the dog, an average of 21.9% of the administered radiolabel was excreted in the urine and 62.3% in the faeces during a 7-day period. Of the tissues analysed, an average of 3.2% of the radiolabel remained in the liver, and blood and muscle accounted for averages of 3.0 and 2.3%, respectively, of the dose. Free and conjugated PCP were found in the urine of dogs; no PCA or tetrachlorohydroquinone (TCH) were found. In dog faeces, PCP and a trace of polar material were observed; no PCA was excreted in dog faeces. In the miniature pig, an average of 25.8% of the administered radiolabel was excreted in the urine and 32.0% in the faeces during a 2-wk period. An average of 4.4% of the radiolabel was found in the liver, 8.8% in the blood, 7.1% in the muscle and 6.4% in the fat. In pig urine, PCP and conjugated PCP were the only metabolites observed; no PCA or TCH was found. Pig faeces contained a trace of unchanged PCA; PCP and polar metabolites were also found. Since pig tissues retained a sizeable residue 2 wk after a single dose of PCA, various agents were used in an attempt to decrease the tissue level of radiolabel in pigs; anion exchange resin was found to be the most effective.

A simple and sensitive high-performance liquid chromatography (HPLC) method for the determination of asarone in rabbit plasma has been developed. Up to 0.1 mL of plasma containing asarone was deproteinated by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected into a Nucleosil 7C18 column using acetonitrile-water-triethylamine (55:45:0.1 v/v, pH 5.4-5.5, adjusted with orthophosphoric acid) as the mobile phase and UV detection at 257 nm, followed by UV spectrum identification (between 200 and 380 nm) with a photodiode array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of asarone in rabbit, after 5, 10, or 20 mg kg-1 intravenous administration. Rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. The plasma disposition at each dose fitted well to a two-compartment open model and the terminal disposition became much slower as the dose was increased, suggesting a nonlinear dose-dependent plasma asarone disposition.

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.

A single treatment with 5-HT uptake inhibitors potentiates the hypermotility in mice produced by the MAO-inhibitor nialamide. The effect of nialamide on motility was studied in mice after 4 weeks of feeding with a normal diet and diets containing various concentrations of the 5-HT uptake inhibitors chlorimipramine and femoxetine. Chronic treatment with the two substances enhanced the motor effects of nialamide about equally, which indicates a preservation of the neuronal 5-HT uptake inhibition during such treatment. The effect of chlorimipramine and femoxetine was obtained at plasma levels equivalent to or lower than the steady-state plasma concentrations found in patients treated with two 5-HT uptake inhibitors. Determination of decreased blood 5-HT after the 4 weeks of treatment was used as an in vivo test for inhibition of 5-HT uptake into platelets. Femoxetine was a much weaker depletor of blood 5-HT than chlorimipramine. These results indicate that blockade of neuronal 5-HT uptake is obtained at lower doses of femoxetine than blockade of 5-HT uptake into platelets. In contrast, chlorimipramine presumably inhibits 5-HT uptake into neurons and platelets at about the same dose.

A method is described for the determination of the antioxidant 3-tert.-butyl-4-hydroxy-anisole in rat plasma using high-resolution capillary gas chromatography-mass spectrometry with selective ion monitoring. Following the addition of the isomer 2-tert.-butyl-4-hydroxy-anisole, used as an internal standard, extraction was made with n-hexane and the extract derivatized with heptafluorobutyric anhydride. The gas chromatographic separation was carried out on a SE-52 fused silica capillary column and the derivatized 3-tert.-butyl-4-hydroxyanisole and its isomer detected by recording the intensities of their common fragment ion at m/e 361. The sensitivity of the method allowed the antioxidant to be measured in 0.1-ml rat plasma samples down to a level of 10 ng/ml with a high degree of specificity and accuracy. The method has been applied to a preliminary pharmacokinetic study in rats after oral dosage.

A procedure has been developed for the gas chromatographic determination of a structurally new 5HT-uptake inhibitor with antidepressant properties (FG 4963) in plasma and urine. The method involves an extraction from alkaline solution with n-pentane, and a quantitative determination by gas chromatography using an internal standard and a nitrogen-sensitive detector. The binding of FG 4963 to glass during storage and evaporation necessitates the addition of a structurally similar compound to the vials used for collection of the blood and urine, in order to compete for the binding sites of the glass. The method is suitable for pharmacokinetic and clinical studies, the sensitivity being ca. 5 ng/ml in plasma and 1 ng/ml in urine and the precision being 10-15% for concentrations greater than 10 ng/ml.

4-Hydroxyanisole (p-methoxyphenol) has been used in the treatment of malignant melanomas. A simple, sensitive, and specific, method for its determination by liquid chromatography with electrochemical detection (LCEC) is described. Vanillin (4-hydroxy-3-methoxybenzaldehyde) was used as an internal standard.

A GC/Ms method is described for the determination of the antioxidant 2-t-butyl-4-methoxyphenol (BHA) in plasma using Sep-Pak C18 cartridges for extraction. Picogram amounts of BHA could be detected in rat plasma with a high degree of specificity, accuracy and significant time and material saving.