Sensitive high-performance liquid chromatographic method for the determination of 2-phenylethylamine in human urine

A new sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of 2-phenylethylamine (PEA) in human urine. The analytical procedure involved a simple extraction of the analyte from urine, followed by precolumn derivatisation of the sample with o-phthalaldehyde. The HPLC separation was performed under isocratic conditions using an Erbasil S C₁₈ (250 × 4.0 mm I.D., particle size 3 μm) reversed-phase column. The limit of quantification was 0.5 ng of PEA/ml of urine. The method showed good linearity, accuracy and precision data in the concentration range 0.5–200 ng/ml of urine. The method was successfully applied to the determination of PEA urinary excretion in Parkinsonian patients after oral administration of the monoamine oxidase B (MAO-B) inhibitor, selegiline.     

Simultaneous quantitative analysis of N-acylethanolamides in clinical samples

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d₄-AEA as the internal standard. The method is linear up to 100 ng/ml with a limit of quantitation of 50 pg/ml for AEA and 100 pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.     

High-pressure liquid chromatographic determination of 13-cis-retinoic acid and all-trans-retinoic acid in human plasma

A simple and selective method is described for the determination of 13-cis-retinoic acid and all-trans-retinoic acid in plasma. The assay involves lyophilization of the sample, extraction of the residue with methanol, and separation of the retinoids by reverse-phase high-pressure liquid chromatography. The procedure can detect as little as 0.05 μg of either compound. It is capable of being readily modified to determine the amount of retinoic acid in other tissues as well as tissue concentrations of other retinoids. Using this assay, the absorption and disappearance from human plasma of an oral dose of 13-cis-retinoic acid have been followed.     

Biogenic amine degradation by homogenates of the bulb mite,Rhizoglyphus echinopus (Fumouze and Robin) (Acari: Astigmata: Acaridae)

Whole homogenates of bulb mites rapidly metabolized 2-phenylethylamine (PEA) but were appreciably less active against tryptamine, 5-hydroxytryptamine, and dopamine; no degradation of octopamine was detected. The rate of PEA degradation by bulb mites was dependent upon both substrate and homogenate concentrations. PEA degradation was inhibited by pargyline (pI₅₀, 6.7), tranylcypromine (pI_{50 6.2}), and harmaline (pI_{50 4.1}), but not by 5-chloro-2,4-dimethoxyformanilide. These results suggested that PEA metabolism by bulb mite homogenates was catalyzed mainly by Type B monoamine oxidase.Contribution from the Missouri Agricultural Experiment Station, Columbia, MO. Journal Series No. 9777     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Simultaneous extraction and separation of trace amines of biological interest

A method is described for the simultaneous extraction and separation of the trace amines 2-phenylethylamine, m-tyramine, p-tyramine, p-octopamine, normetanephrine, and 3-methoxytyramine. The method involves acetylation in aqueous solution, specific hydrolysis of phenolic acetate groups, derivatization with trifluoroacetic anhydride and analysis on a gas chromatograph equipped with an electron-capture detector. Analyses utilizing both packed glass columns and glass capillary columns are described.The method possesses the potential for quantitative as well as qualitative analysis, with one or more of the following amines employed as internal standards: benzylamine, 3-phenylpropylamine, tranylcypromine, and 2-(4-chlorophenyl)ethylamine.     

Simultaneous analysis of diphenylmethoxyacetic acid,a metabolite of diphenhydramine,and its deuterium-labeled stable isotope analog in ovine plasma and urine

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([²H₁₀]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography—electron-impact mass spectrometry (GC—EI-MS) analysis of DPMA and [²H₁₀]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-μl aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [²H₁₀]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [²H₁₀]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [²H₁₀]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [²H₁₀]DPMA in biological samples obtained from sheep (e.g. plasma and urine).     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Gas chromatographic–mass spectrometric analysis of perillyl alcohol and metabolites in plasma

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1′-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC–MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC–MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH₂Cl₂ extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC–MS using NH₃ chemical ionization. POH-d₃ was used as the internal standard for POH while ¹³C-PA-d₂ was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1–2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m² p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane—isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound α₁-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

Monoamine Oxidase Activities in Catfish (Parasilurus Asotus) Tissues

Abstract

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For both tissues, the V_max values were highest with 5-HT and lowest with PEA. The K_m value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 μM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.     

Improved sample preparation method for selected persistent organochlorine pollutants in human serum using solid-phase disk extraction with gas chromatographic analysis

An improved solid-phase extraction (SPE) method was developed to isolate and concentrate trace levels of selected POPs (persistent organochlorine pollutants) in human serum prior to GC–MS in SIM mode or GC–ECD quantitation. The extraction involves denaturation of serum proteins with formic acid, SPE using C₁₈ Empore™ disk cartridges, followed by elimination of lipid interferences using a sulfuric acid wash of the eluate. Use of the SPE disk improved assay throughput and gave a cleaner analytical matrix compared with previously reported solid-phase and liquid–liquid extraction techniques. The extraction method provided consistent recoveries at three fortification levels using ¹³C₁₂ PCB 149 as internal standard. Recoveries ranged from 48 to 140% for organochlorine pesticides (6.25, 12.5 and 25 ng/ml) and 71 to 126% for polychlorinated biphenyls (0.625, 1.25 and 2.5 ng/ml).     

Quantitation of indoleacetic Acid conjugates in bean seeds by direct tissue hydrolysis

Gas chromatography-selected ion monitoring-mass spectral analysis using [¹³C₆]indole-3-acetic acid (IAA) as an internal standard provides an effective means for quantitation of IAA liberated during direct strong basic hydrolysis of bean (Phaseolus vulgaris L.) seed powder, provided that extra precautions are undertaken to exclude oxygen from the reaction vial. Direct seed powder hydrolysis revealed that the major portion of amide IAA conjugates in bean seeds are not extractable by aqueous acetone, the solvent used commonly for IAA conjugate extraction from seeds and other plant tissues. Strong basic hydrolysis of plant tissue can be used to provide new information on IAA content.     

Further evidence for a role of 2-phenylethylamine in the mode of action of delta 9-tetrahydrocannabinol

In rabbits, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) increased the recovery of labeled 2-phenylethylamine (PEA) from brain following its intraventricular administration. Δ⁹-THC also enhanced the excitatory effect of iontophoretic PEA on cortical unit potentials. Although Δ⁹-THC induced sedation in mice, the subsequent injection of reserpine induced transient excitement. Low doses of PEA, which do not significantly alter the behavior of mice, induced marked excitement in mice pretreated with Δ⁹-THC. In mice treated with pargyline, Δ⁹-THC induced excitement (instead of sedation); this excitement was increased by PEA and reduced by phenylethanolamine. These results suggest that Δ⁹-THC inhibits the disposition of PEA. Since endogenous PEA may be one of the adrenergic ergotropic modulators, it may play a role in the euphoriant effect of marihuana.     

A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes

A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.     

A highly sensitive and rapid fluorometric assay for UDP-glucuronyltransferase using 3-hydroxybenzo (a) pyrene as substrate.

A rapid and highly sensitive fluorometric assay for UDP-glucuronyltransferase (EC 2.4.1.17) has been devised using 3-hydroxybenzo(a)pyrene as substrate. The sensitivity of the procedure is based on (a) the high coefficient of fluorescence of the product, benzo(a)pyrene-3-glucuronide, and (b) the very low background which is obtained by an efficient differential extraction of substrate and product and their widely differing fluorescence characteristics in alkaline solution. As little as 5–10 pmol of product can be determined. The procedure involves essentially a single extraction and transfer step. The method may be applicable in measuring transferase activity in a few micrograms of tissue protein or of cultured cells as well as in the routine processing of large numbers of samples. Some of the properties of glucuronyltransferase activity directed toward 3-hydroxybenzo(a)pyrene are described such as kinetic constants and the sensitivity of the reaction to detergents and organic solvents.     

Trifluoromethyl-modified dipeptides by ZrCl4-promoted aza-Henry reactions

Chiral (R)-1-phenylethylamine was successfully employed in a tandem aza-Henry addition–reduction reaction to give chiral β-nitro α-trifluoromethyl amines. A subsequent coupling reaction with N-Boc-protected amino acids leads to obtain optically pure CF₃-modified dipeptides carrying two different N-protecting groups. These peptidomimetic units are characterized by the presence of the [CH(CF₃)NH] group as mimetic of the natural [CONH] peptidic bond and can be used for the synthesis of more complex CF₃-modified peptides after selective deprotection of one of the two amine functions. 2D NMR spectral analyses were employed to determine the absolute configurations of all newly synthesized chiral compounds.     

A sensitive method for quantitation of gamma-hydroxybutyric acid and gamma-butyrolactone in brain by electron capture gas chromatography.

A new and sensitive method for the quantitation of γ-hydroxybutyric acid (GHB) and its lactone precursor γ-butyrolactone (GBL), has been developed and successfully utilized to determine the endogenous content of these compounds in a single rat brain. The method involves conversion of endogenous GHB into GBL and extracting the GBL with CHCl₃. The concentrated CHCl₃ extract is treated with BF₃ methanol reagent to produce methyl γ-hydroxybutyrate. Introduction of electron capturing groups was accomplished by further reacting the methyl γ-hydroxybutyrate with heptafluorobutyric anhydride in the presence of pyridine. Prior to quantitation by electron capture gas chromatography, the sample was cleaned up by thin layer chromatography (tlc) using a preabsorbent plate which removed many extraneous peaks as well as CHCl₃ used as the solvent. The effciency of the procedure was evaluated by carrying [1-¹⁴C]GBL through the derivatization. This indicated that about 15% of the starting labeled GBL was converted to the final electron capturing product. δ-Valerolactone was used as an internal standard.     

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).