Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (Kt) of 108, 93, and 117 μM and maximal velocities (Vmax) of 19, 10, and 21 nmol min−1 mg of protein−1 for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.
The rdpA and sdpA genes encode two enantioselective α-ketoglutarate-dependent dioxygenases catalyzing the initial step of microbial degradation of the chiral herbicide (R,S)-2-(2,4-dichlorophenoxy)propionate (R,S-dichlorprop). Primers were designed to assess abundance and transcription dynamics of rdpA and sdpA genes in a natural agricultural soil. No indigenous rdpA genes were detected, but sdpA genes were present at levels of approximately 103 copies g of soil−1. Cloning and sequencing of partial sdpA genes revealed a high diversity within the natural sdpA gene pool that could be divided into four clusters by phylogenetic analysis. BLASTp analysis of deduced amino acids revealed that members of cluster I shared 68 to 69% identity, cluster II shared 78 to 85% identity, cluster III shared 58 to 64% identity, and cluster IV shared 55% identity to their closest SdpA relative in GenBank. Expression of rdpA and sdpA in Delftia acidovorans MC1 inoculated in soil was monitored by reverse transcription quantitative real-time PCR (qPCR) during in situ degradation of 2 and 50 mg kg−1 of (R,S)-dichlorprop. (R,S)-Dichlorprop amendment created a clear upregulation of both rdpA and sdpA gene expression during the active phase of 14C-labeled (R,S)-dichlorprop mineralization, particularly following the second dose of 50 mg kg−1 herbicide. Expression of both genes was maintained at a low constitutive level in nonamended soil microcosms. This study is the first to report the presence of indigenous sdpA genes recovered directly from natural soil and also comprises the first investigation into the transcription dynamics of two enantioselective dioxygenase genes during the in situ degradation of the herbicide (R,S)-dichlorprop in soil.
A reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for determination of aminoflavone (AF) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 mL of plasma with 1.0 mL ethyl acetate containing 50 ng/mL of the internal standard zileuton. The analytes were separated on a Waters X-Terra™ MS C18 column using a mobile phase consisting of methanol/water containing 0.45% formic acid (70:30, v/v) and isocratic flow at 0.2 mL/min for 6 minutes. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the AF concentration range of 5–2000 ng/mL in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL for AF in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully applied to characterize AF plasma concentration-time profile in the cancer patients in a phase I trial.
Aminoflavone; High performance liquid chromatography; Mass spectrometry; LC-MS/MS; Pharmacokinetics
A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of ABT-888 and its major metabolite (M8) in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.25 ml of plasma with 10μl of 1M NaOH and 1.0 ml ethyl acetate containing 50 ng/ml of the internal standard zileuton. The analytes were separated on a Waters XBridge C18 column using a gradient mobile phase consisting of methanol/water containing 0.45% formic acid at the flow rate of 0.2 ml/min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the ABT-888 and M8 concentration ranges of 1-2000 ng/ml in human plasma. The lower limits of quantitation (LLOQ) were 1 ng/ml for both ABT-888 and M8 in human plasma. The accuracy and within- and between-day precisions were within the generally accepted criteria for bioanalytical method (<15%). This method was successfully employed to characterize the plasma concentration-time profile of ABT-888 after its oral administration in cancer patients.
ABT-888; PARP inhibitor; High performance liquid chromatography; Mass Spectrometry; LC-MS/MS; Pharmacokinetics
A liquid chromatography-tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue was developed and fully validated. Methanol was used to precipitate proteins in brain tissue. Bendamustine and internal standard (chlorambucil) were separated with reverse phase chromatography on a C-18 column with a gradient of water and 95% methanol in 0.1% formic acid. Positive mode electrospray ionization was applied with selected reaction monitoring to achieve 5 ng/ml lower limits of quantitation in mouse brain tissue. The calibration curve for bendamustine in mouse brain was linear between 5 and 2000 ng/ml. The within- and between- batch accuracy and precision of the assay were within 15% at 10, 100 and 1000 ng/ml. The recovery and matrix effect of bendamustine in mouse brain tissue ranged from 41.1% to 51.6% and 107.4% to 110.3%, respectively. Validated the method was then applied to quantitate bendamustine in an animal study. Results indicate the assay can be applied to evaluate bendamustine disposition in mouse brain tissue. This assay will be applied in the future to detect and quantify bendamustine in human brain tissue samples.
Bendamustine; Liquid Chromatography-Mass Spectrometry; LC-MS/MS; Brain
A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was developed for the determination of berberine, palmatine and jatrorrhizine in rat plasma. Target compounds, together with the internal standard (metronidazole), were extracted from rat plasma samples by protein precipitation with acetonitrile-methanol (1:2, v/v). Chromatography was carried out using a C18 column (150 × 4.6mm, 5μm) under isocratic elution with water (containing 0.3% formic acid)-acetonitrile (30:70, v/v). The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in positive ionization mode. The method was linear over the concentration range of 0.2-100 ng/mL for all components. The intra- and inter-day precision values were less than 14.7% and the deviations were within ± 9.0%. The validated method was applied to the comparative pharmacokinetic studies of berberine, palmatine and jatrorrhizine after oral administration of Rhizoma coptidis and Zuojinwan. The results indicated that the pharmacokinetics of berberine, palmatine and jatrorrhizine were significantly different between different groups.
Rhizoma coptidis; Zuojinwan preparation; Liquid chromatography tandem mass spectrometry; Pharmacokinetics
In the title compound, the herbicide bifenox, C14H9Cl2NO5, the dihedral angle between the dichlorobenzene and nitrobenzene rings is 78.79 (14)°. In the crystal, C—H⋯O hydrogen bonds give rise to a three-dimensional network structure in which there are both a π–π interaction [ring centroid separation = 3.6212 (16) Å] and a C—Cl⋯π interaction [Cl⋯ring centroid = 3.4754 (8) Å]. In addition, short Cl⋯Cl contacts [3.3767 (11) and 3.3946 (11) Å] are present.
A simple, sensitive, and reliable analytical method is developed for the rapid determination of fumonisin B1 and fumonisin B2 in corn by high-performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (LC–ESI-MS–MS). Fumonisin B1 and fumonisin B2 are extracted from corn with methanol–water (3:1, v/v) by means of ultrasonic extraction, and directly injected into an LC–MS–MS system after centrifugation. Fumonisin B1 and fumonisin B2 are separated on a Zorbax Eclipse XDB-C18 column with a solution of methanol–water–formic acid as the mobile phase. The method is validated with respect to linearity, accuracy, precision, specificity, and stability. Moreover, the method was applied to real samples and demonstrated to be suitable for the determination of fumonisin B1 and fumonisin B2 in corn. The total time required for the analysis of one sample was ∼30 min.
A rapid, sensitive and specific analytical method based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of thalidomide concentration in human plasma. The analyte and internal standard were extracted by liquid-liquid extraction with ether-dichloromethane (3:2, v/v) and separated on a TC-C18 column using methanol-10 mM ammonium acetate-formic acid (60:40:0.04, v/v/v) as the mobile phase at a flow rate of 0.9 ml/min. The detection was performed using an API 4000 triple quadrupole mass spectrometer in the positive electrospray ionization (ESI) mode and completed within 3.0 min. The multiple reaction monitoring (MRM) transitions were m/z 259.1→84.0 for the analyte and m/z 195.9→138.9 for temozolomide. The calibration curve exhibited a linear dynamic range of 2–1500 ng/ml (r>0.9991). The intra-and inter-day precisions (as relative standard deviation; RSD) were 6.8–13.5% and 4.3–5.0% respectively and the accuracy (as relative error; RE) was 2.0–3.5%. The recoveries and matrix effects were satisfactory in all the biological matrices examined. This method was successfully used in a pharmacokinetic study of thalidomide in healthy male volunteers receiving an oral administration of a 200-mg dose.
human plasma; thalidomide; multiple reaction monitoring; liquid chromatography-tandem mass spectrometry
A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of ε-acetamidocaproic acid (AACA), the primary metabolite of zinc acexamate (ZAC), in rat plasma by using normetanephrine as an internal standard. Sample preparation involved protein precipitation using methanol. Separation was achieved on a Gemini-NX C18 column (150 mm × 2.0 mm, i.d., 3 μm particle size) using a mixture of 0.1% formic acid-water : acetonitrile (80 : 20, v/v) as the mobile phase at a flow rate of 200 μl/min. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 4.0 min, and the calibration curves of AACA were linear over the concentration range of 20~5000 ng/ml in rat plasma. The coefficient of variation and relative error at four QC levels were ranged from 1.0% to 5.8% and from −8.4% to 6.6%, respectively. The present method was successfully applied for estimating the pharmacokinetic parameters of AACA following intravenous or oral administration of ZAC to rats.
Zinc acexamate (ZAC); ε-Acetamidocaproic acid (AACA); Liquid chromatography tandem mass spectrometry (LC-MS/MS); Rat; Pharmacokinetics
Folate metabolism, which is responsible for one-carbon transfer reactions in critical cellular processes including thymidine biosynthesis, is among the most important targets of antibiotic and anticancer drugs. Analysis of intracellular folates is complicated by three different types of folate modification: oxidation/reduction, methylation, and polyglutamylation. Here we present a method for quantifying the full diversity of intracellular folates by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method begins with folate extraction using −75°C methanol:water, with ascorbic acid and ammonium acetate added to prevent folate interconversion. The extract is then separated using hydrophilic interaction chromatography with an amino column, ionized by positive mode electrospray, and analyzed on a triple quadrupole instrument using multiple reaction monitoring. The method has been used to profile the folate pools in Escherichia coli and Saccharomyces cerevisiae, with absolute levels of selected folates in E. coli measured by spiking extracts of cells fed uniformly 13C-glucose with purified, unlabeled folate standards. An isotope-ratio-based approach has been applied to study the effects of trimethoprim, a clinically important antibiotic that blocks bacterial dihydrofolate reductase. In addition to causing the expected increase in oxidized and decrease in reduced folates, trimethoprim triggered a dramatic and previously unrecognized shift towards shorter polyglutamate chain lengths. This finding highlights the potential for analysis of the full spectrum of cellular folates by MS/MS to unveil novel biological phenomena.
Two novel genes, rdpA and sdpA, encoding the enantiospecific α-ketoglutarate dependent dioxygenases catalyzing R,S-dichlorprop cleavage in Delftia acidovorans MC1 were identified. Significant similarities to other known genes were not detected, but their deduced amino acid sequences were similar to those of other α-ketoglutarate dioxygenases. RdpA showed 35% identity with TauD of Pseudomonas aeruginosa, and SdpA showed 37% identity with TfdA of Ralstonia eutropha JMP134. The functionally important amino acid sequence motif HX(D/E)X23-26(T/S)X114-183HX10-13R/K, which is highly conserved in group II α-ketoglutarate-dependent dioxygenases, was present in both dichlorprop-cleaving enzymes. Transposon mutagenesis of rdpA inactivated R-dichlorprop cleavage, indicating that it was a single-copy gene. Both rdpA and sdpA were located on the plasmid pMC1 that also carries the lower pathway genes. Sequencing of a 25.8-kb fragment showed that the dioxygenase genes were separated by a 13.6-kb region mainly comprising a Tn501-like transposon. Furthermore, two copies of a sequence similar to IS91-like elements were identified. Hybridization studies comparing the wild-type plasmid and that of the mutant unable to cleave dichlorprop showed that rdpA and sdpA were deleted, whereas the lower pathway genes were unaffected, and that deletion may be caused by genetic rearrangements of the IS91-like elements. Two other dichlorprop-degrading bacterial strains, Rhodoferax sp. strain P230 and Sphingobium herbicidovorans MH, were shown to carry rdpA genes of high similarity to rdpA from strain MC1, but sdpA was not detected. This suggested that rdpA gene products are involved in the degradation of R-dichlorprop in these strains.
A rapid method to determine fexofenadine concentrations in human plasma using protein precipitation in 96-well plates and liquid chromatography-tandem mass spectrometry was validated. Plasma proteins were precipitated with acetonitrile containing the internal standard fexofenadine-d6, mixed briefly, and then filtered into a collection plate. The resulting filtrate was diluted and injected onto a Phenomenex Gemini C18 (50 × 2.0 mm, 5 micron) analytical column. The mobile phase consisted of 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (35:65, v/v). The flow rate was 0.2 ml/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization and high resolution multiple reaction monitoring mode (H-SRM). The linear standard curve ranged from 1 to 500 ng/ml and the precision and accuracy (intra- and inter-run) were within 4.3% and 8.0%, respectively. The method has been applied successfully to determine fexofenadine concentrations in human plasma samples obtained from subjects administered a single oral dose of fexofenadine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving fexofenadine.
Fexofenadine; fexofenadine-d6; protein precipitation; LC-MS; human plasma
Liquid chromatography-electrospray ionization-mass spectrometry and liquid chromatography-tandem mass spectrometry methods were developed to simultaneously determine the concentrations of angiotensin (Ang) II, Ang 1-7, Ang III and Ang IV in biological samples. The samples were extracted with C18 solid-phase extraction cartridges, and separated by a reverse-phase C18 column using acetonitrile in water with 0.1 % formic acid as a mobile phase. Angiotensin peptides were ionized by electrospray and detected by triple quadrupole mass spectrometry in the positive ion mode. (M+3H)3+ and (M+2H)2+ ions were chosen as the detected ions in the single ion recording (SIR) mode for LC-MS. The limits of detection (S/N=3) using SIR are 1 pg for Ang IV and 5 pg for Ang 1-7, Ang III and Ang II. Multiple reaction monitoring (MRM) mode was used for LC-MS/MS. The limits of detection (S/N=3) using MRM are 20 pg for Ang IV and 25 pg for Ang 1-7, Ang III and Ang II. These methods were applied to analyze angiotensin peptides in bovine adrenal microvascular endothelial cells. The results show that Ang II is metabolized by endothelial cells to Ang 1-7, Ang III and Ang IV with Ang 1-7 being the major metabolite.
Angiotensin II; mass spectrometry; liquid chromatography; electrospray ionization; adrenal cortex
Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization-mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid that severely interfered with sample analysis. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are composed of three components; clusters of trifluoroacetic acid, clusters of mass 159 and iron. Formation of these ions is inhibited by removing trifluoroacetic acid from the mobile phases and using formic acid in its place, replacing the stainless steel union with a titanium union or by adding a small blank fused silica capillary column between the chromatography column and the electrospray tip via a stainless steel union without any adverse effects to chromatographic separation, peak broadening or peptide identifications.
Electrospray ionization; Liquid Chromatography-Mass Spectrometry; Peptides; Iron complex
Three mathematical models were proposed to describe the effects of sorption of both bacteria and the herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) on the biological degradation rates of 2,4-D in soils. Model 1 assumed that sorbed 2,4-D is not degraded, that only bacteria in solution are capable of degrading 2,4-D in solution, and that sorbed bacteria are not capable of degrading either sorbed or solution 2,4-D. Model 2 stated that only bacteria in the solution phase degrade 2,4-D in solution and that only sorbed bacteria degrade sorbed 2,4-D. Model 3 proposed that sorbed 2,4-D is completely protected from degradation and that both sorbed and solution bacteria are capable of degrading 2,4-D in solution. These models were tested by a series of controlled laboratory experiments. Models 1 and 2 did not describe the data satisfactorily and were rejected. Model 3 described the experimental results quite well, indicating that sorbed 2,4-D was completely protected from biological degradation and that sorbed- and solution-phase bacteria degraded solution-phase 2,4-D with almost equal efficiencies.
Yakuchinone A has a plethora of beneficial biological effects. However, the pharmacokinetic (PK) data of yakuchinone A still remain unknown so far. Furthermore, the quantification of yakuchinone A in biological samples has not been reported in the literature. Therefore, in the present study we aimed to develop a new method for the fast, efficient and accurate assessment of yakuchinone A concentration in plasma, as a means for facilitating the PK evaluation of yakuchinone A.
A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the determination of yakuchinone A in rat plasma. Mass spectrometric and chromatographic conditions were optimized. Plasma samples were pretreated by protein precipitation with methanol. LC separation was performed on a Phenomenex Luna C18 column with gradient elution using a mobile phase consisting of methanol–water containing 0.5 mM formic acid (HCOOH) at a flow rate of 0.28 mL/min. ESI-MS spectra were acquired in positive ion multiple reaction monitoring mode (MRM). The precursor-to-product ion pairs used for MRM of yakuchinone A and yakuchinone B were m/z 313.1 → 137.0 and 311.2 → 117.1, respectively. Low concentration of HCOOH reduced the ion suppression caused by matrix components and clearly improved the analytical sensitivity. Yakuchinone A showed good linearity over a wide concentration range (r > 0.99). The accuracy, precision, stability and linearity were found to be within the acceptable criteria. This new method was successfully applied to analyze the rat plasma concentration of parent yakuchinone A after a single oral administration of SuoQuan capsules. Low systemic exposure to parent yakuchinone A was observed.
The proposed method is sensitive and reliable. It is hoped that this new method will prove useful for the future PK studies.
Yakuchinone A; LC-MS/MS; Matrix effects; HCOOH; Pharmacokinetic study; SuoQuan capsules
A new method for the simultaneous determination of celecoxib, erlotinib, and its active metabolite desmethyl-erlotinib (OSI-420) in rat plasma, by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionization mode, was developed and validated. Protein precipitation with methanol was selected as the method for preparing the samples. The analytes were separated on a reverse-phase C18 column (50mm×4.6mm i.d., 3μ) using methanol: 2 mM ammonium acetate buffer, and pH 4.0 as the mobile phase at a flow rate 0.8 mL/min. Sitagliptin and Efervirenz were used as the internal standards for quantification. The determination was carried out on a Theremo Finnigan Quantam ultra triple-quadrupole mass spectrometer, operated in selected reaction monitoring (SRM) mode using the following transitions monitored simultaneously: positive m/z 394.5→278.1 for erlotinib, m/z 380.3→278.1 for desmethyl erlotinib (OSI-420), and negative m/z −380.1→ −316.3 for celecoxib. The limits of quantification (LOQs) were 1.5 ng/mL for Celecoxib, erlotinib, and OSI-420. Within- and between-day accuracy and precision of the validated method were within the acceptable limits of < 15% at all concentrations. The quantitation method was successfully applied for the simultaneous estimation of celecoxib, erlotinib, and desmethyl erlotinib in a pharmacokinetic study in Wistar rats.
Celecoxib; Erlotinib; Desmethyl erlotinib; OSI-420; LC-MS/MS; Bioanalytical
A sensitive and specific liquid chromatography electrospray ionisation–tandem mass spectrometry method for determination of new non-imidazole histamine H3 receptor antagonist 1-[3-(4-tert-butylphenoxy)propyl]piperidine (DL76) in rat serum has been developed and validated. Chromatography was performed on a XBridge™ C18 analytical column (2.1 × 30 mm, 3.5 µm, Waters, Ireland) with gradient elution using a mobile phase containing acetonitrile and water with an addition of 0.1% of formic acid. Detection was achieved by an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer. Electrospray ionization (ESI) was used for ion production. The limit of detection in the SRM mode was found to be 0.5 ng mL−1. The limit of quantification was 1 ng mL−1. The precision and accuracy for both intra- and inter-day determination of DL76 ranged from 1.65 to 15.09% and from 88.74 to 113.43%. The results of this analytical method validation allow to carry out pharmacokinetic studies in rats. The method was used for the pilot study of the pharmacokinetic behavior of DL76 in rats after intravenous administration.
Column liquid chromatography; Tandem MS detection; DL76 compound; Histamine H3 receptor antagonist; Rats serum
A molecularly imprinted polymer (MIP), obtained by precipitation polymerisation with 4-vinylpyridine as the functional monomer, ethylene glycol dimethacrylate as cross-linker, and bisphenol-A (BPA) as template, was prepared. The binding site configuration of the BPA-MIP was examined using Scatchard analysis. Moreover, the behaviour of the BPA-MIP for the extraction of several phenolic compounds (bisphenol-A, bisphenol-F, 4-nitrophenol, 3-methyl-4-nitrophenol) and phenoxyacid herbicides such as 2,4-D, 2,4,5-T and 2,4,5-TP has been studied in organic and aqueous media in the presence of other pesticides in common use. It was possible to carry out the selective preconcentration of the target analytes from the organic medium with recoveries of higher than 70%. In an aqueous medium, hydrophobic interactions were found to exert a remarkably non-specific contribution to the overall binding process. Several parameters affecting the extraction efficiency of the BPA-MIP were evaluated to achieve the selective preconcentration of phenols and phenoxyacids from aqueous samples. The possibility of using the BPA-MIP as a selective sorbent to preconcentrate these compounds from other samples such as urine and river water was also explored.
molecularly imprinted polymer; precipitation polymerisation; Scatchard analysis; bisphenol-A; phenolic compounds; phenoxyacid herbicides
The title compound, NH4
−·0.5H2O, was prepared by the reaction of 2-(2,4-dichlorophenoxy)acetic acid and ammonia in water at 367 K. The molecular structure and packing are stabilized by N—H⋯O and O—H⋯O intermolecular hydrogen-bond interactions.
Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.
Octadecenyl thiophosphate (OTP), a synthetic analog of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid-liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1→95.0 and 403.1→95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.
Octadecenyl thiophosphate (OTP); lysophosphatidic acid (LPA); liquid chromatography/tandem mass spectrometry (LC-MS/MS); pharmacokinetics
Mecamylamine is a nicotine antagonist under investigation in combination with nicotine replacement for smoking treatment.
A simple, rapid and reliable liquid chromatography tandem mass spectrometry (LCMSMS) method was developed and validated for quantifying nicotine, cotinine, trans-3′-hydroxycotinine, norcotinine and mecamylamine in human urine. Chromatography was performed on a Synergi PolarRP column with a gradient of 0.1% formic acid and 0.1% formic acid in acetonitrile at 0.25 ml/min with an 8-min total runtime. Analytes were monitored by positive mode electrospray ionization and multiple reaction monitoring mass spectrometry.
Linear dynamic ranges were 1–500 ng/ml for nicotine and norcotinine, 0.5–500 ng/ml for trans-3′-hydroxycotinine, 0.2–500 ng/ml for cotinine, and 0.1–100 ng/ml for mecamylamine; correlation coefficients were consistently greater than 0.99, and all calibrator concentrations were within 20% of target Extensive endogenous and exogenous interferences were evaluated. At 3 concentrations spanning the linear dynamic range of the assay, mean extraction efficiencies from urine were 55.1–109.1% with analytical recovery (bias) 82.0–118.7% and total imprecision of 0.7–9.1%. Analytes were stable for 24 h at room temperature, 72h at 4°C, 72h in autosampler at 15°C and after three freeze/thaw cycles.
This method is useful for monitoring mecamylamine, nicotine and nicotine metabolites in smoking cessation and other clinical nicotine research.
Mecamylamine; Nicotine; Cotinine; trans-3′-Hydroxycotinine; Norcotinine; Urine; LCMSMS
Airway inflammation characterizing asthma and other airway diseases may be monitored through biomarker analysis of exhaled breath condensate (EBC). In an attempt to discover novel EBC biomarkers, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was used to analyze EBC from ten control non-asthmatics and one asthmatic individual for five fatty acid metabolites: 9,12,13-trihydroxyoctadecenoic acid (9,12,13-TriHOME), 9,10,13-TriHOME, 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 12-hydroxyeicosatetraenoic acid (12-HETE), and 12(13)-epoxyoctadecenoic acid (12(13)-EpOME). The method was shown to be sensitive, with an on-column limit of quatitation (LOQ) in the pg range (corresponding to pM concentrations in EBC), and linear over several orders of magnitude for each analyte in the calibrated range. Analysis of EBC spiked with the five fatty acid metabolites was within 81%–119% with only a few exceptions. Endogenous levels in EBC exhibited intra- and inter-assay precision of 10%–22%, and 12%–36%, respectively. EBC from the healthy subjects contained average analyte levels between 15 and 180 pM with 12-HETE present above the LOQ in only one of the subjects at a concentration of 240 pM. Exposure of the asthmatic subject to allergen led to increased EBC concentrations of 9,12,13-TriHOME, 9,10,13-TriHOME, 12,13-DiHOME, and 12(13)-EpOME when compared to levels in EBC collected prior to allergen exposure (range =40–510 pM). 12,13-DiHOME was significantly increased (Student's t-test, p < 0.05). In conclusion, we have developed a new HPLC-ESI-MS/MS method for the analysis of five fatty acid metabolites in EBC, which are potential biomarkers for asthma monitoring and diagnosis.
Breath analysis; eicosanoids; exhaled breath condensate (EBC); inflammation; liquid chromatography—mass spectrometry