An Improved Micropropagation of Arnebia hispidissima (Lehm.) DC. and Assessment of Genetic Fidelity of Micropropagated Plants Using DNA-Based Molecular Markers

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3–4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog’s (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l^?1 6-benzylaminopurine and 0.1 mg l^?1 IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50?±?0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2–4 mm) of shoots was treated with 300 mg l^?1 of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85–90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.     

Determination of Shikimic Acid in Fruits of Illicium Species and Various Other Plant Samples by LC–UV and LC–ESI–MS

A simple and specific analytical method for the quantitative determination of shikimic acid from the methanol extract of the fruits of Illicium species and from various plant samples was developed. The LC–UV separation was achieved by reversed-phase chromatography on a C18 column with potassium dihydrogen phosphate and methanol as the mobile phase. In the LC–MS method, the separation was achieved by a C12 column using water and acetonitrile, both containing 0.1% acetic acid as the mobile phase. The methods were successfully used to study the percentage compositions of shikimic acid present in nine species of Illicium and various other plant samples. The detector response was linear with concentrations of shikimic acid in the range from 1.0–500.0 μg mL^?1 by LC–UV and 100–1000 ng mL^?1 by LC–MS. Mass spectrometry coupled with electrospray ionization interface is described for the identification of shikimic acid in various plant samples. This method involved the use of the [M-H]^? ions of shikimic acid at m/z 173.0455 (calculated mass) in the negative ion mode with extractive ion monitoring.     

Comparison of phenolic content and antioxidant activity of Actaea racemosa L. and Actaea cordifolia DC

Actaea racemosa L. is used as a component of drugs or dietary supplements to alleviate the menopause symptoms. Its biological activity is associated with the presence of phenolic compounds. In our work, the analysis of isoflavones and phenolic acids – caffeic acid (CA), ferulic acid and isoferulic acid (iFA) – both free and bonded in two species of Actaea, was conducted using HPLC-PAD technique. Moreover, the antioxidant effect of extracts from different parts of the investigated plants was determined on the basis of DPPH assay. Significant variation of CA and iFA content was observed. The highest content of CA was found in A. racemosa, while Actaea cordifolia contained the highest amount of iFA. Isoflavones were not found in the investigated plants. The antioxidant activity assay showed the high free radical-scavenging ability of the extracts obtained from different parts of the plant.     

Analysis of Isopropyl 3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoate, a Metabolite of Danshensu, from Salvia miltiorrhiza, in Rabbit Plasma

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a metabolite of Danshensu, from Salvia miltiorrhiza, has been proved to have potential as a novel drug for regulation of vasomotor activity in small-resistance vascular circulation. In this presentation we report a new specific method for analysis of IDHP in rabbit plasma. Plasma samples were pretreated with 1.5% formic acid in acetonitrile to remove the protein, and the resulting supernatant was extracted with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with 15.0% acetonitrile in 0.3% aqueous formic acid (pH 2.2) as mobile phase. Multiple-reaction-mode ion-trap mass spectrometry was selected for accurate analysis of IDHP. The calibration plot was linear in the range 0.1–200.0 ng mL^?1 for plasma samples. The detection limit was 0.02 ng mL^?1. Intra-day and inter-day coefficients of variation were <13.0% and intra-day and inter-day accuracy was within ±8.0% of known concentrations. Finally, the method was used to investigate the pharmacokinetics of IDHP in rabbits; the results indicated IDHP was eliminated rapidly after oral administration.     

A Simple and Sensitive LC Method for Quantification of Cefteram in Human Plasma

A simple and sensitive liquid chromatographic method was developed for quantification of cefteram in human plasma. Amoxicillin was used as an internal standard. The present method used protein precipitation for extraction of cefteram from human plasma. Separation was carried out on a reversed-phase C₁₈ column. The column effluent was monitored by UV detection at 262 nm. The mobile phase was a mixture of methanol and water containing 0.3% v/v triethylamine and 0.6% v/v glacial acetic acid (35:65:0.3:0.6 v/v) at a flow rate of 0.30 mL min^?1. The column temperature was 20 °C. This method was linear over the range of 47.5–4,750.0 ng mL^?1 with determination coefficient greater than 0.99. The mean extraction recovery of cefteram and IS was ≥76.82 and ≥76.49%, respectively, and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for a pharmacokinetic study of cefteram in human.     

Advancing understanding of actinide(iii) (Ac,Am, Cm) aqueous complexation chemistry

The positive impact of having access to well-defined starting materials for applied actinide technologies – and for technologies based on other elements – cannot be overstated. Of numerous relevant 5f-element starting materials, those in complexing aqueous media find widespread use. Consider acetic acid/acetate buffered solutions as an example. These solutions provide entry into diverse technologies, from small-scale production of actinide metal to preparing radiolabeled chelates for medical applications. However, like so many aqueous solutions that contain actinides and complexing agents, 5f-element speciation in acetic acid/acetate cocktails is poorly defined. Herein, we address this problem and characterize Ac³⁺ and Cm³⁺ speciation as a function of increasing acetic acid/acetate concentrations (0.1 to 15 M, pH = 5.5). Results obtained via X-ray absorption and optical spectroscopy show the aquo ion dominated in dilute acetic acid/acetate solutions (0.1 M). Increasing acetic acid/acetate concentrations to 15 M increased complexation and revealed divergent reactivity between early and late actinides. A neutral Ac(H₂O)₆₍₁₎(O₂CMe)₃₍₁₎ compound was the major species in solution for the large Ac³⁺. In contrast, smaller Cm³⁺ preferred forming an anion. There were approximately four bound O₂CMe¹⁻ ligands and one to two inner sphere H₂O ligands. The conclusion that increasing acetic acid/acetate concentrations increased acetate complexation was corroborated by characterizing (NH₄)₂M(O₂CMe)₅ (M = Eu³⁺, Am³⁺ and Cm³⁺) using single crystal X-ray diffraction and optical spectroscopy (absorption, emission, excitation, and excited state lifetime measurements).

Actinide complexation from aqueous acetic acid/acetate buffered solutions is described. The number of water ligands was directly correlated with the acetate concentration and characterized by X-ray absorption and optical spectroscopy.     

Analysis of Biologically Active Constituents in Centella asiatica by Microwave-Assisted Extraction Combined with LC–MS

Centella asiatica (L.) Urban is a traditional herbal medicine used in Asiatic countries, and is commonly used to treat various wounds, leprosy, tuberculosis and lupus diseases. In this work, a new method based on microwave assisted extraction followed by liquid chromatography–diode array detection–electrospray ionization multistage tandem mass spectrometry analysis has been developed for the identification and quantification of biologically active constituents in C. asiatica, including asiatic acid, asiaticoside and madecassoside. The separation was performed on an Agilent Eclipse C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) with gradient elution of acetonitrile and 0.1% aqueous acetic acid within 50 min. Detection was performed at 205 nm. The calibration curves showed good linearity (r ² > 0.9992). The limits of detection ranged from 1.2 to 1.6 μg mL^?1. The intra- and inter-day precision was less than 3% and the recovery of the assay was in the range of 95.4–106.8%. The method was successfully applied to the quantification of the three constituents in different samples of C. asiatica. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of C. asiatica. The samples were also analyzed on a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry system to confirm the identification results.     

LC–MS–MS Analysis of Mirtazapine in Plasma, and Determination of Pharmacokinetic Data for Rats

A sensitive LC–MS–MS method with electrospray ionization has been developed for analysis of mirtazapine in rat plasma. After addition of diazepam as internal standard, liquid–liquid extraction was used to produce a protein-free extract. Chromatographic separation was achieved on a 150 × 4.6 mm, 5 μm particle, ODS column with 84:16 (v/v) methanol–water containing 0.1% ammonium acetate and 0.01% glacial acetic acid as mobile phase. LC–MS–MS was performed in selected-ion-monitoring (SIM) mode using target fragment ions m/z 195.09 for mirtazapine and m/z 192.80 for the IS. Calibration plots were linear over the range of 0.516–618.8 ng mL^?1. The lower limit of quantification was 0.516 ng mL^?1. Intra-day and inter-day precision were better than 12.6 and 8.8%, respectively. Mean recovery of mirtazapine from plasma was in the range 87.41–90.06%; average recovery was 88.40% (RSD 3.95%). Significant gender differences between mirtazapine pharmacokinetic data were observed in this study.     

Rapid identification, by use of the LTQ Orbitrap hybrid FT mass spectrometer, of antifungal compounds produced by lactic acid bacteria

Fungal contamination of food causes health and economic concerns. Several species of lactic acid bacteria (LAB) have antifungal activity which may inhibit food spoilage fungi. LAB have GRAS (generally recognised as safe) status, allowing them to be safely integrated into food systems as natural food preservatives. A method is described herein that enables rapid screening of LAB cultures for 25 known antifungal compounds associated with LAB. This is the first chromatographic method developed which enables the rapid identification of a wide range of antifungal compounds by a single method with a short analysis time (23 min). Chromatographic separation was achieved on a Phenomenex Gemini C18 100A column (150 mm?×?2.0 mm; 5 μm) by use of a mobile-phase gradient prepared from (A) water containing acetic acid (0.1%) and (B) acetonitrile containing acetic acid (0.1%), at a flow rate of 0.3 µL min^?1. The gradient involved a progressive ramp from 10–95% acetonitrile over 13 min. The LC was coupled to a hybrid LTQ Orbitrap XL fourier-transform mass spectrometer (FTMS) operated in negative ionisation mode. High mass accuracy data (<3 ppm) obtained by use of high resolution (30,000 K) enabled unequivocal identification of the target compounds. This method allows comprehensive profiling and comparison of different LAB strains and is also capable of the identification of additional compounds produced by these bacteria.     

A Validated, Stability-Indicating, LC Method for Rabeprazole Sodium

We present a simple and reliable method for simultaneous determination of voriconazole and its main metabolite resulting from N-oxidation (UK-121,265), in human plasma. The work-up procedure used acetonitrile and potassium salts to precipitate plasma proteins. No internal standard was used. The chromatographic system used a LiChroCART^® 250-4 cartridge packed with LiChrospher^® 100 RP-8 (diameter particules, 5 μm). The UV monochromatic detector was set on 260 nm. The mobile phase consisted of a 60/40 (v/v) mixture of acetonitrile and water. The flow rate was 1 mL min^?1. The retention times for voriconazole and its metabolite were 8.98 and 4.02 min respectively, and total run time was 12 min. The linearity of the method was investigated from 0.31 to 10.0 mg L^?1; the lowest limit of quantification was 0.30 mg L^?1. Precision ranged from 2.41% to 6.32% for voriconazole and 0.80% to 11.6% for the N-oxide voriconazole metabolite. Accuracy was between 93.0% and 101% for voriconazole and 90.0% and 101% for the N-oxide voriconazole metabolite. This rapid and accurate method could be interesting to investigate metabolite/voriconazole ratio with respect to CYP2C19 genetic status and CYP3A4 activity changes.     

A Validated Chiral LC Method for the Separation and Quantification of (S,R,S)-Enantiomer and (R,R,R)-Isomer of Aprepitant

A new and accurate chiral liquid chromatographic method has been developed for the separation and quantification of (S,R,S)-enantiomer (unwanted enantiomer) and (R,R,R)-isomer (key intermediate) of aprepitant in bulk drug and formulation samples of apprepitant. The elution time was approximately 20 min using an immobilized amylose-based chiral stationary phase (Chiralpak-IA). The mobile phase was n-hexane and ethanol (90:10, v/v) and was delivered at a flow rate of 1.0 mL min^?1. Detection was carried out with a wavelength set to 220 nm. The resolution factor between enantiomers was found to be greater than five. Limit of detection for both (S,R,S) enantiomer and (R,R,R) isomer of aprepitant was 0.035 µg, and limit of quantification for both (S,R,S) enantiomer and (R,R,R) isomers of aprepitant was 0.1 µg, for a 10 µL injection. The developed method showed excellent linearity (r > 0.999) for both isomers. When the method was applied to bulk drug samples and in pharmaceutical formulations recoveries were obtained ranging from 97.2 to 103.1%. Aprepitant sample solutions were found to be stable when characterized over a period of 48 h.     

N-(1-Naphthyl)ethylenediamine, a New UV Labeling Reagent Used for LC Determination of Valproic Acid in Human Plasma

A high performance liquid chromatography method is presented for the determination of valproic acid levels in human plasma. The method was based on pre-column derivatization using N-(1-naphthyl)ethylenediamine as a new labeling agent. The calibration curve was linear in the investigated concentration range between 0.1 and 100 μg mL^?1 and showed good accuracy and reproducibility. The assay provided a limit of quantification of 0.1 μg mL^?1 for valproic acid and a limit of detection of 10 ng mL^?1, respectively. The presented method was successfully applied to the determination of valproic acid levels in plasma after oral administration of 600 or 800 mg of sodium valproate.     

Enantiomeric Separation of Moxifloxacin and Its (R,R)-Isomer by Ligand-Exchange Chiral Chromatography

A new stereospecific LC method for the separation and quantification of moxifloxacin and its (R,R)-enantiomer in bulk drug was developed and validated by ligand-exchange liquid chromatography on a reversed phase column using aqueous mobile phase containing the chiral reagent l-isoleucine-Cu(II). The UV detector was operated at 293 nm. The flow rate of mobile phase was set at 0.9 mL min^?1. The achiral ODS column offers good separation of the two enantiomers in less than 20 min. The test concentration was 1,000 μg mL^?1 in the mobile phase. This method was capable of detecting the (R,R)-enantiomer of moxifloxacin up to 0.1 μg mL^?1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with the (R,R)-enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the (R,R)-enantiomer in bulk drug samples ranged from 98.1 to 104.4%. The test solution was found to be stable in the mobile phase for 48 h after preparation.     

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C₁₈ column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL^?1 for zofenopril and 2–1,800 ng mL^?1 for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.     

A novel chemiluminescence reaction system for the determination of lincomycin with diperiodatonickelate(IV)

A sensitive and high selective chemiluminescence (CL) method was developed for the determination of lincomycin in acid medium using diperiodatonickelate as a reagent. The mechanism leading to luminescence is discussed by comparing the spectra of fluorescence and CL. Relative CL intensity is linear in the range from 8.0 ng mL^?1 to 1.0 µg mL^?1, the limit of detection is 2.5 ng mL^?1 (3σ), and the relative standard deviation is 4.0% at 0.1 µg mL^?1 of lincomycin (n?=?7). The method was successfully applied to the determination of lincomycin in injections, human urine, and in serum samples.     

A Validated LC Method for the Determination of Chiral Purity of (1S)-6,11-Dioxo-1,2,3,4,6,11-Hexahydropyridazino[1,2-b]Phthalazine-1-Carboxylic Acid: A Key Intermediate of Cilazapril

A simple and accurate normal phase liquid chromatographic method was developed for the determination of chiral purity of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, S-enantiomer used as key intermediate in the manufacturing of cilazapril bulk drug. Chromatographic separation between (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, and its opposite enantiomer (1R)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid, R-enantiomer was achieved using a Chiralpak AD-H column using a mobile phase containing hexane, isopropyl alcohol and tri-fluoro acetic acid (80:20:0.1 v/v/v). The resolution between the two enantiomers was found to be more than 3.2. The limit of detection (LOD) and limit of quantitation (LOQ) of the R-enantiomer was 0.15 and 0.5 μg mL^?1, respectively, for 10 μL injection volume. The percentage recoveries of the R-enantiomer ranged from 96.5 to 105.3 in the bulk samples of (1S)-6,11-dioxo-1,2,3,4,6,11-hexahydropyridazino[1,2-b]phthalazine-1-carboxylic acid. The test solution and mobile phase was observed to be stable up to 24 h after the preparation. The developed method was validated as per International Conference on Harmonization guidelines in terms of LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

Simple, Sensitive, High-Throughput Method for the Quantification of Mitragynine in Rat Plasma Using UPLC-MS and Its Application to an Intravenous Pharmacokinetic Study

A simple, sensitive and rapid ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed and validated for the quantification of mitragynine in rat plasma using amitriptyline hydrochloride as an internal standard. Sample preparation involved a one-step liquid?Cliquid extraction using methyl t-butyl ether. Mitragynine was separated on an Acquity UPLC^? BEH HILIC column using isocratic elution with a mobile phase of 10 mM ammonium formate buffer containing 0.1% formic acid:acetonitrile (15:85, v/v). At a flow rate of 0.2 mL min^?1, the retention time of mitragynine was found to be 1.3 min. Ionization was performed in the positive ion electrospray mode. The selected mass-to-charge (m/z) ratio transition of mitragynine ion [M + H]⁺ used in the selected ion recording (SIR) was 399.1. The calibration curve was found to be linear over a concentration range of 1?C5,000 ng mL^?1 (r = 0.999) with a lower limit of quantification (LLOQ) of 1 ng mL^?1. Intra- and inter-day assay variations were found to be less than 15%. The extraction recoveries ranged from 85?C93% at the three concentrations (2, 400 and 4,000 ng mL^?1) in rat plasma. This method was successfully used to quantify mitragynine in rat plasma following intravenous administration of the compound.     

A novel solid-phase extraction method for separation and preconcentration of zirconium

This work assesses the use of modified natural clinoptilolite as an adsorptive material for separation and preconcentration of trace amounts of zirconium ions. A simple, rapid and economical method was developed for the preconcentration of trace amounts of zirconium in aqueous medium using 1-(2-pyridylazo)-2-naphthol as a complexing agent. Effect of sample pH, flow rate of sample and elution solutions, breakthrough volume and interference of several ions were studied. Determination of zirconium was made by ICP-AES technique. The sorption was quantitative in the pH range from 3.0 to 4.0, whereas quantitative desorption occurred instantaneously with 2 mol L^?1 hydrochloric acid. Linearity was maintained between 0.05 and 9.0 μg mL^?1. Relative standard deviations range from ±0.9% to ±2.3% (n?=?5). The detection limit was 0.1 ng mL^?1. Because of good recovery (>97%), this method is suitable for preconcentration and determination of zirconium in effluents containing trace amount of zirconium.     

LC-MS-MS Determination of (S,R)-Penehyclidine in Rat Plasma

A sensitive and selective method for determination of (S,R)-penehyclidine in rat plasma by liquid chromatography-tandem mass spectrometry is described. The procedure employed the use of an internal standard (I.S.) and a simple protein precipitation step. The method developed was linear from 0.1 to 100 ng mL^?1, with a sensitivity of 0.1 ng mL^?1 as the lower limit of quantification. The intra- and inter-day assay accuracy (relative error) was within 8.27% and precision (RSD) was below 6.7%. It was successfully applied to pharmacokinetic studies of (S,R)-penehyclidine in rat plasma.     

Development and Validation of a Sensitive LC–Tandem-MS Method for the Quantitative Determination of Picroside II in Rat Plasma

A rapid and sensitive method for the quantitative determination of picroside II in rat plasma was developed and validated using liquid chromatographic separation with tandem mass spectrometric detection. The analytes of interest were extracted from rat plasma samples by ethyl acetate after acidification with 1.0% acetic acid solution. Chromatographic separation was achieved on a Hypersil GOLD column (50 × 2.1 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile–0.1% formic acid solution (30:70, v/v) at a flow rate of 0.2 mL min^?1. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI). The calibration curve was linear in the concentration range of 1.00–400 ng mL^?1 in rat plasma, with a 1.00 ng mL^?1 lower limit of quantification (LLOQ). Satisfactory results were achieved for intraday repeatability [relative standard deviation (RSD) = 6.4–12.4%] and inter-day precision (RSD = 6.8–14.7%). The accuracy in terms of relative error ranged from ?2.1 to 10.0%. The extraction recoveries of picroside II and icariin (internal standard) were 80.0 and 89.3%, respectively. The developed method was successfully employed to determine picroside II plasma concentrations after oral administration to Wistar rats.