Preparative LC-MS Research Articles

Bioactivity of a dihydroxy flavone produced by Penicillium EU0013 against the wilt pathogen of tomato

Bioassay-guided isolation of a dihydroxy flavone from the cell extract of Penicillium EU0013 was carried out using preparative LCMS and the structure was interpreted using 1D and 2D NMR spectra. The fungus was fermented in three different nutrient media i.e. Glucose Nutrient Broth, Glucose Peptone Yeast Broth, and Yeast Extract Broth and the cell extract was obtained using organics. Comparative LCMS profiles of the fungus confirmed the enhanced production of dihydroxy flavone in GPYB media. In this study, the pathogenicity of the dihydroxy flavone is reported for the first time against Fusarium oxysporum f.sp. lycopersici, wilt causing pathogen of a tomato plant. In MIC experiments, the compound inhibited the pathogen at 80 µg mL−1. Molecular docking studies of the compound with Fol tomatinase showed five binding interactions. These docking studies may help explore dihydroxy flavones as a lead for developing new fungicides to prevent wilt disease in tomatoes.

Identification of unknown impurities J, RRT 2.2, 2.4, 2.6 and 3.4 in tetralysal® capsules

Tetralysal® is a Galderma oral drug product (DP) marketed for the treatment of acne. Tetralysal® is sold in capsules containing either 150 mg or 300 mg of the drug substance. In the British Pharmacopoeia monograph for Lymecycline Capsules, the impurities already specified in the drug substance (A-G), visible in the European Pharmacopoeia 〈1654〉, are also specified together with an unidentified impurity at RRT 1.6 (Impurity J). Based on both monographs Galderma has focused on characterizing most of specified and unspecified impurities to better understand the stability and degradation processes of the formulation. In this manuscript, through both formal synthesis, preparative LCMS and formal degradation studies, we are the first group to confirm the structural identities of 5 unidentified impurities (Impurity J (RRT 1.6), RRT 2.2, 2.4, 2.6 and 3.4), conditions which exacerbate the formation of all 5 impurities and response factors for RRT 2.2, 2.6 and 3.4.

A comparative phytochemical study of nine Lauraceae species by using chemometric data analysis.

The diversity of secondary metabolites of individual plants results from multiple enzymatic processes in planta and various environmental factors, such as temperature, moisture, and soil conditions. Chemical composition analysis of plants can lead to a new method to understand relationship among comparable plants along with biological classification such as genetic and anatomical method. In this study, the chemical diversity of nine different Lauraceae species was investigated, and the plant samples were chemically analyzed and classified. Multivariate analysis methods, such as PLS-DA, were used to select important metabolites distinguishing the nine Lauraceae species. The selected metabolites were identified through preparative LC-MS or MS/MS fragment pattern analysis. In addition, the chemical dendrogram for the nine Lauraceae species was interpreted through molecular network analysis and compared with the genetic dendrogram. This approach enabled us to compare the complete chemical compositions of multiple plant samples to identify relationships among plants.

Identification of coffee compounds that suppress bitterness of brew

Untargeted LC–MS flavoromic profiling was utilized to identify compounds that suppress bitterness perception of coffee brew. The chemical profiles of fourteen brew samples and corresponding perceived bitterness intensities determined by descriptive sensory analysis were modeled by orthogonal partial least squares (OPLS) with good fit (R2Y > 0.9) and predictive ability (Q2 > 0.9). Ten chemical markers that were highly predictive and negatively correlated to bitter intensity were subsequently purified by multi-dimensional preparative LC–MS to conduct sensory recombination testing and/or confirm compound identifications by NMR. Three of the ten compounds evaluated, namely 4-caffeoylquinic acid, 5-caffeoylquinic acid, and 2-O-β-d-glucopyranosyl-atractyligenin were identified as bitter modulators in coffee, and significantly decreased the perceived bitterness intensity of the brew.

Potential application of oat-derived ceramides in improving skin barrier function: Part 1. Isolation and structural characterization.

The impaired epidermal barrier and skin dryness in chronic skin conditions such as atopic dermatitis, psoriasis and aged skin are associated with the depletion of ceramides (CERs) in the stratum corneum. Previously, the beneficial effects of phyto-CERs, mainly from wheat and rice, in replenishing the depleted epidermal CERs and restoring the skin barrier have been shown. However, very few efforts have been made to exploit CERs from other plants for dermal applications. In an attempt to explore alternative plant source of CERs, glucosylceramides (GlcCERs) were isolated from the lipid extract of Ethiopian oat grain (Avena abyssinica). The GlcCER species were separated on a reversed phase HPLC and the structure of individual GlcCERs were identified by tandem MS with atmospheric pressure chemical ionization interface. The glycosidic linkage of the GlcCERs was cleaved by acid treatment and the predominant CERs species were isolated using column chromatography and preparative LC-MS. Further structural characterization of the CERs was made by HR/ESI-MS and NMR analyses. All the detected oat-derived GlcCER species consisted of C18 dihydroxy sphingoid bases amide-linked with α-hydroxylated saturated fatty acids (C16-C24). The two predominant GlcCER species consisted of sphingenine (d18:1) amide-linked to hydroxypalmitic acid (h16:0) and hydroxyarachidic acid (h20:0). The molecular formulae of the two major CERs assigned by HR/ESI-MS were identical to the ones identified by LC/APCI-MS/MS. The structural information was also supported by 1H, 13C, 1H COSY NMR and HMBC spectral analyses. The amount of GlcCERs in oat grain, quantified by HPTLC, was found to be 193.5mg/kg. The results indicated the similarity of oat CERs with commercial plant CERs (with comparable GlcCER content) suggesting its potential as source of CERs for oral (as dietary supplements) as well as topical applications.

Pharmacokinetic study of salvianolic acid A in beagle dog after oral administration by a liquid chromatography–mass spectrometry method: A study on bioavailability and dose proportionality

Ethnopharmacological relevanceSalvianolic acid A (SAA) is one of the main water-soluble components isolated from Salvia miltiorrhiza Bunge. Pharmacological researches revealed that it had various curative activities after oral and intravenous administration, including beneficial effects on diabetes and its complications, cardioprotective effect, anti-platelet aggregation, and so on. However, there is no report regarding the pharmacokinetics of SAA in beagle dogs after oral administration up to now. Aim of the studyTo study the pharmacokinetics of different doses of SAA in beagle dogs and figure out the absolute bioavailability and dose proportionality of SAA after oral administration. Materials and methodsMale and female beagle dogs were orally administered SAA 5, 10 and 20mg/kg randomly. The plasma drug concentration was detected by a rapid, sensitive and reproducible liquid chromatography–mass spectrometry (LC–MS) method. The pharmacokinetic parameters were calculated from plasma concentration–time data using the DAS pharmacokinetic software Data Analysis System Version 3.0 program. ResultsAfter single-dose oral administration of SAA, the mean peak plasma concentration (Cmax) values for groups treated with 5, 10 and 20mg/kg doses ranged from 14.38 to 38.18µg/L, and the mean area under the concentration–time curve (AUC(0–t)) values ranged from 38.77 to 130.33 (µg/L⋅h). SAA showed lack of dose proportionality over the dose range 5–20mg/kg, based on the power model. However, the increase in systemic exposure with dose appeared linear. The absolute bioavailability was calculated to range from 1.47% to 1.84%. ConclusionThe pharmacokinetic properties of SAA in beagle dogs after oral administration were characterized as rapid oral absorption, quick clearance, and poor absolute bioavailability. Systemic exposure exhibited lack of dose proportionality over the dose range 5–20mg/kg. Furthermore, a readily preparative LC–MS method was demonstrated in this study for the research of traditional Chinese medicine.

Efficient Three-Step One-Pot Synthesis of a Novel 2,3,5-Substituted Pyrazine Library

The partnership between rational synthesis design and mass-triggered preparative LCMS is a powerful one, capable of furnishing very large libraries in a selective manner in a very short space of time. Herein, we communicate one example of possibly a perfect marriage between the synthetic chemistry and the subsequent purification method employed, affording a ∼1000-member library supplying 50 mg on average of final compound in less than a month.

Liquid chromatography–mass spectrometry-based quantification of steroidal glycoalkaloids from Solanum xanthocarpum and effect of different extraction methods on their content

A new liquid chromatography–mass spectrometry (LC–MS)-based method coupled with pressurized liquid extraction (PLE) as an efficient sample preparation technique has been developed for the quantification and fingerprint analysis of Solanum xanthocarpum. Optimum separations of the samples were achieved on a Waters MSC-18 XTerra column, using 0.5% (v/v) formic acid in water (A) and acetonitrile (ACN):2-propanol:formic acid (94.5:5:0.5, v/v/v) (B) as mobile phase. The separation was carried out using linear gradient elution with a flow rate of 1.0 mL/min. The gradient was: 0 min, 20% B; 14 min, 30% B; 20 min, 30% B; 27 min, 60% B and the column was re-equilibrated to the initial condition (20% B) for 10 min prior to next injection. The steroidal glycoalkaloids (SGAs) which are the major active constituents were isolated as pure compounds from the crude methanolic extract of S. xanthocarpum by preparative LC–MS and after characterization were used as external standards for the development and validation of the method. Extracts prepared by conventional Soxhlet extraction, PLE and ultrasonication were used for analysis. The method was validated for repeatability, precision (intra- and inter-day variation), accuracy (recovery) and sensitivity (limit of detection and limit of quantitation). The purpose of the work was to develop a validated method, which can be used for the quantification of SGAs in commercialized S. xanthocarpum products and the fingerprint analysis for their routine quality control.

LC and LC-MS study of stress decomposition behaviour of isoniazid and establishment of validated stability-indicating assay method

Isoniazid was subjected to different ICH prescribed stress conditions of thermal stress, hydrolysis, oxidation and photolysis. The drug was stable to dry heat (50 and 60 °C). It showed extensive decomposition under hydrolytic conditions, while it was only moderately sensitive to oxidation stress. The solid drug turned intense yellow on exposure to light under accelerated conditions of temperature (40 °C) and humidity (75% RH). In total, three major degradation products were detected by LC. For establishment of stability-indicating assay, the reaction solutions in which different degradation products were formed were mixed, and the separation was optimized by varying the LC conditions. An acceptable separation was achieved using a C-18 column and a mobile phase comprising of water:acetonitrile (96:4, v/v), with flow rate and detection wavelength being 0.5 ml min −1 and 254 nm, respectively. The degradation products appeared at relative retention times (RR T) of 0.71, 1.34 and 4.22. The validation studies established a linear response of the drug at concentrations between 50 and 1000 μg ml −1. The mean values (±R.S.D.) of slope, intercept and correlation coefficient were 35,199 (±0.88), 114,310 (±4.70) and 0.9998 (±0.01), respectively. The mean R.S.D. values for intra- and inter-day precision were 0.24 and 0.90, respectively. The recovery of the drug ranged between 99.42 and 100.58%, when it was spiked to a mixture of solutions in which sufficient degradation was observed. The specificity was established through peak purity testing using a photodiode array detector. The method worked well on application to marketed formulation of isoniazid, and a fixed-dose combination containing isoniazid and ethambutol HCl. It was even extendable to LC-MS studies, which were carried out to identify the three degradation products. The m/ z values of the peaks at RR T 0.71 and RR T 1.34 matched with isonicotinic acid and isonicotinamide, respectively. The product appearing at RR T 4.22 was isolated using preparative LC-MS, and turned out to be a yellow compound that was identified as isonicotinic acid N′-(pyridyl-4-carbonyl)-hydrazide based on mass, FTIR and 1H/ 13C NMR spectral data. The same was indicated to be responsible for discolouration of isoniazid bulk drug substance and formulations, which is a familiar problem. The mechanism of formation of the said compound is outlined.

Preparative LC−MS Purification: Improved Compound-Specific Method Optimization

One of the remaining challenges in providing effective preparative LC-MS purification is balancing throughput and compound purity. We describe here an approach to optimizing preparative LC-MS methods that provides significantly better chromatographic resolution and, hence, better compound purity than generic preparative LC methods consuming the same amount of time. This approach is easier to implement, is more rugged, and permits significantly greater flexibility than previously reported approaches. The instrument configurations and protocols presented here are specifically tailored for open access support, but the basic approach is equally suitable and effective in high-throughput situations.

A solution-phase combinatorial synthesis of selective dopamine D4 ligands.

Utilizing combinatorial synthesis and a preparative LC-MS automated chromatography system we have prepared and purified a library of 4-[2-(1,2,4-oxadiazolyl)]piperidines that were designed to be novel and selective dopamine D4 ligands. In one round of synthesis we identified N-4-chlorobenzyl-4-[2-(3-(2-thienyl)-1,2, 4-oxadiazolyl)]piperidine with a Kd of 5 nM for the human D4 receptor.

Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif.

Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.

New developments in automated PrepLCMS extends the robustness and utility of the method for compound library analysis and purification

New developments in the high throughput purification of combinatorial libraries by automated preparative LC-MS is presented. To facilitate high speed purifications at the multimilligram level, short columns operated at ultra high flow rates were incorporated. In order to match the linear velocity of the short analytical columns for high speed separations (operated at 4.0 mL/min), it was required to operate the preparative columns at flow rates in excess of 70 mL/min. For chromatographically well-behaved compounds, analytical LC-MS analyses and preparative LC-MS analyses could be achieved in as little as 5 min. For compounds exhibiting poor chromatographic peak shapes and/or for compound mixtures requiring higher resolution separations, slightly longer preparative LC-MS analysis times were required (8-10 min/sample).