Mass propagation of Juniperus procera Hoechst. Ex Endl. From seedling and screening of bioactive compounds in shoot and callus extract.

BACKGROUND: Juniperus procera Hoechst. ex Endl. is a medicinal tree in Saudi Arabia, primarily in the Enemas region, but it is locally threatened due to die-back disease and difficulties regarding seed reproduction (seed dormancy and underdeveloped embryonic anatomy, and germination rate < 40%). Hence, the alternative methods for reproduction of Juniperus procera are really needed for conservation and getting mass propagation for pharmaceutical uses. RESULTS: In this manuscript, we articulated the successful in vitro shoot multiplication and callus induction of J. procera by using young seedling as explants and detected an important antibacterial and antitumor product. Explants were grown on different types of media with the supplement of different combinations of Plant Growth Regulators (PGRs) at different concentrations. The best media for shoot multiplication was Woody Plant Media (WPM) supplemented with PGRs (0.5 µM of IAA and 0.5 µM BAP or 0.5 µM IBA and 0.5 µM BAP). Whereas for callus induction and formation Woody Plant Media (WPM) with the addition of PGRs (0.5 µM 2,4-D and 0.5 µM BAP) was better than the Chu Basal Salt Mixture (N6), Gamborg's B-5 Basal Medium (B5), and Murashige and Skoog media. The possibility of multiplication of J. procera in vitro creates significant advantages to overcome the difficulties of seeds dormancy for the reproduction of plants, conservation of trees, and getting mass propagation material for pharmaceutical studies. The shoot and callus extract of J. procera was detected using gas chromatography-mass spectrometry analysis and revealed more than 20 compounds related to secondary metabolites, which contained antibacterial and antitumor agents, such as ferruginol, Retinol, and Quinolone as well as confirmed by Direct Analysis in Real Time, Time of Flight Mass Spectrometry (DART-ToF-MS). Podophyllotoxin (PTOX) was detected in callus material by HPLC with sigma standard and confirmed by DART-ToF-MS and UV spectra. CONCLUSION: We successfully conducted in vitro shoot multiplication and callus induction from J. procera seedlings using WPM and a different combination of PGRs and, detected an important antibacterial and antitumor product such as ferruginol and podophyllotoxin. According to our findings, J. procera has become a new natural source of novel bioactive compounds.

Rapid detection of tert-butoxycarbonyl-methamphetamine by direct analysis in real time time-of-flight mass spectrometry.

PURPOSE: Phenethylamines constitute the majority of drug-related arrests in Japan. Recently, the smuggling of tert-butoxycarbonyl (t-Boc)-protected phenethylamines has become of increasing concern, because of the difficult identification of these masked substances. METHODS: In this study, a rapid and accurate method for the detection of t-Boc-methamphetamine (t-Boc-MP) by direct analysis in real time-time-of-flight-mass spectrometry (DART-TOF-MS) was developed. The efficiency of the method was evaluated by comparison with conventional gas chromatography-MS (GC-MS) and liquid chromatography-TOF-MS (LC-TOF-MS) techniques. RESULTS: During GC-MS analysis of t-Boc-MP, MP was generated in the injection port, which can lead to an analytical error. In the LC-TOF-MS spectrum, fragment ions were detected, which were generated by McLafferty rearrangement in the ion source. On the other hand, in the DART-TOF-MS analysis of t-Boc-MP, pyrolysis could be suppressed by using a micro-syringe injection method, and the fragment ions generated by McLafferty rearrangement were still observed. Moreover, protonated t-Boc-MP could be detected. CONCLUSIONS: Hence, DART-TOF-MS provides a rapid and accurate method for the detection of t-Boc-MP, allowing suppression of the pyrolysis reaction and identification of both fragment ions and protonated t-Boc-MP. To our knowledge, this is the first report for detecting t-Boc-MP by MS techniques.

A comparison between DART-MS and DSA-MS in the forensic analysis of writing inks.

Ambient ionization mass spectrometry is gaining momentum in forensic science laboratories because of its high speed of analysis, minimal sample preparation, and information-rich results. One such application of ambient ionization methodology includes the analysis of writing inks from questioned documents where colorants of interest may not be soluble in common solvents, rendering thin layer chromatography (TLC) and separation-mass spectrometry methods such as LC/MS (-MS) impractical. Ambient ionization mass spectrometry uses a variety of ionization techniques such as penning ionization in Direct Analysis in Real Time (DART), and atmospheric pressure chemical ionization in Direct Sample Analysis (DSA), and electrospray ionization in Desorption Electrospray Ionization (DESI). In this manuscript, two of the commonly used ambient ionization techniques are compared: Perkin Elmer DSA-MS and IonSense DART in conjunction with a JEOL AccuTOF MS. Both technologies were equally successful in analyzing writing inks and produced similar spectra. DSA-MS produced less background signal likely because of its closed source configuration; however, the open source configuration of DART-MS provided more flexibility for sample positioning for optimum sensitivity and thereby allowing smaller piece of paper containing writing ink to be analyzed. Under these conditions, the minimum sample required for DART-MS was 1mm strokes of ink on paper, whereas DSA-MS required a minimum of 3mm. Moreover, both techniques showed comparable repeatability. Evaluation of the analytical figures of merit, including sensitivity, linear dynamic range, and repeatability, for DSA-MS and DART-MS analysis is provided. To the forensic context of the technique, DART-MS was applied to the analysis of United States Secret Service ink samples directly on a sampling mesh, and the results were compared with DSA-MS of the same inks on paper. Unlike analysis using separation mass spectrometry, which requires sample preparation, both DART-MS and DSA-MS successfully analyzed writing inks with minimal sample preparation.

Rapid screening of abused drugs by direct analysis in real time (DART) coupled to time-of-flight mass spectrometry (TOF-MS) combined with ion mobility spectrometry (IMS).

Increasing in cases involving drugs of abuse leads to heavy burden for law enforcement agencies, exacerbating demand for rapid screening technique. In this study, atmospheric pressure ionization technologies including direct analysis in real time (DART) ion source coupled to a time-of-flight mass spectrometer (DART-TOF-MS)as well asdopant-assisted positive photoionization ion mobility spectrometry (DAPP-IMS) without radioactivity were utilized together as the powerful analytical tool for the rapid screening and identification of 53 abused drugs.The limits of detection (LOD) were 0.05-2µg/mL when using DART-TOF-MS and 0.02-2µg when using DAPP-IMS which could satisfy the actual requirement in forensic science laboratory. The advantages of this method included fast response, high-throughput potential, high specificity, and minimal sample preparation. A screening library of reduced mobility (K0), accurate mass of informative precursor ion ([M+H]+) and fragment ions was established respectively by employing a bench-top DAPP-IMS and TOF-MS in-source collision induced dissociation (CID) mode. Then the standardized screening procedure was developed with criteria for the confirmation of positive result. A total of 50 seized drug samples provided by local forensic laboratory we reanalyzed to testify the utility of the method. This study suggests that a method combing DART-TOF-MS and DAPP-IMS is promising for the rapid screening and identification of abused drugs with minimal sample preparation and absence of chromatography.

Validation of Direct Analysis Real Time source/Time-of-Flight Mass Spectrometry for organophosphate quantitation on wafer surface.

Microelectronic wafers are exposed to airborne molecular contamination (AMC) during the fabrication process of microelectronic components. The organophosphate compounds belonging to the dopant group are one of the most harmful groups. Once adsorbed on the wafer surface these compounds hardly desorb and could diffuse in the bulk of the wafer and invert the wafer from p-type to n-type. The presence of these compounds on wafer surface could have electrical effect on the microelectronic components. For these reasons, it is of importance to control the amount of these compounds on the surface of the wafer. As a result, a fast quantitative and qualitative analytical method, nondestructive for the wafers, is needed to be able to adjust the process and avoid the loss of an important quantity of processed wafers due to the contamination by organophosphate compounds. Here we developed and validated an analytical method for the determination of organic compounds adsorbed on the surface of microelectronic wafers using the Direct Analysis in Real Time-Time of Flight-Mass Spectrometry (DART-ToF-MS) system. Specifically, the developed methodology concerns the organophosphate group.

Rapid screening and distribution of bioactive compounds in different parts of Berberis petiolaris using direct analysis in real time mass spectrometry.

Berberis petiolaris Wall. ex G. Don, an unexplored medicinal plant belonging to the family Berberidaceae, is a large deciduous shrub found in Western Himalaya between 1800-3000 m. Chemical profiling of fruit, leaf, root and stem was done by direct analysis in real time mass spectrometry followed by multivariate analysis for discrimination among the plant parts. The bioactive compounds, including magnoflorine, berberine, jatrorrhizine, thalifendine/berberrubine, demethyleneberberine, reticuline, 8-oxoberberine, N-methyltetrahydroberberine, tetrahydropalmatine, tetrahydroberberine and palmatine, were identified by their exact mass measurement and the corresponding molecular formula of each compound. A comparative study of distribution pattern for all these bioactive alkaloids showed qualitative and quantitative variations in different parts of B. petiolaris. Principal component analysis clearly discriminated each part of B. petiolaris plant.

Rapid screening and distribution of bioactive compounds in different parts of Berberis petiolaris using direct analysis in real time mass spectrometry / 药物分析学报

Berberis petiolaris Wall. ex G. Don, an unexplored medicinal plant belonging to the family Berberidaceae, is a large deciduous shrub found in Western Himalaya between 1800-3000 m. Chemical profiling of fruit, leaf, root and stem was done by direct analysis in real time mass spectrometry followed by multivariate analysis for discrimination among the plant parts. The bioactive compounds, including magnoflorine, berberine, jatrorrhizine, thalifendine/berberrubine, demethyleneberberine, reticuline, 8-oxoberberine, N-methyltetrahydroberberine, tetrahydropalmatine, tetrahydroberberine and palmatine, were identified by their exact mass measurement and the corresponding molecular formula of each compound. A comparative study of distribution pattern for all these bioactive alkaloids showed qualitative and quantitative variations in different parts of B. petiolaris. Principal component analysis clearly dis-criminated each part of B. petiolaris plant.

Extraction-free in situ derivatisation of timosaponin AIII using direct analysis in real time TOF/MS.

INTRODUCTION: Direct analysis in real time (DART) TOF/MS has been used for mass information of various non-polar phytochemicals in raw material with no sample preparation. However, low ionisation efficiency for polar compounds including glycosides limits its extensive use in the field of phytochemical analysis. OBJECTIVE: In order to develop a direct analysis method for polar glycosides using in situ derivatisation, which improves ionisation efficiency of hydrophilic glycosides. METHOD: Anemarrhena Rhizoma was used as a model plant targeting on Timosaponin AIII utilising a Dip-It module. Permethylation was applied to the powdered raw material with tetramethylammonium hydroxide in front of a DART ion source. Also, DART TOF/MS combined with permethylation was applied to timosaponin AIII standard solution to obtain the limit of detection (LOD). RESULTS: In situ methylation of timosaponin AIII and Anemarrhena Rhizoma raw material were successfully used to ionise the glycoside. The LOD was found to be in the range of 2.4-4.8 ng for permethylated timosaponin AIII and this level is four times higher than the range of the underivatisation analysis. Direct analysis of permethylated timosaponin from Anemarrhena Rhizoma was also successfully performed. CONCLUSION: A simple and quick derivatisation method with tetramethylammonium hydroxide was developed for the direct identification of a hydrophilic saponin from the plant tissue. Better ionisation efficiency conferred by in situ permethylation enabled ionisation of whole molecules of timosaponin AIII from the plant tissue. This simple analytical method will provide a solution to reduce tedious sample preparation steps, not only for non-polar but also hydrophilic natural products directly from the tissue.

Direct Analysis in Real Time Mass Spectrometry (DART-MS) Analysis of Skin Metabolome Changes in the Ultraviolet B-Induced Mice.

Ultraviolet (UV) radiation is a major environmental factor that leads to acute and chronic reactions in the human skin. UV exposure induces wrinkle formation, DNA damage, and generation of reactive oxygen species (ROS). Most mechanistic studies of skin physiology and pharmacology related with UV-irradiated skin have focused on proteins and their related gene expression or single- targeted small molecules. The present study identified and analyzed the alteration of skin metabolites following UVB irradiation and topical retinyl palmitate (RP, 5%) treatment in hairless mice using direct analysis in real time (DART) time-of-flight mass spectrometry (TOF-MS) with multivariate analysis. Under the negative ion mode, the DART ion source successfully ionized various fatty acids including palmitoleic and linolenic acid. From DART-TOF-MS fingerprints measured in positive mode, the prominent dehydrated ion peak (m/z: 369, M+H-H2O) of cholesterol was characterized in all three groups. In positive mode, the discrimination among three groups was much clearer than that in negative mode by using multivariate analysis of orthogonal partial-least squares-discriminant analysis (OPLS-DA). DART-TOF-MS can ionize various small organic molecules in living tissues and is an efficient alternative analytical tool for acquiring full chemical fingerprints from living tissues without requiring sample preparation. DART-MS measurement of skin tissue with multivariate analysis proved to be a powerful method to discriminate between experimental groups and to find biomarkers for various experiment models in skin dermatological research.

Direct Analysis in Real Time Mass Spectrometry (DART-MS) Analysis of Skin Metabolome Changes in the Ultraviolet B-Induced Mice

Ultraviolet (UV) radiation is a major environmental factor that leads to acute and chronic reactions in the human skin. UV exposure induces wrinkle formation, DNA damage, and generation of reactive oxygen species (ROS). Most mechanistic studies of skin physiology and pharmacology related with UV-irradiated skin have focused on proteins and their related gene expression or single- targeted small molecules. The present study identified and analyzed the alteration of skin metabolites following UVB irradiation and topical retinyl palmitate (RP, 5%) treatment in hairless mice using direct analysis in real time (DART) time-of-flight mass spectrometry (TOF-MS) with multivariate analysis. Under the negative ion mode, the DART ion source successfully ionized various fatty acids including palmitoleic and linolenic acid. From DART-TOF-MS fingerprints measured in positive mode, the prominent dehydrated ion peak (m/z: 369, M+H-H2O) of cholesterol was characterized in all three groups. In positive mode, the discrimination among three groups was much clearer than that in negative mode by using multivariate analysis of orthogonal partial-least squares-discriminant analysis (OPLS-DA). DART-TOF-MS can ionize various small organic molecules in living tissues and is an efficient alternative analytical tool for acquiring full chemical fingerprints from living tissues without requiring sample preparation. DART-MS measurement of skin tissue with multivariate analysis proved to be a powerful method to discriminate between experimental groups and to find biomarkers for various experiment models in skin dermatological research.