Influence of LED light spectra on in vitro somatic embryogenesis and LC-MS analysis of chlorogenic acid and rutin in Peucedanum japonicum Thunb.: a medicinal herb.

BACKGROUND: Peucedanum japonicum Thunb, an important medicinal herb is reported to possess pharmacological properties such as anti-obesity, anti-oxidant, anti-inflammatory, anti-bacterial, anti-diabetic and anti-platelet aggregation. The present study aimed to develop an in vitro plant regeneration system of P. japonicum via somatic embryogenesis and to analyse chlorogenic acid and rutin contents in a few commercially available plant products of P. japonicum in Japan and Taiwan markets, and tissue culture plants derived from somatic embryos. RESULTS: Induction of somatic embryogenesis could be achieved when root derived calli after three subcultures were transferred from Murashige Skoog's salts and vitamins (MS basal) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1-5 mg/L) to a medium with abscisic acid (ABA) (0.5-4 mg/L), or exposed to eight different light spectra provided by light-emitting diode (LED) sources. Concentrations of ABA and LED light spectra had an influence on number of somatic embryos induced and proliferation of callus. Development of secondary somatic embryos and conversion of embryos to plantlets was achieved on a medium with ABA, or their exposure to red or blue lights in a special incubation chamber. Four months old tissue culture plants derived from somatic embryos showed significantly higher levels of chlorogenic acid (10.5 mg/g dw) compared to commercial product sold in Japanese market (0.55 mg/g dw). However, rutin was absent in tissue culture plants in contrast to commercial sample (0.33 mg/g dw). CONCLUSION: In this report, we describe in vitro plant regeneration system in P. japonicum via somatic embryogenesis and production of chlorogenic acid in tissue culture plants. The present study has application in further tissue culture propagation of elite plant material with high chlorogenic acid content, and identification of high yielding plants with the LC-MS method.

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress.

Glycine betaine (betaine) has the highest cellular osmoprotective efficiency which does not accumulate in most glycophytes. The biosynthetic pathway for betaine in higher plants is derived from the oxidation of low-accumulating metabolite choline that limiting the ability of most plants to produce betaine. Halophilic methanoarchaeon Methanohalophilus portucalensis FDF1(T) is a model anaerobic methanogen to study the acclimation of water-deficit stresses which de novo synthesize betaine by the stepwise methylation of glycine, catalyzed by glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase. In this report, genes encoding these betaine biosynthesizing enzymes, Mpgsmt and Mpsdmt, were introduced into Arabidopsis. The homozygous Mpgsmt (G), Mpsdmt (S), and their cross, Mpgsmt and Mpsdmt (G × S) plants showed increased accumulation of betaine. Water loss from detached leaves was slower in G, S, and G × S lines than wild-type (WT). Pot-grown transgenic plants showed better growth than WT after 9 days of withholding water or irrigating with 300 mM NaCl. G, S, G × S lines also maintained higher relative water content and photosystem II activity than WT under salt stress. This suggests heterologously expressed Mpgsmt and Mpsdmt could enhance tolerance to drought and salt stress in Arabidopsis. We also found a twofold increase in quaternary ammonium compounds in salt-stressed leaves of G lines, presumably due to the activation of GSMT activity by high salinity. This study demonstrates that introducing stress-activated enzymes is a way of avoiding the divergence of primary metabolites under normal growing conditions, while also providing protection in stressful environments.

Gramella planctonica sp. nov., a zeaxanthin-producing bacterium isolated from surface seawater, and emended descriptions of Gramella aestuarii and Gramella echinicola.

A Gram-stain negative, strictly aerobic, zeaxanthin-producing, rod-shaped, non-spore-forming bacterial strain which is motile by gliding, designated CC-AMWZ-3(T), was isolated from surface seawater off coastal Kending, Taiwan. Strain CC-AMWZ-3(T) was found to share 93.3 % and 96.0-92.4 % pairwise 16S rRNA gene sequence similarity to Gramella echinicola KMM 6050(T) and other Gramella species, respectively, and formed distinct phyletic lineage during phylogenetic analysis. The major fatty acids were identified as C16:0, iso-C15:0, anteiso-C15:0, C16:1 ω6c and/or C16:1 ω7c and iso-C17:1 ω9c and/or C16:0 10-methyl. Polar lipids were found to include phosphatidylethanolamine, six unidentified lipids and three unidentified aminolipids. The DNA G+C content was determined to be 40.6 mol%. Menaquinone-6 was the sole respiratory quinone identified and triamine-sym-homospermidine was the predominant polyamine. Based on the polyphasic characteristics that are in line with those of Gramella species, in addition to distinguishing phylogenetic and phenotypic features, strain CC-AMWZ-3(T) appears to represent a novel species of the genus Gramella, for which the name Gramella planctonica sp. nov. (type strain CC-AMWZ-3(T) = JCM 18807(T) = BCRC 80553(T)) is proposed. In addition, emended descriptions of the species Gramella aestuarii and Gramella echinicola are also proposed.

Sphingomicrobium astaxanthinifaciens sp. nov., an astaxanthin-producing glycolipid-rich bacterium isolated from surface seawater and emended description of the genus Sphingomicrobium.

A Gram-stain-negative, rod-shaped, strictly aerobic, flagellated and non-spore-forming marine bacterium designated strain CC-AMO-30B(T) was isolated from coastal surface seawater, Taiwan. Strain CC-AMO-30B(T) synthesized astaxanthin [40 µg (g dry weight)(-1)] and formed reddish-orange-coloured colonies on marine agar (Difco 2216). The strain showed highest pairwise 16S rRNA gene sequence similarity to Sphingomicrobium lutaoense CC-TBT-3(T) (96.4%) followed by other members of the family Sphingomonadaceae (<94%) and established a discrete phyletic lineage associated with the former. The polar lipid profile constituted a remarkable number of unidentified glycolipids (GL1-8), in addition to diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and two unidentified lipids (L1-2). The major fatty acids (>5% of total fatty acids) were C(18:1)ω7c/C(18:1)ω6c (summed feature 8), C(16:1)ω7c/C(16:1)ω6c (summed feature 3), C(18:1) 2-OH, methyl C(18:1)ω7c, C(17:1)ω6c and C(16 : 0). DNA G+C content was 70.6%; major respiratory quinone was ubiquinone Q-10; predominant polyamine was the triamine sym-homospermidine. Chemotaxonomic evidence including characteristic glycolipid profile, presence of significant amounts of C(18:1) 2-OH and absence of typical hydroxylated fatty acids such as C(14:0) 2-OH, C(15:0) 2-OH and C(16:0) 2-OH in considerable amounts, accompanied by phylogenetic distinctiveness and several other phenotypic features support the classification of strain CC-AMO-30B(T) as a representative of a novel species within the genus Sphingomicrobium for which the name Sphingomicrobium astaxanthinifaciens sp. nov. is proposed; the type strain is CC-AMO-30B(T) ( =JCM 18551(T) =BCRC 80465(T)).

The determination of flavonoids in Wikstroemia indica C. A. Mey. by liquid chromatography with photo-diode array detection and negative electrospray ionization tandem mass spectrometry.

RATIONALE: Flavonoids in the medicinal plant Wikstroemia indica C. A. Mey. are present in trace amounts and found in complex matrices. An efficient and sensitive method is necessary for the rapid identification of such biomolecules. METHODS: Flavonoids were extracted with methanol via ultrasonic-assisted extraction and analyzed by liquid chromatography with photo-diode array detection and tandem mass spectrometry. The extract was analyzed and compounds were identified using negative electrospray ionization data-dependent tandem mass spectrometry. RESULTS: The results confirmed the presence of three flavonoid compounds, seven biflavonoid compounds, and one coumarin-like compound, daphnoretin, in the extracts of different plant parts of W. indica. The method detection limit was evaluated down to 5 µg/g using kaempfol as a reference standard. CONCLUSIONS: The proposed method offers a rapid and reliable analysis for the determination of flavonoids in medicinal plants.

Determination of cyromazine and melamine in chicken eggs using quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction coupled with liquid chromatography-tandem mass spectrometry.

A rapid and sensitive method has been developed for the simultaneous detection of cyromazine and melamine in chicken eggs using the quick, easy, cheap, effective, rugged and safe (QuEChERS) method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimal extraction solvent for the liquid-liquid extraction was 5 mL of acetonitrile with a 0.1 M hydrochloric acid aqueous solution (99.5:0.5, v/v). The extract was cleaned with 0.5 g of anhydrous magnesium sulfate and 10 mg of graphitized carbon black. The analysis of cyromazine and melamine was accomplished by combining the use of an anion exchange LC column with tandem mass spectrometry in the positive electrospray ionization mode with selected reaction monitoring mode (SRM). The detection limits were 1.6 ng g(-1) for cyromazine and 8 ng g(-1) for melamine, and the quantitation limits were 5.5 ng g(-1) for cyromazine and 25 ng g(-1) for melamine. The recoveries of cyromazine and melamine in the spiked egg samples were 83.2% and 104.6%, respectively, with an relative standard deviation (RSD) of less than 18.1%. The intra-day and inter-day precisions, represented by the RSD, ranged from 1.5% to 8.8% and 6.8% to 14.3%, respectively. The proposed method was tested by analyzing chicken eggs from the markets and from the veterinary medicine laboratory. The concentrations of cyromazine and melamine detected in these samples were in the range of 20-94 ng g(-1). The results demonstrated that the QuEChERS method combined with LC-MS/MS is a simple, rapid and inexpensive method for the analysis of cyromazine and melamine in eggs.

Liquid chromatography incorporating ultraviolet and electrochemical analyses for dual detection of zeranol and zearalenone metabolites in mouldy grains.

BACKGROUND: Zeranol (Z) is a semi-synthetic mycotoxin that is used in some countries as a growth-promoting agent in livestock. In view of the known oestrogenic actions by Z and certain Z analogues, significant concerns exist with regard to the presence of Z residues in human foods and the potential for untoward effects, including carcinogenicity within the reproductive system. In order to confirm that foods are free from harmful Z residues, regulators need a quick and reliable analytical method that can be used for routine confirmation of Z-positive samples identified by enzyme-linked immunosorbent assay (ELISA) screening. In this study the authors have developed and validated a simple and rapid high-performance liquid chromatography method incorporating ultraviolet (UV) absorbance (wavelength 274 nm) and electrochemical (EC) dual-mode detection for simultaneous determination of Z-related mycotoxins produced from mouldy grain matrices, including rice, soybean and corn flakes. RESULTS: Recoveries for all analytes were around 80% and the limits of detection ranged from 10 to 25 ng mL(-1) for UV and from 50 to 90 ng mL(-1) for EC detection with good accuracy and reproducibility. Differential profiles and occurrence rates of Z, ß-zearalenol, ß-zearalanol and α-zearalenol in naturally moulded grain matrices were observed, indicating different metabolite patterns and possibly grain-specific effects of mycotoxin exposure for humans and animals. The strength of this dual detection method lies in its selectivity characterised by a carbon screen-printed electrode such that aflatoxin interference is precluded. CONCLUSION: The combined dual detection technique affords quick and reliable semi-confirmative and quantitative information on multiple types of Z analogues in mouldy grains without the necessity of using expensive mass spectrometry. The method is considered a superior supplement to ELISA, which only screens total Z immunoreactivity.

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the ß-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.

Differential heat stability of amphenicols characterized by structural degradation, mass spectrometry and antimicrobial activity.

Heat stability of amphenicols and the relationship between structural degradation and antimicrobial activity after heating has not been well investigated. Florfenicol (FF), thiamphenicol (TAP), and chloramphenicol (CAP) were heated at 100 degrees C in water, salt water, soybean sauce and chicken meat for up to 2h. Degradation and antimicrobial activity of the compounds was evaluated using capillary electrophoresis (CE) with UV-DAD spectrometry, minimum inhibitory concentration (MIC) assay, and gas chromatography with electron impact ionization mass spectrometry (GC-EI-MS). Heat stability of amphenicols in matrices was ranked as water> or =salt water>soybean sauce>meat, suggesting that heat degradation of amphenicols was accelerated in soybean sauce and was not protected in meat. Heat stability by drug and matrices was ranked as FF>TAP=CAP in water, FF=TAP>CAP in salt water, TAP> or =FF=CAP in soybean sauce, and TAP> or =FF=CAP in meat, indicating differential heat stability of amphenicols among the 3 drugs and in different matrices. In accordance with the less than 20% degradation, the MIC against Escherichia coli and Staphylococcus aureus did not change after 2h heating in water. A 5-min heating of amphenicols in water by microwave oven generated comparable percentage degradation to boiling in water bath for 30 min to 1h. Both CE and GC-MS analysis showed that heating of FF produced TAP but not FF amine as one of its breakdown products. In conclusion, despite close similarity in structure; amphenicols exhibited differential behavior toward heating degradation in solutions and protein matrices. Although higher degradations of amphenicols were observed in soybean sauce and meat, heating treatment may generate product with antimicrobial activity (FF to TAP), therefore, heating of amphenicol residues in food cannot always be assumed safe.

Study of the release of gallic acid from (-)-epigallocatechin gallate in old oolong tea by mass spectrometry.

Liquid chromatography combined with multiple-stage mass spectrometry (LC/MS(n)) was used to study the pathway of the release of gallic acid (GA) from epigallocatechin gallate (EGCG) in infusion of old oolong tea. The possibility of releasing GA from EGCG in old tea preparations was supported by an in vitro observation of GA degraded from EGCG under heating conditions mimicking the drying process. Negative electrospray ionization with the data-dependent mode of MS(n) was used to study the formation pathway of GA in old oolong tea. The MS(n) data show that GA was released from the dimer of EGCG, not directly degraded from EGCG.

Purge-assisted headspace solid-phase microextraction combined with gas chromatography-mass spectrometry for determination of chlorophenols in aqueous samples.

A simple, economical and very effective method is demonstrated for simultaneous determination of 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol, in aqueous samples, by using purge-assisted headspace solid-phase microextraction (PA/HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In the new method, purging the sample enhances the removal of the trace chlorophenols without derivatization from the matrices to the headspace. Extraction parameters including extraction temperature, purge gas flow rate and extraction time were systematically investigated. Under optimal conditions, the relative standard deviations (RSDs) were 4-11% at 50 pg/mL and 5-14% at 5 pg/mL, respectively. The recoveries were in the range of 83-114%. Detection limits were determined at the fg level. These results indicate that PA/HS-SPME provides a significant contribution to highly efficient extraction of semi-volatile CPs, especially for pentachlorophenol, which has the smallest Henry's constant and large octanol-water partitioning coefficient. In addition, the proposed method was successfully applied to the analysis of chlorophenols in landfill leachate. New perspectives are opened for headspace extraction of relatively low vapor pressure compounds in complex matrices.

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine.

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.

Hair analysis for methamphetamine, ketamine, morphine and codeine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography.

We established a capillary electrophoretic method with high sensitivity and specificity for testing hair taken from addicts. After pretreatment of hair sample, the cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was used to test for the presence of abused drugs in human hair. These drugs include morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, an uncoated fused-silica capillary (40 cm, 50 microm I.D.) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, followed by high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s). Electrokinetic injection (10 kV, 600 s) was used to load samples and to enhance sensitivity. Stacking steps and separations were performed at -20 kV with detection at 200 nm, using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to the level of picogram per milligram hair. During method validation, calibration plots were linear (r > or = 0.999) over a range of 0.15-80 ng/mg hair for MA and K, 0.3-30 ng/mg hair for C and 0.5-50 ng/mg hair for M. The limits of detection were 50 pg/mg hair for MA and K, 100 pg/mg hair for C and 200 pg/mg hair for M (S/N=3, sampling 600 s at 10 kV). Our method was applied for analysis of real hair samples taken from addicts. The addicts' specimens were also analyzed by LC-MS, and showed good coincidence of results. This method has proven feasible for application in detecting trace levels of abused drugs in forensic analysis.