Improved chromatographic fingerprints for facile differentiation of two Ganoderma spp.

This paper addresses a comprehensive and comparative study of six phytochemical extraction methods for triterpenes from the fruiting body of Ganoderma spp. Quantitative analysis of extracts was performed by HPLC with photodiode array detection. In general, pressurized liquid extraction and microwave-assisted extraction under optimized conditions produce better yields, and the former also significantly reduces the total time of extraction and manipulation of a sample, as well as the amount of solvent used in comparison with conventional soxhlet, reflux, ultrasonic, and methanol-CO(2) supercritical fluid extractions. Based on the improved extraction protocol, the fingerprinting profiles for two species of Lingzhi were established using the consistent chromatographic features of 12 authentic samples. Eleven common peaks of ganoderic/ganoderenic acids were identified using LC-ESI-MS-MS. These specific triterpene groups were adopted as chemical markers for Lingzhi. Using chemometric analysis, the developed fingerprinting was successfully applied to differentiate between the two species under the Ganoderma genus and is applicable as a method for quality evaluation of this valuable medicinal fungus and its related proprietary products.

Determination of sophoricoside in rat plasma by HPLC and its application to pharmacokinetic studies.

A simple, sensitive, selective and reproducible reversed-phase HPLC method was developed for the determination of sophoricoside in rat plasma after intravenous administration. Naringin was successfully used as internal standard (IS) for calibration. The chromatographic separation was accomplished on a reversed-phase C(18) column using acetonitrile-methanol-0.08% phosphoric acid (8:29:63, v/v/v) as mobile phase with a flow rate of 1.0 ml/min, with UV detection at 260 nm. Plasma samples were injected into the HPLC system after precipitating protein directly by methanol. Good linearity was achieved in the range of 0.0240 approximately 48.0 microg/ml (R(2)=0.9989). The limit of detection (LOD) and limit of quantification (LOQ) of this method were 0.0075 microg/ml and 0.0240 microg/ml, respectively. The absolute recoveries of sophoricoside from plasma were 95.8%, 93.2%, 98.0% at concentrations of 0.0240, 1.92, 15.0 microg/ml. The intra-day and inter-day variabilities were 3.39%~5.78% and 2.17%~4.72%, respectively. The developed method was successfully applied to the pharmacokinetic study of sophoricoside after intravenous administration of 2.5, 10 and 20 mg/kg in rats.

Quality evaluation of Evodia rutaecarpa (Juss.) Benth by high performance liquid chromatography with photodiode-array detection.

A simple, sensitive and accurate HPLC-DAD method was developed for simultaneous determination of wuchuyuamide-I, quercetin, limonin, evodiamine and rutaecarpine in Evodia rutaecarpa that has been widely used as one of the traditional Chinese medicines (TCMs). Chromatographic separations were performed on a reverse-phase C(18) column with the gradient elution of acetonitrile-water and the simultaneous detection at five wavelengths. Good linear behaviors over the investigated concentration ranges were observed with the values of r higher than 0.999 for all the analytes. The recoveries measured at three levels varied from 98.77 to 102.36%. The validated method was successfully applied for the simultaneous determination of the five chemical constituents in 36 batches of samples collected from different regions or time that were investigated and authenticated as E. rutaecarpa (Juss.) Benth. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to differentiate and classify the samples based on the contents of the five characteristic constituents. The total contents of evodiamine and rutaecarpine in different samples were calculated and the blending method proposed was demonstrated to be very useful in saving resources and in guiding rational herb use.

[Determination of isorhamnetin in Hippophae rhamnoides Linn from West Sichuan plateau using near infrared diffuse reflectance spectroscopy].

The objective of this study was to develop a method for the determination of isorhamnetin in Hippophae rhamnoides Linn from West Sichuan plateau using near infrared diffuse reflectance spectroscopy. Applying the method of mixing with SiO2, the near infrared spectra (NIS) with the range of 12 000-4 000 cm(-1) were recorded for the Hippophae rhamnoides Linn containing isorhamnetin with the content of 0.1%-0.8%. Calibration models were established using the PLS (partial least squares). Different spectra pretreatments methods were compared. The study showed that spectral information can be extracted thoroughly by constant offset elimination (COE) pretreatments method with the correlation coefficient (r2) of 0.739 8, SEC of 0.107 (standard deviation of the calibration sets) and SEP of 0.073 (standard deviation of the prediction sets). The results indicate that near infrared diffuse reflectance spectroscopy is more rapid and convenient than conventional methods.

Analysis of volatile components of Curcuma sichuanensis X. X. Chen by gas chromatography-mass spectrometry.

Volatile components of Curcuma sichuanensis X. X. Chen growing in Sichuan, China, were extracted by steam distillation and analyzed by using gas chromatography and gas chromatography-mass spectrometry. A total of 44 volatile essential oil components were identified in the extract of C. sichuanensis X. X. Chen, representing 87.1% of the total integrated chromatographic peaks. The major compounds were found to be epi-curzerenone (26.9%), germacrone (12.4%), isocurcumenol (9.7%), beta-elemene (6.4%) and curzerene (6.2%). The results of semi-quantitative analysis indicated that levels of total sesquiterpene fraction (85.4%) were more than 55 times higher than those of monoterpene components (1.5%).

[Determination of citrinin in red kojic by HPCE].

OBJECTIVE: To establish an instant determination method of citrinin in red kojic by high performance capillary electrophorphores for the first time. METHOD: Red kojic was extracted with the mixtrue of Toluene and ethyl acetate (70:30). Separation was carried out in an uncoated fused silica capillary (50 microm x 45.0 cm). Meanwhile, a running voltage 15 kV, 20 mmol L(-1) borax buffer with 10.0 mmol L(-1) sodium deoxycholate (pH 9.3) and a UV detector at 212 nm were adopted. RESULT: Regression equation of citrinin was Y=9434 + 16781X (r =0.990), The lower limit of quantification (S/N > or =3) was 0.5 mg mL(-1). The assay coefficients of variation ranged from 98.8% to 101.1%. The intra and inter recovery ranged from 0.83 to 1.54% and from 1.86 to 5.09%. Twenty samples were determined with the method. CONCLUSION: The method is proved to be simple, rapid and accurate, and it can be used to determine citrinin in red kojic.

Enantioselective determination of dencichine in rabbit plasma by high-performance liquid chromatography-electrospray mass spectrometry.

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C(18) cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-L-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect D-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for D-dencichine and 8 ng/ml for L-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 microg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.

[Analysis of dencichine by HPLC with pre-column derivatization].

OBJECTIVE: To establish a reversed-phase high performance liquid chromatorgraphy (RP-HPLC) method for detecting the dencichine in Panax notoginseng extracts and drug preparations. METHOD: Dencichine was extracted with the borate buffer (pH 9. 18) and the clear supernatant was used for the derivatization. Pre-column derivatization was performed using 9-fluorenylmethyl chloroformate (FMOC) to form derivatives. The mobile phase consisted of methanol and 0. 05 mol x L( -1) NaH2 PO4 (48: 52) (pH adjusted to 7.4 with NaOH solution) in a flow rate of 1.0 mL m min(-1). The ultraviolet (UV) detection wavelength was set at 262 nm. RESULT: The linearity was demonstrated over a wide range of concentration from 1.76 mg L(-1) to 352 mg x L(-1) for dencichine. The detection limit was determined to be 60 microg x L(-1). The derivative was stable and the derivatization agent did not influence the measurement of dencichine. The average recovery rate was 95. 3% and the relative standard derivation (RSD) was 1. 7%. The method was used to determine dencichine in different P. notoginseng extracts and drug preparations. CONCLUSION: This method is simple, fast and sensitive, suitable for determining the dencichine in P. notoginseng extracts and drug preparations as well as for the study of the dencichine metabolism in vivo.

[An experimental study of cellular mechanic properties of intestinal epithelial cells by micropipette aspiration].

OBJECTIVE: To further understand the mechanic properties of the Intestinal Epithelial cells (IEC). METHODS: The viscoelastic properties of the IEC-6 were determined by micropipette aspiration. RESULTS: The elastic coefficient K1 was 436.21 +/- 89.64 dyn/cm2 and K2 was 298.37 +/- 75.16 dyn/cm2. The visous coefficient micro was 15.94 +/- 4.93 dyn/(sec x cm2). CONCLUSION: The elastic component reflects the degree of initial deformation of the IEC and makes a notable impact on the deformation of the IEC. The viscous component reflects the time dependency of the deformation of the IEC and influences the velocity of the IEC deformation.

[Study on the determination of piperazinylethylestrone].

OBJECTIVE: To establish an HPLC method and a non-aqueous titration for the determination of piperazinylethylestrone drug substance, and an HPLC method for the determination of piperazinylethylestrone in dog plasma. METHODS: Anhydrous acetic acid as solvent, 0.1 mol/L perchloric acid as titrant, crystal violet solution as indicator to establish non-aqueous titrations and ODS column as stationary phase, methanol and a mixture of 0. 025 mol/L sodium phosphate monobasic and 0.02 mol/L sodium dedecyl sulfate (80:20) [adjusted with phosphoric acid to a pH (4.8 +/- 0.1)] as mobile phase, 220 nm as detective wavelength to establish HPLC-UV method for determination of piperazinylethylestrone drug substance; FMOC-CL as a derivatization reagent, FD as a detector to establish an HPLC method with derivatization and column switching for determination of piperazinylethylestrone in dog plasma. RESULTS: The RSD of non-aqueous titrations within-day and between-day were 0.28% and 0.21%, respectively; the established HPLC-UV method had good linearity and precision within the range of 1.001-5.005 microg of piperazinylethylestrone, the detection limit was 4 ng (S/N=3); the linearity of HPLC method with derivatization and column switching was within the range of 8.4-420 ng/ml, the detection limit was 1 ng (S/N=3). The clean-up recoveries were from 77.24% to 83.10%, and the method recoveries were 98.33%-103.3%. The RSD of within-day and between-day were less than 7.7% and 7.3%, respectively. CONCLUSION: The above three methods are simple, accurate and precise for the determination of piperazinylethylestrone drug substance and for the determination of piperazinylethylestrone in dog plasma.

Tentative identification, quantitation, and principal component analysis of green pu-erh, green, and white teas using UPLC/DAD/MS.

Tea (Camellia sinensis L.), an important drink and a natural medicine for thousands of years, contains many health beneficial compounds. Growing season, geographical region, and fermentation methods create many variations in tea compositions, which contribute to each tea's uniqueness. In this study, a simple, rapid, and efficient ultra-performance liquid chromatography (UPLC) method combined with diode array detector (DAD) and mass spectroscopic (MS) detection and chemometrics analysis was used to analyse three different types of teas (green pu-erh, green tea, white tea). Using the developed method, 68 compounds were identified and 54 were quantified based on retention times, UV spectra, and MS spectra by referencing to available standards and data in the literatures. The results showed the chemical differences between the tested teas. Principal component analysis (PCA) was applied to classify and distinguish between tea samples.

[Determination of dencichine in sanchi using liquid chromatography on porous graphitic carbon column].

A simple reversed-phase high performance liquid chromatographic method was developed for the determination of dencichine in sanchi on a porous graphitic carbon (PGC) column. The effects of different buffers, buffer concentrations, pH values of mobile phase and mobile phase compositions as well as column temperatures on the retention of dencichine were studied. The retention mechanism of dencichine on a porous graphitic carbon column seems to be mainly hydrophobic interaction according to the study. The chromatographic method developed was used to determine dencichine in sanchi. The calibration curve was linear in the range of about 0.22 - 4.4 microg (r = 0.999 9) and the recovery for the extract was 99.5% with the relative standard deviation of 2.2% (n = 9).

Evolutionary and biomedical insights from the rhesus macaque genome.

The rhesus macaque (Macaca mulatta) is an abundant primate species that diverged from the ancestors of Homo sapiens about 25 million years ago. Because they are genetically and physiologically similar to humans, rhesus monkeys are the most widely used nonhuman primate in basic and applied biomedical research. We determined the genome sequence of an Indian-origin Macaca mulatta female and compared the data with chimpanzees and humans to reveal the structure of ancestral primate genomes and to identify evidence for positive selection and lineage-specific expansions and contractions of gene families. A comparison of sequences from individual animals was used to investigate their underlying genetic diversity. The complete description of the macaque genome blueprint enhances the utility of this animal model for biomedical research and improves our understanding of the basic biology of the species.

[Study on subcritical water extraction of baicalin from Radix Scutellariae coupled to high performance liquid chromatographic analysis].

A method for the extraction of baicalin from Radix Scutellariae with subcritical water coupled to high performance liquid chromatographic analysis was established. The effects of various parameters, which included the pressure, temperature, extraction time, and the particle size of the plant material as well as the solvent/sample ratio on the yield were investigated. Compared to the conventional organic solvent extraction method, the subcritical water extraction method showed shorter handling time and lower solvent consumption without waste producing. The optimal conditions of extraction of baicalin from Radix Scutellariae with subcritical water were 0.15 - 0.18 mm particle size, solvent/sample ratio of 0.2 mL/mg, 5 MPa, 130 degrees C for about 10 min. This technique seems very promising for the extraction of liposoluble substances from plant materials.

[Monitoring of the residue of fosthiazate in water samples using solid-phase extraction coupled with gas chromatography/mass spectrometry].

Solid-phase extraction (SPE) coupled with gas chromatography/mass spectrometry (GC/MS) was used to determine the fosthiazate residue in water samples. The water samples were first filtered through cellulose filters (0.45 microm pore size). A 100 mL volume of filtered water, in which 1 mL of methanol has been added, was then passed through a pre-conditioned 3 cm C18 cartridge at a flow-rate of 1.5 mL/min. Elution was performed by 1 mL of methanol. The eluant was finally dried under reduced pressure for solvent evaporation. The volume was quantitatively adjusted to 0.5 mL with methanol. The analysis was carried out on GC/MS. The mass spectrometer was operated in selected ion monitoring (SIM) mode. According to mass spectrum of fosthiazate, three selected ions at m/z of 126, 195, 283, respectively, were monitored for identification and quantification. High sensitivity and selectivity were achieved by using this method. The limit of detection for fosthiazate in water samples was determined to be 56.4 ng/L. The linearity was demonstrated over a wide range of concentrations covering from 0.282 to 141 microg/L. The recoveries were more than 85.5% and the relative standard deviations for the overall procedure were less than 4.42%. The fosthiazate residue was detected in the water samples from a pool near cropland where fosthiazate was used. The results demonstrate the suitability of the SPE-GC/MS approach for the analysis of fosthiazate in water.