Glycosylated platycosides: identification by enzymatic hydrolysis and structural determination by LC-MS/MS.

In this study, enzymatic hydrolysis and chemometric methods were utilized to discriminate glycosylated platycosides in the extract of Platycodi Radix by LC-MS. Laminarinase, whose enzymatic activity was evaluated using gentiobiose and laminaritriose, was a suitable enzyme to identify the glycosylated platycosides. The laminarinase produced deapi-platycodin D and platycodin D from the isolated deapi-platycoside E and platycoside E through the loss of two glucose units by enzymatic reaction, respectively. After hydrolyzing a crude extract by laminarinase, the reconstructed total ion chromatogram generated by a chemometric technique sorted peaks of deglycosylated platycosides easily. Structural information of the glycosylated isomers was revealed through fragment ions generated by the sodiated C0ß ion corresponding to reduced disaccharides in the positive MS(4) spectra. Characteristic fragment ions of Glc-(1→6)-Glc moieties were observed through ring cleavages of (0,2)A0ß, (0,3)A0ß, and (0,4)A0ß, whereas Glc-(1→3)-Glc moieties produced only (0,3)A0ß ions. Lithium-adducted platycosides allowed more detailed structural analysis of glycosidic bond cleavage corresponding to Y1ß and B1ß in addition to ring cleavage.

Comparison of GC/MS and LC/MS methods for the analysis of propofol and its metabolites in urine.

Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) were compared for their capacity to metabolite identification, sensitivity, and speed of analysis for propofol and its metabolites in urine samples. Acidic hydrolysis, liquid-liquid extraction (LLE), and trimethylsilyl (TMS) derivatization procedures were applied for GC/MS analysis. The LC/MS analysis used a simple sample pretreatment based on centrifugation and dilution. Propofol and four metabolites were successfully analyzed by GC/MS following TMS derivatization. One compound, di-isopropanolphenol was tentatively characterized as a new metabolite observed for the first time in human urine. The TMS derivatization greatly improved the chromatographic properties and detection sensitivity, especially for hydroxylated metabolites. The lower limits of quantitation (LLOQ) of propofol were about 325 and 0.51 ng/mL for the GC/MS scan mode and selected ion monitoring (SIM) mode, respectively. In addition, five conjugated propofol metabolites were successfully analyzed by LC-MS/MS in negative ion mode. The detection sensitivity for these conjugated metabolites could be greatly enhanced by the addition of triethylamine to the mobile phase without any loss of LC resolution capacity. The LLOQs of propofol-glucuronide (PG) were about 1.17 and 2.01 ng/mL for the LC-MS-selected ion monitoring (SIM) and multiple reaction monitoring (MRM) mode, respectively. Both GC/MS and LC/MS methods sensitively detected nine metabolites of propofol and could be used to provide complementary data for the reasonable propofol metabolism study. Urinary excretion profiles for propofol and its metabolites following administration to human were suggested based on the total ion chromatograms obtained by GC/MS and LC/MS methods, respectively.

Identification of structurally diverse alkaloids in Corydalis species by liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Alkaloids with significant therapeutic effects are the main active constituents of Corydalis (C.) species. There are several kinds of alkaloids in C. species associated with diverse alkaloid metabolism in plants, but they are rarely identified. This study aimed to identify diverse alkaloids in C. species by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). METHODS: Several types of alkaloids were extracted from C. species using ultrasonication with 70% CH(3)OH, and the extract was partitioned at pH 2 and 12. Separation of alkaloids was achieved by C18 high-performance liquid chromatography (HPLC), and MS/MS analysis was conducted by electrospray ionization triple-quadrupole mass spectrometry. For further confirmation, LC/Fourier transform ion cyclotron resonance (FTICR)-MS was used to obtain accurate mass data and gas chromatography (GC)/MS combined with trimethylsilyl derivatization was applied for identification of the minor alkaloids. RESULTS: Thirty-three alkaloids among three different C. species were successfully separated and identified by LC/ESI-MS/MS and LC/FTICR-MS. Structural assignment of individual alkaloids was performed according to MS/MS spectral patterns. For further confirmation, accurate mass data of alkaloids by LC/FTICR-MS were obtained within 5 ppm and the GC/MS data for the trimethylsilyl alkaloids were also obtained. Among 33 alkaloids identified from this study, 13 alkaloids were reported for the first time in the investigated C. species. CONCLUSIONS: The LC/ESI-MS/MS technique was effective in obtaining structural information and yielded diagnostic ions for diverse alkaloids. Based on the identified 33 alkaloids, marker compounds were suggested for the three C. species with different geographic origins. This study may also be useful for elucidating unknown alkaloids in herbal medicines.

Antiproliferative effects of Dangyuja (Citrus grandis Osbeck) leaves through suppression of constitutive signal transducer and activator of transcription 3 activation in human prostate carcinoma DU145 cells.

Although Dangyuja (Citrus grandis Osbeck) exhibits anti-inflammatory and anticancer activities, its molecular targets and pathways, especially in human prostate cancer cells, are not fully understood. In this study, the antiproliferative effect of Dangyuja leaves through the signal transducer and activator of transcription (STAT) 3 signaling pathway was investigated in human prostate carcinoma DU145 cells. The solvent fractions (n-hexane, chloroform, ethyl acetate, and n-butanol) were obtained from a crude extract (80% methanol extract) of Dangyuja leaves. We first found that the chloroform fraction of Dangyuja leaves (DCF) was the most cytotoxic against DU145 cells. DCF inhibited constitutive STAT3 activation through blocking upstream Janus-like kinase 2 and c-Src. Consistent with STAT3 inactivation, DCF down-regulated the expression of STAT3 target genes, including bcl-2, bcl-xl, and cyclin D1; this correlated with the suppression of proliferation, the accumulation of cell cycle at the sub-G(1) phase, and the induction of apoptosis. Furthermore, DCF exerted a relatively minor effect on the growth of human prostate noncancerous RWPE-1 cells. Nobiletin, a major active constituent of DCF, could induce apoptosis via the suppression of constitutive STAT3 activation. Overall, our results indicate that the anti-inflammatory and anticancer activities previously assigned to DCF may be mediated partially through the suppression of the STAT3 signaling.

Development and optimization of a method for the separation of platycosides in Platycodi Radix by comprehensive two-dimensional liquid chromatography with mass spectrometric detection.

Comprehensive two-dimensional chromatography (LCxLC) using combinations of two columns (C(18) x CN and C(18) x NH(2)) was employed with electrospray (ESI) mass spectrometry to analyze platycosides from root extract. Based on the capability of the C(18), CN and NH(2) columns to separate the platycosides, the orthogonality in two-dimensional space according to each combination of columns was predicted from the correlation coefficients between the retention times of the 17 compounds separated by the independent CN and C(18) columns, and NH(2) and C(18) columns. The expected distribution of the peaks was also compared with the two-dimensional plots obtained by practical separation in an LCxLC system. The increased peak capacities using C(18) x NH(2) allowed three minor components and five isomers of the platycosides to be newly separated, which were not identified with 1D-LC using the individual C(18) column, whereas the combination of C(18) x CN did not result in any improvement of the separation performance.