[Fingerprinting analysis of non-saponins from water-soluble part and determination of dencichine from Panax notoginseng, P. ginseng and P. quinquefolium based on liquid chromatography coupled with charged aerosol detection].

This work describes the holistic fingerprinting method based on liquid chromatography coupled with charged aerosol detection(CAD) to profile non-saponin from water-soluble parts and determination of dencichine in Panax ginseng(PG), P. quinquefolium(PQ) and P. notoginseng(PNG). Sample extraction was carried out by water with ultra sonication for 30 min, which was eluted by Retain PEP for further analysis. The analysis was performed on a Hypercarb of porous graphitized carbon(3.0 mm×150 mm, 3 µm) column with acetonitrile and 0.1% perfluoropentanoic acid as mobile phase at a flow rate of 0.8 mL·min~(-1). Temperature of evaporator and nitrogen pressure for CAD were set at 50 ℃and 60.1 psi(1 psi≈6.895 kPa), respectively. As a result, dencichine and other polar components had a good performance on resolution and retention. The correlation coefficient(R~2) of dencichine was 0.998 2 in the concentration from 0.019 2 to 0.48 µg·mL~(-1). Limit of quantitation calculated by signal to noise of 10 was 7.4 ng·mL~(-1), and the recovery ranged from 95.52% to 102.7%. Chemical profile of the water-soluble part from PG, PQ and PNG was similar holistically, while the relative content for dencichine and other partial components varied significantly. The proposed method was used for characteristic of chemical profiling for non-saponin from water-soluble part, and determination of dencichine in PG, PQ and PNG.

Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata.

Echinocandin resistance in Candida glabrata poses a serious clinical challenge. The underlying resistance mechanism of a pan-echinocandin-resistant C. glabrata isolate (strain L74) was investigated in this study. FKS mutants carrying specific mutations found in L74 were reconstructed by the Alt-R CRISPR-Cas9 system (Fks1 WT/Fks2-E655K, strain CRISPR 31) and site-directed mutagenesis (strain fks1Δ/Fks2-E655K). Sequence analysis of strain L74 revealed a premature stop codon W508stop in FKS1 and an E655K mutation preceding the hotspot 1 region in FKS2. Introduction of the Fks2-E655K mutation in ATCC 2001 (strain CRISPR 31) conferred a modest reduction in susceptibility. However, the same FKS2 mutation in the fks1Δ background (strain fks1Δ/Fks2-E655K) resulted in high levels of resistance to echinocandins. Glucan synthase isolated from L74 was dramatically less sensitive to micafungin (MCF) relative to ATCC 2001. Both FKS1/FKS2 transcript ratios and Fks1/Fks2 protein ratios were significantly lower in L74 and fks1Δ/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P <0.05). Mice challenged with CRISPR 31 and fks1Δ/Fks2-E655K mutants failed to respond to MCF. In conclusion, the high-level of echinocandin resistance in the clinical isolate of C. glabrata L74 was concluded to result from the combination of null function of Fks1 and the point mutation E655K in Fks2.

Simultaneous quantitation of five Panax notoginseng saponins by multi heart-cutting two-dimensional liquid chromatography: Method development and application to the quality control of eight Notoginseng containing Chinese patent medicines.

Current China Pharmacopoeia (ChP) standards employ diversified and case-dependent assay methods to evaluate the quality of different Chinese patent medicines (CPMs) that contain Panax notoginseng as the monarch drug. These conventional, HPLC-based approaches, utilizing a complex sample preparation procedure, can easily result in low analytical efficiency and possible component loss. Here, a "monomethod-heterotrait matrix" (MHM) strategy is proposed, that is, developing a universal multi heart-cutting two-dimensional liquid chromatography (MHC-2D-LC) approach that facilitates the simultaneous quantitation of five P. notoginseng saponins (noto-R1, Re, Rg1, Rb1, and Rd) in eight different CPMs. The MHC-2D-LC system was constructed on a dual-gradient liquid chromatography instrument equipped with a Poroshell SB C18 column and a Zorbax SB-Aq column for respective (1)D and (2)D separation. Method validation was performed in terms of specificity, linearity (r(2) and F-test), intra-/inter-day precision (0.4-7.9%), stability (1.2-3.9%), and recovery (90.2-108.7%), and the LODs and LOQs (loaded masses) of the five analytes varied between 4.0-11.0ng and 6.0-33.0ng, respectively. The validated MHC-2D-LC approach was subsequently applied to quantify the five saponins in thirty batches of different CPMs. The method demonstrated superiority over the current ChP assay methods in respect of specificity (avoiding co-elution), resolution (Rs>1.5), sample preparation (easy-to-implement ultrasonic extraction without repeated re-extraction), and transfer rate (minimum component loss). This is the first application of an MHC-2D-LC method for the quantitative assessment of the constituents of CPMs. The MHM approach represents a new, strategically significant methodology for the quality control of CPMs that involve complex chemical matrix.

[Rapid determination of four components in Guizhi Fuling capsule with online two-dimensional liquid chromatography].

To establish the online two-dimensional liquid chromatography by using double gradient liquid chromatography system and UV detector, in order to simultaneously determine the content of paeoniflorin, paenol, amygdaloside and cinnamic acid. A pump of the two-dimensional liquid chromatography was adopted as the one-dimensional separation pump. C18 (3.0 mm x 150 mm, 3 microm) was used as the analytical column, with acetonitrile as the organic phase and 0.08% phosphoric acid + 0.08% triethylamine as the aqueous phase for gradient elution at the flow rate of 0.5 mL x min(-1). Another pump of the two-dimensional liquid chromatography was adopted as the two-dimensional separation pump. PAII C18 was used as the analytical column, with acetonitrile as the organic phase and 20 mmol, pH 3.0 monopotassium phosphate as the aqueous phase for gradient elution at the flow rate of 0.8 mL x min(-1). The detection wavelengths were set at 218, 230, 275 nm by using wavelength time-switching program. The linearity range of paeoniflorin, amygdaloside, paeonol and cinnamic acid were 5.55-222 (r = 0.999 7), 6.6-264 (r = 0.999 8), 3.3-132 (r = 0.999 5) and 0.315-12.6 mg x L(-1) (r = 0.999 7), respectively. The average recoveries of the four components were between 96.12% and 103.9%. The experiment proved that this method was so rapid and accurate in determination results that it could be used for evaluating drug quality.