A strategy for the enrichment and characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom.

A new strategy combined gold-coated magnetic nanocomposites assisted enrichment with mass spectrometry was developed for the characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom. In this work, core-shell nanocomposites were synthesized by the seed-mediated growth method and used for the enrichment of snake venom proteins containing disulfide bonds. A total of 3545 tryptic digested peptides derived from 96 venom proteins in Naja atra venom were identified. The venom proteins comprised 14 toxin families including three-finger toxins, phospholipase A2 , snake venom metalloproteinase, cobra venom factor, and so forth. Extra 16 venom proteins were detected exclusively in the nanocomposites set, among which 11 venom proteins were from the three-finger toxins family. In the present study, the proposed simple and efficient protocol replaced the tedious and laborious technologies commonly used for pre-separating crude snake venom, suggesting widely implementation in low-abundance or trace disulfide bond-contained proteins or peptides characterization.

[Quality evaluation of fried Glycyrrhizae Radix et Rhizoma pieces by HPLC fingerprint and multicomponent quantitative analysis].

To establish the HPLC fingerprint and multi-component determination method of fried Glycyrrhizae Radix et Rhizoma pieces. HPLC analysis was performed on Thermo Acclaim ~(TM)120 C_(18) column(4.6 mm×250 mm, 5 µm). Acetonitrile-0.1% phosphoric acid aqueous solution was taken as the mobile phase for gradient elution. The flow rate was 1 mL·min~(-1),the column temperature was maintained at 30 ℃, and the detection wavelength was 237 nm and 360 nm. The similarity of 15 batches of fried Glycyrrhizae Radix et Rhizoma pieces was higher than 0.849, and 17 common peaks were identified. Liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, isoliquiritigenin and glycyrrhizic acid were identified; among them, the mass fractions of Liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, glycyrrhizic acid were were 0.519%-3.058%, 0.227%-0.389%, 0.070%-0.439%, 0.038%-0.173%, 1.381%-4.252%, respectively. According to the cluster analysis, the 15 batches of decoction pieces were classified into three categories; principal component analysis screened out four principal components, with the cumulative variance contribution rate of 86.630%, indicating that the principal components contained most information of original data. Partial least squares discriminant ana-lysis marked 6 differential components in the decoction pieces. The established fingerprint and multicomponent determination are stable and reliable, and can provide a reference for the quality control of Radix Glycyrrhizae Radix et Rhizomae and fried Glycyrrhizae Radix et Rhizoma pieces.

Simultaneous determination of sulfur compounds from the sulfur pathway in rat plasma by liquid chromatography tandem mass spectrometry: application to the study of the effect of Shao Fu Zhu Yu decoction.

A sensitive, accurate, and time-saving approach was developed for the simultaneous quantification of eight sulfur compounds in the sulfur pathway, which could reflect the status of an organism, including oxidative stress, signal transduction, enzyme reaction, and so on. In order to overcome the instability of highly reactive sulfhydryl compounds, N-ethylmaleimide derivatization was adopted to effectively protect sulfhydryl-containing samples. Using isotope-labeled glutathione (GSH-13C2, 15N), the validated method was demonstrated to offer satisfactory linearity, accuracy, and precision. Separation was done by UHPLC, using a BEH amide column. Accordingly, 0.1% formic acid acetonitrile was selected as the precipitant. A tandem mass spectrometer was coupled to the chromatographic system and afforded a detection limit of 0.2 ng/mL. Good linearity was maintained over a wide concentration range (r2 > 0.994), and the accuracy was in the range of 86.6-114% for all the studied compounds. The precision, expressed in RSD%, ranged from 1.1% to 9.4% as intraday variability and less than 13% as interday precision for all of the analytes. The approach was applied to study the potential therapeutic mechanism of a well-known traditional Chinese medicine, Shao Fu Zhu Yu decoction. The results suggested that Shao Fu Zhu Yu decoction might protect against oxidative damage by increasing the concentrations of sulfhydryl compounds. Graphical abstract An approach to quantitatively determining sulfur compounds in the sulfur pathway simultaneously wasestablished and applied to the study of the effect of Shao Fu Zhu Yu decoction.

[Tissue Distribution of Alkaloids from Coptidis Rhizoma in Rats Determined by UPLC-MS].

Objective: To develop an UPLC-MS method for simultaneous determination of alkaloids from Coptidis Rhizoma in rat tissues, and to study the tissue distribution of alkaloids from Coptidis Rhizoma in rats. Methods: The samples were extracted with ethyl acetate, and analyzed by UPLC-MS with acetonitrile-0. 2% formic acid solution in a gradient elution mobile phase, the flow rate was 0. 2m L/min. Tetrahydropalmatine was used as an internal standard. The mass spectrometer was operated in selected reaction monitoring( SIM) mode with positive electrospray ionization, the transition were m/z 191. 904 /118. 973( noroxyhydrastinine), m/z 335. 877 /308. 072( 8-ocoptisine),m/z 351. 94 /294. 554( palmatine chloride),m/z 335. 94 /262. 112( epiberberine), m/z 337. 94 /322. 422( columbamine), m/z 319. 904 /292. 037( coptisine), m/z 355. 977 /192. 036( tetrahydropalmatine),m/z 335. 94 /320. 036( berberine hydrochloride),m/z 351. 94 /321. 995( oxyberberin), m/z 337. 94 /322. 949( jatrorrhizine respectively). Results: Excellent linearity was observed in all alkaloids in their linear range( r & 0. 9901). The RSD of precision of the developed method was less than 15%,and the accuracy and stability were less than ± 15%,the extraction recovery was 72. 1% ~ 82. 9% with RSD less than 15%. Coptisine,epiberberine,berberine,jatrorrhizine,columbamine,palmatine were widely distributed in rat tissues. Noroxyhydrastinine,8-ocoptisine,oxyberberin could only be determined in liver and heart or kidney. Conclusion: The established method is simple and accurate. Satisfactory results are obtained with applying this method to the tissue distribution study of alkaloids from Coptidis Rhizoma.

Application of the stochastic resonance algorithm to the simultaneous quantitative determination of multiple weak peaks of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry.

The stochastic resonance algorithm (SRA) has been developed as a potential tool for amplifying and determining weak chromatographic peaks in recent years. However, the conventional SRA cannot be applied directly to ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS). The obstacle lies in the fact that the narrow peaks generated by UPLC contain high-frequency components which fall beyond the restrictions of the theory of stochastic resonance. Although there already exists an algorithm that allows a high-frequency weak signal to be detected, the sampling frequency of TOFMS is not fast enough to meet the requirement of the algorithm. Another problem is the depression of the weak peak of the compound with low concentration or weak detection response, which prevents the simultaneous determination of multi-component UPLC/TOFMS peaks. In order to lower the frequencies of the peaks, an interpolation and re-scaling frequency stochastic resonance (IRSR) is proposed, which re-scales the peak frequencies via linear interpolating sample points numerically. The re-scaled UPLC/TOFMS peaks could then be amplified significantly. By introducing an external energy field upon the UPLC/TOFMS signals, the method of energy gain was developed to simultaneously amplify and determine weak peaks from multi-components. Subsequently, a multi-component stochastic resonance algorithm was constructed for the simultaneous quantitative determination of multiple weak UPLC/TOFMS peaks based on the two methods. The optimization of parameters was discussed in detail with simulated data sets, and the applicability of the algorithm was evaluated by quantitative analysis of three alkaloids in human plasma using UPLC/TOFMS. The new algorithm behaved well in the improvement of signal-to-noise (S/N) compared to several normally used peak enhancement methods, including the Savitzky-Golay filter, Whittaker-Eilers smoother and matched filtration.

[Study on HPLC fingerprint chromatograms of Arisaematis Rhizoma].

The fingerprint chromatograms of Arisaematis Rhizoma were established by HPLC. The analysis was performed on a Lichrospher C18 (4.6 mm x 200 mm, 5 microm) column with acetonitrile-water (containing 0.1% acetic acid) as mobile phase at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 270 nm, and the column temperature was 30 degrees C. The similarities of the fingerprint chromatograms were calculated over 0.9 between 11 batches of Arisaematis Rhizoma samples by analyzing 14 common peaks with adenosine as reference substance. However, their fingerprint chromatograms were significantly different from those of Pinellia pedatisecta and P. ternate. Adenine, hypoxanthine, xanthine, uridine, guanosine, adenosine, schaftoside, and isoschaftoside were identified by comparing the retention times and their ultraviolet spectra. The method is repeatable, exclusive and can be used for identification and evaluation of Arisaematis Rhizoma.

[Analysis of flavonoids in platycladi cacumen by UPLC-MS].

OBJECTIVE: To investigate the flavonoids in Platycladi Cacumen. METHOD: The constituents in Platycladi Cacumen were determined by UPLC-MS. A Waters BEH C18 column (2.1 mm x 150 mm, 1.7 microm) was used with a gradient elution of methanol-water containing 0.2% formic acid. The mass spectrometer equipped with electrospay ionization source was used as defector and operated in data was collected under the negative ion modes. RESULT: Eleven constituents were identified. CONCLUSION: In this study, the main flavonoids in Platycladi Cacumen were separated by UPLC, and identified through the information of mass number and UV spectra. With the information of MS/MS from flavonoids, the modes of breaking up flavonoids were discussed. It is an accurate and effective method which can be applied for the constituent identification of Platycladi Cacumen.

Novel computer-assisted separation prediction strategy for online-enrichment-HPLC-FLD in simultaneous monitoring of bisphenols in children's water bottles.

An on-line enrichment-liquid chromatography-fluorescence detection (LC-FD) method was developed for simultaneous determination of nine bisphenols (BPs). In this process, we predicted the separation based on an in-house developed software allowing for calculating both retention time (tR) and half-peak width (W1/2) of the solute by mobile phase fraction (φ) under gradient conditions. The proposed strategy was applied to separation prediction of BPs with high accuracy. Under the optimized conditions, good linearity was obtained with the correlation coefficients (R2) ranging from 0.998 to 1.000. The recoveries in spiked samples were 91.3-110.7% with the intra-day and inter-day relative standard deviation ranging 0.4-9.6% and 0.5-10.2%, respectively. The limits of detection and quantification were 0.13-66.7 ng L-1 and 0.40-200 ng L-1. The developed approach was used to monitor the nine BPs in 28 children's water bottles. The developed method provides an effective way for monitoring bisphenols in other similar matrix.

[Common and variant peak ratios in IR fingerprint of Cacumen platycladi with dual-index sequence analysis].

To establish a new method to analyze IR fingerprint, which is in line with the characteristic of traditional Chinese medicine, two indexes, common peak ratio and variant peak ratio, were applied and their values were calculated by means of sequential analysis, in which each Cacumen platycladi sample's IR fingerprint spectra were set up and the common peak ratio sequences were arranged in the order of size in comparison with other samples. The analytical results showed that samples G1 and G8 from the same region, and G4, G2 and G6 from the closer regions were the most similar samples with higher common peak ratio (> or = 90.0%) and lower variant peak ratio (< or = 11.1%). However, the samples G10, G3, G4 and G5 from the closer regions collected in different years, and G2 and G7 from the farther regions,were of significant disparity with common peak ratio less than 50% and variant peak ratio larger than 50%. As a result, the method could be used to distinguish Cacumen platycladi of different areas and batches. The dual index sequential analysis enables us to distinguish two or more herb's IR fingerprints, is a new method to analyze IR fingerprint spectra, and can be used in line with the characteristics of traditional Chinese medicine.

[Determination of nine bisphenols in children's water bottle by online enrichment coupled with high performance liquid chromatography-fluorescence detection].

A method was developed for the rapid determination of bisphenol A (BPA) and eight structural analogs in children's plastic water bottles by online enrichment coupled with high performance liquid chromatography-fluorescence detection (HPLC-FLD). The correlation coefficients of the nine bisphenols were greater than 0.998. The limits of detection (LOQs) ranged from 0.13 ng/L to 66.7 ng/L. The recoveries ranged from 90.7% to 112.4% (RSD<11.3%, n=6). This method was applied to monitor nine bisphenols in children's water bottles. The results showed that except 4,4'-(9H-fluoren-9-ylidene)bisphenol (BPFL), all the remaining eight bisphenols were detected in different water bottles. The amounts of bisphenols leached increased with the increase of soaking time, and decreased after washing several times at 100℃. The proposed strategy is rapid, sensitive, reliable and eco-friendly, and is suitable for the simultaneous analysis of new bisphenols in water samples.

FÖrster resonance energy transfer (FRET)-based biosensors for biological applications.

FÖrster resonance energy transfer (FRET)-based biosensors have achieved great success for biological applications. However, what is not extensively appreciated is the growing role as versatile FRET biosensors within a similar biological context. This review provides a brief introduction of recent advances in principle, the designing strategies and kinds of applications of FRET biosensors. For each FRET biosensor, the appropriate background and fabrication is explained before studying their related applications. The prominent roles of nanomaterials are present in the development of more sensitive and specific FRET biosensors. Finally, the challenges and outlooks of FRET biosensors are emphasized.

Chemical profiling of Banxia-Baizhu-Tianma decoction by ultra-fast liquid chromatography with tandem mass spectrometry.

Banxia-Baizhu-Tianma decoction (BBTD) is a compound formulae of traditional Chinese medicine (TCM), which has been clinically used for treatments of neural vertigo, hypertension and epilepsy with a long history. In this study, with an ultra-fast liquid chromatography coupled with quadrupole time of flight mass spectrometry (UFLC-Q-TOF-MS) method, a total of 88 components in BBTD were identified by the accurate masses and fragmentation pathways including 19 flavonoids, 8 lactones, 12 triterpenoids, 10 phenolics, 14 amino acids, 13 nucleobases and nucleosides, 7 organic acids, and 5 other compounds. In addition, under the same chromatographic conditions, we developed an ultra-fast liquid chromatography coupled with quadrupole linear ion trap mass spectrometry (UFLC-Q-TRAP-MS) method to simultaneously quantify 20 bioactive components in multiple-reaction monitoring (MRM) mode. The assay method was validated in terms of linearity, precision, repeatability, recovery and was successfully applied for determination of 12 batches of BBTD. We hope that this study work would help to reveal the chemical profiling and provide a valuable and reliable approach for quality evaluation and even efficacy material basis study of BBTD.

[Simultaneous determination of six estrogens in dairy products by high performance liquid chromatography-triple quadrupole-ion trap mass spectrometry coupled with matrix solid-phase dispersion and solid phase extraction].

Matrix solid-phase dispersion (MSPD) and solid-phase extraction (SPE) were respectively developed for the pretreatment six estrogens in milk powder and liquid milk. It was implied that MSPD was suitable for the treatment of milk powder, while SPE was suitable for liquid milk treatment. Based on the optimized pretreatment procedures, the method for the simultaneous determination of the six estrogens in different dairy products was established by high performance liquid chromatography-triple quadrupole-ion trap mass spectrometry (HPLC-Q-TRAP-MS). The proposed method provided low limits of detection (LODs, 0.01-0.05 mg/L) and limits of quantification (LOQs, 0.05-0.10 mg/L), wide linearity range of 0.1-200 mg/L (except for estriol of 0.1-20 mg/L) with excellent correlation coefficients (R2)> 0.99. The average recoveries of the six estrogens in milk powder pretreated by MSPD ranged from 71.8% to 106.0% with RSD of 1.6%-9.2% (n=3), while the corresponding average recoveries in liquid milk pretreated by SPE ranged from 70.3% to 108.4% with RSD of 2.0% and 11.0% (n=3) with spiking levels of 1.0, 5.0, and 10 mg/kg, respectively. This sensitive and reliable method meets the demand for the analysis of trace estrogen residues in complex matrices.

Determination of donepezil hydrochloride (E2020) in plasma by liquid chromatography-mass spectrometry and its application to pharmacokinetic studies in healthy, young, Chinese subjects.

A sensitive, simple, and specific liquid chromatographic method coupled with electrospray ionization-mass spectrometry for the determination of donepezil in plasma is developed, and its pharmacokinetics in healthy, male, Chinese is studied. Using loratadine as the internal standard, after extraction of the alkalized plasma by isopropyl alcohol-n-hexane (3:97, v/v), solutes are separated on a C(18) column with a mobile phase of methanol-acetate buffer (pH 4.0) (80:20, v/v). Detection is performed with a time-of-flight mass spectrometer equipped with an electrospray ionization source operated in the positive-ionization mode. Quantitation of E2020 is accomplished by computing the peak area ratio (donepezil [M+H](+) m/z 380-loratadine [M+H](+) m/z 383) and comparing them with the calibration curve (r = 0.9998). The linear calibration curve is obtained in the concentration range 0.1-15 ng/mL. The limit of quantitation is 0.1 ng/mL. The mean recovery of E2020 from human plasma is 99.4% +/- 6.3% (ranging 93.4-102.6%). The inter- and intraday relative standard deviation is less than 15%. After an oral administration of 5 mg E2020 to 20 healthy Chinese volunteers, the main pharmacokinetic parameters of E2020 are as follow: T(max), 3.10 +/- 0.55 h; t((1/2)), 65.7 +/- 12.8 h; C(max), 10.1 +/- 2.02 ng/mL; MRT, 89.4 +/- 13.4 h; and CL/F, 9.9 +/- 4.3 L/h.

[Determination of donepezil in human plasma by HPLC-MS].

AIM: To establish a sensitive and specific liquid chromatography-mass spectrometry (time-of-flight) [LC-MS (TOF)] method for the determination of donepezil in human plasma after an oral administration of 5 mg donepezil hydrochloride tablet. METHODS: Alkalized plasma was extracted with isopropanol-n-hexane (3:97) and loratadine was used as internal standard. Solutes were separated on a C18 column with a mobile phase of methanol-acetate buffer (pH 4.0) (80:20). Detection was performed on a time-of-flight mass spectrometry equipped with an ESI interface and operated in positive-ionization mode. Donepezil quantitation was realized by computing the peak area ratio (donepezil-loratadine) (donepezil m/z 380[M + H]+ and loratadine m/z 383[M + H]+) and comparing them with calibration curve (r = 0.9998). RESULTS: The linear calibration curve was obtained in the concentration range of 0.1-15 micrograms.L-1. The detection limit of donepezil was 0.1 microgram.L-1. The average recovery was more than 90%. The intra- and inter-run precision was measured to be below 15% of RSD. CONCLUSION: The method is sensitive, simple and rapid, so, it can meet the need of the studies on the pharmacokinetics and bioavailability of donepezil.

[Qualitative and quantitative analysis of the amino acids in Rhizoma Arisaematis by ultra high performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography].

A method for the identification and determination of the polar amino components without ultraviolet activity in traditional Chinese medicines was developed. With Rhizoma Arisaematis as the object of this study, using pre-column derivatization with phenyl isothiocyanate (PITC) as the derivatization reagent, compounds were separated and identified on a C18 column (100 mm x 2.1 mm, 3.5 µm) by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). A total of 20 components, including 18 amino acids and 2 amine compounds were identified. Furthermore, after the optimization of the derivatization conditions, 15 amino acids were determined by high performance liquid chromatography (HPLC) on Diamonsil C18 column (250 mm x 4.6 mm, 5 µm), detected at 254 nm and gradiently eluted by acetonitrile and 0. 05 mol/L ammonium acetate-acetic acid (pH 6. 5) as the mobile phases. The results of methodological study demonstrated that the method can meet the requirements of the determination. All calibration curves expressed good linearity: Glu, Try in the range of 2-100 mg/L, Arg in the range of 6-300 mg/L, others in the range of 0. 8-40 µg/L, with the correlation coefficients ≥ 0. 999 5. The average recovery of this method was among 95%-105% and the RSD was less than 3%. The developed method was successfully applied to quantitative determination of amino compounds in 12 batches of Rhizoma Arisaematis samples. The method is simple, sensitive, accurate, and can be used for rapid identification and determination of amino components in traditional Chinese medicines.

[Analysis of flavonoids and components of stems & leaves of Ranunculus ternatus Thunb. using ultra performance liquid chromatography-quadrupole tandem time of flight mass spectrometry].

In order to explore the applications of liquid chromatography-mass spectrometry (LC-MS) technology in the rapid identification of components of Chinese herbal medicines and natural products, reference flavones were used as precursors, and the stems and leaves of medicinal plant Ranunculus ternatus Thunb. were used as the objects. Ultra performance liquid chromatography with a diode array detector-electrospray ionization quadrupole tandem time of flight mass spectrometry (UPLC/DAD-ESI/Q-TOF MS) was also used to analyse the characteristics of flavonoid homologues and flavonoid isomers. The results showed that ultraviolet (UV) absorption and MS/MS spectra of C-glycosyl were distinct from O-glycosyl. There was also a correlation between glycosidation positions and the retention times, MS/MS fragments and their relative abundances. When applied it to analyse the alcohol extract of stems and leaves of Ranunculus ternatus Thunb. and their acid hydrolysis solution, 22 flavonol glycosides and 3 flavonoid aglycones were identified. The method is simple, convenient and exercisable.

[Determination of nucleosides in Rhizoma Pinelliae by high performance liquid chromatography].

A high performance liquid chromatographic (HPLC) method was established to determine nucleosides in Rhizoma Pinelliae, which is a dried stem tuber of Pinellia pedatisecta Schott in Pinellia plant belonging to Araceae family and has multiple efficiencies about down-bear counterflow and check vomiting, eliminating dampness and phlegm, etc. The separation of adenine, hypoxanthine, xanthine, uridine, thymine, adenosine and guanosine was achieved on a Lichrospher C18 column (150 mm x 4.6 mm, 5 microm) with the detection at 254 nm and gradient elution by acetonitrile-water containing 0.1% formic acid as the mobile phase. The linear ranges were from 1.6 mg/L to 50 mg/L for adenine, hypoxanthine, xanthine, uridine and guanosine, while from 1.2 mg/L to 40 mg/L for thymine and adenosine with correlation coefficients above 0.999 5. The average recoveries were between 98.9% and 101.2% with the relative standard deviations below 3%. The results of methodological study demonstrated that the method met the requirements of the determination. The nucleosides in Rhizoma Pinelliae from different districts were determined. The method is convenient and accurate with good reproducibility and can be used to evaluate the quality of Rhizoma Pinelliae.

Sensitized luminescent terbium nanoparticles: preparation and time-resolved fluorescence assay for DNA.

A highly luminescent terbium nanoparticle as the biolabel based on the sensitization of a dye molecule was prepared. The luminescent complexes included in the particles were composed of a quinolone-based dye molecule as the light-energy transfer donor and a polyaminocarboxylate-based chelator with excellent water-solubility and a high binding constant for lanthanides. The structure of two functional entities in the single molecule made the complex highly luminescent in aqueous solution. Silica nanoparticles containing terbium complexes were prepared by the reverse microemulsion method. Such a terbium nanoparticle is as bright as about 340 free terbium complexes, and it has a 1.5-ms fluorescence lifetime that enables it to be used in the time-resolved fluorescence assays. The conjugate of the nanoparticle with oligonucleotide was prepared and used to carry out a DNA sandwich hybridization assay based on magnetic microbeads as solid-phase carrier. The experimental results showed that the detection sensitivity with the nanoparticles is more than 100-fold as high as that with dye Fluorescein isothiocyanate (FITC) molecules.