Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.     

Simultaneous determination of six major stilbenes and flavonoids in Smilax china by high performance liquid chromatography

A simple, sensitive and specific HPLC method was developed for simultaneous determination of the six major active constituents in Smilax china, namely taxifolin-3-O-glycoside (1), piceid (2), oxyresveratrol (3), engeletin (4), resveratrol (5) and scirpusin A (6), respectively. The samples were separated on an Aglient Zorbax XDB-C18 column with gradient elution of acetonitrile and 0.02% phosphoric acid (v/v) at a flow rate of 1.0 ml/min and detected at 300 nm. The six target compounds were completely separated within 35 min. All calibration curves showed good linearity (r2>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.7%. The recoveries were between 93.7 and 103.0%. The method was successfully applied to the analysis of six constituents in 15 commercial samples of S. china. The results indicated that the developed HPLC assay was readily utilized as a quality control method for S. china.     

Comparison of capillary electrophoresis and high performance liquid chromatography for determination of flavonoids in Achillea millefolium

Flavonoids represent an important bioactive component in Achillea millefolium. The comparison of the most commonly used analytical methods for the identification and quantification of flavonoids, capillary electrophoresis (CE) and high performance liquid chromatography (HPLC), is presented. The methods were optimized and validated. Using a 20 mM borate buffer with 30% (v/v) of methanol (pH 9.3) in the CE analysis and a gradient elution with water-acetonitrile mobile phase in the HPLC analysis, sufficient separation of the analytes was achieved. A relatively high injection volume in the CE analysis (30 mbar x 30s) enabled low limit of detection (LOD) (0.3-0.7 mg/L). Repeatability of both methods was acceptable (relative standard deviation of peak area were <6%). Additionally, the amount of flavonoids in a real sample of the dried herbal drug was determined.     

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.     

Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometric procedure for qualitative and quantitative analyses of nortriterpenoids and lignans in the genus Schisandra

An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS) procedure is designed for the first simultaneous analysis of nortriterpenoids and lignans in Schisandra samples. The method consists of three individual mass spectrometric experiments, including the full scan MS, MS/MS experiment and in-source collision induced dissociation (CID) MS/MS, which enable the identification of diagnostic fragmentation pathways of nortriterpenoids and lignans. As such, a total of 6 nortriterpenoids and 10 lignans were unequivocally identified, and one nortriterpenoid and 20 lignans were tentatively identified from different Schisandra samples within 12.5 min. In addition, 6 nortriterpenoids and 10 lignans were quantified in 48 samples of S. chinensis and S. sphenanthera using an extract ion chromatogram (XIC) of the full scan MS experiment. Dataset obtained from UPLC-MS was processed with principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to compare the difference between the two Schisandra species.     

Qualitative and quantitative analysis of Fructus Corni using ultrasound assisted microwave extraction and high performance liquid chromatography coupled with diode array UV detection and time-of-flight mass spectrometry

The first successful combination of ultrasound assisted microwave extraction (UAME) with liquid chromatography analysis is described for the quality evaluation of Fructus Corni, a commonly used traditional Chinese medicine (TCM). Due to their multifarious biological activities, seven representative bioactive constituents (two phenolics and five iridoids) were chosen as targets for the quality assessment. The chromatographic separation was performed on a C18 Aq column with gradient elution using methanol and aqueous solution containing 0.2% acetic acid. The quantitative method developed was validated and successfully applied to determine the seven markers in 12 batches of Fructus Corni extract from various habitats. Significant variations were demonstrated in the contents of seven compounds. Further 13 components were tentatively identified by online TOF mass analysis.     

Optimization and validation of a high performance liquid chromatography method for rapid determination of sinafloxacin,a novel fluoroquinolone in rat plasma using a fused-core C18-silica column

A novel, simple and rapid high performance liquid chromatographic method has been developed and validated for the determination of sinafloxacin, a new fluoroquinolone, in rat plasma using 96-well protein precipitation, fused-core C₁₈-silica column (4.6 mm × 50 mm, 2.7 μm) packed with a new solid support, which is made of 2.7 μm particles that consist of a 1.7 μm solid core covered with a 0.5 μm thick shell of porous silica.The chromatographic separation was achieved with a mobile phase of 20:80 (v/v) of acetonitrile and phosphate buffer (pH = 3.0) at a flow rate of 1 ml min⁻¹. Fluorescence detection was employed with λ_ex 295 nm and λ_em 505 nm. Lomefloxacin was used as internal standard (IS). The total analysis time was as short as 3 min. The method was sensitive with a limit of detection (LOD) of 2 ng ml⁻¹, with good linearity (R² = 0.9996) over the linear range of 5–500 ng ml⁻¹. The intra-day and inter-day precision was less than 5.8% and accuracy ranged from 100.3% to 103.5% for quality control (QC) samples at three concentrations of 10, 50 and 400 ng ml⁻¹.The fused-core C₁₈-silica column method offered high sample throughput, low injection volume and low consumption of organic solvents. The method was successfully employed in the pharmacokinetic study of sinafloxacin formulation product after tail vein injection to healthy rats.