Han Young Eom

Comparison between evaporative light scattering detection and charged aerosol detection for the analysis of saikosaponins

Saikosaponins are triterpene saponins derived from the roots of Bupleurum falcatum L. (Umbelliferae), which has been traditionally used to treat fever, inflammation, liver diseases, and nephritis. It is difficult to analyze saikosaponins using HPLC-UV due to the lack of chromophores. Therefore, evaporative light scattering detection (ELSD) is used as a valuable alternative to UV detection. More recently, a charged aerosol detection (CAD) method has been developed to improve the sensitivity and reproducibility of ELSD. In this study, we compared CAD and ELSD methods in the simultaneous analysis of 10 saikosaponins, including saikosaponins-A, -B(1), -B(2), -B(3), -B(4), -C, -D, -G, -H and -I. A mixture of the 10 saikosaponins was injected into the Ascentis Express C18 column (100 mm x 4.6 mm, 2.7 microm) with gradient elution and detection with CAD and ELSD by splitting. We examined various factors that could affect the sensitivity of the detectors including various concentrations of additives, pH and flow rate of the mobile phase, purity of nitrogen gas and the CAD range. The sensitivity was determined based on the signal-to-noise ratio. The best sensitivity for CAD was achieved with 0.1 mM ammonium acetate at pH 4.0 in the mobile phase with a flow rate of 1.0 mL/min, and the CAD range at 100 pA, whereas that for ELSD was achieved with 0.01% acetic acid in the mobile phase with a flow rate at 0.8 mL/min. The purity of the nitrogen gas had only minor effects on the sensitivities of both detectors. Finally, the sensitivity for CAD was two to six times better than that of ELSD. Taken together, these results suggest that CAD provides a more sensitive analysis of the 10 saikosaponins than does ELSD.

Species discrimination of Radix Bupleuri through the simultaneous determination of ten saikosaponins by high performance liquid chromatography with evaporative light scattering detection and electrospray ionization mass spectrometry

A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b(1), -b(2), -b(3), -b(4), -c, -d, -g, -h, and -i. These compounds were chromatographed on an Ascentis(®) Express C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50°C drift tube temperature and 3.0 bar nebulizer gas (N(2)) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of four Bupleurum species, namely B. falcatum, B. chinense, B. sibiricum and the poisonous B. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents in B. falcatum, B. chinense, and B. longiradiatum, while one major saikosaponin (saikosaponin-c) was not identified from B. sibiricum. In addition, no saikosaponin-b(3) was detected in B. longiradiatum samples, indicating that the toxic B. longiradiatum may be tentatively distinguished from officially listed Bupleurum species (B. falcatum and B. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely related Bupleurum species.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the determination of goserelin in rabbit plasma

A rapid and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC-ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis((R)) HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100microL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0mmx150mm, 5microm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile=85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250microL/min. The LC-MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor-product ion transitions consisted of m/z 635.7-->m/z 607.5 for goserelin and m/z 424.0-->m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1-20ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6mg of goserelin in healthy white rabbits.

Highly sensitive electromembrane extraction for the determination of volatile organic compound metabolites in dried urine spot.

Electromembrane extraction coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for determination of ten volatile organic compound metabolites in dried urine spot samples. The dried urine spot approach is a convenient and economical sampling method, wherein urine is spotted onto a filter paper and dried. This method requires only a small amount of sample, but the analysis sometimes suffers from low sensitivity, which can lead to analytical problems in the detection of minor components in samples. The newly developed dried urine spot analysis using electromembrane extraction exhibited improved sensitivity and extraction, and enrichment of the sample was rapidly achieved in one step by applying an electric field. Aliquots of urine were spotted onto Bond Elut DMS cards and dried at room temperature. After drying, the punched out dried urine spot was eluted with water. Volatile organic compound metabolites were extracted from the sample through a supported liquid membrane into an alkaline acceptor solution inside the lumen of a hollow fiber with the help of an electric potential. The optimum extraction conditions were determined by using design of experiments (fractional factorial design and response surface methodology). Satisfactory sensitivity was achieved and the limits of quantification (LOQ) obtained were lower than the regulatory threshold limits. The method was validated by assessing the linearity, precision, accuracy, recovery, reproducibility, stability, and matrix effects. The results were acceptable, and the developed method was successfully applied to biological exposure monitoring of volatile organic compound metabolites in fifty human urine samples.

Rapid chiral separation of racemic cetirizine in human plasma using subcritical fluid chromatography-tandem mass spectrometry

A method for fast chiral separation of cetirizine and quantitation of levocetirizine in human plasma using subcritical fluid chromatography with tandem mass spectrometry was developed and validated. The chromatographic separation was performed using a Chiralpak IE column (2.1 mm×150 mm, 5 μm) with an isocratic elution of CO2/organic modifier (55/45, v/v) at a flow rate of 0.85 mL/min. The organic modifier was composed of water/methanol (5/95, v/v). The makeup flow was optimized at water/methanol (10/90, v/v) and 0.2 mL/min. The most influential parameters on the separation of cetirizine affecting resolution, retention time and sensitivity were selected by fractional factorial design. The 3 selected factors were optimized by response surface methodology. Tandem mass spectrometry was used at electrospray ionization, positive ion mode, and multiple-reaction monitoring mode. Isotope-labeled cetirizine-d4 was used as the internal standard. The sample preparation of human plasma was conducted by solid phase extraction of hydrophilic-lipophilic balance (HLB) type. The developed method was validated for selectivity, linearity, precision, accuracy, recovery, limit of quantitation (LOQ), and limit of detection (LOD). The real human plasma samples were analyzed and the pharmacokinetic results were compared with results of previous research. The developed method was found to be reliable based on the similarity between the results of the current and previous methods. The chiral separation for cetirizine and economic feasibility were compared with those of previous studies using normal phase-HPLC or reversed phase-HPLC. The established analytical method could be successfully applied to pharmacokinetic study with reduction in the analysis time and costs.

Simultaneous determination of benzene, toluene, ethylbenzene, and xylene metabolites in human urine using electromembrane extraction combined with liquid chromatography and tandem mass spectrometry

For the first time, electromembrane extraction combined with liquid chromatography and tandem mass spectrometry was applied for the determination of urinary benzene, toluene, ethylbenzene, and xylene metabolites. S-Phenylmercapturic acid, hippuric acid, phenylglyoxylic acid, and methylhippuric acid isomers were extracted from human urine through a supported liquid membrane consisting of 1-octanol into an alkaline acceptor solution filling the inside of a hollow fiber by application of an electric field. Various extraction factors were investigated and optimized using response surface methodology, the statistical method. The optimum conditions were established to be 300 V applied voltage, 15 min extraction time, 1500 rpm stirring speed, and 5 mM ammonium acetate (pH 10.2) acceptor solution. The method was validated with respect to selectivity, linearity, accuracy, precision, limit of detection, limit of quantification, recovery, and reproducibility. The results showed good linearity (r(2) > 0.995), precision, and accuracy. The extract recoveries were 52.8-79.0%. Finally, we applied this method to real samples and successfully measured benzene, toluene, ethylbenzene, and xylene metabolites.

Classification of the medicinal plants of the genus Atractylodes using high-performance liquid chromatography with diode array and tandem mass spectrometry detection combined with multivariate statistical analysis

Analytical methods using high-performance liquid chromatography with diode array and tandem mass spectrometry detection were developed for the discrimination of the rhizomes of four Atractylodes medicinal plants: A. japonica, A. macrocephala, A. chinensis, and A. lancea. A quantitative study was performed, selecting five bioactive components, including atractylenolide I, II, III, eudesma-4(14),7(11)-dien-8-one and atractylodin, on twenty-six Atractylodes samples of various origins. Sample extraction was optimized to sonication with 80% methanol for 40 min at room temperature. High-performance liquid chromatography with diode array detection was established using a C18 column with a water/acetonitrile gradient system at a flow rate of 1.0 mL/min, and the detection wavelength was set at 236 nm. Liquid chromatography with tandem mass spectrometry was applied to certify the reliability of the quantitative results. The developed methods were validated by ensuring specificity, linearity, limit of quantification, accuracy, precision, recovery, robustness, and stability. Results showed that cangzhu contained higher amounts of atractylenolide I and atractylodin than baizhu, and especially atractylodin contents showed the greatest variation between baizhu and cangzhu. Multivariate statistical analysis, such as principal component analysis and hierarchical cluster analysis, were also employed for further classification of the Atractylodes plants. The established method was suitable for quality control of the Atractylodes plants.

Determination of residual arsenic compounds in chicken muscle by ultra-performance liquid chromatography coupled with ultraviolet detection after pre-column derivatization with toluene-3,4-dithiol.

A simple and sensitive derivatization method using toluene-3,4-dithiol as a derivatization reagent for the simultaneous analysis of seven arsenic compounds (roxarsone, nitarsone, p-arsanilic acid, o-arsanilic acid, phenylarsonic acid, phenylarsine oxide, and mono-methylarsonic acid) in chicken muscle was developed and validated by ultra-performance liquid chromatography coupled with ultraviolet detection (UPLC-UV). The structure of the derivatized arsenic compounds was confirmed by liquid chromatography-ion trap mass spectrometry or gas chromatography-mass spectrometry. Optimization of the derivatization reaction conditions was carried out by investigating the influence of reagent concentration, buffer or additive acids, temperature, and time. The optimized conditions were a derivatization reagent concentration of 20mg/mL with 0.05mol/L HCl as an additive acid at 60°C for 15min. In this study, baseline separation of arsenic compounds could be achieved within 13min, except for phenylarsonic acid and phenylarsine oxide whose derivatized products are equal. The developed method was successfully validated and applied to 12 chicken muscle samples from Korean districts and other countries.