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.

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.

Determination of robenidine residues in chicken muscle by high performance liquid chromatography with ultraviolet detection.

A simple, robust and reliable method for the determination of residual robenidine in chicken muscle using high performance liquid chromatography with ultraviolet (UV) detection was developed and validated according to the Codex Alimentarius Commission guidelines. Chicken muscle was extracted by acetonitrile/formic acid (98:2, v/v) and defatted with hexane. Analytes were isocratically separated on a Luna C18 column (4.6 × 150 mm, 5 µm) using 70 % methanol in water containing 0.1 % trifluoroacetic acid at a flow rate of 1.0 mL/min at 30 °C. UV detection was performed at 312 nm. The method was validated by assessing performance parameters including selectivity, linearity, limit of quantification (LOQ), precision, accuracy, recovery, stability and robustness. A calibration curve that was constructed over 0.05-0.5 µg/g showed correlation coefficients of more than 0.999. The intra- and inter-day precisions (as coefficient of variation) were 1.45-3.32 and 2.63-4.99 %, respectively. The intra- and inter-day accuracies were 99.4-105.3 and 98.3-101.6 %, respectively. The recoveries were in the range of 76.6-81.8 % and the LOQ was 0.05 µg/g. The developed method showed suitable performance for the determination of robenidine residues in chicken muscle.

Dispersive liquid-liquid microextraction based on solidification of floating organic droplets followed by high performance liquid chromatography for the determination of duloxetine in human plasma.

A novel dispersive liquid-liquid microextraction method based on solidification of floating organic droplets (DLLME-SFO) technique was developed for the determination of duloxetine in human plasma samples by high performance liquid chromatography with fluorescence detection (HPLC-FLD). During the extraction procedure, plasma protein was precipitated by using a mixture of zinc sulfate solution and acetonitrile. After the protein precipitation step, duloxetine in an alkaline sample solution was quickly extracted by DLLME-SFO with 50 µL of 1-undecanol (extractant). Disperser was unnecessary because the small amount of remaining acetonitrile, which acts as a protein precipitating reagent, was also employed as a disperser; therefore, organic solvent consumption was reduced as much as possible. The emulsion was centrifuged and then fine droplets were floated to the top of the sample solution. The floated droplets were solidified in an ice bath and easily transferred. Various DLLME-SFO parameters such as extractant type, extractant amount, ionic strength, pH and extraction time were optimized. The chromatographic separation of duloxetine was carried out using ethanol as mobile phase. Validation of the method was performed with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), and recovery. Calibration curves for duloxetine showed good linearity with correlation coefficients (r²) higher than 0.99. The method showed good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% (LOQ: less than 20%) at all concentrations. The recovery was carried out following the standard addition procedure with yields ranging from 59.6 to 65.5%. A newly developed environmentally friendly method was successfully applied to the pharmacokinetic study of duloxetine in human plasma and was shown to be an alternative green approach compared with the conventional solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME) techniques.

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.

Simultaneous determination of chromones and coumarins in Radix Saposhnikoviae by high performance liquid chromatography with diode array and tandem mass detectors.

Methods using high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) were developed and validated for the simultaneous determination of 5 chromones and 6 coumarins: prim-O-glucosylcimifugin (1), cimifugin (2), nodakenin (3), 4'-O-ß-d-glucosyl-5-O-methylvisamminol (4), sec-O-glucosylhamaudol (5), psoralen (6), bergapten (7), imperatorin (8), phellopterin (9), 3'-O-angeloylhamaudol (10) and anomalin (11), in Radix Saposhnikoviae. The separation conditions for HPLC-DAD were optimized using an Ascentis Express C18 (4.6 mm×100 mm, 2.7 µm particle size) fused-core column. The mobile phase was composed of 10% aqueous acetonitrile (A) and 90% acetonitrile (B) and the elution was performed under a gradient mode at a flow rate of 1.0 mL/min. The detection wavelength was set at 300 nm. The HPLC-DAD method yielded a base line separation of the 11 components in 50% methanol extract of Radix Saposhnikoviae with no interfering peaks detected. The HPLC-DAD method was validated in terms of linearity, accuracy and precision (intra- and inter-day), limit of quantification (LOQ), recovery, and robustness. Specific determination of the 11 components was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. This HPLC-MS/MS method was also validated by determining the linearity, limit of quantification, accuracy, and precision. Quantification of the 11 components in 51 commercial Radix Saposhnikoviae samples was successfully performed using the developed HPLC-DAD method. The identity, batch-to-batch consistency, and authenticity of Radix Saposhnikoviae were successfully monitored by the proposed HPLC-DAD and HPLC-MS/MS methods.