Jeong-Rok Youm

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.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations.

Determination of metoprolol enantiomers in human urine by GC-MS using (−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride as a chiral derivatizing agent

SummaryA method for the assay of R-(+)- and S-(−)- metoprolol in human urine has been developed using gas chromatography-mass spectrometry. The method involved purification by liquid-liquid extraction and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide to form an O-silyl ether, followed by subsequent chiral derivatization with (−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride to form diastereomeric amide. The reaction was rapid and the diastereomeric derivatives were well resolved. Quantitation was performed by selected-ion monitoring of fragment ions of the diastereomers in electron impact ionization mode. No racemization was found during the reaction. The detection limit was 0.5 ng·mL−1. The intra-day variation ranged between 0.38 and 7.86% in relation to the measured concentration and inter-day variation was 2.26–8.06%. The method has been applied to the determination of R-(+)-and S-(−)- metoprolol in human urine from healthy volunteers dosed with racemic metoprolol tartrate.

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.

Enantioselective determination of cetirizine in human plasma by normal-phase liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

A highly sensitive and enantioselective method has been developed and validated for the determination of levocetirizine [(R)-cetirizine] in human plasma by normal-phase liquid chromatography coupled to tandem mass spectrometry with an atmospheric pressure chemical ionization (APCI) interface in the positive ion mode. Enantioselective separation was achieved on a CHIRALPAK AD-H column using an isocratic mobile phase consisting of a mixture of n-hexane, ethyl alcohol, diethylamine, and acetic acid (60:40:0.1:0.1, v/v/v/v). Levocetirizine-D(8) was used as an internal standard (IS). Levocetirizine and the IS were detected by multiple-reaction monitoring (MRM). Mass transitions of analyte and IS were m/z 389.2→201.1 and 397.2→201.1, respectively. Under optimized analytical conditions, a baseline separation of two enantiomers and IS was obtained in less than 11 min. Samples were prepared by a simple two-step extraction by protein precipitation using acetonitrile followed by liquid-liquid extraction with a n-hexane-dichloromethane mixture (50:50, v/v). The standard curve for levocetirizine was linear (r(2)>0.995) in the concentration range 0.5-300 ng/mL. Recovery was between 97.0 and 102.2% at low, medium, and high concentration. The limit of quantification (LOQ) was 0.5 ng/mL. Other method validation parameters, such as precision, accuracy, and stability, were very satisfactory. Finally, the proposed method was successfully applied to the study of enantioselective oral pharmacokinetics of levocetirizine in healthy Korean volunteers.

Chiral separation of β2-agonists by capillary electrophoresis using hydroxypropyl-α-cyclodextrin as a chiral selector

Enantiomers of five racemic β2-agonists were investigated by capillary electrophoresis employing a hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects of the concentration of HP-β-CD added to the background electrolyte and of the pH of the buffer on the effective mobility and resolution of the studied compounds were examined. Very good resolution was achieved for terbutaline and clenbuterol; salbutamol and bambuterol was able to be partially resolved. Enantioselectivity and resolution were influenced by the concentration of the HP-β-CD, buffer composition and pH.

Bioequivalence of Atorva Tablet ® to Lipitor Tablet ® (Atorvastatin 20 mg)

The present study describes the evaluation of the bioequivalence of two atorvastatin tablets, Lipitor (Pfizer, reference drug) and Atorva (Yuhan, test drug), according to the guidelines of Korea Food and Drug Administration (KFDA). Forty-nine healthy male Korean volunteers received each medicine at the atorvastatin dose of 40 mg in a crossover study with a two weeks washout interval. After drug administration, serial blood samples were collected at a specific time interval from 0-48 hours. The plasma atorvastatin concentrations were monitored by an high performance liquid chromatography -tandem mass spectrometer (LC-MS/MS) employing electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode. The total chromatographic run time was 4.5 min and calibration curves were linear over the concentration range of 0.1-100 ng/mL for atorvastatin. The method was validated for selectivity, sensitivity, linearity, accuracy and precision. (the area under the plasma concentration-time curve from time zero to 48hr) was calculated by the linear log trapezoidal rule method. (maximum plasma drug concentration) and (time to reach ) were complied trom the plasma concentration-time data. Analysis of variance was carried out using logarithmically transformed and . No significant sequence effect was found for all of the bioavailability parameters indicating that the crossover design was properly performed. The 90% confidence intervals of the ratio and the ratio for Atorva / Lipitor were and , respectively. These values were within the acceptable bioequivalence intervals of . Based on these statistical considerations, it was concluded that the test drug, Atorva was bioequivalent to the reference drug, Lipitor .

Sensitive Determination of Pinaverium Bromide in Human Plasma by LC-ESI-MS/MS : Applicability to Oral Bioavailability Determination

A sensitive method for quantification of pinaverium bromide in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI-MS/MS). Glimepiride was used as internal standard. Pinaverium bromide and internal standard in plasma sample were extracted using tert-butylmethylether(TBME). A centrifuged upper layer was then evaporated and reconstituted with mobile phase of acetonitrile-5 mM ammonium formate (80/20, pH 3.0). The reconstituted samples were injected into a reversed-phase column. Using MS/MS with multiple reaction monitoring (MRM) mode, pinaverium and glimepirde were detected without severe interference from human plasma matrix. Pinaverium produced a protonated precursor ion at m/z 510.3 and a corresponding product ion at m/z 228.9. Internal standard produced a protonated precursor ion at m/z 491.5 and a corresponding product ion at m/z 352.0. Detection of pinaverium bromide in human plasma was accurate and precise, with limit of quantitation at 0.5 ng/ml. The method has been successfully applied to bioavailability study of pinaverium bromide tablet in Korean healthy male volunteers. Pharmacokinetic parameters such as were calculated.