• Proceedings of the Korean Society of Toxicology Conference
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• 2006.11a
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• pp.65-74
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• 2006

Modem drug discovery requires rapid pharmacokinetic evaluation of chemically diverse compounds for early candidate selection. This demands the development of analytical methods that offer high-throughput of samples. Naturally, liquid chromatography / tandem mass spectrometry (LC-MS/MS) is choice of the analytical method because of its superior sensitivity and selectivity. As a result of the short analysis time(typically 3-5min) by LC-MS/MS, sample preparation has become the rate- determining step in the whole analytical cycle. Consequently tremendous efforts are being made to speed up and automate this step. In a typical automated 96-well SPE(solid-phase extraction) procedure, plasma samples are transferred to the 96-well SPE plate, internal standard and aqueous buffer solutions are added and then vacuum is applied using the robotic liquid handling system. It takes only 20-90 min to process 96 samples by automated SPE and the analyst is physically occupied for only approximately 10 min. Recently, the ultra-high flow rate liquid chromatography (turbulent-flow chromatography)has sparked a huge interest for rapid and direct quantitation of drugs in plasma. There is no sample preparation except for sample aliquotting, internal standard addition and centrifugation. This type of analysis is achieved by using a small diameter column with a large particle size(30-5O ${\mu}$m) and a high flow rate, typically between 3-5 ml/min. Silica-based monolithic HPLC columns contain a novel chromatographic support in which the traditional particulate packing has been replaced with a single, continuous network (monolith) of pcrous silica. The main advantage of such a network is decreased backpressure due to macropores (2 ${\mu}$m) throughout the network. This allows high flow rates, and hence fast analyses that are unattainable with traditional particulate columns. The reduction of particle diameter in HPLC results in increased column efficiency. use of small particles (<2 urn), however, requires p.essu.es beyond the traditional 6,000 psi of conventional pumping devices. Instrumental development in recent years has resulted in pumping devices capable of handling the requirements of columns packed with small particles. The staggered parallel HPLC system consists of four fully independent binary HPLC pumps, a modified auto sampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The process of Bioanalytical Method Validation is required by the FDA to assess and verify the performance of a chronlatographic method prior to its application in sample analysis. The validation should address the selectivity, linearity, accuracy, precision and stability of the method. This presentation will provide all overview of the work required to accomplish a full validation and show how a chromatographic method is suitable for toxirokinetic sample analysis. A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method developed to quantitate drug levels in dog plasma will be used as an example of tile process.

• Analytical Science and Technology
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• v.34 no.1
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• pp.9-16
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• 2021

In the group of commonly prescribed β-blocker drugs, one of the enantiomers is generally relatively more active than the others. This study aims to develop a technique for the chiral analysis of select β-blockers based on proton nuclear magnetic resonance (1H-NMR) spectrometry. (S)-2-Tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid ((S)-TBMB) was synthesized and utilized as a chiral derivatizing agent. Pure β-blocker enantiomers were isolated from racemates by semi-preparative liquid chromatography prior to derivatization. The reaction time and concentration of (S)-TBMB were controlled to improve the derivatization procedure. No racemization was found during the analysis. High-performance liquid chromatography (HPLC) analysis was also performed for comparative purposes. High agreement between the NMR and HPLC methods was achieved in the determination of (R)-metoprolol in a standard solution of the (S) isomer.

• Korean Journal of Pharmacognosy
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• v.54 no.1
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• pp.38-43
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• 2023

A simple and accurate assay was developed for the quantitative analysis of 1-deoxynojirimycin (1-DNJ) derived from the silkworm (Bombyx mori). Normal-phase high-performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD) and a hydrophilic interaction liquid chromatography column was used. Various parameters were applied to optimize the analysis method. The limits of detection and quantification of 1-DNJ were 2.97 × 10^-3 and 9.00 × 10^-3 mg/mL, respectively. The calibration curve showed good linearity results. The concentration range and the r² value were 0.0625-1.0 mg/mL and 0.9997, respectively. The accuracy test demonstrated a significantly high recovery rate (89.95-103.22%). The relative standard deviation was ≤ 1.00%. Thus, a method for the accurate identification and quantitative analysis of 1-DNJ in silkworms was developed. Moreover, in this procedure, the process of derivatization of 1-DNJ, which was required in previous experiments, could be eliminated. This technique may be actively utilized for the development of pharmaceuticals and health functional foods using 1-DNJ.

• Journal of the Korean Chemical Society
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• v.33 no.1
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• pp.46-54
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• 1989

The extraction method and quantitative analysis for the fat-soluble vitamins present in food stuffs and vitamin products have been investigated. The simultaneous separation and analysis of the vitamins by reverse phase high performance liquid chromatographic method was conducted using an isocratic elution with methanol : water (95 : 5) eluent on a Novapak $C_{18}$ column. The detection of vitamins was achieved by a variable wavelength UV detector. To improve the detection sensitivity detection wavelengths were set at the highest absorption bands such as 330, 265, 285, and 290nm for the respective vitamins. The analysis for the fat-soluble vitamins was finished within 40 minutes. Alkaline hydrolysis and enzymatic hydrolysis were investigated for the sample preparation; and liquid-liquid extraction and liquid-solid extraction were attempted for the extraction of vitamins. Both hydrolysis methods were turned out to be appropriate for the analysis for vitamins A, D, and E, while for the analysis of vitamin K the enzymatic hydrolysis method demonstrated better results. Diethyl ether, pentane, and n-hexane were found to give higher recovery for the liquid-liquid extraction and silica cartridge for the liquid-solid extraction.

• Archives of Pharmacal Research
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• v.17 no.5
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• pp.337-342
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• 1994

Carbohydrate analysis is important in studying structure and activity of complex polysaccharides. New analytical method was applied to get an information on the composition of polysaccharides showing antitumor activity. Monosaccharides were labeled with 7-amino-1, 3-naph-thalenedisulfonic acid (7-AGA) by reductive amination and separated by HPLC. Five kinds of polysaccharides from Basidiomycetes were hydrolyzed and analyzed in combination with electrophresis and HPLC. At the same time, alditol acetate derivatives were prepared and analyzed by gas chromatography. Two different techniques using different derivatization methods showed very similar results. The monosaccharides from Coriolus versicolor and Cordyceps militaris were glucose and galactose. Phellinus linteus composed of glucose, glactose, mannose, arabinose and fucose. The HPLC method with fluorescence detector was very sensitive compared to other methods.

• FOCUS: LIFE SCIENCE
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• no.1
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• pp.3.1-3.7
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• 2024

The article discusses the critical role of chromatography in the analysis and purification of proteins in biopharmaceuticals, emphasizing the importance of comprehensive characterization for ensuring their safety and efficacy. It highlights the use of Avantor® ACE® HPLC columns for the separation and purification of proteins, focusing on the analysis of intact proteins using reversed-phase liquid chromatography (RPLC) with fully porous particles. This article also details the application of different mobile phase additives, such as TFA and formic acid, and emphasizes the advantages of using type B ultra-pure silica-based columns for efficiency and peak shape in biomolecule analysis. Additionally, it addresses the challenges of analyzing intact proteins due to slow molecular diffusion and introduces the concept of solid-core (or superficially porous) particles, emphasizing their benefits over traditional porous particles for the analysis of therapeutic proteins. Furthermore, it discusses the development of Avantor® ACE® UltraCore BIO columns, specifically designed for the high-efficiency separation of large biomolecules, such as proteins, and demonstrates their effectiveness in achieving high-resolution separations, even for higher molecular weight proteins like monoclonal antibodies (mAbs). In addition, it underscores the complexity of analyzing and characterizing intact protein biopharmaceuticals, requiring a range of analytical techniques and the use of wide-pore stationary phases, operated at elevated temperatures and with relatively shallow gradients. It highlights the comprehensive range of options offered by Avantor® ACE® wide pore columns, including both fully porous and solid-core particles, bonded with a variety of complementary stationary phase chemistries to optimize selectivity during method development. The use of ultrapure and highly inert base silica is emphasized for enabling the use of lower concentrations of mobile phase modifiers without compromising analyte peak shape, particularly beneficial for LC-MS applications. Then the article concludes by emphasizing the significance of reversed-phase liquid chromatography and its compatibility with mass spectrometry as a valuable tool for the separation and analysis of intact proteins and their closely related variants in biopharmaceuticals.

• Analytical Science and Technology
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• v.34 no.4
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• pp.180-190
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• 2021

This study compared the efficacy of chiral GC and chiral HPLC for the analysis of nicotine. To develop a suitable dispersive liquid-liquid microextraction (DLLME) method, the following parameters were optimized: pH, extraction solvent, dispersive solvent, type and quantity of salt, and laboratory temperature. The validation of the method was carried out by the established HPLC method. The LODs were 0.11 ㎍/mL and 0.17 ㎍/mL for the (S)- and (R)- enantiomers, respectively. The LOQs were 0.30 ㎍/mL and 0.44 ㎍/mL, respectively. The optimal calibration range was between 0.30-18 ㎍/mL and 0.44-4.40 ㎍/mL, respectively, and the correlation coefficient (r²) was 0.9978-0.9996. The intra-day accuracy was 79.9-110.6 %, and the intra-day precision was 1.3-12.0 %. The inter-day accuracy was 87.8-108.0 %, and the inter-day precision was 4.0-12.8 %. E-liquid and biological fluids (urine and saliva) were analyzed using the established method.

• Journal of Ginseng Research
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• v.43 no.4
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• pp.508-516
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• 2019

Background: Ginsenosides are not only the principal bioactive components but also the important indexes to the quality assessment of Panax ginseng Meyer. Their contents in cultivated ginseng vary with the growth environment and age. The present study aimed at evaluating the significant difference between 36 cultivated ginseng of different cultivation areas and ages based on the simultaneously determined contents of 14 ginsenosides. Methods: A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometer (MS) method was developed and used in the multiple reaction-monitoring (MRM) mode (HPLC-MRM/MS) for the quantitative analysis of ginsenosides. Multivariate statistical analysis, such as principal component analysis and partial least squares-discriminant analysis, was applied to discriminate ginseng samples of various cultivation areas and ages and to discover the differentially accumulated ginsenoside markers. Results: The developed HPLC-MRM/MS method was validated to be precise, accurate, stable, sensitive, and repeatable for the simultaneous determination of 14 ginsenosides. It was found that the 3- and 5-yr-old ginseng samples were differentiated distinctly by all means of multivariate statistical analysis, whereas the 4-yr-old samples exhibited similarity to either 3- or 5-yr-old samples in the contents of ginsenosides. Among the 14 detected ginsenosides, Rg1, Rb1, Rb2, Rc, 20(S)-Rf, 20(S)-Rh1, and Rb3 were identified as potential markers for the differentiation of cultivation ages. In addition, the 5-yr-old samples were able to be classified in cultivation area based on the contents of ginsenosides, whereas the 3- and 4-yr-old samples showed little differences in cultivation area. Conclusion: This study demonstrated that the HPLC-MRM/MS method combined with multivariate statistical analysis provides deep insight into the accumulation characteristics of ginsenosides and could be used to differentiate ginseng that are cultivated in different areas and ages.

• Natural Product Sciences
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• v.9 no.2
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• pp.45-48
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• 2003

The content of chiisanoside in the Acanthopanax Species was determined by reversed-phase high performance liquid chromatographic method. Chiisanoside was separated from the other components in the plant extracts using Zorbax 300 SB $C_{18}$ column with gradient elution of acetonitrile. Identification of chiisanoside was carried out by comparison in the LC/MS spectrum of separated peak from extract with that of standard. By HPLC analysis in this experiment, Acanthopanax species could be classified into two groups based upon the content of chiisanoside-one with low concentration of chiisanoside, such as A. senticosus and A. koreanum, and another with high concentration of chiisanoside, such as A. senticosus f. inermis, A. Divaricatus var. albeofructus, and A. chiisanensis.

• Analytical Science and Technology
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• v.37 no.4
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• pp.211-219
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• 2024

This study presents a sensitive and reliable method for determining tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC) residues in shrimp samples. A two-step process involving liquid-liquid extraction (LLE) followed by solid-phase extraction (SPE) was developed prior to HPLC analysis. The target analytes were effectively extracted using EDTA/McIlvaine buffer (pH 4.0): methanol (80:20, %v/v), with subsequent clean-up using a C18 SPE cartridge. HPLC separation was conducted on a C18 column (250 mm × 4.6 mm i.d., 5 ㎛) at 30 ℃, using 0.01 % trifluoroacetic acid (A) and acetonitrile (B) as the mobile phase. A gradient elution protocol was applied, transitioning from 85(A):15(B) %v/v to 70(A):30(B) %v/v at 7 min, with a 5 min hold, followed by adjustment to 85(A):15(B) %v/v for 13-14 min. The detection was performed using photodiode array (PDA) at 365 nm with a flow rate of 1.0 mL/min. The calibration curves exhibited good linearity within a concentration range of 0.4-6.0 ㎍/mL (R² > 0.995). The limits of detection (LOD) for TC, OTC, and CTC in shrimp were 0.034, 0.029, and 0.021 ㎍/mL, respectively. The limits of quantitation (LOQ) for TC, OTC, and CTC were found to be 0.114, 0.097, and 0.071 ㎍/mL, respectively. Recoveries of TC, OTC, and CTC from spiked shrimp samples ranged from 91.0 % to 95.5 %, 92.4 % to 97.2 %, and 93.3 % to 96.6 %, respectively. This method was successfully applied to the determination of TC, OTC, and CTC residues in shrimp samples sourced from various local markets.