• Natural Product Sciences
• /
• v.18 no.1
• /
• pp.39-46
• /
• 2012

In Korea, the leaves of Sonchus brachyotus (Compositae), an edible mountainous vegetable, are traditionally used to treat hepatitis and hemorrhage and are known to have diuretic action. The aqueous ethanolic extract of this plant was selected in our screening experiment using the peroxynitrite ($ONO_2^-$)-scavenging assay, and the present study was performed to qualitatively and quantitatively identify the active compounds from S. brachyotus and validate the present high-permormance liquid chromatography (HPLC) coupled with ultraviolet absorption detection method based on accuracy, precision and repeatability. Five phenolic substances including the main compound, luteolin $7-O-{\beta}-D$-glucuronopyranoside, as well as chlorogenic acid, luteolin 7-O-rutinoside, luteolin $7-O-{\beta}-D$-glucopyranoside, and luteolin, were found in the aqueous ethanolic extract of S. brachyotus. In the HPLC validation experiment, the linearity of the four compounds was established by $R^2$ values of more than 0.999 within the test ranges, and the recovery rate ranged from 98.2 - 105.3%. Luteolin 7-O-glucuronide was a predominant compound (143 mg/g of extract and 18.3 mg/g of the dry weight of plant material) with a potent peroxynitrite-scavenging effect ($IC_{50}$, $1.02{\pm}0.08{\mu}M$). Luteolin and its three glycosides together with chlorogenic acid were qualitatively and quantitatively determined using an HPLC method validated in the present study.

• YAKHAK HOEJI
• /
• v.55 no.1
• /
• pp.64-68
• /
• 2011

In this study, a high performance liquid chromatography-diode array detector method was established, for simultaneous determination of three compounds, berberine, palmatine and geniposide in Hwangryunhaedok-tang, To develop and validate method, $C_{18}$ column (5 ${\mu}M$, 4.6 mm${\times}$250 mm) was used with gradient mobile phase, water containing 0.1% trifluoroacetic acid (TFA) and MeOH at the column temperature of $30^{\circ}C$. UV wavelength was set at 230 and 280 nm. Validation of the chromatography method was evaluated by linearity, precision and accuracy test. Calibration curve of standard components showed good linearity ($R^2$ > 0.9999). The limits of detection (LOD) and limits of quantification (LOQ) varied from 0.05 to 0.17 ${\mu}g/ml$ and 0.15 to 0.53 ${\mu}g/ml$, respectively. The relative standard deviations (RSDs) data of intra-day and inter-day test were in less than 2.99% and 1.90%, respectively. The results of the accuracy test were in the range of 98.36 to 102.52% with RSDs values 0.32 to 1.98%. The results of validation indicated that this method was a very accurate and sensitive assay.

• Biomolecules & Therapeutics
• /
• v.9 no.4
• /
• pp.291-297
• /
• 2001

The bioequivalence of two triflusal products was evaluated with 20 healthy volunteers following single oral dose according to the guidelines of Korea Food and Drug Administration (KFDA). Trisa $l^{R}$ capsule (Whanin Pharm. Corp., Korea) and Disgre $n^{R}$ capsule (Myung-In Pharm. Corp., Korea) were used as test product and reference product, respectively. Both products contain 300 mg of trifusal. One capsule of test product or reference product was orally administered to the volunteers, respectively, by randomized two period crossover study (2$\times$2 Latin square method). Blood samples were taken at predetermined time intervals for 4 hours and the determination of trifusal was accomplished using semi-microbore HPLC equipped with automated column switching system. The analytical method with HPLC was validated according to the Bioanalytic Method Validation guideline by F7A prior to determining the plasma samples. The pharmacokinetic parameters (AU $C_{0-4h}$ $C_{max}$ and $T_{max}$) were calculated and ANOVA test was utilized for statistical analysis of parameters. As a result of the assay validation, the limit of quantification of trifusal in human plasma by current assay procedure was 50 ng/ml using 500 $\mu$l of plasma. The accuracy of the assay was from 97.76% to 116.51% while the intra-day and inter-day coefficient of variation of the same concentration range was less than 15%. Average drug concentration at the designated time intervals and pharmacokinetic parameters calculated were not significantly different between two products (p>0.05). The difference of mean AU $C_{olongrightarrow4hr}$, $C_{max}$, and $T_{max}$ between the two products (2.92, 4.39, and -2.44%, respectively) were less than 20%. The power (1-$\beta$) and treatment difference ($\Delta$) for AU $C_{olongrightarrow4hr}$ and $C_{max}$ were more than 0.8 and less than 0.2, respectively. Although the power for $T_{max}$ was under 0.8, $T_{max}$ of the two products was not significantly different from each other (p>0.05). These results satisfied the criteria of KFDA guideline for bioequivalence, indicating the two products of triflusal were bioequivalent.quivalent.ent.ent.

• Journal of Pharmaceutical Investigation
• /
• v.22 no.1
• /
• pp.63-68
• /
• 1992

An HPLC procedure with UV detection has been developed for the quantitation of flurbiprofen released into isopropyl myristate used as the receptor phase in an in vitro membraneless drug diffusion cell. The drug and the internal standard (oxaprozin) were extracted from isopropyl myristate with a mixture of dimethylsulfoxide:methanol:water (2:1:1) and quantitated using a reverse phase $C_{18}$ column. The chromatograms were completely free from interfering peaks, and the relative retention times of flurbiprofen and the internal standard were 4.9 and 6.8 min, respectively. Calibration plots were linear over the concentration range of $1-200\;{\mu}g/ml$ of flurbiprofen with correlation coefficients, all higher than 0.99. The mean intra-day precision and accuracy among three replicate sets of the assay in a day were 4.26 and 4.52%, respectively, whereas the mean inter-day precision and accuracy were 3.35 and 3.64%, respectively. The mean recovery of the drug was 92.5% over the calibration range. The method was simple, reliable and accurate for the quantitation of flurbiprofen in unpurified isopropyl myristate.

• Journal of Animal Science and Technology
• /
• v.57 no.4
• /
• pp.13.1-13.5
• /
• 2015

As an alternative to radioimmunoassay a simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for androstenedione quantification in plasma of Karan Fries bulls using second antibody coating technique. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was performed to analyze androstenedione directly in $40{\mu}l$ of bull plasma. The androstenedione standards ranged from 0.20 to 200 pg/$40{\mu}l$/well and the sensitivity of the assay was 5 pg/ml plasma. Serially diluted bull plasma containing high endogenous androstenedione showed good parallelism with bovine androstenedione standard curve. Intra- and inter-assay coefficients of variation (CV) were found to be 8 and 9%, respectively. Peripheral plasma androstenedione concentrations determined in young and adult bull samples ranged between 104-990 pg/ml and 184-2040 pg/ml, respectively.

• KSBB Journal
• /
• v.21 no.3
• /
• pp.212-219
• /
• 2006

The purpose of this study was to develop a assay system of host cell-derived residual proteins in final pharmaceutical products. Accurate and simple assay system for host cell-derived proteins(HCPs) is very important test item in pharmaceutical qualification control. In this study, methods for quantification of residual HCPs in recombinant anti-GPIIbIIIa antibody were developed using a process-specific immunoligand assay which was based on the Enzyme linked Immunosorbent assay(ELISA) system. The assay had a detection limit of 10.8 ng/ml of HCPs with a product concentration of 1 mg/ml. The practical implication of these results is that the developed ELISA system can be used for HCPs qualification control and this system will be applicable to develop another ELISA system of different antibody drug.

• Analytical Science and Technology
• /
• v.31 no.4
• /
• pp.143-148
• /
• 2018

Currently, the Korean Pharmacopoeia (KP XI) recommends HPLC and potentiometric titration (which is less specific than HPLC) for the determination of triflusal content in capsules and raw materials, respectively. Additionally, the British Pharmacopoeia (BP 2017) and European Pharmacopoeia (EP 8.0), which include a monograph for triflusal in raw materials only, describe a titration method for the assay. The latest version of the United States Pharmacopoeia (USP 39) and Japanese Pharmacopoeia (JP 17) still have not published monographs for triflusal and its preparations. To improve the specificity and efficacy of the assay, we present an HPLC method to determine triflusal content in both raw materials and capsules. The proposed method was validated in accordance with the requirements of the International Conference on Harmonization. A good linear relationship was achieved for triflusal in the range of $200-1250{\mu}g/mL$ with a coefficient of determination of approximately 0.9996. The relative standard deviations (RSDs) of inter- and intraday precision were 0.73-1.12 % and 0.34-0.51 %, respectively. The recovery percentage of triflusal was in the range of 98.80-101.31 %. Because its system suitability, intermediate precision, and robustness were satisfactory, this method could be suitable for determining triflusal content in raw materials and capsules.

• CELLMED
• /
• v.2 no.3
• /
• pp.26.1-26.5
• /
• 2012

Adverse side-effects and lack of scientific validation of some chemotherapeutic agents prevent the use of many traditional medicines claimed to have anti-cancer effects. Ethanolic extract of Conium maculatum has long been used in traditional and alternative systems of medicine including homeopathy for the treatment of glandular enlargements, cancerous tumours or hard lumps of testicles, prostate, ovaries, breasts and/ or uterus, particularly in the breast. However, if and how it acts still remains scientifically unknown. This study aims to test if Conium extract (CE), used as mother tincture of Conium in homeopathy, has demonstrable anti-cancer potentials without having much cytotoxicity in normal cells. Cytotoxicity of the drug was tested by conducting MTT assay on both normal (peripheral blood mononuclear cells) and HeLa cells. We also evaluated DNA fragmentation and DNA damage by DAPI and diphenylamine assay. The LDH activity assay was done to evaluate the percentages of apoptosis and necrosis. ROS accumulation also was evaluated to pin-point the actual events of apoptosis. Administration of drug clearly demonstrated its anti-cancer potentials as evidenced by the DNA damage analysis. The ROS activity also increased in case of the CE treated cells. LDH data revealed that the mode of cell death was mainly apoptotic and not necrotic. CE appears to induce apoptosis of cancer cells through ROS mediated pathway, and has negligible cytotoxicity against normal cells.

• Toxicological Research
• /
• v.21 no.1
• /
• pp.1-14
• /
• 2005

Transgenic mouse models have been introduced and accepted by regulatory bodies as an alternative to carcinogenicity assay models to predict and evaluate chemical carcinogens. The recent research outcomes in transgenic mouse models have made progressive advances in the understanding of chemical carcinogenesis and the evaluation of potential human carcinogens. However, these models still remain to be insufficient assay systems although the insufficiencies have been recognised and are being resolved. Based on up to date information from literature, this review article intends to understand currently accepted transgenic mouse models, issues arising from study design, interpretation of the study, results of validation project and their cancer prediction rate, and further perspectives of cancer assay models from the regulatory view point.

• Parasites, Hosts and Diseases
• /
• v.54 no.3
• /
• pp.329-334
• /
• 2016

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.