Effect of noise on morphological structure and functions of rat liver / 环境与职业医学

Background Noise can cause not only auditory system injury, but also liver damage. However, the biomarkers and pathological mechanism of noise-induced liver injury are not clear yet. Objective To observe the effect of noise on the morphological structure and functions of rat liver. Methods A total of 30 Wistar rats were randomly divided into a normal control group, a low noise exposure group [(95 dB sound pressure level (SPL)], and a high noise exposure group (105 dB SPL). After 30 days of noise exposure, blood was collected, and livers were harvested and fixed. The pathological changes of livers were observed. The levels of biochemical indicators of liver function, blood glucose, and blood lipid were measured. Serum metabolites were detected by ultra-high-pressure liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Differential metabolite markers and metabolic pathways were identified. Results Compared with the control group, the body weight gain decreased in the low noise group and the high noise group after noise exposure (P<0.001, P<0.05). The pathological results showed that noise caused the rat livers’ morphological and structural damage at various degrees, and damage of the high noise exposure group was more serious. Compared with the control group, the serum levels of aspartate aminotransferase, albumin, and glycosylated serum protein in the low noise exposure group were increased (P<0.05), but the total bile acid level was decreased (P<0.05). The serum levels of alanine aminotransferase, aspartate aminotransferase, albumin, triglyceride, low density lipoprotein, and glycosylated serum protein in the high noise group exposure were increased (P<0.05), but the glucose level was decreased (P<0.05). In the serum metabolomics analysis, 11 differential metabolites were screened out in the low noise exposure group, which were mainly enriched in 3 pathways (thiamine metabolism, primary bile acid biosynthesis, and bile secretion) related to liver metabolism. Four differential metabolites were screened out in the high exposure noise group, which were mainly enriched in four significantly different metabolic pathways (insulin signaling pathway, non-alcoholic fatty liver disease, bile secretion, and insulin secretion). All the metabolic pathways involved in bile acid secretion and metabolism. Conclusion Nosie exposure can not only damage the liver structure of rats, but also affects the metabolism functions of liver. The mechanism may be related to bile acid secretion metabolic pathway.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark / Caracterização fitoquímica, atividade antioxidante e antibacteriana do extrato hidroetanólico de cascas do caule de Anadenanthera peregrina

The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-1. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físicoquímica, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência (HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark / Caracterização fitoquímica, atividade antioxidante e antibacteriana do extrato hidroetanólico de cascas do caule de Anadenanthera peregrina

The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-¹. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físico química, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência(HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Phytochemical characterization, and antioxidant and antibacterial activities of the hydroethanolic extract of Anadenanthera peregrina stem bark

Abstract The Brazilian Cerrado biome consists of a great variety of endemic species with several bioactive compounds, and Anadenanthera peregrina (L.) Speg is a promising species. In this study, we aimed to perform phytochemical characterization and evaluate the antioxidant and antibacterial activities against Staphylococcus aureus and Escherichia coli of the hydroethanolic extract of A. peregrina stem bark. The barks were collected in the Botanical Garden of Goiânia, Brazil. The hydroethanolic extract was obtained by percolation and subjected to physicochemical screening, total phenolic content estimation, high-performance liquid chromatography (HPLC) fingerprinting, and antioxidant (IC50 values were calculated for the 2,2-diphenyl-1-picrylhydrazyl assay - DPPH) and antibacterial activity determination. The pH of the extract was 5.21 and density was 0.956 g/cm3. The phytochemical screening indicated the presence of cardiac glycosides, organic acids, reducing sugars, hemolytic saponins, phenols, coumarins, condensed tannins, flavonoids, catechins, depsides, and depsidones derived from benzoquinones. The extract showed intense hemolytic activity. The total phenolic content was 6.40 g GAE 100 g-1. The HPLC fingerprinting analysis revealed the presence of gallic acid, catechin, and epicatechin. We confirmed the antioxidant activity of the extract. Furthermore, the extract did not inhibit the growth of E. coli colonies at any volume tested, but there were halos around S. aureus colonies at all three volumes tested. These results contribute to a better understanding of the chemical composition of A. peregrina stem bark and further support the medicinal applications of this species.

Resumo O bioma Cerrado brasileiro apresenta em uma grande variedade de espécies endêmicas com diversos compostos bioativos, e Anadenanthera peregrina (L.) Speg é uma espécie promissora. Neste estudo, objetivamos realizar a caracterização fitoquímica e avaliar as atividades antioxidantes e antibacterianas contra Staphylococcus aureus e Escherichia coli do extrato hidroetanólico de cascas do caule de A. peregrina. As cascas foram coletadas no Jardim Botânico de Goiânia, Brasil. O extrato hidroetanólico foi obtido por percolação e submetido a triagem físico-química, estimativa de conteúdo fenólico total, impressão digital por cromatografia líquida de alta eficiência (HPLC) e determinação da atividade antioxidante (valores de IC50 foram calculados para o ensaio 2,2-difenil-1-picril-hidrazil) e antibacteriana. O pH do extrato foi de 5,21 e a densidade foi de 0,956 g/cm3. A triagem fitoquímica indicou a presença de glicosídeos cardíacos, ácidos orgânicos, açúcares redutores, saponinas hemolíticas, fenóis, cumarinas, taninos condensados, flavonóides, catequinas, depsídios e depsidonas derivados de benzoquinonas. O extrato mostrou intensa atividade hemolítica. O conteúdo fenólico total foi de 6,40 g de GAE 100 g-1. A análise por impressão digital por HPLC revelou a presença de ácido gálico, catequina e epicatequina. Confirmamos a atividade antioxidante do extrato. Além disso, o extrato não inibiu o crescimento de colônias de E. coli em nenhum volume testado, mas houve halos em torno das colônias de S. aureus nos três volumes testados. Estes resultados contribuem para uma melhor compreensão da composição química da casca de A. peregrina e apoia ainda mais as aplicações medicinais desta espécie.

Simultaneous determination of anti-diabetic drugs

A novel reverse phase, isocratic HPLC method is described to separate five anti-diabetic drugs i.e., glimepiride, metformin, sitagliptin, rosiglitazone and pioglitazone. Nucleosil C18 analytical column was used as stationary phase, while mobile phase consisted of acetonitrile:phosphate buffer: methanol (40/40/20, v/v) pH 2.0. Effluent was monitored at a flow rate 1 mL/min and detected at wavelength of 240 nm. This research produced excellent chromatography over a wide concentration range of 25-10000 ng/mL. Sepprated and well resolved quantifiable peaks were obtained and test results were linear in this range. Correlation coefficient of more than 0.9990 was witnessed as well as Low %RSD values i.e., maximum 2.0% documented excellent precision of the method. Good recoveries from pharmaecutical (99-101%), urine and plasma samples (>96%) in a range of concentrtion granted very good linearity, accuracy and precision. The projected method has satisfactory applications in quality control of these molecules as well as quantification of these molecules in urine and plasma samples.

Análise de limite de galactosamina em hexosaminas totais em amostras injetáveis de heparina sódica suína / Galactosamine limit analysis in total hexosamines in injectable porcine sodium heparin samples

Introdução: A pesquisa, uma das vertentes da vigilância sanitária, tem sua importância justificada pela busca de respostas aos diversos prejuízos relativos à saúde. A heparina, produto biológico com propriedades anticoagulantes e antitrombóticas, esteve relacionada com eventos adversos entre 2007 e 2008. Diante do ocorrido, os compêndios oficiais atualizaram a monografia para matéria-prima. No entanto, há uma deficiência de monografias para avaliação do produto final. Objetivo: Propor método físico-químico de análise de limite de galactosamina em hexosaminas totais a partir do produto acabado de heparina sódica suína. Método: Foi desenvolvido método analítico a partir da cromatografia líquida de alta eficiência por troca iônica e detecção amperométrica, com as devidas avaliações de adequação do sistema para posterior análise de três amostras de heparina sódica suína, as quais foram previamente submetidas ao protocolo de preparo de amostra através da coluna Micro Bio-Spin™. Resultados: As amostras foram comparadas com a solução de adequação do sistema e matéria-prima de heparina sódica suína, sendo possível detectar presença de galactosamina em uma das três amostras analisadas em quantidade inferior ao limite estipulado pela Farmacopeia Americana. Conclusões: Conclui-se que o método é eficiente para análise do produto acabado e, por isso, será sugerido à Farmacopeia Brasileira.

Introduction: Research, one of the core areas of health surveillance, has its importance justified by its search for answers to various health problems. Heparin, a biological product with anticoagulant and antithrombotic properties, has been related to adverse events between the years 2007­2008. Because of that, the official compendiums updated the monograph for raw material. However, there is a lack of monographs that evaluate the final product. Objective: The goal of this study is to propose a physicochemical method of analysis of the limits of galactosamine in total hexosamine from the finished product of porcine sodium heparin. Method: We developed an analytical method from the ionexchange high performance liquid chromatography with amperometric detection, with the appropriate evaluations of the system suitability for posterior analysis of three samples of porcine sodium heparin previously submitted to the sample preparation protocol through the Micro Bio-Spin column. Results: The samples were compared to the solution of the system suitability and raw material of porcine sodium heparin. We could detect the presence of galactosamine in one of the three analyzed samples in lower amounts than the limit stipulated by the American pharmacopeia. Conclusions: We concluded that the aforementioned method is efficient for the analysis of the finished product and that is the reason why it will be suggested to the Brazilian pharmacopeia.

A comparative study of two separate analytical techniques for the simultaneous determination of diclofenac sodium and diacerein from combined dosage form

ABSTRACT Diclofenac sodium (DS) and diacerein (DC) have emerged as a potential combination therapy for the treatment of knee osteoarthritis. Therefore a validated analytical method is essential for the simultaneous estimation of both from combined dosage form. A ratio derivative spectrophotometric and a chromatographic technique have been developed for the simultaneous determination of DS and DC. The quantification was done at 263.00 nm for DC and 304.50 nm for DS in the first method, whereas 257 nm for DC and at 274 nm for DS for LC-DAD analysis in chromatographic method using acetate buffer and methanol as the mobile phase at a flow-rate 0.50 mL/min. Both of these methods are found to be linear in the concentration range under study with r2 value 0.999 and 0.996 for DS and DC respectively in ratio derivative spectroscopy and 0.998 and 0.999 for DS and DC respectively in LC-DAD study. Both of these methods are found to be accurate and precise, though greater robustness and precision is observed with chromatographic analysis over the ratio derivative spectroscopy. Statistically there was no significant difference between proposed ratio derivative spectrophotometric and LC-DAD methods.

Determination of hesperidin and liquiritin in Bailong Jieyu granule by high-performance liquid chromatography / 第二军医大学学报

Objective To determine the contents of the hesperidin and liquiritin in Bailong Jieyu granule with high-performance liquid chromatography (HPLC). Methods HPLC was performed with octadecylsilane chemically bonded silica as filler using the Agilent SB-ZOBAX C18 Column (250 mm×4.6 mm, 5 μm). Hesperidin was detected with the mobile phase consisting of acetonitrile and 0.1% of phosphoric acid solution (22:78), with column temperature being 30 °C, the detection wavelength being 283 nm, and the injection volume being 10 μL. Liquiritin was determined with acetonitrile-0.1% of phosphoric acid solution (18:82) as mobile phase, with column temperature being 30 °C, the detection wavelength being 276 nm, and the injection volume being 10 μL. Results Accuracy, repeatability and stability of HPLC for determination of hesperidin and liquiritin were in line with the relevant requirements. The linear relationship of hesperidin was good within the range of 35.64-178.20 μg/mL (r=0.999 8), with the average recovery rate being 99.30%, and relative standard deviation (RSD) being 1.0% (n=9). The good linear range of liquiritin was 13.55-216.80 μg/mL (r=0.999 9), with the average recovery rate being 98.60% and RSD being 1.5% (n=9). Conclusion HPLC method was fast, simple, stable and reliable, and it can be used for the quality control of Bailong Jieyu granule.

Pharmacokinetics of tilmicosin in healthy pigs and in pigs experimentally infected with Haemophilus parasuis

A comparative in vivo pharmacokinetic (PK) study of tilmicosin (TIL) was conducted in 6 crossbred healthy pigs and 6 crossbred pigs infected with Haemophilus (H.) parasuis following oral administration of a single 40 mg/kg dose. The infected model was established by intranasal inoculation and confirmed by clinical signs, blood biochemistry, and microscopic examinations. Plasma TIL concentrations were determined by a validated high-performance liquid chromatography method with ultraviolet detection at 285 nm. PK parameters were calculated by using WinNonlin software. After TIL administration, the main PK parameters of TIL in healthy and H. parasuis-infected pigs were as follows: Area under the concentration-time curve, maximal drug concentration, half-life of the absorption phase, half-life of the distribution phase, and half-life of the elimination phase were 34.86 ± 9.69 vs. 28.73 ± 6.18 µg · h/mL, 1.77 ± 0.33 vs. 1.67 ± 0.28 µg/mL, 2.27 ± 0.45 vs. 2.24 ± 0.44 h, 5.35 ± 1.40 vs. 4.61 ± 0.35 h, and 43.53 ± 8.17 vs. 42.05 ± 9.36 h, respectively. These results of this exploratory study suggest that there were no significant differences between the PK profiles of TIL in the healthy and H. parasuis-infected pigs.

Systemic absorption of 0.1% triamcinolone acetonide as topical application in management of oral lichen planus.

Context: Topical corticosteroids are the treatment of choice for oral lichen planus (OLP) due to its potential anti‑inflammatory effect. However, chronic nature of OLP often requires long‑term and frequent applications, exposing patients to local and systemic side effects. Aim: To detect the systemic absorption of 0.1% triamcinolone acetonide (TAC) through the oral mucosa of patients with OLP. Subjects and Methods: This was a pilot pharmacokinetic study carried out in the Department of Oral Medicine and Radiology in collaboration with the Department of Toxicology, over 10 months. A total of twenty patients with OLP were included and advised to apply 0.1% TAC 3 times/day for 2 weeks and 2 times/day for next 2 weeks. Blood samples were obtained on the first and second visits and analyzed for triamcinolone using High pressure liquid chromatography (HPLC). Statistical Analysis Used: Paired t‑test was done to compare visual analog scale (VAS) score for burning sensation at the first and second visits, statistically significant if P < 0.05. The baseline demographic data were analyzed using descriptive statistics. Results: Paired t‑test was done to compare VAS score for burning sensation at the first and second visits, which turned to being statistically significant (P = 0.001). Although HPLC is an established method for the detection of TAC, none of the study populations showed evidence of steroid (TAC) in the blood sample during 4 weeks of treatment duration. Conclusions: 0.1% triamcinolone is a relatively safe drug to be used with no systemic absorption in the standard dose regimen for oral lichen palnus.

An HPLC method for determination of ebracteolatain A content in the root of Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata / 第二军医大学学报

Objective To establish a method for determination of ebracteolatain A content in the root of traditional Chinese medicine white Langdu, including Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata. Methods An HPLC-DAD method was created for determination at the following condition:the column was Agilent Zorbax SB-C18 (4.6 mm×150 mm, 5 μm);mobile phase was acetonitrile:0.1% formic acid in water=55:45(V:V), isocratic elution, the flow rate was 1.0 mL·min-1, the temperature of column was 25°C, the detection wavelength was set at 290 nm, the injection volume was 20 μL, and the running time was 20 min. Results Ebracteolatain A was separated from the interference in the baseline, and the linear range was 4.545-227.3 μg·mL-1, with the linear correlation being 0.9999 for ebracteolatain A. The result of intra-day and inter-day precisions were both within 2%(n=3), and the average recovery was (99.14±3.4)%(n=6). The content of ebracteolatain A in two batches of Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata were (56.73±1.09) μg/g, (18.98±2.11) μg/g and (235.2±2.4) μg/g (n=6), respectively. Conclusion The present method is simple, rapid, accurate and convenient for determination of ebracteolatain A in the root of traditional Chinese medicine white Langdu.

Evaluation of Analytical Performance of the D-100 Hemoglobin Testing System for Hemoglobin A1c Assay

BACKGROUND: The hemoglobin A1c (HbA1c) level is widely used to diagnose and monitor glycaemic control in people with diabetes mellitus, and various methods are used for its determination. The D-100 hemoglobin testing system (Bio-Rad Laboratories, USA) is a fully automated, high-throughput glycohaemoglobin analyzer based on an ion-exchange high-performance liquid chromatographic method. Here, we evaluated the analytical performance of a newly developed HbA1c analyzer. METHODS: Precision, linearity, and comparison to the Variant II Turbo analyzer (Bio-Rad Laboratories, USA) were evaluated according to the Clinical Laboratory Standards Institute guidelines. Carryover, bias from the value assigned by the HbA1c Network Laboratory of Korea Centers for Disease Control and Prevention, and the vulnerability to interference by hemoglobin variants frequently found in Korea were also assessed. Statistical analyses were performed using Excel 2010 (Microsoft Co., USA) and MedCalc ver. 14.12.0 (MedCalc Software bvba, Belgium). RESULTS: The coefficients of variation for repeatability and within-device precision were less than 1.08% in National Glycohaemoglobin Standardization Program (NGSP) unit and less than 1.68% in international system of unit at all three levels. The calibration curve was linear, with R²=0.996 in the range of 4.6% to 15.4% in NGSP unit. The results highly correlated with those produced by Variant II Turbo (r=0.998). The 95% confidence interval for differences from the assigned values was -3.3% to 2.9%. No significant interferences of haemoglobin variants were observed except for Hemoglobin Yamagata. CONCLUSIONS: The D-100 hemoglobin testing system showed excellent precision, linearity, and good correlation with the Variant II Turbo analyzer and agreement with the assigned values. Therefore, its analytical performance is satisfactory for diabetes diagnosis and treatment monitoring.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin

Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical–chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (t(R) = 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone.

HPLC for simultaneous determination of naringin, hesperidin, neohesperidin, acteoside and liquiritin in Jinlongshe oral solution / 第二军医大学学报

Objective To establish an HPLC method for simultaneous determination of five effective components (naringin, hesperidin, neohesperidin, acteoside and liquiritin) in the Jinlongshe oral solution. Methods SunFire™ C18 (4.6 mm×250 mm, 5 μm) column was used. The mobile phase was acetonitrile (B) and 0.2% formic acid (A), with gradient elution program as follow: 0-10 min, 10%-19% B; 10-32 min, 19% B. The flow rate was 1.0 mL/min, and the temperature was at 40℃. The injection volume was 10 μL, and the detection wavelength was set at 285 nm. Results There were good linearities for concentrations of naringin, hesperidin, neohesperidin, acteoside and liquiritin within the range of 2.00-60.00, 2.12-63.60, 1.68-50.40, 2.00-60.00 and 2.00-60.00 μg/mL (r≥0.999 9), respectively. RSD of the intra-day and inter-day precisions of the five components were less than 2.30% and 3.82%, respectively. RSD of the stability in 24 h was less than 4.47%. The average recoveries were 95.11%, 93.73%, 96.39%, 98.02% and 95.12%, with the RSD being 2.08%, 4.05%, 4.02%, 6.01% and 4.56%, respectively. Conclusion The present method has been proven to be convenient, accurate, sensitive and with good reproducibility, and can be applied for the quality control and assessment of Jinlongshe oral solution.

Pharmacokinetics of Danhong injection in normal and cold-coagulation and blood-stasis rats / 第二军医大学学报

Objective To establish an HPLC method for simultaneous analysis of danshensu (DSS), protocatechuic acid (PA) and hydroxysafflor yellow A (HSYA) in plasma samples, and to study the pharmacokinetics of DSS, PA and HSYA in Danhong Injection in normal and cold-coagulation and blood-stasis model rats. Methods The cold-coagulation and blood-stasis rat models (n=6) were made by continuous stimulation with ice water for 20 days; another 6 normal rats served as controls. The concentrations of DSS, PA and HSYA in the plasma were determined by RP-HPLC (0. 2% formic acid water[A]-methanol [B], gradient elution, wavelength detection: 280 nm [0-40 min] and 402 nm [40-60 min]) at 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, and 90 min after administration of Danhong Injection via the tail vein. The pharmacokinetic parameters were calculated with DAS 3. 0 software. Results DSS and PA had an open two compartment model and HSYA had an open three compartment model. Compared with the normal groups, the model group had significantly increased maximum plasma concentration (Cmax) of DSS, PA and HSYA, the distribution half-life time (t1/2a) of DSS, and elimination half-life time (t1/2a, t1/2γ), area under curve (AUC), and apparent volume (V) of PA and HSYA (P1/2a) of PA and HSYA and area under curve (AUC) of DSS(P<0. 05). Conclusion DSS has a lower distribution and bioavailability under the condition of cold-coagulation and blood-stasis, while PA and HSYA show a faster distribution, slower elimination, increased apparent volume and bioavailability, indicating a better clinical effect.

HPLC in determination of hypoxanthine, xanthine, uridine and uracil in Shanghai Pheretima and Pheretima aspergillum (E. Perrier) / 第二军医大学学报

Objective To establish an effective method for the determining hypoxanthine, xanthine, uridine and uracil in earthworm in Shanghai Pheretima and Pheretima aspergillum (E. Perrier), contributing to quality control of the medicinal material. Methods Hypoxanthine, xanthine, uridine and uracil were extracted from the earthworms with 0.9% NaCl by ultrasonic and determined by HPLC. The chromatographic conditions were: SORBAX SB-Aq column (250 mm×4.6 mm, 5 μm, Aglient Co.,Ltd), 5 mmol/L KH2PO4 (pH 2.9) as the mobile phase with a flow rate of 1 mL/min,the detection wavelength was 254nm, the column temperature was set at 30℃, and the injection volume was 10 μL. Hypoxanthine, xanthine, uridine and uracil were determined by HPLC at the same time. Results The linear range was 0.5-100 μg (r=0.999 9) for hypoxanthine, with the average recovery being 99.37%, RSD=1.36% (n=6). The linear range was 0.5-100 μg (r=0.993 1) for xanthine, with the average recovery being 91.57%, RSD=1.40% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uridine, with the average recovery being 95.31%, RSD=1.64% (n=6). The linear range was 0.5-100 μg (r=0.999 9) for uracil, with the average recovery being 100.21%, RSD=1.98% (n=6). Conclusion The current method is reproducible and has satisfactory recovery, and it can be used to determine hypoxanthine, xanthine, uridine and uracil in earthworm medicinal material.

HPLC in determination of effective contents and related substances in clopidogrel hydrogen sulfate produced by 3 manufacturers / 第二军医大学学报

Objective To establish a high performance liquid chromatography(HPLC) method for quantitative determination of S-clopidogrel and its related substance R-clopidogrel in clopidogrel tablets, and to compare their contents between clopidogrel tablets produced by three different manufacturers. Methods An ULTRON ES-OVM column (4.6 mm × 150 mm,5 μm) was used in this study. The mobile phase consisted of a mixture of acetonitrile and 0.01 mol/L potassium dihydrogen phosphate solution (20:80). The flow-rate was 1 mL/min, with the column temperature being 17°C and the UV detection wave lenth being 220 nm. Results S-clopidogrel and its related substance R-clopidogrel were in good linear relationship within 50-117 mg/L and 1.52-30.04 mg/L, respectively (both r=0.999 8). The precision of our method was satisfactory and the average recoveries were 99.7% and 98.8%, respectively. The test solution remained stable for 12 h. S-clopidogrel and R-clopidogrel in clopidogrel tablets produced by three manufacturers were in line with the quality requirements of pharmacopoeia. Conclusion Our method is accurate and has good specificity. It can be used for the quality control of clopidogrel tablets.

HPLC-DAD for simultaneous determination of 20 colorants in hair dyes / 第二军医大学学报

Objective: To develop a high-performance liquid chromatography (HPLC)-diode array detector (DAD) method for determination of 20 colorants in hair dyes, so as to improve the method for determining oxidative hair dyes in Hygienic Standard for Cosmetics. Methods: After ultrasonic extraction with ethanol-water, samples were analyzed by HPLC. The separation was achieved on an Agilent Zorbax Bonus-RP column (4.6mm × 250mm, 5μm) using a gradient mobile phase system of A: acetonitrile and B: 4mmol/L PBS (pH = 6.0) containing 0.1 % 1-octanesulfonic acid sodium salt solution at a flow rate of 1 mL/min. The gradient elution program was as follows: 0-24 min, 10% A; 24-26 min, 10%-40% A; 26-45 min, 40% A; 45-47 min, 40%-80% A; 47-52 min, keep 80% A unchanged. The detection wavelength was set at 280 nm, the temperature of column was 15°C, and the injection volume was 5 μL. Results: All the components were separated at baseline within 50 min. Within the concentration range of 10.0-500.0 mg/L, the peak areas and concentrations of colorants had good linear relationship, with the linear correlation coefficients being higher than 0.999. The intra-day and inter-day precisions were within 4% and the average recoveries ranged from 81.90% to 119.88%. Conclusion: The present method is simple, rapid and accurate and is suitable for determination of colorants in oxidative hair dyes.

Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies.

Introduction: Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC) is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. Materials and Methods: 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR)-based allele-specific Amplification Refractory Mutation System (ARMS). Molecular analysis for the sickle cell mutation was done by standard methods. Results: The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23%) of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Discussion and Conclusion: Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS) because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

Optimization of extracting conditions for berberine, magnolol and emodin from simplified recipe of Ruyijinhuangsan / 第二军医大学学报

Objective To establish a method for simultaneous determination of berberine, magnolol and emodin in the blend powder of Cortex Phellodendri Chinensis, Cortex Magnoliae Officinalis and Radix et Rhizoma Rhei, so as to provide evidence for preparation technique and quality control of simplified recipe of Ruyijinhuangsan. Methods High-performance liquid chromatography (HPLC) was used for simultaneous determination of berberine, magnolol and emodin at the following conditions: the column was ACE.5C18-AR, mobile phase was A: triethylamine, B: methanol. The flow rate was 1.0 mL/min, the temperature of column was 30°C, the detection wavelengths were set at 294 nm and 345 nm, and the injection volume was 10 μL. Using the yield ratio of three constituents as the assessment indices, we optimized the extraction technology by single factor test and orthogonal test L9 (34). Results The calibration curves were linear within the range of 0.292-292 mg/L for berberine (r2=0.999 8), 0.292-292 mg/L for magnolol (r2=0.999 7), and 0.256-256 mg/L for emodin (r2=0.999 5), with the average recovery rates being 98.26% (RSD 2.53%, n=6), 105.17%(RSD 2.06%, n=6), and 100.39% (RSD 2.60%, n=6), respectively. The results of precisions were (n=6) 0.23%, 0.17%, 0.18%, respectively. The sample solutions were stable within 12 h. The single factor experiment analyzed ultrasonic duration and times, concentration of alcohol, the amount of solvent and the concentration of HCl. The results showed that, with the increase of ultrasonic duration, times and the alcohol concentration, the yield ratio of the three consitituents was increased.Conclusion The optimum options for extracting is as follows: ultrasonic extraction 3 times by 95% alcohol containing 0.1% HCl, with the volume at 35 mL, 30 min each time.