Development and validation of LC-MSMS method for the analysis of amlodipine and perindopril in tablet dosage form

Introduction: Amlodipine (AMLO) and Perindopril (PER) have been combined in one tablet dosage form which has been proven to be an effective and well tolerated antihypertensive. Many analytical methods were applied for the analysis of AMLO and PER in this combination but most of the methods are time and money consuming. A new sensitive, rapid, and specific LC- MS/MS analytical methods for the analysis of AMLO and PER was developed and validated. Chromatographic conditions for the best results were optimized using design of experiment method. 4 TH PHARMACEUTICAL RESEARCH CONFERENCE 2018 Challenging The Inquisitive Minds: Frontier Of The Future 24 – 26 August 2018 | Cyberjaya, Malaysia Method: The instrument used was Waters Aquity UPLC, and the column was Acquity BEH C18 (50 mm × 2.1 mm, 1.7 µm). The mobile phase consisted of solvent A which was 0.1% formic acid in water and solvent B which was ACN. The injected volume was 10 µl with a flow rate of 0.4 ml/min. samples were eluted following gradient program over 5 min. The column temperature was maintained at 35 °C. The detection was performed using Xevo TQD MS with ESI in positive mode. The method was validated according to ICH guidelines. Result: The results showed that the retention times of AMLO and PER were 1.65 min and 1.78 min respectively. Method was linear for AMLO and PER over the range of 5-15 ng/ml with R 2 of 0.9988 and 0.9991 respectively. The accuracies of AMLO and PER were in the range of 98.1% -100.19%. LOD and LOQ were 50.28 and 152.78 pg/ml for AMLO respectively and 16.86 and 51.1 pg/ml for PER respectively. The developed and validated method was applied for the quantification of AMLO and PER at tablet dosage form which is available at the market with satisfactory results. Conclusion: It can be concluded that this method is highly sensitive, rapid, selective and it can be used for the routine analysis of AMLO and PER in QC laboratories

2013 年 9 月 第 11 卷 第 5 期 Chin

[ABSTRACT] The anticoagulant effect of leech saliva was traditionally employed in the treatment of diabetes mellitus complications such as peripheral vascular complications. This study was carried out to examine the effect of leech saliva extract (LSE) on blood glucose levels in alloxan-induced diabetic rats. First, LSE was collected from leeches which were fed on a phagostimulatory solution. Second, total protein concentration was estimated using the Bradford assay. Third, diabetic rats were injected subcutaneously (sc) with LSE at doses of 500 and 1 000 µg·kg 1 body weight (bw). Other diabetic rats were injected sc with insulin at doses of 10 and 20 U·kg 1 bw. Another group was injected simultaneously with LSE (250 µg·kg 1 bw) and insulin (10 U·kg 1 bw). Fasting blood glucose (FBG) concentrations were monitored during a study period of eight hours at regular intervals. Findings showed that both doses of LSE resulted in a significant and gradual decrease in FBG starting from 10%−18% downfall after two hours of injection reaching the maximal reduction activity of 58% after eight hours. Remarkably, LSE was sufficient to bring the rats to a near norm-glycemic state. The high dose of insulin induced a severe hypoglycemic condition after 2−4 h of injection. The lower dose was able to decline FBG for 2−6 h in rats which became diabetic again after 8 h. On the other hand, the concurrent injection of low doses of LSE and insulin produced a hypoglycemic effect with all rats showing normal FBG levels. Taken together, these findings indicated that the subcutaneous injection of LSE of the medicinal Malaysian leech was able to provide better glycemic control compared with insulin. Moreover, the synergism between LSE and insulin suggests that LSE could be utilized as an adjuvant medication in order to reduce insulin dosage or to achieve better control of blood glucose

Reliability of graphite furnace atomic absorption spectrometry as alternative method for trace analysis of arsenic in natural medicinal products

Purpose: To evaluate the comparative efficiency of graphite furnace atomic absorption spectrometry (GFAAS) and hydride generation atomic absorption spectrometry (HGAAS) for trace analysis of arsenic (As) in natural herbal products (NHPs).Method: Arsenic analysis in natural herbal products and standard reference material was conducted using atomic absorption spectrometry (AAS), namely, hydride generation ASSAAS (HGAAS) and graphite furnace (GFAAS). The samples were digested with HNO3–H2O2 in a ratio of 4:1 using microwave-assisted acid digestion. The methods were validated with the aid of the standard reference material 1515 Apple Leaves (SRM) from NISTResults: Mean recovery of three different samples of NHPs, using HGAAS and GFAAS, ranged from 89.3 - 91.4 %, and 91.7 - 93.0 %, respectively. The difference between the two methods was insignificant. A (P= 0.5), B (P=0.4) and C (P=0.88) Relative standard deviation (RSD) RSD, i.e., precision was 2.5 - 6.5 % and 2.3 - 6.7 % using HGAAS and GFAAS techniques, respectively. Recovery of arsenic in SRM was 98 and 102 % by GFAAS and HGAAS, respectively.Conclusion: GFAAS demonstrates acceptable levels of precision and accuracy. Both techniques possess comparable accuracy and repeatability. Thus, the two methods are recommended as an alternative approach for trace analysis of arsenic in natural herbal products.Keywords: Arsenic, Graphite furnace atomic absorption spectrometer (GFAAS), Hydride generation atomic absorption spectrometer (HGAAS), Natural herbal product

Chemosensetizing and cardioprotective effects of resveratrol in doxorubicin- treated animals

BACKGROUND: Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer drug used in the treatment of variety of cancers .Its use is limited by its cardiotoxicity. The present study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of mammary carcinoma (Ehrlich ascites carcinoma) to the action of DOX and at the same time its protective effect against DOX-induced cardiotoxicity in rats. METHODS: Ehrlich ascites carcinoma bearing mice were used in this study. Percent survival of tumor bearing mice was used for determination of the Cytotoxic activity of DOX in presence and absence of RSVL. Uptake and cell cycle effect of DOX in tumor cells in the presence of RSVL was also determined. Histopatholgical examination of heart tissues after DOX and/or RSVL therapy was also investigated. RESULTS: DOX at a dose level of 15 mg/kg increased the mean survival time of tumor bearing mice to 21 days compared with 15 days for non tumor-bearing control mice. Administration of RSVL at a dose level of 10 mg/kg simultaneously with DOX increased the mean survival time to 30 days with 70% survival of the tumor-bearing animals. RSVL increased the intracellular level of DOX and there was a strong correlation between the high cellular level of DOX and its cytotoxic activity. Moreover, RSVL treatment showed 4.8 fold inhibition in proliferation index of cells treated with DOX. Histopathological analysis of rat heart tissue after a single dose of DOX (20 mg/kg) showed myocytolysis with congestion of blood vessels, cytoplasmic vacuolization and fragmentation. Concomitant treatment with RSVL, fragmentation of the muscle fiber revealed normal muscle fiber. CONCLUSION: This study suggests that RSVL could increase the cytotoxic activity of DOX and at the same time protect against its cardiotoxicity

Starvation Time and Successive Collection Effects on Leeches Saliva Collection Quantity and Proteins Quality and Quantity in Wet Season

The salivary gland secretion of the haematophagous animals, leeches, has attracted the attention of therapists since the extreme old ages due to its wide range of medical properties. Thus, many researches have been done to develop and optimize new methods to collect leech saliva with high quality and quantity. In the present study, we aimed to evaluate the effects of starvation period and repeated collection on the quality and quantity of leech saliva extract LSE and its contents of proteins during the rainy season. Protein recovery in the LSE was also studied after first collection. It was found that leeches are able to produce protein-containing saliva whenever fed during the whole study period of 18 weeks with varied protein concentrations. The results showed that the highest protein concentrations (105-91 μg/mL) were produced after 12-15 weeks of starvation. The results of successive collection showed that leeches are able to produce proteins and peptides whenever they suck the solution after first collection with some varies in the concentrations. The concentrations varied between 0 and 72% of the initial concentration. Gel electrophoresis results showed absence for some bands when the concentrations are too low. Also the results showed that leeches are able to recover about 42% of their initial proteins concentration within four weeks of starvation after first feeding. The gel electrophoresis results showed the closeness between the first and second collections. To conclude, all test factors (starvation period, successive collection and recovery test) were shown to have an important impact on protein concentration of leech saliva and therefore its medicinal affectivity. The mentioned results are reported for the first time and they open the gate for further studies

Development and validation of bioanalytical method for the analysis of amlodipine in human plasma with optimized extraction method using design of experiment

SUMMARY. Amlodipine is antihypertensive drug belonging to calcium antagonist group. This study aims to apply experimental design for the optimization of extraction method of amlodipine from human plasma for higher and constant recovery. A chromatographic method involving HPLC (High Performance Liquid Chromatography) with DAD (Diode Array Detector) was developed and validated for the determination of amlodipine in human plasma with cetirizine as internal standard. Sample preparation for the extraction of amlodipine and cetirizine (internal standard) from plasma was optimized using DOE (Design of Experiment). Three different parameters namely solvent type, solvent volume and pH were monitored at different level for the optimization of sample preparation that would produce best recovery of the analytes. For this purpose, three level full factorial design, was conducted. The recovery of amlodipine and internal standard was determined using the developed and pre validated HPLC-DAD method with an Agilent 1100 HPLC system. Chromatographic separation of the analytes was obtained using phenomenix (150 × 4.5 mm, 5 μm) C18 column. The mobile phase consists of 35:65 v/v of ACN: 0.3% triethylamine with pH adjusted at 3 using phosphoric acid. Detection of analyte was conducted using a diode array detector at 237 nm wavelength. Design Expert 10.0 software was used to interpret all data obtained from the optimization study. For the parameters solvent and solvent volume, acetonitrile and 1 mL was found to be the optimum values required to extract amlodipine and internal standard while pH had no effect on the optimum recovery. The recovery of amlodipine and internal standard was 80-88 and 85-99%, respectively. RESUMEN. Amlodipina es un fármaco antihipertensivo que pertenece al grupo de antagonistas del calcio. Este estudio tiene como objetivo aplicar el diseño experimental para la optimización del método de extracción de amlodipina del plasma humano para una recuperación más alta y constante. Se desarrolló y validó un método cromatográfico que involucra HPLC (cromatografía líquida de alta resolución) con DAD (detector de arreglo de diodos) para la determinación de amlodipina en plasma humano con cetirizina como patrón interno. La preparación de la muestra para la extracción de amlodipina y cetirizina (patrón interno) del plasma se optimizó usando DOE (diseño de experimento). Se controlaron tres parámetros diferentes, a saber, el tipo de disolvente, el volumen del disolvente y el pH a diferentes niveles para la optimización de la preparación de la muestra que produciría la mejor recuperación de los analitos. Para este propósito, se realizó un diseño factorial completo de tres niveles. La recuperación de amlodipina y patrón interno se determinó usando el método HPLC-DAD desarrollado y prevalidado con un sistema Agilent 1100 HPLC. La separación cromatográfica de los analitos se obtuvo usando una columna fenix (150 × 4.5 mm, 5 μm) C18. La fase móvil consiste en 35:65 v/v de ACN: 0.3% de trietilamina con pH ajustado a 3 usando ácido fosfórico. La detección del analito se realizó usando un detector de red de diodos a una longitud de onda de 237 nm. Se utilizó el software Design Expert 10.0 para interpretar todos los datos obtenidos del estudio de optimización. Para los parámetros, se encontró que el solvente acetonitrilo y el volumen de solvente de 1 mL eran los valores óptimos requeridos para extraer amlodipina y el estándar interno, mientras que el pH no tuvo efecto sobre la recuperación óptima. La recuperación de amlodipina y el estándar interno fue 80-88 y 85-99%, respectivamente

Development and validation of new RP-HPLC method for the determination of gliclazide in tablet dosage form

A new sensitive and economic analytical method for the analysis of gliclazide in tablet dosage form was developed and validated using commercial HPLC instrument with modern ultra-performance liquid chromatography column. Samples were analysed using Agilent 1100 Series RP-HPLC equipped with a binary pump and auto injector. Samples were injected into Xselect HSS C18 XP column (2.1 × 100 mm, 2.5 μm), 2.5 μm) and the mobile phase consisted of triethylamine buffer 0.3% (pH = 3 adjusted using phosphoric acid) and acetonitrile in the ratio of 50:50 v/v. The flow rate was 0.4 mL/min and the DAD de- tector was used for the detection and the wavelength was 229 nm. The method was validated according to ICH guidelines. The retention time for gliclazide peak was 3.437 ± 0.41 min with the total run time of 6 min with total solvent consumption of 2.4 mL per run. The method was found linear over the range 0.1-10 μg/mL with coefficient of determination R2 of 0.9997. The recovery was 98.09-100.19 %, and the method showed high precision and repeatability. Degradation pathway of gliclazide was studied following differ- ent types of stress degradation which are base, acid, UV, and oxidization. All validated parameters were in the acceptable range of ICH requirements. In conclusion, a new, rapid, sensitive, and economic method was developed and validated for the analysis of gliclazide in tablet dosage form. RESUMEN. Se desarrolló un nuevo método analítico sensible y económico para el análisis de gliclazida en forma de dosificación de comprimidos y se validó usando HPLC con una columna moderna de cromatografía líquida de ultra rendimiento. Las muestras se analizaron utilizando un equipo RP-HPLC Agilent Serie 1200 equipado con bomba cuaternaria y autoinyector. Las muestras se inyectaron a una columna de agua C18 HSS (2,1 × 100 mm, 2,5 μm) y la fase móvil consistió en tampón de trietilamina 0,3% (pH = 3 ajustado usando ácido fosfórico) y ace- tonitrilo en la relación 50:50 v/ v. El caudal era de 0,4 mL/ min y se utilizó un detector UV y la longitud de onda fue de 229 nm. El método fue validado de acuerdo con las pautas de ICH. El tiempo de retención para el pico de gliclazida fue de 3,437 ± 0,41 min con el tiempo total de ejecución de 6 min y un consumo total de disolventes de 2,4 mL por ciclo. El método se encontró lineal en el intervalo de 0,1-10 μg/mL con un coeficiente de determi- nación R2 de 0,9997. La recuperación fue de 98,09-100,19%, y el método mostró alta precisión y repetibilidad. La vía de degradación de gliclazida se estudió después de diferentes tipos de degradación del estrés que son base, ácido, UV y oxidación. Todos los parámetros validados estaban dentro del rango aceptable de los requisitos de ICH. En conclusión, se desarrolló y validó un nuevo método rápido y sensible para el análisis de gliclazida en forma de tableta

Characterization and optimization of lyophilization and storage conditions of Leech saliva extract from the tropical leech Hirudinaria manillensis

The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract (LSE) was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized (solid state) and non-lyophilized (liquid state) were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20 °C as the optimum temperature. Samples stored at ambient temperature and +4 °C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light

Comparative water quality study between peat coagulant treated and untreated model water bodies

Locally invented coagulant from Malaysian peat soil was effective for the clarification of lake and river water. Study was performed on stagnant lake water using model water bodies treated with peat coagulant. Better water quality was found in peat coagulant treated lake water compared to the lake water without peat coagulant treatment. The Dissolved Oxygen (DO) level recovered to the original value within two to three weeks in peat coagulant dosed tank. For the control tank the DO level was as low as 0.8 mg/L and the DO level never reached the original value even after sixty days of monitoring. The other water quality parameters like Chemical Oxygen Demand (COD), nutrients (P and N) and suspended solids were lower than those of the control tank

In vivo study of the pharmacokinetic effect of amlodipine and gliclazide

It was found that there is kind of pharmacodynamic interaction between calcium channels blockers and sulfonylurea. The addition of amlodipine (AMLO) to gliclazide (GLZ) course caused significant decrease in glucose lowering effect of GLZ. The mechanism of AMLO-GLZ interaction is not well understood. There is a possibility that this interaction can be a result of pharmacokinetic interaction of both drugs; therefore, this study was designed to investigate the pharmacokinetic interaction between AMLO and GLZ in animal model. The pharmacokinetic interaction was evaluated using Sprauge Dawly rates as they were divided into three groups. The controle group received saline and the gliclazide groupe received gliclazide orally in the dose of 1.44 mg/kg, while the Amlo-Gliclazide groupe has received amlodipine and gliclazide orally with a dose of 0.09 mg/kg and 1.44 mg/kg respectively. After dosing, animals were anesthetized and the blood was collected using retro orbital blood collection method after 1, 2, 4, 6, 8, 12, 24 hours of the dose. A new and novel LC-MS/MS method was developed and validated for the determination of GLZ in and rat plasma. The results showed that the pharmacokinetic parameters such as Cmax (ng/ml), Tmax (hr) were not altered significantly by AMLO administration. However, the administration of AMLO lead to significant increase (p = 0.000672) in AUC. In conclusion the plasma GLZ concentration increased when it was co administered with AMLO, while Cmax and Tmax remain unchanged. This study suggests that AMLO might reduce GLZ elimination