Validation of R/S-Warfarin Analysis Method in Human Blood Plasma Using HPLC with Fluorescence Detection and its Application to Patient Samples.

Context: Warfarin is extensively used for venous thromboembolism and other coagulopathies. In clinical settings, warfarin is administered as a mixture of S-warfarin and R-warfarin, and both enantiomers are metabolized by multiple cytochrome P450 enzymes into many hydroxylation metabolites. Validation of analysis method and estimation of warfarin plasma levels are important, especially in narrow-index drugs such as warfarin. Aims: This study aimed to obtain a validated method for analyzing warfarin in patient plasma according to the European Medicines Agency (EMA) guidelines. Materials and Methods: A total of 77 patients were enrolled in this study. Five millimeters of venous blood was collected using sodium ethylenediaminetetraacetic acid (EDTA) tubes for pharmacokinetic analysis. Samples were prepared by the protein precipitation technique using acetonitrile. The optimum conditions for the analysis of warfarin in human plasma were tested using fluorescence detector (FLD) high-performance liquid chromatography (HPLC) with Chiralcel OD-RH column (4.6 × 150 mm i.d., 5 µm), Chiralcel OD-RH guard column (4.0 × 10 mm, 5 µm), and a column temperature of 45°C. The mobile phase used was acetonitrile: phosphate buffer pH 2 (40:60), with an isocratic flow rate of 1 ml/min and an injection volume of 20 µl. Excitation and emission wavelengths were 310 and 350 nm (warfarin) and 300 and 400 nm (griseofulvin). The retention time of griseofulvin was 6-7.5 minutes; R-warfarin was 10-11.5 minutes; and S-warfarin was 14-16 minutes. Results: The result of this validation obtained the optimum conditions. This method yielded the limit of detection (LOD) values of 0.0674 ppm (R-warfarin) and 0.0897 ppm (S-warfarin). The limit of quantification (LOQ) values were 0.225 ppm (R-warfarin) and 0.298 ppm (S-warfarin). Linearity existed at concentrations of 0.2-3 ppm with the line equation y = 0.0705x + 0.0704 with R² = 0.978 for R-warfarin and y = 0.0513x + 0.0297 with R² = 0.9924 for S-warfarin. At least 75% of the seven concentrations met the reverse concentration requirements, which were below ± 15%. This method met the requirements of accuracy and precision within and between runs, selectivity, and carryover where the %RSD and %diff values were below ± 15%. The mean plasma concentrations of R-warfarin and S-warfarin were found to be 0.76 ± 1.87 (SD) µg/ml and 0.59 ± 0.81 (SD) µg/ml, respectively. The mean standard dose group plasma concentration from the analysis of 77 samples was 0.68 ± 0.61 µg/mL for R-warfarin and 0.52 ± 0.42 µg/mL for S-warfarin. Conclusions: Based on these results, this analytical method can be declared valid and can be used for sample measurement in warfarin pharmacokinetic studies.

Towards halal pharmaceutical: Exploring alternatives to animal-based ingredients.

Halal is a crucial concept for Muslim consumers regarding consumed products, including pharmaceutical ingredients, which are essential in modern medicine. To address the issue of using porcine-sourced ingredients in pharmaceuticals, it is essential to search for halal alternatives derived from poultry, animal by-products from meat processing, marine sources, and plants. However, the complexity of this problem is further compounded by the rapid advances in innovation and technology, which can lead to adulteration of ingredients derived from pigs. Other challenges include the sustainability of alternative materials, management of waste or by-products practice, halal awareness, certification, government policies, religious adherence of consumers, food suppliers, marketers, and purchasing of products. The importance of halal and non-halal problems, specifically in the context of pharmaceutical materials, is still rarely discussed, including alternatives derived from poultry, animal by-products, marine sources, and plants. Due to the increasing global population, there is a growing need to increase awareness and concern among Muslim consumers for halal products, including pharmaceuticals. Therefore, this research aimed to investigate the importance of halal and non-halal issues in pharmaceutical ingredients, the potential impact on the Muslim community, as well as opportunities and challenges in the search for alternative ingredients.

Validation of bioanalytical method for quantification of Vitamin K2 (MK-4) in human plasma by high-performance liquid chromatography-ultraviolet.

Vitamin K can reduce warfarin's anticoagulant action, causing a variance in response among individuals taking warfarin. Vitamin K comes in two forms, namely Vitamin K1 (phylloquinone) and K2 (menaquinones). Menaquinone-4 (MK-4) is a kind of Vitamin K2 found in meat and dairy products. Analysis of MK-4 levels in human plasma is very useful for patients who receive warfarin therapy. High-performance liquid chromatography (HPLC) can be used for warfarin's bioanalysis, and it must be validated. The purpose of this study was to validate the bioanalytical method for quantification of Vitamin K2 (MK-4) in human plasma according to the 2019 European Medicines Agency (EMA) guideline. Vitamin K2 (MK-4) was extracted using acetonitrile. HPLC with an ultraviolet detector at 245 nm, using a T3 column set at 30°C and an isocratic mobile phase containing methanol: phosphate buffer (95:5) at pH 3, a flow rate of 1 mL/min was used in this study. The warfarin concentration of 0.5-3 µg/mL was used. About 5.50%-17.42% and 6.18%-8.74%, respectively, were the average ranges of percentage coefficient of variation and percentage difference. There was no response at the analyte's retention time in the six blank plasmas and at the analyte's retention time in the blank after the injection of upper limit of quantification, indicates that the procedure was very selective and did not result in any carryover. This bioanalytical method fulfills the parameters of selectivity, accuracy, precision, and carryover based on the 2019 EMA guidelines.

Comparative Study of In Situ Gel Formulation Based on the Physico-Chemical Aspect: Systematic Review.

In recent years, in situ gel delivery systems have received a great deal of attention among pharmacists. The in situ gelation mechanism has several advantages over ointments, the most notable being the ability to provide regular and continuous drug delivery with no impact on visual clarity. Bioavailability, penetration, duration, and maximum medication efficacy are all improved by this mechanism. Our review systematically synthesizes and discusses comparisons between three types of in situ gelling system according to their phase change performance based on the physicochemical aspect from publications indexed in the Pubmed, ResearchGate, Scopus, Elsevier, and Google Scholar databases. An optimal temperature-sensitive in situ gelling solution must have a phase change temperature greater than ambient temperature (25 °C) to be able to be readily delivered to the eye; hence, it was fabricated at 35 °C, which is the precorneal temperature. In a pH-sensitive gelling system, a gel develops immediately when the bio-stimuli come into contact with it. An in situ gelling system with ionic strength-triggered medication can also perhaps be used in optical drug-delivery mechanisms. In studies about the release behavior of drugs from in situ gels, different models have been used such as zero-order kinetics, first-order kinetics, the Higuchi model, and the Korsmeyer-Peppas, Peppas-Sahlin and Weibull models. In conclusion, the optimum triggering approach for forming gels in situ is determined by a certain therapeutic delivery application combined with the physico-chemical qualities sought.

Evaluation of adverse drug reaction in patients warfarin therapy.

This research aimed to understand the adverse drug reaction (ADR) in heart disease outpatients who were administered warfarin at a hospital in Bandung city. The research was conducted using a cross-sectional design with an observational approach. Subsequently, data were collected from 74 patients who met the inclusion criteria. The causality assessment was made by the Naranjo Algorithm and the incidence of bleeding was classified based on the Bleedscore™. The result showed that the most common ADR were nausea, dizziness, stomach ache, ecchymosis, petechiae, bleeding in the mouth, melena, etc. Furthermore, the INR value was the most significant factor in the incidence of ADR. It was 6.445 using a value of P = 0.001 or a confidence interval of 95%. The most common side effect of warfarin in cardiac outpatients was superficial bleeding, followed by internal bleeding (melena). The INR value is the most significant factor in measuring the incidence of ADR.

Validation of warfarin enantiomer analysis method in plasma using high-performance liquid chromatography fluorescence detector.

Warfarin (WF) is an anticoagulant commonly used for thromboembolism-related diseases. This study aims to assess the pharmacokinetic profile of WF. The stereospecific interaction of S-and R-WF requires quantification of the enantiomer to determine the pharmacokinetic profile. The analysis method of the enantiomers in plasma is developed using an HPLC fluorescence detector with a Chiralcel OD-RH column (4.6 mm × 150 mm i.d., 5 m) and a Chiralcel OD-RH guard column (4.0 mm × 10 mm, 5 m). The separation is conducted using isocratic with acetonitrile mobile phase: Phosphate buffer, pH 2.00 (40:60 v/v), column temperature 40°C, flow rate 1 mL/min, injection volume 50 L. WF is measured at an excitation wavelength of 310 nm and emission of 350 nm. This method results in limit of detection (LOD) values of 18.6 ng/mL and limit of quantitation (LOQ) of 62.01 ng/mL for R-WF and LOD values of 18.61 ng/mL and LOQ of 62.04 ng/mL for S-WF. The results showed a linearity in concentration between 100 and 2500 ng/mL with r 2 = 0.9969 and r 2 = 0.9991 for R-and S-WF. The validation requirements of selectivity, accuracy, and precision for within and between run with a value of <15% for % relative standard deviation and % diff were achieved. This method can be used in the sample measurement of WF pharmacokinetic studies.

Mesenchymal Stem Cell Secretome for Dermatology Application: A Review.

Secretome, also known as conditioned medium, is a secreted molecule from mesenchymal stem cells (MSCs) that has a variety of biological activities that can be used in various therapies, especially on the skin applications. A lack of conventional therapies makes secretome as a promising alternative therapy. The presence of growth factors, cytokines, and extracellular vesicles including microvesicles and exosomes in secretome has been widely reported, which serves in improving the proliferation and migration of cells to help in skin regeneration. Therefore, we were able to optimize the use of this secretome in a well-needed special review related to its work in addressing various skin problems. So, in this article, we discussed the benefits and biological activity of secretome on the skin application. This review was compiled based on the approval of several sites, such as Scopus, PubMed, Science Direct, and Google Scholar with the terms "MSC secretome for skin," "secretome for skin," "secretome dermatology," "secretome conditioned medium for skin," "secretome conditioned medium for skin wound," "secretome conditioned medium for aging," "secretome conditioned medium for hair growth," and "secretome conditioned medium for psoriasis." A total of 215 articles were collected for selection, of which 90 articles were used. Based on the results, it was concluded that secretome has a variety of useful activities to regenerate and repair tissue damage that have not been used on the skin, such as for wound healing, photoprotection, promotion of hair growth, psoriasis treatment, and other application as antimicrobial.

Characteristic Comparison of an Intraoral Thin Film Containing Astaxanthin Nanoemulsion Using Sodium Alginate and Gelatin Polymers

Objectives: The present study was conducted to compare the characteristics of a thin film containing an astaxanthin-loaded nanoemulsion (TFANE) using two kinds of natural polymers, namely sodium alginate and gelatin. Materials and Methods: An astaxanthin nanoemulsion was prepared by using the self-nanoemulsifying method, followed by incorporation into a polymer matrix system by the solvent casting method to form TFANE. A characteristic comparison between the sodium alginate and gelatin matrix systems was carried out by comparing the physical and mechanical film properties. At the end of the study, in vitro dissolution tests were also assessed. Results: An intraoral film with good physical and mechanical properties containing an astaxanthin-loaded nanoemulsion was developed successfully using a natural polymer matrix system. The film, made from a gelatin matrix system containing an astaxanthin nanoemulsion, was more flexible and harder than films made from a sodium alginate matrix system, where all of the films have ideal characteristics for intraoral delivery. The dissolution test results showed that, with both sodium alginate and gelatin, more than 90% of the drug was released at 15 minutes. Conclusion: Gelatin as a natural polymer appears to be promising for the preparation of an intraoral thin film delivery system.

The influence of Javanese turmeric (Curcuma xanthorrhiza) on the pharmacokinetics of warfarin in rats with single and multiple-dose studies.

CONTEXT: Co-administration between warfarin (WF) and Curcuma xanthorrhiza Roxb. (Zingiberaceae) (CX) is found in Indonesian patients and need to be evaluated. OBJECTIVE: This study assesses the effect of concomitant administration of CX extract on the pharmacokinetics of WF in rats. MATERIALS AND METHODS: Wistar rats were divided into 4 groups (n = 6) and administered with 2% Pulvis Gummi Arabicum (PGA, control), fluconazole (FZ, 6 mg/kg), CX-1 (6 mg/kg) or CX-2 (18 mg/kg BW) for 7 days. For the single-dose study, at the 8th day, WF (1 mg/kg) was administered to all groups and blood samples were taken from 0.25 to 72 h. For the multiple-dose study, daily dose of WF was administered to all groups of rats and at the 7th to 9th day, the rats were treated with PGA, CX-1, CX-2 and FZ. Blood samples were withdrawn daily at 4 h after administration of WF from the 1st to 11th day. RESULTS: The area under the curve (AUC) of R- and S-WF in the CX-2 group was a significantly higher value compared to the control (77.54 vs. 35.27 mg.h/L for R-WF and 316.26 vs. 40.16 mg.h/L for S-WF; p < 0.05; Kruskal-Wallis method). The CX-2 administration also caused the increasing in the concentration level of R-WF (16%) and S-WF (27%) from the 7th to 9th day of administration. DISCUSSION AND CONCLUSIONS: The CX administration in a higher dose caused alteration on WF pharmacokinetics suggesting the need for clinical evaluation of the interaction between CX and WF.

Isoflavones in Soybean as a Daily Nutrient: The Mechanisms of Action and How They Alter the Pharmacokinetics of Drugs.

Soybeans [Glycine max (L.)] are a good source of isoflavones. The main isoflavone components of soybean are daidzein, genistein, and glycitein. World soybean production is very high. Because of its pharmacological activity, soy isoflavone intake over a long period of time may result in interactions with the drugs. This review summarizes soy isoflavone-drug interactions based on the pharmacokinetic parameters. Soy isoflavones have pharmacokinetic interactions with celecoxib, theophylline, paclitaxel, midazolam, imatinib, carbamazepine, valproic acid, repaglinide, omeprazole and danofloxacin. This is due to the changes in the area under the curve, maximum serum concentration, time that a drug is present at the maximum concentration in serum, clearance and half-life of the drugs when delivered together with soy isoflavones. The mechanisms of pharmacokinetic interactions occurs through the inhibition/induction of drug metabolizing cytochrome P450 (CYP450) enzymes such as CYP3A4, CYP2A1, and CYP2C9 or through the inhibition of drug transporters such as P-glycoprotein and breast cancer resistance protein. Thus, the consumption of soybean, soy isoflavones or soy products with drugs needs to be reconsidered.

Photo-Based Nanomedicines Using Polymeric Systems in the Field of Cancer Imaging and Therapy.

The use of photo-based nanomedicine in imaging and therapy has grown rapidly. The property of light in converting its energy into different forms has been exploited in the fields of optical imaging (OI) and phototherapy (PT) for diagnostic and therapeutic applications. The development of nanotechnology offers numerous advantages to overcome the challenges of OI and PT. Accordingly, in this review, we shed light on common photosensitive agents (PSAs) used in OI and PT; these include fluorescent and bioluminescent PSAs for OI or PT agents for photodynamic therapy (PDT) and photothermal therapy (PTT). We also describe photo-based nanotechnology systems that can be used in photo-based diagnostics and therapies by using various polymeric systems.

In situ ophthalmic gel forming systems of poloxamer 407 and hydroxypropyl methyl cellulose mixtures for sustained ocular delivery of chloramphenicole: optimization study by factorial design.

BACKGROUND: Conventional drug delivery systems have some major drawbacks such as low bioavailability, short residence time and rapid precorneal drainage. An in situ gel drug delivery system provides several benefits, such as prolonged pharmacological duration of action, simpler production techniques, and low cost of manufacturing. This research aims to get the optimum formula of chloramphenicol in situ gel based on the physical evaluation. METHODS: The effects of independent variables (poloxamer 407 and hydroxypropyl methyl cellulose (HPMC) concentration) on various dependent variables (gelling capacity, pH and viscosity) were investigated by using 32 factorial design and organoleptic evaluation was done with descriptive analysis. RESULTS: The optimized formula of chloramphenicol in situ gel yielded 9 variations of poloxamer 407 and HPMC bases composition in % w/v as follows, F1 (5; 0.45), F2 (7.5; 0.45), F3 (10; 0.45), F4 (5; 0.725), F5 (7.5; 0.725), F6 (10; 0.725), F7 (5; 1), F8 (7.5; 1), F9 (10; 1). The results indicated that the organoleptic, pH, and gelling capacity parameters matched all formulas (F1-F9), however, the viscosity parameter only matched F3, F6, F8, and F9. Based on factorial design, F6 had the best formula with desirability value of 0.54, but the design recommended that formula with the composition bases of poloxamer 407 and HPMC at the ratio of 8.16 % w/v and 0.77 % w/v, respectively, was the optimum formula with a desirability value of 0.69. CONCLUSION: All formulas have met the Indonesian pharmacopoeia requirements based on the physical evaluation, especially formula 6 (F6), which was supported by the result of factorial design analysis.

Epidermal growth factor in sacran hydrogel film accelerates fibroblast migration.

Epidermal growth factor (EGF) accelerates epidermal regeneration, and it is widely studied as a wound-healing agent. However, the special carrier for the topical administration of EGF is urgently needed to deliver EGF on the wound site. In a preceding study, sacran hydrogel film (Sac-HF) showed a possible use as a dressing material for wound healing, as well as a good capability as a drug carrier. In the current study, we prepared Sac-HF containing EGF (Sac/EGF-HF) and then characterized their physicochemical properties, including thickness, swelling ratio, degradability, tensile strength, and morphology. In addition, we have also conducted thermal and crystallography studies using differential scanning calorimetry (DSC) and X-ray diffraction, respectively. Furthermore, we investigated the in vitro influence of Sac/EGF-HF on cell migration using a fibroblast cell line. Morphology study confirmed that the casting method used for the film preparation resulted in a homogeneous film of Sac/EGF-HF. Furthermore, EGF significantly increased the thickness, tensile strength, and degradability of Sac/EGF-HF compared to Sac-HF. Sac/EGF-HF had a lower swelling ability compared to Sac-HF; this result corroborated the tensile strength result. Interestingly, X-ray diffraction and DSC results showed that Sac/EGF-HF had an amorphous shape. The in vitro studies revealed that Sac/EGF-HF induced the fibroblast migration activity. These results conclude that Sac/EGF-HF has the potential properties of HF for biomedical applications.

Apoptosis-mediated antiproliferation of A549 lung cancer cells mediated by Eugenia aquea leaf compound 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone and its molecular interaction with caspase receptor in molecular docking simulation.

In a previous study, 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (ChalcEA) isolated from the leaves of Eugenia aquea was reported to inhibit proliferation of the breast adenocarcinoma MCF7 cell line and to promote apoptosis via activation of poly(adenosine diphosphate-ribose) polymerase protein. The present study aimed to evaluate the inhibitory effect of ChalcEA on the proliferation of A549 lung cancer cells using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, and to examine the ability of ChalcEA to induce apoptosis through activation of the caspase cascade signaling pathway in a western blotting assay. The results revealed that ChalcEA inhibited proliferation of the A549 lung cancer cell lines in a time- and dose-dependent manner with IC50 values of 25.36 and 19.60 µM for 24 and 48 h treatments, respectively. Western blot analysis indicated that ChalcEA exerted its anti-proliferative effects by promoting apoptosis via the activation of caspase-9 and caspase-3. Based on in silico results, ChalcEA with the binding energy of -6.53 kcal/mol could compete better than 4-methyl benzenesulfonamide (-6.43 kcal/mol) as an inhibitor of caspase-3 (PDB: 2XYG). ChalcEA has potential since it has three hydrophobic features. These results provided a basis for further study of ChalcEA as an active compound for anticancer therapeutics.

Antidiabetic activity of thin film containing astaxanthin-loaded nanoemulsion using carboxymethylcellulose sodium polymer on alloxan-induced diabetic rabbit.

The present study was conducted to evaluate the potency of thin film containing astaxanthin-loaded nanoemulsion (FDT-As-NE) in lowering blood glucose levels on alloxan-induced diabetic rabbit (ADR). Astaxanthin nanoemulsion (As-NE) was prepared using self-nanoemulsifying method, followed by incorporated into the carboxymethylcellulose sodium matrix polymer using a solvent casting method to form a thin film. The evaluation of FDT-As-NE was performed by chemical, physical, and mechanical characterizations. The administration of thin film was done by an intraoral route. New Zealand albino rabbits were induced with alloxan to get experimental diabetic animals. The antidiabetic activity was carried out in three groups of treatment. Group I was ADR treated by FDT-As-NE, Group II was ADR treated by pure astaxanthin, while Group III was normal control. The measurement of fasting means blood glucose levels was carried out in 0 days (before treatment) and after 14 days of treatment. The histopathological analysis of the pancreas was also examined. Data were statistically evaluated using Kruskal-Wallis statistical test. P < 0.05 was considered statistically significant. FDT-As-NE had good physical and mechanical characteristics that suitable for intraoral administration. Group I reduced elevated blood glucose levels compared to Group II (P < 0.01). Histopathological examination of pancreatic tissue for a Group I showed the normal condition of pancreatic ß-cell, suggesting the absence of any pathological lesions. These results suggest that thin film containing astaxanthin-loaded nanoemulsion administered by an intraoral route potentially useful for reducing glucose levels.

Sacran Hydrogel Film Containing Keratinocyte Growth Factor Accelerates Wound Healing by Stimulating Fibroblast Migration and Re-epithelization.

Regenerative therapy with keratinocyte growth factor (KGF) is a novel therapeutic approach for treatment of chronic wounds. However, KGF cannot be used directly to the wound site due to its physicochemical instability. In previous study, sacran, a natural megamolecular polysaccharide, showed potential properties as a biomaterial for hydrogel film in wound healing. In this study, we fabricated sacran hydrogel film containing KGF (Sac/KGF-HF) and evaluated the effects of Sac/KGF-HF on fibroblasts migration and re-epithelialization process. We successfully prepared a homogenous and -amorphous Sac/KGF-HF by a casting method. In addition, Sac/KGF-HF had a high swelling ratio and flexibility. Sac/KGF-HF promoted a migration process of NIH3T3 cells and improved wound healing ability in mice with a percentage of wound closure reaching 90.4% at 9 d. Interestingly, the addition of KGF in Sac-HF considerably increased the number of epithelial cells compared to control, which is important in the re-epithelialization process. It could be concluded that KGF in Sac-HF has the potential for promoting Sac-HF abilities in wound healing process.

Anticalculi Activity of Apigenin and Celery (Apium graveolens L.) Extract in Rats Induced by Ethylene Glycol-Ammonium Chloride.

OBJECTIVE: Kidney stones (nephrolithiasis) is one of the kidney diseases in the form of stones that contain crystal and organic matrix components. It is one of the most common diseases of the urinary tract. Calcium stone is the most important type of stone (80%) found in the case of kidney stones. Celery (Apium graveolens L.) is a plant rich in flavonoids, which can break down calcium crystals. Apigenin is considered to be one of the main flavonoids because of its presence and abundance in celery. This research aimed to compare the anticalculi effect of apigenin with that of celery extract. MATERIALS AND METHODS: Wistar albino rats were given ethylene glycol 0.75% (vol/vol) and ammonium chloride 2% (wt/vol) orally for 7 days in all groups to induce hyperoxaluria and Rats treated by Apigenin at doses 1.2, 2.4, and 4.8 mg/kg of rat body weight and celery extract at doses of 200, 400, and 600 mg/kg of rat body weight as anticalculi. Measurements of calcium levels in the kidneys and urine of rats was obtained using atomic absorption spectroscopy. Data obtained were statistically analyzed with the IBM SPSS by ANOVA Method version 21.0 probability value < 0.05 was considered significant. RESULT: The results showed that both apigenin and celery extracts caused kidney stone to decay. From the data Apigenin and celery showed that calcium level in urine there were significant differences (p value < 0.05) in treated group from negative control group but calcium level in kidney there were not significant differences (p value > 0.05). CONCLUSION: Celery extract has better ability to break down kidney stones than apigenin.

Lactobacillus plantarum IS-10506 probiotic administration increases amlodipine absorption in a rabbit model.

OBJECTIVE: Probiotics are beneficial in human health. In this study, we investigated the effect of probiotics on absorption of amlodipine, a dihydropyridine calcium antagonist used in the treatment of angina and hypertension, in a rabbit model. METHODS: Lactobacillus plantarum IS-10506 probiotic was administered for 14 days to male New Zealand rabbits. Blood samples were collected before and after probiotic supplementation. Amlodipine (10 mg) was then administered to all groups. Blood samples from a marginal vein were withdrawn at 5, 15, 30, 60, and 120 minutes to determine amlodipine concentrations in rabbit plasma. RESULTS: Amlodipine concentrations in the L. plantarum IS-10506 group were 4.95 ± 1.22, 8.71 ± 0.69, and 12.48 ± 2.53 ng/ml, and those in the control group were 1.69 ± 0.31, 3.89 ± 1.23, and 7.17 ± 1.85 ng/ml at 30, 60, and 120 minutes, respectively after administration of amlodipine. Amlodipine concentrations in the L. plantarum IS-10506 group were significantly higher than those in the control group at 30, 60, and 120 minutes after amlodipine administration. CONCLUSION: Our results suggested that supplementation of L. plantarum IS-10506 significantly increases amlodipine plasma concentrations in rabbits.

Development and validation of simple simultaneous analysis for amlodipine and glibenclamide by nonderivatization high-performance liquid chromatography-fluorescence.

Studies have shown that about 65% of diabetics have hypertension. Treatment for diabetic patients with hypertension is usually given a combination of drugs such as amlodipine (AML) and glibenclamide (GLI). The aim of this study was to develop and validate the simple simultaneous analysis method for separation of AML and GLI using high-performance liquid chromatography (HPLC) with fluorescence detector without derivatization. The arrangement of isocratic and gradient methods, mobile phase compositions, and flow rates to develop and validate the simple simultaneous analysis method for separation of AML and GLI by nonderivatization HPLC fluorescence was done. Optimum condition was obtained using an RP 18 (125 mm × 4 mm, i.d., 5 µm) and guard column RP 18 (4 mm × 4 mm, i.d., 5 µm) with mobile phase composition containing acetonitrile and phosphate buffer pH 3.0 using a 20:80 gradient condition at flow rate 1.0 ml/min measured at 361 nm for λ excitation and 442 nm for λ emission for AML and 235 nm for λ excitation and 354 nm for λ emission for GLI. The analysis of AML and GLI demonstrated a valid result with r 2 value 0.999, recoveries were 100.04% and 99.14% relative standard deviations were 0.508% and 0,797%, respectively, detection limits were 0.055 and 0.104 µg/ml, and quantification limits were 0.166 and 0.316 µg/ml, respectively. An accurate method of separation for AML and GLI using HPLC with fluorescence detector without derivatization has been validated.