THE EFFECT OF ANTICOAGULANT TYPES ON THE IN VITRO ANALYSIS OF CLOPIDOGREL IN HUMAN PLASMA USING LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

Objective: The aim of this study was to optimize and validate a plasma clopidogrel analysis method using liquid chromatography tandem-massspectrometry.Methods: Plasma samples were analyzed using a BEH C18 column (1.7 μm; 100 mm×2.1 mm), the mobile phase was 0.1% formic acid in acetonitrile(30:70, v/v). The flow rate was 0.2 mL/min, with a column temperature set to 35°C, an injection volume of 5 μL, an analysis time of 4 min, andirbesartan as the internal standard. Aliquots were obtained by liquid-liquid extraction using ammonium acetate and diethyl ether. The stability andpeak area ratio of the respective plasma area responses were evaluated using ANOVA.Results: No significant differences (p>0.05) were observed between anticoagulants regarding analyte stability. However, the peak area ratioshowed significant differences (p<0.05) between the anticoagulants. The accuracy and precision of the analysis with citrate, heparin, andethylenediaminetetraacetic acid (EDTA) plasma met the quality requirements, and a linear calibration curve was created with concentrations rangingfrom 0.02 to 5.0 ng/mL.Conclusion: The results showed that improved analysis of clopidogrel was achieved using citrate or heparin plasma compared with EDTA plasma

NOVEL TRANSETHOSOME CONTAINING GREEN TEA (CAMELLIA SINENSIS L. KUNTZE) LEAF EXTRACT FOR ENHANCED SKIN DELIVERY OF EPIGALLOCATECHIN GALLATE: FORMULATION AND IN VITRO PENETRATION TEST

Objective: This study aimed to formulate a transethosome cream (TEC) to increase skin penetration of epigallocatechin gallate (EGCG) in green tealeaf extract and evaluate their physicochemical characteristics and skin penetration capacity.Methods: Transethosomes were prepared through thin-layer hydration method in three formulations (F1−F3) with different Span 80 and ethanol concentrations.Transethosome morphology was characterized using transmission electron microscopy, particle size, polydispersity (PDI), and zeta potential using a particlesize analyzer and entrapment efficiency (EE). Penetration was tested using an in vitro Franz diffusion cell with female Sprague-Dawley rat skin as themembrane.Results: Transethosome F2 containing green tea extract equivalent to 3% EGCG, 4% Lipoid P30, 0.75% Span 80, and 30% ethanol had the bestcharacteristic including spherical shape, smallest particle size (35.35 nm), 0.319 PDI, zeta potential of −29.97±3.05 mV, and highest EE of45.26%±8.15%. TEC permitted greater flux than non-TEC (first phase: 60.56±4.52 vs. 25.69±0.83 μg•cm−2•h−1 and second phase: 23.13±1.38 vs.7.36±1.59 μg•cm−2•h−1).Conclusion: Transethosome can increase the skin penetration of green tea leaf extract

FORMULATION OF A CREAM CONTAINING ETHOSOMAL GREEN TEA (CAMELLIA SINENSIS L. KUNTZE) LEAF EXTRACTS FOR IMPROVED DERMAL PENETRATION

Objective: This study aimed to formulate the epigallocatechin gallate (EGCG) from green tea into ethosomes and measure the resulting increases inskin penetration using a rat model.Methods: Ethosomes were formulated using ethanol concentrations of 25% (F1), 30% (F2), and 35% (F3), and those with favorable characteristicswere then incorporated into a cream for the determination of penetration into Franz diffusion cells.Results: We showed that the formulation F3 had the best spherical morphology, a Z-average value of 73.01 nm, a polydispersity index of 0.26, azeta potential of −47.77±3.93 mV, and the highest percentage of drug entrapment (49.46%±0.62%) compared with the other formulas. The totalcumulative EGCG penetration from the resulting ethosomal cream was 905.75±49.47 μg/cm2, whereas that from the cream containing green tea leafextract was only 413.92±52.83 μg/cm2.Conclusion: These data indicate a higher penetration of EGCG from ethosomal green tea cream than from cream containing non-ethosomal green teaextract

TRANSFERSOMAL GEL CONTAINING GREEN TEA (CAMELLIA SINENSIS L. KUNTZE) LEAVES EXTRACT: INCREASING IN VITRO PENETRATION

Objective: The aim of this study was to increase penetration of EGCG from green tea leaves extract (Camellia sinensis L. Kuntz) through the skin by formulating them into a transfersomal gel.Methods: Transfersomes were prepared by thin-layer hydration method, with different concentration of the extract that equivalent to 1% (F1), 1.5% (F2), and 2% (F3) EGCG. Transfersomes formula with good characteristics would be incorporated into a gel formulation. A gel without transfersomes prepared as a control of comparison. Both of gels were evaluated their physicochemical properties. In vitro penetration test using Franz diffusion cell with the skin of female Sprague-Dawley rats was also performed.Results: The results showed that F1 had the best physicochemical properties. F1 had a spherical shape, Dmean volume at 107.82±0.44 nm, polidispersity index at 0.07±0.01, zeta potential at -40.3±0.10 mV, and entrapment efficiency at 63.16±0.65%. Cumulative amount of EGCG penetrated from transfersomal and non-transfersomal gel were were 1302.63±20.67 μg/cm2 and 414.86±4.40 μg/cm2, resepctively (P<0.05). Flux penetration of transfersomal and non-transfersomal gel were was 57.594±0.91 μg/cm2.h and 36.144±1.22 μg/cm2.h, respectively.Conclusion: It can be concluded that transfersomal gel could increase the in vitro penetration of EGCG from green tea leaves extract compared to non-transfersomal one.Â

FACTORS AFFECTING LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY ANALYSIS OF 6-MERCAPTOPURINE AND 6-THIOGUANINE IN DRIED BLOOD SPOTS

Objective: This study aimed to determine the effects of the method of internal standard addition, spotting volume, paper type, and sample storagetemperature on 6-mercaptopurine, and 6-thioguanine on liquid chromatography tandem-mass spectrometry (LC-MS/MS) bioanalysis methods usingdried blood spot (DBS).Methods: Blood samples were spotted on CAMAG DBS paper and a Perkin Elmer 226 sample collection device (paper) and extracted into methanolcontaining 5-fluorouracil as an internal standard. The separation was performed on a water acquity ultra high-performance LC BEH Amide 1.7 μm(2.1 mm×100 mm) column with a mobile phase of 0.2% formic acid in water - 0.1% formic acid in acetonitrile methanol with gradient elution at aflow rate of 0.2 mL/min.Results: The step at which the internal standard was added (blood, spot on DBS card, or extraction solution) affected the chromatogram. Differencesin paper types and blood volumes significantly affected (p<0.05) the percent coefficient of variation, whereas differences in blood hematocritsignificantly affected the peak area ratio.Conclusion: The method of internal standard addition affected the chromatograms in this study. The best chromatogram was observed whenthe Internal Standard was added to the extracting solution. The card type also affected the analysis, so it is recommended to use the same card duringsample analysis

Untargeted LC-QTOF-MS/MS Based Metabolomic Profile Approach of Bacterial Ferment Lysates and Skin Commensal Bacterial Cocktail Ferment Lysates

Microbial therapy has been increasingly developed in the medical and health fields and has triggered advances in the process of formulating skincare products. The skin microbiota becomes the target in the development of active ingredients to produce an optimal effect in the balance of its composition which leads to its usefulness in maintaining skin health and providing protection. Postbiotic bacteria can maintain homeostasis of the skin microbiome so that it has the potential to be used as an active ingredient in Active Pharmaceutical Ingredient (API) in skincare products and have broad benefits due to its various active substances. The aim of this study was to examine the metabolites profile contained in the bacterial fermented lysate fraction, which is also served as a marker in identifying the metabolite variations of the lysate fractions and their API dosage forms. Ferment lysate API preparations were prepared in the form of freeze dried and spray dried. The metabolite profile analysis was carried out using the untargeted LC-QTOF-MS/MS metabolomic approach and multivariate analysis. Result revealed 30 differential features of the putative metabolites, and by performing metabolites annotationfor their bioactivities through intensive literature research, such as antimicrobial, antioxidant, and anti-inflammatory, we elucidated these compounds are discovered in dry form of lysates

Pemanfaatan Nanoteknologi dalam Sistem Penghantaran Obat Baru untuk Produk Bahan Alam

Sejak dahulu banyak ekstrak dari bahan alam yang secara empiris dimanfaatkan untuk pengobatan. Ekstrak-ekstrak tersebut digunakan karena mengandung senyawa bioaktif yang dapat memberikan efek farmakologis. Isolat dari ekstrak tersebut diuji baik secara in vitro maupun in vivo untuk mengetahui efek dan bioavailabilitas dalam tubuh secara ilmiah. Namun demikian diperkirakan lebih dari 40% senyawa bahan alam memiliki kelarutan yang rendah di dalam air atau bahkan memberikan toksisitas yang tinggi. Kelarutan yang rendah di dalam air serta kurangnya kemampuan permeabilitas menembus barrier absorpsi dapat mempengaruhi bioavailabilitas senyawa bahan alam di dalam tubuh. Tidak hanya itu, bioavailabilitas suatu senyawa juga sangat dipengaruhi oleh stabilitas terhadap pH lambung dan kolon, metabolisme oleh mikroflora normal dalam saluran pencernaan, absorpsi melalui dinding usus, mekanisme aktif pompa efflux dan metabolisme lintas pertama. Solusi dari permasalahan tersebut adalah dengan mengembangkan sistem penghantaran obat yang dikenal dengan sistem penghataran obat baru (novel drug delivery system). Sistem penghantaran obat baru merupakan suatu sistem penghantaran obat yang lebih modern dengan cara mengontrol pelepasan obat sehingga aktivitas farmakologis menjadi lebih baik. Pembuatan sediaan berbasis teknologi baru ini dapat menjadi alternatif dalam pembuatan produk herbal dan diharapkan bioavailabilitas produk herbal dalam tubuh menjadi lebih baik sehingga dapat memberikan efek terapi yang lebih baikNatural products have been known to have a major role in maintaining health. Some studies reported that they demonstrated various pharmacological activities and provided a lead compound or drug candidate. Isolated compounds from the extracts have been studied both in vitro and in vivo to determine their effects and bioavailability in the body. However, more than 40% natural products have low solubility in water, or even give a high toxicity. Low solubility in water and the lack of ability to penetrate the absorption barrier may affect the bioavailability in the body. Furthermore, the bioavailability of a compound is also influenced by the stability of drugs in the pH of the stomach and colon, metabolism by normal microflora in the digestive tract, absorption through the intestinal wall, efflux pumps mechanism and first-pass metabolism. Solution to overcome these problems is by developing a drug delivery system known as a novel drug delivery system (NDDS). The NDDS is a more modern system to control the release of drugs so that it will give better pharmacological activity. Making dosage forms based on this novel technology can be an alternative for manufacturing herbal products. It can increase their bioavailability in the body so that it can provide a better therapeutic effect

MICROENCAPSULATION OF GRAPE SEED OIL (VITIS VINIFERA L.) WITH GUM ARABIC AS A COATING POLYMER BY CROSSLINKING EMULSIFICATION METHOD

Objective: Grape seed oil (GSO) from Vitis vinifera L. is a liquid vegetable oil which has been used mainly for its linoleic acid content. However, there are many efforts to convert the liquid form of the oil into a solid form due to the instability under storage condition. The aim of this study was to convert GSO into the solid microcapsules by emulsion crosslinking method with gum arabic as a coating polymer.Methods: The GSO was formulated with gum arabic in the ratios of 1:2, 1:3, 1:4, and 1:5. Gum arabic solution was emulsified with GSO using Span 80 and glutaraldehyde. The emulsion was dropped into a beaker glass of isopropyl alcohol to form microcapsules. The microcapsules were dried at 70 °C. Then, they were characterized in terms of morphology, particle size, swelling index, water content, and entrapment efficiency.Results: The produced microcapsules of GSO showed white yellowish color and spherical shape. The particle size of F1, F2, F3 and F4 microcapsules were 69 μm, 82 μm, 125 μm, and 131 μm, respectively. The water content of the F1–F4 ranged from 4.37±0.34 to 5.70±0.92% and swelling indexes were ranged from 5.54±0.01 to 5.94±0.04. The value of entrapment efficiency of F1, F2, F3, and F4 were 17.33±0.603, 20.73±0.678, 34.22±1.195, and 67.15±2.019%, respectively.Conclusion: The results of this investigation showed that GSO could be converted into the solid spherical microcapsules by emulsion crosslinking method using gum arabic. Taken together, this study has provided the most promising formulation of GSO microcapsules for further production in pharmaceutical industry

METHOD DEVELOPMENT AND VALIDATION OF CEFOPERAZONE AND SULBACTAM IN DRIED BLOOD SPOTS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY PHOTODIODE ARRAY DETECTOR

Objective: The primary purpose of this research was to develop a simple, precise, fast, and accurate method for measuring cefoperazone and sulbactam simultaneously in dried blood spots (DBS) using HPLC PDA. Methods: A simplified analytical method for quantifying cefoperazone and sulbactam in DBS samples using a High-Performance Liquid Chromatography photodiode array detector with isocratic elution was developed and validated. The best chromatographic conditions were obtained by using a reversed-phase column (250 x 4.6 mm; 5 mm); phosphate buffer 10 mM pH 3.2–acetonitrile (83:17, v/v) as a mobile phase; a flow rate of 1.0 ml/min; a column temperature of 35 °C; a photodiode array detector at 210 nm, and cefuroxime as internal standard. Samples were prepared by liquid-liquid extraction with 100 mL hydrochloric acid 0.5 mol/L and 1000 mL ethyl acetate, evaporated with nitrogen and reconstituted with 100 mL phosphate buffer–acetonitrile (4:1). Results: The total chromatography run time was 15 min, and the elution times for sulbactam, cefoperazone, and IS (cefuroxime) were 3.46, 10.221, and 6.987 min, respectively. A linear response function was established at 0.5-30 mg/ml with (r) 0.995 for sulbactam and 2.5-250 mg/ml with (r) 0.999 for cefoperazone in dried blood spots. The lower limit quantification (LLOQ) concentration of sulbactam 1 mg/ml and cefoperazone were 5 mg/ml. Conclusion: This method has successfully fulfilled the validation requirement referring to the 2011 EMA and 2018 FDA guidelines