Ultrafast liquid chromatographic analysis of erdosteine in human plasma based on fluorimetric detection and precolumn derivatization with 4-bromomethyl-7-methoxycoumarin: Application to pharmacokinetic studies.

In this study, a new analytical method for erdosteine (ERD) in plasma based on high-performance liquid chromatography and a fluorimetric detector, is presented. Precolumn derivatization of ERD with 4-bromomethyl-7-methoxy coumarin (BrMmC) and dibenzo-18-crown-6-ether as a reaction catalyst led to the production of a fluorescent compound. ERD was monitored by fluorescence with an excitation wavelength λext. = 325 nm and emission wavelength λem. = 390 nm. Optimum reaction conditions were carefully studied and optimized. A chromatographic procedure was performed using a C18 column of 150 × 4.6 mm and 3 µm particle size and a mobile phase consisting of methanol:acetonitrile:water (30:30:40, v/v/v) under a flow rate of 0.5 ml min-1 . A calibration plot was established covering analyte concentration range 0.2-3.0 µg ml-1 ; the detection limit was 0.015 µg ml-1 and quantification limit was 0.05 µg ml-1 . Mean recovery was 87.33% and relative standard deviation was calculated to be less than 4.4%. The developed method was successfully used to determine pharmacokinetic preparations of ERD subsequent to administration of a 900 mg dose capsule to a healthy 40-year-old woman volunteer.

Investigation of cell death mechanism and activity of esculetin-loaded PLGA nanoparticles on insulinoma cells in vitro.

AIM: The purpose of this study was to prepare targeted cancer therapy formulation against insulinoma INS-1 cells and to study its effect on cell death with related mechanisms in vitro. METHODS: Polylactide-co-glycolide (PLGA) nano-micelles were used for preparation of esculetin nano-formulation (nano-esculetin). The cells were treated with nano-esculetin and free esculetin. Apoptotic and necrotic cell death percentages, cell proliferation, ATP and GTP reductions and insulin levels were investigated on insulinoma INS-1 cells for both free and nano-esculetin formulations. RESULTS: About 50 mg of PLGA was able to carry 20 mg esculetin in 20 ml of formulation. The obtained optimized formulation was 150 nm, with 92% encapsulation efficiency and a slow-release behaviour was observed during release studies. Nano-esculetin bearing 25, 50 and 100 µg esculetin and free esculetin in equivalent doses successfully decreased cell viability. The prevailing cell death mechanism was necrosis. Along with cell proliferation, intracellular insulin and the ratio of ATP and GTP were decreased even with 12.5, 25 and 50 µg esculetin bearing nano-formulation and its equivalent free esculetin. CONCLUSIONS: The results revealed that esculetin is able to show its anti-tumor afficacy after loading to PLGA nano-micelles and nano-encapsulation intensifies its cytotoxic activity in vitro. Current study shows that esculetin and its nano formulations are promising agents in treatment of insulinoma.

Evaluation of residual monomer release and toxicity of self-adhesive resin cements.

The aim of this study was to evaluate the amount of leached residual monomers from self-adhesive resin cements and evaluate their toxicity in-vitro. A total of 60 disk-shaped specimens (5 mm in diameter and 0.5 mm in thickness) were prepared from each cement (RelyX U200, SpeedCEM, G-Cem) (n=20). Specimens were immersed in artificial saliva and the amount of released monomers [urethane dimethacrylate (UDMA) and triethyleneglycol dimethacrylate (TEGDMA)] was identified. Then, the cytotoxicity and genotoxicity effect on cells were evaluated using the defined amounts of released monomers from cements. The highest monomer release was detected in G-Cem (p<0.05). The highest cytotoxicity value was identified from SpeedCEM (p<0.01) and the highest genotoxicity values were calculated from RelyX U200 (p<0.05). Released UDMA and TEGDMA from self-adhesive resin cements induced cytotoxicity and genotoxicity effect on cells.

Development and validation of a HPLC method for the determination of benzo(a)pyrene in human breast milk.

A simple analytical procedure was developed for the quantitation of benzo(a)pyrene in human breast milk using solid phase extraction (SPE) combined with high performance liquid chromatography. Before the chromatographic process, SPE, including C18 functional groups in silicagel cartridges, was conducted for sample preparation. A C18 column (100×4.6 mm id, 3 µm particle size) was used with acetonitrile:water (80:20) as the mobile phase at a flow rate 1mL/min at 30°C. Fluorimetric detection was performed for excitation and emission at 290 and 406 nm, respectively. It was observed that the calibration curve was linear over the range of 0.5-80 ng/mL. The limit of detection and limit of quantitation were found to be 0.5 and 1.07 ng/mL, respectively. Intraday and interday relative standard deviation values were less than 5.15%. Moreover, the newly developed method provides a fast, simple, cost effective, and sensitive assay to detect an important carcinogen substance, benzo(a)pyrene, in human breast milk.

Current HPLC Methods for Assay of Nano Drug Delivery Systems.

In nano drug formulations the mechanism of release is a critical process to recognize controlled and targeted drug delivery systems. In order to gain high bioavailability and specificity from the drug to reach its therapeutic goal, the active substance must be loaded into the nanoparticles efficiently. Therefore, the amount in biological fluids or tissues and the remaining amount in nano carriers are very important parameters to understand the potential of the nano drug delivery systems. For this aim, suitable and validated quantitation methods are required to determine released drug concentrations from nano pharmaceutical formulations. HPLC (High Performance Liquid Chromatography) is one of the most common techniques used for determination of released drug content out of nano drug formulations, in different physical conditions, over different periods of time. Since there are many types of HPLC methods depending on detector and column types, it is a challenge for the researchers to choose a suitable method that is simple, fast and validated HPLC techniques for their nano drug delivery systems. This review's goal is to compare HPLC methods that are currently used in different nano drug delivery systems in order to provide detailed and useful information for researchers.

Spectrofluorimetric determination of tobramycin in human serum and pharmaceutical preparations by derivatization with fluorescamine.

A simple, sensitive and selective spectrofluorimetric method has been developed for the determination of tobramycin (TOB) in human serum and pharmaceutical preparations. The method is based on the reaction between the primary amino group of TOB and fluorescamine in borate buffer, pH 8.5, to give a highly fluorescent derivative which is measured at 469 nm after excitation at 388 nm. The fluorescence intensity was directly proportional to the concentration over the range 300-1500 ng/mL, with a limit of detection of 65 ng/mL and limit of quantitation of 215 ng/mL. All variables were investigated to optimize the reaction conditions. The method was validated according to International Conference on Harmonization guidelines in terms of specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. Good recoveries were obtained ranging from 97.4 to 100.64%, indicating that no interference was observed from concomitants usually present in pharmaceutical dosage forms. The method was successfully, applied for the analysis of the drug substance in its pharmaceutical preparations and spiked serum samples.

Development of an HPLC-UV Method for the Analysis of Drugs Used for Combined Hypertension Therapy in Pharmaceutical Preparations and Human Plasma.

A simple, rapid, and selective HPLC-UV method was developed for the determination of antihypertensive drug substances: amlodipine besilat (AML), olmesartan medoxomil (OLM), valsartan (VAL), and hydrochlorothiazide (HCT) in pharmaceuticals and plasma. These substances are mostly used as combinations. The combinations are found in various forms, especially in current pharmaceuticals as threesome components: OLM, AML, and HCT (combination I) and AML, VAL, and HCT (combination II). The separation was achieved by using an RP-CN column, and acetonitrile-methanol-10 mmol orthophosphoric acid pH 2.5 (7 : 13 : 80, v/v/v) was used as a mobile phase; the detector wavelength was set at 235 nm. The linear ranges were found as 0.1-18.5 µ g/mL, 0.4-25.6 µ g/mL, 0.3-15.5 µ g/mL, and 0.3-22 µ g/mL for AML, OLM, VAL, and HCT, respectively. In order to check the selectivity of the method for pharmaceutical preparations, forced degradation studies were carried out. According to the validation studies, the developed method was found to be reproducible and accurate as shown by RSD ≤6.1%, 5.7%, 6.9%, and 4.6% and relative mean error (RME) ≤10.6%, 5.8%, 6.5%, and 6.8% for AML, OLM, VAL, and HCT, respectively. Consequently, the method was applied to the analysis of tablets and plasma of the patients using drugs including those substances.

A new HPLC method with fluorescence detection for the determination of memantine in human plasma.

A sensitive and selective HPLC method with fluorometric detection was developed for the determination of memantine in human plasma and applied to a pharmacokinetic study. Memantine was precolumn derivatized with 9-fluorenylmethyl chloroformate, and the fluorescent derivative was separated on an RP C18 column using a mobile phase composed of acetonitrile-10 mM orthophosphoric acid containing 1 mL/L triethylamine with gradient elution. The method was based on the measurement of the derivative using fluorescence detection at 310 nm with excitation at 260 nm. The calibration curve was linear over the range 1.0-50.0 ng/mL. LOD and LOQ were found to be 0.3 and 1.0 ng/mL, respectively. Intraday and interday RSD values were less than 3.39%.

A review of the liquid chromatographic methods for the determination of biogenic amines in foods.

Biogenic amines (BAs) are biologically active molecules which have aliphatic (putrescine, cadaverine, spermine, spermidine), aromatic (tyramine, phenylethylamine) or heterocyclic (histamine, tryptamine) structures. They can be detected in raw and processed foods which are formed and degraded through several pathways during the metabolic processes of animals, plants and microorganisms. The identification and quantitation procedures of BAs in food samples are very important, because BAs are considered as the indicators of food quality and freshness. The determination of BAs are commonly achieved by separation techniques such as high-performance liquid chromatography (HPLC), gas chromatography (GC) and capillary electrophoresis (CE). In this article, analysis of BAs in foods were reviewed from 2007 to present.

Spectrofluorimetric methods for the determination of gemifloxacin in tablets and spiked plasma samples.

Two new, sensitive and selective spectrofluorimetric methods have been developed for the determination of gemifloxacin (GFX) in tablets and spiked plasma samples. Gemifloxacin, as a primary amine compound, reacts with 7-chloro-4-nitrobenzofurazon (NBD-Cl) (for method A) and fluorescamine (for method B) which are a highly sensitive fluorogenic reagents used in many investigations. For method A, the reaction product was measured spectrofluorimetrically at 516 nm with excitation at 451 nm. The reaction proceeded quantitatively at pH 8.5, 80 °C in 7 min. For method B, the method was based on the reaction between GFX and fluorescamine in borate buffer solution of pH 8.5 to give highly fluorescent derivatives that were measured at 481 nm using an excitation wavelength of 351 nm. The fluorescence intensity was directly proportional to the concentration over the range 40-200 ng mL(-1) and 100-1,200 ng mL(-1) for method A and B, respectively. Successful applications of the developed methods, for the drug determination in pharmaceutical preparations and spiked plasma samples, were performed.