Current Pharmaceutical Analysis - Volume 11, Issue 4, 2015

Background: Cellular viability studies are important in many different fields of cell biology research and are based on estimates of live and dead cells from a specific cellular population. Recent progress in technology has brought important advances in the analysis of cellular viability, mainly reducing the time of analysis and sample preparation; however, the costs for equipment and reagents still limit the feasibility of conducting those studies. The purpose of the present study was to use free software, non-specific dyes, and light microscopy to establish a reproducible image analysis test and to compare the results with an established method. Method: 3T3 fibroblast cell line was exposed to different concentrations of dimethylsulfoxide (DMSO) for 24 h, and cellular viability was estimated by the proposed technique (image analysis performed with free software) and with the methylthiazol-tetrazolium salt (MTT) assay. A Bland-Altman analysis was applied to compare the methodologies. Results: Image analysis by CellProfiler®/CellProfiler® Analyst was compared to MTT by Bland-Altman test, and the two methodologies were considered equivalent. Cellular viability decreased in a dose-dependent manner with increasing doses of DMSO, and both methodologies were capable of distinguishing between live and dead cells, producing comparable results (with bias = 1.3550) evaluated by the Bland-Altman analysis. Conclusion: The proposed image analysis can be used as a simple, rapid, and low-cost technology for high throughput analysis of live-dead cell differentiation.

A simple LC-MS/MS method has been advanced for measuring ciprofloxacin, using liquidliquid extraction of plasma with ether and methylene chloride (7/3, v/v) using ofloxacin as an internal standard (IS). The analytes were detected by HPLC using an isocratic mobile phase composed of methanol and 5 mM aqueous ammonium acetate (pH 3.0, 35/65, v/v) on a Luna phenyl-hexyl (Phenomenex 2.0 x 50 mm, 5 μm) column. The eluate was analyzed by MS/MS in the MRM mode using the transitions of the respective (M+H)+ ions (m/z 332.1→231.1 and m/z 362.1→261.1) for determination of ciprofloxacin and IS, respectively. The standard calibration curves indicated good linearity within the range of 0.05-5.0 μg/ml (r2=0.997, 1/x2 weighting). The lower limit of quantification (LLOQ) was 0.05 μg/ml. The retention times of ciprofloxacin and IS were 1.79 and 1.51 minutes, respectively. In addition, no significant impurities were found that interfered with the analysis. Acceptable precision and accuracy were obtained for the concentration range of the standard curve. The validated method was successfully applied to bioequivalence and pharmacokinetic studies of two formulations of ciprofloxacin (single 250 mg doses) in 24 healthy Korean volunteers.

A simple liquid chromatography mass spectrometric method with direct urine injection analysis was developed and validated for the identification and quantification of gammahydroxybutyric acid (GHB), gamma-butyrolactone (GBL), 1,4-butanediol (1,4-BD), methylenedioxyn- methylamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-Nethylamphetamine (MDEA), ketamine (KET) and norketamine (Nor-KET). GHB-d6, GBL-d6, MDMA-d5, and KET-d4 were used as internal standards (IS). The method has the advantage of one step sample preparation which includes an appropriate dilution and filtration before its injection into the LC/MS system. The protonated ions of the analytes were detected and quantified using an ESI probe operating in positive mode and selected ion monitoring (SIM). The linear range was between 8-20 μg/mL for GHB, GBL and 1,4-BD, 50- 1000 ng/mL for MDEA, KET and Nor-KET and 250-1000 ng/mL for MDMA and MDA, providing square correlation coefficient (r2) ≥ 0.98. Interday and intraday errors were found to be ≤ 13.53%. The applicability of the proposed direct injection LC/MS method was confirmed with the analysis of ten authentic samples initially tested for amphetamines through a preliminary immunoassay screening. In addition, eight samples were assayed for KET and Nor-KET and six samples for GHB, GBL and 1,4-BD. The method is simple, accurate and possesses better specificity comparing to the immunoassay techniques, which make it suitable for screening purposes. It also presents the same or lower cost comparing to the respective immunoassay kit, where available. Toxicological laboratories may use the proposed method, overcoming thus the appeared false positive results (amphetamines) or the absence of immunoassay kits (GHB, GBL, 1,4-BD, KET, Nor-KET), avoiding also the use of increased cost instrumentation such as a liquid chromatography tandem mass spectrometry.

An ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS)-based chemical fingerprinting and three-markers (protocatechuic acid, catechin, and epicatechin) determination method has been developed for the quality control of the roots of Indigofera stachyoides. The optimal chromatographic condition was achieved on a Synergi Hydro-RP (2.00 mmx100 mm, 2.5 μm) column with linear gradient elution using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid as the mobile phase. The flow rate was 0.3 mL/min, the injection volume was 3 μL, and the column temperature was set at 35°C. The method is based on a full-scan negative electrospray ionization mass spectrometry (ESI-MS) at m/z 50~1000, with the capillary voltage of 2800 V, nebulizing gas (N2) pressure of 1.2 Bar, drying gas (N2) flow rate of 8.0 L/min and gas temperature of 200°C. MS quantification was based on the high resolution extracted ion chromatogram of the target ion with deviation set at ± 0.005. Satisfactory repeatability, stability and precision were observed for the chemical fingerprint. For quantification, excellent linear behavior over the investigated concentration range was observed with regression coefficient (R2) > 0.9976 for all analytes. The RSDs of intra-day and inter-day precision are less than 1.7%. Recoveries were in the range of 98.4~104.8%. Quantitative analysis results showed that contents of the three common compounds differed significantly among different batches of the roots of I. stachyoides from different growing areas in Guizhou Province. The method was reliable, accurate, and repeatable, and can be applied to the routine quality assessment of I. stachyoides.

Doxepin is a tricyclic antidepressant drug that has proven effective anesthetic and analgesic activity in oral mucositis when administered as a topical oral rinse. Aimed to study alternative doxepin formulations for buccal application, we have developed three different quantitative methods to determine doxepin. Each method was specifically developed for its intended use: in vitro release, ex vivo permeation or in vivo studies analyzing plasma. Simple, rapid and easy to perform UV-vis spectrophotometry analysis was chosen for doxepin quantitation in the in vitro release studies with artificial membranes. A reversedphase HPLC method with UV-vis detection using a C18 column was developed for the quantitative determination of doxepin permeated across porcine buccal mucosa. Finally, to determine doxepin plasma concentrations in pigs, a HPLCcoupled with tandem mass spectrometer analytical method with simple sample preparation was developed. The proposed methods were validated according to ICH guidelines with respect to specificity, linearity, accuracy and precision. Therefore, the developed methods were found to be precise, accurate and selective which is suitable for the estimation of doxepin pharmacokinetics and biopharmaceutics parameters.

The current study entails the development and validation of a high-performance liquid chromatographic method for quantitative estimation of isotretinoin present in bulk, pharmaceutical dosage forms as well as in human plasma and skin as biological matrix. The method employed reversed phase chromatography employing an RP-C18 column with an isocratic mobile phase consisting of acetonitrile, methanol and water (50:45:5 v/v) set at a flow rate of 0.8 ml/min. The analyte detection was done by ultraviolet detection (UV) at 355 nm. The pH of the mobile phase was adjusted to 4.5 ± 0.01 using acetic acid. The isotretinoin was extracted from plasma by liquid-liquid extraction method. A sharp peak with peak asymmetry value of 1.16 was observed for isotretinoin at a retention time of 5.2 ± 0.01 min. The regression equation was linear over concentration range between 0.1-100 μg/ml and regression co-efficient was found to be 0.999 with good accuracy and precision. The limit of detection (LOD) and quantification (LOQ) were found to be 0.006 and 0.018 μg/ml, respectively. The wide range of linearity, accuracy, short retention time and isocratic elution imply that the method is suitable for routine estimation of isotretinoin in bulk, pharmaceutical dosage forms and human plasma with good accuracy and precision.

Schizophrenia is a lifelong debeliating illness requiring extended treatment with antipsychotic agents. A novel atypical antipsychotic agent like olanzapine is required for a longer period of time to prevent relapses. Non-adherence to therapy is a very common and severe problem in these patients. Adherence to therapy can be improved by prescribing depot injectable formulations in such patients to significantly reduce the dosage frequency. PLGA based in situ gel implant was successfully developed on the principle of solvent exchange and evaluated for its in vitro release and in vivo performance in rats. The value of n of all formulations fell within the range of 0.51 to 0.83 which indicates the release pattern of Case II. The Cmax for formulation F2, F3 and F4 was 121.53, 81.64 and 54.39 ng/ml, respectively. Both in vitro release and in vivo pharmacokinetic data indicate that such formulations can be applied for at least one month sustained release with tolerable initial burst release. Olanzapine PLGA based in situ gel implant can be a potential candidate for depot injectable formulation intended to provide sufficient plasma levels for at least 30 days.

A novel multi-templates molecularly imprinted polymer (MIP), using three saponins mixture (notoginsenoside R1 (R1), ginsenoside Rg1 (Rg1) and ginsenoside Rb1 (Rb1)) as the template, was synthesized by precipitation polymerization and used as the solid-phase extraction (SPE) sorbent. The synthesis was optimized by using different functional monomers, polymerization solvents, cross-linkers, and molar ratios of template:functional monomer:cross-linker. The resulting MIPs were evaluated by transmission electron microscope (TEM) and adsorption experiments, exhibiting uniform spherical morphology, high adsorption capacity and rapid kinetics for the rebinding of template. The adsorption capacity of the imprinted polymers to the template was more than 2.0-folds that of non-imprinted polymers (NIPs). MIPs were successfully applied to the SPE for the extraction of R1, Rg1 and Rb1 from the Panax notoginseng herb extract. Mean recoveries of R1, Rg1 and Rb1 determined by HPLC (highperformance liquid chromatography) were in the range of 80.7–82.4% with relative standard deviations (RSDs) less than 3.2%. These results reveal that the multi-templates MIPs as SPE sorbent have good applicability to selectively extract a group of saponins from complex samples.

Obesity and its related complications are one of the world’s alarming preventable causes of tumor , diabetes and cardiovascular diseases. Whereas, medicinal herbs are potential source of therapeutic drugs. The present study was focused on bioactive secondary metabolites of methanolic extract from whole plant Hartmannia rosea G. Don. The new compound(1), quercetin 3-O-β-Dallopyranoside- 3″, 6″- diacetate (1), and two known compounds, ursolic acid (2) and gallic acid (3), were isolated for the first time from Hartmannia rosea G. Don. The structure of isolated new compound (1), quercetin 3-O-β-D-allopyranoside- 3″, 6″- diacetate was elucidated by using chromatographic and modern spectroscopic techniques. The cytotoxic activity, anticancer and anti- obesity effects of compound (1) were evaluated against normal ,(3T3-NIH) and Human Pancreatic cancer cell line (PSN-1). The anti- adipogenic effect on 3T3-L1 cell line was also evaluated. The tested compound (1) showed no cytotoxicity against normal cells (3T3-NIH) but has moderate activity against PSN-1 (Human Pancreatic cancer cell line) at a higher dose. However, it showed a pronounced inhibitory effect against adipogenesis of 3T3-L1 adipocytes. The inhibition of the formation of mature adipocytes indicated that quercetin 3-O-β-D- allopyranoside- 3″, 6″-diacetate (1) from Hartmannia rosea G. Don. has potential anti-obesity effects and may be a better alternate of chemical and biochemical agents used to control obesity and related chronic diseases. However its exact mode of action will further be undertaken.