Current Pharmaceutical Analysis - Volume 11, Issue 3, 2015

Currently more and more high-throughput biotechnologies have been armed by immunologists to explore the pathological mechanism of different autoimmune diseases. The new wave of approaches for the integrated understanding of human immune system based on high-throughput methods, corresponding mathematical and computational tools are increasing. This paper described the combination tendency of multiple methods to investigate the whole immune process at different levels including transcription, translation and post-translation modification, using microarray, Luminex and CyTOF as an example. When proper high-throughput technologies were used in combination, there will be more comprehensive recognition on immune system. The application of such techniques will promote deeper and more integrated study in the immunization field, and become a promising study strategy in this field.

Somatostatin (SST), a cyclic polypeptide, exerts a wide range of biological effects. As a result, decreased SST in plasma or tissues usually plays an important role in pathogenesis of various disorders. Exogenous supplement of SST or its analogues has been applied as an effective medical intervention in many diseases. The quantification of SST in plasma or tissues may provide a more rational application of SST in clinical practice. Currently, several methods for SST quantification have been established. The advantages and drawbacks of these methods are reviewed for clinical selection or further study. Moreover, necessary collection, extraction and preservation of samples for SST quantification are also recommended. Surface plasmon resonance (SPR) is a label-free optical detection technique for measuring biomolecular interactions in real-time. Among the several quantitative methods, an SPR-based immunoassay is suggested as a potential method for real-time SST quantification for clinical practice.

The vasodilatation effects of four components in Baige capsule were investigated on isolated cerebral basilar arteries and mesenteric arteries model of rats. In order to inspect the potential mechanism, the SH-SY5Y cellular membrane chromatography (CMC) was established to specially recognize the ligand which could interact with N-methyl-D-aspartate receptor (NMDA). Frontal chromatography and competitive binding chromatography were applied to investigate the interaction of four main components in Baige capsule with this receptor. MK-801 (an antagonist of NMDA receptor) was used as the displacer, ketamine hydrochloride, puerarin, daidzein, imperatorin, and isoimperatorin was used as the ligand. The results of vasodilatation test suggested that imperatorin and isoimperatorin could effectively relax both of the cerebral basilar arteries and mesenteric arteries. The CMC study showed that imperatorin and isoimperatorin could competitively displace MK-801. The molecular docking model of the NMDA receptor indicated that imperatorin and isoimperatorin showed a similar docking behavior as MK-801. Overall, the studies demonstrated that imperatorin and isoimperatorin had obvious vasodilatation effect and might be prospective candidates as new NMDA receptor antagonists.

A rapid and highly sensitive ultra-performance liquid chromatographic (UPLC) method was developed for analysis of docetaxel trihydrate in human plasma employing the analytical Quality by Design (QbD) approach. The quality target method profile (QTMP) was defined and critical analytical attributes (CAAs) earmarked. Unlike the chromatographic methods reported in literature including the official compendiums focusing on selecting buffer species as mobile phase mixture, acetonitrile and water (60:40 %v/v) was explored as the simpler solvent system along with rational understanding of the method performance through systematic approaches. Risk assessment and factor screening studies helped in identifying mobile phase ratio and injection volume as the critical method parameters (CMPs), followed by method optimization employing face-centred cubic design (FCCD). Search for optimum chromatographic conditions was carried out through numerical desirability function and graphical optimization. Method validation studies were also performed indicating high linearity, accuracy, precision, sensitivity of the method, along with adequate stability of the docetaxel trihydrate in human plasma. The studies successfully demonstrate the utility of analytical QbD approach for the development of a novel and validated bioanalytical method for estimation of docetaxel trihydrate in human plasma with enhanced method performance.

The fluoroquinolones have broad-spectrum activity, good absorption in gastrointestinal canals and superior tissue penetration. Because of these properties, they have been used to treat a wide range of infections. In this study high performance, liquid chromatography and microbiological methods have been developed for the determination of Gemifloxacin (GEM) in pharmaceutical preparations and human plasma. Microbiological assay was performed using Kirby Bauer disk diffusion method according to Clinical and Laboratory Standards Institute criteria, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as test microorganisms. Different concentrations of GEM were absorbed into antimicrobial susceptibility blank disks. Mueller Hinton Agar plates were inoculated with each microorganism that was adjusted to the turbidity of Mc Farland 0.5 standard separately and dried antibiotic disks were placed on the surface of agar. Plates were incubated and inhibition zone diameters were measured. The chromatographic separation was performed using Waters X-Bridge RP-18 column with an isocratic mode. The separation was carried out at 25 °C. All necessary validation parameters and system suitability test results were obtained. Also, interday and intraday studies were realized. RSD % values were reported to show the precision of the method. The proposed and validated methods were applied for the analysis of GEM in pharmaceutical formulations and human plasma for both methods.

The HPLC enantiomeric separation of thirteen flavanones was accomplished in the normalphase mode using six polysaccharide-based chiral stationary phases namely, Chiralcel® OD-H, Chiralcel ® OD, Chiralcel® OJ, Chiralpak® AD, Chiralpak® IA and Chiralpak® IB and various n- alkane/ alcohol mobile phases. The enantioseparation of flavanone and its eight mono substituted derivatives is reported including four methoxylatedflavanones in positions; 4’, 5, 6 and 7, respectively, and four hydroxylated in positions 2’, 4’, 6 and 7, respectively. Furthermore, the enantioseparation of the two aglyconeshesperetin, naringenin and the diasteriomeric separation of their flavanone glycosides hesperidin and naringin were achieved. The chiral recognition mechanism of each stationary phase is suggested based on the chemical structure and conformation of the chiral selector.

An UPLC-MS method was developed and validated for determination of cyclosporine A (CsA) in whole blood samples. Hemolysed and protein precipitated blood samples were centrifuged and the supernatant was transferred for analysis. Mobile phase comprising of methanol (A) and 3mM ammonium acetate buffer (0.1% formic acid) (B) was used in gradient mode at a flow rate of 350 µl/min. CsA and Cyclosporine D (CsD) were eluted on Acquity UPLC®BEH C18 1.7µm, 2.1x50mm Column. Retention time for CsA and CsD was 1.73 and 1.84 min, respectively. TQD-MS was operated under positive electrospray ionization mode (ESI+). The adducts of CsA (m/z 1225.1) and CsD (m/z 1239.1) were measured in single ion recording (SIR) mode. TQD-MS parameters were: cone voltage 92, capillary voltage 3.6 (kV), source temperature 150°C, desolvation temperature 350°C, desolvation gas 600 L/h, low mass resolution 15, high mass resolution 14 and ion energy 1.0. The method was simple, precise (%CV < 9.0), and accurate over the linearity range of 250ng/ml-5µg/ml. Lower limit of quantification for CsA was 250ng/ml. Extraction recovery of CsA at lower and higher quality control samples was about 35 - 40%. The method was successfully employed for pharmacokinetics studies of CsA.

The objective of current study was to improve solubility of poorly water-soluble antimalarial drug named dihydroartemisinin via the development of binary complexes of DHA with hydroxypropyl-β-cyclodextrin(HP-β-CD), ternary complexes of DHA-(HP-β-CD)-Palmitic acid and DHA-(HP-β-CD)-PVPK30 (PolyvinylpyrrolidoneK30). These complexes were prepared in various drug to excipients ratios by physical mixing and freeze dried (FD) methods. Characterization was performed by powder X-ray diffraction patterns, Scanning Electron Microscopy (SEM), Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), solubility test, and dissolution studies. Aqueous solubility of binary complexes of DHA increased up to 27 folds, 36 folds in DHA-(HP-β-CD)-palmitic acid and 216 folds in DHA-(HP-β-CD)-PVPK30 ternary complexes. The highest dissolution rate was observed to be 60 times improved for ternary FDCs of DHA-(HP-β-CD)-PVPK30 as compared to pure DHA. The crystallinity of DHA was decreased in physical mixtures while XRD patterns of freeze dried complexes (FDCs) showed least number of peaks having low intensity along with more displaced angles. The SEM images of PMs showed cavities and FDCs exhibited reduced particle size in binary and ternary systems. ATR-FTIR spectra of binary and ternary complexes revealed bonding interactions among DHA, HP-β-CD, palmitic acid and PVPK30. It can be concluded that binary complex formation of DHA with hydroxypropyl-β-cyclodextrin(HP-β-CD) and ternary complexes of DHA-(HP-β-CD)-Palmitic acid and DHA- (HP-β-CD)- polyvinylpyrrolidone K30 is a suitable approach to enhance solubility of DHA.

A novel microemulsion electrokinetic chromatography (MEEKC) method for the simultaneous separation and quantitative determination of phenylpropanoid glycosides in Rhodiola L. was developed. The effects of many parameters influencing separation characteristics were optimized. Retention factor met rosarin