Drug analyses by capillary electrophoresis and gas chromatography-mass spectrometry

Chiral separation of enantiomers of ten β-blockers is examined by using cyclodextrin-mediated capillary zone electrophoresis. The separation is based on the differential affinity in the formation of inclusion complex of the andyte with the cyclodextrin which leads to a discrepancy in the electrophoretic mobility of the free and complexed enantiomers. Results show that many factors will affect the chiral separation, including the buffer strength, concentration and type of β-cyclodextrin, and type of capillary. In general, higher ionic strength and C-l8 coated capillary give higher resolution of enantiomers of β-blockers. Moreover, three chemically modified β-cyclodextrins - heptakis-(2,6-di-O-methyl)-, hydroxypropyl-, and hydroxyethyl- β-cyclodextrins exhibit higher enantioselectivity for these β-blockers. Higher concentration of β-cyclodextrin is required for hydrophilic β-blockers, whilst hydrophobic β-blockers require low concentration of β-cyclodextrin for optimum chiral separation. In this investigation, enantiomers of alprenolol, atenolol, isoproterenol, oxprenolol, pindolol and propranolol are resolved. Partial resolution is obtained for nadolol, metoprolol and labetalol. Acebutolol is not enantioseparated by all means. Compared with chiral GC and HPLC, CD-mediated CZE possesses many assets including low consumption of exotic and expensive chiral selector, simple instrumentation and ease of automation and its use as a new alternative method in chiral separation will increase rapidly in the near future. In another part of my research, a selective and sensitive method in analysing artemisinin and dihydro-artemisinin is developed. By measuring their gas chromatographic decomposition products using gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM), the concentration of their parent compounds present in plasma can be indirectly determined. Human blood plasma, with triphenylmethanol (internal standard) added, is extracted by n-chlorobutane. The extract is then analysed by GC/MS. Artemisinin and dihydro-artemisinin are first pyrolysed into compounds 1 and 2 respectively in the GC injector at 260 ℃ and separated by the capillary column (30mx0.25μm film thickness HP-5MS) in a HP5890 Series II Gas Chromatograph equipped with HP7673 autosampler. [Figure 1 and 2 are obmitted here.] Higher sensitivity is obtained by selected ion monitoring. Ion groups of 152, 180, 210; 166, 165, 151; and 260, 154 amu are monitored for compounds 2, 1 and the internal standard triphenylmethanol respectively. Good linearity is attained for both compounds (1 and 2) in the concentration range of O-lOOOng/ml, with the correlation coefficient (r) greater than 0.999. The extraction recoveries([plus and minus]SD) of both 20ng/ml of QHS and DHQHS are 109.2[plus and minus]17.9% and 98.5[plus and minus]2.3% (n=4) respectively. The intra-and inter-day precisions of QHS at either 20ng/ml or 200ng/ml are found to be less than 3% and 7%CV respectively. The accuracy is within 3%. Similarly, for DHQHS, the intra- and inter-day repeatability at both 20ng/ml and 200ng/ml are less than 7%CV. The accuracy is found to be within 6%. By the present method, the limits of quantitation for QHS and DHQHS are found to be 2ng/ml and 4nglml respectively in l-ml plasma. Although artemether and arteether do interfere in the analysis of dihydro-artemisinin, no interference is observed by the endogenous contaminants from the plasma and another antimalarial drug, quinine, in the analyses of artemisinin and dihydro-artemisinin in human blood plasma. The concentration of artemether in plasma is determined by a similar approach. It is performed by measuring its gas chromatographic decomposition product using on-column gas chromatography/mass spectrometrykelected ion monitoring (on-column GC/MS/SIM). Quantitation of this compound can thus reflect the amount of artemether present in plasma samples. The human blood plasma, with arteether (internal standard) added, is extracted by n-chlorobutane. The extract is pyrolysed in the capillary column and separated by the same column (28mx0.25mm i.d.0.25μm film thickness HP-5MS connected with lmxO.53mm i.d. deactivated retention gap in front of it) in HP5890 Series II Gas Chromatograph equipped with HP7673 autosampler. For achieving a lower detection limit, selected ion monitoring is employed. Ions of 138 amu are monitored for both pyrolysed products of artemether and arteether. In the concentration of 0-3OOng/ml artemether in plasma, the linearity is excellent with the correlation coefficient (r) greater than 0.9999. Moreover, the extraction recovery is high (>95%). Both intra- and inter-day precisions are less than 5% CV for the concentrations of 2, 3, 4, and 2OOng/ml and accuracy is within 10% for the concentrations of 3, 4, and 200ng/ml. This method has the limit of quantitation of lng/ml for the sample size of l-ml plasma and it shows a high selectivity as the endogenous contaminant, dihydro-artemisinin(a major metabolite of artemether in plasma), artemisinin and quinine do not interfere in the analysis. The methods developed for analysing these antimalarial drugs can be utilized in human pharmacokinetics studies, especially in the low nanogram level of drugs in plasma

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field