It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF‐7 cells was carried out. Dietary administered CLO caused the dose‐dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl‐2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase‐3 and ALDH1 expression increase after high‐dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro‐apoptotic effects of CLO extract in MCF‐7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase‐7, Bcl‐2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.

A well established method of direct injection of larger than conventional sample volumes ranging from 0.1 mL to 10 mL in HPLC is the injection valve method in which a loop of tubing is totally or partially filled with sample. Recent HPLC pumps have a flow-rate setting accuracy of +/- 1-2% over a flow-rate range from 0.1 mL/min to 10 mL/min and the flow stability is 0.2% or less. Quarternary low pressure gradient pumps are widely available and used, but all their hydraulic lines are seldom utilised. The idea of using one line of a common commercial HPLC quaternary low-pressure pump for direct on-column injection (pumping) of large sample volumes ranging from 1 mL to 100 mL was tested. This approach was evaluated during practical work on the development of an RP-HPLC method of determination of residual atrazine and hydroxyatrazine. In lysimetric environmental experiments hydroxyatrazine was formed in situ in a soil column by hydrolysis of atrazine. The results proved the applicability of this approach not only in experiments with model mixtures of analytes at microg/L levels in solutions. Analysis of 20 mL of soil leachates and extracts of soil samples containing atrazine and hydroxyatrazine at the 10 microg/kg level (in dry soil) revealed that good figures-of-merit were preserved, even in the presence of a large excess of humic substances.

The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.

The idea of utilization of one hydraulic line of a common commercial HPLC pump for direct on-column sample pumping injection of large sample volumes, 20 mL, was further investigated with the aim to develop multicomponent pesticides trace residues HPLC method in gram soil samples. Target pesticides group involve asulam, atrazine, 2,4-D, PCA, propazine, simazine, 4-chloro-2-methylphenoxyacetic acid, 2-(4-chloro-2-tolyloxy) propionic acid, chlortoluron, metoxuron, epoxiconazole. The results proved the applicability of this approach in experiments with mixtures of analytes at low ng/mL levels. Analysis of 20 mL of soil leachates and extracts of fortified soil samples containing these pesticides at the 10-50 ng/g level (in dry soil) revealed good figures of merit, also in the presence of large excess of humics. LODs achieved by detection at 220 nm evaluated from calibration runs of spiked soil extracts by Hubaux et al. method ranged from 5-12 ng per injected volume. For 20 mL large volume injection it represents 0.25-0.6 ng/mL of diluted soil extract, or 2.5-6 ng/mL of crude extract, or 6-5 ng/g dry soil. Recoveries of pesticides at concentration levels approaching half of maximum allowable concentration of pesticides in soil (100 ng/g) ranged from 85 to 98% with acceptable reproducibility, except asulam and metoxuron.

Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid chromatography hyphenated with mass spectrometry, Journal of Pharmaceutical and Biomedical Analysishttp://dx.doi.org/10. 1016/j.jpba.2017.05.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Highlights New HILIC-HPLC-MS method was developed for determination of TPMT activity  HILIC-HPLC-MS combination was approved by excellent performance parameters  HILIC-HPLC-MS was compared to traditional RP-HPLC-UV as well as advanced RP-HPLC-MS  Conventional approaches were surpassed in terms of selectivity, LOD, analysis time  HILIC-HPLC-MS is favorable for routine assay of 6-MMP/6-MP ratio in RBC lysates List of abbreviations6-MMP -6-methylmercaptopurine, 6-MP -6-mercaptopurine, 6-TG -6-thioguanine, AZA -azathioprine, Cl-P -6-chloropurine, DTT -dithiothreitol, IBD -inflammatory bowel diseases, RBC -red blood cells, SAM -S-adenosyl-L-methionine, TGN -thioguanine nucleotides, TPMT -thiopurine-S-methyl transferase. IndroductionThiopurine drugs, namely azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are immunosuppressive agents that are, due to their low cost and high effectiveness, widely used in the treatment of inflammatory bowel diseases (IBD), but also in other autoimmune diseases, some hemopoetic disorders and some solid organ transplant [1][2][3][4][5]. However, the occurrence of adverse drug reactions limits the use of these drugs. Up to 15 % of IBD patients discontinue this type of treatment due to adverse events that include nausea, skin reactions, pancreatitis, hepatotoxicity or myelotoxicity [6,7].Thiopurine drugs are prodrugs, so their biological activity is preceded by extensive metabolism. Briefly, after absorption, AZA is metabolized in the liver to 6-MP which then can be metabolized to active thioguanine nucleotides (TGN) and to inactive methylated product 6-methylmercaptopurine (6-MMP) by the thiopurine-S-methyl transferase (TPMT) enzyme [8]. TPMT (EC 2.1.1.67), a key enzyme involved in the thiopurine drug metabolism, is subject to common genetic polymorphism that leads to trimodial distribution of the TPMT activity in population [9]. Individuals with lower TPMT activity might have an increased risk of developing thiopurine-induced myelosuppression. On the contrary, individuals with high TPMT activity have lower concentrations of active TGN metabolites resulting in reduced therapeutic efficacy of the drug [10]. In addition, high TPMT activity leads to accumulation of the hepatotoxic methylated metabolites [11]. Therefore, some scientific committees advise to determine the TP...