A facile and sensitive HPLC-MS/MS method for quantification of 3,3'-diindolylmethane in plasma samples.

Indole-3-carbinol is the subject of ongoing biomedical research owing to its potential antiatherogenic, anticarcinogenic and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3'-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/ml concentration range. Consequently, the development of highly sensitive analytical methods to determine DIM in various biological samples is an urgent issue. In this study, an HPLC-MS/MS method was established for the quantification of DIM in human plasma. The developed method was validated according to the European Medicines Agency guidelines. Sensitivity, selectivity, accuracy and precision were good, allowing DIM quantification in the concentration range of 5-500 ng/ml. The limit of detection and the lower limit of quantification were 1 and 5 ng/ml, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were extracted from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C18 column (50 × 2.0 mm, 4 µm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid; 85:15, v/v) was delivered at a flow rate of 0.20 ml/min. DIM and the IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to healthy female Russian volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).

Quantification of a promising JNK inhibitor and nitrovasodilator IQ-1 and its major metabolite in rat plasma by LC-MS/MS.

Background: IQ-1 is a promising c-Jun-N-terminal kinase inhibitor and nitrovasodilator. An LC-MS/MS method was validated to determine IQ-1 isomers and major metabolite IQ-18 in rat plasma. Materials & methods: The analytes were extracted using ethyl acetate. The chromatographic separation was performed on a C8 column (150 × 4.6 mm, 5 µm) under acetonitrile-water (5 mM ammonium formate buffer, pH 2.93) gradient elution. Multiple reaction monitoring was used for MS/MS detection in the positive ion mode. Results: The method was fully validated over the range of 0.1-400 ng/ml (Z-isomer), 0.9-3600 ng/ml (E-isomer), 5.0-4000 (IQ-18). Conclusion: This method has been successfully applied to pharmacokinetic studies of IQ-1 and IQ-18 in rats after a single oral dose of IQ-1 (50 mg/kg).

Quantitative determination of new anti-inflammatory drug indomenthyl and its metabolite in rabbit plasma by HPLC-MS/MS.

Background: Indomenthyl is an innovative anti-inflammatory drug with a high analgesic activity. Indomenthyl releases indomethacin under the influence of neutrophil esterases in the inflammation focus. Methodology/results: This research is aimed at developing a highly sensitive method for the quantitative determination of indomenthyl and its active metabolite indomethacin in rabbit plasma by HPLC-MS/MS. Protein precipitation and extraction with acetonitrile were used for analyte isolation from plasma according to the QuEChERS principle. The target quantitative ion pairs m/z were respectively 496.4 → 358.0 for indomenthyl, 358.0 → 139.5 for indomethacin, and 340.1 → 202.1 for the IS. Conclusion: The calibration curve was linear over the range 0.1-1000 ng mL-1. The technique was applied to the pharmacokinetic study at a dose of 25 mg kg-1 to rabbits.

Effects of Simvastatin on the Metabolism of Fatty Acids in Combined Secondary Prevention of Coronary Heart Disease: Dosage and Gender Differences between the Effects.

BACKGROUND: Statins are currently used for secondary prevention of Coronary Heart Disease (CHD), as the lipid-lowering therapy with them is proven safe and effective. OBJECTIVE: The purpose of this research is to investigate the dose-dependent effect of statins used for secondary prevention of coronary heart disease, as well as mechanisms of quantitative and qualitative changes in lipoproteins, fatty acids and cholesterol in the blood and tissues of people of both sexes. METHODS: In a clinical trial (n=125, of which 89 patients belong to group 1 and 36 to group 2) and an experiment on laboratory animals (n = 100), simvastatin reduced the total level of fatty acids in blood plasma, when given in the amount that was within the therapeutic dose range. RESULTS: This effect was achieved through a drug-induced improvement in the capacity of hepatic cells to absorb Low-density (LDL) and Very-low-density (VLDL) lipoproteins. CONCLUSION: Considering the formation of saturated fatty acids, statin performed better in males. With Omega-3 polyunsaturated fatty acids involved, changes in lipoproteins, cholesterol and fatty acids (liver and myocardium) were similar to those caused by small doses of a statin drug. Effects of the combination of bisoprolol and acetylsalicylic acid were completely different from those caused by the use of statin.

Features of Pharmacodynamics of the Anticoagulant Dabigatran in Secondary Thrombophilia.

One of the crucial risk factors for development of severe postthrombotic disease (PTD) is the recurrence of deep vein thrombosis (DVT). New opportunities for pharmacological thromboprophylaxis of secondary thrombophilia were associated with the direct thrombin inhibitor-Dabigatran (Pradaxa; Boehringer Ingelheim, Germany). We aimed to investigate the daily pharmacodynamics of dabigatran in healthy volunteers and patients with PTD. Treatment with dabigatran in patients with PTD having chronic chronometric hypercoagulation and structural hypocoagulation before the administration of the drug is fraught with excessive anticoagulation and a high risk of clinically significant bleeding. In patients with PTD with detected chronometric and structural hypercoagulability before taking a direct thrombin inhibitor, treatment with dabigatran is fraught with possible inadequate anticoagulation and a high risk of clinically significant relapses of thromboses. According to our data, markers of risk of hemorrhagic complications under Dabigatran are the thromboelastography indicators lying within the reference values of the healthy before the administration of the drug: fibrin-platelet clot formation, maximum amplitude of TEG; total lytic activity of blood, and thrombodynamic potential index . Monitoring the effects of the targeted anticoagulant demonstrated the need for correction of dosage and discrete use of the drug in prevention and treatment for thrombohemorrhagic complications in this category of patients. The results of the study prove the efficiency of the therapy with dabigatran and "behavior" of hemostatic potential in patients being taken into account and controlled. Therapy may be long term but requires dynamic monitoring of patients with timely dose adjustment to achieve and maintain the target level of hemostatic potential.

Lower Urinary Tract Symptoms in Metabolic Syndrome in Young Adults.

BACKGROUND AND OBJECTIVE: Metabolic syndrome (MS) is widespread among middle age people and presents an acute issue in preventive cardiology. A list of conditions associated with MS is quite long and it is constantly growing. Despite the data, described in scientific literature, on general pathogenetic mechanisms, the conditions associated with androgynous status, are not included into the register of MS associated nosologies. Such association is identified in men older than 60 years old and is explained by age related hypoandrogenaemia. However, the issue of occurrence rate of lower urinary tract syndromes (LUTS) in young men with MS and their association of androgen levels remains open. METHODS: 62 European men aged from 25 to 40 (30 patients with MS and 32 conditionally healthy persons) were examined. Apart from generally accepted methods of physical and instrumental examination, evaluation of hormonal status (insulin, testosterone, dehydrotestosteron), IPSS-QOI testing and transrectal USI of prostate and bladder were performed. RESULTS: Revealed a high frequency of increasing the size and volume of the prostate gland, the number of points on the IPSS questionnaire, corresponding to the initial manifestation of hyperplastic prostate diseases on the background of insulin resistance and normal androgen levels raises questions about the search for new pathogenetic links of the metabolic syndrome with the processes of induction of prostate growth. CONCLUSION: Modern standards of examination of patients with MS do not include routine methods for the detection of prostate diseases. The above data raise questions about the need for further study in young patients with verified MS, including IPSS-QOI questionnaire surveying and TRUS.