Development and validation of HPLC-MS/MS technique for the quantitative determination of a new derivative of sydnonymine and its metabolites in rabbit plasma

Introduction. The study of the systemic exposure of a new original drug is an essential part of its preclinical study. 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide is a new selective carbonic anhydrase II inhibitor for the treatment of open-angle glaucoma. Methods for the quantitative determination of this compound and its N-hydroxy- and N-acetyl metabolites in the plasma of laboratory animals have not been previously developed.Aim. Development and validation of a method of quantitative determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and its metabolites N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M1) and N-acetyl-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide (M2) in rat and rabbit blood plasma by HPLC-MS/MS.Materials and methods. Protein precipitation by methanol was applied for sample preparation. 5-[2-(morpholine-4-carbonyl)-1,3-oxazole-5-yl]-thiophene-2-sulfonamide was used as an internal standard. A 5 % aqueous solution of ascorbic acid was added to the plasma samples at volume ratio 1 : 2 to prevent decomposition of N-hydroxy-5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide. The combination of sodium fluoride and potassium oxalate was selected as an anticoagulant. Chromatographic separation was performed on Zorbax Eclipse Plus C18 column (150 × 3.0 mm, 3.5 µm) with Zorbax Eclipse Plus C18 pre-column (12.5 × 2.1 mm, 5.0 µm) using a mobile phase based on a 0.1 % aqueous solution of formic acid and methanol. Mass spectrometric detection was carried out in the MRM mode using electrospray ionization in negative polarity. The method was tested during a pharmacokinetic study of a 1 % ocular suspension of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide on 6 Wistar rats. Blood samples were collected before administration, as well as 30 min, 1 h, 1 h 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 144 h, 216 h after administration. The non-compartment approach was used for calculation pharmacokinetic parameters.Results and discussion. The developed method has been validated in parameters of selectivity, calibration curve, accuracy and precision, matrix effect, dilution integrity, carry over, reinjection reproducibility, stability. The analytical range of determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide in plasma was 10–4000 ng/ml, M1 – 1.0–400.0 ng/ml, M2 – 0.1–40.0 ng/ml. The selected combination of anticoagulant and stabilizer solution allows storage of plasma samples in freezing chamber for 28 days.Conclusion. The developed method has been fully validated and confirmed its suitability for quantitative determination of 5-[5-(trifluoromethyl)-1,2-oxazole-3-yl]-furan-2-sulfonamide and its metabolites in the blood plasma of laboratory animals. The method has been successfully used for pharmacokinetic study of 1 % ocular suspension of the drug.

Introduction: 4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (ODASA) is a new pharmacologically activecompound, which is capable of reducing intraocular pressure by inhibiting carbonic anhydrase II. It is necessary to calculate the pharmacokinetic parameters of this compound and its metabolites in blood plasma of laboratory animals during a preclinical study. Aim: Evaluation of pharmacokinetic parameters of ODASA and its metabolites in rat bloodplasma after instillation of ocular suspension and its intraperitoneal administration. Materials and Methods: The study was conducted on 2 groups of Wistar rats, each including 6 individuals. The firstgroup underwent instillation of 1% ocular suspension of ODASA of 20 µL into each eye (1.6 mg/kg). The drug was injected intraperitoneally at the same dose to the second group. Blood sampling was performed before the administration,0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72 h, 96h, 120h, 144h, 192h, 240h, 288h after the administration. Plasma was immediately stabilized by 10% solution of ascorbic acid and frozen to a temperature no higher than -70°C. The sampleswere analyzed using the HPLC-MS/MS method. Chromatographic separation was performed on a Kinetex Phenyl Hexylcolumn (50*4.6 mm, 2.6 microns) in a gradient mode. Detection was carried out in the MRM mode using electrosprayionization. Results: The developed method was validated in the range of 2-2000 ng/mL for ODASA and 4-[5-(hydroxymethyl)-1,3,4-oxadiazole-2-yl]-benzenesulfonamide (M1) and 0.5 -500.0 ng/mL for N-hydroxy-4-(5-methyl-1,3,4-oxadiazole-2-yl)-benzenesulfonamide (M2). The size of the maximum plasma concentration after instillation of ODASA into eyes was 349.85±62.50 ng/mL, in M1 – 30.91±6.00 ng/mL and in M2 – 2.70±0.62 ng/mL (M±SEM). The half-life time of ODASA after ocular administration was 46.4±3.8 h, M1 – 70.0±14.3 h, and M2 - 36.5±15.2 h (M±SEM). The relative bioavailability of ODASA compared with injection was 81.03%. Conclusion: The performed validation of the method guaranteed the accuracy of the obtained results. The activesubstance has a high relative bioavailability after instillation into eyes. M1 is a major metabolite, and M2 is a minormetabolite. The studied compounds had a long half-life.

Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection : Application to pharmacokinetic studies in sheep

ABSTRACTRapid and comprehensive qualitative and quantitative analytical procedures are crucial in 24/7 emergency toxicology (ET) to support diagnosis and treatment of acute intoxications and to monitor their progression and efficacy of detoxification strategies. This study aimed to develop the simultaneous qualitative and quantitative analysis of 62 drugs, as well as seven active metabolites in human blood plasma using an automated liquid chromatography (LC)‐linear ion trap mass spectrometry (MS) screening system. Sample preparation was conducted by liquid–liquid extraction, and plasma concentrations were determined using an electronically stored 5‐point calibration. Validation was performed according to international guidelines and recommendations for ET including selectivity, carry‐over, accuracy, precision, and matrix effects. Finally, applicability was evaluated using case samples and proficiency tests. The method demonstrated selectivity for all analytes, with no significant carry‐over or matrix effects. Accuracy and precision recommended for ET could be fulfilled for all tested analytes, except for 10 analytes. Patient plasma samples were analyzed and compared with results obtained by reference LC‐tandem MS or gas chromatography‐MS methods. Furthermore, the applicability of the method could be demonstrated. It provides a fast, robust, and reliable blood plasma screening for 69 analytes and an additional quantification of 59 analytes relevant in ET. The use of an electronically stored 5‐point calibration and a simplified “push and print solution” allows for straightforward assessment of blood plasma levels of the analytes.

The aim of this work was to study the content of non-esterified fatty acids in plasma and liver of rabbits with acute arginine pancreatitis and its correction by linseed oil. The experiment was carried out on male rabbits breed gray giant with live weight 3.8–4.0 kg. The material for the study was sampled from blood and liver. Lipids from plasma and liver were extracted with a mixture of chloroform and methanol. After that the non-esterified fatty acids were isolated and methylated. Methyl esters of fatty acids were studied by the gas-liquid chromatography with the chromatograph ''Chrom 5'' (Prague,CzechRepublic). We have found that the content of non-esterified fatty acids decreases in the blood plasma and liver of rabbits with the acute arginine pancreatitis. It takes place at the expense of a reduce of saturated fatty acids with odd and even number of carbon atoms in a chain, monounsaturated fatty acids of the families ω-7 and ω-9 and polyunsaturated fatty acids of the families ω-3 and ω-6. That may indicate a greater use of non-esterified fatty acids for energy metabolism and esterification of lipids. We suppose that this is a consequence of the probable increase in content of non-esterified and esterified cholesterol in the rabbits’ blood plasma. Those processes provoke the cholesterol deposits in blood vessels and therefore cardiovascular diseases. We tried to influence on the processes by addition of linseed oil to the rabbits diet. We have found that in the linseed oil-fed rabbits the content of non-esterified fatty acids decreases at the expense of saturated fatty acids with odd and even number of carbon atoms in a chain and monounsaturated fatty acids of the families ω-7 and ω-9 inblood plasma and liver of the rabbits with acute arginine pancreatitis. Furthermore the levels of non-esterified polyunsaturated fatty acids of ω-3 family increase in the rabbits’ plasma and liver. As this takes place the ratio of non-esterified polyunsaturated fatty acids of ω-3 family to non-esterified polyunsaturated fatty acids of ω-6 family increased. The increase of non-esterified linolenic acid content in the rabbits’ blood plasma is apparently a result of a greater intake of linseed oil with food. In turn, the greater intensity of linolenic acid transformation in long-chain and unsaturated derivatives caused the increase of non-esterified docosapentaenoic and docosahexaenoic acids levels. Therefore, feeding with linseed oil led to normalizations of both the effective use of non-esterified fatty acids for energy processes and the level of esterified cholesterol in the blood plasma of rabbits with acute arginine pancreatitis.

Aim: to adapt the HPLC-MS/MS technique to determining valproic acid and its metabolites in blood plasma for drug therapy monitoring.Materials and Methods: The chromatographic assay was run using an Agilent 1260 Infinity II chromatograph with a Phenomenex synergi Fusion analytical column 4 μm-C18 2×50 mm. The mobile phase consisted of 0.1% ammonium acetate in distilled water and 0.1% ammonium acetate in methanol (10:90 v/v, 0.5 ml/min). The multiple ions monitoring (MIM) mode was used for mass- spectrometric detection of valproic acid at m/z = 143.1, with the negative ion mode. The method was found applicable over the range from 1 mcg/ml to 200 mcg/ml of valproic acid. For the mass spectroscopy detection of valproic acid metabolites, the multiple reaction monitoring (MRM mode) was used. MS identifications of 2-propyl-4-pentanoil-β-О-glucuronide; 2-propyl-4-pentenoic acid, 3-hydroxy-2- propylpentanoic acid, 4-hydroxy-2-propylpentanoic acid, 2-propylglutaric acid and 3- oxo-2-propylpentanoic acid in the negative ion mode were carried out at m/z 319.2→143.2; m/z 140.1→140.1; m/z 159.1→101; m/z 159.1→123.1; m/z 173→129.1 and m/z 157.05→11, respectively. The method was sensitive over the range from 10 ng/ml to 500 ng/ml of the tested compounds.Results: The developed technique allows for determining valproic acid and its metabolites in a single sample; thus, the preliminary stage of separate sample preparation can be omitted, which increases the informative value of the assay without increasing its cost.Conclusion: This innovative methodology for the quantification of valproic acid and its metabolites in the blood plasma is expected to facilitate the individual approach to the treatment of patients with epilepsy, thereby increasing the efficacy and safety of the pharmacotherapy.

The effects of co-ingested lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats were investigated. Plasma extracts of rats that had been fed onion-containing diets for one and two weeks were analyzed by HPLC with electrochemical detection after a treatment with sulfatase/beta-glucuronidase. Almost all of the quercetin metabolites in the plasma were sulfate/glucuronide conjugates of quercetin and isorhamnetin. More than 4.6% (w/w) of soybean oil in the diets significantly enhanced the accumulation of quercetin metabolites in the plasma. Fish oil and beef tallow increased this to an extent similar to that with soybean oil, and lecithin was more effective than the other three lipids. Two emulsifiers, sodium caseinate and sucrose fatty acid ester, also showed an enhancing effect on the accumulation of quercetin metabolites. These results indicate that co-ingested lipids and emulsifiers could enhance the bioavailability of quercetin glucosides in onion.

Metabolic disorders of the mother adversely affect early embryo development, causing changes in maternal metabolism and consequent alterations in the embryo environment in the uterus. The goal of this study was to analyse the biochemical profiles of embryonic fluids and blood plasma of rabbits with and without insulin-dependent diabetes mellitus (DT1), to identify metabolic changes associated with maternal diabetes mellitus in early pregnancy. Insulin-dependent diabetes was induced by alloxan treatment in female rabbits 10 days before mating. On day 6 post-coitum, plasma and blastocoel fluid (BF) were analysed by ultrahigh performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) (Metabolon Inc. Durham, NC, USA). Metabolic datasets comprised a total of 284 and 597 compounds of known identity in BF and plasma, respectively. Diabetes mellitus had profound effects on maternal and embryonic metabolic profiles, with almost half of the metabolites changed. As predicted, we observed an increase in glucose and a decrease in 1,5-anhydroglucitol in diabetic plasma samples. In plasma, fructose, mannose, and sorbitol were elevated in the diabetic group, which may be a way of dealing with excess glucose. In BF, metabolites of the pentose metabolism were especially increased, indicating the need for ribose-based compounds relevant to DNA and RNA metabolism at this very early stage of embryo development. Other changes were more consistent between BF and plasma. Both displayed elevated acylcarnitines, body3-hydroxybutyrate, and multiple compounds within the branched chain amino acid metabolism pathway, suggesting that lipid beta-oxidation is occurring at elevated levels in the diabetic group. This study demonstrates that maternal and embryonic metabolism are closely related. Maternal diabetes mellitus profoundly alters the metabolic profile of the preimplantation embryo with changes in all subclasses of metabolites.

Liker B., R. Bozac. I. Husdzinovic. S. Muzic. B. Bio~ina, A. Tumpic, V. Rupic: Activity of Some Enzymes in the Blood Plasma ofHylla Rabbits Fed Various Proportions of Substrate from the Production of the Plellrotlls pulmonarius Mushroom. Acta vet. Bmo 1998,67: 27-36. The nutritive effect of different amounts of substrate from the production of Pleurotus pulmonarills on the activity of AST, ALT, LD, CK, OOT and AP in the blood plasma of rabbits, after a nine weeks experimental fattening. was investigated. Sixty male Hyla rabbits, divided in four groups equal in number. one control (C) and three experimental groups (EI' E2, E3), were included in experiment. The control group (C) was fed the standard fodder mix without substrate. Added to the standard mix, the groups E!. E2 and E3 received various proportions, 10%,20%, and 30%, respectively, of the substrate left from the production of Pleurotus pulmonarius. Different proportions of added substrate did not affect the activity of AST and OOT, respectively. whose values (x±s.d.) in the control group (C) amounted to 32.49 ± 12.8 V.I'! and 6.14 ± 1.35 V.I'!, respectively. At the same time the activity of ALT was altered by experimental diets. The activity of ALT in the control group (C) amounted to 28.0±8.81 V.I·I, whereas in the E3 group it was significantly greater (P<0.05) than in the group C and E2, i.e. 38.86± 11.95 V.I· l . Vnder the conditions of experimental feeding the activity of AP was greater too: in the control group (C) it amounted to 107 ± 42.5 V.I·I, but in the group E3 it was significantly greater (P<0.05) and amounted to 158.7 ± 27.7 V.I'!. On the contrary, the activity of CK is decreased during the experimental feeding (P<0.07). and in the control group (C) it amounted to 2044 ± 647 V.I'!, in the E3 group to 1479.6 ± 927 V.I'!. The activity of lactate dehydrogenase (LD) generally reflected the composition of fattening diets and is inversely proportionate to the share of substrate added. The highest values ofLD activity was found in group C (94.57±24.78 V.I'!), and the lowest was in group E3 (63.49± 14.12 V.I' I). The difference was significant compared to the C and EI' groups (P<O.OI) and to the E, (P<0.05) group. The results indicate -B 1 hypovitaminosis and the development of compensatory metabolic acidosis. Nutrition. substrate of mushrooms, aspartate aminotransferase lAST), alanine aminotransferase (ALT). lactate dehydrogenase ILD), creatine kinase (CK). gamma-glutamyltransferase (GGT), alkaline phosphatase (AP) J hypovitaminosis. metabolic acidosis

Recently, scientific approaches to the classification of the antioxidant system by the mechanism of free radical peroxidation have emerged. This classification of antioxidants is based on the metabolic link to which the action of the antioxidant itself is directed. Therefore, the study of the effect of vitamin and mineral supplements on the indicators of antioxidant protection in farm animals is becoming increasingly important. The aim of this work was to study the effect of different doses of the vitamin-mineral supplement "Tekro" on the biochemical parameters of the New Zealand White breed rabbits. Scientific and economic experience on the topic of work and production verification were carried out on clinically healthy New Zealand White rabbits. The animals were fed with complete feed. Access to food and water was free. To conduct research on the farm, 4 groups of animals were formed: 1 control group, whose animals consumed the basic diet (RR); 2 research – OR with the content of vitamin and mineral supplements (VMS) "Tekro" 3.0%; 3 research – OR with the content of VMS "Tekro" 3.5%; 4 research – OR with a content of VMS "Tekro" 4.0%. The activity of antioxidant enzymes and the content of ceruloplasmin in the blood plasma and liver of rabbits were determined on the 45-th, 60-th, 75-th and 90-th days. In the blood plasma of rabbits from the control and experimental groups, a change in the activity of antioxidant defense enzymes was established. The obtained research results showed that long-term use of the vitamin-mineral supplement "Tekro" significantly positively influenced the antioxidant defense system of the animal body. The established changes in the indicators of antioxidant protection indicate the tension of balancing the prooxidant-antioxidant system of young rabbits. In particular, this phenomenon can be explained by the age-related characteristics of the formation of the antioxidant system of the growing organism of the New Zealand white breed rabbits under the influence of the vitamin-mineral supplement "Tekro". This phenomenon may be evidence of the stabilization of antioxidant processes in the blood plasma of rabbits of the experimental groups as a result of the complex effect of the optimal dose of biologically active substances of the vitamin and mineral supplement. Prospects for further research consist in a detailed study of the effect of various doses of the vitamin-mineral supplement "Tekro" on protein metabolism in the rabbit's body.

High-performance liquid-chromatographic determination of 5-aminosalicylic acid and its metabolites in blood plasma

Background Statins are used to treat and prevent cardiovascular diseases (CVDs) by reducing the total serum cholesterol concentration. Unfortunately, dose-related side effects and sub-optimal response, attributed to non-adherence amongst others, were described. Therefore, a fast and sensitive liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for adherence testing and therapeutic drug monitoring of all currently marketed statins and their active metabolites in human blood plasma should be developed, validated and tested for applicability. Methods Atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, as well as ortho- and para-hydroxy-atorvastatin, lovastatin hydroxy acid and simvastatin hydroxy acid were included and several internal standards (IS) tested. Validation was performed according to the guideline of the European Medicines Agency including selectivity, carry-over, accuracy, precision, matrix effects, dilution integrity and analyte stability. Finally, applicability was tested using 14 patient samples submitted for regular toxicological analysis. Results Due to an analytical interference of atorvastatin-d5, diazepam-d5 and pentobarbital-d5 were chosen as IS for positive and negative ionization mode, respectively. All statins and metabolites fulfilled the validation acceptance criteria except for fluvastatin, which could not be quantified reliably and reproducibly, most probably due to instability. Analyses of human plasma samples revealed concentrations of statins and metabolites below the reference plasma concentrations in the case of eight patients. However, nothing was known concerning patients' adherence and time between intake and sampling. Conclusions An LC-HRMS/MS method for identification and quantification of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin and four active metabolites was successfully developed and applicability demonstrated.

The objective of the research - to determine the dynamics of endothelial dysfunction markers among patients with pulmonary hypertension on the background of chronic obstructive pulmonary disease under the influence of treatment.Material and methods. The results of the study are based on data from a comprehensive survey of 170 patients with chronic obstructive pulmonary disease (COPD) aged from 40 to 65 years, 123 of which had pulmonary hypertension and 47 ones had no pulmonary hypertension. Also 31 almost healthy people were examined on an outpatient basis. Patients with pulmonary hypertension on a background of COPD were divided into two subgroups of observations depending on the treatment. The first subgroup was used as the background therapy and the second subgroup - background therapy with the roflumilast. Markers of endothelial function were determined during screening and after 12 months of follow-up.Results. The median level of endothelin-1 in the group of patients with pulmonary hypertension on the background of COPD was 3.17 [2.19 ; 4.14] fmol/ ml and was significantly higher, both against the value of 1.78 [1.25 ; 2.18] fmol/ml in the group of COPD patients without pulmonary hypertension (p &lt; 0.05), and 10.2 times higher than versus the level of 0.31 [0.19 ; 0.36] fmol/ml in the group of healthy individuals (p&lt;0.05). The amount of nitrogen oxide metabolites (NO2+NO3) was significantly lower (by 15.8 %) in the group of patients with pulmonary hypertension on the background of COPD versus the value of 22.00 [21.00 ; 23,00] mmol/l in the group of COPD patients without pulmonary hypertension, and is 37 % lower than in the group of healthy individuals, where the level of this indicator was 26,00 [25,00 ; 28,00] mmol/l (p&lt;0.05). Then a correlation analysis was performed. Significant relationships were determined between the following indicators: duration of COPD and NO3 level (R = -0.29, p = 0.001); duration of COPD and NO2+ NO3 level (R - 0.26, p = 0.003); mean pulmonary artery pressure and endothelial-1 level (R = +0.70, p = 0.001); mean pulmonary artery pressure and NO2 level (R = -0.59, p = 0.001); mean pulmonary artery pressure and NO2+ NO3 (R = -0.50, p = 0.001). The level of endothelin-1 and nitrogen oxide metabolites in the blood plasma of patients was comparable between the first and second subgroups of observation during screening (p &gt; 0.05). After 12 months, there was a statistically significant decrease of Δ1% = -57.53 % endothelin-1 to 1.87 [1.44; 2.36] fmol/ml in the first subgroup and Δ2% = -82.34 % to 1.62 [1.37; 1.92] fmol/ml in the second subgroup. After 12 months of therapy in both subgroups, the level of total nitrogen oxide metabolites in blood plasma significantly increased Δ1% = 10.00 % to 21.00 [18.00 ; 23.00] mmol/l in the basic therapy subgroup, and Δ2% = 19.05 % to 24.00 [22.00; 26.00] mmol/l in the roflumilast subgroup, (p &lt; 0.05). A statistically significant difference in endothelin-1 levels and the amount of nitrogen oxide metabolites between subgroups was determined after 12 months of treatment.Conclusions. Among patients with COPD, endothelial dysfunction occurs, characterized by an increase in the concentration of endothelin-1 and a decrease in nitrogen oxide metabolites in the blood plasma. An increase in the level of endothelin-1 in blood plasma has a direct correlation with the value of the mean pulmonary artery pressure. Background therapy among patients with pulmonary hypertension on the background of COPD improves the state of endothelial function, with increased nitrogen oxide metabolites in the blood plasma. When added to background therapy, roflumilast significantly reduced the concentration of endothelin-1 in blood plasma.

Emergency toxicology analysis – Method development for the straightforward determination of total 70 drugs and metabolites in human blood plasma by LC-MSn

Rapid, high-throughput, and sensitive screening of herbal product metabolites in blood plasma using 2D carbon microfiber fractionation coupled with quadrupole time-of-flight high-resolution mass spectrometry: Huangkui capsule insights.