A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.

Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its metabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN) from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples. A method employing a rapid and simple liquid-liquid extraction followed by reversed-phase (C18) liquid chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeatability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 % ± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ± 17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 % for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN. The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples. In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and 5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN 150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the metabolites were transferred from mother to fetus with a slight time delay.

Poisoning of a dog with the explosive pentaerythrityl tetranitrate.

A three-year-old male Labrador retriever was presented at the Clinic of Internal Medicine, University of Zagreb, Croatia. The owner reported that the dog was ataxic, and this was evident by its markedly unsteady, swaying gait. The dog also had difficulty rising and fell several times while trying to stand. It had come into contact with the explosive, pentaerythrityl tetranitrate, while training to detect explosives. The following clinical symptoms were observed: bradycardia, depression, mild disorientation and a broad-based stance. The dog had conscious proprioceptive deficits in the hindlimbs, but cranial nerve function was normal except for miosis. Ion scan analysis of the dog's serum after evaporation of the current phase by mass spectroscopy revealed the presence of fragments that are characteristic of pentaerythrityl tetranitrate. The aim of the present case report was to identify pentaerythrityl tetranitrate poisoning and describe the clinical signs of pentaerythrityl tetranitrate poisoning in dogs. To the authors' knowledge, there are no published scientific articles on pentaerythrityl tetranitrate poisoning in dogs.

[Quantitative gas chromatography-mass spectrometry determination of pentaerythrityl tetranitrate metabolites pentaerythrityl trinitrate, pentaerythrityl dinitrate and pentaerythrityl in human plasma]. / Quantitative gaschromatographische/massenspektrometrische Bestimmung der Pentaerithrityltetranitrat-Metaboliten Pentaerithrityltrinitrat, Pentaerithrityldinitrat und Pentaerithritylmononitrat in Humanplasma.

Assay methods based on gas chromatography/mass spectroscopy (GC-MS) may be used to measure PE1N (pentaerithrityl mononitrate, CAS 1607-00-7), PE2N (pentaerithrityl dinitrate, CAS 1607-01-8) and intermediate pentaerithrityltrinitrate (PE3N, CAS 1607-17-6) in human plasma. Based on this method a simplified method to quantify the metabolites of PETN (pentaerithrityl tetranitrate, CAS 78-11-5, Pentalong) is described. In the present study a consistent method to extract the metabolites of human plasma and following derivatisation is described. Since PE1N can be quantified up to 150 ng/ml, PE2N and PE3N up to 30 ng/ml in human plasma, a dilution of the plasma samples can be avoided. The mean recovery rate is not so high as in other described methods, and inaccuracy is about 10%. Therefore a calibration range between 0.2-30 ng/ml of PE2N and 1-150 ng/ml of PE1N has to be considered. The described method offers an alternative and applicable option to quantify the PETN-metabolites and elucidate their part as NO-donors.

Quantitative determination of pentaerythrityl tetranitrate and its metabolites in human plasma by gas chromatography/mass spectrometry.

Up to now, there has been no data available on the pharmacokinetics of pentaerythrityl tetranitrate (PETN, CAS 78-11-5) and its metabolites, pentaerythrityl-trinitrate (PE-tri-N), pentaerythrityl-dinitrate (PE-di-N), pentaerythrityl-mononitrate (PE-mono-N) in human plasma. Therefore, in order to determine PETN and its metabolites in plasma sensitive and highly selective GC/MS methods had to be developed and validated. PETN and its metabolite PE-tri-N were validated in the concentration range 50 pg/ml to 10 ng/ml. Isosorbide dinitrate (ISDN) was used as the internal standard and the analytes were extracted with dichloromethane from the plasma. The mass spectrometric tests were carried out using chemical ionization in the negative mode (NlCl) with the application of ammonia as a reagent gas. The nitrate ion m/z 62 was determined in the analytes and internal standard. The accuracy of the mean of the quality control samples during the three days (between days) was between 100 and 110% (PETN), as well as 90 and 106% (PE-tri-N). After an oral application of 100 mg PETN in a pilot study, unchanged PETN and PE-tri-N was measured in plasma. Both metabolites PE-di-N and PE-mono-N were validated at the concentration range of 0.25 ng/ml to 25 ng/ml plasma. After extraction, these analytes were derivatized with BSTFA (N,O-bis[trimethylsilyl]trifluoro-acetamide). The applied internal standard was isosorbide-5-mononitrate (IS-5-MN). The mass spectrometric tests were carried out in the same manner as for PETN and PE-tri-N with chemical ionization in the NlCl mode. The detected masses were m/z 324 for PE-di-N, m/z 351 for PE-mono-N and m/z 217 for IS-5-MN. The accuracy of the mean of the quality control samples during 5 days were between 104 and 107% (PE-di-N) and 102 and 106% (PE-mono-N). The maximum concentration of these analytes in the subject samples were on the average all over 5 ng/ml plasma after the oral administration of 100 mg PETN.

In vivo effects of pentaerythrityl-tetranitrate and isosorbide-5-mononitrate on the development of atherosclerosis and endothelial dysfunction in cholesterol-fed rabbits.

We wished to determine whether long-term treatment with organic nitrovasodilators has pharmacological effects on the development of atherosclerotic lesions and endothelial dysfunction in cholesterol-fed rabbits. For 15 weeks, six groups of 9 New Zealand White rabbits received a standard diet, which contained no admixture, pentaerythrityl-tetranitrate (PETN 6 mg/kg body weight/day), or isosorbide-5-mononitrate (ISMN 2 mg/kg body weight/day). In the other three groups, the same diets were further enriched with cholesterol (0,75%). Four rings of thoracic aorta were used for tension studies; these rings and the aortas from the aortic arch to bifurcation were then fixed in formol and stained with Sudan IV to determine the area of luminal atherosclerotic lesions by a computerized laser-scanning approach. The cholesterol diet increased plasma levels of cholesterol from 69.8 +/- 10.4 to 907.1 +/- 85.5 mg/dl. A similar result was obtained in the group receiving PETN/cholesterol, but the group fed ISMN/cholesterol showed a significantly higher plasma level of cholesterol (1,165 +/- 81.4 mg/dl). Plasma levels of PETN metabolites were still detectable by gas chromatography/mass spectrometry after a 24-h in vivo washout period. The cholesterol diet induced a pronounced degree of atherosclerotic lesions in the aortic arch and the thoracic and abdominal aorta: 73.3 +/- 1.9, 46.3 +/- 2.5, and 49.6 +/- 3.6%, respectively. Additional treatment with PETN resulted in a reduction of this atherosclerotic area to 58.6 +/- 2.05% (p < 0.0001), 34.7 +/- 1.98% (p < 0.01), and 39.3 +/- 3.06% (p < 0.05). In contrast, ISMN had no significant effect on this parameter. The cholesterol diet also induced an endothelial dysfunction as indicated by the diminished vasorelaxation induced by acetylcholine (ACh). Treatment with PETN completely inhibited the development of endothelial dysfunction, whereas ISMN had no effect. In the three groups receiving a cholesterol diet, an increased extent of aortic lesions significantly correlated with increased endothelial dysfunction measured in the same preparations. The long-term treatment with PETN did not affect the vasorelaxing potency of PETN in aortic rings, and similar results were obtained in the case of ISMN. We conclude that long-term treatment with doses of PETN, which do not promote the development of in vitro vascular nitrate tolerance, may protect against atherosclerosis and endothelial dysfunction. This novel, yet unknown pharmacodynamic quality of nitrovasodilators like PETN may contribute to their long-term efficacy in coronary artery disease but may also imply new therapeutic indications in the future.

Pathophysiological significance of the global and regional left-ventricular ejection fraction (LV-EF) on ischaemic heart disease patients at rest and during ergometric exercise load (from radiocardio-cyclography (RKG-RCG) to parametric amplitude and phase scan).

As early as in the graphical RKG-RCG period a close inverse correlation could be established between the LV-EF and the serum myoglobin level during the acute course of myocardial infarction, in 10 patients in repeated follow up examinations. Corr. coeff. -0.91, p less than 0.01. In the mid seventies it could be shown by RKG-RCG, in 15 IHD patients with angina pectoris that the decrease of the basal LV-EF during ergometric load reflected the severity of IHD, compared with the increasing LV-EF tendency of 15 normal subjects. This fact could be verified on 19 middle age males (mean age, 41 years) by 99mTc RBC gamma camera ventriculography, i.e. that under modest load (100 W ergometry) a more than 10% decrease was a non-specific sign of main branch or three-vessel coronary heart disease. So in this extreme case our nuclear stethoscope-like RKG-RCG method alone may be satisfactory for staging and screening of coronary ischaemic heart disease (IHD) patients. All the 11 normal subjects belonged to the load-reaction group with more than 5% LV-EF increase, while the extensive anterior and inferior scar patients reacted without exception with more than 10% deficit (their basal LV-EF value was already under 45%). Supported by data in the literature in the comparison of load ECG and coronarography and two-step load, we could gain more refined data, but in accordance with the one-step load on the same patients. As regards the reproducibility of our global LV-EF investigations with gamma camera computer program Supersegams, it was within 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of the in vitro degradation of [14C] pentaerythritol tetranitrate in rat and human blood with an improved thin-layer radiochromatographic procedure.

Improvements were made on a reported thin-layer radiochromatographic assay for the determination of [14C]pentaerythritol tetranitrate (PETN) and its metabolites in whole blood, using methanol instead of dioxane as the extracting solvent. Recovery of total radioactivity for the entire work-up procedure was greater than 90%, and the distribution of PETN and its metabolites in degraded blood samples was found to be reproducible. This modified method appeared simpler and yielded better recovery of radioactivity than the literature method. In vitro metabolism of [14C]PETN in rat and human blood was examined by incubation of the drug with fresh blood at 37 degrees C for 60 min. In rat blood, the half-life of PETN degradation was about 15 min producing the trinitrate, dinitrate and mononitrate metabolites. Human blood was also capable of degrading PETN in vitro, but at a lower rate than rat blood, yielding only the trinitrate metabolite in quantifiable amounts during the incubation period. Equilibrium of PETN between plasma and red blood cells was observed within 1 min after PETN addition to both rat and human blood. The apparent plasma/red blood cells partition ratios of PETN were 1.1 and 1.7 for rat and human blood, respectively. PETN degradation was approximately ten times slower in rat plasma than in rat blood, suggesting that enzymes in erythrocytes are important for PETN metabolism in rat whole blood.

Measurement of plasma concentrations of vasodilators and metabolites by the TEA Analyzer.

The determination of therapeutic levels of glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate (PETN), and their metabolites in human plasma was conducted using the TEA Analyzer interfaced to a high-performance liquid chromatograph (HPLC-TEA). Chromatographic separation of these compounds was accomplished under isocratic HPLC conditions. The sensitivity of the instrument was established to be 0.1 ng for each of the three nitrate esters. At the 5 ng ml-1 plasma fortification level, the precision of the method gives mean relative standard deviations (RSD) of +/- 10.2 per cent, +/- 6.5 per cent, and +/- 5.9 per cent for GTN, ISDN, and PETN, respectively. The recoveries for the nitrate esters and their metabolites from human plasma over the range 1-50 ng ml-1 are presented. The specific and sensitive HPLC-TEA method developed in this study provides a reliable and rapid assay for the routine analysis for metabolism or bioavailability studies of nitrate esters and their metabolites in plasma of blood, with a detection limit as low as 1 ng ml-1.

A study of the plasma levels of pentaerythritol mononitrate following administration of pentaerythritol tetranitrate in combination with meprobamate and diphenhydramine.

The systemic absorption of meprobamate, diphenhydramine and pentaerythritol tetranitrate (PETN) has been demonstrated following oral administration of a formulation containing all three drug substances to human volunteers. A study undertaken in dogs has also been made of the pharmacokinetics of the major nitrated metabolite of PETN when the parent drug is administered with and without meprobamate and diphenhydramine. Pentaerythritol mononitrate shows a six-fold increase in both peak plasma concentrations and area under the 0-12 hour plasma concentration-time curve when PETN is co-administered with a combination of meprobamate, diphenhydramine and nicotinic acid. No such increase is apparent when either meprobamate or diphenhydramine is excluded from the dose. Further increases in pentaerythritol mononitrate plasma levels and AUC 0-12 h are observed when all of the drugs are administered as the formulated coated tablet (VisanoCor).

Blood levels of the metabolites of glyceryl trinitrate and pentaerythritol tetranitrate after administration of a two-step preparation.

Blood levels and urinary excretion rates of glyceryl trinitrate- pentaerythritol tetranitrate, and their less nitrate containing metabolites have been determined in ten human volunteers after a single dose of a two- step preparation containing glyceryl trinitrate and pentaerythritol tetranitrate. Blood levels accounted for peak levels of about 40% of the glyceryl trinitrate and 0.4% of the pentaerythritol tetranitrate metabolites, respectively. Within the first 24 h post administration 22% of the glyceryl trinitrate and 19% of the pentaerythritol tetranitrate were excreted as nitrate metabolites, chiefly in form of conjugates. The determinations were obtained by gas chromatography on extremely inactive columns and electron capture detection by means of derivatives.

High-performance liquid chromatographic determinaton of pentaerythritol in plasma.

A sensitive analysis of pentaerythritol in plasma has been devised, based on the formation of its tetra-p-methoxybenzoate derivative and high-performance liquid chromatography employing an ultraviolet photometric detector. The method permits analysis of pentaerythritol in the ppm range.

Pentaerythritol tetranitrate and metabolites in rat plasma.

Intact pentaerythritol (PE) tetranitrate and all seven of its metabolites were present in blood withdrawn from the hearts of rats dosed by gavage with 14-C-PE tetranitrate (10 mg/kg). The plasma half-life (T 1/2) of PE tetranitrate was 2 hours which is far longer than the rat T 1/2 values of all other organic nitrates in clinical use. PE trinitrate, the obligatory metabolite of PE tetranitrate now in clinical trial, establishes much higher plasma levels and has a longer T 1/2 (3 hours) than its parent drug. PE trinitrate glucuronide acts as a reservoir for its aglycone and remains in blood for 48 hours after PE tetranitrate administration.