Optimisation of the enantiomeric separation of 12 2-aminotetralin analogues using Chiral AGP high-performance liquid chromatography by simultaneous factorial design.

A method for the simultaneous optimisation of mobile phase composition for the resolution of pairs of enantiomers of 12 2-aminotetralin analogues is presented. The selectivity necessary to discriminate between 12 analytes was obtained by using mass selective detection. The ability to examine more than a few analytes at a time extends the otherwise limited applicability of a factorial design strategy to the rapid development of chiral assays.

Synthesis of glycosides in which the aglycon is an N-(hydroxymethyl)amino-1,3,5-triazine derivative.

The synthesis of analogues of the anti-tumour drug 2-[N-(hydroxymethyl)methylamino]-4,6-bis(dimethylamino)-1,3,5-triazine (HMPMM) in which the OH or a dimethylamino group is replaced by a carbohydrate has been explored. Triazinyl beta-glycosides were readily prepared by reaction of sugars with trimethyl-triazinylammonium salts. These were made with one or two methylamino groups on the triazine for reaction with formaldehyde to give the cytotoxic NMeCH2OH group. However, reaction of the triazinyl glycosides with formaldehyde gave complex intractable mixtures. When the carbohydrate portion was changed to the fully protected 2,3,4,6-tetra-O-acetyl glucose a good yield of the 2-[N-(hydroxymethyl)methylamino]-4-(dimethylamino)-1,3,5-triazin-2 -yl tetra-O-acetyl beta-glucoside was obtained. However, de-acetylation using sodium methoxide also removed the N-CH2OH group. We are investigating protection of the base-sensitive N-CH2OH group as trialkylsilyl and benzyl ethers and are looking at de-acetylation methods that are more selective. We have prepared glycosides in which the sugar is joined through the oxygen of the NMeCH2OH group. Coupling of acetobromoglucose with HMPMM catalysed by silver salts was not successful. Although methyl and cyclohexyl derivatives of HMPMM may be produced in high yields by reaction of HMPMM with methyl and cyclohexyl alcohols under acidic catalysis, production of glycosides in this way gave poor yields. MNDO calculations on reactions of HMPMM helped us devise improved reaction conditions for the condensation of 2,3,4,6-tetra-O-acetyl glucose with HMPMM and its derivatives. The best procedure to generate one of the target glycosides is to react 2,3,4,6-tetra-O-acetyl glucose and formaldehyde with 2-methylamino-4,6-bis(dimethylamino)-1,3,5-triazine. The beta-glycoside product was de-acetylated using potassium carbonate in dry methanol.

Development of chiral high performance liquid chromatographic assays for the enantiomers of 8-hydroxy-(di-n-propylamino)tetralin and three 8-keto-pyrrole-substituted analogues.

Two chiral HPLC methods using protein-based stationary phases for the analysis of 8-hydroxy-(di-n-propylamino)tetralin and three 8-keto-pyrrole analogues are presented. Efficient solid-phase extraction enabled quantification of 0.02 microgram ml-1 of the 8-keto-pyrrole analogues from 500 microliters rat hepatocyte suspensions, and 0.2 microgram ml-1 to be measured from 50 microliters of rat plasma using UV detection at 315 nm.

Stereoselective metabolism of two keto-pyrrol analogues of 8-hydroxy-2-dipropyl-aminotetralin by freshly isolated rat hepatocytes.

In vitro metabolism models have been used to determine the relative metabolic stability of novel 2-aminotetralin analogues for the treatment of CNS diseases. Few of these new compounds had been produced as stereochemically pure materials and the achiral analytical techniques, used initially, measured the average metabolic clearance of the two enantiomers of the racemic mixtures. A chiral HPLC assay, using a Chiral AGP column, was developed for two of these racemic analogues and was used to measure the clearance of the enantiomers from suspensions of freshly isolated rat hepatocytes. Robust separations were obtained for both compounds and a number of metabolic products. The enantiomers of only one analogue were subject to different rates of metabolism. The extent of the difference was dependent upon the initial starting concentration of the incubation. The identity of certain metabolites was investigated using LC/MS. The enantio-selectivity appears to have arisen from the restricted hydroxylation of one analogue compared to that of the other.

High-performance liquid chromatographic determination of spectinomycin in swine, calf and chicken plasma using post-column derivatization.

An HPLC method for the determination of spectinomycin in swine, calf and chicken plasma at 0.1 microgram/ml or higher is described. The clean-up is based upon ion-pair solid-phase extraction on a High Hydrophobic C18 column treated with sodium dioctyl sulfosuccinate. After elution with methanol, spectinomycin is chromatographed on a Spherisorb SCX column using 0.1 M sodium sulphate solution (pH 2.6)-acetonitrile (80:20, v/v) as mobile phase. Fluorescence detection is at an excitation wavelength of 340 nm and an emission wavelength of 460 nm after post-column oxidation with sodium hypochlorite followed by derivatization with o-phthaldialdehyde. Mean recoveries were 99 +/- 2% (n = 6), 99 +/- 2% (n = 7) and 104 +/- 2% (n = 6) for swine, calf and chicken plasma, respectively, at the 0.1 microgram/ml level.

The development and validation of a high performance liquid chromatography (HPLC)/tandem mass spectrometry assay for fenticonazole in human plasma and comparison with an HPLC-UV method.

A method is described for the determination of fenticonazole in human female plasma. The method utilizes high performance liquid chromatography coupled to atmospheric pressure positive-ion chemical ionization triple quadrupole mass spectrometry. Multiple reaction monitoring is employed for selectivity and sensitivity which enables quantification over the range 0.5-20 ng mL-1 with acceptable precision and accuracy. A comparison is made with an existing HPLC-UV assay and the utility of the technology of combined liquid chromatography and tandem mass spectometry for subnanogram per mL assays is discussed.

The geometry of N-hydroxymethyl compounds. Part 3: Geometries of N-(hydroxymethyl)amides related to reactivities.

Using MNDO calculations, ground state structures of 11 N-(hydroxymethyl)amides were predicted to have pyramidal nitrogens and an s-Z conformation. The results compare favourably with ab initio calculations and with X-ray data except that MNDO tends to predict twist angles around partial double bonds which are too large. A correlation was found between N-CH2OH bond length and the logarithm of the half-life under physiological conditions. MNDO ground state structures for five N-(hydroxymethyl)amide anions were calculated to have s-E conformations and their N-CH2OH bond lengths also correlated with the half-lives of the N-(hydroxymethyl)amides. These correlations may be valuable in assessing potential anti-tumour activity.

The geometry of N-hydroxymethyl compounds. Part 2: Crystal structures of 1-(4-carbethoxyphenyl)-3-hydroxymethyl-3-methyltriazene, N-hydroxymethylpentamethylmelamine and N-hydroxymethylbenzamide.

Of the three N-hydroxymethyl compounds in the title, the first two have pronounced antineoplastic activity while the latter is biologically inactive. Crystals of the triazene have monoclinic symmetry with a = 8.540(1), b = 6.346(4), c = 22.460(5)A, beta = 98.75(2) degrees, and space group P21/c. The melamine forms disordered crystals of orthorhombic symmetry with a = 11.957(3), b = 17.267(3), c = 5.769(3)A. Of the symmetry elements in the observed space group Pnma, a mirror plane bisects the average molecule, implying that the hydroxymethyl group has equal probability of lying either side of this plane. Crystals of the benzamide show orthorhombic symmetry with a = 10.045(6), b = 7.763(3), c = 19.409(8)A, and space group Pbca. All three compounds are intermediates along biochemical demethylation pathways. The observed N-CH2OH distances, which are 1.469(5), 1.452(4), and 1.438(4)A respectively for the three compounds, correlate with the stability of this bond as measured by half-life. It is suggested that the correct degree of lability is important for biological activity, short strong bonds being too unreactive and excessively long ones being too unstable.

Pharmacokinetics of atracurium and its metabolites in patients with normal renal function, and in patients in renal failure.

The plasma pharmacokinetic profiles of atracurium and its derivatives, laudanosine and monoquaternary alcohol, were studied in six patients with renal failure after a bolus dose of atracurium 0.3-0.4 mg kg-1. The pharmacokinetics of the derivatives only were studied in a group of four normal patients receiving atracurium 0.3 mg kg-1. Measurements of plasma and urine concentrations were performed by high pressure liquid chromatography. Pharmacokinetics of atracurium were not significantly different in the renal failure group when compared with those obtained in a previous study on six normal patients. Although 2-10% of the dose was recovered in the urine of normal patients as unchanged atracurium, and 3-4% as laudanosine, renal failure produced no significant differences in plasma pharmacokinetics, with mean plasma elimination half-lives of 20 min for atracurium, 234 min for laudanosine and 39 min for quaternary alcohol.

Clinical experience and plasma laudanosine concentrations during the infusion of atracurium in the intensive therapy unit.

Twenty patients in the intensive care unit received an infusion of atracurium to permit mechanical ventilation. The duration of infusion ranged from 38 to 219 h and the average rate of infusion during the study was 0.76 mg kg-1 h-1. In 14 patients an increase in atracurium requirement occurred within the first 72 h of the infusion. Recovery from neuromuscular blockade after a prolonged infusion was sufficiently rapid to avoid pharmacologically induced reversal. In six patients maximum plasma concentrations of laudanosine were 1.9-5 micrograms ml-1, and there was no evidence of cerebral excitation.

Effect of a failure of energy supply on adenine nucleotide breakdown in placentae and other fetal tissues from rat and guinea pig.

The effects of ischaemia on adenylate energy charge of tissues from fetal rats and fetal guinea pigs were measured. Adult rat and guinea-pig tissues, as well as human placentae, were also studied. The largest differences observed were between the fetuses from different pregnant animals (P = 4.74 X 10(-15). Reductions in energy charge in placentae were slower than in other defined fetal tissues, especially brain. In the rat, an immature species at birth, greater 'stability' was observed in placentae of 14 days of gestation than near term at 20 days of gestation. As contrast, in the guinea pig, a mature species at birth, there was no difference in 'stability' in placenta or other fetal tissues between about 40 days of gestation and near term, about 60 days of gestation. In addition to these tissue and maturity effects in the fetus, it has been confirmed that fetal tissues are more 'resistant' than adult tissues to failures of energy supply. Concentrations of adenosine, uridine, guanosine and cytidine nucleotides in placenta show similar patterns in rats and guinea pigs. Fetal liver contains more uridine nucleotides and brain more cytidine nucleotides. It is suggested that the placenta retains an early fetal ability to maintain itself during ischaemia; this might be advantageous during parturition. Possible endocrine and other mechanisms 'damping' fetoplacental metabolism are linked with a discussion of the large maternal effect.

Studies of the mode of action of antitumor triazenes and triazines. 6. 1-Aryl-3-(hydroxymethyl)-3-methyltriazenes: synthesis, chemistry, and antitumor properties.

1-Aryl-3-(hydroxymethyl)-3-alkyltriazenes [ArN = NN(CH3)CH2OH] have been synthesized by diazonium coupling to the carbinolamine (RNHCH2OH), generated in situ from the alkylamine and formaldehyde mixtures. The (hydroxymethyl)triazene structure has been confirmed by IR, NMR, and mass spectral analysis and also by the preparation of a crystalline benzoate derivative. The mass spectra of the (hydroxymethyl)triazenes suggest that they fragment by loss of formaldehyde to give the methyltriazene, which is also the product of hydrolysis in solution. The degradation of the (hydroxymethyl)triazenes in solution has been followed by UV spectroscopy and by HPLC analysis, and the half-lives were determined under a variety of conditions. The half-lives of the corresponding methyl- and (hydroxymethyl)triazenes are very similar. Both methyl- and (hydroxymethyl)triazenes decompose on silica plates during TLC analysis to give products consistent with known diazo-migration reactions. The (hydroxymethyl)triazenes have pronounced antitumor activity against the TLX5 tumor in vivo; in vivo-in vitro bioassay experiments suggest that the (hydroxymethyl)triazenes exert their in vivo antitumor activity via the degradation product, the alkyltriazene.

Ratio of the concentration of hypoxanthine to creatinine in urine from newborn infants: a possible indicator for the metabolic damage due to hypoxia.

The ratio of the urinary concentrations of the ATP metabolite, hypoxanthine, to that of creatinine was determined in normal newborn infants. An increase in this ratio reflects high hypoxanthine excretion and thus ATP breakdown. The ratio can be determined on random urine samples, thus simplifying sampling. Urinary changes are persistent; abnormalities are detectable on the second day of life after intrapartum hypoxia. Preliminary results suggest that this ratio on a sample during the second day of life could 'diagnose' intrapartum hypoxia and might therefore quantitatively assess those obstetric 'risk factors' believed to operate through hypoxia.

Purine transport and metabolism in man: the effect of exercise on concentrations of purine bases, nucleosides and nucleotides in plasma, urine, leucocytes and erythrocytes.

1. After decreasing muscle ATP by a 2 min period of intense exercise, we have studied purine metabolism by using high-pressure liquid chromatography. 2. A major increase in hypoxanthine concentration in plasma and urine was found with increases in xanthine and, in plasma, inosine. Erythrocyte hypoxanthine rose with the level in plasma, but there was no corresponding rise in IMP, the first intracellular metabolite of hypoxanthine. No rises in uridine or urate were found in plasma. 3. Plasma adenosine did not rise and fall significantly after exercise, but a small rise and fall in adenine nucleotide concentrations in plasma was found. 4. Running, swimming and games, which tended to be at the weekend, were associated with a rise in hypoxanthine and xanthine excretion; exercise was probably the cause of the higher excretion during the day than at night. Such activities do not produce changes in concentrations of ATP in muscle, although turnover must rise. 5. The results are consistent with widespread purine exchange between tissues and a 'circulating hypoxanthine pool'.

Intrapartum hypoxia: the association between neurological assessment of damage and abnormal excretion of ATP metabolites.

A series of 29 newborn infants had been studied after intrapartum hypoxia defined as meconium aspiration, an Apgar score of less than or equal to 6 at 5 min or a peripheral blood pH of 7.2 or less after resuscitation. Two independent sets of techniques were used; one concerned with the critical system in hypoxic damage, the central nervous system, the other assessing the central biochemical events in hypoxia. Both sets of data were assembled, then graded separately and only then combined. In this way detailed neurological assessment has been combined with measurement of urinary excretion of the ATP metabolites, hypoxanthine and xanthine. The essential metabolic consequence of hypoxia is a reduction in the synthesis of the energy currency of cells, ATP. This is associated with an outflow of ATP metabolites from cells. The extent of neurological damage was related to the magnitude of the hypoxanthine and xanthine excretion; neither were closely related to the initial blood pH. Infants who were normal neurologically had normal oxypurine excretion. Infants with neurological abnormalities for less than 48 h had lower excretion than those who were abnormal for more than 48 h. The duration of abnormal oxypurine excretion after an acute episode of hypoxia was studied in two infants with respiratory distress and in two other infants with apnoeic attacks. Severe hypoxia was followed by abnormal oxypurine excretion for at least 40 h after an acute episode. It is justifiable to suggest that abnormalities of oxypurine excretion should indicate intrapartum hypoxia in newborn infants. This excretion should also quantify the metabolic damage.

Activities of enzymes involved in purine metabolism and some related adenine nucleotide concentrations of leucocytes in renal failure.

We have studied purine metabolism in mononuclear and polymorphonuclear cells from uraemic patients using microradiochemical enzyme assays and high-pressure liquid chromatography. In mononuclear cell lysates the mean activities of adenosine deaminase (EC 3.5.4.4) and 5'-nucleotidase (EC 3.1.3.5) were significantly diminished. The activities of adenylate kinase (EC 2.7.4.3), purine nucleoside phosphorylase (EC 2.4.2.1), adenine phosphoribosyltransferase (EC 2.4.2.7), and hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were not significantly different in the two groups. The activities of adenosine deaminase and adenine phosphoribosyltransferase were reduced in the polymorphonuclear cell lysates. No clear differences emerged in the concentration of adenine nucleotides in the mononuclear cells. The significance of these changes, which are less marked than those in erythrocytes, is discussed with reference to the immunodeficiency associated with uraemia.