Development and validation of an LC-MS/MS method for simultaneously determining doxapram and keto-doxapram in human serum.

AIM: Doxapram, a respiratory stimulant, is used to treat apnea. A reliable method of determining doxapram in blood is required for monitoring purposes. RESULTS: Doxapram, keto-doxapram (active metabolite) and propranolol (internal standard) were extracted from human serum by protein precipitation and plate filtration. Molecular ions were generated by electrospray ionization in positive ion mode, and the ions were analyzed using a triple-quadrupole mass spectrometer. The calibration curves were linear from 20 to 5000 ng/ml. The method was validated and the selectivity, reproducibility and stability met the acceptance criteria. CONCLUSION: An LC-MS/MS method was successfully developed for determining doxapram and keto-doxapram in human serum. The method can be used to monitor doxapram and keto-doxapram concentrations in blood.

Doxapram metabolism in human fetal hepatic organ culture.

The biotransformation of doxapram, a respiratory stimulant was studied with use of explants from human fetal livers (n = 15 fetuses) obtained from therapeutic abortions (gestational age, 10 to 20 weeks). Explants were cultured in Leibowitz medium and the media from cultured samples were collected before and at 3, 6, 12, and 24 hours after incubation with 2.5, 5.0, and 10 micrograms/ml doxapram. The concentrations of doxapram and its metabolites (AHR 0914, an analog of doxapram, AHR 5955 or ketodoxapram, and AHR 5904) were measured by high pressure liquid chromatography. Explant histopathology and alkaline phosphatase activity showed no direct toxic effects of the drug on liver tissue. The fastest rate of doxapram metabolism occurred during the first 3 hours of incubation (198 +/- 73.3, 438 +/- 63.3, and 538 +/- 62 ng/mg/hr liver protein at doxapram concentrations of 2.5, 5.0, and 10.0 micrograms/ml, respectively). At 3 hours of incubation, the amount of doxapram metabolized (nanogram per milligram of liver protein) was significantly higher (p less than 0.01) at doxapram concentrations of 10.0 (1616 +/- 186) and 5.0 microgram/ml (1315 +/- 190) than at 2.5 micrograms/ml (594 +/- 220). The oxidative pathway producing keto-doxapram, or AHR 5955 and AHR 5904, is more active than the de-ethylation producing the analog of doxapram AHR 0914. Data indicate substantial metabolism of doxapram by the human fetal lives.

Pharmacodynamic effects and pharmacokinetic profiles of keto-doxapram and doxapram in newborn lambs.

Keto-doxapram (keto-dox), an oxidative metabolite of doxapram, is a possible ventilatory stimulating agent. Our study characterizes its ventilatory properties, pharmacodynamic effects, and pharmacokinetic profile, and those of its parent compound, doxapram. Two groups of five awake, unsedated, newborn lambs (2- to 6-d old) received, respectively, i.v. infusions of keto-dox or doxapram (2.5 mg/kg) over a period of 1 min. Ventilatory parameters were continuously recorded before and for 1 h after the drug infusion. The pharmacokinetic profiles of both drugs were determined from blood samples collected serially before and after drug injection. Both drugs stimulated ventilation. Keto-dox increased baseline minute ventilation by 46 +/- 6.1% and 27.8 +/- 8.1% (p less than 0.002) at 1 and 5 min, respectively, an effect that decreased after 5 min of infusion. Doxapram increased minute ventilation by 57 +/- 9% (p less than 0.002) at 1 min, and by 48 +/- 7% at 5 min, but its effect lasted for 20 min after injection. Compared with the effects of keto-dox, this doxapram increase was significantly higher (p less than 0.02). Also, doxapram, but not keto-dox, caused an increase in systolic blood pressure (from 110 +/- 3.5 to 118 +/- 3.4 mm Hg at 10 min, p less than 0.01), as well as a change in neuro-behavior. Both drugs exhibited a biexponential decay curve, characterized by a short alpha and a longer beta t1/2, but keto-dox has a faster elimination rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Ventilatory response to keto-doxapram in intact and carotid body denervated lambs.

We aimed to investigate the role of the carotid bodies in the ventilatory response to keto-doxapram, and whether this response was dose-dependent; we studied two group (n = 5 per group) of awake, intact and carotid-body denervated (CBD) lambs, aged 10-15 days. At 20 min intervals, they received 0.5, 1.0, 2.5 and 5.0 m g/kg mg/kg of keto-doxapram as an intravenous bolus infusion. Plasma keto-doxapram was measured (HPLC). Ventilation was recorded via a face mask and a pneumotachograph. In intact lambs, an immediate dose-dependent increase in minute ventilation (V1) was observed. At 2 min, VI increased from baseline by 125 +/- 28, 212 +/- 49, 378 +/- 41 and 637 +/- 92 mol.kg 1.min 1 (mean +/- SE, P less than 0.01) corresponding to the foregoing incremental doses. A significant correlation was observed between the peak VI and the corresponding plasma keto-doxapram concentrations (r = 0.73, P less than 0.0003). In CBD lambs, VI increased significantly less than in intact animals. In conclusion, early ventilatory response to keto-doxapram depends mainly on intact carotid bodies, and the effect is dose-dependent.

Influence of charge and polarity on the redox potentials of high-potential iron-sulfur proteins: evidence for the existence of two groups.

We have investigated the HiPIPs from Ectothiorhodospira vacuolata (iso-1 and iso-2), Chromatium vinosum, Rhodocyclus gelatinosus, Rhodocyclus tenuis (strain 2761), Rhodopila globiformis, and Rhodospirillum salinarum (iso-2) by direct electrochemistry. Using a glassy carbon electrode with a negatively charged surface, direct, unpromoted electrochemistry is possible with the positively charged HiPIPs. With the negatively charged HiPIPs, the positively charged and flexible bridging promoter poly(L-lysine) is required. The stability of the response can be improved by morpholin, aspartate, tryptophan, or 4,4'-dipyridyl. These "stabilizers" prevent the blocking of the electrode by denatured protein. The redox potential of 500 mV found for R. salinarum iso-2 is the highest HiPIP potential reported. The presence of histidines in the sequence does not per se predict a pH-dependent redox potential. Only C. vinosum and R. gelatinosus HiPIPs show a weak but significant pH dependence with a difference of 35 mV between the low- and the high-pH form and maximum slopes of -20 mV/unit. The dependence of the midpoint potential on temperature and on ionic strength varies over the different HiPIPs. The dependence of the potentials on square root of I cannot be fully explained by the Debye-Hückel theory because the linearity exceeds the limiting concentration and only small negative slopes are observed (o to -28 mV/square root of M) Combination of the sequences, the optical spectra, the overall charges, and the redox thermodynamics suggests that existence of two groups of HiPIPs. One group consists of Chromatium-like HiPIPs with redox potentials between 300 and 350 mV, modulated only by the solvation of the cluster. The second group is formed by Ectothiorhodospira-like HiPIPS with potentials between 50 and 500 mV, modulated by the overall charge of the peptide (25 mV/unit) and by the solvation of the cluster.