Simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram in plasma using liquid chromatography-tandem mass spectrometry.

A simple and specific UPLC-MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto-doxapram. The internal standard was fentanyl-d5 for all analytes. Chromatographic separation was achieved with a reversed-phase Acquity UPLC HSS T3 column with a run-time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli-Q ultrapure water or in methanol with a total flow rate of 0.4 mL min-1 . A plasma volume of only 50 µL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto-doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run-time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run.

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.

[Analysis of 37 drugs in whole blood by HPLC after solid phase extraction].

OBJECTIVE: To develop a specific, sensitive, reproducible SPE-HPLC method for the determination of 37 drugs in whole blood. METHODS: With the doxapram as internal standard, Oasis column was used to extract drugs from whole blood. Two kinds of mobile phases were used in this study. Separations were achieved by a LiChrospher 100 RP-C18 (250 mm x 4.0 mm x 5 microm) column kept at 50 degrees C, the DAD detector was set at 230 nm and 250 nm. RESULTS: The limit of detection were 1-30 ng/mL. The method showed excellent linearity and the linear correlation coefficient was > or =0.997 98. The relative standard deviation for between-day and within-day assay were <10%. CONCLUSION: The method is effective, simple, reliable and has been used in real cases.

Doxapram only slightly reduces the shivering threshold in healthy volunteers.

We determined the effects of doxapram on the major autonomic thermoregulatory responses in humans. Nine healthy volunteers were studied on 2 days: control and doxapram (IV infusion to a plasma concentration of 2.4 +/- 0.8, 2.5 +/- 0.9, and 2.6 +/- 1.1 microg/mL at the sweating, vasoconstriction, and shivering thresholds, respectively). Each day, skin and core temperatures were increased to provoke sweating, then reduced to elicit peripheral vasoconstriction and shivering. We determined the sweating, vasoconstriction, and shivering thresholds with compensation for changes in skin temperature. Data were analyzed with paired t-tests and presented as mean +/- sd; P < 0.05 was considered statistically significant. Doxapram did not change the sweating (control: 37.5 degrees +/- 0.4 degrees C, doxapram: 37.3 degrees +/- 0.4 degrees C; P = 0.290) or the vasoconstriction threshold (36.8 degrees +/- 0.7 degrees C versus 36.4 degrees +/- 0.5 degrees C; P = 0.110). However, it significantly reduced the shivering threshold from 36.2 degrees +/- 0.5 degrees C to 35.7 degrees +/- 0.7 degrees C (P = 0.012). No sedation or symptoms of panic were observed on either study day. The observed reduction in the shivering threshold explains the drug's efficacy for treatment of postoperative shivering; however, a reduction of only 0.5 degrees C is unlikely to markedly facilitate induction of therapeutic hypothermia as a sole drug.

QT interval lengthening in premature infants treated with doxapram.

OBJECTIVE: Doxapram, routinely used in premature infants treated for apnea of prematurity unresponsive to methylxanthines, has been related to cardiac conduction disorders. This study was designed to evaluate doxapram cardiac and general tolerance and its relationship to drug plasma concentrations in very premature infants. METHODS: Forty infants (mean +/- SEM, 28.9 +/- 0.3 weeks of gestation) who were given intravenous doxapram, 0.5 to 1 mg/kg per hour, at 15.9 +/- 2.4 days of life were evaluated prospectively. Electrocardiograms were monitored before and during the first 3 days of treatment. QT interval corrected for heart rate (QTc) longer than 440 ms was regarded as clinically pertinent, given that it is considered a significant risk of conduction disorder leading to torsades de pointes and sudden death. Other side effects were recorded. Toxic plasma concentration of doxapram and ketodoxapram was set at >4 mg/L. RESULTS: A statistically significant but moderate lengthening of QTc interval has been observed from 394 +/- 4 ms before doxapram to 409 +/- 4 ms at 48 and 72 hours of treatment (P =.0065). For 6 patients, QTc interval became longer than 440 ms without any other rhythm or conduction disorder. Digestive disorders were observed in 20 infants but 9 presented with concomitant septicemia. No relationship was found between presence or absence of adverse effects and drug plasma concentrations. CONCLUSION: Our study enlightened the lengthening effect of doxapram on QTc interval in premature infants with a risk of exceeding the 440 ms threshold that is considered life-threatening. This finding emphasizes the need for electrocardiogram follow-up when using doxapram in neonates.

[A comparative study about the therapeutic effect of theophylline and doxapram in apnoeic disorders]. / Vergleichende Untersuchung zur Wirkung von Theophyllin und Doxapram bei Apnoen.

The objective of this study was to prove the superiority of doxapram compared to theophylline therapy in apneas of prematurity in very low birth weight infants. Therefore all VLBW infants (gestational age < 35 weeks) were randomized if they had in a 2 hours-interval more than 2 apneas, 4 bradycardias or 4 oxygen desaturations. They received either theophylline (loading dose 5 mg/kg b. w., 3 mg/kg b. w. bid) or doxapram by continuous infusion of 0.5 mg/kg/h. Apneas, bradycardias and desaturations were recorded from the trend analysis of our monitoring system over the first 3-days and a 7 days period and compared statistically (Mann-Whitney U-test). Plasma levels of both drugs and a polysomnographic recording were obtained during steady state conditions in parallel to a behavioral observation according to Prechtl. The recorded events were again compared using the Mann-Whitney U-test. Twenty patients were treated with theophylline, 14 with doxapram. In 9 patients of each group we could perform a polysomnography and behavioral observation. The incidence of apneas, bradycardias and desaturations in a 7 days-interval was not significantly different between both groups. Analyzing the first 3 days of treatment, however, we could detect a significantly lower rate of apneas in the doxapram group (2.5 apneas compared to 7 in the theophylline group, p < 0.037). In the polysomnographic recording and in our behavioral observations we could not record any significant differences between both groups. Therefore we can conclude that theophylline and doxapram are comparable in the treatment of apneas of prematurity, however, doxapram is superior to theophylline in reducing the rate of apneas in the first 3 days of treatment.

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.

Low-dose doxapram therapy in premature infants and its CSF and serum concentrations.

The efficacy of low-dose doxapram therapy (0.2 mg/kg/h) in combination with methylxanthines was evaluated in 20 premature infants with idiopathic apnea unresponsive to methylxanthines alone, and in 13 premature infants with secondary apnea. The serum concentrations of doxapram and, in some infants, the simultaneous cerebrospinal fluid and serum concentrations were measured, and the correlation between cerebrospinal fluid and serum concentrations in the postnatal period was determined. The following results were obtained: 1) In idiopathic apnea of prematurity, low-dose doxapram therapy was as effective as a dose of 1.0-2.5 mg/kg/h and the side effects were few, mild, and reversible. 2) In premature infants over seven days of age, serum concentrations of doxapram were almost stable but were significantly lower than in infants within the first six days of life. 3) The ratio of the cerebrospinal fluid to serum doxapram concentration was 0.48 +/- 0.13 (mean +/- SD). There was a good correlation between cerebrospinal fluid and serum concentrations (r = 0.933, p less than 0.001). The initial doxapram dose can be set as low as 0.2 mg/kg/h in very young premature infants with idiopathic apnea of prematurity unresponsive to methylxanthines.

Clinical and physiological responses to prolonged nasogastric administration of doxapram for apnea of prematurity.

We hypothesized that enteral doxapram would effectively treat apnea of prematurity without the appearance of major side effects. Of 16 infants, 10 (BW 1,520 +/- 102 g) received doxapram alone and 6 (BW 1,020 +/- 35 g) received doxapram plus theophylline. Apneas decreased from 16.7 +/- 1.9 to 2.1 +/- 0.6 in infants receiving doxapram alone, and from 38.2 +/- 4.4 to 7.9 +/- 2.2 apneas/24 h in those receiving doxapram plus theophylline. This was associated with an increase in alveolar ventilation, a shift of the ventilatory response to CO2 to the left, and no change in the immediate ventilatory response to 100% oxygen. Side effects included premature teeth buds corresponding to the lower central incisors, prevalence of occult blood in stool and necrotizing enterocolitis. The findings suggest that doxapram effectively controls apnea when given enterally, but should be used cautiously because of potentially harmful side effects.

A blinded, randomized, placebo-controlled trial to compare theophylline and doxapram for the treatment of apnea of prematurity.

A blinded, randomized, placebo-controlled trial was conducted to evaluate the effectiveness of theophylline and doxapram therapy in 31 infants with significant apnea of prematurity. Of 10 infants, two had a short-term response to placebo, 8 of 10 infants to theophylline, and 7 of 11 infants to doxapram (placebo vs treatment with theophylline or doxapram: p = 0.01). The two infants who initially responded to placebo remained responsive for the duration of the study. Of the eight infants in whom treatment with placebo failed, five were randomly assigned to receive theophylline, for a total of 15 infants treated with theophylline, and two of the eight were randomly assigned to receive doxapram, for a total of 13 infants treated with doxapram; the remaining infant required tracheal intubation. Of the 15 infants randomly assigned to receive theophylline, seven responded for the duration of the study; of the eight infants who did not respond to treatment with theophylline, five responded to doxapram, one responded to a combination of theophylline and doxapram, and two remained resistant to treatment. Of the 13 infants randomly assigned to receive doxapram four responded for the duration of the study; of the nine who did not respond to doxapram, seven responded to theophylline, one responded to a combination of theophylline and doxapram, and one remained resistant to treatment. This study demonstrates that although therapy with theophylline or doxapram is associated with a significant short-term reduction in the incidence of apnea compared with that in placebo-treated infants, the long-term response to treatment is frequently incomplete and is not sustained more than 1 week.

Effects of caffeine and doxapram perfusion on local cerebral glucose utilization in conscious rats.

The quantitative autoradiographic 2-[14C]deoxyglucose method was used to measure the effects of a continuous infusion of the respiratory stimulants, caffeine or doxapram, 18 mg/kg per h, on local cerebral glucose utilization in the adult male rat. Local cerebral glucose utilization was measured in 54 cerebral structures from different systems. Caffeine induced widespread increases in energy metabolism, resulting in a significant increase in glucose utilization in 25 structures out of the 54 studied. These increases were distributed within all systems studied, sensory, extrapyramidal motor, limbic and hypothalamic systems. In addition, caffeine induced a non-significant, 10-15%, increase in local cerebral glucose utilization in central respiratory areas. Doxapram infusion did not change the rates of glucose utilization in any of the structures. The rates of local cerebral glucose utilization were significantly lower after doxapram than after caffeine exposure in five cerebral areas, among which were three central respiratory areas. The results confirm the absence of side-effects of doxapram as compared to caffeine when used as respiratory stimulant, especially in neonates. These results also favor a preferentially central action of caffeine on respiratory areas and a more peripheral action of doxapram on chemoreceptors, at least at therapeutic levels.

Effect of oral doxapram on morphine-induced changes in the ventilatory response to carbon dioxide.

A double-blind crossover volunteer trial has been carried out to determine if oral doxapram reduces the respiratory depression caused by morphine 0.12 mg kg-1 i.m. Doxapram was given to the subjects 90 min before the morphine and significantly reduced the displacement of the ventilatory response to carbon dioxide caused by the morphine. This occurred despite the fact that the plasma concentrations of doxapram were significantly lower when the subject had been given morphine than when a placebo injection had been administered. Doxapram alone in doses of 300 mg and 600 mg did not alter significantly the respiratory variables measured in this study.

Serum doxapram and respiratory neuromuscular drive in normal man.

To investigate the means by which doxapram affects the control of ventilation, ventilatory function and P0.1 have been related to serum doxapram concentration during a 45-min infusion of doxapram hydrochloride in 7 healthy, conscious subjects under normoxic conditions. Serum doxapram concentrations increased during the infusion: 1.88, 2.48, 3.42, and 3.97 micrograms/ml after 5, 10, 30 and 45 min, respectively. The majority of significant changes in the measurements from the baseline were observed at 30 and 45 min: VE, VT, P0.1, P0.1/end-tidal CO2 tension, VT/Ti and blood pressure were increased, and end-tidal CO2 tension was decreased. No significant changes in Pdimax, Ti/Ttot, VE/P0.1, and P0.1/(VT/Ti) were observed. A correlation was observed between the % increases in P0.1 and VE and doxapram concentration, and between VE and P0.1. The doxapram-induced increase in VE appears to be caused by increased neural drive. It is related to the serum drug concentration in the conscious subject.

Pharmacokinetics of doxapram in idiopathic apnea of prematurity.

Pharmacokinetics of doxapram were determined in 13 infants with idiopathic apnea of prematurity uncontrolled by aminophylline and caffeine. Doxapram was maintained for 72-96 h at a constant infusion rate of 2-2.5 mg/kg/h. Plasma doxapram levels were measured by gas liquid chromatography. The infants studied had a birth weight of 1,247 +/- 240 g (mean +/- SD), a gestational age of 29.4 +/- 2 weeks and were 9.9 +/- 6 days old. Steady-state plasma doxapram levels reached by all infants averaged 5.8 +/- 1.8 mg/l. Half-life was 6.6 +/- 5.7 h, plasma clearance 0.44 +/- 0.1 litres/kg/h, and calculated volume of distribution 4 +/- 2.7 litres/kg. Doxapram controlled apnea successfully in 12 of 13 babies. A significant fall in PaCO2 and reduction in the rate of apnea was seen within 6-8 h with corresponding doxapram levels of 3.7 +/- 1.8 mg/l. The factors influencing the pharmacokinetics of doxapram in newborns are presently unknown.

Dose-response relationship of doxapram in the therapy for refractory idiopathic apnea of prematurity.

Eighteen infants with idiopathic apnea of prematurity refractory to therapeutic levels of aminophylline were treated with incremental doses of doxapram beginning at 0.5 mg/kg/h. Continuous recording of heart rate, thoracic impedance, and transcutaneous PO2 demonstrated that 47% of the infants satisfied objective response criteria at the lowest dose, 53% responded at 1.0 mg/kg/h, 65% at 1.5 mg/kg/h, 82% at 2.0 mg/kg/h, and 89% at the highest allowed dose of 2.5 mg/kg/h. The mean serum doxapram concentration at the response dose was 2.9 +/- 1.3 micrograms/mL, and all infants who responded had levels greater than 1.5 micrograms/mL. BP was significantly elevated at doses higher than 1.5 mg/kg/h (P less than .05). Minute ventilation significantly increased and PCO2 significantly decreased as the doxapram dosage was increased (P = .02). Terminal elimination half-life was 9.9 +/- 2.9 hours. When doxapram is used for treatment of refractory neonatal apnea the starting dosage should be no more than 0.5 mg/kg/h.