Liquid chromatographic determination of the plasma concentrations of cefotaxime and desacetylcefotaxime in plasma of critically ill patients.

A method for the simultaneous determination of cefotaxime (CTX) and desacetylcefotaxime (DES) in plasma was developed, using acetonitrile protein precipitation and high-performance liquid chromatography (HPLC) with UV-detection at 285 nm. Desacetylcefotaxime was also analysed after conversion in highly acidic medium to its lactone form (DES-lactone). The chromatographic separation was performed on a C18 Aqua column. The lower limit of quantitation was 1 microg/ml for CTX and 0.5 microg/ml for DES and DES-lactone, using 25 microl of plasma samples. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of > or =0.990. The within-day and between-day precisions were <12% (n = 18) for the two products and the accuracy was between 88 and 101%. The developed HPLC method was applied for CTX and DES determination in plasma samples of critically ill patients after continuous intravenous infusion of CTX.

Development and validation of a high-performance liquid chromatographic method for the determination of gamma-hydroxybutyric acid in rat plasma.

A method for the determination of gamma-hydroxybutyric acid (GHB) in rat plasma was developed using solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with UV detection. GHB was isolated from plasma using strong anion-exchange SPE columns. The chromatographic separation was performed on a C18 Aqua column. The lower limit of quantification was 10 microg/ml using 60 microl of plasma. The linearity of the calibration curves was satisfactory as indicated by correlation coefficients of >0.990. The within-day and between-day precision were <10% (n=24), the accuracy was nearly 101%. Plasma concentrations in rats after GHB infusion determined by HPLC-UV were compared with the corresponding concentrations determined with a validated gas chromatographic-mass spectrometric method by orthogonal distance regression. A good correlation was observed and a t-test indicated no significant differences from 0 and 1 for the intercept and slope, respectively.

Relationship between gamma-hydroxybutyrate plasma concentrations and its electroencephalographic effects in the rat.

In view of the potential interest in an objective parameter for the depth of coma in intoxications with the recreational drug gamma-hydroxybutyrate (GHB), we have studied the relationship between the plasma concentrations and the electroencephalographic (EEG) changes induced by GHB in the rat. Fifteen rats randomly received either 150 (n = 3), 200 (n = 6) or 300 mg kg(-1) (n = 6) GHB over 5 min, followed by a supramaximal dose of 450 mg kg(-1) over 5 min at the end of the experiment. Plasma concentrations were determined with HPLC. The EEG was continuously recorded and the amplitude in the 15.5-30 Hz frequency band was quantified using aperiodic analysis. The plasma concentration-time profiles were fitted to a two-compartment model with Michaelis-Menten elimination. The pharmacokinetic parameters Vmax, Km and the apparent volume of distribution (Vd) proved to be independent of the dose and the mean pooled values were Vmax 2068 +/- 140 microg min(-1) kg(-1), Km 58 +/- 16 microg mL(-1) and Vd 476 +/- 12 mL kg(-1). The EEG amplitude in the 15.5-30 Hz frequency band displayed a monophasic inhibition and the effect-plasma concentration curve showed hysteresis. This hysteresis between EEG effect and plasma concentrations was minimized by simultaneous calculation of hypothetical effect-site concentrations and fitting the effect vs effect-site concentration curve to a sigmoid inhibitory Emax model. The descriptors of this Emax model (Emax, EC50, k(e,0), gamma and E0) were independent of the dose with an equilibration half-life t1/2k(e,0) of 5.6 +/- 0.3 min (mean value of the pooled results of the 5-min treatment groups). To investigate the origin of this hysteresis, a dose of 600 mg kg(-1) GHB was infused over either 45 or 60 min each in three animals. The hysteresis was much less pronounced with 45 min than with 5 min and was absent with 60-min infusions. This indicated that the hysteresis was due to a distribution delay between the central compartment and the effect site. This study showed that the concentration-effect relationship of GHB could be characterized in individual rats using aperiodic analysis in the 15.5-30 Hz frequency band.

Influence of hypovolemia on the pharmacokinetics and the electroencephalographic effect of propofol in the rat.

BACKGROUND: Hypovolemia decreases the dose requirement for anesthetics, but no data are available for propofol. As it is impossible to study this in patients, a rat model was used in which the influence of hypovolemia on the pharmacokinetics and pharmacodynamics of propofol was investigated. METHODS: Animals were randomly allocated to either a control (n = 9) or a hypovolemia (n = 9) group, and propofol was infused (150 mg x kg(-1) x h(-1)) until isoelectric periods of 5 s or longer were observed in the electroencephalogram. The changes observed in the electroencephalogram were quantified using aperiodic analysis and used as a surrogate measure of hypnosis. The righting reflex served as a clinical measure of hypnosis. RESULTS: The propofol dose needed to reach the electroencephalographic end point in the hypovolemic rats was reduced by 60% (P < 0.01). This could be attributed to a decrease in propofol clearance and in distribution volume. Protein binding was similar in both groups. To investigate changes in end organ sensitivity during hypovolemia, the electroencephalographic effect versus effect-site concentration relation was studied. The effect-blood concentration relation was biphasic, exhibiting profound hysteresis in both hypovolemic and control animals. Semiparametric minimization of this hysteresis revealed similar equilibration half-lives in both groups. The biphasic effect-concentration relation was characterized by descriptors showing an increased potency of propofol during hemorrhage. The effect-site concentration at the return of righting reflex was 23% (P < 0.01) lower in the hypovolemic animals, also suggesting an increased end organ sensitivity. CONCLUSIONS: An increased hypnotic effect of propofol occurs during hypovolemia in the rat and can be attributed to changes in both pharmacokinetics and end organ sensitivity.

Trovafloxacin concentrations in airway fluids of patients with severe community-acquired pneumonia.

The penetration of trovafloxacin (TVA), 200 mg once daily, into the airways of 17 patients with severe pneumonia was studied. The mean (standard deviations are given in parentheses) steady-state TVA concentrations, 2 h after the last intake, were 3.1 (0.3) mg/liter in induced sputum (n = 8), 3.2 (1.1) mg/liter in bronchial secretions (n = 9), 3.2 (0.9) mg/liter in bronchoalveolar lavage fluid (n = 10), and 4.9 (1.4) mg/liter in epithelial lining fluid (n = 11).

Effects of smoking, CYP2D6 genotype, and concomitant drug intake on the steady state plasma concentrations of haloperidol and reduced haloperidol in schizophrenic inpatients.

The effects of smoking, CYP2D6 genotype, and concomitant use of enzyme inducers or inhibitors on the steady state plasma concentrations of haloperidol (HAL) and reduced haloperidol (RHAL) were evaluated in 92 schizophrenic inpatients. All but three of these patients received concomitant medication, in many cases with drugs potentially interacting with HAL. Of the 92 patients, 63 were treated orally with HAL in a daily dose of 0.4 to 50 mg; 29 patients were treated intramuscularly with a daily equivalent dose of HAL decanoate (expressed as HAL) of 1.8 to 17.9 mg. A wide interindividual variation in HAL dose and in steady state plasma concentrations of HAL and RHAL was observed. In the patients treated orally, the daily oral dose was about 4 times higher and the dose-normalized HAL (but not RHAL) plasma concentrations were significantly lower in smokers (n = 40) than in nonsmokers (n = 23) (p < 0.01). The dose-normalized RHAL (but not HAL) plasma concentrations and the RHAL/HAL ratio were significantly higher in poor metabolizers (PMs) than in extensive metabolizers (EMs). There was a trend toward an effect of potentially interacting drugs (inducers or inhibitors) on dose, dose-normalized HAL and RHAL plasma concentrations, and the RHAL/HAL ratio. In the patients treated intramuscularly, the dose-normalized HAL (but not RHAL) plasma concentrations were significantly lower in smokers than in nonsmokers, but no differences in doses were observed. This naturalistic study of modest sample size in a polymedicated population shows an effect of smoking and CYP2D6 genotype (and to a lesser extent, of interacting drugs) on the kinetics of HAL.

Relationship between etomidate plasma concentration and EEG effect in the rat.

PURPOSE: The effect-plasma concentration relationship of etomidate was studied in the rat using electroencephalographic changes as a pharmacodynamic parameter. METHODS: Etomidate was infused (50 mg/kg/h) in chronically instrumented rats (n=6) until isoelectric periods of 5 s or longer were observed in the electroencephalogram (EEG). The EEG was continuously recorded during the experiment and frequent arterial blood samples were taken for determination of etomidate plasma concentrations. The changes observed in the raw EEG signal were quantified using aperiodic analysis in the 2.5-7.5 Hz frequency band. The return of the righting reflex was used as another parameter of anesthesia. RESULTS: A mean dose of 8.58+/-0.41 mg/kg needed to be infused to reach the end point of 5 s isoelectric EEG. The plasma concentration time profiles were most adequately fitted using a three-exponential model. Systemic clearance, volume of distribution at steady-state and elimination half-life averaged 93+/-6 ml/min/kg, 4.03+/-0.24 l/kg and 59.4+/-10.7 min respectively. The EEG effect-plasma concentration relationship was biphasic exhibiting profound hysteresis. Semi-parametric minimization of this hysteresis revealed an equilibration half-life of 2.65+/-0.15 min, and the biphasic effect-concentration relationship was characterized nonparametrically by descriptors. The effect-site concentration at the return of the righting reflex was 0.44+/-0.03 microg/ml. CONCLUSIONS: The results of the present study show that the concentration-effect relationship of etomidate can be characterized in individual rats using aperiodic analysis in the 2.5-7.5 Hz frequency band of the EEG. This characterization can be very useful for studying the influence of diseases on the pharmacodynamics of etomidate in vivo.

The influence of hemorrhagic shock on the pharmacokinetics and the analgesic effect of morphine in the rat.

The influence of hemorrhagic shock (removal of 30% of the blood volume) on the pharmacokinetics and the analgesic effect of morphine was investigated in conscious rats. Plasma concentrations of morphine after a bolus injection (5 mg/kg) are higher in the shock animals, which is attributed to a small decrease in clearance (-22%; P > 0.05) and a significant decrease in distribution volume (-33%; P < 0.05) of the drug. The areas under the plasma concentration-time curve of the metabolite morphine-3-glucuronide (M3G) are significantly higher (+237%; P < 0.01) in the shock rats, which is probably explained by a decreased distribution and renal excretion. The analgesic effect of morphine was evaluated using the tail-flick test during a continuous infusion (10 mg/kg/h) with measurement of the plasma concentrations of morphine and M3G. Data from these experiments show higher plasma concentrations of morphine (+33%; P < 0.05) and M3G (+66%; P > 0.05) during shock, and a significantly increased analgesic effect (+43%; P < 0.05). Our data suggest that the increased analgesic effect of morphine during hemorrhagic shock can most likely be explained by pharmacokinetic changes resulting in higher morphine concentrations.

Comparison of two high-performance liquid chromatographic methods for monitoring plasma concentrations of haloperidol and reduced haloperidol.

Two high-performance liquid chromatographic methods for monitoring haloperidol (HAL) and reduced haloperidol (RHAL) plasma concentrations were compared. In one method ultraviolet detection and a C18 column were used (UV method); in the other method electrochemical detection and a CN-column were used (EC method). Both methods are accurate and precise. For plasma samples spiked with HAL or RHAL, an excellent correlation was observed between the concentrations of HAL and RHAL found with both methods (r < or = 0.99, p < 0.01). However, for plasma obtained from patients treated with HAL the correlation between the two methods was poor (r > or = 0.71, p < 0.01). The main reason for the discrepancy between the two methods is probably interference of comedications or their metabolites, mostly in the EC method. Although the quantitation limit of the UV method (2 ng/ml for HAL and RHAL) is higher than that of the EC method (0.5 ng/ml for HAL and RHAL), the UV method is to be preferred for monitoring plasma levels in psychiatric patients because there is less interference from comedication.

Variability of morphine disposition during long-term subcutaneous infusion in terminally ill cancer patients.

OBJECTIVE: To study the plasma concentrations of morphine and its glucuronides to assess the intra- and interindividual variability of the disposition of morphine administered by subcutaneous infusion in cancer patients. METHODS: Blood samples were taken repeatedly in eight patients with severe cancer pain who were being treated with morphine (60-3000 mg per day) via chronic (8-160 days) subcutaneous infusion. Venous blood samples were collected at least weekly and, when possible, on 3 consecutive days after dose adaptation or any other major change in the patients' treatment. Concentrations of morphine and its glucuronides in plasma were measured after solid-phase extraction using a validated high-performance liquid chromatography assay. The stability of the morphine solutions was determined by repeated measurement of the concentrations of morphine and its degradation products in the solutions. RESULTS: The morphine concentration in the infusion solutions remained unchanged during storage and infusion. The plasma concentrations of morphine and its glucuronides were within the ranges reported in the literature. There was, as expected, a large interindividual variability: from patient to patient, the mean of the normalised plasma concentrations ranged from 0.3 ng.ml(-1).mg(-1) to 0.8 ng.ml(-1).mg(-1) for morphine, from 1.0 ng.ml(-1).mg(-1) to 3.1 ng.ml(-1).mg(-1) for morphine-6-glucuronide and from 6.8 ng.ml(-1).mg(-1) to 24.3 ng.ml(-1).mg(-1) for morphine-3-glucuronide. Intraindividual variability was also important. The residual standard deviation of the mean normalised plasma concentrations calculated for each patient ranged from 26% to 56% for morphine, from 20% to 51% for morphine-6-glucuronide and from 20% to 49% for morphine-3-glucuronide. The normalised plasma concentrations of morphine and its glucuronides did not increase with dose or time, and no explanation for the pronounced pharmacokinetic intraindividual variability was found. CONCLUSION: During subcutaneous infusion of morphine, there is a large intra- and interindividual variability of the morphine disposition which could be of clinical relevance.

Measurement of cefuroxime in human bronchoalveolar lavage fluid by high-performance liquid chromatography after solid-phase extraction.

A sensitive and selective method for the determination of cefuroxime in bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography (HPLC) with UV detection at 280 nm after solid-phase extraction with C18 cartridges was developed. A Waters symmetry C18 column was used and the mobile phase was acetonitrile-0.05 M ammonium phosphate buffer (pH 3.2) (15:85, v/v). The method enabled the determination of cefuroxime at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 5-100 ng/ml for 400 microliters of BAL. The intra- and inter-assay coefficient of variations for 10, 40 and 80 ng/ml were between 5.3 and 8.9%. Analytical recoveries were between 92.7 and 106.2%. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1 using 400 microliters of BAL. The method was successfully used for the analysis of BAL fluid from patients after oral administration of 500 mg cefuroxime axetil twice daily.

Characterization of the cytochrome P450 isoenzymes involved in the in vitro N-dealkylation of haloperidol.

AIMS: The present study was carried out to identify the cytochrome P450 isoenzyme(s) involved in the N-dealkylation of haloperidol (HAL). METHODS: In vitro studies were performed using human liver microsomes and c-DNA-expressed human P450 isoforms. N-dealkylation of HAL was assessed by measuring the formation of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP). RESULTS: There was a tenfold variation in the extent of CPHP formation amongst the nine human liver microsomal preparations. The CPHP formation rates as a function of substrate concentration, measured in three livers, followed monophasic enzyme kinetics. Km and Vmax values ranged respectively from 50 to 78 microM and from 180 to 412 pmol mg-1 min-1 CPHP formation rates in the nine liver preparations were significantly correlated with dextromethorphan N-demethylase activity (a marker of CYP3A4 activity), but not with the activity of dextromethorphan O-demethylase (CYP2D6), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). Ketoconazole, an inhibitor of CYP3A4, inhibited competitively CPHP formation (Ki=0.1 microM), whereas sulphaphenazole (CYP2C9), furafylline (CYP1A2) and quinidine (CYP2D6) gave only little inhibition (IC50 > 100 microM). CPHP formation was, moreover, enhanced by apha-naphtoflavone, an effect common to CYP3A4 mediated reactions. Anti-CYP3A4 antibodies strongly inhibited CPHP formation, whereas no inhibition was observed in the presence of CYP2D6 antibodies. Among the recombinant human CYP isoforms tested, CYP3A4 exhibited the highest activity with respect to CPHP formation rate, with no detectable effect of other CYP isoforms (CYP1A2, CYP2D6 and CYP2C9). HAL inhibited dextromethorphan O-demethylase (CYP2D6) with IC50 values between 2.7 and 8.5 microM, but not (IC50 > 100 microM) dextromethorphan N-demethylase (CYP3A4), phenacetin O-deethylase (CYP1A2) or tolbutamide hydroxylase (CYP2C9). CONCLUSIONS: These results strongly suggest that the N-dealkylation of HAL in human liver microsomal preparations is mediated by CYP3A4.

High performance liquid chromatography determination of dextromethorphan and its metabolites in urine using solid-phase extraction.

A high performance liquid chromatography assay coupled with fluorescence detection was developed for the determination of dextromethorphan and its metabolites in urine. The products and the internal standard, pholcodine, were separated on an Alltima C18, 5 microns column (250 x 4.6 mm), using a mobile phase containing sodium dodecyl sulphate (1 mM) in a mixture of acetonitrile-sodium dihydrogen phosphate (0.01 M) 40.5:59.5, v/v) (pH* = 2.5). A novel solid-phase extraction procedure with strong cation exchange, non end-capped, Isolute SCX cartridges allows good recovery of the products (mean 85% or more). For all analytes, the assay is sensitive (LOQ 25 ng ml-1, using 200 microliters urine), reproducible (RSD < 15%) and accurate (< 15% deviation of the nominal value) over the range evaluated. This method can be used to measure dextromethorphan and its metabolites to phenotype individuals as poor or extensive metabolizers of drugs metabolized by CYP2D6.

Severe diltiazem poisoning with intestinal pseudo-obstruction: case report and toxicological data.

This case report concerns a 30-year-old man who survived a 4.2 g diltiazem overdose. He sustained vasoplegic shock with a junctional escape rhythm which required high doses of norepinephrine and epinephrine. Among other complications, ileus with paralytic intestinal pseudo-obstruction developed on day three. Cecal distention was demonstrated by abdomen computed tomodensitometry. The ileus resolved on day seven following the poisoning. Diltiazem plasma concentrations were determined during the first three days. The possible role of other medications, activated charcoal and sufentanil, is noted.

Capillary gas chromatographic determination with nitrogen-phosphorus detection of the calcium antagonist nicardipine and its pyridine metabolite M-5 in plasma.

A capillary gas chromatographic method with nitrogen-phosphorus detection for the simultaneous determination of nicardipine and its pyridine metabolite M-5 was developed. The method involves extraction of the plasma with hexane-methylene chloride (1:1, v/v), followed by evaporation of the organic phase. The extract is injected into a fused-silica capillary column coated with cross-linked 5% phenyl-methylsilicone. A temperature gradient (85-285 degrees C) is applied and the two products and the internal standard can be separated within 22 min. The limit of detection is 0.5 ng/ml for both products. The method is suitable for pharmacokinetic studies in humans.

Influence of endotoxin on the stereoselective pharmacokinetics of oxprenolol, propranolol, and verapamil in the rat.

The influence of endotoxin-induced inflammation on the enantioselective pharmacokinetics of propranolol, oxprenolol, and verapamil, which bind to alpha 1-acid glycoprotein, was studied in the rat. The racemic mixtures were given orally. In the control animals, for propranolol and oxprenolol, the plasma concentrations of the (R)-enantiomer were higher than those of the (S)-enantiomer, while for verapamil the reverse was true. Protein binding and intrinsic clearance are the main factors responsible for this enantioselectivity. After endotoxin treatment, for the three drugs tested the plasma concentrations and the plasma binding of both enantiomers were significantly increased. This effect was more pronounced for (R)-propranolol, (R)-oxprenolol, and (S)-verapamil than for their respective antipodes. The enantioselective effect of endotoxin on the plasma concentrations of the drugs studied seems mainly due to the enantioselective increase in binding to alpha 1-acid glycoprotein.

Metabolism and hepatotoxicity of N,N-dimethylformamide, N-hydroxymethyl-N-methylformamide, and N-methylformamide in the rat.

The metabolism and hepatotoxicity of N,N-dimethylformamide (DMF) and two of its metabolites, N-hydroxymethyl-N-methylformamide (HMMF) and N-methylformamide (NMF) were evaluated over a 4-day period in rats. DMF toxicity was dose dependent and delayed toxicity after the administration of a high DMF dose (13.7 mmol/kg) in comparison to a lower dose (4.1 mmol/kg) was observed. Treatment of rats with 13.7 mmol/kg DMF, HMMF, or NMF showed i) that DMF is more toxic than HMMF or NMF, and ii) that hepatotoxicity occurs later for DMF than for HMMF or NMF. Analysis of serum and urine samples demonstrated that DMF is first metabolized to HMMF, which is then partially converted to NMF. After HMMF administration, NMF was found both in serum and in urine. The time course of DMF and HMMF toxicity in relation to NMF formation fitted the hypothesis that the hepatotoxicity of DMF and HMMF is mediated via NMF. The degree of hepatotoxicity after HMMF and NMF treatment is similar. However, the degree of DMF hepatotoxicity is much higher than in the case of NMF or HMMF. The role of NMF as an obligatory intermediate in DMF and HMMF hepatotoxicity is discussed.

Chiral high-performance liquid chromatographic determination of oxprenolol in plasma.

A sensitive, stereospecific high-performance liquid chromatographic assay for oxprenolol enantiomers in rat plasma was developed, using a chiral derivatization agent. Racemic oxprenolol and the internal standard (racemic propranolol) are extracted with dichloromethane after alkalinization of the plasma. Quantitation of R(+)- and S(-)-oxprenolol is based on derivatization with the chiral agent S(-)-1-(1-naphthyl)-ethyl isocyanate, followed by chromatographic separation on a C18 reversed-phase column, with fluorometric detection (excitation at 226 nm, emission at 333 nm). The assay is reproducible as judged by a coefficient of variation of less than 17.5% for both enantiomers at all concentrations used. Preliminary experiments in the rat demonstrate that the method is sufficiently sensitive for pharmacokinetic studies in that species.

Simultaneous determination of N,N-dimethylformamide, N-monomethylformamide and N-hydroxymethyl-N-methylformamide in rat plasma by capillary gas chromatography.

A capillary gas chromatographic method with nitrogen-phosphorus detection for the simultaneous quantitative determination of N,N-dimethylformamide, and its two metabolites, N-monomethylformamide and N-hydroxymethyl-N-methylformamide, in rat plasma has been developed. The method involves a single extraction step with ethyl acetate-acetone (4:1, v/v). The extract is injected into a fused-silica capillary column coated with Carbowax 20M. A temperature gradient (65-110 degrees C) is applied, and the three products can be separated within 10 min. The quantitation limits, using 25 microliters of rat plasma, for N,N-dimethylformamide, N-monomethylformamide and N-hydroxymethyl-N-methylformamide are 0.4, 0.4 and 2 micrograms/ml, respectively. This method is suitable for toxicokinetic studies in rats.

Stereoselective pharmacokinetics of atenolol in the rat: influence of aging and of renal failure.

The pharmacokinetics of R- and S-atenolol after intravenous administration of racemic atenolol were studied in 3-, 12- and 24-month-old rats and in 3-month-old rats with renal failure induced by uranyl nitrate. In all age groups, the area under the plasma concentration-time curves is higher for R- than for S-atenolol; volume of distribution, total clearance and renal clearance are lower for R-atenolol than for S-atenolol, but the differences are small. In function of age there is for both enantiomers a significant increase in AUC, due, at least in part, to a decreased renal clearance; the effect of aging is not stereoselective. In rats with renal failure, the AUC of both enantiomers increases, due mainly to a decrease in renal clearance, but to a lesser degree also to a decrease in nonrenal clearance. For both enantiomers, the volume of distribution decreases and the half-life increases in the uraemic rats. The total amount of both enantiomers excreted in the urine is decreased in the rats with renal failure. There are no stereoselective effects of treatment of the rats with uranyl nitrate.