Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection.

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography-ultraviolet (HPLC-UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0-2000 pg/ml) and midazolam (range 0-400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (<15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography-mass spectrometry (GC-MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC-UV system.

Validation of an improved reversed-phase high-performance liquid chromatography assay with reductive electrochemical detection for the determination of artemisinin derivatives in man.

For the determination of artemisinin (ART) and analogs, a reversed-phase high-performance liquid chromatography method using reductive electrochemical detection (ED) was set up with some important modifications as compared to previously published assays. A different technique of deoxygenating resulted in a factor 2-3 lower background current. A Spectroflow 400 liquid chromatograph in combination with a Triathlon autoinjector coupled to a Decade electrochemical detector was used. The detector was operated in the reductive mode as a closed system under chromatography grade helium to exclude any access of oxygen. The Decade has a glassy carbon electrode and a reference Ag/AgCl electrode. Infrequent electropolishing was required implicating a very stable system. By increasing acetonitril or lowering the pH of the mobile phase, the various derivatives could be determined in the same chromatogram. The assay was validated using artemether (ATM) and dihydroartemisinin (DHA) as test substances. In the concentration range seen in people after usual doses (5 to 220 ng/ml), the assay performs with adequate accuracy and precision. The interassay and intraassay precision are < 6% for ATM. For DHA, the interassay and intraassay precision are < 9%. The accuracy expressed as the deviation from the expected concentration varies from -1% to +4.5% for the intraassay ATM-determinations and from +1% to +6.3% for the interassay measurements. For DHA, the accuracy is somewhat less, varying from -0.3% to -9.5% for the intraassay measurements and -0.6% to +2.6% for the interassay measurements. The reproducibility of the assay, measured over a time period of 3 months, is good for ATM and DHA with an interassay precision of < 18% in 70 repetitive samples and an accuracy varying from -0.6% to +7.6%. In a cross-check with two other reference laboratories who used comparable methods of determination, a strong correlation (correlation coefficient > 0.98) was achieved. The method was applied in a study in which artemether was administered orally to healthy white subjects. We consider high-performance liquid chromatography with electrochemical detection an accurate and precise method for quantitative determination of artemisinin derivatives in pharmacokinetic studies.

First high-performance liquid chromatography assay of formoterol concentrations in the low-picogram-per-milliliter range.

A method for the assay of plasma concentrations of the long-acting beta 2-adrenoceptor agonist, formoterol is described. This method, in which high-performance liquid chromotography is used with electrochemical detection, enables, for the first time, pharmacokinetic and pharmacodynamic research with this drug. After column extraction of plasma samples, the eluent is injected on the chromatographic system. Retention times of formoterol and the internal standard, bromo-formoterol, were 5.6 and 10 min, respectively. Our results show that this is a sensitive and reproducible method with a very low limit of detection (20 pg/ml), which creates the possibility of measuring concentrations in a range achievable in humans.

Debrisoquine phenotype and the pharmacokinetics and beta-2 receptor pharmacodynamics of metoprolol and its enantiomers.

The metabolism of the cardioselective beta-blocker metoprolol is under genetic control of the debrisoquine/sparteine type. The two metabolic phenotypes, extensive (EM) and poor metabolizers (PM), show different stereoselective metabolism, resulting in apparently higher beta-1 adrenoceptor antagonistic potency of racemic metoprolol in EMs. We investigated if the latter also applies to the beta-2 adrenoceptor antagonism by metoprolol. The drug effect studied was the antagonism by metoprolol of terbutaline-induced hypokalemia. By using pharmacokinetic pharmacodynamic modeling the pharmacodynamics of racemic metoprolol and the active S-isomer, were quantitated in EMs and PMs in terms of IC50 values, representing metoprolol plasma concentrations resulting in half-maximum receptor occupancy. Six EMs received 0.5 mg of terbutaline s.c. on two different occasions: 1) 1 hr after administration of a placebo and 2) 1 hr after 150 mg of metoprolol p.o. Five PMs were studied according to the same protocol, except for a higher terbutaline dose (0.75 mg) on day 2. Blood samples for the analysis of plasma potassium, terbutaline, metoprolol (racemic, R- and S-isomer), and alpha-hydroxymetoprolol concentrations were taken at regular time intervals, during 8 hr after metoprolol. In PMs, metoprolol increased the terbutaline area under the plasma concentration vs. time curve (+67%). Higher metoprolol/alpha-hydroxymetoprolol ratios in PMs were predictive for higher R-/S-isomer ratios of unchanged drug. There was a difference in metoprolol potency with higher racemic metoprolol IC50 values in PMs (72 +/- 7 ng.ml-1) than EMs (42 +/- 8 ng.ml-1, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)