Regula Theurillat

Monitoring of tricyclic antidepressants in human serum and plasma by HPLC: characterization of a simple, laboratory developed method via external quality assessment

A reversed-phase high performance liquid chromatography (HPLC) method for the determination of plasma and serum levels of amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI) is described. The assay is based upon single step liquid/liquid extraction of these compounds using hexane at pH 11 (recovery between 92 and 105%), a Nova-Pack C-18 HPLC cartridge column, a mobile phase composed of a phosphate buffer with 50% (v/v) acetonitrile and about 0.2% (v/v) diethylamine (final pH: 8) and solute detection at 242 nm. Using 1 ml of plasma or serum and econazole as internal standard, drug levels between 20 and 400 ng ml(-1) (about 60-1450 nM) were found to provide linear calibration graphs. For drug concentrations in the range of 70-120 ng ml(-1) (about 240-430 nM), intraday and interday imprecisions (n = 5) were determined to be < 6.0, and < 15%, respectively. Data reported include those gathered over a 3-year period during which this assay was employed for therapeutic drug monitoring and clinical toxicology. The performance of the laboratory developed assay was assessed via analysis of monthly samples provided by an external quality control scheme.

Ocular Distribution of Intravenously Administered Lipid Formulations of Amphotericin B in a Rabbit Model

ABSTRACT Little is known about the ocular penetration of amphotericin B (AMB) and its lipid formulations, the current drug of choice in fungal endophthalmitis. The ocular distribution of AMB lipid complex (ABLC), liposomal AMB (L-AMB), and AMB deoxycholate (D-AMB) was studied in a rabbit model. D-AMB (1 mg/kg of body weight/day), ABLC (5 mg/kg/day), or L-AMB (5 mg/kg/day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7 consecutive days after induction of unilateral uveitis by intravitreal injection of endotoxin. AMB concentrations in aqueous humor, vitreous humor, and plasma were determined by high-pressure liquid chromatography 16 h after administration of a single dose or 24 h after the last of seven doses. After single-dose administration, L-AMB achieved at least eightfold-higher AMB concentrations in the aqueous of inflamed eyes than ABLC or D-AMB (1.21 ± 0.58 μg/ml versus 0.14 ± 0.04 and 0.11 ± 0.09 μg/ml, respectively). At that time point no drug was detectable in the vitreous. After 7 days of treatment, the concentration of AMB in the vitreous was higher after treatment with L-AMB (0.47 ± 0.21 μg/ml) than after treatment with ABLC (0.27 ± 0.18 μg/ml) and D-AMB (0.16 ± 0.04 μg/ml). Similarly, AMB concentration in the aqueous was higher after repeated doses of L-AMB (0.73 ± 0.43 μg/ml) than after repeated doses of ABLC (0.03 ± 0.02 μg/ml) or D-AMB (0.13 ± 0.06 μg/ml). No AMB was detected in noninflamed eyes. Following systemic administration, AMB distribution to the eye is inflammation dependent and occurs sequentially, first to the aqueous and then to the vitreous. Compared to D-AMB and ABLC, L-AMB reaches higher drug concentrations in both ocular compartments.

Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro.

Ketamine, a phencyclidine derivative, is used for induction of anesthesia, as an anesthetic drug for short term surgical interventions and in subanesthetic doses for postoperative pain relief. Ketamine undergoes extensive hepatic first-pass metabolism. Enantioselective capillary electrophoresis with multiple isomer sulfated β-cyclodextrin as chiral selector was used to identify cytochrome P450 enzymes involved in hepatic ketamine and norketamine biotransformation in vitro. The N-demethylation of ketamine to norketamine and subsequently the biotransformation of norketamine to other metabolites were studied via analysis of alkaline extracts of in vitro incubations of racemic ketamine and racemic norketamine with nine recombinantly expressed human cytochrome P450 enzymes and human liver microsomes. Norketamine was formed by CYP3A4, CYP2C19, CYP2B6, CYP2A6, CYP2D6 and CYP2C9, whereas CYP2B6 and CYP2A6 were identified to be the only enzymes which enable the hydroxylation of norketamine. The latter two enzymes produced metabolic patterns similar to those found in incubations with human liver microsomes. The kinetic data of ketamine N-demethylation with CYP3A4 and CYP2B6 were best described with the Michaelis-Menten model and the Hill equation, respectively. This is the first study elucidating the individual enzymes responsible for hydroxylation of norketamine. The obtained data suggest that in vitro biotransformation of ketamine and norketamine is stereoselective.

Determination of γ-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

Abstract γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 m M nicotinic acid (compound for indirect detection), 3 m M spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ , respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→ m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ .

Effects of a low dose infusion of racemic and S-ketamine on the nociceptive withdrawal reflex in standing ponies

OBJECTIVE To investigate the effect of plasma concentrations obtained by a low dose constant rate infusion (CRI) of racemic ketamine or S-ketamine on the nociceptive withdrawal reflex (NWR) in standing ponies. STUDY DESIGN Prospective, blinded, cross-over study. ANIMALS Six healthy 5-year-old Shetland ponies. METHODS Ponies received either 0.6 mg kg(-1) racemic ketamine (group RS) or 0.3 mg kg(-1) S-ketamine (group S) intravenously (IV), followed by a CRI of 20 microg kg(-1)minute(-1) racemic ketamine (group RS) or 10 microg kg(-1)minute(-1) S-ketamine (group S) for 59 minutes. The NWR was evoked by transcutaneous electrical stimulation of a peripheral nerve before drug administration, 15 and 45 minutes after the start of the bolus injection and 15 minutes after the end of the CRI. Electromyographic responses were recorded and analysed. Arterial blood was collected before stimulation and plasma concentrations of ketamine and norketamine were measured enantioselectively using capillary electrophoresis. Ponies were video recorded and monitored to assess drug effects on behaviour, heart rate (HR), mean arterial blood pressure (MAP) and respiratory rate. RESULTS The NWR was significantly depressed in group RS at plasma concentrations between 20 and 25 ng mL(-1) of each enantiomer. In group S, no significant NWR depression could be observed; plasma concentrations of S-ketamine (9-15 ng mL(-1)) were lower, compared to S-ketamine concentrations in group RS, although this difference was not statistically significant. Minor changes in behaviour, HR and MAP only occurred within the first 5-10 minutes after bolus drug administration in both groups. CONCLUSION Antinociceptive activity in standing ponies, demonstrated as a depression of the NWR, could only be detected after treatment with racemic ketamine. S-ketamine may have lacked this effect as a result of lower plasma concentrations, a more rapid metabolism or a lower potency of S-ketamine in Equidae so further investigation is necessary.

Therapeutic drug monitoring of lamotrigine using capillary electrophoresis: Evaluation of assay performance and quality assurance over a 4-year period in the routine arena

The performance of a capillary zone electrophoresis (CZE)-based assay for lamotrigine (LAMO) in human plasma and serum with complete internal and external quality assurance over an extended period of time is reported. The assay, originally reported by Shihabi and Oles [J. Chromatogr. B 683 (1996) 119], is based upon protein precipitation by acetonitrile and analysis of an aliquot of the acidified supernatant and was adopted in our laboratory for routine use with multi-level internal calibration on different commercial instruments. Evaluation of the calibration and control data of 103 sets of analysis and data from four years of external quality assurance based upon analysis of four-monthly sera containing LAMO and eight other anticonvulsants in sub-therapeutic, therapeutic or toxicological concentration levels revealed the robustness of the CZE-based assay and its suitability for therapeutic drug monitoring of LAMO in a routine setting. CZE data obtained in single determinations were found to compare well with the spike values and the mean of HPLC data determined in 50-70 laboratories. Furthermore, the gathered data were evaluated retrospectively using single-level internal calibration. When applied with caution, this approach was determined to produce slightly higher but otherwise equivalent drug concentrations. For the 4 years of routine operation with external quality control, the reported laboratory ranking was between 19 (out of 67 participating laboratories) and 43 (69). This is the first account of a CZE-based drug assay with complete external quality assessment.

Therapeutic drug monitoring of albendazole: Determination of albendazole, albendazole sulfoxide, and albendazole sulfone in human plasma using nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoretic method (NACE) for the fast determination of plasma levels of albendazole (ABZ), albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) is described. The assay is based upon liquid/liquid extraction of these compounds using dichloromethane at pH 10.2 (recovery between 63 and 98%), followed by a NACE separation performed within 8 min employing a 0.036 M borate buffer (apparent pH 9.9) in a mixture of methanol and N‐methylformamide (1:3) and on‐column absorbance detection at 280 nm. Using 0.5 mL of plasma and extract reconstitution in 200 μL N‐methylformamide, drug levels between 1.0—10 μM were found to provide linear calibration graphs. Intraday and interday imprecisions evaluated from peak area ratios (n = 5) were < 10% and < 12%, respectively. Corresponding imprecisions of detection times (n = 5) were < 1% and < 6%, respectively. The limit of detection (LOD) for ABZ, ABZSO and ABZSO2 was 8 × 10−7 M. The reliability of the method developed was verified via analysis of 45 plasma samples obtained from patients treated with ABZ. Good agreement was obtained between the levels of ABZSO and those determined by routine HPLC. ABZ was found to be undetectable in all patient samples, whereas the levels of ABZSO2 were below or close to LOD.

Therapeutic drug monitoring of antiepileptics by capillary electrophoresis. Characterization of assays via analysis of quality control sera containing 14 analytes.

Quality assurance is an important aspect in therapeutic drug monitoring (TDM). Capillary electrophoresis (CE) assays for determination of (i) ethosuximide via direct injection of serum or plasma, (ii) lamotrigine after protein precipitation by acetonitrile and analysis of an aliquot of the acidified supernatant, and (iii) carbamazepine and carbamazepine-10,11-epoxide after solute extraction followed by analysis of the reconstituted extract are characterized via analysis of a large number of commercial quality control sera containing up to 14 analytes (9 of them are anticonvulsants) in sub-therapeutic, therapeutic and toxicologic concentration levels. CE data obtained in single determinations are shown to compare well with the spike values and the mean of data determined in other laboratories using immunoassays and/or high-performance liquid chromatography, values that are reported by the external quality control scheme. Carbamazepine and ethosuximide drug levels are also shown to agree well with those determined in our departmental drug assay laboratory using automated immunoassays. The presented data reveal the effectiveness of assay assessment via analysis of quality control sera and confirm the robustness of the assays for TDM in a routine setting.

Determination of fluconazole in human plasma by micellar electrokinetic capillary chromatography with detection at 190 nm

The determination of fluconazole (Diflucan) in human plasma by micellar electrokinetic capillary chromatography (MECC) with on-column UV absorption detection at 190 nm from primary, deproteinized and extracted plasma samples is discussed. Direct injection of plain plasma or of the supernatant after protein precipitation with acetonitrile is shown to permit the determination of fluconazole drug levels of > 5 micrograms/ml only. With liquid-liquid extraction employing dichloromethane, the detection limit is about 1 microgram/ml. After extraction using disposable solid-phase C18 cartridges and 1 ml of plasma, however, drug levels as low as 100 ng/ml can be determined unambiguously. Calibration graphs between 0.125-25.0 micrograms/ml (seven data points) are shown to be linear, with a regression coefficient r > 0.999. for fluconazole plasma levels of 5 micrograms/ml, intra-day and inter-day imprecisions (n = 10) are about 2 and 5%, respectively. Using the same solid-phase extraction procedure, 44 fluconazole plasma levels that were determined by MECC are shown to agree well with those obtained by HPLC and elucidated pharmacokinetic data compare well with those found in the literature. The advantages of using MECC instead of HPLC for the determination of fluconazole plasma levels and pharmacokinetics are the high resolution efficiency, low-cost capillary columns and the small consumption of inexpensive and environmentally friendly chemicals.

Plasma levels of a low-dose constant-rate-infusion of ketamine and its effect on single and repeated nociceptive stimuli in conscious dogs

This study quantitatively investigated the analgesic action of a low-dose constant-rate-infusion (CRI) of racemic ketamine (as a 0.5 mg kg(-1) bolus and at a dose rate of 10 microg kg(-1) min(-1)) in conscious dogs using a nociceptive withdrawal reflex (NWR) and with enantioselective measurement of plasma levels of ketamine and norketamine. Withdrawal reflexes evoked by transcutaneous single and repeated electrical stimulation (10 pulses, 5 Hz) of the digital plantar nerve were recorded from the biceps femoris muscle using surface electromyography. Ketamine did not affect NWR thresholds or the recruitment curves after a single nociceptive stimulation. Temporal summation (as evaluated by repeated stimuli) and the evoked behavioural response scores were however reduced compared to baseline demonstrating the antinociceptive activity of ketamine correlated with the peak plasma concentrations. Thereafter the plasma levels at pseudo-steady-state did not modulate temporal summation. Based on these experimental findings low-dose ketamine CRI cannot be recommended for use as a sole analgesic in the dog.