Ross L.G. Norris

Sensitive high-performance liquid chromatographic assay with ultraviolet detection of methadone enantiomers in plasma

Methadone is being prescribed increasingly as an analgesic in palliative medicine. R-Methadone has been shown to be responsible for most of the pharmacological activity of this drug. Despite that in most countries it is administered as the racemate. Few assay methods for the enantiomers are available; e en fewer can determine accurately the low concentrations of enantiomers required to undertake pharmacokinetic studies in patients taking the drug in analgesic doses. We present here an HPLC method used to determine concentrations of the specific enantiomers of methadone as low as 5.0 ng/ml with adequate precision and accuracy. The mean R/S ratio of the plasma concentrations was 0.80 +/- 0.05 (n = 3 samples) in one patient taking 25-27.5 mg daily and 1.21 +/- 0.12 (n = 6 samples) in another taking 10-20 mg daily. In the second patient, concentrations of the enantiomers ranged between 5.8 and 25.9 ng/ml. Tricyclic antidepressants did not interfere with the assay but dextropropoxyphene did. Its presence could be detected by dual wavelength monitoring.

A reliable high-performance liquid chromatography assay for high-throughput routine cyclosporin A monitoring in whole blood.

We report here a reliable high-performance liquid chromatography-ultraviolet assay for routine assay of cyclosporin A (CsA) in whole blood using solid-phase extraction. This assay is linear, between 20 and 2,000 μg/L, with correlation coefficients >0.998 for five consecutive standard curves. All coefficients of variation (CV) were <8% at CsA concentrations of 45, 480, and 1,800 μg/L, with the exception of the between-day CV at 45 μg/L, which was <15%. The relative accuracy of the method is >94% at 45, 480, and 1,800 μg/L. The mean recoveries for CsA and cyclosporin D (internal standard) were 38.2 ± 4.8% (n = 45) and 40.1 ± 6.7% (n = 45), respectively. This method has proven to be reliable and robust in a high-throughput therapeutic drug monitoring laboratory.

Effect of disopyramide on left ventricular performance: the relationship of free and total concentrations of the drug and of its mono-N-dealkylated metabolite to noninvasive indices of function.

Summary We used M-tnode echocardiography and recordings of systolic time intervals to follow changes in left ventricular systolic function of 10 normal subjects during administration of 200 mg oral disopyramide every 8 h. Left ventricular function was significantly depressed (peak rate of change of dimension −17%, p < 0.001; mean velocity of circumferential fiber shortening −15%, p < 0.01; percent fractional shortening −16%, p < 0.05) for as long as 7 h after a dose. These changes could be correlated with those in plasma concentrations of free and total disopyramide, and of its mono-N-dealkylated metabolite (change in peak rate of change of dimension versus levels of disopyramide plus metabolite, r = −0.41, p < 0.03; changes in ratio preejection time/ejection time versus levels of disopyramide and metabolite, r = 0.62, p < 0.002).

Assessment of a two-step high-performance liquid chromatographic assay using dual-wavelength ultraviolet monitoring for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol in human serum or plasma

The technique of dual-wavelength monitoring was used to verify the purity of high-performance liquid chromatographic (HPLC) peaks quantified as 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. The data obtained show the need for a second HPLC step prior to quantitation. Potential inaccuracy arising from inadvertent collection of radio-labelled decomposition products was assessed. Between-day coefficients of variation were 7.3, 5.0 and 3.6%, respectively for 11.3 (n = 12), 17.1 (n = 14), and 32.9 (n = 8) ng/ml of 25-hydroxycholecalciferol. For 25-hydroxyergocalciferol, these values were 6.4 and 3.8% for 11.1 (n = 12) and 20.1 (n = 8) ng/ml concentrations, respectively. Comparison of total 25-hydroxycalciferol with a competitive protein binding assay was made. The comparison produced a correlation coefficient (r) of 0.94 and a relationship of y = 1.03x + 3.3. Four of the samples contained more than 10 ng/ml of 25-hydroxyergocalciferol and the results are consistent with the reported 100% cross-reactivity of the competitive binding protein method for 25-hydroxyergocalciferol and 25-hydroxycholecalciferol. A simple regeneration procedure is also described which enables Sep-Pak C18 cartridges to be reused up to eighteen times. Samples may be stored at -18 degrees C for upto several months before assay and either serum or plasma may be used.

Intravenous Disopyramide in Acute Myocardial Infarction: A Haemodynamic and Pharmacokinetic Study

We studied the haemodynamic and pharmacokinetic effects of intravenous disopyramide phosphate in 12 patients (average age, 59 years) with proven trans-mural myocardial infarction, whose symptoms began less than 12 h prior to the study. The aim was to assess the effects of intravenous disopyramide (2 mg/kg given over 5 min) on cardiac index (CI), left ventricular filling pressure (LVFP), heart rate (HR), mean systemic arterial blood pressure (BP), and systemic vascular resistance (SVR) for 60 min after administration of the drug. Both total and free concentrations of disopyramide in the plasma were also measured. A significant elevation (p < 0.01) of LVFP (estimated indirectly as pulmonary artery end-diastolic pressure) occurred and persisted through the 1-h evaluation period. There was a small but significant (p = 0.02) initial fall in CI and a rise in SVR (p = 0.05). No significant changes occurred in HR or BP. Serum concentrations of disopyramide reached recommended therapeutic concentrations. There was no significant correlation of the changes in cardiac variables from pretreatment values with total serum concentrations, but the free concentration of disopyramide in plasma correlated better with cardiac effect, and the relationships of the free concentration of disopyramide to the changes in LVFP and in SVR from pretreatment values were significant (p < 0.05). In two patients studied in detail, there was evidence of dose-dependent protein binding of disopyramide.

Comparison of high-performance liquid chromatography and monoclonal fluorescence polarization immunoassay for the determination of whole-blood cyclosporin A in liver and heart transplant patients

Summary Conflicting conclusions have been drawn from comparisons of high-performance liquid chromatography (HPLC) and the Abbott Diagnostics monoclonal fluorescence polarization immunoassay (mFPIA) for cyclosporin. The aim of this study was to compare whole blood cyclosporin A (CsA) concentrations measured by both mFPIA and HPLC in liver and heart transplant patients. One hundred and twenty-four liver and 62 heart transplant patient samples were assayed by both methods. Assay imprecision for both methods during the studies was <7% over the range 150–800 μg/L. At an HPLC-determined concentration of 100 μg/L, mFPIA overestimated CsA by 60% (liver) and 77% (heart). At 300 μg/L, the overestimation was 40% (liver) and 45% (heart). On this basis, the mFPIA is not interchangeable with HPLC.

A modified assay for cyclosporin in blood using solid-phase extraction with high-performance liquid chromatography

A modified procedure for measuring cyclosporin in whole blood by high-performance liquid chromatography is described and evaluated for clinical use. Sample preparation uses solid-phase extraction cartridges that can be reused. Life of the reverse-phase analytical column exceeds 1,000 injections at 70°C. Cyclosporins A and D (internal standard) elute after 5.6 and 7.6 min, respectively. Calibration plots are linear from 50 ng/ml to at least 2,000 ng/ml. Within-day and between-day imprecision is less than 9% (coefficient of variation). Minimum measurable concentration is 50 ng/ml.

Determination of unbound fraction of disopyramide in plasma: A comparison of equilibrium dialysis, ultrafiltration through dialysis membranes and ultrafree anticonvulsant drug filters

A simple, rapid ultrafiltration technique for determination of free drug concentration in plasma is described and compared with equilibrium dialysis and ultrafiltration through dialysis membranes. When used for disopyramide protein binding studies, this method requires only 1 ml of plasma and up to 20 samples may be filtered simultaneously in 20-40 min. Commercially available Ultrafree anticonvulsant drug filters are used, these are attached to 2 ml leur tip syringes, which provide the pressure gradient for filtration. Compared to equilibrium dialysis this technique is far quicker and permits protein binding to be measured at the drug concentration in the original plasma. Ultrafiltration through dialysis membranes was found to be more tedious and time-consuming than it was through Ultrafree filters. Adsorption of disopyramide from the plasma sample and protein leakage were also problems with this method. Leakage of protein did not occur with either Ultrafree filters or equilibrium dialysis. With the Ultrafree method, recovery of 14C-labeled disopyramide in buffer at 1.1 micrograms/ml and 8.4 micrograms/ml was 87% and 89% respectively. In carefully controlled experiments, a comparison of the Ultrafree method with equilibrium dialysis and ultrafiltration gave comparable values for the free fraction of the drug for total concentrations from 0.3-8 microgram/ml disopyramide.

The influence of vancomycin concentration and the pH of plasma on vancomycin protein binding

A review of numerous studies of the protein binding of vancomycin suggests major discrepancies among their results. The reported percent protein binding of vancomycin varies from 0% to 98%. The influence of pH and concentration on the protein binding of vancomycin was investigated in this study. There was a significant difference (p < 0.001) in percent protein binding in vancomycin-spiked plasma samples across the pH range of 7.0-8.0. There was no significant difference (p > 0.05) in percent protein binding in vancomycin-spiked plasma samples across the concentration range of 2-80 mg/L. It is likely that some of the variation reported to date may be due to a lack of control of pH during the measurement of protein binding of vancomycin.

Measurement of dothiepin and its major metabolites in plasma by high-performance liquid chromatography

This paper describes a reversed-phase high-performance liquid chromatographic method which will simultaneously measure dothiepin and its three major metabolites (northiaden, northiaden-S-oxide and dothiepin-S-oxide) in plasma using trimipramine as internal standard. Sample preparation involved a basic extraction using diethyl ether followed by an acid back-extraction. The method we report is linear over the range 50-1000 ng/ml (r = 0.999), for all analytes. Total imprecision is less than 11% (coefficient of variation) and accuracy is greater than 94% (n = 20). Recovery of analytes varied considerably from 51.7% for northiaden-S-oxide to 90.2% for dothiepin-S-oxide.