Tacrolimus (FK506): validation of a sensitive enzyme-linked immunosorbent assay kit for and application to a clinical pharmacokinetic study.

Tacrolimus (FK506) is a macrolide immunosuppressant approved for the prophylaxis of organ rejection in liver transplant. Immunoassays of low intra- and interday variability and high sensitivity are necessary to adequately characterize terminal elimination phase concentrations in pharmacokinetic studies. A new ELISA kit for the quantitation of tacrolimus in human whole blood has been validated for use in pharmacokinetic studies. Methanol sample extracts were dried and reconstituted in a horseradish peroxidase (HPR)-FK506 conjugate solution. The reconstituted samples and mouse anti-FK506 were added to a microplate, precoated with secondary antibody, and incubated, FK506 and the HPR-FK506 conjugate competed to bind with anti-FK506, which was immobilized by binding to the secondary antibody. Unbound FK506 was washed away, and substrate was added for color development. Once the reaction was stopped with 2 N H2SO4, the plate was read at 450 nm. The linear range was 0.5-60 ng/ml, with a limit of quantitation of 0.5 ng/ml. Interday precision and accuracy were < or = 10.4% C.V. and < or = 3% R.E. for quality control samples. The lack of interference from endogenous compounds was established by parallelism and recoveries of FK506 from six lots of control matrix. Cross-reactivity against the metabolites and analogs were not performed because the kit monoclonal antibody was from the same source as Kobayashi et al (1). The utility and sensitivity of the kit present a good method for the quantitation of tacrolimus in blood from pharmacokinetic studies. The method is robust and has been used to assay tacrolimus in several thousand whole blood samples by multiple analysts.

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection.

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile-methanol-water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-microns column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1-100 ng/ml for butorphanol and hydroxybutorphanol, and 2-200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.

Bioanalytical automatlon: history and future plans.

Bioanalysis is determining the concentration of drugs and metabolites in biological fluids (i.e. plasma and urine). During the past 15 years tremendous advances in bioanalysis, for example HPLC, auto injectors, data collection systems and robotics has enabled the productivity of the bioanalyst to increase but it still requires considerable manual intervention. This paper describes the rationale, the justification and the plans Bristol-Myers Squibb has to completely automate the entire bioanalytical process.

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma.

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 microns column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10-1000 ng/ml (0.012-1.17 microM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.

Effect of food on the bioavailability of gepirone in humans.

A randomized two-period crossover study was conducted in 20 healthy male volunteers to assess the effect of food on the pharmacokinetics of gepirone (BMY-13805) and its metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP) after a single 20-mg dose of gepirone either after fasting or after consumption of a standard high-fat breakfast. There was a 1-week washout period between treatments. Plasma samples were obtained predose and at specified time points after dosing and analyzed for gepirone and 1-PP content by a specific gas chromatographic-mass spectrometric method. Food did not significantly affect gepirone maximum peak plasma concentration (Cmax) and half-life (t1/2). The mean gepirone Cmax was 16.98 +/- 8.12 ng/mL (fed) and 18.73 +/- 10.30 ng/mL (fasted), with mean t1/2 of 3.32 +/- 1.84 hours (fed) and 2.94 +/- 0.90 hours (fasted). Food significantly increased the mean area under the curveinf (AUCinf) from 55.26 +/- 35.74 ng.hour/mL (fasted) to 75.69 +/- 42.79 ng.hour/mL (fed), and the mean residence timeinf (MRTinf) from 4.31 +/- 0.78 hours (fasted) to 5.37 +/- 1.21 hours (fed). The median time to maximum plasma concentration (tmax) for gepirone was also significantly increased in the presence of food, 2.0 hours, versus 0.75 hours in the absence of food. For 1-PP, food had no affect on Cmax, t1/2, or AUCinf. Mean t1/2 for 1-PP in the presence and absence of food was 6.06 +/- 1.75 and 5.76 +/- 1.75 hours, respectively. MRTinf, however, was increased significantly from 9.32 +/- 2.68 hours (fasted) to 10.53 +/- 2.89 hours (fed).(ABSTRACT TRUNCATED AT 250 WORDS)

The site of gastrointestinal absorption of gepirone in humans.

This study was conducted in seven healthy male subjects and was performed over four sessions with a 1-week washout between sessions. It was designed to compare the bioavailability of an oral 20-mg gepirone dose (treatment 1) with that obtained after application of the same dose by gastric intubation to the distal (treatment 2) and proximal (treatment 3) regions of the small intestine, and after 4 consecutive 5-mg gepirone doses given orally at hourly intervals (treatment 4). Serial blood samples were taken over 24 hours after dose after each treatment. Plasma concentrations of gepirone and 1-(2-pyrimidinyl)-piperazine (1-PP), a metabolite of gepirone, were quantitated by gas chromatography-mass spectrometry. Mean gepirone time to reach peak concentration (tmax) after treatments 1, 2, and 3 ranged between 0.57 and 1.07 hours. There were no significant differences between sites and treatments for gepirone t1/2, which ranged between 2.8 and 3.3 hours. The mean gepirone maximum peak plasma concentration (Cmax) was significantly higher (P less than .05) after treatment 2 (12.92 +/- 7.24 ng/mL) compared with treatment 1 (6.79 +/- 3.54 ng/mL) or treatment 3 (6.33 +/- 2.26 ng/mL). Gepirone area under the curve (AUCinf) was also significantly higher (P less than .05) after treatment 2 (29.83 +/- 17.42 ng.h/mL) compared with treatment 1 (18.07 +/- 6.10 ng.h/mL) or treatment 3 (17.74 +/- 7.69 ng.h/mL). There were no significant differences in gepirone AUCinf between treatments 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic method for the determination of nefazodone and its metabolites in human plasma using laboratory robotics.

A quantitative analytical method, using high-performance liquid chromatography and ultraviolet detection, has been established for the determination of nefazodone (NEF) and its metabolites, m-chlorophenylpiperazine (mCPP),p-hydroxynefazodone (PHN), and hydroxynefazodone (HO-NEF), in human plasma. The fully automated, robotic procedure consisted of addition of internal standard (aprindine), extraction with butyl chloride, followed by phase separation, organic phase evaporation, reconstitution of the residue, and injection onto the chromatographic system. The limits of detection for NEF, mCPP, PHN, and HO-NEF were 5, 1, 10, and 5 ng/ml, respectively, at a signal-to-noise ratio of 4. The method had a linear range of 10-1000 ng/ml for NEF and HO-NEF, 20-2000 ng/ml for PHN, and 2.5-250 ng/ml for mCPP. Correlation coefficients of 0.996 or greater were obtained during validation and study sample analysis.

Simultaneous quantitation of buspirone and 1-(2-pyrimidinyl)piperazine in human plasma and urine by capillary gas chromatography-mass spectrometry.

Buspirone and a buspirone metabolite, 1-(2-pyrimidinyl)piperazine (1-PP), are extracted from matrix using C18 extraction columns. The metabolite and its internal standard (d4-1-PP) are derivatized with pentafluorobenzoyl chloride to the corresponding amides. The 1-PP derivatives, buspirone and the buspirone internal standard (5-fluorobuspirone) are co-chromatographed. Chromatography and detection are performed using capillary gas chromatography with a fused-silica column and selected-ion monitoring-mass spectrometry. Linear range of the standard curves in plasma is 0.1-14 ng/ml for buspirone and 0.2-25 ng/ml for 1-PP with lower limits of quantitation of 0.1 and 0.2 ng/ml, respectively. In urine the linear range of the standard curves is 0.2-14 ng/ml for buspirone and 8-500 ng/ml for 1-PP with lower limits of quantitation of 0.2 and 8.0 ng/ml, respectively. Intra-assay accuracies were within 14% for buspirone and 1-PP in plasma and urine. Intra-assay precision was within 12% for both compounds in both matrices.

Liquid chromatographic procedure for the quantitative analysis of carboplatin in beagle dog plasma ultrafiltrate.

A specific and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of carboplatin (JM-8) in dog plasma ultrafiltrate. Plasma ultrafiltrate samples were generated using Amicon Centrifree micropartition systems or Amicon Centriflo cones, and injected onto a microBondapak NH2 column. The mobile phase consisted of acetonitrile:methanol:0.005 M sodium perchlorate, pH 2.4 (77-75:13-15:10, v/v/v); the flow rate was 1.5 mL/min. Detection was performed by monitoring UV absorbance of the column effluent at 229 nm. Carboplatin eluted between 9.5 and 11.0 min. The internal standard, JM-10, eluted between 11.0 and 13.0 min. The peak height ratio of carboplatin:internal standard versus carboplatin concentration was linear over a range of 0.2 to 20.0 micrograms/mL. The limit of quantitation was 0.2 microgram/mL. The intra-assay precision of this method, as measured by percent relative standard deviation (%RSD), was within 12% for the theoretical concentrations 0.5, 5.0, and 50.0 micrograms/mL. Accuracies were within 11%. The results of the validation procedures indicated that this procedure was accurate and specific.

The disposition of carboplatin in the beagle dog.

Carboplatin was administered i.v. to four groups of three male beagle dogs at doses of 3, 6, 12, and 24 mg/kg (60-580 mg/m2). Plasma samples were obtained at appropriate times and protein-free plasma ultrafiltrates (PU) were generated with Amicon Centrifree micropartition systems. Urine was collected at 24-h intervals for 96 h. PU and urine samples were analyzed for carboplatin by HPLC and for total platinum by atomic absorption spectrophotometry. Carboplatin accounted for about 90% of the free platinum in plasma. The Cmax and AUCinf values for carboplatin and for free platinum increased linearly with dose. The terminal elimination half-life and mean residence times for carboplatin and free platinum were each about 1 h. Total-body clearances for carboplatin (5.6 l/h per m2) and free platinum (5.1 l/h per m2) were constant over the dose range studied, as were the respective volumes of distribution (5.7 and 5.0 l/m2). A mean of 46% of the dose was excreted as carboplatin in 24-h urine; and by 72 h, 70% of the platinum administered was excreted in the urine. Free platinum was cleared by both renal and non-renal processes. These results show that a dose of carboplatin is rapidly excreted in the urine and that carboplatin and plasma-free platinum exhibit linear pharmacokinetics in the beagle dog.

The disposition of carboplatin in ovarian cancer patients.

Carboplatin was given as a 30-min infusion to 11 ovarian cancer patients at doses of 170-500 mg/m2. The ages, weights, and creatinine clearances (Clcr) ranged from 44 to 75 years, from 44 to 74 kg, and from 32 to 101 ml/min, respectively. Plasma, plasma ultrafiltrate (PU), and urine samples were obtained at appropriate times for 96 h and were analyzed for platinum. The PU and urine were also analyzed for the parent compound by HPLC. In patients with a Clcr of about 60 ml/min or greater, carboplatin decayed biexponentially with a mean t1/2 alpha of 1.6 h and a t1/2 beta of 3.0 h. The mean (+/- SD) residence time, total body clearance, and apparent volume of distribution were 3.5 +/- 0.4 h, 4.4 +/- 0.85 l/h, and 16 +/- 3 l, respectively. Cmax and AUCinf values increased linearly with dose, and the latter values correlated better with the dose in mg than in mg/m2. No significant quantities of free, ultrafilterable, platinum-containing species other than the parent compound were found in plasma, but platinum from carboplatin became protein-bound and was slowly eliminated with a minimal t1/2 of 5 days. The major route of elimination was excretion via the kidneys. Patients with a Clcr of 60 ml/min or greater excreted 70% of the dose as the parent compound in the urine, with most of this occurring within 12-16 h. All of the platinum in 24-h urine was carboplatin, and only 2%-3% of the dosed platinum was excreted from 48 to 96 h. Patients with a Clcr of less than about 60 ml/min exhibited dose-disproportional increases in AUCinf and MRT values. The latter were inversely related to Clcr (r = -0.98). Over a dose range of 300-500 mg/m2, carboplatin exhibited linear, dose-independent pharmacokinetics in patients with a Clcr of about 60 ml/min or greater, but dose reductions are necessary for patients with mild renal failure.

Comparison of the pharmacokinetics of AMSA and AMSA-lactate in patients with acute nonlymphoblastic leukemia.

A pharmacokinetic study was performed in 13 adult patients with acute nonlymphoblastic leukemia to compare two formulations of 4'-(9-acridinylamino)-methanesulphone-m-ansidide (AMSA): the original formulation, AMSA-NCL, and a water-soluble lyophilized formulation, AMSA-lactate (Bristol Myers, Syracuse, N.Y. USA). Initially, the patients received either AMSA-NCL or AMSA-lactate, 75-90 mg/m2 daily, for 3-7 days as a 1-h infusion. Eight patients subsequently crossed over to receive the other formulation. Plasma samples for drug determination were collected during the first 3 days. A new method for determination of AMSA is described. Acidified plasma samples containing an internal standard were extracted with hexane, then made alkaline, whereafter, AMSA was extracted with ethylacetate. Extracts were reconstituted in absolute ethanol and analyzed by high-pressure liquid chromatography (HPLC) using a reverse-phase C-18 column and UV detection at 254 nm. There were no clear differences in clinical effects and toxicity between the two formulations. Patients with the highest total area under the drug concentration-versus-time curves (AUCs) for plasma concentrations versus time had significantly lower nadir for white blood cell count, suggesting a relation between plasma levels and bone marrow toxicity for AMSA. The pharmacokinetics showed a biphasic elimination for both formulations. The mean terminal elimination half-life of AMSA-NCL and AMSA-lactate was 7.1 and 6.3 h, respectively, and the mean volume of distribution was 105 and 99 L/m2, respectively. No significant differences in the pharmacokinetics comparing days 1 and 3 were seen.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatographic method for the determination of etoposide in plasma using electrochemical detection.

A quantitative analytical method has been established for the determination of a semi-synthetic epipodophyllotoxin, etoposide, in plasma. The method employs reversed-phase high-performance liquid chromatography and electrochemical detection. Sample preparation consisted of extraction with 1,2-dichloroethane followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Observed recoveries were 76.8 and 87.5% for 50 and 500 ng/ml, respectively. The method had a linear range of 10-1000 ng/ml. Correlation coefficients of 0.997 or greater were obtained during validation experiments and study sample analysis.

Liquid chromatographic procedure for the quantitative analysis of megestrol acetate in human plasma.

A simple, sensitive, and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of megestrol acetate in human plasma. An internal standard, 2,3-diphenyl-1-indenone, was added to 0.5 mL of plasma followed by extraction with hexane. The residue remaining after evaporation of hexane was reconstituted in methanol and injected onto a mu-Bondapak C18 column. The column was eluted with acetonitrile:methanol:water:acetic acid (41:23:36:1), and the eluant was monitored at 280 nm. Megestrol acetate and the internal standard eluted at 6-7 and 12-14 min, respectively. The peak height ratio (megestrol acetate/internal standard) versus plasma concentration was linear over a range of 10-600 ng of megestrol acetate/mL of plasma, and the limit of detection was 5 ng/mL. The mean intra- and interassay accuracies were within 3% of the actual values. The mean intra- and interassay precision, as estimated by RSD, were 4 and 6%, respectively. Constituents in human plasma and megestrol, a possible degradation product, did not interfere in the assay. The procedure was applied to the analysis of plasma samples from subjects receiving 40 mg of Megace q.i.d.

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.

The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca2+ transport. The absorbance and fluorescence responses of 3,3'-diethyl-2,2'-indodicarbocyanine and oxonol VI during ATP-dependent Ca2+ transport are influenced by the effect of accumulated Ca2+ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca2+ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca2+, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulum in vivo and in vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca2+ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca2+ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca2+ translocation. The potential generated during Ca2+ transport is rapidly dissipated by passive ion fluxes across the membrane.