Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase.

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface.

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0-50.0 ng/ml for nicotine and 10.0-500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil CI8 column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were < or = 6.42% relative standard deviation (R.S.D.) and < or = 11.8% relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls, (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were < or = 6.34% R.S.D. and < or = 7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.

Stereospecific determinations of (+/- )-DU-124884 and its metabolites (+/- )-KC-9048 in human plasma by liquid chromatography.

(+)-DU-124884 is a 5-HT1-like receptor agonist under investigation for drug development. A sensitive, stereospecific LC method was developed for the analysis of (+)-DU-124884, its optical isomer (-)-DU-124884 and their N-dealkylated metabolites, (+/- )-KC-9048, in human plasma. A plasma sample was treated with triethylamine in methanol and the proteins were precipitated by acetonitrile. The supernatant was evaporated to dryness under nitrogen. The analytes and internal standard (acebutolol) formed diastereomers with (S)-(+)-1-(1-naphthyl)ethyl isocyanate immediately. The diastereomers formed were extracted into diethyl ether. They were completely resolved from each other and from matrix peaks on a Microsorb silica column with a mobile phase of methanol-chloroform-hexane (8:12:80, v/v/v) in a run time of 26 min. Detection was by fluorescence with excitation wavelength at 320 nm and emission wavelength at 440 nm. The linearity range is 0.1-200 ng ml-1 (r > 0.99). The limit of quantitation is 0.1 ng ml-1 and the detection limit is 0.02 ng ml-1 (signal-to-noise ratio = 3). The interday precision and accuracy of quality control samples were 5.5-7.6% RSD (relative standard deviation) and 0 to+4% bias for (+)-DU-124884, 5.5-7.9% RSD and 0 to +4% bias for (-)-DU-124884, 4.5-6.5% RSD and -7 to 0% bias for (+)-KC-9048 and 4.5-7.5% RSD and -7 to 0% bias for (-)-KC-9048. Consistent recovery from different lots of human plasma, parallelism of the method, stabilities of on-system, reinjection, bench-top, freeze-thaw cycles and sample storage were established.

Ganciclovir absolute bioavailability and steady-state pharmacokinetics after oral administration of two 3000-mg/d dosing regimens in human immunodeficiency virus- and cytomegalovirus-seropositive patients.

Oral ganciclovir has recently been approved for use in long-term maintenance therapy in the treatment of cytomegalovirus (CMV) retinitis in immunocompromised patients. Although oral ganciclovir at a dose of 3,000 mg/d is moderately less effective than intravenous (i.v.) ganciclovir maintenance therapy (5 mg/kg as a 1-hour i.v. infusion every 24 hours), convenience and practicality make oral maintenance therapy desirable. Two dosing regimens--1,000 mg three times daily (TID) and 500 mg every 3 hours (six times daily)--have been shown to be efficacious. Eighteen human immunodeficiency virus- and CMV-seropositive patients participated in a three-way, open-label, crossover study to evaluate the steady-state pharmacokinetics and absolute bioavailability of the two oral regimens compared with the i.v. regimen. Sixteen patients completed the study and received ganciclovir as a single 5-mg/kg i.v. infusion over 1 hour, 500 mg orally every 3 hours while awake (six times daily) for 3 days, and 1,000 mg TID orally for 3 days. Blood samples were obtained over a 24-hour period after the single i.v. dose and on day 3 of the oral dosing regimens. Mean peak serum concentrations were 8.27, 1.02, and 1.18 micrograms/mL for the i.v. and oral regimens, respectively. Twenty-four-hour area under the curve (AUC) for the oral regimens--500 mg every 3 hours and 1,000 mg TID--were 15.9 and 15.4 micrograms.h/mL, respectively, as compared with a total AUC of 22.1 micrograms.h/mL for the single i.v. dose. The absolute bioavailabilities for the two oral regimens were 8.84% and 8.53%, respectively. The extent of ganciclovir absorption, peak concentrations, and average concentration at steady state were not statistically different between the two oral regimens. The peak-to-trough concentration ratio (Cmax:Cmin) was greater for the 1,000-mg TID regimen than for the regimen of 500 mg every 3 hours (5.35 vs 3.81 [P < 0.01]). Both oral regimens resulted in concentrations in the range of the concentration that inhibits 50% of most human CMV isolates. Because both oral regimens provide equivalent absorption, the 1,000-mg TID regimen may be preferred for the convenience and potentially greater compliance associated with fewer daily doses.

Surrogate biochemical markers: precise measurement for strategic drug and biologics development.

More efficient drug and biologics development is necessary for future success of pharmaceutical and biotechnology companies. One way to achieve this objective is to use rationally selected surrogate markers to improve the early decision-making process. Using typical clinical chemistry methods to measure biochemical markers may not ensure adequate precision and reproducibility. In contrast, using analytical methods that meet good laboratory practices along with rational selection and validation of biochemical markers can give those who use them a competitive advantage over those who do not by providing meaningful data for earlier decision making.

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma.

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 microliters of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C18 column with a mobile phase of methanol-0.1 M potassium phosphate monobasic-water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1-100 micrograms/ml (r > 0.999). The limit of quantitation was designed as 1 microgram/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and -2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 micrograms/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and -0.9 to -0.4% R.E. Recovery of methocarbamol was 91.4-100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C18 columns.

Pharmacokinetics of esmolol and ASL-8123 in renal failure.

The effect of renal function on the pharmacokinetics of esmolol, an ultra-short-acting beta-adrenergic blocker, and its major metabolite, ASL-8123, was examined in six healthy control subjects, six patients maintained on hemodialysis, and six patients on continuous ambulatory peritoneal dialysis (CAPD). In addition, the impact of hemodialysis and CAPD on removal of esmolol and ASL-8123 was determined. Multiple blood, urine, and dialysate samples were collected during a 72-hour period and assayed for esmolol and ASL-8123 by HPLC. The pharmacokinetic disposition of esmolol was not significantly altered by renal failure. Mean (+/- SD) total body clearance for esmolol was 171.4 +/- 69.8, 249.8 +/- 176.3, and 265.3 +/- 143.1 ml/min/kg for the control, hemodialysis, and CAPD patients, respectively. Mean elimination half-life (t1/2) was 7.2 minutes in control subjects compared with 7.1 and 8.0 minutes for the hemodialysis and CAPD groups, respectively. The apparent volume of distribution of esmolol did not differ significantly among the three groups. ASL-8123 was shown to accumulate in patients with renal failure, as evidenced by a mean maximum blood concentration of 42.8 +/- 12.2 micrograms/ml in the control group compared with 76.1 +/- 23.9 and 87.1 +/- 20.4 micrograms/ml in the hemodialysis and CAPD groups, respectively (p less than 0.05). The elimination t1/2 of ASL-8123 was prolonged in patients with renal failure, averaging more than 42 hours compared with only 4 hours in the control subjects. Approximately 20% of the esmolol dose as ASL-8123, was removed by either hemodialysis or CAPD, contributing minimally to the elimination of the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of esmolol in hepatic disease.

Esmolol is an intravenous beta blocker with a short duration of action. The pharmacokinetics of esmolol and its acid metabolite, ASL-8123, were studied in nine patients who had stable, biopsy-proved Laennec's cirrhosis and in three normal volunteer controls. Kinetics were determined after a four-hour continuous infusion of esmolol at a rate of 200 micrograms/kg/min. Blood samples were collected during the infusions and at frequent intervals thereafter. The parameters studied were the steady state concentration, the total body clearance, the elimination half-life, the area under the curve, and the volume of distribution. No significant differences in any of these parameters were detected between control subjects and those with hepatic disease, for either esmolol or its acid metabolite. It is concluded from this study that Laennec's cirrhosis does not cause any change in the pharmacokinetics of esmolol or its major metabolite. Therefore, adjustments in dosage of esmolol are not required for patients with Laennec's cirrhosis.

Pharmacokinetics and pharmacodynamics of flestolol, a new short-acting, beta-adrenergic receptor antagonist.

The pharmacokinetics and pharmacodynamics of flestolol, a new short-acting, beta-adrenergic receptor antagonist, were examined in nine healthy subjects after a constant intravenous infusion of 5 micrograms/kg/min for 72 hours. Flestolol blood levels were determined by high-performance liquid chromatography. In all subjects, flestolol blood concentration attained steady state 30 minutes after initiation of infusion. The mean +/- standard deviation steady-state concentration of flestolol was 31.1 +/- 12.0 ng/mL. The elimination half-life averaged 7.2 minutes. The mean +/- standard deviation total body clearance was 181 +/- 66 mL/min/kg. The apparent volume of distribution and the area under the curve averaged 1.89 L/kg and 2.23 micrograms-hr/mL, respectively. Flestolol did not cause any significant change (P greater than .05) in the heart rate or systolic or diastolic blood pressure from the baseline. Flestolol significantly (P less than .05) attenuated the isoproterenol-induced increase in heart rate and systolic blood pressure and decrease in diastolic blood pressure in comparison with baseline. The average maximum reduction in isoproterenol tachycardia was in the range of 63% to 79% during flestolol infusion. There was a rapid recovery from beta blockade after termination of flestolol infusion; the recovery averaged 96% 20 minutes after the infusion was stopped. We conclude that flestolol exhibits a very short half-life and is cleared mainly by extrahepatic routes. It is an effective beta blocker and possesses a short duration of action.

Effects of intensive granulocyte donation on donors and yields.

Thirteen HLA-identical bone marrow donors served as the sole source of daily granulocyte transfusions for respective marrow recipients during the period of severe neutropenia between transplantation and engraftment. They experienced 12 to 29 (median, 17) daily, continuous flow centrifugation leukapheresis procedures using hydroxyethyl starch, but no corticosteroids, with little serious difficulty. No immediate clinical reactions occurred in 90 percent of 228 procedures. Mild citrate reactions were noted in 9 percent, and only two procedures (0.8%) were discontinued due to severe reactions. Ten donors (77%) answered a questionnaire mailed weeks later, and six reported transient, late clinical adverse effects. Five had moderate dermatologic problems; one had minimal hypertension requiring no therapy. Donors were monitored daily for laboratory abnormalities while donating granulocytes. Hemoglobin concentration and platelet counts remained stable (autologous red cell transfusions had been given). Blood leukocyte counts gradually fell (p less than 0.05), particularly after 10 or more daily granulocyte donations, and this fall was associated with a decrease of about 33 percent in leukocyte yields. No attempts were made to improve yields by giving higher doses of hydroxyethyl starch or by corticosteroid stimulation. With primary emphasis on donor safety, it seems feasible for a few compatible donors to provide prolonged granulocyte transfusion support for designated patients. However, diminishing leukocyte yields may result from intensive, repeated leukapheresis.

Liquid-chromatographic analysis for esmolol and its major metabolite in urine.

We describe a simple, reproducible liquid-chromatographic method for determination of esmolol (a short-acting beta blocker) and its major metabolite in human urine. Esmolol is extracted from urine at a pH of 8.4 into methylene chloride; the more polar metabolite remains in the aqueous phase. We then measure esmolol with a muBondapak C18 column and measure ultraviolet absorbance at 229 nm; the metabolite is analyzed with a Spherisorb phenyl column, with absorbance measured at 280 nm. The average extraction recoveries of esmolol and the metabolite were 95 and 92%, respectively. Standard curves were linear and reproducible for esmolol from 0.025 to 5 mg/L and for the metabolite from 1 to 250 mg/L. Within-day CVs for both compounds were less than 6%.

Metabolism and urinary excretion of esmolol in humans.

The urinary excretion patterns of esmolol, a short-acting beta blocker, and its major metabolite were investigated in eight healthy men after intravenous infusion of 50, 100, 200, and 300 micrograms/kg/min of esmolol for six hours and 150 micrograms/kg/min for 24 hours. Esmolol and the metabolite concentrations in urine were determined by high-performance liquid chromatography. The mean urinary recoveries of the unchanged drug were 0.64%, 0.67%, 0.69%, 0.77%, and 0.98% after the 50, 100, 150, 200, and 300 micrograms/kg/min dose, respectively. Recovery of the metabolite was independent of dose, and the overall mean recovery accounted for 73% of administered dose. The results of this study indicate that esmolol is extensively metabolized, and the extent of the metabolism is not dose related in the dosage range used. The renal route plays a very minor role in the elimination of the drug but is important for the elimination of the metabolite.

Pharmacokinetics of flestolol in man: preliminary data.

The steady state pharmacokinetics of flestolol, a short acting beta-adrenoceptor blocking agent, were studied in six healthy subjects following intravenous infusion of six different doses; 18, 24, 35, 50, 75 and 100 micrograms kg-1 min-1, for 60 min. Steady state blood levels increased linearly with dose for all six subjects. The overall mean half-life was 6.5 min. The total body clearance averaged 208 ml min-1 kg-1 indicating significant extrahepatic metabolism of the drug. There were no significant changes in the half-lives or the total body clearances after the six doses, suggesting that the kinetics of flestolol are linear over the dose range studied.

Hetastarch: an overview of the colloid and its metabolism.

Hetastarch, ethoxylated amylopectin, has found clinical utility as a plasma volume expansion agent, a sedimenting agent during pheresis, and a pump priming fluid. Hetastarch is a complex mixture of derivatized amylopectin molecules of various molecular sizes. The derivatization causes resistance to enzymatic hydrolysis, therefore, allowing hetastarch sufficient vascular residence time to be an effective vascular osmotic agent. This has led to its use as a volume expander and to its consequent use as a pump priming fluid. The metabolism of hetastarch proceeds through alpha-amylase hydrolysis of glycosidic bonds, yielding molecules small enough for renal clearance, but does not result in complete hydrolysis. Hence, glucose is not a significant product of hetastarch metabolism. Metabolism proceeds at such a rate that volume expansion is seen for 24-36 hours with a maximum effect (100-172 percent of the infused volume) occurring shortly after infusion. Ninety percent of the dose is eliminated with a half-life of about 17 days.

Pharmacokinetics of high molecular weight hydroxyethyl starch in dogs.

The elimination of high molecular weight hydroxyethyl starch (HMW-HES) was determined in dogs. . Eight dogs were divided into two groups; one of which was bled 25 ml/kg. Both groups were given 25 ml/kg of 6% HMW-HES solution over a 30 min period. There was no significant difference in plasma concentration vs time profiles between the two groups. The biological half-life estimated between days 7-28, averaged 7.5 and 8.4 days in control and bled dogs, respectively. The renal clearance of HMW-HES diminished considerably in both groups during the 7 day period. Despite large interanimal variations in renal clearance, there was not a significant difference between groups.

Pharmacokinetics of hydroxyethyl starch in normal subjects.

To determine the elimination of high-molecular-weight hydroxyethyl starch (HES, Mw 450,000) in normal subjects, ten volunteers were given 500 ml 6% HES solution by intravenous infusion, and serial blood and urine samples were collected for nonglucose total carbohydrate determination. On the average, 46 and 64 per cent of the dose was excreted in the urine within two and eight days, respectively. The plasma concentration declined rapidly during the first week after infusion. The average terminal half-life was 17 days during the first 42 days, which accounted for elimination of about 90 per cent of the dose. The remainder was eliminated with a terminal half-life of 48 days determined between days 42 and 83 of the study. As expected, the infusion of HES resulted in plasma volume expansion over a 48-hour period during which time levels of nonglucose carbohydrates were above 3.5 mg/ml. HES is metabolized by alpha-amylase in the body. During the first 48 hours after infusion of HES, plasma alpha-amylase activity was significantly increased over control. Concomitantly, alpha-amylase activity in urine was also elevated but not significantly so.