Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids.

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC-MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 microg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d6-DDVP I.S. and MS detection. The LOQ of DDVP was 1 microg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at -20 degrees C or -80 degrees C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at -20 degrees C and after 23 months of storage at -80 degrees C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.

Pharmacokinetics of a once-daily oral dose of moxifloxacin (Bay 12-8039), a new enantiomerically pure 8-methoxy quinolone.

The pharmacokinetics, safety, and tolerability of oral moxifloxacin, a new 8-methoxy quinolone, were assessed in a randomized, double-blind, placebo-controlled study in which healthy male and female volunteers received either 400 mg of moxifloxacin once daily (n = 10) or a placebo once daily (n = 5) for 10 days. Plasma moxifloxacin concentrations on days 1 and 10 were measured by high-performance liquid chromatography and fluorometric detection. Standard pharmacokinetic parameters were estimated by noncompartmental methods. Natural logarithmic estimates for each pharmacokinetic variable of each subject were analyzed by a two-way analysis of variance. Hematology, blood chemistry, vital signs, and adverse events were monitored, and electrocardiograms (ECG) were performed. Plasma moxifloxacin concentrations of predicted therapeutic relevance were achieved in this study. For day 1, the mean maximum concentration of drug in serum (C(max)) and the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) were 3. 4 mg/liter and 30.2 mg. h/liter, respectively. Corresponding means on day 10 were 4.5 mg/liter and 48 mg. h/liter, respectively. On day 10, the mean elimination half-life was approximately 12 h. Plasma moxifloxacin concentrations exceeded the MIC for Streptococcus pneumoniae throughout the 24-h dosing period. The day 1 and day 10 mean AUC/MIC ratios were 121 and 192, respectively, and the mean C(max)/MIC ratios were 13 and 18, respectively. Moxifloxacin was well tolerated; no clinically relevant changes in the standard laboratory tests, vital signs, or ECG were observed. Pharmacokinetic parameters demonstrated linearity, and estimates of pharmacokinetic/pharmacodynamic ratios (AUC/MIC and C(max)/MIC) indicate that the regimen of 400-mg once daily should be effective for treating a variety of infections. Moxifloxacin was found to be safe and well tolerated in healthy volunteers when it was given as a single daily 400-mg dose for 10 days.

Quantitative analysis of HBY 097 and its metabolites in human serum and urine by HPLC.

Two HPLC methods were developed: one for the quantitation of HBY 097 reverse transcriptase inhibitor and its metabolites M2 and M3 in human serum, and one for the quantitation of metabolite M5 in urine. The HPLC procedure for the quantitation of HBY 097 and its metabolites M2 and M3 in human serum involved protein precipitation with acetonitrile followed by automated on-line trace enrichment. The HPLC procedure for the analysis of metabolite M5 in urine involved enzymatic hydrolysis of urine with beta-glucuronidase to convert metabolite M5 (glucuronide of M3) to M3. Reverse phase chromatographic separation with gradient elution. UV detection at 335 nm, and internal standard were used to quantitate analytes in both procedures. The lower quantitation limits were 25 ng ml-1 for HBY 097 and metabolites M2 and M3 in serum, and 0.5 microgram ml-1 for the metabolite M5 in urine measured as metabolite M3 after hydrolysis. The HBY 097 and metabolite M3 concentrations were specific but metabolite M2 was semi-specific because the two diastereomers of M2 were not resolved by the present chromatographic procedure. Both procedures were applied to the quantitation of HBY 097 and its metabolites in serum and urine of HIV positive patients who were enrolled in a clinical study of drug safety and pharmacokinetics.

Determination of LTE4 in human urine by liquid chromatography coupled with ionspray tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatographic/ionspray tandem mass spectrometric (LC/ionspray MS/MS) method was developed and validated to quantitate leukotriene E4 (LTE4) in human urine. This method involves solid-phase extraction with Empore membrane disks to isolate LTE4 and its internal standard, LTE4-d3, from the urine stabilized with antioxidant and metal ion chelating agent. The reconstituted extracts were analyzed by LC/ionspray MS/MS in the selected reaction monitoring (SRM) mode. The assay has a lower level of quantitation (LOQ) of 50 pg ml-1 for LTE4 based on 5 ml aliquots of urine. The calibration graphs were linear from 50 pg ml-1 to 10 ng ml-1 for LTE4 extracted from urine. The inter- and intra-assay precision (RSD) and accuracy (DEV) did not exceed 11% and 8% at any level of the calibration standards and quality control (QC) samples, respectively. The recovery of LTE4 using the Empore disk solid-phase extraction technology was independent of LTE4 concentration in human urine. The overall extraction recovery for LTE4 was 72% (RSD = 2.14%).

Ciprofloxacin pharmacokinetics in burn patients.

Many drugs exhibit altered pharmacokinetic parameters in burn patients. We prospectively evaluated the pharmacokinetics of ciprofloxacin in eight burn patients with active infections. Each patient received a 400-mg dose of ciprofloxacin intravenously (i.v.) every 8 h, with each dose infused over 1 h by using a rate control device. Blood samples for analysis of plasma ciprofloxacin concentrations, determined by high-performance liquid chromatography, were obtained immediately predose, at the end of the infusion, and 1, 2, 3, 4, 5, 6, and 7 h after the end of the infusion. Urine was collected from 0 to 2, 2 to 4, and 4 to 8 h following the same dose, and an aliquot was saved for determination of the ciprofloxacin concentration. Urine was also collected for 24 h prior to this dose for measurement of creatinine clearance (CLCR). Pharmacokinetic parameters were estimated by noncompartmental analysis. Mean maximum and minimum plasma ciprofloxacin concentrations were 4.2 +/- 1.1 and 0.70 +/- 0.55 microgram/ml, respectively. Mean values for clearance (CL), renal clearance (CLR), volume of distribution, terminal elimination rate constant, half-life (t1/2), and area under the concentration-time curve (AUC) were 29.1 +/- 17.5 liters/h, 13.5 +/- 10.1 liters/h, 1.75 +/- 0.41 liters/kg, 0.222 +/- 0.098 h-1, 4.5 +/- 3.9 h, and 20.7 +/- 16.6 micrograms.h/ml, respectively. CL was higher and t1/2 was shorter than noted in previous studies of acutely ill, hospitalized patients. A good correlation was noted between creatinine clearance CL(CR) and both total ciprofloxacin CL (r = 0.85) and CLR (r = 0.84). A moderate inverse correlation was noted between percent body surface area burned and total ciprofloxacin CL (r = -0.55). An AUC/MIC ratio above 125 SIT-1 (where SIT is serum inhibitory titer), which has been strongly correlated with clinical response and time to bacterial eradication, was achieved in five of eight patients (63%) with a MIC of 0.25 microgram/ml. At a ciprofloxacin dosage of 400 mg i.v. every 12 h, an AUC/MIC ratio above 125 SIT-1 would have been achieved in only two of eight patients (25%). We conclude that ciprofloxacin CL is highly variable, but generally increased, in burn patients compared with that in acutely ill, general medical and surgical patients. Because of an increase in CL, a ciprofloxacin dosage of 400 mg i.v. every 8 h is more likely to produce the desired response in burn patients than the same dose given every 12 h.

HPLC analysis of ciprofloxacin and ciprofloxacin metabolites in body fluids.

An improved high-performance liquid chromatography (HPLC) procedure for the analysis of ciprofloxacin and three of its metabolites in plasma, serum and urine samples was developed. The previously published HPLC procedure described the isocratic separation of ciprofloxacin and three ciprofloxacin metabolites in urine samples on a polystyrene-divinylbenzene reverse-phase column followed by quantitation using a UV detector. The present procedure involved the same chromatographic separation, but is also applicable to the analysis of plasma and serum as well as urine samples, and quantitation was based on fluorometric detection after postcolumn induction of fluorescence instead of UV detection. The post-column induction of fluorescence was necessary because the M2 and M3 metabolites of ciprofloxacin have relatively weak native fluorescence, and induction enhanced the fluorometric signals of metabolites M2 and M3 forty-four-fold and eleven-fold, respectively. The observed enhancement of fluorescence may be attributed to the partial conversion by UV light of metabolites M2 and M3 to metabolite M1 which has intense native fluorescence. The lower quantitation limits of ciprofloxacin and metabolites M1, M2 and M3 were 0.05 micrograms ml-1, 0.01 micrograms ml-1, 0.05 micrograms ml-1, and 0.5 micrograms ml-1, respectively. All four analytes were quantitated using one isocratic elution of either plasma or serum supernatant after the precipitation of proteins or the isocratic chromatography of diluted urine samples.

LC separation and induced fluorometric detection of rivastatin in blood plasma.

An LC procedure suitable for quantitative analysis of pg ml-1 concentrations of the HMG-CoA reductase inhibitor rivastatin in blood plasma was developed. The procedure involves an extraction step, chromatography on an ODS column, and fluorometric detection of a post-column photolytic decomposition product that was isolated and identified. The achieved quantitation limit (25 pg ml-1) facilitated analysis of relatively low rivastatin concentrations in plasma that were observed after 100-300 micrograms oral doses of rivastatin. At 25 pg ml-1 concentration the RSD ranged from 3.6 to 13.5% and mean deviation from the nominal value was 8.0%; at 8 ng ml-1 the RSD range was 0.7-3.6% while the mean deviation was -1.8%. The concentrations obtained with the LC procedure were compared to the concentrations obtained with a specific but less sensitive capillary GC method and a radioimmunoassay (RIA) procedure. Concentrations obtained with the HPLC and GC procedures agreed within experimental error; the RIA concentrations were about 30% higher.

Nitrendipine in human plasma and breast milk.

To assess the disposition of the dihydropyridine calcium antagonist, nitrendipine, in lactating mothers, we studied three breast-feeding women to determine simultaneous plasma and breast milk concentrations of nitrendipine and its inactive pyridine analog metabolite after both a single 10 mg oral dose and 5 days of continuous therapy (20 mg per day). Nitrendipine was excreted in breast milk at peak concentrations ranging from 4.3 to 6.5 ng/ml 1-2 h after acute dosing while its inactive pyridine metabolite ranged from 6.9 to 11.9 ng.ml-1. After 5 days of dosing, Cmax remained in the same range and the breast milk/whole plasma concentration ratio for nitrendipine was 0.2 to 0.5. On the fourth day of continuous dosing, average concentrations of nitrendipine from 24-h collections of the milk were 1.1 to 3.8 ng.ml-1. Thus, nitrendipine and its metabolite are excreted in very low concentrations in human breast milk. Based on a maternal dose of 20 mg daily, a newborn infant would ingest an average of 1.7 micrograms of nitrendipine per day, or a relative dose of 0.095%.

The effects of age and race on nitrendipine pharmacokinetics and pharmacodynamics.

The pharmacokinetics and pharmacodynamics of a single 20-mg dose of nitrendipine (NTP) were studied in four groups of subjects (n = 9 per group). Group 1 were young white normotensive males, Group 2 were elderly white hypertensive males, Group 3 were black hypertensive males aged 40-55 years, and Group 4 were white hypertensive males aged 40-55 years. All other medications were withdrawn prior to NTP dosing. NTP was given in the morning 1 h after breakfast. Plasma samples for NTP assay were collected at predetermined times up to 48 h after dosing. Blood pressure was monitored before dosing, and at 0.5, 1, 3, 5, 12, and 24 h postdose. Pharmacokinetic parameters were found to be dependent on age. The area under the curve for Groups 1, 2, 3, and 4 was 50.5 +/- 19.4, 186 +/- 120, 107 +/- 49, and 88 +/- 43 ng h/ml, respectively (p less than 0.05). Corresponding values of elimination half-life were 9.9 +/- 1.3, 20 +/- 6.5, 13.3 +/- 6.1, and 15.9 +/- 8.0 h (p less than 0.05). Both diastolic and systolic blood pressures were significantly reduced from the baseline value in Groups 2, 3, and 4, with diastolic pressure remaining significantly lower than baseline at 24 h postdose. Based on the increased plasma levels and slower elimination of NTP in the elderly, as well as the measured blood pressure lowering, once daily dosing of NTP may be appropriate in some patients.

Bioequivalence and metabolism of nitrendipine administered orally to healthy volunteers.

Relative bioavailability of 5-, 10-, and 20-mg nitrendipine tablets was determined in a four-way crossover bioequivalence study involving 22 normal male volunteers. Liquid suspension of nitrendipine was used as a reference. Plasma and urine samples collected during each study period were assayed by high performance liquid chromatographic and capillary gas chromatographic (GC) procedures for nitrendipine and the nitrendipine pyridine metabolite. Four other more polar urinary nitrendipine metabolites were also analyzed in urine by the GC procedure, which involved diazomethane esterification. Although relative bioavailability of the tablets ranged from 58.0 to 69.9%, there was no statistically significant difference in the area under the curve among the doses. Since 35 to 43% of both the liquid and tablet doses was recovered in the urine of volunteers, excretion of urinary metabolites appears to be independent of the dosage form. However, a rank-order correlation between the relative tablet bioavailability and cumulative amounts of excreted metabolites was observed. Nitrendipine, its pyridine metabolite, and the glucuronide conjugates were also detected in the urine, but the amount of nitrendipine and its pyridine metabolite did not exceed 0.1% of dose, whereas the glucuronides accounted for about 8% of the dose.

Steady-state pharmacokinetics of nitrendipine in hepatic insufficiency.

As a calcium antagonist, nitrendipine will be used in the treatment of various diseases in patients with hepatic insufficiency, and it is important to know if they require modified dosing schedules. In this study, six patients with biopsy-confirmed cirrhosis and six age/sex-matched normal healthy subjects were given 10 mg nitrendipine as a single dose on day 1 and 10 mg nitrendipine every 12 h from day 3 through the first dose on day 8. Blood levels of nitrendipine were determined to confirm the attainment of steady state and evaluate the pharmacokinetics in each group. Nitrendipine concentrations were consistently higher in the hepatic group. On day 1, the maximum concentration (Cmax) in the normals was 4.67 +/- 2.60 ng/ml and in the hepatic patients 16.87 +/- 9.42 ng/ml. On day 8, these values were 8.60 +/- 8.82 ng/ml and 27.37 +/- 8.56 ng/ml, respectively. The time to Cmax was not significantly different in the two groups. The elimination half-life was only slightly prolonged from 15.29 +/- 7.25 h in the normals to 19.57 +/- 4.28 h in the hepatic impairment group. This resulted in a marked increase in the area under the concentration-time curve from 28.71 +/- 28.92 ng . h/ml for the normals to 119.56 +/- 34.39 ng . h/ml for the hepatic patients on day 1 and similar results on day 8. Trough levels at steady state were expectedly higher in the hepatic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas and liquid chromatographic analyses of nimodipine calcium antagonist in blood plasma and cerebrospinal fluid.

Gas (GC) and liquid chromatographic (LC) assay procedures were developed for analysis of nimodipine (1,4-dihydropyridine calcium antagonist, BAY e 9736) in blood plasma at low nanogram concentration levels. To avoid decomposition during gas chromatography, nimodipine was oxidized to nimodipine pyridine (P) analogue before it was chromatographed on the OV-17 column and quantitated using an electron-capture detector. In contrast, the LC procedure involved chromatographic separation and quantitation of the underivatized nimodipine and of the endogenous P analogue using a 3-micron Spherisorb ODS column and UV detection. The same plasma extract and three alternative internal standards were used for both assays. Taking into consideration the fact that the GC assay result includes endogenous P analogue as well as nimodipine, good correlation between GC and LC assay data was obtained. Comparison of the results observed with the two procedures confirmed the accuracy of each procedure and provided an alternative when one of the assay results was subject to patient plasma constituent interference. The LC assay was also used for analysis of the demethylated metabolites of nimodipine. To detect sub-nanogram concentrations of nimodipine in cerebrospinal fluid a combined LC-GC procedure using an LC clean-up step and a GC quantitation step was also developed. The above GC and LC procedures were used to obtain preliminary pharmacokinetic data.

Radioreceptor and high-performance liquid chromatographic assays for the calcium channel antagonist nitrendipine in serum.

Radioreceptor and high-performance liquid chromatographic (HPLC) assays for nitrendipine were developed and applied to the analysis of serum samples. The HPLC assay required both extraction and concentration of the serum samples, whereas the radioreceptor assay involved only direct dilution of the serum. The HPLC assay, in contrast to the radioreceptor assay, can be used for detection and quantitative analysis of biologically inactive metabolites. The radioreceptor assay is based on the competition between nitrendipine in serum and [3H]nitrendipine for high-affinity binding sites on cardiac membranes. Forty-two serum samples were obtained from five volunteers, and the HPLC and radioreceptor assay results were compared. A correlation coefficient of 0.98 was found between the results of the two assays within the nitrendipine serum concentration range of 1 to 20 ng/ml. No significant levels of active metabolites or any other interferences were found. The radioreceptor assay provides a specific and sensitive alternative to HPLC; it is rapid and inexpensive and with minor modifications may be applicable to most presently available Ca2+ channel antagonists.

Trace analysis of the MIF analogue pareptide in blood plasma by high-performance liquid chromatography and short-wavelength excitation fluorometry.

A high-performance liquid chromatographic procedure was developed and applied to analysis of the pharmacologically active MIF analogue pareptide in human plasma. The procedure involves formation of a fluorescent 7-chloro-4-nitrobenzyl-2-oxa-1,3-diazole (NBD-Cl) pareptide derivative followed by separation of the NBD derivative from plasma components on a 30-cm microparticle octadecylsilane bonded column. The separated derivative was quantitated using a short-wavelength excitation fluorometric detector. The detection limit of pareptide in plasma samples was 5 ng or 17 pmoles per ml of plasma. In the absence of plasma, the corresponding on-column detection limit was 0.5 pmoles.

High-speed liquid chromatographic analysis of dapsone and related compounds.

A nonaqueous solvent absorptive support system and an aqueous solvent reversed-phase support liquid chromatographic system for analysis of dapsone and related compounds were investigated. The absorptive support system was more suitable for analysis of dapsone in raw materials, formulations, and tissue residues. The suitability was judged by the relative selectivity, efficiency, precision, and sensitivity of the systems. The adsorptive support system was used for the analysis of trace amounts of raw material impurities and dapsone metabolites. Coupling fluorometric detection to the chromatographic system yielded a 10-pg on-column detection limit for dapsone; the UV detection limit was 250 pg.