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C. Pyrgaki, S. J. Bannister, L. Gera, J. G. Gerber, and J. Gal. Stereoselective determination of the epimer mixtures of itraconazole in human blood plasma using HPLC and fluorescence detection. Chirality 23: 495-503 (2011).

Itraconazole is an antifungal drug widely used in a variety of fungal infections, which have become a significant public-health problem in recent decades. Itraconazole is a chiral drug consisting of two diastereoisomeric racemates, i.e., four stereoisomers. Data in the literature suggests that stereochemistry may play a significant role in the action and disposition of the drug and therefore stereoselective analytical methods for the determination of the drug in biological fluids are needed for the elucidation of that role. We report a stereoselective HPLC method that incorporates solvent extraction, the use of an internal standard, two chiral stationary phases in series, and fluorescence detection. The procedure is enantioselective and partially diastereoselective and provides the concentrations in blood plasma of the two epimer mixtures 2R,4S,2’R/2R,4S2’S and 2S,4R,2’R/2S,4R,2’S, respectively, each of which is a combination of the two epimers that differ in the configuration at the sec-butyl group. The analytical method has suitable sensitivity, recovery, precision, and accuracy. Analysis of the plasma of a human subject six hours after the oral administration of a single 200-mg dose of itraconazole showed a 3.4-fold difference between the concentrations of the epimer mixtures. The method has certain advantages over the published alternative procedure that uses LC-MS.

M. S. Alexander, M. M. Kiser, T. Culley, J. R. Kern, J. W. Dolan, J. D. McChesney, J. Zygmunt, and S. J. Bannister. Measurement of paclitaxel in biological matrices: high-throughput liquid chromatographic-tandem mass spectrometric quantification of paclitaxel and metabolites in human and dog plasma. Journal of Chromatography B 785: 253-261 (2003).

A GLP-validated, sensitive and specific LC–MS–MS method for the quantification of paclitaxel and its 6-α and 3’-p-hydroxy metabolites is presented. A 0.400 ml plasma aliquot is spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1 ml methyl-tert.-butyl ether. The ether is evaporated and the residue is reconstituted in 130 ml of 30% aqueous acetonitrile (ACN) containing 0.1% trifluoroacetic acid. Isocratic HPLC analysis is performed by injecting 50 µL of the reconstituted material onto a 50x2.1 mm C18 column with an ACN–water–acetic acid (50:50:0.1) mobile phase at 200 µL/min flow. Detection is by positive ion electrospray followed by multiple reaction monitoring of the following transitions: paclitaxel (854>509 u), 6-α-hydroxy paclitaxel (870>525 u), 3’-p-hydroxy paclitaxel (870>509 u) and internal standard (860.509 u). Quantification is by peak area ratio against the 13C6 internal standard. The method range is 0.117–117 nM (0.1–100 ng/ ml) for paclitaxel and both metabolites using a 0.400 ml human or dog plasma sample. Analysis time per sample is less than 5 min.

N. K. Jagota, S. J. Bannister, R. B. Poser, and J. T. Stewart. HPLC Determination of Utibapril and its Diacid FPL 63674XX in Rodent Laboratory Diet Using Selective Extraction and Gradient Elution Chromatography. Journal of Liquid Chromatography 14: 2979-2991 (1991).

Utibapril is a novel thiadiazoline that is currently under investigation as an antihypertensive agent. The major degradation product, a diacid FPL 63674XX, is biologically active. An HPLC method has been developed to simultaneously determine both utibapril and its diacid in rodent laboratory diet used to dose animals in long term toxicology studies. The method is based on liquid-solid extraction of the compounds followed by direct injection of the extract. Gradient elution chromatography is utilized to better separate the diacid from interferences. Recovery of utibapril and the diacid from the lab diet was found to be 97.6±0.44% and 99.7±2.8%, respectively (n =3) at 0.43%w/w levels. The separation is achieved on a octadecylsilane column using a flow rate of 0.75 ml/min and a column temperature of 70°C. The UV detector was set at 260 nm. A linear and step gradient program was established using mobile phases consisting of 0.05 M phosphate buffer, pH 2.5 and 40:60 0.05 M phosphate buffer, pH 2.5 - acetonitrile. An injection volume of 20p1 was utilized and the chromatographic run time was set at 38 min. The assay method shows linearity for utibapril over a 0.25 - 2% w/w range (r = 0.9998, n = 5) and for the diacid over a 0.0043 - 0.43% w/w range (r = 0.9960, n = 5). Accuracy and precision for both compounds were in the 1-5% range over the linear ranges specified above. The method was applied to an actual rodent diet sample formulated to contain 0.43% w/w utibapril.

S. J. Bannister, V. P. Houser, J. D. Hulse, J. C. Kisicki, and J. Rasmussen. Evaluation of the potential for interactions of paroxetine with diazepam, cimetidine, warfarin, and digoxin. Acta Psychiatrica Scandinavica 80: 102-106 (1989).

This work was undertaken to assess the probability of significant drug-drug interactions of paroxetine with drugs likely to be co-administered that may affect the disposition of paroxetine or whose disposition may be affected by paroxetine.

L. A. Sternson, K. C. Marsh, S. J. Bannister, and A. J. Repta. Detection systems for assay of antineoplastic platinum complexes. Analytical Proceedings 20: 366-368 (1983).

S. J. Bannister, L. A. Sternson, and A. J. Repta. Evaluation of reductive amperometric detection in the liquid chromatographic determination of antineoplastic platinum complexes. Journal of Chromatography B: Biomedical Sciences and Applications 273: 301-318 (1983).

The usefulness of reductive electrochemical detection at mercury drop electrodes has been determined for platinum complexes separated by solvent-generated anion-exchange high-performance liquid chromatography. Both current-sampled dropping mercury and hanging mercury drop electrodes (DME and HMDE) provide significant advantages over UV absorbance and off-line non-flame atomic absorption detection. The effects of chromatographic and polarographic parameters on analytical system performance have been investigated. By raising the detector cell temperature, the detector response to cis-dichlorodiammineplatinum(II) (DDP) can be shifted anodically to 0.0 V vs. Ag/AgCl, thereby increasing detector selectivity for this compound. The noise-limited minimum detectable quantities of DDP with DME and HMDE are 1.8 ng and 70 pg injected, respectively. DDP can be determined in untreated urine at levels below 100 ng/ml.

S. J. Van der Wal, S. J. Bannister, and L. R. Snyder. Automated Analysis of Acetaminophen and Caffeine in Serum Using the FAST-LC System: Contributions to Assay Imprecision in Procedures Based on HPLC with Sample Pretreatment. Journal of Chromatographic Science 20: 260-265 (1982).

Two procedures are described for the fully automated analysis of several therapeutic drugs in serum, using HPLC with on-line pretreatment (solvent extraction) of the sample. The FAST-LC system (Technicon Instruments) was used for the assay of mixtures of 1) acetaminophen, theophylline, and/or caffeine, or 2) phenylethylmalonamide, primidone, phenobarbital, carbamazepine epoxide, phenytoin, and/or carbamazepine. The rate of sample analysis was 15/hr for the theophylline group of drugs and 12/hr for the six anticonvulsants. The precision of resulting assays was about 3% (CV), and only 75μl of samples was required. The precision of resulting assays, in terms of a previously reported model, is also discussed.

C. M. Riley, L. A. Sternson, A. J. Repta, and S. J. Bannister. Intact cisplatin in urine following intravenous infusion. Journal of Pharmacy and Pharmacology 34: 826-826 (1982).

Although the clinical pharmacokinetics of intact cisplatin in plasma have been investigated (Patton et al 1982), and recently information on total platinum excreted in urine has been reported (Gill et a1 1981), there are no data available on the elimination of intact cisplatin in urine. In view of the use and effectiveness of cisplatin in the treatment of bladder cancer, information of this type appeared to be desirable. Consequently, this study was undertaken and preliminary findings are presented here.

S. J. Bannister, S. Van der Wal, J. W. Dolan, and L. R. Snyder. Liquid-chromatographic analysis for common tricyclic antidepressant drugs and their metabolites in serum or plasma with the Technicon FAST-LC system. Clinical Chemistry 27: 849 (1981).

We describe a single procedure for assay of seven tricyclic antidepressant drugs and metabolites in serum or plasma: protriptyline, nortriptyline, amitriptyline, desmethyldoxepin, doxepin, desipramine, and imipramine. With the Technicon "FAST-LC" system, samples are aspirated directly into the unit and pretreated via double extraction; the concentration of each drug is then determined by "high-performance" liquid chromatography. Final chromatograms are monitored at 205 nm, at analysis rates of 7.5 samples/h. Concentration and absorbance are linearly related for each drug from 0 to 1400 micrograms/L. Day-to-day CVs averaged 5 to 6% for each drug, and there is good correlation of FAST-LC values with those obtained by gas-chromatographic methods. Total sample volume is 750 microliters.

J. W. Dolan, S. Van der Wal, S. J. Bannister, and L. R. Snyder. On-line liquid-chromatographic analysis for drugs in serum with the Technicon "FAST-LC" system: performance data for theophylline and for four commonly used anticonvulsants and their metabolites. Clinical Chemistry 26: 871 (1980).

We describe a new instrument for use in assay of therpeutic drugs in serum by "high-performance" liquid chromatography, the "FAST-LC" system (Technicon). Serum samples are aspirated directly into the unit, extracted with solvent, and the evaporated and redissolved extract is injected onto a chromatographic column. We illustrate the performance of the system by assays in serum for theophylline and four anticonvulsants (primidone, phenobarbital, phenytoin, and carbamazepine) plus two of their active metabolites (phenylethylmalonamide and carbamazepine epoxide). For theophylline, final chromatograms are monitored at 270 nm, at analysis rates of 10/h. Concentration and absorbance are linearly related from 0 to 130 mg of theophylline per liter. For the anticonvulsants, chromatograms are monitored at 200 nm, at analysis rates of 7.5/h. The six individual determinations are each linear beyond the therapeutic range. For both drug panels, day-to-day CV's were 4 to 6%. Results correlate well with those by enzyme immunoassay. A total sample volume of 150 microL is required.

S. J. Bannister, J. Stevens, D. Musson, and L. A. Sternson. High-performance liquid chromatographic analysis of emetine after oxidative activation to a fluorescent product. Journal of Chromatography A 176: 381-390 (1979).

A clinically useful analytical method is described for monitoring plasma levels of emetine. The drug is initially extracted from plasma with dichloromethane (0.3 volumes). The extract can be analyzed directly by paired-ion reversed-phase high-performance liquid chromatography to levels of 500 ng/ml of plasma by spectrophotomeric monitoring of column effluent. For analysis of emetine at lower concentrations, the dichloromethane extracts are subjected to mild mercuric acetate oxidation prior to separation, thereby converting emetine to a fluorescent product. Spectrofluorometric monitoring of the column effluent readily extends the sensitivity of the assay to 10 ng of emetine/ml of plasma. At these levels measurements can be made with a precision of ±4%.

S. J. Bannister, L. A. Sternson, and A. J. Repta. Urine analysis of platinum species derived from cis-dichlorodiammineplatinum(II) by high-performance liquid chromatography following derivatization with sodium diethyldithiocarbamate. Journal of Chromatography A 173: 333-342 (1979).

A clinically useful method is described for the quantitative analysis of platinum species derived from cis-dichlorodiammineplatinum(II) in urine. The drug and its biodegradation products are derivatized directly in urine by reaction with sodium diethyldithiocarbamate (DDTC) to form a common product, a 2:1 DDTC-platinum adduct. This complex is stable and can be quantitatively extracted into 0.1 volumes of chloroform. An aliquot of the chloroform layer is then subjected to high-performance liquid chromatography on a μBondapak CN column and the eluent monitored spectrophotometrically at 254 nm. At this wavelength the DDTC-platinum adduct has a molar absorptivity of 43,000, and platinum levels of 25 ng/ml of urine can be detected with a precision of ±2.5% and an accuracy of ±4%.

R. J. Belt, K. J. Himmelstein, T. F. Patton, S. J. Bannister, L. A. Sternson, and A. J. Repta. Pharmacokinetics of non-protein-bound platinum species following administration of cis-dichlorodiammineplatinum (II). Cancer Treatment Reports 63: 1515 (1979).

The pharmacokinetics of non-protein-bound platinum species derived from cis-dichlorodiammineplatinum(II) (cis-platinum) was studied under a variety of dosing conditions. Following rapid infusions (15-minute) of cis-platinum at 100 mg/m2, the unbound drug declined in a biphasic mode with a mean terminal half-life of 48 minutes. The mean beta-phase half-life after a 6-hour infusion of the same dose of cis-platinum was 26 minutes. Urinary excretion of filterable platinum was substantially greater after a 6-hour infusion than after a 15-minute injection. Concomitant administration of mannitol appeared to result in higher peak plasma concentrations and decreased urinary excretion of unbound platinum species but did not alter the terminal half-life. Renal impairment was associated with extremely high plasma levels of filterable platinum but did not affect other pharmacokinetic parameters. Preliminary data on the distribution of cis-platinum to ascitic fluid are also presented.

S. J. Bannister, Y. Chang, L. A. Sternson, and A. J. Repta. Atomic absorption spectrophotometry of free circulating platinum species in plasma derived from cis-dichlorodiammineplatinum (II). Clinical Chemistry 24: 877 (1978).

We describe a method of analysis for free circulating platinum species derived from cis-dichlorodiammineplatinum(II) in blood plasma. Protein-bound and free platinum species were separated from each other by centrifugal ultrafiltration. Platinum in the ultrafiltrate was converted to a cationic complex by reaction with ethylenediamine, and the product was collected on paper impregnated with cation-exchange resin, where it could be stored indefinitely without loss. The platinum was eluted from the disk with 5 mol/liter hydrochloric acid, and an aliquot of this solution was then analyzed by flameless atomic absorption spectrophotometry. The overall analytical recovery of platinum was 80 +/- 2%. The minimum quantity of cis-dichlorodiammineplatinum detectable was 35 microgram/liter of plasma at the 99% confidence level. Detector response was linearly related to drug concentration in the range from 80 microgram to 290 mg of Pt per liter of plasma. Reaction variables were made optimal, so as to yield maximum sensitivity and reproducibility (+/- 2%) consistent with minimal sample transfers and manipulations.

T. F. Patton, K. J. Himmelstein, R. Belt, S. J. Bannister, L. A. Sternson, and A. J. Repta. Plasma levels and urinary excretion of filterable platinum species following bolus injection and iv infusion of cis-dichlorodiammineplatinum (II) in man. Cancer Treatment Reports 62: 1359 (1978).

S. J. Bannister, L. A. Sternson, A. J. Repta, and G. W. James. Measurement of free-circulating cis-dichlorodiammineplatinum (II) in plasma. Clinical Chemistry 23: 2258 (1977).

Dichlorodiammineplatinum(II) is an anti-neoplastic agent that is currently undergoing clinical evaluation. We describe an analytical method for monitoring the free drug (or its breakdown products) in plasma. The method is able to distinguish between free and protein-bound drug. Plasma samples are deproteinized by centrifugal ultrafiltration. The platinum in the ultrafiltrate is converted to a cationic species by reaction with ethylenediamine and then collected on paper impregnated with cation-exchange resin. This process concentrates the samples, increases the stability of the platinum compounds (by removing the compound from solution), and places the sample in a uniform matrix of minimum thickness, which maximizes detection capabilities. Platinum was measured directly on the ion-exchange disks by X-ray fluorescence. The detection limit for free drug is 240 microgram/liter of plasma at the 3s level and fluorescence intensity is linearly related to drug concentration in the range from 570 to 5700 microgram/liter.

L. A. Sternson, F. Hincal, and S. J. Bannister. Gas chromatographic analysis of acetophenone oxime and its metabolites. Journal of Chromatography A 144: 191-200 (1977).

A gas-liquid chromatographic (GLC) method has been developed for monitoring the metabolic reduction of acetophenone oxime or oxidative metabolism of the corresponding amine, α-methylbenzylamine in liver homogenates. The oxime, amine, N-hydroxy-α-methylbenzylamine and acetophenone are quantitatively determined after GLC separation of components with temperature programming on an SP-2401-DB-coated column. The first three compounds were silylated with N,O-bis(trimethylsilyl)-acetamide prior to chromatographic analysis to enhance the stability and improve the chromatographic properties of these components. The effluent gas was monitored with flame ionization detection, and permitted quantitaion of components at sub-μg/ml levels with reproducibility between injections of ±2%. The optimal composition of enantiomeric mixtures of (R, S)-α-methylbenzylamines formed during metabolic reduction of acetophenone oximes were determined by conversion to diastereomeric amides and subsequent GLC analysis.

L. A. Sternson, A. W. Sternson, and S. J. Bannister. A differential pulse polarographic assay for O-methylation of catechols by catechol-O-methyltransferase. Analytical Biochemistry 75: 142-152 (1976).

A new method is described for monitoring the enzyme-catalyzed O-methylation of norepinephrine (or other catechol substrate) by catechol-O-methyltransferase. Norepinephrine and normetanephrine combine with bis-2-(ethylhexyl)hydrogen phosphate at pH 7.4 to form an ion pair which is quantitatively extracted with an immiscible organic solvent by an application of partition chromatography. The mixture of catechol substrate and O-methylated products are then simultaneously determined in 0.5 m H2SO4 solution by monitoring their oxidation at a carbon paste electrode by a differential pulse polarographic technique.

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