Donald J. Mangold

HPLC Assay for S-2-(3-Aminopropylamino)ethyl Phosphorothioate (WR 2721) in Plasma

Abstract A specific HPLC assay has been developed for determination of the radioprotective drug WR 2721. The method is based on precolumn derivatization of plasma with fluorescamine, separation with a C-18 cartridge and detection by fluorescence. An external standard was used for calibration, and values were adjusted based upon recovery of added 14C-labeled WR 2721. WR 2721 had a retention time of about 13 minutes using a mobile phase of acetonitrile/water (22:78), 0.01 M in dibutylammonium phosphate, at a flow rate of 2 mL/min. Sensitivity of the assay was characterized to 2 μg/mL, and detector response was linear over the range of 2 to 1100 μg/mL. The assay requires 90 μL of plasma and has a total chromatography time of about 45 minutes. 2-(3-Aminopropylamino)ethanethiol (WR 1065) and bis- [2- (3-aminopropylamino)ethyl]disulfide (WR 33278), metabolites of the drug, and a variety of primary amines were shown not to interfere with the assay. Suitability of this assay for pharmacokinetic studies was demons...

A method for the combined measurement of ethiofos and WR-1065 in plasma: application to pharmacokinetic experiments with ethiofos and its metabolites.

An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 micrograms/mL (0.75 to 186 mumol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120-150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 micrograms/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 micrograms/mL, 0.23 microM) 2-3 hours postinfusion. Clearances averaged 43.5 +/- 13.4 (SD) mL min-1 kg-1 and half-lives observed in the 20-60 minute postinfusion period were 8-15 min.

HPLC assay for 2-(3-aminopropylamino)ethanethiol (WR-1065) in plasma☆

A high pressure liquid chromatography (HPLC) plasma assay for WR-1065 is described which is both precise (coefficient of variation less than 5%) and accurate (% average deviation less than or equal to 6.1) throughout the concentration range from 1 to 500 micrograms/mL of plasma. The analyte is separated by HPLC and detected with a thiol specific electrochemical transducer cell. The detector response is linear over the ranges 1 to 10 micrograms/mL (R2 = 0.995), 10 to 100 micrograms/mL (R2 = 0.995), and 100 to 500 micrograms/mL (R2 = 0.974). The absolute retention times for WR-1065 and WR-1729 are 9 and 12 minutes, respectively. The assay uses 100 microL of plasma and requires a total chromatography cycle time of 40 minutes. The method has been found suitable for the determination of WR-1065 in plasma from a beagle dog after i.v. administration of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721).

An improved HPLC assay for S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) in plasma

An HPLC assay is presented for the detection and quantitation of the radioprotective drug S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721, ethiofos) present in plasma. Improved selectivity and a 40-fold increase in sensitivity have been demonstrated over the method previously reported by this laboratory. Using precolumn derivatization with fluorescamine and S-3-(4-aminobutylamino)propyl phosphorothioate (WR-80855, a homolog of WR-2721) as the internal standard, drug levels of 0.05 to 1000 micrograms/mL were determined with excellent precision (CV less than or equal to 5% over the concentration range). An isocratic mobile phase of acetonitrile/ethanol/water (16:7:77) modified with 0.01 M tetrabutylammonium phosphate eluted the drug and the internal standard from the C-18 reverse-phase column in 23 minutes and 26 minutes, respectively. Detector response was linear over the entire range. The assay uses 150 microL of plasma and requires a total chromatography time of about 50 minutes. The method was found suitable for pharmacokinetic studies in a preliminary experiment with a beagle dog in which no interferences due to plasma constituents or drug metabolites were observed.

Measurement Of Ethiofos (Wr 2721) In Plasma: Preliminary Pharmaco-Kinetics in the Beagle

Abstract A specific, sensitive high performance liquid Chromatographie method for ethiofos [S-2-(3-aminopropylamino)ethyl phosphorothioate, WR 2721, I] in plasma is described. The detection limit is 0.05 (μg/mL (0.23 (μM). Application of the method to the development of pharma-cokinetic parameters following IV administration of the drug to beagle dogs is demonstrated. Presented are pharmacokinetics of unchanged drug in plasma on 10-min constant-rate infusion of 150 mg/kg to two dogs, two studies in each dog. Following the cessation of drug infusion the plasma concentration versus time profile was best described by a two-compartment pharmacokinetic model. Mean pharmacokinetic parameters were: terminal elimination half-life=16.0 min, volume of central compartment=120 mL/kg and clearance=11.0 mL/min/kg.

The Disposition of Ethiofos (WR-2721) in the Isolated Perfused Rat Liver

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.

A method for the measurement of total drug convertible to cysteamine: application to pharmacokinetic experiments with ethiofos

Abstract An analytical method has been developed for the quantification of cysteamine (2-aminoethanethiol) in plasma. A reductive sample pretreatment is used to convert disulfide-bound cysteamine to the free thiol which is subsequently separated by HPLC and detected by electrochemical detection (LCEC). The method was developed to follow drug disposition after administration of ethiofos (WR-2721, S-2-(3-aminopropylamino) ethyl phosphorothioic acid) and WR-1065 (2-(3-aminopropylamino)ethanethiol). Standard calibration curves were linear over the range 0.01- to 25.0 /μg/mL (0.130-324 μM) and a minimum detectable quantity of 0.01 μg/mL was calculated at a signal-to-noise ratio of 3. Assay precision over the same range averaged 2% (coefficient of variation) and relative recovery, used as a measure of accuracy, was 100Z. Stability of cysteamine in plasma, relative to an internal standard (2-aminopropanethiol, WR-186) was good; stored samples were found to contain an average of 94.6 ± 10.4z of the original cyste...