Development of a microfluidic-chip system for liquid-phase microextraction based on two immiscible organic solvents for the extraction and preconcentration of some hormonal drugs.

In the present study, for the first time, an on-chip liquid phase microextraction (LPME) coupled with high performance liquid chromatography was introduced for the analysis of levonorgestrel (Levo), dydrogesterone (Dydo) and medroxyprogesterone (Medo) as the model analytes in biological samples. The chip-based LPME set-up was composed of two polymethyl methacrylate (PMMA) plates with microfabricated channels and a microporous membrane sandwiched between them to separate the sample solution and acceptor phase. These channels were used as a flow path for the sample solution and a thin compartment for the acceptor phase, respectively. In this system, two immiscible organic solvents were used as supported liquid membrane (SLM) and acceptor phase, respectively. During extraction, the model analytes in the sample solution were transported through the SLM (n-dodecane) into the acceptor organic solvent (methanol). The new set-up provided effective and reproducible extractions using low volumes of the sample solution. The effective parameters on the extraction efficiency of the model analytes were optimized using one variable at a time method. Under the optimized conditions, the new set-up provided good linearity in the range of 5.0-500µgL(-1) for the model analytes with the coefficients of determination (r(2)) higher than 0.9909. The relative standard deviations (RSDs%) and limits of detection (LODs) values were less than 6.5% (n=5) and 5.0µgL(-1), respectively. The preconcentration factors (PFs) were obtained using 1.0mL of the sample solution and 20.0µL of the acceptor solution higher than 19.9-fold. Finally, the proposed method was successfully applied for the extraction and determination of the model analytes in urine samples.

Control of illegal medroxyprogesterone acetate-application in veal calves by residue analysis in adipose tissue using HPLC/RIA methods.

Procedures for the determination of medroxyprogesterone acetate (MPA) in adipose tissue collected at time of slaughter allowing the control of MPA application to veal calves are described. Screening radioimmunoassays after sample clean-up were sufficient for a first survey of MPA treatments in livestock herds. Validation of all positive samples was performed by two-dimensional (silica gel diol phase and RP-18 phase) HPLC/RIA immunograms. Megestrol acetate and melengestrol acetate with cross-reactivities of 31% and 0.3% respectively were clearly separated by the RP HPLC. With an absolute detection limit of 4 pg MPA/tube (90% relative binding) negative control samples did not exceed 6 pg/tube, equivalent to 6 pg/g fat in the validating method. Seventeen days after intramuscular (i.m.) injection of 24 mg MPA only 32 pg MPA/g fat were found, while i.m. injection of 60 mg MPA and a waiting period of 19 days resulted in 2700 pg MPA/g fat. After feeding two calves 20 micrograms MPA per head daily for 1 week followed by 200 micrograms MPA per head daily for 2 weeks 359 and 468 pg MPA/g fat were measured. In plasma as well as in adipose tissue more than 80% of the whole immunoreactive material was MPA itself, without indications for the presence of cross-reacting MPA metabolites as confirmed by HPLC/RIA immunograms. Based on day of slaughter ratios of accumulation of MPA from plasma into fat of MPA-fed veal calves were 52 and 72 respectively. In urine MPA was only detectable a few days after injection; as compared to a plasma concentration of 950 pg MPA/ml the amount in urine was only 37 pg MPA/ml and also 325 pg unidentified MPA-equivalents/ml.

Disposition of medroxyprogesterone acetate and drug metabolism activity.

The role of drug-metabolizing enzyme activity on the disposition of medroxyprogesterone acetate (MPA) was investigated in male rats. The metabolizing enzyme system was induced by phenobarbital (PB) or inhibited by SKF 525A. Plasma concentrations of unmetabolized MPA and the amounts of MPA-related residues in various tissues were higher in the SKF 525A and slightly lower in the PB treated animals than in the controls. The disappearance of MPA-related substances from the lung, skeletal muscle and brain was slow, and in the lung there was even an increase in the amount of radioactivity in the PB and saline treated rats at 24 hr. Almost equal amounts of radioactivity were excreted in the urine and intestine in the control rats, while the induction of drug metabolism enhanced the excretion in the urine and its reduction enhanced elimination in the intestinal tract within 24 hours. The findings demonstrate that plasma and tissue levels of MPA and its elimination route are influenced by the drug metabolism activity.

The metabolic fate of medroxyprogesterone acetate in the baboon.

PIP: The metabolic fate of medroxyprogesterone acetate (6alpha-methyl-17a lpha-acetoxyprogesterone; MAP)was studied in intact baboons and in those with bile fistulas. The steroid moiety was labeled with tritiated hydrogen at positions 1 and 2 and the 17alpha-acetate with carbon-14, thus affording the opportunity to ascertain the loss of the 17-acetoxy group and the fate of both labels. Following the iv administration of labeled MAP only a small percentage (less than 15%) of the administered dose was recovered in the urine in 7 hours in intact baboons, as well as in the urine of baboons with biliary fistulas. Higher amounts of radioa ctivity were excreted in the bile (approximately 25%), amounting to almost double the percentage excreted into the urine. The similarity in the urinary excretion of radioactivity in intact and biliary fistula animals indicates that, even though a substantial biliary excretion of the labeled MAP occurred, the amount involved in an enterohepatic circulation is probably small. Glucosiduronates were the predominant conjugates, both in the urine and bile. The loss of the 17alpha-acetate group appeared to be rather extensive, ranging 30-70% among different co njugated and unconjugated metabolities of MAP. The degree of in vivo hydrolysis of the axial 17alpha-acetate of MAP, though extensive appeared to be of a significantly lesser magnitude than that exhibited toward the equatorial 3beta and 17beta acetate groups of labeled ethynodiol diacetate injected into baboons. The deacetylation of the 17alpha-acetate in MAP was similar to that observed in humans given the drug. Oxygenation of MAP at positions 1 and/or 2 appeared to be rather minimal (less than 5%).^ieng

Parenteral use of medroxyprogesterone acetate as an antifertility agent in the bitch.

PIP: 10 kg unbred Beagle and mongrel bitches were used in each of 4 experiments (exp). 6 alpha-methyl-17 alpha-acetoxyprogesterone (MAP or medroxyprogesterone acetate) was prepared as a sterile aqueous suspension (50 mg/ml) and was used in exps 1, 2 and 3. Tritiated MAP was used in exp 4. In exp 1, 7 bitches were given 500 mg and 7 were given 50 mg subcutaneously (sc). 6 controls were used. In exp 2, 50, 100 or 250 mg were given sc, intramuscular (im) or intraperitoneal (ip) to 4 dogs at each dose level and route of administration. In exp 3, 12.5 or 25 mg was given either sc or im in 4 groups of 8 bitches each. In exp 4, a single 50 mg mixture of MAP and tritiated MAP was given to each of 3 dogs: 1 sc, 1 im, and 1 ip. Results of exp 1 showed those on 500 mg had first posttreatment estrus an average of 504 days (range of 247-730) after injection. Those on 50 mg averaged 329 days (range of 205-429). Breeding resulted in 6 live pups/litter for the controls 5.2 for those on 50 mg, and 4 for those on 500 mg. In exp 2, results showed prolonged cycling in those injected sc (295 days to estrus), while those injected im reached estrous in 225 days and those injected ip in 148 days. In exp 3, 3 on 25 mg sc had a 6 month estrous delay while 4 did when injected im. 3 of 16 given 12.5 mg sc and im had a 6 month delay in cycling. Exp 4 showed ip treated dogs had estrus 148 days after treatment, 225 days for im, and 295 days for sc. Ip treated dogs whelped 5 pups; im 7 pups; and sc, 5 pups. 7 months lapsed before nearly all of the 50 mg sc administered dose was excreted. An additional 2.5 months lapsed before estrus occurred in this dog.^ieng