Plasma Cortisol in the Horse,Diurnal Rhythm and Effects of Exogenous Acth

Peripheral blood plasma Cortisol concentration and its diurnal variation was measured in 4 horses. Mean concentration of Cortisol during 24 hrs. was 42 ng/ml (s ± 20 ng/ml). Peak values occurred at 6 a.m. and the lowest values were observed at about 6 p.m. (mean 65 ng/ml and 20 ng/ml, respectively). Long-acting ACTH at a dose of 150 i.u. was given by intramuscular injection to the 4 horses. Peak Cortisol concentrations markedly exceeding the prestimulation level were obtained between 2 and 4 hrs. after injection. During the immediate 24 hrs. after these peaks, the mean Cortisol level was markedly lower and the cyclic variation out of phase with the basal diurnal pattern. After a gradual adjustment during the second postinjection day, no differences could be seen between the 2 patterns on day 3 after injection.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.     

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Turnover rate of anti-D IgG injected during pregnancy.

Anti-D IgG was injected into 15 Rh-negative women in the 28th week of gestation and into three non-pregnant women. The uptake of anti-D after the intramuscular injections was calculated by measuring the concentration of antibody in the plasma with an autoanalyser. The biological half life and the catabolic rate of anti-D IgG were calculated according to a compartmental model. The recovery in vivo of anti-D was an average 24% in the non-pregnant women and 21% in the pregnant women. The half life of anti-D were 24 and 21 days, respectively. With a dose of 125 micrograms the plasma anti-D concentration was less than 1 ng/ml at about 10 weeks after the injection. With double the dose the concentration at delivery was at least 1 ng/ml. Although 250 micrograms of anti-D IgG seems to be effective when given in the 28th weeks of gestation, the great individual variations in uptake and recovery rates will lead to occasional cases of Rh-immunisation during pregnancy despite all routine regimens.     

Sensitive gas chromatographic determination of trifluoperazine in human plasma

Plasma trifluoperazine levels of patients taking a single 20-mg dose of trifluoperazine were measured by a sensitive and linear method. The low detection limit of 0.1 ng/ml plasma was obtained through use of a highly sensitive nitrogen—phosphorus detector combined with an efficient extraction method. Recovery of trifluoperazine added to human plasma was 96%. Data are presented on the stability of trifluoperazine in refrigerated human plasma.     

Determination of artemether and its metabolite,dihydroartemisinin, in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode

An analytical method for the determination of artemether (A) and its metabolite dihydroartemisinin (DHA) in human plasma has been developed and validated. The method is based on high-performance liquid chromatography (HPLC) and electrochemical detection in the reductive mode. A, DHA and artemisinin, the internal standard (I.S.), were extracted from plasma (1 ml) with 1-chlorobutane—isooctane (55:45, v/v). The solvent was transferred, evaporated to dryness under nitrogen and the residue dissolved in 600 μl of water-ethyl alcohol (50:50, v/v). Chromatography was performed on a Nova-Pak CN, 4 μm analytical column (150 mm×3.9 mm I.D.) at 35°C. The mobile phase consisted of pH 5 acetate—acetonitrile (85:15, v/v) at a flow-rate of 1 ml/min. The analytes were detected by electrochemical detection in the reductive mode at a potential of −1.0 V Intra-day accuracy and precision were assessed from the relative recoveries (found concentration in % of the nominal value) of spiked samples analysed on the same day (concentration range 10.9 to 202 ng/ml of A and 11.2 to 206 ng/ml of DHA in plasma). The mean recoveries over the entire concentration range were from 96 to 100% for A with C .V. from 6 to 13%, from 92% to 100% for DHA (α-tautomer) with C .V. from 4 to 16%. For A, the mean recovery was 96% at the limit of quantitation (LOQ) of 10.9 ng/ml with a CV of 13%. For DHA, the mean recovery was 100% at the LOQ of 11.2 ng/ml with a CV of 16%.     

Determination of a cyclooxygenase II inhibitor in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of a cyclooxygenase II (COX-II) inhibitor (3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5'-dimethyl-5H-furan-2-one, I) in human plasma, in two concentration ranges of 0.1-20 and 5-1000 ng/ml, are described. Following liquid-liquid extraction, the residue, after evaporation of the organic phase to dryness, was reconstituted in acetonitrile (20 l) and part of the extract (1 l) was analyzed by GC/MS/SIM. The drug (I) and internal standard (II) were separated on a 25 mx0.2 mm capillary column with HP Ultra 1 (100% dimethylpolysiloxane, 0.33 m) phase and analyzed by MS/SIM monitoring ions at m/z 237 and 282 for I and II, respectively. The standard curve was linear within the lower concentration range of 0.1-20 ng/ml and the lower limit of quantification (LLOQ) in plasma was 0.1 ng/ml. Intraday coefficients of variation (CV, n=5) were 8.9, 4.2, 5.7, 3.1, 1.9, 1.9, and 4.4% at 0.1, 0.2, 0.5, 1.0, 5.0, 10, and 20 ng/ml, respectively. The standard curve was also linear within the higher concentration range of 5-1000 ng/ml and the LLOQ in plasma was 5 ng/ml. Intraday coefficients of variation (CV, n=5) were all below 9% at all concentrations within the standard curve range. The accuracy for I in human plasma was 91-112% and the recovery of I and II was greater than 70% at all concentrations within both standard curve ranges. The details of the assay methodology are presented.     

Plasma growth hormone, insulin-like growth factor-I, and milk production responses to exogenous human growth hormone-releasing factor analogs in dairy cows

Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5 ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmunoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3 ng/ml at about 5 h from 1.4-1.7 ng/ml at 0 h (just before injection), and returned to the level before injection after 10-12 h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6 h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4 ng/ml at 20 h from 43.7-46.4 ng/ml at 0 h. The IGF-I concentration at 24 h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2 h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline injected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows.     

Sensitive method for the simultaneous measurement of fluphenazine decanoate and fluphenazine in plasma by high-performance liquid chromatography with cuolometric detection

A highly sensitive and specific high-performance liquid chromatographic method with coulometric detection was developed for the simultaneous assay of fluphenazine decanoate and fluphenazine in plasma. The extraction and sample clean-up procedures are simple, rapid to execute, yet yield chromatograms relatively free of any interference from endogenous plasma constituents, such that the extraordinary sensitivity of the coulometric detector can be exploited fully. This is the first analytical procedure for the simultaneous determination of fluphenazine decanoate and fluphenazine. The detection limits for both fluphenazine decanoate and fluphenazine were 0.1 ng/ml plasma and the limits of quantitation were 0.25 ng/ml plasma. Standard curves from 0.25 to 10 ng/ml were linear with coefficients of variation <10%. The method was applied to measure plasma levels of fluphenazine decanoate and fluphenazine in patients under medication with 25–50 mg biweekly intramuscular (i.m.) injections of fluphenazine decanoate. It was possible to monitor the plasma levels of fluphenazine in all cases. Fluphenazine decanoate was present in measurable concentration in the plasma of 4 out of 5 patients who received biweekly i.m. injections of 50 mg fluphenazine decanoate. In a pilot experiment with a dog, the method was used to follow fluphenazine decanoate and fluphenazine plasma levels up to 13 days, at least, after i.m. single dose (10 mg/kg).     

Prolactin secretion in cattle as affected by haloperidol and α-Methyl-p-Tyrosine

Prolactin (PRL) secretion in response to i.v. injection of different doses of α-Methyl-p-Tyrosine (αMT) and haloperidol (HAL) was studied in one cow and three bulls. HAL was tested at doses of 0.033, 0.1, and 0.33 mg/kg body weight (BW), and αMT was tested at doses of 0.1, 1.0, 10, and 30 mg/kg BW. Blood was collected via an indwelling catheter into the external jugular vein, and plasma PRL was analysed by radioimmunoassay. Dose-related increases in plasma PRL concentrations were observed after administration of both αMT and HAL. Peak PRL concentrations after injection of HAL at a low, medium, and high dose were 22, 45, and 70 ng/ml, respectively. Peak PRL concentrations after injection of increasing doses of αMT were 3.0, 10, 40 and 70 ng/ml. HAL (0.1 mg/kg BW) and αMT (10 mg/kg BW) were administered intravenously to four heifers during summer and winter. Response to both drugs, as measured by changes in PRL secretion, was greater in summer than winter. Peak plasma PRL levels after HAL injection were 327 ± 58 ng/ml in June and 149 ± 27 ng/ml in January, whereas peak levels after αMT were 166±29 and 60±9 ng/ml, respectively. Basal PRL secretion was also greater in summer than winter. The results of this study demonstrate that PRL in peripheral plasma of cattle is increased in response to administration of antidopaminergic drugs and that this increase is greater during the summer than the winter.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Production and characterization of antibody against diacetoxyscirpenol.

Antibodies against diacetoxyscirpenol (DAS) were obtained from rabbits after immunizing them with hemisuccinate or hemiglutarate derivatives of DAS conjugated to bovine serum albumin (BSA). DAS-hemiglutarate-BSA was found to be a much better immunogen than DAS-hemisuccinate-BSA. Competitive radioimmunoassay revealed that the antisera obtained from rabbits after immunization with DAS-hemiglutarate-BSA showed high specificity toward DAS. The concentrations causing 50% displacement of radioactive DAS by unlabeled DAS, 4-monoacetoxyscirpenol (MAS), and 15-MAS were found to be 1.5, 130, and 300 ng per assay, respectively. Thus, the cross-reactivities for 4-MAS and 15-MAS are ca. 87 and 300 times weaker than that of DAS. Practically no cross-reaction (less than 5% displacement) was observed when deoxynivalenol, T-2 toxin, deoxyverrucarol, and scirpentriol were tested at a concentration of 2,000 ng/ml.     

Treatment of patients with cardiovascular disease with L-4F, an apo-A1 mimetic, did not improve select biomarkers of HDL function

L-4F, an apolipoprotein A-I (apoA-I) mimetic peptide (also known as APL180), was administered daily by either intravenous (IV) infusion for 7 days or by subcutaneous (SC) injection for 28 days in patients with coronary heart disease in two distinct clinical studies. L-4F was well tolerated at all doses tested. Despite achieving plasma levels (mean maximal plasma concentration of 2,907 ng/ml and 395 ng/ml, following IV infusion and SC injection, respectively), that were effective in previously published animal models, treatment with L-4F, as assessed by biomarkers of HDL function such as HDL-inflammatory index (HII), and paraoxonase activity, did not improve. Paradoxically, there was a 49% increase in high-sensitivity C-reactive protein (hs-CRP) levels after seven IV infusions of 30 mg L-4F (P < 0.05; compared with placebo) and a trend for hs-CRP increase in subjects receiving 30 mg SC injection for 28 days. In a subsequent, ex vivo study, addition of L-4F at concentrations of 150, 375, or 1,000 ng/ml to plasma from subjects prior to L-4F treatment resulted in significant dose-dependent HII improvement. In conclusion, in vivo L-4F treatment, delivered by either SC injection or IV infusion, did not improve HDL functional biomarkers despite achieving plasma levels that improved identical biomarkers ex vivo and in animal models.     

Plasma progesterone concentrations derived from the administration of exogenous progesterone to ovariectomized mares.

Six ovariectomized mares were divided into 3 groups to determine the effects of exogenous progesterone in oil and repositol progesterone on plasma progesterone concentrations. Progesterone in oil was administered in 7 daily injections in Exp. I. Progesterone concentrations were not maintained greater than 1.0 ng/ml for 24 h with 50 mg/day. However, they remained greater than 1.0 ng/ml during the last 4 days of 100 mg/day and greater than 1.5 ng/ml throughout the injection sequence of 200 mg/day. Repositol progesterone was administered on Days 1 and 7 in Exp. II. At 500 mg, progesterone concentrations peaked in 6 h but returned to near 1.0 ng/ml in 2 days. At 1000 mg and 2000 mg, plasma progesterone was maintained at approximately 2.0 and 4.0 ng/ml respectively for 7 days after injection on Day 1 and was 1.5 and 3.5 ng/ml respectively, 11 days after injection on Day 7. An indication of a cumulative effect on plasma progesterone was observed following repeated dosages of both progesterone in oil and repositol progesterone.     

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

Effects of an agonist of gonadotrophin releasing hormone in cattle. I. Hormone concentrations and oestrous cycle length

Four trials were completed to study the effects of a single intramuscular injection of 5 μg of an agonist of gonadotrophin releasing hormone (Hoe 766) on plasma concentrations of LH and progesterone, and on oestrous cycle length in normally cycling dairy cows.The first trial (four cows) showed that a mid-cycle injection of Hoe 766 temporarily increases plasma LH from less than 5 ng/ml to over 20 ng/ml within 2.5 h. Average plasma progesterone concentrations ranged from 4.8 to 7.0 ng/ml compared to 3.3 ng/ml in the control animal.The second trial (22 cows) showed that an injection of Hoe 766 on Cycle Day 3, 6 or 9 (Oestrus = Cycle Day 0) increased average plasma progesterone concentrations during Cycle Days 13, 14 and 15 by 1.2 ng/ml. Each of three cows injected on Cycle Day 16 maintained plasma concentrations above 3.9 ng/ml until Cycle Day 19 and corpus luteum (CL) size was maintained until Cycle Day 21. Except for the group of cows injected on Cycle Day 3, all other groups had temporarily reduced concentrations of plasma progesterone when sampled 24 h after Hoe 766 administration.The third trial (216 cows) showed that a single injection of Hoe 766 made between Cycle Day 1 and 10 did not alter oestrous cycle length (21.5 vs 21.3 days). In contrast, in the fourth trial (371 cows), a single injection of Hoe 766 between Cycle Days 12 to 16 altered the distribution of cycle lengths of 17–29 days, the average cycle length and the incidence of ovulation without detected oestrus. Compared to matched control cows, fewer Hoe 766-treated cows were detected in oestrus (73.9% vs 90%), or had cycle lengths of less than 20 days (4.7% vs 22.2%). These effects were most pronounced among cows injected on Cycle Day 16 when only 51.7% were detected in oestrus and their average cycle length was 24.1 days.These effects were not due to the formation of a secondary CL. Rather, the injection of Hoe 766 stimulated CL function and appeared to prevent or delay normal luteolysis when administered from Cycle Day 12.     

Oral bioavailability of active principles from herbal products in humans. A study on Hypericum perforatum extracts using the soft gelatin capsule technology.

The oral absorption of two known active principles of Hypericum perforatum, namely hyperforin and hypericin, was studied in an open, single dose, two-way, randomized, cross-over study involving 12 healthy subjects (six males and six females). Alcoholic Hypericum extract (300 mg, containing 5% hyperforin and 0.3 % hypericin) was administered in the morning after 12 hours fasting. The formulation was administered as softgel capsules containing, inter alia, soya oil together with the herbal extract. A second standard formulation in two piece hard gelatin capsules was also used for comparison purposes. Blood was sampled from the subjects at different times after drug administration and the plasma was analysed according to published analytical methods for the determination of hyperforin and hypericin. Peaks of plasma concentration, Cmax of hyperforin were 168.35 ng/ml +/- 57.79 for the soft gelatin formulation (CV=34.32, n=12) and 84.25 ng/ml +/- 33.51 for the hard gelatin capsule (CV=39.77, n=12). The Tmax values for hyperforin were 2.50 h +/- 0.83 for the soft gelatin formulation compared to 3.08 h +/- 0.79 for the reference formulation, whereas the total AUC were respectively 1482.7 h x ng/ml +/- 897.13 and 583.65 h x ng/ml +/- 240.29. As for hypericin, plasma levels were detectable in approximately half of the subjects treated. However also in this case the soft gelatin capsules exhibited a higher individual absorption when compared with the corresponding data for the hard gelatin capsules.     

Opioid action on luteinizing hormone secretion in newborn pigs: paradoxical effect of naloxone

German Landrace piglets, 6-7 days of age, received either saline (9 males, 8 females), 0.5 mg naloxone/kg body weight (7 males, 7 females), 2.0 mg naloxone/kg (7 males, 8 females) or 0.5 mg DADLE (potent leu-enkephalin analog)/kg (7 males, 7 females) through a catheter inserted into the jugular vein 2-4 days previously. Male or female piglets were allocated randomly, within litter, to the different experimental groups. Blood samples were withdrawn for a period of 240 min at 10-min intervals for the first 60 min following injection and at 20-min intervals for the rest of the test period. Piglets were separated from their mother via a detachable wall and were allowed to suckle every 50 min. DADLE failed to alter plasma levels of LH in both males and females. Naloxone induced a significant (P less than 0.01) decrease in LH concentrations in females 10 to 60 min after injection (saline: 2.3 +/- 0.2 ng/ml plasma (SEM); 0.5 mg naloxone/kg: 1.0 +/- 0.2 ng/ml plasma and 2 mg naloxone/kg 1.2 +/- 0.4 ng/ml plasma). In males low doses of naloxone reduced plasma LH levels 10 to 40 min after injection (saline: 2.0 +/- 0.3 ng/ml plasma and 0.5 ng naloxone/kg: 1.1 +/- 0.3 ng/ml), whereas a decrease in plasma LH levels occurred 80 to 140 min after injection of high doses of naloxone (saline: 2.1 +/- 0.2 ng/ml and 2 mg naloxone/kg: 1.0 +/- 0.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Medroxyprogesterone acetate in human serum

Conjugates of medroxyprogesterone acetate (MPA) in human serum are investigated using chromatography and techniques (equilibrium dialysis, gel filtration, and polyacrylamide gel electrophoresis) previously described for studying the binding of MPA. 17 serum samples were obtained from 7 women at various times after the intramuscular injection of 150 mg Depo-Provera. Mean concentration of MPA in the unconjugated fraction of serum was 3.9 mg/ml (range 0.8-10.7 ng/ml); in the conjugated fraction, the value was 2.7 ng/ml (range 0.6-11.4 ng/ml), a mean value of 81.7% (range 18.4-286%) of that in the unconjugated fraction. The conjugate appears to be mainly a glucuronide since solvolysis released only small amounts of MPA. MPA metabolites were also detected in blood. The MPA levels in blood measured by radioimmunoassay were generally lower when serum was extracted with an organic solvent rather than when the assay was carried out directly in the serum. This finding suggests the presence in blood of either MPA in a conjugated form or metabolites interacting with the antiserum which were not extracted by the solvents used. Equilibrium dialysis showed that undiluted plasma bound 85.8% of triated hydrogen-MPA; with increasing dilution of the plasma, the amount of bound triated hydrogen-MPA decreased. The apparent association constant calculated according to the method of Vermeulen and Verdonck was 2.6 x 10 4 1/mol. MPA appeared to be loosely bound to albumin in blood but there was no specific binding protein for the steroid. MPA conversion to the glucuronide may be 1 of the factors regulating the level of the unconjugated but presumably biologically active steroid in blood.