A radioimmunoassay for deoxycorticosterone in human peripheral plasma using sephadex LH-20 chromatography

An accurate, precise, sensitive and relatively simple radioimmunoassay method for the measurement of deoxyocorticosterone (DOC) in human plasma is described. A hexane pre-extraction Is carried out when the sample contains a high progesterone level. A Sephadex LH-20 column (45 × 0.9 cm, in the system dichloromethane:methanol, 98:2) provides adequate purification before radioimmunoassay. However, it has been observed that progesterone inteference is likely in plasma of pregnant females. A specific antibody was generated against DOC-3-oxime coupled to bovine albumin. The Intra and inter assay precision yielded a coefficient of variation of 12.4% for five samples and 27% for 17 samples. The accuracy was checked by measuring recovery of DOC added to pre-extracted plasma (98.5 ± 12.4 (S.D.)%). The sensitivity (10 pg) and blank values (1.5 ng/100 ml) are satisfactory. The normal plasma DOC level obtained (6.4 ± 4.4 ng/100 ml, n = 14) is in agreement with previously reported values.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

Developmental patterns of plasma dehydroepiandrosterone sulfate and testosterone in male pigs

The relationship between plasma levels of dehydroepiandrosterone sulfate (DHAS) and testosterone (T) was determined by radioimmunoassays in growing and adult pigs. Seven young males were bled at 2-weekly intervals between 1 and 47 weeks of age and two adult boars were cannulated for short-term studies. Plasma samples were extracted with methylene chloride and T was isolated by Celite chromatography. DHAS was assayed directly in the aqueous phase.Dehydroepiandrosterone occurred predominantly (89.7 ± 10.6%) as the sulfoconjugate in boar plasma (n = 50). Plasma DHAS was undetectable in castrated males (n = 2). At 1 week of age, mean levels (± S.D.) of DHAS and T were 5.0 ± 3.0 ng/ml and 0.15 ± 0.10 ng/ml, respectively; and they rose to small peaks of 16.0 ± 2.0 ng/ml and 0.63 ± 0.10 ng/ml at 3 weeks. At 7 weeks, the levels of DHAS and T increased gradually from 10.0 ± 6.7 and 0.11 ± 0.10 ng/ml to 27.0 ± 6.6 and 1.84 ± 0.61 ng/ml at 19 weeks. There followed a marked increase to 4.90 ± 3.30 ng/ml at 21 weeks for T and a less abrupt rise to 44.0 ± 9.3 ng/ml at 23 weeks for DHAS. The mean levels remained high from then onwards, fluctuating between 24.0 ± 8.7 and 54.5 ± 5.0 ng/ml for DHAS and between 1.73 ± 0.86 and 4.43 ± 1.26 ng/ml for T. Episodic fluctuations were noted in two boars during hourly collection for 24 h, with mean levels of 9.0 ± 4.9 and 50.0 ± 10.4 ng/ml for DHAS, and 1.76 ± 0.83 and 3.26 ± 0.63 ng/ml for T, respectively.For all ages of males, plasma DHAS and T levels were highly correlated (r = 0.95) with greater concentrations of DHAS in all samples. Although individual differences in steroid profiles were noted, concentrations for DHAS and T showed almost parallel increases at puberty and corresponding fluctuations in adult boars. It is suggested that plasma DHAS determinations provide a simple, sensitive assessment of androgen production in the male pig.     

Radioimmunoassay for etiocholanolone

A sensitive and reliable radioiinmunoassay for serum unconjugated etiocholanolone (3α-hydroxy-5β-androstan-17-one) is reported. The antiserum was obtained from rabbits by immunization of etiocholanolone-17-(O-carboxy-methyl) oxime [CMO] -bovine serum albumin [BSA]. Two ml of serum with ³H-etiocholanolone added for recovery was extracted with ether, and etiocholanolone was separated from cross-reacting steroids by Sephadex LH-20 column chromatography.The mean recovery after extraction and chromatography was 80.7 ± 6.8(S.D.)%. The sensitivity of the assay was less than 40 pg. The intraassay and interassay coefficients of variation were 9.2% and 10.9%, respectively. The mean of serum unconjugated etiocholanolone concentration determined by the present method was 0.39 ± 0.10 (S.D.) ng/ml (n = 50) in normal men and 0.36 ± 0.08 (S.D.) ng/ml (n = 20) in women in the follicular phase of the menstrual cycle.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Radioimmunoassay of plasma 20 alpha-dihydroprogesterone

A radioimmunological method is described and evaluated for the determination of 20α-dihydroprogesterone (20α-hydroxypregn-4-en-3-one) in peripheral venous plasma (0.1 – 2.O ml) of men and women. The antiserum is highly specific. The concentration (mean ± S.D., ng/ml plasma) in a group of healthy men was 0.19 ± 0.08; in women in follicular phase, 0.28 ± 0.11; in luteal phase, 2. 19 ± 1.19; and during pregnancy (24th week to term) in the range 8.44– 40.94.     

Plasma Testosterone and Testosterone Binding Affinities in Men with Impotence,Oligospermia, Azoospermia,and Hypogonadism

Mean plasma testosterone levels (± S.D.), using Sephadex LH-20 and competitive protein binding, were 629 ± 160 ng/100 ml for a group of 27 normal adult men, 650 ± 205 ng/100 ml for 27 impotent men with normal secondary sex characteristics, 644 ± 178 ng/100 ml for 20 men with oligospermia, and 563 ± 125 ng/100 ml for 16 azoospermic men. None of these values differ significantly. For 21 men with clinical evidence of hypogonadism the mean plasma testosterone (± S.D.), at 177 ± 122 ng/100 ml, differed significantly (P < 0·001) from that of the normal men.The mean testosterone binding affinities (as measured by the reciprocal of the quantity of plasma needed to bind 50% of ³H-testosterone tracer) were similar for normal, impotent, and oligospermic men. Though lower for azoospermic men the difference was not significant (P >0·1). For 12 of the 16 hypogonadal males the testosterone binding affinity was normal, but raised binding affinities, similar to those found in normal adult females or prepubertal boys (about twice normal adult male levels), were found in four cases of delayed puberty. These findings help to explain why androgen therapy is usually useless in the treatment of impotence.     

Evaluation of progesterone and 20-oxo-progestagens in the plasma of Asian (Elephas maximus) and African (Loxodonta africana) elephants

The corpus luteum of African elephants produces high amounts of 5α-reduced progesterone metabolites (5α-pregnane-3,20-dione and 5-α-pregnane-3α-ol-20-one), whereas progesterone itself is quantitatively less important, and plasma levels of progesterone during the estrous cycle in elephants are considerably lower than those of other mammals. The objective of this study was to compare the concentration of progesterone in plasma of Asian and African elephants as determined by specific progesterone assays with those of total immunoreactive progestagens containing a 20-oxo-group (20-oxo-P). These metabolites were determined by an enzyme immunoassay using an antibody against 5-α-pregnane-3β-ol-20-one, 3HS:BSA. Plasma of non-pregnant Asian (n = 4) and African (n = 4) elephants was collected at weekly intervals for periods of 8–15 months and at random intervals during pregnancy in one Asian elephant. High-performance liquid chromatography separation of plasma samples of both species demonstrated that in the 20-oxo-P assay, 5α-pregnane-3,20-dione makes up ˜60% of the total immunoreactive material. The progesterone and 20-oxo-P values during the estrous cycle showed a parallel pattern and were significantly correlated (P < 0.001; Asian: r = 0.80; y = 3.76 × –0.10; African: r = 0.75; y = 2.66 × –0.08). Progesterone and 20-oxo-P values in Asian and African elephants were <0.15 ng/ml during the follicular phase (weeks –4 to 0) of the estrous cycle; progesterone values during the luteal phase (weeks 2–9) were 0.60 ± 0.03 and 0.53 ± 0.03 ng/ml, and the 20-oxo-P values were 2.19 ± 0.16 and 1.48 ± 0.12 ng/ml, respectively. The 20-oxo-P values of the pregnant animal, although slightly higher, were comparable to those of non-pregnant elephants during the luteal phase. Total immunoreactive 20-oxo-P values are about three times higher than those of progesterone during the luteal phase, and 5α-pregnane-3,20-dione is the major immunoreactive 20-oxo-P in the plasma of Asian and African elephants. Zoo Biol 16:403–413, 1997. © 1997 Wiley-Liss, Inc.     

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 mM phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 mM phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear (r²≥0.999) over the concentration range 10–1000 ng/ml.     

Radioimmunoassay of plasma androsterone

A method is described and evaluated for the determination of androsterone in peripheral venous plasma (1.0 ml) from men and women. The procedure involves addition of labelled internal standard and extraction with diethyl ether. Aliquots (10 %) are removed for radioimmunoassay. An antiserum to androsterone-17-carboxymethyl oxime-bovine serum albumin and tritiated androsterone complete the system. The practical systematic errors have been determined by replicate analyses. The range of values (mean ± S.D.) in plasma from 40 healthy men are 54 ± 32 ng/100 ml, and the corresponding values for women 46 ± 28.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Blood leukotriene levels during the acute asthma attack in children

Leukotrienes (LT) have been proposed to be important mediators in the etiology of the acute asthma attack (AAA). We therefore studied blood LT levels in 18 children having AAA. Heparinized blood samples were obtained before and after treatment with epinephrine injections and/or metaproterenol inhalations in the emergency room. The samples were acidified and subjected to Sep-pak chromatography. Reverse phase high performance liquid chromatography (RP-HPLC), ultraviolet (UV) spectroscopy and bioassay on guinea pig ileum were used to identify the LT based on comparison to data produced by standard synthetic LT samples. Radioimmunoassay (RIA) was used to further confirm the presence of LT LT C, D and E were detected in the plasma of children having AAA. Only LT C levels were significantly elevated over control values. The mean blood LT C level of control patients was 1.6 ± 1.2 nanograms per milliliter (ng/ml, mean t SEM) while that of the asthma patients was 73.8 ± 18.2 ng/ml prior to treatment. After emergency room treatment the asthma patients had a mean blood LT C level of 22.5 ± 11.7 ng/ml. Lowered levels of LT C accompanied improved clinical condition of the patients. This finding indicates that the AAA in children is associated with elevated blood levels of LT C.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

Seasonal changes in the concentration of estrogens and testosterone in the plasma of the stallion

A blood sample was taken from each of 15 stallions at monthly intervals for 14 consecutive months. Plasma concentrations of estrogens and testosterone were measured by radioimmunoassay methods. Estrogens in peripheral blood were present in much higher amounts than testosterone and were principally in a water-soluble, solvolyzable form (> 98%). The major component in the solvolyzed extracts behaved chromatographically as estrone. The mean plasma level (± S.E.) of estrogens averaged across months was 52.9 ± 4.5 ng ml^?1. Individual stallions showed considerable month-to-month variation; for example, single monthly samples ranged from 29.5 to 160.6 ng ml^?1 for the stallion with the highest single value.The highest mean monthly concentration was 69 ± ng ml^?1 in May, and plasma levels were < 40 ng ml^?1 during November and December. For the 11 Thoroughbred stallions in the study, the mean concentrations of estrogens were 73 ± 5.8 ng ml^?1 for May to July and 45 ± 4.1 ng ml^?1 for November to January (P > 0.001).The mean monthly concentrations (± S.E.) of testosterone ranged from 0.22 ± 0.05 to 0.90 ± 0.14 ng ml^?1, and individual samples ranged from < 0.02 to 2.8 ng ml^?1 of plasma. While the highest mean level of testosterone was seen in September, there was a significant difference (P < 0.01) between the values in the breeding season (May–July, 0.73 ± 0.07 ng ml^?1) and the non-breeding season (November–January, 0.38 ± 0.08 ng ml^?1). No marked seasonal changes were observed, however, in testosterone levels in several stallions. It was concluded that plasma estrogen levels may provide a more sensitive index of endocrine function of the testes in the stallion.     

Salivary progesterone for the assessment of the ovarian function in the capuchin monkey (Cebus apella)

Salivary and plasma progesterone were measured in normally cycling (n=10) and castrated (n=4) femaleCebus monkeys (Cebus apella). During the follicular phase, progesterone levels in saliva ranged between 0.05 and 1.40 ng/ml and in the luteal phase they increased to between 0.22 and 4.70 ng/ml. These values represented on average 6.5 and 3.2% of those values measured in plasma, for the follicular and luteal phases, respectively. The regression analysis of the steroid concentrations in both fluids showed a highly significant correlation (r=0.8985,n=180,P<0.0001). Ovariectomized monkeys had consistently low salivary (0.37±0.02 ng/ml) and plasma (4.70±0.25 ng/ml) progesterone, showing a low, but significnat, correlation coefficient (r=0.2592,n=58,P=0.047). The ratio of plasma/salivary progesterone was significantly higher in the luteal phase (31.09±1.65) than in the follicular phase (23.06±2.26) and in castrated monkeys (16.00±1.38). The free fraction of progesterone constituted 5.3±0.2% of the total plasma progesterone during the follicular phase and 3.3±0.1% during the luteal phase. Ovariectomized monkeys showed a significantly higher percentage of free progesterone in plasma (7.7±0.1%). In contrast, free progesterone made up 64.4 and 70.9% of the total salivary progesterone for the follicular and luteal phases, respectively. The proportion of free progesterone in castrated animals was within the range observed in cycling animals. We suggest that the levels of progesterone in the saliva of capuchin monkey follow a pattern similar to that for plasma progesterone, reflecting the free steroid fraction. Thus, the measurement of such steroid in saliva may offer a valuable alternative to plasma determinations for the assessment of the ovarian function inCebus and probably other New World monkey species.     

Plasma prolactin in the periparturient sow

A homologous radioimmunoassay was used for measurement of porcine prolactin in blood plasma collected from sows during the periparturient period. The assay was able to detect prolactin over a range of 0.5 to 7.0 ng/assay tube. There was no significant cross reaction with growth hormone, luteinizing hormone, or follicle stimulating hormone at amounts up to 10⁵ ng/assay tube while porcine ACTH gave 30% binding at 10⁴ ng. Prolactin was not detected in plamsa from a hypophysectomized pig or 2 ergocryptine-treated sows when 100 μ l plasma were assayed. Prolactin concentration in plasma was then measured in 14 periparturient sows within a period extending from 7 days before farrowing to 7 days after farrowing. Samples were collected at 15 min intervals between 1330 and 1630 h each day. However, prolactin assays were done only on the even-numbered samples (30 min interval). Plasma prolactin concentrations (ng/ml, $\text{X}\text{± SEM}$) were 23.7 ± 2.0 on days ?7 to ?5 prepartum, began to rise by day ?3 prepartum (42.5 ± 5.9), and peaked at 127.5 ± 17.6 on day 1 prepartum. By day 3 postpartum, prolactin concentrations in plasma had decreased to 80.5 ± 12.6 and further declined to 51.6 ± 4.6 on day 7 postpartum. The mean prolactin concentration in plasma for all pigs on days ?1 to +2 was 116.8 ± 13.8. This mean concentration for days ?1 to +2 was different (P < 0.025) from the mean prolactin concentration for the period both prior and subsequent to these days (?8 to ?2 and +3 to +8 days).     

Soluble CD23 in Human Immunodeficiency Virus Type 1 Infected Patients

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10⁶ cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m±S.D. = 1.0 ±0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m±S.D. = 2±1.33, n = 9) and some of the patients of group IV (AIDS) (m±S.D. = 1.3 ±1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m±S.D. = 0.9±0.33), in group II patients (n = 17) from 0 to 3 U/ml (m±S.D. = 0.92±0.83) and in group IV patients (n =73) from 0 to 2.9 U/ml (m±S.D. = 1.15±0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-C1 and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B). Elevated values of sCD23 were detected even in patients with low counts of CD4⁺ T cells and CD8⁺ T cells in their peripheral blood. sCD23 has numerous activities including control of IgE synthesis and cytokine-like properties. Our results show a disarray of sCD23 in HIV-infected patients which could be involved in drug reactions, allergic manifestations and the IgE-level increase. Further investigations should attempt to define the role of sCD23 in clinical manifestations of HIV infection.     

Myocardial uptake of thiopental enantiomers by the isolated perfused rat heart

Myocardial uptake of thiopental enantiomers by an isolated perfused rat heart preparation was examined after perfusion with protein-free perfusate. Outflow perfusate samples were collected at frequent intervals for 20 min during single-pass perfusion with 10 μg/ml racemic thiopental (washin phase) and for another 45 min during perfusion with drug-free perfusate (washout phase). (+)- and (−)-thiopental concentrations were assayed by chiral high-performance liquid chromatography. Heart rate, perfusion pressure, and electrocardiogram were also monitored. During the washin phase, there was no significant difference between the mean values of the equilibration rate constants of (+)- and (−)-thiopental enantiomers (0.44 ± 0.07 min⁻¹ and 0.43 ± 0.09 min⁻¹, respectively, P > 0.05). Mean volumes of distribution of (+)- and (−)-thiopental enantiomers were similar (6.34 ± 1.20 and 6.45 ± 1.29 ml/g for the washin phase and 7.22 ± 0.71 and 7.47 ± 0.81 ml/g for the washout phase, respectively, P > 0.05). This indicates that tissue accumulation of thiopental enantiomers in the isolated perfused rat heart was not stereoselective. Uptake of thiopental by the heart was perfusion flow rate-limited and independent of capillary permeability. These findings suggest that myocardial tissue concentration of racemic thiopental should be an accurate predictor of myocardial drug effect. © 1996 Wiley-Liss, Inc.     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.