A sensitive solid phase enzymeimmunoassay for testosterone in plasma and saliva.

A sensitive, solid phase enzymeimmunoassay suitable for determining testosterone concentrations in samll aliquots of plasma (20 microliter) and saliva (200 microliter) has been developed. A solid phase antiserum raised against a testosterone-11 alpha-hemisuccinate/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. The "enzyme label" was a covalently linked testosterone/horseradish peroxidase conjugate. The assay had a lower limit of sensitivity of 4pg/assay tube and satisfied accepted criteria of specificity and precision. Testosterone concentrations determined by enzyme-immunoassay were in excellent agreement not only with a gas liquid chromatography/mass spectrometry procedure (r=0.96, n=12) but also with the radioimmunoassay in routine use (r=0.95, n=12). The EIA can therefore replace RIA in both the small clinical laboratory and high throughput service centres for determining plasma and salivary testosterone concentrations. In normal males salivary testosterone concentrations reflected circulating steroid levels and indicated the possibility of assaying saliva rather than plasma in clinical studies.     

A sensitive enzymeimmunoassay with a fluorimetric end-point for the determination of testosterone in female plasma and saliva

A fluorimetric enzymeimmunoassay has been developed having the sensitivity (50O fg/assay tube) required for determining testosterone concentrations in female plasma and saliva samples. The assay featured a solid-phase antiserum raised against an llα-hydroxytestosterone-11-hemisuccinate bovine serum albumin conjugate, an llα-hydroxytestosterone-11-hemisuccinate horseradish peroxidase conjugate as the “enzyme label” and p-hydroxyphenylacetic acid as the substrate for the development of fluorescence. Specificity was ensured by “extracting” testosterone from samples with a solid-phase anti testosterone-3-¦O-carboxymethyl¦-oxime serum. The assay was shown to satisfy accepted validation criteria providing results in good agreement with routine radioimmunoassay procedures in both plasma (r > 0.98, n = 28) and saliva (r > 0.99, n = 28). In saliva samples collected at 2 hourly intervals by normal healthy women (n = 5) testosterone concentrations showed a well defined circadian rhythm: the mean testosterone concentration in early morning samples (174 pmol/litre) fell by 83% in late evening collections. In healthy female volunteers (n = 7), mean salivary testosterone concentrations in samples collected daily throughout one complete cycle ranged from 5O to 218 pmol/litre. Following dexamethasone administration testosterone concentrations in plasma fell by approximately 50% and salivary concentrations were undetectable after one hour. This enzymeimmunoassay may be useful in studies of female infertility.     

A sensitive and specific enzymeimmunoassay for serum testosterone.

A very sensitive enzymeimmunoassay for testosterone was developed using testosterone-penicillinase conjugate and an antibody to testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin. The specificity of the assay was demonstrated by the fact that estradiol-17 beta, estrone, estriol, progesterone, 17 alpha-hydroxy-progesterone, dehydroepiandrosterone, androstenedione, cortisol and cortisone were ineffective in crossreacting with testosterone while dihydrotestosterone was 8 times less crossreactive as compared to testosterone. The minimum detectable amount of testosterone was 10-15 pg per assay tube. Intra-assay and inter-assay coefficients of variation for samples containing 0.3-6ng/ml of testosterone were 6-8% and 8-10%, respectively. A high degree of correlation (r = 0.97) was observed between serum testosterone values obtained by enzymeimmunoassay and radioimmunoassay. The levels of testosterone in the sera of normal men and women and those in hypogonadal males following stimulation with human chorionic gonadotropin determined by this enzymeimmunoassay appear similar to those reported by other investigators.     

Salivary and plasma cortisol response to adrenocorticotropin administration in pigs

Two experiments were conducted to determine the responsiveness of salivary and plasma cortisol to acute (i.v.), depot (i.m.) and chronic (repeated i.m.) adrenocorticotropin (ACTH) administration in swine. In Experiment 1, barrows (castrated pigs) were assigned to one of three injection treatments: (1) saline i.m. (SHAM1, n=2); (2) 0.75 IU/kg BW ACTH in saline i.v. (ACUTE, n=2); (3) 2.25 IU/kg BW ACTH in gel i.m. (DEPOT, n=3). Total cortisol concentrations were determined for concurrent saliva and blood samples. Correlations between salivary and plasma cortisol within treatments were: SHAM1, r=0.60; ACUTE, r=0.58; DEPOT, r=0.79. In Experiment 2, barrows were assigned to one of two injection treatments: (1) gel i.m. (SHAM2, n=3); (2) 2.25 IU/kg BW ACTH in gel i.m. (CHRONIC, n=4). The injections occurred every 6 h for a total of eight injections. Concurrent saliva and blood samples were obtained every 3 h for 72 h followed by an increasing sampling interval until day 6. Overall correlations between salivary and plasma cortisol were: SHAM2, r=0.30 and CHRONIC, r=0.61. Experiment 1 found that the relationship between salivary and plasma cortisol was stronger during longer (DEPOT) than shorter (ACUTE) ACTH stimulation. Experiment 2 found a strong relationship between the two measurements during chronic ACTH stimulation, but that relationship weakened after ACTH stimulation ceased.     

Salivary cortisol for monitoring adrenal activity during marathon runs

In non-elite male runners (n = 8), changes in adrenal activity were monitored by measurement of salivary cortisol in samples collected at 4-mile intervals during marathon runs. These changes were compared with those in similarly timed samples collected on rest days. Immediately prior to the Cardiff marathon, at 09.00 h, mean salivary cortisol concentrations (21.5 nmol/l) were higher than those in similarly timed rest day samples (14.9 nmol/l). Cortisol concentrations increased during the marathon, and although values at 25 miles were high (79.4 nmol/l), maximum values (87.9 nmol/l) were observed in samples collected 30 min after completion of the run. Some Cardiff marathon runners also participated in the Bristol marathon (n = 4) and a non-competitive event (n = 3). The changing pattern in secretory activity was similar in all events. The easy collection of saliva without cessation of exercise is ideal for monitoring the hormonal response to exercise.     

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.     

Solid phase method for measurement of the binding capacity of testosterone-estradiol binding globulin in human serum

A solid phase method for measuring the binding capacity of serum testosterone-estradiol binding globulin (TeBG) is described and compared with other methods. TeBG, a glycoprotein, is adsorbed from serum or plasma onto a solid phase matrix of concanavalin-A, a carbohydrate-specific adsorbent. The TeBG binding capacity is determined by Scatchard analysis of the binding of radioactive testosterone at physiologic pH, in standard test tubes, and without the addition of albumin. Transcortin binding of testosterone is inhibited by the addition of cortisol.The levels of TeBG binding capacity determined with this solid phase method showed an excellent correlation with levels determined by procedures using equilibrium dialysis (with added cortisol) or ammonium sulphate precipitation. The serum TeBG binding capacity was 0.798±0.064 (mean±SE) μg/100 mL in men (n=32), 1.06±0.13 in women (n=10), 2.18±0.19 in women taking oral contraceptives (n=4), 6.2±2.9 in hyperthyroid women (n=2), and 11.6±3.1 in pregnant women (n=5). The serum TeBG binding capacity determined in heparinized plasma did not differ from that determined in serum. The within-assay variation is 9.6% and the between-assay variation is 11.2%.This solid phase method for measurement of serum TeBG binding capacity is simple, precise, and reproducible, and gives values which correlate well with those determined by other methods.     

Evaluation of the BRAHMS KRYPTOR thyroglobulin "mini-recovery" test in thyroid healthy subjects

The aim of the work was to compare the automated thyroglobulin (Tg) assay on the automated BRAHMS KRYPTOR platform (hTG KRYPTOR) to the established BRAHMS Tg Plus immunoradiometric assay for the measurement of Tg levels and regular Tg recovery rates and to assess a recovery test using a low Tg concentration of 10 μg/l ("mini-recovery") in samples with a native Tg level of <10 μg/l. Tg levels and recovery rates, as well as the mini-recovery, were determined in 208 serum samples from thyroid-healthy patients using both assays. The reference ranges for the Tg-Plus assay are 2.0-51.0 μg/l for Tg levels and 81.5-108% for recovery rates at 100 μg/l. The reference ranges for hTG KRYPTOR are 2.4-47.8 μg/l for Tg, 83.3-110.4% for a conventional recovery with 80 μg/l in Tg levels ≥ 10.0 μg/l (n=121) and 94.4-122.9% for the mini-recovery with Tg <10.0 μg/l (n=87). The correlation between the Tg-Plus and hTG KRYPTOR is excellent for Tg (r2=0.95; p<0.001), but not significant for recovery rates. Tg levels determined using the KRYPTOR Tg assay are clinically comparable to the conventional Tg-Plus assay. New features of the KRYPTOR assay such as the ability to perform a "mini-recovery" still require further study before clinical use.     

Further evidence for the usefulness of the salivary testosterone radioimmunoassay in the assessment of androgenicity in man in basal and stimulated conditions

Since correct assessment of testicular function and androgenic status in humans requires multiple sampling, a sensitive and accurate radioimmunoassay (RIA) of testosterone (T) was established for male and female saliva samples. This easily collected biological fluid, which contains nonprotein-bound T, may represent an attractive alternative or a complement to total plasma T assays. In saliva samples from 5 normal males, a clear circadian rhythm was observed, and morning concentrations (135 +/- 31 pg/ml) were significantly higher (p less than 0.02) than evening samples (85 +/- 23 pg/ml). In 11 normal females, morning saliva levels were 12.8 +/- 1.8 pg/ml. The levels of T in male saliva, in response to both exogenous T administration (100 mg i.m.) and HCG stimulation (2 X 2,000 IU i.m.), accurately reflected the changes observed in plasma T, and the magnitude of increase in T levels was clearly greater in saliva than in plasma samples during the intramuscular administration of the long-acting T preparation. In males, significant correlations were observed between salivary and plasma T concentrations in morning samples (r = 0.61, p less than 0.01), following HCG stimulation (r = 0.89, p less than 0.05) and during T administration (r = 0.87, p less than 0.05). In women, the correlation at 8 a.m. was also significant (r = 0.82, p less than 0.05).     

Salivary cortisol in low dose (1 microg) ACTH test in healthy women: comparison with serum cortisol

To date, a single report has appeared on the use of salivary cortisol for adrenal function testing with a low dose ACTH, although 1 microg has become preferred as a more physiological stimulus than the commonly used 250 microg ACTH test. Our present study was aimed to obtain physiological data on changes of free salivary cortisol after 1 microg ACTH stimulation. This approach was compared with the common method based on the changes of total serum cortisol. Intravenous, low-dose ACTH test was performed in 15 healthy women (aged 22-40 years) with normal body weight, not using hormonal contraceptives, in the follicular phase of the menstrual cycle. Blood and saliva for determination of cortisol were collected before ACTH administration and 30 and 60 min after ACTH administration. Basal concentration of salivary cortisol (mean +/- S.E.M., 15.9+/-1.96 nmol/l) increased after 1 microg ACTH to 29.1+/-2.01 nmol/l after 30 min, and to 27.4+/-2.15 nmol/l after 60 min. The differences between basal and stimulated values were highly significant (p<0.0001). The values of salivary cortisol displayed very little interindividual variability (p<0.04) in contrast to total serum cortisol values (p<0.0001) A comparison of areas under the curve (AUC) related to initial values indicated significantly higher AUC values for salivary cortisol than for total serum cortisol (1.89+/-0.88 vs. 1.22+/-0.19, p<0.01). Correlation analysis of serum and salivary cortisol levels showed a borderline relationship between basal levels (r=0.5183, p=0.0525); correlations after stimulation were not significant. Low-dose ACTH administration appeared as a sufficient stimulus for increasing salivary cortisol to a range considered as a normal adrenal functional reserve.     

Development and application of a direct radioimmunoassay for aldosterone in saliva

A previously described direct radioimmunoassay for plasma aldosterone has been modified to enable direct measurement of the steroid in saliva. The specificity of the method has been demonstrated by assay after high pressure liquid chromatographic purification of saliva extracts. Assay of matched plasma and saliva samples taken from normal subjects during unrestricted and controlled sodium intakes, either under basal conditions or while undergoing ACTH stimulation or dexamethasone suppression, confirms that salivary aldosterone values provide a good reflection of levels in plasma. Mean salivary aldosterone values are approximately one-third of those in plasma. Sampling immediately upon waking appears to provide reliable values for salivary aldosterone, and the potential application of this technique to the screening of hypertensive patients is discussed.     

Determination of human thrombin-antithrombin III complex by enzyme immunoassay

We have developed a specific and sensitive ELISA for the measurement of the TAT in human plasma. The assay follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The anti-thrombin antibody population used for coating was purified by immunoadsorption on immobilized prothrombin and thrombin, respectively. Antithrombin III antibodies were conjugated with peroxidase. Plasma samples containing TAT were incubated in polystyrene tubes coated with anti-thrombin antibodies; after washing, peroxidase-conjugated antithrombin III antibodies were added and bound enzyme activity was subsequently measured using o-phenylenediamine. The assay was calibrated with definite concentrations (2.0 to 60 micrograms/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance at 492 nm against TAT concentrations revealed a linear correlation (r = 0.98). A reference range from 0.85 to 3.0 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT was found up to 7-13 micrograms/l. Patients with septicemia associated with a consumption coagulopathy (n = 10) showed markedly increased TAT values (greater than or equal to 10 micrograms/l). From these data it can be concluded that measurement of TAT might be a parameter for detection of a latent clotting pathway activation.     

Comparison of plasma and urinary phytoestrogens in Japanese and Finnish women by time-resolved fluoroimmunoassay

Time-resolved fluoroimmunoassays (TR-FIA), with europium labeled phytoestrogens as tracers, were developed for the quantitative measurement of genistein, daidzein and enterolactone in plasma and urine for the purpose of screening large populations and studies on possible correlation between the values in biological fluids and the risk of western diseases. The mean values of the three phytoestrogens in plasma as determined by TR-FIA were similar to those obtained by gas chromatography-mass spectrometry (GC-MS). The urinary excretion levels of total individual phytoestrogens were higher than those obtained by GC-MS, with the exception of the daidzein values. However, comparing the assay results obtained by the present method and those obtained by GC-MS, a strong correlation was evident (r = 0.87 - 0.99, p < 0.001). We measured plasma levels of genistein, daidzein and enterolactone in 111 healthy Japanese women The mean and median levels of genistein were 406.8 and 306.3 nmol/l, respectively, and those of daidzein were 118.4 and 76.8 nmol/l, respectively. These levels are higher than those reported for Americans and Western Europeans. Isoflavone intake as calculated from dietary records (genistein: mean, 86.5 mircomol/day and daidzein: mean, 57.4 micromol/day) was correlated with the plasma concentrations observed (genistein: r = 0.287, p < 0.01 and daidzein: r = 0.313, p < 0.01). Plasma enterolactone levels were low in Japanese women (mean, about 10 nmol/l). The levels of urinary excretions of genistein, daidzein were also measured and it was found that, in the majority, the levels ranged between 5-25 and 5-50 micromol/24 h, respectively. In contrast, healthy Finnish women showed very low values of isoflavones (below 10 nmol/l in plasma (n = 87) and below 0.6 micromol/24 h in urine (n = 126) for both compounds) and high levels of enterolactone in both plasma and urine (plasma: mean, 25 nmol/l and urine: majority range, 1-7 micromol/24 h).     

Salivary testosterone concentrations in prepubertal and pubertal males: comparison with total and free plasma testosterone

Salivary testosterone (T) levels in male children and adolescents were measured and compared with plasma T. Salivary concentrations correlated well with plasma total T (r = 0.72) and even better with free plasma T (r = 0.89) in subjects with plasma T levels of pubertal or adult levels (greater than 1.0 nmol/l). In subjects with prepubertal or low plasma T (less than 1.0 nmol/l), there was neither a correlation with plasma total, nor with free T. In hCG tests (responder and nonresponder), salivary T reflected plasma levels faithfully. The results suggest that salivary T, which is suitable for repeated sampling, is a good marker of T secretion in pubertal males.     

Enhanced chemiluminescent immunoassay for aldosterone

A solid phase immunoassay for aldosterone using enhanced chemiluminescent detection has been developed. Monoclonal antibodies against aldosterone were used for the immune reaction and compared with polyclonal antibodies. Uniform Protein A coated polystyrene tubes were used as solid phase for the monoclonal antibody and second (anti-rabbit) antibody coated tubes for the polyclonal antibody. Horseradish peroxidase was covalently linked to aldosterone as enzyme label. Optimum conditions were established for the generation and measurement of the luminescent reactions using luminol, p-iodophenol as enhancer and hydrogen peroxide. The advantages of this assay are the high sensitivity with a detection limit of 100fg/tube, the prolonged luminescence signal with a simplification of the measurement (simpler detectors, external start pipetting) and the short measure time with the possibility of repeated measurement. The coefficients of variation were 4.2%–7.3% in the concentration range 140–1180 pmol/l. The assay showed a significant correlation (r = 0.91) with the ELISA. The aldosterone concentrations in plasma and saliva of patients with Conn's syndrome were significantly increased, and in patients with Addison's disease were found near the detection limit.     

Relationship between canine mucosal and serum immunoglobulin A (IgA) concentrations: serum IgA does not assess duodenal secretory IgA

A double-sandwich enzyme immunoassay method was developed for determination of serum immunoglobulin A (S-IgA) and mucosal secretory immunoglobulin A (sIgA) in duodenal brush samples obtained via endoscopy and the relationship between enteric mucosal sIgA, salivary sIgA and S-IgA in dogs was examined. Twenty healthy dogs underwent routine endoscopy. A brush sample from the duodenal mucosa was obtained and washed in PBS, with a serum sample being taken concurrently. A saliva sample was collected from twelve of these dogs. S-IgA and sIgA with total protein concentrations in the duodenal washings and saliva samples were determined. A significant negative correlation (r = -0.64, P = 0.0059) was found between duodenal sIgA/protein ratios and S-IgA concentrations. Saliva sIgA/protein ratios did not correlate with sIgA/protein ratios of duodenal samples. The method described here allows for direct assessment of duodenal IgA; therefore indirect measures based on serum IgA or salivary IgA can be avoided. In addition, these indirect measures appear to be poor indicators of duodenal sIgA competence in dogs.     

High constitutional nitrate status in young cattle.

Nitrate or nitrite can be ingested or endogenously produced from nitric oxide. They can cause intoxication and are of general concern for health because they relate to various diseases. Our goal was to study ontogenetic and nutritional effects on the nitrate+nitrite (NOx-) status in cattle, particularly calves. NOx- concentration in blood plasma, cerebrospinal fluid, saliva, and urine was measured based on nitrate conversion by added nitrate reductase to nitrite, which was then determined by the Griess reaction. Concentrations of nitrate were the result of the difference between NOx- and nitrite values. Nitrate in blood plasma, saliva and urine was > or =97% and in cerebrospinal fluid of calves was approximately 35% of NOx-. Preprandial plasma NOx- in calves born after shortened or normal lengths of pregnancy (277 and 290 days) was 470 and 830 micromol/l, respectively, decreased within 4-7 days to 40-60 micromol/l, remained in this range up to 4 months, was < or =5 micromol/l in heifers and no longer measurable in 3-8-year-old cows. Cerebrospinal NOx- in 8-day-old calves was 14 micromol/l and approximately 11-fold lower than in blood plasma. Salivary NOx- decreased postnatally from 600 to 200 micromol/l at 2 days and to 25 micromol/l at 4 weeks. Urinary NOx- excretion decreased from 125 or 16 micromol/l per kg x 24 h in 5-day-old calves to 45 or 8 micromol/kg x 24 h between 10 and 115 days of life and was undetectable in urine of heifers and cows. Feeding neonatal calves no or variable amounts of colostrum, delaying colostrum intake by 24 h after birth or feeding at different feeding intensity had no effect on the NOx- status. In conclusion, the high plasma, salivary and urinary NOx- concentrations especially in newborn calves, ingesting but insignificant amounts of nitrite or nitrate, indicated marked endogenous formation of nitrate, which decreased with age. The high nitrate status may contribute to enhanced susceptibility of young calves to exogenous nitrite+nitrite ingestion.     

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.     

Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA_359−379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10⁻³ μg/ml and a limit of quantification of 24.5 × 10⁻³ μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.     

Simultaneous liquid chromatographic analysis for carbamazepine and carbamazepine 10,11-epoxide in plasma and saliva by use of double internal standardization

High-performance liquid chromatography (HPLC) was used for simultaneous quantitation of carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZ-EP) in plasma and saliva. Because concentrations of CBZ can greatly exceed those of CBZ-EP after single doses, two internal standards, lorazepam and N-desmethyldiazepam were added to all samples. Following extraction with chloroform, the components are separated on a μBondapak CN column with a mobile phase composed of 30% acetonitrile in water. Total chromatography time is 10 min. Concentrations of CBZ and CBZ-EP as low as 18 and 56 ng/ml, respectively, can be detected using 0.5 ml of plasma or saliva. The maximum within-day and day-to-day coefficients of variation for both compounds are 6.3 and 7.0%, respectively. Specificity of the method was supported by a significant correlation (r = 0.99) between assay results of the present method and those of a previously published HPLC assay. Application of the method to protein binding and salivary measurements in a single-dose CBZ disposition study is demonstrated.