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A method has been developed for quantification of bromhexine in plasma using gas chromatography mass spectrometry with selected ion monitoring. A deuterium labelled analogue was synthesized and used as the internal standard. To evaluate the gas chromatographic electron capture detection method described earlier, 23 plasma samples have been analysed by both techniques. Although a good correlation was shown, selected ion monitoring was superior to the electron capture detection method for levels below 3 ng ml-1. The mass spectrometric method has also been used to set up a pharmacokinetic study of bromhexine in horses. Urine extracts were subjected to gas chromatographic mass spectrometric analysis to identify the different metabolites of bromhexine. Three groups of metabolites were detected and identified.

It is well known that nikethamide (N,N-diethylnicotinamide, CoramineR) is metabolized very rapidly to nicotinamide. Hence, there is difficulty in proving that nikethamide has been used as a doping substance because nicotinamide is a normal physiological metabolite in the organism as well as a vitamin preparation. However, an intermediate metabolite (N-ethylnicotinamide) was found by us in the urine of horses treated with CoramineR. This was characterized by gas chromatography/mass spectrometry, and synthesized and identified as being N-ethylnicotinamide. The excretion and metabolism of nikethamide after intramuscular injection in the horse was followed using quantitative gas chromatography of urine extracts over a period of several hours and the results of these experiments are reported. Changes in urinary pH had no significant effect upon either the metabolism or rate of excretion of the drug.

A high pressure liquid chromatographic (HPLC) method for measuring the theobromine content in cocoa husks, pelleted food and horse urine is described. Starting with 2 ml of urine, concentrations of 500 ng/ml could easily be detected. When feed containing 38.4 mg of theobromine was given twice daily to horses for 21/2 days, two days were needed after the last intake before the theobromine concentrations fell below the threshold value of 2 µg/ml. The time at which the peak excretion rate occurred varied from 2 to 12 h after the last administration, while the excretion rate seemed to be dependent on the urinary flow. Theobromine could not be detected in plasma after administration in this way.

The possibilities, applications and limitations of the electron capture detector (ECD) in human and equine doping analysis are considered. As many stimulants and/or their metabolites possess and amino group the introduction of halogen containing groupings by acylation results in improved chromatographic performance almost combined with enhanced sensitivity and selectivity when using the ECD. Several examples of the confirmation of doping agents like amphetamines, ephedrine, phenylbutazone, pemoline and procaine by GC/ECD are given.

Massage of the seminal vesicles and ampullae was carried out 68 times during 35 diuresis experiments on 6 sexually mature rams. The animals had received a hydrating dose of water. In 52 cases (76%) definite inhibition of water diuresis was observed accompanied by increased concentrations of chloride, sodium and potassium in the urine. Ejaculation was observed after 42 stimuli (61%).The influence of coitus on water diuresis was studied on 5 rams in 24 experiments. In 17 cases out of 48 (35%) coitus caused definite inhibition of water diuresis together with increased concentrations of chloride, sodium and potassium in the urine. These antidiuretic responses were weaker than those observed after massage of the seminal vesicles and ampullae. Evidence of ejaculation was obtained after coitus on 37 occasions (77%).The antidiuretic action of physiological doses of oxytocin was very low. The inhibition of urine flow induced by massage of the seminal vesicles and ampullae or coitus could be imitated by intravenous injection of physiological doses of ADH. The mean antidiuretic effects in response to massage were matched by injection of 3–4 mu. Pitressin. The mean antidiuretic effects induced by coitus were equivalent to 2 mu. Pitressin.It is concluded that there is a release of ADH in response to massage of the seminal vesicles and ampullae. There is some evidence that a similar phenomenon occurs during coitus.

Cross-circulation experiments were carried out using either two lactating ewes (six experiments) or one sexually mature ram and one lactating ewe (seven experiments). Under local anaesthesia an anastomosis was established by means of two plastic tubes between a jugular vein of one animal (A) and a jugular vein of the second animal (B) in each pair. The other jugular veins were clamped.Vaginal distension performed in ewe A by means of an inflated balloon often caused a sharp rise of pressure in the udder cisterns of ewe B after a minimum latency of 30 sec. Many pressure curves could be obtained successively in the same preparation by repeating the vaginal distension stimulus.Massage of the seminal vesicles and ampullae of the ram often caused a sharp pressure rise in the udder of the ewe after a minimum lag period of 30 sec. This effect could be obtained several times with the same preparation, but considerable individual variation was observed between the different rams used. Massage of the seminal vesicles and ampullae induced emission in many cases but not always. Intravenous injection of a synthetic oxytocin, Syntocinon (500–1000 mu.), in the ram or distension of the vagina in the ewe failed to elicit emission.Pressure responses were induced by injection of suitable doses of Syntocinon into the plastic tube leading to the ewe in which the cistern pressure was recorded, after a latency of approx. 30 sec. It may be concluded that vaginal distension in the ewe causes the discharge of a hormone from the head. A similar phenomenon occurs in the ram after massage of the seminal vesicles and ampullae. These effects are not abolished by atropine. It is postulated that this hormone is oxytocin in both sexes. The amount of hormone released after one stimulus was usually equivalent to 40–100 mu. oxytocin in the ram and 20–50 mu. oxytocin in the ewe.