Effect of ethanol on the ratio between testosterone and epitestosterone in urine.

The testosterone/epitestosterone weight ratio in urine is used to detect cases of doping when an athlete has treated himself with exogenous testosterone. When this ratio exceeds 6, it is considered evidence of testosterone doping. We show here that intake of ethanol can affect this ratio. Ingestion of 110-160 g of ethanol, about 2 g per kilogram body weight, increased the ratio between testosterone and epitestosterone in urine from 1.14 +/- 0.07 to 1.52 +/- 0.09 in four healthy male volunteers. The increase ranged from 30% to 90% in the different subjects studied (mean 41%). In cases where doping with testosterone is suspected, the possibility should be considered that at least part of an observed increased testosterone/epitestosterone ratio in urine is ascribable to previous ingestion of ethanol.

Isotope dilution--mass spectrometry of thyroxin proposed as a reference method.

We describe a highly accurate and specific method based on isotope dilution--mass spectrometry for assay of thyroxin in serum. A fixed amount of 2H2-labeled thyroxin is added to a fixed amount of serum. Thyroxin is isolated from the serum by an extraction--solvent-distribution method, converted into the N,O-bis(trifluoroacetyl)methyl ester, and subjected to combined gas chromatography--mass spectrometry. The amount of thyroxin is determined from recordings at m/e 799 and m/e 801. These two ions correspond to the M-184 peak in the mass spectrum of the derivative of unlabeled and 2H2-labeled thyroxin, respectively. With this derivative and with the specific gas-chromatographic conditions used, the "memory" effects observed in previous work were absent. The accuracy of the method was checked by analyses of serum samples without addition of internal standard and by different recovery experiments. The intra-assay variation (CV) was less than 2%, the interassay variation (CV) less than 3.5%. We suggest that this method may be used as a Reference Method. Results by the present method were compared with those by a commercial radioimmunoassay. To our knowledge this represents the first time that radioimmunoassay of thyroxin has been evaluated with a more nearly accurate method. There was excellent agreement between results by the two methods (r = 0.997). We conclude that radioimmunoassay of serum thyroxin is sufficiently accurate for use in clinical work.

Validation of radioimmunoassay for estradiol-17 beta by isotope dilution-mass spectrometry and by a test of radiochemical purity.

Isotope dilution-mass spectrometry (ID-MS) was used as a reference method to determine the concentration of estradiol-17 beta (E2) in five different plasma pools (concentrations ranging from 0.040 to 65 nmol/l). The same plasma pools were also subjected to radioimmunoassay (RIA) using five different antisera of largely varying specificity. With the best antiserum (E), a direct RIA apparently gave accurate results (i.e. results statistically indistinguishable from those obtained by ID-MS) at all levels except the lowest one (0.040 nmol/l). It was shown, however, that the apparent accuracy of this RIA to some extent could be due to a lowering effect of lipids in the serum masking a lack of specificity of the antibodies. With the least specific antiserum (A), accurate results were obtained only after chromatography. However, in the assay of the lowest concentration of E2 with this antiserum there was a significant overestimation, even after chromatography. The other three antisera (B, C, D) of average quality gave accurate results in assays of plasma diethyl ether extracts in various numbers of the plasma pools tested, depending on their intrinsic specificity. This specificity was not correlated with the cross-reaction reported for individual antisera. ID-MS is difficult to use in most laboratories. We demonstrate here that the validity of a RIA may in this case be assessed by a relatively simple method, the test of radiochemical purity (RP-test). This test is based on the measurements of specific activity (e.g. dpm/pg) in small consecutive fractions of the chromatographic zone which is usually employed for the RIA.

Assay of the major bile acids in serum by isotope dilution-mass spectrometry.

A specific method for determination of cholic, chenodeoxycholic and deoxycholic acid in serum based on deuterium-labelled standards and selected ion monitoring is described. The coefficient of variation of the method as calculated from replicate determinations was in the range 1.4-3.3% for all three bile acids. Recovery experiments were in accord with the high accuracy expected for this type of assay. In analyses of sera from 23 healthy subjects, the following concentrations were obtained in the fasting state: cholic acid, 0.29 +/- 0.81 mumol/l; chenodeoxycholic acid, 0.18 +/- 0.32 mumol/l; deoxycholic acid, 0.77 +/- 0.38 mumol/l (mean +/- SD). The method may be suitable as a reference method and in experiments with a high need for accuracy.

Identification of thyroxine in human breast milk by gas chromatography-mass spectrometry.

In several previous publications it has been reported that human breast milk contains significant amounts of T4. Great differences in concentrations were found by different authors. In all these publications T4 was assayed by RIA, and it seems probable that the varying results obtained are due to varying specificity of the antibodies used in the assay. Definite demonstration of the presence of T4 in breast milk requires a more specific method than RIA. In the present work we have used combined gas chromatography-mass spectrometry. T4 was isolated from human breast milk by combined anion- and cation-exchange chromatography. The purified material was treated with HCl in methanol and trifluoroacetic acid anhydride and was analyzed by single-ion monitoring of the ions at m/e 983, m/e 870, and m/e 799, major ions in the mass spectrum of the N,O-bis(trifluoroacetyl) methyl ester of T4. Significant peaks confirmed the presence of T4 in human milk. The estimated concentration was less than 10 ng/ml. Attempts were made to quantitate the T4 by selected ion monitoring of the ions at m/e 870 and m/e 822 after addition of monobromotriiodothyronine as internal standard. According to this latter method, the concentration of T4 in four samples of centrifuged breast milk was less than 4 ng/ml. The possible physiological significance of the finding is discussed.

Simultaneous determination of prednisolone and cortisol in serum by HPLC and by isotope dilution--mass spectrometry.

A reference method for the simultaneous assay of cortisol and prednisolone by isotope dilution-mass spectrometry has been developed. A fixed amount of 2H4-labelled cortisol is added to a fixed amount of serum. The mixture is then extracted, converted into methoxime-trimethylsilyl derivative, and subjected to analysis by combined gas chromatography-mass spectrometry. The ions at m/e 603, m/e 605 and m/e 609 are followed through the gas chromatography. These ions correspond to the M-31 ions in the mass spectrum of methoxime-trimethylsilyl ether derivative of prednisolone, unlabelled cortisol and 2H4-cortisol, respectively. With the use of a standard curve, the concentration of prednisolone can be calculated from the ratio between the peak at m/e 603 and the peak at m/e 609 and the concentration of cortisol can be calculated from the ratio between the peak at m/e 605 and the peak at m/e 609. The coefficient of variation was about 2% with respect to determination of prednisolone and about 3% with respect to determination of cortisol. This method was compared with a routine method based on high-performance liquid chromatography (HPLC) The coefficient of variation was 2-3% with respect to determination of cortisol and 1-3% with respect to determination of prednisolone. There was a good correlation between the isotope dilution method (x) and the HPLC method (y). In the assay of serum prednisolone the regression analysis gave the following equation: y = 1.02x -69 nmol/l, r = 0.99. In the assay of serum cortisol, the regression equation was: y = 1.07x-14 nmol/l, r = 0.95. It is concluded that the HPLC-method, used for routine determination of cortisol and prednisolone, has an accuracy sufficient for its intended use.

Phenytoin excretion in human breast milk and plasma levels in nursed infants.

Phenytoin excretion into human breast milk was studied in six nursing women with epilepsy. The average ratio between the areas under the plasma (and milk) concentration versus time curves (AUC) was 0.13. There was a good (r = 0.97) correlation between the mean plasma and milk concentrations of phenytoin, and an even better relation (r = 0.99) between the AUC for phenytoin in plasma and the mean milk concentration. The ratio between unconjugated and conjugated 4-OH-phenytoin (the main metabolite) in plasma was 0.08-0.09. The corresponding ratio in milk was considerably higher. The present data do not argue against breast feeding during phenytoin therapy, not even when weighed against the potential risks for toxicity of the parent compound. Only two of six infants had a measurable, yet very low plasma concentration of phenytoin. The calculated body weight--related doses of phenytoin secreted into milk will be less than 5% of the dose to infants and small children.

Accuracy of some routine method used in clinical chemistry as judged by isotope dilution-mass spectrometry.

Serum from patients was pooled, filtered, dispensed, and frozen. This pooled specimen was used for accuracy control in 64 participating laboratories in Sweden. Mean values ("state-of-the-art" values) were obtained for creatinine, cholesterol, glucose, urea, uric acid, and cortisol. These values were compared with values obtained with highly accurate reference methods based on isotope dilution-mass spectrometry. Differences were marked in the case of determination of creatinine and cortisol. Concerning the other components, the differences between the state-of-the-art value and the values obtained with the reference methods were negligible. Moreover, the glucose oxidase and the oxime methods for determination of glucose and urea were found to give significantly lower values than the hexokinase and urease methods, respectively. We conclude that methods with a higher degree of accuracy are required for routine determination of creatinine and cortisol.

Phenytoin and IgA concentrations in plasma and saliva in epileptic children.

The concentrations of immunoglobulin A (IgA) and phenytoin were determined in 36 epileptic children with a mean age of 11 years. There was a good correlation between the plasma and saliva concentrations of phenytoin (r = 0.94). The concentration of phenytoin and IgA showed little variation during the dosage interval. The phenytoin treated children did not differ with respect to the concentrations of IgA in saliva in comparison to the controls.

Use of isotope dilution--mass spectrometry for accuracy control of different routine methods used in clinical chemistry.

Serum from patients was pooled, filtered, dispensed and frozen. The serum pool obtained was used for accuracy control in twenty-one participating laboratories in the Stockholm area. Mean values (state of art values) were obtained for creatinine, cholesterol, glucose, urea and uric acid. These values were compared with values obtained with highly accurate reference methods, based on isotope dilution--mass fragmentography. The most marked difference was found in the case of determination of creatinine. It is suggested that immediate access to such a well-defined human serum might decrease the need for conventional interlaboratory control programmes.

The use of mass fragmentography in the evaluation of routine methods for glucose determination.

A highly specific and accurate mass fragmentographic reference method for determination of serum glucose is described. A fixed amount of hepta-deuterated glucose is added to a fixed amount of serum. The mixture is lyophilized, converted into the penta-trimethylsilyl-methyloxime derivative and subjected to analysis with a combined gas chromatograph - mass spectrometer equipped with a MID-unit (multiple ion detector). The amount of unlabeled glucose was determined from the ratio between recordings at m/e 319 and 323. The two ions used correspond to the base peak in the mass spectrum of derivative of unlabeled and hepta-deuterium labeled glucose, respectively. The relative standard deviation of the method was 1.9%. The method was compared with different enzymatic methods based on the hexokinase reaction and the glucose oxidase reaction. Of the different methods tested, a glucose oxidase method with determination of maximal rate of consumption of oxygen gave results most close to the results of the reference method.