[en] Methods have been developed for quantitative analysis of commonly abused drugs in physiological fluids using gas chromatography/chemical ionization mass spectrometry. The methods are being evaluated in volunteer analytical and toxicological laboratories, and analytical manuals describing the methods are being prepared. The specific drug and metabolites included in this program are: Δ⁹-tetrahydrocannabinol, methadone, phencyclidine, methaqualone, morphine, amphetamine, methamphetamine, mescaline, 2,5-dimethoxy-4-methyl amphetamine, cocaine, benzoylecgonine, diazepam, and N-desmethyldiazepam. The current analytical methods utilize relatively conventional instrumentation and procedures, and are capable of measuring drug concentrations as low as 1 ng/ml. Various newer techniques such as sample clean-up by high performance liquid chromatography, separation by glass capillary chromatography, and ionization by negative ion chemical ionization are being investigated with respect to their potential for achieving higher sensitivity and specificity, as well as their ability to facilitate simultaneous analysis of more than one drug and metabolite. (Auth.)

[en] A mass fragmentographic assay of 25-hydroxy vitamin D₃ has been developed. [26-²H₃]-labelled vitamin D₃ is used as internal standard. A fixed amount of the standard is added to a fixed amount of serum or incubation mixture. 25-Hydroxy vitamin D₃ is extracted and the 3-t -butyldimethylsilyl derivation of 25-hydroxy vitamin D₃ is prepared. The latter is purified by means of thin layer chromatography. A trimethylsilyl group is introduced in position 25 prior to analysis by gas chromatography/mass spectrometry. The molecular ion at m/e 586 for unlabelled and m/e 589 for deuterium labelled 3-t-butyldimethylsilyl/25-trimethylsilyl derivative of 25-hydroxy vitamin D₃ are used in the analysis. The assay is designed to determine a few nanograms of 25-hydroxy vitamin D₃. It has been used for the determination of 25-hydroxy vitamin D₃ in blood serum. The mean value for 25-hydroxy vitamin D₃ obtained from 12 healthy men and women was 21 ng/ml. The relative standard deviation of the method was about 3 %. The assay has also been used to determine the rate of 25-hydroxylation of vitamin D₃ in mitrochondrial fractions of rat liver. (Auth.)

[en] For quantitative determination of known metabolites from the biological sample by direct chemical ionization mass spectrometry (CI-MS), the method of internal standard using stable isotopically labelled analogs appears to be the method of choice. In the case where stable isotope ratio determinations could not be applied, and alternative quantification can be achieved using non-labelled external or internal standards and a calibration curve (sum of peak height per a given number of scans versus concentration). The technique of computer monitoring permits display and plotting of ion current profiles (TIC and SIC) or spectra per a given number of scans or a given range of mass per charge. Examples are given in areas of clinical application and the quantitative data show very good agreement with the conventional chromatographic measurements. (Auth.)

[en] Two areas of mass spectral study related to biogenic amine metabolism are presented: The use of electron capture negative ion chemical ionization mass spectrometry for the quantitation of melatonin and other indole amines, and general synthetic procedures useful for the synthesis of deuterated diazomethane and deuteromethylated catechols. The factors determining instrumental sensitivity in negative ion chemical ionization are discussed, and the enhancement of the primary ion beam using magnetic fields is described. Quantitation of human plasma melatonin at the parts per trillion or pg/ml level has been demonstrated and is routinely performed as a selected ion monitoring assay. (Auth.)

[en] For quantitative determination of stable iosoptically labelled metabolic substrates in patients who had been fed deuterium labelled amino acids (50 mg per kg body wt.), the appearance and level of deuterated substrates and metabolites in the body fluids could be rapidly followed using direct mass spectrometry (DMS) without chromatographic separation in a time course tests. The per cent of deuteration could be calculated from (peak height of labelled) X 100 / peak height of (labelled + unlabelled) without the use of an internal standard. To determine the amino acid concentration of both the labelled and the unlablled species present in the sample, the addition of the same amino acid but differently labelled is required. If a differently labelled amino acid is not available for use as an internal standard, it is still possible to determine the concentration of the labelled amino acid. The technique is rapid and specific and the method is useful for the diagnosis and study of metastable defects. (Auth.)

[en] Radio-gas chromatography (RGC) and gas chromatography-mass spectrometry (GC-MS) were used to identify estrogen and progesterone metabolites. The RGC enables the identification of metabolites of labelled precursors (³H)-estradiol-17β and (¹⁴C)-progesterone were used as precursors. The GC-MS analytical technique with mass fragmentography, offers the interest of using unlabelled precursors at physiological levels. The identification of metabolites was based on obtaining the mass spectrum or the compiled fragmentogram on the basis of the most characteristic fragment ions. More over, several metabolites can be quantified on the same fragmentogram. Results on the metabolism of estradiol-17β and progesterone by the hepatic tissue of guinea pigs are given. (Auth.)

[en] Gas chromatography - mass spectrometry (GC-MS) was employed along with selected ion monitoring (SIM) to measure an isotope effect in the metabolic conversion of tremorine to oxotremorine using rat liver microsomes in vitro. The isotope effect Ksub(H)/Ksub(D) was measured by estimating the rate of formation of d₂- and d₄-oxotremorine from isotope subsituted tremorine (1,4-dipyrrolidino-2-butyne), specifcally substituted at positions 2 and 5 in one of the pyrrolidine rings. The ratios of the two labelled variants of the metabolite were calculated from standard curves representing known amounts of the labelled metabolites which were synthesized to a specific substitution of 98.4 % ²H. With phenobarbital induced microsomes, the enzymic reaction, which appears to be cytochrome P-450 mediated, showed an isotope effect of 2.2+-0.05 at saturating substrate concentrations. Similar apparent Ksub(M) values (approximately 40 μm) were obtained for the formation of the two labelled oxotremorine variants. (Auth.)

[en] In an attempt to develop quantitative mass spectrometry toward a definitive method for organic constituents, each step of the GC-MS procedures have been subjected to critical evaluation of their effects on the ratio measurement between corresponding fragments, using isotope dilution technique and glucose and glycerol labelled with ²H and ¹³C. The results show that in particular ion source settings strongly influence the fragment ratio measurements. The data suggest that in high precision quantitative analyses the use of relative measurements (standard curves) should be used. By optimizing all parameters a coefficient of variation between parallels close to 0.3% and with no known inaccuracy was obtained as shown for glucose. (Auth.)

[en] The Pickup and Mc Pherson isotope dilution theory has been applied to the twin-ion mass fragmentometric quantification of 5α-androstanediol di-trimethylsilyl ethers. The influence of the interference between the natural and the labeled compounds at the selected masses is discussed for four examples using different ²H synthesized molecules. It appears that the carrier-internal standard and the sample interferences are critical. In a reference method where high accuracy is required, the functions of internal standard and carrier must be allocated to two different molecules: a labeled analog (carrier) and a non labeled homolog (internal standard), so that the quantification of a well-defined sample may be carried out by single ion detection. (Auth,.)

[en] The analytical procedure for the measurement of steroid hormones consists of the following steps: (a) addition of a ¹⁴C-labelled steroid to the serum sample; (b) extraction of the ¹⁴C-labelled and of the non-labelled steroid by the use of an organic solvent; (c) purification of the steroid fraction by column chromatography on Sephadex LH-20 on Lipidex-5000; (d) formation of a derivative; (e) combined gas chromatography - mass spectrometry. During gas chromatography the mass spectrometer continuously records two m/e - values corresponding to the labelled and the non-labelled steroid; quantitative determination is performed by comparing the peak heights of the steroid to be determined and of the labelled internal standard. In the present investigation the technique of ID-MS has been applied to the specific and accurate determination of oestriol, oestradoil-17β, testosterone, progesterone, aldosterone and cortisol in human serum. The accuracy of the method is based on the high specificity of mass spectrometry and on the exact control of recovery employing the principle of isotope dilution. Therefore, the analytical procedures described here may be recommended as definitive methods in clinical chemistry. (Auth.)