Mass fragmentography. Identification of chlorpromazine and its metabolites in human blood by a new method

Chlorpromazine and some of its metabolites have been identified in human blood with the combination of gas chromatography and mass spectrometry. A new method, mass fragmentography has been elaborated. It is based upon a continuous recording of up to three mass numbers characteristic of a single substance or a group of compounds. With this technique both a high sensitivity and a high selectivity which can be changed according to wish are achieved. Compounds are identified by their retention times and the fact that all mass numbers are represented in characteristic relative intensities. Refocusing on other characteristic fragments and/or the molecular ion confirms the identity. Through repeated refocusing “a partial mass spectrum” of a compound can be established even when the amounts present are too small for the scanning of a complete spectrum.With this method chlorpromazine, and its desmethylated and didesmethylated metabolites have been identified in plasma. The latter two metabolites were obtained also after treatment of the plasma with β-glucuronidase, as was 2-chlorophenothiazinylpropionic acid, which was found in quantities that allowed the scanning of a complete mass spectrum. In red blood cells the two desmethylated metabolites could be identified with mass fragmentography only after treatment with β-glucuronidase. The use of the method is discussed, particularly with regard to blood levels of drugs as related to therapeutic effects and side effects.     

Steroid t-butyldimethylsilyl ethers as derivatives for mass fragmentography.

The rates of formation, chromatographic properties and mass spectral characteristics of various steroid t-butyldimethylsilyl ($\text{t}$-BDMS) ethers are examined and their application to mass fragmentography discussed.     

Endogenous steroid concentrations in human breast tumours determined by high-resolution mass fragmentography (Short Communication)

A pilot study of the endogenous steroid concentrations in human breast tumours was performed. The technique of high-resolution molecular-ion monitoring during combined g.l.c.-mass spectrometry was used to determine oestrone, oestradiol-17beta and oestriol in concentrations above 1ng/g wet wt. of tissue, and dehydroepiandrosterone, testosterone, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) and 3beta-hydroxy-5alpha-androstan-17-one in concentrations exceeding 5ng/g, in extracts of five primary breast tumours.     

An immunocytochemical method for assaying oestrogen receptors in breast cancers. A comparison with the steroid binding assay

The presence of oestrogen receptors was studied in 105 human breast carcinomas using monoclonal antibodies (Abbott ER-ICA kit). The oestrogen receptors of neoplastic cells were semiquantitatively measured and correlations were made to receptor values determined by a dextran-coated charcoal (DCC) steroid binding assay and to histological grade. Immunoreactive cells were found in about 2/3 of the tumours. Usually only a fraction of the cells within each tumour were immunoreactive, and the staining intensity varied among different cells. In general, well differentiated tumours had a greater proportion of immunoreactive cells than poorly differentiated ones. In most cases (65/98) a good agreement was found between the ER-ICA method and the DCC assay. However, in 33 cases discrepancies were demonstrated.     

Plate diffusion assay as a rapid method for dosimetry of mutagens.

This paper presents a method for determining mutagenic concentrations of chemicals by using an agar diffusion assay. The method is based on the linear relationship between the amount of chemical placed at the center of the dish and the radius of the mutagenic zone. A brief theoretical discussion and experimental data confirming this relationship are given. Alkylating agents and mycotoxins were used to test the system. This method can be used to follow up decreased mutagenic potencies of solutions of unstable mutagens and to follow the production of mutagenic substances throughout fermentation.     

Plate diffusion assay as a rapid method for dosimetry of mutagens.

In the feces of conventional rats, the amount of omega-muricholic and hyodeoxycholic acids vary according to the diet. To understand this phenomenon, we investigated the bacterial formation of these bile acids. The present paper reports the first isolation, from conventional rat feces, of a strain of Clostridium group III which transforms beta-muricholic acid, the main bile acid in germfree rats, into omega-muricholic acid.     

Gas—liquid chromatography reference method for the assay of urinary creatinine

The gas—liquid chromatographic measurement of urinary creatinine described in this paper employs methylation, the use of a diethylene glycol succinate stationary phase, an internal L-hydroxyproline standard and a temperature of 180°C. The technique, which is specific and reproducible, is shown to be a reference method providing more precise and reliable results than a conventional colorimetric method. In addition, it can be used as a routine method because of its simplicity.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.     

Spin assay as a general method for studying plasma protein binding. Bilirubin-albumin binding.

A mutant of the Chinese Hamster Ovary cell line, CHO-K1, has been isolated which is resistant to killing by 25-hydroxy cholesterol in the absence of cholesterol. There is no effect on acetate incorporation into cholesterol by 25-hydroxy cholesterol in the mutant under conditions in which incorporation is inhibited in the parent cell. The mutant also appears to be defective in the regulation of cholesterol synthesis by serum choleserol.     

Testosterone as biotransformation product in steroid conversion byMycobacterium sp.

Summary Testosterone production byMyc. sp. NRRL B-3683 is discussed. The unexpected finding that testosterone is not formed by single reduction of 17-keto group of 4-androstene-3,17-dione (AD) but by a double reduction of both 17-keto group and 1–2 doble bound of 1,4-androstadiene-3,17-dione (ADD) is presented.