John Roboz

Determination of α-keto acids as silylated oximes in urine and serum by combined gas chromatography-mass spectrometry☆

Abstract Nine α-keto acids in serum and urine were analyzed by gas chromatography as their oxime-trimethylsilylated derivatives, following oxime formation with hydroxylamine in pyridine and silylation with BSA containing 10% TMCS. The oxime-TMS derivatives were identified by both low- and high-resolution mass spectrometry. Recoveries from urine were greater than 90% and from serum greater than 80%. Loads of valine, leucine and isoleucine are shown to result in increased serum concentrations of corresponding α-keto acids in healthy adults. In a child with maple-syrup-urine disease under treatment by dietary restriction of the branchedchain amino acids, administration of leucine results in a large excretion of α-keto isocaproic acid and a less striking excretion of α-keto-β-methylvaleric and α-keto isovaleric acid. The excretion of the branched-chain α-keto acids is reported in two patients with maple-syrup-urine disease before and after institution of dietary treatment. Nine α-keto acids and at least 15 phenolic acids and related compounds can be determined in a single analysis using the method described. The ‘metabolic profile’ thus obtained may be used in screening for abnormalities in the metabolism of amino acids. The gas Chromatographic analysis alone is adequate for screening; when an abnormal quantity of a constituent is found, mass spectrometry is used for confirmation of identity.

Mass fragmentographic determination of pyrazinamide and its metabolites in serum and urine

A combined gas chromatographic-mass spectrometric technique is described for the simultaneous determination of pyrazinamide and its two main metabolites, pyrazinoic acid and 5-hydroxypyrazinoic acid. Serum (200 microliter) is deproteinized and evaporated to dryness; urine (20 microliter) is evaporated. The crude residues are silylated and selected ions are monitored in the chemical-ionization mode with isobutane as both chromatographic carrier and reagent gas. The sensitivity is 10 ng/ml for pyrazinamide and pyrazinoic acid and 20 ng/ml for the 5-hydroxy metabolite in a single analysis. Nicotinic acid and nicotinamide are internal standards. Both unchanged drug and metabolites were identified and quantified in the serum and urine of human subjects.

Determination of neuraminidase-suseeptible and total N-acetylneuraminic acid in cells by selected ion monitoring

Abstract N-acetylneuraminic acid (NANA) is released from glycoproteins by neuraminidase or acid hydrolysis and quantified by monitoring the protonated molecular ions of fully silylated NANA ( m e = 814 ) and neuraminic acid β-methylglycoside (internal standard, m e = 714 ) in a gas chromatograph—mass spectrometer system using isobutane ionization. Detection limit is 200 pg (0.6 pmol, underivatized weight) of NANA injected. In biological samples 5 ng (15 pmol) of NANA can be detected in 50 μl of hydrolysate. Only 1 to 50 μl of hydrolysate is needed, sample preparation is simple, NANA is positively identified in every analysis, 2-deoxy carbohydrates and other sialic acids do not interfere, only free NANA is determined, and the internal standard increases reliability. The NANA content of neuraminidase-treated human leukemic cells was on the order of 0.3μ mol 10 9 cells. NANA was quantified using as few as 5 × 104 cells, in contrast to the conventional colorimetric (thiobarbituric) technique which requires 2.5 × 107 cells.

Comparison of the interaction of antineoplastic aminoanthraquinone analogs with DNA using competitive fluorescence polarization

1,4-dihydroxy-5-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione one (NSC 287836) and 1,4-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione diacetate (NSC 287513) have shown activity against solid tumors and are now in Phase I clinical trials. Fluorescence polarization was used to determine the extent of inhibition of the binding of acridine orange to DNA (Richardson, Boboz, Holland, Res. Comm. Chem. Pathol. Pharmac. 27, 497, 1980). Displacement of 50% of acridine orange from calf thymus DNA was obtained with 0.18 micro M of NSC 287836 while 0.52 micro M of NSC 287513 was needed to displace an equivalent amount of acridine orange. NSC 287513 showed preference for polynucleotides of high adenine + thymine content while NSC 287836 did not. Analogs lacking both hydroxyethylaminoethyl-amino side chains did not displace acridine orange.

Mass Spectrometry in Clinical Chemistry

Publisher Summary This chapter discusses the nature of mass spectra and the functional elements of mass spectrometers. The currently employed analytical techniques—such as the combined gas chromatography-mass spectrometry (GC/MS) and ionization techniques—are also reviewed. The “medical-mass spectrometers” technology has advanced to routine clinical use. The most important areas of its applications are multibed monitoring in intensive care and trauma units, monitoring during surgery, and physiological testing and diagnosis. The spark-source-mass spectrometry is useful for the comprehensive survey of all the elements at ultratrace levels in a single analysis. Additive, subtractive, and synergistic interactions can be studied by this method. The GC/MS-computer technique is well suited for the multicomponent analysis of endogenous metabolites in body fluids. This technique—when chemical ionization and fragmentography are available—is ideally suited for monitoring the individual drug levels and for detecting, identifying, and quantifying the drugs and metabolites in pharmacological studies.

Characterization of digoxigenin derivatives of human coagulation factors VIIa and X by electrospray mass spectrometry and enzyme kinetics.

Digoxigenin ester (Dig) derivatives of coagulation factors VIIa and X facilitate staining studies to localize tissue factor activity in human atherosclerotic plaques. The larger the number of attached Dig units the easier it is to form highly visual stains. Electrospray ionization was used to characterize the Dig derivatives, as a function of excess derivatization reagent, to determine the optimal derivatives, i.e. the highest number of Dig units attached with the product still retaining enzymatic activity. The enzymatic activity of factor VIIa derivatized at 50-fold Dig excess (with 28, 34, 48 or 52 Dig units attached, masses to 79 kDa) remained the same as that of native factor VIIa. In contrast, the enzymatic activity of factor X derivatives diminished above 15-fold Dig excess (15 Dig units attached, mass 65.6 kDa). The distribution of derivatized lysine, histidine, arginine, tryptophan and tyrosine residues was estimated for several possible configurations.

Incorporation of 15N from Glycine into Uric Acid in Gout: A Follow-Up Study

Over-incorporation of 15N-labeled glycine into uric acid indicates over-production of uric acid by de novo purine biosynthesis. This metabolic aberration, though considered to be inborn (3), may be modified by changing of life style, aging and long-term therapy. In the patient under study, the protracted use of allopurinol seems to have played the most important role. Aging contributed to a certain extent, and changing life style was the least significant factor.

Mass Fragmentographic Quantification of Phosphonoacetic Acid in the Blood of Monkeys Infected with Herpesvirus Saimiri

Herpesvirus Saimiri° was isolated from a degenerating squirrel monkey kidney culture in 1968 (1). HVS induces leukemia and/or lymphoma when inoculated into a variety of nonhuman primates (2). The induced lymphoma appears to be analogous to those involved in EBV and Burkitt’s lymphoma in humans, thus it is a good model for evaluating antiviral agents used alone or in combination with chemoimmunotherapy. In 1973 it was reported that phosphoacetic acid (PAA) and its disodium salt has an inhibitory effect on the in vitro and/or in vivo replication of several DNA-containing viruses which are assumed to be oncogenic, including HVS (3, 4). PAA inhibits the synthesis of late viral products by interfering with the activity of virus-associated DNA polymerase (5).