Robert Schaffer

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Abstract We describe a manual fluorometric method for the quantitation in protein solutions of total free amino compounds, expressed as norleucine. A trichloroacetic acid deproteinization step is employed, and o-phthalaldehyde, buffered with phosphate at pH 9,2, is used as the fluorogenic reagent. The method is linear, reproducible, and rapid. Recoveries of amino acids added to serum are quantitative. Sensitivity is in the picomole range. Results on unselected patient sera are diseussed.

Reverse-Phase Liquid Chromatographic Analysis of the Acid-Catalyzed Rearrangement of Nadh, Separation of New Products and an Analysis of the First Two Reactions of the Rearrangement

Abstract The previously unresolved products of acid-catalyzed rearrangement of NADH have been separated into seven peaks by liquid chromatography on microparticle ODS. The peak with the second highest retention volume was identified as O2,6-B-cyclotetrahydronicotinamide adenine dinucleotide. Based on the order of appearance and disappearance of the peaks, and on their production in the presence of glyceraldehyde-3-phosphate dehy-drogenase (EC 1.2.1.12), four of the peaks have been assigned to specific products in the reaction sequence originally proposed by Oppenheimer and Kaplan [Biochemistry, 13, 4675, 1974]. The three remaining peaks (in addition to AMP) are previously unidentified products of the acid-catalyzed rearrangement of 6-B-cyclotetrahydronicotinamide adenine dinucleotide. The first step in the rearrangement of NADH to 6-B-cyclotetrahydronicotinamide adenine dinucleotide was shown to be dependent on the pH and ionic strength of the buffer, but neither the first product nor any other product in...

Reverse Phase Liquid Chromatographic Analysis of the Impurities in Nicotinamide Adenine Dinucleotides

Abstract Commercial preparations of several different nicotine adenine dinucleotides were examined by liquid chromatography on an octadecylsilane column (Margolis, S., et al., Clin. Chem., 22, 1322, 1976). Seven impurity peaks were detected in NADP+, eight in NADPH, and five in NAD+. The estimated purity of NADP+ from different commercial suppliers varied from 89 to 95 percent. For NADPH the purity ranged from 77.5 to 96 percent and for NAD+ from 90 to 93.5 percent. Preparations of NAD+ contained AMP, ADPR, nicotinamide, and two unidentified impurities. The impurities found in NADP+ and NADPH preparations did not correspond to compounds that we could identify. Four of the impurity peaks found in NADPH form under acidic storage conditions. Five of the impurity peaks observed in NADP+ and three of the impurity peaks in NAD+ form as products of alkali-catalyzed rearrangements.

Standard Reference Materials: SRM 900, antiepilepsy drug level assay standard

Recognition of the efficacy of monitoring the concentrations of therapeutic drugs in the blood of patients has revealed many needs for standardization of the laboratory tests used for such monitoring. The National Bureau of Standards was asked to provide a Standard Reference Material (SRM) consisting of three serum samples, each to contain four antiepilepsy drugs at different concentrations. The four drugs are phenobarbital , phenytoin, primidone, and ethosuximide. The SRM would fill a basic role for the achievement of accurate analysis to help ensure the reliability of analyses for these drugs. The needs that had to be fulfilled to produce the SRM included: (1) analytical criteria for purity of the drugs; (2) serum to be used as a matrix for the drugs; (3) techniques for achieving homogeneity and stability of the SRM; and (4) two independent, highly accurate analytical methods for the certification. This document describes development of methods and procedures used to produce and certify the SRM.