M.J.Medina Hernández

Formation and instability of o-phthalaldehyde derivatives of amino acids

o-Phthalaldehyde reacts with amino acids in the presence of a thiol to give highly fluorescent 1-alkyl-thio-2-alkyl-substituted isoindoles. However, the instability of the derivatives limits the general utility of the reaction. Mechanistic descriptions of isoindole formation and degradation proposed in the last years, which permit a better understanding of the factors affecting isoindole stability, are presented. The use of alternative thiols and o-phthalaldehyde-like reagents is also reviewed.

Studies on the formation and stability of isoindoles derived from amino acids, o-phthalaldehyde and N-acetyl-L-cysteine

Abstract A kinetic-spectrophotometric study is performed on the formation and degradation of the isoindole derivatives of amino acids with o -phthalaldehyde and N -ace-tyl- l -cysteine. The experimental and structural factors which affect the formation and stability of the compounds are considered, and the results are compared with those obtained for mercaptoethanol. N -Acetyl- l -cysteine derivatives are highly stable, not requiring a strict control of the time of reaction as in the case of mercaptoethanol.

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.

Spectrophotometric determination of N-acetylcysteine in drug formulations with o-phthalaldehyde and isoleucine

A spectrophotometric procedure is proposed for the assay of N-acetylcysteine (NAC) in the range 0.5–49 µg ml–1, based on the reaction of the thiol group with o-phthalaldehyde and isoleucine at pH 9.5 (λmax.= 335 nm). The procedure was applied successfully to the determination of NAC in several pharmaceutical formulations. The recoveries ranged from 98 to 106%, with relative standard deviations of less than 1.5%. The method is free from interferences and is simple, rapid and easy to automate.

Determination of total free amino acids with o-phthalaldehyde and N-acetyl-l-cysteine

Abstract A spectrophotometric procedure is proposed for the determination of total free amino acids after reaction with o-phthalaldehyde and N-acetyl- l -cysteine, using isoleucine as the reference. The procedure was applied to the analysis of five samples of widely different composition. Recoveries were 98–105%.

Evaluation of the proteolysis degree with the o-phthalaldehyde/N-acetyl-L-cysteine reagent

SummaryThe o-phthalaldehyde/N-acetyl-L-cysteine (OPA-NAC) reagent is applied to the spectrophotometric evaluation of the proteolytic activity of enzymes. The high stability of the OPA-NAC isoindoles makes a strict control of the time of reaction unnecessary. A mathematical expression is proposed to calculate proteolysis degrees, where the absorbance decrease of the OPA-NAC derivative of the protein itself during the hydrolysis process is taken into account. The method is applied to bovine serum albumine, caseine, lysozyme, lactoglobuline and protamine sulphate as substrates, and pronase, papaine, trypsin and chymotrypsin as enzymes.

Some observations on the automation by flow injection analysis of the spectrophotometric determination of amino acids and proteins witho-phthalaldehyde

Automation by flow injection analysis with Spectrophotometric detection of the determination of total amino acids and proteins witho-phthalaldehyde is not straightforward. The use of spectrophotometry, instead of spectrofluorimetry, and of N-acetyl-L-cysteine, instead of the conventional mercaptoethanol is advantageous because of the lower variability of absorptivities with respect to fluorescence yields, and the larger stability of the derivatives. Under adequate working conditions and with leucine as reference, the procedure can be used for the evaluation of total amino acids. A similar procedure is proposed for the analysis of proteins in a sample. Limits of detection are ≈ 1 × 10−5M for amino acids, and ≈ 1 × 10−6M for proteins, respectively.

Spectrophotometric measurement of pH gradients in continuous-flow systems

Abstract In the presence of an acid-base indicator and using fast diode-array spectrophotometric scans, pH gradients in the spectrophotometric cell can be measured at the same time as any other colorimetric flow-injection (FI) determination is performed. The effects of refractive index changes, adsorption-desorption processes at the tube walls and association of the indicator species with the system to be investigated are considered. Effective correction for the first two effects is demonstrated. Indicators with different molecular structures and polarities gave similar pH gradients, suggesting adequate accuracy for most potential applications. Application to the detection and correction of systematic and random errors in pH-sensitive FI procedures is demonstrated using the o -phthalaldehyde- N -acetyl- l -cysteine method for bovine serum albumin.

Spectrophotometric determination of cystine by formation of an o-phthalaldehyde/N-acetyl-l-cysteine derivative

Abstract Cystine reacts with o -phthalaldehyde (OPA) in the absence and presence of a thiol compound to yield different compounds. The use of N -acetyl- l -cysteine as thiol leads to the formation of two derivatives, likely simple and double isoindoles, where the disulfide bond remains unbroken. In contrast, mercaptoethanol gives rise to the reduction of the amino acid to form a cysteine derivative. Obtaining cystine isoindoles makes it possible to spectrophotometrically determine the amino acid after Chromatographic separation and is further evidence of the large stabilization effect produced by N -acetyl- l -cysteine in the formation of OPA-thiol derivatives.

Catalytic—enthalpimetric determination of diethyldithiocarbamate (DDTC) and nickel(II) using the iodine—azide reaction

Abstract An enthalpimetric method for the determination of diethyldithiocarbamate (DDTC) based on the catalysis of the iodine—azide reaction is proposed. The behaviour of those metal ions which inhibit the catalytic activity of DDTC by complex formation is also studied, and a simple and rapid procedure for the determination of trace amounts of nickel(II) is described. The relative standard deviations are within 2 and 3% for concentration levels of 2×10 −5 M DDTC and 2×10 −6 M nickel(II), respectively.