M.C.García Alvarez-Coque

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.

Direct injection of physiological fluids in micellar liquid chromatography

Abstract Micellar liquid chromatography (MLC), which uses mobile phases of surfactants above the critical micellar concentration, provides a solution to the direct injection of physiological samples by solubilizing the protein components, and coating the analytical column with surfactant monomers to avoid clogging. A review showing the advantages and limitations of this technique over other chromatographic techniques used in drug analysis, working protocols, and examples of application is presented. The possibility of direct sample introduction simplifies and greatly expedites the treatments with reduced cost, improving the accuracy of the procedures. Surfactant monomers and micelles appear to displace drugs bound to proteins, releasing them for partitioning to the stationary phase. The versatility of MLC encompasses the wide range of drug classes normally monitored, such as analgesics, anticancer drugs, antidepressants, bacteriostats, β-blockers, bronchodilators, catecholamines, diuretics and steroids, among others. Analytical procedures have been developed in urine, plasma, serum and cow milk samples. Most of them utilize sodium dodecyl sulphate as surfactant and a C 18 column. UV detection is usual, but enhanced detection has been reported by measuring the absorbance in the visible region of drug derivatives formed precolumn, and with a variety of other techniques, such as fluorimetry, amperometry, inductively coupled plasma–mass spectrometry and immunoassay. Column-switching with on-line surfactant-mediated sample clean-up is shown as an attractive enrichment technique, which expands the practical use of MLC beyond the singular dimensional chromatographic process.

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.

Determination of anabolic steroids in pharmaceuticals by liquid chromatography with a microemulsion of sodium dodecyl sulfate and pentanol as mobile phase

The Chromatographic behaviour of fourteen anabolic steroids using micellar mobile phases has been studied. Anabolic steroids are strongly associated with the non-modified alkyl chains of the C18 stationary phase. However, appropriate retention times were obtained with a microemulsion of 0.1 M sodium dodecyl sulfate (SDS) and 7% pentanol as mobile phase (capacity factors, k′ < 14). A Chromatographic procedure for the determination of anabolic steroids in pharmaceuticals is proposed. The sample preparation step is simple, since the pharmaceuticals were easily dissolved in the SDS medium. The accompanying compounds were eluted at the head of the chromatogram due to the large eluent strength of the mobile phase. The values found usually agreed with those declared by the manufacturers and were reproducible.

Quantitation of hydrophobicity in micellar liquid chromatography

Abstract Micellar liquid chromatography (MLC) is shown to be a promising technique for measuring the hydrophobicity of solutes. The presence of micelles has a profound effect on the chromatographic characteristics of reversed-phase columns. The linear relationships between the logarithm, log k , of the retention factor and such diverse properties as the number of carbon atoms in homologous series, octanol–water partition coefficients and solvatochromic parameters, which are observed in conventional reversed-phase liquid chromatography (RPLC), are not usually valid in MLC. For series of compounds exhibiting a wide range of hydrophobicity, k itself is linearly related to these properties. The reasons for this behavior, and the advantages of MLC over conventional RPLC in the quantitation of hydrophobicity, are discussed. The values of k in mobile phases extrapolated to zero micelle concentration are shown to give a better measure of hydrophobicity than k with 100% water in conventional RPLC. The ability of MLC to characterize bioactive compounds is also discussed. A single MLC retention parameter can describe the bioactivity of phenols, while three conventional structural descriptors are needed to achieve a similar correlation. The correlation between the retention in MLC and the sites of action of diuretics within the nephron provides another interesting example of its mimetic value for physiological systems, where hydrophobic and electrostatic interactions, and kinetic phenomena are important.

Reduction of Convective Low-Frequency Noise in Thermal Lens Spectrometry

The construction and optimization of a thermal lens spectrometer with coaxial pump/probe configuration is reported. The low-frequency noise of convective origin is studied in detail, and it is demonstrated that a horizontal slit, instead of a pinhole, can be used as a simple way of reducing the noise to a negligible level without losing sensitivity.

Determination of Amino Acids by Micellar High-Performance Liquid Chromatography and Pre-column Derivatization with O-Phthalaldehyde and N-Acetyl-L-cysteine

Abstract Micellar liquid chromatography of proteic primary amino acids with pre-column derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine was studied, using mobile phases containing a short-chain alcohol. The modification of pH gave a large variation of the retention as a result of the protonation of the carboxylate group of amino acids. Maximum resolution and adequate retentions were achieved with a 0.05 M sodium dodecyl sulphate/3% propanol mobile phase at pH 3. The reproducibility was lower than 1.0% at a 1 × 10−4 M concentration level and between 0.6 and 2.2% for 1 × 10−6 M. The determination of glycine, lysine, methionine and threonine in pharmaceutical formulations gave recoveries, with respect to the values declared by the manufacturers, in the 90–105% range.

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.

Modelling of the elution behaviour in hybrid micellar eluents with different organic modifiers

Abstract The elution behaviour in reversed-phase liquid chromatography (RPLC), with mobile phases containing micelles, can be described according to a distribution pattern among three phases: bulk water, micellar pseudophase and stationary phase. Several mechanistic equations based on this pattern, previously used to predict the retention in hybrid mobile phases of sodium dodecyl sulphate and propanol, were checked with several organic modifiers and a group of solutes of diverse polarity. The modifiers were the alcohols 1-propanol, 1-butanol and 1-pentanol, and other organic solvents widely used in conventional RPLC such as acetonitrile and tetrahydrofuran. New models were also proposed to take into account diverse interactions of the modifiers with the micelles and the stationary phase. In spite of the differences in hydrophobicity of the three alcohols studied, the behaviour of the probe solutes in their presence was optimally described with the same equation, with relative mean fitting errors below 2%. The inclusion of the distribution of 1-butanol and 1-pentanol between bulk water and micelles in the equations produced a further increase in the accuracy. This distribution also explained the small deviations from linearity observed in the plots of 1/ k vs. micellar concentration, in the presence of pentanol. The more complex behaviour of the probe solutes in micellar eluents containing acetonitrile and tetrahydrofuran required a model with a larger number of parameters to obtain errors below 3.5%.