Previous HPLC Research Articles

Highly‐Sensitive Determination of 6‐Mercaptopurine and Its Metabolites by Electrochemical Reductive Desorption Measurements

AbstractThe electrochemical detection of 6‐mercaputopurine (6‐MP), which is one of the most well‐known drugs of antimetabolites, and its analogues was studied. The presented method is based on chemisorption (accumulation)/desorption processes of drugs onto/from the Au electrode surface. Using this method, we succeeded in detecting 6‐MP even in very diluted solution (concentration of 6‐MP: 1×10−8 M). The lower detection limit of 6‐MP was considerably improved compared with the previous HPLC studies by using our proposed system. Desorptive potentials of 6‐MP‐metabolites and its analogues were different from those of 6‐MP. This provides the possibility of simultaneous detection of 6‐MP and its typical metabolites.

Activation and inhibition of Candida rugosa and Bacillus-related lipases by saturated fatty acids, evaluated by a new colorimetric microassay.

Research on lipase inhibitors could help in the therapy of diseases caused by lipase-producing microorganisms and in the design of novel lipase substrate specificities for biotechnology. Here we report a fast and sensitive colorimetric microassay that is low-cost and suitable for high-throughput experiments for the evaluation of lipase activity and inhibition. Comparison of Candida rugosa activity and inhibition with previous HPLC results validated the method, and revealed the importance of the reaction mixture composition. The assay was used to evaluate the effect of saturated fatty acids on Bacillus-related lipases. Cell-bound esterases were strongly inhibited by fatty acids, suggesting a negative feedback regulation by product, and a role of these enzymes in cell membrane turnover. Bacillus subtilis LipA was moderately activated by low concentrations of fatty acids and was inhibited at greater concentrations. LipB-like esterases were highly activated by myristic and lauric acids and were only slightly inhibited by high capric acid concentrations. Such an activation, reported here for the first time in bacterial lipases, seems to be part of a regulatory system evolved to ensure a high use of carbon sources, and could be related to the successful adaptation of Bacillus strains to nutrient-rich environments with strong microbial competition.

A spectrophotometric method to assay epoxide hydrolase activity

The Aspergillus niger epoxide hydrolase activity was assayed by spectrophotometric using ( rac) p-nitrostryrene oxide (pNSO) as substrate. Both the substrate (pNSO) and the reaction product, p-nitrostryrene diol (pNSD), had a strong absorbance in UV at 280 nm. The assay was based on the measure of the pNSD absorbance of the water phase after extraction of the non-reacted pNSO with a solvent. Among the five solvents tested, chloroform was selected since it extracted more than 99% of the epoxide and only 32% of the produced diol. This extraction yield was independent of the diol and epoxide concentrations and it was fairly reproducible. Using different enzyme amounts, the reaction kinetics were linear for the first 10 min corresponding to degrees of conversion less than 5% for the epoxide. Two controls were run simultaneously, one with the substrate alone (epoxide hydrolysis and non-complete extraction) and one with the enzyme alone (enzyme absorbance at 280 nm). The resulting ΔOD/min was linear with the amount of enzyme added within a large range from 2 to 80 μg of the EH preparation. The new spectrophotometric assay correlates well with the previous HPLC assay and could be used routinely for an easy and fast evaluation of EH activity. The kinetic parameters of ( rac) pNSO hydrolysis by A. niger epoxide hydrolase could be easily determined and K M (1.1 mM) compared well with that previously reported (1.0 mM).

Optimization of HPLC method for stability testing of bacitracin

A stability indicating HPLC assay for bacitracin has been developed and validated. The assay is based on a gradient elution, reversed phase column and UV diode array detection. On the basis of our previous analytical work several additional systematic HPLC tests for optimization of analytical method were performed. In order to achieve the highest selectivity of HPLC method, tests were conducted with extremely complex samples -- zinc bacitracin feed grade as food additive for animals. The influence of pH of mobile phase and type of columns on chromatographic separation of active (A, B(1) and B(2)) and inactive (F) polypeptide components of bacitracin were investigated in detail. It was found also that the peak B(1) comprises three and the peak F two subunits -- probably isomers. The obtained analytical procedure proved to be very selective and effective for the simultaneous determination of active polypeptide A, B(1) and B(2), impurities, known and unknown degradation products and ballast material.

Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry

Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis. Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L). The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.

Study of the metabolism of spiramycin in pig liver

A major metabolic pathway of spiramycins in pig liver is described. This biochemical reaction involves l-cysteine – a common amino acid present in most animal tissues – which reacts with the aldehyde function of the antibiotic forming a thiazolidine ring. This transformation of spiramycin derivatives drastically increased their polarity. A preliminary HPLC method enabling the quantitation of each metabolite in the range 0.5 μg/g of liver tissue is proposed. Spiramycin S is used as an internal standard while extraction procedures take into account the physico-chemical properties of the thiazolidine moieties. By comparison, previous HPLC methods underestimated the exact amount of antibiotic residues because these metabolites were not extracted from the studied tissues.

Separation of tetracyclines by high-performance capillary electrophoresis and capillary electrochromatography

Separations of various tetracycline mixtures by high-performance capillary electrophoresis (HPCE) and a new form of electrochromatography (CEC) are compared. The new CEC method involves etching the inner wall of the capillary surface with an appropriate reagent (ammonium dihydrogen fluoride) in order to produce a significant increase in surface area. The etched surface is then modified by a silation/hydrosilation reaction sequence to first produce a hydride intermediate which is then further reacted to attach a C 18 moiety. The bare and hydride capillaries are tested under HPCE conditions while the C 18 capillary functions in the CEC mode. The effects of pH and the presence of an organic modifier (methanol) are also studied. Detection limits (<10 μg/ml) are comparable to previous HPLC and HPCE results. Resolutions for mixtures which simulate real analytical problems are equal to or better than those reported for separations on polymeric and diol columns by HPLC and in earlier studies by HPCE and MECC.

Plasma and Urine Salsolinol in Humans: Effect of Acute Ethanol Intake on the Enantiomeric Composition of Salsolinol

The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R-enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S-enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.

Structural and Dynamic Characterization of Pro Cis/Trans Isomerization in a Small Cyclic Peptide

We describe the structure of a small cyclic peptide Cys-Leu-Pro-Arg-Glu-Pro-Gly-Leu-Cys containing two non-vicinal prolines. A previous HPLC analysis showed two well-resolved peaks at low temperatu...

Improved HPLC method for the simultaneous measurement of allantoin and uric acid in plasma.

Uric acid is generated by the oxidation of purines, but no enzyme is present to oxidize it further. It is present in plasma at a concentration approaching 500 J.lIIlollL and has been proposed as an important antioxidant. 1 In agreement with this proposal experiments in vitro have shown the protective effect of uric acid against oxidative damage. The first and major product of uric acid oxidation is allantoin, and it has been suggested that allantoin may be useful as a 'marker' of free radical reactions in vivo.2,3 However, methods currently used for the analysis of allantoin in body fluids are tedious and complicated, and since allantoin is very polar' it is difficult to obtain resolution from other serum constituents that have a similar polarity on a reverse-phase HPLC column. In the method of Grootveld and Halliwelluric acid is first analysed by HPLC, and allantoin is then measured in a separate procedure which involves initial separation and fraction collection by HPLC, hydrolysis and derivatization of allantoin, and finally separation of the derivatives by HPLC. We have developed a unique chromatographic assay to determine uric acid and allantoin simultaneously in plasma ultrafiltrate which is a significant improvement on previous HPLC methods.

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of the tumor marker 1-methylinosine in human urine

A highly sensitive enzyme-linked immunoassay (ELISA) was developed to detect and quantify the tumor marker, 1-methylinosine (m 1I), in human urine. The rabbit antisera was highly specific for m 1I with negligible or no inhibition by other nucleosides excreted into urine. Using the competitive ELISA, nanogram amounts of m 1I were easily measured directly in urine. The assay agreed with our previous hplc analysis of m 1I in urine for identifying those individuals with chronic myelogenous leukemia. Thus, this assay should greatly facilitate the quantitation of m 1I as a tumor marker.

Highly sensitive determination of PLP in human plasma with HPLC method.

To investigate the validity of using HPLC procedure with the fluorescence detection for the estimation of the pyridoxal 5'-phosphate (PLP) concentration in human blood plasma, we modified the conditions of our previous HPLC method for PLP. By changing the pH of the mobile phase (pH 5.5) and by altering the detection wavelengths in fluorescence (excitation at 322 nm and emission at 417 nm), both the improved sensitivity for pyridoxic acid 5'-phosphate and elimination of interfering extraneous peaks were attained. We reported the total vitamin B6 content of healthy adult volunteers obtained by this HPLC method.

Preparative and analytical separation of amygdalin and related compounds in injectables and tablets by reversed-phase HPLC and the effect of temperature on the separation.

Previous HPLC procedures for amygdalin and related compounds in injectables and tablets were either time consuming or produced inadequate separations of D-amygdalin and its epimer. A study of the effects of temperature on the separation resulted in development of an HPLC method for amygdalin and some related compounds, using water as the mobile phase at 15 degrees C. Multimilligram quantities of amygdalin and related compounds were separated by this preparative procedure. The aqueous mobile phase allows the compounds to be recovered by simple lyophilization of the sample after elution. This permitted the carbon-13 NMR spectrum of the isolated aglyconic epimer of amygdalin to be reported for the first time. D-amygdalin, its L-mandelonitrile epimer (D-epiamygdalin), their hydrolysis products (the epimeric amides and epimeric acids), and the sugar gentiobiose were separated by the method.

A rapid method for the simultaneous determination of the major metabolites of sulphasalazine in plasma

In the course of recent studies on the disposition of sulphasalazine in patients with large bowel disease we have developed a rapid, sensitive HPLC method for the determination of 5 aminosalicylate (AS), sulphapyridine (SP) and their acetylated metabolites (AcAS and AcSP). The advantage of the present assay over previous HPLC assays is that it measures all the metabolites simultaneously and consequently is more rapid.