High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

Determination of amoxicillin in plasma samples by capillary electrophoresis

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C₁₈ cartridges followed by elution with water–methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 μg l⁻¹ by the FASI technique.     

Anhydrous tert-pentanol as a novel media for the efficient enzymatic synthesis of amoxicillin

The efficient enzymatic synthesis of amoxicillin using anhydrous tert-pentanol as a novel media has been demonstrated for the first time. p-OH-Phenylglycine methyl ester (HPGM) was selected as the activated acyl donor due to its good solubility in organic solvents. The screening results of 21 organic solvents showed that solvents with either strong polarity or poor substrate solubility were unfavorable. Remarkable catalytic activity of the immobilized penicillin acylase (IPA) from Escherichia coli was retained in tert-pentanol, and high yield could be obtained. Effects of various parameters such as acyl donor, water content or cosolvents of tert-pentanol, substrate concentration, temperature, etc., on the enzymatic synthesis of amoxicillin in tert-pentanol were investigated systematically. The best reaction medium, the optimal temperature, initial concentration of 6-APA and HPGM and concentration of enzyme were tert-pentanol, 15 °C, 100, 200 mM and 20 IU/mL, respectively. Under the optimal conditions, the yield of amoxicillin was as high as 88% after a reaction time of 20 h.     

Development of a localized surface plasmon resonance-based gold nanobiosensor for the determination of prolactin hormone in human serum

A localized surface plasmon resonance immunoassay has been developed to determine prolactin hormone in human serum samples. Gold nanoparticles were synthesized, and the probe was prepared by electrostatic adsorption of antibody on the surfaces of gold nanoparticles. The pH and the antibody-to-gold nanoparticle ratio, as the factors affecting the probe functions, were optimized. The constructed nanobiosensor was characterized by dynamic light scattering. The sensor was applied for the determination of prolactin antigen concentration based on the amount of localized surface plasmon resonance peak shift. A linear dynamic range of 1–40 ng ml⁻¹, a detection limit of 0.8 ng ml⁻¹, and sensitivity of 10 pg ml⁻¹ were obtained. Finally, the nanobiosensor was applied for the determination of prolactin in human control serum sample.     

Optimization of the trace element determination by ICP-MS in human blood serum

The ICP-MS for simultaneous trace element determination in human blood has prevailed as the most suitable methodology for clinical aims because of its rapidity, detection limits and minimal sample quantity needed for analysis.As the proteic matrix is high, it is necessary to fine-tune the ICP-MS Agilent 7500i with autosampler CETAC ASX-500 and ISIS System connected, and further we have to the sample pre-treatment in order to obtain good results.The study of the results shows that the best pre-treatment for blood serum samples consists of a basic treatment by 1/10 dilution with a solution of EDTA and NH₄OH, with a detection limit of the order of μg/L and a reduction of the necessary patient sample volume to 2 mL.     

Optimization of the determination of 4-aminopyridine in human serum and urine by column liquid chromatography

Two convenient reversed-phase column liquid chromatographic procedures are described for the determination of 4-aminopyridine in human serum and urine. A 0.5-ml aliquot of serum after the addition of a 0.5-ml solution of 4-(aminomethyl)pyridine in 0.1M Na₂HPO₄ as the internal standard is passed through a 1-ml BondElut C₁₈ silica extraction column. The column is selectively washed to remove acidic, neutral and weakly basic compounds. The desired compounds are eluted with a 0.3-ml aliquot of 35% perchloric acid-methanol (1:100, v/v). A 10-μl aliquot of the eluate is infected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₁₈ silica particles that is eluted at ambient temperature with a mobile phase containing octanesulfonic acid as the ion-pairing agent. The peaks are monitored at 263 nm. A 0.25-ml aliquot of urine or 0.5 ml of serum is mixed with N-propionylprocainamide as the internal standard and subjected to benzoylation by Schotten Baumann reaction. The reaction mixture is adjusted to pH 5.5–6 and extracted with a BondElut C₁₈ extraction column. An aliquot of the eluate is chromatographed at ambient temperature with a mobile phase containing tetramethylammonium perchlorate. The peaks are monitored at 278 nm.     

Application of enzyme field-effect transistors for determination of glucose concentrations in blood serum

Glucose-sensitive enzyme field effect transistors (ENFETs) modified by an additional Nafion membrane have been developed and used for diluted blood samples analysis. The ENFET was used in the linear portion of the calibration curve up to 1.5 mM glucose in a model solution, which corresponds with up to 60 mM glucose in the undiluted samples (dilution 1:40). The high linearity of the Grans curve (factor of linearity is 1.03) obtained by the method of standard additions indicates the high precision of analysis. Glucose concentrations in different blood serum samples determined by ENFETs were compared with those measured by the commercial analyzer 'Eksan-G' and colorimetric method ('Diagluc' enzymatic kit), and good correlation between these methods was revealed. The high reproducibility and operational stability of the biosensor developed were demonstrated.     

High-performance liquid column and thin-layer chromatographic determination of human serum glibenclamide at therapeutic levels

For glibenclamide bioavailability studies in serum, high-performance liquid column and thin-layer chromatographic methods were introduced. Both methods are specific, accurate and sensitive with detection limits of at least 5 ng of glibenclamide per ml of serum. Detection is performed in the ultraviolet at wavelengths of 200 nm for liquid chromatography or 300 nm for thin-layer chromatography.Serum levels determined by either method correlated well with those determined by an already existing radioimmunoassay. Some pharmacokinetic data were computed using a one-compartment open model.     

Polymorphism of blood serum transcortin during column chromatography

Serum protein bound progestins, androgens, estrogens, and cortexolone, were fractionated on a number of chromatographic systems. Contrary to earlier suggestions of a homogeneous unit by competition binding and Scatchard analysis, polymorphism and heterogeneity in the molecular nature of the corticosteroid binding globulin (CBG) were evident with progesterone on Sephadex A-25 columns. Components in the 55 000 and 67 000 molecular weight regions were obtained with cortexolone, estradiol, progesterone and testosterone on Ultrogel columns. Separation on DEAE-cellulose-52 resin revealed a fraction in the low ionic prewash followed by another, highly charged entity eluted only with 0.06 M phosphate buffers. Thus, under these conditions, serum protein bound sex steroids eluted in the same position as transcortin glucocorticoid complexes. These results are presented as a caution against the universal use of association dissociation assays to study steroid ligand binding and biological response. The techniques here detailed may fruitfully be employed to explore the hydrodynamic characteristics of protein ligand interactions. In addition, they support the model, earlier proposed with tissue specific steroid receptors, that calls for the various subunits of the ligand as a prerequisite for the expression of all grades of agonist and antagonist activity of a given hormone.     

Cations in the human blood serum

The review summarizes data (more than 450 references) on concentration of human serum cations (Na+, K+, Ca2+, and Mg2+) and human blood serum osmolality depending on age, diverse physiological and pathological states, and action of physiologically active substances. There are summarized data of many thousand measurements of physicochemical parameters of the blood serum, the mean values of osmolality and cation concentrations in healthy people are calculated. The values are kept at a stable level throughtout the entire life since the moment of birth; in many cases they are maintained by regulatory systems within the normal limits and during various physiological and pathological states. There are formulated the main types of the states characterized by deviations from norm of physicochemical parameters of the internal medium fluids.     

Stabilization of reduced streptolysin-o for the determination of antistreptolysin in blood serum

Summary In the stabilization of streptolysin-O by adding 0.1% of sodium hydrosulphite and 0.25% of hydroquinone, followed by storing in deep-freeze in small quantities, we hope to have found a solution of one of the tricky problems of rheumatism serology. Part of a Research Programme made possible by a grant from the “Utrechtse Stichting voor Rheumatiekbestrijding”. Most of the titrations were done by MissS. van Rossem.