Simultaneous determination of F-β-alanine and β-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

F-β-Alanine and β-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-β-alanine and β-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-β-alanine and β-alanine in the HPLC system were approximately 0.3 and 0.7 pmol, respectively. The procedure proved to be very reproducible with intra-assay RSDs and inter-assay RSDs being less than 8%. The usefulness of the method was demonstrated by the analysis of the F-β-alanine and β-alanine concentrations in plasma and urine samples from tumor patients treated with S-1 (Tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate in a molar ratio of 1:0.4:1).     

Extractionless high-performance liquid chromatographic method for the simultaneous determination of piroxicam and 5′-hydroxypiroxicam in human plasma and urine

A simple and rapid (extractionless) high-performance liquid chromatographic method with UV detection, at 330 nm, was developed for the simultaneous determination of piroxicam and its major metabolite, 5′-hydroxypiroxicam, in human plasma and urine. Acidified plasma and alkali-treated urine samples are used and naproxen is added as internal standard. The separation is performed at 40°C on a C₁₈ Spherisorb column with acetonitrile-0.1 M sodium acetate (33:67, v/v, pH 3.3) as mobile phase. The retention time is 2.2 min for 5′-hydroxypiroxicam, 2.6 min for piroxicam and 3.2 min for naproxen. The detection limit is 0.05 μg/ml using a 100-μl loop.     

Capillary liquid chromatographic analysis of fat-soluble vitamins and β-carotene in combination with in-tube solid-phase microextraction

A capillary liquid chromatography (CLC) system with UV/vis detection was coupled with an in-tube solid-phase microextraction (SPME) device for the analysis of fat-soluble vitamins and β-carotene. A monolithic silica-ODS column was used as the extraction medium. An optical-fiber flow cell with a long light path in the UV/vis detector was utilized to further enhance the detection sensitivity. In the in-tube SPME/CLC system, the pre-condition of the extraction column and the effect of the injection volume were investigated. The detection limits (LOD) for the fat-soluble vitamins and β-carotene were in the range from 1.9 to 173 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The relative standard deviations of migration time and peak area for each analyte were less than 5.0%. The method was applied to the analysis of fat-soluble vitamins and β-carotene contents in corns.     

High-performance liquid chromatographic determination of (S)- and (R)-propanolol in human plasma and urine with a chiral β-cyclodextrin bonded phase

The determination of propanolol enantiomers in microsamples of human plasma and urine by HPLC using a chiral stationary phase is described. After extraction from 200 μl of plasma or urine with racemic alprenolol as internal standard (I.S.), the enantiomers are separated on a β-cyclodextrin column with a polar organic mobile phase and determined by fluorescence detection. The retention times of I.S. and propranolol enantiomers are about 12–13 min and 16–18 min, respectively. Peak resolutions are 1.4 for I.S. and 2.2 for propranol. The use of alprenolol as I.S. improves significantly the coefficients of variation (C.V.: 0.6–4.2%). Sensitivity is approximately 1.5 ng/ml per propranolol enantiomer. The assay is applied to pharmacokinetic studies of racemic propranolol in human biological fluids. The (S)-propranolol levels are always higher than the (R)-antipode concentrations in plasma and urine.     

High-performance liquid chromatographic determination of phenol, 4-nitrophenol, β-naphthol and a number of their glucuronide and sulphate conjugates in organ perfusate

This paper describes a simple and more sensitive reversed-phase HPLC method for the quantification of phenol, 4-nitrophenol and β-naphthol and some of their glucuronide and sulphate conjugates in aqueous solution and liver perfusate buffer. Methanol-water mobile phases with ion-pairing agents for each phenolic group are detailed. The assay showed good recovery, accuracy and precision and is suitable for the quantification of these phenolic compounds in liver perfusion experiments.     

Fast liquid chromatographic–mass spectrometric determination of pharmaceutical compounds

We present fast LC–MS–MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid–liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC–MS–MS analysis.     

High-performance liquid chromatographic determination of the β2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization

A sensitive high-performance liquid chromatographic method has been developed for the determination of the β₂-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid–liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher^® 100 RP 18 and a LiChrospher^® RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.     

Analysis of naltrexone and its metabolite 6-ß-naltrexol in serum with high-performance liquid chromatography

ABSTRACT: BACKGROUND: Naltrexone has been proven to be an effective treatment option for the treatment of alcohol dependency. In this article we introduce a reliable and simple method developed for the simultaneous determination of naltrexone and 6-beta-naltrexol in human serum by using high-performance liquid chromatography (HPLC). FINDINGS: Liquid-liquid extraction with butyl acetate from basic solutions (pH 9) was chosen for extraction with nalorphine as an internal standard (IS). Analytes were back-extracted from organic solvent into perchloric acid. The acid extract was chromatographed by HPLC with a reverse-phase ODS-column and electrochemical detector. The mobile phase was a NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium hydrogen sulphate as ion-pair reagents. The recovery of the extraction method was 48 % for naltrexone and 75 % for 6-beta-naltrexol. The limit of quantification was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-beta-naltrexol. The analysed concentrations of naltrexone differed from the theoretic concentrations by 0.7 to 2.3 % and those of 6-beta-naltrexol by 2.6 %. The relative standard deviation of within-day assay was from 0.9 to 5.7 % for naltrexone and from 0.8 to 4.2 % for 6-beta-naltrexol; for the between-day assay it was 5.7 % and 4.2 %, respectively. CONCLUSIONS: Our results indicate that the developed method is suitable for determination of naltrexone and 6-beta-naltrexol in human serum.     

Biosynthesis,characterisation and direct high-performance liquid chromatographic analysis of gemfibrozil 1-O-β-acylglucuronide

Gemfibrozil 1-O-β-acylglucuronide was purified from the urine of a volunteer administered gemfibrozil, and an isocratic reversed-phase HPLC method was developed for its direct measurement. Quantitation of gemfibrozil and gemfibrozil 1-O-β-acylglucuronide was carried out from plasma, following extraction from acidified specimens into ethyl acetate, on a 5-μm CN reversed-phase column with a mobile phase (pH 3.5) containing acetonitrile, tetrabutylammonium sulphate and distilled water, using fluorescence detection at 284 nm excitation and 316 nm emission. Calibration curves were linear for both compounds over a concentration range of 0.1 to 40 mg/l, with intra-assay coefficients of variation <5% at concentrations of 20.0, 2.0 and 0.2 mg/l, and inter-assay coefficients of variation <10%. No degradation of gemfibrozil 1-O-β-acylglucuronide was detected as a result of the analytical procedure. However, a preliminary application of the method indicates that gemfibrozil acylglucuronide is chemically unstable undergoing intra-molecular rearrangement and hydrolysis under physiological conditions.     

Attempt at Affinity Labeling of α- and β- Amylases by α- and β-d-Glucopyranosides and α- and β-Maltooligosaccharides with 2,3-Epoxypropyl Residue as Aglycone: Specific Inactivation of β-Amylases

Among 2,3-epoxypropyl α-d-glucopyranoside and 2,3-epoxypropyl α-maltooligosaccharides and the β-anomers, 2,3-epoxypropyl α-d-glucopyranoside (α-EPG) strongly inactivated the β-amylases [EC 3.2.1.2] of sweet potato, barley, and Bacillus, cereus, in addition to soybean β amylase [J. Biochem., 99, 1631 (1986)]. However, none of the compounds used inactivated any α-amylases [EC 3.2.1.1] of porcine pancreas, Aspergillus oryzae, or Bacillus amyloliquefaciens. Irreversible incorporation of ¹⁴C-labeled α-EPG into β-amylases was stoichiometric, i.e., one α-EPG per active site of the enzyme was bound, and the inactivations were almost complete. The results suggest that α-EPG is an affinity labeling reagent selective for β-amylase. Slow inactivations by the other compounds were also observed, depending on the difference of source of β amylase.     

Isocratic high-performance liquid chromatographic measurement of optimal 5α-steroid reductase activity in Hep-G2 cells

Measurement of 5α-reductase activity usually involves quantitation of the radiolabelled products of [³H]testosterone. Recently, however, it has been claimed that the activity of 5α-reductase is masked by the activities of 17β-hydroxysteroid dehydrogenase and 3-ketosteroid reductase. Therefore in determining 5α-reductase activity in Hep-G2 cells, we have monitored the concentration of androstenedione to ensure that the conditions for measurement of optimum enzyme activity are maintained. Using a polar (cyano) bonded-phase column and hexane—isopropanol (9:1, v/v) as eluent, the ratio of relative retention times (methyl lithocholate used as the reference standard) of the closest peaks, dihydrotestosterone and estradiol, was 1.2, whilst the highest inter-assay coefficient of variation was 2.7%. Therefore this technique appears suitable for the evaluation of 5α-reductase in cell and tissue samples.     

High-performance liquid chromatographic–tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS–MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC–MS–MS. Linearity was given in the tested range of 50–5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50–5000 ng/ml was 1.47–6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37°C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.     

High-performance liquid chromatographic analysis of β-phenylethylamine for the estimation of in vivo protein synthesis

A rapid, sensitive, and automated reversed-phase liquid chromatographic method was developed for the analysis of phenylalanine as β-phenylethylamine, for the measurement of in vivo protein synthesis. β-Phenylethylamine was derivatized with o-phthaldialdehyde (OPA) to form a fluorescent derivative that was successfully measured in tissue cell fluids and hydrolysates as the decarboxylation product of phenylalanine. The system was extremely sensitive enabling the accurate determination of 0.5 pmol in biological samples. Analysis time was less than 11 min, so that 130 samples can be analysed per day. The method eliminates the need for time-consuming column extraction procedures. This method offers substantial advantages over existing methods for the isolation and determination of β-phenylethylamine.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Determination of the α,β-adrenoceptor blocker YM-09538 in plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of the α,β-adrenoceptor blocker 5-{1-hydroxy-2-[2-(o-methoxyphenoxy)ethylamino]ethyl}-2-methylbenzenesulphonamide hydrochloride (YM-09538) in plasma, using 5-di-n-butylaminonaphthalene-1-sulphonyl chloride as a reagent for fluorescence labelling, is described. The detection limit is 20 ng/ml, which is sensitive enough to determine YM-09538 plasma levels after the oral administration of effective doses to dogs and humans.