Detection of oxilofrine in plasma and urine by high-performance liquid chromatography with electrochemical detection and comparison with gas chromatography-mass spectrometry

A high-performance liquid chromatographic (HPLC) method with electrochemical detection for the determination of oxilofrine [1-(4-hydroxyphenyl)-2-methylaminopropanol] in human plasma and urine (before and after cleavage of the metabolic conjugates) is described. Isolation from biological fluids is performed batchwise by weak acid cation exchange. Separation of plasma and urine components is achieved on a reversed-phase C18 column as an ion pair with heptanesulphonic acid. For amperometric detection the potential of the electrode was set at 0.95 V versus an Ag/AgCl reference electrode. The detection limit for oxilofrine in plasma is 1 ng/ml and in urine 12.5 ng/ml at a signal-to-noise ratio of 2.0 using 1.0 ml of plasma and 0.02 ml of urine. The method was compared with a gas chromatographic-mass spectrometric method and showed a good concordance for plasma (r = 0.996) and urine (r = 0.994). With the HPLC method it is also possible to determine related sympathomimetic drugs, e.g., etilefrine, norefenefrine or octopamine, after a slight modification of the mobile phase.     

Determination of promethazine in human plasma by automated high-performance liquid chromatography with electrochemical detection and by gas chromatography-mass spectrometry

A highly specific and sensitive method using automated high-performance liquid chromatography with electrochemical detection (HPLC-ED) and a method using gas chromatography-mass spectrometry (GC-MS) have been developed for the quantitative determination of promethazine in plasma. The lowest detectable concentration by HPLC-ED is 0.1 ng/ml of plasma and by GC-MS 0.5 ng/ml of plasma. The HPLC-ED method incorporates a valve switching unit to prevent all of the electroactive impurities from entering the electrode compartment, thus maintaining the sensitivity of the detector for the analyses of large numbers of samples. The GC-MS method incorporates the highly specific selected-ion monitoring technique. Plasmas derived from healthy subjects each given a single 50-mg oral dose of promethazine were analyzed by both HPLC-ED and GC-MS. The two methods compare favorably with a correlation coefficient of 0.92 and a slope of 1.059. While both methods are suitable for studying single-dose pharmacokinetics of promethazine, the automated HPLC-ED method has a decided advantage in being more sensitive and suitable for unattended overnight analyses of the large number of samples encountered in pharmacokinetic studies. The specificity of the HPLC-ED method is demonstrated by comparison to the GC-MS analysis of biological samples.     

Highly sensitive assay for gamma-glutamyltranspeptidase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.     

Determination of butylated hydroxytoluene in food samples by high-performance liquid chromatography with ultraviolet detection and gas chromatography/mass spectrometry

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and compared with a gas chromatography/mass spectrometry (GC/MS) method for determining butylated hydroxytoluene (BHT) in foodstuffs as a result of migration from plastic packaging. Similar extraction procedures were used in both methods. BHT was quantitated using an external standard in the HPLC method and an internal standard in the GC/MS method. Both methods presented good linearity (r(2) > or = 0.9917) and low detection limits. Recoveries obtained with the HPLC method (chicken meat, 95.8%, and Gouda cheese, 83.9%) were better than with the GC/MS method (chicken meat, 85.6%, and Gouda cheese, 71.3%).     

Method for the determination of gamma-L-glutamyl-L-dihydroxyphenylalanine and its major metabolites L-dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the analysis of gamma-L-glutamyl-L-dihydroxyphenylalanine (gludopa) and its major metabolites L-dihydroxyphenylalanine (L-DOPA), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is described. High sensitivity is achieved with a multi-cell coulometric detector utilising the specific electrochemical properties of gludopa (limit of detection 10 pg on-column). The retention time of gludopa was both pH-dependent and sensitive to negatively charged ion-pairing agents. An alumina-based solid-phase sample preparation technique with dihydroxybenzylamine as internal standard is described for plasma and urine (limit of detection 40 pg/ml) and an ultrafiltration technique is described for tissues (limit of detection 1-10 ng/g). After treatment with 50 mg/kg gludopa, in excess of twenty separate catecholic metabolic peaks can be detected in rat urine, whereas in humans after 9 mg/kg the only catechols detected were L-DOPA, dopamine and DOPAC.     

Highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection

A highly sensitive assay for choline acetyltransferase activity by high-performance liquid chromatography with electrochemical detection was devised. This assay method is based on the separation of acetylcholine and choline on a Develosil Ph-5 reversed-phase column (a phenyl column), followed by their enzymatic conversion to hydrogen peroxide through post-column reaction with acetylcholinesterase and choline oxidase. The sensitivity of the system is excellent and 5 pmol of acetylcholine enzymatically formed could be detected. The linearity between the peak height and the amount of acetylcholine was observed over the range of 5 pmol to 5 nmol. Some enzymatic properties were investigated by using a soluble fraction of bovine caudate nucleus as enzyme. The Michaelis constants of the enzyme for choline and acetyl coenzyme A were 0.3 mM and 0.03 mM, respectively. The enzyme exhibited the maximum activity over the pH range 7.4-9.5. The regional distribution of choline acetyltransferase activity in rat brain was examined. The order of the activity from the highest to the lowest agreed with the reported brain distribution of the enzyme: striatum, pons plus medulla oblongata, cerebral cortex, thalamus plus hypothalamus, olfactory bulb and cerebellum.     

Determination of serotonin, melatonin and metabolites in gastrointestinal tissue using high-performance liquid chromatography with electrochemical detection

In this paper we show a simple isocratic chromatographic method for the detection of serotonin and its precursors and metabolites from various types of gastrointestinal tissue. The paper measures for the first time basal measurements of melatonin in the gastrointestinal tract, which has recently been shown to be released from the musosal lining of the gut. Tissue samples were stable following sample preparation in either 0.1 m perchloric acid or mobile phase. Analysis was carried out using a mobile phase consisting of 10% acetonitrile-90% acetate acid buffer pH 4.0 with 2 mm decane-sulfonic acid sodium salt at a column temperature of 50 degrees C. Electrochemical detection was utilized at a potential of +850 mV vs Ag/AgCl reference electrode at 10 microA full-scale deflection. The detection limit of 5-HT and melatonin was 241 and 308 nm respectively for a 10 microL injection. As a result of the method optimization, total analysis was reduced to 30 min. Accurate responses of the tissue samples following sample preparation could be obtained following a week after storage at -80 degrees C. This method is capable of preparing and analysing of samples from all regions of the gastrointestinal tract.     

Identification of phenprocoumon metabolites in human urine by high-performance liquid chromatography and gas chromatography-mass spectrometry

The oral anticoagulant phenprocoumon is eliminated in urine mainly as the glucuronide conjugate to an extent of 20% of the dose. The urine from patients undergoing phenprocoumon therapy was investigated and the following metabolites were isolated and identified: 7-hydroxyphenprocoumon as the main component, and 4'-hydroxyphenprocoumon and 6-hydroxyphenprocoumon as conjugates. They were characterized by high-performance liquid chromatography and, after methylation, by gas chromatography-mass spectrometry.     

Sensitive analysis of blood for amodiaquine and three metabolites by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method using oxidative electrochemical detection has been developed for selective and sensitive quantification of the antimalarial drug amodiaquine and three of its metabolites in the blood of dosed individuals. The method requires only one extraction step and has detection limits of 1 ng/ml for amodiaquine and its metabolites desethylamodiaquine and bisdesethylamodiaquine and 3 ng/ml for 2-hydroxydesethylamodiaquine. Minor modification of the mobile phase preserves the chromatographic separation and allows ultraviolet spectroscopic detection, which, although appreciably less sensitive, permits monitoring of levels of amodiaquine and the three metabolites in blood and urine samples if an electrochemical detector is unavailable. Levels of amodiaquine and the three metabolites were determined for two volunteers undergoing a nine-week chemoprophylactic regimen in connection with travel to a malarious area. Data are included to compare the in vitro antimalarial activities against three strains of Plasmodium falciparum of amodiaquine and the three metabolites considered.     

Determination of vinclozolin metabolites in human urine by high-performance liquid chromatography and electrochemical detection

A sensitive and reliable method to assess occupational exposure to vinclozolin based on biomonitoring principles has been elaborated. The conditions for pretreating the human urinary samples were chosen in such a way that vinclozolin metabolites containing the intact 3,5-dichloroaniline (3,5-DCA) moiety are completely degraded into this amine by means of basic hydrolysis. After addition of 3,4-DCA as an internal standard, steam distillation and extraction, the analysis is carried out by high-performance liquid chromatography and electrochemical detection. The determination limit is 5 g 3,5-DCA/l urine. The method turned out to be sensitive enough to quantify not only occupational but also nutritional excretions of 3,5-DCA containing metabolites to some extent. Interpreting these results, which are verified by an independent method, it must be considered that in addition to vinclozolin some further crop protection agents are also based on the 3,5-DCA moiety.     

Determination of vinclozolin metabolites in human urine by high-performance liquid chromatography and electrochemical detection

A sensitive and reliable method to assess occupational exposure to vinclozolin based on biomonitoring principles has been elaborated. The conditions for pretreating the human urinary samples were chosen in such a way that vinclozolin metabolites containing the intact 3,5-dichloroaniline (3,5-DCA) moiety are completely degraded into this amine by means of basic hydrolysis. After addition of 3,4-DCA as an internal standard, steam distillation and extraction, the analysis is carried out by high-performance liquid chromatography and electrochemical detection. The determination limit is 5 microg 3,5-DCA/l urine. The method turned out to be sensitive enough to quantify not only occupational but also nutritional excretions of 3,5-DCA containing metabolites to some extent. Interpreting these results, which are verified by an independent method, it must be considered that in addition to vinclozolin some further crop protection agents are also based on the 3,5-DCA moiety.     

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.