Determination of morphine and norlaudanosoline in murine brain regions by dispersive liquid-liquid micro-extraction and liquid chromatograpy-electrochemical detection.

Morphine can be synthesized endogenously by mammals from dopamine via the intermediate norlaudanosoline. Previously, both compounds have been detected separately in whole brains of mice and brain regions of rats, and in urine of humans. Here, we report a novel method for the analysis of both compounds in single murine brain regions. Initially, a variant of dispersive liquid-liquid microextraction was established by using methanol as an extractant, cyclohexane as solvent, and tributylphosphate as disperser. The extraction method was applied to murine brain regions homogenized with perchloric acid while the subsequent detection was carried out by HPLC with electrochemical detection. In the thalamus of C57Bl/6J mice (n = 3, male, age 4-8 months), morphine and norlaudanosoline could be detected at levels of 19 ± 3.9 and 7.2 ± 2.3 pg/mg, respectively. Overall, we provide a novel method for the simultaneous extraction and detection of both morphine and norlaudanosoline in single murine brain regions.

A new rapid method for the analysis of catecholic tetrahydroisoquinolines from biological samples by gas chromatography/mass spectrometry.

A new method for the analysis of tetrahydroisoquinolines (TIQ) from biological materials is described. The method combines extraction and acylation of the TIQs from urine in a one-step procedure followed by analysis by gas chromatography/mass spectrometry. By this extractive derivatization method, a decrease of the extraction time, to speed up sample preparation in order to minimize oxidative side reaction, has been achieved. The detection limits for the analysis of salsolinol and salsolin in urine were 10 fmol/ml sample volume. Tetrahydropapaveroline could only be detected in spiked urine samples with a detection limit of 1 pmol/ml urine. The method is not only restricted to urine samples, but can also be applied to a variety of tissue types, including blood components.

Chiral separation of diltiazem, trimetoquinol and related compounds by micellar electrokinetic chromatography with bile salts.

The separation of optically isomeric diltiazem hydrochloride, trimetoquinol hydrochloride and related compounds by micellar electrokinetic chromatography was investigated employing four bile salts as chiral surfactants. The chiral separation of diltiazem hydrochloride and timetoquinol hydrochloride was successfully achieved by use of sodium taurodeoxycholate under neutral conditions, although enantiomers of carboline derivatives A and B and 2,2'-dihydroxy-1,1'-dinaphthyl were resolved with all the bile salts under conditions from neutral to alkaline. The chiral separation of diltiazem-related compounds was affected by the structure of the samples in addition to the effects of bile salt structures and pH of the buffer solutions. Application to the optical purity testing of trimetoquinol hydrochloride by the area percentage method is described. A possible chiral separation mechanism is briefly mentioned.

A method for determination of subpicomole concentrations of tetrahydropapaveroline in rat brain by high-performance liquid chromatography with electrochemical detection.

A sensitive and selective method for detection of tetrahydropapaveroline (THP) in rat brain has been developed. The procedure employs a multiple-stage separation scheme that selectively isolates THP from rat brain tissue and utilizes the sensitivity and resolution of reversed-phase high-performance liquid chromatography with electrochemical detection to provide an analysis with high specificity for THP. The mean (+/- SD) recovery of THP from rat brain homogenates, fortified at levels ranging from 0.25 to 3.0 pmol per whole brain, was 43.4 +/- 3.5%. The concentration of THP in brains of rats pretreated with L-dopa was 0.44 +/- 0.14 (SD) pmol per gram. The limit of detection of THP was approximately 0.1 pmol (0.03 ng) per gram brain.

Precise GC/MS assays for salsolinol and tetrahydropapaveroline: the question of artifacts and dietary sources and the influence of alcohol.

The use of GC/MS with appropriate deuterated internal standards has enabled the development of highly precise assays for salsolinol and THP. The deuterated standards we have prepared for the present assays are entirely free of contamination by non-deuterated species. The extraction and analytical techniques used in these studies do not lead to artifactual formation of either salsolinol or THP due to dopamine cyclization since neither alkaloid was detected in a variety of samples that contained high concentrations of dopamine. Examples are tomato, certain urine samples, rat brain samples including hypothalami from animals treated with pharmacological doses of l-dopa. While we have found little analytical support, at least in urine, for significant in vivo formation of salsolinol from endogenous dopamine and acetaldehyde, there is clear evidence that dietary sources of salsolinol contribute to its detection in biological samples. Whatever the origin of alkaloids such as salsolinol and THP in mammalian samples, their potential for affecting behavioural and physiological systems merit their continued study.

Estimation of the in vivo concentration of salsolinol and tetrahydropapaveroline in rat brain after the administration of ethanol.

Tetrahydroisoquinolines (TIQs) are not only pharmacologically active, but their intraventricular infusion evokes alcohol drinking in rats. It is not known if salsolinol or tetrahydropapaveroline (THP), the two most commonly studied TIQs, spontaneously form in vivo in brain during chronic or acute consumption of ethanol. A theoretical calculation based on a steady-state assumption was made in order to estimate what could be the upper limit of these compounds in rat brain. The model used was that the rate of formation was equal to the rate of disappearance. The latter term is the rate constant for metabolism times the TIQ concentration. The former was estimated for salsolinol from data obtained from human urine while for THP it was simply the second order rate equation. The values obtained for both concentrations were ca 1 ng/g-brain tissue.

Relationship between 3,4-dihydroxyphenylacetaldehyde levels and tetrahydropapaveroline formation.

It was demonstrated that DOPAL, the aldehyde derived from the action of MAO on dopamine, can condense with dopamine to form THP. This compound is of special interest, since it is an intermediate in the biosynthesis of morphine.