In vivo monitoring of norepinephrine and its metabolites in skeletal muscle

Although skeletal muscle sympathetic nerve activity plays an important role in the regulation of vascular tone and glucose metabolism, relatively little is known about regional norepinephrine (NE) kinetics in the skeletal muscle. With use of the dialysis technique, we implanted dialysis probes in the adductor muscle of anesthetized rabbits and examined whether dialysate NE and its metabolites were influenced by local administration of pharmacological agents through the dialysis probes. Dialysate dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured as two major metabolites of NE. The skeletal muscle dialysate NE, DHPG and MHPG were 11.7+/-1.2, 38.1+/-3.2, and 266.1+/-28.7 pg/ml, respectively. Basal dialysate NE levels were suppressed by tetrodotoxin (Na(+) channel blocker, 10 microM) (5.1+/-0.6 pg/ml), and augmented by desipramine (NE uptake blocker, 100 microM) (25.8+/-3.2 pg/ml). Basal dialysate DHPG levels were suppressed by pargyline (monoamine oxidase blocker, 1mM) (24.3+/-4.6 pg/ml) and augmented by reserpine (vesicle NE transport blocker, 10 microM) (75.8+/-2.7 pg/ml). Basal dialysate MHPG levels were not affected by pargyline, reserpine, or desipramine. Addition of tyramine (sympathomimetic amine, 600 microM), KCl (100 mM), and ouabain (Na(+)-K(+) ATPase blocker, 100 microM) caused brisk increases in dialysate NE levels (200.9+/-14.2, 90.6+/-25.7, 285.3+/-46.8 pg/ml, respectively). Furthermore, increases in basal dialysate NE levels were correlated with locally administered desipramine (10, 100 microM). Thus, dialysate NE and its metabolite were affected by local administration of pharmacological agents that modified sympathetic nerve endings function in the skeletal muscle. Skeletal muscle microdialysis with local administration of a pharmacological agent provides information about NE release, uptake, vesicle uptake and degradation at skeletal muscle sympathetic nerve endings.     

Effect of lithium on norepinephrine metabolic pathways

We investigated lithium-induced changes in norepinephrine (NE) catabolism. NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenyl glycol (DHPG), ions such as lithium (Li(+)), magnesium (Mg(2+)), and potassium (K(+)) were measured in rat plasma and cerebral cortex using an HPLC method with electrochemical detection for amines. The results obtained with a group of rats treated by lithium chloride (2 mmol/kg/IP) were compared with a control group receiving sodium chloride (2 mmol/kg/IP). Animals were killed at different times over a period of six hours in the morning following salt administration to minimize possible chronobiological effects. There are two pathways leading to MHPG formation: way A, without DHPG, and way B, with DHPG. In plasma and cerebral cortex of lithium treated rats, way A catabolism seems to be preferential. Lithium increases Mg(2+) and K(+) plasma levels. These results suggest that lithium may increase inactivation of NE and decrease NE available for adrenergic receptors.     

Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography-Mass Fragmentography

Abstract: A gas chromatographic-mass fragmentographic (GC-MF) procedure is described for the simultaneous quantitation of 3,4-dihydroxyphenyl-ethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl-trifluoroacyl esters, using [²H₂]DHPG and [²H₃]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.     

Dialysate dihydroxyphenylglycol as a window for in situ axoplasmic norepinephrine disposition

To examine basal axoplasmic norepinephrine (NE) kinetics at the in situ cardiac sympathetic nerve ending, we applied a dialysis technique to the heart of anesthetized cats and performed the dialysate sampling with local administration of a pharmacological tool through a dialysis probe. The dialysis probe was implanted in the left ventricular wall, and dihydroxyphenylglycol (DHPG, an index of axoplasmic NE) levels were measured by liquid chromatogram-electrochemical detection. Control dialysate DHPG levels were 161+/-19 pg/ml. Pargyline (monoamine oxidase inhibitor, 1 mM) decreased the dialysate DHPG levels to 38+/-10 pg/ml. Further alpha-methyl-para-tyrosine, omega-conotoxin GVIA, desipramine (NE synthesis, release and uptake blockers) decreased the dialysate DHPG levels to 64+/-19, 106+/-15, 110+/-22 pg/ml, respectively. In contrast, reserpine (vesicle NE transport inhibitor, 10 microM) increased the dialysate DHPG levels to 690+/-42 pg/ml. Thus, NE synthesis, metabolism and recycling (release, uptake and vesicle transport) affected basal intraneuronal NE disposition at the nerve endings. Measurement of DHPG levels through a dialysis probe provides information about basal intraneuronal NE disposition at the cardiac sympathetic nerve endings. Yohimbine (alpha(2)-adrenoreceptor blocker, 10 microM) and U-521 (catechol-O-methyltransferase blocker, 100 microM) did not alter the dialysate DHPG levels. Furthermore, there were no significant differences in the reserpine induced DHPG increment between the presence and absence of desipramine (10 microM) or alpha-methyl-para-tyrosine (100 mg/kg i.p.). These results may be explained by the presence of two axoplasmic pools of NE, filled by NE taken up and synthesized, and by NE overflow from vesicle. The latter pool of NE may be closed to the monoamine oxidase system in the axoplasma.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Plasma 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers.

The usefulness of the plasma concentrations of two major metabolites of norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release was investigated. The potent alpha 2-adrenoceptor agonist, dexmedetomidine, induced only about 15% maximal reductions in the metabolite concentrations, in spite of almost total inhibition of neuronal NE release, as evidenced by 90% reductions in plasma NE concentrations. Similarly, administration of the alpha 2-adrenoceptor antagonist atipamezole was followed by only small increases in plasma DHPG and no change in MHPG levels, in spite of almost six-fold, albeit short-lasting, increases in plasma NE. In contrast, a single dose of the reversible monoamine oxidase type A (MAO-A) inhibitor moclobemide reduced plasma DHPG levels by 78% and MHPG levels by 51%. It is concluded that the plasma concentrations of DHPG and MHPG are largely determined by intraneuronal, MAO-A-dependent metabolism of NE, and do not accurately reflect acute alterations in neuronal NE release. The concentration of NE in venous plasma is clearly a more sensitive indicator of alpha 2-adrenoceptor-mediated regulation of NE release.     

Interspecies differences in the metabolism of brain norepinephrine to glycol metabolites

Using a highly sensitive and specific gas chromatography-mass spectrometric assay, the glycol metabolites of norepinephrine (NE), 3,4-dihydroxyphenylethyleneglycol (DHPG) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were determined simultaneously in brain and body fluids of several mammalian species, including humans. Highest molar ratios of DHPG to MHPG were found in rat brain (1.20), a species in which these glycol metabolites were primarily conjugated. In mouse, guinea pig, hamster, monkey, and human brain, DHPG and MHPG were mostly unconjugated, and DHPG concentrations were about 30–60% of the respective MHPG levels. In dog cortex, MHPG occurred predominantly as conjugates, whereas DHPG could only be detected in its unconjugated form. In all species studies, highest DHPG and MHPG concentrations occurred in hypothalamus followed, in general, by midbrain and brainstem whereas cerebral cortex, caudate and cerebellum had the lowest values. These results demonstrate substantial differences in the degree of conjugation and relative abundance of brain DHPG compared to MHPG between the rat and other animal species studied.     

High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study

High performance liquid chromatographic (HPLC) methods were validated for the determination of aripiprazole (OPC-14597, Abilify) in rat plasma and brain. Separation was by Nova-pak phenyl column; flow rate, 1.0 ml/min; mobile phase, acetonitrile-methanol-20 mM sodium sulfate-acetic acid (27:25:48:1, v/v/v/v); UV detection at 254 nm. Reproducibility in plasma and brain showed excellent precision (within 7.8 and 10.6%) and accuracy (96.0-102.4% and 99.0-108.7%) with calibration curve ranges 10.0-2000 ng/ml and 30.0-6000 ng/g, respectively. Validated HPLC methods were successfully applied to pharmacokinetic study of aripiprazole in rats, demonstrating brain concentrations after oral administration five times higher than plasma concentrations.     

Rat Brain Norepinephrine Metabolism: Substantial Clearance Through 3,4-Dihydroxyphenylethyleneglycol Formation

To assess whether the metabolic clearance of rat brain norepinephrine (NE) through 3,4-dihydroxyphenylethyleneglycol (DHPG) formation is quantitatively comparable or greater than through 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) production, we studied the accumulation rates of conjugated DHPG and MHPG following probenecid administration in whole brain as well as in several brain regions. Administration of increasing doses of probenecid (100-500 mg/kg, i.p.) 1.5 h before sacrifice produced a dose-dependent increase of conjugated DHPG and MHPG levels. The maximum increment of these conjugated metabolites occurred at a dose of 300 mg/kg or higher. During the first hour following probenecid administration (300 mg/kg, i.p.), rat brain conjugated DHPG and MHPG levels accumulated linearly at a rate of 646 and 319 pmol/g/h, respectively. With the probenecid technique, the estimated appearance rates of conjugated DHPG significantly exceeded those of conjugated MHPG in hypothalamus, midbrain, brainstem, hippocampus, and cerebral cortex. These results clearly indicate that under resting conditions, formation and efflux of conjugated DHPG is the major route of metabolic clearance of rat brain NE.     

Relative Importance of 3-Methoxy-4-Hydroxyphenylglycol and 3,4-Dihydroxyphenylglycol as Norepinephrine Metabolites in Rat, Monkey, and Humans

Abstract: A gas chromatographic-mass spectrometric assay, which allowed simultaneous measurement of 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), was used to show that the concentration of MHPG in primate CNS far exceeded that of DHPG and that both metabolites were mainly in the unconjugated form. In rat brain, DHPG concentration was generally higher than that of MHPG, and both existed predominantly as conjugates. Rat and primate plasma contained more MHPG than DHPG. In plasma of primates but not of rats, higher proportions of the metabolites were conjugated, compared to those in brain. Significant correlations existed between MHPG and DHPG in rat brain, monkey brain, human plasma, and both monkey CSF and plasma. In monkeys, a significant CSF-plasma correlation was found for MHPG, but not for DHPG. Acute administration of piperoxane raised rat brain MHPG and DHPG concentration; desipramine prevented this rise in DHPG, but not in MHPG. Desipramine alone decreased DHPG, but not MHPG, concentration. Piperoxane increased monkey brain MHPG, but not DHPG, concentration. These data suggest that DHPG is a valuable metabolite to measure when assessing norepinephrine metabolism in the rat. Under certain conditions, measurement of rat brain MHPG and DHPG may provide information concerning the site of norepinephrine metabolism. However, in primates the importance of monitoring DHPG, in addition to MHPG, is uncertain.     

Simultaneous measurement of monoamines, their metabolites and 2,3- and 2,5-dihydroxybenzoates by high-performance liquid chromatography with electrochemical detection. Application to rat brain dialysates

A reversed-phase chromatographic method with electrochemical detection was developed for the simultaneous determination of 2,3- and 2,5-dihydroxybenzoates, indicators of in vivo hydroxyl free radical formation, monoamines (NE, DA, 5-HT) and their metabolites (MHPG, DOPAC, HVA, 3MT, 5-HIAA). Linearity was observed from 10 pg to 10 ng injected. Reproducibility is correct (C.V. about 9%) except for 3MT and 5-HT. The limit of detection for almost all products was about 20 pg injected on the column. An application of this method in the study of the neurotoxicity of high pressure oxygen in rat is described. The limit of quantification for all compounds was 5 ng/ml except for HVA (10 ng/ml). Some basal levels DA, 5-HT, 5-HIAA, HVA, DOPAC, 3MT, 2,5-DHBA and 2,3-DHBA in microdialysates coming from striatum of normoxic restrained rats are given.     

The effect of desmethylimipramine on the metabolism of norepinephrine

Eleven normal volunteers were given an acute and two chronic doses of desipramine (DMI). The plasma norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), and dihydroxyphenylglycol (DHPG) concentrations were measured before and during drug administration. DMI reduced plasma concentrations of MHPG by 13% and DHPG by 17%. After two weeks of drug administration, the MHPG/NE ratio was reduced, and there was a significant negative correlation with the concurrent drug concentration. These results suggest that DMI: (1) reduces the turnover of NE; and (2) diminishes the oxidative deamination of NE. In addition, the drug concentration response relationship indicates that the effects of uptake inhibition may not be maximal until concentrations in the apparent therapeutic range are achieved.     

Development and validation of a LC/MS/MS method for quantifying the next generation calcineurin inhibitor, voclosporin, in human whole blood

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Relationship between hypothalamic noradrenergic neuronal activity and serum 3-methoxy-4-hydroxyphenylethylene glycol in the rat

It has been suggested that the circulating levels of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), a major end metabolite of noradrenaline (NA), may provide an index of central NA neuronal activity. The aim of this study was to examine in the rat the relationship between serum MHPG and hypothalamic NA neuronal activity during basal conditions, and when hypothalamic NA neuronal activity was stimulated or suppressed. Hypothalamic NA neuronal activity was assessed from the concentrations of the primary neuronal NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG), MHPG, and the DHPG/NA and MHPG/NA ratios. Following 2-deoxyglucose (2DG) and cysteamine administration, hypothalamic NA neuronal activity and serum MHPG rose significantly. In contrast, hypothalamic NA neuronal activity and serum MHPG fell significantly in gentled rats. Serum MHPG correlated significantly with hypothalamic DHPG and the DHPG/NA ratio in control rats, and with hypothalamic DHPG, MHPG, and the DHPG/NA and MHPG/NA ratios in gentled, 2DG- and cysteamine-treated rats. In the latter two groups, serum MHPG also correlated significantly with serum glucose, which is itself closely related to hypothalamic NA neuronal activity. These studies demonstrate a significant relationship between serum MHPG and hypothalamic NA neuronal activity in the rat, so that serum MHPG provides an index of hypothalamic NA neuronal activity in the rat.     

Determination of bencycloquidium bromide in rat plasma by liquid chromatography-electrospray ionization-mass spectrometry

A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) assay for the determination of bencycloquidium bromide (BCQB) in rat plasma was firstly developed and validated. After addition of 1-ethyl-bencycloquidium bromide as an internal standard (I.S.), the plasma samples were deproteinized with methanol and the supernatant was assayed by LC-ESI-MS. Chromatographic separation was achieved with a Hanbon Lichrospher 5-C18 column. The mobile phase consisted of methanol-40 mM ammonium acetate buffer-formic acid (75:25:0.25, v/v/v) and delivered at the flow rate of 1.0 ml/min. LC-ESI-MS was carried out on a single quadrupole mass spectrometer using electrospray ionization (ESI) and positive selected-ion monitoring (SIM). Target ions were monitored at [M](+)m/z 330.2 for BCQB and [M] (+)m/z 344.2 for I.S. Calibration curve was linear over the range of 3-1500 ng/ml. The lower limit of quantification (LLOQ) was 3.0 ng/ml. The intra- and inter-run relative standard deviations (R.S.D.%) of the assay were less than 7.1 and 12.3%, respectively. The accuracy determined at the concentrations of 3.0, 100.0, 500.0 and 1500 ng/ml for BCQB were within +/-15.0%. The established method has been applied successfully to study the pharmacokinetics of BCQB in rats after intranasal administration.     

Postmortem Stability of Brain 3-Methoxy-4-Hydroxyphenylethyleneglycol and 3,4-Dihydroxyphenylethyleneglycol in the Rat and Mouse

Abstract: To assess the postmortem stability of brain 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and 3,4-dihydroxyphenylethyleneglycol (DHPG) levels, groups of rats and mice were killed by cervical dislocation and left at either 21° or 4°C for intervals of up to 24 h until removal and freezing of whole brain. Whole brain free and total MHPG and DHPG levels were determined simultaneously by gas chromatography-mass fragmentography (GC-MF). By 2 h after death, statistically significant decrements occurred in rat brain free DHPG (20%), total MHPG (21%), and total DHPG (11%) at 4°C, but free MHPG increased significantly (50%) compared with controls. At 21°C, rat brain total MHPG increased compared with controls at 2 h (15%) but decreased at 4 h (15%) and 8 h (15%), whereas free MHPG levels were increased at these times. Although brain total and conjugated DHPG levels showed little change, free DHPG levels were reduced at all times. In mouse brain no significant changes occurred in free MHPG and DHPG by 24 h at 4°C. At 21°C, mouse brain DHPG levels decreased whereas MHPG concentrations increased over the 8-h period of study. These findings demonstrate the occurrence of significant postmortem time- and temperature-dependent changes in brain MHPG and DHPG concentrations and indicate caution in the interpretation of changes in these metabolites in studies employing human postmortem brain tissue.     

Biogenic amines and their metabolites in mouse brain tissue: development, optimization and validation of an analytical HPLC method

A simple and fast HPLC method based on an isocratic, reversed-phased ion-pair with amperometric end-point detection for simultaneous measurement of noradrenergic (MHPG/NA and A), dopaminergic (DOPAC, HVA/DA) and serotonergic (5-HIAA/5-HT) compounds in mouse brain tissue was developed. In order to improve the chromatographic resolution (Rs) with an acceptable total analysis time, experimental designs for multivariate optimization of the experimental conditions were applied. The optimal conditions for the separation of the eight neurotransmitters and metabolites, as well as two internal standards, i.e., DHBA and 5-HMT, were obtained using a mixture of methanol–phosphate–citric buffer (pH 3.2, 50 mM) (9:91, v/v) containing 2 mM OSA as mobile phase at 32 °C on a microbore ALF-115 column (150 mm × 1.0 mm, 3 μm particle size) filled with porous C₁₈ silica stationary phase. In this study, a two-level fractional factorial experimental design (½ 2^K) was employed to optimize the separation and capacity factor (k′) of each molecule, leading to a good separation of all biogenic amines and their metabolites in brain tissue. A simple method for the preparation of different bio-analytical samples in phosphate–citric buffer was also developed. Results show that all molecules of interest were stabilized for at least 24 h in the matrix conditions without any antioxidants. The method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). The acceptance limits were set at ±15% of the nominal concentration. The method was found accurate over a concentration range of 4–2000 ng/ml for MHPG, 1–450 ng/ml for NA, 1–700 ng/ml for A, 1–300 ng/ml for DOPAC, 1–300 ng/ml for 5-HIAA, 1–700 ng/ml for DA, 4–2800 ng/ml for HVA and 1–350 ng/ml for 5-HT. The assay limits of detection for MHPG, NA, A, DOPAC, 5-HIAA, DA, HVA and 5-HT were 2.6, 2.8, 4.1, 0.7, 0.6, 0.8, 4.2 and 1.4 pg, respectively. It was found that the mean inter- and intra-assay relative standard deviations (RSDs) over the range of standard curve were less than 3%, the absolute and the relative recoveries were around 100%, demonstrating the high precision and accuracy, and reliability of the analytical method described to apply in routine analysis of biogenic amines and their metabolites in brain tissue.     

Simple and rapid determination of serotonin and catecholamines in biological tissue using high-performance liquid chromatography with electrochemical detection

Using the CNS of Lymnaea stagnalis a method is described for the rapid analysis of neurotransmitters and their metabolites using high performance liquid chromatography coupled with electrochemical detection. Tissue samples were homogenised in ice-cold 0.1 M perchloric acid and centrifuged. Using a C(18) microbore column the mobile phase was maintained at a flow rate of 100 microl/min and consisted of sodium citrate buffer (pH 3.2)-acetonitrile (82.5:17.5, v/v) with 2 mM decane-sulfonic acid sodium salt. The potential was set at +750 mV versus Ag|AgCl reference electrode at a sensitivity of 50 nA full scale deflection. The detection limit for serotonin was 11.86 ng ml(-1) for a 5 microl injection. Preparation of tissue samples in mobile phase reduced the response to dopamine and serotonin compared with perchloric acid. In addition it was found that the storage of tissue samples at -20 degrees C caused losses of dopamine and serotonin. As a result of optimising the sample preparation and mobile phase the total time of analysis was substantially reduced resulting in a sample preparation and assay time of 15-20 min.     

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.