Determination of serotonin in whole blood, platelet-rich plasma, platelet-poor plasma and plasma ultrafiltrate

The important biogenic amine, serotonin (5HT), was determined in whole blood, platelet-rich plasma (PRP), platelet-poor plasma (PPP), and plasma ultrafiltrate after simple deproteinization. Following ion-pair chromatography on standard or narrow-bore reverse-phase columns, 5HT and the internal standard (N-methylserotonin-NMS) were detected by fluorometry with absolute detection limits of 2-4 pg. Levels obtained for whole blood and PRP were in agreement with previous methods. However, mean (+/- SD) values obtained for platelet-poor plasma (PPP) of 578 +/- 277 pg/ml (N = 7) were approximately 3-fold lower than the lowest previous reports. We also report the first determination of 5HT in plasma ultrafiltrate, having observed mean levels of 387 +/- 222 pg/ml (N = 7).     

Simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat plasma using HPLC with on-line solid-phase extraction

A HPLC method with on-line solid phase extraction (SPE) and DAD detection was developed for the simultaneous determination of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rat (SHR) plasma. Plasma samples (100 μL) were injected directly onto a CAPCELL MF C(8) SPE column. High-abundance proteins and most matrixes in plasma were removed by on-line SPE technology, while nitrendipine and hydrochlorothiazide trapped on the SPE column were effectively separated on a C(18) analytical column. The column temperature was maintained at 20°C. The optimal detection wavelength was 237 nm for NTDP and 271 nm for HCTZ. The total analytical run time was 34 min. The proposed method was linear over the range 5-500 ng mL(-1) for nitrendipine and 10-1000 ng mL(-1) for hydrochlorothiazide. The lower limit of detection (LLOD) was 0.5 and 0.6 ng mL(-1) for nitrendipine and hydrochlorothiazide, respectively. The sensitivity and precision of the method were within acceptable limits during validation period. The method was successfully used to investigate the pharmacokinetic characteristics of nitrendipine and hydrochlorothiazide in spontaneously hypertensive rats.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

On-line coupling of solid-phase extraction and capillary electrophoresis for the determination of cefoperazone and ceftiofur in plasma

We present a method for determining two cephalosporins (cefoperazone and ceftiofur) in plasma by on-line solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) with a T-split interface. Using this interface, a part of the SPE elution plug containing the cephalosporins is injected while the rest of the sample is flushed to waste. SPE was carried out using a C(18) micro-precolumn and the cephalosporins presented good retention properties with breakthrough volumes above 1 ml. Using a desorption volume of 426 nl of acetonitrile, recoveries were 75 and 90%, for cefoperazone and ceftiofur, respectively. The resulting elution volume was about 1.8 microl. A deproteinization step was included prior to SPE for the analysis of plasma samples with recoveries of 90 and 57% for cefoperazone and ceftiofur, respectively. With UV detection at 254 nm, linear relationships between the injected concentration and peak area was measured between 10 and 500 ng ml(-1) for standards, and 200 and 1500 ng ml(-1) for plasma samples. Intra-day (n=5) and inter-day (n=5) peak area repeatability were lower than 12% RSD. The detection limits obtained for spiked plasma (100 ng ml(-1) cefoperazone and ceftiofur) are sufficient for applying the method to pharmacokinetic studies.     

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-l-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons - from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.     

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.     

Measurement of free and bound 5-hydroxytryptophan in plasma by liquid chromatography with electrochemical detection

A new method for the measurement of concentrations of 5-hydroxytryptophan (5HTP) in plasma is described. This method, which uses high-performance liquid chromatography with electrochemical detection, was used to measure 5HTP in plasma of patients with the carcinoid syndrome, and also in the plasma of rats after injection of 5HTP (30 mg/kg). A significant proportion of 5HTP was bound to macromolecules in plasma both in the rats and in the patients.     

Determination of cefaclor in human plasma by a sensitive and specific liquid chromatographic-tandem mass spectrometric method

A sensitive and specific liquid chromatographic-tandem mass spectrometric method is described for the determination of cefaclor in human plasma. The plasma samples were treated by two sample preparation procedures, i.e. protein precipitation (PPT) and solid-phase extraction (SPE). The pretreated samples were analyzed on a C(18) HPLC column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization (ESI) was employed as the ionization source. The analyte and internal standard ampicillin (for PPT) or cefetamet (for SPE) were detected by use of selected reaction monitoring (SRM) mode. The lower limit of quantitation obtained as a result of the PPT procedure was 100 ng/ml. The intra- and inter-run precision, calculated from quality control (QC) samples was less than 12% for cefaclor. The accuracy as determined from QC samples was within +/-3% for the analyte. The SPE procedure could provide the lower limit of quantitation of 2 ng/ml. The precision and accuracy were measured to be below 7.1% and between -3.6% and 1.1%, respectively, for all QC samples. The method was applied for the evaluation of the pharmacokinetic profiles of cefaclor sustained-release formulation.     

Serotonin-containing epithelial cells in rat duodenum. II. Quantitative study of the effect of 5HTP administration

Summary The effect of 5-hydroxytryptophan (5HTP) administration on serotonin (5HT)-containing epithelial cells in rat duodenum was investigated quantitatively using three-dimensional morphometry to determine cell density and HPLC to measure 5HT and 5HTP concentrations. The results are interpreted in terms of the amine precursor uptake and decarboxylation (APUD) capacity of the cells. After administration of 5HTP, no significant change was observed in the density of 5HT-fluorescent epithelial cells in the duodenal region examined. Moreover, no evidence could be obtained that the concentration of 5HT in duodenal villi was increased after 5HTP administration, despite a highly significant increase in serum 5HTP and 5HT levels. These results indicate that no cells in the duodenal epithelium have the ability to decarboxylate exogenously administered 5HTP and convert it to 5HT under physiological conditions.     

Serotonin-containing epithelial cells in rat duodenum. II. Quantitative study of the effect of 5HTP administration

The effect of 5-hydroxytryptophan (5HTP) administration on serotonin (5HT)-containing epithelial cells in rat duodenum was investigated quantitatively using three-dimensional morphometry to determine cell density and HPLC to measure 5HT and 5HTP concentrations. The results are interpreted in terms of the amine precursor uptake and decarboxylation (APUD) capacity of the cells. After administration of 5HTP, no significant change was observed in the density of 5HT-fluorescent epithelial cells in the duodenal region examined. Moreover, no evidence could be obtained that the concentration of 5HT in duodenal villi was increased after 5HTP administration, despite a highly significant increase in serum 5HTP and 5HT levels. These results indicate that no cells in the duodenal epithelium have the ability to decarboxylate exogenously administered 5HTP and convert it to 5HT under physiological conditions.     

Pharmacological profile of a model for central serotonin receptor activation

The head shake response in rats after systemic administration of the serotonin (5HT) precursor 5-hydroxytryptophan (5HTP) was pharmacologically characterized and shown to be a useful animal model to quantify brain 5HT receptor activation. The behavior occurred in a dose-dependent manner after injection of 5HTP and the 5HT agonist quipazine. Head shakes were also observed after injection of L-tryptophan, 5-methoxydimethyltryptamine and fenfluramine. The 5HT antagonists cyproheptadine and metergoline were potent blockers of the response. Xylamidine, a peripheral 5HT antagonist, had no effect on head shaking. Inhibition of 5HT uptake with fluoxetine potentiated the head shake response after 5HTP. Manipulation of central cholinergic or GABAergic mechanisms did not alter 5HTP-induced shakes. Alpha-noradrenergic receptor blockade had no significant effect on head shakes. However, desmethylimipramine was equipotent with methysergide as an antagonist of the behavior. Beta-noradrenergic receptor blockade had no specific effect on 5HTP head shakes. Concomitant dopamine receptor activation with SK&;F 38393 did not affect head shakes but the neuroleptics chlorpromazine and pimozide reduced the number of head shakes after 5HTP. The H₁ receptor antagonist pyrilamine had no effect on head shakes. It is concluded that 5HTP-induced head shakes in rats is a quantitative model of brain 5HT receptor activation which is particularly sensitive to 5HT antagonists.     

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography–electron-capture mass spectrometry

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC–ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene–divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2–25, 0.4–40, 4–200 and 4–200 pg g⁻¹ plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo-o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93–107%. The repeatability of the method was in the range 4–30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g⁻¹ plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.     

Acute effects of L-tryptophan on tryptophan hydroxylation rate in brain regions (hypothalamus and medulla) of rainbow trout (Oncorhynchus mykiss)

Levels of 5-hydroxytryptophan (5-HTP) in brain regions (hypopthalamus and medulla) of rainbow trout were analysed by HPLC-EC 0, 10, 30, and 40 min after intraperitoneal administration of different doses of L-tryptophan (Trp) (0, 12.5, and 25 mg. kg(-1) body weight) in fish treated with 3-hydroxybenzylhydrazine (NSD1015; 75 mg. kg(-1)). The results show that, in control fish, 5-HTP levels in hypothalamus (58.03 +/- 6.36 pg. mg(-1) brain tissue) were significantly higher than those observed in medulla (28.04 +/- 4.32 pg. mg(-1) brain tissue). Basal tryptophan hydroxylation rates (after 0 mg. kg(-1) Trp administration) were 0.42 +/- 0.07 pg 5-HTP. mg(-1). min(-1), and 0.63 +/- 0.24 pg 5HTP. mg(-1). min(-1), for hypothalamus and medulla respectively. On the other hand, the results demonstrate that L-tryptophan administration induced significant increases in the rate of tryptophan hydroxylation, both in hypothalamus and medulla. These findings indicate that, in a way similar to that observed in mammals, brain tryptophan hydroxylase is unsaturated by its substrate (tryptophan) under normal physiological conditions. J. Exp. Zool. 286:131-135, 2000.     

Sensitive determination of ranitidine in rabbit plasma by HPLC with fluorescence detection

A sensitive high-performance liquid chromatographic method for determination of ranitidine (RAN) in rabbit plasma is described. The method is based on liquid-liquid extraction, labeling with dansyl chloride and monitoring with fluorescence detector at 338nm (ex)/523nm (em). Plasma samples were extracted with diethyl ether alkalinized with 1M sodium hydroxide. Ephedrine HCl (EPH-HCl) was used as internal standard. Both, RAN and EPH were completely derivatized after heating at 60 degrees C for 10min in sodium bicarbonate solution (pH 9.5). The derivatized samples were analyzed by HPLC using Agilent Zorbax Extended C18 column (150mmx4.6mm i.d.) and mobile phase consists of 48% acetonitrile and 52% sodium acetate solution (0.02M, pH 4.6). The linearity of the method was in the range of 0.025-10microg/ml. The limits of detection (LOD) and quantification (LOQ) were 7.5+/-0.18 and 22.5+/-0.12ng/ml, respectively. Ranitidine recovery was 97.5+/-1.1% (n=6; R.S.D.=1.8%). The method was applied on plasma collected from rabbits at different time intervals after oral administration of 5mg/kg ranitidine HCl.     

Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.     

Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma

A high throughput preparation method for the determination of trileptal (oxcarbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in human plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 μl) containing the compounds to be analysed involved solid-phase extraction (SPE) on Empore C₁₈ 96-well SPE plates. Eluates from the plate were injected onto a reversed-phase column (Hypersil C₁₈, 3 μm) with UV detection at 210 nm. Detector response was linear over the ranges 0.2–10, 0.1–200 and 0.1–20 μmol/l, for OXC, MHD and DHD, respectively, with relative standard deviations from 1 to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of quantitation were 0.2, 0.1 and 0.1 μmol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concentrations obtained using the 96-well method and the previous method using a traditional SPE method with a 50 mg C₁₈ cartridge. About a threefold increase in sample throughput and a twofold decrease of plasma volume required for the assays, were the main advantages obtained from the previous method. The method was applied for the determination of 3000 plasma samples from clinical studies.     

Cerebral serotonin and the hypothalamo-hypophyseal-adrenal system of the rat during aging]

The role of brain serotonin (5HT) on the hypothalamus-pituitary-adrenal system (HPAs) under basal condition and after injections of p-chlorophenylalanine (pCPA) and L-5-hydroxytryptophan (L-5HTP) has been studied in 6, 12 and 28 month old male Wistar rats. Four experimental groups were made for each age: control, saline, injected with pCPA (250 mg/kg i.p.) and L-5HTP (200 mg/kg i.p.), the effects being valued 2 hours after L-5HTP administration and 24 hours after pCPA injection. In all groups the plasmatic ACTH, the corticosterone levels as well as the simultaneous changes of the 5TH content tryptophan hydroxylase activity in whole brain were estimated two hours after the L-5HTP injection and 24 hours after that of pCPA. Significant changes are not found in the plasmatic ACTH and corticosterone values with respect to age under basal condition. Nevertheless, the response of HPAs differs with the age after pCPA or L-5HTP injection. The ACTH and corticosterone levels augment by L-5HTP and decrease by pCPA in all age groups, but this corresponding increase or decrease was less marked in the older rats. The 5HT content as tryptophan hydroxylase activity in brain decreased in old animals. pCPA and L-5HTP determine, respectively, high falls and rise of 5TH values, these changes being more intense for pCPA in old rats and for L-5HTP in young and mature animals. The tryptophan hydroxylase activity is decreased by pCPA as L-5HTP injections.(ABSTRACT TRUNCATED AT 250 WORDS)