A simple, rapid and sensitive method for simultaneous determination of rivastigmine and its major metabolite NAP 226-90 in rat brain and plasma by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry

A simple and sensitive reversed-phase liquid chromatography coupled with electrospray-mass spectrometry was developed and validated for the simultaneous determination of rivastigmine, a cholinesterase inhibitor, and its major metabolite NAP 226-90 in rat plasma and brain homogenates. Rivastigmine and NAP 226-90 were extracted from plasma and brain by ethyl acetate and, after drying under nitrogen, re-dissolved in acetonitrile and separated isocratic by HPLC on a C(18) column and quantified by single ion monitoring mass spectrometer. The mean (+/-SD) extraction efficiency for rivastigmine in plasma and brain was 93 +/- 2 and 95 +/- 2% (n = 5) of NAP 226-90 in a drug range of 10-100 pmol/mL or pmol/g. The method proved to be linear within the tested range (regression coefficient, r = 0.9999, n = 5). Intra- and inter-day precision coefficients of variation and accuracy bias were acceptable (within 15%, n = 5) over the entire range for both compounds using plasma or brain samples. The limits of quantification were 0.5 pmol/mL plasma and 2.5 pmol/g brain for rivastigmine and 1 pmol/mL plasma and 5 pmol/g brain for NAP 226-90, respectively. The analytical technique was used to determine the concentrations of rivastigmine and its metabolite NAP 226-90 in rat plasma and brain after oral drug administration. The concentrations of the parent drug and its major metabolite were compared to a pharmacodynamic parameter, the ex vivo inhibition of acetylcholinesterase.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

Simultaneous determination of testosterone and androstadienone (sex attractant) in human plasma by gas chromatography-mass spectrometry with high-resolution selected-ion monitoring

Androsta-4,16-dien-3-one (androstadienone) and androst-4-en-3-one-17 beta-ol (testosterone) in healthy human plasma were simultaneously determined under several experimental conditions by gas chromatography-mass spectrometry with high-resolution selected-ion monitoring. Internal standards were [2,2,4,6,6-2H5]androstadienone and [2,2,4,6,6-2H5]testosterone. Samples were extracted with an Extrelut column, purified using Lipidex 5000 and converted into hydroxime-trimethylsilyl derivatives for determination. Physiological concentrations of androstadienone and testosterone found in eleven healthy men were 2.05 +/- 0.74 and 18.6 +/- 4.9 pmol/ml in plasma (mean +/- S.D.), respectively. No correlation was observed between these steroid concentrations.     

Residues and metabolism of 19-nortestosterone laurate in steers.

The illegal use of 19-nortestosterone (19NT; 4-estren-17 beta-ol-3-one; nandrolone) and its esters in livestock, for growth promotion purposes, has been widely reported in the European Union. The target residues for surveillance of abuse in bovine urine and bile samples are 17 alpha- and 17 beta-19NT, although this choice of target residues is not based on in vivo radiotracer biotransformation data. In this study, four steers were administered [3H2]- and [2H3] 17 beta-19NT laurate (2 mg kg-1 body mass) by intramuscular injection and blood, urine, faeces and bile samples were taken for 30 d until slaughter, after which tissues were sampled for total residue analysis. Total plasma radiolabelled residues reached a maximum of 56.3 +/- 15.9 pmol ml-1 at 36 h and were still appreciable (13.3 +/- 1.6 pmol ml-1) 30 d after treatment. Throughout the study period, total residue concentrations in bile (about 2-16 nmol ml-1), urine and faeces (0.5-3 nmol ml-1 or g-1) were higher than in other tissues sampled at slaughter. At slaughter there was evidence of residue accumulation in pigmented eye tissue (33.1 +/- 6.1 pmol g-1) and in white (13.4 +/- 3.4 pmol g-1) and black hair (28.9 +/- 8.9 pmol g-1). Evaluation of radio-HPLC profiles of urine and bile extracts generally indicated that 19NT and 19NT laurate residues were present in relatively small amounts among a complex mixture of metabolites. GC-MS analysis of glucuronidase-hydrolysed bile extracts indicated that the major metabolites were 5 beta-estrane-3 alpha, 17 alpha-diol, 5 alpha-estrane-3 beta, 17 alpha-diol. 5 alpha-estran-3 alpha-ol-17-one (norandrosterone) and estra-1,3,5(10)-triene-3,17 alpha-diol (17 alpha-estradiol).     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

Development of an HPLC method for the determination of salidroside in beagle dog plasma after administration of salidroside injection: application to a pharmacokinetics study

A simple RP-HPLC method was established for the determination of salidroside in dog plasma. Salidroside is one of the most active ingredients of Rhodiola L. The method had within-run precision values in the range of +/- 2.3 to +/- 9.1% (n = 5) and between-run precision in the range of +/- 3.2 to +/- 9.8%. A simple protein precipitation for salidroside extraction was processed using ACN at precipitant-to-plasma volume ratio (P-P ratio) of 3:2. The extraction recoveries of salidroside at seven concentrations were higher than 63.2%. There was a linear relationship between chromatographic area and concentration over the range of 0.83-520 microg/mL for salidroside in plasma (R = 0.9926). The LOQ (S/N = 10) of the method was 0.83 microg/mL. The method was applied in a study of the pharmacokinetics of salidroside injection in six beagle dogs. The major pharmacokinetic parameters of C(max), AUC(0-24), AUC(0-infinity), and t(1/2) of salidroside in beagle dogs after i.v. administration of a single 75 mg/kg (5 mL/kg) dose were 96.16 +/- 8.59 microg/mL, 180.3 +/- 30.6 microg h/mL, 189.3 +/- 32.1 microg h/mL, and 2.006 +/- 0.615 h, respectively.     

A liquid chromatographic/tandem mass spectroscopic method for quantification of the cyclic peptide melanotan-II. Plasma and brain tissue concentrations following administration in mice

Melanotan-II (MT-II), a synthetic analogue of the natural melanocortin peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), is well known for the anorexic effects it elicits in rodents. These effects are, at least partly, associated with agonistic action on the centrally located melanocortin receptors, MC3R and MC4R. Whether MT-II exerts this effect via brain penetration still remains unclear. In order to address this question we administered MT-II in rodents at efficacious doses and then employed a sensitive methodology for the determination of MT-II in plasma and brain samples. MT-II was extracted from mouse plasma and brain tissue by acetonitrile precipitation followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The described assay improved significantly previously reported MT-II levels of quantification in rat plasma and brain. The lower limits of quantification (LLOQs) of 0.5 ng/mL and 2.5 ng/g were obtained in 50 microL plasma and 100 microL brain homogenate, respectively. The calibration curve was linear over the concentration range of 0.5-500 ng/mL for plasma and 2.5-250 ng/g for brain tissue. The method was successfully applied in measuring levels of MT-II in plasma and brain tissue following intraperitoneal (ip) administration of 1 mg/kg of peptide in mice. Following administration of MT-II, clearance from plasma was rapid. The sensitivity of the assay allowed the determination of low concentrations of MT-II (11.4 +/- 5.5 ng/g) in brain homogenate at 30 min after dosing. However, the brain concentrations when compared with the high plasma levels of MT-II at the same time point confirmed the low penetrability of the peptide in mouse brain.     

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.     

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.     

Liquid chromatography-mass spectrometric method for determination of the non-imidazole H3-receptor antagonist UPR1056 in rat plasma

The non-imidazole H3 receptor antagonist UPR1056 was dosed in plasma samples from rats individually administered with a single i.p. dose of 1.25 mg/kg by means of a newly validated HPLC-MS method. UPR1056 was extracted from rat plasma by protein precipitation with acetonitrile and was separated by linear gradient elution, employing water and methanol both additioned with 0.05% trifluoroacetic acid as mobile phases. UPR1056 was detected in MS using an electrospray ion source operating in positive ion mode. Acquisition was performed in single ion monitoring mode at m/z=349.3. The method was validated over the concentration range of 17.43-1743 ng/mL (50-5000 pmol/mL). Within- and between-run precision for the low, mid and high quality controls (QC) levels were 6.75% or less and accuracy ranged from 95.8 to 107.6%. The lower limit of quantification was 17.43 ng/mL. The analysis of the time course of UPR1056 concentrations over the 24-h period revealed a C(max) of 1147 ng/mL after 2 h from peripheral administration of the non-imidazole H(3)-receptor antagonist, with a prolonged elimination half-life of over 9 h.     

Simultaneous determination of angiotensins II and 1-7 by capillary zone electrophoresis in plasma and urine from hypertensive rats

We describe the procedure developed for the simultaneous detection and quantification of angiotensin II and angiotensin-(1-7), by capillary zone electrophoresis with UV detection by photodiode-array, at a wavelength of 200 nm, in the plasma and urine from hypertensive rats. Optimal separation was achieved with a 100 mM boric acid + 3 mM tartaric acid + 10 fM gold (III) chloride electrolyte solution at pH 9.80. The applied voltage was 30 kV and the capillary temperature was kept constant at 20 °C. The method was over the concentration range of 0.01-500 pmol/mL. All determination coefficients were higher or equal to 0.9985. Limits of detection and quantification for angiotensin II were 0.0110 pmol/mL (S/N = 3) and 0.0195 pmol/mL (S/N = 5), respectively. While for angiotensin-(1-7), the limits were 0.0112 pmol/mL (S/N = 3) and 0.0193 pmol/mL (S/N = 5), respectively. The present method offers a time-saving way to simultaneous determination of angiotensin II and angiotensin-(1-7), since it can be completed in 10 min, compared to other methodologies reported in the literature for capillary electrophoresis and liquid chromatography, which require more than 1 h for analysis of complex matrices, such as plasma and urine. The procedure is illustrated by experiments that quantify simultaneously angiotensin II and angiotensin-(1-7) in plasma and urine from hypertensive and normotensive rats, with and without antihypertensive treatment. The levels of angiotensin II and angiotensin-(1-7) detected in the experimental model, resulted in a recovery of 99.00-106.01% and a reproducibility of less than 10%. The proposed analytical method is a use full tool for the simultaneous detection of angiotensin II and angiotensin-(1-7) implicated in vascular remodeling in pathologies such as hypertension.     

Atmospheric pressure photoionization applied to quantitation of cyproterone acetate in human plasma

Cyproterone acetate [6-chloro-1beta,2beta-dihydro-17alpha-hydroxy- 3'H-cyclopropa(1,2)-pregna-1,4,6-triene-3,20-dione acetate] is a powerful antiandrogen used in the treatment of women suffering from disorders associated with androgenization such as hirsutism and acne. A fast, sensitive, and robustness method is developed for the determination and quantitation of cyproterone acetate in human blood plasma by liquid chromatography coupled with tandem mass spectrometry. Cyproterone acetate is extracted from 0.2 mL human plasma by liquid-liquid extraction. The method has a chromatographic run of 4.5 min, using a C18 analytical column (100- yen 2.1-mm i.d.), and the linear calibration curve over the range is linear from 1 to 500 ng/mL (r2 > 0.994). The between-run precision, based on the relative standard deviation replicate quality controls, is 96.2% (3 ng/mL), 97.5% (120 ng/mL), and 99.1% (400 ng/mL). The between-run accuracy was +/- 2.7%, 3.1%, and 4.8% for the previously mentioned concentrations, respectively. The method is employed in a bioequivalence study of two tablet formulations of cyproterone acetate (100 mg).     

Determination of 3',4',5',5,7-pentamethoxyflavone in the plasma and intestinal mucosa of mice by HPLC with UV detection

In a preliminary experiment 3',4',5',5,7-pentamethoxyflavone (PMF) inhibited adenoma development in Apc(Min) mice, a model of the human heritable condition familial adenomatous polyposis. An HPLC method for tricin was modified and validated to permit measurement of PMF in mouse plasma and intestinal mucosa. HPLC analysis was carried out on a Hypersil-BDS C(18) column with detection at 324 nm and tricin as internal standard. The assay was linear in the range of 100-2000 ng/mL plasma and 1.0-40 microg/mL mucosa. PMF in plasma was efficiently extracted using solid-phase columns. In the case of mucosa organic solvent protein precipitation displayed satisfactory accuracy and precision. The assay recovery at low, medium and high concentrations was between 85 and 103% for both biomatrices, with a relative standard deviation of <15%. The lower limits of quantitation for plasma and mucosa were 100 ng/mL and 1.0 microg/mL, respectively. This method allowed measurement of PMF steady-state median concentrations in plasma (1.08 nmol/mL, n = 11; 10th and 90th percentiles: 0.633 and 2.385 nmol/mL) and mucosa (108.5 nmol/g, n = 9; 10th and 90th percentiles: 38.9 and 164.4 nmol/g) in mice which had received PMF (0.2%, w/w) with their diet.     

Alkaloids from some amaryllidaceae species and their cholinesterase activity

Alkaloid extracts of four Amaryllidaceae species were studied with respect to their acetylcholinesterase and butyrylcholinesterase inhibitory activity and alkaloid patterns. Twenty-one alkaloids were determined by GC/MS, and seventeen of them identified from their mass spectra and retention times. The GC/MS analysis of the alkaloid extract of Nerine filamentosa is the first phytochemical investigation of this species. Promising erythrocytic acetylcholinesterase inhibitory activity was demonstrated by the alkaloid extracts of Narcissus poeticus var recurvus, Nerine filifolia and N. filamentosa (IC(50,HuAChE) = 6.0 +/- 0.1 microg/mL; IC(50,HuAChE) = 18.5 +/- 0.8 microg/mL, IC(50,HuAChE) = 21.6 +/- 1.1 microg/mL). The most potent inhibitory activity against serum butyrylcholinesterase was shown by extracts of Sternbergia lutea and Nerinefilamentosa (IC(50,HuBuChE) = 3.7 +/- 0.1 microg/mL; IC(50.HuBuChE) = 13.0 +/- 0.7 microg/mL).     

Determination and pharmacokinetic study of bergenin in rat plasma by RP-HPLC method

A validated reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the determination of bergenin in rat plasma. Bergenin in rat plasma was extracted with methanol, which also acted as a deproteinization agent. Chromatographic separation of bergenin was performed on a C(18) column, with a mobile phase of methanol-water (22:78, v/v) at a flow-rate of 0.8 mL/min and an operating temperature of 40 degrees C, and UV detection was set at 220 nm. The calibration curve was linear over the range 0.25-50 microg/mL (r = 0.9990) in rat plasma. The limit of quantification was 0.25 microg/mL using a plasma sample of 100 microL. The extraction recoveries were 83.40 +/- 6.02, 81.49 +/- 2.40 and 72.51 +/- 2.64% at concentrations of 0.5, 5 and 50 microg/mL, respectively. The intra-day and inter-day precision and accuracy were validated by relative standard deviation (RSD%) and relative error (RE%), which were in the ranges 3.74-9.91 and -1.6-8.0%. After intravenous administration to rats at the dose of 11.25 mg/kg, the plasma concentration-time curve of bergenin was best conformed to a two-compartment open model. The main pharmacokinetic parameters indicated that bergenin exhibited a wide distribution and moderate elimination velocity in rat.     

Reversed-phase high-performance liquid chromatography and amperometric detection of 3-O-methyl isoprenaline sulphate: application to studies on the presystemic metabolism of d-isoprenaline in man

A selective method for the determination of 3-O-methyl isoprenaline sulphate in human urine and blood plasma has been developed using reversed-phase high-performance liquid chromatography with amperometric detection. The sulphoconjugate was subjected to acidic hydrolysis and the liberated 3-O-methyl isoprenaline was isolated by organic extraction and conventional cation exchange. An internal standard of 3-O-methyl isoetharine was synthesized from commercially available isoetharine and used to correct for recovery losses. The assay was shown to be linear over the range 5 ng/ml to 20 micrograms/ml with a limit of detection of 2 ng/ml. The reliability of the analytical method was examined together with its applicability to in-vivo studies in man.     

Sensitive assay for catecholamines in pharmaceutical samples and blood plasma using flow injection chemiluminescence analysis.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis was described for the determination of three catecholamines: dopamine, adrenaline and dobutamine, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. Under the optimized conditions, the calibration graphs relating the increase of CL intensity to the concentration of the analytes were linear. The present method allows for the determination of dopamine, adrenaline, and dobutamine over the range of 1.0 x 10(-10) - 1.0 x 10(-7) g/ml. The relative standard deviations for measurements (n=11) of dopamine, adrenaline and dobutamine were 2.9, 2.3 and 1.8% when the concentrations of three catecholamines were at 1.0 x 10(-9) g/ml, respectively. The detection limits of the method were 2.0 x 10(-11) g/ml dopamine, 1.0 x 10(-11) g/ml adrenaline and 4.0 x 10(-11) g/ml dobutamine. The method was successfully applied to the determination of three catecholamines in pharmaceutical samples and blood plasma.     

High-performance liquid chromatographic analysis of hippuric acid in human blood plasma

A method has been developed for the isocratic high-performance liquid chromatographic analysis of hippuric acid in human blood plasma. After the addition of an internal standard (3-methoxysalicylic acid), plasma samples (1 ml) were made alkaline and extracted stepwise with methylene chloride and ethyl acetate. The detection limit was 50 pmol of hippuric acid per ml of plasma. The concentrations of hippuric acid in plasma from house painters (n = 8), with long-term exposure to solvent vapours from alkyd paints, were in the range 1-21 nmol/mol (median 11 nmol/ml). These values were statistically significantly higher than those for controls (n = 9): 2-8 nmol/ml (median 3 nmol/ml).     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans

A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C(18) column and detected by ultraviolet absorbance at 239 nm. The use of methanol-water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a fl ow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0-300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4-104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 +/- 14.3 ng/mL at 3.1 +/- 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 +/- 83.1 ng.h/mL.