Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection

The formation of the hydroxyl free radical (HFR) can be quantified indirectly, by measuring two products of the hydroxylation of salicylic acid, 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB). In this study, we used reversed-phase high-performance liquid chromatography with electrochemical (coulometric) detection to measure 2,3- and 2,5-DHB levels in human platelets. The limits of detection of the method were 10 and 5 fmol on column for 2,3-DHB and 2,5-DHB, respectively. We tested the technique by measuring increases in dihydroxybenzoate levels after exposure of platelets to experimentally induced oxidative stress. Then, we measured platelet levels of 2,3- and 2,5-DHB in patients with Parkinson’s disease, under therapy with l-DOPA, and in normal subjects. We also measured platelet concentrations of l-DOPA and its major metabolite, 3-O-methyldopa (3-OMD). Parkinsonian patients showed increased levels of both 2,3- and 2,5-DHB. Platelet levels of 2,3-DHB were positively correlated with platelet levels of l-DOPA and 3-OMD. The technique we describe proved simple and extremely sensitive and may represent a useful tool for the study of oxidative stress in humans.     

Free and conjugated plasma catecholamines, DOPA and 3-O-methyldopa in humans and in various animal species

The aim of the present study was to determine the extent to which plasma catecholamines are conjugated in different animals compared to man and how widespread is the presence of dihydroxyphenylalanine (DOPA) and 3-methoxy-4-hydroxyphenylalanine (3-OMD) in plasma among the different animal species. Free and conjugated norepinephrine, epinephrine, and dopamine were measured in plasma in humans and in several animal species (dog, rat, Gunn rat, cat, rabbit, guinea pig, African green monkey, young pig, calf, and one American black bear) using HPLC with electrochemical detection. The same technique was used to measure free and conjugated DOPA and 3-OMD in plasma of man, dog, rat, Gunn rat, calf, and American black bear. Human plasma contains the highest concentration of total (free and conjugated) catecholamines (46.1 pmole/ml), while low concentrations (below 15 pmole/ml) were observed in unstressed rats, calves, cats, and young pigs. In man, 95.3% of total plasma catecholamines were conjugated. The extent to which plasma catecholamines were conjugated varied greatly between animal species. The conjugated fraction expressed as percentages of the total catecholamines is lowest in the young pig (4.7%) and highest in the bear (100%). Conjugated dopamine was present in the plasma of all species, varying between 3% of the total catecholamine pool in young pig to 90% in dog. Conjugated norepinephrine was also present in plasma of all species except in unstressed rats with access to food. Conjugated epinephrine was detected only in cat and rat. Free DOPA and 3-OMD were present in plasma of all tested species with especially high levels of 3-OMD being present in dog. Conjugated DOPA and 3-OMD were not consistently found in any species. Our results indicate that man, dog, bear, and African green monkey are particularly good catecholamine conjugators and that young pig, guinea pig, rabbit, and calf are poor conjugators.     

Modified high-performance liquid chromatography with electrochemical detection method for plasma measurement of levodopa, 3-O-methyldopa, dopamine, carbidopa and 3,4-dihydroxyphenyl acetic acid

Plasma measurements of levodopa and its major metabolites including dopamine and 3-O-methyldopa have been limited by cumbersome methods and poor sensitivity within relatively narrow ranges of plasma levels. We now report a modification of an HPLC method that permits concomitant measurements of a wide range of concentrations of levodopa, dopamine (DA), carbidopa, 3-O-methyldopa (3-OMD) and 3,4-dihydroxyphenyl acetic acid (DOPAC) from one HPLC injection. The recoveries ranged from 77 to 107% with an intra-day precision around 5% (CV) and inter-day CV's about 10-20%. This validated method will simplify pharmacokinetic studies of levodopa and its metabolites for mechanistic studies or therapeutic clinical monitoring which play a crucial role in development of strategies to prolong motor benefits from individual doses and reduce involuntary movements called dykinesias.     

Determination of the renin inhibitor Ro 42-5892 in human plasma by automated pre-column derivatization, reversed-phase high-performance liquid chromatographic separation and electrochemical detection after post-column irradiation

The renin inhibitor Ro 42-5892 has been found to be very potent, thereby necessitating a sensitive assay method for the evaluation of its pharmacokinetics in man. We report here the development of a very sensitive and selective HPLC assay for the analysis of this compound in human plasma. Ro 42-5892 was extracted from plasma with dichloromethane, derivatized with 2,4-dinitrofluorobenzene and then chromatographed on a Novapak C₁₈ column (150 × 3.9 mm I.D.) with acetic acid buffer (pH 7)-acetonitrile (100:85). Detection was performed by irradiation at 254 nm, followed by electrochemical oxidation at 550 mV. The extraction recovery of Ro 92-5 from human plasma (mean 102%) was quantitative. With this method a limit of quantitation of 0.3 ng/ml was achieved. The assay was linear up to 5 ng/ml, had acceptable inter-assay precision (12.2%) and accuracy (9.3%) and was successfully tested for selectivity. This assay was successfully applied to over 250 samples from a pharmacokinetic study in hypertensive patients.     

Assay methodology for the quantitation of unbound ertapenem,a new carbapenem antibiotic,in human plasma

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).     

Assay of coenzyme Q(10) in plasma by a single dilution step

A new method is described for determining coenzyme Q(10) (CoQ(10)) in plasma. The method is based on oxidation of CoQ(10) in the sample by treating it with para-benzoquinone followed by extraction with 1-propanol and direct injection into the HPLC apparatus. This method achieves a linear detector response for peak area measurements over the concentration range of 0.05-3.47 microM. Diode array analysis of the peak was consistent with CoQ(10) spectrum. Supplementation of the samples with known amounts of CoQ(10) yielded a quantitative recovery of 96-98.5%; the method showed a level of quantitation of 1.23 nmol per HPLC injection (200 microl of propanol extract containing 33.3 microl of plasma). A correlation of r = 0.99 (P < 0.0001) was found with a reference electrochemical detection method. Within run precision showed a CV% of 1.6 for samples approaching normal values (1.02 microM). Day-to-day precision was also close to 2%.     

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.     

Liquid chromatographic method for detection and quantitation of STI-571 and its main metabolite N-desmethyl-STI in plasma, urine, cerebrospinal fluid, culture medium and cell preparations

An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.     

INHIBITION OF CATALASE IN MESENCEPHALIC CULTURES BY l-DOPA AND DOPAMINE

Catalase activity in cell cultures of fetal rat mesencephalon was decreased by 42 and 50%, respectively, after exposure to l-3,4-dihydroxyphenylalanine (l-DOPA, 100 μM) or dopamine (100 μM) for 48 h. Catalase activity was also decreased 21% by 10 μM hydroquinone. Ascorbic acid (200 μM), an agent that suppresses the autoxidation of l-DOPA and dopamine, blocked the anti-catalase effect of l-DOPA, but not that of dopamine. Inhibitors of the A and B forms of monoamine oxidase (20 μM clorgyline plus 20 μM pargyline) had no effect on the anti-catalase action of either l-DOPA or dopamine. The latter results suggest that products of the oxidative deamination of dopamine by monoamine oxidase are not involved in the suppression of catalase activity. However, autoxidation reactions of l-DOPA may play a role since ascorbate suppressed the anti-catalase effect of l-DOPA. On the contrary, the basis for the failure of ascorbate to similarly block the anti-catalase effect of dopamine is uncertain. l-DOPA and dopamine (25 μM) also inhibited crystalline catalase in solution after incubation for 1 h at neutral pH (40–50% inhibition). Inhibition was blocked by 0.45 M ethanol, indicating a need for autoxidation and the formation of compound II, which is an enzymatically inactive form of catalase. The ability to model the enzyme inhibition in purely chemical experiments indicates a probable mechanism for loss of enzymatic activity in cell cultures. Inhibition of catalase may contribute to cell damage during incubation of cultures with l-DOPA, dopamine, or other autoxidizable compounds. Copyright © 1996 Elsevier Science Ltd     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

Determination of an investigational HIV integrase inhibitor in human plasma using high performance liquid chromatography with tandem mass spectrometric detection

An HPLC-MS/MS assay for the determination of an HIV integrase inhibitor, 5-(1,1-dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (I) in human plasma has been developed and validated. Compound I and a stable isotope labeled internal standard (II) were isolated from 0.5 mL plasma samples by solid phase extraction using an Ansys SPEC C-8 96-well plate. Extracts were separated on a Hypersil BDS C-18 HPLC column (3.0 mmx50 mm, 3 microm) with a mobile phase consisting of 25 mM ammonium formate pH 3.0:acetonitrile (60:40) vol%/vol% pumped at 0.5 mL/min. A Sciex API 365 mass spectrometer equipped with an atmospheric pressure chemical ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 431-->109 (I) and m/z 437-->115 (II) used for quantitation. The assay was validated over the concentration range of 10-5000 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was 69%. The intra-day accuracy of the assay was within 4% of nominal and intra-day precision was better than 4% C.V. Following a 200 mg dose of the compound administered to human subjects, concentrations of I ranged from 21.1 to 1500 ng/mL in plasma samples collected up to 12 h after dosing. Inter-day accuracy and precision results for quality control samples run over a 3-month period alongside clinical samples showed mean accuracies of within 6% of nominal and precision better than 3.5% C.V.     

Determination of etoposide in human plasma and leukemic cells by high-performance liquid chromatography with electrochemical detection

This paper describes a high-performance liquid chromatographic method with electrochemical detection for the determination of etoposide levels in plasma, total and non-protein bound concentration, and in leukemic cells. The precision for between-runs (n=6) was 7.0, 4.9, and 9.5%, the accuracy was 3.7, 7.1 and 6.3%, and within-runs precision (n=6) was 3.9, 2.9 and 5.1% for total plasma, non-protein bound plasma fraction and leukemic cells, respectively. The correlation coefficients (R²) were 1.00 for all calibration curves. These assays have been applied to analyze samples from one patient with acute myelogenous leukemia during 24 h after i.v. infusion of etoposide (100 mg/m²).     

Determination of A 3,4-diaminopyridine in plasma by liquid chromatography with electrochemical detection using solid-phase extraction

In order to quantify a small amount of a drug, 3,4-diaminopyridine (3,4-DAP), in animal plasma samples, an analytical method was developed. It involved an extraction of 3,4-DAP and phenylephrine, used as internal standard (IS), from plasma with solid-phase extraction (SPE) on C18 cartridges. This analytical method is a hyphenated technique based on high-performance liquid chromatography with electrochemical detection (HPLC-EC) whose purpose is to obtain first a sensitive method and second a satisfying separation between 3,4-DAP and phenylephrine. The analytical method is accurate, specific, and linear between 10 and 500 g of 3,4-DAP per litre. The recovery of 3,4-DAP is estimated at 70.8% with a 95% confidence interval of (66.0 -75.6%). Intermediate precision was evaluated on three quality control samples; the intra-day precision was estimated at 13.5, 9.1, 7.8% and the inter-day precision at 17.9, 8.4, 9.3%. The limit of quantification of the method was evaluated at 10 g l-1. First toxicokinetic parameters determined on dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg-1 were: Cmax=395.7 microg l-1; Tmax =15 min; t1/2=113.6 min; Clearance/F=16.8 ml kg-1 min-1 and Vd/F=2.7 l kg -1.     

The Role of 3-<Emphasis Type="Italic">O</Emphasis>-Methyldopa in the Side Effects of <Emphasis Type="SmallCaps">l</Emphasis>-dopa

Long-term treatment of l-dopa for Parkinson’s disease (PD) patients induces adverse effects, including dyskinesia, on–off and wearing-off symptoms. However, the cause of these side effects has not been established to date. In the present study, therefore, 3-O-methyldopa (3-OMD), which is a major metabolite of l-dopa, was tested to determine whether it plays a role in the aforementioned adverse effects. The effects of 3-OMD on the dopaminergic nervous system in the brain were investigated, by examining behavioral, biochemical, and cellular changes in male Sprague–Dawley rats and catecholamine-producing PC12 neuronal cells. The results revealed that the intracerebroventricular (icv) injection of 1 μmol of 3-OMD impaired locomotor activities by decreasing movement time (MT), total distance (TD), and the number of movement (NM) by 70, 74 and 61%, respectively. The biochemical analysis results showed that a single administration of 1 μmole of 3-OMD decreased the dopamine turnover rate (DOPAC/DA) by 40.0% in the rat striatum. 3-OMD inhibited dopamine transporter and uptake in rat brain striatal membranes and PC12 cells. The subacute administration of 3-OMD (5 days, icv) also significantly impaired the locomotor activities and catecholamine levels. 3-OMD induced cytotoxic effects via oxidative stress and decreased mitochondrial membrane potential in PC12 cells, indicating that 3-OMD can damage neuronal cells. Furthermore, 3-OMD potentiated l-dopa toxicity and these toxic effects induced by both 3-OMD and l-dopa were blocked by vitamin E (α-tocopherol) in PC12 cells, indicating that 3-OMD may increase the toxic effects of l-dopa to some extent by oxidative stress. Therefore, the present study reveals that 3-OMD accumulation from long-term l-dopa treatment may be involved in the adverse effects of l-dopa therapy. Moreover, l-dopa treatment might accelerate the progression of PD, at least in part, by 3-OMD.     

High-performance liquid chromatography with electrochemical detection applied to the analysis of 3,4-dihydroxymethamphetamine in human plasma and urine

Metabolic activation in the disposition of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") has been implicated in some of its pharmacological and toxicological effects, with the major metabolite 3,4-dihydroxymethamphetamine (HHMA) as a putative toxicant through the formation of thioether adducts. We describe the first validated method for HHMA determination based on acid hydrolysis of plasma and urine samples, further extraction by a solid-phase strong cation-exchange resin (SCX, benzenesulfonic acid), and analysis of extracts by high-performance liquid chromatography with electrochemical detection. The chromatographic separation was performed in an n-butyl-silane (C4) column and the mobile phase was a mixture of 0.1 M sodium acetate containing 0.1 M 1-octanesulphonic acid and 4 mM EDTA (pH 3.1) and acetonitrile (82:18, v/v). Compounds were monitored with an electrochemical cell (working potentials 1 and 2, +0.05 and +0.35 V, respectively, gain 60 microA). A mobile phase conditioning cell with a potential set at +0.40 V was connected between the pumping system and the injector. Calibration curves were linear within the working concentration ranges of 50-1000 microg/L for urine and plasma. Limits of detection and quantification were 10.5 and 31.8 microg/L for urine and 9.2 and 28.2 microg/L for plasma. Recoveries for HHMA and DHBA (3,4-dihydroxybenzylamine, internal standard) were close to 50% for both biological matrices. Intermediate precision and inter-day accuracy were within 3.9-6.5% and 7.4-15.3% for urine and 5.0-10.8% and 9.2-13.4% for plasma.     

Development and validation of a method to determine the unbound paclitaxel fraction in human plasma

Paclitaxel is pharmaceutically formulated in a mixture of Cremophor EL and ethanol (1:1, v/v). The unbound fraction of the anticancer drug paclitaxel in plasma is dependent on both plasma protein binding and entrapment in Cremophor EL micelles. We have developed a simple and reproducible method for the quantification of the unbound paclitaxel fraction in human plasma. Human plasma was spiked with [3H]paclitaxel and [14C]glucose (unbound reference) and incubated at 37 degrees C for 30 min. Plasma ultrafiltrate was prepared by a micropartition system (MPS-1) and collected in a sample cup containing 100 microl of plasma to prevent the loss of paclitaxel due to adsorption. The radionuclides were separated after combustion of the biological samples using a sample oxidizer and the radioactivity was determined by liquid scintillation counting. The unbound fraction of paclitaxel was calculated by dividing the ratios of 3H and 14C in plasma ultrafiltrate and in plasma. The method was thoroughly validated using human plasma spiked with pharmacologically relevant concentrations of paclitaxel (10-1000 ng/ml) and Cremophor EL (0.25-2.0%). The method was precise, with a within-day precision ranging from 3.9 to 11.0% and a between-day precision ranging from 5.8 to 13.1%. In patient plasma with low serum albumin values containing 1% of Cremophor EL, the unbound fraction appeared to be significantly higher than that in plasma with normal albumin values. The determination of the unbound fraction of paclitaxel proved to be stable during a 10-week storage at -20 degrees C. Furthermore, the assay was applicable in patient samples. This assay can be used to determine the unbound fraction of paclitaxel in plasma. Moreover, its design should allow the determination of the unbound concentrations of other hydrophobic drugs.     

Role of catecholaminergic mechanisms in regulating the autogenic periodic motor activity of rat pups

In 1-, 2- to 3-, 7- to 8-, and 10-day old intact and spinal rat puppies, studies have been made of the effect of l-DOPA (100 mg/kg, intraperitoneally) on autogenic periodic motor activity in the gastrocnemius muscle. In 1- to 3-day old pups, strong stimulating effect was observed up to a prolonged continuous activity for 5-10 or even more minutes. This effect decreases with age. Traces of the inhibitory effect are observed at early stages in the form of total decrease of the activity. The inhibitory effect increases with age. In all spinal animals, at the background of a decreased activity, stimulating effect of l-DOPA was predominantly observed. Age peculiarities of the effect of l-DOPA are discussed in relation to ontogenetic development of catecholaminergic innervation in rats. It is suggested that stimulating effect of l-DOPA is associated with its action on the descending noradrenergic system, whereas the inhibitory one is mediated by the brain structures.     

Chiral analysis of butaclamol enantiomers in human plasma by HPLC using a macrocyclic antibiotic (vancomycin) chiral stationary phase and solid phase extraction

An enantioseparation of the antipsychotic drug butaclamol in human plasma by high-performance liquid chromatography (HPLC) with solid phase extraction is presented. The separation was achieved on the vancomycin macrocyclic antibiotic chiral stationary phase (CSP) Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol : glacial acetic acid : triethylamine (100:0.2:0.05, v/v/v) at a flow rate of 0.5 ml/min. The detection wavelength was 262 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of butaclamol samples from plasma. The method was validated over the range of 100-3,000 ng/ml for each enantiomer concentration (R(2) > 0.999). Recoveries for (+)- and (-)-butaclamol were in the range of 94-104% at the 300-2,500 ng/ml level. The method proved to be precise (within-run precision ranged from 1.1-2.6% and between-run precision ranged from 1.9-3.2%) and accurate (within-run accuracies ranged from 1.5-5.8% and between-run accuracies ranged from 2.7-7.7%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 ng/ml and 50 ng/ml, respectively.     

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.     

Detection of antibodies to HIV in homologous sets of plasma, urine and oral mucosal transudate samples using rapid assays in Tanzania

The aim of this study was to evaluate the performance of commercially available anti-HIV assays when testing plasma, urine and oral mucosal transudate (OMT) samples for the presence of antibodies to HIV. Homologous sets of plasma, urine and oral mucosal transudate specimens were collected from 288 hospitalized patients in northern Tanzania and tested for antibodies to HIV using a routine enzyme immunoassay (Recombinant 3rd Generation EIA, Abbott) and two rapid assays (Testpack HIV-1/HIV-2; Abbott and SUDS HIV-1, Murex). Incubation times and/or sample volumes when testing OMT or urine were increased as compared to those recommended for plasma. The corresponding plasma specimens from all repeatedly reactive samples and samples presenting discordant results were subjected to confirmational testing by an HIV-1/2 Western blot. A total of 15.3% (44/288) of the plasma samples were anti-HIV-1 positive by Western blot. The sensitivity using plasma was 100% by all assays, 69.7-97.7% using urine, and 92.7-100% using oral transudate specimens. The sensitivity of both rapid assays was excellent and higher than the EIA when testing OMT. Specificities ranged from 98.8-100% for plasma, 99-100% for urine and were 100% by all assays using oral samples. The results obtained using oral mucosal transudate specimens and rapid assays were at least comparable to those obtained with plasma, while the use of urine specimens produced suboptimal sensitivities with two of the three assays. The testing of alternative body fluids for antibodies to HIV is yet another strategy that may be applicable, particularly in developing countries.