MR angiographic and sonographic indications for endarterectomy

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C8 columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water-phosphate buffer (pH 2.5)-acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 microg/cm2 and of SA from 0.1 to 5 microg/cm2 in tape and to quantities of ASA 0.1 to 2 microg/ml and 1 to 50 microg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 microg/cm2) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 microg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100-200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Functionally null mutations in patients with the cblG-variant form of methionine synthase deficiency

A column-switching liquid chromatographic method is described for the simultaneous determination of aspirin and salicylic acid in human plasma. Blood samples are taken into chilled tubes containing a fluoride anticoagulant, and the plasma is isolated by centrifugation. Following a simple acidification step, a 200 microL aliquot of the sample is injected directly onto the HPLC system. The C-18 extraction column is washed with acidified water for 2 min, after which time the compounds are removed by back-flushing directly onto the analytical column (C-8 Nucleosil, 5 microns, 250 mm x 4.6 mm). The flow rate through both columns is 1 mL/min, and the analytes are quantified by measurement of their UV absorbance at 225 nm. The mobile phase is a mixture of water-methanol-acetonitrile-orthophosphoric acid (650:200:150:1 v/v/v/v). The method is linear in the concentration ranges 0.10-5.00 micrograms/mL for aspirin and 0.25-15.00 micrograms/mL for salicylic acid. Both compounds have a limit of quantitation of 0.10 microgram/mL and a limit of detection of 0.04 microgram/mL. Extensive stability tests have been carried out, and validation studies reveal the method to be reproducible and repeatable. Excellent recoveries from plasma obviate the need for an internal standard. The procedure is easier to execute and requires less sample handling than methods currently described in the literature. It has been successfully applied to the investigation of the levels of aspirin and salicylic acid in a healthy, nonfasting volunteer following a 600 mg oral dose of aspirin.     

Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C18 column (250 x 4.6 mm I.D.) using 10 mM phosphoric acid-acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 micrograms/ml and the detection limits were 0.06 micrograms/ml for indomethacin and 0.08 micrograms/ml for mefenamic acid, for 50-microliters plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Genetic polymorphisms in glutathione S-transferase mu and theta, N-acetyltransferase, and CYP1A1 and risk of gliomas

The simultaneous determination of betamethasone dipropionate (BD) and salicylic acid (SA) in both ointment and topical solution was developed using high-performance liquid chromatography (HPLC). The method was standardized using a LiChrospher 100 RP-18 (125 x 4 mm, 5 microns) column, acetonitrile-tetrahydrofuran-acetic acid 1% (25:20:55 v/v), apparent pH 3.3, as mobile phase, and UV detection at 254 nm. The peak area response versus concentration was linear in a concentration range from 5.0 to 50.0 micrograms/ml of BD and from 20.0 to 200.0 micrograms/ml of SA. The correlation coefficients were 0.9997 for BD and 0.9987 for SA, and the relative standard errors of estimates were 1.38% for BD and 3.27% for SA. The coefficient of variation and the recovery average were, respectively, 0.41-1.15% and 100.09% for BD, and 0.57-0.95% and 99.79% for SA.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithiocarbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 microl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150 x 3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 microg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 microg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 microg/ml in plasma (500 microl) using a 1/y weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ratio values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 microg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat

A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml(-1) using 1 ml of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile-aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 microg ml(-1). Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 microg ml(-1) and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 microg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9-2.1 and 1.1-6.4%, respectively, at concentrations ranging between 5 and 200 microg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

The dense granule antigen, GRA2 of Toxoplasma gondii is a glycoprotein containing O-linked oligosaccharides

A high-performance liquid chromatographic method for the determination of DRF-2189, using troglitazone as internal standard, is described. A dichloromethane-ethyl acetate solvent mixture (6:4, v/v) was used as the extraction solvent. A Kromasil C18 column with a mobile phase consisting of 0.05 M phosphate buffer-acetonitrile-methanol (22.5:37.5:40) (pH 5.0) was used at a flow-rate of 1.0 ml/min. The eluate was monitored by using fluorescence detection with excitation and emission wavelengths at 292 nm and 325 nm, respectively. Ratio of peak area of analyte to internal standard was used for quantification of plasma samples. Using this method, the absolute recovery of DRF-2189 from rat plasma was >95% and the limit of quantitation was 50 ng/ml. The intra-day relative standard deviation (R.S.D.) ranged from 1.74 to 7.24% at 1 microg/ml and 1.86 to 3.83% at 10 microg/ml. The inter-day R.S.D.s were 8.34 and 4.91% at 1 and 10 microg/ml, respectively. The method was applied to measure plasma concentrations of DRF-2189 in pharmacokinetic studies in Wistar rats.     

Radiographic analysis of regenerated bone around endosseous implants in the canine using recombinant human bone morphogenetic protein-2

A selective and sensitive HPLC method was developed for the determination of U-39968E in rat plasma. The assay involved solid-phase extraction of the analyte and the internal standard and precolumn derivatization with cyclohexane-1,3-dione reagent before injection on to the HPLC column. The samples were chromatographed on a Spherisorb S5 CN column (25 cm x 4.6 mm i.d.) with a mobile phase containing acetonitrile-trifluoroacetic acid-water (17:0.2:83, v/v/v) at a flow rate of 1.5 ml min-1. The column eluent was monitored by flourescence detection with excitation at 272 nm and emission at 320 nm. The assay is linear over the range 4-759 ng ml-1. The relative standard deviation at the limit of quantification, 4 ng ml-1, was 7.1%. This method was successfully applied to the determination of U-89968E in rat plasma during pharmacokinetic studies.     

Monitoring of tricyclic antidepressants in human serum and plasma by HPLC: characterization of a simple, laboratory developed method via external quality assessment

A reversed-phase high performance liquid chromatography (HPLC) method for the determination of plasma and serum levels of amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI) is described. The assay is based upon single step liquid/liquid extraction of these compounds using hexane at pH 11 (recovery between 92 and 105%), a Nova-Pack C-18 HPLC cartridge column, a mobile phase composed of a phosphate buffer with 50% (v/v) acetonitrile and about 0.2% (v/v) diethylamine (final pH: 8) and solute detection at 242 nm. Using 1 ml of plasma or serum and econazole as internal standard, drug levels between 20 and 400 ng ml(-1) (about 60-1450 nM) were found to provide linear calibration graphs. For drug concentrations in the range of 70-120 ng ml(-1) (about 240-430 nM), intraday and interday imprecisions (n = 5) were determined to be < 6.0, and < 15%, respectively. Data reported include those gathered over a 3-year period during which this assay was employed for therapeutic drug monitoring and clinical toxicology. The performance of the laboratory developed assay was assessed via analysis of monthly samples provided by an external quality control scheme.     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C18 reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 microl of whole blood or plasma sample. Calibration curves were linear (r2=0.99) over a 1-10 microg/ml concentration range and intra- and inter-day precision were between 4-11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Effects of protraction mechanics on the midface

A sensitive enantioselective high-performance chromatographic (HPLC) method was developed validated to determine low levels of (-)-R and (+)-S-albuterol in plasma. Baseline resolution was achieved by using a teicoplanin-based chiral stationary phase with a polar organic mobile phase consisting of methanol/ acetonitrile/glacial acetic acid/diethylamine, 40:60:0.3:0.2, (v/v/v/v) and a flow-rate of 1.0 ml/min. Enantioselectivity (alpha) equaled 1.18 and resolution (RS) equaled 1.8. By using fluorescence detection maximized at 230 and 310 nm for excitation and emission, respectively, concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Initially, the method was applied to plasma samples from a small single-dose inhalation study of racemic albuterol in a human volunteer and, later, to in vivo samples from a canine inhalation study of the single enantiomer, (-)-R-albuterol. Results from the canine study showed that no chiral inversion of (-)-R-albuterol occurs in the dog.     

Anti-tumor gene therapy

A high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of pyrimethamine and sulphadoxine in human plasma. After an automated liquid-solid extraction on a C8 cartridge, the compounds are separated on a C18 column by isocratic elution; the mobile phase is methanol-acetonitrile-water (10:25:65, v/v/v) with triethylamine (1%) and adjusted to pH 5.6 with phosphoric acid. The eluent is monitored with an ultraviolet detector at 240 nm. The limit of quantification was 10 ng/ml for pyrimethamine and 22 microg/ml for sulphadoxine. No chromatographic interferences can be detected from endogenous compounds, other anti-malarial drugs or major drugs used for the treatment of children. Sulphadimethoxine is used as an internal standard. The method is accurate and precision is good with relative standard deviations lower than 6%. The chromatographic procedure takes 11 min. The method is comparatively rapid, simple, sensitive and can be used for therapeutic drug monitoring, clinical and pharmacokinetic studies.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 microl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

Chromatographic studies on the isolation of peroxydisulphate oxidation products of primaquine

Eight compounds from peroxydisulphate oxidation of primaquine were fractionated on Bio-Gel P-2 column using water as an eluent. A HPLC method employing acetonitrile-methanol-1 M perchloric acid-water (30:7:1:95, v/v) as a mobile phase at 1.0 ml/min on microBondapak reversed-phase column and UV detection at 254 nm was developed for the separation and identification of different oxidation products of primaquine. A combination of Bio-Gel chromatography with reversed-phase HPLC was found to be the most suitable analytical technique for the semipreparative isolation of various products formed from the oxidation. Two oxidation products that were isolated had three or four times higher gametocytocidal activity as compared to primaquine.     

High-performance liquid chromatographic assay for a new fasciolicide agent, alphaBIOF10, in biological fluids

This paper describes a high-performance liquid chromatographic method with ultraviolet absorbance detection at 304 nm for the determination of 6-chloro-5-(1-naphthyloxy)-2-methylthio benzimidazole (alphaBIOF10) -- a new fasciolicide agent -- and its sulphoxide (SOalphaBIOF10), in plasma and urine. It requires 2 ml of biological fluid, an extraction using Sep-Pak cartridges, and methanol for drug elution. Analysis is performed on a microBondapak C18 (10 microm) column, using methanol-acetonitrile-water (40:30:30, v/v) as the mobile phase. Results showed that the assay is sensitive: 7.2 ng/ml for alphaBIOF10 and SOalphaBIOF10 in plasma and 3.6 ng/ml for both compounds in urine. The response was linear between 0.195 and 12.5 microg/ml. Maximum intra-day coefficient of variation was 5.3%. Recovery obtained was 97.8% for both alphaBIOF10 and SOalphaBIOF10. In urine, recovery was 99.6% and 93.1% for alphaBIOF10 and SOalphaBIOF10 respectively. The method was used to perform a preliminary pharmacokinetic study in two sheep and was found to be satisfactory.     

Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection

Two high-performance liquid chromatography (HPLC) methods were developed for the determination of trecetilide in plasma samples. Differing only in the addition of a derivatization step and different detection wavelengths, the two methods encompassed a wide concentration range. In both methods, plasma samples (0.1 ml) with added internal standard were applied to solid-phase extraction discs containing a non-polar/strong cation mixed-phase, washed and eluted with an acetone-acetonitrile triethylamine mixture. The eluate was evaporated to dryness, and either reconstituted and directly injected onto an HPLC column or first derivatized with 1-naphthyl isocyanate before HPLC analysis. In both methods, the separation was performed isocratically on a cyano analytical column utilizing a mobile phase composed of acetonitrile-pH 7.9 phosphate buffer (70:30, v/v). The column effluent was monitored by fluorescence detection at 290/345 nm (with derivatization) or 235/320 nm (without derivatization). The limits of detection and quantitation of the assay were 0.57 and 1.9 ng/ml, respectively, when derivatization was used, or 4.3 and 14 ng/ml, respectively, without derivatization.     

Determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis-3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with LiChrospher 100 RP-18 (5 microm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, with a quantification limit of 1 ng ml(-1). The mean recoveries from human plasma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1-500 ng ml(-1)). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at -20 degrees C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.     

Mitochondrial DNA mutations in multiple symmetric lipomatosis

We describe an analytical technique for measuring residues of imidacloprid, a relatively new and highly active insecticide, in water and soil using high-performance liquid chromatography (HPLC). All analyses were performed on reversed-phase HPLC with UV detection at 270 nm using a mobile phase of acetonitrile-water (20:80, v/v). Fortified water samples were extracted with either solid-phase extraction (SPE) or liquid-liquid extraction methods. A detection limit of 0.5 microgram/l was achieved using the SPE method. The imidacloprid residues in soils were extracted with acetonitrile-water (80:20, v/v), and the extract was then evaporated using a rotary evaporator. The concentrated extract was redissolved in 1 ml of acetonitrile-water (20:80, v/v) prior to analysis by reversed-phase HPLC. A detection limit of 5 micrograms/kg was obtained by this method which is suitable for analysis of environmental samples. Accuracy and precision at 10 and 25 micrograms/kg soil samples were 85 +/- 6% and 82 +/- 4%, respectively.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).