Quantitative determination of domperidone in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and selective analytical method for the determination of domperidone in rat plasma is described. The procedure involves liquid-liquid extraction followed by reversed-phase high-performance chromatographic analysis with fluorometric detection at 282 nm for excitation and 328 nm for emission. The detection limit was 1 ng ml(-1) using 1 ml of plasma. This assay procedure should be useful for the pharmacokinetic study of domperidone in small animals such as rats.     

Sensitive determination of nitrotyrosine in human plasma by isocratic high-performance liquid chromatography

A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [32P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.     

Sensitive and selective determination of methylenedioxylated amphetamines by high-performance liquid chromatography with fluorimetric detection

A rapid, sensitive and selective liquid chromatographic method with fluorimetric detection was developed for the separation and quantification of four methylenedioxylated amphetamines without interference of other drugs of abuse and common substances found in illicit tablets. The method was validated by examining linearity, precision and accuracy as well as detection and quantification limits. Methylenedioxylated amphetamines were quantified in eight tablets from illicit drug seizures and results were quantitatively compared to HPLC-UV analyses. To demonstrate the better sensitivity of the fluorimetric detection, methylenedioxylated amphetamines were analyzed in serum after a liquid-liquid extraction procedure and results were also compared to HPLC-UV analyses.     

Determination of plasma and serum homocysteine by high-performance liquid chromatography with fluorescence detection

Determination of homocysteine in plasma or serum is becoming an important diagnostic procedure. Accurate, rapid and low cost methods for measuring homocysteine are therefore required. We have improved an HPLC method and made it suitable for clinical application. The total homocysteine in plasma consists of free homocysteine (i.e., reduced plus oxidized homocysteine in the non-protein fraction of plasma) and protein-bound homocysteine. The method consists of the following steps: reduction of the sample with tri-n-butylphosphine, precipitation of proteins with trichloroacetic acid (10%) and derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. The derivatives are separated by reversed-phase high-performance liquid chromatography followed by fluorescence detection. The concentrations (mean +/- S.D.) of total homocysteine in plasma from 77 normal subjects, 44 male and 33 female adults, were 8.4 +/- 2.15 and 7.1 +/- 1.18 mumol/l, respectively. Serum concentrations were 8.8 +/- 2.6 mumol/l in males and 7.6 +/- 1.5 mumol/l in females.     

Determination of 7alpha-hydroxycholesterol in dog plasma by high-performance liquid chromatography with fluorescence detection

One of the groups at highest risk of anal cancer is homosexual and bisexual men. Like cervical cancer, anal cancer is associated with human papillomavirus (HPV) infection. Anal HPV infection was characterized in a study of 346 human immunodeficiency virus (HIV)-positive and 262 HIV-negative homosexual and bisexual men. Anal HPV DNA was detected in 93% of HIV-positive and 61% of HIV-negative men by polymerase chain reaction. The spectrum of HPV types was similar in HIV-positive and HIV-negative men, with HPV-16 the most common type. Infection with multiple HPV types was found in 73% of HIV-positive and 23% of HIV-negative men. Among HIV-positive men who were positive by hybrid capture for group B HPV types (16/18/31/33/35/39/45/51/52/56/58) or group A types (6/11/42/43/44), lower CD4 cell levels were associated with higher levels of group B DNA (P = .004) but not group A DNA. These data suggest increased replication of the more oncogenic HPV types with more advanced immunosuppression.     

Simultaneous determination of urinary catecholamines and 5-hydroxyindoleamines by high-performance liquid chromatography with fluorescence detection

OBJECTIVE: The relationship between bronchopulmonary dysplasia (BPD) and neurodevelopmental outcome after extracorporeal membrane oxygenation (ECMO) has not been extensively reported. We compared the outcomes in a large series of infants with and without BPD after ECMO. STUDY DESIGN: Hospital charts and follow-up records of 145 infants treated with ECMO (1985 through 1990) were reviewed. Complete long-term respiratory and follow-up outcome data were available in 64 infants. BPD occurred in 17 survivors; the remaining 47 did not have BPD. RESULTS: Babies with BPD were more likely to have had respiratory distress syndrome. Mean (+/- SD) age at ECMO initiation was later for the BPD group (127+/-66 vs 53+/-39 hours, p < 0.001), and the duration of ECMO treatment was longer (192+/-68 vs 119+/-53 hours, p < 0.001). Bayley Scales of Infant Development scores at <30 months were lower in infants with BPD (p < 0.001), as were three of four Mullen Scales of Early Learning scores (> or = 30 months, p < 0.001 or p = 0.01). At 57+/-16 months 11 (64%) patients with BPD had mild neurologic disabilities, and 3 (18%) had severe disabilities. At a similar age (53+/-16 months, p = NS) 16 (34%) patients without BPD had mild disabilities, whereas 2 (4%) had severe disabilities (p < 0.01). CONCLUSIONS: The occurrence of BPD after ECMO is associated with adverse neurodevelopmental outcome. Patients with BPD after ECMO merit close long-term follow-up.     

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.     

Determination of norfloxacin in human plasma and urine by high-performance liquid chromatography and fluorescence detection

A method for the determination of norfloxacin in human plasma and urine is described. Plasma samples were deproteinized using acetonitrile. The supernatant was analysed by C18 HPLC. Fluorescence detection at an excitation wavelength of 300 nm and an emission wavelength of 450 nm was utilized. The assay was validated in the concentration range of 31 to 2507 ng/ml when 0.5-ml aliquots of plasma were handled. The intra-day precision of the spiked quality control samples ranged from +/- 0.37 to +/- 4.14% in plasma (concentration range: 70.3-2109.2 ng/ml) and from +/- 0.51 to +/- 1.56% in urine (concentration range: 7.5-299.4 micrograms/ml). The intra-day accuracy obtained for norfloxacin in the quality control samples ranged from -5.18% to -9.47% in plasma and from -10.56% to - 5.91% in urine. The assay has been used to support human pharmacokinetic studies.     

Determination of avermectins in plasma at nanogram levels using high-performance liquid chromatography with fluorescence detection

An analytical method is described for the determination of the avermectins in plasma based upon high-performance liquid chromatography of fluorescent derivatives of these compounds. The analyte is isolated by adsorption chromatography on Florisil, dehydrated in an acetic anhydride-pyridine mixture, and the fluorophore is further separated by chromatography on silica gel in advance of introduction into a reversed-phase system. This method, which can be applied to samples containing as little as 0.2 ng drug per ml, has an accuracy of 5% mean relative error and a precision of 8% relative standard deviation. A study and discussion of several factors which affect the analytical reaction are included.     

Determination of heptylphysostigmine in plasma by high-performance liquid chromatography with electrochemical detection

P0, the major structural protein of peripheral myelin, is a homophilic adhesion molecule with a single immunoglobulin (Ig) domain, which contains a single N-linked glycosylation site and two cysteines. We have previously reported four different mutations of the myelin P0 gene in four families of Charcot-Marie-Tooth neuropathy type 1 (CMT1). In this study we found a new mutation of the myelin P0 gene in a small family of CMT1. The affected persons had an A - to - G substitution of nucleotide 245 of the myelin P0 gene in one allele, leading to a cysteine substitution for tyrosine82 in the extracellular Ig-domain. An additional cysteine in the extracellular domain may form a disulfide bond and cause an inappropriate change in the tertiary structure of the functional Ig-domain of P0.     

Determination of total cysteamine in human serum by a high-performance liquid chromatography with fluorescence detection

A convenient, reliable and rapid method for determination of total cysteamine in human plasma by high-performance liquid chromatography with fluorescence detection is reported. This assay involves reduction of samples with dithiothreitol, derivatization of total cysteamine by addition of monobromobimane and protein precipitation by perchloric acid. The calibration curve was linear in the range 2-150 nmol ml-1 and the detection limit was 0.5 nmol ml-1. This method was successfully applied for a pharmacokinetic study of three cysteamine derivatives in healthy volunteers without any interference from coexisting substances.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 x 2.1 mm I.D., 5 microns Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate-acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 microliters sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean +/- S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 +/- 1.2% and 91.4 +/- 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Stability and determination of aflatoxins by high-performance liquid chromatography with amperometric detection

A method based on reversed-phase high-performance liquid chromatography (RP-HPLC) with amperometric detection with a glassy carbon electrode at a constant potential of 1.4 V is reported for the separation and identification of aflatoxins B1, B2, G1 and G2 in a model mixture. The chromatography was performed on a PAH-Baker column with a ternary mobile phase containing methanol, acetonitrile and aqueous LiClO4 electrolyte. Aflatoxin G1 showed the highest electroactivity in the compound series studied. Calibration curves of aflatoxins G1 and B2 were linear up to 0.2 and 0.3 mmol/l, respectively. Sensitivity varied between 7 and 10 ng for the different aflatoxins. The combination of different HPLC detectors in the analysis of these compounds was applied to investigate the stability of aflatoxins G1 and B2.     

Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection

This unusual pathology has not been described in the medical literature of the last ten years. A 39-year-old patient, affected by unilateral cryptorchidism, on the right side, and congenital inguinal hernia, reached the operating theatre suffering from occlusive intestinal syndrome, due to a clogged hernial sac. This clog was caused by a retracting testicle which in turn stopped the ileal ansa from slipping back in to the peritoneum. Through this case we can underline the excursus of such pathology, which isn't very frequent in the adult but can, nevertheless create a fairly serious pathology, often leading to neoplan.     

Simultaneous determination of granisetron and its 7-hydroxy metabolite in human plasma by reversed-phase high-performance liquid chromatography utilizing fluorescence and electrochemical detection

A highly sensitive and selective high-performance liquid chromatographic method was developed for the determination of granisetron and its active metabolite, 7-hydroxygranisetron (7OH-G) in human plasma. Granisetron is a selective 5-hydroxytryptamine receptor antagonist used in the treatment of cytotoxic drug-induced emesis. The method involves isolation of granisetron, 7OH-G and the internal standards from plasma by solid-phase extraction prior to reversed-phase ion-pair chromatographic separation on an octyl silica column with subsequent quantification of analytes simultaneously either with electrochemical (7OH-G) or fluorescence (granisetron) detectors which are placed in series. The recovery of granisetron and 7OH-G from human plasma was quantitative. Using 1 ml of plasma, the limits of quantification for granisetron and 7OH-G were 0.1 and 0.25 ng/ml, respectively. Linear responses in analyte/internal standard peak-area ratios were observed for analyte concentrations ranging from 0.1 to 50 ng/ml plasma. Precision and accuracy were within 13% across the calibration range for both granisetron and 7OH-G. The method was sufficiently sensitive, accurate and precise to support pharmacokinetic studies for granisetron and 7OH-G, in both normal and patient populations.     

Determination of PNU 157706, a new dual inhibitor of 5alpha-reductase, in rat plasma by high-performance liquid chromatography with ultraviolet detection

The beta2-glycoprotein I (beta2GPI)-binding properties of five murine monoclonal antibodies immobilized as capture antibodies were studied using surface plasmon resonance detection. The monoclonal antibody with the fastest dissociation kinetics (6F3) was selected for the development of an immunoaffinity chromatography procedure, assuming that its behaviour would be similar in both systems since the covalent coupling chemistries involved amino groups in both cases. Under our experimental conditions of a fast one-step procedure, beta2GPI was purified to homogeneity from human plasma with a yield of about 50%. Beta2GPI was eluted under fairly mild conditions, either at low pH or at high pH. The immunoadsorbent was used five times without any apparent loss of binding capacity. The immunopurified protein showed similar binding to cardiolipin-coated polystyrene wells as beta2GPI purified by conventional methods. However, differences in the pattern of immunoreactivity in relation to the purification procedure were observed by surface plasmon resonance using the monoclonal antibody with the highest association kinetics (9G1) immobilized on the sensor surface.     

Selective method for plasma quantitation of the stereoisomers of a new aminotetralin by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method is described for the quantitation in plasma of the four stereoisomers of a new aminotetralin, (SRR, RSS)(SRS, RSR)-5,6-dimethoxy-2-[3'-(p-hydroxyphenyl)-3'-hydroxy-2'- propyl]aminotetralin (CHF 1255, internal code). After liquid-liquid extraction of the drug, separation was obtained after chiral derivatization with R-(+)-alpha-methylbenzyl isocyanate. The selective derivatization of the amino group was obtained by controlling the pH of the reaction medium at 7.5. The reaction was quantitative after a period of 16 h. The structures of the urea derivatives were confirmed by proton nuclear magnetic resonance spectroscopy and high-performance liquid chromatography with mass spectrometric detection. The use of an electrochemical detector, operating in the oxidative mode, allows the quantitation in plasma of all four urea derivatives at the nanogram level. The method was demonstrated to be precise, reproducible and applicable to pharmacokinetics studies after administration of the two epimeric racemates.     

Simultaneous determination of guanine, uric acid, hypoxanthine and xanthine in human plasma by reversed-phase high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm-3, pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid, 1-5000 mumol dm-3; guanine, 0.5-2000 mumol dm-3; hypoxanthine, 0.1-500 mumol dm-3 and xanthine, 0.5-5000 mumol dm-3. The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.     

Simultaneous determination of phospholipid hydroperoxides and cholesteryl ester hydroperoxides in human plasma by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the simultaneous determination of phosphatidylcholine hydroperoxides (PCOOH) and cholesteryl ester hydroperoxides (CEOOH). Lipid hydroperoxides (LOOH) were quantitatively extracted from human plasma with a mixture of n-hexane and ethyl acetate, and separated by column-switching high-performance liquid chromatography using one aminopropyl column and two octyl columns followed by chemiluminescence detection. LOOHs could be completely separated from each other and detected at picomole levels. The results of method validation tests were satisfactory. This method was then applied to determine LOOH in normal human plasma; the levels of PCOOH and CEOOH found were 36.0+/-4.0 nM (mean+/-S.D., n=6) and 12.3+/-3.1 nM (mean+/-S.D., n=6), respectively.