Determination of famotidine in human plasma by high performance liquid chromatography with column switching

While functional magnetic resonance imaging (fMRI) is now used widely for demonstrating neural activity-related signals associated with perceptual, motor, and cognitive processes in humans, to date this technique has not been developed for use with nonhuman primates. fMRI in monkeys offers a potentially valuable experimental approach for investigating brain function, which will complement and aid existing techniques such as electrophysiology and the behavioral analysis of the effects of brain lesions. There are, however, a number of significant technical challenges involved in using fMRI with monkeys. Here, we describe the procedures by which we have overcome these challenges to carry out successful fMRI experiments in an alert monkey, and we present the first evidence of activity-related fMRI signals from monkey cerebral cortex.     

Determination of nimesulide and hydroxynimesulide in human plasma by high performance liquid chromatography

Two specific methods for the simultaneous determination of nimesulide, a non steroidal anti-inflammatory drug, and its hydroxylated metabolite in human plasma are described. Adopting a high performance liquid chromatographic (HPLC) system with UV detection (230 nm), the compounds, extracted from plasma in acidic medium, were separated on ODS columns under gradient conditions, using a phosphate buffer solution and methanol as mobile phase. For each method column length, gradient rate and composition were appropriately selected. The limit of quantitation was 25 ng/mL for both compounds. The two methods were validated by intra day assays at three concentration levels and applied in kinetic studies in healthy volunteers, during which inter-day assays were carried out confirming their feasibility.     

Paediatric reference values for urinary catecholamine metabolites evaluated by high performance liquid chromatography and electrochemical detection

This report summarizes our experiences with the subjective visual vertical (SVV) as a clinical neuro-otological tool. In the SVV test, patients have to orient a dim light bar in an otherwise dark surrounding earth-vertical, using a remote-control. Normal subjects in an upright position did not deviate more than 2 degrees from true vertical. After vestibular neurectomy, the SVV was consistently tilted by some 12 degrees toward the affected ear. Smaller tilts (approximately 7 degrees) of the SVV occurred in patients with spontaneous peripheral vestibular diseases. This shift in SVV disappeared within weeks to months, similar to the spontaneous nystagmus. After stapes surgery slight deviations of the SVV towards the unoperated ear were seen in about 20% of the patients, indicating a slight irritation of the otolith organs. Assessed in an upright position, the SVV thus may be regarded as reflecting tonic otolithic input differences between the two ears. Asymmetries in the shifts of the SVV induced by roll tilts of the gravito-inertial vector by eccentric rotations of the subject have been proposed as a test for otolithic sensitivity. In our studies such asymmetries in the shifts of the SVV could not be induced by 26 degrees or 90 degrees roll tilts of subjects towards the affected or healthy ears. A simple clinical test to reveal unilateral otolithic sensitivity (comparable to an otolithic "caloric test") thus still has to be found.     

Effect of quercetin on chemiluminescence of human platelets induced by arachidonic acid

Arachidonic acid (AA)-induced platelet chemiluminescence (CL) was measured with a lumiphotometer. Quercetin remarkably inhibited the CL, the IC50 of quercetin was 3 mumol.L-1. When quercetin plus aspirin, which inhibits only cyclooxygenase, was added, the inhibitory rate of platelet-CL obviously increased (P < 0.01). On the other hand, the quercetin had a scavenging effect on superoxide anion radical using alkaline sodium dithionite solution generation. The IC50 was 20.9 mumol.L-1. In addition, superoxide dismutase of 0.1 mg.ml-1 inhibited the platelet-CL by 97.8%, while mannitol, a hydroxyl radical scavenger, only by 43.3% at a concentration of 80 mg.ml-1. These results suggest that the mechanism of the inhibiting AA-induced platelet-CL by quercetin was associated with scavenging the superoxide anion radical directly and with inhibiting the cyclooxygenase.     

Rapid simultaneous analysis of prostaglandin E2, 12-hydroxyeicosatetraenoic acid and arachidonic acid using high performance liquid chromatography/electrospray ionization mass spectrometry

Arachidonic acid (AA) can be metabolized to a variety of lipid mediators including prostaglandins (PGE), and hydroxyeicosatetraenoic acids (HETE) by cyclooxygenase, lipoxygenase and cytochrome P450-dependent monooxygenase enzymatic pathways. Traditional experimental procedures to quantify these lipid mediators require purification, often by high performance liquid chromatography (HPLC), prior to derivatization for gas chromatography/mass spectrometry (GC/MS) analysis. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE2, 12-HETE, and AA by HPLC/electrospray ionization mass spectrometry on cultured human dermal fibroblast supernatants. Extension of the method to analyse 5-HETE and 15-HETE was investigated. The advantages of this method include minimal sample preparation and elimination of the problem associated with thermal stability for GC/MS analysis. A detection limit of 20pg on column for PGE2 and 5pg on column for 12-HETE and AA was determined.     

The effect of thrombin on the uptake and transformation of arachidonic acid by human platelets

Washed human platelets take up arachidonic acid from plasma and incorporate the fatty acid into the major classes of complex lipids. Thrombin impairs net incorporation. It activates endogenous phospholipases which liberate arachidonic acid from phospholipids. As a consequence of thrombin induced aggregation platelets release arachidonic acid intermediates formed by the action of platelet fatty acid cyclooxygenase and by platelet fatty acid lipoxygenase. Cyclooxygenase, but not lipoxygenase, is inhibited by aspirin and indomethicin. Analysis of the pathways of arachidonic acid metabolism may furnish new insight into platelet function and into disorders of primary hemostasis.     

Monitoring serum concentrations of clomipramine and metabolites: fluorescence polarization immunoassay versus high performance liquid chromatography

This study compared fluorescence polarization immunoassay (FPIA) with a high performance liquid chromatographic (HPLC) method from the point of view of their applicability to therapeutic drug monitoring of patients treated with clomipramine alone. Blood was withdrawn from 20 depressed inpatients (54 +/- 14 years) under steady state conditions. The FPIA determined total tricyclic antidepressant (TCA) concentrations with day to day variability below 11%. The automated HPLC method separated clomipramine, N-desmethylclomipramine, 8-hydroxyclomipramine and 8-hydroxydesmethylclomipramine with interassay coefficients of variance below 12%. The concentrations measured by FPIA were similar to HPLC results. Total TCA concentrations measured by FPIA and the sum of clomipramine and desmethylclomipramine measured by HPLC correlated significantly (r = 0.780 and p < 0.01). However, 40% of individual FPIA determinations yielded results that differed by more than 50% from the HPLC concentrations. Changes in clinical rates were related only to TCA serum concentrations that had been analyzed by HPLC. It is concluded that the semiquantitative FPIA is unsuitable for therapeutic drug monitoring in patients under clomipramine treatment, whereas the differential analysis of clomipramine and metabolites by HPLC is informative and can be used to improve the antidepressant drug treatment.     

Thromboxane A2 stimulates mitogen-activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A2 mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of 32P to a specific substrate for MAPK. U46619 also liberated [3H] arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [3H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca2+ ions. This is a first demonstration that TXA2 activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

High performance liquid chromatography mobile phase composition optimization for the quantitative determination of a carboxylic acid compound in human plasma by negative ion electrospray high performance liquid chromatography tandem mass spectrometry

A systematic investigation was undertaken to study the effects of varying concentrations of additives in the acetonitrile/water high performance liquid chromatography mobile phase, especially formic acid and ammonium formate, on the negative ion electrospray response of a carboxylic acid compound. The study showed that the response progressively decreased with increase in the formic acid concentration. While such a decrease in the response could be qualitatively explained by the decrease in the concentration of the ionized form of the carboxylic acid compound due to the lower pH of the mobile phase, the change in response was not as large as expected from the change of the concentration of the ionized form. The response also progressively decreased with increase in the ammonium formate concentration but the decrease cannot be explained by the change in the pH of the mobile phase. Although the best negative ion electrospray response was obtained with a water/acetonitrile mobile phase that contained no additives at all, the retention time of the analyte was not found to be adequately reproducible on repeated injections. Thus, this mobile phase was deemed unacceptable for practical, routine use. Comparing formic acid against ammonium formate, the former was preferable since it caused a smaller attenuation of the negative ion response. Equally important was the fact that addition of formic acid had the desirable effect of maintaining a reasonably high capacity factor (k') for the analyte even at a relatively high acetonitrile concentration. A concentration of 1 mM formic acid in the mobile phase was large enough to achieve the reproducible elongated retention time for the analyte, with a loss in the analyte response of about 60% only. It should be noted that the sensitivity achieved with the 1 mM formic acid mobile phase, in which the carboxylic acid is expected to be about 10% in the ionized form, is about 9 times better than the sensitivity achieved in the 1 mM ammonium formate mobile phase, in which the carboxylic acid is expected to be about 99% in the ionized form.     

超高效液相色谱法测定甲基磺酸锡电镀液中甲基磺酸

准确测定甲基磺酸锡电镀液中甲基磺酸的含量对镀锡板稳定生产和电镀液老化评价有着重要意义。采用BEH Amide色谱柱(3.0×150mm,1.7μm),以0.010mol/L乙酸铵乙腈溶液-0.010mol/L乙酸铵水溶液(体积比为80∶20)为流动相,控制流速为0.40mL/min,建立了超高效液相色谱法测定甲基磺酸锡电镀液中甲基磺酸含量的方法。干扰试验表明:甲基磺酸锡电镀液中其他共存组分对甲基磺酸的测定均没有干扰。在甲基磺酸质量浓度为1.00~10.00mL/L范围内,甲基磺酸质量浓度的对数与其峰面积的对数呈线性关系,线性相关系数为0.999 6。将实验方法应用于甲基磺酸锡电镀液实际样品中甲基磺酸的测定,测得结果的相对标准偏差(RSD,n=11)为0.79%~1.2%,加标回收率为99%~102%。按照甲基磺酸锡电镀液配方体系配制甲基磺酸锡电镀液合成样品,并采用实验方法进行测定,测定值与理论值相符。     

Determination of nalidixic acid and its two major metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography

This paper describes a precise and sensitive method for analysis of nalidixic acid and its two major metabolites in plasma and urine following the oral administration of a therapeutic dose in humans. After addition of an internal standard (oxolinic acid), 1-ml samples of plasma or urine are extracted at acidic pH with chloroform. The extracts are purified by re-extraction with sodium hydroxide solution and then chloroform. The final extracts are evaporated to dryness, reconstituted in mobile phase and injected into a high-performance liquid chromatograph equipped with RP-8 column and UV detector operating at 254 nm. The limit of sensitivity of the method is lower than 0.5 micrograms/ml of plasma or urine for each compound. The applicability of the method to pharmacokinetic studies of nalidixic acid in humans is demonstrated.     

Quantitative analysis of ceramide molecular species by high performance liquid chromatography

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen for various neoplastic cells, including neoplastic bronchial epithelia. METHODS: Immunoreactive hepatocyte growth factor/scatter factor (HGF/SF) was measured in extracts prepared from 129 nonsmall cell lung carcinoma (NSCLC) specimens, using an enzyme-linked immunosorbent assay. These specimens represented 5 cases of solitary/localized bronchioloalveolar cell carcinoma (BAC), 4 cases of diffuse/infiltrative BAC, 90 cases of non-BAC adenocarcinoma, 25 cases of squamous cell carcinoma, and 5 cases of large cell carcinoma. RESULTS: The mean concentration of immunoreactive HGF/SF was more than 19-fold higher in tissue extracts from diffuse-type BAG (265.0 +/- 110.2 ng/100 mg protein) than in those from solitary-type BAC (13.9 +/- 15.9, P < 0.005), non-BAC adenocarcinoma (13.8 +/- 14.9, P < 0.001), squamous cell carcinoma (13.2 +/- 14.4, P < 0.001), or large cell carcinoma (11.2 +/- 6.5, P < 0.005). When immunohistochemical staining for HGF/SF was performed, intense HGF/SF staining was uniformly observed in diffuse-type BAC tumor cells, but not in solitary-type BAC. CONCLUSIONS: Although BAC is included as a subtype of adenocarcinoma in the World Health Organization classification, diffuse-type BAC should be considered a distinct biologic entity, at least in terms of HGF/SF expression, from solitary-type BAC or non-BAC adenocarcinoma. In addition, the solitary and diffuse forms of BAC are known to be associated with different prognoses; for the latter, the prognosis is much poorer than for the former. The results of this study may at least partly explain this difference in prognosis.     

Biological monitoring of exposure to chlorpyrifos by high performance liquid chromatography

Sixteen patients with hypogonadotropic hypogonadism received gonadotropin replacement therapy. Two patients treated with HCG alone showed induction of spermatogenesis 2 and 12 months after the start of treatment. Three subjects receiving combination therapy showed sperm appearance 6-28 months after treatment. The patients showing sperm appearance, whose testicular volume was > or = 4 ml, showed a higher sperm count and impregnated their partners, although no relationship was found between pretreatment testicular volume and sperm appearance. The response to HCG test correlated with sperm appearance after gonadotropin therapy. Sperm appearance was not observed in any subject except for one who showed no response to luteinizing hormone-releasing hormone (LH-RH) test and none of the patients without response of FSH to LH-RH demonstrated any induction of spermatogenesis. In conclusion, the responses to LH-RH test and possibly to HCG test could predict the induction of spermatogenesis after gonadotropin replacement therapy, and a large testicular volume is associated with post-treatment fertility.     

Determination of miloxacin and metabolites in human serum and urine by high-pressure liquid chromatography

A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin.     

索氏抽提-柱层析分离-高效液相色谱法测定煤沥青中多环芳烃

以甲苯为溶剂对煤沥青进行索氏抽提,抽提液经过有机滤膜过滤,过滤后的抽提液用二氯甲烷溶解,将样品溶液用硅胶柱分离,然后用体积比为1∶1的石油醚与甲苯混合液以5.0mL/min的流量进行淋洗,将淋洗液旋干后用乙腈定容至10mL,利用ZORBAX Eclipse PAH柱以不同体积比的乙腈-水体系为流动相对样品溶液进行梯度洗脱,建立了煤沥青中16种多环芳烃(PAHs)的高效液相色谱分离检测方法。结果表明,16种PAHs的线性范围为0.50~20mg/L,相关系数(r)不小于0.999,检出限为0.04~0.33μg/L,按照实验方法对湘钢煤沥青实际样品中16种PAHs进行测定,测得结果的相对标准偏差(RSD,n=6)为0.20%~3.5%,回收率为97%~109%。采用实验方法分别测定湘钢、涟钢两种不同煤沥青中16种PAHs的含量,测得结果与湘钢、涟钢两公司提供的推荐值基本一致。结果表明,每1kg湘钢煤沥青中16种致癌性PAHs质量为107.9g,即质量分数为10.79%;每1kg涟钢煤沥青中16种致癌性PAHs质量为104.1g,即质量分数为10.41%;其中苯并[a]芘分别为11.86g/kg和13.82g/kg,即质量分数为1.186%和1.382%。     

Determination of free bile acids in pharmaceuticals by thin layer chromatography and high performance liquid chromatography

High-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and thin layer chromatography with flame ionization detection (TLC-FID) have been applied to the separation of five main free bile acids present in humans: cholic (CA), chenodeoxycholic (CDCA), deoxycholic (DCA), lithocholic (LCA) and ursodeoxycholic (UDCA) acid. HPLC separation was performed on Biospher Si 100 column using a mixture of n-heptane, isopropanol, ethylacetate, methanol and glacial acetic acid as a mobile phase. All the compounds were separated in less than 12 minutes by using a gradient elution mode. TLC-FID separation was performed on S-II Chromarods with a mixture of isooctane, ethylacetate and glacial acetic acid as a mobile phase. HPLC-ELSD method was applied to the determination of CDCA and UDCA in pharmaceuticals and their purity control when LCA, DCA and CA were considered as impurities.