A fundamental and clinical study of ferritin 'Eiken' radioimmunoassay kit

We have measured serum ferritin level using double antibody radioimmunoassay kit (Eiken ICL) and evaluated the characteristics of the kit and clinical usefulness. Satisfactory results were observed in standard curve, reproducibility, dilution and recovery test. In clinical evaluation, we have measured in normal subjects and patients with various diseases. The range in normal males and females were 13.0-158.7 ng/ml and 7.3-73.0 ng/ml, respectively. Serum ferritin level was elevated in patients with hepatoma, biliary cancer, lung cancer and other malignant diseases. Measurement of serum ferritin value would be useful in the monitoring of cancer patients.     

A validated HPLC assay to monitor riluzole plasma or serum concentrations in patients with amyotrophic lateral sclerosis

A specific, accurate and precise high-performance liquid chromatographic assay was developed for the determination of riluzole, a drug used to treat patients with amyotrophic lateral sclerosis. Samples were treated by extraction with dichloromethane followed by reversed-phase chromatography with ultraviolet detection at 260 nm. Preset validation criteria were met from 20 to 2000 ng/mL with a linear response curve. Extraction recovery of riluzole was 65-76%. The accuracy of the method was 102-103%. Intra- and inter-day coefficients of variation were in the ranges 2.8-4.9% and 1.8-9.7%. A detection limit of 5 ng/mL was found. Determination of concentrations in serum and plasma resulted in similar results below 500 ng/mL. At higher values a matrix effect cannot be excluded. This presented method can be used to monitor plasma or serum levels in ALS patients treated with riluzole.     

Determination of the catechol-O-methyltransferase inhibitor Ro 40-7592 in human plasma by high-performance liquid chromatography with coulometric detection.

A sensitive and specific high-performance liquid chromatographic method has been developed to measure the catechol-O-methyl-transferase (COMT) inhibitor 3,4-dihydroxy-4'-methyl-5-nitrobenzophenone (Ro 40-7592) in human plasma. The compound and the internal standard were extracted from plasma at pH 2 with n-butyl chloride-ethyl acetate (95:5, v/v). The extract was chromatographed on a reversed-phase column (Hypersil ODS, 5 microns) using a mixture of phosphate buffer (0.05 M, pH 2), methanol and tetrahydrofuran (45:55:5, v/v/v) as the mobile phase. Long-retained components were removed from the system by means of a simple column-switching system. Quantification of the catechol-O-methyltransferase inhibitor was performed by means of coulometric detection (0.1 V). The limit of quantification was about 1 ng/ml, using a 1-ml specimen of plasma. The recovery from human plasma was greater than 88%. The mean inter-assay precision was 5.3% in the range 2.5-1000 ng/ml. Linearity of the standard curve was obtained in the concentration range 2.5-500 ng/ml. The catechol-O-methyltransferase inhibitor was stable in human plasma when stored for six months at -20 degrees C and for 24 h at room temperature. The practicability of the new method was demonstrated by the analysis of more than 400 plasma samples from a tolerance study performed in human volunteers.     

Solid phase radioimmunoassay for testosterone in human serum using antibodies coupled to magnetizable cellulose

Summary A simple user-friendly, solid phase radioimmunoassay for testosterone in human serum based on magnetic particles is described. IgG fractions precipitated from polyclonal testosterone antiserum were coupled to magnetizable cellulose particles using carbonyl diimidazole. The prepared antibody solid phase was stable for one year when stored at 4 °C. The optimized assay involves the incubation of 50 ml of testosterone standards (0.3-10 ng/ml), 100 ml of magnetizable cellulose particle coupled antibody suspension and 100 ml of ¹²⁵I-testosterone derivative for 4 hours at 37 °C. At the end of the incubation, the tubes were placed on a magnetic rack for 10 minutes after the addition of wash buffer and decanted. The sensitivity of the assay is 0.2 ng/ml. The intra-assay variation was <15% throughout the assay range. The recovery varied from 88 to 115%. On dilution of samples having high levels of testosterone, linearity ranged between 87 and 125%. Correlation coefficient of 0.978 was obtained when the developed solid phase assay was compared to the earlier established liquid phase assay.     

Determination of L-753,037 in human plasma by liquid chromatography/turbo ionspray tandem mass spectrometry.

A liquid chromatographic/turbo ionspray tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of L-753,037, a potent endothelin receptor antagonist currently under development for the treatment of cardiovascular diseases, in human plasma. L-753,037 is extracted from 0.5 ml of human plasma using liquid-liquid extraction and analyzed by LC/MS/MS with a turbo ionspray interface. Method validation results showed that this method is very sensitive, reliable, selective and reproducible. L-753,048, an ethoxy analogue of L-753,037, was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 50 pg ml(-1) with a linear calibration range of 0.05-50 ng ml(-1). The intra-day precision and accuracy (n = 5) were measured to be below 10% relative standard deviation (RSD) and between 97.4 and 102.8% of the nominal values, respectively, for all calibration standard concentrations within the calibration curve range. The inter-day precision and accuracy (n = 3 days, 5 replicates per day) were measured to be below 6.5% RSD and between 99.3 and 102.0% of the nominal values, respectively, for all quality control concentrations. The extraction recovery was determined to be approximately 99% on average. The analyte was found to be stable in plasma through three freeze-thaw cycles, for at least 4 h at ambient temperature and for up to 40 days under -20 degrees C freezer storage conditions. The analyte was also shown to be stable for at least 24 h in the reconstitution solution at room temperature and for up to 3 days as a dried extract at 4 degrees C. Additional variations in plasma concentration of the analyte due to the use of different sources of plasma were also evaluated.     

A rapid and selective LC method for simultaneous determination of cyclosporin a and its major metabolite (AM1) in human serum at room temperature

A simple and highly selective isocratic reverse-phase high performance liquid chromatography (RP-LC) method at room temperature is developed in order to determination of Cyclosporine A (CyA) and its major metabolite (AM1) in serum samples of kidney transplanted patients. The method uses a phenyl column stationary phase, acetonitrile–water–methanol 47:50:3 as mobile phase and 215 nm detector wavelength, at room temperature. The solid phase extraction procedure using cyano disposable extraction column was carried out to separtate the CyA and AM1 with recovery 99±6 and 98±10, respectively. A linear correlation was found at the range of 40–1000 ng ml⁻¹ for CyA and 25–500 ng ml⁻¹ for AM1. The average intra and inter-day variations were 5.03 and 7.89% for CyA, 5.92 and 8.12% for AM1, respectively. The detection limit of 20 ng ml⁻¹ was found for CyA and 12.5 ng ml⁻¹ for AM1. Also, the clinical application of the method using serum concentration against time profile from kidney transplantated patients is reported.     

Determination of ondansetron and its hydroxy metabolites in human serum using solid-phase extraction and liquid chromatography/positive ion electrospray tandem mass spectrometry

Ondansetron and its hydroxylated metabolites were determined in human serum using solid-phase extraction (SPE) and liquid chromatography/positive ion electrospray tandem mass spectrometry. Pyrimethamine was used as the internal standard. The analytes were eluted from the SPE cartridge using 2 x 1 ml of methanol containing 0.5% triethylamine, evaporated under vacuum and the residue was reconstituted in the mobile phase. The liquid chromatographic separation was achieved on a silica column using a mobile phase of aqueous 20 mM ammonium acetate (pH 4.7)-acetonitrile (85 : 15, v/v) at a flow-rate of 0.4 ml min(-1). The method was linear over the range 1-500 ng ml(-1) for ondansetron and each of the metabolites in human serum. The intra-day accuracy was better than 9.1% and the precision was <10.3%; the inter-day accuracy was better than 9.5% and the precision was <12.6%. The limit of detection was 250 pg ml(-1) based on a signal-to-noise ratio of 3. The absolute recovery from serum for all analytes was >90%.     

RP-HPLC测定兔血清和脑脊液中甲硝唑含量

 报告了用RP-HPLC测定免血清和脑脊液中甲硝唑浓度的方法，采用ZorbaxC8柱，NaH2PO4水溶液（0.0075mol／L）-甲醇（4:1，PH4.0）为流动相，氢氯噻嗪为内标，检测波长320um。线性范围0.5～80mg／L，最低检出浓度80μg／L（信噪比为3）。     

Simultaneous Determinations of Cinobufagin and Its Metabolites by Reverse-Phase High Performance Liquid Chromatography in Rat Serum and Urine

Abstract

A high-performance liquid chromatography was developed for simultaneous determinations of Cinobufagin (CB) and its metabolites deacetylcinobufagin, 3-epideacetyl-cinobufagin, 3-ketodeacetylcinobufagin, 3-epicinobufagin and 3-ketodeacetylcinobufagin in serum and urine of rat. The biological samples were extracted with diethylether-ethylacetate (4:1v/v) in the presence of bufalin as the internal standard. Recoveries for CB and all other compounds were in the range of 80.8%–100.2%. Excellent resolution was obtained by reversed-phase chromatography on a 4.6 mm I.D.×150 mm ODS column using acetonitrile:water (50:50 v/v) as the mobile phase at a flow-rate of 0.7 ml/min with UV detector at 300 nm. Standard curve data revealed linearity over a range of 10–500 ng per ml serum and urine. The detection limits of CB and its metabolites were less than 10 ng/ml. Coefficients of variation for the analysis were less than 10. CB and its some metabolites were observed in     

Immunochemical measurement and immunohistochemical detection of membrane-associated placental tissue protein 1

Using the avidin-biotin binding system, an enzyme immunoassay procedure was developed to measure the membrane-associated placental tissue protein 1 (MP1) in serum. The standard curve covered the range from 10 to 1000 ng/ml of MP1. The intra- and inter-assay coefficient of variations (C.Vs) were less than 5 and 10%, respectively. Recoveries of MP1 added to serum ranged between about 96 and 101%. The MP1 serum level was over 10 and under 112 ng/ml in non-pathological men, and under 240 ng/ml in non-pathological women. The MP1 level in the ovulatory phase was higher than in other phases of the menstrual cycle. In pregnancies during 6-39 weeks, the MP1 level ranged from 10 to 540 ng/ml, and it increased during the third trimester of gestational age. In benign gynecologic diseases, the MP1 concentration in serum ranged from 10 to 215 ng/ml. The MP1 levels in benign diseases were compared with those in ovarian malignancies, in endometrial carcinoma, and in uterine cervical cancer. The immunohistochemical location of MP1 was detected in the cell membrane of ovarian cystadenocarcinoma.     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

反相高效液相色谱法测定实验动物血液和脑组织中川芎嗪

 用反相高效液相色谱法测定了川芎药材、实验动 物血液和脑匀浆中的川芎嗪,方法简便快速。在270 nm检测波长下,不经浓缩可检测动物体 内1 mg/L的川芎嗪,川芎嗪在5～500 mg/L范围内具有良好的线性关系,线性相关系数为0.99 9。川芎嗪的加标回收率为98%～103％。川芎提取物样品、处理后的动物血清和脑匀浆样品具 有较好的稳定性,室温下可放置1星期。     

Solid phase inclusive immunoradiometric assay for human chorionic gonadotropin using monoclonal antibodies

Summary A simple, one step, inclusive immunoradiometric assay for human chorionic gonadotropin (hCG) employing monoclonal antibodies is described. Commercially available monoclonal antibodies from various commercial sources were screened. Identified “detection” antibody was radiolabeled with ¹²⁵I and the selected “capture” antibody was chemically coupled to magnetizable cellulose to form a solid phase. In the procedure developed, standard/sample, radiolabeled antibody and capture antibody were incubated together for 3 hours at room temperature with shaking. After incubation, the bound complex was quantitated for the associated radioactivity. The analytical sensitivity observed was 1.0 mIU/ml with a wide concentration range up to 1000 mIU/ml of hCG. “High dose hook” of the developed assay was observed beyond 2000 mIU/ml. Results showed that the developed assay had a good precision: intra-assay CV less than 8%, inter-assay CV less than 10% and good analytical recovery of 97-109%. The clinical samples analyzed by the developed procedure showed a good correlation with that of the commercial kit (r = 0.92; y = 0.99x+0.51).     

Clinical evaluation of serum neuron-specific enolase levels in patients with lung cancer

Serum neuron-specific enolase (NSE) levels were studied in 105 patients with malignant neoplasms (lung cancer 38, others 67), 13 patients with various benign diseases and 7 healthy adults. The mean serum NSE level in adult control subjects was 7.4 +/- 0.8 ng/ml, and cut off level was decided 10 ng/ml. Serum NSE levels were elevated in 14/38 (37%) of patients with lung cancer and in 14/67 (21%) of patients with the other malignant neoplasms. In patients with benign diseases, serum NSE level was elevated only in one patient with pituitary adenoma. In 7 patients with small cell lung cancer, the positive rate was higher (86%) than in those with non-small cell lung cancer (26%), and serum NSE levels were higher than 25 ng/ml except one case. There was no correlation between serum NSE and CEA (carcinoembryonic antigen) levels in patients with small cell lung cancer, also in patients with lung cancer. The measurement of serum NSE level seemed to be useful for diagnosis in patients with small cell lung cancer.     

Determination of fluorescent diamidines in plasma of experimental animals by high-performance liquid chromatography

The fluorescent diamidines (E)-2,2'-vinylenedi-1-benzo [b] furane-5-carboxamidine dihydrochloride (I) and 2-[2-(6-amidinoindole-2-yl)-(E)-vinyl]-1-benzofurane-5-ca rbo xamidine dihydrochloride (II) were determined in the plasma of experimental animals by high-performance liquid chromatography with a mobile phase of methanol-water (60:40, v/v) containing 0.005 M octanesulphonic acid and 0.003 M dimethyloctylamine. Samples were prepared by precipitation of plasma proteins with methanol-perchloric acid. Quantitation was performed by measuring the peak heights after monitoring the native fluorescence. The assay was linear over the range 5-750 ng/ml for I and 5-500 ng/ml for II, with limits of determination of 2.5 ng/ml for I and 1.5 ng/ml for II. Coefficients of variation were below 10% at all concentrations studied.     

Simultaneous determination of centbutindole and its hydroxy metabolite in serum by high-performance liquid chromatography.

A high-performance liquid chromatographic assay has been developed and validated for the determination of centbutindole and its hydroxy metabolite in serum. The method involves extraction of serum samples with diethyl ether at pH greater than 8, back-extraction into 0.5 M hydrochloric acid and finally again with diethyl ether after addition of 2 M potassium hydroxide. Separation was accomplished by reversed-phase high-performance liquid chromatography on a cyano column with an acetonitrile-phosphate buffer system. The recovery of centbutindole and its metabolite was always greater than 80%. Calibration curves were linear over the concentration range 0.25-5 ng/ml for centbutindole and 0.05-1 ng/ml for the hydroxy metabolite. Although the lower limit of detection was 0.1 ng/ml for centbuntindole and 0.02 ng/ml for the hydroxy metabolite, the reliable limits of quantitation were 0.25 and 0.05 ng/ml, respectively, using 4 ml of serum.     

8-Methoxypsoralen and long-wave ultraviolet A inhibit the release of proinflammatory mediators and cytokines from human Fc epsilon RI+ cells: an in vitro study

The in vitro effects of 8-MOP (concentrations of 20, 100 and 500 ng/ml) alone or in combination with UVA on mediator release from human basophils and skin mast cells (HSMC), activated with immunological and non-immunological stimuli, were investigated. With respect to basophils activated with anti-IgE serum, the results of this study show that: (i) 8-MOP alone inhibits histamine, LTC(4), IL-4 and IL-13 release concentration dependently with a maximal effect at 500 ng/ml (a concentration not reached in vivo); and (ii) UVA irradiation (5 J/cm(2)), after 8-MOP incubation, enhances this inhibitory effect on all released mediators, but for IL-4 and IL-13 the percentage inhibition is also significant for the 8-MOP concentrations (20-100 ng/ml) employed in vivo during PUVA treatment. Moreover, histamine release from basophils activated with non-immunological stimuli (FMLP and A23187) is inhibited by 8-MOP, alone or in combination with UVA. With respect to the HSMC activated with anti-IgE serum, the results show that: (i) 8-MOP alone reduces histamine release concentration dependently; and (ii) this inhibitory effect is enhanced by UVA irradiation (5 J/cm(2)). Histamine release from HSMC activated with A23187 is not modified either by 8-MOP alone or by 8-MOP plus UVA.     

Simultaneous and high-throughput quantitation of urinary tetranor PGDM and tetranor PGEM by online SPE-LC-MS/MS as inflammatory biomarkers

Quantitation of urinary tetranor PGDM or tetranor PGEM (tPGDM and tPGEM) in the past was performed separately using off-line SPE LC-MS/MS methods. The manual SPE procedure is generally time-consuming and cost-ineffective. In addition, simultaneous quantitation of tPGDM and tPGEM is favorable yet very challenging because of the similar chemical structures and identical MRM transitions. This work describes the development and validation of a high-throughput online SPE-LC-MS/MS method, allowing simultaneous and high-throughput measurement of tPGDM and tPGEM in human urine. The reportable range of the assay was 0.2-40 ng/ml for tPGDM and 0.5-100 ng/ml for tPGEM. Intra- and inter-assay precision and accuracy determined using quality control samples were all within acceptable ranges (% CV and % Bias < 15%). Tetranor PGDM was stable under all tested conditions while tPGEM was stable at 4 °C and after three F/T cycles but not stable at room temperature for 24 h (recovery below 80%). The assay was applied to measure urinary tPGDM and tPGEM among healthy volunteers, smokers and COPD patients. Significantly higher urinary levels of both tPGDM and tPGEM were observed in COPD patients than those of non-smoking healthy volunteers. These results demonstrated that the high-throughput online SPE-LC-MS/MS assay provides sensitive, reproducible and accurate measurement of urinary tPGDM and tPGEM as biomarkers for assessing inflammatory diseases such as COPD.     

A fully validated isotope dilution HPLC-MS/MS method for the simultaneous determination of succinylcholine and succinylmonocholine in serum and urine samples

A high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous detection of succinylcholine (SUX) and its metabolite succinylmonocholine (SMC) in serum and urine is presented. For internal standardization using isotope dilution, the deuterated compounds SUX-d(18) and SMC-d(3) were employed. Full validation was performed according to international guidelines. Solid-phase extraction (SPE) of acidified samples was accomplished using Strata-X polymeric reversed phase cartridges together with heptafluorobutyric acid (HFBA) as ion-pairing reagent. Separation was achieved within 13 min on a Phenomenex Synergi Hydro RP C18 column (4 microm, 150 x 2 mm) using a gradient of 5 mM ammonium formate buffer pH 3.5 and acetonitrile.To ensure the method's applicability in forensic as well as clinical toxicology, the specific demands of both research fields were taken into account, and the method was thus validated for a low and high concentration range. For both serum and urine as sample matrix, the validation revealed good intraday and interday precisions, consistently ranging below 15% for the lowest and below 10% for elevated concentrations. Accuracy was likewise good and never exceeded 10%. Extraction recovery was excellent, ranging between 88.1 and 103.9% for SUX and SMC in both tested matrices. Matrix effects were significant, the otherwise optimized extraction and detection methods, however, allowed for a very satisfactory sensitivity of the described method: For serum, the limits of detection and quantitation were determined to be 1.9 and 6.0 ng/ml for SUX, as well as 2.5 and 8.6 ng/ml for SMC, respectively; for urine, the corresponding values were established to be 1.4 and 4.0 ng/ml (SUX), as well as 1.5 and 4.9 ng/ml (SMC).The presented method was successfully applied to authentic samples of two forensic cases investigated in the institute of forensic medicine in Bonn, allowing the diagnosis of SUX intoxications.     

A validated method for the determination of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma using solid-phase extraction and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry

A liquid chromatographic-mass spectrometric method for the simultaneous determination of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma was developed and validated. Analytes and deuterated internal standards were extracted from human plasma using solid-phase extraction and analyzed by liquid chromatography/atmospheric pressure chemical ionization-mass spectrometric detection with selected ion monitoring (SIM). Limits of detection and quantification were 1.0 and 2.5 ng/ml, respectively, for all analytes. Linearity ranged from 2.5 to 500 ng/ml of human plasma using a weighting factor of 1/x; correlation coefficients for the calibration curves were > 0.99. Intra- and inter-assay precision and accuracy were < 15.0%. Recoveries were 108.2-110.8% nicotine, 95.8-108.7% cotinine, 90.5-99.5% trans-3'-hydroxycotinine, and 99.5-109.5% norcotinine. The method was also partially validated in bovine serum, owing to the difficulty of obtaining nicotine-free human plasma for the preparation of calibrators and quality control (QC) samples. This method proved to be robust and accurate for the quantification of nicotine, cotinine, trans-3'-hydroxycotinine, and norcotinine in human plasma collected in clinical studies of acute nicotine effects on brain activity and on the development of neonates of maternal smokers.