Level of Human Growth Hormone (hGH) in Urine Determined by A Highly Specific and Sensitive Sandwich Enzyme Immunoassay

Abstract

Levels of human growth hormone (hGH) in urine samples were determined by a highly specific and sensitive sandwich enzyme immunoassay, which had little cross-reaction with human luteinizing hormone, human follicle-stimulating hormone, human thyroid-stimulating hormone and human prolactin. The volume of urine samples used was 0.05 ml. Urine hGH levels in normal subjects, aged less than 2 yr, 2-5 yr, 6-15 yr and 28-35 yr (male) were 78-113, 19-51, 7.8-25 and 1.1-5.2 ng/g of creatinine, respectively. In dwarfism patients aged 6-15 yr, urine hGH levels were < 0.2-6.0 ng/g of creatinine.     

Ultrasensitive determination of human growth hormone (hGH) with a disposable electrochemical magneto-immunosensor

In this paper, an electrochemical magneto-immunosensor for the detection of human growth hormone (hGH) is described for the first time. The immunosensor involves the use of tosyl-activated magnetic microparticles (TsMBs) to covalently immobilize a monoclonal mAbhHG antibody. A sandwich-type immunoassay with a secondary pAbhGH antibody and anti-IgG labelled with alkaline phosphatase (anti-IgG-AP) was employed. TsMBs–mAbhGH–hGH–pAbhGH–anti-IgG-AP conjugates were deposited onto the surface of a screen-printed gold electrode using a small neodymium magnet, and electrochemical detection was performed by square-wave voltammetry upon the addition of 4-aminophenyl phosphate as the AP substrate. All the variables involved in the preparation of immunoconjugates and in the immunoassay protocol were optimized. A calibration curve for hGH was constructed with a linear range between 0.01 and 100 ng/mL (r?=?0.998) and a limit of detection of 0.005 ng/mL. This value is nearly three orders of magnitude lower than that obtained using surface plasmon resonance (Treviño et al., Talanta 78:1011–1016, 2009). Furthermore, good repeatability, with RSD?=?3% (n?=?10) at the 1-ng/mL hGH level, was obtained. Cross-reactivity studies with other hormones demonstrated good selectivity. The magneto-immunosensor was applied to the analysis of human serum spiked with hGH at the 4- and 0.1-ng/mL levels. Mean recoveries of 96?±?6% and 99?±?2%, respectively, were obtained.     

Fluorophotometric Determination of Glucuronic Acid Using the Fluorescence-Reaction with N-Benzyl-2-Naphthylamine

Abstract

First, the fluorescence-reactions between glucuronic acid (D-glucuronolactone) as an uronic acid and N-benzyl-2-naphthylamine(NBNA) or N-benzyl-1-naphthylamine(NB-1-NA) as a secondary naphthylamine were systematically investigated in acetic acid medium. Secondly, NBNA-glucuronic acid fluorescence-product that has a maximum emission wavelength(Em) at 508 nm with excitation wavelength(Ex) at 468 nm was used for a selective assay of glucuronic acid; the calibration curve was linear in the range from 5 ng/ml to 2.0 μg/ml glucuronic acid, and the recovery test in normal urine was satisfactory (98.4–102.5%).     

A Unique Solid Phase Extraction Column for Isolation of 11-Nor-Δ-9-Tetrahydrocannabinol-9-Carboxylic Acid in Human Urine

Abstract

A sensitive, specific, qualitative, and quantitative extraction procedure followed by an hplc assay of 11-nor-Δ-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from human urine is developed. Using a new, “mixed mode”, bonded silica gel solid phase extraction (SPE) column, the analyte was selectively isolated from the urine component. Following extraction, the presence of THC-COOH was confirmed and quantitated by a UV detector on a Varian 15cm C18 column using 35:65 v/v 50 mM phosphoric acid:acetonitrile at a flow rate of 1.5 mL/min. The limit of detection was 10 ng/mL at a signal to noise ratio of 2.5. The method showed linearity in the 10–300 ng/mL range (r=0.999) with good precision (RSD 1.4%) and accuracy (87% absolute recovery).     

High Performance Liquid Chromatoraphic Determination of R-831, A New Antiallergic Agent,in Dogs and Humans

Abstract

A simple, selective and sensitive HPLC method has been developed to measure R-831 levels in dogs and humans. It is an internal standard technique with a single step extraction and one wash. Samples are chromatographed on a reversed-phase system with ultraviolet detection. The lowest detectable concentration for plasma is 25 ng R-831/ml with a 1 ml sample and the linear range is 25–8000 ng R-831/ml. The lowest detectable concentration for urine is 250 ng R-831/ml with a 0.1 ml sample and the linear range is 250–8000 ng R-831/ml. This method has been used to quantitate levels of R-831 in bioavailability and toxicity studies in dogs, and in pharmacokinetic and efficacy studies in humans.     

A New Method for the HPLC Analysis of Pt(II) in Urine

Abstract

An analytical method has been developed to measure Pt(II) in urine via derivatization and UV or HPLC analysis. A measured quantity of urine is heated briefly with diethyl ammonium diethyl-dithiocarbamate, and the resulting Pt(Et₂NCS₂)₂ is extracted into a measured volume of chloroform. Concentrations of Pt(II) are determined by UV absorption at 346 nm or by reverse phase HPLC analysis. The detection limit for Pt(II) as its dithiocarbamate is ～ 1 ng by HPLC; the concentration limit for HPLC analysis by direct extraction was ～ 25 ng/ml. Chromatographic response was linearly related to Pt(II) concentration over the range 100-4, 000 ng/ml; dilution of more concentrated samples has extended this range to at least 30, 000 ng/ml. This method has been applied to the analysis of Pt(II) in the urine of patients who have received cis-dichlorodiamniineplatinum(II) (CDDP) chemotherapy.     

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     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

The Determination of a New Trifluorinated Quinolone,Fleroxacin, Its N-Demethyl,and N-Oxide Metabolites in Plasma and Urine by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method is described for the determination of the new fluoroquinolone Ro 23–6240 and its N-demethyl and N-oxide metabolites in plasma and urine. The three substances were extracted from aqueous solution with dichloromethane/isopropanol containing sodium dodecyl sulphate. After evaporation and reconstitution, samples were analysed on a reversed-phase column using ion pair chromatography and fluorescence detection. The limit of quantification was 10–20 ng/ml (RSD 4%) using a 0.5 ml plasma sample, and the inter assay precision was 3–10% over the concentration range 50 ng/ml to 20 μg/ml. Recovery from plasma was 81% (RSD 10%) over the range 10 ng/ml to 5 μg/ml. The method has been applied successfully to the analysis of several thousand samples from human pharmacokinetic studies. Care has to be taken to avoid exposure of samples to direct sunlight, and the use of opaque vessels for sample storage and handling is recommended.     

Fluorometric Determination of WR-148, 946[α-DI-n-Butylaminomethyl-2,6-bis(Trifluoromethylphenyl)-4-Pyridine Methanol Hydrochloride] in Biological Fluids

Abstract

A fluorometric procedure for WR-148,946 is reported based upon a fluorophore formed in 9 N sulfuric acid. A study of various acidic, neutral, and basic media is presented to establish sulfuric acid as the solvent system needed for maximum fluorescence intensity. A profile of various normal solutions of sulfuric acid showed that 9 N gave highest fluorescence readings. The fluorophore in 9 N sulfuric acid is postulated to be an “ene” formed from dehydration of the drug. Fluorescence is linear over the range 2–1000 ng/ml of sample with a quantum yield of 0.45. Application of the procedure to the analysis of WR-148, 946 in human plasma and urine gave an accuracy of 2.5–6.2%.     

Determination of the New Ace-Inhibitor Quinapril and Its Active Metabolite Quinaprilate in Plasma and Urine by High-Performance Liquid Chromatography and Pre-Column Labelling for Fluorescent-Detection

Abstract

A sensitive and selective method for the determination of quinapril and its active metabolite quinaprilate in human plasma and urine is described. The method is based on isolation using C18 Bond Elut cartridges, pre-column derivatization with 9-anthryldiazo-methane and purification of the reaction mixture on CBA columns followed by reversed-phase high performance liquid chromatography with fluorometric detection. Calibration curves were linear between 20 ng and 1000 ng/ml of plasma (100-2000 ng for urine) for both substances, the lower limit of detection being 5-10 ng/ml.

The present assay procedure has been applied to monotoring plasma and urine concentrations in several pharmacokinetic studies in humans.     

Reverse Phase HPLC Determination of Azq in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method employing a simple sample preparation procedure and utilizing an internal standard was developed to measure the new antitumor agent AZQ in biological fluids. A single chloroform extraction gave drug recoveries of greater than 88% from plasma, urine and CSF in the range of expected physiological concentrations (20–800 ng/ml). Isocratic reverse phase HPLC with UV detection at 340 nm resulted in a limit of quantisation of 5 ng/ml although smaller amounts of the drug could be detected. This assay was successfully applied to determine the single dose plasma pharmacokinetics of AZQ in rats. The potential of this method for determining AZQ disposition and pharmacokinetics in human subjects was demonstrated by analysis of patient CSF.     

A high selective and sensitive liquid chromatography–tandem mass spectrometry method for quantization of BPA urinary levels in children

A selective and highly sensitive liquid chromatography–tandem mass spectrometry method has been developed and validated for determination of Bisphenol A (BPA) in human urine using labeled d₆-BPA as internal standard. BPA was purified from human urine by affinity chromatography on solid extraction AFFINIMIP® Bisphenol A cartridges, based on molecularly imprinted polymers. After purification, the samples were analyzed on a Phenomenex Kinetex 100?×?4.6 mm, 2.6 μm particle PFP reversed-phase HPLC column, coupled to a triple quadrupole mass spectrometer by an electrospray ion source. Analyses were performed in the multiple reaction monitoring mode and negative ionization; the product ions at 133.2 and 212.1?m/z for BPA and at 138.2 and 215.0?m/z for d₆-BPA were monitored to assess unambiguous identification. The linearity of the detector response was verified in human urine over the concentration range 0.100–200 ng/mL. The detection limit was calculated as 0.03 ng/mL and the limit of quantification of the method is 0.10 ng/mL. This LC/ESI-MS/MS method was in-house validated evaluating specificity, trueness, within-day and between-days precision. The mean recoveries of BPA from spiked urine samples were higher than 94 % and good reproducibility (relative standard deviations?≤?8.1 %) was observed. The developed method was applied to a pilot study involving 105 children, aged from 6 to 14 years (16 normal weight and 89 obese children), from the Regione Campania (Southern Italy). The aim of this study was to determine the concentrations of BPA in urine of children and possible correlations with childhood obesity.     

Analysis of Indenolol In Biological Fluids By High Performance Liquid Chromatography

Abstract

The analysis of indenolol in plasma and urine is described. The method involves extraction of the drug from plasma or urine using chloroform at basic pH. The separation was performed on CN column using methanol and 0.01M potassium dihydrogen phosphate solution 50:50. The efficiency of extraction was 97%. Minimum detectable amount by fluorescence was 20 ng/ml.     

Optimization of solid phase microextraction procedure for determination of paraquat using reduction process

Headspace solid phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry was developed for the determination of paraquat in urine. The volatile product resulting from reduction of paraquat by sodium borohydride–nickel chloride was used for HS-SPME. The calibration curve was linear from 10–1000 ng/mL and the limit of detection was 0.1 ng/mL. The optimized methods were validated using 500 and 750 ng/mL concentrations of paraquat in the spiked urine samples. The recoveries obtained in this study were 98.2% for intra-day (n = 6) and 99.2% for inter-day (n = 6), respectively, with RSD lower than 1.1%.     

Pyrocatechol-1-Aldehyde 2-Benzothiazolylhydrazone As Reagent for the Spectrofluorimetric Determination of Nanogram Amounts of Gallium in Urine and Blood Serum

Abstract

A method is developed for the spectrofluorimetric determination of 1–80 ng.ml^?1 of gallium with pyrocatechol-1-aldehyde 2-benzothiazolylhydrazone, in a 50% (v/v) DMSO-Water medium at apparent pH 4.0 (monochloracetic/monochloracetate buffer). Λ_ex = 400nm, Λ_em = 504 nm (corrected). Interferences have been evaluated and the method applied to the determination of gallium in human urine and blood serum samples.     

Revised HPLC Determination of Atenolol in Plasma and Urine

Abstract

An HPLC method for analysis of atenolol in human plasma and urine is presented. Based on alkaline extraction, acid backextraction and reverse phase ion-pair chromatography this method is quite specific for atenolol. For a 0.5 ml plasma sample the sensitivity ranges from 20 ng/ml in fasted healthy volunteers to 50 ng/ml in various groups of patients. A sensitivity in urine of 1.0 mcg/ml was sufficient for all samples studied. As presented this method has been used in several clinical pharmacokinetic studies involving hundreds of samples.     

Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS

The aim of this work was to synthesize morphine‐3‐O‐sulfate and morphine‐6‐O‐sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC‐MS/MS method. Morphine‐6‐O‐sulfate and morphine‐3‐O‐sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed‐phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5–500 ng/mL for morphine‐3‐O‐sulfate and 4.5–454 ng/mL for morphine‐6‐O‐sulfate in plasma. In urine, the measuring range was 50–5000 ng/mL for morphine‐3‐O‐sulfate and 45.4–4544 ng/mL for morphine‐6‐O‐sulfate. The intra‐assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine‐3‐O‐sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine‐6‐O‐sulfate, while no detectable morphine‐6‐O‐sulfate was found in plasma samples.     

Determination of exemestane and 17-hydroxyexemestane by high-performance liquid chromatography coupled with tandem mass spectrometry and high-resolution mass spectrometry

An approach was developed for determining and confirming the presence of exemestane and its metabolite 17-hydroxyexemestane in urine. It is based on the application of high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-MS/MS) and atmospheric pressure chemical ionization high-resolution mass spectrometry (HPLC-HRMS). To detect hydroxyexemestane, the analysis of the hydrolyzed fraction of urine is preferable. The recovery rates of exemestane and 17-hydroxyexemestane were 83 and 91%, respectively. The detection limits were 1 ng/mL for HPLC-MS/MS and 2.5 ng/mL for HPLC-HRMS. In spite of a considerable effect of ionization suppression, the sensitivity and selectivity of the determination are affected by the selection of the optimal detection conditions in HPLC-MS/MS and by the high accuracy of mass determination in mass spectrometry with orbitrap detection, enabling resolution at a level of 5 ppm. The procedures can be used for screening and confirmatory analysis.     

Simultaneous quantitative and qualitative analysis of aliskiren,enalapril and its active metabolite enalaprilat in undiluted human urine utilizing LC‐ESI‐MS/MS

The benefit–risk ratio of combined blocking by the direct renin inhibitor aliskiren and an angiotensin‐converting enzyme inhibitor (e.g. enalapril) on the renin–angiotensin–aldosterone system is discussed. No method was available for simultaneous determination of both drugs in urine. A novel sensitive method for simultaneous quantification in undiluted human urine was developed which enables systematic pharmacokinetic investigations, especially in poorly investigated populations like children. Matrix effects were clearly reduced by applying solid‐phase extraction followed by a chromatographic separation on Xselect^TM C₁₈ CSH columns. Mobile phase consisted of methanol and water, both acidified with formic acid. Under gradient conditions and a flow rate of 0.4 mL/min the column effluent was monitored by tandem mass spectrometry with electrospray ionization. Calibration curves were constructed in the range of 9.4–9600 ng/mL regarding aliskiren, 11.6–12000 ng/mL for enalapril and 8.8–9000 ng/mL for enalaprilat. All curves were analyzed utilizing 1/x²‐weighted quadratic squared regression. Intra‐run and inter‐run precision were 3.2–5.8% and 6.1–10.3% for aliskiren, 2.4–6.1% and 3.9–7.9% for enalapril as well as 3.1–9.4% and 4.7–12.7% regarding enalaprilat. Selectivity, accuracy and stability results comply with current international bioanalysis guidelines. The fully validated method was successfully applied to a pharmacokinetic investigation in healthy volunteers. Copyright © 2014 John Wiley & Sons, Ltd.