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.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface

A novel electrochemical immunosensor for the determination of carcinoma antigen 125 (CA125) was developed by means of immobilizing CA125 antibody (anti-CA125) on gold nanoparticles (Au) and thionine (Thi)-modified carbon paste interface. To avoid the leak of hydrophilic gold nanoparticles and thionine from carbon paste interface, the Au-Thi-modified carbon paste electrodes (CPEs) were first treated in the mixture solution containing 10% HNO₃ and 2.5% K₂Cr₂O₇ for 1.5 min at +1.5 V to make the carbon surface with -COOH groups, which can react with -NH₂ groups on the thionine molecule, in the meantime, gold nanoparticles were absorbed on the thionine surface. Subsequently, CA125 antibodies were assembled onto the surface of gold nanoparticles. The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy (FTIR) and UV-vis absorption spectroscopy. The performance and factors influencing the performance of the immunosensor were studied in detail. A direct electrochemical immunoassay format was employed to detect CA125 antigen based on the current change before and after the antigen-antibody reaction. The current change was proportional to CA125 concentration ranging from 10 to 30 U/ml with a detection limit of 1.8 U/ml (at 3δ). The immunosensors were used to analyze CA125 in human serum specimens. Analytical results of clinical samples show that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

Abstract

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 – 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10^?4M NaH₂PO₄ and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0–100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.     

Simultaneous Quantitation of Minoxidil and Tretinoin in Magistral and Pharmaceutical Preparations by First Derivative Spectrophotometry

Abstract

A first derivative spectrophotometry method has been developed for the simultaneous quantitation of minoxidil and tretinoin. The method is based on measuring the first derivative signals (D₁) of minoxidil and tretinoin at 290 and 351 nm, respectively, without any interference from each other, or any other coexisting materials. Beer's law was valid over the concentration range 2–10 μg/ml of minoxidil and 0.25–1.25 μg/ml of tretinoin. The proposed method has been applied successfully to the determination of some magistral and pharmaceutical preparations. Relative standard deviations for the assay of both drugs were less than 0.95%.     

Determination of Formaldehyde in Waste Water by Lucigenin Chemilummescence

Abstract

A chemiluminescence analysis has been developed for the determination of formaldehyde based on its inhibition of the chemiluminescence reaction of lucigenin-C10^?-H₂o₂. The method is sensitive, convenient and selective with a detection limit of 0.05ng/ml. The linear dynamic range is 1.0ng/ml to 0.1 μg/ml. The variation coefficient of ten determinations for 2.Ong/ml formaldehyde is 1.2%. Applications to the trace determination of formaldehyde in industrial waste waters are discussed.     

Phenanthroline method for quantitative determination of surface carboxyl groups on carboxylated polystyrene particles with high sensitivity

This study developed a phenanthroline method for quantitative determination of surface carboxyl groups on carboxylated polystyrene (PS‐COOH) particles based on the coordination between the carboxyl groups and Fe²⁺. The ratio of the carboxyl groups, which is determined by conductometric titration method, to Fe²⁺ coordinated with the particles, which is determined by phenanthroline method, is 4.7, i.e. n_COOH = 4.7 × Δn_Fe²⁺. The Lambert–Beer law is obeyed in the range of 0–60 × 10^?9 mol/ml for the amount of surface carboxyl on the particles. The detection limit of the method is 2 nmol COOH/ml. The average standard deviation of the experiments is 4.4%. The relative deviation of the data obtained with this method is lower than 7% compared with that obtained with the conductometric titration method. The weight of the sample necessary for phenanthroline method is only about 0.1% of that necessary for conductometric titration method. It has been demonstrated that the phenanthroline method is suitable for quantitative determination of low amount of surface carboxyl groups on PS‐COOH particles due to its high sensitivity. Copyright © 2009 John Wiley & Sons, Ltd.     

Kinetic Flow Injection Spectrophotometric Determination of Nitrite by its Catalytic Effect on the Oxidation of Chlorophosphonazo-pN by Bromate

Abstract

A flow injection kinetic method has been developed for the determination of nitrite, based on its catalytic effect on bromate oxidation of chlorophosphonazo-pN in H₂SO₄ medium. The reaction is followed spectrophotometrically by measuring the decrease in the absorbance at 551 nm. The sampling frequency was 83 h^?1. The calibration curve was linear between 0.050 and 1.00 μg/ml, and the detection limit was 0.018 μg/ml. The proposed method was applied to the determination of nitrite in waters and soil with satisfactory results.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Liquid Chromatography–Electrospray Ionization–Mass Spectrometric Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Pharmacokinetic Study

Abstract

A rapid, convenient, and sensitive liquid chromatography–electrospray ionization–mass spectrometry method was developed and validated for the quantification of hydrochlorothiazide in human plasma. The samples were first spiked with the internal standard, and the analyte was then extracted with ethyl acetate. The chromatographic separation was achieved on a C₁₈ column by using water–acetonitrile (68:32, v/v) as mobile phase. The method was linear within the range of 2.5–200 ng/ml. The lower limit of quantification was 1.0 ng/ml. Finally, the validated method was successfully applied for the evaluation of the pharmacokinetic profiles of hydrochlorothiazide in healthy male Chinese volunteers.     

A Method for the Determination of Methionine in Human Serum by High-Performance Liquid Chromatography with Electrochemical Detection

Abstract

We have found remarkable enhancement of electrochemical response for the analysis of methionine by use of a glassy carbon electrode preanodized in a 0.2 M phosphate buffer (KH₂PO₄?KOH, pH 6.5) at +1.9 V vs. Ag/AgCl for 2 min. On the basis of this finding, we have developed a method for the determination of methionine in human serum by reversed-phase high-performance liquid chromatography with electrochemical detection using the electrochemically pretreated glassy carbon electrode. The minimum detectable quantity of methionine has been found to be about 1 ng.     

Determination of Propylthiouracil in Human Breast Milk by Direct Injection High Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic(HPLC) method was developed for the assay of propylthiouracil in human breast milk. After filtration with membran filter(Molcut II), the eluent was injected into a liquid chromatogaph equipped with C₁₈ precolumn and analytical column in series according to column switching techniques. This method is sufficiently sensitive for most pharmacokinetic studies in human breast milk. The concentration of propylthiouracil was linear over the 50 – 5000ng/ml range. The recovery and the coefficient of variation was 92.0 – 100.6% and 1.6 – 2.9%, respectivery. This assay has the advantages of specificity, simplicity and reproducibility for the measurement of propylthiouracil in human breast milk.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Determination of Naloxone Hydrochloride in Dosage form by High-Performance Liquid Chromatography

Abstract

A new, sensitive and rapid method for the determination of naloxone hydrochloride as drug in dosage entity and form using HPLC has been developed. Authentic naloxone hydrochloride was used to establish a calibration curve. A linear relationship was obtained for concentrations ranging from 10 μg/ml to 50 μg/ml. The column used was C₁₈, Micropak MCH-10 (monomeric) and the mobile phase was acetonitrile : 0.01 M KH₂PO₄ (70 : 30) at a flow rate of 2 ml/min. Retention time for naloxone hydrochloride was 3.3 minutes. The proposed method has been proved accurate and precise compared to other pharmacopoeia methods of assay for naloxone hydrochloride.     

Functional Magnetic Nanoparticles for Clinical Application: Electrochemical Immunoassay of Hepatitis B Surface Antigen and α-Fetoprotein

This work reports an efficient method to quantify the Hepatitis B surface antigen and α-fetoprotein in human serum using a functional magnetic nanoparticle-assisted sandwich-type electrochemical immunoassay. The Fe ₃ O ₄ magnetic nanoparticles were first modified with carboxyl functional groups to permit stable bioconjugation to the amine groups of most biological targets. The primary antibodies were then covalently stained on the surface of the functional magnetic nanoparticles, followed by the analyte and secondary antibodies, resulting in a sandwich-type (antibody-antigen-antibody/enzyme) immune complex. The secondary antibodies were labeled with horseradish peroxidase for the catalytic oxidation of 2-aminophenol to yield electrochemically reducible molecules. The separation using an external magnetic field guaranteed fast and reliable purification and enrichment of analytes. Quantitative analysis was performed upon representative clinical targets: Hepatitis B surface antigen and α-fetoprotein in human serum. The detection limits were 0.06 ng/mL for the former and 0.5 ng/mL for the latter, which were about 10 times lower than values obtained by conventional enzyme-linked immunosorbent assays. The reported method may be adopted as a general strategy for the sensitive and selective determination of additional proteins and biological molecules.     

A Fluorimetric Method for the Determination of Atmospheric Sulphur Dioxide With 2′, 7′—Dichlorofluorescein∗

Abstract

A rapid, simple and sensitive fluorimetric method has been developed for the determination of atmospheric sulphur dioxide with dichlorofluorescein as fluorogenic reagent (λ_ex = 505 nm, λ_em = 520 nm) at pH 4.0–6.0. A linear calibration curve was obtained in the range 0.01–0.40 μg SO₂/25 ml. The detection limit is 0.01 μg SO₂/25 ml. Nitrogen dioxide does not interfere with the method. The method was successfully applied to the determination of atmospheric sulphur dioxide.

     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

A Fluorescence Synergistic Method for the Determination of Stilboestrol

Abstract

A simple, highly sensitive and selective fluorescence synergistic method has been developed for rapid determination of stilboestrol with Triton × — 100 (λ_ex = 318nm, λ_em = 415nm) at pH 8.80 ～ 10.20. The calibration graph is linear over the range of 0.0 ～ 30.0μg/10ml. The detection limit is 0.03μg/ml stilboestrol. The method has been successfully applied for the determination of stilboestrol in tabellae stilboestrol and injectio stilboestrol with satisfactory results. It can also be applied for the determination of stilboestrol in body fluid of special patients.     

A Fluorimetric Method for the Determination of Cyanide with Fluorescein and Iodine∗

Abstract

A rapid, simple and sensitive fluorimetric method has been developed for the determination of cyanide with fluorescein as fluorogenic reagent (λ_ex = 494 nm, λ_em = 514 nm) at pH 6.0–7.0. A linear calibration curve was obtained in the range 0.004–2.0 μg CN^?/25 ml. The detection limit is 0.004 μg CN^-/25 ml. The method was successfully applied to the determination of cyanide in waste water.

     

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.