Special Report: Standard Practice of Liquid Size-Exclusion Chromatography - SEC (GEL Permeation Chromatography - GPC

Abstract

A reversed-phase ion-pair liquid chromatographic method was applied to the analysis of bambuterol hydrochloride chemical reference substance (CRS) and tablets. The purity evaluation of the reference substance was performed with amperometric, UV, diode array and mass spectrometric detection. The total amount of impurities found was less then 0.1%.

For the determination of bambuterol hydrochloride in tablets the method reproducibility was 0.9% and the recovery was in the range 99.8–100.7%. Detection limits for conceivable degradation products were about 1 ng. The suitability of different column packing materials was investigated.     

Determination of Vitamin D3 and 25-Hydroxyvitamin D3 Using Liquid Chromatography with Amperometric Detection

Abstract

The electrochemical behaviour of vitamin D₃ and 25-hydroxyvitamin D₃ (25-OH D₃) in a high performance liquid chromatography system using amperometric detection is described. Separation is carried out using a C18 reversed-phase column and the optimum mobile phase was a 0.1 M LiClO₄ solution in methanol-water (97:3, v/v) at a flow rate of 1.25 ml/min. 25-OH D₃ and vitamin D₃ were eluted with good resolution at retention times of 3 and 6 minutes respectively, and determined by amperometric detection with a glassy carbon electrode at + 1.050 V (vs Ag/AgCl). Calibration graphs for both substances showed good linearity when amounts of vitamin D₃ between 18 and 312 ng and 27 and 412 ng of 25-OH D₃ were injected. Detection limits of 8 ng (vitamin D₃) and 25 ng (25-OH D₃); relative standard deviations of 3.2% (vitamin D₃) and 5.8% (25-OH D₃) were obtained.     

HPLC Assay of a Leukotriene D4 Antagonist,in Pharmaceutical Preparations and Biological Samples with UV and Fluorescence Detection

Abstract

Analytical methodology has been developed to assay L-660, 711, 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thio)propionic acid to assess drug stability in pharmaceutical preparations and drug concentration in plasma samples. Reversed-phase HPLC chromatography with UV detection at 232 nm and 300 nm was used to analyze drug content and assess stability in pharmaceutical formulations. In addition to UV, fluorescence detection with 300 nm and 400 nm as the excitation-emission wavelength pair was used to enhance sensitivity for biological samples with low drug concentrations. The latter system had a detection limit of 1–5 ng/ml for a 25 μl injection. It was used to monitor drug concentration in plasma following intrapulmonary, intravenous and oral administration.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection.     

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.     

Comparison of amperometric and UV-spectrophotometric monitoring in the HPLC analysis of pesticides

Summary Studies have been carried out to compare different detection techniques including amperometric detection in the reductive and oxidative mode and UV detection following the HPLC separation of pesticides. By the example of 4,6-dinitro-o-cresol (DNOC), 2-sec-butyl-4,6-dinitrophenol (Dinoseb) and 2-tert-butyl-4,6-dinitrophenol (Dinoterb), it is shown that the electrochemical detection exhibits higher sensitivities than the UV-technique. The detection limits are in case of the oxidative amperometric technique 0.1 ng for all pesticides, in the reductive mode 0.3 ng for DNOC and 1 ng for Dinoseb and Dinoterb. By UV-monitoring the detection limits were found to be 2 ng for DNOC and 24 ng for Dinoseb and Dinoterb.     

Determination of Organic Peroxides by High Performance Liquid Chroma-Tography with Electrochemical Detection

Abstract

The application of electrochemical detection to the high performance liquid chromatographic determination of organic peroxides has been studied. The use of a buffered mobile phase was found to be critical to the successful analysis of samples containing hydroperoxides. Using amperometric detection, mixtures of peroxide containing compounds were readily determined. The sensitivity of the amperometric detector was in the one nanogram range for both benzoyl peroxide and cumene hydroperoxide. Polar-ographic detection was found to be a highly reproducible method for the analysis of samples containing peroxides as components of mixtures in the range of 5-2000 ng. The peroxide containing compounds determined in this manner were t-butyl hydroperoxide, cumene hydroperoxide, and 13-hydroperoxy-9(Z)-11(E)-octadecadien-oic acid. The polarographic detection system was used to obtain observed half-wave potentials for the peroxides under different chromatographic conditions. These observations correlated closely with literature results on the polarography of these compounds.     

The Direct Electrochemical Detection of Amino Acids at a Platinum Electrode in an Alkaline Chromatographic Effluent

Abstract

It is the general experience that most organic compounds including amino acids do not produce reversible or even quasi-reversible anodic waves at a Pt electrode under conditions of conventional cyclic voltammetry. Furthermore, amperometric detection of these compounds at a constant electrode potential is not successful because of the accumulation of adsorbed reaction products and/or an oxide film at the electrode surface. However, it is observed that a Pt electrode surface is cleaned quite effectively of adsorbed organic molecles and radicals simultanaeously with the anodic formation of the oxide layer. This oxidation of adsorbed organic species is concluded to be electrocatalyzed by PtOH formed as the first step in the production of the oxide layer (PtO). A pulsed-potential waveform applied at a frequency of ca. 1 Hz is demonstrated to provide direct amperometric detection of adsorbed amino acids at a Pt electrode. Satisfactory analytical precision (i.e., < 3% rel. std. dev.) results because the waveform reproducibly generates the catalytically active surface state at the Pt electrode. Both primary and secondary amino acids are determined with satisfactory detection limits: e.g., ca. 13 ng for glycine, 7 ng for phenylamine and 23 ng for hydroxyproline in 50-μL samples. Analytical response is concluded to depend on the adsorption isotherm of the amino acid being detected. Hence, the calibration plot of I/I_peak vs. 1/C^D is linear for low surface coverages. Results are shown for amperometric detection of a synthetic mixture of amino acids by anion-exchange chromatography using NaOH as the eluent and supporting electrolyte.     

Comparative Study of the Determination of Parabens in Shampoos by Liquid Chromatography with Amperometric and Coulometric Detection

The simultaneous determination of four para‐hydroxybenzoic acid esters (parabens) in shampoos was studied by liquid chromatography (LC) with amperometric (LC‐AD) and coulometric (LC‐CD) detection. The parabens were separated on an ODS C18 reversed column by isocratic elution with a mobile phase based on methanol‐0.1 M acetic acid (60 : 40%, v/v) with 0.02 M NaClO₄ at a flow rate of 0.8 mL min^?1. The limit of detection (S/N>3) for the analytes was in the 15–25 pg (injected mass) range at an applied potential of 1.20 V vs. Ag/AgCl using the LC‐AD and in the 2–3 pg range at a potential of 0.790 V vs. Pd using the LC‐CD. The peak ratio of the internal standard peak (IS: 4‐hydroxybenzoic acid sec‐butyl ester) versus the analyte peak was found to be related to the amount injected from 0.1 ng to 100ng (r=0.996–0.999) with the LC‐AD and from 0.050 ng to 100 ng range (r=0.999–1.000) with the LC‐CD. The relative standard deviation (RSD, n=10) was comprised between 1.8 to 3.5% by LC‐AD ( 5 ng injected) and between 2.0 to 2.4% by LC‐CD (0.5 ng injected). The determination of four most used parabens in ten different shampoos was successfully realized.     

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     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

Expert system for pharmaceutical analysis. I. Selection of the detection system in high-performance liquid chromatographic analysis: UV versus amperometric detection

The usefulness of amperometric detection in pharmaceutical analyses was investigated for different groups of drugs. The UV response at 254 nm and that at the absorption maximum of the solute were compared with the electrochemical signal obtained. The minimum detectable concentration (nanograms on-column) of each substance is reported for the three different detection systems. This comparison was performed for 72 drugs (local anaesthetics, antipyretics, tricyclic antidepressants, sulphonamides, sex hormones, beta-adrenoceptor blocking agents, phenothiazines, alkaloids, diuretics and penicillins). The median limit of detection of the amperometric detector (see definition in the text) is 1.0 ng on-column and the median gain in sensitivity, compared with UV detection is 22.5.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

Detection of Phomenone by High-Performance Liquid Chromatography in Tomato Plants Infected by Phoma Destructiva Plowr

Abstract

Identification and quantitative determination of phomenone in ethyl acetate extracts from Tomato plants infected by Phoma destructiva Plowr., was carried out by high-performance liquid chromatography with UV detection, and ethanol-water (30/70, v/v) as mobile-phase on Perkin-Elmer RP-18/10 stainless column, at 20°C and 0.9 ml min^?1 flow rate. Detection limit was 5.5 ng, with standard deviation of ^± 3%, and retention time of 6.22 min. Analysis of extracts from spiked tomato fruits shows the same parameters. The method appears to be adequate for detection and quantitation of phomenone in contaminated tomato leaves and fruits using a pre-column packed with Lichroprep RP-18/25–40 μm.     

Rapid Determination of Bupropion in Human Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) technique has been developed for the determination of bupropion hydrocloride (Bup) in human plasma, using a reversed-phase method, with UV detection at 250 nm.

The internal standard 5-(P-methylphenyl)-5-phenylhydantoin (MPPH), was used as an aid to quantitation. The plasma was deprotemized with acetonitrile and the clear supernatant was directly injected in the chromatographic system. The lower limit of quantitation was 5.0 ng/ml using only 100 μl of plasma sample.

Linear regression analysis for the calibration plots obtained on five different days over a two-week period for the the two ranges used (10–250 ng/ml and 250–2000 ng/ml) in plasma indicated excellent linearity and reproducibility. The mean recovery of spiked Bup in plasma samples over the concentrations studied was found 96.5 ± 3.14%.

The method revealed that more than 30% of Bup was lost when the supernatant was stored at room temperature for 24 hrs.     

Amperometric Enzyme Sensor-Based Enzyme Immunoassay for Factor VIII-Related Antigen

Abstract

The coupling of an enzyme immunoassay for factor VIII-related antigen with a commercial glucose oxidase based amperometric sensor permits the determination of 1.6 to 16 ng of factor XIII-related antigen in human plasma. Further pure amperometric sensors or amperometric enzyme sensors for determination of the main marker-enzymes of enzyme immunoassays are described.     

An Amperometric Biological Toxic Hydrazine Sensor Based on Multiwalled Carbon Nanotubes and Iron Tetrasulfonated Phthalocyanine Composite Modified Electrode

Hydrazines are well‐known for their diverse biological properties but especially for their toxicity. An amperometric hydrazine sensor was developed at multi‐walled carbon nanotubes (MWCNT) and iron tetrasulfonated phthalocyanine (FeTsPc) composite modified electrode for the first time. The TEM and UV‐Vis spectroscopy results revealed the successful formation of MWCNT/FeTsPc composite. Compared with the response of MWCNT and FeTsPc modified electrodes, the MWCNT/FeTsPc composite showed enhanced oxidation current response with lower overpotential for hydrazine. Under optimum conditions, the amperometric i–t response of hydrazine was linear in the concentration range from 100 nM L^?1 to 3 μM L^?1 with the detection limit of 7.6 nM L^?1. The response time of hydrazine was found as 6 s with a high sensitivity of 7.615 μA/μM L^?1 cm^?2.     

Electrochemical Detection of Retinoids Using Normal Phase HPLC

Abstract

Non-aqueous electrochemical (EC) detection of 13-cis-retinoic acid, all-trans retinoic, acitretin and vitamin A palmitate in non-aqueous solvents are reported.

Non-aqueous (EC) detection allows for normal-phase chromato-graphy of these compounds prior to detection. The normal phase system used a mobile phase of HEX/THF/AcOH for the separation of all four compounds. The stationary phase was either silica or PVA-sil. The lipophilic salts, t-butylammoniumtetrafluoroborate or t-butyl-ammoniumhexafluorophosphate necessary for EC detection were added post-column.

The limit of detection (LOD) for EC detection of these compounds is approximately 1 ng on column compared with an LOD by UV absorption of 2 ng on column.

The linear detection for these compounds with the EC detector was about two orders of magnitude.     

Separation and Determination of Clenbuterol,Cimaterol and Salbutamol by Capillary Electrophoresis with Amperometric Detection

Capillary electrophoresis with amperometric detection was applied to determine some β₂‐agonists, such as clenbuterol, cimaterol and salbutamol in this paper. The working electrode used was a 0.3 mm diameter carbon disk electrode. In this work, the pH 6.0–6.4 borax‐potassium dihydrogen phosphate was used as running buffer (150 mmol/L), 10 kV as the separation voltage and 1.05 V (vs. Ag/AgCl, 3 mol/L KCl) as the detection potential. Under the optimum conditions, the analytes were baseline separated within 16 min. Linear range for cimaterol, clenbuterol and salbutamol was 1.0–2000, 2.0–2000 and 1.0–2000 ng/mL, respectively. The detection limits (define as 3σ/k) were 0.5, 1.0 and 0.4 ng/mL for cimaterol, clenbuterol and salbutamol, respectively. The developed method has been applied to determine these three analytes in feed and urine by standard addition. The mean recoveries for these three analytes ranged from 89.0% to 102.0%.     

A simplified method for levoglucosan quantification in wintertime atmospheric particulate matter by high performance anion-exchange chromatography coupled with pulsed amperometric detection

Levoglucosan, a tracer for the assessment of the biomass burning contribution to atmospheric particulate matter (PM) concentrations, was determined by means of high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). In this work we propose a modification in the instrumental set-up aiming at an improvement in the detector response by adding NaOH after chromatographic separation to increase the pH. The comparison between this technique and the gas chromatography/mass spectrometry (GC/MS) method commonly used showed good agreement. Repeatability is 4.8% RSD, limits of detection for pevoglucosan, mannosan and galactosan are in the range 0.001–0.002 µg mL^?1 in solution, corresponding to 3–4 ng m^?3 for 24 m³ of air sampled. PM10 samples were characterised for levoglucosan and for organic and elemental carbon contents. The preliminary results reported here for five sites in the Lombardy region (Northern Italy) are, as far as we know, the first data on levoglucosan contribution to OC in Italy. The levoglucosan concentrations observed in Lombardy vary in the range 173–963 ng m^?3 with an average levoglucosan-C to OC ratio ranging from 1.5% to 2.5%.