High-performance Liquid Chromatography System Research Articles

Selective determination of ubiquinone in human plasma by HPLC with chemiluminescence reaction based on the redox cycle of quinone

Ubiquinone is an important biologically active compound in the living body. The determination of ubiquinone in human plasma is useful for the investigation of bioavailability of ubiquinone and for early diagnosis of several diseases. Therefore, we developed a high-performance liquid chromatography (HPLC) with chemiluminescence detection method for the analysis of ubiquinone in plasma samples. The method is based on luminol chemiluminescence detection of super oxide anion that is generated by the redox cycle reaction between ubiquinone and dithiothreitol. The HPLC system involved an octyl column with a mobile phase of methanol. Ubiquinone eluted from the column was mixed with dithiothreitol and luminol solutions simultaneously, and generated chemiluminescence was monitored by chemiluminescence detector. The calibration curve for standard ubiquinone solution was linear from 0.09 to 43.2μg/mL (0.45-216ng on column) with the correlation coefficient of 0.999, and the detection limit (S/N = 3) was 26ng/mL (130pg on column). Using the proposed HPLC method, the peak of ubiquinone in human plasma could be clearly detected on the chromatogram without any interference from plasma components.

Metabolomic approach based on liquid chromatography coupled to high resolution mass spectrometry to screen for the illegal use of estradiol and progesterone in cattle

Administration of hormonal compounds as growth promoters in livestock farming was banned by Council Directive 93/22/EC, however, this kind of substances are sometimes reported within the framework of European monitoring residue plans. Various analytical methods have been previously developed to screen for their misuse, and they are now especially efficient for monitoring the illegal administration of synthetic and semi-synthetic hormones. Nevertheless, proving an exogenous administration of hormones from natural origin (i.e. estradiol-17β or progesterone) still remains a challenging task for European authorities. As a result of their origin, these target compounds are indeed always present in the analytical matrix, and because the concentration levels of natural steroids are extremely variable from one animal to another, the establishment of reference thresholds appears very difficult. During this preliminary study, metabolomic data was acquired on a high performance liquid chromatography system coupled to high resolution mass spectrometer (HPLC–LTQ-Orbitrap). Serum samples were collected from dairy cows treated or not with sex steroid hormones commonly employed in animal husbandry: estradiol-17β (or its ester estradiol benzoate) and progesterone. After appropriate data processing and multivariate statistical analyses (OPLS-DA), it was possible to highlight significant metabolic modifications in serum consecutively to the administration of estradiol and/or progesterone. Those differences were used to build predictive models able to suspect illegal administration of these hormones in cattle. Potential biomarker candidates of estradiol and/or progesterone were pointed out, that remains to be structurally elucidated.

Photocatalytic behavior of ZnO and Pt-incorporated ZnO nanoparticles in phenol degradation

Nanostructured Pt–ZnO composite thick films were prepared using the composite nanoparticles synthesized through Triton X-100 polymer assisted thermolysis of zinc acetate. It has been observed that only a fraction of added Pt incorporates into the ZnO lattice and the rest segregates over the ZnO nanoparticle surface to form metallic nanoclusters. Morphology, crystallinity, and optical properties of the ZnO and Pt–ZnO nanostructures have been studied. Photocatalytic behavior of the samples for phenol degradation process has been studied in a high performance liquid chromatography system. It has been observed that though the phenol degradation rate is higher for pure ZnO nanostructures, Pt–ZnO nanostructures act as selective catalyst, producing only one intermediate product and dissociating it faster than ZnO.

Determination of Oxyclozanide in Beef and Milk using High-Performance Liquid Chromatography System with UV Detector

This study was developed and validated for the determination of oxyclozanide residue concentrations in beef and commercial milk, using high-performance liquid chromatography system. Oxyclozanide was successfully separated on a reverse phase column (Xbridge-C18, 4.6×250 mm, 5 µm) with a mobile phase composed of acetonitrile and 0.1% phosphoric acid (60:40, v/v%). This analytical procedure involved a deproteinization process using acetonitrile for beef and 2% formic acid in acetonitrile for commercial milk, dehydration by adding sodium sulfate to the liquid analytical sample, and a defatting process using n-hexane; after these steps, the extract was exposed to a stream of nitrogen dryness. The final extracted sample was dissolved in the mobile phase and filtered using a 0.45 µm syringe filter. This method had good selectivity and recovery (70.70±7.90-110.79±14.95%) from the matrices. The LOQs ranged from 9.7 to 9.8 µg/kg for beef and commercial milk. The recoveries met the standards set by the CODEX guideline.

A simple and easy method for determination of meloxicam in rat muscle and plasma

The aim of this paper is to provide a simple and easy method for determination of meloxicam in rat muscle and plasma, based on dissolution of tissue samples with SolvableTM and measurement with a column-switching high performance liquid chromatographic system. After tissue and plasma samples were extracted with SolvableTM and methanol, samples were injected into a precolumn (Hypersil ODS) in 0.05 M phosphate buffer (pH 3), and after clean-up and enrichment the analytes were transferred to an analytical column (YMC Pack Pro C18) by backflushing with 0.05 M phosphate buffer (pH 6)-methanol (60:40, v/v). The eluate was monitored with a UV-detector set at 360 nm. Coefficients of determination (r2) for muscle and plasma were both more than 0.999. The limits of quantification (LOQ) in muscle and plasma were 50 ng/g and 20 ng/mL, respectively. The accuracy and precision were achieved in the range of 50-2500 ng/g in muscle and 20-2500 ng/mL in plasma. Our method could be successfully applied to evaluate the pharmacokinetics of topically administrated meloxicam.

Estimation of Pharmacokinetics of Propofol in Indian Pateints by HPLC Method

A quick and sensitive reversed phase high performance liquid chromatography (HPLC) method has been developed in Indian surgical patients to determine the concentration of Propofol In human plasma. Propofol can be isolated from human plasma by adding 1ml precipitating solution which consists of acetonitrile and perchloric acid (67:33) mixture, which also contains dibutylpthalate (1mg/ml) as an internal standard. The sample is mixed for two minute on a vortexer. The plasma substance precipitated by acetonitrile and perchloric acid are further separated by centrifugation. The supernatant is directly injected into the HPLC system with the help of autosampler. The analysis was carried out using column 250 × 4.6 mm column packed with10-μm Spherisorb reversed phase octadecyl silane particles (C 18 ). The 500ml of mobile phase (67:33:0.04) consisted of 335ml of acetonitrile and 165ml of distilled water and 200μl of acetic acid maintaining the pH 4.0.The flow rate of the mobile phase was 1.5ml/ min. propofol was monitored by a UV detector at a 270nm wavelength. The limit of detection of propofol (in human plasma) was found to be 0.0001μg/ml while limit of quantification was found to be 0.001μg/ml for a 20ul injection volume.

Determinação de 2,5-hexanodiona em urina empregando cromatografia líquida de alta eficiência, após derivatização com 2,4-dinitrofenil-hidrazina

A method for quantifying urinary 2,5-hexanedione was optimized and validated. Urine samples were hydrolyzed and derivatized with 2,4-dinitrophenylhydrazine. The analyte was separated in a high performance liquid chromatography system with a diode array detector, using a C18 column (150 x 4.6 mm, p.d. 5 µm) and a mobile phase composed of phosphate buffer pH 2.3:acetonitrile (40:60, v/v), at a flow rate of 1 mL/min. The chromatograms were monitored at 334 nm. Retention time was 7.3 minutes. Main validation parameters were: coefficient of determination: 0.9994, accuracy: 96 to 107%; intra-assay precision (RSD): 3.08 to 6.72%; inter-assay precision (RSD): 2.54 to 8.17% and limit of quantitation of 0.19 µg/mL.

α, β-DCP-LA Selectively Activates PKC-ε and Stimulates Neurotransmitter Release with the Highest Potency among 4 Diastereomers

Background/Aims: We have been probing bioactivities of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a linoleic acid derivative with cyclopropane rings instead of cis-double bonds, using a racemic modification. Racemic DCP-LA contains possible 4 diastereomers. We, therefore, separately synthesized DCP-LA diastereomers such as α α-, α, β-, β,α-, and β, β-DCP-LA and assessed the effects of each diastereomer on protein kinase C (PKC) activity and transmitter release. Methods: PKC activity under the cell-free conditions and in PC-12 cells, and glutamate, dopamine, and serotonin released from rat brain slices were assayed with a high performance liquid chromatography (HPLC) system. Results: Of 4 diastereomers α, β-DCP-LA selectively and directly activated PKC-ε, with the highest potency. α β-DCP-LA stimulated release of glutamate, dopamine, and serotonin from rat hippocampal, striatal, and hypothalamic slices, respectively, under the control of PKC, possibly PKC-ε, and α7 nicotinic ACh receptors, with the highest potency among 4 diastereomers. Conclusion: α, β-DCP-LA serves as a selective and direct activator of PKC-ε, to stimulate transmitter release by targeting α7 nicotinic ACh receptors. This suggests that α, β-DCP-LA could be developed as a promising drug for treatment of not only dementia but neurodegenerative diseases and psychiatric disorders due to reduction/deficiency of neurotransmitters.

高速液体クロマトグラフィー法を利用したビアフィリング硫酸銅めっき用添加剤の濃度分析

Recently, via-filling plating used build-up processes has been an active topic of study. Generally, polyethylene glycol 4000 (PEG4000) has been used as a carrier with a wide concentration range of 100-1000 ppm. In addition, Bis-(sodium sulfopropyl)-disulfide (SPS) is used as a brightener with a very narrow concentration range of 1-10 ppm. The concentration of additives is considered to affect via-filling plating strongly. However, no simple analytical method has been reported for these additives. This study analyzes the concentrations of PEG4000 and SPS in a simulated plating bath. These additives were analyzed using a high-performance liquid chromatography (HPLC) system incorporating boron-doped diamond (BDD) as the electrochemical detection electrode material. Results show that PEG4000 and SPS in the simulated plating bath were detectable in separation. The correlation coefficients between added concentration and peak currents were 0.988 for PEG4000 and 0.999 for SPS.

The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures.

Perspectives on Method Validation: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

<b>Perspectives on Method Validation</b>: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

Liquid chromatographic estimation of (S) - glycidyl butyrate in (R) - glycidyl butyrate

Aim:The present study was undertaken out of a commercial need for the synthesis of Linezolid with impurity limits within the specification.Materials and Methods:(R)-Glycidyl butyrate (RGB) is raw material for the synthesis of Linezolid drug substance. This RGB contains (S)-(+)-Glycidyl butyrate (SGB) and SGB appears in same concentration in the final Active Pharmaceutical Ingredient. So, a normal phase high-performance liquid chromatography (HPLC) method has been developed to determine the SGB level in the raw material. RGB and SGB were separated using an HPLC system equipped with quaternary gradient pumps on a Daicel chiralpak AD-H (250 × 4.6 mm) column with a mobile phase consisting 2.0 ml of ethanol in 1 000 ml of n-hexane. A 0.5 ml/minute flow rate and a 10 μl injection volume was used and the compounds were detected at 215 nm.Results:Method validation parameters demonstrated the same to be reliable, reproducible, and accurate one.Conclusion:Thus, the present study may be used for regular quality control of RGB to improve commercial feasibility for the synthesis of Linezolid.

Comparative study on kynurenic acid production in the rat striatum by tryptophan enantiomers: An in vivo microdialysis study

Kynurenic acid (KYNA), an endogenous antagonist of N-methyl-D-aspartate and α(7) nicotinic acetylcholine receptors, is one of the L-tryptophan (Trp) metabolites. To compare the level of KYNA produced in the striatum of rats after independent administration of L-Trp and D-Trp, rats were intraperitoneally administered L-Trp and/or D-Trp (100 mg/kg), and a microdialysis (MD) probe was implanted in the striatum. The KYNA level in the MD samples was determined using the column-switching high-performance liquid chromatography system. KYNA levels in the MD samples increased by approximately twofold in rats that were administered D-Trp or L-Trp; this result suggests that just as L-Trp, D-Trp was also metabolized to KYNA in the striatum. Additionally, 30 min before the administration of D-Trp, rats were administered 3-methyl pyrazole-5-carboxylic acid (MPC) (50 mg/kg), which is a specific inhibitor of D-amino acid oxidase (DAAO). Pretreatment with MPC suppressed striatal KYNA production; this result suggests that DAAO, encoded by one of the susceptible genes for schizophrenia, may contribute to the production of KYNA from D-Trp in the striatum of rats.

Hypocholesterolemia is associated negatively with hemolysate lipid peroxidation in sickle cell anemia patients

The oxidative stress levels in plasma and hemolysate and cholesterol levels in plasma of sickle cell anemia patients, carriers and controls were evaluated. A total of 40 cases-17 patients, 13 carriers and 10 controls-were involved in the study. Plasma and hemolysate malondialdehyde (MDA) were detected via thiobarbituric acid reaction with a fluorimetric detector by high-performance liquid chromatography system. Plasma cholesterol was determined by enzymatic colorimetric method. Mean MDA levels of SCA patients were higher than those of the carriers' and healthy children's both in plasma and in hemolysate (P<0.005). The mean plasma and hemolysate MDA levels were 25.3±1.6nmol/l and 86.7±19.3nmol/l in patients, 19.1±0.8nmol/l and 54.1±10.8nmol/l in carriers and 19.6±0.8nmol/l and 56.8±9.3nmol/l in healthy children. Mean plasma total cholesterol levels were 92.1±19.1mg/dl in patients, 116.2±23.3mg/dl in carriers and 126.6±16.4mg/dl in controls (P<0.005). There was a significant negative correlation of -0.520 between hemolysate MDA and plasma cholesterol levels in patients (P<0.05). The degree of correlation increased up to -0.782 (P=0.008) in the patients with HbSS phenotype. This negative correlation between MDA and cholesterol may imply a potential association between oxidative stress and hypocholesterolemia in sickle cell anemia.

Quantitative analysis of Hb Bart’s in cord blood by capillary electrophoresis system

It has long been recognized that the presence of hemoglobin (Hb) Bart's in newborn's blood is associated with α-thalassemia. However, the automated high-performance liquid chromatography or low-performance liquid chromatography system is unable to quantify the amount of Hbs Bart's and H, which are eluted at the retention time close to 0min. This study used automatic capillary electrophoresis (CE) system to diagnose various types of α-thalassemia in 587 cord blood samples, including 429 normal α-globin genotype, 120 cases of thalassemia with one α-globin gene defect, 34 cases with two α-globin genes defect, and four cases with three α-globin genes defect. The result showed that the level of Hb Bart's in cord blood was increased accordingly with the increasing numbers of the defective α-globin genes. In addition, Hb Bart's level at 0.2%, as measured by CE, can be used as a cut-off point for α-thalassemia diagnosis in newborns.

HPLC assay method for ceftibuten in plasma and its application to pharmacokinetic study

The purpose of this study was to validate a reliable analytical method for pharmacokinetic study of ceftibuten in human plasma by high performance liquid chromatography (HPLC) system with UV detection. Ceftizoxime was used as the internal standard. After plasma sample was precipitated with acetonitrile and dichloromethane, the supernatant was directly injected into the HPLC system. Separation was performed on a Capcell Pak C18 UG120 column (4.6 mm × 250 mm, 5 μm particles) with a mobile phase of acetonitrile/50 mM ammonium acetate (5: 95, v/v) and UV detection at a wavelength of 262 nm. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The lower limit of quantification was 0.5 hg/mL of ceftibuten using 0.5 mL of plasma. The calibration curve was linear in concentration range of 0.5–30 μg/mL (r 2 = 0.9998). The mean accuracy was 96–102%. The coefficient of variation (precision) in the intra- and inter-day validation was 0.9–3.9 and 0.9–2.4%, respectively. The pharmacokinetics of ceftibuten was evaluated after a single oral administration of 400 mg to healthy volunteers. The AUC0–9 h, c max, T max, and T 1/2 were 86.6 ± 12.7 μg h/mL, 18.4 ± 1.5 μg/mL, 2.63 ± 0.83 and 2.65 ± 0.41 h, respectively. The method was demonstrated to be highly reproducible and feasible for pharmacokinetic studies of ceftibuten in eight volunteers after oral administration (400 mg as ceftibuten).

Constructing a LabVIEW-Controlled High-Performance Liquid Chromatography (HPLC) System: An Undergraduate Instrumental Methods Exercise

In this laboratory exercise, students develop a LabVIEW-controlled high-performance liquid chromatography system utilizing a data acquisition device, two pumps, a detector, and fraction collector. The programming experience involves a variety of methods for interface communication, including serial control, analog-to-digital conversion, and instrument control via digital input−output. The assignment can be set up as a class project or students can be rotated through the different tasks as a part of the course.

Determination of Six Bioactive Compounds in Buyang Huanwu Decoction by High Performance Liquid Chromatography Coupled with Electrospray Mass Spectrometry

A high performance liquid chromatography (HPLC) coupled with an electrospray mass spectrometry (ESI-MS) method was developed for the determination of six bioactive compounds in a Buyang Huanwu decoction (BYHWD), which was composed of seven spices of Traditional Chinese Medicines (TCMs). The separations were performed on a Shim-pack (VP-ODS) C18 analytical column (5 μm, 250 × 4.6 mm ID, Shimadzu, Kyoto, Japan) with the column temperature maintained at 30°C. A linear gradient elution of A (0.1% formic acid solution) and B (100% acetonitrile) was used at a flow rate of 0.8 mL/min. The six compounds in BYHWD were identified by an Agilent-1100 HPLC system with a photodiode array detector coupled with an LC/MSD Trap SL electrospray ion mass spectrometer, and the contents of these compounds were determined by a Shimadzu 20A HPLC system coupled with a LCMS-2010EV quadrupole mass spectrometer. The standard calibration curves were linear with regression coefficient r2 > 0.99. The intra-day and inter-day precisions of this method were evaluated with the relative standard deviation values (less than 2.06% and 2.88%, respectively). The recoveries of the six investigated compounds exceeded 96%. This method was successfully used to determine the six target compounds in BYHWD.

Development and Validation of a High-Performance Liquid Chromatography Method for Determination of Cefquinome Concentrations in Sheep Plasma and Its Application to Pharmacokinetic Studies

Cefquinome has a broad spectrum of antibacterial activity and was developed especially for use in animals. A simple and sensitive high-performance liquid chromatography (HPLC) method with UV-visible detection for quantification of cefquinome concentrations in sheep plasma was developed and validated. Separation of cefquinome from plasma components was achieved on a Phenomenex Gemini C(18) column (250 mm by 4.6 mm; internal diameter [i.d.], 5 μm). The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid in water and was delivered at a rate of 0.9 ml/min. A simple and rapid sample preparation involved the addition of methanol to 200 μl of plasma to precipitate plasma proteins followed by direct injection of 50 μl of supernatant into the high-performance liquid chromatography system. The linearity range of the proposed method was 0.02 to 12 μg/ml. The intraday and interday coefficients of variation obtained from cefquinome were less than 5%, and biases ranged from -3.76% to 1.24%. Mean recovery based on low-, medium-, and high-quality control standards ranged between 92.0 and 93.9%. Plasma samples were found to be stable in various storage conditions (freeze-thaw, postpreparative, short-term, and long-term stability). The method described was found to be readily available, practicable, cheap, rapid, sensitive, precise, and accurate. It was successfully applied to the study of the pharmacokinetics of cefquinome in sheep. This method can be very useful and an alternate to performing pharmacokinetic studies in the determination of cefquinome for clinical use.

A low cost universal photoelectrochemical detector for organic compounds based on photoelectrocatalytic oxidation at a nanostructured TiO 2 photoanode

A photoelectrochemical detector (PECD) was developed for determination of organic compounds in flow injection analysis (FIA) and high performance liquid chromatography (HPLC) based on the extraordinary oxidation power of nanostructured TiO 2 photoanodes under UV illumination. The PECD is a simple, small, and compact photoelectrochemical cell consisting of low cost components such as a TiO 2 nanostructured photoanode and a UV-LED. Compared with conventional FIA and HPLC selective detectors, such as UV–Vis detector, electrochemical detector and fluorescent detector, the PECD has the advantage of being low cost, non-selective and sensitive. Using the FIA mode, the analytical principle of the PECD was demonstrated by detecting a large variety of organic compounds, such as sugars, amino acids, alcohol, straight chain carboxylic acids, and aromatic carboxylic acid. The PECD was subsequently coupled with a typical FIA and HPLC system for the determination of sugars. For the application in FIA, under the optimum experimental conditions, the linear ranges for glucose and sucrose were 25–600 μM and 25–500 μM, respectively, with the same detection limit of 10 μM. When the PECD was coupled with a HPLC system, the linear range for both glucose and sucrose was 7.5–200 mM with the same detection limit of 1 mM under the optimal experimental conditions.