Gradient Reversed-phase High-performance Liquid Chromatography Research Articles

Improved Analytical Extraction and Clean-up Techniques for the Determination of PAHs in Contaminated Soil Samples

Development and optimisation of fast, efficient, quantitative, economic and environmentally friendly analytical extraction techniques for the extraction clean up, and pre-concentration in the quantification of 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs) in contaminated soils have been carried out. Three different extraction methods (Soxhlet, ultra sonication and mechanical shaking) were investigated on a low-level PAH soil certified reference material (CRM131-100) and the results were compared to determine the technique with the highest extraction efficiency. The clean up and pre-concentration procedures were optimized using both the conventional method (i.e. column packing with silica gel) as well as the solid phase extraction (SPE). Chromatographic conditions for the separation of PAHs using High Performance Liquid Chromatography (HPLC) using UV-DAD and fluorimetric detection with programmed excitation and emission wavelengths were also optimized. Six different extraction solvents: acetone, cyclohexane, 2- propanol, methanol, acetonitrile and dichloromethane, were tested to select the most suitable solvent for the extraction of the 16 PAHs from the certified soil reference material. Acetonitrile, dichloromethane and tetrahydrofuran were also tested as eluants for the optimisation of SPE clean up. The optimized ultrasonic extraction procedure utilizing four 15 minutes extraction cycles at 50 oC and SPE clean up with tetrahydrofuran: acetonitrile (1:1) and subsequent separation by gradient reversed phase HPLC with fluorimetric detection extracted the PAHs from the certified reference material with recoveries ranging from 63.6 % to over 100 % .

Identification, synthesis and quantification of process-related impurities in auraptene

Impurities in chemically synthesized auraptene, an active pharmaceutical ingredient (API), were detected by a gradient Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) method. Molecular weights and major product ions of these chemical compounds were determined by Liquid Chromatography/Triple Quadrupole (LC–MS/MS) analysis. Structural assignments were presumed as umbelliferone (Imp-I), (E)-6,7-dihydroxy-3,7-dimethyl-2-octene-umbelliferone (Imp-II), (E)-6,7-epoxy-3,7-dimethyl-2-octene-umbelliferone (Imp-III) and 4-methylauraptene (Imp-IV). The impurities were authentically synthesized, confirmed by Nuclear Magnetic Resonance spectroscopy (NMR) and Infrared spectroscopy (IR), and subsequently used as reference samples in routing HPLC system suitability testing for method specificity and detectability. Method specificity was further verified by forced degradation studies. The developed method was validated for characterization of impurities in synthesized auraptene according to the guidelines of the International Conference on Harmonization (ICH) in our laboratory.

Development and Validation of an HPLC Method for the Simultaneous Analysis of 23 Selected Drugs Belonging to Different Therapeutic Groups in Human Urine Samples

We have developed and validated a new and reliable gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method with a diode array detector (DAD) for the simultaneous separation and determination of 23 frequently prescribed selected drugs belonging to different therapeutic groups in human urine samples. For the drugs listed below, this method of analysis for human urine was also successfully applied to determine urine concentrations of these drugs in samples from treated patients: enalapril (ENA), paracetamol (PAR), sotalol (SOT), dipyrone (DIP), vancomycin (VAN), captopril (CAP), fluconazole (FLU), cefazolin (CEF), metoprolol (MET), aspirin (ASP), ticlopidine (TIC), prednisolone (PRE), propranolol (PRO), digoxin (DIG), sildenafil (SIL), furosemide (FUR), dexamethasone (DEX), carvedilol (CAR), ketoprofen (KET), nifedipine (NIF), terbinafine (TER), acenocoumarol (ACE) and spironolactone (SPI). Separation of the analytes was achieved by RP-HPLC-DAD with a mobile phase composed of acetonitrile, methanol and 0.05% trifluoroacetic acid in water using a gradient elution program. Good linear relationships over the investigated concentration ranges were observed with values of r2 higher than 0.998 for all of the drugs. The intra-day and inter-day precisions of this method were evaluated with RSD values less than 4.26 and 5.42%, respectively. The relative recoveries of the 23 investigated compounds ranged from 93.60 to 106.00% with RSD values less than 4.46%. An expanded uncertainty budget was constructed for all investigated drugs in human urine samples.

Changes of Isoflavone Profile in the Hypocotyls and Cotyledons of Soybeans during Dry Heating and Germination

A gradient reversed-phase high-performance liquid chromatography (HPLC) method has been developed to be suitable for the separation and determination of 12 isoflavones in soybeans. Profiles of daidzein, genistein, glycitein, and their malonyl-, acetyl-, and nonconjugated beta-glycosides were determined in cotyledons and hypocotyls of soybeans as affected by dry heating and germination. The results showed that the compositions and concentrations of isoflavones were remarkably different in the two parts of soybeans, and hypocotyls contained a much higher content of isoflavones than cotyledons (e.g., 7.8-fold higher). In hypocotyls, daidzein and its glycoside conjugates (59.6%) were the most abundant isoflavones, being followed by glycitein (26.6%) and genistein series (13.8%). In cotyledons, genistein and its glycoside conjugates (61.9%) were the main isoflavones, being followed by daidzein series (38.1%), and no glycitein series was found. Both hypocotyls and cotyledons contained remarkably high amounts of malonylglycosides (69.1 and 69.4%, respectively) and beta-glycosides (27.1 and 25.4%), and only a very small quantity of aglycones (3.8 and 5.2%) and no acetylglycosides were detected. Acetylglycosides and beta-glycosides were the thermal decarboxylation and deesterification products, respectively, of malonylglycosides, which were thermally unstable. The relative rates of decarboxylation and deesterification reactions were different in cotyledons and hypocotyls at different temperatures. During the process of germination, beta-glycosides decreased, and malonylglycosides and aglycones increased, and then, malonylglycosides were the major fractions in germinating soybeans. Interestingly, the present study occasionally found a significant circadian change between malonylglycosides and aglycones with a nocturnal increase of aglycones and decrease of malonylglycosides during germination, and even aglycones became the most abundant forms at night. However, this mechanism is yet to be investigated.

Gradient HPLC separation of dehydroepiandrosterone (DHEA) from its metabolites and biological congeners: role of tetrahydrofuran in the chromatographic mechanism

A three-step gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the separation of dehydroepiandrosterone (DHEA), its sulfate ester (DHEA-S), its three C7-oxidized metabolites (7alphaOH-DHEA, 7betaOH-DHEA, 7-keto-DHEA), and its biosynthetic congeners (androstenedione, testosterone, estradiol, pregnenolone). This new method allows the quantitative characterization of DHEA metabolism and biosynthetic transformation under given physiological, pathological, or therapeutically influenced circumstances. Tetrahydrofuran probably acts as a proton acceptor coadsorbent, while isopropanol behaves as a proton donor during the separation of testosterone, estradiol, and the stereoisomers of 7-OH-DHEA.

A reversed‐phase high‐performance liquid chromatography method for bovine serum albumin assay in pharmaceutical dosage forms and protein/antigen delivery systems

Bovine serum albumin (BSA) is among the most widely used proteins in protein formulations as well as in the development of novel delivery systems as a typical model for therapeutic/diagnostic proteins and the new versions of vaccines. The development of reliable and easily available assay methods for quantitation of this protein would therefore play a crucial role in these types of studies. A simple gradient reversed-phase high-performance liquid chromatography with ultra-violet detection (HPLC-UV) method has been developed for quantitation of BSA in dosage forms and protein delivery systems. The method produced linear responses throughout the wide BSA concentration range of 1 to 100 micro g/mL. The average within-run and between-run variations of the method within the linear concentration range of BSA were 2.46% and 2.20%, respectively, with accuracies of 104.49% and 104.58% for within-run and between-run samples, respectively. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.5 and 1 microg/mL, respectively. The method showed acceptable system suitability indices, which enabled us to use it successfully during our particulate vaccine delivery research project.

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%).

The application of gradient reversed-phase high-performance liquid chromatography to the p Ka and [formula omitted] determination of polyprotic analytes

The purpose of this work was to propose a theoretical model of the combined pH/organic modifier gradient in reversed-phase high-performance liquid chromatography (RP HPLC) with special emphasis on its applicability to polyprotic analytes. The model was developed and approximated to be useful for a data set comprising organic modifier gradients obtained at different pH changes and different gradient durations. It was evaluated regarding its ability to describe experimental data. The chromatographic p K a and lipophilicity parameter, log k w , were obtained by fitting to the proposed model and comparing to the literature values.

Evaluation of ergosterol and its esters in the pileus, gill, and stipe tissues of agaric fungi and their relative changes in the comminuted fungal tissues

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the rapid determination of free ergosterol, ergosteryl esters, and ergocalciferol. The HPLC method was used to evaluate the distribution of ergosterol and ergosteryl esters in the different parts (stipe, pileus, and gills) of the agaric fungi, Agrocybe aegerita, Termitomyces albuminosus, and Lentinus edodes, and the relative changes of free and esterified ergosterols during the degradation of ergosterol in the comminuted fungal tissues. The results showed that total ergosterol levels and the relative abundances of free to esterified ergosterols were different among the various species and in the different parts of these agaric fungi. The results also indicated that ergosteryl esters were more stable than free ergosterol. While the content of free ergosterol markedly decreased, substantial amounts of ergosteryl esters remained for a long period, and even an increase in the contents of ergosteryl esters was also found in some comminuted fungal tissues. Therefore, it is possible that free ergosterol in the cell membrane of the dead fungal hyphae undergoes degradation or esterification, by which excess free ergosterol may be removed, and stored in cytosolic lipid particles. It is suggested that free ergosterol (not total ergosterol) should be used as a biomarker for fungal biomass.

Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization.

We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor α (ERα) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17β-estradiol, 17α-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERα. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERα. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence.

Distribution of Nucleosides and Nucleobases in Edible Fungi

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of seven nucleosides, adenosine, cordycepin (3'-deoxyadenosine), cytidine, guanosine, thymidine, uridine, and inosine, and five nucleobases, adenine, cytosine, thymine, uracil, and hypoxanthine in Cordyceps sinensis, Cordyceps militaris, Ganoderma lucidum, Agrocybe aegerita, Termitornyces albuminosus, and Lentinus edodes. The results showed that total nucleoside and nucleobase contents ranged from 0.14 to 26.57 mg/g dry matter in these fungi. The higher total nucleoside and nucleobase levels (>10 mg/g dry matter) were found in the gills and the pilei of A. aegerita and T. albuminosus, and the gills of L. edodes. The lower levels (<1 mg/g dry matter) were detected in the stipe and the pileus of G. lucidum. The results indicated that A. aegerita, T. albuminosus, and L. edodes had much higher contents of nucleosides and nucleobases than C. sinensis, C. militaris, and G. lucidum. It is notable that the hymenophore tissues, which contained the spore-producing cells, such as the gills for A. aegerita, T. albuminosus, and L. edodes, the tubes for G. lucidum, and the perithecia for C. sinensis, were found to have considerably higher amounts of total nucleosides and nucleobases as compared to other parts of these fungi.

Simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method : Application to human urine samples

We have developed and described a highly sensitive, accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle in human urine samples. After pre-column derivatization with ortho-phthaldialdehyde (OPA) reagent containing 3-mercaptopropionic acid (3MPA), the fluorescent derivatives were separated by a gradient elution and detected by fluorescence measurement at 338 nm (excitation) and 455 nm (emission). l-Arginine (ARG) and its metabolites: l-glutamine (GLN), N G-hydroxy- l-arginine (NOHA), l-citrulline (CIT), N G-monomethyl- l-arginine (NMMA), l-homoarginine (HARG), asymmetric N G, N G-dimethyl- l-arginine (ADMA), symmetric N G, N G′-dimethyl- l-arginine (SDMA), l-ornithine (ORN), putrescine (PUT), agmatine (AGM), spermidine (SPERMD) and spermine (SPERM) were extracted in a cation-exchange solid-phase extraction (SPE) column and after derivatization separated in a Purospher ® STAR RP-18e analytical column. The calibration curves of analysed compounds are linear within the range of concentration: 45–825, 0.2–15, 16–225, 12–285, 0.1–32, 15–235, 0.1–12, 0.1–12, 10–205, 0.02–12, 0.1–24, 0.01–10 and 0.01–8 nmol mL −1 for GLN, NOHA, CIT, ARG, NMMA, HARG, ADMA, SDMA, ORN, PUT, AGM, SPERMD and SPERM, respectively. The correlation coefficients are greater than 0.9980. Coefficients of variation are not higher than 6.0% for inter-day precision. The method has been determined or tested for limits of detection and quantification, linearity, precision, accuracy and recovery. All detection parameters of the method demonstrate that it is a reliable and efficient means of the comprehensive determination of ARG and its 12 main metabolites, making this approach suitable for routine clinical applications. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.

Distribution of free and esterified ergosterols in the medicinal fungus Ganoderma lucidum

The fruiting bodies, spores, and lipid from the spores of Ganoderma lucidum have been widely used for medicinal purpose in China. Ergosterol content may be a suitable marker for evaluating the quality of ganoderma spore and ganoderma spore lipid (GSL) products. A gradient reversed-phase high-performance liquid chromatography method was developed for the simultaneous determination of free and esterified ergosterols in G. lucidum. The contents of free and esterified ergosterols in the different parts (the stipe, pileus, tubes, and spores) of G. lucidum and GSL were determined. The results showed that total ergosterol levels in the stipe, pileus, tubes, and spores of G. lucidum were between 0.8 and 1.6 mg/g. The relative abundances of free to esterified ergosterol were different in the different parts of G. lucidum. The spores and the tubes, the hymenophore tissue that contains the spore-producing cells, have a considerably higher percentage of ergosteryl esters (41.9 and 39.7% of total ergosterol) in comparison with the pileus and stipe tissues (3.6 and 6.2%).

Identification, isolation and characterization of potential degradation product in risperidone tablets

One unknown impurity (degradation product) present at a level below 0.1% in the initial samples increased to a level of 0.5% in 6 M/40 °C/75% RH stability samples of risperidone tablets was detected by gradient reverse-phase high-performance liquid chromatography (HPLC). This impurity was isolated using reverse-phase preparative liquid chromatography. Based on the spectral data the structure of this impurity is characterized as 3-[2-[4-[6-fluoro-1,3-benzoxazol-2-yl]piperidin-1-yl]ethyl]-2-methyl-6,7,8,9-tetrahydro-4 H-pyrido[1,2- a] pyrimidin-4-one. Structural elucidation of this impurity by spectral data ( 1H NMR, 13C NMR, DEPT, MS and IR), formation and mechanism has been discussed in detail.

Online Biochemical Detection of Glutathione-S-Transferase P1-Specific Inhibitors in Complex Mixtures

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.

High-performance liquid chromatography assay for the determination of the HIV-protease inhibitor tipranavir in human plasma in combination with nine other antiretroviral medications

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay has been developed and validated for the simultaneous quantitative determination of tipranavir with nine other antiretroviral drugs in plasma. A liquid–liquid extraction of the drugs in tert-butylmethylether (TBME) from 200 μL of plasma is followed by a reversed phase gradient HPLC assay with UV detection at 210 nm. The standard curve for the drug was linear in the range of 80–80,000 ng/mL for tipranavir; 10–10,000 ng/mL for nevirapine, indinavir, efavirenz, and saquinavir; and 25–10,000 ng/mL for amprenavir, atazanavir, ritonavir, lopinavir, and nelfinavir. The regression coefficient ( r 2) was greater than 0.998 for all analytes. This method has been fully validated and shown to be specific, accurate and precise. Due to an excellent extraction procedure giving good recovery and a clean baseline, this method is simple, rapid, accurate and provides excellent resolution and peak shape for all analytes. Thus this method is very suitable for therapeutic drug monitoring.

Simultaneous determination of free ergosterol and ergosteryl esters in Cordyceps sinensis by HPLC

The caterpillar-shaped Chinese medicinal mushroom Cordyceps sinensis consists of the fruiting body (CsA) and the host caterpillar (CsB). Ergosterol is a principal sterol in fungi and can indicate the level of mycelia of C. sinensis. Ergosterol is present in two forms, as free ergosterol and esterified ergosterol, which have different physiological functions. The relative abundances of free to esterified ergosterol are different among the various species. In the present study, a gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of free and esterified ergosterol in CsA and CsB of C. sinensis. The results showed that CsA and CsB had similar ergosterol compositions, but the level of ergosteryl esters in CsB was much higher than that in CsA, indicating that CsA and CsB might be in different growth phase or have different physiological functions for the growth and multiplication of C. sinensis.

Determination of Ergosterol in Ganoderma Spore Lipid from the Germinating Spores of Ganoderma lucidum by High-Performance Liquid Chromatography

A gradient reversed-phase high-performance liquid chromatography (HPLC) method was developed for the separation and determination of free ergosterol in ganoderma spore lipid (GSL) extracted from the sporoderm-broken germinating spores of Ganoderma lucidum. Sodium hydroxide in methanol was added for the hydrolysis of ergosteryl esters to determine the total content of ergosterol in GSL by HPLC. A 0.04 M concentration of sodium hydroxide in reaction mixtures was appropriate for the complete hydrolysis of ergosteryl esters without a significant loss of ergosterol during saponification. In addition, the ergosterol content in four commercial GSL softgel supplements from four different firms was determined. The results showed that the ergosterol content in these samples had significant differences. Ergosterol content may be a suitable marker for evaluating the quality of GSL products.

Determination of selectivity differences for basic compounds in gradient reverse phase high performance liquid chromatography under high pH conditions by partial least squares modelling

The retention behaviour of compounds in a chromatographic system is believed to be multivariate by nature, i.e. many physico-chemical properties of an analyte can influence its retention. Principal component analysis (PCA) and partial least squares (PLS) can therefore be particularly useful tools for visualising, exploring and modelling the complex interactions between solutes and the mobile and stationary phase. PCA allows the relationships between compounds (the observations) and their retention parameters (the variables) to be visualised in usually just two or three dimensions. PLS can be used to model quantitative structure-retention relationships (QSRRs) and may lead to better understanding of retention and selectivity changes in chromatographic systems. The objective of the study was to investigate the chromatographic behaviour of basic compounds under optimised gradient conditions using octadecyl high performance liquid chromatography (HPLC) columns designed for high pH separations. Three pharmaceutical mixtures were analysed by linear gradient reverse phase HPLC (RP-HPLC) at high pH using ammonia as a pH modifier, and methanol and/or acetonitrile as the organic modifier. The separations were carried out on three octadecyl columns: Waters XTerra MS C18, Agilent Zorbax Extend C18 and Thermo Hypersil-Keystone BetaBasic-18. Multivariate PCA and PLS modelling were employed to explore and explain the differences in selectivity between the chromatographic systems studied when the basic compounds were analysed under the high pH conditions. The interactions between the analytes and the mobile–stationary phases were described by relating the compound molecular descriptors with the selectivity of each chromatographic system. The selectivity differences between the chromatographic systems were identified.

Determination of endogenous glycosaminoglycans derived disaccharides in human plasma by HPLC: Validation and application in a clinical study

SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of -8.92 to 5.61% and -16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.