Column-switching HPLC System Research Articles

Measurement of 8-hydroxyguanine as an oxidative stress biomarker in saliva by HPLC-ECD

IntroductionOxidative stress leads to many kinds of diseases. Currently, urinary 8-hydroxydeoxyguanosine (8-OHdG) is widely measured as an oxidative stress biomarker. There is a specific advantage if saliva can be used as the sample to measure the oxidative stress biomarker, because saliva is much easier to collect than urine. In this study, we investigated the measurement of 8-hydroxyguanine (8-OHGua) as an oxidative stress marker in saliva, by a column switching HPLC system equipped with an electrochemical detector (HPLC-ECD).FindingsThe 8-OHGua in saliva could be detected as a single peak by HPLC-ECD. The average level of 8-OHGua in saliva was 3.80 ng/mL in ordinary, non-smoking subjects. The salivary 8-OHGua levels of smokers were significantly higher than those of non-smokers.ConclusionsSalivary 8-OHGua may be a useful noninvasive and promising oxidative stress biomarker.

Development of a Fully-automated On-line Oxidation Column-switching HPLC System for the Determination of Endogenous Melatonin in Human Clinical Samples.

A fully-automated on-line oxidation column-switching HPLC system has been developed for the determination of endogenous melatonin in human plasma and saliva. This HPLC system consists of four processes. In the first step, melatonin is fractionated using a reversed-phase C4 column (Proteonavi, 75 mm × 1.0 mm i.d.). In the second step, the obtained melatonin fraction is on-line collected, and oxidized to a highly-fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), by mixing with hydrogen peroxide under alkaline conditions. In the third step, the produced 6-MOQMA is concentrated, and the oxidation reagents are removed using an alkaline resistive reversed-phase column, Asahipak ODP (35 mm × 1.0 mm i.d.). In the final fourth step, the 6-MOQMA is determined by a microbore-ODS column packed with ultrafine particles (CAPCELL PAK C18 IF, 250 mm × 1.0 mm i.d.). The limit of detection of melatonin using this system is about 200 amol/injection, and the determination of endogenous melatonin in a small volume of human physiological fluids, such as 100 μL of plasma and 300 μL of saliva, was successfully accomplished.

Characterization of insulin-loaded liposome using column-switching HPLC

We evaluated the drug-encapsulation state of insulin (INS)-loaded liposome using a novel column-switching HPLC system that can automatically separate unloaded drug from encapsulated drug by hydrophobic interaction. When the INS-loaded liposome was dispersed in water (pH 7.4), the encapsulation efficiency (EE) obtained by the column-switching HPLC system was consistent with that obtained by a conventional ultracentrifugation method. However, the INS-loaded liposome dispersed in 0.1% acetic acid (pH 3.3) showed disagreement between the EEs obtained by both methods. Considering the results of particle size, zeta potential, and transmission electron microscope (TEM) observations, we hypothesized that the column-switching HPLC method was able to distinguish INS adsorbed onto the liposome surface from the encapsulated INS, although an ultracentrifugation method precipitated the adsorbed INS onto the liposome surface along with the encapsulated INS. Therefore, the novel column-switching HPLC system may be a more accurate and useful technique for characterization and optimization of the INS-loaded liposome formulation.

Determination of Miglitol by Column-Switching Ion-Pair HPLC with Tris(2,2'-bipyridine)ruthenium(II)-Electrogenerated Chemiluminescence Detection.

We have developed a highly sensitive, simple method for the quantitative determination of miglitol in standard serum samples using column-switching ion-pair HPLC with tris(2,2'-bipyridine)ruthenium(II)-electrogenerated chemiluminescence detection. The serum samples were directly injected into a column-switching HPLC system with a Shim-pack MAYI-SCX precolumn to remove the serum matrix. Chromatographic separation of miglitol was achieved on a TSKgel ODS 100-V column using a mobile phase containing sodium 1-octanesulfonate as an ion-pair reagent. The detection and quantification limits of miglitol were 3 and 10 ng/mL, respectively. The calibration curve for miglitol in the serum samples showed good linearity (r(2)=0.9997) in the range of 10-2500 ng/mL. The recovery rate of miglitol from the serum samples was more than 94% as calculated from blank serum samples spiked with miglitol 50, 100, 500, 1000, and 2000 ng/mL. Therefore, this method can be applied to routine therapeutic monitoring of miglitol in serum samples.

8-Hydroxyguanine in Urine and Serum as an Oxidative Stress Marker: Effects of Diabetes and Aging

8-Hydroxydeoxyguanosine (8-OH-dG) is the most extensively analyzed oxidative stress marker. Recently, 8-hydroxyguanine (free base: 8-OH-Gua) has been recognized as an oxidative stress marker. To verify the usefulness of 8-OH-Gua, the 8-OH-dG and 8-OH-Gua levels in the urine and the 8-OH-Gua levels in the serum of type 2 diabetic model animals, db/db mice, were measured as oxidative stress markers by a column switching HPLC-system coupled to an electrochemical detector. The urinary 8-OH-Gua and 8-OH-dG levels in db/db mice (7-26 weeks old) were significantly higher than those in control (db/m+) mice. The 8-OH-Gua levels in the serum of the db/db mice were also about 2-fold higher than those in the control mice at 26 weeks of age. In addition, the urinary levels of 8-OH-dG and 8-OH-Gua increased with age (9-26 weeks). A significant positive correlation was obtained between the 8-OH-dG and 8-OH-Gua levels in urine. Although no difference was observed in the 8-OH-dG levels in the liver and kidney DNA between the diabetic and control mice, these results suggested that urinary 8-OH-dG and free base 8-OH-Gua in urine or serum may be good biomarkers of oxidative stress.

Development of Liposomal Anticancer Drugs

Liposomes are drug delivery systems that can alter the pharmacokinetic properties of compounds. The adverse effects of anticancer agents are a limiting factor for cancer chemotherapy, therefore, liposomal formulations have the potential to improve the therapeutic efficacy of anticancer agents by enhancing their accumulation in tumors and reducing non-selective distribution to normal tissues, which is known as the enhanced permeability and retention effect. To develop a liposomal anticancer agent as a drug product, its formulation must be designed to ensure its quality until it is administered to patients and to exert maximum potency in clinical use rather than in animal experiments. The chemical stability and physicochemical stability of the ingredients are key factors in the design of liposomal formulations. Drug release rates are critical factors in the therapeutic efficacy of liposomal drug products because the encapsulated drug has no pharmacological activity, and only released drug can exert antitumor/toxic activities. Liposomes should maintain the drug in a stable state in the circulation and then promptly release it after accumulation in the target tissue in order to achieve a sufficient drug concentration. To understand the profile of the formulation and to guarantee the quality of drug product, a reliable analytical method that can determine the released and encapsulated drugs in biological fluids is required. Simple online solid phase extractions of the released and encapsulated drugs using a column-switching HPLC system meet the requirements and this system enables accurate in vitro release testing and in vivo pharmacokinetic evaluation. This review introduces the process of liposomal drug product development from various viewpoints.

Rapid determination of the encapsulation efficiency of a liposome formulation using column-switching HPLC

The feasibility of a rapid automated method for determination of the encapsulation efficiency (EE) of a liposome formulation using a column-switching HPLC system was confirmed by employing several types of liposome formulations containing doxorubicin (DXR). A suspension of DXR liposome was injected directly into an online solid-phase extraction (SPE) system comprising a Diol SPE column and an ODS SPE column connected in series. Free (not encapsulated) DXR was trapped on the Diol SPE column, whereas encapsulated DXR was eluted without interaction. The eluted encapsulated DXR was trapped on the ODS SPE column after being extracted from the inner phase of the liposome by mixing with an organic solvent. Trapped free and encapsulated DXR were eluted sequentially and analyzed separately by gradient HPLC. The time taken by this automated method was only 25min, whereas conventional methods such as ultracentrifugation are time consuming and labor intensive. Validation results and comparison with ultracentrifugation suggested that our method was sufficiently accurate and sensitive to be used to evaluate EE of a liposome formulation without complicated pretreatment.

Retention properties of macroporous spongy monolith and its application for concentration of polyaromatic hydrocarbons

We report the fundamental chromatographic retention properties of spongy monolith that was previously reported as a novel separation medium for the effective pretreatment of environmental pollutants. According to the detailed examination on liquid chromatographic evaluations using hydrophobic, hydrophilic, and polyaromatic compounds as solutes, the spongy monolith consisted of poly(ethylene-co-vinyl acetate) and having macroporous co-continuous structures showed the selective retention ability for polyaromatic hydrocarbons (PAHs), especially coplanar compounds, whereas lower selectivity was observed for typical hydrophobic and hydrophilic compounds compared with the commonly used C18 medium. Moreover, we demonstrate the effective preconcentration of benzo[a]pyrene using a spongy monolith as a pretreatment column of an online column switching HPLC system.

Rapid and Simple Determination of Vitamin A and Vitamin E in Human Plasma by Column-Switching High-Performance Liquid Chromatography

In this contribution, the rapid and simple determination of vitamins A and E, and vitamin E acetate as internal standard in human plasma by high-performance liquid chromatography with a column-switching technique was proposed. Restricted Access Material - RAM column 25 x 4 mm containing 25 μm C18 alkyl-diol silica support was integrated into lab-made column-switching HPLC system. A monolithic column Chromolith Performance RP-18e, 100 x 4.6 mm, (Merck) was used for simple separation of vitamins in gradient mode. The mobile phase I: methanol – 20 mM water solution of dodecyl sulfate sodium salt (30:70, v/v) was used for protein matrix elution, flow rate 1.3 ml min-1. Valve switching time was determined to 4 minutes after sample injection. The gradient elution of mobile phase II: methanol – water 90:10 (v/v) → 100% methanol, flow rate 1.0 ml min-1 was used as optimal condition of vitamins separation in relatively short time. Analysis time from raw sample to peaks evaluation was less than 15 min. UV detection was carried out at wavelength 285 nm. The recoveries of vitamins from spiked human plasma were 102.5% for vitamin A and 91.4% for vitamin E. Limits of detection were 0.24 µmol l-1 and 0.84 μmol l-1 for vitamin A and E, respectively.

Presence and origin of large amounts of d-proline in the urine of mutant mice lacking d-amino acid oxidase activity

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of D-proline (D-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of D-amino acids in vivo, a mutant mouse strain lacking D-amino acid oxidase activity (ddY/DAO(-) mouse) was used. In the brain, relatively large amounts of D-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of D-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO(-) mice excreted large amounts of D-Pro in their urine (433 nmol/mL, 20 times that of L-Pro). The origin of D-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary D-Pro amount. Concerning the dietary origin, a notable amount of D-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the D-Pro is produced in the mice. Age-dependent changes in the urinary D-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO(-) mice were found to excrete large amounts of D-Pro in the urine constantly throughout their lives.

High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.

Uniformly sized molecularly imprinted polymer for atropine and its application to the determination of atropine and scopolamine in pharmaceutical preparations containing Scopolia extract

A uniformly sized molecularly imprinted polymer (MIP) for atropine has been prepared. The MIP was prepared using 2-(trifluoromethyl) acrylic acid and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and thermal polymerization method. The selectivity factor, which is defined as the ratio of the retention factors ( k) on the molecularly imprinted and non-imprinted polymers, k imprinted/ k non-imprinted, was 2.2 for atropine on the MIP. The obtained MIP was applied for the determination of tropane alkaloids (atropine and scopolamine) in a commercial gastrointestinal drug by a column-switching HPLC system, consisting of an MIP material as a pre-column, and a conventional cation-exchange analytical column. An interference peak was observed at the retention time of atropine derived from pre-column. However, since the peak area was less than 0.5% the peak area of atropine of a standard solution under the analytical conditions of this study (0.2 μg of atropine was loaded), this interference was negligible in the determination of atropine. On the other hand, no interference peak was observed at the retention time of scopolamine. Calibration curves of atropine and scopolamine showed good linearity in the range of 0.02–0.9 μg/ml ( r = 0.9999) and 0.003–0.09 μg/ml ( r = 0.9998), respectively. The mean recoveries of atropine and scopolamine from a placebo pharmaceutical preparation sample were 98.9 and 99.9%, respectively. The intra-day precision (measured by relative standard deviation, R.S.D. (%)) of both ingredients was less than 2.0%. The optimized column-switching system was applied successfully to the determination of atropine and scopolamine in a commercial gastrointestinal drug.

Novel methylcellulose-immobilized cation-exchange precolumn for on-line enrichment of cationic drugs in plasma

We developed a novel methylcellulose-immobilized strong cation-exchange (MC-SCX) precolumn for direct analysis of drugs in plasma. MC-SCX consists of silica gel with a methylcellulose outer-surface and a 2-(4-sulfophenyl) ethyl phase inner-surface. The MC-SCX precolumn was evaluated by direct analysis using pyridoxine, atenolol and sulpiride spiked in plasma, using a column-switching HPLC system. Each drug was retained and enriched on MC-SCX using an acidic mobile phase, which resulted in good linearity, sufficient reproducibility, intra- and inter day precision, and accuracy in analytical ion-pair LC with trifluoroacetic acid. The analytical methods for model drugs were applied to pharmacokinetics of atenolol and sulpiride in rats.

Determination of rat blood S- d-lactoylglutathione by a column-switching high-performance liquid chromatography with precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A method for the determination of S- d-lactoylglutathione (SLG), an intermediate metabolite of the glyoxalase system, in rat blood is described. After hemolysis and deproteinization of 30 μl of rat blood, SLG in the blood was determined by a column-switching HPLC system with precolumn fluorescence derivatization with a fluorogenic reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Calibration curves for the determination of SLG showed a good linearity ( r 2>0.999) over the range of 20–100 pmol SLG spiked in rat blood samples. The accuracy was in the range of 97–104% (20–100 pmol SLG spiked in rat blood sample, N=4). The detection limit was 8.12 fmol, and the quantitation limit was 27.07 fmol, respectively. The intra- and interday coefficients of variance were 4.63% ( N=5) and 9.98% ( N=5), respectively. The concentrations of SLG in whole blood from male Wistar–Kyoto rats (12 weeks old) were 3.48 ± 0.78 μM ( N=4, mean ± SE). In streptozotocin-induced diabetic rats, the concentration of SLG was significantly increased, approx 5-fold, compared with normal rats, suggesting that the metabolic flux of the glyoxalase system increases in red blood cells during hyperglycemia.

Preparation of uniformly sized molecularly imprinted polymers for phenolic compounds and their application to the assay of bisphenol A in river water.

Uniformly sized molecularly imprinted polymers, which can recognize bisphenol A (BPA), have been prepared by a multi-step swelling and polymerization method using BPA or a structurally related analogue of BPA [p-t-octylphenol (OP) or p-t-butylphenol (BP)] as the template molecule, 4-vinylpyridine as the functional monomer, and ethylene glycol dimethacrylate as the cross-linker. The BP-imprinted polymer showed higher molecular recognition ability for BPA than the OP-imprinted polymer. The BPA- and BP-imprinted polymers were applied for the assay of a trace amount of BPA in river water using column-switching HPLC with fluorescence detection: A BPA-imprinted polymer was used for removal of BPA from the pretreatment eluent as the trap column, and a BP-imprinted polymer was used for selective pretreatment and enrichment of BPA in river water as the pretreatment column. The calibration graph, constructed from peak area data plotted versus BPA concentration, was linear with a correlation coefficient of >0.999 in the concentration ranges of 25-1000 ppt. The limit of quantitation was 25 ppt with a 5-ml injection. The column-switching HPLC system was successfully applied for the assay of BPA in river water.

Restricted access media-molecularly imprinted polymer for propranolol and its application to direct injection analysis of beta-blockers in biological fluids.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for propranolol (PRP) has been prepared for direct injection analysis of beta-blockers in biological fluids. First, the MIP for PRP was prepared using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and added directly to the MIP for PRP after 4 h from the start of polymerization. Then further polymerization was carried out for 20 h. The obtained RAM-MIP for PRP showed excellent molecular recognition ability for PRP, good ones for alprenolol (ALP) and pindolol, and fair ones for other beta-blockers. The RAM-MIP was applied for direct injection analysis of ALP enantiomers in a rat plasma sample by a column-switching HPLC system using a beta-cyclodextrin phenylcarbamate-bonded silica column as the analytical column. The calibration graph, constructed from peak area versus each ALP enantiomer concentration, was linear with a correlation coefficient of > 0.999 over the concentration ranges of 12.5-250 ng ml(-1). The limit of quantitation was 12.5 ng ml(-1) with a 50 microl injection. This method could be applicable for the assay of ALP enantiomers at the therapeutic plasma levels, and have wide applicability for the assay of beta-blockers in biological fluids.

Determination of d-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography

A column-switching chiral HPLC system for the determination of minute amounts of d-Ala in mammalian tissues has been established. d-Ala and its l-enantiomer are purified as a dl mixture on a micro-ODS column after precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and are introduced to a chiral column to determine each enantiomer. The calibration curve of d-Ala spiked into a rat cerebellum sample is linear from 5 to 5000 fmol with a correlation coefficient of 1.0000. The lower limit of quantitation of d-Ala is 5 fmol ( S/ N=5). Within-day and day-to-day precisions of spiked d-Ala (15 fmol) are 3.9 and 4.8% (R.S.D), respectively. With this system, the anatomical distribution of free d-Ala in the rat central nervous system and periphery has been investigated. Among the 22 examined tissues of the rat, the highest amount of d-Ala has been observed in the anterior pituitary gland ( 86.4±9.9 nmol/g wet tissue), and the second highest amount has been observed in the pancreas ( 29.2±5.0 nmol/g wet tissue). Postnatal and day–night changes in d-Ala amounts in the anterior pituitary gland have also been studied. The amount of d-Ala is highest at 6 weeks of age and significantly decreases with age, and the amount of d-Ala is significantly higher during the daytime than during the nighttime.

Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100×4.6 mm I.D., 10 μm particle size and 120 Å pore size) to an octadecyl Hypersil column (150×4.6 mm I.D., 5 μm particle size and 120 Å pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 μl) of the appropriated standard solutions were added to each culture tube containing an 180 μl of plasma and a solution of 0.30 m M tetrabuthylammonium phosphate (60 μl). They were vortexed for 15 s and then 290 μl were transferred to autosampler vials. Aliquots (250 μl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 μl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).

Automated column-switching high-performance liquid chromatography system for quantifying N-methyl- d- and - l-aspartate

The occurrence and biological significance of the d-amino acids, N-methyl- d-aspartate (NMDA) and N-methyl- l-aspartate (NMLA), have been recently studied in a variety of living organisms. In this study, we established a highly sensitive and reliable fluorometric HPLC system for determining levels of N-methyl-aspartate (NMA). The system comprises fluorescent derivatization of NMA with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and two chromatographic steps: one that separates NMA from other primary amino acids in reverse-phase mode and another that enantioseparates NMDA and NMLA in a normal-phase mode. These two steps are linked by an automated column-switching system. A simple pretreatment step with o-phthalaldehyde to remove primary amino acids that can interfere with sensitivity is also described. The detection limit for NMDA is as low as 5 fmol and the correlation between peak heights and concentrations between 5 fmol and 1 pmol is satisfactory ( r=0.999). Following sample preparation and separation using the column-switching HPLC system, more than 80% of NMDA was recovered from rat liver homogenates spiked with NMDA. This method was employed to determine the levels of NMDA in tissues from bivalves and the results obtained were consistent with the values reported previously.

Measurement of bisphenol A in human urine using liquid chromatography with multi-channel coulometric electrochemical detection

Environmental exposure levels of bisphenol A (BPA) in human were investigated by measuring BPA glucuronide (BPA-G) in urine. After enzymatic hydrolysis of glucuronide substances in urine, BPA was extracted with diethyl ether. The extract was analyzed using a column-switching HPLC system employing a C 8 and a C 18 column with multi-channel coulometric electrochemical detection (ECD). The sensitivity and selectivity provided with redox mode of ECD allowed measurement of low level BPA in hydrolyzed urine. The quantification limit of BPA-G in urine was 0.2 ng/ml. RSDs of the intraassay precision were less than 3% and recoveries of the method were over 96% when analyzing BPA spiked urine samples (1.0 and 10 ng/ml). In a group of 48 women students, the level of BPA-G in urine ranged from 0.2 to 19.1 ng/ml with a median concentration of 1.2 ng/ml. Normalized against urinary creatinine, BPA-G ranged from 0.1 to 11.9 ng/mg creatinine with a median of 0.77 ng/mg creatinine.