Ion-pair Reversed-phase Liquid Chromatographic Separation of Oligonucleotides.

Therapeutic oligonucleotides have recently been approved in the United States, the EU, and Japan. Hence, the analysis of oligonucleotides is an important topic in drug development. Liquid chromatographic techniques are commonly used for purity verification and the determination of oligonucleotides. In ion-pair reversed-phase separation, several parameters, such as the pore size of the stationary phase, mobile phase additives, and column temperature, were investigated using three types of oligonucleotides (18, 19, and 20 mer). All of the investigated parameters could influence the separation, and they are expected to be useful for optimizing oligonucleotide separation.

A simple and rapid measurement method of encapsulation efficiency of doxorubicin loaded liposomes by direct injection of the liposomal suspension to liquid chromatography.

A simple and rapid chromatographic measurement method for determining doxorubicin (DOX) encapsulation efficiency (EE) into PEGylated liposomes using nanoparticle exclusion chromatography (nPEC) was developed. In this work, Doxil® and two PEGylated liposomes spiked with DOX were employed as model liposomes, and unencapsulated DOX was measured by high performance liquid chromatography with diode-array detector using an N-vinylpyrrolidone modified nPEC column without any sample pretreatment. Only 5 µL of an intact liposomal suspension and 3 min analysis time were required for the determination of the quantity of unencapsulated DOX. The method was validated in terms of linearity, accuracy, precision, and recovery in the range from 0.00-1.0 mg/mL (corresponding to 100-50% EE). Applicability of the method was confirmed using the measurement of time-dependent DOX loading% into the liposomes with the remote loading method using an ammonium sulfate gradient. Furthermore, it was found that the peak area of DOX-loaded liposomes in the chromatogram was proportional to DOX EE%. As this simple and rapid analytical method can measure the EE precisely, it is expected that this method will be applicable to the in-process control of liposome preparation manufacturing and the quality control of the liposome drug products.

Extraction of catecholamines from urine using a monolithic disk-packed spin column and high-performance liquid chromatography-electrochemical detection.

A simple method for the determination of catecholamines (norepinephrine, epinephrine, and dopamine) in urine is described. The analytes were extracted from urine by extraction using a monolithic silica disk-packed spin column. The spin column has a phenylboronate moiety, which forms a stable negatively charged complex with cis-hydroxyl groups of catecholamines. The extraction recoveries of catecholamines were above 92.2%. The extracted catecholamines were separated on a reversed-phase column followed by electrochemical detection using a boron-doped diamond electrode. The limits of detection for the catecholamine were 0.2 ng mL-1. The developed method was successfully applied for the determination of catecholamines in urine samples.

Use of folded micromachined pillar array column with low-dispersion turns for pressure-driven liquid chromatography.

In this study, we show for the first time that the separation efficiency of a pillar array column under pressure-driven liquid chromatography (LC) conditions can be improved using a separation channel with low-dispersion turns. The pillar array column was fabricated by reactive ion etching of a silicon substrate. With the low-dispersion-turn geometry, a column with a length and width of 110 mm and 400 microm, respectively, could be fabricated on a 20 x 20 mm microchip. Under nonretained conditions, the solute bands obtained for fluorescent compounds remained almost unchanged even after passing through the low-dispersion turns; however, significant skewing of the solute bands was observed in the case of constant-radius turns. Two coumarin dyes were well resolved under reversed-phase conditions, and a maximum theoretical plate number of 8000 was obtained. Successful separation of the fluorescent derivatives of six amino acids was achieved in 140 s. These results indicated that the separation efficiency of microchip chromatography could be significantly improved using a long separation channel with low-dispersion turns.

Simultaneous determination of dopamine and 3,4-dihydroxyphenylacetic acid in mouse striatum using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography.

The measurement of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) is useful for an index of dopamine turnover. We developed a simultaneous determination method for dopamine and DOPAC with high-performance liquid chromatography-fluorescence detection. Mixed-mode reversed-phase and cation-exchange column (CAPCELL PAK CR column) which contained C18 silica particles and sulfonic acid cation-exchange particles was used for the separation of 3,4-dihydroxy-l-phenylalanine, catecholamines (norepinephrine, epinephrine, and dopamine) and DOPAC. The mobile phase was optimized for factors such as pH and counter ion concentration. With a mobile phase of 15 mM sodium acetate buffer (pH 4.5), separation was achieved within 22 min. The developed method was applicable to the determination of dopamine and DOPAC in mouse striatum. The concentrations of dopamine and DOPAC in mouse striatum were 4.98+/-0.66 and 1.00+/-0.11 microM, respectively (n=10).

Determination of selenomethionine by high-performance liquid chromatography-fluorescence detection coupled with on-line oxidation.

A simple and sensitive determination method for selenomethionine (Se-Met) using an HPLC-fluorescence detection system coupled with an on-line electrochemical reactor has been developed. NBD-F (4-fluoro-7-nitro-2,1,3-benzoxadiazole) was used as the fluorescent derivatization reagent for Se-Met. NBD-Se-Met was separated from NBD-derivatives of 22 other amino acids within 35 min. Applying an optimized oxidation potential enhanced the fluorescence intensity of NBD-Se-Met 10-fold. The calibration curve was linear in the range of 300 fmol to 30 pmol with a correlation coefficient of 0.997. Detection limit (S/N = 3) was calculated to be 50 fmol, which is comparable to that of inductively coupled plasma mass spectrometry. This simple and sensitive method should be useful for the determination of Se-Met in physiological samples, such as serum or urine.

Iodine-131-metaiodobenzylguanidine therapy with reduced-intensity allogeneic stem cell transplantation in recurrent neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood, and iodine-131-metaiodobenzylguanidine (MIBG) therapy is a new approach for grade IV neuroblastoma. We describe the case history of a 3-year-old girl with recurrent neuroblastoma who received MIBG therapy with reduced-intensity allogeneic stem cell transplantation (RIST) because of an extensive bone marrow involvement. The post-transplant course was uneventful and complete chimerism was obtained. Neither acute nor chronic graft-versus-host disease (GVHD) was observed. The patient remained in remission for 3 months after RIST until the second relapse. MIBG therapy combined with RIST warrants further trials.

Development of diffuse large B cell lymphoma during the maintenance therapy for B-lineage acute lymphoblastic leukemia.

Non-Hodgkin lymphoma (NHL) is a very rare complication of acute lymphoblastic leukemia (ALL). A Japanese boy presented with B-lineage ALL at the age of 2.5. He was treated with chemotherapy for standard-risk ALL. While he was receiving maintenance treatment 2 years and 9 months after the diagnosis of ALL, diffuse large B cell lymphoma (DLBL) was diagnosed from a biopsy of an abdominal mass. DLBL was treated by surgical resection followed by chemotherapy for 6 months. The patient has been free from the recurrence of ALL or DLBL for 16 months after the development of DLBL.

Determination of NG,NG-dimethyl-L-arginine in rat plasma and dimethylarginine dimethylaminohydrolase activity in rat kidney using a monolithic silica column.

A fast, simple and sensitive column-switching high-performance liquid chromatography (HPLC)-fluorescence detection method was developed on a monolithic silica column for the determination of N(G),N(G)-dimethyl-L-arginine (ADMA), which is an endogenous nitric oxide synthase inhibitor. After fluorescence derivatization of plasma samples or homogenized tissues with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the samples were injected into the HPLC system. The NBD-derivatized ADMA was trapped on a cation-exchange column and separated within 15 min on a monolithic silica column. The detection limit for ADMA was 36 nM (250 fmol per injection) when the signal-to-noise ratio was 3. A good linearity for calibration curve for ADMA was observed within the range of 140 nM (1.0 pmol per injection) - 140 microM (1.0 nmol per injection) using N(G)-monomethyl-L-arginine (L-NMMA) as an internal standard. The proposed method was used for the quantitative determination of ADMA in rat plasma. The concentrations of ADMA in rat plasma were 0.82+/-0.05 microM (n=4). Furthermore, the method developed was applied to determine dimethylarginine dimethylaminohydrolase (DDAH) enzyme activity in rat kidney, which was assayed by measuring the amount of ADMA metabolized by the enzyme.

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole.

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.

Alterations of plasma and cerebrospinal fluid glutamate levels in rats treated with the N-methyl-D-aspartate receptor antagonist, ketamine.

It has been reported that the repeated administration of a sub-anesthetic dose of an N-methyl-D-aspartate receptor antagonist, ketamine, can produce an animal model of schizophrenia. Since no information is available on the alterations of the amino acid levels in ketamine-treated rats, we investigated the amino acid composition in the plasma and cerebrospinal fluid of rats that were repeatedly administered with ketamine for 5 consecutive days (30 mg/kg/day). The plasma and cerebrospinal fluid amino acid compositions in the fifth week after cessation of repeated ketamine administration were determined by highperformance liquid chromatography with fluorescence detection using a pre-column fluorescence reagent, i.e. 4-fluoro-7nitro-2,1,3-benzoxadiazole. Among the amino acids investigated in the present study, the level of plasma glutamic acid increased significantly (p < 0.05), while that of the cerebrospinal fluid glutamic acid decreased significantly in the ketamine-treated rats as compared with these levels in control rats injected with saline (p < 0.05, n = 7). These alterations in the glutamic acid level in the plasma and cerebrospinal fluid resemble those in schizophrenic patients, suggesting that ketamine-treated rats may be a useful model for performing research on the pathophysiology of schizophrenia.

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent.

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.