BUNSEKI KAGAKU Abstracts

Determination of selenomethionine (SeMet) in a selenium-enriched yeast standard material (SELM-1, National Research Council of Canada, NRC) was carried out, and extraction methods were investigated. SeMet in SELM-1 was determined by using a hyphenated instrument of HPLC, and an inductively coupled plasma-three dimensional quadrupole mass spectrometer (HPLC-ICP3DQMS), and two different isotope dilution analysis methods (species-unspecific IDA and species-specific IDA). Two different IDA methods were evaluated by comparing the analytical results. It was confirmed that enzymatic hydrolysis with using Lipase VII (Sigma Chemicals, St, Luis, MO, USA) and Protease, Type XIV (Sigma Chemicals, St, Luis, MO, USA) for 24 hours was appropriate for the extraction of SeMet. Our analytical values of SeMet for species-unspecific IDA (3040 ± 77 μg g⁻¹) and species-specific IDA (3154 ± 82 μg g⁻¹) showed agreements with the certified value (3448 ± 146 μg g⁻¹) within 4σ and 2σ, respectively. It is concluded that species-specific IDA should be used when a stable isotope enriched compound could be synthesized.

A simple and highly sensitive visual speciation analysis for As^III and As^V based on solid-phase extraction to a membrane filter has been developed. The colorless complex, which was formed through a reaction between As^III and sodium dibenzyldithiocarbamate (DBDTC), was adsorbed quantitatively. The As^III-DBDTC complex on a membrane filter was converted to a yellowish Cu^II-DBDTC complex by passing through a solution containing Cu^II ion. The concentration of As^III was determined by a visual comparison with the color standard. The visual detection limit was 2 μg dm⁻³. The calibration curve assessed with the reflection spectrometric response was linear in the concentration range 0 – 20 μg dm⁻³ (r = 0.998). The detection limit was 0.2 μg dm⁻³ in 5 cm³ of the sample solution. The concentration of As^V was determined similarly after a chemical reduction of As^V to As^III by using L-cysteine. Interference of heavy metal ions was removed by filtration of the metal-DBDTC complex at pH 7.0 prior to extracting the As^III-DBDTC complex. The proposed method was applied to the determination of As^III and As^V in river-water samples.

According to the Japanese Industrial Standard (JIS) K 0102, we must use a 4-aminoantypyrine (4-AA) batchwise method to determine the quantity of the phenol index after distillation. However, the conventional distillation method must use a large-scale evaporator system, which is time-consuming. In the present work, a miniature evaporator system for phenol index was designed and developed. Also, a sequential injection analysis (SIA) system was proposed for determining the phenol index based on 4-AA spectrophotometry. A calibration curve obtained by the optimized SIA system was linear with a correletion coefficient of 0.998 in the range of 10 μg L⁻¹ – 10 mg L⁻¹. The limit of detection (3σ) was 0.62 μg L⁻¹, and the limit of quantitation (10σ) was 2.0 μg L⁻¹. An SIA peak for phenol was obtained every 75 sec. The performance of the proposed miniature evaporator system was evaluated by distilling standard phenol solutions, followed by the spectrophotometric SIA method. The sample volume for distillation could be reduced to be as small as 35 mL (approximately 1/7 compared with the current JIS procedure) with good recovery. The proposed SIA method coupled with a miniature evaporator system was applicable for determining the phenol index in wastewater.

Although natural water, such as river water and hot spring water can be analyzed for calcium directly by ICP-AES or ICP-MS after appropriate dilution, atomic absorption spectrophotometry (AAS) is still the major analytical method. Standard solutions or the water samples are generally acidified by hydrochloric acid for AAS, but the solutions are acidified by nitric acid for ICP-MS. Consequently, nitric acid is recently more common acid for acidification than hydrochloric acid. In the present work, calcium was determined by atomic absorption spectrophotometry in the presence of lanthanum or strontium as an interference inhibitor of silicon or aluminum. Nitric acid inhibited calcium absorption, which was evident in the presence of lanthanum or strontium. In contrast, hydrochloric acid showed only little inhibition with calcium absorption. Upon increasing the concentration of lanthanum over 2000 mg dm⁻³, in contrast with strontium, decreased the interference of calcium absorption by nitric acid.

A new anodic stripping voltammetry (ASV) using a microporous membrane electrode has been developed for the on-line monitoring of trace Cu^II in concentrated alkaline solutions. Microporous membrane electrodes were prepared by plating gold onto membrane filters made of polypropylene (PP). By SEM observations, it was confirmed that the membrane filter was modified by gold fine particles. The microporous membrane electrodes were set in a penetrated-type electrochemical flow-cell, and Cu^II was deposited while the sample solution passed through the electrode. The concentration of Cu^II was estimated with the anodic stripping peak currents. The calibration curve showed linearity within a range of 0 – 25 μg L⁻¹ Cu^II in a 48% KOH solution. The proposed method will become a useful technique for an on-line monitoring system for trace Cu^II, which has been in urgent demand by the semiconductor manufacturing.

We have developed a new separation-oriented derivatization method based on the affinity of the interaction between the pentafluorophenyl (PFP) group for the HPLC determination of native fluorescent non-steroidal anti-inflammatory drugs (NSAIDs). This method involves the derivatization of NSAIDs with pentafluorobenzyl bromide and the separation of derivatives using a PFP-modified stationary phase column. In this method, the PFP-derivatized NSAIDs were selectively retained on the PFP column, followed by fluorescence detection, whereas the derivatives were not retained on the ODS-, phenyl-, and perfluoroalkyl-modified HPLC columns. The detection limits (signal-to-noise ratio of 3) of the NSAID standards were found to be 44 – 84 fmol on column for a 20 μL injection, and the repeatability of this method was within 7.1% as relative standard deviation. We show that the proposed method can be used to analyze NSAIDs in human plasma samples without the interference of endogenous matrices.

We examined the determination of cortisol and cortisone in human saliva with dispersive liquid-liquid microextraction (DLLME) coupled to HPLC/UV detection. In this study, by adding a mixture of acetonitrile (disperser solvent) and 1,1,2,2-tetrachloroethane (extraction solvent) into the sample, DLLME was performed. The resulting sedimented phase was injected into a reversed-phase HPLC column using a mixture of water, acetonitrile, and trifluoroacetic acid as a mobile phase for isocratic elution, and the analytes were determined with an ultraviolet detector at 254 nm. The enrichment factors of this method were ca. 20 and 40 for cortisol and cortisone, respectively, and the limits of detection were 0.30 ng mL⁻¹ and 0.20 ng mL⁻¹, respectively. We showed that the proposed method can be used to analyze trace amounts of cortisone in human saliva samples with sufficient sensitivity.

Diabetes is a major cause of chronic kidney disease, and oral antidiabetic drugs are the mainstay of therapy for most patients with Type 2 diabetes. Type 2 diabetes is typically associated with insulin resistance and the dysfunction of insulin-secreting pancreatic beta-cells. Addressing these defects often requires therapy with a combination of differently acting antidiabetic agents. A potential novel combination in development brings together a DPP-4 inhibitor with the pioglitazone into a fixed-dose single-tablet combination. The former component acts mainly to increase prandial insulin secretion; the latter improves insulin sensitivity. Ultra-high-speed HPLC has become increasingly popular in analytical research fields. This novel analytical system provides fast and efficient chromatographic separation over a wide range of flow rate and pressure. In this study, we investigated the analysis of active ingredients in combination drugs using an ultra-high-speed HPLC system equipped with a photo-diode array detector. The HPLC system utilized in this study was a LaChrom Ultra system (Hitachi, Tokyo, Japan), equipped with a L-2455U diode array detector. A LaChrom Ultra C18 (particle size; 2 μm) was used as the analytical column. Rapid and simultaneous analysis of active ingredients in combination drugs was achieved.

Isoprenoids are intermediates of cholesterol biosynthesis, and are also related to Ras prenylation associated with cell proliferations. Recently, an inhibitory effect of bisphosphonate on the biosynthesis of isoprenoids has been noted as a target of an anti-cancer drug. There were small amounts of isporenoids in the biological fluid; therefore, a highly sensitive determination method of isoprenoids by LC/MS/MS is required. On the other hand, LC/MS/MS requires one to remove any suppressed ionization substance, e.g., phospholipids, from the matrix before analysis. We tried to separate isoprenoids from phospholipids using Hybrid SPE^TM-Phospholipid (HybridSPE) solid-phase extraction to determine isoprenoids, and then applied to determine them in a HepG2 cell culture medium. We could effectively separate isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP) from phospholipids. Also, this method was successfully applied to determine the total amount of IPP and DMAPP in a cell culture medium. Bisphoshonates, inhibitors of farnesyl-pyrophosphate (FPP) synthetase, increase IPP and DMAPP in a cell culuture medium with a dose dependence. Functional evaluation of bisphosphonates could be performed by determining IPP and DMAPP.

We developed a capillary zone electrophoresis method with direct UV detection for the determination of inorganic anions (Cl⁻, NO₂⁻, and NO₃⁻), organic acids (oxalic acid, citric acid, and malic acid), and amino acids (aspartic acid and glutamic acid) in the common ice plant (Mesembryanthemum crystallinum L.). As the background electrolyte, a 20 mmol L⁻¹ disodium hydrogenphosphate dodecahydrate solution adjusted to pH 10.6 was used with the addition of 0.001% (w/v) hexadimethrine bromide to reverse electroosmotic flow. The detection responses for the above analytes were improved by 3.5 – 580 fold in terms of the limits of detection (LODs, S/N = 3) in comparison with conventional indirect UV detection. The LODs for the analytes were 0.040 – 2.9 mg L⁻¹. The values of the relative standard deviation (RSD, n = 4, intra-day) of the migration time and the peak area were, respectively, 0.081 – 0.43% and 0.28 – 9.3% when extract from the common ice plant was analyzed. Using the proposed procedure, Cl⁻, NO₂⁻, NO₃⁻, and oxalic acid in the common ice plant were detected within 9 min; citric acid, malic acid, aspartic acid, and glutamic acid were detected within 12 min.

The section of liquid chromatography in “General Tests, Processes, and Apparatus” has been revised in The Japanese Pharmacopoeia, Sixteenth Edition. In the case of using photodiode array detection (PDA), a new comment was added such that highly specific identification can be achieved by confirming the identity of the chemical structure of the component and that of an authentic specimen, in addition to the identity of their retention times. Thus, in this paper we describe a rapid and effective identification and quantification of the components of some combination tablets using the PDA detector. In the case of the ECARD^® combination tablet, which contains Candesartan cilexetil and Hydrochlorothiazide, the analysis time was 15 min using conventional HPLC. The analysis time was cut down to under 2.5 min by using ultra-high-speed liquid chromatography (ultra-high-speed LC). In addition, the separated peaks that were detected by PDA detector exhibited the same spectra as those measured by a UV-VIS detector. Therefore, rapid and effective identification and quantification could be carried out by using the ultra-high-speed LC-PDA detector system. We think that the present system is of use for the analysis of various combination drugs and multi-component systems.

A one-drop solvent concentration method with a compact evaporator could be used for HPLC analysis to determinate trace levels of Ni(II), Co(II) and Cu(II) ions in an aqueous solution after complexation with sodium diethyldithiocarbamate (DDTC). The ethyl acetate phase, in which metal complexes were extracted by solvent extraction, was transferred to a centrifuge tube, and was added to a micro volume of dimethyl sulfoxide (DMSO). Successful preconcentration of metal complexes into the DMSO phase was accomplished by removing ethyl acetate with a compact evaporator in a water bath (40°C). A four hundred-fold concentration was achieved within 15 min. A portion of the residual DMSO phase was used for a determination by HPLC. The proposed analysis showed both high reproducibility and high sensitivity. The detection limits (3σ) for Ni(II), Co(II) and Cu(II) ions reached at 8.7 × 10⁻⁸ M, 6.0 × 10⁻⁸ M and 2.0 × 10⁻⁸ M, respectively. The relative standard deviation (n = 5) at 5.0 × 10⁻⁷ M was 4.2%, 2.0% and 1.0%, respectively.