Tomoaki Hino

Purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin by high-speed countercurrent chromatography.

Curcuminoids are substances of great interest because of their important pharmacological activities, particularly anti-inflammatory, anticarcinogenic, and anti-Alzheimers activities. In this study, we report the first procedure and effect of processing for the high, efficient, and useful purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric powder. Purification involves high-speed countercurrent chromatographic (HSCCC) separation of these curcuminoids using a simple two-phase solvent system composed of n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v). The HSCCC-fractionated effluent peaks indicated that the peak resolutions were 1.7 between curcumin and demethoxycurcumin and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded turmeric powder. These purified substances were analyzed by liquid chromatography-tandem mass spectrometry with scan and daughter scan negative modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (>98% purity). Moreover, the antioxidant effect of curcuminoids was measured by a 1,1-diphenyl-2-picrylhydrazil assay. The order of antioxidant activity was purified curcumin > purified demethoxycurcumin > purified bisdemethoxycurcumin > turmeric powder. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin can be used for various evaluations of their pharmacological activities.

An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation

In the field of pharmaceutical and biomedical analysis of peptides, a rapid on-line detection and identification for a methodology have been required for the discovery of new biological active products. In this study, a high-speed counter-current chromatography with electrospray mass spectrometry (HSCCC/ESI-MS) was developed for the on-line detection and purification of polypeptide antibiotics of enramycin-A and -B. The analytes were purified on HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile solvent of two-phase system composed of n-butanol/hexane/0.05% aqueous trifluoroacetic acid solution (43/7/50, V/V/V), and detected on an LCMS-2010EV quadrupole mass spectrometer fitted with an ESI source system in positive ionization following scan mode (m/z 100-2000). The HSCCC/ESI-MS peaks indicated that enramycin-A (major m/z 786 [M+3H](3+) and minor m/z 1179 [M+2H](2+)) and enramycin-B (major m/z 791 [M+3H](3+) and minor m/z 1185 [M+2H](2+)) have the peak resolution value of 2.9 from 15mg of loaded enramycin powder. The HSCCC collected amounts of the peak fractions were additionally 4.3mg (enramycin-A), and 5.9mg (enramycin-B), respectively. These purified substances were analyzed by LC/ESI-MS with scan positive mode. Based on the LC/ESI-MS chromatograms and spectra of the fractions, enramycin-A and -B were estimated to be over 95% purity. The overall results indicate that this approach of HSCCC/ESI-MS is a powerful technique for the purification and identification of bioactive peptides.

Simultaneous determination of avermectins in bovine tissues by LC-MS/MS

Analytical method for the simultaneous quantification of avermectins (AVMs), abamectin B1a, abamectin 8,9-Z isomer B1a, emamectine benzoate B1a, emamectine benzoate 8,9-Z isomer B1a, ivermectin, eprinomectin B1a, doramectin and moxidectin in bovine tissues (muscle, liver and fat) was developed by LC-MS/MS in electrospray positive ion mode. The separation was achieved on a short TSK-GEL ODS 100V column with the mobile phase consisting of acetonitrile and aquatic 0.1 mM ammonium formate containing 0.1% formic acid v/v at a flow rate of 0.2 mL/min with gradient elution. Liquid-liquid extraction with isooctane was used for the sample extraction/preparation of analytes in bovine samples. The linearity of the calibration curves was excellent in matrix-matched standards, and yielded the coefficients (r(2)=0.997-0.999, range from LOQ to 500, 1000 or 5000 ng/g) of determination of the target analytes. Recoveries were in the range of 87.9-99.8% with associated precision values (within-day: 1.5-7.4%, n=6, and between-day: 1.5-8.4% for 3 days) for repeatability and reproducibility. LC-MS/MS method has been proven to be highly efficient and suitable for the simultaneous determinations of eight AVMs in bovine tissue samples.

Screening Assay for Metal-Catalyzed Oxidation Inhibitors Using Liquid Chromatography−Mass Spectrometry with an N-Terminal β-Amyloid Peptide

Production of microregional catalytic reactive oxygen species (ROS) by metal-binding amyloid-beta (Abeta) peptides mediates the neurotoxicity of Alzheimers disease, and inhibitors of this activity may be of therapeutic value. No current analytical methods target specific ROS inhibitors produced by metal-binding peptides. We report a screening assay for metal-catalyzed oxidation (MCO) inhibitors based on liquid chromatography-mass spectrometry (LC-MS) with a model N-terminal Abeta peptide (Abeta(1-6)). When subjected to MCO by Cu(II)/ascorbic acid, singly and doubly charged Abeta(1-6) molecules were observed at m/z 729.2 and 364.8 and m/z 685.3 and 343.3, respectively, corresponding to a decrease in mass of 45 and 89 Da compared with the model peptide. In contrast, H(2)O(2) did not modify the Abeta(1-6) peptide. Modified peptides were characterized by a specific MCO of Abeta(1-6), which contains both His and N-terminal Asp residues. LC-MS detection of the modified peptides allowed us to identify antioxidants that inhibit MCO of Abeta(1-6). MCO of the model peptide was inhibited by curcumin, but not dibutylhydroxytoluene, carotene, tocopherol, estradiol or nicotine, revealing a clear difference between curcumin and other antioxidants. This novel assay may allow for the identification of antioxidants that protect against MCO of peptides and proteins related to degenerative diseases.

Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d5 fragment ions at m/z 180→105 and 185→110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.

LC-MS/MS and centrifugal ultrafiltration method for the determination of novobiocin in chicken, fish tissues, milk and human serum

We present a rapid and simple method for detecting novobiocin in biologic samples using a methanol-based extraction of the tissue matrix and liquid chromatography with electrospray tandem mass spectrometry (LC-ESI-MS/MS) on positive mode. The sample, prepared using centrifugal ultrafiltration with 5.0% SDS, was directly injected into the LC-MS/MS. Chromatographic separation was performed on a TSK-GEL ODS 100 V column using 0.5% formic acid in water/methanol. The method was validated according to the Japanese Maximum Residue Limits recommendations. Detection was linear over a range of 5-100 ppb matrix solution (r>0.998). Novobiocin recovery values from chicken (0.05 ppm) and fish tissues (0.05 ppm), milk (0.08 ppm), and human serum (0.05 and 0.01 ppm) samples ranged from 71+/-1 to 95+/-2%.

Quantification of N-acetyl-seryl-aspartyl-lysyl-proline in hemodialysis patients administered angiotensin-converting enzyme inhibitors by stable isotope dilution liquid chromatography–tandem mass spectrometry

We developed a sensitive, selective and accurate method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) to determine N-terminal thymosin-β peptides of Ac-SDKP and Ac-ADKP in human plasma samples. Quantification of Ac-SDKP and Ac-ADKP was performed using solid phase extraction (SPE) based on C(18), reversed phase LC separation, and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) and Ac-ADKP-d(7) were synthesized for the internal standards. These MRM monitoring ions were m/z 488→129 (quantitative ion)/226 for Ac-SDKP, m/z 496→137 for Ac-SDKP-(13)C(6), (15)N(2), m/z 472→129 (quantitative ion)/226 for Ac-ADKP, and m/z 479→129 for Ac-ADKP-d(7), respectively. Lower limit of quantitation (LLOQ) of Ac-SDKP and Ac-ADKP was 0.1ng/mL in human plasma. Recovery values were ranged from 94.7% to 106.3% for inter- (RSD: 0.6-3.5%) and intra- (RSD: 0.4-4.9%) day assays. Plasma Ac-SDKP levels were significantly higher in hemodialyzed subjects treated with angiotensin-converting enzyme inhibitors of enalapril (27.3±24.6ng/mL, n=10) and trandolapril (12.3±16.9ng/mL, n=18) than healthy (0.4±0.2ng/mL, n=7) and hemodialyzed subjects (0.6±0.2ng/mL, n=34). This analytical method would be useful to measure N-terminal thymosin-β peptides in human plasma for the clinical study.

Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry Method for the Determination of Bicozamycin in Milk

Abstract A fast and simple method for the quantitative determination of bicozamycin (BCM) in milk samples by hydrophilic interaction liquid chromatography with electrospray tandem mass spectrometry (HILIC-ESI-MS/MS) and centrifugal ultrafiltration (CUF) was presented. The milk sample was extracted with an acetonitrile/water (4/1, v/v) and CUF procedure. After preparation, the sample solution was directly injected onto the HILIC-MS/MS. Chromatographic separation of the components was performed on a TSK-GEL NH2 column using 50 mM ammonium acetate in water (pH 4.0 adjusted with acetic acid) and acetonitrile. The mass spectrometer was operated in the negative ESI-MS/MS mode (m/z 301 → 209). The LOD and LOQ were 2.5 ng/mL (25 pg) and 5 ng/mL, respectively. This method was validated according to the Japanese maximum residue limit (0.1 µg/g) of BCM. The matrix matched calibration of BCM was linear over the calibration range from 50 to 500 ng/g (r2 = 0.999). Recovery values were from 82.6 to 109.9% (RSD 7.5%, n = 27). The present method could be applied to measure BCM in milk samples.

Separation of Major Safflowers from Carthamus Yellow using High‐Speed Countercurrent Chromatography

Abstract High‐speed countercurrent chromatography (HSCCC) has been successfully applied to the separation of safflower A (SF‐A) and safflower B (SF‐B) from Carthamus yellow, Carthamus tinctorius L. A 25 mg quantity of Carthamus yellow was separated using a two‐phase solvent composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.5% aqueous trifluoroacetic acid solution (2/2/1/5, V/V). The total separation time was 6 hr with the total elution volume of 720 mL. HSCCC fractions were analyzed using LC/MS/MS with scan and daughter scan modes, and DAD monitored by wide absorbance from 200 to 500 nm. The separation yielded 1.5 mg of SF‐A (95%>purity) and 1.1 mg of SF‐B (95%>purity). These results present a successful application of HSCCC to the preparative purification of major safflowers.

A strategy for high-speed countercurrent chromatography purification of specific antioxidants from natural products based on on-line HPLC method with radical scavenging assay

We have proposed a novel and first strategy of high-speed countercurrent chromatography (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in materials are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of target peaks would be investigated for the two-phase solvent system, and purified by HSCCC. In this study, the specific antioxidants in red cabbage, perilla and elderberry pigments were evaluated by on-line HPLC with DPPH radical scavenging assay, and purified by HSCCC technique. Specific antioxidants could be rapidly pinpointed in complex mixtures by on-line HPLC with DPPH radical scavenging assay. Then, the optimal two-phase solvent systems were investigated using these HPLC peaks. Finally, the purification of these nine antioxidants form three mixtures were performed by HSCCC. Using mass spectrometric analysis, these antioxidants were confirmed to cyanidin-based anthocyanin from red cabbage and elderberry pigments, and luteolin-based flavones from perrilla pigment. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.