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A high-performance liquid chromatography with electrochemical detection (HPLC-ECD) system was developed for the simultaneous determination of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) in human plasma. In the present HPLC-ECD system, EPA, DHA, and AA were separated using a reverse-phase C30 column and detected based on the voltammetric reduction of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ). Chromatographic peak areas were proportional to the concentration of EPA, DHA, and AA from 0.75 μM to 0.1 mM (r > 0.998). The concentrations of EPA, DHA, and AA in plasma from healthy human subjects after overnight fasting were determined, and the ratio of EPA to AA was obtained by the present HPLC-ECD method, which required 40 μL of human plasma and a simple procedure of sample preparation using diethyl ether extraction. Moreover, changes in EPA, DHA, and AA concentrations in a human subject were monitored before and after fish oil supplement administration by the present HPLC-ECD system.

A sensitive method for the electrochemical determination of synephrine (SYN) by hydrophilic interaction liquid chromatography (HILIC) has been developed. Optimal chromatographic separation and high sensitive determination by HILIC with electrochemical detection (HILIC-ECD) was achieved using a sulfobetaine-type zwitterionic monolith column (100×1.02 mm, i.d.), a mixture of 10 mM sodium phosphate (pH 4) and acetonitrile (20 : 80, v/v) as mobile phase, and a glassy carbon working electrode which was applied with a potential at +1.0 V vs. Ag/AgCl. The chromatographic peak height of SYN was proportional to the concentration from 5.0 µg/L to 1.0 mg/L (r=0.999). The detection limit of SYN (S/N=3) was 3.7 pg on the column. Moreover, the present HILIC-ECD could be applied to the accurate and precise determination of SYN in Aurantii nobilis Pericarpium. In conclusion, we have demonstrated that an ECD is one of useful detection methods applicable to HILIC.

A method for the determination of ergosterol, brassicasterol, and cholesterol has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). Using an ODS column and a mobile phase of acetonitrile-water (95:5, v/v), we found that the peak area was linearly related to the amount of sterols injected, ranging from 1.5 to 100 μmol/L (r ≥ 0.998), except for cholesterol (2.5–400 μmol/L). Repeated injection showed that the relative standard deviation (RSD) at 50 μmol/L of sterols was less than 7.1% (intra-day, n = 6) and 13% (inter-day, n = 3). Ergosterol and brassicasterol in rat serum spiked at four concentration levels (16, 25, 100, 250 μmol/L) was determined by the internal standard (IS) method using campesterol as an IS with the recovery of more than 88.8% and the RSD (n = 5) of less than 12.5%. Practical applications: This method has been applied to determine the serum ergosterol and brassicasterol concentration in spontaneously hypertensive rats stroke-prone (SHRSP rats) fed a high-ergosterol diet containing 1% ergosterol. The results indicate that no apparent peak for ergosterol was observed, whereas the serum brassicasterol concentration was increased by the ergosterol-feeding. Brassicasterol was confirmed by electrospray ionization triple quadrupole tandem mass spectrometry after derivatization to picolinyl esters. In addition, brassicasterol-d1 was detected after oral administration of ergosterol-d1 to SHRSP rats by ion-trap time-of-flight mass spectrometry. Because SHRSP rats are known as an experimental animal model of phytosterolemia, it has been expected that ergosterol is absorbed and converted to brassicasterol. In summary, the current method provides an experimental tool to help understand ergosterol absorption and metabolism. A method for the determination of ergosterol, brassicasterol, and cholesterol has been developed by UPLC-UV. We found that a high-ergosterol diet induces the elevation of serum brassicasterol in SHRSP rats.

When 3,5-di-t-butyl-1,2-benzoquinone (DBBQ) is electrochemically reduced in an unbuffered solution, the addition of small amounts of acid to the solution is found to give rise to a new peak (termed the prepeak) at a more positive potential than the original reduction potential of DBBQ. In addition, the prepeak current heights of DBBQ are proportional to the added acid concentration. From these findings, we utilized the voltammetric behaviors of DBBQ to develop an analytical method for the determination of titratable acidity in food and beverage samples. Moreover, a portable voltammetric sensor (weight, 100 g; power, two AAA batteries), that implements this analytical method, was developed in order to provide an on-site analytical device. To show the applicability of this portable sensor, the determination of titratable acidity in fruit juice, wine, Japanese sake, and shochu was performed. The results obtained by the portable sensor showed a good correlation with those from official potentiometric titration using 0.1 mol/L NaOH. In conclusion, the present sensor required a reduced sample volume and measurement time for an assay compared with conventional potentiometric titration, consequently showing potential for quality assessment in a wide range of food and beverage analyses.

A highly sensitive and simple method for determining cryptotanshinone (Cry), tanshinone I (Tan I), and tanshinone IIA (Tan IIA) in Salvia miltiorrhiza was developed using micro HPLC with electrochemical detection (µHPLC-ED). The tanshinones were extracted using an ultrasonic method, with methanol as the extractant. The separation was carried out on a C18 (150 mm × 1.0 mm i.d., 3 µm particle size) analytical column. The mobile phase was acetonitrile-water-formic acid mixture (52:48:0.6, v/v/v) solution. The flow rate and the temperature of the column were set at 30 µL/min and 35°C. The applied potential was set at −0.4 V vs. Ag/AgCl. The peak heights for Cry, Tan I, and Tan IIA were found to be linearly related to the amounts injected, ranging from 0.145 µmol/L to 3.88 µmol/L, 0.226 µmol/L to 3.01 µmol/L, and 0.149 µmol/L to 3.98 µmol/L, respectively. The RSD values of intra-day precision and repeatability were not more than 3.0%, and the recovery of three analytes were ranged from 96.7% to 97.5%. The resu...

This paper puts forward a time and material-saving method for evaluating the repeatability of area measurements in gradient HPLC with UV detection (HPLC-UV), based on the function of mutual information (FUMI) theory which can theoretically provide the measurement standard deviation (SD) and detection limits through the stochastic properties of baseline noise with no recourse to repetitive measurements of real samples. The chromatographic determination of terbinafine hydrochloride and enalapril maleate is taken as an example. The best choice of the number of noise data points, inevitable for the theoretical evaluation, is shown to be 512 data points (10.24 s at 50 point/s sampling rate of an A/D converter). Coupled with the relative SD (RSD) of sample injection variability in the instrument used, the theoretical evaluation is proved to give identical values of area measurement RSDs to those estimated by the usual repetitive method ( n = 6) over a wide concentration range of the analytes within the 95% confidence intervals of the latter RSD. The FUMI theory is not a statistical one, but the “statistical” reliability of its SD estimates ( n = 1) is observed to be as high as that attained by thirty-one measurements of the same samples ( n = 31).

A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.

A capillary liquid chromatography with UV detection (CLC-UV) system has been developed for determining platinum-based antitumor drugs (e.g., cisplatin, carboplatin, and nedaplatin) in plasma based on the pre-column derivatization of platinum with N,N-diethyl dithiocarbamate (DDTC). The chelated platinum separation was carried out on a capillary column (Inertsil ODS-3, 150 mm × 0.3 mm i.d., 3 µm) using an acetonitrile-water mixture (8:2, v/v) as a mobile phase that flowed at 5.0 µL/min. Detection was carried out by absorbance at 254 nm. Chromatographic peak height was found to be linearly related to the spiked concentration of nedaplatin in the blank control plasma from 5.0 ng/mL to 15 µg/mL (r(2)>0.998). The repeatability (n=5) of the chromatographic peak height for 2.5 µg/mL nedaplatin was 2.6% relative standard deviation (R.S.D.). The CLC-UV system, which required only 20 µL of plasma sample, was applied to the determination of total and free form platinum-based antitumor drugs in plasma after injection into rats. The recovery rates (n=5) of total and free form nedaplatin in plasma were 98% and 99%, respectively, and these repeatability were 2.4% R.S.D. and 3.1% R.S.D., respectively. In addition, the recovery rates (n=5) of total and free form carboplatin in plasma were 99% and 99%, respectively, and these repeatability were 2.9% R.S.D. and 0.24% R.S.D., respectively. The concentration-time profiles of total and free form nedaplatin in rat plasma were monitored to determine the pharmacokinetic parameters.

For the performance assessment of an electrochemical flow cell using a boron-doped diamond (BDD) electrode in high-performance liquid chromatography with electrochemical detection (HPLC-ECD) to determine cholesterol, the standard deviation (SD) of area measurements in chromatographic baseline noise (AMCBN) was evaluated based on the function of mutual information (FUMI) theory without repetitive chromatographic measurements. After the selection of suitable components for the electrochemical flow cell in HPLC-ECD, the detection limit of cholesterol was 0.04 pmol at a detection potential of +2.2 V vs. an Ag/AgCl electrode. The present HPLC-ECD was applied to determine cholesterol in various meats.

The Tf2CH-CH2 functionality is introduced to a wide range of phenols or phenol ethers ortho to the hydroxy or ether group.

A novel electrochemical detection coupled with high-performance liquid chromatography (HPLC) was developed for determining theophylline in plasma samples. On a voltammogram of acetonitrile containing trolox (TrOH), theophylline and LiClO 4 , a new oxidation peak (termed a prepeak) appeared at a potential less positive than that for the oxidation peak of TrOH without theophylline. Since the prepeak current linearly increased with the theophylline concentration, the electrochemical detection was carried out by measuring the prepeak current height at a constant potential in a flow-type thin-layer cell. HPLC using a silica gel column and a mobile phase of acetonitrile was conducted for the separation of the theophylline. The eluate from the column was mixed with a solution of 1.5 mmol L −1 TrOH and 20 mmol L −1 LiClO 4 in acetonitrile. The chromatographic peak height for theophylline at a detection potential of +1.1 V vs. Ag/AgCl was found to be linearly related to the concentrations injected, ranging from 2.0 to 100 μmol L −1 ( r > 0.999). The detection limit of the concentration of theophylline was 0.55 μmol L −1 . The present method, which required only 10 μL of plasma sample, was applied to the determination of theophylline in rat plasma. The concentration–time profile of theophylline in rat plasma was monitored to determine pharmacokinetic parameters.

A simple method for the detection of oxysterols in oxidatively modified LDL (Ox-LDL) has been developed using MALDI-TOF MS. To identify the ion peaks of oxysterols, seven major oxysterols in Ox-LDL (7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β-epoxycholesterol, 25-hydroxychokesterol, (25R)-26-hydroxycholesterol), and cholesta-3,5-dien-7-one were analyzed by MALDI-TOF MS. Among these oxysterols, 7-ketocholesterol, a very abundant oxysterol in Ox-LDL, was found to show a characteristic peak of [M + H] + at m/z 401. Cholesta-3,5-dien-7-one, which is known as a degradation product of 7-ketocholesterol upon saponification of Ox-LDL, gave a major peak of [M + H] + at m/z 383. In contrast, other oxysterols showed similar peak patterns at m/z 367 and 385. These results were applied to the analysis of Ox-LDL by MALDI-TOF MS after saponification and hexane-extraction, detecting ion peaks at m/z 367, 383, 385, and 401. This MALDI-TOF MS method has a potential as a simple tool to show the presence of oxysterols in Ox-LDL without derivatization and chromatographic separation.

Cholesterol has been shown to be electrochemically oxidized at a carbon electrode to form cholesta-4,6-dien-3-one in acetonitrile [Hosokawa, Y. Y.; Hakamata, H.; Murakami, T.; Aoyagi, S.; Kuroda, M.; Mimaki, Y.; Itoh, A.; Morosawa, S.; Kusu, F. Electrochim. Acta , 2009 , 54 , 6412–6416]. To further expand on this reaction in the field of organic synthesis, the electrolysis conditions of cholesterol were optimized on a laboratory synthetic scale. Using a flow-through column electrolysis system, cholesta-4,6-dien-3-one was efficiently produced at the applied potential of 1.9 V versus Ag/AgCl with a flow rate of 2.5 mL/min, providing a green tool for the synthesis of cholesta-4,6-dien-3-one.

Cholesterol was shown to be oxidized at the glassy carbon electrode in an acetonitrile–2-propanol mixture and this oxidation reaction was applied to the determination of serum total cholesterol by high-performance liquid chromatography with electrochemical detection (K. Hojo, H. Hakamata, A. Ito, A. Kotani, C. Furukawa, Y.Y. Hosokawa, F. Kusu, J. Chromatogr. A 1166 (2007) 135–141). To gain insight into the detection mechanisms of cholesterol, an electrolytic product of cholesterol was collected and characterized by infrared spectroscopy, one- and two-dimensional nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The three techniques, together with comparisons of literature spectral data, confirmed the formation of cholesta-4,6-dien-3-one. The conversion of cholesterol to cholesta-4,6-dien-3-one, a four-electron, four-proton electrochemical process, has been proposed as an electrochemical oxidation mechanism of cholesterol in acetonitrile.

A simple method for the determination of oxysterols was developed by semi-micro high-performance liquid chromatography with electrochemical detection (semi-micro HPLC–ECD). Semi-micro HPLC–ECD was established using a C30 microbore column, acetonitrile containing 50 mmol/L LiClO 4 as a mobile phase, and an applied potential at +2.8 V versus Ag/AgCl. The current peak height was linearly related to the amount of sterol injected from 12.5 to 250 pmol ( r > 0.999) with a relative standard deviation (RSD) of less than 2.9% ( n = 6). This method was applied to the determination of seven oxysterols in oxidatively modified low-density lipoprotein (Ox–LDL). Oxysterols were determined with a recovery of more than 78.0% and an RSD of less than 2.9% ( n = 6) except for 7-ketocholesterol. 7-Ketocholesterol was determined as a sum of intact 7-ketocholesterol and its degradation product on saponification, cholesta-3,5-dien-7-one, with a recovery of 98.0% and an RSD of 2.5% ( n = 6). From these results, the current method enabled the simultaneous determination of seven oxysterols without any derivatization, providing a useful tool for the assessment of oxysterol contents in Ox–LDL.

A simple method for determining short-chain fatty acids (SCFAs) in rat and human feces was developed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). A two-channel HPLC-ECD system was fabricated using an ion exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solutions of 0.1 mM HClO(4) and of ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and a quinone solution, respectively. Peak areas for lactic, acetic, propionic, butyric, isovaleric, and valeric acids at a detection potential of -0.9 V vs. an Ag/AgCl electrode showed a linear relationship with the acid amount in the range 0.1 to 40 nmol. Standard acids at 4 nmol were determined ten times with relative standard deviations (RSD) of less than 2.0%. The analytical results of healthy human feces were measured within 35 min. RSD (n = 5) in all SCFAs were less than 2.7%, and recoveries of SCFAs were more than 92%. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes, and it has the potential for clinical and biomedical applications.

The optimum conditions of the mobile phase for HPLC with electrochemical detection (HPLC-ECD) were selected from among solvents of different grades with the standard deviation (SD) of area measurements based on FUMI (function of mutual information) theory as a criterion. In HPLC-ECD for determining baicalin and baicalein using a phosphoric acid-methanol-water mixture (0.5:60:40, v/v/v) as a mobile phase, the effects of three grades of phosphoric acid, of five grades of methanol, and of seven grades of water on the measurement precision were examined. The mixture, if characterized by the minimum SD of measurements, was selected as the optimal mobile phase. This selection method saves considerable amounts of chemicals and experimental time, and would be a useful exploration technique for the routine check and troubleshooting of HPLC-ECD.

A novel technique for measuring the concentration rate of a redox compound at an electrode/solution interface was developed using slab optical waveguide (SOWG) spectroscopy. The electrolysis of 5 mmol dm −3 ferroin in 1 mol dm −3 potassium nitrate solution was carried out on an indium-tin oxide (ITO) electrode and plastic-formed carbon (PFC) electrode by cyclic voltammetry, using each spectroelectrochemical cell in SOWG spectroscopy. Their SOWG spectra were obtained every 0.05 mV during the potential sweep between +0.4 and +1.4 V for the ITO electrode and between +0.4 and +1.2 V for the PFC electrode. To obtain the concentration profiles near the electrode during the cyclic voltammetry of ferroin, the SOWG spectra were converted to concentration ratios at given locations, and these ratios were plotted against a penetration depth of evanescent wave. The theoretical derived concentration–distance profiles during CV were experimentally verified by the present technique.

A simple method is described for the determination of azaperone and its metabolite, azaperol, in animal tissues by high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Chromatography was performed using an ODS column, an acetonitrile-0.025% aqueous diethylamine mixture (2:3, v/v) as a mobile phase and UV detection at 250 nm. Peak heights were found linearly related to the concentrations injected from 0.05 to 2 microg/mL (r>0.999). Azaperone and azaperol spiked into several animal tissues were solubilized in 1 mol/L NaOH, extracted with hexane, transferred to 0.1 mol/L H(2)SO(4) and re-extracted with hexane in a mild basic condition. Recoveries of both compounds from 12 types of samples (swine muscle, swine adipose tissue, swine liver, bovine muscle, bovine adipose tissue, bovine liver, poultry muscle, poultry adipose tissue, poultry liver, bovine milk, poultry egg, and salmon muscle) were more than 72%. The lower limit of quantification of was 0.025 microg/g. Azaperone and azaperol at 0.1 microg/g were confirmed by LC/MS. In conclusion, we found this method is both simple and useful for the determination of azaperone and azaperol in a variety of animal tissues for food safety and veterinary applications.

The voltammetric behavior of trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) in the presence of amino acids was studied at a plastic formed carbon working electrode in an unbuffered dimethylsulfoxide solution containing 50 mmol L−1 LiClO4. A well-defined oxidation peak was observed on the linear potential sweep voltammogram of trolox at 0.6 V vs. Ag/AgCl. Following the addition of amino acid (such as alanine, arginine, asparagine, glutamic acid, glutamine, glycine, histidine, homocysteine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) to the solution, a new peak (termed a prepeak) appeared on the voltammogram at more negative potentials than the original oxidation peak of trolox. The half-peak potential (Ep/2) of the prepeak shifted to a more negative value accompanied by a decrease in the pKb1 of the added amino acid. A plot of the Ep/2 of the prepeak against the pKb1 shows an approximately linear line with a slope of 31 mV p K b 1 − 1 and correlation coefficient (r) of 0.709. Since a plot of the Ep/2 of the prepeak against the pH at the isoelectric point (pI) shows a linear line with a slope of 19 mV pI−1 and r of 0.958, amino acid would contribute to the proton transfer process in this electrode reaction not as a conjugated base but as an amphoteric zwitterion under the unbuffered condition. The prepeak height was proportional to the amino acid concentration ranging from 0.01 to 0.2 or 0.3 mmol L−1 with an r of more than 0.997. The present method has a possibility for the electrochemical determination of amino acids.

Rapid and unambiguous identification of three degradation products (DP-1, DP-2 and DP-3) found in heat-stressed loxoprofen sodium adhesive tapes (Loxonin tapes) was achieved by LC-MS and dynamic pressurized liquid extraction (PLE)-solid-phase extraction (SPE) coupled to LC-NMR without complicated isolation or purification processes. The molecular formulae of the degradation products were determined by accurate mass measurements and product ion analyses and on-line hydrogen/deuterium (H/D) exchange experiments provided information about changes in the degradation of loxoprofen. To compensate for the low sensitivity of NMR, on-line dynamic PLE-SPE was employed and higher concentrations of degradation products trapped on the SPE column were afforded in a shorter time than they would be in such time-consuming sample preparations as pre-concentration after extraction. The loop-storage procedure was used in the LC-NMR analysis to allow the acquisition of the (1)H spectra of the three degradation products in one chromatographic run without affecting the peak separation and to avoid the carry-over of previously eluted DP-1 of high concentration by washing the NMR detection cell prior to the measurement of the DP-2 spectrum. Based on the resulting (1)H NMR spectra in combination with the MS results, DP-1 was successfully identified as an oxidation product having an oxodicarboxylic acid structure formed by the cleavage of the cyclopentanone ring of loxoprofen, DP-2 as a cyclopentanone ring-hydroxylated loxoprofen and DP-3 as a loxoprofen l-menthol ester.

An unknown degradation product (DP-1) increased in olmesartan medoxomil (OLM) tablets stored at 40 °C/75% r.h., reaching 0.72% after 6 months. The molecular weight and fragment information obtained by LC–MS suggested that DP-1 was a dehydrated dimer of olmesartan (OL) and the presence of ester carbonyl group was indicated by solvent-elimination LC–IR analysis. LC– 1 H NMR confirmed the structure of DP-1 as an esterified dimer of OL. Rapid and accurate identification of the degradation product was achieved by the complementary use of HPLC hyphenated techniques without complicated isolation or purification processes.

Application of the HPLC hyphenated techniques of LC-two-dimensional (2D) NMR using pre-concentration column trapping and LC-MS was demonstrated by the identification of two major degradation products, DP-1 and DP-2, in stressed commercial tablets of amlodipine maleate. The molecular formulas were estimated by LC-MS. Sample pre-concentration by column trapping was conducted to obtain adequate 2D-NMR signals by reducing the peak widths of the degradation products and making sure that the maximum amount of each component was inside the flow cell for NMR detection. Double-quantum filtered correlation spectroscopy (DQF-COSY) was applied to identify DP-1 as beta-N-lactosylamlodipine by suppressing the residual water signal without affecting the sample signal and by measuring the coupling constant of the lactose anomeric proton. Heteronuclear multiple bond coherence spectroscopy (HMBC) was applied to characterize DP-2 as an aspartic acid derivative of amlodipine by detecting long-range CH correlations. The chemical structures of the degradation products could be successfully elucidated unambiguously without an isolation process.

A measurement uncertainty estimation in the determination of ethyl aminobenzoate by diazotization titration and HPLC with UV detection (HPLC-UV) was carried out. Fishbone diagrams for determining the ethyl aminobenzoate purity by diazotization titration and HPLC-UV were prepared for the identification of factors that affect measurement uncertainties. In the case of diazotization titration, combined standard uncertainties for titers of a sodium nitrite solution used in the standardization and determination were larger than that for other factors. In the case of the HPLC, combined standard uncertainties for the chromatographic peak areas of ethyl aminobenzoate in standard and sample solutions were larger than that for other factors. A determination of the ethyl aminobenzoate purity in the bulk powder was also carried out using diazotization titration and HPLC-UV. The results concerning the ethyl aminobenzoate purity by diazotization titration and HPLC-UV were 100.5% (RSD = 0.26%, n = 6) and 100.6% (RSD = 0.36%, n = 6), respectively, indicating that both analytical results were essentially identical. The estimated combined standard uncertainties were within the 95% confidence interval of SD, which were obtained by the above-mentioned repetitive measurements.

A simple method for determination of mosapride citrate and its metabolite, des-p-fluorobenzyl mosapride (M-1), in equine muscle, liver, kidney, adipose tissue and intestine by liquid chromatography–tandem mass spectrometry has been developed. (±)-4-Amino-5-chloro-2-ethoxy-N-[[4-(2-chlorobenzyl)morpholinyl]methyl]benzamide was used as an internal standard. The analytes and internal standard were spiked and extracted from tissues by acetonitrile. The chromatographic separation was performed on a reversed-phase TSK-GEL SUPER ODS column with a mobile phase of acetonitrile–0.05% (v/v) formic acid containing 5 mmol/L nonafluoropentanoic acid (2:3, v/v). The method exhibited a large linear range from 0.0005 to 0.2 μg/mL for both mosapride citrate and M-1 (r > 0.9976). In the intra-day assay (n = 5), the relative standard deviations (RSDs) ranged from 1.1 to 7.8% for mosapride citrate and 1.6 to 7.2% for M-1. In the inter-day assay (n = 3), the RSDs ranged from 1.0 to 13% for mosapride citrate and 0.8 to 11% for M-1. The extraction recovery at 1.28 μg/g of mosapride citrate from equine tissues ranged from 97 to 107%. The lower limit of quantification for mosapride citrate was found to be 0.004 μg/g. Stability studies were carried out at different storage conditions. The method reported is reliable, precise, and accurate and it has the capacity to be used for determination of mosapride citrate and its metabolite in tissue samples.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.

Various acids contained in food show signicant effects on both taste and aroma. Acids also inuence the ability of microorganisms to grow, so some, such as citric acid, lactic acid, or phosphoric acid, are used as common food additives for preservation or pH control. Therefore, the determination of acidity and acid content is important for the taste and quality assessment of food, regardless of whether acids exist in the original foods or in the additives. For example, fats and oils tend to decompose slowly upon storage in contact with the atmosphere and to release their fatty acid constituents, resulting in a lowering of their quality. Thus, a quality and freshness assessment of fats and oils can be made based on the determination of their free fatty acid content. In the case of beverages such as coffee and fruit juice, various organic acids are natural ingredients. pH control agents and acidulants are food additives used to achieve the stability and/ or avor of foods. Even though the content of each acid is small, the acid amount has a signicant effect on the taste and aroma of the beverages. Therefore, an acid assay is quite important for processing and quality control in the manufacturing and preservation of foods and beverages.

We have proposed an assessment methods to estimate the measurement relative standard deviation (RSD) of chromatographic peaks in quantitative HPLC for herbal medicines by the methodology of ISO 11843 Part 7 (ISO 11843-7:2012), which provides detection limits stochastically. In quantitative HPLC with UV detection (HPLC-UV) of Scutellaria Radix for the determination of baicalin, the measurement RSD of baicalin by ISO 11843-7:2012 stochastically was within a 95% confidence interval of the statistically obtained RSD by repetitive measurements (n = 6). Thus, our findings show that it is applicable for estimating of the repeatability of HPLC-UV for determining baicalin without repeated measurements. In addition, the allowable limit of the "System repeatability" in "Liquid Chromatography" regulated in a pharmacopoeia can be obtained by the present assessment method. Moreover, the present assessment method was also successfully applied to estimate the measurement RSDs of quantitative three-channel liquid chromatography with electrochemical detection (LC-3ECD) of Chrysanthemi Flos for determining caffeoylquinic acids and flavonoids. By the present repeatability assessment method, reliable measurement RSD was obtained stochastically, and the experimental time was remarkably reduced.

To summarize our electroanalytical research in the biomedical field over the past 43 years, this review describes studies on specular reflection measurement, redox potential determination, amperometric acid sensing, HPLC with electrochemical detection, and potential oscillation across a liquid membrane. The specular reflection method was used for clarifying the adsorption of neurotransmitters and their related drugs onto a gold electrode and the interaction between dental alloys and compound iodine glycerin. A voltammetric screening test using a redox potential for the antioxidative effect of flavonoids was proposed. Amperometric acid sensing based on the measurement of the reduction prepeak current of 2-methyl-1,4-naphthoquinone (VK3) or 3,5-di-tert-buty1-1,2-benzoquinone (DBBQ) was applied to determine acid values of fats and oils, titrable acidity of coffee, and enzyme activity of lipase, free fatty acids (FFAs) in serum, short-chain fatty acids in feces, etc. The electrode reactions of phenothiazines, catechins, and cholesterol were applied to biomedical analysis using HPLC with electrochemical detection. A three-channel electrochemical detection system was utilized for the sensitive determination of redox compounds in Chinese herbal medicines. The behavior of barbituric acid derivatives was examined based on potential oscillation measurements.

For the determination of seven caffeoylquinic acids [neochlorogenic acid (NcA), cryptochlorogenic acid (CcA), chlorogenic acid (CA), caffeic acid (CfA), isochlorogenic acid A (Ic A), isochlorogenic acid B (Ic B), isochlorogenic acid C (Ic C)] and two flavonoids [luteolin 7-O-glucoside (LtG) and luteolin (Lt)], a three-channel liquid chromatography with electrochemical detection (LC-3ECD) method was established. Chromatographic peak heights were proportional to each concentration, ranging from 2.5 to 100 ng/mL for NcA, CA, CcA, and CfA, and ranging from 2.5 to 250 ng/mL for LtG, Ic B, Ic A, Ic C, and Lt, respectively. The present LC-3ECD method was applied to the quantitative analysis of caffeoylquinic acids and flavonoids in four cultivars of Chrysanthemum morifolium flowers and their sulfur-fumigated products. It was found that 60% of LtG and more than 47% of caffeoylquinic acids were lost during the sulfur fumigation processing. Sulfur fumigation showed a destructive effect on the C. morifolium flowers. In addition, principle component analyses (PCA) were performed using the results of the quantitative analysis of caffeoylquinic acids and flavonoids to compare the "sameness" and "differences" of these analytes in C. morifolium flowers and the sulfur-fumigated products. PCA score plots showed that the four cultivars of C. morifolium flowers were clearly classified into four groups, and that significant differences were also found between the non-fumigated C. morifolium flowers and the sulfur-fumigated products. Therefore, it was demonstrated that the present LC-3ECD method coupled with PCA is applicable to the variation analysis of different C. morifolium flower samples.

Determination of hesperidin contents in Pericarpium Citri Reticulatae was performed by a simple extraction with methanol and semi-micro high-performance liquid chromatography with electrochemical detection (muHPLC-ECD). Chromatography was performed using a microbore octadecylsilica (ODS) column, methanol-water-phosphoric acid (40:60:0.5, v/v/v), as a mobile phase and applied potential at +0.9 V versus Ag/AgCl. Peak heights were found linearly related to the concentrations of hesperidin injected 9.16 ng/ml to 3.06 microg/ml (r>0.999). The detection limit (S/N=3) was 3.06 ng/ml (15.3 pg). Hesperidin of 305 ng/ml was detected with a relative standard deviation (R.S.D.) of 0.79% (n=5). Hesperidin in Pericarpium Citri Reticulatae was extracted with methanol, diluted with the mobile phase, and injected into the muHPLC-ECD for determination. The hesperidin content of Pericarpium Citri Reticulatae from four different districts in China were determined with R.S.D. of 3.59%, 2.29%, 2.36%, and 2.32% (n=5), respectively. Recoveries of hesperidin from the four Pericarpium Citri Reticulatae sources were 100.3%, 99.83%, 100.7%, and 100.6%, respectively. This method is useful for the determination of hesperidin in Pericarpium Citri Reticulatae, and especially so for instances when samples are sparse.

A method is proposed to detect the abnormal situations of people's health in case of the unexpected outbreak of a disease by monitoring the daily variations in the prescriptions at a pharmacy. The abnormal situations are defined as the situations which are not included in the majority (99.9%) of the normal situations. An epidemic probably caused by infectious micro-organisms in a terrorist attack is taken as an example. The drugs for the typical symptoms are monitored: influenza anti-viral agent and common cold drug. This paper demonstrates that the border between the normal and abnormal situations corresponds to the detection limit which is a fundamental concept in analytical chemistry.

A highly sensitive method for determining baicalin and baicalein in rat plasma was developed using micro HPLC with electrochemical detection (muHPLC-ED). Peak heights for baicalin and baicalein were found to be linearly related to the amounts injected, ranging from 2.2 pg to 4.5 ng (r = 0.997) and from 1.4 pg to 2.7 ng (r = 0.997), respectively. The detection limits (signal/noise ratio = 3) of the current method for baicalin and baicalein were 0.89 and 0.54 pg, respectively. The concentrations of baicalin, baicalein, and their conjugates in rat plasma after oral administration of 2.0 mg/kg of Saiko-keishi-to (TJ-10) were determined. The glucuronide and sulfate forms of baicalin and baicalein in plasma were hydrolyzed enzymatically using beta-glucuronidase and sulfatase, respectively, and the hydrolyzed solutions were extracted with a 0.1-mol/L phosphoric acid-ethyl acetate mixture (1:1, v/v). Based on the time courses of the concentrations of the free and conjugated forms of baicalin and baicalein in rat plasma after oral administration of Saiko-keishi-to, the pharmacokinetic parameters of C(max), t(max), and AUC(0-6 h) were obtained.

Eighty-one prescription drugs were divided into three groups according to the strength of the auto-correlation (strong, weak or none) of daily variations in the drug supply amounts at a pharmacy. The power spectral density and auto-correlation function were used as an indicator for the classification. Sixty-four drugs fell into the no auto-correlation group, 15 drugs into the weak auto-correlation group and 2 drugs into the strong auto-correlation group. Interestingly, our grouping using auto-correlation as a criterion was consistent with the target-oriented classification (standard commodity classification of Japan).

Slab optical waveguide (SOWG) spectroscopy was carried out for an in situ observation of ferroin and ferriin formed at an electrode/solution interface during electrolysis on an indium-tin oxide (ITO) electrode. A new spectroelectrochemical cell for SOWG spectroscopy was developed to observe in situ absorption spectra of species in the vicinity of the ITO electrode surface. A cyclic voltammogram (CV) of 5 mmol·dm−3 ferroin in 1 mol·dm−3 potassium nitrate aqueous solution was obtained as the potential swept from +1.4 to +0.4 V and then from +0.4 to +1.4 V. Furthermore a cathodic and anodic peaks appeared at +0.82 and +0.92 V, respectively. The SOWG spectra were obtained every 0.05 mV during the sweep. The SOWG spectra at the potential of +0.4 and +1.4 V were identical to the UV-VIS spectra of ferroin and ferriin, respectively. The concentration profiles near the electrode during cyclic voltammetry of ferroin were discussed based on the absorbance at 510 and 650 nm on SOWG spectra. Furthermore, in situ real-time monitoring of absorption during potential step experiment of ferroin was made at various incident angles from 70 to 77° using SOWG spectroscopy.

A weak base present in an unbuffered solution containing α-tocopherol (α-T) was found to give rise to a new peak (termed as the oxidation prepeak) on the voltammogram of α-T at a potential less positive than that for the oxidation peak of α-T. The half-peak potential of the oxidation prepeak for a weak base shifted to a positive value along with an increase in pKb of the base. On voltammograms of flunitrazepam, nitrazepam, bromazepam, chlordiazepoxide, and medazepam each in an acetonitrile solution containing 3 mM α-T and 50 mM LiClO4, the prepeak heights were proportional to their concentration. Based on these findings, a novel approach to the sensing of such weak bases by means of linear sweep voltammetry and controlled potential electrolysis using a flow-type electrochemical cell is proposed. Moreover, an electrochemical detection method for high-performance liquid chromatography (HPLC) was developed for determining benzodiazepines.

A new spectroelectrochemical cell for slab optical waveguide (SOWG) spectroscopy was developed in order to observe in situ an electrode/electrolyte interface for bulk electrolysis. The new SOWG spectroelectrochemical cell has been evaluated by simultaneous electrochemical-absorption experiments of methylene blue (MB) using cyclic voltammetry (CV) and SOWG spectrometry. CV was performed in the SOWG spectroelectrochemical cell using indium tin oxide (ITO) coated glass as the working electrode, platinum wire as the counter electrode and a silver/silver chloride electrode (Ag/AgCl) as the reference electrode. Based on the CV and SOWG spectrometric data, it was found that the SOWG spectra showed the MB spectra on the electrode surface selectively and that SOWG with the cell would be useful as a tool for in situ study of an electrode/electrolyte interface. Using this cell, the effects of the supporting electrolytes, NaNO 3 , KNO 3 , CH 3 COONa, and CH 3 COOK on the absorbance of MB were examined at the potential of +0.8 V versus Ag/AgCl. The decrease in MB absorbance by nitrate ions was greater than that of acetate ions. Therefore the competitive adsorption of nitrate ions was stronger than that of acetate ions. Thus, the decrease in absorbance of MB in the presence of anions demonstrates the competitive adsorption of anions. These results show that the extent of specific adsorption of electrolytes was observed by measuring the SOWG absorbance intensity of MB.