Determination of Eicosapentaenoic, Docosahexaenoic, and Arachidonic Acids in Human Plasma by High-Performance Liquid Chromatography with Electrochemical Detection.

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.

Theoretical repeatability assessment without repetitive measurements in gradient high-performance liquid chromatography.

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 (10.24s 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).

Repeatability Assessment by ISO 11843-7 in Quantitative HPLC for Herbal Medicines.

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.

[Development and application of electroanalytical methods in biomedical fields].

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.

Quantitative comparison of caffeoylquinic acids and flavonoids in Chrysanthemum morifolium flowers and their sulfur-fumigated products by three-channel liquid chromatography with electrochemical detection.

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.

The effect of hyperglycemia on the pharmacokinetics of valproic acid studied by high-performance liquid chromatography with electrochemical detection.

The effects of hyperglycemia on the pharmacokinetics of valproic acid (VPA) were examined by time-concentration profiles of plasma VPA accompanied with blood glucose (BG) changing. In addition, time-concentration profiles of plasma free fatty acids (FFAs) were also obtained to examine the interaction between VPA and FFAs in vivo. For the experiments in vivo, normal rats, given multiple doses of maltose orally, and diabetic rats, which were made to maintain hyperglycemia, were used. Plasma VPA and FFA were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) systems based on the reduction of quinone for the selective determination of acids, respectively. BG was determined by pocket-size glucose meter. The maximum plasma concentrations (Cmax) of VPA in normal rats given multiple doses of maltose orally and in diabetic rats were remarkably decreased in comparison with those in the control rats. From the present study, it was shown that the metabolism of plasma VPA is accelerated under hyperglycemia. Moreover, we also found that VPA was preferentially metabolized in comparison with the plasma FFA in vivo.

Simultaneous determination of various bioactive redox components in Shuang-Huang-Lian preparations using a novel three-channel isocratic elution liquid chromatography with electrochemical detection system.

A novel three-channel isocratic elution high performance liquid chromatography with electrochemical detection (3LC-ECD) system was developed for the simultaneous determination of various bioactive redox components in Shuang-Huang-Lian (SHL) preparations. This 3LC-ECD system consists of three isocratic elution flow ways, two switching valves, four columns, and three electrochemical detectors. Through alternately rotating switching valves to change the elution flow way, six caffeoylquinic acids, four flavonoids, and one phenylethanoid glycoside compound were simultaneously determined within 70 min by the present 3LC-ECD method, in which three compounds of protocatechuic aldehyde, propyl gallate, and kaempferol were used as internal standards. The results demonstrated that the present 3LC-ECD method has achieved desired linearity (r>0.999), precision (relative standard deviation <2.5%), accuracy (recovery, 95.6-103.6%), and high sensitivity (limit of detection, 0.11-0.90 ng/mL). In addition, we successfully determined the contents of these 11 bioactive redox components in 14 batches of SHL oral liquid and 12 batches of SHL lyophilized powder for injection produced by different manufacturers in China.

Development of an electric charge-tunable micro-column for capillary liquid chromatography.

An electric charge-tunable micro-column (CTMC) was developed as a precolumn for reversed-phase capillary chromatography using conductive carbon fibers as the stationary phase. The carbon fibers, which constitute one electrode, are packed into a heat-shrinkable tube, which is attached to a stainless-steel column body. The mobile phase flows into CTMC through a stainless-steel tube that acts as a counter electrode. The retention times for hydrophobic and ionic compounds are controlled by the applied potential. The characteristics, such as the electrolysis efficiency and shift in retention factor, were evaluated. As a case study, estrogens in spiked beef were analyzed by capillary chromatography. With no applied potential, the resolution between the unknown peak and estriol and the recoveries of estriol were 0.81 and 65.5%, respectively. These values improved to 1.2 and 78.8%, respectively, upon applying +500 mV. These results indicate that a high-resolution capillary chromatography system can be achieved with CTMC.

Three-channel column-switching high-performance liquid chromatography with electrochemical detection for determining bioactive redox components in Salvia miltiorrhiza.

Three-channel column-switching high-performance liquid chromatography with electrochemical detection (3LC-3ED), which consists of three flow ways, two switching valves, four columns, and three electrochemical detectors, was developed for determining various redox components in Salvia miltiorrhiza with high sensitivity. In this study, the analytes were divided into three groups as follow: Group I [salvianic acid A (Danshensu) sodium salt (DSS), protocatechuic acid (PA), protocatechuic aldehyde (PAD), and caffeic acid (CA)], Group II [rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SAB)], and Group III [cryptotanshinone (Cry), tanshinone I (Tan I), and tanshinone IIA (Tan IIA)]. By rotating each switching valve to change the elute flow way, the components in Groups I, II, and III were directed into the oxidative (+0.7 V), another oxidative (+0.7 V), and reductive (-0.2V) detection channels, respectively. Chromatographic peaks of components in Groups I, II, and III appeared within 69, 53, and 60 min, respectively, in each channel. The detection limits of DSS, PA, PAD, CA, RA, LA, SAB, Cry, Tan I, and Tan IIA were 4.2, 2.8, 10, 10, 3.9, 3.8, 3.0, 132, 119, and 109 fmol, respectively, thus the sensitivity by the present 3LC-3ED is superior to those of previously reported gradient LC-ED and LC with UV detection. As an application, the phenolic acid contents and tanshinones in nine batches of S. miltiorrhiza were successfully determined.

Capillary liquid chromatography with UV detection using N,N-diethyl dithiocarbamate for determining platinum-based antitumor drugs in plasma.

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.

An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells.

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, ß-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.

Simultaneous determination of serum lathosterol and cholesterol by semi-micro high-performance liquid chromatography with electrochemical detection.

A simple method has been developed for the simultaneous determination of lathosterol and cholesterol by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Lathosterol was found to be electrochemically oxidized and its current peak height was linearly related to the amount of lathosterol injected, ranging from 0.15 µmol/L to 300 µmol/L (r=0.995). Similar results were obtained with cholesterol from 15 µmol/L to 600 µmol/L (r=0.995). The separation was carried out with an ODS column, acetonitrile containing 30 mmol/L lithium perchlorate as a mobile phase, and an applied potential at +2.8 V vs. Ag/AgCl. The detection limit (S/N=3) of lathosterol as well as cholesterol was 0.03 µmol/L (0.15 pmol). Total lathosterol in control human and rat serum was determined by the present method with a recovery of more than 95.8% and an RSD (n=5) of less than 7.3%. The present method was applied to an experiment with rats to examine the effect of lathosterol feeding. There were no significant changes in serum lathosterol or cholesterol levels in rats fed with a high-lathosterol diet for six days. Therefore, we found this method to be both simple and useful for the simultaneous determination of lathosterol and cholesterol in serum.

Column switching high-performance liquid chromatography with two channels electrochemical detection for high-sensitive determination of isoflavones.

Column switching HPLC with electrochemical detection (HPLC-ED), which consists of one pre-column and two electrochemical detectors subsequent to each analytical column, called HPLC-2ED, has been developed for determining isoflavones (daidzin, genistin, daidzein, and genistein) with high sensitivity. In the present HPLC-2ED, the eluted daidzin and genistin from the pre-column were separated on an analytical column using a methanol-water-phosphoric acid mixture (30:70:0.5) as the mobile phase (MP), and daidzein and genistein were separated on another analytical column using a methanol-water-phosphoric acid mixture (50:50:0.5). The way of the elute flow from the pre-column was changed by rotating the switching valve at 17 min. The difference in retention times of genistein between isocratic HPLC-ED and HPLC-2ED was 52.2 min. The detection limit (S/N=3) per column injection (5 microL) of genistein was 0.5 pg. The sensitivity by the present method is superior to that of previously reported gradient HPLC-ED for the determination of isoflavones.

Determination of ethoxyquin by high-performance liquid chromatography with fluorescence detection and its application to the survey of residues in food products of animal origin.

An analytical method using HPLC with fluorescence detection (HPLC-FL) has been developed and applied for the survey of residue levels of ethoxyquin in a variety of food products of animal origin. HPLC was performed using a silica octadecylsilane column, butylhydroxytoluene-acetonitrile-water (0.05 + 800 + 200, v/v/v) mobile phase, and detection at excitation and emission wavelengths of 370 and 415 nm, respectively. HPLC/MS was used to confirm whether a chromatographic peak was ethoxyquin. The LOQ of the foods was 0.01 microg/g, except for pig fat and cow's milk, and the RSD (n=6) at 0.1 microg/mL of the standard solution was 1.12%. The accuracy of the calculated data of the standard solution was within the range of 94.0 to 101.2%. Recoveries of ethoxyquin from the food products of cattle, pigs, chickens, and salmon were more than 71.0% with an RSD of < 9.3%, except for chicken liver at different concentration levels, including the lower LOQ, the maximum residue limit (MRL), and in some tissues, twice the MRL. Residue levels of ethoxyquin in 33 commercially available food products of animal origin that were purchased on the west side of the Tokyo metropolitan area were surveyed. Contents of ethoxyquin residues in three chicken fat samples by the HPLC-FL method were 0.08, 0.03, and 0.04 microg/g, all less than the MRL (5 microg/g).

Determination of serum cholestanol by semi-micro high-performance liquid chromatography with electrochemical detection.

A simple and sensitive method that dose not require derivatization for determining cholestanol has been developed using HPLC with electrochemical detection (HPLC-ECD). The current peak height was linearly related to the amount of cholestanol injected, ranging from 1 to 200 muM (r = 0.999). The detection limit (S/N = 3) of cholestanol was 0.23 muM (1.2 pmol). Total cholestanol in control human and mouse serum was determined by the present method with a recovery rate of more than 90% and an RSD (n = 5) of less than 7.3%. Further, this method was successfully applied to monitor experimental hypercholestanolemia in mice fed a high-cholestanol diet, an animal model of cerebrotendinous xanthomatosis (CTX). In conclusion, we found this method to be both simple and useful for the determination of cholestanol in serum, helping in the diagnosis of CTX.

[DPPH radical scavenging activities of 31 flavonoids and phenolic acids and 10 extracts of Chinese materia medica].

OBJECTIVE: To investigate DPPH radical scavenging activities of 31 flavonoids and phenolic acids and 10 extracts of Chinese materia medica. METHOD: The antioxidant activities of the above samples were evaluated by a DPPH method, the half-wave oxidation potentials (E1/2) of the 31 compounds were determined by an HPLC-CoulArray method, at the same time, phenolic contents of the the total compounds in the 10 extracts of Chinese materia medica were analyzed by Folin-Ciocalteu method. RESULT: The 31 compounds showed a 50% inhibition of DPPH radical in the concentration range of 6.7-3 500 micromol x L(-1), in which (-) -EGCg demonstrated the strongest activity with the IC50 value of 6.7 micromol x L(-1). The E1/2 of 31 compounds spanned a wide potential range of more than 0.6 V. Myricetin had the lowest E1/2 value (20 mV) whereas apigenin and vitexin-2"-O-rhamnoside had the highest E1/2 value (620 mV). Among the 10 herb extracts, having 82% phenolic acid, tea extract showed the strongest DPPH radical scavenging activity with the IC50 value of 0.011 7 mg x mL(-1) whereas safflower demonstrated the weakest DPPH radical scavenging activity with the IC50 value of 1.250 mg x mL(-1), in which only 7% phenolic acids was tested. CONCLUSION: The DPPH radical scavenging activities of the 31 compounds were founded to be related to their chemical structures, such as the number and position of hydroxyl groups. And a qualitative relationship was found between DPPH radical scavenging activities and E1/2 values of the 31 compounds, the lower the E1/2 values, the higher the DPPH radical scavenging activities. A quantitative relationship was obtained to describe the DPPH radical scavenging activity of the herb extracts: Y = 7.779X(-0.48), r = 0.929 5, where Y stands for the concentration for 50% inhibition of DPPH radical, and X stands for the concentration of total phenolic compaunds, namely the extracts with higher content of flavonoids and phenolic acid exhibited the stronger DPPH radical scavenging activity.

Determination of short-chain fatty acids in rat and human feces by high-performance liquid chromatography with electrochemical detection.

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.

Selection of the optimal solvent grade for the mobile phase in HPLC with electrochemical detection based on FUMI theory.

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.