Electrochemical Analysis for Total Alkalinity of Water by the Measurement of Cathodic Prepeak of Quinone Caused by Surplus Acid.

In this study, an electrochemical analysis, coupled with the concept of back neutralization titration and the voltammetric determination of surplus acid, is proposed for determining the total alkalinity of water samples. When linear sweep voltammetry of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) with H2SO4 in a water and ethanol (44 : 56, v/v) mixture was carried out using a bare glassy carbon working electrode, a cathodic prepeak of DBBQ caused by H2SO4 was observed on the voltammogram at a more positive potential than when compared with the original cathodic peak of DBBQ. When similar voltammetry was carried out in the presence of Na2CO3 and H2SO4, the cathodic prepeak height of DBBQ was decreased with an increase in the Na2CO3 concentration. The decrease of the cathodic prepeak height of DBBQ was found to be linearly related to the Na2CO3 concentration ranging from 0.025 to 2.5 mM (r2 = 0.998). The total equivalent concentrations of inorganic bases in samples of mineral water and tap water were determined, and then the results were converted to the total alkalinities of the water samples (mg/L CaCO3). The total alkalinities of the water samples determined by the present electrochemical analysis were essentially the same compared with those by the neutralization titration method. From these results, we were able to demonstrate that the present electrochemical analysis with accuracy and precision could be applied to determine the total alkalinity, which is one of the indicators to examine water quality. The present electrochemical analysis would contribute to achieving the sustainable development goals (SDGs) of #6 and #14.

Supercritical fluid chromatography with post-column addition of supporting electrolyte solution for electrochemical determination of tocopherol and tocotrienol isomers.

A supercritical fluid chromatography with electrochemical detection system was developed for the simultaneous determination of tocopherol and tocotrienol isomers. The supercritical fluid chromatography with electrochemical detection system was connected with an additional pump to create a flow path to add a supporting electrolyte solution. The supporting electrolyte solution was mixed with a mobile phase in a post-column fashion, enabling the independent control of the separation and detection. After optimization of the measurement conditions, vitamin E isomers and an internal standard substance (2,2,5,7,8-pentamethyl-6-hydroxychroman) were separated within 30 min using a mixture of supercritical carbon dioxide and methanol (99:1, v/v) as a mobile phase and a cyanopropyl column (4.6 mm inner diameter × 250 mm length, 5 µm). For the electrochemical detection, methanol containing 1.0 mol/L ammonium acetate was used as a supporting electrolyte solution, and the applied potential was set at +0.8 V. This analytical method showed good linearity (5-100 µg/mL) and repeatability (less than 2.5% relative standard deviation, n = 6) and was applicable to the determination of tocopherol and tocotrienol isomers in nutrition supplements.

Determination of Phenolic Compounds in Beverages by Three-Flow Channel Isocratic HPLC with Electrochemical Detections Using a Column-Switching Technique.

For the quantitative analysis of phenolic compounds in beverage samples, a three-flow channel isocratic HPLC with electrochemical detection (3LC-ECD) system was devised using a column-switching technique. Phenolic compounds with significantly different hydrophobicity (the range of calculated log P: -0.77 to 3.02) were simultaneously measured to draw three chromatograms by the 3LC-ECD; the peaks of gallic acid (GA), protocatechuic acid (PCA), and gallocatechin (GC) were observed at electrochemical detector 1 (D1) within 42 min, the peaks of procyanidin B3 (B3), epigallocatechin (EGC), catechin (C), epicatechin (EC), procyanidin B2 (B2), ethyl gallate (Eg), and epigallocatechin gallate (EGCg) were observed at D2 within 50 min, and the peaks of epicatechin gallate (ECg), gallocatechin gallate (GCg), catechin gallate (Cg), propyl gallate (Pg), and resveratrol (RVT) were observed at D3 within 70 min. The relationships between the phenolic compound concentrations and their chromatographic peak heights gave good linearity with correlation coefficients of more than 0.998. The detection limits of the phenolic compounds by the 3LC-ECD ranged from 0.6 to 3.0 µg/L. Further, the phenolic compound concentrations of commercially available teas and wines were determined with a relative standard deviation (RSD) of less than 4.9% (n = 6), and their recoveries ranged from 91 to 109%. These results indicate that the 3LC-ECD system provided an accurate, precise, and specific determination of the phenolic compounds in beverages without affecting the matrices derived from these samples.

Emerging Separation Techniques in Supercritical Fluid Chromatography.

Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, two-dimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.

Chemometric Evaluation of Repeatability Using the Autocorrelation Method in High-Performance Liquid Chromatography with Ultraviolet Detection.

The mixed random processes of the first order autoregressive process (AR(1)) and white noise have been proved to provide a good approximation of baseline noise in a variety of analytical instruments, and may therefore be useful for estimating precision profiles. This study aims to examine a recently proposed autocorrelation method for estimating three noise parameters involved in the mixed processes (two for AR(1) and one for white noise) of HPLC, which can then be used to calculate the precision profile. This chemometric method was applied to repeatability evaluations of estriol determination using HPLC with UV detection (HPLC-UV). The relative standard deviations (RSDs) of peak area measurements for 5.0 mg/L estriol were observed to be 1.42% for the autocorrelation method and 1.63% for actual repeated measurements of real samples (n = 6). The theoretical RSDs of the autocorrelation method fell within the 95% confidence intervals of the repeated measurements. It is found that the noise parameters are obtained from real chromatographic baseline via the autocorrelation method. Moreover, the instrumental detection limit of estriol based on ISO 11843 was obtained from the precision profile (plot of RSD of measurements against concentration). This is the first paper to describe the autocorrelation method is a practically useful technique for evaluating the precision profile of HPLC-UV analyses without recourse to the repeated measurements of real samples.

Assessment of Repeatability in Supercritical Fluid Chromatography with Electrochemical Detection Based on the ISO 11843 Part 7.

The present study proposes a method for the assessment of repeatability in supercritical fluid chromatography with electrochemical detection (SFC-ECD), based on the ISO 11843 part 7 (ISO 11843-7:2018) which can theoretically provide detection limits and standard deviation (S.D.) through the stochastic properties of baseline noise without repetitive measurements of real samples. On the baseline noise of SFC-ECD, large-amplitude and periodic noises with less than 0.05 Hz were observed, and the power spectrum of the baseline noise showed 1/f fluctuation (f = frequency). It was found that the present power spectrum analysis, according to the law of error propagation, can provide suitable noise parameters to calculate S.D. of baseline noise and a relative S.D. (RSD) of peak area by ISO 11843-7. The chromatographic determinations of α-, ß-, γ- and δ-tocopherol have been taken as examples. In the present SFC-ECD, the RSDs of peak areas for α-, ß-, γ- and δ-tocopherol obtained by ISO 11843-7 were within 95% confidence intervals of the RSD of them obtained by repetitive measurements (n = 6). Thus, we found that ISO 11843-7 is applicable to the assessment of repeatability in SFC-ECD for determining tocopherols without repetitive measurements.

2-(Pyridinium-1-yl)-1,1-bis(perfluoroalkylsulfonyl)ethan-1-ide: A Practical Reagent for Synthesis of Strongly Acidic 1,1-Bis(perfluoroalkylsulfonyl)alkanes.

On mixing (Rf SO2 )2 CH2 (Rf =perfluoroalkyl), paraformaldehyde, and substituted pyridines, a three-component reaction proceeded smoothly to give unusual zwitterions bearing both pyridinium and stabilized carbanion moieties in good to excellent yields. Of these, 2-fluoropyridinium derivatives rapidly dissociated in acetonitrile to give equilibrium mixtures of the zwitterions and (Rf SO2 )2 C=CH2 /2-fluoropyridine, as confirmed by detailed variable-temperature NMR studies. The dynamic behavior of such 2-fluoropyridinium compounds allows them to be used as shelf-stable, easy-to-handle sources of (Rf SO2 )2 C=CH2 . With these reagents, strongly acidic carbon acids (Rf SO2 )2 CHR were synthesized, which served as a new type of acid catalysts. Moreover, C-C bond-forming reactions with a ketene silyl acetal proceeded efficiently with Tf2 C=CH2 generated in situ.

3-Nitro-2-pyridinesulfenates as Efficient Solution- and Solid-Phase Disulfide Bond Forming Agents.

In this paper, a new disulfide-forming agent based on the finding that alkoxy 3-nitro-2-pyridinesulfenates (Npys-OR) can oxidize thiol groups is reported. Methyl 3-nitro-2-pyridinesulfenate (Npys-OMe), which is easily prepared from 3-nitro-2-pyridinesulfenyl chloride in a one-step reaction and has a reduction peak potential (Epc ) of -0.541 V versus Ag/AgCl, produces the cyclic nonapeptide oxytocin from its linear form in good yield (92 %) with minimal oligomer formation. Npys-OMe in the solid phase also demonstrated excellent results in oxytocin synthesis. Other disulfide-containing peptides, such as α-human atrial natriuretic peptide and α-conotoxin ImI, were also successfully synthesized. During these syntheses, no side reactions of methionine (Met) and tryptophan (Trp) residues or the S-acetamidomethyl (Acm) protecting group were detected. These results suggested that Npys-OMe or its solid-phase analog provides a new strategy for regioselective disulfide bond formation to assist the synthesis of complex disulfide-rich peptides.

A flow-through column electrolytic cell for supercritical fluid chromatography.

A novel flow-through column electrolytic cell was proposed as a detector to obtain current signals for supercritical fluid chromatography. The electrochemical cell consisted of two electrodes and its holder, and a working and a counter electrode were fabricated from 192 carbon strings, which were composed of 400 carbon fibers of 10 µm in diameter filled into a heat-shrinkable tube. These electrodes were placed in the center of a holder made from polyether ether ketone blocks and they were separated by polytetrafluoroethylene membrane filters. To evaluate the sensitivity of this cell, a standard solution of ferrocene was injected into the supercritical fluid chromatography system connected to the electrolytic cell. The ferrocene was eluted through a silica gel column using a mixture of a mobile phase of supercritical CO2 and a modifier of methanol containing ammonium acetate. The current peak area of ferrocene correlated to the ferrocene concentration in the range of 10-400 µmol/L (r = 0.999). Moreover, the limit of detection on the column estimated from a signal-to-noise ratio of 3 was 9.8  × 10-13  mol.

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.

Psychological Barriers to the Use of Opioid Analgesics for Treating Pain in Patients With Advanced Recurrent Cancer: A Multicenter Cohort Study.

Background: We aimed to gain insight into psychological barriers toward initiation of strong opioid analgesic use in patients with advanced recurrent cancer. Methods: This study included 46 patients who were prescribed with opioid analgesics for advanced recurrent cancer. The primary outcome was psychological barriers assessed using the Japanese version of the Barriers Questionnaire-II (JBQ-II). The secondary outcomes were psychological changes and pain relief one week after the induction of strong opioid analgesics. Results: The mean age of participants was 63.6 years. Furthermore, 26.1% had an Eastern Cooperative Oncology Group (ECOG) performance status of ≥3. The mean JBQ-II total score was 1.97 (95% confidence interval: 1.75-2.19). At the initiation of opioid therapy, there was no difference in the total scores between the baseline and one week later. Nevertheless, there was a significant difference in the subscale "disease progression" score (mean 2.97 vs. 2.59, difference in means 0.38, standard error 0.16, p = 0.026). Personalized Pain Goal (PPG) was achieved in about half of the participants, and a trend toward a higher score in the subscale "harmful effects" (concern about adverse events) was observed in those who did not achieve PPG. Conclusion: This study showed that patients with advanced recurrent cancer have psychological barriers to opioid induction. The relationship between the presence of psychological barriers before and after induction of opioid analgesics and the speed of pain improvement was determined. The results may provide fundamental information for prospective intervention studies to develop individualized education programs for patients with psychological barriers to opioids.Clinical Trial Registration Number UMIN000042443.

Gradient elution of hydroxyacetophenones by supercritical fluid chromatography with electrochemical detection.

Linear gradient elution supercritical fluid chromatography with electrochemical detection was developed using hydroxyacetophenones as analytes. Separation was carried out with a diol column (4.6 mm id × 250 mm length, 5 µm) as a stationary phase and a mixture of supercritical carbon dioxide and methanol as a mobile phase, where the ratio of carbon dioxide and methanol was changed from 99:1 (v/v) to 60:40 (v/v). For the electrochemical detection, methanol containing 1.0 mol L-1 ammonium acetate was used as a supporting electrolyte solution and + 1.2 V was applied to the electrochemical cell. We compared the performance of the present method to isocratic elution supercritical fluid chromatography, and the repeatability, linearity, and detection capability all showed better analytical parameters in the gradient elution. As such, we found that gradient elution supercritical fluid chromatography can achieve the faster separation and save resources compared to isocratic elution. Thus, the present method may contribute to the development of green analytical methods.

Electrochemical determination of free amino nitrogen in aged lean beef by the measurement of surplus acid after neutralization.

In this study, an electroanalytical method, which is based on the concept of acid-base back titration and the electrochemical sensing of acids by means of the reduction of quinone, is proposed for determining free amino nitrogen (FAN) in lean beef samples. To perform the electrochemical determination of FAN in beef samples, the following voltammetric behaviors of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) in the presence of acid have been applied: (1) A cathodic prepeak height of DBBQ caused by HCl (iH) is proportional to the HCl concentration in the electrolyte solution. (2) iH is linearly decreased with an increase in amino acids concentration in the electrolyte solution after the amino acids are neutralized by excess HCl. By the present electroanalytical method, FAN in a lean beef sample was determined to be 4.70 mgN g-1 with a repeatability of 3.4% relative standard deviation (n = 5). This result by the present electroanalytical method was in good agreement with a colorimetric method using a ninhydrin reagent. Moreover, the present electroanalytical method was able to monitor increases of FAN in lean beef samples during wet-aging. These results demonstrate the present electroanalytical method could help in the development of an on-site analysis for estimating aged beef samples via the determination of FANs.

Determination of oxalic acid in herbal medicines by semi-micro hydrophilic interaction liquid chromatography coupled with electrochemical detection.

In this study, the determination of oxalic acid in herbal medicines was performed by using a hydrophilic interaction liquid chromatography coupled with electrochemical detection (HILIC-ECD) method. A semi-micro column packed with amide-silica particles and an acetonitrile-30 mM phosphate buffer (pH 6.8) mixture (65:35, v/v) were used as the stationary and mobile phases, respectively, in the HILIC-ECD. A potential of + 1.1 V vs. Ag/AgCl was applied to a glassy carbon working electrode. The ratio of the peak height of oxalic acid to that of the internal standard (synephrine) was proportional to the concentration of 0.45 µg L-1 to 1.8 mg L-1 with a correlation coefficient of 0.999. The detection limit (signal-to-noise ratio, S/N = 3) of oxalic acid was 0.17 µg L-1. By the HILIC-ECD, the oxalic acid content in crude drugs and Kampo medicine extract granules (Zingiberis Rhizoma Processum, Pinelliae Tuber, Sho-seiryu-to, Hange-shashin-to, etc.) were determined with less than 2.9% relative standard deviation (RSD, n = 6), and their recoveries were more than 88.7% with less than 3.3% RSD (n = 6). In conclusion, we demonstrated that the HILIC-ECD performed measurements that were quite selective, accurate, and precise for the determination of oxalic acid in herbal medicines.

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.

Chemometric evaluations of repeatability and detection limit in high-performance liquid chromatography with electrochemical detection.

The function of mutual information (FUMI) theory proposes that it is possible to obtain a relative standard deviation (RSD) of the peak area of an analyte from baseline noises and a signal on a single chromatogram when the analyte concentrations are proportional to their peak areas. In this study, we demonstrate that the FUMI theory using noise parametrization by the difference method is applicable for the evaluations of repeatability and detection limit (DL) in high-performance liquid chromatography with electrochemical detection (HPLC-ECD). HPLC-ECD for determining vincristine (VCR) was taken as an example, and VCR was detected on a glassy carbon surface at +0.8 V vs. Ag/AgCl. In the comparisons of precision profiles (plots of RSD of peak area against concentrations of an analyte), the stochastically estimated RSD (N = 1) by the FUMI theory fell within the 95% confidence intervals of statistically estimated RSD (N = 6) by repetitive measurements. Furthermore, the DL obtained by the FUMI theory (3.3×SD) was almost the same value as that by the signal-to-noise ratio (S/N) = 3. Moreover, we introduced a manual method for signal-noise resolution to obtain noise parameters from a chromatogram with low-pass Bessel filter processing. In conclusion, our results show that the FUMI theory using noise parametrization by the difference method can be applied to the evaluations of repeatability and DL in HPLC-ECD without and with a low-pass Bessel filter.

Effects of digital processing on repeatability assessment of a multiple reaction monitoring liquid chromatography-tandem mass spectrometry system by ISO 11843-7.

ISO 11843 part 7 (ISO 11843-7) can provide a standard deviation (SD) of area measurements of a target peak through the stochastic behaviors of instrumental noises. The purpose of this study is to demonstrate that ISO 11843-7 can be applied to assess repeatability in an isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS) system without repetitive measurements. The relative standard deviation (RSD) of the peak area of ergosterol picolinyl ester, which was used as an example, on a multiple reaction monitoring (MRM) chromatogram was determined by ISO 11843-7. The RSD by ISO 11843-7 (N = 1) was within a 95% confidence band of the RSD by repetitive measurements (N = 6). Moreover, the effects of digital smoothing, such as moving average, were also examined on the repeatability assessment in LC-MS/MS by ISO 11843-7. From the results of the comparisons of the RSDs obtained by ISO 11843-7 and the repetitive measurements, it was shown that suitable RSDs of the peak area were obtained from the smoothed MRM chromatograms by the moving average for narrow data point windows (e.g., one-sixth of the peak width). In conclusion, the utility of repeatability assessment based on ISO 11843-7 has been expanded for the validation of an LC-MS/MS system.

Effects of sampling rate and noise filter processing on repeatability assessment in UHPLC with ultraviolet detection based on the ISO 11843-7.

The present study examines whether short measurement time and noise filter processing in an ultra-high-performance liquid chromatography with ultraviolet detection (UHPLC-UV) contribute to limitations for repeatability assessment based on the ISO 11843 part 7 (ISO 11843-7), which can stochastically provide a measurement standard deviation (SD) caused by baseline noise (SB). In this study, ergosterol was used as an example in UHPLC-UV analysis. From the results of power spectrum analysis of baseline noise, 1024 consecutive digital data points provided a suitable SB. Thus, it was found that an SB can be obtained from about 1 min of baseline when a chromatogram was recorded at sampling rate of 20 points s-1 in the present UHPLC-UV system. The relative SDs (RSDs) of the peak area obtained by the ISO 11843-7 were within 95% of the confidential intervals of the RSDs obtained by repetitive measurements, indicating the ISO 11843-7 is applicable to estimate repeatability in a UHPLC-UV system. In a similar way, we found that the RSD of the peak area obtained from a chromatogram with noise filter processing in UHPLC-UV could also be estimated by the ISO 11843-7. In conclusion, we experimentally demonstrate that short measurement time and noise filter processing are not limitations for repeatability assessment based on the ISO 11843-7.

Simultaneous determination of deuterium-labeled ergosterol and brassicasterol in stroke-prone spontaneously hypertensive rats by ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

A previous study has shown that brassicasterol-d1 was detected in the serum of stroke-prone spontaneously hypertensive rats after oral administration of ergosterol-d1. To quantitatively evaluate the serum concentration of brassicasterol-d1, an ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the simultaneous determination of picolinyl ester-derivatized ergosterol-d1 and brassicasterol-d1. The separation was performed on an ODS column (Waters Acquity UPLC BEH C18) with a mobile phase consisting of methanol and water containing 0.1% acetic acid (95/5, v/v). Linear calibration curves in the presence of the serum were obtained in a concentration range of 0.04-8 µg mL-1. Recovery rates of 95.6-119% were obtained with an RSD (n = 6) of less than 7.5%. The method was applied to the determination of time-concentration curves of ergosterol-d1 and brassicasterol-d1 in stroke-prone spontaneously hypertensive rats, showing a pharmacokinetic profile of ergosterol-d1 where the peak serum concentration of brassicasterol-d1 was 3-fold higher than that of ergosterol-d1.