Toshihide Ihara

Practical guide for accurate quantitative solution state NMR analysis

A practical guide to experimental conditions such as statistical variation of signal intensity on quantitative solution state nuclear magnetic resonanace (NMR) analysis is discussed and presented. Statistical analysis showed that there is a relationship between a targeted precision and practical pulse intervals. The bandwidth of the audio filter needs to be set so that all the signals of interest fall into 80% of the centre part of the filter. When fulfilling these conditions, the relative standard deviation (RSD) of a signal area in repeated experiments can be estimated by determining the signal-to-noise ratio (SN) of a single spectrum. When the SN reached 1000, the RSD became constant with increasing SN. With such a condition, accuracy better than 1% should be obtained with quantitative NMR.

Determination of ethanol in alcoholic beverages by high-performance liquid chromatography-flame ionization detection using pure water as mobile phase.

A method for the determination of ethanol in alcoholic beverages by high-performance liquid chromatography-flame ionization detection (HPLC-FID) was developed. An FID system could be directly connected to an HPLC system using pure water as a mobile phase. In a durability test using triacontylsilyl (C30)-silica gel stationary phase for 96 h, no significant change in the retention time of four alcohol compounds was observed. So the HPLC separation of alcoholic beverages was carried out on the C30-silica gel stationary phase. On application to the analysis of six kinds of alcoholic beverages, ethanol could be determined accurately by the proposed method.

HPLC/PDA determination of carminic acid and 4-aminocarminic acid using relative molar sensitivities with respect to caffeine

ABSTRACT To accurately determine carminic acid (CA) and its derivative 4-aminocarminic acid (4-ACA), a novel, high-performance liquid chromatography with photodiode array detector (HPLC/PDA) method using relative molar sensitivity (RMS) was developed. The method requires no analytical standards of CA and 4-ACA; instead it uses the RMS values with respect to caffeine (CAF), which is used as an internal standard. An off-line combination of 1H-quantitative nuclear magnetic resonance spectroscopy (1H-qNMR) and HPLC/PDA was able to precisely determine the RMSs of CA274nm/CAF274nm and 4-ACA274nm/CAF274nm. To confirm the performance of the HPLC/PDA method using RMSs, the CA and 4-ACA contents in test samples were tested using four different HPLC-PDA instruments and one HPLC-UV. The relative standard deviations of the results obtained from five chromatographs and two columns were less than 2.7% for CA274nm/CAF274nm and 1.1% for 4-ACA274nm/CAF274nm. The 1H-qNMR method was directly employed to analyse the CA and 4-ACA contents in test samples. The differences between the quantitative values obtained from both methods were less than 5% for CA and 3% for 4-ACA. These results demonstrate that the HPLC/PDA method using RMSs to CAF is a simple and reliable quantification method that does not require CA and 4-ACA certified reference materials.

Establishment of an analytical method for accurate purity evaluations of acylcarnitines by using quantitative 1H NMR spectroscopy

Recently, it has become possible to examine metabolism abnormalities by detecting increases in specific acylcarnitines in blood tests of newborn babies using tandem mass spectrometer. However, acylcarnitine standard solutions with metrological traceability to the International System of Units (SI) for accurate calibration of tandem mass spectrometer are not yet available worldwide. In this study, we examined a quantitative 1H NMR procedure for obtaining accurate and SI-traceable purity evaluations of acylcarnitines having different numbers of carbon atoms as each raw material for their standard solutions. In particular, the solvent composition and measurement temperature were optimized to reduce the influence of water signal overlapping on analyte signals. It was found that, when the signal of that 1H which directly bound to the asymmetric carbon of the acylcarnitine is the target signal, it was possible to reduce overlapping of the water signal on the target signal by using deuterium oxide as a solvent. On the other hand, in the case of an acylcarnitine that was poorly soluble in deuterium oxide, it was possible to reduce overlapping of the water signal on the target signal by adding an appropriate amount of deuterium oxide to methanol-d4 in which the acylcarnitine had high solubility. At this time, the optimum mixing volume ratio of methanol-d4/deuterium would be 80:20. The overlapping of the water signal could be also reduced when the measurement temperature was 15xa0°C to 40xa0°C. When the measurement temperature was an around room temperature (in this study, 25xa0°C), fine shimming could be performed easily. Therefore, the optimum temperature would be 25xa0°C, because fine shimming was essential to quantify any signal area accurately. Finally, this study confirmed that accurate values with SI traceability could be obtained at about 1xa0% or less expanded uncertainty for five kinds of acylcarnitines.

Extended internal standard method for quantitative 1H NMR assisted by chromatography (EIC) for analyte overlapping impurity on 1H NMR spectra

We devised a novel extended internal standard method of quantitative 1H NMR (qNMR) assisted by chromatography (EIC) that accurately quantifies 1H signal areas of analytes, even when the chemical shifts of the impurity and analyte signals overlap completely. When impurity and analyte signals overlap in the 1H NMR spectrum but can be separated in a chromatogram, the response ratio of the impurity and an internal standard (IS) can be obtained from the chromatogram. If the response ratio can be converted into the 1H signal area ratio of the impurity and the IS, the 1H signal area of the analyte can be evaluated accurately by mathematically correcting the contributions of the 1H signal area of the impurity overlapping the analyte in the 1H NMR spectrum. In this study, gas chromatography and liquid chromatography were used. We used 2-chlorophenol and 4-chlorophenol containing phenol as an impurity as examples in which impurity and analyte signals overlap to validate and demonstrate the EIC, respectively. Because the 1H signals of 2-chlorophenol and phenol can be separated in specific alkaline solutions, 2-chlorophenol is suitable to validate the EIC by comparing analytical value obtained by the EIC with that by only qNMR under the alkaline condition. By the EIC, the purity of 2-chlorophenol was obtained with a relative expanded uncertainty (ku202f=u202f2) of 0.24%. The purity matched that obtained under the alkaline condition. Furthermore, the EIC was also validated by evaluating the phenol content with the absolute calibration curve method by gas chromatography. Finally, we demonstrated that the EIC was possible to evaluate the purity of 4-chlorophenol, with a relative expanded uncertainty (ku202f=u202f2) of 0.22%, which was not able to be separated from the 1H signal of phenol under any condition.

Determination of Hesperidin and Monoglucosylhesperidin Contents in Processed Foods Using Relative Molar Sensitivity Based on 1 H-Quantitative NMR

We designed an off-line combination of HPLC/photodiode array detector (PDA) and 1H-quantitative NMR (1H-qNMR) to estimate the relative molar sensitivity (RMS) of an analyte to a reference standard. The RMS is calculated as follows: a mixture of the analyte and the reference is analyzed using 1H-qNMR and HPLC/PDA. The response ratio of the analyte and the reference obtained by HPLC/PDA is then corrected using the molar ratio obtained by 1H-qNMR. We selected methylparaben (MPB), which is a certified reference material, as the reference standard and hesperidin (Hes) and monoglucosylhesperidin (MGHes) as analytes, and the RMSs of Hes283 nm/MPB255 nm and MGHes283 nm/MPB255 nm were determined as 1.25 and 1.32, respectively. We determined the contents of Hes and MGHes in processed foods by the conventional absolute calibration method and by the internal standard method employing the RMS values with respect to MPB. The differences between the values obtained with the two methods were less than 2.0% for Hes and 3.5% for MGHes.

Application of post-column reaction GC for accurate and direct determination of musty odor substances in standard solution

By using post-column reaction GC, a method for accurate and direct characterization of musty odor substances (2-methylisoborneol and geosmin) in a candidate standard solution has been developed. Because the GC system gives a response depending on the number of carbon atoms constituting the organic compound by combustion and conversion to methane, naphthalene and diethyl phthalate, purity of which had been accurately assessed, were used as reference materials for calibration. To verify the method, concentrations of 2-methylisoborneol and geosmin in the sample determined by the method were compared with the concentrations calculated based on gravimetric mixing that is one of the general methods for the characterization of the organic standard solutions. The analytical results of each analyte obtained by the two methods agreed within a deviation of about 0.5xa0% or less.