Yuzo Nishizaki

Determination of PAHs in Solution with a Single Reference Standard by a Combination of 1H Quantitative NMR Spectroscopy and Chromatography

We have applied a combination of 1H quantitative NMR spectroscopy (1H-qNMR) and chromatography (GC or LC) to establish reliable analytical methods (qNMR/GC and qNMR/LC) for organic compounds. In this method, a reference standard is used as an internal standard for both 1H-qNMR and chromatography to estimate relative molar sensitivity (RMS) for analytes. The RMS values are calculated from the molar ratios between analytes and the reference standard obtained by 1H-qNMR; and the response ratio between them obtained by chromatography. Concentrations of analytes in the organic solution can be simultaneously determined from the RMS and amount of the reference standard added in the sample solution. This analytical method is an innovative one because only one reference standard with International System of Units (SI)-traceable property value, purity, or concentration, is necessary to determine accurate concentrations of multiple organic components in organic solutions, without the respective certified reference standards for various analytes. To verify this method, a certified reference material, NIST SRM 1647f, was used. Among the 16 polycyclic aromatic hydrocarbons (PAHs) included in NIST SRM 1647f, naphthalene and benzo[a]pyrene were selected as analytes for this method, using 1,4-bis(trimethylsilyl)benzene-d4 as the reference standard. Each quantitative value obtained by qNMR/GC and qNMR/LC agreed with each certified value within its expanded uncertainty.

Development of a Novel Method for Quantifying Quassin and Neoquassin in Jamaica Quassia Extracts Using the Molar Absorption Coefficient Ratio

A novel HPLC-based method employing molar absorption coefficient ratios to 4-hydroxybenzoic acid (4HBA) was developed for the determination of quassin and neoquassin in Jamaica quassia extract, which is used as a food additive in Japan. Based on comparisons of quantitative NMR (qNMR) spectra and HPLC chromatograms of an artificial mixture of quassin, neoquassin, and 4HBA, the molar absorption coefficient ratios of quassin and neoquassin to 4HBA were determined as 0.84 and 0.85, respectively. Quassin and neoquassin were quantified in food additives by qNMR and HPLC based on molar absorption coefficient ratios using 1,4-bis(trimethylsilyl)benzene-d4 and 4HBA as internal standards, respectively. The differences in quantitation values between qNMR and HPLC analyses were below 1.2%. Our proposed novel HPLC-based quantitation method employing the molar absorption coefficient ratios is a reliable tool for determining levels of quassin and neoquassin in food additives and processed foods.

Determination and purification of sesamin and sesamolin in sesame seed oil unsaponified matter using reversed-phase liquid chromatography coupled with photodiode array and tandem mass spectrometry and high-speed countercurrent chromatography.

In Asian countries, sesame seed oil unsaponified matter is used as a natural food additive due to its associated antioxidant effects. We determined and purified the primary lignans sesamin and sesamolin in sesame seed oil unsaponified matter using reversed-phase liquid chromatography coupled with photodiode array and tandem mass spectrometry and high-speed countercurrent chromatography. Calibration curves showed good correlation coefficients (r2 > 0.999, range 0.08 and/or 0.15 to 5 μg/mL) with a limit of detection (at 290 nm) of 0.02 μg/mL for sesamin and 0.04 μg/mL for sesamolin. Sesame seed oil unsaponified matter contained 2.82% sesamin and 2.54% sesamolin, respectively. Direct qualitative analysis of sesamin and sesamolin was achieved using quadrupole mass spectrometry with positive-mode electrospray ionization. Pure (>99%) sesamin and sesamolin standards were obtained using high-speed countercurrent chromatographic purification (hexane/ethyl acetate/methanol/water; 7:3:7:3). An effective method for determining and purifying sesamin and sesamolin from sesame seed oil unsaponified matter was developed by combining these separation techniques for standardized food additives.

Preparation of a Ammonia-Treated Lac Dye and Structure Elucidation of Its Main Component

Lac dye and cochineal extract contain laccaic acids and carminic acid as the main pigments, respectively. Both laccaic acids and carminic acid are anthraquinone derivatives. 4-Aminocarminic acid (acid-stable carmine), an illegal colorant, has been detected in several processed foods. 4-Aminocarminic acid is obtained by heating cochineal extract (carminic acid) in ammonia solution. We attempted to prepare ammonia-treated lac dye and to identify the structures of the main pigment components. Ammonia-treated lac dye showed acid stability similar to that of 4-aminocarminic acid. The structures of the main pigments in ammonia-treated lac dye were analyzed using LC/MS. One of the main pigments was isolated and identified as 4-aminolaccaic acid C using various NMR techniques, including 2D-INADEQUATE. These results indicated that ammonia-treatment of lac dye results in the generation of 4-aminolaccaic acids.

Single reference quantitative analysis of xanthomonasin A and B in Monascus yellow colorant using high-performance liquid chromatography with relative molar sensitivity based on high-speed countercurrent chromatography

Monascus yellow (MY) is a natural yellow food coloring. The main components from MY are xanthomonasin A (XA) and xanthomonasin B (XB) for natural yellow colorant of food additives. However, few chromatographic assays of XA and XB exist in food additive products because of unavailable standards for calibration curves. In this study, the single reference (SR) quantitative analysis of XA and XB in MY product is proposed by high-performance liquid chromatography with photodiode array detection (HPLC/PDA) using relative molar sensitivity (RMS). Moreover, high-speed countercurrent chromatography (HSCCC) purification with 1H quantitative NMR (qNMR) evaluation is necessary to separate the two analytes for the RMS to be demonstrated. For HSCCC separation, the biphasic solvent system (hexane/ethyl acetate/methanol/0.1% formic acid in water, 1/5/1/5) was used to obtain XA and XB fractions that were subjected to qNMR for the determination of their contents in each test solution. Using these solutions and SR solution of carbazochrome acid (CBZ), the RMS of XA and XB are calculated from slopes ratios of calibration curves (three ranges from 0 to 177 μM for XA and 0-126 μM for XB, r2 > 0.998). The averaged RMS of XA/CBZ and XB/CBZ were 8.75 ± 0.07 and 14.8 ± 0.26, respectively. The concentrations of XA and XB in MY can be determined from RMS, peak area and content of CBZ added in the samples; the concentrations were found to be 7.26 μmol/g and 2.53 μmol/g, respectively. The performance of HPLC/PDA using RMS was compared with an absolute calibration curve method. This developed HPLC/PDA using RMS is simple and reliable quantification that does not require native XA and XB standards based on HSCCC purification and qNMR evaluation.

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.

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 (k = 2) 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 (k = 2) 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.