Taichi Yamazaki

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.

Quantitative Nuclear Magnetic Resonance Spectroscopy Based on PULCON Methodology: Application to Quantification of Invaluable Marine Toxin, Okadaic Acid.

ERETIC2 (Electronic Reference To access In vivo Concentrations 2) based on PULCON (Pulse Length–based Concentration determination) methodology is a quantitative NMR (qNMR) using an external standard. The performance of the PULCON method was assessed using maleic acid (MA). Quantification of the diarrhetic shellfish toxin and okadaic acid by PULCON was successfully consistent with that obtained by a conventional internal standard method, demonstrating that the PULCON method is useful for the quantification of invaluable marine toxins without any contaminations by an internal standard.

Development of High-purity Certified Reference Materials for 17 Proteinogenic Amino Acids by Traceable Titration Methods

To ensure the reliability of amino acid analyses, the National Metrology Institute of Japan of the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed high-purity certified reference materials (CRMs) for 17 proteinogenic amino acids. These CRMs are intended for use as primary reference materials to enable the traceable quantification of amino acids. The purity of the present CRMs was determined based on two traceable methods: nonaqueous acidimetric titration and nitrogen determination by the Kjeldahl method. Since neither method could distinguish compounds with similar structures, such as amino acid-related impurities, impurities were thoroughly quantified by combining several HPLC methods, and subtracted from the obtained purity of each method. The property value of each amino acid was calculated as a weighted mean of the corrected purities by the two methods. The uncertainty of the property value was obtained by combining measurement uncertainties of the two methods, a difference between the two methods, the uncertainty from the contribution of impurities, and the uncertainty derived from inhomogeneity. The uncertainty derived from instability was considered to be negligible based on stability monitoring of some CRMs. The certified value of each amino acid, property value with uncertainty, was given for both with or without enantiomeric separation.

Metal free columns for determination of deoxynucleotide monophosphate by liquid chromatography/mass spectrometry and application to oligonucleotide

We have developed a highly sensitive method for the analysis of deoxynucleotide monophosphates (dNMPs), which involves the use of liquid chromatography/mass spectrometry (LC/MS) and a new metal-free column. The new column solves the problem that the phosphate group in dNMPs interacts with the metal portion of the device or column. After optimization of the analytical conditions, the limits of detection (LODs) of dNMPs were from 5.4ng/g to 6.3ng/g. Those values were 10 times lower than the LODs of previous methods. We applied the method to the determination of the base composition and the quantification of 20-mer oligonucleotide. Despite use of a very small sample amount of 14.5ng, we were able to determine the base composition, and the result was consistent with theoretical values. We were also able to quantify the mass fraction of oligonucleotide with 8.2% expanded uncertainty (k=2). By means of the developed method, we were able to analyze dNMPs with high sensitivity as well as determine the base composition and quantify the mass fraction of oligonucleotide despite use of a small amount of sample.

Purity Determination of Acetaldehyde in an Acetaldehyde Certified Reference Material.

Acetaldehyde is regulated as a toxic substance in various fields, and the method for monitoring or analysis of acetaldehyde is important. However, handling is difficult because of the high reactivity and low boiling point of acetaldehyde. Therefore, a reference material for high purity acetaldehyde with high accuracy was not available. Although the measuring method of acetaldehyde as a reagent is published in the Japanese Industrial Standard (JIS) where the specification of acetaldehyde purity is more than 80%, the analytical method described in JIS is not enough for an accuracy purity determination method. In this research, the high precision purity determination method for development of a certified reference material (CRM) of acetaldehyde was examined. By controlling the volatility and reactivity of acetaldehyde, we established the purity determination method of acetaldehyde with a relative standard uncertainty of less than 0.3%. Furthermore, this method was applied to develop a high purity acetaldehyde CRM with an expanded uncertainty of 0.005 kg kg(-1) (k = 2).

Evaluation of perfluorooctanoic acid purity based on potentiometric titration

Reliable purity evaluation of perfluorooctanoate (PFOA), surfactants, is desired for accurate quantification because purity is essential for the preparation of calibration solutions. To meet this requirement, homogenized PFOA monohydrate was prepared for the improvement of repeatability, and was diluted in aqueous NaOH for measures of poor solubility on titration analyses, which is commonly used for purity evaluation of chemicals. Purity based on the titration technique was determined by subtracting the mass fractions of impurities related to PFOA analyzed by using a LC/MS from those of purity expressed as acids analyzed by neutralization potentiometric titration using a potentiometric titrator. To validate the obtained result, another analytical result based on the mass balance technique was obtained by subtracting the mass fractions of every impurity. From the results in this study, this titration technique can help evaluating the reliable purity of analytes similar to PFOA.

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.

Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification

Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) with formic acid hydrolysis was established for the accurate quantification of λDNA. The over-decomposition of nucleobases in formic acid hydrolysis was restricted by optimizing the reaction temperature and the reaction time, and accurately corrected by using deoxynucleotides (dNMPs) and isotope-labeled dNMPs as the calibrator and the internal standard, respectively. The present method could quantify λDNA with an expanded uncertainty of 4.6% using 10fmol of λDNA. The analytical results obtained with the present method were validated by comparing with the results of phosphate-base quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed good agreement with each other. We conclude that the formic acid hydrolysis/LC-IDMS method can quantify λDNA accurately and is promising as the primary method for the certification of DNA as reference material.

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.

Interlaboratory comparison of liquid chromatography-tandem mass spectrometry quantification of diarrhetic shellfish toxins in scallop midgut glands

An interlaboratory comparison (ILC) was organized as a measure of the analytical competency in the liquid chromatography-tandem mass spectrometry quantification of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in scallop midgut gland samples. The test sample was prepared using boiled midgut glands of naturally contaminated scallops with DTX1 and its esters by spiking with OA, and homogeneity and stability of this test sample was assessed to be appropriate. Twenty laboratories participated in the ILC based on the Japanese official testing method; they submitted two sets of analytical concentrations of target analytes along with the details of their analytical protocols. For assessing these data, assigned values were established from another ILC where ten participants quantified the target analytes by the standard addition method. The mean analytical results of the former ILC showed good agreement with the assigned values, and the corresponding relative reproducibility standard deviations met the criterion of CODEX STAN 292. Meanwhile, the results of more than half of the participants were out of the uncertainty range of the assigned values; these participants were encouraged to investigate their protocols to improve their analytical capability.