Development of 3ß,4α-dihydroxy-5α-androstan-17-one standard solution for doping analyses.

Doping analyses are essential for sporting events because some athletes might use prohibited substances to win games. To obtain reliable results from doping analyses, it is important to use both reliable standard solutions and validated analytical methods at accredited laboratories. Among the focused compounds related to prohibited substances listed by the World Anti-Doping Agency, we developed a certified reference material (CRM) for 3ß,4α-dihydroxy-5α-androstan-17-one (DHAS), a metabolite of formestane that is used to conceal prohibited anabolic steroids, in methanol solution (NMIJ CRM 6212-a). To develop a CRM traceable to the International System of Units (SI), we newly applied different analytical methods with an SI-traceable internal standard for quantitative NMR (qNMR) instead of mass balance approach because this CRM solution was required to develop rapidly using a limited amount of high-purity DHAS. One method was gravimetric blending using the purity of DHAS powder evaluated by both qNMR and a combination of qNMR and high-performance liquid chromatography (HPLC), and the other was direct quantification of the DHAS mass fraction in the candidate solution CRM by both qNMR and qNMR/HPLC. Because the values obtained by gravimetric blending and direct quantification of the mass fraction were comparable, the arithmetic mean was applied to obtain the certified value. Considering homogeneity and stability according to ISO Guide 35: 2017, the certified values with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were (135.2 ± 9.5) µg/g for the mass fraction and (107.0 ± 7.5) µg/ml for the mass concentration.

Reliable purity assay of highly hygroscopic trichloroacetic acid for the development of high-purity reference material of NMIJ CRM 4074-a.

Trichloroacetic acid is known as one of the harmful disinfection byproducts with chlorine of tap water and is regulated according to legally binding standards in Japanese Drinking Water Quality Standards. We developed a high-purity trichloroacetic acid reference material, NMIJ CRM 4074-a, with certified purity as a traceability source of standard solution supplied under the Japan Calibration Service System (JCSS). As trichloroacetic acid is hygroscopic, water could be the main impurity. Although all impurities in the sample can be possibly detected by the freezing point depression method (FPD), it was unclear for trichloroacetic acid whether water was detected by FPD owing to evaporation of water from the sample during fusion. Therefore, we confirmed that water in trichloroacetic acid was detected as an impurity by FPD. The procedure was validated from an increment of purity by FPD due to reduction of water content and an agreement of purity by FPD with those by neutralization titrimetry (NT) and mass balance approach (MBA), both methods were based on different measurement principles from FPD. The certified value was determined to be (0.999 ± 0.003) kg kg-1 from the purity assay by FPD and NT, and uncertainties due to the homogeneity and stability of the CRM were included in the expanded uncertainty. The reliability of the certified value was verified by the agreement of purities by FPD, NT, and MBA.

Quantification of 23 Volatile Organic Compounds with a Single Reference Material Using Post-column Reaction Gas Chromatography Combined with a Stainless-steel Heating Furnace.

We built a heating furnace using stainless-steel instead of aluminum in gas chromatography combined with an oxidation/reduction system; it increased the oxidation temperature to 650°C. At 600°C, it completely oxidized five organochlorine compounds. This system was applied to a standard solution of 23 volatile organic compounds. The analytical results of 20 hydrocarbon and organochlorine compounds showed good agreement with the expanded uncertainty (k = 2) of the reference values. Three organobromine compounds obtained values higher than the reference; this was investigated further.

Determination of Mogroside V in Luohanguo Extract for Daily Quality Control Operation Using Relative Molar Sensitivity to Single-Reference Caffeine.

Mogroside V is one of the characteristic and effective components of luohanguo extract, a food additive used as a sweetener in Japan as per Japan's Standards and Specifications for Food Additives (JSFA; 9th ed.). JSFA stipulates that the quantitative determination for mogroside V content in luohanguo extract applies HPLC using analytical standard mogroside V. However, no mogroside V reagents with proven purities are commercially available. Therefore the current JSFA determination method is not particularly suited for daily quality control operations involving luohanguo extract. In this study, we applied an alternative quantitative method using a single reference with relative molar sensitivity (RMS). It was possible to calculate the accurate RMS by an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/variable-wavelength detector (VWD). Using the RMS of mogroside V to a commercial certified reference material grade caffeine, the mogroside V contents in luohanguo extracts could be determined using HPLC/VWD without analytical standard mogroside V. There was no significant difference between the mogroside V contents in luohanguo extracts determined using the method employing single-reference caffeine with the RMS and using the JSFA method. The absolute calibration curve for the latter was prepared using an analytical standard mogroside V whose purity was determined by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative determination of mogroside V in luohanguo extract and can be used as an alternative method to the current assay method in JSFA.

Use of Relative Molar Sensitivity as a Specific Value for Evaluating Heptaoxyethylene Dodecyl Ether Concentrations in Methanol Solution.

Relative molar sensitivity (RMS) determined using quantitative 1H NMR and HPLC with a refractive index (RI) detector was applied as a specific value for quantifying the levels of heptaoxyethylene dodecyl ether (HOEDE), a typical non-ionic surfactant, in methanol solutions. RMS was robust against changes of the analytical conditions (i.e., RI cell temperature, acetonitrile content in the mobile phase, HPLC system). Furthermore, the obtained HOEDE concentrations using a previously evaluated RMS were comparable to those obtained using a reference method for over 1 year.

Enhancing the accuracy of measurement of small molecule organic biomarkers.

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.

Determination of perillaldehyde in perilla herbs using relative molar sensitivity to single-reference diphenyl sulfone.

Perillaldehyde (PRL) is one of the essential oil components derived from perilla plants (Perilla frutescens Britton) and is a characteristic compound of the traditional medicine "perilla herb ()" listed in the The Japanese Pharmacopoeia, 17th edition (JP17). HPLC using an analytical standard of PRL has been used to quantitatively determine the PRL content in perilla herb. However, PRL reagents have been reported to decompose easily. In this study, we utilized an alternative quantitative method using on a single reference with relative molar sensitivity (RMS) based on the results of experiments performed in two laboratories. It was possible to calculate the exact RMS using an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/photodiode array (PDA) detector (or an HPLC/variable-wavelength detector [VWD]). Using the RMS of PRL to the single-reference compound diphenyl sulfone (DFS), which is an inexpensive and stable compound, the PRL content in the perilla herb could be determined using HPLC/PDA or HPLC/VWD without the need for the analytical standard of PRL. There was no significant difference between the PRL contents of perilla herb determined using the method employing the single-reference DFS with RMS and using the JP17 assay, the calibration curve of which was generated using the analytical standard of PRL with adjusted purity measured by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative assays of perilla herb and can be an alternative method for the current assay method defined in the JP17.

Relative molar sensitivities of carnosol and carnosic acid with respect to diphenylamine allow accurate quantification of antioxidants in rosemary extract.

We have been developing a high-performance liquid chromatography/photodiode array (HPLC/PDA) employing relative molar sensitivities (RMSs) and adopted it to the accurate quantification of carnosol (CL) and carnosic acid (CA) which are the antioxidants in rosemary extract. The method requires no references of CL or CA and instead uses RMSs with respect to diphenylamine (DPA) whose certified reference material is available from a reagent manufacturer. The molar and response ratios of the analytes to the reference in an artificial mixture of them were determined using 1H-quantitative nuclear magnetic resonance spectroscopy (1H-qNMR) and HPLC/PDA at a wavelength of 284 nm under isocratic condition, respectively, and then RMSs were calculated to be 0.111 for CL/DPA and 0.0809 for CA/DPA as averaged values in three HPLC-PDA instruments. The RMS values varied by up to 1.1% as relative standard deviation. To evaluate the performance of HPLC/PDA with the RMSs, the CL and CA contents in rosemary extracts were determined using DPA as a reference. The CL and CA contents were compared with those determined using calibration curves of CL and CA obtained by HPLC measurement of standard solutions prepared from their reagents whose absolute purities were determined using 1H-qNMR. The differences between the two methods for CL and CA were ≤3% as relative error. This chromatographic method with RMSs allows a simple and reliable quantification when reference of the analyte is unavailable.

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

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

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.

Pseudo-Infected Red Blood Cell Beads as Positive Control for Cell Microarray Chip-Based Detection of Plasmodium-Infected RBCs.

The cell microarray chip is a polystyrene plate with 20,944 microchambers, and it is used to detect red blood cells (RBCs) infected with the causative agent of malaria, Plasmodium. Plasmodium-infected red blood cells (iRBCs) stained with a nuclear staining dye (SYTO 21) form a monolayer on the bottom of the microchambers, and about 130 RBCs are accommodated in each such microchamber of the chip. The iRBCs in the RBC monolayer (containing 2.7 million RBCs) can be identified using a fluorescence detector, and the infection rate can be calculated by counting the number of fluorescent-positive RBCs. This diagnostic device is highly sensitive and hence advantageous for early diagnosis of malaria infections in endemic areas. However, a standard positive control for Plasmodium-infected RBCs is required to ensure that the reagents and detectors of these cell microarray chips are working efficiently in remote endemic areas. Here, we introduce "pseudo-iRBC beads," which consist of a mixture of DEA beads mimicking RBCs and DEA beads coated with nucleic acids mimicking nuclei of the parasite. These beads can be stained with SYTO 21, applied onto the cell microarray chip to form a monolayer, and detected using the fluorescence detector in the same way as iRBCs. Therefore, the introduction of pseudo-iRBC beads as a positive control ensures unbiased malaria diagnoses with the cell microarray chip device in remote endemic areas.

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.

Simultaneous Direct Determinations of Na, Mg, K, Ca, P, and S in Biodiesel Fuel by ICP-QMS/QMS after Xylene Dilution: Development and Application of a High-throughput Method for a Homogeneity Assessment of a Candidate Reference Material.

Inductively coupled plasma tandem quadrupole mass spectrometry (ICP-QMS/QMS) measurements after xylene dilution were investigated as a method for determining the elements (Na, Mg, K, Ca, P, and S) in a biodiesel fuel (BDF) candidate reference material (RM). Optimizations were respectively carried out for the following parameters to obtain the best performance for measurements: O2 flow rate (additional gas to the carrier gas) to ensure complete combustion of the xylene solvent in the plasma, plasma power to obtain lower background signal intensities for Na and K, O2 flow rate (reaction cell gas) to remove any spectral interference with the S, H2 flow rate so as to remove spectral interference with Ca. After optimization, the lower detection limits of Na, Mg, K, Ca, P, and S were 0.0004, 0.00004, 0.0003, 0.00012, 0.00005, and 0.002 mg kg-1, respectively. Typical relative standard deviations were 2.1, 2.0, 1.7, 1.1, 2.5, and 2.5% for Na, Mg, K, Ca, P, and S, respectively, where the elemental concentrations in the BDF sample were, respectively, ca. 1 mg kg-1 each for Na, Mg, K and Ca, ca. 2 mg kg-1 for P, and ca. 6 mg kg-1 for S. The established method was applied to the homogeneity assessment of a candidate RM of BDF made from palm oil. The relative uncertainties of the homogeneity were 0.3, 0.4, 0.6, 0.3, 1.6, and 0.6% for Na, Mg, K, Ca, P, and S, respectively.

Difference between Consensus Value of Participants' Results and Isotope-Dilution Mass Spectrometric Results in Proficiency Testing for Pesticide Residues in Husked Wheat.

Proficiency testing was organized by the National Metrology Institute of Japan (NMIJ) as a measure of analytical competency in the quantification of pesticide residues in husked wheat powder. Seventy-one participants submitted analytical concentrations of the target pesticides (diazinon, fenitrothion, malathion, and etofenprox) along with details of the analytical method employed. Two types of assigned values were obtained for each target pesticide, i.e., the participants' analytical results and the results obtained by NMIJ based on isotope-dilution mass spectrometry (IDMS). The former values were lower than the latter due to the incomplete recovery yield of the target pesticides during the pretreatment process. The discrepancy between the two assigned values was particularly pronounced for malathion because of the longer duration of water-soaking used for the test samples. Two corresponding types of z-scores were then calculated to evaluate the analytical performance of the participants, where the z1-score indicates the performance of a participant relative to all participants, and the z2-score indicates the relative deviation of the analytical results of the participant from the IDMS value.

Certification of reference materials for the determination of alkylphenols.

Certified reference materials (CRMs) are playing an increasingly important role in national and international standardizing activities. In Japan, primary standard solutions for analyses of endocrine disrupters are supplied under the national standards dissemination system named the Japan Calibration Service System (JCSS). For the traceability on reference materials used for preparation of the primary standard solutions based on the JCSS, the National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed and certified high-purity reference materials of alkylphenols as NMIJ CRMs, such as 4-n-nonylphenol, 4-tert-octylphenol, 4-n-heptylphenol, 4-tert-butylphenol, and 2,4-dichlorophenol. Thereafter, it is essential to determine the alkylphenols by using these solutions based on the JCSS for environmental monitoring and risk assessments because analytical values obtained by using the solutions can ensure the reliability and traceability of the chemical analyses.

Proficiency testing for determination of pesticide residues in soybean: comparison of assigned values from participants' results and isotope-dilution mass spectrometric determination.

Proficiency testing (PT) for the determination of pesticide residues in soybean samples was organized by the National Metrology Institute of Japan (NMIJ). The candidate certified reference material, NMIJ CRM 7509-a, that was prepared from the raw soybeans containing target pesticides (diazinon, fenitrothion, chlorpyrifos, and permethrin) was used as the test sample. Forty participants submitted two sets of analytical results along with the details of the analytical method and conditions they applied. Two types of assigned values were established for each target pesticide: one was derived from the analytical results of the participants, and the other was provided from the analytical results by isotope-dilution mass spectrometry (IDMS). The latter values were 7.4-16% higher than the former values, plausibly because the analytical values from the IDMS measurements were not affected by the recovery ratio of the target pesticides during the analytical process. Thus, two kinds of z-scores were calculated for individual participants using the corresponding assigned values: one (z1-score) showed the relative performance score for the present PT and the other (z2-score) could be used for evaluation of the trueness of their analytical methods.

Evaluation of the performance of 57 Japanese participating laboratories by two types of z-scores in proficiency test for the quantification of pesticide residues in brown rice.

A proficiency test for the analysis of pesticide residues in brown rice was carried out to support upgrading in analytical skills of participant laboratories. Brown rice containing three target pesticides (etofenprox, fenitrothion, and isoprothiolane) was used as the test samples. The test samples were distributed to the 57 participants and analyzed by appropriate analytical methods chosen by each participant. It was shown that there was no significant difference among the reported values obtained by different types of analytical method. The analytical results obtained by National Metrology Institute of Japan (NMIJ) were 3 % to 10 % greater than those obtained by participants. The results reported by the participant were evaluated by using two types of z-scores, that is, one was the score based on the consensus values calculated from the analytical results of participants, and the other one was the score based on the reference values obtained by NMIJ with high reliability. Acceptable z-scores based on the consensus values and NMIJ reference values were achieved by 87 % to 89 % and 79 % to 94 % of the participants, respectively.

A certified urea reference material (NMIJ CRM 6006-a) as a reliable calibrant for the elemental analyses of amino acids and food samples.

We examined the reliability of a certified reference material (CRM) for urea (NMIJ CRM 6006-a) as a calibrant for N, C, and H in elemental analyzers. Only the N content for this CRM is provided as an indicative value. To estimate the C and H contents of the urea CRM, we took into account the purity of the urea and the presence of other identified impurities. When we examined the use of various masses of the calibrant (0.2 to 2 mg), we unexpectedly observed low signal intensities for small masses of H and N, but these plateaued at about 2 mg. We therefore analyzed four amino acid CRMs and four food CRMs on a 2-mg scale with the urea CRM as the calibrant. For the amino acid CRMs, the differences in the analytical and theoretical contents (≤0.0026 kg/kg) were acceptable with good repeatability (≤0.0013 kg/kg in standard deviation; n = 4). For food CRMs, comparable repeatabilities to those obtained with amino acid CRMs (≤0.0025 kg/kg in standard deviation; n = 4) were obtained. The urea CRM can therefore be used as a reliable calibrant for C, H, and N in an elemental analyzer.

Development of soybean certified reference material for pesticide residue analysis.

A soybean certified reference material for pesticide residue analysis was developed by the National Metrology Institute of Japan. Three organophosphorus (diazinon, fenitrothion, chlorphyrifos) and one pyrethroid (permethrin) pesticides were sprayed on soybeans three times before harvest. These soybeans were freeze pulverized, homogenized, bottled, and sterilized by γ-irradiation to prepare the candidate material. Three isotope-dilution mass spectrometric methods that varied in terms of the solvents used for extraction of the target pesticides, the clean-up procedure, and the injection techniques and columns used for quantification via gas chromatography/mass spectrometry were applied to the characterization. Each target pesticide was quantified by two of these analytical methods, and the results were in good agreement. Homogeneity and stability assessment of the material demonstrated that the relative standard uncertainties due to the inhomogeneity and the instability for an expiry date of 55 months were 1.89-4.00% and 6.65-11.5%, respectively. The certified pesticide concentrations with expanded uncertainties (coverage factor k=2, approximate 95% confidence interval) calculated using the results of the characterization and the homogeneity and stability assessment were 21.7 ± 3.2 µg/kg for diazinon, 88 ± 21 µg/kg for fenitrothion, 11.1 ± 3.2 µg/kg for chlorpyrifos, and 20.1 ± 4.3 µg/kg for permethrin (as the sum of the constituent isomers).

Variation in concentration of perfluorooctanoic acid in methanol solutions during storage.

Perfluoroalkyl carboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) have been widely recognized as persistent environmental contaminants. For accurate quantification of PFCAs, their stability in calibration solutions is important because they are criteria of quantification. To examine stability of PFCAs in methanol, we monitored PFOA and its related compounds around 4 years. Interestingly, perfluorooctanoate was varied randomly, and methyl perfluorooctanoate (MePFOA) and methyl formate were observed when perfluorooctanoate decreased. Moreover, no detection of both methyl esters was in methanol solutions immediately after preparation. In each of prepared methanol solution of perfluorohexanoic, perfluoroheptanoic, and perfluorononanoic acids, their corresponding methyl esters and methyl formate were observed. Furthermore, MePFOA was observed even in the solutions stored around 4 months and thereafter MePFOA increased with increase in methyl formate. Therefore, PFCAs including PFOA should be used immediately after preparation when methanol is used as a solvent.