Quantitative 31P-NMR for the Purity Determination of the Organophosphorus Compound Brigatinib and Its Method Validation.

The spectrum of 31P-NMR is fundamentally simpler than that of 1H-NMR; consequently identifying the target signal(s) for quantitation is simpler using quantitative 31P-NMR (31P-qNMR) than using quantitative 1H-NMR (1H-qNMR), which has been already established as an absolute determination method. We have previously reported a 31P-qNMR method for the absolute determination of cyclophosphamide hydrate and sofosbuvir as water-soluble and water-insoluble organophosphorus compounds, respectively. This study introduces the purity determination of brigatinib (BR), an organophosphorus compound with limited water solubility, using 31P-qNMR at multiple laboratories. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were selected as the reference standards (RSs) for 31P-qNMR and 1H-qNMR, respectively. The qNMR solvents were chosen based on the solubilities of BR and the RSs for qNMR. CD3OH was selected as the solvent for 31P-qNMR measurements to prevent the influence of deuterium exchange caused by the presence of exchangeable intramolecular protons of BR and PAA on the quantitative values, while CD3OD was the solvent of choice for the 1H-qNMR measurements to prevent the influence of water signals and the exchangeable intramolecular protons of BR and PAA. The mean purity of BR determined by 31P-qNMR was 97.94 ± 0.69%, which was in agreement with that determined by 1H-qNMR (97.26 ± 0.71%), thus indicating the feasibility of purity determination of BR by 31P-qNMR. Therefore, the findings of this study may provide an effective method that is simpler than conventional 1H-qNMR for the determination of organophosphorus compounds.

[Quantitative Determination Method of Aconitum Monoester Alkaloids Using Relative Molar Sensitivity (RMS) for the Assay in the Japanese Pharmacopoeia].

Recently, a novel quantitative method using relative molar sensitivity (RMS) was applied to quantify the ingredients of drugs and foods. An important development in this regard can be observed in the Japanese Pharmacopoeia (JP) 18, where the quantification of perillaldehyde, an unstable compound, in crude drug "Perilla Herb," was revised to incorporate the RMS method. In this study, the primary objective was to improve the tester safety and reduce the amount of reagents used in the JP test. To achieve this, the quantification of three toxic Aconitum monoester alkaloids (AMAs) was explored using the RMS method, employing a single reference compound for all three targets. These AMAs, namely benzoylmesaconine hydrochloride, benzoylhypaconine hydrochloride, and 14-anisoylaconine hydrochloride, which are the quantitative compounds of Kampo extracts containing Aconite Root (AR), were quantified using the reference compound benzoic acid (BA). Reliable RMS values were obtained using both 1H-quantitative NMR and HPLC/UV. Using the RMS of three AMAs relative to the BA, the AMA content (%) in commercial AMAs quantitative reagents were determined without analytical standards. Moreover, the quantitative values of AMAs using the RMS method and the calibration curve method using the three analytical standards were similar. Additionally, similar values were achieved for the three AMAs in the Kampo extracts containing AR using the RMS and the modified JP18 calibration curve methods. These results suggest that the RMS method is suitable for quantitative assays of the Kampo extracts containing AR and can serve as an alternative to the current method specified in the JP18.

Quantitative 31P-NMR for Purity Determination of Sofosbuvir and Method Validation.

Quantitative 1H-NMR (1H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the 31P nucleus because of the simplicity of its signals and previously reported 31P-qNMR in D2O. Here we report 31P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d4 (1,4-BTMSB-d4) were used as reference standards for 31P-qNMR and 1H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31P-qNMR was 100.63 ± 0.95%, whereas that determined by 1H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T2 relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d6 (DMSO-d6), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (DSS-d6) as reference standards for 31P-qNMR and 1H-qNMR, respectively. SOF purities determined by 31P-qNMR and 1H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by 31P-qNMR agreed with the established 1H-qNMR values, suggesting that an aprotic solvent is preferable for 31P-qNMR because it is unnecessary to consider the effect of deuterium exchange.

Purity Determination of Cyclophosphamide Hydrate by Quantitative 31P-NMR and Method Validation.

Recently, quantitative NMR (qNMR), especially 1H-qNMR, has been widely used to determine the absolute quantitative value of organic molecules. We previously reported an optimal and reproducible sample preparation method for 1H-qNMR. In the present study, we focused on a 31P-qNMR absolute determination method. An organophosphorus compound, cyclophosphamide hydrate (CP), listed in the Japanese Pharmacopeia 17th edition was selected as the target compound, and the 31P-qNMR and 1H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH2PO4) or O-phosphorylethanolamine (PEA) as the reference standard for 31P-qNMR and sodium 4,4-dimethyl-4-silapentanesulfonate-d6 (DSS-d6) as the standard for 1H-qNMR. Condition 1: separate sample containing CP and KH2PO4 for 31P-qNMR or CP and DSS-d6 for 1H-qNMR. Condition 2: mixed sample containing CP, DSS-d6, and KH2PO4. Condition 3: mixed sample containing CP, DSS-d6, and PEA. As conditions 1 and 3 provided good results, validation studies at multiple laboratories were further conducted. The purities of CP determined under condition 1 by 1H-qNMR at 11 laboratories and 31P-qNMR at 10 laboratories were 99.76 ± 0.43 and 99.75 ± 0.53%, respectively, and those determined under condition 3 at five laboratories were 99.66 ± 0.08 and 99.61 ± 0.53%, respectively. These data suggested that the CP purities determined by 31P-qNMR are in good agreement with those determined by the established 1H-qNMR method. Since the 31P-qNMR signals are less complicated than the 1H-qNMR signals, 31P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate 1H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.

Molecular Structure of Gardenia Blue Pigments by Reaction of Genipin with Benzylamine and Amino Acids.

Genipin was reacted with benzylamine and several amino acids to prepare gardenia blue (GB). The time-course of GB formation with benzylamine was monitored by high-performance liquid chromatography (HPLC), liquid chromatography time-of-flight mass spectrometry (LC-TOFMS), and 1H and 13C NMR measurements. In this experiment, we determined the molecular structures of some intermediates using accurate masses and additional NMR techniques such as heteronuclear multiple bond correlation (HMBC). GBs with amino acids (GB-AAs) were characterized by both liquid and solid-state NMR measurements. Interestingly, many significant peaks appeared in the solid-state NMR spectra, although the 13C NMR spectra from solution samples did not show any distinct peaks. Therefore, we determined that GB-AAs had an alternating copolymer structure composed of methyne and 5H-2-pyrindine, which was substituted by amino acids at N atom and linked with methyne at 5 and 7 positions. To confirm this molecular structure, the pyrolysis gas chromatography-mass spectrometry (GC-MS) measurement of GB-AAs was carried out, and 5H-2-pyrindine and its methyl derivatives were formed as main pyrolysis products from the polymer chains.

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.

Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

Absolute Purity Determination of a Hygroscopic Substance, Indocyanine Green, Using Quantitative NMR (qNMR).

Quantitative NMR (qNMR) is applied to determine the absolute quantitative value of analytical standards for HPLC-based quantification. We have previously reported the optimal and reproducible sample preparation method for qNMR of hygroscopic reagents, such as saikosaponin a, which is used as an analytical standard in the assay of crude drug section of Japanese Pharmacopoeia (JP). In this study, we examined the absolute purity determination of a hygroscopic substance, indocyanine green (ICG), listed in the Japanese Pharmaceutical Codex 2002, using qNMR for standardization by focusing on the adaptation of ICG to JP. The purity of ICG, as an official non-Pharmacopoeial reference standard (non-PRS), had high variation (86.12 ± 2.70%) when preparing qNMR samples under non-controlled humidity (a conventional method). Additionally, residual ethanol (0.26 ± 0.11%) was observed in the non-PRS ICG. Next, the purity of non-PRS ICG was determined via qNMR when preparing samples under controlled humidity using a saturated sodium bromide solution. The purity was 84.19 ± 0.47% with a lower variation than that under non-controlled humidity. Moreover, ethanol signal almost disappeared. We estimated that residual ethanol in non-PRS ICG was replaced with water under controlled humidity. Subsequently, qNMR analysis was performed when preparing samples under controlled humidity in a constant temperature and humidity box. It showed excellent results with the lowest variation (82.26 ± 0.19%). As the use of a constant temperature and humidity box resulted in the lowest variability, it is recommended to use the control box if the reference ICG standard is needed for JP assays.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

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.

Spiroketalcarminic Acid, a Novel Minor Anthraquinone Pigment in Cochineal Extract Used in Food Additives.

Cochineal extract prepared from the scale insect Dactylopus coccus (American cochineal) has been used as a natural red dye for food, cosmetics, and pharmaceuticals. The major pigment in cochineal extract is carminic acid (CA), an anthraquinone glucoside, and several minor pigments have been previously reported. Our investigation aimed at establishing the safety of cochineal dye products using ultra performance liquid chromatography-photo diode array-electrospray ionization-time of flight (UPLC-PDA-ESI-TOF)/MS found an unknown minor pigment, spiroketalcarminic acid (1), in three commercial cochineal extract samples; cochineal extract used in food additives, carmine that is an aluminum salt of cochineal extract used as natural dye, and a research reagent of CA. The purification of 1 from cochineal extract involved sequential chromatographic techniques, including preparative reversed-phase HPLC. Two dimensional (2D)-NMR and mass analyses established the structure of 1 to be a novel anthraquinone with an unusual 6,5-spiroketal system instead of the C-glucosyl moiety of CA. The absolute stereochemistry of the spiroketal moiety in 1 was determined by nuclear Overhauser effect spectroscopy (NOESY) correlations and optical rotation. No data corresponding to 1 had previously been reported for extracts of dried cochineal insects and traditional art products dyed with cochineal extract, indicating that 1 is likely produced during the preparation of commercial cochineal extract.

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.

[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 of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Method for the determination of natural ester-type gum bases used as food additives via direct analysis of their constituent wax esters using high-temperature GC/MS.

Natural ester-type gum bases, which are used worldwide as food additives, mainly consist of wax esters composed of long-chain fatty acids and long-chain fatty alcohols. There are many varieties of ester-type gum bases, and thus a useful method for their discrimination is needed in order to establish official specifications and manage their quality control. Herein is reported a rapid and simple method for the analysis of different ester-type gum bases used as food additives by high-temperature gas chromatography/mass spectrometry (GC/MS). With this method, the constituent wax esters in ester-type gum bases can be detected without hydrolysis and derivatization. The method was applied to the determination of 10 types of gum bases, including beeswax, carnauba wax, lanolin, and jojoba wax, and it was demonstrated that the gum bases derived from identical origins have specific and characteristic total ion chromatogram (TIC) patterns and ester compositions. Food additive gum bases were thus distinguished from one another based on their TIC patterns and then more clearly discriminated using simultaneous monitoring of the fragment ions corresponding to the fatty acid moieties of the individual molecular species of the wax esters. This direct high-temperature GC/MS method was shown to be very useful for the rapid and simple discrimination of varieties of ester-type gum bases used as food additives.

Absolute quantitation of stevioside and rebaudioside A in commercial standards by quantitative NMR.

The extract prepared from the leaves of Stevia rebaudiana BERTONI (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A. Highly purified stevia extracts have become popular worldwide as a natural, low-calorie sweetener. They contain various types of steviol glycosides, and their main components are stevioside and rebaudioside A. The content of each steviol glycoside is quantified by comparing the ratios of the molecular weights and the chromatographic peak areas of the samples to those of stevioside or rebaudioside A standards of the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA) and other specifications. However, various commercial standard reagents of stevioside and rebaudioside A are available. Their purities are different and their exact purities are not indicated. Therefore, the measured values of stevioside and rebaudioside A contained in a sample vary according to the standard used for the quantification. In this study, we utilized an accurate method, quantitative NMR (qNMR), for determining the contents of stevioside and rebaudioside A in standards, with traceability to the International System of Units (SI units). The purities of several commercial standards were determined to confirm their actual values.

[Examination of original plant of mulberry bark extract, a natural food additive, based on composition of the constituents].

Mulberry bark extract, a natural food additive, is described as a "root bark extract from Morus bombycis" (Japanese name: Yamaguwa) in the Notice (1996) relating to existing food additives used in Japan. The results of analyses by LC/UV and LC/MS suggested that the Mulberry bark extract products that were tested were actually made from the root bark of Morus alba (Japanese name: Maguwa) or its hybrid species, because the compositions of the constituents in the products are more similar to those in the extracts of the dried root bark of M. alba and hybrid species that are cultivated in Japan than to those of M. bombycis. In addition, the constituents of the food additive products were different from those of the natural medicine Mori Cortex products ('Souhakuhi' in Japanese) made from the root bark of mulberry grown in China, and which is described as being derived from M. alba in the Japanese pharmacopoeia. These results were also corroborated by Principal Component Analysis using the peak areas of LC/MS analysis as explanatory variables. After this study, it was decided that Mulberry bark extract is one of the existing food additives that should be excluded from the list this year in Japan.

[Absolute quantitation of quercetin and the glycosides in natural food additives by quantitative NMR].

We are developing a simple absolute quantitation method for organic compounds, by means of quantitative nuclear magnetic resonance (qNMR), with traceability to the International System of Units (SI units). The qNMR method was applied to the absolute quantitation of rutin, isoquercitrin and quercetin in natural food additives, rutin (extract), enzymatically decomposed rutin extract and quercetin, and those compounds as commercial reagents. In this study, 1,4-bis-(trimethylsilyl)benzene-d(4) (1,4-BTMSB-d(4)) whose purity was precisely evaluated on the basis of metrology, was newly used as a qNMR reference material, to be added to the sample solution as an internal standard. The contents of quercetin and quercetin glycosides were calculated from the ratio of the signal intensities of each aromatic proton at the 2' position of the three compounds (these are observed at different chemical shifts) to the eighteen protons of the six methyl groups on 1,4-BTMSB-d(4) used as a qNMR reference material. Rapid and simple qNMR method with only one step process was carried by using 1,4-BTMSB-d(4). It was demonstrated that the purities of rutin, isoquercitrin and quercetin can be separately determined by qNMR without the need for a separation process or reference materials for all the target compounds.