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 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.

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.

The antiviral effects of baloxavir marboxil against influenza A virus infection in ferrets.

BACKGROUND: Baloxavir marboxil (BXM), the oral prodrug of baloxavir acid (BXA), greatly reduces virus titers as well as influenza symptoms of uncomplicated influenza in patients. OBJECTIVES: To investigate the pharmacokinetic profiles of BXA and its efficacy against influenza A virus infection in ferrets. METHODS: Ferrets were dosed orally with BXM (10 and 30 mg/kg twice daily for 1 day), oseltamivir phosphate (OSP) (5 mg/kg twice daily for 2 days) or vehicle to measure the antiviral effects of BXM and OSP. The pharmacokinetic parameters of BXA was determined after single oral dosing of BXM. RESULTS: The maximum plasma concentrations of BXA were observed at 1.50 and 2.00 hours with the two BXM doses, which then declined with an elimination half-life of 6.91 and 4.44 hours, respectively. BXM at both doses remained detectable in the plasma in ferrets, which may be due to higher stability in liver microsomes. BXM (10 and 30 mg/kg twice daily) treatment at Day 1 post-infection (p.i.) reduced virus titers by ≥3 log10 of the 50% tissue culture infective doses by Day 2, which was significantly different compared with vehicle or OSP. Body temperature drops over time were significantly greater with BXM than with vehicle or OSP. Significant reduction in virus titers was also demonstrated when BXM was administrated after symptom onset at Day 2 p.i. compared with vehicle and OSP, although body temperature changes largely overlapped between Day 2 and Day 4. CONCLUSIONS: The results highlight the rapid antiviral action of BXM with post-exposure prophylaxis or therapeutic dosing in ferrets and offer support for further research on prevention of influenza virus infection and transmission.

Characterization of heme-coordinating histidyl residues of an engineered six-coordinated myoglobin mutant based on the reactivity with diethylpyrocarbonate, mass spectrometry, and electron paramagnetic resonance spectroscopy.

A genetically engineered porcine myoglobin triple mutant (H64V/V68H/H93A) (VHA-Mb) contains 6 non-axial His residues (His24, His36, His48, His81, His82, and His119) besides two candidate axial His residues (His68 and His97). Although previous resonance Raman study on the ferric VHA-Mb were not conclusive for its coordination structure, present EPR parameters of the ferric VHA-Mb were consistent with bis-imidazole coordination of His68/His97. We further investigated the reactivity of these possible His ligands with diethylpyrocarbonate (DEPC) to clarify the coordination structure and their protonation states in ferric form. We found that the non-axial His residues were easily modified with a low concentration of DEPC based on UV spectral changes and MALDI-TOF-MS analyses. On the other hand, the two candidate axial His ligands were protected from the modification due to a limited steric exposure of their imidazoles to solvent, the Fe(3+)-N(epsilon2) coordination bond, and the protonation of N(delta1) by forming a hydrogen bond with their immediate surroundings. However, once N-carbethoxylation occurred at N(epsilon2) of His97, resulting in a disruption of the heme Fe(3+)-N(epsilon2) coordination bond, it facilitated the second N-carbethoxylation to take place at N(delta1) of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. These phenomena were consistent with the bis-His68/His97 coordination. Further, these were not observed at all for cytochrome b(561), a transmembrane di-heme containing protein responsible for the ascorbate-specific transmembrane electron transfer, where only a specific N(delta1)-carbethoxylation of axial His occurred at a low concentration of DEPC, leading to an inhibition of the electron acceptance from ascorbate without a release of the heme. These distinct results might be related to a specific physiological mechanism being operative at the cytosolic heme center of cytochrome b(561).