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.
Quantitative 1 H-NMR ( 1 H-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 31 P nucleus because of the simplicity of its signals and previously reported 31 P-qNMR in D2O. Here we report 31 P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-BTMSB-d4 were used as reference standards for 31 P-qNMR and 1 H-qNMR, respectively, in methanol-d4. The purity of SOF determined by 31 P-qNMR was 100.63 ± 0.95%, whereas that determined by 1 H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the 31 P signal of PAA and SOF were 3.38 ± 2.39 Hz 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, DMSO-d6, was used as the dissolving solvent with PAA and DSS-d6 as reference standards for 31 P-qNMR and 1 H-qNMR, respectively. SOF purities determined by 31 P-qNMR and 1 H-qNMR were 99.10 ± 0.30% and 99.44 ± 0.29%, respectively. SOF purities determined by 31 P-qNMR agreed with the established 1 H-qNMR values, suggesting that an aprotic solvent is preferable for 31 P-qNMR because it is unnecessary to consider the effect of deuterium exchange.
Recently, quantitative NMR (qNMR), especially 1 H-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 1 H-qNMR. In the present study, we focused on a 31 P-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 31 P-qNMR and 1 H-qNMR results were compared under three conditions with potassium dihydrogen phosphate (KH 2 PO 4 ) or O-phosphorylethanolamine (PEA) as the reference standard for 31 P-qNMR and DSS-d 6 as the standard for 1 H-qNMR. Condition 1: separate sample containing CP and KH 2 PO 4 for 31 P-qNMR or CP and DSS-d 6 for 1 H-qNMR. Condition 2: mixed sample containing CP, DSS-d 6 , and KH 2 PO 4 . Condition 3: mixed sample containing CP, DSS-d 6 , 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 1 H-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 31 P-qNMR are in good agreement with those determined by the established 1 H-qNMR method. Since the 31 P-qNMR signals are less complicated than the 1 H-qNMR signals, 31 P-qNMR would be useful for the absolute quantification of compounds that do not have a simple and separate 1 H-qNMR signal, such as a singlet or doublet, although further investigation with other compounds is needed.
Bioconversion using microorganisms and their enzymes is an important tool in many industrial fields. The discovery of useful new microbial enzymes contributes to the development of industries utilizing bioprocesses. Streptomyces sp. EAS-AB2608, isolated from a soil sample collected in Japan, can convert the tetrahydrobenzotriazole CPD-1 (a selective positive allosteric modulator of metabotropic glutamate receptor 5) to its hydroxylated form at the C4-(R) position. The current study was performed to identify the genes encoding the enzymes involved in CPD-1 bioconversion and to verify their function. To identify gene products responsible for the conversion of CPD-1, we used RNA sequencing to analyze EAS-AB2608; from its 8333 coding sequences, we selected two genes, one encoding cytochrome P450 (easab2608_00800) and the other encoding ferredoxin (easab2608_00799), as encoding desirable gene products involved in the bioconversion of CPD-1. The validity of this selection was tested by using a heterologous expression approach. A bioconversion assay using genetically engineered Streptomyces avermitilis SUKA24 Δsaverm3882 Δsaverm7246 co-expressing the two selected genes (strain ES_SUKA_63) confirmed that these gene products had hydroxylation activity with respect to CPD-1, indicating that they are responsible for the conversion of CPD-1. Strain ES_SUKA_63 also showed oxidative activity toward other compounds and therefore might be useful not only for bioconversion of CPD-1 but also as a tool for synthesis of drug metabolites and in optimization studies of various pharmaceutical lead compounds. We expect that this approach will be useful for bridging the gap between the latest enzyme optimization technologies and conventional enzyme screening using microorganisms. Key points• Genes easab2608_00800 (cyp) and easab2608_00799 (fdx) were selected by RNA-Seq.• Selection validity was evaluated by an engineered S. avermitilis expression system.• Strain ES_SUKA_63 showed oxidative activity toward CPD-1 and other compounds.
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