Pharmacognosy Institute & Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois Chicago, IL 60612, United States.
NMR Solutions Ltd., Kuopio 70110, Finland.
J Pharm Biomed Anal. 2024 Dec 15;251:116390. doi: 10.1016/j.jpba.2024.116390. Epub 2024 Aug 7.
This study introduces a new NMR-based methodology for identification (ID) and quantification (purity, strength) assays of widely used amino acids. A detailed analysis of four amino acids and their available salts was performed with both a high-field (600 MHz) and a benchtop (60 MHz) NMR instrument. To assess sensitivity constraints, samples for H NMR analysis were initially prepared using only 10 mg of analyte and 1 mg of maleic acid (MA) as an internal calibrant (IC) and secondary chemical shift reference. The characteristic dispersion of the peak patterns indicating the presence or absence of a counterion (mostly chloride) was conserved at both high and low-field strength instruments, showing that the underlying NMR spectroscopic parameters, i.e., chemical shifts and coupling constants, are independent of the magnetic field strength. However, as the verbal descriptions of H NMR spectra are challenging in the context of reference materials and pharmaceutical monographs, an alternative method for the identification (ID) of amino acids is proposed that uses C NMR patterns from multiplicity-edited HSQC (ed-HSQC), which are both compound-specific and straightforward to document. For ed-HSQC measurements, the sample amount was increased to 30 mg of the analyte and several acquisition parameters were tested, including t increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1 ppm was achieved for the C chemical shifts from 1D C NMR spectra (150 MHz) vs. those extracted from ed-HSQC (15 MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by H qNMR (abs-qHNMR) at both 600 and 60 MHz. At high field, both IC and relative quantitations were performed, however, with the low-field instrument, only the IC method was used. The results showed that the analyzed reference material candidates were generally highly pure compounds. To achieve adequately low levels of uncertainty for such high-purity materials, the sample amounts were increased to 100 mg of analytes and 10 mg of the IC and replicates were analyzed for selected amino acids.
本研究介绍了一种新的基于 NMR 的方法，用于广泛使用的氨基酸的鉴定（ID）和定量（纯度、强度）分析。使用高场（600 MHz）和台式（60 MHz）NMR 仪器对四种氨基酸及其可用盐进行了详细分析。为了评估灵敏度限制，最初仅使用 10mg 分析物和 1mg 马来酸（MA）作为内标（IC）和二级化学位移参比物来制备用于 1 H NMR 分析的样品。在高场和低场强度仪器上均保留了指示反离子（主要是氯离子）存在或不存在的峰型特征色散，表明基础 NMR 光谱参数，即化学位移和偶合常数，与磁场强度无关。然而，由于在参考材料和药物专论的背景下，1 H NMR 光谱的描述具有挑战性，因此提出了一种替代的氨基酸鉴定（ID）方法，该方法使用多重编辑 HSQC（ed-HSQC）的 13 C NMR 图谱，该图谱既具有化合物特异性，又易于记录。对于 ed-HSQC 测量，将样品量增加到 30mg 分析物，并测试了多个采集参数，包括脉冲程序中的 t 增量、扫描次数和重复时间。从 1D 13 C NMR 光谱（150 MHz）与从 ed-HSQC（15 MHz 谱线）提取的 C 化学位移之间达到了极好的一致性，偏差 <0.1 ppm。最后，通过在 600 和 60 MHz 下进行 1 H qNMR（abs-qHNMR）对所有氨基酸候选参考物质样品进行定量。在高场，同时进行 IC 和相对定量，然而，对于低场仪器，仅使用 IC 方法。结果表明，分析的参考材料候选物通常是高纯度化合物。为了对这种高纯度材料达到足够低的不确定度水平，将样品量增加到 100mg 分析物和 10mg IC，并对选定的氨基酸进行了重复分析。
