Identification by HSQC and quantification by qHNMR innovative pharmaceutical amino acid analysis.
J Pharm Biomed Anal
; 251: 116390, 2024 Aug 07.
Article
en En
| MEDLINE
| ID: mdl-39190935
ABSTRACT
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 (600â¯MHz) and a benchtop (60â¯MHz) NMR instrument. To assess sensitivity constraints, samples for 1H NMR analysis were initially prepared using only 10â¯mg of analyte and 1â¯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 1H 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 13C 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 30â¯mg of the analyte and several acquisition parameters were tested, including t1 increments used in the pulse program, number of scans, and repetition time. Excellent congruence with deviations <0.1â¯ppm was achieved for the 13C chemical shifts from 1D 13C NMR spectra (150â¯MHz) vs. those extracted from ed-HSQC (15â¯MHz traces). Finally, all samples of amino acid candidate reference materials were quantified by 1H qNMR (abs-qHNMR) at both 600 and 60â¯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 100â¯mg of analytes and 10â¯mg of the IC and replicates were analyzed for selected amino acids.
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01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
J Pharm Biomed Anal
Año:
2024
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Article