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As a new synthetic molecule, no certified reference standard was available to be applied as a response factor using traditional chromatographic techniques, such as high-performance liquid chromatography (HPLC) or gas chromatography (GC).
At RSSL, we have extensive experience using nuclear magnetic resonance (NMR) spectroscopy as a method of determining the purity of new synthetic small molecules. Although the technique is commonly used for structure elucidation of small organic compounds, NMR has a huge potential to provide quantitative information, even within complex mixtures.
Strengths of NMR include an intrinsically linear response meaning there is no requirement for a calibration curve or conditioning prior to measurements, making it suitable for automation and allowing high-throughput analysis. Furthermore, only a small amount of sample is required (as little as 2 mg). This was a factor we had to take into consideration as only a very limited amount of the sample was available for analysis. If required, method verification or validation can be performed, depending on client’s requirement.
NMR can perform both relative and absolute quantitation. For the determination of sample purity, we used the absolute quantitation approach, which involves the direct measurement of the actual amount of target analyte within a sample. Absolute quantitation methods require the use of a known amount of a reference standard with known structure and purity, which can be co-dissolved with the analyte in the same test solution (internal reference standard) or prepared separately (external reference standard). For high accuracy analysis, the internal reference method is preferred.
The appropriate internal reference standard was selected during careful planning regarding solubility and chemical compatibility (Figure 1). As the reference standard and test sample were co-dissolved, a similar solubility profile must be observed. Deuterated solvents are required for NMR analysis. Fortunately, deuterated versions of commonly used laboratory solvents are widely available (e.g., deuterium oxide, chloroform, methanol). Additionally, the reference standard must not react with the analyte of interest and overlap between the quantifying signals of the sample and reference must be avoided.
RSSL has over 25 years’ experience in the use of NMR spectroscopy as a valuable analytical technique in the life sciences sector. In a GxP compliant environment, RSSL’s capabilities include a 400 MHz JEOL ECA and a 600 MHz Bruker AVANCE NEO NMR spectrometers. We have worked collaboratively with clients from a variety of industries such as pharmaceuticals, organic synthesis and drug discovery, dietary and food supplements, polymers and biomaterials, providing custom solutions to meet their requirements.
We routinely use qNMR experiments for the purity determination of pharmaceutical products and its active pharmaceutical ingredients. More recently, qNMR has shown to be particularly valuable in the analysis of new synthetic molecules and new psychoactive substances where no commercial reference standards are available.
Despite being a versatile and robust technique, a few considerations are paramount to generate accurate results when performing a qNMR experiment. A summary of the main considerations followed is highlighted in the figure below.
Our ability at RSSL to adapt an existing approach to solve a specific client’s need produced quick and reliable results. This project was the beginning of a long-term relationship where the client has asked RSSL to assess the purity of many other new small molecules used in the development of new pharmaceutical drugs.
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