The development of an efficient mass balance approach for the purity assignment of organic calibration standards

Abstract

The purity determination of organic calibration standards using the traditional mass balance approach is described. Demonstrated examples highlight the potential for bias in each measurement and the need to implement an approach that provides a cross-check for each result, affording fit for purpose purity values in a timely and cost-effective manner. Chromatographic techniques such as gas chromatography with flame ionisation detection (GC-FID) and high-performance liquid chromatography with UV detection (HPLC-UV), combined with mass and NMR spectroscopy, provide a detailed impurity profile allowing an efficient conversion of chromatographic peak areas into relative mass fractions, generally avoiding the need to calibrate each impurity present. For samples analysed by GC-FID, a conservative measurement uncertainty budget is described, including a component to cover potential variations in the response of each unidentified impurity. An alternative approach is also detailed in which extensive purification eliminates the detector response factor issue, facilitating the certification of a super-pure calibration standard which can be used to quantify the main component in less-pure candidate materials. This latter approach is particularly useful when applying HPLC analysis with UV detection. Key to the success of this approach is the application of both qualitative and quantitative (1)H NMR spectroscopy.

Production of certified reference materials for the sports doping control of the REV‐ERB agonist SR9009

“…Upon ashing both candidate materials returned residue levels below the 2 mg/g limit of quantification (LOQ), a value of zero being assigned to the non‐volatile residue content with a standard uncertainty of 1.1 mg/g based on a rectangular probability distribution between zero and the LOQ . The low levels of water, solvent, and inorganics were further supported by elemental microanalysis which returned measures of carbon, hydrogen, nitrogen, and sulfur which matched the expected content in line with the molecular formula of the main component.…”

“…The purity of each candidate material was assessed as a mass fraction (mg/g) using a range of complementary traditional analytical techniques comprising the so‐called mass balance approach . Water content assessed by Karl Fischer coulometric titration confirmed low levels of water (< 3 mg/g) in each sample.…”

“…By 1 H NMR the hydrochloride salt of amine 1 was shown to contain trace amounts (0.5 mg/g) of the recrystallization solvent ethanol, while diamine 2 was found to contain TBME and hexane at 1 mg/g and 1.8 mg/g, respectively. In the case of amine 1 hydrochloride integration of the resonance at 4.31 ppm, including 13 C satellites, and the ethanol resonances at 1.2 ppm and 3.6 ppm provided a measure of the relative mass fraction of each component . For diamine 2 the terminal methyl group was used along with the respective solvent peaks.…”

“…With the evidence available we cannot rule out the possibility that the measure of imine 6 is to some extent biased by degradation of diamine 2 in solution. Accordingly, it was considered prudent to assign a conservative uncertainty to this measure of the imine using a rectangular probability distribution centered at the measured value and extending to zero and twice the measured value, the resulting uncertainty (1.2 mg/g) dominating the standard uncertainty assigned to the organic purity estimate . This, in part, contributed to the larger uncertainty at the 95% confidence interval being assigned to diamine 2 compared to amine 1 hydrochloride (Table ).…”

“…Confirmation of identity was achieved using nuclear magnetic resonance (NMR), infrared spectroscopy, and electrospray mass spectrometry. Certified purity values were assigned as a mass fraction using the mass balance approach, ensuring metrological traceability to the international standard unit for mass (kg) …”