RSSL utilised liquid chromatography-mass spectrometry (LC-MS), specifically a quadrupole time-of-flight (QTOF) accurate mass instrument, to investigate the mass ion that may be related the unknown impurity. This sensitive analytical technique provides accurate mass ion data, which can provide the molecular weight of a compound, a proposed molecular formula and with the use of MS/MS fragmentation, structural information of a compounds.
Figure 1: UV Chromatograms of control and client’s sample
Following client chromatography, the client’s method was further developed to be compatible with LC-MS and injected with both the control and the impurity containing sample. Figure 1 shows the UV chromatograms (230 nm) of the control and the sample with the impurity respectively. Ibuprofen is seen at about 18.19 minutes in both chromatograms, and the impurity is seen at about 14.19 minutes (relative retention to ibuprofen of 0.79), which was consistent with the client chromatography.
RSSL selected an accurate LC-MS instrument, as the addition of this detector to the client’s UV analytical method provides the ability to analyse an HPLC injected sample by both UV and MS simultaneously and generate highly accurate mass spectrometry data of the observed UV peaks.
Analysis of the MS data at the retention time of the unknown impurity produced the spectra seen in Figure 2. The mass spectrometry analysis in this instance was performed in negative mode to observe any negatively charged ions. Figure 2 highlighted several mass ions, but careful interpretation determined that mass ion m/z 188.9510 was the deprotonated peak of the impurity compound. Additionally, analysis of the isotopic pattern gave an indicative pattern for the presence of 2 chlorine atoms. The most abundant ion observed was determined to be a sodium adduct dimer ion (2M+Na-2H)-, which further confirmed the impurity had a molecular weight of 189.9583 Daltons.
Figure 2: Unknown purity spectra
To confirm the identity of the impurity using the mass spectrometry data produced, a standard of DCBA was purchased. This standard was analysed using the same LC-MS method developed to determine the identification of the impurity, which allowed the mass spectra to be directly compared.
Figure 3 shows both the impurity and DCBA mass spectra. The resulting mass spectra displays the same mass ions and a very similar profile, thus confirming the impurity as DCBA. The standard also eluted at the same retention time as the impurity, providing further confirmation (Figure 4).
Figure 3
Figure 4
Accurate mass spectrometry within an LC-MS configuration is a well-suited instrument to investigate the identification and structural elucidation of an unknown species. Furthermore, the highly sensitivity capability of this instrument allows for barely detectable impurities to be analysed.
Our team of experts employ a range of robust analytical techniques to identify and elucidate impurities quickly and reliably. Find out more about our impurity isolation and investigation service now.
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