Mass spectrometry (MS) is a technique that can analyze a variety of chemical and biological compounds. An enormous growth seen in the field of MS hyphenation techniques in last few decades and is still ongoing. Nowadays, high performance liquid chromatography (HPLC) and other liquid or gas-phase separation techniques coupled to mass spectrometry are routinely used in analytical laboratories as a research and quality tool. Modern mass analyzers have powerful features for the structural elucidation; for example high resolving power, high mass accuracy, tandem mass spectrometry. A numerous reviews, research articles and books on mass spectrometry technology are available to explicate its principles, instrumentation and interpretation of MS and MS/MS spectra including basic rules, workflow, challenges and perspective. However, interpretation of full scan (MS) and tandem mass spectrum (MS/MS) is still remain difficult due to several probable structures, neutral losses, fragments with the almost similar m/z values and multiple fragmentation pathways.1-10 Therefore, we made an attempt to provide a step by step rational and simplified workflow for the interpretation of mass spectra acquired by atmospheric pressure ionization (API) techniques; electrospray ionization (ESI), also for other soft ionization technique such as atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry (MS/MS) spectra generated using collision-induced fragmentation. The workflow for interpretation of MS and MS/MS spectra are derived from the rules given in reference text books, literature and the rational approaches proposed by experienced researchers.11-14
INTERPRETATION OF MS AND MS/MS SPECTRA
The hyphenation of tandem mass spectrometers with modern chromatography instruments, for the identification small molecules and their impurities is became an essential tool for research laboratories. The MS/ MS spectra provide distinctive fragment patterns which in turn help in providing the structural information. In mass spectrometric analysis, the most essential task is to understand the mass spectra i.e. interpretation of the spectral data. Basics for mass spectrometric interpretation; nitrogen rule, accurate mass, types of ions, adducts, isotopic abundance, monoisotopic molecular weight, charge migration, cleavages, cyclization rearrangements and even odd electron rule need to be reminisce, to efficiently apply the workflows (Figure 1 & Figure 2) for the interpretation of acquired MS and MS/MS spectra of small organic molecules. To interpret the MS spectral data such as protonated and deprotonated ions formed during atmospheric ionization of analytes in ion source as well as mass difference of most common ion types calculated and presented in Table 1 for the quick reference of researchers. Table 1 may be quite informative, which may further help in the identification the molecular ion m/z value. Subsequently to classify fragments, reference table imbedded in Figure 2, will certainly helpful for assigning the right elemental composition and structural fragments to m/z values of fragments. Especially nitrogen rule is very helpful to assign a correct elemental composition for given mass number in case of nitrogenous molecules i.e. “a compound with an odd molecular weight will have an odd number of nitrogen and compounds with an even molecular weight will have either no nitrogen or an even number of nitrogen atoms”. Nitrogen rule in combination with Odd-Electron (OE) and Even-Electron (EE) rule for fragment ions interpretation is given in Table 2, which is extremely helpful to assign the correct elemental composition to fragment ions m/z values.
The systematic workflow is essential for identification of unknowns in new drugs substances, to investigate the suspicious, discover the unknowns and then add to the routine analysis. Identification of molecular ion peak for trace level of impurities is very essential to assign molecular weight of analyte and its elemental formula. Flow chart with various rational steps for the identification of molecular ion peak “M” in MS spectra was established and presented in Figure 1. The rational steps are summarized in a few words; select at least 2-3 most intense m/z values from MS spectra. Then, refer Table 1 to verify the mass difference with nearby m/z values and identify adducts, neutral loss and correlate adducts for the identification of molecular ion m/z value. Note down m/z value of [M+H] + and propose monoisotopic molecular weight (M) of analyte. Refer Table 1 to verify the molecular ion (m/z value) with cluster ions [2M+H]+ or [2M-H]- and adducts. Also, verify the isotopic abundance i.e. the presence elements such as chlorine, bromine and / sulfur, etc and also verify for multiple charged ions. Then, propose elemental composition, verify with nitrogen rule and accurate mass. Probable structure can be proposed based on the verified elemental composition. Further to verify with MS/MS information.
For the interpretation of MS/MS spectra, a flowchart containing various rational steps is presented in Figure 2. The rational steps are summarized as: first prepare the fragmentation table as shown in Figure 2 and then assign the common neutral losses (H2O, NH3, COO, CO, HCN and other neutral losses), Then propose the element composition and verify with the rules EE/OE rule, nitrogen rule and mass accuracy. In case of unknown impurity or metabolite, identify the modification by comparing with parent compound; verify the modification by using fragmentation pattern and possibility of formation. Propose a fragmentation pathway, based on charge migration, bond cleavages, charge retention sites, cyclization and rearrangements.
MS technology has been used an exploratory analytical technique from several decades in the research arena of chemistry. In fact, HPLC coupled with MS has become a powerful analytical tool for the identification of drugs, especially small drug molecules and their impurities. In this technical review, interpretation workflows were designed for interpretation of MS and MS/MS spectral data (Figure 1 and Figure 2) surely beneficial for readers. Notably, adduct ions information presented in Table 1 is very useful for identification of ions generated by ESI and APCI ion source. The information and the complex approach existed in the literature is gathered and presented in Figure 1 and Figure 2 in a simple way, which is easy to apply for the interpretation. The established workflow is efficient and can be applied for structure verification studies and for structure elucidation studies of small drug molecules and their impurities.
|m/z of fragment ion||Odd Electron Ions||Even Electron Ions|
|Even||No Nitrogen / Even Nitrogen||Odd Nitrogen|
|Odd||Odd Nitrogen||No Nitrogen / Even Nitrogen|