ReviewCritical practical aspects in the application of liquid chromatography–mass spectrometric studies for the characterization of impurities and degradation products
Graphical abstract
Introduction
The definition of quality of pharmaceuticals has changed in recent times. From previous focus on purity, a greater emphasis is being paid today on impurities (IMPs), degradation products (DPs), etc. The characterization of such trace to minor analytes is not only important during drug and product development, but also from the perspective of regulatory approvals. Fortunately, the advent of a variety of sophisticated hyphenated techniques has made this task much simpler [1], against the time-consuming exercise involving isolation and enrichment of targeted compounds to milligram quantities, followed by acquisition of the spectral (mass, UV, NMR and IR) and elemental (CHN) data. In particular, liquid chromatography–mass spectrometry (LC–MS) is a technique of choice among all other hyphenated techniques due to its sensitivity and ease of use. Also, it carries potential in itself to provide unequivocal characterization of the structure of most trace to minor components, except enantiomers and epimers. A variety of LC–MS instruments are available today, which vary in (i) atmospheric pressure ionization (API) sources, like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and/or atmospheric pressure photo ionization (APPI); and (ii) analyzers, like quadrupole, ion trap, time of flight (TOF), Orbitrap, etc. Usually ESI and APCI interfaces along with ion trap, TOF and Orbitrap analyzers are more useful for providing qualitative information on component structures.
The unequivocal structure elucidation of unknown analytes by LC–MS techniques is possible through the strategy put forth by us earlier [1], [2]. It entails the generation of molecular ion and fragment spectra of both the drug and its related substances; determination of their accurate and exact masses, elemental formula, ring plus double bonds and number of nitrogens; determination of the number of labile hydrogens through hydrogen/deuterium (H/D) exchange mass studies; MSn study on molecular ions and fragments, and establishment of fragmentation pathways, followed by their comparison. Introductory details on each of these steps were provided by us in our previous publication [1]. The strategy has been duly validated in our laboratories and even by other researchers by application to the characterization of process impurities [3], drug degradation products [4], [5], [6], drug–drug interaction products [7], [8], drug-excipient interaction products [9], and metabolites of a variety of drugs [10], [11].
The purpose of this review is to critically address practical issues at each step of the proposed strategy meant to characterize IMPs/DPs by LC–MS tools. To our experience, straight forward characterization of IMPs/DPs is possible in most cases. However, challenges arise at times with respect to ionization of the analyte, achieving optimum signal sensitivity, problem in identification of the molecular ion, absence of characteristic fragment ions relative to the drug, appearance of unusual fragments, and difficulty in establishing fragmentation pathways. These aspects are duly discussed with examples.
Section snippets
Strategy for the characterization of impurities/degradation products by mass spectrometric approaches
The strategy for the characterization of IMPs/DPs by LC–MS, proposed by us earlier [1], [2], is shown in an expanded form in Fig. 1. While mass investigations on the drug are carried out in a direct infusion mode, parallel studies are conducted on IMPs/DPs after achieving their separation on a LC column. In the latter case, optimization of the mobile phase parameters to obtain resolution between the analytes with maximum mass sensitivity is an additional step. In both cases, it becomes prudent
Practical issues in sample preparation, method development and instrument settings
LC–MS tools are sophisticated and complex. Therefore, successful generation of data from them requires good knowledge and clarity on practical aspects. Critical practical issues and tips with respect to sample preparation, method development and instrument settings are discussed below.
Acquisition and analyses of mass spectra
Acquisition of mass data and analyses are subsequent critical aspects. They also have multiple practical intricacies.
Compilation of experimental and derived data
Systematic organization of the experimental data for the drug and its IMPs/DPs in a table format assists in easy interpretation and also comprehensive review. An example is given in Table 8.
Establishing fragmentation pathways
Once the whole data, like accurate mass, MSn, isotopic abundance and H/D exchange are collected immaculately by taking care of all the practical aspects, and also after data are organized systematically in the tabulated form, the next step is to lay down the fragmentation pathway of the drug and its IMPs/DPs. The successful construction of fragmentation pathways first for the drug and then for the postulated IMPs/DPs provides justification to the structures envisaged. There are many other
Comparison of fragmentation pathways of drug and impurities/degradation products
In a very simple case, the proposed structure of IMP/DP is easily substantiated if its spectrum shows similar fragmentation lines and same neutral losses as the drug. An example of this is shown in Fig. 22, which portrays fragmentation pathway of pioglitazone and it's DP. The ESI-MS/TOF fragmentation spectra of both the drug and its DP in positive ionization mode are shown in Fig. 23. As apparent from figure a, the drug (m/z 357) fragmented into a total of nine fragments. MS2 studies revealed
Confirmation of the proposed structures through mechanistic explanation
Once the structure is proposed through mass data and justified through mass fragmentation, the final structure shall be substantiated through mechanistic explanation. While proposing the mechanism, one must duly consider reaction/stress conditions under which specific IMP/DP was generated. Under these conditions, it should be possible to explain the formation of the specific IMP/DP mechanistically, normally through standard reaction mechanisms described in the literature [155], [156], [157]. In
Limitations of mass spectrometry for unequivocal characterization of unknown structures
Although mass spectrometry itself has potential to elucidate structure unequivocally, in rare situations it may be difficult to assign structures to unknown IMPs/DPs from the available mass data. This may be the case when multiple structures are proposed after critical review of the LC–MS data. We encountered a few situations of this type, for example, during characterization of a DP of ezetimibe, three alternate structures were possible, as given in Fig. 25. Mass data justified all the three
Conclusion
The above discussion clearly highlights that it is at every step of the use of mass tools that one needs to be careful of practical aspects involved in acquisition and interpretation of mass results for the purpose of characterization of IMPs/DPs. It is hoped that the discussion will help the new practitioners to achieve the task confidently. This may be true even of characterization of other minute components, like drug–drug interaction products, drug-excipient interaction products,
Acknowledgment
The grant of INSPIRE fellowship, by Department of Science and Technology, Government of India, to one of the authors (M.N.) is duly acknowledged.
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- 1
These authors contributed equally.
- 2
Present address: Biocon Bristol Myers-Squibb Research and Development Center (BBRC), Biocon Park, Bengaluru, India.