4 Qualitative and quantitative methods of impurities

The original quantification of impurities mainly relied on chemical methods, such as volumetric method. So far, some scholars have used this method to determine impurities in drugs. 64 With the rapid development of various detection methods, the combined of chromatography and various detectors has become the main means of impurity analysis today. Quantitative methods for impurities in many pharmacopoeias can be divided into an external standard method with impurity reference standards, the principle component self-control method with correction factors, and the principle component self-control method without correction factors. 65 The most ideal quantitative method is an external standard method with impurity reference standards, but it is difficult to obtain impurity reference standards in the early stage of impurity research, so this method has certain limitations on the quantitative research of impurities. The principle component self-control method without correction factors also has many shortcomings. When the UV detector is used to quantify unknown impurities, the response of UV detectors for impurities and sample are also different, due to the difference of chemical structure between impurities and impurities, and between impurities and samples. Even the impurities do not have UV absorption. Some mass-type detectors can reduce the response difference between compounds when using the principle component self-control method without correction factors, and they are also widely used in the detection of impurities. Such as Evaporative Light Scattering Detector (ELSD), 66Charged Aerosol Detector (CAD), 67 Refractive Index (RI), 68 Chemical Lumines-cent Nitrogen-specific Detector (CLND). 69 Although the quantitative capabilities of these detectors are strong, their qualitative capabilities are weak, especially for the direct qualitative determination of unknown substances, which requires the use of known reference materials or related chromatographic qualitative reference data (retention time) for qualitative identification. This also highlights the advantages of Mass Spectrometer (MS) and Nuclear Magnetic Resonance spectrometer (NMR) in impurity qualitative analysis. Fig. 3 shows the general process of impurity characterization.

4.1 Mass Spectroscopy

Because the combination of MS and various chromatograms integrates the high separation capability of chromatography with the high sensitivity of mass spectroscopy, Liquid Chromatography-Mass Spectroscopy (LC-MS) has become the preferred technique for drug impurity analysis. The advantages of MS for impurity analysis are the identification of known impurities and the structure derivation of unknown impurities. The application of high-resolution mass spectroscopy can not only distinguish compounds with very similar molecular weights, but also determine the elemental composition of impurities. After that, the general chemical structure of the unknown compound can be determined by deriving the fragmentation patterns of impurities. Zhu et al.70 used LC-MS to study the fragmentation patterns of impurities in sodium drug substance and eye drops, deduced the chemical structures of impurities, confirmed the structure with 1D and 2D NMR data, and achieved the structures characterization of 2 unknown impurities and 6 unknown degradation products and a plausible mechanism for the formation of the degradation products was also proposed. Hertzler et al. 71 study fragmentation patterns and fragmentation pathways of paromomycin impurities based on UHPLC/MS/MS and the literature of other structurally related aminoglycoside compounds, and made reasonable suggestions for the storage methods of drugs.
A summary of the fragmentation patterns of similar drugs is helpful to quickly analyze the structure of impurities in the drug. However, this method is only an auxiliary function for structure identification, and it is impossible to sure the position and spatial configuration of some groups in the structure. NMR is required to further characterize the impurities. 72 Mass spectroscopy fragment information can be clarified through many databases, 73 but there is no comprehensive mass spectroscopy fragment database for impurity research.
The difference between MS and other detectors is not only in the structural analysis of impurities, but also in that it can detect very small amounts of impurities, which greatly improves the safety of drugs containing low levels of highly toxic impurities. Isotope internal standard and multiple reaction monitoring (MRM) mode are unique features of mass spectroscopy quantification. Isotope as an internal standard quantification method can reduce the accidental errors caused by separate injection of standards, 74 but this method has certain limitations because the standard of impurities is not easy to obtain, and it is mostly used to determine known impurities, such as mycotoxins. 75 The MRM mode can realize the simultaneous, exclusive, sensitive and rapid quantitative detection of dozens of similar impurities with different concentration levels by detecting specific ions of the target compound. 76 It is very conducive to the simultaneous quantitative determination of low concentration and multiple structurally similar impurities in complex systems. 77
In response to the problem that the mobile phase for LC - MS can not contain non-volatile salts, so that many chromatographic methods cannot be directly converted to LC-MS methods. Some researchers also use column switching technology to introduce the mobile phase containing non-volatile salts into the desalted chromatographic column through an on-off valve and then enter the MS for analysis. 78 In the case of low response impurity, deriving and adding alkali metals solve this problem. Wijk et al. 79 chose 1-(pyridin-4-yl) piperidine 4-carboxylate (BPPC) as a new, selective pre-column derivatization reagent to obtain reagent related fragmentation of the whole reagent as well as a side group of the reagent when analyzing potential genotoxic compounds.
MS detectors are also commonly used for the detection of elemental impurities. Inductively coupled plasma-mass spectroscopy (ICP - MS) can analyze almost all elemental impurities. ICP - MS has the advantages of fast determination speed, low detection limit, wide detection range, and simultaneous determination of multiple elements. 80Zheng et al. 81 established an ICP - MS method to determinate 24 elemental impurities of the ICH guidelines in ubenimex API after direct dissolution in diluted acid solution and successfully applied to the elemental impurities determination in 3 batches of ubenimex API from different factories. There are many analytical methods for impurity elements. Such as colorimetry, Flame Absorption Spectroscopy (FAAS), 82 Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP - AES), 83X-Ray Fluorescence Spectroscopy (XRFS), 84 and Atomic Fluorescence Spectrometry (AFS). Their advantages and disadvantages are shown in Table 2. In contrast, ICP - MS relies on the advantages of the MS detector are more suitable for trace and ultra-trace element analysis.

4.2 Nuclear Magnetic Resonance spectroscopy

Nuclear magnetic resonance spectroscopy technology can play an important role in the analysis of impurities in drugs, not only qualitatively or quantitatively. MS and its combined technology can deduce the structure of impurities, but it can not obtain the exact chemical structure of impurities, while NMR can provide comprehensive structural information (planar structure, relative structure, three-dimensional structure) and is also an accepted method to determine the identification structure of organic compounds. If NMR analyzes the structure of a compound, the sample needs to reach the mg level and the purity of the sample is very high. However, the sensitivity of NMR is low, which is also a huge challenge to the structure analysis of impurities. There are also some researchers who use preparative HPLC to enrich the impurities in the samples, so that the amount of impurities reaches the detection line of NMR. 85-87 However, the amount of impurities in the sample is very low. If it is directly extracted and separated from the medicine, the preparation is difficult and the cost is high. Some researchers increase the content of impurities in the sample through forced degradation experiments to reduce the workload of preparation.88 There are also researchers who obtain a large amount of impurities through synthesis methods, 89-91but this method needs to derive the structure of the impurities, then design the synthesis route, and finally verify the synthesized impurities. It has many steps, takes a long time, and the success rate is not high.
Follow the idea of LC - MS. The combination of liquid phase and NMR will also become a possibility. However, the hyphenation of LC - NMR has a little limitation on impurity analysis, because the accumulation time of each chromatographic peak is too short to obtain NMR spectra of minor impurities and the stopped-flow technique leading to peak diffusion in the column during accumulation. 92 LC - NMR analysis can be conducted with a cryogenic probe to cool the radiofrequency coil and preamplifier, leading to a reduction in the thermal noise and an increase in the detection sensitivity. 93 In addition, ultra-high field magnets (> 800 MHz) can be used in LC-NMR to improve the detection sensitivity, but it has been little progress over the past decade. 94 Takashi et al.95 constructed a UHPLC - NMR system to concentrate chromatographic peaks. In the UHPLC - NMR system, the magnetic field strength was increased, a cryogenic probe was used to improve the sensitivity, and the loop-storage technique was used to suppress diffusion. The schematic diagram of UHPLC-NMR system is shown in Fig. 4. The sensitivity is higher than an ultra-high field magnet (800 MHz) and a probe.
In the early stage of drug research, it is often difficult to have standards for impurities, especially unknown impurities, but NMR can accurately quantify impurities without reference materials. The qNMR quantitative technology is based on the fact that the area of the nuclear magnetic resonance spectrum signal is proportional to the number of excited atoms in the sample to achieve the quantitative goal.96 The qNMR quantitative method is divided into external standard method and internal standard method. The result of external standard method is greatly affected by the instrument, so the internal standard method is usually used for quantification.97 Commonly used internal standards are benzoic acid, maleic acid and fumaric acid. The structure and content of the internal standard should be known, and the response signal of the internal standard should be well separated from the response signal of the impurity to be measured without overlapping. Naoki et al.98 also propose a novel extended internal standard method of qNMR assisted by chromatography (EIC) that accurately quantifies 1H signal areas of samples, when the chemical shifts of the impurity and samples signals overlap completely, and used 2-chlorophenol and 4-chlorophenol containing phenol as an impurity as examples in which impurity and samples signals overlap to validate and demonstrate the method. Liu et al. 99 used q NMR and HPLC - UV to determine ten impurities of cefazolin and provided the relative response factors of ten impurities. Made up for the defect the principle component self-control method without correction factors in the determination of related substances. Compared with LC - UV that commonly used for impurity analysis, qNMR does not require complicated pre-separation processing. For the determination of drug content without ultraviolet absorption in molecular structure, or corresponding reference substance, qNMR is also a very suitable method.100