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