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\begin{document}

\title{Research progress on the impurities identification and determination in
pharmaceuticals}



\author[1]{wenlong Li}%
\author[1]{Yuru Chen}%
\author[1]{Zhiyong Zhang}%
\author[1]{Xin Gao}%
\author[1]{Xuting Guo}%
\affil[1]{Tianjin University of Traditional Chinese Medicine}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
\textbf{Rationale:} Impurities refer to any substances that affect the
purity of pharmaceuticals. Controlling impurities has always been a
significant concern during drug development. Impurities impact the
drug's purity, diminish the efficacy of pharmaceuticals, and alter their
appearance, physical, and chemical properties. Additionally, impurities
can compromise the stability of pharmaceuticals and elevate their toxic
and side effects. \textbf{Methods:} Impurity source analysis is the
basis of drug impurity control. To clarify the source of impurity can
optimize the synthesis process, prescription process, packaging and
storage conditions of the pharmaceuticals, and control the impurity
within a reasonable limit to achieve the ultimate goal of impurity
research. \textbf{Results:} Analysis method is a means to obtain
impurity information, and diversified analysis methods are also possible
for effective control of different types of impurities. At present,
there are relatively many quantitative studies on impurities, but there
are still some challenges for the structure analysis of impurities,
especially trace impurities. \textbf{Conclusion:} The research progress
of drug impurity control and evaluation from the sources of various drug
impurities and impurity analysis techniques was reviewed in this
article, with the aim of providing references for the related research.%
\end{abstract}%



\sloppy


\textbf{Research progress on the impurities identification and
determination in pharmaceuticals}

Yuru Chen\textsuperscript{a\#}, Zhiyong Zhang\textsuperscript{a\#}, Xin
Gao\textsuperscript{a}, Xuting Guo\textsuperscript{a}, Wenlong
Li\textsuperscript{a, b, c}11\textsuperscript{*} Corresponding author.
Tel.: +86 22 59596814; Fax: +86 22 59596814.\emph{E-mail address:
wshlwl@tjutcm.edu.cn} (W. Li).

\textsuperscript{a} College of Pharmaceutical Engineering of Traditional
Chinese Medicine, Tianjin University of Traditional Chinese Medicine,
Tianjin, 301617, PRC;

\textsuperscript{b} State key Laboratory of Component-based Chinese
Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin,
301617, PRC;

\textsuperscript{c} Haihe Laboratory of Modern Chinese Medicine,
Tianjin, 301617, PRC

\textbf{Rationale:} Impurities refer to any substances that affect the
purity of pharmaceuticals. Controlling impurities has always been a
significant concern during drug development. Impurities impact the
drug's purity, diminish the efficacy of pharmaceuticals, and alter their
appearance, physical, and chemical properties. Additionally, impurities
can compromise the stability of pharmaceuticals and elevate their toxic
and side effects.

\textbf{Methods:} Impurity source analysis is the basis of drug impurity
control. To clarify the source of impurity can optimize the synthesis
process, prescription process, packaging and storage conditions of the
pharmaceuticals, and control the impurity within a reasonable limit to
achieve the ultimate goal of impurity research.

\textbf{Results:} Analysis method is a means to obtain impurity
information, and diversified analysis methods are also possible for
effective control of different types of impurities. At present, there
are relatively many quantitative studies on impurities, but there are
still some challenges for the structure analysis of impurities,
especially trace impurities.

\textbf{Conclusion:} The research progress of drug impurity control and
evaluation from the sources of various drug impurities and impurity
analysis techniques was reviewed in this article, with the aim of
providing references for the related research.

\textbf{Keywords:} Impurities in pharmaceuticals; Impurity source
identification; Separation and purification techniques; Qualitative and
quantitative methods

\section*{1 Introduction}

{\label{introduction}}

As the public pays more and more attention to the safety of drugs, the
study of impurities has become the key to drug quality control.
International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH), U.S. Food and Drug
Administration (FDA) and other institutions have issued various
guidelines for impurities. Among them, the ICH was the first to
formulate guidelines on impurities. In 1995 and 1996, guidelines for
``Impurities in New Drug Substances'' and ``Impurities in New Drug
Products'' were promulgated and revised in subsequent years by the ICH.
In the ICH Q3A(R2) guideline, impurities are divided into organic
impurities, elemental impurities and residual solvents according to
their physical and chemical properties, and the limits of organic
impurities are specified based on the permitted daily exposure. If the
impurity content is greater than the reporting limit, the impurity needs
be reported in the test report. If the impurity content is greater than
the identification limit, the chemical structure of the impurity must be
confirmed. If the impurity content exceeds the quality control limit,
it's necessary to clarify the safety of the impurity, or reduce the
impurity content below the quality control limit through process
optimization and other ways. \textsuperscript{1} Q3C
\textsuperscript{2}and Q3D \textsuperscript{3} also give detailed
guidelines for residual solvents and elemental impurities, and they
provide guidance on the content and limits of impurity studies for drug
declaration. The U.S. FDA also promulgated ``Guidance for Industry Drug
Product Chemistry, Manufacturing, and Controls Information'' in 1998,
\textsuperscript{4}and provided clear technical requirements for the
study of impurities.

Impurities are any substances that affect the purity of drugs.
Impurities that are related to the quality of drugs not only affect the
purity of medicines and reduce the efficacy, but also affect the safety
of human medication. For example, \selectlanguage{greek}β-\selectlanguage{english}lactam antibiotics have the
characteristics of strong bactericidal activity and wide indications,
but the polymer impurities of \selectlanguage{greek}β-\selectlanguage{english}lactam antibiotics can quickly cause
hypersensitivity reactions, especially lactam antibiotics for
injection.\textsuperscript{5} Chiral impurities that cause adverse
reactions are also frequently reported. Li et al. \textsuperscript{6}
study the toxicity of the chiral compound muscone that is an active
ingredient in natural musk. The chiral compound muscone can be divided
into s-muscone and r-muscone. The research results show both of them are
acutely toxic to zebrafish embryos, but s-muscone has higher toxicity at
the same concentration. It's necessary to synthesize the chiral isomer
of muscone to ensure the safety of medication. Yi et al.
\textsuperscript{7}reported the genotoxicity of impurities in the
rabeprazole that was used to treat gastrointestinal diseases. The silico
analysis that used the Derek and Sarah software showed that the impurity
2-{[}{[}4-(3-methoxy propane)-3-methyl -2-pyridyl{]} methiony
l{]}-1H-benzimidazole has a warning structure, and it is proposed that
this impurity should be controlled within the limit of 0.01\%. It can be
seen that genotoxic impurities are extremely important to the safety of
drugs, and some regulations on genotoxic impurities have also been
promulgated. The M7 guidelines for genotoxic impurities promulgated by
ICH in 2013,\textsuperscript{8} ``guidline on the limits of genotoxic
impurities'' promulgated by the European Medicines Agency (EMA) in
2007\textsuperscript{9} and the FDA promulgated ``Guidance for Industry,
Genotoxic and Carcinogenic Impurities in Drug Substances and Products:
Recommend Approaches''in 2008 to put forward clear requirements for
genotoxic impurities. \textsuperscript{10}

Nowadays, the research concept of impurity has shifted from the concept
of purity and limit to the direction of impurity profile. The concept of
impurity profile is to understand the source, content, structure,
physical and chemical properties, biological activity of each impurity
in the drug, and to give a reasonable quality control limit after
integrating all information. Impurity source analysis is the basis of
drug impurity control. \textsuperscript{11, 12} To clarify the source of
impurities has important guiding significance for optimizing the
synthesis process, prescription process, packaging and storage
conditions of drugs. Analysis method is a instrument to obtain impurity
information. Choosing a suitable analysis method can ensure the
effective detection of impurities. Diversified analysis methods also
make it possible to effectively control different types of impurities.
The following introduces the sources of impurities in various drugs, and
focuses on the analysis techniques used in the impurity research
process.

\section*{2 Sources of impurities in various
drugs}

{\label{sources-of-impurities-in-various-drugs}}

The ICH guidelines clearly stipulate that the source of impurities in
drugs that exceed the identification limit must be clarified. Impurity
source analysis is the basis for drug impurity control. Identifying the
source of impurities can optimize the synthesis process, prescription
process, packaging and storage conditions of the drug. Analyzing the
actual impurity generation pathway to optimize drug production, storage,
and transpoFrtation by inferring the potential impurities that may be
generated, so as to achieve the purpose of impurity control. Fig. 1
shows the main sources of impurities.

\subsection*{2.1 Sources of impurities in synthetic
drugs}

{\label{sources-of-impurities-in-synthetic-drugs}}

The impurities of synthetic drugs can be divided into process impurities
and degradation products according to their sources. In the production
process of synthetic drugs, some starting materials, intermediates and
by-products can all be regarded as impurities. \textsuperscript{13,
14}Unreasonable prescription process design, reaction between excipients
and Active Pharmaceutical Ingredients (API) also produce some
impurities. The type and amount of such impurities are often determined
by the optimization level of process parameters. Angelo et
al.\textsuperscript{15} studied the effects of excipients and pH on
dimer impurities of the penicillin. Fig. 2 shows the degradation
pathways of penicillin. After penicillin 1 is degraded to penicillic
acid 2, the pH value of the solution is increased and the degradation of
other products is further accelerated. Experiments show that the
addition of sodium citrate that was used to change the pH of the
solution can inhibit the formation of dimer impurities, but the addition
of EDTA have not much effect on the dimer content. Vossen et al.
\textsuperscript{16}optimized the formulation process of amlodipine
liquid formulations that treatment the hypertension in children and
adolescents through stability tests to avoid the formation of
precipitation during storage.

The oxidation, hydrolysis, polymerization, isomerization and other
reactions of API can also introduce new impurities during the storage
and transportation of medicines because of various environmental
changes, such as: temperature, humidity and light,
\textsuperscript{17}and unreasonable packaging selection can also cause
the polymer or element impurities of the packaging to penetrate into the
drug.\textsuperscript{18} Such impurities are called degradation
products and usually determined the degradation pathway through
stability tests and forced degradation tests. \textsuperscript{19} The
forced degradation test is that the drug is destroyed by high
temperature, high humidity, strong light irradiation, acid hydrolysis,
alkali hydrolysis and oxidation, \textsuperscript{20} which can obtain a
large amount of impurity information in a short time and provide
guidance for the packaging and storage conditions of drugs to avoid and
reduce the generation of drug degradation products.

Some elemental impurities are introduced when the catalysts or reagents
added intentionally during the synthesis of drugs. Of course, it is not
ruled out that the introduction of elemental impurities is related to
contact packaging and metal devices that are not resistant to acid and
alkali. \textsuperscript{21} The toxicity of elemental impurities cannot
be ignored. For example, acute and chronic exposure to cadmium can cause
damage to the reproductive system, kidney, liver, bone, lung,
cardiovascular and other tissues in the body, and can suppress immunity
and cause teratogenesis. \textsuperscript{22} Although copper is an
essential element for the human body, a high concentration can also
cause changes in the body, such as: lipid metabolism, neuronal
activity.\textsuperscript{23} In addition, the element impurities in the
drugs have no effect on curing diseases, and may also catalyze the
degradation of active pharmaceutical ingredients, so as to cause side
effects and adverse reactions. The ICH Q3D categorizes element
impurities into three categories based on their permitted daily exposure
and their likelihood of occurrence in pharmaceuticals and puts forward
control requirements for them. \textsuperscript{3} Similar regulations
were also issued by the current United States Pharmacopoeia (USP) in
general chapter \textless{}231\textgreater{},
\textless{}232\textgreater{} and \textless{}233\textgreater{} and
European Pharmacopoeia (EP).\textsuperscript{24}

\subsection*{2.2 Sources of impurities in natural
drugs}

{\label{sources-of-impurities-in-natural-drugs}}

The ICH guidelines limit the types of drugs, and only provide a
illustrative decision tree for the supervision of impurities in
synthetic drugs. Some biopharmaceuticals (vaccines, cell metabolites,
plasma, plasma products, etc.) and natural drugs are not applicable.
Only some pharmacopoeias provide standards for the quality control of
impurities. The natural drugs come from a wide range of sources, and the
quality of natural drugs from different places is also quite different.
It is likely to be mixed with some improper origins and incorrect
medicinal parts to introduce some impurities. Li et
al.\textsuperscript{25} used Fourier transform near-infrared
spectroscopy combined with chemometrics analysis to identify the
authenticity of Rhodiola from four different base sources and provide a
valuable reference for the safety and effectiveness of clinical
application of Rhodiola. In addition, some single-component preparations
that extracted and separated from natural drugs will introduce some
impurities that similar chemical structures and properties due to
insufficient production technology. \textsuperscript{26} The natural
drugs can absorb or accumulate heavy metal elements from the natural
environment during the growth process, and pesticide is used. The
impurity inspections of natural drugs preparations also focus on harmful
elements,\textsuperscript{27} pesticide residues \textsuperscript{28}
and mycotoxins. \textsuperscript{29} Nan et al.
\textsuperscript{27}discussed the content and proportion of heavy metals
Pd, Cu, As, Cd, Hg in different tissues of peony medicinal materials and
in different months, and the results showed that the content of
different species of heavy metal can change during the growing period of
plants and the total content of heavy metals can migrate from different
tissues. It is pointed out that it is more scientific and reasonable to
analyses the species of heavy metals during growing period of plant
medicine.

\subsection*{2.3 Sources of impurities in
biopharmaceuticals}

{\label{sources-of-impurities-in-biopharmaceuticals}}

Biopharmaceuticals are different from general chemicals. The active
ingredients of biopharmaceuticals are generally proteins, peptides,
nucleic acids, enzymes, hormones, etc. They are more sensitive to
factors such as humidity, temperature, pH, and light. It is prone to
degradation changes such as oxidation, aggregation or
fission.\textsuperscript{30} Biopharmaceuticals will face a complex
multi-phase system that contains microbial cells, metabolites, unused
culture medium, etc in the production process, so the concentration of
the target product is very low and the impurity content is high. It is
more difficult to separate the active ingredients and impurities without
destroying the activity of the target product. \textsuperscript{31} If a
drug with higher purity is required, the separation steps required will
increase, and the yield of the drug will decrease accordingly. Some new
extraction and separation methods have been proposed to solve this
challenge. Ga\selectlanguage{ngerman}ëlle et al. \textsuperscript{32} studied the hydrophilic
nanohydrogel particles that are used for the extraction and purification
of recombinant proteins. Zhang et al. \textsuperscript{33} report a
bifurcated continuous field-flow fractionation chip for high-yield and
high-throughput nucleic acid extraction and purification and increase
the nucleic acid extraction rate compared with commercial equipment. The
risk of elemental impurities being introduced in biopharmaceuticals is
very low. \textsuperscript{3} Because the production of
biopharmaceuticals does not require metal ions as catalysts or reagents,
the elements impurities that added to the culture medium are also trace
amounts and will not accumulate, and the purification process of
biopharmaceuticals (extraction, separation, etc.) can also remove the
introduced elements impurities.

\section*{3 Separation methods for
impurities}

{\label{separation-methods-for-impurities}}

According to the impurities source, it is a big challenge to completely
remove the impurities in the medicine. In addition, the analysis method
of impurities not only needs detect the known impurities of the drug,
but also needs effectively detect the potential impurities in the
impurity spectrum concept. Therefore, choosing appropriate analytical
methods that accurately distinguish and quantify impurities is a key
link in the quality control of drugs. Some detectors can detect
impurities without separation. For example, quantitative Nuclear
Magnetic Resonance Spectroscopy (qNMR) \textsuperscript{34} has been
described completely in the qualitative and quantitative methods
section. However, it is not feasible for some detectors to directly
perform qualitative and quantitative analysis of impurities in drugs.
Through separation technology, a single compound can be obtained before
detection to ensure the accuracy and authenticity of the detection
result. Some separation methods are shown in Table 1.

\subsection*{3.1 Liquid Chromatography}

{\label{liquid-chromatography}}

\textbf{3.1.1 Reversed Phase-High Performance Liquid Chromatography}

Nowadays, Reversed Phase-High Performance Liquid Chromatography (RP -
HPLC) separation technology is the main method of impurities separation
in pharmaceuticals. \textsuperscript{35-37} Some new mobile phases and
stationary phases have also been studied and applied for the separation
of various complex samples. For example, ionic liquids can be used as
mobile phase additives to significantly improve the separation effect of
components, suppress tailing of chromatographic peaks and shorten
analysis time by inhibiting the harmful effect of free residuals of
silanol groups in the stationary phase. \textsuperscript{38} High
temperature liquid chromatography technology can increase the resolution
of chromatographic peaks and the analysis speed by increasing the
temperature of the chromatographic column. Edgar et
al.\textsuperscript{39} used high temperature liquid
chromatography-tandem mass spectroscopy to determine nine high-intensity
sweeteners in a variety of drink samples, and consume only 0.85 m L of a
green organic solvent (ethanol). There are also some scholars who
introduce quality-by-design principles when they establish analysis
methods to obtain the best analysis method, so as to save analysis time
and mobile phase consumption. \textsuperscript{40}

\textbf{3.1.2 Hydrophilic Interaction Liquid Chromatography}

Hydrophilic Interaction Liquid Chromatography (HILIC), which has a
complementary effect with reverse chromatography, but is also different
from normal phase chromatography, can retain strongly polar hydrophilic
compounds. \textsuperscript{41} HILIC does not use a more toxic mobile
phase. The characteristic of HILIC is that its stationary phase is
bonded with various polar functional groups. \textsuperscript{42} Mohit
et al. \textsuperscript{43} used HILIC - UV to achieve identification
and quantification of five potential genotoxic impurities, and compared
the separation effect of zorbax silica column, ZIC-HILIC column and
nitrile-HILIC column. The zorbax silica column can give the most ideal
analysis results. Marta et al. \textsuperscript{44} developed an
alternative direct chiral HPLC method to separate the (R)-and (S)-
ramosetron on a chlorinated cellulose-based chiral stationary phase in
HILIC mode. A new method provided to analyze the enantiomer of
ramosetron.

\textbf{3.1.3 Mixed mode column}

In order to satisfy the separation of complex samples, some emerging
chromatographic columns have been developed and utilized. Mixed mode
chromatographic columns combine different fillers and bonded functional
groups on the same chromatographic column to separate compounds with
large differences. At present, there are many applications of mixed mode
chromatography columns in impurity separation.
\textsuperscript{45-47}Size Exclusion Chromatography (SEC) is an
analytical method that realizes separation based on the relative
relationship between pore size and molecular weight.
\textsuperscript{48} It is suitable for the separation of substances
with different molecular weights, especially for biological products.
With the increase of repeating units, high molecular weight compounds
also have great differences in their physical and chemical properties,
such as solubility, ionic bonding capacity. When reverse chromatography
is used to separate compounds with large molecular weights, these
differences may cause precipitation and adsorption, which may cause the
chromatographic peak to broaden and fall below the detection line, or it
may not be eluted. \textsuperscript{49}Lin et al. \textsuperscript{50}
proposed a size-exclusion chromatography on a reversed-phase column that
separated trace high molecular weight species with a variety of
functional groups from high concentration of small molecule substance
and solve a majority of high-MW impurity cases in active pharmaceutical
ingredient. reversed-phase chromatography-size-exclusion chromatography
is the stationary phase of reverse chromatography in the mode of size
exclusion chromatography.\textsuperscript{51} Because of its high
sensitivity and wide solvent compatibility, it can potentially become a
method to detect the high molecular weight impurity.

There are many liquid chromatography method for separating impurities in
drugs. Haddad's team studied the impact of Ion Chromatography (IC) with
universal detectors \textsuperscript{52} and organic
solvent\textsuperscript{53} on the analysis of pharmaceutical
impurities. In addition, the application of two-Dimensional Liquid
Chromatography\textsuperscript{54} (2D-LC) and Ultrahigh Performance
Liquid Chromatography \textsuperscript{55} (UPLC) facilitates the
separation of complex samples and improves the efficiency of impurity
analysis. Of course, these impurities separation methods can only be
combined with suitable detectors to achieve greater benefits.

\subsection*{3.2 Thin layer
chromatography}

{\label{thin-layer-chromatography}}

Thin-layer chromatography (TLC) also plays a key role in the separation
and determination of impurities because of economic
benefits.\textsuperscript{56-58} After the sample is separated, the TLC
image can be obtained by a flatbed scanner, and then processed by image
analysis software to calculate the area or gray value of the
characteristic spots on the TLC thin-layer plate for quantitative
analysis. Bagcinele et al.\textsuperscript{59} developed a manual Thin
Layer Chromatography (TLC) to separate a mixture of all lipopeptide
families with other peptide and lipid impurities and validated for the
semi quantitative determination of the surfactin lipopeptide family.
Precision would be comparable to that of estimation via HPTLC, and the
analytical method reduces the measurement cost, time and consumption of
organic reagents. Eglal et al.\textsuperscript{60} is the first use of
TLC method for the determination of aspirin, omeprazole and the impurity
of aspirin (salicylic acid). TLC method is eco-friendly and greener when
compared to the already reported method.

\subsection*{3.3 Other separation
methods}

{\label{other-separation-methods}}

There are many methods for separating impurities in drugs. In the
separation of chiral impurities, Supercritical Fluid Chromatography
(SFC) is also a good choice. Kalíková et al. \textsuperscript{61} used
SFC to analyze 21 chiral compounds with different physical and chemical
properties on a cellulose 3s-(3,5-dimethylphenylcarbamate)-based chiral
stationary phase. It has the best separation effect on \selectlanguage{greek}β - \selectlanguage{english}receptor
blockers, and achieve better separation in a shorter time than HPLC.
Henrik et al. \textsuperscript{62} used Capillary Electrophoresis (CE)
method to determine dapoxetine hydrochloride and its enantiomeric
impurities and the result was comparable to the data of an
enantioselective HPLC method. Enea et al. \textsuperscript{63} also gave
a detailed review of structurally related compounds in Gas
Chromatography (GC) separation of medicines.

\section*{4 Qualitative and quantitative methods of
impurities}

{\label{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. \textsuperscript{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. \textsuperscript{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),
\textsuperscript{66}Charged Aerosol Detector (CAD), \textsuperscript{67}
Refractive Index (RI), \textsuperscript{68} Chemical Lumines-cent
Nitrogen-specific Detector (CLND). \textsuperscript{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.

\subsection*{4.1 Mass Spectroscopy}

{\label{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.\textsuperscript{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. \textsuperscript{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. \textsuperscript{72} Mass spectroscopy fragment information
can be clarified through many databases, \textsuperscript{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, \textsuperscript{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. \textsuperscript{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. \textsuperscript{76} It
is very conducive to the simultaneous quantitative determination of low
concentration and multiple structurally similar impurities in complex
systems. \textsuperscript{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. \textsuperscript{78} In
the case of low response impurity, deriving and adding alkali metals
solve this problem. Wijk et al. \textsuperscript{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.
\textsuperscript{80}Zheng et al. \textsuperscript{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), \textsuperscript{82} Inductively Coupled
Plasma-Atomic Emission Spectroscopy (ICP - AES),
\textsuperscript{83}X-Ray Fluorescence Spectroscopy (XRFS),
\textsuperscript{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.

\subsection*{4.2 Nuclear Magnetic Resonance
spectroscopy}

{\label{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. \textsuperscript{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.\textsuperscript{88} There are also researchers who obtain a
large amount of impurities through synthesis methods,
\textsuperscript{89-91}but 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. \textsuperscript{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. \textsuperscript{93} In addition,
ultra-high field magnets (\textgreater{} 800 MHz) can be used in LC-NMR
to improve the detection sensitivity, but it has been little progress
over the past decade. \textsuperscript{94} Takashi et
al.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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.\textsuperscript{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. \textsuperscript{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.\textsuperscript{100}

\section*{5 Conclusions}

{\label{conclusions}}

At present, there are relatively many quantitative studies on
impurities, but there are still some challenges for the structure
analysis of impurities, especially trace impurities. The ICH guidelines
clearly point out that it is necessary to analyze the structure of
impurities exceeding a certain content in the quality control of drugs.
Although the sensitivity of MS can reach the microgram level, there are
still some problems in the identification of unknown impurity
structures. Because MS only deduces the approximate chemical structure
based on the fragmentation law. The structural characterization of
impurities also needs to rely on more spectral information, such as NMR,
infrared spectroscopy. However, the sensitivity of NMR spectroscopy is
poor, and the structural characterization of trace impurities is still a
huge challenge. Due to the small amount of impurities in the sample, it
is not easy to obtain a large amount of impurities by preparative HPLC.
The impurity can be enriched by forced degradation and directed
synthesis, so that the impurities in the sample can reach the detection
line and purity, but this method does not solve all the impurity
problems. Improving the sensitivity of NMR and the system of LC - NMR
may be solutions to the detection of trace impurities, and it is also
the direction that many researchers are working on.

In many cases, the quantification of unknown impurities lacks standard
products, so that absolute quantification of impurities cannot be
achieved. Some researchers have also established a method of impurity
reference substance database \textsuperscript{101} to solve this
problem. The database provides qualitative parameters of impurity
(chromatographic retention time, UV/IR, MS and NMR) and quantitative
parameters of impurity (principal component response factors). It is
also a good choice to solve the accurate quantification of impurities.

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{Acknowledgements}

We are extremely grateful to the financial support of Tianjin Science
and technology project (No. 20ZYJDJC00090).



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\end{enumerate}

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