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. 45-47Size Exclusion Chromatography (SEC) is an analytical method that
realizes separation based on the relative relationship between pore size
and molecular weight. 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. 49Lin et al. 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.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 52 and organic solvent53 on the analysis of pharmaceutical impurities. In
addition, the application of two-Dimensional Liquid Chromatography54 (2D-LC) and Ultrahigh Performance Liquid
Chromatography 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.
3.2 Thin layer
chromatography
Thin-layer chromatography (TLC) also plays a key role in the separation
and determination of impurities because of economic benefits.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.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.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.
3.3 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. 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 β - receptor
blockers, and achieve better separation in a shorter time than HPLC.
Henrik et al. 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. 63 also gave
a detailed review of structurally related compounds in Gas
Chromatography (GC) separation of medicines.