**Table 3.** *Multi-dimensional LC analysis in pharmaceutical products (trace and chiral analysis).*

*Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

their pharmacokinetic profiles. The method consists of a gradient RPLC method on a C18 column coupled with a stereoselective, isocratic, RPLC method on polysaccharide chiral column, and the result explained in **Figure 1** [36].

Another instructive example is the combination of achiral and chiral separations in a single mLC–LC separation of warfarin and hydroxywarfarin isomers, where the first dimension resolves the majority of the analytes and the second dimension completes the job. The first dimension has a chiral stationary phase, whereas the second dimension has an achiral separation. This indicates that neither achiral nor chiral separation is sufficient to resolve the mixture. When the same separation modes are used in a multiple heart-cutting 2D-LC separation, the achiral reversed-phase separation used in the second dimension is highly complementary to the chiral separation used in the first dimension. These provide enough selectivity to quickly resolve those components of the sample that remains unresolved after passing through the 1D column [29, 34].

Another popular pharmaceutical application is comprehensive LC in stress-testing studies for different pharmaceutical products. The API's and intrinsic stability of other drug products should be assessed, and the degradation mechanisms should be disclosed. Stress testing, also called forced degradation study, helps to establish the intrinsic stability of the API. Stress testing includes a number of experiments, such as the effect of temperature, humidity, oxidation, photolysis, or hydrolysis at different pH values, as outlined by the World Health Organization [33, 35, 36, 52].

The 1D-LC analysis of a strongly stressed (temperature, UV irradiation, and organic solvent) omeprazole tablet demonstrates this notion. One has the impression that in this study, the omeprazole peak is corresponding to only one product, the API, but recording a mass spectrum revealed that many solutes are co-eluting in the peak. Increasing resolution by utilizing longer columns or smaller particles is not a viable option because there is not enough peak capacity to resolve the solutes in the primary peak (zero resolution). Although choosing another stationary phase is an option, the risk of other peaks now overlapping is realistic. By far, the best solution is to combine different selectivities in an LC × LC combination even if the two mechanisms are comparable, for example, RPLC × RPLC but using different stationary and mobile phases [13, 35, 36].

#### **Figure 1.**

*Plasma concentration-time profile of (A) paracetamol (B) S and R-ketorolac, and (C) simultaneous analysis of both drugs after IV administration based on achiral-chiral 2D-LC.*

*Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

Different mobile phase compositions with different selectivities were used in the first- and second-dimension columns. The study revealed more than 50 spots, with the omeprazole peak resolved in the second dimension into four distinct products, and its mass spectrum is also recorded. The conditions developed in this 2D-LC approach can be considered more or less generic for detection of impurities in APIs, at least when eluting under reversed phase LC conditions. In this robust and repeatable method, good finds were also achieved for analyses of metoclopramide, acetaminophen, diclofenac, ibuprofen, and lidocaine [32–34, 53–56].

#### **4.2 Traditional medicines (TMs)**

Regardless of the fact multi-dimensional chromatography is applicable to many traditional medicines, its application in traditional Chinese medicine is quite well researched. Traditional Chinese medicines are garnering attention in modern pharmaceutical institutes as a valuable resource for medication development. TCMs are extremely complex mixes having hundreds, if not thousands of components of various structures and concentrations, with only a few compounds responsible for specific pharmacological activity and/or toxicity. As a result, the use of latest analytical techniques is critical for the elucidation of the composition and quality control of TCMs [42, 43, 57, 58].

For the analysis of TCMs, a number of two-dimensional LCs have been designed with different column. Mostly, commercial columns with various separation techniques, such as strong cation-exchange chromatography or reverse phase liquid chromatography, have been coupled. Such coupled separation mode may reveal much more information on components due to a specific interaction with these biomacromolecules of traditional medicines. In most of the cases, the two columns have been combined off-line, which makes the process simpler. However, a few are truly comprehensive (LC × LC). *Ligusticum chuanxiong, Angelica sinensis, Swertia franchetiana,* and *Lonicera caprifolium* are some of the Chinese traditional medicines that are separated and identified by multi-dimensional liquid chromatography. Analysis of a traditional Chinese medicine using a two-dimensional cyano octadecyl silyl system with an eight port valves with two sample loops and UV APCI MS detection also gave over 52 components [40–42, 57, 58].

#### **4.3 Lipidomics**

The wide and complex lipid composition in biological samples requires MDLC methodologies to sufficiently separate the lipids prior to MS or other detector characterization. This lipid separation usually demonstrated with normal phase, reversed phase, and silver ion chromatography. The application of MDLC in complex field of lipidomics is becoming of increasing significance. One typical example of these lipidomics application is analysis of egg yolk by 2D high-performance liquid chromatography-mass spectrometry for phosphatidylcholine. Phosphatidylcholine is the main phospholipid present in egg yolk. For characterization of the fatty acids composition in phosphatidylcholines molecules in egg yolk, an off-line LC × LC-ESI-MS/MS (preparative C18 column) method using a triple quadrupole mass spectrometry was used. The study identified phosphatidylcholines, which contains unsaturated fatty acids from both omega-3 and 6 groups. Phosphatidyl ethanolamine, lysophosphatidylglycerol, sphingomyelin, lysophosphatidylethanolamine, lysophosphatidylcholine, and glycerophosphorylcholine were also among the 13 phospholipid fractions evaluated by this method [53, 54, 59–62].

#### **4.4 Proteomics**

MDLC has been used very successfully in proteomics for about two decades. These commonly comprehensive type separations, variant unique to peptide analysis, are known as multi-dimensional protein identification technology (MudPIT). Mass spectrometry has been widely used with as the most common detector. MDLC coupled with mass spectrometry is becoming increasingly important in proteome research owing to its high speed, high resolution, and high sensitivity. Recent proteomics technologies offer excellent separation and enormous data-gathering capabilities in the discovery of peptides and proteins, particularly disease-specific biomarkers in serum, plasma, urine, tissue, and other biological samples. The performance of multidimensional chromatography separation techniques compatible with MS, which are commonly used in proteomics applications, is summarized in the **Table 4** [45, 46, 53].

The first applications of multi-dimensional liquid chromatography for environmental research were performed at the end of the twentieth century. This application was known as column switching mode, which is widely employed for the separation and analysis of pesticides. Pol and coworkers published the first studies on the use of a comprehensive two-dimensional liquid chromatography system, termed LC–LC, for the analysis of environmental samples in 2006. This analysis showed acidic compounds present in atmospheric aerosols were separated using a multi-dimensional chromatographic system for the first time, proving the capabilities of LC–LC systems to separate intricate mixtures that would be too difficult for 1D-LC [67].

One of the environmental contaminants is endocrine-disrupting compounds (EDCs) containing chemicals and hormones having endocrine-disrupting activity. This compound becomes critical emerging contaminants due to their presence in environmental waters and worries about probable detrimental adverse effects to wild life and humans. A study done in Czech Republic was used multi heart-cutting two-dimensional liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry method for the determination of endocrine-disrupting compounds in water. Twenty real samples collected from the Loucka and the Svratka rivers were analyzed, and compounds were found in all Svratka samples (9.7–11.2 ng l−1 for -estradiol, 7.6–9.3 ng l−1 for estrone, and 24.6–38.7 ng l−1 for bisphenol A) [64, 65, 68].


*SAX: Strong Anion Exchange, SCX: Strong Cation Exchange HILIC: Hydrophilic Interaction. Liquid Chromatography, HEK 293 T cells: Human embryonic kidney 293 cells.*

#### **Table 4.**

*Performance of various off-line 3D-LC systems in protein analysis.*

*Perspective Chapter: Multi-Dimensional Liquid Chromatography - Principles and Applications DOI: http://dx.doi.org/10.5772/intechopen.104767*

Another novel multi-dimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative screening analysis and characterization of wastewater sample. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-μm stationary phase provide a fast second-dimensional separation and a sufficient sampling frequency without a first-dimensional flow stop. To seek data for a suspected target screening of a wastewater sample, 99 substances were added in the reference mix [62, 66].

Another novel method was developed utilizing LC × LC-ESI-TOF coupled MS for the determination of organic acids in atmospheric aerosols. They analyzed methanolic extracts of filters containing atmospheric aerosols and expected to find mainly polar organic compounds such as aliphatic, aromatic, and substituted carboxylic acids. Because of the combined knowledge of the elution pattern and the sensitive and accurate mass spectrum data, this innovative perspective liquid chromatography (LC-LC-TOF coupled MS) is proven to be an appropriate method for screening undiscovered acidic compounds [67, 69, 70].

#### **4.5 Recent advances and future perspectives**

Over the last decades, multi-dimensional liquid chromatography techniques have been extensively exploited with the latest significant improvements in terms of instrumental setup and availability of novel stationary phases. Nowadays, robust and full-featured instrumentations are available from most LC manufactures. Miniaturization and downscaling, **s**witching valves as well as suitable software offer the possibility to adapt two-dimensional procedures and instrumentations will certainly continue in the future, and consequently, a significant rise of 2D LC systems is expected in several research fields [71].

Another new development in this area is Nano 2D-HPLC. 2D online nano-LC/ MS was developed that substituted the inserted salt step gradient with an optimized semi-continuous pumped salt gradient, and also, 8-Isoprostaglandin F2α was measured from human urine. A microchip-based nano-HPLC was also used. So it will be the most applicable technique especially in the chiral resolution in the future [72–75].

Currently, a multi-dimensional liquid chromatography also gives different opportunity to those challenging areas for the analysis. Recently, major developments are seen and attracted significant interest toward complex in the analysis of small, more complex molecules and biological products. For example, biopharmaceuticals such as monoclonal antibody (mAbs), interferons/cytokines, and vaccines are recently analyzed by the advanced instrumental technology of multi-dimensional liquid chromatography [76].

#### **5. Conclusion**

Multi-dimensional liquid chromatography where a complex mixture is separated by facing different dimension is useful in several fields, especially in-depth analysis of proteome and lipidomics, environmental chemicals, food and pharmaceuticals industries. It boosts separation power, peak resolution, and reproducibility while increasing system complexity. The primary reason for multi-dimensional separations is that they

provide a more effective and efficient method of generating high-peak capacities, and hence allowing for more comprehensive resolution of complicated mixtures. Separation and characterization of complicated mixtures are critical in a wide variety of fields that demand considerable separation power. Multi-dimensional separations envisaged that better understanding and application of multi-dimensional separations would open up opportunities for meaningful analyses of extremely complex samples and allowing the regular analysis of thousands of constituents from a single sample in a single run.

## **Author details**

Esayas Tesfaye1,2, Tadele Eticha2 , Ariaya Hymete2 and Ayenew Ashenef2,3\*

1 Department of Pharmacy, College of Health and Medical Sciences, Dilla University, Dilla, Ethiopia

2 Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

3 Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa), College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia

\*Address all correspondence to: ayenew.ashenef@aau.edu.et

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Multi-Dimensional Liquid Chromatography: Principles and Applications DOI: http://dx.doi.org/10.5772/0*

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*Perspective Chapter: Multi-Dimensional Liquid Chromatography: Principles and Applications DOI: http://dx.doi.org/10.5772/0*

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#### **Chapter 4**
