Multidimensional liquid chromatography-mass spectrometry for metabolomic and lipidomic analyses

Accepted Manuscript Multidimensional liquid chromatography-mass spectrometry for metabolomic and lipidomic analyses Wangjie Lv, Xianzhe Shi, Shuangyuan Wang, Guowang Xu PII:

S0165-9936(18)30199-7

DOI:

https://doi.org/10.1016/j.trac.2018.11.001

Reference:

TRAC 15302

To appear in:

Trends in Analytical Chemistry

Received Date: 10 May 2018 Revised Date:

31 October 2018

Accepted Date: 1 November 2018

Please cite this article as: W. Lv, X. Shi, S. Wang, G. Xu, Multidimensional liquid chromatographymass spectrometry for metabolomic and lipidomic analyses, Trends in Analytical Chemistry, https:// doi.org/10.1016/j.trac.2018.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Multidimensional

liquid

chromatography-mass

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spectrometry for metabolomic and lipidomic analyses

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Wangjie Lv a, b, #, Xianzhe Shi a, #, Shuangyuan Wang a, Guowang Xu a, *

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a

CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China

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University of Chinese Academy of Sciences, Beijing 100049, China

#

These authors equally contributed to this work

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* Address correspondence to:

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Prof. Dr. Guowang Xu, CAS Key Laboratory of Separation Science for Analytical

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Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,

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Dalian 116023, China. Tel. / Fax: 0086-411-84379530. E-mail: [email protected].

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Abstract As important components of omics, metabolomics and lipidomics are dedicated

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to detecting as many metabolites and lipids as possible, respectively, in biological

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samples. Because the physicochemical properties of metabolites and lipids greatly

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differ, the comprehensive analysis of metabolomics and lipidomics using one

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dimensional liquid chromatography-mass spectrometry is extremely difficult.

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Benefited

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multidimensional liquid chromatography (MDLC) has been considered a powerful

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approach for the analysis of complex samples. In this review, the construction modes

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and modulation modes of MDLC and its applications mainly in last five years in the

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field of metabolomics and lipidomics analysis are summarized in details. In addition

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to common interfaces (i.e., heart-cutting, stop-flow and comprehensive mode with a

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storage loop and trap column), the novel construction modes (i.e., selective

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comprehensive and pulsed-elution 2DLC, stop-flow 3DLC) and modulation modes

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(e.g., vacuum evaporation interface and evaporation membrane modulation etc.) are

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also introduced.

combination

of

different

separation

mechanisms,

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Keywords: multidimensional liquid chromatography, metabolomics, lipidomics,

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metabolic profiling, stop-flow

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Contents

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1.

Introduction………………………………………………………………….

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State of the art of MDLC…………………………………………………....

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2.1 Construction modes of MDLC……………………………………………...

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2.2 Modulation modes of 2DLC………………………………………………..

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3.

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3.1 Heart-cutting 2DLC………………………………………………………...

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3.2 Stop-flow 2DLC……………………………………………………………

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3.3 Comprehensive 2DLC and selective comprehensive 2DLC……………….

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3.4 3DLC………………………………………………………………………

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4.

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4.1 Heart-cutting 2DLC………………………………………………………..

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4.2 Stop-flow 2DLC…………………………………………………………...

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4.3 Comprehensive 2DLC……………………………………………………..

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Conclusions and perspective………………………………………………

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Acknowledgements…………………………………………………………....

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References……………………………………………………………………..

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MDLC applications in metabolomics……………………………………..

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MDLC applications in lipidomics……………………………………………18

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1. Introduction Metabolomics is committed to the qualitative and quantitative analysis of all

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small molecular metabolites (<1500 Da) in specific biological samples. Lipidomics,

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as a part of metabolomics, aims to systematically study all lipids in a biological

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system, ranging from their structures and functions to their interactions [1]. The latest

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updated Human Metabolome Database (HMDB) has already recorded 114,100

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compounds, including 23,746 metabolites and 90,354 lipids [2]. These metabolites

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exhibit a wide range of diversity in their structures. As a result, their physicochemical

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properties (e.g., polarity, charged state, and acidity or basicity) and tandem mass

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(MS/MS) spectra are different from each other. Lipids are primarily the esters of

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long-chain fatty acids with alcohols and their derivatives. They have many important

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functions in energy deposition, the formation of cellular membranes, and cellular

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signalling pathways. Based on the work of Fahy, lipids can be classified into eight

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categories:

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glycerophospholipids (GP), sphingolipids (SP), saccharolipids (SL), sterol lipids (ST),

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prenol lipids (PR), respectively [3, 4]. Each category also consists of numerous

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classes and subclasses. Unlike those of metabolites, the MS/MS spectra of lipids are

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predictive and in-silico libraries can be used. According to the records of LipidBlast

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software, 119,200 lipids from 26 lipid classes and a total of 212,516 MS/MS spectra

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are currently available [5].

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polyketides

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Liquid chromatography coupled with mass spectrometry (LC-MS) has hitherto

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become one of the most extensively used platforms for the global component profiling

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due to its good resolving power, high sensitivity and excellent repeatability [6, 7]. The

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three most commonly used liquid chromatography techniques are reversed-phase

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liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and 4

ACCEPTED MANUSCRIPT normal-phase liquid chromatography (NPLC). RPLC achieves separation based on

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differences in hydrophobicity, so all compounds except for highly polar metabolites

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can be well separated using the RPLC mode. Generally, metabolomics methods use

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acetonitrile or methanol and water as mobile phases, while lipidomics methods use

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iso-propanol, acetonitrile and water as mobile phases. In addition, the hydrophobicity

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of a lipid is closely related to the numbers of carbon and double bonds contained in its

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fatty acid chains, which in turn makes its retention time present a certain degree of

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regularity [8, 9]. As complementary tools to RPLC, HILIC and NPLC yield

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satisfactory separation towards (highly) polar metabolites (e.g., amino acids, organic

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acids, polyamines and sugars) [10-12]. Lipids can also be separated by HILIC and

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NPLC based on the polarities of their polar head groups, thus allowing separation at

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the level of lipid classes to be attained [13-19].

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When utilizing one-dimensional LC (1DLC) such as RPLC, HILIC or NPLC to

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separate these complex metabolites and lipids, we can only make use of one property

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of these substances. Therefore, each LC mode has its own application scope and

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limitations. For example, water-soluble and highly polar metabolites, which co-elute

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close to the dead time of RPLC, can be finely isolated by HILIC and NPLC and vice

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versa for non-polar compounds. The serious co-elution phenomenon can cause some

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negative effects, such as ion suppression and the obscured detection of low abundance

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compounds, which are harmful for the following qualitative and quantitative

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processes. To resolve these issues and improve their coverage, multiple injection

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analyses have been performed by employing different complementary LC modes [20].

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However, this strategy not only wastes time and energy but also produces many

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redundant data. Moreover, many isomers with similar structures are unable to be

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resolved by 1DLC.

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Multidimensional

liquid

chromatography

(MDLC)

techniques,

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two-dimensional liquid chromatography (2DLC), can inherently integrate different

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separation mechanisms, including RPLC, NPLC and HILIC. Therefore, MDLC is

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considered to be a powerful technique for complex sample analysis due to its high

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resolution and large peak capacity [21, 22]. Over the past decade, many new

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construction modes and modulation modes have been developed and employed to

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build MDLC, which have enhanced its flexibility and universality [22, 23]. Technical

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advances in MDLC have greatly extended its application fields from foods to

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traditional Chinese medicine, polymers, environmental and life sciences [24-28]. Due

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to its strong separation power, MDLC coupled with mass spectrometry (MDLC-MS)

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has exhibited obvious superiority in the enhanced resolution and coverage of

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metabolomes and lipidomes compared with 1DLC [29, 30]. Comprehensive

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metabolomics and lipidomics analyses can promote not only a deeper understanding

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of the biochemical mechanisms of metabolism-associated diseases but also contribute

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to the discovery of biomarkers for disease diagnosis, prognostic monitoring, and

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therapeutic interventions.

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Several recent reviews mainly focusing on the general fundamentals [22, 31],

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hardware design [23, 32] and applications of this technique in food analysis [24] and

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pharmaceutical analysis [27] were published. However, until now, few reviews have

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described the use of MDLC in lipidomics and metabolomics. Because of the

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complexity and properties of omics samples, MDLC usually employs an electrospray

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ionization-mass spectrometer as the detector (e.g., triple-quadrupole MS for targeted

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analysis and quadrupole-time-of-flight (Q-TOF) for non-targeted analysis). There

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have already been many reviews of MS applications in metabolomics [33, 34] ; thus,

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this review will not discuss the progress that has been made in mass spectrometry due

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to space limitations. The main aim of this review is to summarize the present state of

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the art of MDLC and the main applications of MDLC-MS in the field of lipidomics

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and metabolomics analyses, in the last five years.

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2. State of the art of MDLC

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2.1 Construction modes of MDLC

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Here, the construction modes refer to the physical ways in which MDLC

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separation is performed. The most widely used construction modes of MDLC include

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heart-cutting 2DLC and comprehensive 2DLC. Heart-cutting 2DLC, which is the

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earliest and simplest form, is mainly used to enhance the separation of targeted

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components. Usually, the first dimension (1D) is performed to pre-separate one or

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several targeted components from other interfering matrices, and only targeted

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fractions are transferred to the second dimension (2D) for further separation. The

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superiority of heart-cutting 2DLC is in 2D to enable the fine separation of interesting

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regions from 1D, and both dimensions are generally carried out under conventional

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LC conditions, especially the flow rate, which is very suitable for the detection of

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low-abundance components and the purity analysis of pharmaceutical components

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[35]. For comprehensive 2DLC, all fractions from 1D are transferred into 2D for

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further analysis, which is suitable for the non-targeted and comprehensive analysis of

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complex samples to obtain as much information as possible. Every 1D peak should be

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cut into at least 3 or 4 portions to remain the resolution of 1D in light of the previous

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pioneering work of Murphy, Schure and Foley [36]. As a result, the first dimension

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generally uses a long and microbore column and a lower flow rate (e.g. 10-50

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µL/min); in contrast, the second dimension uses a short and thick column and a higher

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flow rate (e.g. 1-5 mL/min) to achieve rapid separation. Some measures have been

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ACCEPTED MANUSCRIPT taken to improve the orthogonality of the 2DLC system, such as using shift and

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segment gradient elution mode in the second dimension [37, 38] and different solvent

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compositions and modifiers [39]. However, some primary challenges remain with

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respect to comprehensive 2DLC analyses, e.g., undersampling due to inadequate cuts,

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peak deterioration due to solvent incompatibility and decreases in sensitivity due to

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dilution. In addition, the LC conditions of two dimensions are also influenced and

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restricted by each other. Therefore, the practical peak capacity of comprehensive

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2DLC is often far less than the theoretical value. As an ingenious combination of

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comprehensive 2DLC and multi-heart cutting (MHC) 2DLC, selective comprehensive

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2DLC (sLC × LC) was designed; this method can capture and transiently store more

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than six fractions from each 1D peak and then serially transfer these fractions into a

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2

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sacrificed due to the adequate sampling of 1D peaks, while more efficient 2D

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separation is obtained at a longer analysis time. Since the advent of sLC × LC, this

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equipment has become increasingly mature, and commercialized instruments are

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currently available [40-43].

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D column for further analysis [40]. As a result, the resolution of 1D separation is not

On the other hand, the contradictory relationship between the 1D sampling time

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and the 2D analysis time poses a great challenge with the respect to the establishment

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of 2DLC, particularly for the comprehensive mode. Thus, the 1D sampling time

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should be sufficiently short to cut the 1D peak into adequate fractions, while the 2D

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analysis time should be sufficiently long to ensure satisfactory resolution. As a readily

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associated strategy, the stop-flow 2DLC pattern was developed, in which the 1D flow

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was stopped while the 2D analysis was in progress. In this way, both dimensions do

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not affect each other and can be operated under conventional LC conditions. However,

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the total analysis time considerably increases with the increasing stop-flow time. In

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ACCEPTED MANUSCRIPT addition to stopping the 1D flow rate, Nielsen’s group presented a proof of concept

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study to improve the flexibility of two dimensions by using pulsed elution in the first

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dimension. In this 2DLC, many pulses were used to elute the 1D fractions, while the

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first dimension was situated in a no-elution state with weak-strength eluent between

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the pulses, which made the first dimensional analysis almost stagnant [44]. Therefore,

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the pulsed elution mode not only ensures the sufficient sampling of the 1D peak but

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also improves the 2D resolution. Accordingly, the flexibility and peak capacity of this

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2DLC were significantly increased. Unfortunately, some weakly retained analytes

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could be lost during the no-elution time.

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Compared with 2DLC, three-dimensional liquid chromatography (3DLC)

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theoretically possesses a higher resolution and larger peak capacity. However, the

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construction of online 3DLC is quite difficult considering the irreconcilable

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relationship between each dimensional sampling time and analysis time. Recently, an

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online 3DLC was developed by coupling 1D pre-separation with a conventional

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comprehensive 2DLC in stop-flow mode [45]. The flow scheme of the 3DLC system

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is shown in Figure 1. Complex samples were initially divided into several fractions by

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the 1D pre-separation based on their different features. Then, each fraction was

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transferred into the following LC × LC part for further analysis. In theory, the total

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peak capacity of the 3DLC system is the sum of peak capacities of the multiple 2DLC.

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The 3DLC system can thus provide an alternative option for analysing very complex

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samples.

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In practice, heart-cutting, stop-flow, comprehensive and selective comprehensive

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2DLC are the most frequently used construction modes of MDLC; these modes are

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summarized and shown in Figure 2.

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2.2 Modulation modes of 2DLC Modulation is the core process of 2DLC separation, which represents the

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collection of 1D fractions and their effective transfer to a 2D column for further

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separation. A simple modulation consisting of a switching valve and two storage loops

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is one of the most widely used modulators for comprehensive 2DLC (see Figure 2G).

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The volume of these loops depends on the first dimensional flow rate and sampling

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time. Although the operation of this modulation is convenient, the separation in the

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second dimension can be seriously worsened, and peak deterioration will occur when

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the loop volume reaches tens or even hundreds of microliters [46]. Another issue with

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this modulation is the incompatibility of mobile phases (i.e., immiscibility and

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mismatch in elution strength) in the two dimensions [47]. These problems can be

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partly resolved if the loops are replaced by trap columns in which the analytes are

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focused and the solvents from the 1D fractions are removed. Usually, a counter

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make-up flow is added to the 1D effluent for effective trapping [48, 49]. However,

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retention matching may become difficult due to the slight discrepancy between the

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two trap columns and the incomplete re-equilibration of the trap columns. Missing

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some components is apparently inevitable during the capture process. Stoll et al.

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further developed an active solvent modulation (ASM) without additional instrument

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hardware for make-up flow. The ASM technique was executed on a specially-made

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8-port/4-position valve, where two normal sample loops were used to transfer the 1D

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fractions and a bypass connector behind the 2D pump was applied to dilute them prior

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to 2D separation. The fixed solvent modulation (FSM) consisted of a normal

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8-port/2-position valve and two simple tee connectors to connect the 2D pump and 2D

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column. In FSM approach, bypass flow exists throughout the entire 2D analysis. In the

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ASM approach, bypass flow only exists in transfer of 1D fraction to 2D column.

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Compared with FSM, ASM can toggle the diluent flow during each 2D separation

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cycle and thus accelerate the 2D re-equilibration and simplify the elution solvent [50].

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As a result, the peak shape and width of 2D separation were greatly improved and the

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overall sensitivity of the practical 2DLC system was obviously enhanced. Analogously, a vacuum evaporation interface (VEI) [51-53] and airflow-assisted

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adsorption (AAA) interface [54] were designed to solve the serious immiscibility

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existing in the NPLC × RPLC system. The loop in the VEI was heated and connected

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to a vacuum pump to remove the solvents, but some volatile compounds were easily

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lost. Later, the VEI was ameliorated and built based on a 2-position 10-port valve with

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two loops instead of a 2-position 6-port valve with one loop to realize the

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not-stop-flow NPLC × RPLC system [55-57]. The central section of the AAA

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interface was a double enrichment unit and a blowing airflow. This technique included

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three procedures: sweeping eluents from the 1D NPLC into NP enrichment columns

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by nitrogen, transferring analytes from NP enrichment columns to RP enrichment

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columns with the assistance of water make-up flow, and flushing analytes to the 2D

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RP column. Afterwards, a vacuum evaporation assisted adsorption (VEAA) interface

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[58] and a thermal evaporation assisted adsorption (TEAA) interface were further

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developed [59]. The main advantage of the VEAA and TEAA interfaces over the AAA

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interface was that they could rapidly evaporate and remove the NPLC solvent under

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vacuum conditions or heating conditions. Later, Fornells et al. designed an

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evaporation membrane modulation (EMM) based on a membrane evaporation device

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(see Figure 3) [60]. At this interface, the solvent from the 1D effluent was evaporated

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through a porous polytetrafluoroethylene hydrophobic membrane, and the heating

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intensity was automatically adjusted based on the flow rate of the 1D effluent to keep

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the evaporation constant. The peak capacity and sensitivity of 2DLC using the EMM

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were significantly improved compared with those using loop modulation. Thermal modulation is the common modulation interface for comprehensive two

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dimensional gas chromatography. Because low thermal mass resistive heating

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technology was introduced as a modulator, thermal modulation was employed for the

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first time in off-line 2DLC [61]. Here, the temperature-cycled modulator was based

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on the retention of compounds with changing when low temperature for capture to

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high temperature for release. Creese et al. developed a longitudinal on-column

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thermal modulation technique for comprehensive 2DLC. A highly retentive porous

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graphitic carbon (PGC) packed column was used as a modulation column to connect

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the 1D and 2D columns without any switching valves. Through the alternating

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manipulation of a moving heated sleeve and cooled compressed air on the PGC

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column, highly efficient modulation was realized. The sensitivity was increased ten

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times by the longitudinal thermal modulator compared with the valve-based

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modulator [62].

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3. MDLC applications in metabolomics MDLC, which inherently integrates different separation mechanisms, possesses

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two vital merits: improved chromatographic resolution and peak capacity [21, 22].

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Therefore, MDLC has contributed greatly to the detection of a wide range of

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metabolites in the past decade. In this section, the applications of MDLC in metabolic

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profiling analyses are discussed in detail based on specific examples of four different

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construction modes.

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3.1 Heart-cutting 2DLC Heart-cutting 2DLC is an efficient tool for the analysis of a group of targeted 12

ACCEPTED MANUSCRIPT metabolites. Kula et al. utilized heart-cutting 2DLC to determine the secondary

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metabolites in the flowers of L. caerulea [63]. Fifty-one metabolites including

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flavonoids, phenolic acids and iridoids were separated and identified. Yao et al. and

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Gackowski et al. established a similar MHC-2D-RPLC-MS system to quantify five

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panax notoginseng saponins [64] and analyse modified and unmodified nucleosides

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[65], respectively. The latter used a trap column; thus, the detection sensitivity of

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nucleosides was higher. In addition, stable isotope-labelled nucleosides were added

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for reliable identification and quantification. Three modified nucleosides were found

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to have lower contents in colorectal carcinoma tissue than in normal tissue. In

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addition to the frequently used RP column, a molecular imprinted column (MIC),

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which has emerged as a novel sample pretreatment material, can selectively recognize

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and capture specific compounds from complicated matrices. Therefore, online

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heart-cutting 2DLC with MIC as the first dimension can be a powerful technique for

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the detection of metabolites with specific functional groups. For example, estradiol in

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cosmetics [66], clenbuterol residues in biological samples [67] and sulfonylurea

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additive in traditional Chinese medicines [68] have been successfully determined

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using this technique. To achieve a very important application of heart-cutting 2DLC,

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Hamase’s group, on the other hand, focused on the separation of enantiomers by

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2DLC, such as lactate, 3-hydroxybutyrate, citrulline, ornithine, aspartic acid, glutamic

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acid and alanine [69-76]. Usually, a C18 column was used in 1D to isolate targeted

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fractions, and a chiral column was used in 2D to achieve chiral separation. The use of

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enantioselective analysis could ultimately deepen our understanding of pathological

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processes.

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In addition to targeted metabolomics analysis, heart-cutting 2DLC can also be

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used for non-targeted analysis. A typical example was presented in our previous work: 13

ACCEPTED MANUSCRIPT a column-switching HILIC-RPLC system used for the complementary analysis of

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polar and apolar metabolites [77, 78]. The polar metabolites of the sample were

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retained and separated on a HILIC column while the non-retained apolar metabolites

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eluting at dead time were transferred and separated on a RPLC column through a

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heart-cutting interface with a trap column and dilution flow. This 2DLC-MS method

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was employed to investigate the pharmacodynamic effect of ginsenoside Rg3 on the

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metabolome of liver-tumour-bearing rats [79]. A total of 5686 polar and 1808 apolar

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urinary metabolite ion features were detected within a total analysis time of 52 min.

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Among them, 17 biomarker candidates were defined. This 2DLC system significantly

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3.2 Stop-flow 2DLC

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Under stop-flow 2DLC, the 1D flow rate is stopped when 2D analysis is carried

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out. As a result, these two dimensions run independently, and each dimension can use

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the same conditions as those in traditional 1DLC. A 2D flow programming

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counter-current chromatography (CCC) × RPLC approach was developed for the

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separation of toad venom, in which the CCC was performed at a controlled flow rate

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[80]. As a result, the elution time of each fraction in the first dimension was the same

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as the analysis time of the last fraction in the second dimension. Then, Qiu’s group

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constructed an online CCC × RPLC platform based on the heart-cutting and stop-flow

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techniques to preparatively isolate coumarin derivatives from Peucedanum

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praeruptorum Dunn [81]. Using interface integrating trapping columns and make-up

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flow allowed for the efficient capture of targeted compounds during the long-time

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transfer process. Heart-cutting was able to eliminate interfering substances, and

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stop-flow assured that there was sufficient time for 2D separation, leading to higher

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resolution. A total of 16 compounds with high purity and recovery were acquired from

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P. praeruptorum in less than 330 min. Following the initial attempt to use online stop-flow 2DLC strategy for lipid

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profiling (described later in this review) [82], Wang et al. extended this method to the

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separation and identification of triterpenoid saponins in ginseng extract [83]. HILIC

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was used as the first dimension, and RPLC was used as the second dimension. The

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use of trapping column enabled the first dimension to be operated at a conventional

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flow rate. A total of 94 triterpenoid saponins in ginseng were characterized and 19 of

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them were found to be different in ginsengs with different growth years. Ren and

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co-workers showed that the combination of online heat-cutting and stop-flow 2DLC

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enabled both the identification and quantification of 12 major components in tartary

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buckwheat [84].

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3.3 Comprehensive 2DLC and selective comprehensive 2DLC

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In the comprehensive 2DLC technique, the separation selectivity can be

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modulated by using different columns and/or buffers. Willmann et al. developed a

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comprehensive RPLC × RPLC system to identify modified nucleosides from RNA

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metabolism [85]. This analysis involved a Zorbax Eclipse Plus C18 column in the first

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dimension followed by a Zorbax Bonus-RP column in the second dimension. The use

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of water/methanol and water/acetonitrile gradient elution programs in 1D and 2D,

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respectively, improved the chromatographic resolution. Finally 28 modified

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nucleosides in urine samples and 26 modified nucleosides in cell cultures were

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detected using this 2DLC approach. Although the comprehensive RPLC × RPLC

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system is relatively easy to construct, the orthogonality of its two dimensions is not

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good enough. Donato et al. designed a RPLC × RPLC system for the separation of

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wine polyphenols with shift gradient elution in the second dimension to improve the

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orthogonality of the two dimensions [86]. In total 43 polyphenols were resolved and

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identified. In order to further improve the orthogonality, Qiao et al. systematically

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optimized

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methanol/water/formic acid and acetonitrile/water/formic acid were used in 1D

6

(Acquity CSH C18 column) and 2D (Poroshell Phenyl-Hexyl column), respectively.

7

Meanwhile a synchronized gradient elution mode was adopted to enhance the

8

resolution. The RPLC × RPLC counter plot of the licorice ethyl acetate extract is

9

shown in Figure 4. The practical peak capacity reached up to 1329 and the

10

orthogonality reached 79.8%. As a result, 311 phenolic compounds and triterpenoid

11

saponins were detected in licorice within 40 min and 21 selected unknown

12

compounds were tentatively determined by MS.

mobile

phase

system

of

RPLC

×

RPLC

[39],

and

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To reduce the interference of highly abundant components, online heart-cutting

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and comprehensive 2DLC were innovatively coupled together byYe’s group [87].

15

Online heart-cutting played a role in removing five major compounds from Ge-Gen

16

extract. At the same time, comprehensive RPLC × RPLC coupled with

17

mass spectrometry was used to efficiently characterize the retained minor compounds.

18

Due to the removal of major compounds, the detection sensitivity of minor

19

compounds was significantly enhanced. In total, 271 peaks in P. lobata and 254 in P.

20

thomsonii were detected within 35 min.

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As another common and typical comprehensive 2D mode, comprehensive HILIC

22

× RPLC exhibits satisfactory orthogonality and mild incompatibility. The recent work

23

of Villiers’ group demonstrated the power of HILIC × RPLC coupled with HRMS for

24

the elucidation of proanthocyanidins (PACs) in grape seed [88] and the determination

25

of anthocyanins and derived pigments in red wine [89]. Highly structurally diverse 16

ACCEPTED MANUSCRIPT PACs are natural oligomeric and polymeric phenolic compounds. HILIC fractionated

2

PACs based on size before their subsequent isomeric separation by RPLC. Finally, a

3

total of 78 PACs up to degrees of polymerization of 16 and galloylation of 8 were

4

identified based on their retention times in two dimensions and accurate molecular

5

weight. However, the high post-1D flow splitting of 1:24 tremendously reduced the

6

detection sensitivity. After modification, later work comprised a capillary LC device

7

in the first dimension and a UPLC device in the second dimension. Sensitivity was

8

maintained by using a slow 1D flow rate without post-1D flow splitting, but at the cost

9

of longer analysis time. The group-type separation was beneficial to compound

10

identification and 94 anthocyanin-derived pigments were tentatively deduced from

11

one- and six-year-old Pinotage wines. Figure 5 shows the HILIC × RPLC separation

12

of six-year-old Pinotage wines. Campiglia et al. developed a HILIC × RPLC system

13

to analyse a complex polyphenolic extract from an Italian apple cultivar. A total of

14

121 polyphenolic compounds were detected, including multiple polyphenolic classes

15

up to a polymerization degree of 10 [90]. Montero et al. utilized a similar HILIC ×

16

RPLC method coupled with diode array (DAD) and MS to profile licorice metabolites

17

from different locations. A total of 89 different metabolites, including triterpene

18

saponins, glycosylated flavanones andchalcones, were identified, and the obtained

19

unique metabolite profiles could possibly be used to confirm the geographical origin

20

and authenticity of unknown licorice samples [91].

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sLC × LC has gradually been applied to the separation and characterization of

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compounds with similar structures, such as stereoisomers and structural isomers. Van

23

de Schans et al. used the online sLC × LC configuration to analyse isomeric

24

pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PAs-ox) in plants [92].

25

The respective adjustment of the 1D and 2D mobile phases to pH 3.0 and pH 10.0 17

ACCEPTED MANUSCRIPT strengthened the selectivity and resolution of PAs and PAs-ox. Twenty individual PAs

2

and PAs-ox including six pairs of isomers were resolved and visualized. Moreover,

3

comprehensive 2DLC and selective comprehensive 2DLC were combined to fully

4

profile complex samples, such as traditional Chinese medicine. Qiao et al.

5

investigated Gegen-Qinlian Decoction (GQD) on a C18 × Phenyl-Hexyl 2DLC

6

system under acidic 1D mobile phases and alkaline 2D phases with an optimized shift

7

gradient elution [93]. A total of 280 peaks were detected within 42 min, among which

8

125 were characterized. sLC × LC equipped with eleven 40 µL loops enabled the

9

separation and detection of 13 additional minor compounds. In a similar manner,

10

Denzhan Shenmai (DZSM) was separated by a comprehensive C8 × C18 column

11

system with integrated shift and full gradient elution, and a total of 283 compounds,

12

including phenolic acids, flavonoids, saponins and lignans, were detected within 75

13

min [94]. A selective comprehensive C8 × Chiral column system, in which the

14

selectivity of the chiral column adopted in 2D was highly complementary to the

15

achiral analysis in 1D, was further used to separate and characterize 12 pairs of isomer

16

compounds.

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3.4 3DLC

In some cases, resolution and selectivity of 2DLC are not enough, more

22

dimensions are needed. Stoll et al. established a heart-cutting 3DLC method for

23

targeted analysis [95]. Three RP columns (weak cation exchange, carbon clad zirconia

24

(C/ZrO2) and conventional C18 functional groups) with different selectivities were

25

used in the first, second and third dimensions, respectively. Four target analytes at 18

ACCEPTED MANUSCRIPT 1

trace level were highly sensitively detected from various complex matrices including

2

urban wastewater, human urine and river water. Our laboratory also developed an on-line 3DLC method for non-targeted analysis

4

[45], 83 flavonoids were identified from soybean extract, which was nearly 30% more

5

than the corresponding 2D-RPLC analysis. Especially, the isomers and low abundant

6

components were better separated and accurately identified by this 3DLC method.

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Briefly, MDLC-MS is mainly used to increase peak capacity. It is also helpful in

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the resolution enhancement of structurally similar metabolites. Some typical examples

9

of 2DLC methods for metabolomic analysis are listed in Table 1.

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4. MDLC applications in lipidomics

Lipids are vital amphipathic biomolecules, which usually consist of different

13

polar head groups and nonpolar aliphatic chains. Therefore, MDLC is very suitable

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for lipidomics analysis.

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4.1 Heart-cutting 2DLC

Heart-cutting 2DLC can often be used to enhance the resolution of specific lipid

18

classes. Phoshpholipid (PL) comprises glycerophospholipid and sphingolipid in view

19

of their different glycerol skeleton. Glycerophospholipid includes phosphatidylcholine

20

(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol,

21

phosphatidylinositol (PI), cardiolipin. PC is the main components of lipid bilayer

22

structure of the eukaryotic cell membrane and plays an important role in cell

23

membrane fusion, material exchange and information transmission. Sun et al.

24

instructively coupled 2DLC with in-line ozonolysis-mass spectrometry (O3-MS) to

25

elucidate PC structures [96]. This system involved gradient HILIC separation in the

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ACCEPTED MANUSCRIPT first dimension followed by isocratic C18-based RP separation in the second

2

dimension. PL extracts from rat liver were divided into different classes, and only PC

3

was directly eluted to a 2D column and the other PLs were eluted as waste. A total of

4

19 PC molecular species were identified with double bond positions in their two fatty

5

acid chains clearly appointed by the aldehyde ions generated from the in-line

6

ozonolysis reaction. This established method could also be extended to achieve the

7

accurate identification of other types of PL. However a homemade in-line O3-MS was

8

essential, which surely lacked universality and practicability. A heart-cutting

9

HILIC-RPLC approach was also developed for the comprehensive analysis of low

10

abundance ceramides and PCs from mice livers, and a triple-quadrupole MS was used

11

as the detector [97]. The 1D HILIC could distinguish the lipid classes and the 2D

12

RPLC could separate the lipid species of the same class. Benefitting from the reduced

13

matrix effect, the sensitivity of this 2DLC was 2- to 3-fold higher than that of the

14

1DLC.

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In addition to the temporary storage of 1D fractions, another typical execution

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mean of heart-cutting was direct transferring. Byrdwell et al. built a LC2/MS2

17

platform to simultaneously separate polar and non-polar lipids in one injection [98].

18

The 1D NPLC was linked to the LCQ Deca ion trap mass spectrometer, while the 2D

19

RPLC was linked to the TSQ 700 tandem mass spectrometer. Non-polar lipids, mainly

20

including ceramide (Cer), diacylglycerol (DG) and triacylglycerol (TG), were eluted

21

early from serially connected amide columns and immediately redirected into serially

22

connected C18 columns. After the entire sample injection process was finished,

23

separations on the dual parallel LC-MS were performed synchronously. This

24

heart-cutting-based LC2/MS2 device certainly achieved the goal of the simultaneous

25

analysis of non-polar and polar lipids within one injection. The main drawbacks of

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ACCEPTED MANUSCRIPT 1

this method were the demand for two mass spectrometers and the processing of

2

redundant data. Metabolomes and lipidomes have different physicochemical properties; typically,

4

two methods are used to analyse them. Recently Wang et al. established an ingenious

5

heart-cutting 2D RPLC-MS approach to gain information about both metabolomics

6

and lipidomics within a 30-min analytical run [99]. A schematic diagram of this

7

configuration is shown in Figure 6A. Complex samples were first cut into two

8

fractions by the 1D pre-separation column based on their hydrophobicity. The first

9

fraction (metabolome) was eluted directly to one 2D Acquity BEH C18 column for

10

metabolomics analysis. The second fraction (lipidome) was retained on the

11

pre-separation column and then transferred to another 2D Acquity HSS T3 column for

12

lipidomics analysis. The total ion chromatogram for plasma is presented in Figure 6B,

13

and a total of 447 and 289 metabolites and lipids in plasma, respectively, were

14

identified under the positive and negative modes of Q Exactive HF MS. Therefore,

15

this 2DLC method was suitable for large-scale omics analysis. Unfortunately, some

16

non-polar lipids, such as TGs, could not be completely covered due to their

17

indissolubility in the reconstitution solvent (50/50, isopropanol/water), which is the

18

primary limitation of this method. Later, this 2DLC method was successfully

19

employed to discover potential metabolic biomarkers for esophageal squamous cell

20

carcinoma (ESCC) [100]. A total of 120 differential metabolites and lipids including

21

amino acids, free fatty acids, carnitines, choline and PCs were identified, which was

22

helpful for investigating the molecular mechanisms of ESCC progression. By

23

changing the pH of the mobile phases in the 2D parallel analysis, this 2DLC was

24

modified and used to analyse acyl-coenzyme As (acyl-CoAs) within one injection, as

25

acyl-CoAs are pivotal metabolic intermediates that participate in many biological

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ACCEPTED MANUSCRIPT processes [101]. Through a pre-fractionation column, complex acyl-CoAs were

2

divided into two fractions: short-chain acyl-CoAs and medium-/long-chain acyl-CoAs.

3

Further separations were performed on 2D parallel columns with a weakly alkaline

4

mobile phase for short-chain acyl-CoAs and a stronger alkaline mobile phase for

5

medium- and long-chain acyl-CoAs. In total, 90 and 46 acyl-CoAs were identified

6

from mouse liver (see Figure 7) and glioma cells extracts, respectively. This

7

established method was validated to have good repeatability, high sensitivity and a

8

broad linear range.

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4.2 Stop-flow 2DLC

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Nie et al. demonstrated the enormous potential of the VEI-based NPLC-RPLC

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system for lipid profiling [102]. The combination of VEI and stop-flow mode allowed

13

the use of conventional HPLC columns in two dimensions. Samples were cut into five

14

fractions on the 1D NPLC column, and a total of 721 lipids from 12 lipid classes were

15

identified in the rat peritoneal surface layer, in which 32 potential biomarkers of

16

peritoneal dialysis were discovered. However, the difficulties associated with the

17

construction and operation of this system restricted its widespread application.

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HILIC offers a similar chromatographic mechanism as NPLC for lipid analysis

19

but is more compatible with RPLC in the mobile phase. Different stop-flow

20

HILIC-RPLC-MS platforms were constructed for the separation and characterization

21

of lipids by distinguishing the polar head functional groups and fatty acid chains.

22

Dugo et al. developed a stop-flow HILIC × RPLC-MS method to separate

23

phospholipids in biological samples. Although 23 fractions with volumes as large as

24

100 µL were produced by a loop-based interface, neither serious dilution effects nor

25

peak distortion occurred during 2D separation. This was probably due to the use of a

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ACCEPTED MANUSCRIPT moderately polar organic solvent in the HILIC mode, which permitted the

2

phospholipids to be strongly retained at the isocratic conditions employed in the

3

second dimension. Up to 16 and 14 different phospholipids species were identified in

4

cow’s milk and plasma, respectively [103]. In our system, stop-flow 2DLC with a trap

5

column-based interface was employed to comprehensively profile lipids in plasma,

6

thus eliminating the dilution effect [82]. Complex lipids were divided into six

7

fractions based on their polar head groups in the 1D HILIC; then, further separation

8

was performed based on the aliphatic chains in the 2D RPLC. Both dimensions were

9

performed using the same type of analytical conditions (e.g., flow rate) due to the

10

introduction of the trap column and make-up flow. As a result, the detection

11

sensitivity was improved, and 372 lipids were detected in human plasma in positive

12

mode, i.e., 88 more lipids were detected than using 1D RPLC.

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In addition to NPLC and HILIC, strong anion exchange (SAX) was also used to

14

fractionate various lipid classes based on their charged states. Utilizing a homemade

15

capillary SAX column in the first dimension, Bang et al. developed an online 2D

16

capillary SAX- nanoflow RPLC method for lipid profiling [104]. Capillary SAX

17

adopted salt stepwise elution (methanol, 10 mM ammonium acetate (AA), 250 mM

18

AA, 1 M AA), and lipids were divided into four groups based on their different

19

electrical features: neutral, weak anionic, anionic and special lipids. A short C18

20

trapping column was used prior to the 2D capillary RPLC column to retain the desired

21

lipids and remove salts and other impurities. By applying the established method to

22

plasma lipid extracts, 303 lipids from 14 diverse classes were finally identified.

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4.3 Comprehensive 2DLC Recently, a continuous-flow 2D NPLC/RPLC system with a modified VEI 23

ACCEPTED MANUSCRIPT modulation was developed and applied to the plasma lipid research of atherosclerosis

2

patients [55]. Galactosylceramides (GalCs) and glucosylceramides (GluCs) were

3

favourably resolved using this 2DLC method, and the concentrations of GalCs rather

4

than GluCs were found to significantly increase in atherosclerosis patients. This 2D

5

NPLC/RPLC method was also used to investigate benign and malignant breast

6

tumours, p-chlorophenylalanine-treated mice and lacunar infarction patients [56, 105,

7

106]. Similarly, a continuous 2D HILIC/RPLC system with two trap columns was

8

designed by Berkecz et al. and then used for the phospholipid and sphingomyelin

9

analysis of mouse brains. Over 150 lipid species were detected, of which 37 were

10

found to be differentially expressed in mice with anxiety disorders [107]. In contrast

11

to customary practice, an online comprehensive RPLC × HILIC approach was

12

established by using RPLC in the first dimension and HILIC in the second dimension

13

[108]. In total, 143 lipids from 4 categories and 10 classes were detected from human

14

plasma and porcine brain (see Figure 8), which are significantly less than those

15

detected using the aforementioned methods. This is partly due to the serious diluent

16

effect caused by the use of a high flow rate of 5 mL/min in the second dimension and

17

a high splitting ratio of 8:100 prior to mass spectrometry detection. Later, Baglai et al.

18

employed a microbore HILIC column with a relatively low flow rate of 2 mL/min in

19

the second dimension. Thus, a splitting ratio of 1:3 prior to MS detection was adopted,

20

and the sensitivity was improved [109]. More recently, Mondello’s group established

21

an online comprehensive HILIC × RPLC-MS/MS method to obtain the global lipid

22

fingerprinting of Mediterranean mussel. The dilution effect still existed, and a total of

23

226 lipids were determined [110].

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As a large class of complex neutral lipids, triacylglycerides (TGs) widely exist in

25

both plants and animals. Due to their lack of polar head groups, TGs cannot be 24

ACCEPTED MANUSCRIPT analysed using conventional HILIC and NPLC techniques. However, Ag+ HPLC

2

could distinguish the degree of unsaturation of TGs based on the reversible interaction

3

between Ag+ and double bond electrons. Therefore, to elucidate TG as thoroughly as

4

possible, Mondello and Dugo et al. initiated a silver-ion (Ag+) HPLC × non-aqueous

5

RPLC system, with an atmospheric pressure chemical ionization (APCI) MS as the

6

detector [111-113]. This strategy was verified as an efficient tool for profiling TGs in

7

plant oil, milk fat and mouse tissue [114]. More recently, Byrdwell et al. developed a

8

contrary non-aqueous RPLC × Ag+-based UPLC system with four MS instruments to

9

analyse DGs and TGs from seed oils [115]. The first dimension was equipped with

10

two mass spectrometers as detectors. APCI-MS was used for the quantification of

11

tocopherol, while high-resolution ESI-MS was used for the identification of a

12

previously unreported oxidized TG. The 2D silver-ion UPLC coupled with two mass

13

spectrometers (atmospheric pressure photoionization ionization (APPI)-MS and

14

ESI-MS) provided more information about isomers, cis- or trans-double bonds and

15

the sn-1/3 or sn-2 position of TGs. However, the required participation of so many

16

mass spectrometers and other detectors represents a demand that is difficult for most

17

laboratories to meet.

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The abovementioned examples reveal that the aim of 2DLC-MS is to improve the

19

degrees of separation of specific lipid classes or all lipids. Consequently, more lipid

20

species, including isomers, can be favourably resolved. Some typical examples of

21

2DLC methods for lipidomic analysis are listed in Table 2.

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5. Conclusions and perspectives

24

In the past decade, multidimensional liquid chromatography has been greatly

25

improved by advances in construction modes and modulation modes, such as selective 25

ACCEPTED MANUSCRIPT comprehensive 2DLC, pulsed-elution 2DLC, stop-flow 3DLC, active solvent

2

modulation and evaporation membrane modulation etc. These novel construction

3

modes and modulation modes have made MDLC more flexible and ingenious. Its

4

application fields have also been gradually extended to metabolomics and lipidomics

5

analyses. Based on the complexity and properties of biological samples, different

6

construction modes of MDLC have been developed for targeted and non-targeted

7

analyses. In addition, the orthogonality of chromatographic columns and the

8

compositions of mobile phases have also had large effects on the separation

9

performance of MDLC. The main advantages of MDLC are that it provides either

10

wide coverage or enhanced resolution, which contribute to accurate qualitative and

11

quantitative analyses.

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In the future, the combination of different LC modes or other chromatographic

13

modes will display more powerful separation capacities. For example, a novel online

14

comprehensive RPLC × supercritical fluid chromatography (SFC) system with a trap

15

column interface was recently used to separate the phenolic compounds in

16

depolymerized lignin samples [116]. The RPLC × SFC system showed a high degree

17

of orthogonality. In addition, some novel techniques, such as ion-mobility

18

spectrometry (IMS), have made tremendous progress and have displayed great

19

promise for the future analysis of complex samples. After MDLC is coupled with IMS,

20

the resolution power of isomers will be significantly enhanced [117]. This technique

21

will be especially suitable for the separation of metabolites with different functional

22

groups [118-120] or lipid isomers with different double band positions [109, 121]. It

23

can be expected that the coupling of MDLC with other new technologies will play a

24

more important role in metabolomics and lipidomics studies.

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ACCEPTED MANUSCRIPT 1

Acknowledgements

2

This work was financially supported by the National Key Research and

3

Development Program of China (2017YFC0906900) and the foundations (21575142,

4

81472374 and 21435006) from the National Natural Science Foundation of China.

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[105] L. Yang, X. Cui, N. Zhang, M. Li, Y. Bai, X. Han, Y. Shi, H. Liu, Comprehensive lipid profiling of

4

plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers, Analytical

5

and bioanalytical chemistry, 407 (2015) 5065-5077.

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[106] L. Yang, P. Lv, W. Ai, L. Li, S. Shen, H. Nie, Y. Shan, Y. Bai, Y. Huang, H. Liu, Lipidomic analysis of

7

plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid

8

chromatography-quadrupole time-of-flight mass spectrometry, Analytical and bioanalytical chemistry,

9

409 (2017) 3211-3222.

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[107] R. Berkecz, F. Tomosi, T. Kormoczi, V. Szegedi, J. Horvath, T. Janaky, Comprehensive phospholipid

11

and sphingomyelin profiling of different brain regions in mouse model of anxiety disorder using online

12

two-dimensional (HILIC/RP)-LC/MS method, Journal of pharmaceutical and biomedical analysis, 149

13

(2018) 308-317.

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[108] M. Holcapek, M. Ovcacikova, M. Lisa, E. Cifkova, T. Hajek, Continuous comprehensive

15

two-dimensional liquid chromatography-electrospray ionization mass spectrometry of complex

16

lipidomic samples, Analytical and bioanalytical chemistry, 407 (2015) 5033-5043.

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[109] A. Baglai, A.F.G. Gargano, J. Jordens, Y. Mengerink, M. Honing, S. van der Wal, P.J. Schoenmakers,

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Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase

19

separations:

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chromatography-trapped-ion-mobility-mass spectrometry, Journal of chromatography. A, 1530 (2017)

21

90-103.

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[110] P. Donato, G. Micalizzi, M. Oteri, F. Rigano, D. Sciarrone, P. Dugo, L. Mondello, Comprehensive

23

lipid profiling in the Mediterranean mussel (Mytilus galloprovincialis) using hyphenated and

24

multidimensional chromatography techniques coupled to mass spectrometry detection, Analytical and

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bioanalytical chemistry, 410 (2018) 3297-3313.

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[111] L. Mondello, P.Q. Tranchida, V. Stanek, P. Jandera, G. Dugo, P. Dugo, Silver-ion reversed-phase

27

comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection

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in lipidic food analysis, Journal of Chromatography A, 1086 (2005) 91-98.

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[112] P. Dugo, T. Kumm, B. Chiofalo, A. Cotroneo, L. Mondello, Separation of triacylglycerols in a

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liquid

chromatography-mass

spectrometry

vs.

liquid

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Two-dimensional

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ACCEPTED MANUSCRIPT complex lipidic matrix by using comprehensive two-dimensional liquid chromatography coupled with

2

atmospheric pressure chemical ionization mass spectrometric detection, Journal of separation science,

3

29 (2006) 1146-1154.

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[113] P. Dugo, T. Kumm, M.L. Crupi, A. Cotroneo, L. Mondello, Comprehensive two-dimensional liquid

5

chromatography combined with mass spectrometric detection in the analyses of triacylglycerols in

6

natural lipidic matrixes, Journal of chromatography. A, 1112 (2006) 269-275.

7

[114] Q. Yang, X. Shi, Q. Gu, S. Zhao, Y. Shan, G. Xu, On-line two dimensional liquid

8

chromatography/mass spectrometry for the analysis of triacylglycerides in peanut oil and mouse

9

tissue, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences,

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1

895-896 (2012) 48-55.

11

[115] W.C. Byrdwell, Comprehensive Dual Liquid Chromatography with Quadruple Mass Spectrometry

12

(LC1MS2 x LC1MS2 = LC2MS4) for Analysis of Parinari Curatellifolia and Other Seed Oil Triacylglycerols,

13

Anal Chem, 89 (2017) 10537-10546.

14

[116]

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chromatographyxsupercritical fluid chromatography with trapping column-assisted modulation for

16

depolymerised lignin analysis, Journal of chromatography. A, 1541 (2018) 21-30.

17

[117] T.J. Causon, S. Hann, Theoretical evaluation of peak capacity improvements by use of liquid

18

chromatography combined with drift tube ion mobility-mass spectrometry, Journal of chromatography.

19

A, 1416 (2015) 47-56.

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[118] C. Chalet, B. Hollebrands, H.G. Janssen, P. Augustijns, G. Duchateau, Identification of phase-II

21

metabolites of flavonoids by liquid chromatography-ion-mobility spectrometry-mass spectrometry,

22

Analytical and bioanalytical chemistry, 410 (2018) 471-482.

23

[119] X. Zhang, K. Kew, R. Reisdorph, M. Sartain, R. Powell, M. Armstrong, K. Quinn, C.

24

Cruickshank-Quinn, S. Walmsley, S. Bokatzian, E. Darland, M. Rain, K. Imatani, N. Reisdorph,

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Performance of a High-Pressure Liquid Chromatography-Ion Mobility-Mass Spectrometry System for

26

Metabolic Profiling, Anal Chem, 89 (2017) 6384-6391.

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[120] K.L. Arthur, M.A. Turner, A.D. Brailsford, A.T. Kicman, D.A. Cowan, J.C. Reynolds, C.S. Creaser,

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Rapid Analysis of Anabolic Steroid Metabolites in Urine by Combining Field Asymmetric Waveform Ion

29

Mobility Spectrometry with Liquid Chromatography and Mass Spectrometry, Anal Chem, 89 (2017)

M.

Sandahl,

C.

Turner,

Comprehensive

on-line

two-dimensional

liquid

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ACCEPTED MANUSCRIPT 7431-7437.

2

[121] J.E. Kyle, X. Zhang, K.K. Weitz, M.E. Monroe, Y.M. Ibrahim, R.J. Moore, J. Cha, X. Sun, E.S.

3

Lovelace, J. Wagoner, S.J. Polyak, T.O. Metz, S.K. Dey, R.D. Smith, K.E. Burnum-Johnson, E.S. Baker,

4

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry

5

and mass spectrometry, The Analyst, 141 (2016) 1649-1659.

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ACCEPTED MANUSCRIPT 1

Legends

2 3

Fig. 1. Flow scheme of the established 3DLC system. Position A is loading position:

4

1

5

compounds retained on trapping column 1 were eluted and subjected to RPLC ×

6

RPLC analysis. Reproduced from Ref. [45] with permission.

7

RI PT

D effluents were retained on trapping column 1. Position B is elution position:

Fig. 2. Visual comparison of four different construction modes of 2DLC and their

9

chromatograms. Storage means most often used are sample loops or trapping columns.

SC

8

Panels A and B show the details of multiple heart-cutting (MHC) 2DLC in which

11

several 1D fractions are injected into 2D column. Panels C and D show the details of

12

stop-flow 2DLC in which the whole 1D effluent, cut into several fractions, are

13

injected into 2D column. Panels E and F show the details of LC × LC in which the

14

whole 1D effluent is injected into 2D column repeatedly. Panels G and H show the

15

details of sLC × LC in which the selected peaks of 1D effluent are cut and injected

16

into 2D column. Reproduced from Ref. [83] and Ref. [42] with permission.

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Fig. 3. Diagram of the 2DLC system with an evaporative interface. (a) and (b) are

19

chromatogram before (a) and after (b) evaporation, respectively. Reproduced from

20

Ref. [60] with permission.

21

AC C

18

22

Fig. 4. RPLC × RPLC courter plot of licorice ethyl acetate extract. Data were

23

acquired with ultraviolet (UV) detector at 250 nm. Unknown compounds, saponins

24

and phenolic compounds were marked with black, yellow and white characters,

25

respectively. Reproduced from Ref. [39] with permission. 42

ACCEPTED MANUSCRIPT 1 2

Fig. 5. HILIC × RPLC courter plot for the analysis of six-year-old Pinotage wine.

3

Data were acquired with UV detector at 500 nm. Reproduced from Ref. [89] with

4

permission.

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5

Fig. 6. (A) Schematic diagram for simultaneous metabolomics and lipidomics

7

analyses: from sample preparation to chromatographic analysis. (B) Total ion

8

chromatography for plasma analysis using this configuration. Reproduced from Ref.

9

[99] with permission.

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Fig. 7. Extraction ion chromatography of all detected acyl-CoAs in mouse liver.

12

Reproduced from Ref. [101] with permission.

13

Fig. 8. RPLC × HILIC-MS courter plot for the separation of total lipids extracts: (a)

15

human plasma; (b) porcine brain. The abbreviations except these mentioned in text

16

were as follows: lysophosphatidylcholine (LPC); cholesterol (Chol); cholesteryl ester

17

(CE); hexosyl ceramides (HexCer); dihexosyl ceramides (Hex2Cer). Reproduced

18

from Ref. [108] with permission.

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ACCEPTED MANUSCRIPT

Typical applications of 2DLC in metabolomics analysis

five Panax

1

2

D column

D column

Poroshell SB C18,

heart-cutting

comprehensive

2.1 × 100 mm, 2.7 µm

4.6 × 100 mm, 3.5

O/0.25 mL/min

nucleosides in DNA

Kinetics C18,

µm X-select C18 CSH,

gradient/ACN- H2O

from human and

2.1 × 150 mm, 1.7 µm

2.1 × 150 mm, 1.7

porcine tissues minor compounds in

Acquity CSH C18,

µm Poroshell 120

Piermaria

2.1 × 100 mm, 1.7 µm

clenbuterol in pork

homemade MIC, 4.6 × 100

Phenyl-Hexyl , 3.0 × 50 mm, 2.7 µm

mm

coumarone in Peucedanum praeruptorum Dunn triterpenoid sapiens in

COSMOSIL MSII multilayer coiled column

C18, 20.0 × 250 mm TSK-GEL Amide-80, 2.0

stop-flow × 150 mm, 3.0 µm

2.1 × 100 mm, 1.7

AC C

ginseng

Acquity BEH C18,

Detector

mL/min

gradient/MeOH-

30s shift gradient/ ACN-

H2O /0.1 mL/min

H2O /2.5 mL/min

isocratic/ACN- H2O

isocratic/MeOH- H2O /0.8

[64] valve/loop 2-pos 6-port valve/trap

UV+MS

[65] column duo 2-pos 4+10 port

UV+MS

[87] valves/loops 2-pos 10-port

UV

/0.8 mL/min

mL/min

hydrodynamic

gradient/MeOH- H2O /8.0

[67] valve/trap column 2-pos 4+8 port valves/

UV

equilibrium/n-hepta

mL/min

ne-acetone- H2O /gradient/ACN- H2O

gradient/ACN- H2O / 0.25

[81] trap columns 2-pos 8+10 port

MS /0.15 mL/min

mL/min

gradient/MeOH-

54s shifted gradient/ ACN-

Ref.

2-pos 10-port

gradient/MeOH- H2O /0.35

0.25 mL/min

Interface

UV

H2O /2.0 mL/min

C18 column liver and urine

stop-flow

Isocratic+gradient/ACN-

not-ginseng sapiens

heart-cutting

heart-cutting+

gradient/MeOH-H2

D elution system

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heart cutting+

2

D elution system

TE D

heart-cutting

Zorbax SB-As,

1

SC

Samples

EP

MDLC mode

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Table 1

[83] valve/trap column

µm

modified nucleosides comprehensive

in cell culture and urine

Zorbax Eclipse Plus C18, 2.1 × 150 mm, 1.8 µm

Zorbax Bonus-RP, 4.6 × 50 mm, 1.8 µm

duo 2-pos 4-port MS

H2O 0.07 mL/min

H2O 2.5 mL/min

[85] valve/ loops

44

ACCEPTED MANUSCRIPT

Acclaim Mixed-Mode

Acclaim RSLC Polar

from tartary

HILIC-10, 2.1 × 10 mm, 3 µm

wine polyphenols and

Ascentis ES-Cyano, 1.0 ×

both gradient/ACN- H2O

0.5 mL/min

/0.5 mL/min

UV

Advantage II,

stop-flow buckwheat

2-pos 6+10 port gradient/ACN- H2O

2.1 × 150 mm, 2.2 Ascentis Express gradient/ACN- H2O C18, 4.6 ×30 mm, 2.7

impurity substances

250 mm, 5 µm

/0.02 mL/min µm

Acquity CSH C18,

Poroshell 120

2.1 × 100 mm, 1.7 µm

Phenyl-Hexyl,

licorice (phenols)

Develosil Diol-100,

3.0 × 50 mm, 2.7µm Zorbax SB-C18,

PACs from grape

anthocyanins and

4.6 × 50 mm, 1.8 µm

Acquity BEH Amide, 1.0

Phenomena Kinetics

derivatives in red

× 150 mm, 1.7 µm

C18,

wine compounds in toad

Shimadzu silica gel,

2.1 × 50 mm, 1.3 µm Phenomena Kinetics

comprehensive skin

sComprehensive

sComprehensive

H- H2O /25 µL/min

/1.5 mL/min

gradient/ACN- H2O

120s gradient/ACN- H2O

/1 µL/min

/0.86 mL/min

gradient/n-hexane-E

190s gradient/ ACN- H2O /3.5 mL/min

Hypersil Gold As,

µm Acquity BEH C18,

gradient/ACN- H2O

gradient/ACN- H2O /0.7

2.1 × 100 mm, 1.7

AC C

Poroshell 120

2.1 × 100 mm, 1.7 µm Acquity CSH C18,

2.1 × 100 mm, 1.7 µm

Phenyl-Hexyl,

3.0 × 50 mm, 2.7 µm Poroshell 120 Phenyl-Hexyl,

2-pos 8-port MS

[39] valve/loops 2-pos 10-port

MS

[88] valve/loops 2-pos 10-port

UV+MS

[89] valve/loops TEAA inerface/

UV

tOH/0.1 mL/min

Acquity CSH C18,

compounds in GQD

120s gradient/ ACN- H2O

C18, 4.6 × 50 mm, 5

1 × 100 mm, 1.9 µm

+

gradient/ACN-MeO

4.6 × 150 mm, 5 µm

PAs and PAs-ox

comprehensive

H2O /2.0 mL/min

[86] (×2)/loops

30s shift gradient/ACN-

H2O /0.1 mL/min

TE D

comprehensive

1.0 × 250 mm, 5 µm

EP

comprehensive

gradient/MeOH-

[84]

2-pos 6-port valve UV+MS

ACN- H2O /2.5 mL/min

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components in comprehensive

valves/direct transfer+back-fulsh

60s full shifted gradient/

SC

comprehensive

RI PT

major componuds heart-cutting+

[59] trap columns 6-pos 14-port (×2)+

MS

[92]

/0.15 mL/min

mL/min

2-pos 6-port valves/

gradient/ACN- H2O

30s shift gradient/ACN-

2-pos 8-port

/0.1 mL/min

H2O /2.0 mL/min

valve/loops UV+MS

[93]

gradient/ACN- H2O

210-s gradient/ ACN- H2O

6-pos 14-port (×2)

0.1 mL/min

/1.0 mL/min

valves/loops

3.0 × 50 mm, 2.7 µm

45

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gradient/MeOH-

30-s integrated shift & full

2.1 × 100 mm, 3.5 µm

4.6 × 50 mm, 1.8 µm

H2O /0.1 mL/min

/ACN- H2O /2.0 mL/min

compounds in DZSM

2-pos 8-port valve/loops UV+MS

[94]

CHIRALPAK IC-3,

gradient/MeOH-

300s gradient /ACN- H2O

6-pos 14-port (×2)

2.1 × 100 mm, 3.5 µm

4.6 × 150 mm, 3 µm

H2O /0.1 mL/min

/1.0 mL/min

valves/loops

TE D

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SC

Xterra MS C8,

EP

sComprehensive

Eclipse Plus C18,

AC C

+

Xterra MS C8,

RI PT

comprehensive

46

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Typical applications of 2DLC in lipidomics analysis MDLC Samples

1

2

1

Ascentis Express HILIC, 2.1

Ascentis Express C18,

gradient/ACN- H2O

D column

D column

D elution system

heart-cuttin PCs in rat liver

heart-cuttin g

lipids in bovine brain

×150 mm, 2.7 µm

2.1 ×150 mm, 2.7 µm

Caltech adsorb sphere NH2

MetaChem Inertsil ODS-3

(×2), 4.6 × 250 mm, 5 µm

/0.2 mL/min

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g

(×2), 4.6 × 250 mm, 5 µm

2

D elution system

SC

mode

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Table 2

gradient/IPA-n-hexane -EtOH- H2O /0.8

Detector

isocratic/THF-ACN-IPA

Interface

Ref.

2-pos 10-port MS

/0.475 mL/min

[96] valve/direct transfer

gradient/IPA-n-hexane-E

UV+ELSD

2-pos 6-port valve/

tOH/0.8 mL/min

+MS2

direct transfer

[98]

mL/min

Acquity BEH C18, metabolites and

Acquity BEH C8,

g

lipids in plasma

2.1 × 5 mm, 1.7 µm

TE D

heart-cuttin

gradient/ACN- H2O /

2-pos 6+8+10-port

2.1 × 50 mm, 1.7 µm

gradient/ - / -

0.35 mL/min gradient/IPA-ACN- H2O

2.1 × 50 mm, 1.7 µm

/0.3 mL/min

EP

Acquity HSS T3,

MS

valves/ direct

[99]

transfer

Acquity HSS T3, acyl-CoAs in tissues

Acquity BEH C18,

g

and cells

2.1 × 5 mm, 1.7 µm

2.1 × 50 mm, 1.7 µm

AC C

heart-cuttin

Acquity BEH C18,

2-pos 6+8+10 port gradient/ACN- H2O

both gradient/ACN- H2O

/0.3 mL/min

/0.3 mL/min

MS

valves/direct

[101]

transfer

2.1 × 100 mm, 1.7 µm

47

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lipids in rat

gradient/IPA-n-hexane Rx-SIL silica,

gradient/EtOH-H2O/0.3 -EtOH- H2O /0.1

peritoneal surface 2.1 × 150 mm, 5 µm

2.1 × 10 mm, 3.5 µm

mL/min

layers PLs in milk and

mL/min Ascentis Express HILIC, 2.1 ×

Ascentis Express C18,

gradient/ACN-EtOH-

stop-flow 150 mm, 2.7 µm

4.6 × 150 mm, 2.7 µm

lipids in human

Acquity BEH HILIC,

Acquity BEH C8,

H2O/0.1 mL/min

[102] valve/ loop

isocratic/THF-ACN-IPA

2-pos 10-port MS

-H2O/0.9 mL/min

SC

plasma

VEI/2-pos 6-port MS

RI PT

stop-flow

Eclipse Plus C8,

[103] valve/loop

gradient/IPA-ACN-Et

gradient/IPA-ACN- H2O

OH- H2O /0.15 plasma

2.1 × 100 mm, 1.7 µm

2.1 × 100 mm, 1.7 µm

M AN U

stop-flow

2-pos 8+10-port MS

/0.25 mL/min

[82] valve/trap column

mL/min

lipids in human

homemade SAX column, 75

homemade C18 column, 75

stop-flow plasma

µm × 50 mm, 5 µm-100 Å

µm × 70 mm, 3 µm-100 Å

lipids in human

Rx-SIL silica,

Poroshell 120 EC C8,

step elution/EtOH

-salt solution/1 µl/min

gradient/IPA-ACN-EtO

2-pos 6-port valve MS

H- H2O /16 µl/min

[104] (×2)/trap column

TE D

gradient/IPA-n-hexane

comprehens

gradient/EtOH- H2O /0.3

-EtOH- H2O /0.2

ive

plasma

2.1 × 150 mm, 5 µm

2.1 × 50 mm, 2.7 µm

VEI/2-pos 10-port MS

mL/min

[55] valve/loops

mL/min

lipids in human Acquity BEH C18,

Cortecs HILIC,

plasma and ive

1.0 × 150 mm, 1.7 µm

3.0 × 50 mm, 2.7 µm

AC C

porcine brain

EP

comprehens

gradient/IPA-ACN-

TGs in soybean and

homemade Ag+ column,

H2O /0.02 mL/min

linseed oils

1.0 × 150 mm, 5 µm

mm

/5 mL/min

[108] loops

120s gradient/n-hexane-AC

monolithic C18, 4.6 × 100

ive

2-pos 8-port valve MS

ChromolithTM Performance

comprehens

60s gradient/ACN- H2O

2-pos 8-port gradient/IPA-ACN/4

N/ 11 µL/min

MS

[113] valve/loops

mL/min

48

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SC

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ACCEPTED MANUSCRIPT

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EP

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SC

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ACCEPTED MANUSCRIPT

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EP

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SC

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ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights Applications of MDLC in metabolomics and lipidomics analysis are reviewed; Different physical connection ways and modulating modes of MDLC are summarized;

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MDLC provides wide coverage and high resolution for metabolome and lipidome analysis;

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MDLC displayed powerful ability for the separation of isomeric and low abundant metabolites and lipids.