Supercritical fluid chromatography hyphenated to mass ...Supercritical Fluid Chromatography (SFC)...

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Received: 07 27, 2020; Revised: 10 14, 2020; Accepted: 10 15, 2020

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Supercritical Fluid Chromatography Hyphenated to Mass Spectrometry for Metabolomics

Applications

Author: Ruth Gordillo

Affiliation: University of Texas Southwestern Medical Center. Touchstone Diabetes

Center, Dallas, TX

Corresponding Author: Ruth Gordillo, Ph.D. Associate Professor of Internal

Medicine. Touchstone Diabetes Center. Director of UT Southwestern Metabolic

Phenotyping Core.

5223 Harry Hines Blvd. Dallas, TX 75390-8549. Email address:

[email protected]

Running title: Hyphenated MS Techniques for Metabolomics

Article Related Abbreviations: 2D, two dimensional; 2EP, 2-ethylpydidine; ABPR,

automatic back-pressure regulator; APCI, atmospheric pressure chemical ionization;

APLI, atmospheric pressure laser ionization; APPI, atmospheric pressure photo

ionization; BEH, ethylene bridged hybrid; C30, triacontyl; CE, cholesterol ester; Cer,

ceramide; CIS, coordination ion spray; DEA, dietylamine; DG, diacylglyceride; DPA;

docosapentaenoic acid, ELSD, evaporative light scattering detector; EtOH, ethanol;

EPA, eicopentaenoic acid; FFA, free fatty acid; FID, flame ionization detector; FT-

MS, Fourier transform mass spectrometer; HILIC, hydrophilic interaction liquid

chromatography; HLB, hydrophilic-lipophilic balance; IPA, 2-propanol, LDL, low-

density lipoprotein; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPI,

https://doi.org/10.1002/jssc.202000805



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lysophosphatidylinositol; LPS, lysophosphatidylserine; LSER, linear solvation energy

relationship; MeOH, methanol; MG, monoacylglycerol; MRM, multiple reaction

monitoring; NP, normal-phase; ODS, octadecylsilane; otSFC, open tubular

supercritical fluid chromatography; PA, phosphatidic acid; PC, phosphatidylcholine,

pcSFC, packed column supercritical fluid chromatography; PE,

phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS,

phosphatidylserine; PTAD, 4-phenyl-1,2,4-truazikube-3,5-dione; PUFA,

polyunsaturated fatty acids; QqQ, triple quadrupole; Q-TOF, quadrupole time of

flight; R&D, research and development; Sa, sphinganine; SCCO2, supercritical CO2;

SFE, supercritical fluid extraction; SI-pcSFC; silver ion packed column supercritical

fluid chromatography; SM, sphingomyelin; So, sphingosine; So1P, sphingosine-1-

phosphate; SWATH/MS, sequential window acquisition of all theoretical mass

spectra; TGs, TC, tandem column; triacylglycerides; TMS, trimethylsilylation; TFA,

trifluoroacetic acid; TFE, trifluoroethanol; TMSD, trimethylsilyldiazomethane;VLDL,

very-low-density lipoprotein;

Keywords: Biomarkers discovery, metabolomics, supercritical fluid,



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Abstract

While Supercritical fluid chromatography was developed over fifty years ago, it is

only over the past fifteen to twenty years that it has become routinely utilized. Along

with the commercialization of a new generation of instruments, during the last twenty

years supercritical fluid chromatography has improved performance, reliability and

robustness. SFC is fully compatible with mass spectrometric techniques. This review

compiles the application of supercritical fluid chromatography separations coupled to

MS instrumentation for the exploration, profiling and quantitation of metabolites

during the last two decades. The selection of metabolites chosen for this article have

direct applications in preclinical models of disease and clinical applications as

potential biomarkers of disease including lipids, steroid hormones, bile acids, polar

metabolites, peptides, and proteins.



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1. Introduction

Supercritical Fluid Chromatography (SFC) was first introduced in the 1960s [1], but it

was only in the 1980s and 1990s it became an emerging technique [2]. Carbon

dioxide is the main fluid used in SFC because it is relatively inert with a low toxicity, it

is affordable, and its critical parameters are easy to reach (31 °C and 73 bar) [3].

CO2 is a naturally abundant material. Like water, if CO2 can be withdrawn from the

environment, employed in a process, then returned to the environment “clean” no

environmental detriment accrues [4].

The advantages of SFC over GC and LC are summarized as follows: 1. SFC is

applicable to thermally degradable compounds that cannot be analyzed by GC. In

addition, high-boiling compounds can be separated by SFC; 2. The separation

capacity of SFC is much higher than that of HPLC. The viscosity of supercritical CO2

(SCCO2) is similar to gas whereby its diffusivity is higher than liquid facilitating

throughput (shorter analysis times) and compound chromatographic resolution [5, 6];

3. The solvating power of SFC can be changed considerably by controlling the

temperature and pressure, i. e., the elution behavior in a single fluid can be

regulated. SCCO2 is non-polar similar to n-hexane [7]. CO2 is miscible with polar

solvents, such as methanol (MeOH), ACN, ethanol (EtOH) and 2-propanol (IPA)

which can change the polarity of the mobile phase when used as modifier solvents.

Other additives such as acids, bases and even water can be mixed with the

modifiers. These additives also increase the separation efficiency and peak shape by

acting as ion-pairing agents and by covering active sites on the stationary phase,

leading to less tailing and better elution of polar compounds. The latest trend in SFC

is the use of water as an additive in a CO2-MeOH mobile phase to improve peak

shape (at a proportion of 1-5%, miscible in the mobile phase) or to enable the elution



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of very polar compounds (up to 30%, forming a ternary mixture) [8-10]. Modifiers

selection in combination with complementary column selections, from polar to non-

polar stationary phases, unfolds myriad possibilities for optimum compound

separations [6]; 4. In the case of preparative SFC, since CO2 is released as a gas at

normal temperatures and pressures, Thus, solvent elimination is easy, allowing more

accurate component concentration to be determined [1, 11, 12].

SFC resurged in the last decade in parallel with the with the introduction of a new

generation of instruments capable of performing robust, reproducible, reliable, and

quantitative analysis [8, 13].

Similar to what has been observed in conventional LC, these new instruments have

also fostered the development of columns packed with sub-2-µm and sub-3-µm

superficially porous particles specially designed for SFC analysis. None of the

existing analytical techniques can simultaneously separate and measure all the

cellular metabolites due to complexity of cellular metabolome, and therefore, a

combination of analytical techniques must be used. Traditionally NMR, GC-MS and

LC-MS are most often used in metabolomics. SFC, which can increase metabolome

coverage while decreasing cost and analysis time, can provide alternative to other

analytical techniques [14].

2. Instrumentation

SFC can be classified into two main groups: open tubular column SFC (otSFC) and

packed column SFC (pcSFC) [6]. otSFC has a uniform distribution of the stationary

phase on the interior surface of the column and it’s considered “GC-like”. On the

other hand, pcSFC uses a column packed densely and evenly with solid-phase

support and allows the use of a polar solvent as a modifier.



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The first open tubular capillary column SFC was introduced in 1981 [2, 15, 16]. Due

to the technical challenges presented by these systems (incompatibility with

detection techniques, difficulty regulating flow velocity and reaching optimum

pressure, difficult adding modifiers and incompatibility with different detectors),

otSFC rapidly diminished in the early 1990’s.

Before otSFC, all SFC research was performed on packed columns. In 1982, a

Hewlett Packard (HP) HPLC system was modified to operate as an SFC system by

adding a backpressure regulator and other devices. It was shown that SFC gave

higher efficiency with 3, 5 and 10 µm packing materials especially in high flow

velocity region. pcSFC was developed almost independently of otSFC and it

regained popularity once their wider application was demonstrated. A pcSFC is very

similar to an HPLC system. A back pressure regulator is integrated in order to keep

the fluid pressure above the critical pressure and an oven (heater) that keeps the

fluid temperature above the critical temperature [2].

Current design strategies to fluidically couple pcSFC with MS can be divided in two

broad categories- a) split-flow introduction, and b) full-flow introduction (Figure 1)

[17-20].

a) Split-flow introduction. The “MS split after UV with make-up pump” interface has

been commercialized since 2012 by Waters Corporation with the UPC2 system. The

Agilent Technologies system (1260 Infinity II SFC system) offers the possibility to

perform experiments using both “full-flow through BPR with make-up” and “MS split

after UV with make-up pump” configurations depending on the tubing connections,

which can be of interest to use either a split or not. In this configuration, the system

has a UV detector after the column followed by two zero dead volume T-unions. The

up-stream T-union is linked to a make-up pump. Similarly, to the “full-flow through



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ABPR with make-up” configuration, the make-up addition prevents analyte

precipitation after CO2 decompression and can enhance the ionization. The down-

stream T-union is a splitter; one fraction is going to the ABPR and the other one is

directed towards the MS. The flexible regulation of the ABPR governs the split

depending on the mobile phase properties. The split ratio is dynamic and allows a

suitable mobile phase flow rate for the ESI source in order to avoid any dilution effect

which may be deleterious especially with a concentration-like-dependent detector

and maintain good sensitivity. However, even if the split ratio could be predicted

depending on the pressure drop in a given tubing, the use of a passive splitter

prevents any control of the split and an active splitter would increase the system

volume. Moreover, the effluent part going to the MS is not under the control of the

ABPR and the supercritical pressure is only maintained thanks to a restrictor

capillary and depends on its length. This capillary may often be changed in case of

clogging for example and its dimensions are crucial for good reproducibility. This

configuration has been thoroughly studied and it has been found that the mobile

phase decompression was the major contributor to peak broadening rather than the

addition of tubing when comparing SFC-UV and SFC-MS split with make-up pump,

with and without an online UV detector [17-19].

b) Full-flow introduction. The advantage of full-flow is the ability to introduce all or

most of the analyte molecules for detection. The challenge, however, is to design an

interface which is low-volume, robust and does not put any constraint on SFC

method design. In the current market, full-flow is achieved by passing the full

chromatographic flow through an automatic back-pressure regulator (ABPR) with

make-up pump. This design is commercialized since 2015 by Shimadzu Corporation

with the Nexera UC system and by Agilent Technologies (1260 Infinity II SFC



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system). These systems implement a make-up, introduced through a zero dead

volume T-union between the analytical column (or the UV detector, linked serially to

the column) and the ABPR. Then, the ABPR exit is directly connected to the MS

source. For Shimadzu and Agilent, the system uses low dead volume of the ABPR

(ideally < few μL) which allows direct coupling to the MS without need of splitting

while maintaining good chromatographic performances. In most systems, the ABPR

is heated to limit CO2 decompression due to the cooling phenomenon from

supercritical to atmospheric conditions. The make-up also helps avoiding analyte

precipitation, especially at low percentage of modifier and can improve the ionization

being an additional source of protons [17-19].

Full compatibility of SFC with mass spectrometry detection makes it even more

attractive analytical platform for metabolomics [14, 21, 22]. SFC was successfully

used with multiple ionization sources, including ESI [23, 24], atmospheric pressure

chemical ionization (APCI) [23, 25], atmospheric pressure photo ionization (APPI)

[26] and atmospheric pressure laser ionization (APLI) [21].

Tarafder [17], Schadand [18] and Akbal et al. [19], constitute excellent compilations

for extensive and detailed discussion of SFC-MS interfaces configurations in current

instrumentation.

Unlike other non-polar solvents, e.g. hexane or heptane, with which CO2 is often

compared, CO2 is miscible over a broad range of pressures and temperatures, with

liquid organic solvents having wide polarity ranges (MeOH, EtOH, propanols, ACN,

etc.). Modifiers or Co-solvents influence SFC chromatography in multiple ways: (a)

modifying the polarity, hence the solvating power, of the mobile phase; this is the

most important contribution and the effect of modifiers on solvent elution is greater

than any other factors , (b) altering the density of the mobile phase, (c) blocking



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active sites on the stationary phase and inhibiting adsorption, (d) modifying

stationary phase characteristic through sorption; (e) adsorbed modifier molecules

can lead to increase the net volume of the stationary phase, altering the phase ratio;

and (f) selectively solvating polar compounds in the mobile phase forming clusters

with different distribution properties [9]. other non-conventional modifiers like

dichloromethane, Chloroform, Methyl tert-butyl ether, Acetone, Ethyl acetate,

Tetrahydrofuran, Methyl tetrahydrofuran, 2,2,2-Trifluoroethanol, Cyclopentyl methyl

ether, Toluene and N,N-dimethylformamide were used with MeOH at different

proportions to prepare modifier blends for drug candidates which are poorly soluble

in traditional modifiers [9, 27-29].

Many different classes of polar solutes e.g. phenols, polyhydroxy, hydroxyacids,

polyacids, aliphatic amines, and many other drug families could be separated using

additives like citric acid, trifluoroacetic acid, isopropylamine, triethylamine,

ammonium acetate, etc [30]. In the case of polar urinary metabolites, different

modifier additives were chosen based on the desired outcome e.g. ammonium

acetate for more even peak distribution. Ammonium hydroxide for reduction in peak

width, or ammonium formate for reduced peak widths across a wider range of

analytes. The combination of ammonium formate with a small percentage (2% or

5%, v/v) of water resulted in additional gains in signal intensity [31]. The suggested

roles of additives are diverse too: (a) enhancement of solvating power of mobile

phase, (b) ionization suppression and ion-pairing with charged analytes, (c)

modification of stationary phase surface properties by covering active sites, changing

polarity, etc [9].

Stationary phases for pcSFC, like in HPLC, can be broadly divided into (a) chiral and

(b) achiral. However, in SFC, the distinction between these two types are not as



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clearly defined as in HPLC. In HPLC, generally the chiral separation is conducted in

normal-phase mode, whereas the achiral in reversed-phase or HILIC mode. In SFC,

there is no distinction between normal-phase and reversed-phase separations [9].

Chiral chromatography can be carried out with a chiral stationary phase and a mobile

phase composition that could have been used with achiral stationary phases too.

Chiral columns can be also employed for achiral analysis in SFC [32]. Achiral and

chiral columns or columns with different polarities can be connected in series to

create unique selectivity scope[9]. Columns designed to be used in HPLC can be

used in SFC as well. Regarding column geometries, for analytical studies

commercial SFC columns are increasingly being offered in sub-2-µm particles, which

is taking SFC significantly ahead of others for high-throughput analysis. Along with

the use of sub-2-µm particles, use of core-shell or superficially porous particles are

also increasing SFC to achieve similar efficiency but at much higher speed without

higher pressure drops [9, 33, 34]. Chromatographers should be aware that for achiral

analyses, SFC can replace non aqueous RPLC, NPLC an, d HILIC as long as the

studied compounds are soluble in the CO2-rich mobile phase [34]. In an effort to

classify stationary phase chemistry selectivity, West, Lesellier and co-workers have

undertaken extensive studies based on the solvatochromic model (also known as

linear solvation energy relationship, LSER). This approach allows classification of all

stationary phase types using five descriptors (hydrogen-bond donor and acceptor,

dipole-dipole, charge transfer, and solute volume) related to the interaction

capabilities between the stationary phase and various analytes. LSER data for over

75 SFC stationary phases have been reported so far, including alkyl, aromatic, polar,

or HILIC. All these phases have been characterized [35-42]. This classification

allows easy identification of the stationary phases providing the most potential



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orthogonal selectivity [33, 34]. The same group showed that for five stationary

phases packed with sub-2-µm particles and specifically commercialized for SFC

(Waters HSS C18 SB, XSelect CSH Fluorophenyl, BEH (silica), BEH 2EP and BEH

RP18 Shield), changing the particle side from 5 to 1.7 does not have a significant

effect on the interactions [43].

3. Early Applications

While SFC was developed over fifty years ago, the method was not routinely utilized

until the past fifteen or twenty years. At the beginning of its “rebirth” SFC was mainly

used for preparative separations [1, 11]. Chromatographic enantioseparation in

preparative scale is routinely used in pharmaceutical research and development

(R&D) to generate individual enantiomers. SFC has many advantages over HPLC for

these separations, i.e. rapid screening of separation conditions at the analytical

scale, rapid preparative separations, higher purification throughputs, lower solvent

consumption and waste generation and higher product concentrations post

separation. For this particular application, SFC is used coupled to tandem UV and

polarimetric detection for confirming entantioseparation [11]. Although the vast

majority of chiral separations in SFC are achieved on polysaccharide-based

stationary phases, other chiral stationary phases such as Pirkle-type and antibiotic-

based columns are also applicable. The low pressures generated though the

stationary phase in SFC, allow coupling several columns, in order to obtain the

desired selectivity towards more complex mixtures of racemates [44-46].

Due to its capability for separating enantiomeric compounds, SFC-MS has a

tremendous potential for the analysis of drugs in biological samples [47, 48], as well

as in the separation of impurities.



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4. Applications of Supercritical Fluid Chromatography Hyphenated to MS in

Metabolomics.

Free Amino Acids. SFC is widely used in chiral chromatography where

polysaccharide and cyclic oligosaccharides stationary phases are the most versatile

in use. The polarity can be increased by the addition of alcohol to the mobile phase.

The nature and percentage of the alcohol in the mobile phase can change drastically

the selectivity [3, 11]. As in Chiral LC, to predict the best chiral stationary phase and

the best mobile phase is difficult because chiral interactions are very complex. There

have been some attempts to create data bases [49]. But in many cases, a screening

with several columns and modifiers is mandatory [3]. Raimbault et al. published an

analytical method for the separation and analysis of underivatized free amino acids

using SFC coupled to a MS analyzer. This method had two tiers: a) a generic

method allowing satisfactory elution of amino acids and b) resolution of D- and L-

amino acids enantiomeric pairs. They used Chiralpak ZWIX (+) and (-) stationary

phases and unified chromatography joining SFC and HPLC (wide gradient from 10 to

100% of co-solvent in CO2) [50]. Studies comparing the efficiency of ESI and APCI in

the analysis of free amino acids using SFC-MS/MS revealed that MeOH is the

modifier of choice regarding elution strength and ionization efficiency in positive and

negative ion mode. In negative ion mode highest signal responses were observed by

employing NH4OAc as additive, whereby NH4FA and H2O is the additive of choice in

positive ion mode. Generally, ion enhancement as matrix effect were observed using

SFC-APCI-MS/MS, whereby ion suppression was observed for SFC-ESI-MS/MS in

positive ion mode. Generally, for apolar amino acids and the metabolites derived

from the tryptophan pathway SFC-ESI-MS/MS seems to be the ionization technique

of choice. On the other hand for polar amino acids and apolar amino acids with an



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additional heteroatom as well as acidic amino acids SFC-APCI-MS/MS seems to be

the ionization technique of choice [51].

Neurotransmitters. Melatonin and N-acetyl-serotonin were successfully and

reproducible analyzed in human serum samples within 3 minutes on an ethylene

bridged hybrid (BEH) column [52].

Lipids. In the field of lipids SFE has been widely utilized. The critical temperature of

CO2 makes it an ideal solvent for extracting thermally sensitive materials. SFE

involving SCCO2 is also effective for the discovery of new natural compounds

because undesirable hydrolysis, oxidations, degradation and rearrangements do not

occur. As opposed to other organic solvents that can contaminate the product, CO2

can be easily eliminated from the sample by simple expansion to atmospheric

pressure which eliminates the sample concentration process which is usually time

consuming. Hence, SFE has a great application in lipids extractions from food

sources in the industry. Finally, SFE process has a beneficial impact in the

environment since the utilization of contaminant organic extraction solvents is largely

reduced or eliminated [53]. The special features of its mobile phase (polarity) makes

SFC suitable for the separation of hydrophobic compounds and isomers [54].

Lee et al. developed a method for the determination of 10 phospholipid classes by

trimethylsilylation (TMS) derivatization. Derivatization improved peak shape and

sensitivity to phosphatidic acid (PA) phosphatidylinositol (PI), lysophosphatidic acid

(LPA), lysophosphatidylinositol (LPI) and sphingosine-1-phosphate (So1P). The

compounds of interest were separated on an octadecylsilane (ODS) column, and

methanol 0.1% (v:w) ammonium formate was employed as modifier. Derivatization

greatly improved peak shape and chromatographic separation [55]. In a follow up

article, the same group applied trimethylsilyldiazomethane (TMSD) methylation and



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extended the method to phosphatidylserine (PS), lysophosphatidylserine (LPS) and

non-methylated ceramides (Cer), sphingosine (So) and sphinganine (Sa) [56].

Yamada et al. coupled a SFC chromatograph to an orbitrap Fourier transform mass

spectrometer (orbitrap FT-MS). Using an ODS column it was possible to resolve lipid

species according not only to their fatty acyl moiety but also their polar head groups.

Moreover, the method successfully resolved isomeric forms of phosphatidylcholine

(PC) and phosphatidylethanolamine (PE) [57]. In 2005, again Bamba’s group

published a lipidomic study on plasma lipoprotein fractions in myocardial infarction-

prone rabbits using orbitrap high resolution mass spectrometer coupled to a SFC.

Plasma levels of each lipid class was found to be significantly higher in the disease

model group. Elevated levels of plasmalogens and PEs were found in the low-

density liporprotein (LDL) fraction when compared to the very-low-density lipoprotein

(VLDL) fraction in the disease model rabbit [58]. The same authors expanded their

early targeted lipidomic methods by integrating an on-line home-made SFE system

for dried plasma spot disks [59]. 134 phospholipids were extracted and quantified

from 3 µL of dried plasma. SCCO2, because its diffusivity, low viscosity and polarity it

is ideal for the extraction of lipophilic compounds. The on-line SFE-SFC-MS/MS

system was assembled by placing the dry blood spots disks in a mini guard column,

acting as extraction cell. Five column ports in the column oven were utilized as the

extraction cell positions. Extraction was carried out by the mobile phase flowing

through the extraction cell continuously and the extracts were concentrated on top of

the column. After extraction the extraction cell was delinked from the flow path by a

switching valve. Phospholipids were separated on a hydrophilic interaction liquid

chromatography (HILIC) column that has a phosphorylcholine group modifying the

silica body [59]. Oxidized PCs (hydroxy-, peroxy- and hydroperoxy-) were separated



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by SFC using a 2-ethylpyridine normal-phase column. The positional isomers of

hydroperoxides and epoxides were indentified using multiple reaction monitoring

(MRM). The method was applied to the analysis of these lipid species in mouse

livers [60]. In 2002, Sandra et al. published a method for the determination of TGs

using silver ion (SI) pcSFC-MS. A mixture of AN/IPA (6/4; v:v) was used as modifier

and methanol as post column make-up solvent. APCI and coordination ion spray

(CIS) with silver ions were used as ionization modes. The method resulted effective

for the separation of TGs. However, the separation depends on the injection of silver

ions before every analysis, which can affect the reproducibility of analysis.

Additionally, approximately 90 min is required to analyze one sample [61]. High

resolution separation of 20 pairs of TGs isomers was achieved by using a triacontyl

(C30) silica gel reverse phase column and a triple quadrupole (QqQ) mass

spectrometer operating in MRM mode [62]. The use of chromatographic columns

packed with superficially porous particles improve the separation of isomeric pairs of

TGs using ACN as modifier at low temperature [63]. Bamba et al. developed a

method for the analysis of 14 lipid classes including (PC, PE, PI, phospatidylglycerol

(PG), PS, lysophosphatidylcholine (LPC), TG, diacylglycerol (DG), mono- and

diglycodiacylglycerols, Cer, sphingomyelin (SM) and cerebrosides). The detector

was a single quadrupole mass spectrometer. All the lipids were separated on a

cyano column in less than 15 minutes. MeOH with 0.1% (v:w) ammonium formate

was used as modifier and as make-up solvent. A two dimensional (2D) profile was

constructed which enabled a reliable identification of individual TGs. The 2D profile

was constructed with retention time plotted on the X axis and m/z values on the Y

axis and abundance was shown by the density. The method was applied to the

analysis of lipid plant extracts and it demonstrated its usefulness for high-troughput



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determination of lipid profile [64]. In a follow up article, the same group reported the

analysis of TGs in soybean. The different TG subspecies were separated using three

monolithic silica gel ODS columns connected in series. Methanol 0.1% w/v formic

acid was employed as modifier and as post column make-up solvent. The detector

was a single quadrupole mass spectrometer. The same 2D profile was constructed

which enabled a reliable identification of individual TGs. Moreover, the authors

applied programmed cone voltages from 30 to 90 V, each TG ion were assigned DG

and monoacylglycerol (MG) fragments. The free fatty acid (FFA) composition of TGs

could be determined differentiation between isobaric species was achieved [65].

Ashraf et al. reported the separation and quantitation of underivatized FFA by

UHPSFC and a ODS sub-2-µm column coupled to evaporative light scattering

detector (ELSD) and a high resolution quadupole-time-of-flight (Qq-TOF) mass

spectrometry detector. Isobaric FFA with different chain positions of the double

bonds (e.g., 18:3 (Δ6, 9, 12) and C18:3 (Δ9, 12, 15). The lipids were separated

within 7 minutes which is a much faster separation when compared to GC-MS

conventional methods. Moreover, esterification of FFA is not required [66]. One must

recognize the invaluable contribution of Bamba’s group to the field of lipidomics

using SFC-MS. More recently, Takeda et al. from Bamba’s group published a widely-

targeted quantitative lipidomics method using a diethylamine (DEA) column. The

modifier was MeOH with 0.1% (w/v) ammonium acetate. Peak shape of PA, LPA, PS

and LPS were improved by the addition of 5% water to the modifier solution.

Moreover, separation of positional isomers of lysophospholipid species was

achieved. The article includes a MRM library for over 500 lipid species including

FFA, MG, DG, TG, PC, PE, PI, PG, PS, alkyl-acyl PC, alkenyl-acyl PC, alkenyl-acyl

PE and their corresponding lysoforms as well as cholesterol esters (CE) and



17

sphingolipids. The method was applied to the analysis of plasma from rabbit that

were supplemented with eicopentaenoic acid (EPA) during 5 weeks. Lipid molecular

species with an EPA side chain were dramatically increased by approximately 41-

fold, and lipid molecular species with a docosapentaenoic acid (DPA) side chain

were also increased by 5.7 fold as a consequence of the extension of the EPA chain

by the action of elongase-2. On the contrary, the total levels of several lipid classes

with high percentages of incorporation of linoleic acid decreased by EPA

supplementation (LPC, LPE, PC, alkenyl-acyl PC, PE, PI, DG, and TG) as a global

effect due to the reduction of linoleic acid levels (Figure 2) [67]. Lisa and Holcapek

developed a comprehensive lipidomics method using UHPSFC-QTOF-MS on a 1.7

µM BEH column. 436 lipids from 24 different lipid classes were identified and

quantified in 7 minutes. The method was validated with porcine brain extracts (Figure

3) [68]. Al Hamimi et al. performed a comprehensive stationary phase screening for

global lipid profiling by UHPSFC. Diol columns showed improved resolution when

compared with columns having ß-amino alcohol ligands. Non-polar columns

displayed very poor retention of polar lipids. Diol columns provided within lipid class

separation (not achievable with normal-phase-LC (NP-LC) or HILIC) as well as

between lipid class separation (not feasible with reverse-phase-LC (RP-LC)) [69].

The analysis of fifteen estrogen metabolites was achieved in less than ten minutes.

The described HPLC method at that time was almost one-hour long. The analytes

were derivatized with dansyl chloride and two columns connected in series,

cyanopropyl and diol, were used to resolve the metabolites of interest. A QqQ

instrument was use as the detector. The authors report sensitivity level equivalent to

that obtained by HPLC [70]. Quanson et al. reported the high-throughput analysis of

19 endogenous androgenic steroids using unified chromatography. Separation was



18

achieved within 5 minutes on a 1.7 µm BEH 2-ethylpyridine (2-EP) column.

Moreover, the method displayed superior compound resolution and sensitivity when

compared to UHPLC-RP [71]. De Kock et al. published a method for the separation

and quantifications of 19 steroids from the four major classes (estrogens, androgens,

progestogens and corticosteroids). The separation was achieved in 5 minutes on an

Acquity UPC2 BEH column using MeOH/IPA (1:1; v:v) containing 0.1% Formic acid.

0.1% Formic acid in MeOH was used as make-up solvent. Metabolites were

derivatized with methoxyamine hydrochloride which allows the elimination of excess

of reagent by evaporation. The method was applied to the analysis of plasma

samples from breast cancer patients [72]. Separation of both glucuronide and sulfate

steroids was successfully achieved on both BEH and BEH-2EP although the run

times to obtain the separation of both classes of compounds was 40 minutes. To

provide high-throughput analysis of sulfate steroids was performed on the BEH 2-EP

and glucuronide steroids was achieved on the BEH. Both classes of compounds

were separated in less than 9 minutes. The sample preparation performed is based

on a strong-anion-exchange SPE method which enables the efficient separation of

glucuronated and sulfated esteroids, making this approach practical to the

application [73]. The described UHPSFC-MS/MS method was applied to the analysis

of urine samples of animals treated with steroids. UHPFC provided had better

performance in terms of sensitivity and repeatability compared with UHPLC-MS/MS

[73].

SFC separations of structural isomers of carotenoids was successfully achieved with

a monolithic silica column. This column provided an efficient and fast separation.

Moreover, the low viscosity of SFC mobile phase allowed the employment of long

monolithic columns or connecting three monolithic columns achieving excellent



19

compound resolution [74]. The same group published in 2011 a high-throughput and

high-sensitivity profiling system for β-cryptoxanthin (βCX) and β-cryptoxanthin fatty

acid ester (βCXFA) [75]. The separation and quantitation of and βCXFA was

achieved using a polymeric type ODS column. MeOH 0.1% ammonium formate w/v

was used as modifier and as make-up post column solvent. The detector was a QqQ

mass spectrometer operating the ion source in ESI positive mode. In 2012 the same

authors expanded to the analysis of epoxidiced carotenoids and the method was

successfully applied to human plasma samples and low-density lipoprotein samples

[76]. More recently a qualitative and quantitative profiling of carotenoids and

apocarotenoids in intact human blood employing SFE-SFC-MS/MS was carried out

[77]. Zeaxanthin, βCX, β-carotene, and capsanthin, and some apocarotenoids

(carotenoids oxidative and enzymatic cleavage products) were directly extracted and

identified, some of them for the first time. 10 µL of whole blood ere directly loaded

into the available vessel in the SFE instrument (Shimadzu Corporation, JP).

Compound separation was achieved on a C30 150 mm x 4.6 mmx 2.7 µm column

[77].

25 conjugated and unconjugated Bile acid species were resolved and analyzed

using an amide 1.7 µm particle size column and MeOH/water (95:5, v:v) with 0.2%

ammonium formate (w:v) and 0.1% formic acid (v:v) as modifier. The detector was a

QqQ mass spectrometer operating the ion source in ESI negative mode. The method

was applied to the analysis of rodent serum samples with a simple methanol

extraction sample preparation procedure [24].

Oxylipins, including eicosanoids, docosanoids, and octadecanoids, are an important

family of lipids, which are formed by the oxidation of polyunsaturated fatty acids

(PUFA). Berkecz, et al. developed an UHPSFC-ESI-MS method with the ion source



20

operating in negative mode. The optimum separation was achieved with a 1-

aminoantrhacene column. 46 oxylipin standards were screened. UHPSFC enabled

the separation of only 20 isobaric oxylipins while UHPLC provided the baseline

separation of 24 isobaric species. Moreover, the LC method resulted to be

considerably more sensitive [78].

Hexosylceramides consist of both glucosylceramides and galactosylceramides,

which cannot be distinguished by RP-HPLC-MS/MS. Both diastereomers can be

resolved and quantified by SFC-MS/MS on a silica column using MeOH as modifier.

This methodology is used in our Metabolic Phenotyping Core facility at UT

Southwestern Medical Center as part of the services offered at the research

community. This technique was applied to study renal gycosphingolipid metabolism

in lupus nephritis. The analyses were provided by the Medical University of South

Carolina Lipidomics Core Facility. Most of the glucosylceramide species were found

to be significantly elevated, whereas galactosyl species remained unchanged [79].

Gangliosides are a species of glycosphingolipids with one or more sialic acids linked

to a galactose residue or an N-acetylgalactosamine residue in a carbohydrate

moiety.

Gangliosides have complicated structures consisting of hydrophilic oligosaccharide

chains, ceramides as lipophilic fatty acid chains and ionic sialic acid residues.

Variation of either the polar or the ceramide chain generates structural diversity. The

diversity and complexity result in numerous difficulties in separation and analysis.

Recently, gangliosides profiling in swine brain extracts was accomplished by offline

2D SFC on a diol column x RP-LC on a ODS column. The detector was a Q-TOF

mass spectrometer. The different fraction collected by SFC were further analyzed by

LC/MS [80].



21

Vitamins. Bamba’s group reported the simultaneous analysis for water- and fat-

soluble vitamins by unified supercritical fluid chromatography and liquid

chromatography which bridges SFC and LC. The gradient of the mobile phase

started at almost 100% SCCO2 and was completely replaced with 100% methanol at

the end. 17 vitamins were separated in 4 minutes using non-end-capped ODS

column and methanol/water (95:5, v:v) with 0.2% (w:v) ammonium formate [81].

Vitamin D isomeric metabolites were quantified in human plasma samples by SFC-

LC-MS/MS on a 3 µm cellulose column in 6 minutes. Chiral metabolites

23,25(OH)2D3, 24,25(OH)2D3 and 1α,25(OH)2D3 along with the c3-epimer 3-epi-

25OHD3 from 25OHD2, and 24OHD2 from 25OHD2. A more sensitive method

employing metabolite derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD)

was also described [82]. A sensitive high-throughput analysis of vitamin K1 in human

samples was described by Sandvik et al. [83]. The sample preparation was

performed semi-automatically and consisted on a 96-well SPE plate (Oasis

hydrophilic-lipophilic balance (HLB) PRIME). The employed chromatography column

was a sub-2-µm Acquity UPC2 Torus 1-Amino Anthracene (1-AA). Compound

analysis and column re-equilibration was achieved in less than 4 minutes. The

method was successfully applied to clinical plasma samples [83].

Polar metabolites. Akbal et al. demonstrated the practicability of SFC-MS for the

analysis of polar metabolites in urine. 74 metabolites could be detected in a single

analysis separated on a diol column and the use of a metabolomics library. The

combination SFC with high-resolution mass spectrometry becomes particularly

attractive with data independent acquisition mode in particular sequential window

acquisition of all theoretical mass spectra (SWATH/MS) as it enables to obtain

qualitative and quantitative information in a single analysis at the MS2 level. With



22

SWATH/MS, the sample can be re-interrogated at any time. When ultimate

sensitivity is required, these methods could be easily transferred to triple quadrupole

instruments [84].

Simultaneous analysis of hydrophilic and lipophilic substances. In recent years there

have been some efforts to expand SFC-MS to global metabolomics. This application

is where the great potential of SFC in the future resides. Desfontaine et al. used

unified chromatography gradient from 2 to 100% organic modifier in CO2 to cover the

widest possible range of analytes. The investigators covered a wide range of

stationary phases chemistries and found that taking into account the kinetic

performances and upper pressure limit of the SFC system, the most appropriate

stationary phase technology to work with unified gradient was the columns packed

with sub-3-µm superficially porous particles, due to their high efficiency and the

limited generated pressure. In terms of chemistry, the best stationary phases for this

application is HILIC. The use of a sample diluent containing 50% ACN as injection

solvent was fundamental for this application. The authors concluded that for the

mobile phase, a significant amount of salts was necessary to increase solubility,

decrease retention and obtain suitable peak shapes for ionizable species. They

accomplished the simultaneous analysis of 57 metabolic compounds covering a wide

range of chemical classes, such as nucleosides, nucleotides, small organic acids,

small bases, sulfated/sulfonated metabolites, poly-alcohols, lipid related substances,

quaternary ammonium metabolites, phosphate-based substances, carbohydrates

and amino acids (Figure 4) [85]. In a follow up article, the same group expanded this

application to the evaluation of the impact of biological matrices commonly analyzed

in metabolomics, such as urine or plasma using UHPSFC coupled to a high

resolution MS instrument. Only a limited number of compounds displayed matrix



23

effect in both matrices (30% in plasma; 25% in urine). Ion suppression was the main

source of matrix effect for urine. Additionally, the authors examined commercial

mass spectrometry library of compounds containing almost 600 metabolites (Sigma

Mass Spectrometry Metabolite library of Standards, Sigma-Aldrich, St. Louis, MO).

The detection success was of 66%. However, poor performance was found for

phosphorylated metabolites, and nucleotides [86]. Perrenoud et al. demonstrated

that UHPSFC-QTOF-MS is a suitable tool for the analysis of 120 secondary

metabolites from plant extracts. These metabolites are bioactive agents with

pharmacological applications. They selected a library of compounds displaying a

broad range of physical properties (covering more than 18 logP orders of magnitude)

including, alkaloids, amino acids, fatty acids and steroids among others. In addition,

the authors performed an extensive and systematic study on column stationary

phase chemistries [87].

Recently Suzuki et al. used online SFE-SFC to analyze the disease biomarkers in

dried serum spots, with the aim to apply the relative differences in biomarkers to

disease diagnosis. In this study, four hydrophilic metabolites and 17 hydrophobic

metabolites were simultaneously detected within 15 min, and they exhibited

comparable diagnostic performance to the serum analysis using LC-MS/MS [88].

Proteins and peptides. The major limitation of SFC in the separation and analysis of

peptides and proteins is the low solubility of these biomolecules in organic solvents

typically required for SFC-MS [89, 90]. By modifying the SFC solvent with a variety of

polar additives, it is possible to exploit the improved speed, resolution, and normal

phase properties of SFC for the separation of peptides with analysis by ESI-MS.

Solubility problems encountered in RP-HPLC solvents are avoided since SFC is

compatible with MeOH, trifluoroethanol (TFE), chloroform, and many of the other



24

solvents used to dissolve hydrophobic peptides and proteins [89]. Orwa et al.

demonstrated the rapid analysis of gramicidin and other peptides. A commercially

available sample of gramicidin D, a heterogeneous mixture of six components was

analyzed by both HPLC-MS and SFC-MS. The SFC-MS system separated the three

forms of gramicidin (B, C, and A) in under 5 min whereas by HPLC-MS, it was

difficult to achieve any notable separation of the three in less than 30 min. MeOH

with 0.2% water, 0.01 M ammonium acetate, and 0.4% isopropylamine, was initially

found to produce the best separation of the gramicidin, and later it was found that

methanol with 0.5% TFE worked as well [89, 91]. SFC-MS was also applied to

alleviate Hydrogen/Deuterium back exchange in solution phase in proteomic

analyses [92]. In 2006 Zheng et al. screened the elution of polypeptides up to 40

residues using SFC [93]. The polypeptides contained a variety of acidic and basic

residues. Trifluoroacetic acid (TFA), a relatively strong acid, was used as additive in

the CO2/ MeOH mobile phase to suppress ionization of the peptides’ carboxylic acid

groups and to protonate the peptides’ amino groups. TFA acid likely protonates

some fraction of the employed pyridine functional groups on the ethylpyridine

stationary phase. Fronting peak shapes and lower retention were observed when

higher concentrations of TFA acid were used. The authors hypothesized that

electrostatic repulsion between the protonated peptides and some fraction of the

stationary-phase functional groups takes place. Three silica-based stationary

phases: a strong anion exchange, an amino, and an ethylpyridine stationary phase

were investigated. The ethylpyridine was the only one from which the peptides were

eluted. This was probably due to deactivation of the active silanol groups (as the

stationary phase was not end capped) by hydrogen bonding between the pyridine

functional groups on the stationary phase and the silanols, as well as limited access



25

to the silica surface due to steric hindrance caused by the pyridine aromatic ring. The

mobile phase is compatible with mass spectrometry analysis [93]. Membrane

proteins comprise 25-30% of the human genome and play critical roles in a wide

variety of important biological processes [94]. However, their hydrophobic nature has

compromised efforts at structural characterization by both X-ray crystallography and

mass spectrometry. The detergents that are generally used to solubilize membrane

proteins interfere with the crystallization process essential to X-ray studies and

cause severe ion suppression effects that hinder mass spectrometric analysis. In

2008 Zhang et al. reported the use of supercritical fluid chromatography-mass

spectrometry for the separation and analysis of integral membrane proteins and

hydrophobic peptides. It was shown that detergents were rapidly and effectively

separated from the proteins and peptides, yielding them in a state suitable for direct

mass spectrometric analysis [95]. The chromatographic separation was performed

on a SFC cyano-bonded silica, non-end-capped. Different mobile phase modifiers

consisted of either MeOH with 0.5% Trifluoroethanol (TFE), methanol with 0.1%

TFA, or methanol with CHCl3 and 1% aqueous formic acid (4:4:1; v:v:v). Gramicidin

and bacteriorhodopsin were purified from detergents and lipids by SFC, and high-

quality on-line mass spectra were acquired. Photosystem II was also investigated by

SFC-MS, and a total of 16 out of 26 core proteins eluted in 15 min. Enmark et al.

investigated the robustness of SFC separations of the peptide gramicidin, using

either isocratic or gradient elution [96]. The column stationary phase was a pH-stable

hybrid silica column (Kromasil SFC-2.5-XT) and using an eluent containing CO2,

water, and MeOH. The authors reported that in gradient elution, the separation is at

least three times more robust to perturbations than in isocratic elution. Finally, the

methods were transferred to another laboratory. The results of the isocratic method



26

differed greatly between the laboratories, the main reasons for this being differences

in pressure and in the total co-solvent fraction between the systems. For the gradient

separation, the transfer was successful. The results clearly indicate that gradient

elution resulted in a considerably more robust separation system [96].

Supplementary Table 1 summarizes the methods and applications described in

Section 4.

5. Technical considerations Supercritical Fluid Chromatography-MS versus

HPLC-MS

SFC offers several potential advantages over LC regarding complementary

selectivity, which has given SFC great applications in enantioseparations, and rapid

analysis time as well as reduced solvent consumption with an environmental benefit

of using CO2. With modern SFC reliable instrumentation, scientists who are

experienced in the use of HPLC equipment will find that in terms of usability and

application, SFC is very similar. When the two techniques are compared, many

similarities are apparent: both can be used in different modes based on stationary

and mobile phase characteristics, organic modifiers and additives are used to adjust

selectivity, silica based porous or fused core particles can be employed and

separations are run in either gradient or isocratic mode. However, in SFC small

changes in pressure affect fluid density can have a strong effect on the analyte

retention. A backpressure regulator to keep the system pressure stable is a crucial

part of an SFC system. To increase metabolite ionization and sensitivity a make-up

solution can be introduced post column without affecting the chromatography or pH

column stability. One of the most challenging aspects of SFC is the fact that

retention mechanisms are not as well defined in SFC-MS as they are in LC making

the method development more empirical [18].



27

When comparing SFC-MS and LC-MS matrix effect, optimizing the post-column

make-up solution is fundamental [97] it also prevents compound precipitation in the

ion source when using less than 5% of modifier. From supercritical conditions to

gaseous state (room temperature, atmospheric pressure), the CO2 expands around

430 times, contributing to almost 5% of the nebulizing process. In positive mode, the

presence of ammonium ions either in the mobile phase or in the make-up did

significantly increase the MS signal, even at basic apparent pH. The ionization

performance of electrospray is influenced by the acidic buffer power of the carbon

dioxide, and was found to be restricted in the apparent pH range of 3.8–7.2 [97].

Svan et al. conducted an in depth study to determine the differences in matrix effects

using SFC and HPLC. Both chromatographic systems were coupled to a QqQ and a

Q-TOF mass spectrometers operating in ESI in positive and negative mode. A wide

range of matrices were tested using post extraction addition and post column

infusion methods. For LC, 65% of all the tested compounds were enhanced in all the

matrices compared to only 7% for SFC. Polar compounds from water-based samples

such as urine, add additional challenges for SFC [98]. Desfontaine et al. found a

lower susceptibility to matrix effect in SFC versus LC (reverse phase) particularly in

urine when a simple dilute and shoot clean-up procedure was performed. The type of

matrix effect observed in LC-MS/MS was generally a signal enhancement and an ion

suppression for urine and plasma samples, respectively. In the case of SFC-MS/MS,

the type of matrix effect randomly varied according to the analyzed matrix, selected

column and sample treatment [99].

Due to the lower viscosity of the mobile phase in SFC it is a common practice to

connect two or more columns in series. Tandem column (TC)-SFC is an economic

way to combine selectivity from multiple columns and in comparison to the complex



28

instrument setup required for 2D separations, TC-SFC can be conducted using a

conventional SFC instrument. Coupling columns of the same type in series is an

economic way to produce longer columns in the laboratory. Longer columns produce

a higher peak capacity and separation efficiency. When complete separation of all

components on one column type is not achievable, coupling columns with

complementary selectivity offers a potential solution. This approach is also

compatible with tandem coupling of chiral and achiral columns or two or more chiral

columns [100, 101].

6. Conclusions and Future Directions

Supercritical fluid chromatography by age could be considered a mature separation

technique but by knowledge of the separation process it lags well behind gas and

liquid chromatography. Both kinetic optimization and retention mechanisms seem

more complex and interdependent on the system operating conditions resulting in

the lack of a comprehensive model for column design. Most of what is known about

the factors that are responsible for separations is descriptive and at best semi-

empirical in supercritical fluid chromatography and in need of further study [102].

West et al. have recently published a couple of articles evaluating the effects of

common additives on polarity and acidity of the mobile phase [103] and absorption

on the stationary phase [104]. In the last section of this series of papers, the authors

anticipate the examination of the possible effects occurring between the analytes

and additives [104].

Along with the commercialization of a new generation of instruments, SFC gains

improved performance, reliability and robustness, and the on-line coupling SFC

system has been booming recently. So far, SFE and SPE are the two major sample

preparation techniques which have been online coupled with SFC. The SPE-SFC



29

system consists of an SPE module and an SFC module. The configuration of the

SPE-SFC system is quite similar to that of the common online SPE-UHPLC system

because both of them use the valve switching strategy [105]. SFE-SFC-MS or SFE-

SFC-MS/MS could become a fundamental tool for comprehensive lipid profiling [53,

88]. Analysis of drugs and drugs metabolites in dry urine samples is also achievable

with this technique [106, 107]. Recently, Wicker et al. reported a multivariate

approach for on-line SFE-SFC-MS/MS for method development with the aim to

better understand the synergistic relationship between the extraction and separation

processes by focusing on the optimization of extraction parameters for target

analytes with a wide range of physicochemical properties in matrices of variable

retentivity. The authors anticipate to expand their studies including analyte

descriptors (log D or Abraham solvation parameters), different column chemistries

and the examination of the trapping efficiencies of different stationary phases for on-

line coupling of SFE to SFC [108].

SFC has already shown its potential for untargeted metabolomics by coupling the

chromatographer to a high resolution mass spectrometer [6, 12, 65, 87, 88, 109,

110].

Modern and most advanced instrumentation has the capability to perform unified

chromatography which allows to obtain a wide range of solvent polarity, the modifier

composition can range from 0 to 100% when using pcSFC. Applying this approach

may enable the separation and simultaneous analysis of diverse polarity compounds

that are typically analyzed not only with different methods but with different

separation techniques. Unified chromatography continuously shifts the physical state

of the mobile phase without phase separation by controlling temperature and back

pressure. This expands the convergence regions among GC, SFC and LC for



30

metabolite analysis [6]. Indubitably SFC hyphenated to MS is a complementary

analytical technique that has become an essential tool in the metabolomics

laboratory.

In the world of peptides and proteins, the reduced number of publications to date

provide preliminary work and, to this author’s knowledge, applications to the analysis

of biological samples has not been reported [94-96]. This is a field that will require

extensive exploration.

Other area where a major development is foreseen in the near future, is the

implementation of multi-dimensional chromatography systems incorporating SFC as

one of more dimensions [110].

Acknowledgement

The author thanks Dr. Kevin A. Schug, Dr. Christy Gliniak and Dr. Philipp E. Scherer

for useful discussions. RG is supported by US National Institute of Health (NIH) grant

P01 DK088761.

Conflict of Interest: The author declares no conflict of interest.



31

References

[1] Klesper E., Corwin A. H., Turner D. A., High Pressure Gas Chromatography

above Critical Temperatures. J. Org. Chem. 1962, 27, 2.

[2] Saito M., History of supercritical fluid chromatography: Instrumental development.

J. Biosci. Bioeng. 2013, 115, 590-599.

[3] Speybrouck D., Corens D., Argoullon J. M., Screening Strategy for Chiral and

Achiral Separations in Supercritical Fluid Chromatography Mode. Curr. Top. Med.

Chem. 2012, 12, 1250-1263.

[4] Beckman E. J., Supercritical and near-critical CO2 in green chemical synthesis

and processing. J. Supercrit. Fluids 2004, 28, 121-191.

[5] Magalhaes A. L., Vaz R. V., Goncalves R. M. G., Da Silva F. A., Silva C. M.,

Accurate hydrodynamic models for the prediction of tracer diffusivities in supercritical

carbon dioxide. J. Supercrit. Fluid 2013, 83, 15-27.

[6] Taguchi K., Fukusaki E., Bamba T., Supercritical fluid chromatography/mass

spectrometry in metabolite analysis. Bioanalysis 2014, 6, 1679-1689.

[7] Ikushima Y., Saito N., Arai M., Arai K., Solvent Polarity Parameters of

Supercritical Carbon-Dioxide as Measured by Infrared-Spectroscopy. B. Chem. Soc.

Jpn. 1991, 64, 2224-2229.

[8] Haselber R., Pirok, B. W. J., Gargano, A. F. G., Kholer, I., Clinical Metabolomics:

Expanding the Metabolome Coverage Using Advanced Analytical Techniques. LC

GC Eur. 2019, 32, 465-480.

[9] Tarafder A., Metamorphosis of supercritical fluid chromatography to SFC: An

Overview. Trends Analyt. Chem. 2016, 81, 3-10.



32

[10] Desfontaine V., Guillarme D., Francotte E., Novakova L., Supercritical fluid

chromatography in pharmaceutical analysis. J. Pharm. Biomed. Anal. 2015, 113, 56-

71.

[11] Kalikova K., Slechtova T., Vozka J., Tesarova E., Supercritical fluid

chromatography as a tool for enantioselective separation; a review. Anal. Chim. Acta

2014, 821, 1-33.

[12] Matsubara A., Fukusaki E., Bamba T., Metabolite analysis by supercritical fluid

chromatography. Bioanalysis 2010, 2, 27-34.

[13] Dispas A., Jambo H., Andre S., Tyteca E., Hubert P., Supercritical fluid

chromatography: a promising alternative to current bioanalytical techniques.

Bioanalysis 2018, 10, 107-124.

[14] Shulaev V., Isaac G., Supercritical fluid chromatography coupled to mass

spectrometry - A metabolomics perspective. J. Chromatogr. B 2018, 1092, 499-505.

[15] Novotny M., Springton S.R., Peaden P. A., Fjeldsted J., Lee M. L., Capillary

supercritical fluid chromatography. Anal. Chem. 1981, 53, 407-414.

[16] Smith R. D., Fjeldsted J. C., Lee M. L., Direct Fluid Injection Interface for

Capillary Supercritical Fluid Chromatography-Mass Spectrometry. J. Chromatogr.

1982, 247, 231-243.

[17] Tarafder A., Designs and methods for interfacing SFC with MS. J. Chromatogr.

B 2018, 1091, 1-13.

[18] Schad G. J., SFC-MS versus LC-MS Advantages and Challenges.

Chromatography Today 2019.

[19] Akbal L., Hopfgartner G., Hyphenation of packed column supercritical fluid

chromatography with mass spectrometry: where are we and what are the remaining

challenges? Anal. Bioanal. Chem. 2020, 412, 6667-6677.



33

[20] Fujito Y., Hayakawa Y., Izumi Y., Bamba T., Importance of optimizing

chromatographic conditions and mass spectrometric parameters for supercritical

fluid chromatography/mass spectrometry. J. Chromatogr. A 2017, 1508, 138-147.

[21] Klink D., Schmitz O. J., SFC-APLI-(TOF)MS: Hyphenation of Supercritical Fluid

Chromatography to Atmospheric Pressure Laser Ionization Mass Spectrometry.

Anal. Chem. 2016, 88, 1058-1064.

[22] Li F., Hsieh Y., Supercritical fluid chromatography-mass spectrometry for

chemical analysis. J. Sep. Sci. 2008, 31, 1231-1237.

[23] Lisa M., Cifkova E., Holcapek M., Lipidomic profiling of biological tissues using

off-line two-dimensional high-performance liquid chromatography-mass

spectrometry. J. Chromatogr. A 2011, 1218, 5146-5156.

[24] Taguchi K., Fukusaki E., Bamba T., Simultaneous and rapid analysis of bile

acids including conjugates by supercritical fluid chromatography coupled to tandem

mass spectrometry. J. Chromatogr. A 2013, 1299, 103-109.

[25] Pinkston J. D., Wen D., Morand K. L., Tirey D. A., Stanton D. T., Comparison of

LC/MS and SFC/MS for screening of a large and diverse library of pharmaceutically

relevant compounds. Anal. Chem. 2006, 78, 7467-7472.

[26] Cho Y., Choi M. H., Kim B., S. K., Supercritical fluid chromatography coupled

with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass

spectrometry for compound speciation. J. Chromatogr. A 2016, 1444, 123-128.

[27] DaSilva J. O., Coes B., Frey L., Mergelsberg I., McClain R., Nogle L., J. W. C.,

Evaluation of non-conventional polar modifiers on immobilized chiral stationary

phases for improved resolution of enantiomers by supercritical fluid chromatography.

J. Chromatogr. A 2014, 1328, 98-103.



34

[28] Miller L., Use of dichloromethane for preparative supercritical fluid

chromatographic enantioseparations. J. Chromatogr. A 2014, 1363, 323-330.

[29] Lee J., Lee J. T., Watts W. L., Barendt J., Yan T. Q., Huang Y., Riley F., Hardink

M., Bradow J., Franco P., On the method development of immobilized

polysaccharide chiral stationary phases in supercritical fluid chromatography using

an extended range of modifiers. J. Chromatogr. A 2014, 1374, 238-246.

[30] Berger T., Berger B., E. M. R., A Review of Column Developments for

Supercritical Fluid Chromatography. Lc Gc N. Am. 2010, 28, 344-357.

[31] Sen A., Knappy C., Lewis M. R., Plumb R. S., Wilson I. D., Nicholson J. K.,

Smith N. W., Analysis of polar urinary metabolites for metabolic phenotyping using

supercritical fluid chromatography and mass spectrometry. J. Chromatogr. A 2016,

1449, 141-155.

[32] Regalado E. L., Welch C. J., Separation of achiral analytes using supercritical

fluid chromatography with chiral stationary phases. Trends Analyt. Chem. 2015, 67,

74-81.

[33] Perrenoud A. G.-G., Veuthey J.-L., Gillarme D., The use of columns packed with

sub-2 micron particles in supercritical fluid chromatography. Trends Anal. Chem.

2014, 63, 44-54.

[34] Novakova L., Perrenoud A. G., Francois I., West C., Lesellier E., Guillarme D.,

Modern analytical supercritical fluid chromatography using columns packed with sub-

2 micron particles: a tutorial. Anal. Chim. Acta. 2014, 824, 18-35.

[35] West C., Lesellier E., Characterization of stationary phases in subcritical fluid

chromatography by the solvation parameter model I. Alkylsiloxane-bonded stationary

phases. J. Chromatogr. A 2006, 1110, 181-190.



35

[36] West C., Lesellier E., Characterisation of stationary phasese in subcritical fluid

chromatography with solvation parameter model IV - Aromatic stationary phases. J.

Chromatogr. A 2006, 1115, 233-245.

[37] West C., Lesellier E., Tchapla A., Retention characteristics of porous graphitic

carbon in subcritical fluid chromatography with carbon dioxide-methanol mobile

phases. J. Chromatogr. A 2004, 1048, 99-109.

[38] West C., Lesellier E., Effects of modifiers in subcritical fluid chromatography on

retention with porous graphitic carbon. J. Chromatogr. A 2005, 1087, 64-76.

[39] West C., Lesellier E., Characterisation of stationary phases in subcritical fluid

chromatography with the solvation parameter model III. Polar stationary phases. J.

Chromatogr. A 2006, 1110, 200-213.

[40] West C., Khater S., Lesellier E., Characterization and use of hydrophilic

interaction liquid chromatography type stationary phases in supercritical fluid

chromatography. J. Chromatogr. A 2012, 1250, 182-195.

[41] West C., Lesellier E., Characterisation of stationary phases in subcritical fluid

chromatography by the solvation parameter model II. Comparison tools. J.

Chromatogr. A 2006, 1110, 191-199.

[42] West C., Lesellier E., Characterisation of stationary phases in supercritical fluid

chromatography with the solvation parameter model - V. Elaboration of a reduced

set of test solutes for rapid evaluation. J. Chromatogr. A 2007, 1169, 205-219.

[43] Khater S., West C., Lesellier E., Characterization of five chemistries and three

particle sizes of stationary phases used in supercritical fluid chromatography. J.

Chromatogr. A 2013, 1319, 148-159.



36

[44] De Klerck K., Mangelings D., Vander Heyden Y., Supercritical fluid

chromatography for the enantioseparation of pharmaceuticals. J. Pharm. Biomed.

Anal. 2012, 69, 77-92.

[45] Galea C. M., Vander Heyden Y., Mangelings D., in: Poole, C. F. (Ed.),

Supercritical Fluid Chromatography. Elsevier Inc., Cambridge, MA 2017, pp. 345-

379.

[46] Mangelings D., Vander Heyden Y., Chiral separations in sub- and supercritical

fluid chromatography. J. Sep. Sci. 2008, 31, 1252-1273.

[47] Chen J., Hsieh Y., Cook J., Morrison R., Korfmacher W. A., Supercritical fluid

chromatography-tandem mass spectrometry for the enantioselective determination

of propranolol and pindolol in mouse blood by serial sampling. Anal. Chem. 2006,

78, 1212-1217.

[48] Li L., Enantiomeric Determination of Methamphetamine, Amphetamine,

Ephedrine, and Pseudoephedrine usin Chiral Supercritical Fluid Chromatography

with Mass Spectrometric Detection. Microgram J. 2015, 12, 19-30.

[49] Perrin C., Matthijs N., Mangelings D., Granier-Loyaux C., Maftouh M., Massart

D. L., Vander Heyden Y., Screening approach for chiral separation of

pharmaceuticals Part II. Reversed-phase liquid chromatography. J. Chromatogr. A

2002, 966, 119-134.

[50] Raimbault A., Dorebska M., West C., A chiral unified chromatography-mass

spectrometry method to analyze free amino acids. Anal. Bioanal. Chem. 2019, 411,

4909-4917.

[51] Wolrab D., Fruhauf P., Gerner C., Direct coupling of supercritical fluid

chromatography with tandem mass spectrometry for the analysis of amino acids and



37

related compounds: Comparing electrospray ionization and atmospheric pressure

chemical ionization. Anal. Chim. Acta 2017, 981, 106-115.

[52] Wolrab D., Fruhauf P., Gerner C., Quantification of the neurotransmitters

melatonin and N-acetyl-serotonin in human serum by supercritical fluid

chromatography coupled with tandem mass spectrometry. Anal. Chim. Acta 2016,

937, 168-174.

[53] Lee J. W., Fukusaki E., Bamba T., Application of supercritical fluid carbon

dioxide to the extraction and analysis of lipids. Bioanalysis 2012, 4, 2413-2422.

[54] Song S. Y., Liu H. W., Bai Y., Supercritical Fluid Chromatography and Its

Application in Lipid Isomer Separation. J. Anal. Test. 2017, 1, 330-334.

[55] Lee J. W., Yamamoto T., Uchikata T., Matsubara A., Fukusaki E., Bamba T.,

Development of a polar lipid profiling method by supercritical fluid

chromatography/mass spectrometry. J. Sep. Sci. 2011, 34, 3553-3560.

[56] Lee J. W., Nishiumi S., Yoshida M., Fukusaki E., Bamba T., Simultaneous

profiling of polar lipids by supercritical fluid chromatography/tandem mass

spectrometry with methylation. J. Chromatogr. A 2013, 1279, 98-107.

[57] Yamada T., Uchikata T., Sakamoto S., Yokoi Y., Nishiumi S., Yoshida M.,

Fukusaki E., Bamba T., Supercritical fluid chromatography/Orbitrap mass

spectrometry based lipidomics platform coupled with automated lipid identification

software for accurate lipid profiling. J. Chromatogr. A 2013, 1301, 237-242.

[58] Takeda H., Koike T., Izumi Y., Yamada T., Yoshida M., Shiomi M., Fukusaki E.,

Bamba T., Lipidomic analysis of plasma lipoprotein fractions in myocardial infarction-

prone rabbits. J. Biosci. Bioeng. 2015, 120, 476-482.

[59] Uchikata T., Matsubara A., Fukusaki E., Bamba T., High-throughput

phospholipid profiling system based on supercritical fluid extraction-supercritical fluid



38

chromatography/mass spectrometry for dried plasma spot analysis. J. Chromatogr. A

2012, 1250, 69-75 DOI: 10.1016/j.chroma.2012.06.031.

[60] Uchikata T., Matsubara A., Nishiumi S., Yoshida M., Fukusaki E., Bamba T.,

Development of oxidized phosphatidylcholine isomer profiling method using

supercritical fluid chromatography/tandem mass spectrometry. J. Chromatogr. A

2012, 1250, 205-211.

[61] Sandra P., Medvedovici A., Zhao Y., David F., Characterization of triglycerides

in vegetable oils by silver-ion packed-column supercritical fluid chromatography

coupled to mass spectroscopy with atmospheric pressure chemical ionization and

coordination ion spray. J. Chromatogr. A 2002, 974, 231-241.

[62] Lee J. W., Nagai T., Gotoh N., Fukusaki E., Bamba T., Profiling of regioisomeric

triacylglycerols in edible oils by supercritical fluid chromatography/tandem mass

spectrometry. J. Chromatogr. B 2014, 966, 193-199.

[63] Lesellier E., Latos A., de Oliveira A. L., Ultra high efficiency/low pressure

supercritical fluid chromatography with superficially porous particles for triglyceride

separation. J. Chromatogr. A 2014, 1327, 141-148.

[64] Bamba T., Shimonishi N., Matsubara A., Hirata K., Nakazawa Y., Kobayashi A.,

Fukusaki E., High throughput and exhaustive analysis of diverse lipids by using

supercritical fluid chromatography-mass spectrometry for metabolomics. J. Biosci.

Bioeng. 2008, 105, 460-469.

[65] Lee J. W., Uchikata T., Matsubara A., Nakamura T., Fukusaki E., Bamba T.,

Application of supercritical fluid chromatography/mass spectrometry to lipid profiling

of soybean. J. Biosci. Bioeng. 2012, 113, 262-268.



39

[66] Ashraf-Khorassani M., Isaac G., Rainville P., Fountain K., Taylor L. T., Study of

UltraHigh Performance Supercritical Fluid Chromatography to measure free fatty

acids with out fatty acid ester preparation. J. Chromatogr. B 2015, 997, 45-55.

[67] Takeda H., Izumi Y., Takahashi M., Paxton T., Tamura S., Koike T., Yu Y., Kato

N., Nagase K., Shiomi M., Bamba T., Widely-targeted quantitative lipidomics method

by supercritical fluid chromatography triple quadrupole mass spectrometry. J. Lipid.

Res. 2018, 59, 1283-1293.

[68] Lisa M., Holcapek M., High-Throughput and Comprehensive Lipidomic Analysis

Using Ultrahigh-Performance Supercritical Fluid Chromatography-Mass

Spectrometry. Anal Chem 2015, 87, 7187-7195.

[69] Al Hamimi S., Sandahl M., Armeni M., Turner C., Spegel P., Screening of

stationary phase selectivities for global lipid profiling by ultrahigh performance

supercritical fluid chromatography. J. Chromatogr. A 2018, 1548, 76-82.

[70] Xu X., Roman J. M., Veenstra T. D., Van Anda J., Ziegler R. G., Issaq H. J.,

Analysis of fifteen estrogen metabolites using packed column supercritical fluid

chromatography-mass spectrometry. Anal. Chem. 2006, 78, 1553-1558.

[71] Quanson J. L., Stander M. A., Pretorius E., Jenkinson C., Taylor A. E., Storbeck

K. H., High-throughput analysis of 19 endogenous androgenic steroids by ultra-

performance convergence chromatography tandem mass spectrometry. J.

Chromatogr. B 2016, 1031, 131-138.

[72] de Kock N., Acharya S. R., Ubhayasekera S., Bergquist J., A Novel Targeted

Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid

Chromatography Hyphenated to Tandem Mass Spectrometry. Sci. Rep. 2018, 8,

16993.



40

[73] Doue M., Dervilly-Pinel G., Pouponneau K., Monteau F., Le Bizec B., Analysis of

glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid

chromatography hyphenated tandem mass spectrometry. Anal. Bioanal. Chem.

2015, 407, 4473-4484.

[74] Matsubara A., Bamba T., Ishida H., Fukusaki E., Hirata K., Highly sensitive and

accurate profiling of carotenoids by supercritical fluid chromatography coupled with

mass spectrometry. J. Sep. Sci. 2009, 32, 1459-1464.

[75] Wada Y., Matsubara A., Uchikata T., Iwasaki Y., Morimoto S., Kan K., Okura T.,

Fukusaki E., Bamba T., Metabolic profiling of beta-cryptoxanthin and its fatty acid

esters by supercritical fluid chromatography coupled with triple quadrupole mass

spectrometry. J. Sep. Sci. 2011, 34, 3546-3552.

[76] Matsubara A., Uchikata T., Shinohara M., Nishiumi S., Yoshida M., Fukusaki E.,

Bamba T., Highly sensitive and rapid profiling method for carotenoids and their

epoxidized products using supercritical fluid chromatography coupled with

electrospray ionization-triple quadrupole mass spectrometry. J. Biosci. Bioeng. 2012,

113, 782-787.

[77] Zoccali M., Giuffrida D., Salafia F., Giofre S. V., Mondello L., Carotenoids and

apocarotenoids determination in intact human blood samples by online supercritical

fluid extraction-supercritical fluid chromatography-tandem mass spectrometry. Anal.

Chim. Acta 2018, 1032, 40-47.

[78] Berkecz R., Lisa M., Holcapek M., Analysis of oxylipins in human plasma:

Comparison of ultrahigh-performance liquid chromatography and ultrahigh-

performance supercritical fluid chromatography coupled to mass spectrometry. J.

Chromatogr. A 2017, 1511, 107-121.



41

[79] Nowling T. K., Mather A. R., Thiyagarajan T., Hernandez-Corbacho M. J.,

Powers T. W., Jones E. E., Snider A. J., Oates J. C., Drake R. R., Siskind L. J.,

Renal glycosphingolipid metabolism is dysfunctional in lupus nephritis. J. Am. Soc.

Nephrol. 2015, 26, 1402-1413.

[80] Si W., Liu Y. F., Xiao Y. S., Guo Z. M., Jin G. W., Yan J. Y., Shen A. J., Zhou H.,

Yang F., Liang X. M., An offline two-dimensional supercritical fluid chromatography x

reversed phase liquid chromatography tandem quadrupole time-of-flight mass

spectrometry system for comprehensive gangliosides profiling in swine brain extract.

Talanta 2020, 208, 120366.

[81] Taguchi K., Fukusaki E., Bamba T., Simultaneous analysis for water- and fat-

soluble vitamins by a novel single chromatography technique unifying supercritical

fluid chromatography and liquid chromatography. J. Chromatogr. A 2014, 1362, 270-

277.

[82] Jenkinson C., Taylor A., Storbeck K. H., Hewison M., Analysis of multiple

vitamin D metabolites by ultra-performance supercritical fluid chromatography-

tandem mass spectrometry (UPSFC-MS/MS). J. Chromatogr. B 2018, 1087-1088,

43-48.

[83] Sandvik T.A., Husa A., Buchmann M., Lundanes E., Routine Supercritical Fluid

Chromatography Tandem Mass Spectrometry Method for Determination of Vitamin

K1 Extracted from Serum with a 96-Well Solid-Phase Extraction Method. JALM

2017, 637-648.

[84] Akbal L., Hopfgartner G., Supercritical fluid chromatography-mass spectrometry

using data independent acquisition for the analysis of polar metabolites in human

urine. J. Chromatogr. A 2020, 1609, 460449.



42

[85] Desfontaine V., Losacco G. L., Gagnebin Y., Pezzatti J., Farrell W. P.,

Gonzalez-Ruiz V., Rudaz S., Veuthey J. L., Guillarme D., Applicability of supercritical

fluid chromatography - mass spectrometry to metabolomics. I - Optimization of

separation conditions for the simultaneous analysis of hydrophilic and lipophilic

substances. J. Chromatogr. A 2018, 1562, 96-107.

[86] Losacco G. L., Ismail O., Pezzatti J., Gonzalez-Ruiz V., Boccard J., Rudaz S.,

Veuthey J. L., Guillarme D., Applicability of Supercritical fluid chromatography-Mass

spectrometry to metabolomics. II-Assessment of a comprehensive library of

metabolites and evaluation of biological matrices. J. Chromatogr. A 2020, 1620,

461021.

[87] Perrenoud A. G.-G., Guillarme D., Boccard J., Veuthey J. L., Barron D., Moco

S., Ultra-high performance supercritical fluid chromatography coupled with

quadrupole-time-of-flight mass spectrometry as a performing tool for bioactive

analysis. J. Chromatogr. A 2016, 1450, 101-111.

[88] Suzuki M., Nishiumi S., Kobayashi T., Sakai A., Iwata Y., Uchikata T., Izumi Y.,

Azuma T., Bamba T., Yoshida M., Use of on-line supercritical fluid extraction-

supercritical fluid chromatography/tandem mass spectrometry to analyze disease

biomarkers in dried serum spots compared with serum analysis using liquid

chromatography/tandem mass spectrometry. Rapid. Commun. Mass. Spectrom.

2017, 31, 886-894.

[89] Bolanos B., Greig M., Ventura M., Farrell W., Aurigemma C. M., Li H., Quenzer

T. L., Tivel K., Bylund J. M. R., Tran P., Pham C., Phillipson D., SFC/MS in drug

discovery at Pfizer, La Jolla. Int. J. Mass. Spectrom. 2004, 238, 85-97.

[90] Chen R., A Brief Review of Interfacing Supercritical Fluid Chromatography with

Mass Spectrometry. Chromatography Today 2009, 4, 11-13.



43

[91] Orwa J. A., Govaerts C., Roets E., Van Schepdael A., Hoogmartens J., Liquid

chromatography of gramicidin. Chromatographia 2001, 53, 17-21.

[92] Emmett M. R., Kazazic S., Marshall A. G., Chen W., Shi S. D. H., Bolanos B.,

Greig M. J., Supercritical fluid chromatography reduction of hydrogen/deuterium

back exchange in solution-phase hydrogen/deuterium exchange with mass

spectrometric analysis. Anal. Chem. 2006, 78, 7058-7060.

[93] Zheng J., Pinkston J. D., Zoutendam P. H., Taylor L. T., Feasibility of

supercritical fluid chromatography/mass spectrometry of polypeptides with up to 40-

mers. Anal. Chem. 2006, 78, 1535-1545.

[94] Albets B. J. A., Lewis J., Raff M., Roberts K., Walter P., Mollecular Biology of the

Cell. Garland Science, New York 2002.

[95] Zhang X., Scalf M., Westphall M. S., Smith L. M., Membrane protein separation

and analysis by supercritical fluid chromatography-mass spectrometry. Anal. Chem.

2008, 80, 2590-2598.

[96] Enmark M., Glenne E., Lesko M., Langborg Weinmann A., Leek T., Kaczmarski

K., Klarqvist M., Samuelsson J., Fornstedt T., Investigation of robustness for

supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode.

J. Chromatogr. A 2018, 1568, 177-187.

[97] Akbal L., Hopfgartner G., Effects of liquid post-column addition in electrospray

ionization performance in supercritical fluid chromatography-mass spectrometry. J.

Chromatogr. A 2017, 1517, 176-184.

[98] Svan A., Hedeland M., Arvidsson T., Pettersson C. E., The differences in matrix

effect between supercritical fluid chromatography and reversed phase liquid

chromatography coupled to ESI/MS. Anal. Chim. Acta 2018, 1000, 163-171.



44

[99] Desfontaine V., Capetti F., Nicoli R., Kuuranne T., Veuthey J. L., D. G.,

Systematic evaluation of matrix effects in supercritical fluid chromatography versus

liquid chromatography coupled to mass spectrometry for biological samples. J.

Chromatogr. B 2018, 1079, 51-61.

[100] Wang C. T., A.A.; Zhang, Y., in: Poole, C. F. (Ed.), Supercritical Fluid

Chromatography. Elsevier, Cambridge, MA 2017, pp. 153-172.

[101] Wang C., Tymiak A. A., Zhang Y., Optimization and simulation of tandem

column supercritical fluid chromatography separations using column back pressure

as a unique parameter. Anal. Chem. 2014, 86, 4033-4040.

[102] Poole C. F., Stationary phases for packed-column supercritical fluid

chromatography. J. Chromatogr. A 2012, 1250, 157-171.

[103] West C., Melin J., Ansouri H., Mengue Metogo M., Unravelling the effects of

mobile phase additives in supercritical fluid chromatography. Part I: Polarity and

acidity of the mobile phase. J. Chromatogr. A 2017, 1492, 136-143.

[104] West C., Lemasson E., Unravelling the effects of mobile phase additives in

supercritical fluid chromatography-Part II: Adsorption on the stationary phase. J.

Chromatogr. A 2019, 1593, 135-146.

[105] Yang Y., Liang Y., Yang J., Ye F., Zhou T., Gongke L., Advances of

supercritical fluid chromatography in lipid profiling. J. Pharm. Anal. 2019, 9, 1-8.

[106] Hofstetter R., Fassauer G. M., Link A., Supercritical fluid extraction (SFE) of

ketamine metabolites from dried urine and on-line quantification by supercritical fluid

chromatography and single mass detection (on-line SFE-SFC-MS). J. Chromatogr. B

2018, 1076, 77-83.

[107] Sanchez-Camargo A. D. P., Parada-Alonso F., Ibanez E., Cifuentes A., Recent

applications of on-line supercritical fluid extraction coupled to advanced analytical



45

techniques for compounds extraction and identification. J. Sep. Sci. 2019, 42, 243-

257.

[108] Wicker A. P., Tanaka K., Nishimura M., Chen V., Ogura T., Hedgepeth W.,

Schug K. A., Multivariate approach to on-line supercritical fluid extraction

supercritical fluid chromatography - mass spectrometry method development. Anal.

Chim. Acta 2020, 1127, 282-294.

[109] Bamba T., Lee J. W., Matsubara A., E. F., Metabolic profiling of lipids by

supercritical fluid chromatography/mass spectrometry. J. Chromatogr. A 2012, 1250,

212-219.

[110] Jones M. D., Rainville P. D., Isaac G., Wilson I. D., Smith N. W., Plumb R. S.,

Ultra high resolution SFC-MS as a high throughput platform for metabolic

phenotyping: application to metabolic profiling of rat and dog bile. J. Chromatogr. B

2014, 966, 200-207.



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Figure Captions

FIGURE .1. System configuration of SFC/MS interface [20].

FIGURE 2. High-resolution SFC separation of each lipid class using a DEA column.

A) SFC/QqQMS (MRM) chromatograms of internal standard mixture of 19 lipid

classes. B) SFC/MRM chromatograms of lipids in the WHHLMI rabbit plasma [67].



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FIGURE 3. Positive-ion UHPSFC/ESI-MS chromatograms of mixture of lipids class

standards [68].



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FIGURE 4. Proportion of non-detected peaks (red), distorted peaks (orange) and

Gaussian peaks (green) obtained on the seven tested stationary phases. Mobile

phase: CO2+10mM in MeOH/water (95:5; v:v). Mobile phase temperature of 40 °C

and backpressure of 120 bar [85].

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