Trends in Analytical Chemistry 146 (2022) 116486

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Supercritical fluid chromatography for pharmaceutical quality control:

Current challenges and perspectives
Hugues Jambo a, Philippe Hubert a, Amandine Dispas a, b, *
a
University of Liege (ULiege), CIRM, Laboratory of Pharmaceutical Analytical Chemistry, Li ege, Belgium
b
University of Liege (ULiege), CIRM, Laboratory for the Analysis of Medicines, Li
ege, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Quality control is a fundamental and critical activity in the pharmaceutical industry that permits to
Available online 19 November 2021 guarantee the quality of medicines and consequently safeguard the health of the patients. In recent years,
supercritical fluid chromatography has emerged as a robust and suitable analytical technique in several
Keywords: fields thanks to the introduction of modern SFC instruments. The present review aims to give an over-
Supercritical fluid chromatography (SFC) view of the recent advances of the use of SFC in pharmaceutical quality control and highlights current
Mass spectrometry (MS)
challenges and perspectives. SFC instrumentation is presented and published applications are discussed
Pharmaceutical quality control (QC)
from an analytical method lifecycle point of view. At each step regulatory requirements and recom-
Good manufacturing practices (GMP)
Analytical method lifecycle
mendations are also presented to highlight the readiness of modern SFC for pharmaceutical QC.
Validation © 2021 Elsevier B.V. All rights reserved.
Quality by Design (QbD)

1. Introduction the 2010's, highlighted by a continuous increase of publications.

Quality control is a major concern for pharmaceutical com-
panies to guarantee medicines quality, efficacy and safety. As
described in the Good Manufacturing Practices (GMP) guidelines
[1], quality control encompasses all aspects of pharmaceutical
manufacturing, from the control of the drug substance to the
release of the drug product. One of the main objectives of quality
control is to identify and quantify the active substance(s) and to
track impurities (i.e., synthesis/manufacturing impurities and
degradation products). In this context, several analytical tech-
niques, including separation and spectroscopic techniques, are
commonly used. Based on European and US Pharmacopeias, liquid
chromatography is the predominant analytical tool used for QC
analysis. It could also be extrapolated to the overall pharmaceutical
environment where HPLC and UHPLC, with or without MS detec-
tion, remain the most often selected techniques for a broad range of
applications [2].
Throughout history, analytical scientists tried to improve
analytical methods and instruments to provide faster, greener, less
expensive, more efficient and/or more sensitive tools. In this
context, SFC has known an impressive resurgence of interest since
* Corresponding author. Laboratory of Pharmaceutical Analytical Chemistry,
ge, CHU B36, Avenue Hippocrate 15, B-4000, Lie
University of Lie ge, Belgium.
E-mail address: amandine.dispas@uliege.be (A. Dispas).
After more than fifty years as a niche technique with poor robust
equipment, the acetonitrile crisis in 2008 and the worldwide effort
to promote green chemistry led to the development of modern SFC
instrumentation. The marketing of robust and reliable SFC equip-
ment could be considered as the keystone of SFC resurgence,
leading to the advent of modern SFC. The implementation of
modern SFC to the analytical portfolio [3e5] provides to pharma-
ceutical analysts several advantages: (i) versatility regarding the
range of compounds polarity that could be analyzed; (ii) greenness
by using mainly supercritical CO2 as modifier with the significant
reduction of organic solvent in comparison with LC; (iii) high effi-
ciency with short analysis time thanks to kinetic properties of su-
percritical fluids and the possibility to use sub 2-mm particles; (iv)
easy hyphenation with MS detection thanks to the availability of
dedicated interfaces and; (v) easy transferability to preparative
scale separation.
Nevertheless, the implementation of new technologies and/or
new methods for pharmaceutical quality control remains chal-
lenging. Indeed, several regulatory aspects should be carefully
observed and well described to accept new techniques and/or to
modify currently used methods. In this context, the aim of the
present review is to discuss the recent advances of SFC focused on
pharmaceutical analysis, in respect with the current regulatory
requirements.

https://doi.org/10.1016/j.trac.2021.116486
0165-9936/© 2021 Elsevier B.V. All rights reserved.
H. Jambo, P. Hubert and A. Dispas
2. SFC instrumentations
To be presented as a realistic challenger to LC, SFC equipment
performances should be at least equivalent. SFC is generally
described as LC with some technical adaptations. However, the true
story is more complex because SFC instrumentation should be
designed to properly manage a supercritical mobile phase [6]. The
first technical challenge to face is the control of CO2 to deliver an
accurate flow rate. Secondly, the injection of a liquid sample
through a supercritical mobile phase should be performed pre-
cisely. Another difficulty is the regulation of back-pressure and the
proper CO2 decompression at the end of the system. Finally,
adaptation of optical detection with high-pressure detection cell
and dedicated SFC-MS interface were crucial in the domain of
pharmaceutical analysis. The development of robust in-
strumentations specifically designed for SFC with low dead vol-
umes led to the emergence of modern SFC. Nowadays, several
modern SFC instruments are available on the market. At the
analytical scale, different strategies are proposed by the manufac-
turers: dedicated analytical SFC instrumentation (e.g., Waters®,
Jasco®, Agilent®, Shimadzu®), hybrid LC/SFC system (e.g., Agi-
lent®, Shimadzu®), hybrid analytical/semi-prep instrument (e.g.,
Pic-Solution®) and also hyphenated SFE-SFC equipment (e.g., Shi-
madzu®). Moreover, different commercial MS interfaces are avail-
able, mainly based on split and spitless strategies [4]. Obviously,
each SFC (and SFC-MS) configuration presents advantages and
drawbacks. The versatility of some instruments leads also to higher
dead volume for instance. The maximal pressure of the pumps and
the system also varies by the instruments and could be critical for
some applications. In this context, it is really important to select the
suitable instrumentation according to the design qualification re-
quirements and to the objective of future method developments.
3. Instrument qualification
A critical point emphasized in GMP guidelines concerns the
qualification of equipment used for pharmaceutical QC. The
objective of instrument qualification is to verify its performances
with the final perspective to guarantee data quality of measure-
ments and results. In this context, a recent chapter of USP [7]
introduced analytical instrument qualification as the keystone of
data quality. System qualification includes periodic and systematic
functional and performance testing to ensure the compliance of the
system to the specifications. Based on standard operating proced-
ure (SOP), equipment qualification is totally implemented in
pharmaceutical companies and in certified quality control labora-
tories. In this context, any SFC instrumentation used for QC analysis
should be fully qualified according to a SOP. It means also that the
instrument manufacturers should proposed installation and oper-
ational qualification in accordance with the pharmaceutical re-
quirements. The use of instrumentation with available qualification
protocols is also an advantage to speed up the technique imple-
mentation in regulated environment for QC analysis.
4. Analytical method lifecycle in SFC
The literature discussion is articulated around the analytical
method lifecycle, represented in Fig. 1, as it is the main framework
of management for analytical QC methods. Studies published in the
last five years (2016e2021) were considered for this review [8e38].
Scopus database was used (wide search strategy with multiple
combinations of search terms of supercritical fluid chromatog-
raphy/SFC and “quality control”, “quality”, “pharma*“, “drug*“,
“API*“, “excipient*“, “impurity*“, “medicine*“). Around 150 papers
were obtained and evaluated. The focus has been given mainly to
2
Trends in Analytical Chemistry 146 (2022) 116486
papers that cover the whole analytical lifecycle to highlight ready-
made applications that could be implemented in QC laboratories.
Finally, 31 publications reporting QC applications were selected and
among them 24 proposed method validation. An overview of these
relevant publications is presented in Table 1 and Table 2. It should
be noted that for this review, a bias is supposed since pharma-
ceutical industry users (and especially QC departments) do not
always publish their methods. Consequently, we can expect that a
significant number of SFC methods are currently used in QC labo-
ratories but not reported.
4.1. Method development
4.1.1. Traditional approach
The traditional univariate approach is the most commonly used
for QC method development in SFC (Table 1). It is also referred as
One-Factor-At-a-Time (OFAT) or Quality-by-Testing (QbT). Selected
examples are discussed with regards to the method development
choices performed by the authors.
Pandya et al. [11] developed a SFC method for the determination
of four (achiral) anti-diabetic drugs. Interestingly, the separation
was performed on a chiral stationary phase (CSP) (Chiralpak IG).
The use of CSPs for the separation of achiral compounds is an
approach that is increasingly being tested as it has been demon-
strated that, in some cases, it can provide better separation
compared to achiral columns [39]. It merits consideration during
column screening experiments, especially for closely related
compounds.
A comprehensive study on the use of SFC in impurity control
was published by Plachka  et al. [9]. First, a generic screening
approach was developed for the determination of 10 pharmaceu-
tical QC mixtures, each containing an API with its relevant impu-
rities [40]. The optimization, mainly of the gradient, was needed for
some applications before validation. Other separations required
more modifications such as the specific use of a UHPLC-dedicated
column (Acquity UPLC HSS C18 SB) compared to the SFC-
dedicated column that had the same stationary phase (Viridis
UPC2 HSS C18 SB). Indeed, significant increase in retention and
enhanced resolution could be achieved in this configuration and
was attributed to difference in carbon load of the columns (8.5 and
8.0 respectively). The serial coupling of two columns was also
needed to achieve satisfactory resolution in a specific case. These
results highlighted the predominant impact of stationary phase
selection for SFC method development. All the methods developed
were then successfully validated.
It is important to note that a major drawback of OFAT is that a
comprehensive understanding of the method is not achieved
regarding the impact of critical method parameters and their in-
teractions. This can lead to the blind selection of sub-optimal or
even non-robust conditions. The evaluation of robustness could
therefore be required and is deeply recommended when such
development strategy is selected. Robustness reflects the ability of
an analytical method to be unaffected by small and deliberate
changes in the settings. For example, Puppala et al. [17] developed a
method for the determination of R and S-Eliglustat tartrate with its
stereo isomers and degradation impurities. The robustness was
evaluated for the R and S isomers by varying the flow rate (2.7 g/
min or 3.3 g/min), temperature (25 C or 35 C), percentage of co-
solvent (9.0% or 11.0% and pressure (130 or 150 bars). System
suitability results were calculated in each condition and were found
to be within acceptance limits. Robustness can also be deeply
evaluated using design of experiments [22,41]. However, as seen in
Tables 1 and 2, we can observe that it was not systematically
evaluated after OFAT method development, which can represent a
risk for the future use of the method [42]. Several efforts have been
H. Jambo, P. Hubert and A. Dispas
Fig. 1. Analytical method lifecycle. ATP: analytical target profile.
made to improve upon OFAT by developing a more integrated
approach (Quality-by-Design) that can help to perform a robust
optimization during the development step.
4.1.2. Quality-by-design approach
Risk management is a key principle emphasized in pharma-
ceutical guidelines [43e45]. In this context, the implementation of
a risk-based approach for QC analytical methods is also promoted
[46]. This requirement is met by a Quality-by-Design (QbD)
approach, also termed Analytical Quality-by-Design (AQbD) when
applied to analytical methods. Four major steps can be highlighted
for the development of an SFC method following this approach:
- Definition of an analytical target profile (ATP). The purpose and
the required performance of the method are defined. This is a
thorough evaluation that encompasses both qualitative and
quantitative aspects of the method. It is translated in critical
method attributes (CMAs) that must be met with a predefined
specification (e.g. separation criterion of the critical pair 0);
- Determination of critical method parameters (CMPs) (e.g. tem-
perature, pressure, gradient) trough risk assessment and/or
preliminary experiments;
- Robust method optimization trough design of experiments
(DoE) and definition of the design space (DS), also called Method
Operable Design Region (MODR). It is the space where the
multivariate combination of CMPs ensures to meet the specifi-
cations of the CMAs with a pre-specified quality level (e.g. 95%
prediction level). A working point is then selected;
- Continuous monitoring and improvements of the implemented
method trough the definition of a control strategy (CS).
A more comprehensive description of AQbD is given in the
following references [47,48]. It is a systematic approach that per-
mits an increased understanding of the method and guarantees the
robustness of a wide set of analytical conditions inside the MODR.
As seen in Table 1, several SFC studies have successfully adopted
this novel approach in method development.
For example, Andri et al. [25] developed an SFC method for the
quantitative determination of vitamin D3 and three related impu-
rities in drug substance. The approach was focussed on the chro-
matographic separation and thus the selected CMA was the
separation criterion (S), which is defined for a critical pair as the
difference between the end of a first peak and the beginning of the
second peak. A specification of S  0 was set, meaning that a
baseline separation must be achieved for the critical pair. The final
CMPs selected following preliminary experiments were the tem-
perature, the gradient final composition at the end of gradient time
and gradient time. A DoE was performed and retention times were
3
Trends in Analytical Chemistry 146 (2022) 116486
modelled. Finally, the computation of the MODR followed a
multivariate Bayesian model that takes into account the prediction
error and its propagation. In a following study [26], this method
was transferred to MS detection to perform the assay of vitamin D3
in a drug product (oily matrix). Significant changes in instrument
configuration were needed to perform the MS hyphenation which
impacted the separation. A simple adjustment of flow rate to reach
similar pressure than using SFC-UV configuration (while keeping
the same optimized CMPs) permitted to resolve this issue, high-
lighting the method robustness achieved thanks to the AQbD
approach.
This strategy was also successfully used by Schmidtsdorff et al.
for the sensitive detection of nitrosamines in various drug sub-
stances and drug products using SFC-UV-MS/MS. The surrounding
context is further discussed in section 4.4. as it is an important
current issue in the pharmaceutical domain. It is important to
notice that these studies were performed using qualified instru-
ment and validated software as required by the GMP (see section
3). It means that this method may be directly transferred in a
regulated environment for routine analysis. The first study [27]
was focused on two APIs raw material, their impurities and 10
nitrosamines. The selected CMAs were the peak resolution, peak
height and symmetry, signal-to-noise and retention. First,
screening experiments were performed and helped to select a
suitable column (Viridis HSS C18 SB). To increase the separation,
the authors used two columns in series. The optimization was
achieved in several steps and the knowledge acquired at each step
permitted to refine the design space. Multivariate design of ex-
periments and statistical analysis were performed with the help
of a commercial software, Fusion QbD (S-Matrix Corporation,
Eureka, California, USA). It permitted to select in succession i) the
type of modifier and type of additive in the modifier (TFA) and in
the MS make-up (formic acid (FA)) ii) the amount of modifier in
the start gradient, the concentration of TFA in the modifier and the
column temperature iii) the concentration of FA and the flow rate
of the MS make-up solvent. At each step a MODR was computed to
display the best results considering the defined goals for the
CMAs. In the second study [28], the method was extended as a
universal analysis method to cover a broader set of molecules (16
nitrosamines and 8 APIs) and also permit the analysis of drug
products. This required the redefinition of the ATP and redevel-
opment in the context of Analytical Method Lifecycle Manage-
ment [42]. The development time was greatly reduced by
implementing the knowledge gained in the previous study. The
column was changed to a single Supel Carbon porous graphitic
carbon (PGC) and the optimization was performed considering
the multivariate combination of the CMPs: column temperature,
backpressure, initial percentage of modifier in the gradient and
H. Jambo, P. Hubert and A. Dispas Trends in Analytical Chemistry 146 (2022) 116486

Table 1
Overview of SFC methods for pharmaceutical quality control.

Reference SFC System Detection Analytes Matrix Development approach Validation

[8] Waters UPC2 UV Agomelatine and 6 impurities Raw material OFAT ✓

Tablets
[9] Waters UPC2 UV Agomelatine and 6 impurities Raw material OFAT ✓
Abiraterone and 5 impurities Tablets
Atomoxetine and 6 impurities
Dasatinib and 5 impurities
Enzalutamide and 4 impurities
Ezetimibe and 5 impurities
Ticagrelor and 3 impurities
Vardenafil and 6 impurities
[10] Waters UPC2 UV Tocopherol derivatives Tables, capsules, OFAT ✓
MS granulate, drops
[11] Waters SFC Investigator UV Metformin Tablets OFAT ✓
system (semi-prep) Canagliflozin, dapagliflozin, Blank human plasma
empagliflozin
[12] Waters UPC2 UV 7 phytocannabinoids Cannabis medicine OFAT
(Bedrocan®)
[13] Shimadzu Nexera UC UV Itraconazole stereoisomers Raw material OFAT ✓
30AD Series
[14] Shimadzu Nexera UC MS Vitamin D3 Oily drops OFAT
operated in two
dimensions SFCxLC
[15] Two-dimensional UV Confidential API and its related Raw material OFAT
analysis: sLCxSFC impurities
LC: Agilent 1290
Infinity I 2D-LC system
SFC: Agilent 1260
Infinity II SFC System
[16] Waters UPC2 UV Verubecestat Raw material OFAT
[17] Waters UPC2 MS Eliglustat tartrate and 3 stereoisomers Raw material OFAT and robustness ✓
study
[18] Waters SFC Investigator UV Aliskiren, amlodipine, atenolol, Tablets (co- OFAT and robustness ✓
system (semi-prep) candesartan, hydrochlorothiazide, formulations of the study
lisinopril, losartan, metoprolol, drugs)
olmesartan, telmisartan, valsartan
[19] JASCO-2000 MS Ethambutol Tablets OFAT and robustness ✓
Urine study
[20] Waters UPC2 UV Docetaxel and 5 impurities Injection formulations OFAT and robustness ✓
Bortezomib and 7 impurities study
[21] Agilent-Aurora A5 UV Merck research compounds Raw material OFAT and robustness ✓
Fusion SFC (undisclosed) study
Waters UPC2
[22] Waters UPC2 UV 9 phytocannabinoids Resin, inflorescence OFAT and robustness
study
[23] Shimadzu Nexera UC MS Ketamine and metabolites Raw material OFAT and molecular
Drug formulations modelling
(infusion and nasal
spray)
[24] Waters UPC2 UV Luliconazole stereoisomers Raw material OFAT and molecular ✓
Cream docking
[25] Waters UPC2 UV Vitamin D3 and 3 related impurities Raw material Multivariate (AQbD) ✓
[26] Waters UPC2 MS Vitamin D3 Oily formulation AQbD method transfer ✓
to MS detection
[27] Waters UPC2 UV-MS/MS Valsartan, 2 impurities and 1 related Raw material Multivariate (AQbD) ✓ (Limit testing)
substance
Losartan and 11 impurities
10 Nitrosamines
[28] Waters UPC2 MS/MS Metformin, Ranitidine, Pioglitazone, Raw material Multivariate (AQbD) ✓ (Limit testing)
Losartan, Valsartan, Irbesartan, Drug products (exact
Candesartan, Olmesartan, Eprosartan, formulation type not
Telmisartan, Hydrochlorothiazide, mentioned)
Amlodipin, Vidagliptin, Sitagliptin
16 Nitrosamines
[29e32] Waters UPC2 UV Salbutamol sulfate impurities Raw material Multivariate (AQbD) ✓
(Interlaboratory 1 and
2)
Agilent Infinity
(Interlaboratory 2)
Shimadzu Nexera UC
(interlaboratory 2)
[32] Waters UPC2 MS 6 phytocannabinoids Plant Multivariate (AQbD) ✓
17 synthetic cannabinoids
[33] Waters UPC2 UV Isoniazid and impuritites APIs mixture Multivariate (DoE) and ✓
Rifabutin, 25-O-desacetyl rifabutin Commercial product robustness study

4
H. Jambo, P. Hubert and A. Dispas Trends in Analytical Chemistry 146 (2022) 116486

Table 1 (continued )

Reference SFC System Detection Analytes Matrix Development approach Validation

[34] Waters UPC2 UV Multiple pharmaceutical compounds. Capsule (validation Multivariate (DoE) ✓
Quinine sulfate selected for validation study)
study
[35] Waters UPC2 UV Simvastatin, lovastatin, losuvastatin, Raw material Multivariate (DoE) ✓
fluvastatin, atorvastatin, ezetimibe, Tablets
pravastatin, pitavastatin
[36] Waters UPC2 UV Tenofovir disoproxil fumarate and its Raw material Multivariate (DoE) ✓
stereoisomers Tablets
Lamivudine and its stereoisomers
[37] Waters UPC2 UV Drug mixture of API and impurities Raw material Multivariate (DoE and
(undisclosed composition) chromatogram
simulation)
[38] SFC-PICLAB UV-ELSD Ondansetron and its counter-ion Raw material Multivariate (DoE and ✓
(hydrochloride) desirability functions
computation for
column ranking)

Table 2
Overview of validation criteria evaluated for referenced SFC methods.

Reference Guideline Selectivity Linearity LOD LOQ Trueness Accuracy Precision Intermediate Robustness Reproducibility
repeatability precision

[8] ICH ✓ ✓ ✓ ✓ ✓ ✓
[9] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[10] ICH ✓ ✓ ✓ ✓
[11] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓
[13] ICH ✓ ✓ ✓ ✓ ✓
[17] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[18] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[19] ICH / FDA ✓ ✓ ✓ ✓ ✓ ✓
[20] ICH ✓ ✓ ✓ ✓ ✓ ✓
[21] GMP ✓ ✓ ✓ ✓ ✓ ✓
[24] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓
[25] ICH / SFTSP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[26] ICH / SFSTP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[27] ICH (limit testing) ✓ ✓
[28] ICH (limit testing) ✓ ✓
[29e31] ICH / SFSTP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[32] ICH / SFSTP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[33] ICH ✓ ✓ ✓ ✓ ✓ ✓ ✓
[34] ICH / SFSTP ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
[35] ANVISA ✓ ✓ ✓ ✓ ✓
[36] ICH ✓ ✓ ✓ ✓ ✓ ✓
[38] ICH / SFSTP ✓ ✓ ✓ ✓ ✓ ✓ ✓

additive (TFA) concentration. The resulting MODR and represen-
tative chromatogram at the final working point are reported in
Fig. 2.
To conclude this section on AQbD, another interesting advan-
tage is that it also brings flexibility from a regulatory point of view.
Indeed, since the MODR can be described in the marketing
authorization file, method modifications inside it should not
require post-approval changes submission. This strategy is now
fully advisable and is increasingly promoted by the regulators [49].
4.1.3. Other multivariate approaches
Several studies have also adopted a multivariate development
approach using design of experiments as seen in Table 1. They are
most distinguished from AQbD regarding the statistical approach
(no MODR computation) and are therefore discussed distinctly
here. A DoE approach was used by Galea et al. [37] for the sepa-
ration of a drug and its impurities. Following a screening phase, the
column temperature, back-pressure and gradient slope were
simultaneously optimized in fifteen experiments using a face-
centred central composite experiment design. The minimal reso-
lution and number of peak pairs having a resolution smaller than
1.5 were selected as responses. Simple statistical tools such as
5
Microsoft Excel were used to perform data analysis and permitted
to identify the best combinations of factors for the separation. Then,
chromatograms were simulated and compared experimentally, a
good agreement was obtained. In the context of enantioseparation,
Kurmi et al. [36] used a three-step design of experiments for the
simultaneous determination of chiral impurity of lamivudine and
tenofovir disoproxil fumarate in tablets fixed dose combinations.
These approaches proposed a more interesting optimization
strategy than OFAT (4.1.1) but do not fulfil the AQbD requirements.
These approaches could not be considered as proper robust opti-
mization methodology.
4.2. Validation
Method validation is required by regulatory agencies (FDA, EMA,
etc.). To really appreciate its concept and challenges, it is important
to consider the final objective of an analytical method, i.e., routine
analysis: “the objective of validation is to give to the laboratories and
regulatory agencies “guarantees” that every single measure that will
be performed in routine analysis will be close enough to the unknown
true value of the sample to be analyzed” [50]. The validation criteria
to evaluate are defined in guidelines such as ICH Q2(R1) [51] and
H. Jambo, P. Hubert and A. Dispas Trends in Analytical Chemistry 146 (2022) 116486

Fig. 2. Adapted from Ref. [28] with permission from Elsevier. A) Robustness evaluation for the optimized SFC method's design space. Left: Column temperature and the initial
percentage of modifier (“strong solvent”) during the first isocratic step of the gradient method. Right: Concentration of TFA (additive) in the modifier and backpressure. Displayed in
green the resolution of NMor to NEiPA (isobar peak pair) and in blue NDPA to NDiPA (isomers). In red relative peak areas for each single NA, compared to the most intense peak
within this full-factorial experiment, are displayed. Colored areas indicate where the predefined and listed objective is not achieved (note that mixed-colors are possible) and
therefore white areas indicate positive results. Final working point (T) with MODR shown. B) Representative MS/MS chromatogram of a metformin DP sample spiked with all 16
nitrosamines (20 ng/mL) and their retention times (min), obtained on the Supel Carbon column with the final method. The method was validated for limit testing according to ICH
considering the criteria of selectivity and limit of detection (LOD).

depend on the objective of the method which is usually quantita-
tive measurement (assays or impurities testing). More explana-
tions, with a special focus on SFC methods, can be found in a
dedicated reference [52]. As highlighted in Table 1, the majority of
the referenced publications present validation results, showing the
great interest of this demonstration for SFC scientists. Details of the
criteria evaluated in the referenced SFC papers are given in Table 2.
Most of the published methods were properly validated using the
criteria required by the guidelines. The term trueness (expressed as
bias or recovery) has been used in this review to avoid confusion
with the definition of the accuracy criterion (reflecting the total
error, i.e., trueness and precision). Selected papers will be discussed
in the following sections.
4.2.1. Dissociated error validation approach
The design of validation studies is a topic of great interest as it is
seldom addressed in the major guidelines. A classical approach
consists in evaluating each criterion individually and see if they
satisfy the requirements of the regulatory agencies, it means that
each type of error is dissociated. For example, Nova kova et al. [10]
developed a method for the determination of lipophilic vitamins in
dietary supplements from multiple manufacturers and different
dosage forms (drops, capsules, tablets and granulates). The method
was validated for selectivity, linearity, trueness and precision.
Limits of detection (LOD) and quantification (LOQ) were not
6
evaluated as they are not mandatory for assay methods according
to ICH. First, a system suitability test was performed and showed
good results for the repeatability of retention time and peak areas
(<0.70% RSD and <2.0% RSD respectively). Satisfactory results were
obtained for trueness (bias <5.0%) and precision (<5.0% RSD). They
were determined simultaneously in real samples using the stan-
dard addition method at three concentration levels (80%, 100% and
120%).
Petrusevska et al. [33] developed a method for the simultaneous
determination of isoniazid, rifabutin and their related substances in
a new proposed fixed-dose combination. The validation was per-
formed according to ICH. A stability study and filter study were also
performed. However, few observations can be made. The linearity
was evaluated on standard solutions considering the relationship
between the analytical response and the concentrations. This is in
contradiction with ICH requirements which states that it is the
linearity of the results that needs to be demonstrated, expressed as
the relationship between the introduced and calculated concen-
trations. Moreover, the precision and trueness were not evaluated
on the same type of samples and are therefore not linked to
demonstrate the validity of the method.
In general, trueness and precision values in the reported
literature were in accordance with pharmaceutical requirements
for drug substances or drug products, highlighting the good
quantitative performance of SFC. However, for the precision
H. Jambo, P. Hubert and A. Dispas
criteria, the computation of intermediate precision RSD (%) ap-
pears to be inadequate in some papers [11,20]. Indeed, they report
lower values at the intermediate precision level than the repeat-
ability level, which is incoherent since the intermediate precision
also includes the repeatability (as the sum of intra- and inter-
series variances).
4.2.2. Total error validation strategy
A difficulty encountered using the dissociated error approach
concerns the interpretation of the impact of the evaluated (quan-
titative) parameters on the analytical result, especially regarding
the trueness and precision criteria. In this context, a harmonized
approach was proposed by the SFSTP commission: the total error
strategy [53e56]. It is an approach that follows risk management
and is therefore in line with QbD principles. The heart of this
strategy is the accuracy profile which is based on the b-expectation
tolerance interval. It is a predictive tool that allows to assess the risk
associated to the method measurements and is computed from the
estimates of bias and precision (i.e. the total error). An interval is
ultimately defined that predicts that the future results will fall in-
side with a predefined risk level a. A great advantage of the accu-
racy profile strategy is that it is a powerful decision tool that
permits to readily state the validity of a method considering pre-
defined acceptance limits. A more complete description can also be
found in Ref. [52].
Several studies have adopted this approach in SFC method
validation [8e12,23]. Bouchot et al. [38] developed and validated an
SFC-UV-ELSD method for the quantitative determination of
ondansetron (UV detection) and its chloride counter-ion (ELSD
detection) in drug substance. Bromide was used as internal stan-
dard for chloride determination. From the accuracy profile, the
method was valid in the range 80e120% of the target concentration
with acceptance limits set at 10%, which can be considered large for
the determination of an API in raw material. The authors
acknowledged that the equipment used (SFC-PICLAB, analytical and
semi-preparative) had less precise control of the backpressure
which had an impact of the flow entering the ELSD nebulizer. This
technical constraint had a negative impact on the trueness and
especially the precision (up to 4% RSD) of the method. The deter-
mination of the molar ratio of ondansetron versus chloride in
sample solution could still be achieved and was equal to
1.001 ± 0.003, which is in accordance with the theoretical stoi-
chiometry of 1:1.
Total error approach was also used by Plachka  et al. [9] in the
context of the determination of impurities in 10 pharmaceutical
mixtures. This study is interesting as the validation design followed
more of a traditional approach and permitted to assess all the
required validation criteria of ICH, including LOD and LOQ for im-
purities. Then, the generated data were also used to compute ac-
curacy profiles. The validity of the methods was confirmed
considering acceptance limits set at 5%. This serves to make an
interesting observation on validation designs for the total error
methodology which are single “all-in-one” approaches. They are
certainly not more labour intensive or more costly than a tradi-
tional approach validation design and result in a more compre-
hensive understanding of the quantitative performance of the
method, with the evaluation of all criteria required by ICH.
Consequently, the use of the total error strategy should be
promoted.
4.3. Method transfer between laboratories
Method transfer is an important step in the analytical method
lifecycle. Indeed, it is very common that method development and
validation (e.g., from R&D laboratory) are not performed in the
7
Trends in Analytical Chemistry 146 (2022) 116486
same laboratory where the routine analysis will be carried out
(e.g., QC laboratory). Another important aspect in pharmaceutical
quality control remains on the use of normative methods. Indeed,
pharmacopeia (USP, EP, etc.) propose methods that should and
could be used in any laboratory without validation. To propose
such a normative method, the method transferability and the
proper evaluation of reproducibility should be carefully
performed.
There are very few publications of SFC method transfer in the
literature. Khalikova et al. reported the transfer of a method be-
tween two laboratories (in identical operational conditions) for the
quantitative determination of nine sunscreens in cosmetic samples
[57]. A fast separation could be achieved in 2.5 min and the
quantitative performance were demonstrated with the total-error
approach. A complete description is out of the scope of this paper
but good results were obtained for the method transfer between
the two laboratories regarding the reproducibility of retention
times (average value of 95%) and response factors (bias around 2%
calculated by difference (Bland-Altman) plot).
In the context of pharmaceutical quality control, two studies
were published by Dispas et al. regarding the transfer of a method
in extensive interlaboratory trials. The method was geared towards
the quantitative determination of salbutamol sulfate impurities in
raw material. It was first developed following AQbD principles to
separate a total of seven impurities (five known and two unknown)
and the API [29]. Method validation was performed according to
the total error approach for four impurities (B, D, F G). For all im-
purities, results showed that the method was valid in the range
50e150% of the corresponding target concentration (0.3% w/w)
with acceptance limits set at 15%.
After validation, a first interlaboratory study was conducted
[30]. It was a collaborative effort involving 19 laboratories using the
same equipment (Waters UPC2). First, preliminary experiments
were performed to set-up of the method in the different labora-
tories and to verify the selectivity, sensitivity and system repeat-
ability. Then, the quantitative determination of impurity D in
salbutamol sulfate samples (at three concentration levels) was
performed which permitted to estimate the precision of the
method in terms of repeatability (between 2.8% and 3.4%) and
reproducibility (between 7.7% and 10.7%). These results highlighted
the excellent quantitative performance of SFC and demonstrated
the technique to be on par with other chromatographic techniques.
In the second interlaboratory study, the evaluation was
extended to multiple instruments [31]. Three types of instruments
were considered from Waters®, Agilent® and Shimadzu® imple-
mented in 21 participating laboratories. Method adjustments were
necessary to transfer the method to Agilent® and Shimadzu®
systems. Specifically, a variable wavelength detector was needed
for the Agilent® system. For the Shimadzu® system, the modifi-
cation of sample diluent and increase of volume injection were
required to balance the lower sensitivity due to a shorter UV de-
tector optical path. Typical chromatograms obtained on each in-
strument is presented in Fig. 3. Preliminary experiments were then
performed followed by the quantitative determination of impurity
D in the laboratories. In this more complex setup, the reproduc-
ibility RSD (%) was between 15% and 17% which is excellent
considering the multiple variability sources, including the addi-
tional challenge of multiple instrument types. These studies
demonstrated that SFC could be implemented for QC analysis in
multiple laboratories working with different instruments.
4.4. Innovative SFC applications
In this section selected applications will be highlighted and
discussed. The analysis of inorganic ions is novel in SFC and was
H. Jambo, P. Hubert and A. Dispas
Fig. 3. Reproduced from Ref. [31] with permission from Elsevier. Interlaboratory 2. Chromatogram obtained in each laboratory during the familiarization step. Blue Waters in-
strument e Green Agilent instrument e Red Shimadzu instrument.
performed by Bouchot et al. [38]. The aim was to determine the
stoichiometry of a drug and its counter-ion (ondansetron hydro-
chloride) in a single SFC method using dual detection (UV and
ELSD). To the best of our knowledge, the simultaneous (in a single
run) determination of a drug and its counter-ion could not be
performed easily with other separation techniques. They proposed
8
Trends in Analytical Chemistry 146 (2022) 116486
the computation of a Derringer desirability coefficients to rank the
tested columns during preliminary experiments and the 2-EP was
selected. The mobile phase was composed of methanol with water
and diethylamine as additives (MeOH/H2O/DEA e 98/2/0.2 e v/v/
v). Temperature was set to 35 C and pressure at 150 bar. The
method was validated following the total error methodology (see
H. Jambo, P. Hubert and A. Dispas
section 4.2.2). In a first, the authors had previously demonstrated
the analysis of inorganic ions by SFC [58], which is an application
traditionally performed by ion-exchange chromatography. They
followed with this work to demonstrate the applicability to a more
complex molecule. These studies highlight the versatility of SFC as a
technique able to analyse molecules with a wide range of polarity.
The suitability of SFC for the analysis of molecules with low to
medium polarity is already established and researchers are now
increasingly interested in the analysis of very polar (hydrophilic)
compounds, as recently reviewed by Losacco et al. [5]. In this
context, strategies such as unified chromatography (UC) or
enhanced fluidity liquid chromatography (EFLC) are explored. A
comprehensive review on these approaches for the analysis of
biomolecules was recently published by Molineau et al. [59]. UC
and EFLC approaches with MS hyphenation should increase in the
next years to face biopharmaceuticals quality control challenges.
A current hot topic in the pharmaceutical industry concerns
nitrosamines impurities, which are chemical compounds classified
as probable human carcinogens. It began in mid-2018 when they
were unexpectedly detected in some batches of valsartan drug
substance. As the investigation continued, it occurred that it
affected several other products. Regulatory agencies (EMA and FDA)
therefore asked all marketing authorisation holders to conduct a
review of their products on the potential risk of containing nitro-
samine impurities. A detailed timeline of the nitrosamines crisis is
described in Ref. [28] but this issue is still developing to this day and
has impacted the availability of some medicines. In this context,
Schmidtsdorff et al. [27,28] have applied SFC hyphenated to tandem
mass spectrometry (SFC-MS/MS) for the detection of nitrosamines
in drug substances and drug products (see section 4.1.2). The
methods were validated for limit testing in accordance with ICH Q2.
In the second study, sample analysis uncovered nitrosamines
contaminations in ranitidine samples at 10-times above the toler-
ance limit. These studies highlight the readiness of SFC to meet
current challenges in the pharmaceutical industry, as stand-alone
or complementary to more mainstream techniques (LC/GC).
Multidimensional analysis is an approach that is increasingly
used for the separation of complex samples. Iguiniz et al. [15]
evaluated the potential of on-line RPLCxSFC for the comprehen-
sive chiral/achiral analysis of pharmaceutical compounds. The
interface used permitted to collect fractions from the LC dimen-
sion (chiral analysis) using a “Multiple Heart-Cut” (MHC) valve
system that were successively analyzed in the SFC dimension
(achiral analysis). The separation was achieved in 50 min. Peak
intensities were found to be lower (3e4 times) compared to single
dimension SFC, which was attributed to extra-column dispersion
and dilution from the 2D process. These results are promising
with a LOD of 0.5%. Note that the recent Shimadzu system (Shi-
madzu Nexera UC®) can work in two-dimension SFC-LC mode to
carry out on-line sample pre-treatment in the SFC dimension first,
then analysis in LC in the second dimension. This approach was
used by Feng. et al. for the quantitative determination of vitamin
D3 in oily drops [14]. Multidimensional approaches involving SFC
could face some tricky analytical challenges. Nevertheless, the
robustness and quantitative performance of 2D (or more) sepa-
ration deserve deeper investigations.
The analysis of cannabis is another current hot topic. Indeed,
there is substantial evidence of its therapeutic properties and has
thus gathered the attention of the pharmaceutical industry. Several
studies have been published detailing the quality control of
cannabis and cannabis-based products. Mazzoccanti et al. [12]
developed a method for the chemo- and enantio-selective sepa-
ration of seven cannabinoids standards using the so-called
9
Trends in Analytical Chemistry 146 (2022) 116486
“inverted chirality columns approach” (ICCA). The method was
then applied for in the analysis of a medicinal cannabis product
(Bedrocan®) and the enantiomeric excess (ee) of the isomer
( )-Delta9-THC was estimated to be equal to 99.73%. Deidda et al.
[22] developed a method to separate nine cannabinoids in real
cannabis samples (inflorescences and resins). Following OFAT
development, method robustness was evaluated trough design of
experiments. Finally, quantification was performed for five main
cannabinoids in 92 samples during routine analysis using the
developed SFC method in comparison with a reference UHPLC
method. A good agreement of the results between the two methods
was demonstrated using Bland-Altman analysis, highlighting the
suitability of SFC for cannabinoid analysis. Jambo et al. [32] devel-
oped a generic SFC-MS method to detect cannabis adulterations
with synthetic cannabinoids (SC). To this aim, a set of seventeen
representative SCs was assessed to ensure adequate retention and
achieve resolution from natural cannabinoids. Method develop-
ment was performed following AQbD principles. The quantitative
performances were demonstrated by means of method validation
based on the total error approach for the quantification of a selected
SC in fibre type cannabis plant matrix. Cannabinoids and cannabis
products present ideal properties for SFC separation. In this context,
the demonstration of SFC ability should help to replace the usual
technique used in forensic laboratories (i.e., GC) to propose an
easier and faster analysis.
5. Conclusion
Pharmaceutical quality control domain remains really conser-
vative, generally to ensure the respect of all its specific re-
quirements. In this context, the implementation of new techniques
is always performed several years after the demonstration of their
performances in academia and R&D laboratories. In the specific
case of SFC, this technique suffered of bad reputation of non-robust
technique during decades. However, the significant improvements
of SFC instrumentation in the beginning of the 2010's aimed to
propose robust and reliable equipment. Following this technolog-
ical revolution, the demonstration of SFC chromatographic and
quantitative performances has known a real success in different
laboratories, from academia to compagnies. Recent publications
highlighted that SFC could be presented as a real challenger of gold-
standard LC, with suitable quantitative performance and its easy
implementation in several laboratories by means of inter-
laboratory studies. With respect to this literature review, it seems
obvious that SFC will be an important contributor to the current,
next-future and future QC analytical technology panel. The deep
and large implementation in regulated laboratoriess and the
training of analysts and scientists is the next step to accomplish.
Finally, the introduction of SFC for normative methods could be
considered of one of the most important challenge for QC in the
next decade.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
Research grants from the Walloon Region of Belgium and EU
Commission (project FEDER-PHARE) to A. DISPAS are gratefully
acknowledged.
H. Jambo, P. Hubert and A. Dispas Trends in Analytical Chemistry 146 (2022) 116486

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