3-Hydroxypropionaldehyde (3-HPA) quantification by HPLC using a synthetic acrolein-free 3-hydroxypropionaldehyde system as analytical standard

Article abstract of DOI:10.1039/c5ra18274c

HPLC-based quantification of 3-HPA using a synthetic acrolein-free 3-HPA standard obtained from commercially available 1,2,4-butanetriol through a straightforward and easy synthetic process has advantages over previous colorimetric methods of easier and safer implementation, and greater specificity. This HPLC method is very simple to implement in a lab, does not need any extra handling of the sample to be analyzed, and is suitable even in the presence of other aldehydes and 3-HPA derivatives, provided that the latter do not have similar retention times.

Full text of DOI:10.1039/c5ra18274c

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: G. Burgé, A. L.
Flourat, B. Pollet, H. E. Spinnler and F. Allais, RSC Adv., 2015, DOI: 10.1039/C5RA18274C.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 1 of 16
RSC Advances
View Article Online
DOI: 10.1039/C5RA18274C
3-Hydroxypropionaldehyde (3-HPA) quantification by HPLC using
synthetic acrolein-free 3-hydroxypropionaldehyde system as analytical
standard
G. Burgé§1,2,3, A.L. Flourat§1,4,5, B. Pollet2,3, H.E. Spinnler2,3, F. Allais1,2,3*
1 Chaire AgroꢀBiotechnologies Industrielles (ABI) ꢀ AgroParisTech, Fꢀ51100 Reims, France
2
AgroParisTech, UMR 782 Génie et Microbiologie des Procédés Alimentaires (GMPA), Fꢀ78850
ThivervalꢀGrignon, France
3
INRA, UMR 782 Génie et Microbiologie des Procédés Alimentaires (GMPA), Fꢀ78850 Thivervalꢀ
Grignon, France
4 AgroParisTech, Institut JeanꢀPierre Bourgin (IJPB), Fꢀ78026 Versailles Cedex, France
5 INRA, Institut JeanꢀPierre Bourgin (IJPB), Fꢀ78026 Versailles Cedex, France
§ These authors contributed equally to this work.
ABSTRACT HPLCꢀbased quantification of 3ꢀHPA using a synthetic acroleinꢀfree 3ꢀHPA
standard obtained from commercially available 1,2,4ꢀbutanetriol through a straightforward
and easy synthetic process has the advantages over previous colorimetric methods of easier
and safer implementation, and greater specificity. This HPLC method is very simple to
implement in a lab, does not need any extra handling of the sample to be analyzed, and is
suitable even in the presence of other aldehydes and 3ꢀHPA derivatives, provided that the
latters do not have similar retention times.
Keywords
3ꢀHydroxypropionaldehyde, reuterin, chemical synthesis, NMR, HPLC, HESIꢀMS, GCꢀMS
Introduction
3ꢀHydroxypropionaldehyde (aka 3ꢀHPA, βꢀhydroxypropionaldehyde, reuterin) was originally
discovered in 1910 by Voisenet.1 Its formation was observed during bacterial spoilage of wine
by Bacillus amaracrylus (now classified as Paenibacillus polymixa). Voisenet assumed that
3ꢀHPA was produced by dehydration of glycerol, which was later confirmed by Abeles.2 3ꢀ
HPA is a viscous and colorless liquid with a slightly acrid smell. Nowadays, the name reuterin
is used as a synonym for 3ꢀHPA. This molecule exhibits powerful antimicrobial activity
against many pathogenic microorganisms (Gramꢀpositive and Gramꢀnegative, yeast,
protozoa),3 particularly into the gastrointestinal tract4. Minimal inhibitory (MIC) and minimal
bactericidal concentrations (MBC) were determined for several intestinal bacteria, showing
that they were very sensitive to this molecule.5 3ꢀHPA therefore participates, at least in part,
on the L. reuteri probiotic behavior,6 which was confirmed by randomized, doubleꢀblinded,
and placeboꢀcontrolled clinical studies.7 3ꢀHPA may also cause oxidative stress in cells, by
reacting with thiol groups of proteins, enzymes and small molecules. However, the
mechanisms responsible for this toxicity have not been identified yet.8 3ꢀHPA finds also use
as a food preservative and as a therapeutic auxiliary agent in the pharmaceutical industry.9
1

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
RSC Advances
Page 2 of 16
View Article Online
DOI: 10.1039/C5RA18274C
In addition, 3ꢀHPA is a platform chemical of considerable industrial interest as it can be
relatively easily converted into a number of commodity chemicals. It can be dehydrated to
acrolein then oxidized to acrylic acid. 3ꢀHPA can also be reduced to 1,3ꢀpropanediol (1,3ꢀ
PDO) through the Degussa and Shell processes,10 or oxidized to 3ꢀhydroxypropionic acid (3ꢀ
HP) to produce malonic acid. All these molecules are starting materials for polymers and
therefore of great interest for the industrial applications. Given the potential of this building
block, biotechnological production of 3ꢀHPA from glycerol is therefore being intensively
studied11 and commercial production is expected to start in the near future. The
biotechnological production of 3ꢀHPA by Lactobacillus reuteri is initiated during the
stationary growth phase, in the presence of glycerol as substrate.12
In water, 3ꢀHPA system is made of several monomeric and oligomeric components that exist
in a dynamic equilibrium, and depends upon the set conditions (Fig. 1). For example, studies
on the structure of 3ꢀHPA showed that 3ꢀHPA in water is part of a concentrationꢀ and pHꢀ
dependent dynamic system and revealed that at high concentrations (4.9 M), the 3ꢀHPA
system was predominantly composed of 3ꢀHPA dimer along with acrolein and unspecified
HPA derivatives.10
O
HO
A
H2O
OH
OH
OH
O
O
∗
HO
O
HO
O
HO
OH
B 1,1,3-PPT
C Cyclic dimer
D Open dimer
O
O
OH
O∗H
∗
HO
O
O
HO
O
O
O
E trimer
F trimer
Fig. 1 3ꢀHPA system: (A) 3ꢀHPA monomer, (B) 3ꢀHPA hydrate (aka 1,1,3ꢀtrihydroxypropane, 1,1,3ꢀ
propanetriol or 1,1,3ꢀPPT), (C) 3ꢀHPA cyclic dimer (2ꢀ(2ꢀhydroxyethyl)ꢀ4ꢀhydroxyꢀ1,4ꢀdioxane), (D) 3ꢀHPA
open dimer, (E) and (F) 3ꢀHPA trimers
Given the great interest of 3ꢀHPA, analytical methods for quantification of 3ꢀHPA were
developed using MIC (Minimum Inhibitory Concentration, calculated from the highest
dilution showing a complete inhibition of the tested strain),8 colorimetry and HPLC. Although
being one of the most used methods, MIC assay has a major drawback as it only provides
relative 3ꢀHPA concentrations expressed in arbitrary units (units 3ꢀHPA.mLꢀ1). Indeed, there
is a definite advantage for using methods allowing the determination of defined molar 3ꢀHPA
concentrations. To access the latters, colorimetric methods can be used.13ꢀ15 They all consist in
transforming the complex 3ꢀHPA system (monomers and oligomers) into acrolein by
dehydrating 3ꢀHPA, thus preventing the formation of the dynamic system. Acrolein is then
further converted to UV absorbing compounds allowing its quantification through
2

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
Page 3 of 16
RSC Advances
View Article Online
DOI: 10.1039/C5RA18274C
colorimetry. Though they allow the determination of the total amount of 3ꢀHPA (mol.Lꢀ1),
these methods have two main drawbacks. Not only they require extra handling of the samples
and the use of harsh conditions (toxic or harmful reagents), but they also do not distinguish
between 3ꢀHPA and eventual other aldehydes ꢀ such as acrolein ꢀ originally present in the
samples, thus leading to inaccurate values. HPLCꢀbased analytical methods were also
reported,3 however pure 3ꢀHPA (or 3ꢀHPA system) analytical standards being not readily
available from common chemical suppliers, such method requires preparing them before
performing the analyses. Up to now, pure 3ꢀHPA aqueous samples were obtained through
bioconversion of glycerol using Lactobacillus reuteri,3 followed by purification of the
resulting fermentation broth involving semiꢀpreparative HPLC3a or flash chromatography on
silica gel10 to remove contaminants (e.g., glycerol, 1,3ꢀPDO, acrolein). Though effective,
semiꢀpreparative HPLC and silica gel chromatography provide 3ꢀHPA in small quantity or
with relatively low yield (45%), respectively. In view of these considerations, the preparation
of pure, acroleinꢀ and waterꢀfree 3ꢀHPA standard in high yield through a scalable chemical
synthesis under mild conditions appeared as a simpler and faster alternative. The main
objectives of the present work were therefore to (i) design a straightforward and efficient
chemical synthesis of 3ꢀHPA that could be either easily implemented in an analytical
chemistry laboratory, or used by a chemical supplier to produce commercial 3ꢀHPA
standards, (ii) fully characterize pure synthetic compound using 13C NMR, HPLC, GCꢀMS,
LCꢀMS and FTꢀIR, as well as (iii) use it to develop a reliable quantitative analytical method
using HPLC.
EXPERIMENTAL SECTION
Materials and methods.
Chemicals (1,2,4ꢀbutanetriol, sodium periodate, acrolein (>99.0% analytical standard, 0.2%
hydroquinone as stabilizer) were purchased from Aldrich and used as received. Reagent grade
solvents were purchased from ThermoFisher Scientific. Deuterated chloroform (CDCl3) was
purchased from Eurisoꢀtop.
Instrumentation.
Evaporations were conducted under reduced pressure at temperature below 25 °C to avoid 3ꢀ
HPA evaporation. Column chromatography was carried out with an automated flash
chromatography system (PuriFlash 4100, Interchim) and preꢀpacked INTERCHIM PFꢀ30SIꢀ
HP columns (30 µm silica gel).
IR and UVꢀVisible analyses were performed on Cary 60 UVꢀVIS and Cary 630 FTꢀIR from
Agilent technologies, respectively.
HESIꢀMS analyses were performed on a QꢀExactive (ThermoꢀFischer Scientific) at a flow
rate of 5 µL/min in positive mode. The electrospray voltage was set to 3.70 kV, the S lens RF
level was set to 55 V. The sheath auxiliary and sweep gas (nitrogen) flow rates were set to 30,
10 and 0 arbitrary units, respectively. The capillary and heater temperatures were set to 320
3