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Quantitation of Sialic Acids in Infant Formulas by Liquid Chromatography-Mass Spectrometry: An Assessment of Different Protein Sources and Discovery of New Analogues Aaron Wylie, and Wesley F. Zandberg J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01042 • Publication Date (Web): 06 May 2018 Downloaded from http://pubs.acs.org on May 6, 2018
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Journal of Agricultural and Food Chemistry
Quantitation of Sialic Acids in Infant Formulas by Liquid Chromatography-Mass Spectrometry: An Assessment of Different Protein Sources and Discovery of New Analogues Aaron D Wylie1, and Wesley F Zandberg1* 1
The University of British Columbia, Okanagan, Chemistry Department
* Corresponding author contact details: Wesley F Zandberg The University of British Columbia, Department of Chemistry Charles E Fipke Centre for Innovative Research 3247 University Way Kelowna, BC V1V 1V7 Canada
[email protected] 250-807-9821
(t)
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Abstract.
2
Glycosidically-bound, but not free, dietary sialic acids are used for the biosynthesis of new
3
glycoconjugates in humans, making the quantitation of these two forms in infant food sources
4
important, as in neonates the demand for sialic acid may exceed the de novo biosynthetic supply.
5
Here, a rapid high-performance liquid chromatography-mass spectrometry method was
6
developed to identify and quantitate glycosidically-bound and free sialic acids in infant formulas.
7
The sialic acid contents of eight commercially-available infant formulas with varying protein
8
source or manufacturer were investigated. The formula protein sources (whey vs. casein) did not
9
have a large impact on the ratios of free to bound sialic acids, nor did protein hydrolysis or
10
sample form (solid vs. liquid). Hydrolyzed bovine whey protein-based formulas were found to
11
contain the highest amount of the most abundant human sialic acid, 5-N-acetylneuraminic acid
12
(Neu5Ac). O-acetylated Neu5Ac was quantified in all formulas tested and, for the first time, 2-
13
keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn) was detected in several infant formulas.
14 15
Keywords. Sialic acids; infant formulas; milk proteins; mass spectrometry, liquid
16
chromatography
17 18 19 20 21 22 23
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Introduction
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Sialic acids are an important family of anionic, amine-containing monosaccharides
27
composed of a nine carbon backbone and bearing a common α-keto carboxylic acid functional
28
group (Figure 1). Sialic acids are almost exclusively observed glycosidically linked to the non-
29
reducing ends of the oligosaccharides attached to proteins, lipids or lactose, a position in which
30
they are poised to mediate a plethora of biological recognition events linked to both healthy and
31
diseased states. Sialic acids are biosynthesized by all vertebrates, with the most abundant
32
member of this family being 5-N-acetyl-D-neuraminic acid (Neu5Ac). Another common sialic
33
acid species, 5-N-glycolyl-D-neuraminic acid (Neu5Gc), although commonly observed in
34
mammals, is not biosynthesized by humans (Figure S1), although they may acquire it through
35
their diets. To date over 60 sialic acid analogues have been described1 in which Neu5Ac or
36
Neu5Gc cores are elaborated with O-acetyl- (Figure 1B) or O-methyl groups. These
37
modifications have a significant impact on Neu5Ac/Neu5Gc recognition by lectins or hydrolytic
38
enzymes. Other monosaccharides such as 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid
39
(Kdn) and 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo), although not classified as sialic
40
acids per se, nevertheless bear identical α-keto acid moieties (Figure 1B) and, in the case of Kdn
41
share a common biosynthetic origin.
42
Though humans are capable of biosynthesizing Neu5Ac, and related O-acetylated
43
analogues, dietary sources of these monosaccharides are important to human health,1 especially
44
in neonates. The free milk oligosaccharides have received considerable attention in this respect
45
as these have a profound impact on infant health due to their prebiotic capability and other
46
functions.2 Human milk oligosaccharides (HMOs) are, in fact, recognized as a major factor
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responsible for the reduced incidences of disease among breast-fed infants,3–5 prompting efforts
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to supplement formulas with bovine milk-derived analogues.6 Both human milk and infant
49
formulas also contain abundant amounts of glycoproteins which, in formulas, are usually of
50
bovine origin. The sialic acid content of infant formulas has been reported to be less than one
51
quarter the abundance observed in mature human milk, and whereas ca. 70 percent of all sialic
52
acid in human milk is borne by HMOs, an equivalent fraction in formulas has been reported to be
53
glycosidically-bound to glycoproteins.7 Recent evidence has suggested that the oligosaccharides
54
linked to milk proteins may, like HMOs, have prebiotic functions.8 Beyond prebiotic functions,
55
animal studies have indicated that exogenous sialic acids may play roles in developmental
56
processes linked to brain development and neural plasticity, especially in early infancy when the
57
neonatal capacity to biosynthesize Neu5Ac de novo may not completely meet the required
58
levels.1,9
59
Accurate, precise analytical methods for quantitating Neu5Ac, Neu5Gc, and their
60
analogues (Figure 1A) are required if infant formulas are to be more closely matched to human
61
milk in terms of both the content and macromolecular distribution of these carbohydrates.
62
Studies in both people and in animals models10–14 have demonstrated that Neu5Ac and Neu5Gc
63
are more bioavailable when they are glycosidically-bound than when ingested in their free,
64
unconjugated forms which are rapidly excreted in the urine. Reports describing the detection of
65
intact, Neu5Ac-containing HMOs in the blood stream15 corroborate the hypothesis that the
66
bioavailable forms of sialic acid are indeed glycosidically-bound. Although sialylated
67
glycoconjugates are thought to reach the large intestine without significant metabolism, their
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prebiotic functions in this organ may be in direct competition with the necessary intestinal
69
absorption required if these are to serve as sialic acid sources elsewhere in the body. Note that
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the microbial metabolism or recycling of sialic acids requires their prior hydrolysis from
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glycoconjugates by sialidases16–18 or trans-sialidases,19 enzymes that are sensitive to Neu5Ac or
72
Neu5Gc modifications such as O-acetylation.20,21
73
While it is important to determine what fraction of ingested glycoprotein-bound Neu5Ac
74
represents the actual bioavailable amount, with respect to Neu5Gc the converse is also of
75
interest, especially in the case of infants fed formulas derived from bovine milk proteins that are
76
sources of this non-human monosaccharide. The reason for this is that diet-derived Neu5Gc may
77
be recycled via monosaccharide salvage pathways and subsequently incorporated into host
78
tissues13 22 by sialyltransferases that do not distinguish between Neu5Ac and Neu5Gc. However,
79
the human immune system does differentiate between Neu5Ac and Neu5Gc, recognizing
80
Neu5Gc-containing host tissues as antigenic23,24 a phenomenon that may be linked to the
81
etiology of several inflammatory disorders.14 Thus, it is prudent to ensure that infant formulas as
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closely as possible match the sialic acid content of human milk, both in terms of the bioavailable
83
monosaccharides that are desired (Neu5Ac), and those that are not (Neu5Gc).
84
Over the past decades several strategies have been developed for sialic acid quantitative
85
analysis. These include spectroscopic methods that require derivatization with chromogens,25,26
86
or their conversion into fluorogenic quinoxaline-containing analogues after condensation with
87
1,2-diamino-4,5-methylenedioxybenzene (DMB10,13,14,16,27) or 4,5-dimethylbenzene-1,2-diamine
88
(DMBA; Figure 1B).28 These methods alone are unable to distinguish between Neu5Ac and
89
Neu5Gc and thus are most often combined with high-performance liquid chromatography
90
(HPLC) with optical detection,10,16,26,29,30 although mass spectrometry (MS) has also been
91
employed to detect and quantitate either DMB-labelled14,27 or free sialic acids.31 In nearly all
92
studies reported to date concerning the sialic acid content of infant formulas, strong acids (HCl,28
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trifluoroacetic acid (TFA31) or H2SO416,26,29,30) have been used to cleave the acid-labile sialic acid
94
glycosidic bond prior to subsequent derivatization and analysis. However, note that unless care is
95
exercised during the sample preparation10 this procedure is unable to distinguish between bound
96
and free forms of sialic acid; furthermore, these acidic conditions may cleave labile O-acetyl
97
groups preventing the detection of these species, a complication that some researchers10,13,14 have
98
avoided by deliberately saponifying these esters under alkaline conditions prior to sialic acid
99
hydrolysis. In contrast, sialic acid hydrolysis catalyzed by acetic acid (AcOH) has been shown to
100
preserve acid-labile O-acetate esters.27,32,33 More recently,14 both AcOH and TFA have been used
101
under different derivatization strategies (Figure 1C) to differentiate between glycosidically-
102
bound and free Neu5Ac and Neu5Gc (albeit with prior O-acetate saponification).
103
This study combines the Varki group’s hydrolytic procedures14 with the superior
104
derivatization ability of DMBA28 (over the widely used DMB) in order to accurately assess the
105
distribution of these monosaccharides in a range of infant formulas using HPLC-MS. Relevant
106
figures of merit for the MS analysis of DMBA-labelled sialic acids have not yet, to our
107
knowledge, been reported in the literature. By using hydrolytic strategies capable of
108
distinguishing between free and bound sialic acids, while still preserving functionally-relevant
109
O-acetate esters, the following research objectives/questions were addressed:1) Does the
110
Neu5Ac:Neu5Gc ratio in infant formulas vary according to glycoprotein source, i.e. whey vs.
111
casein, or hydrolyzed vs. intact protein? 2) Do the conditions required to produce extensively
112
hydrolyzed, hypoallergenic formulas alter the free vs. bound ratios of sialic acids? Likewise, do
113
liquid and solid formulas vary in this respect? 3) Finally, to what extent do infant formulas
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contain O-acetylated sialic acids or analogues such as Kdn? Our data indicate whey-based
115
formulas contained the highest amount of Neu5Gc while the Neu5Ac:Neu5Gc ratio was lowest
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in casein-derived products. The developed methods, for the first time, permitted the separate
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quantitation of free vs. glycosidically-bound sialic acids and, in addition, permitted the
118
identification of Neu5,9Ac2 and Kdn in infant formulas.
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Materials and Methods
120
Chemicals and General Details.
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otherwise. Methanol (MeOH), trifluoroacetic acid (TFA), acetonitrile (ACN), and 4,5-
122
dimethylbenzene-1,2-diamine (DMBA; also called 4,5-dimethyl-1,2-phenylenediamine) were
123
purchased from Sigma-Aldrich (St. Louis, MO, USA). DMBA was stored at 4 °C. Glacial acetic
124
acid (AcOH, ACS grade) was purchased from EMD Chemicals (Savannah, GA, USA). Formic
125
acid (FA) was purchased from Fluka (Steinheim, Germany). Neu5Ac (≥98%) was purchased
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from Carbosynth (Compton, Berkshire, UK). Neu5Gc ( ≥95%) and Kdo ( ≥97%) were purchased
127
from Sigma-Aldrich. [3-13C]-N-acetylneuraminic acid ([3-13C]-Neu5Ac, 99 atom-%) was
128
purchased from Omicron Biochemicals, Inc. (South Bend, IN, USA). Bovine submaxillary
129
mucin (BSM) was purchased from Calbiochem (San Diego, CA, USA) and stored at 4 °C. Kdn
130
was chemoenzymatically synthesized and kindly provided by Dr. Margo Moore (Simon Fraser
131
University; Burnaby, BC, Canada). Deionized H2O (18 MΩ) was supplied by a Barnstead E-pure
132
water purification system (Thermo Fisher Scientific; Waltham, MA, USA). Infant formulas were
133
purchased from commercial retailers and stored as instructed. All formulas were tested prior to
134
their expiration dates and all except the caprine-based formulas were labelled as “starter
135
formulas”, i.e. intended for infants between 0 and 3 months (Table 2). Discovery™ DSC-18
136
solid phase extraction (SPE) cartridges (1 mL, 50 mg, 50 µm particle size, 70 Å pore size) were
137
purchased from Supelco (Bellefonte, PA, USA) and Strata™ C18-E SPE cartridges (1 mL, 50
138
mg, 55 µm particle size, 70 Å pore size) were purchased from Phenomenex (Torrance, CA,
All solvents used were of HPLC grade unless stated
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USA). Monosaccharides, samples and standard solutions were all stored at -20 °C in the dark
140
unless stated otherwise.
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Standard and Calibration Solutions. Stock solutions of 1, [3-13C]-Neu5Ac (ISTD), 2, 3 and 4
142
were prepared by weighing 10.0 mg of each and dissolving in 18 ΩM H2O to a final stock
143
concentration of 10.0 µg/mL. DMBA solutions (24 mM in either 2 M AcOH or 40 mM TFA)
144
were prepared fresh daily. Qualitative standards of each sialic acid were prepared as follows: 100
145
µL aliquot of each standard was added to 1.5 mL microcentrifuge tubes and mixed with 15 µL of
146
the ISTD solution. Solutions were dried (Savant SPD121P SpeedVac Concentrator) and re-
147
dissolved in 50 µL of the DMBA solution (in 2M AcOH). After brief sonication, solutions were
148
incubated at 60 °C for 1 h in the dark28, cooled on ice, dried, solubilized in 100 µL 30% MeOH
149
(v/v) and subsequently transferred to amber HPLC vials with glass inserts. Six-point calibration
150
solutions were likewise prepared at monosaccharide concentrations of 0.5 - 15 (1, 3, 4) and 0.5 –
151
20 (2) µg/mL and fortified with 15 µL of the ISTD solution. Each calibrant was derivatized with
152
DMBA as outlined above.
153
Sample Preparation. BSM (2 mg), a source of O-acetylated Neu5Ac/NeuGc,10,33 was weighed
154
into a 1.5 mL microcentrifuge tube followed by addition of 500 µL 2 M AcOH. Following
155
dissolution by vortex mixing and sonication, the solution was incubated at 80 °C for 3 h and
156
subsequently cooled on ice.10,14,27,32 The hydrolysate was then centrifuged (15,600 × g, 10 min)
157
and the sialic acid-enriched supernatant retrieved and dried. The DMBA labelling solution (250
158
uL) was added to the dried samples, which were dissolved by brief sonication, and then
159
incubated in the dark (60 °C, 1 h). Following cooling on ice, the solution was purified by C18
160
SPE as follows: the cartridge (Discovery™) was conditioned with aq. 80% ACN/0.1% TFA (1
161
mL) followed by rinsing with H2O (2 x 1 mL). Derivatized sialic acids were loaded, washed with
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H2O (1 mL) and eluted with two sequential aliquots of aq. 50% ACN (0.5 mL). A flow rate of ~1
163
mL/min was maintained. Eluate was then dried in vacuo at ambient temperature, and solubilized
164
in 100 µL HPLC solvent as outlined above.
165
Each formula (~25.0 mg, n = 5 replicates per formula) was accurately weighed and vortexed
166
in 2 M AcOH (1 mL); liquid formula was lyophilized before analysis. An aliquot of each (100
167
µL) was collected, fortified with ISTD (7.5 µL of a 100 µg/mL stock solution), and brought up to
168
a total volume of 1 mL (2 M AcOH). After vortexing, samples were incubated and the
169
hydrolysates centrifuged (vide supra). An aliquot from the middle aqueous layer (200 µL) was
170
retrieved from each sample and dried. To the dried residue was added the DMBA solution (2 M
171
AcOH, 50 µL) followed by sonication and incubation in the dark (60 °C, 1 h). Following cooling
172
on ice, the solutions were brought up to a volume of 200 µL (18 MΩ H2O), purified by C18 SPE
173
(vide supra), dried, and solubilized in 30 % MeOH (100 µL). Sample preparation for the
174
determination of Neu5Gc was performed separately; each formula (~10.0 mg, n = 3) was
175
accurately weighed and directly fortified with ISTD (7.5 µL, 100 µg/mL), brought up to a total
176
volume of 1 mL (2 M AcOH) and prepared as above. Identical procedures were used to
177
quantitate free 1, with the exception that the DMBA labelling reagents were dissolved in 40 mM
178
TFA14 (in the absence of reducing agents) and incubated for 48 h at 4 °C. Note that values for
179
glycosidically-bound sialic acids were determined by taking the difference between the total pool
180
(determined using the AcOH hydrolysis procedure) and the free sialic acid fraction.
181
uHPLC-MS/MS.
182
Technologies, Santa Clara, CA, USA) with a 1290 Infinity binary pump, 1290 Infinity
183
autosampler and a 1290 Infinity column compartment. All DMBA-derivatized sialic acids were
184
analyzed on a Phenomenex Kinetex Biphenyl 100 x 2.1 mm column (2.6 µm particle size, 100 Å
uHPLC was conducted on an Agilent 1290 Infinity system (Agilent
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pore size) at a temperature of 40 °C. Samples were analyzed using an injection volume of 2 µL
186
(QToF-MS) and 10 µL (QToF-MS/MS) at a flow rate of 0.500 mL/min. Mobile phases A and B
187
were set to H2O and MeOH, respectively, each containing 0.1% formic acid. Gradient elution
188
was programmed with a total runtime of 14 min as follows: 0 – 4.2 min, 30 – 36.2% B; 4.2 – 8
189
min, 38% B; 8 – 11 min, 50% B; 11 – 11.1 min, 90% B; 12.1 – 12.2 min, 30% B.
190
Mass spectrometry was conducted on an Agilent Technologies 6530 QToF mass spectrometer
191
with an Agilent Jet Stream electrospray ionization (ESI) source. The spectrometer was set to
192
positive ion mode, source drying gas (N2) temperature 300 °C and flow rate of 11 L/min, sheath
193
gas (N2) temperature 350 °C with a flow rate of 11 L/min, nebulizer pressure of 40 psig, source
194
nozzle voltage 1000 V, and capillary voltage 3500 V. Compounds were identified by Agilent
195
MassHunter’s Find-By-Formula algorithm. QToF-MS spectral acquisition rate was 2 spectra/s,
196
while the QToF-MS/MS spectral acquisition rate was 1 spectra/s for precursor ions and 2
197
spectra/s for product ions. All sialic acids species, except 2, were quantified based on the
198
response ratios of their respective molecular ions against the ISTD, while 2 was quantified by the
199
absolute intensity of the m/z 426.1866 to 213.1028 parent-precursor ion transition.
200 201
Method Performance. Calibration curves were constructed using linear regression. Limit of
202
detections (LOD) and limit of quantitations (LOQ) were assessed by the following equation:
203 204
= ×
(1)
205 206
Where was the standard deviation of n low-level calibrators (n = 20 in all cases) for
207
compound i and k was a scalar (k = 3.3 for LOD and 10 for LOQ determinations). LOD and
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LOQ are reported in units of mass (ng) loaded onto the HPLC column. Method accuracy was
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assessed by fortifying a soybean-based infant formula, a matrix free of any sialic acid species,
210
with 1 µg/mL 1 – 4 (n = 3 for each monosaccharide) and the ISTD; percent recovery values for
211
the different labelling conditions were determined for each monosaccharide by comparison to the
212
MS detector response of controls prepared in water.
213
Data Acquisition and Processing.
214
performed using the MassHunter Workstation software suite (Agilent Technologies), with
215
version numbers as follows: Data Acquisition Workstation (v B.06.01), Qualitative Analysis (v
216
B.07.00) and Quantitative Analysis (v B.07.00). Data processing and statistics were performed
217
using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA).
218
Results and Discussion
219
Method Development and Validation. Commercially-available or synthesized standards 1, 2, 3
220
4, and O-acetylated sialic acids obtained from the weak acid hydrolysis of a well-characterized
221
glycoprotein (BSM) were labelled with DMBA in the absence of the commonly used reducing
222
agents Na2S2O4 and 2-mercaptoethanol.28 As noted by Wang et al.28 these DMBA-labelled
223
derivatives were very stable and could be stored for months in solution (at -20 °C) and
224
subsequently reanalyzed; they also tolerated both acidic and alkaline hydrolysis conditions,
225
which could be used as a secondary means of identifying O-acetylated species33 in the absence of
226
MS detection (data not shown). HPLC columns encompassing a range of different stationary
227
phases—including reverse (superficially porous C18), normal (aminopropyl), graphite
228
(HyperCarb) and biphenyl phases—were initially screened to determine which provided the best
229
analyte resolution, especially for isobaric pairs of O-acetylated species. It was determined that
230
the biphenyl column gave the best resolution all DMBA-labelled sialic acid species, rapidly
uHPLC-QToF data acquisition and processing were
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achieving baseline separation of all isobaric pairs (Figure 2). The biphenyl-based method
232
developed here offers a substantial improvement in analysis time compared to the C18-based
233
separation procedure initially reported,28 reducing retention times for 1 and 2 from 17 and 21
234
min, respectively, to 3.4 and 3.9 min; approximately 2-16,27 to 10-fold14,33 reductions in analysis
235
time were also realized for 1 and 2 labelled with the more commonly employed DMB. As
236
anticipated, the AcOH-catalyzed hydrolysis procedure preserved the O-acetate esters on sialic
237
acids borne by BSM (Figure 2B). These species encompassing 5 – 8 and several sialic acids
238
bearing multiple O-acetyl moieties (nine analogues in total) were initially identified based on
239
their relative retention times, m/z, and their known relative abundances in BSM;33 several of
240
these putative assignments were subsequently confirmed by tandem MS (as discussed below).
241
Having optimized the chromatographic resolution of DMBA-derivatized sialic acids
242
attempts were made to reproduce the AcOH-catalyzed sialic acid hydrolysis27,32 procedure with
243
the DMBA labelling of O-acetyated sialic acids. Likewise, the TFA-catalyzed14 labeling
244
procedures were evaluated with DMBA in the absence of reducing agent additives. While both
245
procedures worked well for pure standards (1 – 4) and for sialic acids produced from model
246
glycoproteins like BSM—which could be easily removed from the DMBA labelling mixture by
247
centrifugation—the infant formulas formed colloidal suspensions that could not be clarified (by
248
centrifugation) prior to DMBA-labelling, a condition that hindered efficient analyte
249
derivatization and that posed a risk to plugging and/or damaging HPLC components. Therefore,
250
unlike many reported procedures for sialic acid analysis using DMB/DMBA where the
251
hydrolysis and labelling are performed sequentially on the same sample (without minimal
252
workup besides precipitation/centrifugation), for the formulas tested here an extra sample
253
preparation step was required. Both solid phase extraction (SPE) and filtration (through a 3 kDa
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cutoff centrifugal filter) proved effective at removing insoluble infant formula components from
255
the sialic acid hydrolysis mixture. The final, optimized procedure involved precipitation of the
256
hydrolysis mixture (by incubation on ice followed by centrifugation) prior to DMBA-labelling
257
after which a solid phase extraction (C18) post-labelling cleanup permitted the removal of AcOH,
258
salts, remaining suspended formula components, and soluble, polar formula ingredients prior to
259
HPLC-MS analysis. Relevant figures of merit for the optimized DMBA-labelling and analysis
260
procedures were determined for the four α-keto acids (1 – 4) for which it was possible to produce
261
external calibration curves, by fortifying these compounds into a soy-based formula naturally
262
free of these monosaccharides (Table 1). On-column picogram (pg) detection and quantitation
263
limits (LOD/LOQs) were obtained for all four standards which, although higher than those
264
reported for the fluorescence-based detection for 1 and 2 (albeit, details on how these were
265
calculated in this instance were not provided28), were nevertheless suitable for the purpose of
266
sialic acid quantitation in infant formulas. Acceptable method precision was ensured through the
267
use of a 13C3-labelled Neu5Ac ISTD. However, while the matched ISTD permitted the accurate
268
quantitation of 1 following the AcOH-hydrolysis and DMBA-labelling method, it proved less
269
effective for other sialic acid analogues (2 – 4). In contrast, near quantitative recoveries of 1 – 4
270
were observed when pairing DMBA with the TFA-catalyzed procedure. While the source of this
271
discrepancy was not analyzed in detail, these data suggest that uncoupling sialic acid hydrolysis
272
(AcOH-catalyzed) and subsequent solvent removal prior to TFA-catalyzed DBMA-derivatization
273
may be the best method if absolute quantitation is desired. Nevertheless, only the AcOH-
274
catalyzed procedure was used for the analysis of glycosidically-bound sialic acids in formula
275
since (i) semi-quantitative analysis permitted us to address all research objectives/hypotheses, (ii)
276
it reduced labelling time from 48 h to 2 h, and (iii) the AcOH method would accommodate a
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one-pot hydrolysis and labelling procedure for matrices less challenging than infant formulas. It
278
should be noted, however, that the TFA-catalyzed labelling procedure for free sialic acids must
279
be used even with filtration (through size exclusion cartridges)10 or SPE of formula samples prior
280
to addition of the DMBA due to the presence of traces of both sialylated bovine milk
281
oligosaccharides34 and gangliosides29 that are contained within the glycoprotein ingredients of
282
infant formulas.
283
Quantitation of Sialic Acids in Infant Formulas. A considerable amount of research has been
284
dedicated to understanding the plethora of bioactive functions filled by milk proteins.35 Based on
285
the knowledge generated by this research, and due to advances made in the technology used to
286
fractionate bovine milk proteins and/or valorize them from byproduct streams,36 the specific
287
protein sources used in infant formulas have gradually shifted from casein-dominant formulas to
288
those containing mixtures of casein and whey,29,36 or only whey.37 Of concern herein was the
289
question of sialic acid content as a function of the glycoprotein source used to prepare various
290
infant formulas (i.e. sialic acid as a bioavailable micronutrient). Accordingly, the formulas
291
chosen for analysis here were a subset of a wide range of commercially-available products in
292
which a single glycoprotein source (either whey or casein) was clearly identified on the products’
293
ingredients label (Table 2).
294
Considerable variability (spanning roughly 40 to 250 mg/100 g formula) in total Neu5Ac
295
(1) content among all bovine milk-based formulas was observed using the developed method
296
(Table 3), consistent with previous literature.16,26,29 All four bovine whey-based formulas
297
analyzed were, at minimum 2.6-fold higher in 1 content than those derived from casein, a trend
298
that has been previously reported for formulas of differing whey/casein ratios.29 When
299
comparing formulas that were only derived from extensively hydrolyzed protein ingredients (C
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300
and D vs. E and F), it was observed that the 1 content in the whey-containing samples exceeded
301
that of the casein-containing products by over five-fold in spite of the fact that the all of the
302
casein-derived products contained slightly higher levels of protein. Interestingly, the amount of
303
glycosidically-bound (and thus presumably bioavailable) 1 detected in whey-based formulas
304
prepared from hydrolyzed proteins was found to be nearly double that observed in the two non-
305
hydrolyzed whey formulas analyzed. It is hypothesized that peptides produced from the
306
proteolytic processing of whey are likely enriched in sialic acid-bearing oligosaccharides as these
307
are known to sterically block protease cleavage sites,38 thus preventing glycosylated whey
308
peptides from extensive degradation and ultimate removal. Relative to the glycosidically-bound
309
levels of 1 there was less variability observed in the free 1 content of each formula and, across all
310
samples analyzed, no correlation (Spearman’s rank correlation, ρ = 0.07) between the
311
glycosidically-bound and free levels of 1 was detected. This suggests that if sialic acids are lost
312
from milk protein ingredients due to the hydrolysis (or any other preparative) procedures, any
313
resulting free sialic acids are not retained in the final products, which, in the case of whey-
314
derived peptides, were actually enriched in total sialic acid content. Finally, little variation was
315
observed in the 1 content of liquid vs. powdered formulas, both prepared from hydrolyzed bovine
316
casein, suggesting that the dehydrating procedure, and/or storage conditions, did not lead to the
317
hydrolysis of sialic acid glycosidic bonds.
318
Quantitation of 2 in infant formulas using the developed method proved to be difficult.
319
For all formulas tested, with the exception of one partially hydrolyzed whey and a soy-based
320
formula, an abundant co-eluting matrix component with m/z = 425.1931 interfered with the
321
generation of extracted mass chromatograms for 2, thereby preventing accurate peak integration
322
(Figure 3). Specifically, the A+1 peak for this interferant could not be mass resolved from the
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323
monoisotopic peak (m/z = 426.1883) for 2. Attempts to chromatographically resolve these
324
compounds were unsuccessful, as were attempts to remove the interferant by C18 SPE and/or
325
filtration either prior to, or after, DMBA-labelling. This interference persisted using both the
326
AcOH- and TFA-catalyzed labelling procedures and, curiously, was observed in commercially-
327
available (from Sigma-Aldrich) bovine whey but not bovine submaxillary mucin (BSM; Figure
328
2B). It was reasoned that tandem MS could be used to resolve this issue, as DMBA-labelled 2
329
and the problematic interferant yielded unique product ion spectra (Figure 3D and 3E,
330
respectively). More specifically, 2 yielded prominent product ions at m/z of 213.1027, 267.1143
331
and 408.1816, each corresponding to a DMBA-containing fragment analogous to the previously
332
reported major product ions for DMB-labelled Neu5Gc;33 importantly, none of these major
333
product ions were observed among the product ions of the interferant. Although method
334
LODs/LOQs were higher using MS/MS detection (Table 1), they were sufficient for establishing
335
the relative values of 2 among all infant formulas tested (Table 3). Taken as a whole, the levels
336
of 2 (2 – 9 mg /100g) detected here were near the 2.0 - 5.2 mg/100 g range reported for six
337
different formulas tested by Lacomba et al.29 although they are higher than the 0.14 – 0.97
338
mg/100 g range reported by Claumarchirant et al.16 It should be noted that the levels of 1
339
quantified in the latter study were also an order of magnitude lower than those reported herein
340
and by Lacomba et al. The source of these discrepancies is currently unclear as both Lacomba et
341
al. and Claumarchirant et al. analyzed whey-based formulas, using DMB-derivatization followed
342
by fluorescence-based quantitation. Chen et al.31 have previously observed that milk originating
343
from animals in different countries contained differing absolute amounts of 2, which may
344
partially explain the differences observed here. In absolute terms, the hydrolyzed whey-based
345
formulas tested here contained the highest levels of 2 among all bovine-based products; however,
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346
these formulas also contained the highest amount of 1 and thus, the 2-to-1 ratio was comparable
347
for all whey-based formulas; indeed, considering only bovine-based products (Table 3, formulas
348
A – F), the 2 content as a fraction of total sialic acids was the highest for the casein-derived
349
formulas. Formulas produced from caprine (goat)-based protein ingredients contained very high
350
levels of 2, exceeding those found in all bovine-based products by a minimum ten-fold. This
351
result was anticipated based on observations that sheep and goat milk,1 dairy products (i.e.
352
cheese),14 and meat14,31 all contain much higher 2 levels than comparable bovine-derived
353
equivalents. It has been hypothesized by Taylor et al.24 that the origin of anti-Neu5Gc
354
antibodies23 in breast-fed infants corresponds with the introduction of solid foods and/or
355
formulas containing sources of this non-human monosaccharide. In vitro, high relative levels of
356
1 tend to lead to a decrease in 2 incorporation into newly biosynthesized glycoproteins39. Should
357
this metabolic competition occur in vivo it would therefore follow that the extensively
358
hydrolyzed bovine whey formulas would provide the lowest amount of bioavailable 2 since these
359
product have the highest 1-to-2 ratio; conversely (and independent of any other health benefits
360
such as reduced risk of milk allergies, etc.) caprine-based formulas contain the highest amounts
361
of total 2 and the lowest amount of biosynthetic completion with 1 to reduce the probability of its
362
biosynthetic incorporation into infant tissues.
363
To the best of our knowledge, this is the first report of mild acid hydrolysis being used to
364
prepare infant formula sialic acids for analysis. Using this procedure, abundant amounts of 9-O-
365
acetylated 1 (i.e. 5) were detected in all infant formulas (Table 3). This assignment was based on
366
the co-retention of this species with the dominant mono-O-acetylated 1 analogue in BSM as well
367
as the distinguishing product ion spectra produced for all sialic acid species (Table 4). Assuming
368
that 5 and 1 have identical MS response factors, the highest amount of 5 was observed in the
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369
extensively hydrolyzed whey formulas and the lowest was observed for the caprine-based
370
products. However, when 5 is expressed as a ratio of the total 1 present all bovine-derived
371
formulas, it exhibits a remarkably consistent 15:1 ratio of 1-to-5. The impact of 1 O-acetylation
372
on its bioavailability is difficult to predict. On the one hand, this modification may protect 5-
373
containing oligosaccharides from microbial sialidases in the infant GI tract, permitting the
374
systemic absorbance of a greater fraction that may be used for the biosynthesis of new
375
glycoconjugates, although human sialidases are also known to be less tolerant of O-acetylated
376
sialic acids.20 Future research is required to establish the metabolic equivalence, or lack thereof,
377
of these differing sialic acid species.
378
The analytical procedures developed here also permitted the detection of Kdn (3) in
379
several (D, F, G and H) of the formulas tested, although the levels were below the method LOQ.
380
This represents the first time 3 has been detected in infant formulas, although Chen et al. have
381
previously noted that this sialic acid-like compound accounts for 12 percent of the total sialic
382
acids in bovine milk, and exceeds 25 percent in milk-derived food products such as cheese and
383
yogurt.31 To date there has been less research concerning the expression and function of Kdn-
384
containing glycoconjugates in humans40 than other more well-known sialic acids (i.e. 1 and 2)
385
and few details are known concerning the regulation of de novo Kdn biosynthesis.41
386
Nevertheless, the ability of some sialyl-transferases to use cytidine-5’monophosphate (CMP)-
387
activated Kdn as a substrate,42 and the noted promiscuity of some Neu5Ac biosynthetic
388
enzymes,43,44 suggests that the salvaging of dietary 3, like 2, is possible and that the functional
389
consequences should be considered, especially as dairy sources of 3 appear to be the major
390
exogenous source31 of this monosaccharide.
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391
MS/MS Analysis of DMBA-Labelled Sialic Acids. MS/MS experiments conducted on DMBA-
392
labelled sialic acid standards including isobaric mono-O-acetylated species produced product
393
ions which could be used to identify the acetate substitution position on these isobaric species
394
(Table 4). A collision cell acceleration voltage of 20 V produced fragments with the greatest
395
amount of structural information; in every case, the most abundant product ions contained the
396
quinoxaline moiety. The product ion with nominal m/z = 267 was observed as the base peak for
397
1, 2 and 7 but was absent in 5, 6 and 9. The lack of this product ion in 5, 6 and 9 may be due to
398
the necessity of free hydroxyls at positions C8 and C9 to result in favorable fragmentation to the
399
proposed m/z = 267 product ion. The closely-eluting isobars 5 and 6 exhibit similar product ion
400
spectra, except that 6 has a lower abundance of the m/z = 297 and [M-H2O+H]+ product ions,
401
attributable its inability to undergo dehydrative cyclization between the hydroxyls of C4 and C8
402
due to the C8-position of the acetate. The assignments reported here are consistent with those
403
reported by Klein et al. (albeit with the DMB label).33
404
In conclusion, a recently introduced sialic acid fluorescent derivatizing reagent, DMBA,28
405
has been extended here to encompass the MS and MS/MS analysis of these monosaccharides.
406
We have demonstrated that sialic acid derivatization using DMBA occurs efficiently under both
407
AcOH-7,26,27 and TFA-catalyzed14 conditions permitting us to quantitate, for the first time, the
408
levels of free vs. glycosidically-bound sialic acids in infant formulas in addition to detecting
409
previously unreported 9-O-acetylated-Neu5Ac. Our data showed a complete lack of correlation
410
between glycosidically-bound and free Neu5Ac levels, suggesting that any of the latter was not
411
produced at the expense of the former during manufacturing and/or storage of these formulas.
412
Our data also indicated that for formulas based on bovine-sourced proteins, the specific protein
413
source affected the Neu5Ac to Neu5Gc ratio, with casein-based formulas containing the lowest
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414
ratio, while hydrolyzed whey-based formulas contained nearly double the bioavailable Neu5Ac
415
as their non-hydrolyzed samples, and quadruple the levels observed in hydrolyzed casein-based
416
products. Our analyses have also demonstrated, for the first time, the presence of Kdn in infant
417
formulas. Finally, the predictable fragmentation patterns of BMBA-labelled sialic acids, like that
418
of their DMB-containing analogues,33 proved essential for the quantitation of Neu5Gc, which in
419
infant formulas was obscured by an abundant matrix component; the product ion spectra
420
recorded here (Figure 4) will be a valuable tool for the discovery of sialic acid analogues often
421
overlooked in food sources and other biological matrices.
422
Funding Sources. This research was supported by funding from the Natural Science and
423
Engineering Research Council of Canada (NSERC; Discovery Grant, 2016-03929).
424
Infrastructure was obtained with the support of the Canada Foundation for Innovation (project
425
number 35246) and the British Columbia Knowledge Development Fund. AW was supported by
426
an Undergraduate Student Research Award (USRA) from NSERC.
427
Supporting Information. The Supporting Information is available free of charge on the ACS
428
publication website. Neu5Ac/Neu5Gc biosynthesis and salvage pathway, product ion spectra,
429
extracted ion chromatogram for one formula sample, and mass spectra of Neu5Gc and
430
interferant.
431
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432
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195–201.
571 572 573 574 575 576 577
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Figure Captions
579
Figure 1. Analysis of sialic acids requires their hydrolysis and derivatization to form
580
quinoxaline-containing species. (A) Sialic acids are a large family of α-keto-acid-containing
581
monosaccharides, all bearing an amine moiety and a nine-carbon backbone, numbered in red.
582
The most abundant sialic acids are Neu5Ac (1) and Neu5Gc (2). Related monosaccharides that
583
are not strictly sialic acids include Kdn (3) and the bacterial Kdo (4). Common modifications to
584
sialic acids (and Kdn) include the attachment of O-acetyl (Ac) moieties to one or more hydroxyl
585
groups. Bovine-derived Neu5Ac (1) is most often mono-O-acetylated at the 9 (5), 8 (6), or 7 (7)
586
hydroxyl groups; likewise, 2 may be O-acetylated, yielding, for example, Neu5Gc,9Ac (8). (B)
587
Acyclic forms of sialic acids may be labelled with aromatic diamines, forming highly
588
fluorescent, quinoxaline-containing derivatives. Recently, stable sialic acid derivatives of 4,5-
589
dimethylbenzen-1,2-diamine (DMBA) have been synthesized in the absence of widely used
590
reducing agents.28 (C) Differing hydrolytic and labelling strategies have been developed to
591
distinguish between free and glyosidically-bound sialic acids.14
592
Figure 2. Sialic acids may be rapidly separated using a biphenyl-functionalized HPLC stationary
593
phase permitting their detection and quantitation by MS. (A) Separation of commercially-
594
available sialic acid standards (1 – 4) and (B) sialic acid species borne on bovine submaxillary
595
mucin (BSM). Note that the isobaric mono-O-acetylated species (5 – 7) were putatively assigned
596
based on their known relative abundances in BSM and their product ion spectra following
597
MS/MS analysis. Di-O-acetylated analogues are labelled Neu5Ac3.
598
Figure 3. An infant formula-specific matrix component prevented accurate 2 quantitation in the
599
absence of tandem mass spectrometry. (A) Extracted ion chromatogram (m/z = 426.1871) and
600
(B) the corresponding mass spectrum at this retention time demonstrating the presence of a
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601
prominent peak at m/z = 425.1931, the A+1 peak of which interfered with accurate integration of
602
2. (C) Mass spectrum of 2 standard demonstrating similarity to that of the interferant. (D)
603
Tandem MS (QToF) yielded a diagnostic product ion spectrum for 2 that differed from (E) the
604
product ion spectrum produced from the interfering species.
605
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Tables Table 1. Figures of merit for sialic acid quantitation by HPLC-MS following DMBA labelling. sialic acid
LOD/LOQa rrt ± sdb (ng) (mg/100g) (min)
Neu5Ac 0.129/0.392c 0.005/0.016 c
regression equatione
r2
recovery (%)f AcOH TFA
y = 0.391x - 0.174 0.999 106 ± 7.2 104 ± 2.1
Neu5Gc
0.159/0.482 9.73/29.5d
0.006/0.019 -0.519 ± y = 0.440x - 0.267 0.996 80 ± 6.5 114 ± 4.0 0.39/1.18 0.008 y = 9.131x + 356 0.995
Kdn
0.095/0.287c
0.004/0.011
-0.616 ± y =0.380x + 0.035 0.991 77 ± 5.3 104 ± 4.2 0.013
Kdo
0.114/0.347c
0.005/0.014
-0.453 ± y =0.454x – 0.322 0.991 58 ± 4.1 105 ± 5.4 0.010
a
LOD/LOQ = limit of detection / limit of quantitation. (ng): on-column; (mg/100g): per 100 g dry formula using reported method. bRelative retention time ± standard deviation (n = 10) with respect to the 13C3-Neu5Ac ISTD; Neu5Ac, which co-eluted with the ISTD had a retention time of 3.91 ± 0.014 min. cQToF-MS method; dQToF-MS/MS method; ey = ISTD-corrected peak area (QToF-MS) or absolute peak area (QToF-MS/MS), x = concentration; fPercent recoveries are reported ± the standard error of the mean (n = 3). AcOH: acetic acid derivatization method; TFA: trifluoroacetic acid derivatization method.
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Table 2. Infant formulas analyzed for sialic acid content.
manufacturer
country of origin
source / protein
A
i
Canada
bovine / whey
10.1
B
ii
Canada
bovine / whey
10.6
C
ii
Canada
bovine / whey
11.8
D
iii
Canada
bovine / whey
11
E
i
Canada
bovine / casein
13.9
F
ii
Canada
bovine / casein
14.1c
G
iv
USA
caprine / whey
15.8
Switzerland
caprine / undefined
n/ad
sample
H
v
protein concentrationa
formb
hydrolyzed
S
no
S
no
S
yes
S
yes
S
yes
L
yes
S
no
S
no
a
Per 100 g dried formula. bS = solid; L = liquid. cBased on a lyophilized sample. dn/a = not available.
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Table 3. Quantitation of glycosidically-bound and free form Neu5Ac with total Neu5Gc and Neu5,9Ac2 in infant formulas of differing glycoprotein sources (mg/100 g dry formula ± standard error from n = 5).
a
Neu5Ac
Neu5Gc
Neu5,9Ac2
bound
free
total
total Neu5Ac / Neu5Gc ratio
A
127 ± 2.2
7.29 ± 0.073
4.09 ± 0.341
32.8 : 1
8.38 ± 0.226
B
104 ± 1.1
5.22 ± 0.029
4.33 ± 0.166
25.2 : 1
6.45 ± 0.104
C
211 ± 11.2
2.35 ± 0.119
9.07 ± 0.512
23.5 : 1
13.3 ± 0.19
D
246 ± 15.7
4.55 ± 0.021
6.67 ± 0.207
37.6 : 1
15.2 ± 0.95
E
38.8 ± 0.51
3.53 ± 0.018
2.07 ± 0.090
20.4 : 1
2.61 ± 0.137
F
36.6 ± 0.62
5.67 ± 0.017
5.07 ± 0.302
8.3 : 1
2.74 ± 0.062
G
46.2 ± 0.83
2.76 ± 0.019
153 ± 2.3
0.3 : 1
1.64 ± 0.114
H
25.7 ± 0.63
2.51 ± 0.028
90.4 ± 1.79
0.3 : 1
1.19 ± 0.058
Formula
totala
Quantitated based on the Neu5Ac regression equation (Table 1).
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Table 4. List of characteristic product ions observed in the QToF MS/MS spectra of mono-Oacetylated Neu5Ac and Neu5Gc species. Proposed fragment structures are consistent with those observed previously for DMB-labeled sialic acids.
DMBA-labelled sialic acid Neu5,9Ac2 Neu5,8Ac2 Neu5,7Ac2 Neu5Gc,9Ac
position of O-Ac C9 C8 C7 C9
major ions (m/z) 279 / 297 279 267 / 297 279 / 297
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Figure Graphics
Figure 1
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Figure 2
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Figure 3
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TOC graphic
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