Quantitation of Sialic Acids in Infant Formulas by Liquid

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Food and Beverage Chemistry/Biochemistry

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)

1 ACS Paragon Plus Environment


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1

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

26

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

48

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

68

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

71

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

82

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

117

quantitation of free vs. glycosidically-bound sialic acids and, in addition, permitted the

118

identification of Neu5,9Ac2 and Kdn in infant formulas.

119

Materials and Methods

120

Chemicals and General Details.

121

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

126

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.

141

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

209

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.

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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

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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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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



Page 16 of 37

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,


Page 17 of 37


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



Page 18 of 37

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.


Page 19 of 37


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



Page 20 of 37

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


Page 21 of 37


432

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434

milk monosaccharide, and its application as a functional food ingredient. Crit. Rev. Food

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glycobiome on the neonate gut microbiota. Annu. Rev. Anim. Biosci. 2015, 3, 419–445.

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Pacheco, A. R.; Barile, D.; Underwood, M. A.; Mills, D. A. The impact of the milk

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Charbonneau, M. R.; O’Donnell, D.; Blanton, L. V.; Totten, S. M.; Davis, J. C. C.;

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Barratt, M. J.; Cheng, J.; Guruge, J.; Talcott, M.; Bain, J. R.; et al. Sialylated milk

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oligosaccharides promote microbiota-dependent growth in models of infant

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undernutrition. Cell 2016, 164, 859–871.

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Matsuki, T.; Yahagi, K.; Mori, H.; Matsumoto, H.; Hara, T.; Tajima, S.; Ogawa, E.;

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Kodama, H.; Yamamoto, K.; Yamada, T.; et al. A key genetic factor for fucosyllactose

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utilization affects infant gut microbiota development. Nat. Commun. 2016, 7, 11939.

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Davis, J. C. C.; Lewis, Z. T.; Krishnan, S.; Bernstein, R. M.; Moore, S. E.; Prentice, A.

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Wang, B.; Brand-Miller, J.; McVeagh, P.; Petocz, P. Concentration and distribution of sialic acid in human milk and infant formulas. Am. J. Clin. Nutr. 2001, 74, 510–515.

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Karav, S.; Le Parc, A.; de Moura, M.L.N; Bell, J.; Frese, S. A.; Kirmiz, N.; Block, D. E.; Barile, D.; Mills, D. A. Oligosaccharides released from milk glycoproteins are selective



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Nöhle, U.; Schauer, R. Uptake, metabolism and excretion of orally and intravenously

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Lewis, A. L.; Lewis, W. G. Host sialoglycans and bacterial sialidases: A mucosal perspective. Cell. Microbiol. 2012, 14, 1174–1182.

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R.; Kakehi, K. Specific distribution of sialic acids in animal tissues as examined by LC -

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performance liquid chromatography. RSC Adv. 2014, 4, 45797–45803.

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Lacomba, R.; Salcedo, J.; Alegría, A.; Barberá, R.; Hueso, P.; Matencio, E.; Lagarda, M.

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acetylneuraminic acid, N-glycolylneuraminic acid and ketodeoxynonulosonic acid levels

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Methods to minimize the loss and migration of O-acetyl groups. Anal. Biochem. 1984,

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Inoue, S.; Kitajima, K.; Inoue, Y. Identification of 2-keto-3-deoxy-D-glycero-D-

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555

humanlung carcinoma cells. J. Biol. Chem. 1996, 271, 24341–24344.

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Go, S.; Sato, C.; Furuhata, K.; Kitajima, K. Oral ingestion of mannose alters the

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Nakata, D.; Münster, A. K.; Gerardy-Schahn, R.; Aoki, N.; Matsuda, T.; Kitajima, K.

560

Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both

561

deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates.

562

Glycobiology 2001, 11, 685–692.

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Lawrence, S. M.; Huddleston, K. A.; Pitts, L. R.; Nguyen, N.; Lee, Y. C.; Vann, W. F.;

564

Coleman, T. A.; Betenbaugh, M. J. Cloning and expression of the human N-

565

acetylneuraminic acid phosphate synthase gene with 2-keto-3-deoxy-D-glycero-D-galacto-

566

nononic acid biosynthetic ability. J. Biol. Chem. 2000, 275, 17869–17877.

567

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Hao, J.; Vann, W. F.; Hinderlich, S.; Sundaramoorthy, M. Elimination of 2-keto-3-deoxy-

568

D-glycero-D-galacto-nonulosonic acid

9-phosphate synthase activity from human N-

569

acetylneuraminic acid 9-phosphate synthase by a single mutation. Biochem. J. 2006, 397,


Page 27 of 37

570


195–201.

571 572 573 574 575 576 577



Page 28 of 37

578

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


Page 29 of 37


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



Page 30 of 37

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.


Page 31 of 37


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.



Page 32 of 37

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).


Page 33 of 37


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


Quantitation of Sialic Acids in Infant Formulas by Liquid

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