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Fingerprinting of Phospholipids Molecular Species from Human Milk and Infant Formula Using HILIC-ESI-IT-TOF-MS and Discriminatory Analysis by Principal Component Analysis Chenyu Jiang, Baokai Ma, Shuang Song, Oi-Ming Lai, and Ling-Zhi Cheong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01393 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 17, 2018

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Journal of Agricultural and Food Chemistry

Fingerprinting of Phospholipids Molecular Species from Human Milk and Infant Formula Using HILIC-ESI-IT-TOF-MS and Discriminatory Analysis by Principal Component Analysis

Chenyu Jianga, Baokai Mab, Shuang Songc*, Oi-Ming Laid,e, Ling-Zhi Cheonga*

a

Department of Food Science and Engineering, School of Marine Science, Ningbo

University, Ningbo 315211,China; b

School of Life and Sciences, Shanghai University , Shanghai, 200444,China.

c

National Institute for Nutrition and Health, Chinese Center for Disease Control and

Prevention, Beijing 100050, China d

Department of Bioprocess Technology, Faculty of Biotechnology & Bimolecular

Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. e

Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,

Malaysia.

_________________________ Corresponding author: Ling-Zhi Cheong, Tel: +86-574-87608368, Fax: +86– (0)574-87608368.

E-mail:

[email protected],

Song

Shuang,

Tel:

+86-10-66237158. Email: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT

2

Phospholipids composition in the milk fat globule membrane (MFGM) fluctuates

3

during entire lactation period in order to suit the growing needs of newborn infants.

4

Present study elucidated and relatively quantified phospholipids molecular species

5

extracted from human milk (HM), mature human milk (MHM) and infant formulas

6

(with or without MFGM supplementation) using HILIC-ESI-IT-TOF-MS system.

7

Principal component analysis was used to clarify the differences between

8

phospholipids composition in HM, MHM and infant formulas. HM and MHM

9

contained high concentrations of sphingomyeline (HM: 107.61µg/mL, MHM: 227.18

10

µg/mL), phosphatidylcholine (HM: 59.96 µg/mL, MHM: 50.77 µg/mL) and

11

phosphatidylethanolamine (PE) (HM: 25.24 µg/mL, MHM: 31.76 µg/mL). Significant

12

concentrations ( 3 months exclusive breastfeeding) and human milk samples

92

(including colostrum, transitional milk and mature milk) were collected from healthy

93

Chinese women in Huangpu city (Guangdong Province). All human milk samples

94

were collected from full expression of one breast using electronic milk pump and

95

while the baby was fed on the opposite breast. This is to assure sample homogeneity

96

as milk quality and quantity have been found to vary during one feeding and whether

97

or not milk is expressed by a pump or by the baby. All the collected human milk

98

samples were stored at -80 °C until further analysis. Written informed consent was

99

obtained from all participants and the study was approved by ethics committee of the

100

National Institute of Nutrition and Food Safety, China CDC. Infant formulas (2 liquid 5



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formula and 2 powder formula) were purchased from local supermarkets in Ningbo,

102

China. PLs standards including phosphatidylinositol (16:0/16:0), phosphatidylglycerol

103

(14:0/14:0), phosphatidylethanolamine (14:0/14:0), phosphatidic acid (14:0/14:0),

104

phosphatidylserine (14:0/14:0), phosphatidylcholine (14:0/14:0), sphingomyelin

105

(d18:1/12:0), lysophosphatidylglycerol (14:0), lysophosphatidylethanolamine (14:0),

106

lysophosphatidylinositol (16:0), lysophosphatidylcholine (17:0) and SPLASH

107

LIPIDOMIX (isotopic internal standard mixture) were purchased from Avanti Polar

108

Lipids (USA). Other reagents and chemicals used were either of HPLC or MS grade

109

(chloroform, methanol, acetonitrile, hexane, ammonium formate and formic acid) and

110

purchased from Sigma Aldrich (USA). Ultra-pure water was obtained from Milli-Q

111

system (Millipore, USA).

112

Milk samples preparation and extraction of PL from milk samples. Infant

113

formula (1 g powder formula) was dissolved in lukewarm water (7.0 mL) before PL

114

extraction. Meanwhile, human milk and liquid formula without prior pretreatment

115

were used directly for PL extraction. PL were extracted from 100 µL of milk samples

116

according to previously described method.27 Three parallel samples were prepared for

117

each milk samples to reduce variation caused by sampling.

118

HILIC-ESI-IT-TOF-MS for separation, identification and quantification of

119

PL in milk samples. HPLC system (LC-20AB, Shimadzu Corporation, Japan)

120

equipped with binary pump, online degasser, autosampler and thermostatic column

121

oven was coupled online to an Ion Trap-Time of Flight mass spectrometer (IT-TOF

122

MS, Shimadzu Corporation, Japan) equipped with an Electrospray Ionization (ESI)

123

source for milk PL separation, identification and quantification.

124

Separation of PL in milk samples. The extracted PL were separated on a

125

CORTECS HILIC Column (2.7 µm, 2.1 mm × 150 mm, Waters, USA). CORTECS 6


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HILIC Columns is a high-efficiency column based on solid-core particle that are

127

designed to retain extremely polar analytes such as phospholipids. It has been reported

128

for excellent separation of plasma phospholipids.28 Separation of PL in milk samples

129

was conducted according to our previously described method.27 Column temperature

130

was set at 40 °C. Eluent A and eluent B were water and acetonitrile/water (95/5),

131

respectively. Both eluents contained 10mM ammonium formate and 0.1% formic acid.

132

Flow rate of the eluent was set at 0.3 mL/min. The gradient elution was as follows: 0

133

min, 100% B; 40 min, 90% B; 40.5 min, 70% B; 50 min, 70% B; 50.5 min, 100% B;

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60 min, 100% B. No significant carryover of lipids was observed in blank injection

135

(10 µL methanol).

136

Identification and quantification of PL in milk samples. Identification and

137

quantification of milk PL were conducted under negative electrospray ionization

138

mode. Instrument parameters were set as follows: Nebulizer gas flow: 1.5 L/min;

139

interface (probe) voltage: 3.5 kV; CDL temperature: 200 °C; heat block temperature:

140

200 °C; detector voltage: 1.70 kV; IT zone vacuum: 1.7×10-2 Pa; Flight tube

141

temperature: 40 °C.

142

MS data were collected in the range of 400-1000 m/z with 30 ms of ion

143

accumulation in ion trap; meanwhile, MS2 data were collected in the range of 100-800

144

m/z with 50 ms of ion accumulation and 50-100% of collision energy. MS3 data were

145

collected in the range of 100-700 m/z with 50 ms of ion accumulation and 50-100% of

146

collision energy. All the data were collected at a loop time of 0.82 s. Extracted milk

147

PL were identified by comparing obtained m/z values with calculated exact masses

148

using LIPID MAPS Structure Database (LMSD).

149

Extracted milk PLs were relatively quantified using internal standard method.

150

Calibration curves were constructed using PL standards in series of concentrations 7



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spiked with constant concentration of SPLASH LIPIDOMIX. Peak area of the

152

internal standards and milk PL were integrated from extracted ion chromatograms

153

(EICs). Final concentration of PL was expressed in mean ± standard deviations

154

(ng/mL).

155

Statistical analysis. Discriminatory analysis of the PL composition from human

156

milk, mature human milk and four different types of infant formulas were analyzed by

157

PCA using IBM SPSS. PCA is a statistical procedure which converts a set of

158

observations of possibly correlated variables into a set of values of linearly

159

uncorrelated variables called principal components (PCs). Each PC is defined by a

160

vector known as the eigenvector of the variance-covariance matrix. The first PC (PC1)

161

expresses the most messages of original variances and the following is the PC2. Much

162

of the total variability in the data set can be expressed by only the first several PCs to

163

entail data reduction. Each loading of variables was used for the contribution of the

164

original variables to the PC.29, 30 To achieve visual discrimination of human milk,

165

mature human milk and four different types of infant formulas, the PCA plots mapped

166

variables (n=161) and eighteen samples through loadings and scores in dimension

167

determined by the significant PCs. The loading plots represent the contribution of

168

important variables to the PCs and the score plots indicate the differences of the milk

169

PL.

170

RESULTS AND DISCUSSION

171

Separation of milk PLs using HILIC-ESI-IT-TOF-MS system. Hydrophilic

172

stationary phase and reversed-phase mobile phase (usually acetonitrile and small

173

amount of water) are commonly employed in HILIC-ESI-IT-TOF-MS system. The

174

polar mobile phase form a water-enriched layer on the surface of the polar stationary

175

phase. This water-enriched stationary layer and organic solvent-enriched mobile phase 8


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have different solvency for PLs resulting in distribution of PLs between these two

177

layers. The water-enriched stationary layer retain hydrophilic molecules; hence, PLs

178

are eluted in order of increasing hydrophilicity of their headgroups.25,

179

elution mode can prevent co-elution of PL molecules with similar m/z values but

180

different head groups which are commonly encountered in reversed phase liquid

181

chromatography (RPLC) that separates PL according to chain length and unsaturation

182

degree of the hydrophobic acyl moiety.33 In addition, unlike normal phase liquid

183

chromatography (NPLC) which uses water-isopropanol-hexane elution systems,

184

HILIC uses reversed-phase eluent system which can ensure good retention stability

185

and ionization efficiency in ESI.27, 33 In terms of detection, MS detection has the

186

advantage over evaporative light-scattering detection (ELSD) technique as it is

187

capable of identifying and quantifying individual molecular species of PLs.34 The

188

aforementioned reasons underlined the advantages of HILIC-ESI-IT-TOF-MS system

189

in accurate identification and quantification of milk PLs as compared to other

190

analytical methods (thin-layer chromatography, 31P nuclear magnetic resonance

191

spectroscopy, etc.)

31, 32

Such

192

Figure 1 shows the total ion chromatograms (TICs) of PLs in human milk, mature

193

human milk and four different types of infant formulas. All the different classes of

194

milk PLs with the exception of LPGs and PEs were efficiently separated and eluted

195

within 40 min. LPGs and PEs have odd and even m/z value and thus can be easily

196

distinguished in mass spectra. As aforementioned, PLs molecular species within the

197

same class were eluted was according to increasing polarity (decreasing acyl chain

198

length and increasing degree of unsaturation) which is in consistent with previously

199

reported findings.31 According to our previous findings,27 buffer modifiers namely a

200

mixture of 0.1% formic acid and 10mM ammonium formate can be used to improve 9



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201

the peak shape of neutral PLs (PEs, PCs, and SMs) and anion PLs (PI, PG, and

202

especially PS). In present study, a satisfactory tailing factor (0.80-1.15) can be

203

achieved by using similar buffer modifiers.

204

Fragmentation principle of milk PLs. Present study found all the characteristics

205

fragments required for structural elucidation of PLs present in human milk, mature

206

human milk and infant formulas can be collected under negative electrospray

207

ionization mode. In our previous study, we have compared the fragmentation of PLs

208

under both positive and negative ionization mode. Although higher intensities (about

209

twice) could be achieved under positive ionization mode, either characteristic

210

fragments or fatty acyl identification fragments was not available under ESI+ mode

211

for most PL species. In contrast, all the necessary information for structure

212

identification could be collected under ESI- mode. Moreover, lower background noise

213

and better baseline separation was observed under ESI- mode. Thus, the negative

214

mode was used for PLs analysis in present work.28 Herein, SM (d18:1/22:0), PC

215

(18:0/18:2) and PE (18:0/18:2) detected most abundantly in human milk and mature

216

human milk; and PA (16:0/18:2) detected most abundantly in infant formulas were

217

selected to elucidate fragmentation principle of PLs under negative electrospray

218

ionization mode.

219

Figure 2A shows the multistage mass spectra of SM (d18:1/22:0). Under the

220

negative ESI mode, SM (d18:1/22:0) at m/z ion 831.65 demonstrated a neutral loss of

221

60.02 between [M+HCOO]- and [M-CH3]-. This could be the characteristic fragment

222

of choline head group. The neutral loss of 322.30 between [M-CH3]- and

223

[M-CH3-R2]- provided information of the fatty acyl moiety attached to sphingosine

224

which can be attributed to the loss of docosanoic acid (C22:0). The remaining fatty

225

acyl moiety could be further calculated from [M-CH3-R2]- fragment by subtracting 10


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the remaining head group which was identified as C18:1. Similar fragmentation law

227

can be applied for PC (18:0/18:2) which also contains a choline headgroup (Figure

228

2B). PC (18:0/18:2) at m/z ion 830.59 also demonstrated a neutral loss of 60.02

229

between [M+HCOO]- and [M-CH3]- indicating characteristic fragment of choline

230

head group. Signals at m/z ions 279.28 in MS3 can be attributed to linoleic acid on

231

sn-2 and should represent [M-CH3-R2]-. Meanwhile, the fatty acyl structure on sn-1

232

could be calculated from [M-CH3-R2]- fragment by subtracting the remaining head

233

group and the glycerol backbone which was identified as C18:0.

234

As for PE (18:0/18:2) at m/z ion 742.53, PE firstly lost one fatty acyl chain

235

between MS and MS2. The neutral loss of 262.22 between MS and MS2 can be

236

attributed to the loss of linoleic acid. Fatty acyl substituent at sn-2 was sterically more

237

favorable to dissociate as compared to that at sn-1 due to the phosphate charge site

238

closer to the fatty acyl moiety at sn-2.35, 36 Therefore, the m/z ion at 480.30 in MS2

239

and the 283.27 in MS3 should represent [M-H-R2+OH]− and [R1−H]− respectively.

240

The neutral loss of 197.05 between [M-H-R2]- and [R1-H] should represents

241

glycerolphosphoethanolamine and could be used as characteristic fragment of PE

242

(Figure 2C). Another PLs with amine-containing head groups, PS also demonstrated

243

similar fragmentation law to that of PE. The characteristic fragment for PS is NL

244

87.03. In addition, [M-H-R2]- is replaced by [M-H-Ser]- and [M-H-Ser-R2]- (Figure

245

S1). Figure 2D shows the multistage mass spectra of PA(16:0/18:2). PA (16:0/18:2) at

246

ions

671.46

produced

[M-H-R2]-

and

[M-H-R2-Glycerol]-

in

MS2.

247

m/z

248

[M-H-R2-Glycerol]- was then used as second precursor ion to produce [R1-H]- in MS3.

249

The neutral loss of water (17.99) between [M+H-R2]- and [M+H-R2-Glycerol]- could

250

be used for characterization of PA. Fragmentation law of other anionic PLs namely PI 11



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251

(Figure S2) and PG (Figure S3) is very similar to that of PA. Except that, a neutral

252

loss of 162.05 was used as a characteristic fragments for PI.

253

Structural elucidation and quantification of PLs in human milk and infant

254

formula. Table S1 shows PLs molecular classes identified and relatively quantified in

255

human milk, mature human milk and infant formulas. A total of 161 types of PLs were

256

detected in human milk, mature human milk and infant formulas. Figure 3 shows the

257

total molecular species within each PL classes in human milk, mature human milk and

258

different infant formulas. Both samples of human milk and mature human milk were

259

found to have a diverse species of PE (45 species) and SM (ranged from 17-20

260

species). Meanwhile, all the different brands of infant formula analyzed contained

261

diverse species of PE (ranged from 26 to 38 species), PC (ranged from 20 to 22

262

species) and SM (ranged from 19 to 22 species). IF 1, IF 2 and IF 4 were found to

263

have more diverse species of lysoPLs and PA (ranged from 28 to 30 species) in

264

comparison to human milk samples (11 species) and IF 3 (17 species).

265

All the different PL classes were relatively quantified according to their

266

corresponding standard curves (RSD of retention time ≤ 0.15%, peak area ratio ≤ 5%).

267

Table 1 shows the calibration equations and R2 values of PL standards and their

268

corresponding internal standards. PLs concentration varied significantly across the

269

different samples of human milk and infant formulas. As shown in Table S1, total PLs

270

concentrations in human milk and mature human milk were found to be 406.45 and

271

228.60 µg/mL, respectively. This is in agreement with values previously reported in

272

cohort studies involving Asian22, 26, 37 and European lactating mothers20. Similar to

273

findings in present study, higher total PLs concentrations in colostrum and transitional

274

milk as compared to mature human milk have been previously reported. It has been

275

postulated that biosynthesis of PLs in mammary gland diminishes during later stages 12


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of lactation resulting in significant decrease in the MFGM thickness.38, 39 Meanwhile,

277

total PLs concentrations in the different infant formulas varied significantly (IL 1:

278

942.75 µg/mL, IL 2: 2274.33 µg/mL, IL 3: 150.48µg/mL; IL 4 – 958.18 µg/mL). The

279

significantly (P

Fingerprinting of Phospholipid Molecular Species ... - ACS Publications

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