Cholesterol Content in Human Milk during Lactation: A Comparative

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Food and Beverage Chemistry/Biochemistry

Cholesterol content in human milk during lactation. A comparative study of enzymatic and chromatographic methods ISLAM J.A. HAMDAN, Luis Manuel Sanchez-Siles, Esther Matencio, Guadalupe Garcia Llatas, and María Jesús Lagarda J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02205 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Journal of Agricultural and Food Chemistry

Cholesterol content in human milk during lactation. A comparative study of enzymatic and chromatographic methods

Islam J.A. Hamdan1, Luis Manuel Sanchez-Siles2, Esther Matencio2, Guadalupe Garcia-Llatas1*, María Jesús Lagarda1 1

Nutrition and Food Science Area, Faculty of Pharmacy, University of Valencia,

Avda. Vicente Andrés Estellés s/n, 46100 – Burjassot (Valencia), Spain. 2

R&D Department, Institute of Infant Nutrition, Hero Group. Avda. Murcia 1, 30820

– Alcantarilla (Murcia), Spain. * To whom correspondence should be addressed (Telephone +34-963543766; Fax +34-963544954; E-mail: [email protected])

Authors’ contribution: Islam J.A. Hamdan contributed with the analytical assays and with data acquisition and interpretation. Guadalupe Garcia-Llatas and María Jesús Lagarda contributed with the conception and design of the study. Guadalupe Garcia-Llatas, María Jesús Lagarda, Luis Manuel Sánchez-Siles and Esther Matencio interpreted the data. All authors collaborated in the drafting and revision of the article, and approved the final version of the manuscript for submission.

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Abstract

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This study validates a gas chromatography (GC) method for determining the sterol

3

profile of human milk (HM) and compares it with an enzymatic-spectrophotometric (E-

4

S) method. Good linearity (r > 0.97) and low limits of detection and quantification were

5

obtained with the GC method (< 1.8 and < 6 µg/100 g of HM, respectively). Suitable

6

intra- and inter-assay precisions (all < 18%) and satisfactory recovery percentages (80-

7

109%) were obtained for both methods. In addition, both methodologies were used to

8

assess cholesterol evolution in HM during lactation, showing a 50% decrease at 6

9

months versus colostrum. The E-S method overestimated cholesterol content by < 20%

10

versus the GC method. The results indicate that both methods may be used by the

11

industry and in research to better understand the differences between the sterol profiles

12

of infant formulas and HM.

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Keywords

14

Breast

15

milk;

cholesterol;

colostrum;

enzymatic-spectrophotometry;

chromatography; sterols.


gas

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INTRODUCTION

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Human milk (HM) is a very complex biological fluid with a composition that

18

changes with the lactation stage, the time of day, within feedings (more carbohydrates

19

in foremilk and more lipids in hindmilk), delivery or gestation length (preterm or term

20

HM), maternal parity, body mass index (BMI), age, diet and residential area, among

21

other factors1-4. These changes allow HM to adjust to the needs of the infant, ensuring

22

correct growth and development, but also make HM very difficult to mimic. For the

23

aforementioned reasons, HM is considered the gold standard for feeding infants up to 6

24

months of age, and exclusive breastfeeding is recommended for infants up to that age5.

25

In HM, lipids are the most variable macronutrient, being dispersed as fat globules

26

formed by a core (rich in triglycerides) surrounded by a membrane known as the milk

27

fat globule membrane (MFGM). The latter in turn is composed of an inner membrane

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(rich in phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and

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cholesterol) and an outer membrane (rich in phosphatidylcholine, sphingomyelin,

30

cholesterol, peptides, glycoproteins, cerebrosides and gangliosides, among other

31

compounds)6. The MFGM of HM is a good source of cholesterol, which is crucial to

32

infant growth, since it is essential for the synthesis of bile acids, hormones, vitamin D

33

and lipoproteins7. Furthermore, cholesterol is needed as a structural component of all

34

cell membranes, and especially as a constituent of myelin during the rapid brain growth

35

period8.

36

The cholesterol content in HM decreases in the course of the lactation stages, and in

37

any case is higher than that found in infant formulas (IFs). Thus, different qualitative

38

and quantitative profiles of cholesterol and other sterols (cholesterol precursors and

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plant sterols) can be found in HM and IFs9-18. This fact causes breast-fed infants to

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consume up to 5 times more cholesterol than formula-fed infants (18.2 versus 3.4 3 ACS Paragon Plus Environment


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mg/kg/day at 20 weeks of age and with a mean body weight of 6.8 kg)19.

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Claumarchirant et al.16 reported similar cholesterol intakes (3.7 mg/kg/day) from IFs in

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infants of the same age and weight.

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This increased intake of cholesterol by breast-fed infants has been postulated as one

45

possible reason why serum cholesterol levels are higher in breast-fed infants than in

46

formula-fed infants, while the opposite occurs in relation to plant sterol levels20-21.

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Several studies have suggested that higher serum cholesterol levels in breast-fed infants

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could prevent cardiovascular diseases in adult life20,22-27 by downregulating hepatic

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hydroxymethyl glutaryl coenzyme A reductase via epigenetic modifications28. However,

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it has been mentioned that further studies are needed to better understand programming

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in the neonatal period and the role of the other sterols present in HM8,29.

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Despite the importance these bioactive compounds may have for the infant, few

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studies have examined the sterol profile (focused mainly on cholesterol and

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desmosterol) in HM (Table 1), and only three publications involve validated

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methodology15,30-31. Chromatographic methods have been the most widely used option

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for the identification and quantification of sterols in HM, particularly gas

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chromatography (GC), given its advantage of being able to establish the complete sterol

58

profile. However, such methods are time consuming and are not suitable for large

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numbers of samples. Enzymatic-spectrophotometric (E-S) methods are faster and less

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expensive, and can also be used for sterol determination - though they are not as specific

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as GC, since the cholesterol content is overestimated. In this regard, cholesterol oxidase,

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used in E-S, oxidizes the 3β-hydroxyl group present in cholesterol and other sterols

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(desmosterol, lathosterol, campesterol, stigmasterol and β-sitosterol). 32-33

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On the other hand, few studies have compared analytical methods for the

65

determination of cholesterol in HM. Harzer et al.12 and Haug & Harzer33 compared the


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cholesterol contents

obtained

by

E-S assay and

high performance

liquid

67

chromatography (HPLC), and described a 20% lower content with the latter technique -

68

this difference being attributed to the non-specificity of E-S. Kamelska et al.34 in turn

69

reported variable contents obtained by attenuated total reflectance-Fourier transform

70

infrared spectroscopy (ATR-FTIR) or GC. Furthermore, Kamelska et al.31 obtained

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higher values with E-S than with GC when direct saponification was applied to HM.

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Due to the abovementioned reasons, the two main objectives of the present study

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were: (a) to validate a GC method for sterol (cholesterol, desmosterol, lathosterol,

74

campesterol, stigmasterol, lanosterol, β-sitosterol) determination in HM and compare it

75

with a proposed E-S method; and (b) to assess the evolution of cholesterol content in

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HM samples from different lactation stages using both techniques. Results obtained in

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this study would facilitate selection of the analytical technique best suited for

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cholesterol determination in HM.

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MATERIALS AND METHODS

80

Reagents

81

Epicoprostanol (5β-cholestan-3α-ol, 96.9% purity) as internal standard (IS),

82

cholesterol (5-cholesten-3β-ol, 99% purity), stigmasterol (5,22-stigmastadien-3β-ol,

83

95% purity), lanosterol (8,24-lanostadien-3β-ol, 99%), lathosterol (5α-cholest-7-en-3β-

84

ol, 99.5%), β-sitosterol (5-stigmasten-3β-ol, 98% purity), N,O-bis-(trimethylsilyl)

85

trifluoroacetamide with 1% trimethylchlorosilane (BSTFA+1% TMCS) and potassium

86

hydroxide (KOH) were purchased from Sigma–Aldrich Chemical (St. Louis, MO,

87

USA).

88

hydroxycholesterol (5-cholestene-3β,7β-diol, 97% purity) and desmosterol (5,24-

89

cholestadien-3β-ol, 98% purity) were purchased from Steraloids Inc. (Newport, RI,

7α-hydroxycholesterol

(5-cholestene-3β,7α-diol,


98%

purity),

7β-


90

USA). Campesterol (24α-methyl-5-cholesten-3β-ol, 96.2% purity) was obtained from

91

Chengdu Biopurify Phytochemicals Ltd. (Sichuan, China).

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Hexane and 2-propanol were acquired from Scharlau Chemie, S.A. (Barcelona,

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Spain). Ethanol absolute grade was purchased from Panreac Química SLU (Barcelona,

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Spain) and pyridine extra dry (99.5% purity) from Acros Organics (Geel, Belgium). The

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E-S kit for cholesterol determination was obtained from Boehringer Mannheim / R-

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Biopharm (R-Biopharm AG, Darmstadt, Germany). Hydrogen (purity >99%, NMH2-

97

500; LNI Schmidlin S.A., Geneva, Switzerland) and nitrogen (N2) (purity >99%, Zefiro

98

35 LC-MS, F-DGS SAS, Evry, France) generators were used.

99

All standards used were of GC grade, and all reagents were of analytical grade.

100

Millipore MilliQ deionized water (Millipore Ibérica S.A., Barcelona, Spain) was used.

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Samples

102

For the determination of analytical parameters, a pool of mature HM was prepared

103

from milk samples obtained from four healthy Spanish donors aged between 25-35

104

years. The donors were non-smokers, well-nourished and without caloric restrictions.

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The volunteer mothers had gestations > 37 weeks and infants with normal weight at

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birth. The study of the evolution of cholesterol content throughout the lactation period

107

was carried out with pools of HM samples obtained from 65 volunteer mothers of term

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infants between 18-40 years of age, as described by Claumarchirant et al.35 These pools

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were obtained from different stages of lactation (colostrum, transition, 1, 3 and 6

110

months) and three cities in Spain (Madrid, Murcia and Valencia). The study protocol

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was approved by the Clinical Research Ethics Committee and Breastfeeding Committee

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of Puerta de Hierro-Majadahonda University Hospital (Madrid, Spain). All subjects

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were informed and gave written consent before inclusion in the study.

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Determination of sterols by GC


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Methodology

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Direct saponification and extraction of the unsaponifiable fraction were carried out

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for sterol determination. Two µg of epicoprostanol (IS) and 3 ml of an ethanol (90%)

118

solution of 2 N KOH were added to 0.5 ml (~ 0.54 g) of HM and placed in a 10 ml

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Pyrex glass tube, followed by heating of the mixture at 80ºC during 45 min in a shaking

120

water bath (~100 rpm, Stuart SBS30)36. Then, once the sample had cooled, MilliQ

121

water and hexane were added (1 ml each), and vortexing (1 min) and centrifugation at

122

3200 rpm, 4°C, 10 min (Eppendorf 5810R) were performed. The hexane phase was

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transferred to a new tube, and extraction with hexane was repeated twice more,

124

collecting all the organic phases. The unsaponifiable fraction was then dried with N2 and

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subjected to derivatization at 65ºC during 1 h in a thermoblock (Stuart SBH200D) with

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200 µl of BSTFA+1% TMCS: pyridine (10:3, v/v)17. The trimethylsilyl ether (TMSE)

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derivatives obtained were dissolved in hexane, filtrated (using a syringe-driven Millex-

128

FH filter unit, 3 ml, 0.45 µm Millipore, Milford, MA, USA), and the solvent was

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evaporated with N2. Each sample was dissolved in 800 µl of hexane for cholesterol and

130

in 40 µl for the determination of other sterols. From each solution, one microliter was

131

injected into a gas chromatography–mass spectrometry system (GC-MS, Trace GC-

132

Ultra ITQ ion trap 900, Thermo Scientific, Waltham, MA, USA) for identification, and

133

into a GC-flame ionization detector (GC–FID, Autosystem XL, Perkin-Elmer,

134

Nortwalk, CT, USA) for quantification purposes. Both instruments were equipped with

135

CP-Sil 8 low bleed/MS (50 m × 0.25 mm × 0.25 µm film thickness) capillary columns

136

(Chrompack-Varian, Middelburg, The Netherlands).

137

In GC-MS, the carrier gas was hydrogen operating at a constant flow of 1 ml/min.

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The temperature of the injector port was 280ºC, and splitless injection mode was

139

applied. The oven was initially programmed at 150 °C maintained during 3 min, heated



140

to 280 °C at a rate of 30 °C/min, and kept during 28 min, then raised to 295 °C at a rate

141

of 10 °C/min. Finally, this temperature was maintained for 10 min. The transfer line

142

temperature was 310 ºC, the ion source temperature was 250 ºC, the scan range was 50-

143

650 m/z, and the filament emission current was -70 eV. Identification of sterols was

144

carried out by comparing their retention times and MS spectra with those of standards

145

and the NIST v.2 library. In GC-FID, hydrogen was also the carrier gas at 0.91 ml/min,

146

the detector port was set at 300 ºC, and the oven and injector conditions were identical

147

to those of GC-MS. Both instrument conditions were slightly modified from Garcia-

148

Llatas et al.37 and Hamdan et al.17

149

Validation

150

Validation was made by studying the analytical parameters of selectivity, linearity,

151

limit of detection (LOD), limit of quantification (LOQ), precision and accuracy,

152

according to the guidelines of the International Conference on Harmonization (ICH) and

153

the Association of Official Analytical Chemists guidelines for the validation of

154

analytical methods38-39.

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Selectivity and linearity

156

Sterol calibration curves were performed with 0.25 ml of HM and without addition

157

of matrix to check any interference from other compounds present in the HM

158

(selectivity assay). For establishing linearity, a minimum of 5 calibration points were

159

prepared with a constant amount of IS (2 µg), with derivatization using the

160

aforementioned method and analysis by GC-FID. Calibration curves were plotted for

161

each sterol, representing the sterol area/IS area ratio versus their respective quantities

162

(µg sterol/µg IS) (see Table 2).

163

Limit of detection (LOD) and limit of quantification (LOQ)


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The LOD and LOQ of the method were determined from the background response of

165

6 method blanks using 0.5 ml of water. They were calculated as three and 10 times (for

166

LOD and LOQ, respectively) the standard deviation (SD) of the response against the

167

slope of the calibration curves for each sterol.

168

Precision

169

Intra- and inter-assay precisions were evaluated by calculating the relative standard

170

deviation (RSD) of the analysis of the sample on two different days.

171

Four aliquots of the sample were analyzed on the same day (intra-assay) and another

172

four aliquots on a different day (inter-assay).

173

Accuracy

174

The accuracy of the method for all sterols was established from recovery assays. The

175

sterol contents of four aliquots of the sample (0.25 ml of HM), and another four spiked

176

aliquots (see Table 3) were determined. The spiked amounts of sterols were also

177

verified in quadruplicate. Recovery (as a percentage) was calculated as: [(µg sterol in

178

spiked aliquots - µg sterol in non-spiked aliquots) x 100] / [µg spiked sterol], and

179

expressed as the mean ± SD.

180

Determination of sterols by E-S

181

Methodology

182

An aqueous solution of 8.02 N KOH (0.8 ml) and 1.2 ml of ethanol were added to 1

183

ml of HM (~1.08 g) in a 10 ml Pyrex glass tube. The mixture was heated at 70ºC during

184

30 min in a shaking water bath (Stuart SBS30)15. After cooling the sample at room

185

temperature, extraction of the unsaponifiable fraction was performed as previously

186

described for the chromatographic method, with different centrifugation conditions

187

(4000 rpm, 4 °C, 5 min). The collected hexane phases were evaporated and dissolved

188

with 1 ml of 2-propanol. This dissolution, referred to as the “sample solution”, was



189

processed with the enzymatic kit according to the instructions of the manufacturer. The

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kit is based on the oxidation of cholesterol to cholestenone, producing hydrogen

191

peroxide. In the presence of catalase, the hydrogen peroxide produced oxidizes

192

methanol to formaldehyde, which in turn reacts with acetyl acetone, forming a yellow

193

lutidine dye in the presence of ammonium ions. The lutidine dye formed (measured at

194

405 nm) is stoichiometric to the concentration of cholesterol (Spectrometer Lambda 2

195

UV/Vis, Perkin Elmer®).

196

Validation

197

Precision and accuracy were assessed according to the guidelines of the ICH38 and

198

AOAC39, whereas the analytical parameters of linearity, LOD and sensitivity were

199

provided by the manufacturer.

200 201 202 203 204

Precision The intra- and inter-assay precisions were established as described above in section 2.3.2. Validation (precision). Accuracy Accuracy was determined as described above in section 2.3.2. Validation (accuracy)

205

using 1 ml of HM.

206

Statistical analysis

207

Normal data distribution and homogeneity of variance were checked using the

208

Shapiro-Wilk and Levene tests, respectively. A two-tailed Student t-test (n-2 degrees of

209

freedom) was performed to evaluate the linearity of the calibration curves, and

210

ANCOVA or Kruskal-Wallis testing for normal and non-normal distribution of data,

211

respectively, was used to compare the slopes of the calibration curves (with and without

212

addition of matrix). To assess significant differences, analysis of variance (ANOVA,

213

with Tukey’s post hoc test) was applied to chromatographic and E-S cholesterol


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214

contents in the course of lactation, and a Student t-test was used to compare cholesterol

215

contents between the two methods40 . For all cases, α = 0.05 was applied.

216

RESULTS AND DISCUSSION

217

Validation of the GC method

218

Selectivity and linearity

219

The total analytical run time was 28 minutes, as can be seen from the chromatogram

220

of the pool of mature HM (Figure 1). The identified sterols (relative retention time with

221

respect to the IS, 18.3 min) were: 7α-hydroxycholesterol (1.08), cholesterol (1.13),

222

desmosterol (1.18), lathosterol (1.21), 7β-hydroxycholesterol (1.27), campesterol (1.28),

223

stigmasterol (1.36), lanosterol (1.44) and β-sitosterol (1.45). Their GC-MS spectra are

224

shown in Figure 2.

225

Table 2 reports the tested amount ranges, the adjusted linear equations, the statistical

226

tests to check the goodness of fit of the calibration curves (without and with matrix),

227

and the matrix interference assay. All correlation coefficients were ≥ 0.98, and the

228

calculated t values were higher than the critical t value (3.18) for all sterols, which

229

reflected good linearity (Table 2).

230

Since a matrix interference was detected for cholesterol, desmosterol, lathosterol and

231

β-sitosterol (p < 0.05) (Table 2), we decided to use calibration curves added with matrix

232

for all sterol quantifications. Few publications have described the use of calibration

233

curves to quantify sterols (only cholesterol and desmosterol) in HM. Ramalho et al.15

234

reported HPLC calibration curves for cholesterol and desmosterol, with r > 0.999 for

235

both compounds. However, these authors did not report any matrix interference assay,

236

though they performed direct saponification of the sample. Kamelska et al.41 developed

237

a calibration curve for cholesterol quantification in IF by GC which was further applied



238

in HM samples31,42. The slope of the curve was 5-fold lower (y=0.1861x+7.78, r = 0.96)

239

than our own.

240

Limit of detection (LOD) and limit of quantification (LOQ)

241

The LOD and LOQ (as ng in assay and µg/100 g of HM) obtained are shown in

242

Table 3. This method allowed the detection and quantification of all studied sterols at

243

concentrations < 2 and < 6 µg/100 g of HM for LOD and LOQ, respectively. The

244

highest LOD and LOQ corresponded to campesterol (1.80 and 5.99 µg/100 g of HM,

245

respectively) and the lowest to stigmasterol (0.26 and 0.87 µg/100 g of HM,

246

respectively). Cholesterol was quantifiable above 1.9 µg/100 g HM. Our LOQs were

247

20-fold lower for cholesterol and 6-fold lower for desmosterol than those reported by

248

Ramalho et al.15 (40 and 20 µg/100 ml, respectively).

249

Precision

250

Intra- and inter-assay precisions (% RSD) are reported in Table 3. Intra-assay

251

precision was lower than 13% for all the sterols analyzed, and ranged from 0.8%

252

(desmosterol) to 12.5% (stigmasterol), while inter-assay precision was lower than 18%

253

for all the sterols analyzed (from 3% for cholesterol to 17.6% for β-sitosterol). The intra

254

and inter-assay precision values comply with those indicated by the AOAC39 for a

255

single laboratory:

Cholesterol Content in Human Milk during Lactation: A Comparative

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