Accepted Manuscript

Determination of Vitamin A and Vitamin E Esters in Infant Formulae and For-

tified Milk Powders by HPLC: Use of Internal Standardisation

David C. Woollard, Anja Bensch, Harvey Indyk, Adrienne McMahon

PII: S0308-8146(15)30079-0
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.10.077
Reference: FOCH 18272

To appear in: Food Chemistry

Received Date: 11 February 2015

Revised Date: 11 September 2015
Accepted Date: 18 October 2015

Please cite this article as: Woollard, D.C., Bensch, A., Indyk, H., McMahon, A., Determination of Vitamin A and
Vitamin E Esters in Infant Formulae and Fortified Milk Powders by HPLC: Use of Internal Standardisation, Food
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.10.077

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 1 Determination of Vitamin A and Vitamin E Esters in Infant Formulae and

2 Fortified Milk Powders by HPLC: Use of Internal Standardisation

4 David C. Woollarda* , Anja Benschb, Harvey Indykc and Adrienne McMahond

5
a
6 R.J. Hill Laboratories Ltd, 1, Clyde Street, Hamilton 3216, New Zealand
b
7 New Zealand Laboratory Services, Auckland, New Zealand
c
8 Fonterra Cooperative Group Ltd, Waitoa, New Zealand
d
9 Wyeth Nutrition, Askeaton, County Limerick, Republic of Ireland

10

11 *Corresponding author. Tel.: +64 223134424.

12 E-mail address: david.woollard@hill-labs.co.nz

13

14 Abstract

15

16 An HPLC method is described using normal phase conditions with an unbonded silica

17 column to determine concentrations of supplementary vitamin A and vitamin E esters

18 and β-carotene in infant formulae. The method utilises selective dual-channel

19 fluoresence for vitamins A and E and visible absorbance for β-carotene. An attribute of

20 the method is the use of retinol propionate, α-tocopheryl propionate and all-E-β-apo-8’-

21 carotenoic acid ethyl ester internal standards to compensate for analytical variations

22 associated with these labile vitamins. Extraction is performed without saponification,

23 with the aid of protease to remove vitamin encaspsulation and facilitate vitamin partition

24 into hydrocarbon solvent. Figures of merit indicate the method is suitable for its

25 intended purpose in the highly regulated infant formula environment, including liquid

26 fomulations. The method is extendable to whole milk powders where total vitamin A

27 content data can be calculated by summing the innate long-chain vitamin A esters with

28 the added esters.

29 1. Introduction

30

31 Methods for the determination of fat-soluble vitamins are widely reported and

32 summarized in many authoritative reviews (De Leenheer, Lambert & Van Bocxlaer,

33 2000; Rucker, Suttie, McCormick & Machlin L, 2001; Eitenmiller, Landen & Ye, 2008).

34 In general, and except for vitamin K, sample preparation for foods including milk

35 products commence with alkaline digestion, where the matrix is disrupted to liberate

36 unsaponifiable materials for subsequent examination, usually by HPLC (DeVries &

37 Silvera, 2002 Eitenmiller et al., 2008; Gentili et al., 2013). However, where the nature of

38 the food allows simplified extraction, the lengthy saponification procedure is avoided to

39 minimise oxidative and manipulative losses of the target analyte. Such alternative

40 methods have been reported for vitamin A in liquid milk (Hite, 2003) and milk powders

41 (Woollard & Woollard, 1984) using total fat extraction and subsequent normal phase

42 liquid chromatography.

43 Endogenous vitamin A exists predominantly in milk and milk products as an array

44 of long-chain fatty-acid retinyl esters (Woollard & Indyk, 1989), whereas the

45 supplementary forms are invariably retinyl palmitate or retinyl acetate, both available

46 commercially in large quantity. Retinyl acetate is essentially absent in milk so this

47 additive can be uniquely determined in a dairy product. Endogenous vitamin E exists in

48 milk almost exclusively as d-α-tocopherol, albeit in low concentration, while the principal

49 additives are synthetic d,l-α-tocopheryl acetate or its naturally-derived equivalent, d-α-

50 tocopheryl acetate. Other vitamin E esters of commercial significance are α-tocopheryl

51 succinate and α-tocopheryl nicotinate but neither is commonly used for infant nutrition.

52 Vegetable oils contain significant amounts of non-esterified d-α- and other tocopherols

53 and tocotrienols, and are common components of infant formulations.

54 Saponification results in the loss of speciation identity of vitamin A and E esters

55 during subsequent HPLC analysis; therefore manufacturers of nutritional products

56 interested in both total vitamin content and concentration of the supplementary forms

57 must select methods that avoid saponification, such as those described by Woollard &
58 Blott (1986), Woollard, Blott & Indyk (1987), Hewavitharana, van Brakel & Harnett M.

59 (1996), Chase, Ye, Stoakes, Eitenmiller & Long (2004) or Chávez-Servín, Castellote &

60 López-Sabater (2006). Reflecting this situation, recent AOAC methods 2012.09

61 (Thompson et al., 2013) and 2012.10 (McMahon, Christiansen, Shine, Loi & Dowell,

62 2013), have focused on normal-phase separation of total lipid extracted from infant and

63 adult formulations. The former uses on-line column switching between dual 3 µm silica

64 columns to create a clean extract for concurrent UV detection (UVD) of retinyl palmitate,

65 retinyl acetate, vitamin E acetate and vitamin E alcohol. The latter procedure employed

66 selective use of UVD at 325 nm and fluorescence detection (FLD) to create efficient

67 detection of the same analytes, with separation achieved on an amine-modified column

68 which allowed gradient elution of co-extracted fats. This method incorporated a papain

69 digestion, which removed encapsulation of fortified vitamins if present in the samples.

70 Following the first reports of fluorescence for vitamin E acetate (Woollard, Blott &

71 Indyk, 1987), the present study improves previous methodology for the quantitation of

72 vitamin A and E esters using modern fluorescence detectors and a 3 µm silica column

73 for efficient separations, coupled with analyte quantitation by the internal standard

74 technique. The use of internal standards for vitamins A, E and β-carotene has been

75 extensively reported for estimation of retinol, α-tocopherol and carotenoid content in

76 serum (Woollard & Woollard, 1984; Nierenberg & Nann, 1992; Epler, Ziegler & Kraft,

77 1993; Aust, Sies, Stahl & Polidori, 2001: Liu Z., Lee H-J., Garofalo F., Jenkins D.J.A. &

78 El-Sohemy A. 2011; Kand’ár et al., 2013) and for cosmetics (Baruffini et al., 1992) and is

79 similarly applied here where surrogate materials for each measurand improved relative

80 recoveries by compensating for manipulative or degradative losses. Thus, a single

81 extraction became possible with a concomitant improvement in analytical throughput. A

82 protease was incorporated in the method to efficiently liberate the embedded vitamins

83 from their encapsulating materials. The described method also facilitated an estimation

84 of total vitamin A content through summation of the added and natural esters.

85

86
87 2. Material and Methods

88

89 2.1 Chromatography

90 A Shimadzu (Nakagyo-ku, Kyoto, Japan) LC20AT Prominence HPLC with

91 LC20AT quaternary pump and SIL-20AHT autosampler was operated in normal phase

92 mode with an Alltech Econosphere 3 µm silica column (150 x 4.6 mm, part number

93 228345), held at 25 oC in a CTO-20AC column oven. Detection used an RF-20A XS

94 dual-wavelength fluorescence detector and a SPD-M20A diode-array detector in series.

95 Channel 1 of the fluorescence detector was set at 325 nm (excitation) and 450 nm

96 (emission) with medium sensitivity (x4 gain) to monitor the retinyl esters, while channel

97 2 was set at 280 nm (excitation) and 330 nm (emission) with medium sensitivity (x4

98 gain) to detect α-tocopheryl acetate. The diode-array detector was used at 450 nm to

99 monitor β-carotene signals but also collected data at other wavelengths, notably 325

100 nm for an alternative vitamin A option. Typical flow-rate of 1.0 mL/ min was used with

101 an injection volume of 10 µL. For enhanced sensitivity, up to 50 µL could be injected

102 without exhibiting excessive band broadening.

103 Other equipment was proven suitable by repeating analyses on an Agilent 1100

104 (Santa Clara, CA, USA) with a single channel programmable fluorescence detector

105 (FLD) and diode-array detector. The configuration was either i) FLD at 280 nm / 330 nm

106 (excitation/emission) for α-tocopheryl acetate with simultaneous absorbance detection at

107 325 nm (retinyl esters) and 450 nm (β-carotene) or, ii) programmed FLD wavelength

108 initially at 325 nm / 450 nm for retinyl esters, changed prior to α-tocopheryl acetate

109 elution, with β-carotene on UVD at 450nm.

110

111 2.2 Other Apparatus

112 Varian Cary 50 UV-vis. spectrophotometer (Palo Alto, CA, USA) with 1cm quartz

113 cuvettes was used to calibrate retinyl propionate (325 nm), tocopherol propionate (284

114 nm) and apo-carotenoic acid ethyl ester (450 nm). Protease incubation was performed

115 at 50 ±3 oC in a Grant water bath with GD100 controller (Chelmsford, UK). Sample
116 clarification was performed in a Gerber centrifuge (Funke-Gerber, Berlin, Germany) at

117 1000 revs/min (~ 350 rcf) and buckets to contain 50 mL polypropylene Centristar®

118 centrifuge tubes (Corning, NY, USA). pH control was done with a Metrohm 867 (Herisau.

119 Switzerland).

120

121 2.3 Reagents

122 Thermostable protease (EC 3.4.21.14), subtilisin A from Bacillus licheniformis was

123 sourced from Megazyme (Bray, County Wicklow, Eire), product code E-BSPRT in 50% glycerol,

124 with >300 tyrosine equivalent U/mL (casein substrate, pH 8).

125 Tris.HCl buffer (1 M) was prepared by dissolving 121 g tris base (Sigma Aldrich, St Louis,

126 MO, USA) in about 800 mL water, then bringing to pH 8.0 with drop-wise addition of

127 concentrated hydrochloric acid. This was made to 1L and diluted to 0.1 M for use.

128 Solvents used were HPLC-grade hexane, ethanol and isopropanol (IPA) from Fisher

129 Scientific (Loughborough, UK). An extraction solution of 50% ethanol in hexane was prepared.

130 To prepare the mobile phase, various volumes of IPA were tested to achieve optimum separation

131 of vitamin A and E esters, the preferred being 600 µL in 1 L hexane. The mobile phase was

132 filtered through anhydrous sodium sulphate (Scharlau, Barcelona, Spain) on a 0.45 µm

133 membrane filter with standard vacuum apparatus thus minimising its humidity and stabilising

134 retention times. The mobile phase was continuously recycled to reduce reagent costs and

135 replaced when retention time or baseline fluctuations were observed.

136 Saturated sodium chloride was prepared by adding 320 g sodium chloride (Merck,

137 Darmstedt, Germany) to 1 L of deionised water, ensuring some solid remained

138 undissolved.

139 Vitamin A propionate (2,500,000 IU/g) was sourced from BASF (part 50096515,

140 Basel, Switzerland) and stored in a freezer under nitrogen gas. A stock solution of about

141 625 IU/mL was prepared by dissolving 25 mg in 100 mL isopropanol (IPA) containing 100

142 – 200 mg butyrated hydroxytoluene crystals (BHT, Sigma Aldrich, St Louis, MO, USA) to

143 minimise vitamin degradation. This was stored in the dark in a freezer (-20 °C) for up to

144 six months and used as internal standard for vitamin A palmitate and vitamin A acetate.
145 It was calibrated, using equation 1, by dilution of 100 µL to 10 mL with subsequent

146 absorbance measurement against IPA at 325 nm. Recalibration was performed on a

147 weekly basis. Calculations were converted to IU/mL to permit unitary (1.00) response

148 factors no matter which retinyl esters were being measured, including the natural long-

149 chain esters.

150

151 Retinyl propionate (IU/mL) = A325 x 104 x 100 (1)

152 1595 x 0.359
153

154 where

155 A325 = observed absorbance at 325 nm (1 cm)

156 1595 = extinction coefficient in IPA at 325 nm

157 104 = conversion of % to µg/mL

158 0.359 = conversion of µg to IU for retinyl propionate

159 100 = dilution for usable absorbance

160

161 Retinyl palmitate and retinyl acetate were obtained from Sigma-Aldrich (St Louis,

162 MO, USA), containing 1,750,000 IU/g and 2,800,000 IU/g, respectively, and prepared in

163 IPA at concentrations similar to retinyl propionate.for peak identity purposes.

164 α-Tocopheryl propionate, internal standard for α-tocopheryl acetate, was not

165 available commercially, so prepared by acylation based on the procedure described by

166 Bonrath and Cirillo (2002): 2.5 g (equivalent to 5.8 mmol) of α-tocopherol (Sigma-

167 Aldrich) was weighed into a 50 mL round-bottomed flask containing 1.5 g (11.5 mmol, bp

168 167 oC) of propionic anhydride (Merck & Co., NJ, USA). 200 µL (2.5 mmol, bp 115 oC)

169 of pyridine (Merck & Co) was added as catalyst and a water-cooled condenser fitted.

170 The flask and condenser were held in a boiling water for 3 hr covered with aluminium foil

171 protection from light. The product was cooled and 20 mL water added. The stoppered

172 flask was shaken and left overnight to hydrolyse excess propionic anhydride to propionic

173 acid. The lower aqueous phase was withdrawn and the oily propionate ester washed

174 twice with 20 mL water. The flask was then left under rotary evaporation (Buchi R3 with
175 V700 pump and F105 chiller, Flawil, Switzerland) at 70 °C under 80 mbar vacuum until

176 dry. The vitamin E propionate was stored in the flask under nitrogen gas at -20 oC until

177 required and was stable for > 1 year under such conditions.

178 A stock solution of α-tocopheryl propionate was prepared by dissolving about 250

179 mg in 100 mL IPA containing 100 – 200 mg BHT, to yield nominally 2.5 mg/mL, and

180 stored for up to six months at -20 oC. It was calibrated, using equation 2, after dilution

181 of 100 µL to 10 mL with IPA and subsequent absorbance measurement against IPA at

182 284 nm. Recalibration was performed on a weekly basis to guard against degradation.

183 α-Tocopheryl propionate (mg/mL) = A284 x 10 x 100 (2)

184 42.3

185 where

186 A284 = observed absorbance at 284 nm (1 cm)

187 42.3 = extinction coefficient in IPA at 284 nm

188 10 = conversion of % to mg /mL

189 100 = dilution to obtain a usable absorbance

190

191 Vitamin E acetate (Sigma-Aldrich) solution was prepared at about the same

192 concentration as vitamin E propionate for peak identity purposes.

193 Pure all-E-β-apo-8’-carotenoic acid ethyl ester (CAS 1109-11-1; CI 40825; E160f),

194 abbreviated here as β-apo-ester, was gifted in vials from DSM (Heerlen, Netherlands),

195 and stored in a freezer until opened. A stock solution of about 1000 µg/mL was

196 prepared by dissolving ~ 100 mg apo-ester in 100 mL dichloromethane containing 100 -

197 200 mg BHT to minimise oxidation, and stored for up to one month at -20 oC. It was

198 calibrated by dilution of 100 µL to 10 mL with IPA and subsequent absorbance

199 measurement against IPA at 449 nm using equation 3. Recalibration was performed

200 weekly to guard against degradation.

201

202 β-apo-Ester (µg/mL) = A449 x 104 x 100 (3)

203 2550
204 where

205 A449 = observed absorbance at 449 nm (1 cm)

206 2550 = extinction coefficient in IPA at 449 nm

207 104 = conversion of % to µg/mL

208 100 = dilution to obtain a usable absorbance

209

210 β-carotene (Sigma-Aldrich) solution was prepared at about the same

211 concentration as β-apo-Ester for peak identity purposes.

212 A mixed internal standard was prepared in an amber 25 mL flask using 10 mL

213 retinyl propionate, 10 mL of α-tocopheryl propionate and 5 mL of apo-ester stock

214 standards, with no further dilution. These ratios suited most applications but were

215 varied as required, taking care to monitor the exact amount of each internal standard

216 added to each sample.

217 A system suitability solution was prepared by mixing 10 mL retinyl propionate, 10

218 mL retinyl acetate, 10 mL retinyl palmitate, 10 mL of α-tocopheryl propionate, 10 mL α-

219 tocopheryl acetate, 5 mL of apo-ester and 5 mL β-carotene stock solutions, to ensure

220 optimal HPLC performance.

221

222 2.4 Extraction

223 20.0 g of milk powder or infant formula was accurately weighed into a 200 mL

224 beaker and 80.0 g warm water (~ 40 oC) added. The mixture was stirred to dissolve the

225 powder and 5.0 g of reconstituted sample (equivalent to 1.0 g powder) weighed into a 50

226 mL polypropylene centrifuge tube. In the case of liquid milk or ready-to-feed

227 formulations, 5.0 g was used and treated the same way.

228 5 mL of 0.1 M Tris.HCl buffer, pH 8 was added to the centrifuge tube followed by

229 20 µL thermostable protease (Megazyme, activity >300 units/mL) and incubated at 50 ±

230 3 oC for 30 min with occasional mixing. After cooling, 200 µL of mixed internal standard

231 solution was accurately added, followed by 20 mL hexane : ethanol mixture (50:50) and
232 mixed for one minute by vortex or shaking. The extraction was completed by adding 5

233 mL saturated sodium chloride and vortex mixing or shaking again for one minute.

234 Phase separation was continued by standing in the dark for 30 – 60 min until

235 complete, otherwise centrifuged for 10 min at ~350 rcf. The upper hexane layer was

236 filtered through a 0.45 µm nylon filter and 2 mL collected in an amber crimp-topped

237 HPLC vial.

238

239 2.5 Chromatography

240 Sample extracts (10 µL) were injected into the HPLC with the eluent monitored

241 simultaneously by FLD and UVD. FLD gain settings were adjusted as required to keep

242 peaks on scale.

243 The usual flow-rate was 1.0 mL/min with the 3 µm, 150 x 4.6 mm ID, column,

244 kept at 25 oC, the low viscosity of hexane producing minimal back-pressure. The flow

245 was raised to 2 mL/ min after the elution of the last peak of interest (α-tocopheryl

246 acetate) and continued for the duration of the chromatogram to elute lipids in readiness

247 for the next injection. Mobile phase recycling was implemented in order to conserve

248 solvent.

249

250 2.6 Calculations

251 The concentrations of each internal standard in the mixed standard were calculated from their

252 concentrations, as determined by equations 1 - 3 (in IU/mL, mg/mL or µg/mL, as stated), multiplied by

253 their proportions in the mixed standard (0.40 for vitamins A and E or 0.20 for β-apo-Ester). The

254 amount of each internal standard added to the samples was calculated by multiplication with 0.2 mL,

255 the typical volume added.

256 This data was used to calculate the vitamin concentrations in reconstituted and liquid samples

257 according to equations 4, 5 and 6:

258

259 Vitamin A esters, IU/100 g = ASMP x A propionate added (IU) x 100 x recon (4)
260 AIS x W (g) x RF
261
262

263 Vitamin E acetate, mg/100 g = ASMP x E propionate added (mg) x 100 x recon (5)
264 AIS x W (g) x RF
265

266

267 β-Carotene, µg/100 g = ASMP x apo-ester added (µg) x 100 x recon (6)
268 AIS x W (g) x RF
269

270 where

271 ASMP = area of sample peak

272 AIS = area of internal standard peak

273 W = weight (g) of reconstituted or liquid sample, typically 5g

274 recon = reconstitution factor for powdered samples = 20 (1 for liquid samples)

275 RF = response factor (see below)

276 100 = conversion to 100g sample

277

278 Response factors used were 1.000 for vitamin A esters, the units (IU) being independent of

279 molecular weights. Response factors, as divisors, of 0.971 were applied to vitamin E acetate and

280 0.985 for β-carotene respectively, based upon literature E1% values in hexane (FAO, 1991; JECFA,

281 2000). Experimentation proved these response factors were valid during the current study.

282 To express vitamin A in weight units, IU/100 g was multiplied by 0.3 to give ug/100 g retinol

283 equivalents. In the case of vitamin E acetate, IU/100 g and mg/100 g are equivalent for synthetic

284 forms.

285

286 2.6 Validation

287 The method was subjected to normal intra-laboratory challenges of precision and

288 accuracy. Repetitive testing of a range of commercial infant formulations yielded relative

289 standard deviations (RSD) that were compared for acceptability using the Horwitz

290 function. Certified Reference Materials (CRM) were used where applicable but were

291 unsuitable in the case of vitamin E because certificates reported total tocopherol, rather
292 than the α-tocopheryl acetate entity. Spiked recovery data were generated for each of

293 the vitamins.

294

295 3 Results and Discussion

296

297 3.1 Background

298 Manufacturers of infant formulae increasingly require data about the level of

299 supplementary vitamins added during the fortification processes. Where these differ

300 structurally from the endogenous forms, and where the analytical process does not

301 modify analyte structure, this aim is possible. Thus, an unambiguous quantitation is

302 possible for the acetate esters of vitamin A and E. In the case of retinyl palmitate and β-

303 carotene, both are innate to milkfat and potentially exist in infant formulae, but

304 estimations of the additive concentrations remain feasible because most infant formulae

305 are milkfat-free and have minimal endogenous levels of either compound.

306 Although not the main purpose of this study, the near absence of retinol itself in

307 milk meant the described method allowed a determination of total vitamin A by

308 summation of all the esters present. Vitamin A exists as a collection of medium- and

309 long-chain esters, in a similar pattern to triglyceride fatty acids (Woollard and Indyk,

310 1989). The identity and variable molecular weight of each ester is not important when

311 concentration units are expressed in international units. Conversions to weight-based

312 units, typically µg/100 g retinol equivalents, were then performed at the conclusion of the

313 calculations.

314 Total vitamin E determinations were not possible because of the presence of

315 considerable free α-tocopherol (and other tocopherol congeners) from those vegetable

316 oils incorporated to replace milkfat, with an estimation of added α-tocopheryl acetate

317 significantly lower than total vitamin E content. Chavez-Servın et al, (2006) used an

318 amine-column to detect α-tocopherol and its congeners using solvent gradients, a

319 strategy not possible in this study where a silica column was used.

320
321 3.2 Selection of internal standards

322 The use of internal standards for vitamins A, E and β-carotene is not new in

323 clinical chemistry where the acetate esters are used for retinol and α-tocopherol in

324 serum (Kand’ár et al., 2013). In the absence of saponification, other esters of vitamins A

325 and E make ideal surrogates for the measurands and when added early in the extraction

326 process, compensate for oxidative and manipulative losses of the labile analytes, while

327 also accounting for chromatographic variables during quantitation.

328 The propionate esters of retinol and α-tocopherol were well resolved with respect

329 to retinyl acetate, retinyl palmitate and α-tocopheryl acetate, thereby supporting their use

330 as internal standards. Retinyl propionate was commercially available, but α-tocopheryl

331 propionate required laboratory preparation by an esterification procedure based on

332 Borath & Cirillo (2002) involving the pyridine-catalysed reaction of α-tocopherol with

333 propionic anhydride. 3 h heating at 100 oC was used rather than using a microwave

334 reactor for 30 min but esterification was complete as demonstrated by the absence of

335 free α-tocopherol during chromatography. An extra water washing step was used to

336 ensure all propionic anhydride was removed. A similar esterification has been reliably

337 demonstrated in the compendial analysis of tocopherol food additives by GC-FID where

338 conversion to propionate esters precedes GC determinations (JECFA, 2000).

339 Commercially available vitamin E esters (succinate and nicotinate) had poor

340 chromatographic properties on silica due to their polar nature so could not be considered

341 as internal standards.

342 Non-polar β-carotene elutes near the void volume on normal-phase columns,

343 allowing a range of hydroxylated homologues to be used for internal standardisation.

344 Many candidate xanthophylls were expensive or highly retained on silica columns which

345 reduced the number of practical choices. β-apo-8’-carotenoic acid ethyl ester, all-E

346 (trans) isomer, is manufactured in large quantity as a feed additive for egg colouration so

347 was chosen for further study, although all-E-β-apo-8’-carotenal (CAS 1107-26-2),

348 available from Sigma-Aldrich, was also found useful.

349 Retinol and its esters have identical molar extinction coefficients (ε) as the ester

350 moiety does not influence the chromophoric conjugated structure, although their weight-

351 based coefficients (E1%) are a function of molecular weight. For simplicity, calculations

352 of vitamin A concentrations were kept in international units (IU), where, like ε, all forms

353 are equal. A response factor of 1.00 was therefore applicable independent of which

354 supplementary ester (acetate or palmitate) or natural ester was being calibrated by the

355 retinyl propionate internal standard.

356 Similarly, α-tocopheryl acetate, the sole measurand for vitamin E, and α-

357 tocopheryl propionate have same molar extinction coefficient. Calculations were in

358 weight units so a response factor corresponding to their molecular weights ratio (0.971)

359 was applied. This response factor was confirmed experimentally from standard

360 solutions, proving the extra carbon in the side chain has no measurable impact on

361 spectral absorptivity. Additionally, the stereoisomeric variation of the α-tocopherol moiety

362 was ignored, since a single chromatographic α-tocopheryl acetate peak was observed

363 irrespective of whether the synthetic (all-rac-α-tocopherol) or naturally-extracted form

364 (RRR-α-tocopherol) was present.

365 Despite the disrupted β-ionone ring of β-apo-ester and lower molecular weight of

366 this C30 carotenoid (460.7 mol/g) compared to C40 β-carotene (536.9 mol /g), it has a

367 similar reported E1% (2550) as β-carotene (2590) in hexane yielding a theoretical

368 response factor of 0.985. There is a small hypsochromic shift of the principle peak in

369 hexane of β-apo-ester (449 nm) compared with β-carotene (453 nm), the net result of

370 one fewer conjugated bond and an extra carbonyl (Schwieter et al., 1969). This had no

371 impact with a detector bandwidth of 8 nm as shown by experiments with authentic

372 standards thus confirming the theoretical response factor could be reliably used.

373

374 3.3 Sample Preparation

375 Studies performed in the 1980’s reported a useful method for testing vitamins A

376 and E in dairy powders using a fast extraction of the fat in a single tube, followed by

377 HPLC with selective fluorescence detection (Woollard & Indyk, 1986; Woollard & Indyk,
378 1989). The current method followed a similar strategy but used advances in HPLC

379 instrumentation and internal standardisation to improve data quality. The original

380 method, exploiting extraction with dimethysulphoxide (DMSO), was modified in the

381 present study with protease digestion to facilitate release of micro-encapsulated vitamins

382 that are increasingly present in modern dry-blended products. The hydrophobic fat-

383 soluble vitamins are embedded at manufacture in a proteinaceous material such as

384 gelatin or gum arabic polysaccharide and therefore required effective release into

385 solution prior to extraction and detection. Gum arabic has a peptide backbone that is

386 susceptible to protease disruption and release of entrapped contents.

387 Dry-blended infant formulations, with their intact encapsulations and

388 heterogenous distribution, prompted use of 20 g samples, easily expandable to larger

389 weights as required. Sample sizes up to 50 g were trialled with concomitant increases in

390 volumes, internal standards and protease but no improvement in precision was noted

391 with the samples tested.

392 Infant formulae manufactured by alternative wet-blended processes have

393 supplemental vitamins added pre-evaporation and therefore have no remaining intact

394 encapsulation. However, for practical purposes 20 g of these homogeneous samples

395 were treated identically to dry-blended products. Besides, protease digestion has a

396 secondary purpose in digesting milk proteins that potentially interfere with phase

397 separation of subsequent hydrocarbon extractions. Similarly, UHT liquid milks and

398 ready-to-drink infant formulations were treated with protease, although vitamin

399 encapsulations were almost certainly absent.

400 The choice of proteolytic enzyme was made based upon the well established

401 properties of alkaline proteases, also called proteinases, from bacillus licheniformis

402 (Ferrero, Castro, Abate, Baigorı & Sińeriz, 1996) and previous knowledge of the dietary

403 fibre test where complete removal of protein is mandatory. Serine protease, Subtilisin A,

404 from Bacillus licheniformis sourced from Megazyme proved to be a convenient and

405 stable protease, although papain was used in AOAC 2012.10 (McMahon et al., 2013).
406 During extraction, a 5 g portion was utilised from the total 100 g digest,

407 equivalent to 1 g of powder. This was extracted into 10 mL hexane, as a 50:50 mixture

408 with ethanol, and salt solution to improve phase separation. Higher boiling solvents,

409 heptane and isooctane, were equally effective extractants, although hexane was easier

410 to reduce in volume for improved sensitivity if required, albeit not routinely needed in this

411 study.

412

413 3.3 Chromatography

414 Unbonded silica columns of various geometry were successfully tested, including

415 5 µm columns, but a 3 µm (150 x 4.6 mm) Alltech column with 80 Å pore size was

416 chosen for subsequent studies. Hewavitharana et al. (1996) successfully used a similar

417 column but with different polar modifiers. Injection volumes were typically 10 µL but

418 could be increased to 30 µL (100 µL for 5 µm columns) without loss of performance.

419 The diameter of the selected column was kept to a maximum to allow increased

420 flow-rates for faster lipid clearance. The mobile phase had low viscosity, allowing flow

421 rates up to 3 mL/min (5 mL/min for 5 µm columns) without excessive back-pressure.

422 This promoted rapid clearance of triglycerides from the column, creating a more stable

423 chromatographic environment. The work of Thompson, Schimpf & Baugh (2013)

424 overcame the same problem through column switching, thereby diverting lipid to waste

425 from a short pre-column. Chromatography was continued here at elevated flow rate until

426 the relatively polar unesterified α-tocopherol had eluted to prevent it being carried over

427 to the next chromatogram. Late eluting β- and γ-tocopherols did not cause interference

428 problems because the peaks were indefinitely broadened.

429 Vitamins A and E both have natural fluorescence, and β-carotene a selective 450

430 nm viewing wavelength, so are amenable to simplified detection in the presence of other

431 lipid material. Co-extracted lipids passed through the detectors without response, and

432 exited the column quickly enough to prevent accumulation and associated

433 chromatographic changes. In the absence of dual-channel fluorescence detection,

434 retinyl esters could be satisfactory detected by UVD at 325 nm with a multi-wavelength
435 detector. However, vitamin E was necessarily detected by fluorescence because UVD

436 at 284 nm experienced extensive interferences.

437 It was important to avoid incursion of water onto the silica column with

438 consequently diminished retention times. As part of this process, the mobile phase was

439 passed through anhydrous sodium sulphate desiccant before use, placed above the

440 0.45 um solvent clarification filter. Refiltration through anhydrous sodium sulphate was

441 also an option to maintain low humidity. To conserve solvents, the mobile phase was

442 recycled for a week, otherwise replaced when retention or baseline shifts were noted.

443 Retention times were highly sensitive to the concentration of the polar modifier

444 isopropanol (IPA), which was necessarily pipetted into hexane in an accurate manner. A

445 number of IPA concentrations were tested over the range 0.04 - 0.08%, with 0.06%

446 shown to give good isomer separations on the selected Alltech column. This was

447 reduced or increased as required in small increments 50 µL increments to retain optimal

448 separation.

449 Chromatographic selectivity for the analytes was also dependent on IPA

450 concentration, in particular, the retention of β-apo-ester rapidly decreased with

451 increasing IPA until it eluted near the retinyl or α-tocopheryl esters. However, the unique

452 FLD detection modes for vitamins A and E esters overcame any potential

453 chromatographic interferences from β-apo-ester, and conversely, neither vitamin

454 absorbed in the visible region at 450 nm. Similarly, at higher IPA concentrations α-

455 tocopheryl acetate also eluted near retinyl propionate, but without spectral interferences.

456 Fig 1 shows the chromatographic output for a typical infant formula extract

457 containing supplementary retinyl palmitate, α-tocopheryl acetate and β-carotene using

458 concurrent dual-channel fluorescence and visible detection. In the latter detection

459 mode, only β-carotene (combined Z (cis) - and E (trans) isomers) and β-apo-ester

460 internal standard are visible. It should be noted that α-carotene, if present, is not

461 separated from β-carotene and calculations will essentially represent total carotenoid. In

462 addition, the xanthophylls lutein, astaxanthin and canthaxanthin did not
463 chromatographically interfere with the non-polar carotenes, although lycopene co-eluted

464 with β-carotene.

465 Fig 2 shows chromatograms of a typical whole-milk powder extract containing

466 supplementary retinyl acetate and α-tocopheryl acetate, with naturally occurring retinyl

467 esters and β-carotene. When geometric Z-isomer (cis) peaks were present they eluted

468 prior to the corresponding all-E (all-trans) isomer peaks and were included to give a total

469 peak area, but without corrections for their lower biopotency.

470

471 3.3 Precision

472 Four commercially available formulations were selected for repeatability studies,

473 each with supplementary α-tocopheryl acetate and β-carotene as judged by label

474 declarations. Two of the formulations contained retinyl palmitate while the other two

475 contained retinyl acetate, the latter also known to be dry-blended. In addition, a

476 vitaminised whole milk powder was repetitively tested for its total vitamin A content. The

477 results are given in Table 1 where repeatability relative standard deviations (RSDr) were

478 observed in the range 3 - 10% across the three vitamins. HorRat values (observed

479 RSD/ calculated RSD from Horwitz equation) are also given that confirm acceptability of

480 the observed RSDr at the observed concentrations. Observed repeatability was equal or

481 better than generally accepted guidance limits for repeatability based on blind duplicates

482 (HorRat 0.3 – 1.3; Horwitz & Albert, 2006). Vitamin A calculations were converted from

483 international units to weight units as required by Horwitz mathematics.

484 Two infant formulae were obtained from a New Zealand manufacturer and tested

485 in duplicate (n=2), twice per week for five weeks (d = 10 pairs), both manufactured by

486 dry-blending of proximates with premixes, containing retinyl palmitate, α-tocopheryl

487 acetate and β-carotene. Table 2 summaries the observed intermediate precision

488 (RSDiR) obtained using standard statistics (Chesher, 2008) and associated HorRat,

489 historically expected to lie 0.5 – 1.5 (Horwitz & Albert, 2006). Re-estimation of RSDr was

490 also performed based on the differences (squared) between duplicates which showed

491 comparability with Table 1.

492

493 3.3 Accuracy

494 To estimate recoveries, each of the three vitamins, prepared in calibrated IPA

495 solution, were spiked at three levels into suitable matrices prior to protease digestion.

496 For vitamin A, suitable blank matrices were available for the palmitate and acetate

497 esters by selection of infant formulae without the targeted analyte. For vitamin E, a

498 whole milk powder without supplementary α-tocopheryl acetate was used, while a fully

499 oil-filled infant formula without fortified carotenoids was used for β-carotene recovery

500 estimates. Table 3 shows 89 -104% recovery with an average 98.5 % across all the

501 vitamins. Each vitamin has a parallel internal standard added to the digest that

502 compensates for potential losses from degradation, volume changes and extraction

503 shortcomings, thereby leaving spectral calibration and dispensing of the internal

504 standards as the main analytical challenges.

505 Two infant formula certified reference materials with vitamin data were available

506 from National Institute of Standards and Technology (NIST). Triplicate (n =3) testing of

507 NIST 1849 and NIST 1849a yielded vitamin A data shown in Table 4. When compared

508 with certified concentrations agreement was good, confirming method accuracy. NIST

509 1849 and NIST 1849a both contained retinyl palmitate ester, with no retinyl acetate, the

510 data being expressed as retinol using relative molecular masses.

511 Direct comparison of vitamin E acetate data with data assigned in NIST 1849

512 was not possible because the latter included total α-tocopherol, most of which was oil-

513 derived and therefore unesterified. However, the NIST 1849 certificate noted that about

514 5 mg /100 g (50 mg/kg) α-tocopherol acetate is expected, which concurs with the

515 current observations as shown in Table 4. NIST 1849a had a certified range for vitamin

516 E acetate.

517 Similarly, a β-carotene value was not assigned in NIST 1849 or NIST 1849a.

518 Although highly variable in NIST 1849 documents, (200 – 1200 µg/100 g, Sharpless et

519 al., 2010), concentrations observed here were 239 µg/100 g (SDr = 35.4 µg/100 g), at

520 the lower end of this range although it is known that β-carotene is particularly unstable.
521 Results obtained for vitamin A and β-carotene in whole milk powder agreed well

522 with published data, although both vitamins are known to be seasonally variable in New

523 Zealand (Indyk, Lawrence & Broda, 1993)

524

525 Conclusion

526

527 In response to the needs of infant formula manufacturers or regulators to determine the

528 quantity and nature of supplementary vitamins A and E esters, a method has been

529 developed using modern HPLC equipment with dual-channel fluorescence detection.

530 Benefits of the method are avoidance of saponification, which allows other esters to be

531 used as internal standards, namely retinyl propionate, and α-tocopheryl propionate. The

532 method was extended to include β-carotene with all-E-β-apo-8’-carotenoic acid ethyl

533 ester as internal standard, although the limitations of its early elution from normal phase

534 columns is acknowledged. The extraction procedure, using simple protease digestion is

535 considerably faster than traditional methods involving alkaline digestion with multiple

536 washing and extractions, and provides adequate precision and recovery. A corollary of

537 the method is the rapid determination of total vitamin A, including natural esters and

538 provitamin A (β-carotene) in milk and milk powders, although the small amount or

539 endogenous retinol is excluded.

540 References

541

542 Aust O., Sies H., Stahl, W. & Polidori M. C. (2001). Review. Analysis of lipophilic

543 antioxidants in human serum and tissues: tocopherols and carotenoids. Journal of

544 Chromatography A, 936, 83–93

545

546 Baruffini A., De Lorenza E., Gandini C., Kitsos M. and Massolini G.(1992). High-

547 performance liquid chromatographic determination of α-tocopheryl nicotinate in cosmetic

548 preparations, Journal of Chromatography, 593, 95-97

549

550 Bonrath W. & Cirillo F (2002). Process for Preparation of Tocol Acylates and Tocopherol

551 Acylates. US patent 6,444,098 B2

552

553 Chávez-Servín J. L., Castellote A. I. & Lóopez-Sabater M. C. (2006). Simultaneous

554 analysis of vitamin A and E in infant milk-based formulae by normal-phase high-

555 performance liquid chromatography- diode array detection using a short narrow-bore

556 column. Journal of Chromatography, 1122, 138 – 143

557

558 Chesher D. (2008). Evaluating assay precision. The Clinical Biochemist Reviews, S23 –

559 S26.

560

561 Chase W. G., Ye L., Stoakes V. C., Eitenmiller R. R. & Long A. R. (2004). An

562 interlaboratory-verified method for the determination of vitamins A and E in milk- and

563 soy-based infant formula by liquid chromatography with matrix solid-phase dispersion

564 extraction. Journal AOAC International, 87, 1173 – 1178

565

566 DeVries J.W. & Silvera K.R. (2002). Determination of vitamins A (retinol) and E (alpha-

567 tocopherol) in foods by liquid chromatography: collaborative study. Journal AOAC International,

568 85, 424 - 434

569

570 De Leenheer A. P., Lambert W.. E. & Van Bocxlaer J. F. editors (2000). Modern

571 chromatographic analysis of vitamins, 3rd edition. Chromatographic Science Series

572 volume 84. Marcel Dekker, New York, Basel. ISBN 0-8247-0316-2

573

574 Eitenmiller R. R., Landen W. O. & Ye L. editors (2008). Vitamin Analysis for the health

575 and food sciences, Second edition. CRC Press, Taylor and Francis Group, Boca Raton,

576 USA, ISBN 0-8493-9771-4

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578 Epler K. S., Ziegler R. G. and Kraft N. E. (1993). Liquid chromatographic method for the

579 determination of carotenoids, retinoids and tocopherols in human serum and in food.

580 Journal of Chromatography, 619, 37 – 48

581

582 FAO (1991). FAO Food and Nutrition Paper 19. Spefications for Identity and Purity.

583 Food and Agricultural Organisation of the Unitech Nations, Rome, Italy, ISBN 92-5-

584 101126-5

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586 Ferrero M. A., Castro G. R. Abate C. M., Baigorı M. D. and Sińeriz F. (1996).

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590 Gentili, A., Caretti, F., Bellante, S., Ventura, S., Canepari, S., & Curini, R. (2013).

591 Comprehensive profiling of carotenoids and fat-soluble vitamins in milk from different

592 animal species by LC-DAD-MS/MS hyphenation. J. Agric. Food Chem., 61, 1628-1639.

593

594 Hewavitharana A. K., van Brakel A. S. & Harnett M. (1996). Simultaneous liquid

595 chromatographic determination of vitamins A, E and β-carotene in common dairy foods.

596 International Dairy Journal, 6, 613-624

597
598 Hite D. (2003). Determination of retinyl palmitate (vitamin A) in fortified fluid milk by

599 liquid chromatography: collaborative study. Journal AOAC International, 86, 375 – 385

600

601 Horwitz, W., & Albert, R. (2006). The Horwitz ratio (HorRat): A useful index of method performance

602 with respect to precision. Journal of AOAC International, 89, 1095 - 1109.

603

604 Indyk H., Lawrence R. & Broda D. (1993). The micronutrient content of bovine whole

605 milk powder: influence of pasture feeding and season. Food Chemistry, 46, 389-396

606

607 JECFA (2000). Compendium of Food Additive Specifications: Addendum 8. Joint

608 FAO/WHO Expert Committee on Food Additives. 55Th session, p 121-127, Geneva,

609 Switzerland, ISSN 0254-4725

610

611 Kand’ár R., Novotná P. & Drábková (2013). Determination of retinol, α-tocopherol,

612 lycopene, and β-carotene in human plasma using HPLC with UV-Vis detection:

613 application to a clinical study, Journal of Chemistry, 2013, 1 – 7

614

615 Liu Z., Lee H-J., Garofalo F., Jenkins D.J.A. & El-Sohemy A. (2011). Simultaneous

616 measurement of three tocopherols, all-trans-retinol, and eight carotenoids in human

617 plasma by isocratic liquid chromatography. Journal of Chromatographic Science, 49,

618 221 – 227

619

620 McMahon, A., Christiansen, S., Shine, L., Loi, C., & Dowell, D. (2013). Simultaneous

621 determination of 13-cis and all-trans vitamin A palmitate (retinyl palmitate), vitamin A

622 acetate (retinyl acetate), and total vitamin E (α-tocopherol and DL-α-tocopherol acetate)

623 in infant formula and adult nutritionals by normal phase HPLC: First action 2012.10.

624 Journal of AOAC International, 96, 1073 -1081.

625

626 Nierenberg D. W. & Nann S. (1992). A method for determining concentrations of retinol,
627 tocopherol, and five carotenoids in human plasma and tissue samples. American

628 Journal for Clinical Nutrition, 56, 4l7 - 426

629

630 Sharpless K. E., Lindstrom R. M., Nelson B. C., Phinney K. W., Rimmer C. A., Sander L.

631 C., Schantz M. M., Spatz R. O., Thomas D. B., Turk G. C., Wise S. A., Wood L. & Yen

632 J. H. (2010). Preparation and characterization of standard reference material 1849

633 Infant/Adult Nutritional Formula. Journal of AOAC International, 93, 1262 – 1274

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635 Schwieter U., Englert N., Rigassi N. and Vetter W. (1969). Physical Organic Methods in

636 carotenoid Research. Pure Applied Chem., 20, 365 - 420

637

638 Rucker R.B., Suttie J. W., McCormick D. B. & Machlin L. (2001). Handbook of vitamins,

639 third edition Marcel Dekker, New York. ISBN 0-8247-0428-2

640

641 Thompson, L. B., Schimpf, K & Baugh, S. (2013). Determination of vitamins A and E in

642 infant formula and adult/pediatric nutritional formula by HPLC with UV and fluorescence

643 detection: First action 2012.09. Journal of AOAC International,., 96, 1407-1413.

644

645 Woollard G. A. & Woollard D. C. (1984). The Determination of Serum Retinol by High

646 Performance Liquid Chromatography. Journal of High Resolution Chromatography and

647 Chromatography Communications, 7, 466-472

648

649 Woollard D. C. & Woollard G. A. (1984). Determination of vitamin A in fortified milk

650 powders using high performance liquid chromatography. New Zealand Journal of Dairy

651 Science and Technology, 16, 99 – 112

652

653 Woollard D. C. & H. Indyk H. (1986). The HPLC analysis of vitamin A isomers in dairy

654 products and their significance in biopotency estimations. Journal of Micronutrient

655 Analysis, 2, 125 – 146

656

657 Woollard D. C. & Blott A.D. (1986). The routine determination of vitamin E acetate in

658 milk powder formulations using high performance liquid chromatography. Journal of

659 Micronutrient Analysis, 2, 97 – 115

660

661 Woollard D. C., Blott A.D. & H. Indyk H. (1987). Fluorometric detection of tocopheryl

662 acetate and it’s use in analysis of infant formulae. Journal of Micronutrient Analysis, 3, 1

663 – 14

664

665 Woollard D. C. & H. Indyk H. (1989). The distribution of retinyl esters in milks and milk

666 products. Journal of Micronutrient Analysis, 7, 35 – 52

667

668

669
670 Fig 1: Chromatograms with dual-channel florescence (plots A and B) and visible
671 detection (plot C) of a typical infant formula extract containing supplementary retinyl
672 palmitate, α-tocopheryl acetate and β-carotene.
673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

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694

695

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698
699 Fig 2: Chromatograms with dual-channel fluorescence (plots A and B) and visible

700 detection (plot C) of a typical whole-milk powder extract containing supplementary

701 retinyl acetate and α-tocopheryl acetate, with naturally occurring retinyl esters and β-

702 carotene.

703

704

705

706

707

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727
728 Table 1: Repeatability studies for vitamin A esters, vitamin E acetate and β-carotene (provitamin A)
729 in manufactured formulations by same-day repetition (n=5)
730
731
Sample Vitamin A Vitamin E β-Carotene
Mean RSDr Mean RSDr Mean RSDr
HorRat HorRat HorRat
ug/100 g % mg/100 g % ug/100 g %
a,c
Formula 1 502 3.09 0.15 5.28 3.95 0.28 118 8.93 0.40
a,d
Formula 2 379 4.56 0.22 7.40 3.38 0.24 81.0 6.36 0.25
b,c
Formula 3 983 5.70 0.33 8.71 3.76 0.28 137 6.60 0.29
b,d
Formula 4 526 3.71 0.18 6.00 2.95 0.20 179 7.16 0.33
WMP 323 5.28 0.25 na na na 141 6.07 0.26
a b c d
Wet-blended; Dry blended; retinyl acetate; retinyl palmitate; na = not applicable

732
733
734

735

736
737 Table 2: Same-day (r) and between-day (iR) precision estimates from ten duplicate between-day
738 analyses
739
Vitamin A Vitamin E β-Carotene
A: Intermediate Precision
Mean RSDiR Mean RSDiR Mean RSDiR
HorRat HorRat HorRat
ug/100 g % mg/100 g % ug/100 g %
Formula 1 542 5.26 0.42 7.54 4.94 0.59 134 8.20 0.54
Formula 2 359 6.29 0.48 4.60 5.89 0.66 87.7 6.49 0.40
B: Repeatability
Mean RSDr Mean RSDr Mean RSDr
HorRat HorRat HorRat
ug/100 g % mg/100 g % ug/100 g %
Formula 1 542 3.67 0.30 7.54 3.08 0.37 134 5.76 0.38
Formula 2 359 3.83 0.29 4.60 3.68 0.41 87.7 4.25 0.26
740
741

742

743
744 Table 3: Recovery of vitamin A esters, vitamin E acetate and β-carotene (provitamin A)
745 spiked into nutritional formula or milk powders
746

Theoretical Observed Recovery %

Retinyl palmitate (IU/100 g) 17a
1 507 510 97.2
2 1014 1021 99.0
3 1521 1567 101.9
Retinyl acetate (IU/100 g) 0a
1 521 527 101.2
2 1042 1058 101.5
3 1563 1516 97.0
α-Tocopheryl acetate (mg/100 g) 0a
1 5.11 5.17 101.2
2 10.22 10.29 100.7
3 15.33 14.88 97.1
β-carotene (ug/100 g) 7a
1 52 61 103.8
2 104 100 89.4
3 156 151 92.3
a
Unspiked (blank) concentration
747
748

749

750
751 Table 4; Vitamin A and E ester results compared with certified reference materials
752 (CRM) from National Institute of Standards and Technology (NIST)
753
754
Observeda Certified Range

Retinol Palmitateb
NIST 1849 1631 (35.5) 1640 1510 – 1770
NIST 1849a 788 (19.2) 768 745 – 791
α-Tocopheryl Acetatec

NIST 1849 5.63 (0.31) 5d na

NIST 1849a 14.8 (0.93) 15.8 14.0 – 17.6
a
Standard deviation in parenthesis; b Vitamin A units in µg/100 g, expressed as
retinol, listed in NIST certificate in mg/kg; c Vitamin E units in mg/100 g, expressed in
NIST certificate in mg/kg; d Information only; na = not applicable
755
756

757

758
759 Highlights for Review

760

761 We developed a fast method for vitamin A, E and carotene additives in dairy powders.

762 We used protease digestion to open the vitamin encapsulation.

763 We avoided saponification to allow use of propionate ester as internal standards.

764 We extended the method to include carotenes with apo-carotenoate internal standard.

765 The method has potential to improve existing AOAC official method 2012.10

766

767

768