Food Chemistry 151 (2014) 225–230

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Development of an analytical protocol for the estimation of vitamin D2

in fortified toned milk

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.11.085

⇑ Corresponding author. Tel.: +91 184 2259156 (O); fax: +91 184 2250042.E-mail address: sumitak123@gmail.com (S. Arora).

Ravinder Kaushik, Bhawana Sachdeva, Sumit Arora ⇑, Balbir Kaur WadhwaDairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, India

a r t i c l e i n f o

Article history:Received 6 August 2012Received in revised form 7 November 2013Accepted 15 November 2013Available online 23 November 2013

Keywords:Fortified milkHPLCVitamin D2 estimation

a b s t r a c t

A simple and rapid method has been developed for the isolation and determination of vitamin D2 in for-tified milk samples by HPLC. The advantage of the proposed method is that sample preparation time isreduced and both crystalline and encapsulated forms of vitamin D2 can be estimated. The developed pro-tocol involves a few extraction steps and requires less amounts of reagents. The HPLC separation of vita-min D2 was carried out on a reverse phase C18 column with photo diode array detector at 254 nm. Someadditional steps were required for extraction of microencapsulated vitamin D2 for breaking down coatingmaterial in comparison to crystalline vitamin D2. The recovery of vitamin D2 in fortified toned milk by theproposed method ranged from 96.46% to 99.05%. Non significant degradation was observed in vitamin D2

during seven days storage and also under light exposure (1485 lux) for 32 h.� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The importance of vitamin D in human diet has been recognizedsince the early 1900’s (Mc Collum, Becker, & Shipey, 1922). VitaminD fortified fluid milk was introduced in 1931 (Olson & Wallis,1935) and has since been a significant source of this vitamin. Vita-min D exists in two forms, namely vitamin D2 (ergocalciferol) andvitamin D3 (cholecalciferol). Vitamin D3 can be obtained from dietor exposure of skin to sunlight. The UV rays of sunlight induce thephotolytic conversion of 7-dehydrocholesterol to provitamin D3

followed by thermal isomerization to vitamin D3 (Holick et al.,1977). Vitamin D2 is synthesized in a similar manner from ergos-terol through sunlight exposure to provitamin D2 which rapidlyisomerizes to vitamin D2 in plant and fungi (Heftmann, 1975). Vita-min D2 and vitamin D3 undergo the same metabolic processesinvolving 25-hydroxylation in the kidney, to produce biologicallyactive metabolites 1,25-(OH)2 D3, respectively. The same dose ofvitamin D2 was as effective as vitamin D3 in maintaining serum25-hydroxyvitamin D levels and did not negatively influence ser-um 25-hydroxy vitamin D3 levels. Therefore, vitamin D2 is equallyas effective as vitamin D3 in maintaining 25-hydroxyvitamin D sta-tus (Holick et al., 2008).

Vitamin D deficiency has long been known to cause rickets(Rajakumar, 2003), but inadequate levels of vitamin D has more re-cently been implicated in a wide variety of diseases (Bischoff-Fer-rari, Giovannucci, Willett, Dietrich, & Dowson-Hughes, 2006),including some types of cancer (Garland, Gorham, Mohr, &

Garland, 2009; Mohr, 2009), cardiovascular disease (Holick, 2004;Zittermann, 2006), diabetes, multiple sclerosis, and others (Gesek& Desmond, 2008; Janssens et al., 2009; Pappa, Bern, Kamin, &Grand, 2008; Shoenfeld, Amital, & Shoenfeld, 2009). Vitamin D2

fortified milk not only increased vitamin D2 absorption but also in-creased calcium, iron and zinc absorption (Kaushik, Sachdeva, Aro-ra, Kapila, & Wadhwa, 2013). Increased melamine content,sunscreen use, clothing and aging can significantly reduce thecutaneous production of vitamin D3. Vitamin D is rare in naturallyoccurring foods with the exception of fatty fish, fish liver oil and fatand liver of some sea mammals (Holick, 1996).

Vitamin D content in cow and human milk is low, ranging from4 to 40 IU (0.1–1.0 lg/L) and which correspond mainly to vitaminD3 and 25-hydroxy-vitamin D3 (Fox & McSweeney, 1998; Muniz,Wehr, & Wehr, 1982). Except milk vitamin D3, other sources ofvitamin D3 are nonvegetarian. In Indian scenario, where most ofthe population is vegetarian, vitamin D2 was used for fortificationof toned milk.

For several years, the determination of vitamin D in foods hadbeen performed by biological assay methods (Kumar & Kaur,2005). These techniques, although specific, were time-consumingand involved a large uncertainty (Johnsson, Halen, Hessel, Nyman,& Thorzell, 1989). AOAC (2005) and Wher and Frank (2004) de-scribed vitamin D analysis using HPLC, but both methods are labo-rious, consume lots of chemicals and sample size is also high.

Almost all methods involved alkaline saponification followed byextraction with an organic solvent. In this sense, hot saponificationhas been applied to milk (Henderson & McLean, 1979). Most of theaforementioned studies made use of ultraviolet (UV) detectors forvitamin D3 determination in dairy products (Muniz et al., 1982).

226 R. Kaushik et al. / Food Chemistry 151 (2014) 225–230

In the present study a simple and rapid analytical protocol hasbeen developed for the estimation of vitamin D2 in fortified tonedmilk. The developed assay was successfully implemented for theanalysis of vitamin D2 in fortified milk samples.

2. Material and methods

2.1. Materials and reagents

Cow and buffalo milk were collected from Cattle yard ofNational Dairy Research Institute (Karnal, India). Crystalline vita-min D2 (40,000,000 IU/g) was procured from Sigma Aldrich (St.Louis, MO, USA) and encapsulated vitamin D2 (100,000 IU/g) wasobtained from DSM Nutritional Products (Singapore). All solventsand reagents employed in this study were HPLC-grade. Water, hex-ane and pyrogallol were purchased from Rankem (New Delhi, In-dia). Acetonitrile, methanol and chloroform were procured fromSigma Aldrich (St. Louis, MO, USA). Potassium hydroxide and HCl(AR grade) were procured from Fisher Scientific (Mumbai, India).Ethyl alcohol (99.9%) was procured from Jiangsu Huaxi Interna-tional (China). Ammonium hydroxide was procured from GlaxoLaboratories (Mumbai, India).

2.2. Preparation of toned milk

Cow milk and buffalo milk were mixed in 1:1 ratio and creamwas removed for the preparation of toned milk. The fat and solidnon fat (SNF) were set to 3.0% and 8.5%, respectively by addingcream/skim milk using Pearson square method. Pearson’s Squarehandily solves the problem of how to mix two solutions of knownpercentages without having to solve sets of simultaneousequations.

2.3. Fortification of milk with crystalline vitamin D2 and encapsulatedvitamin D2

A solution of 100,000 IU/ml was prepared by dissolving 100 mgof crystalline vitamin D2 in 40 ml of melted butteroil and10,000 IU/ml solution was prepared by dissolving 1 g of microen-capsulated vitamin D2 in 10 ml of water. The vitamin D2 solutionswere sonicated for 5.0 min in an ultrasonicator (SN-1, ToshconIndustries, Haridwar, India) for uniform mixing. The vitamin D2

solutions were stored below �20 �C in dark or in low actinic glassvessels. Vitamin D2 was added to milk at the level of 600 IU/L basedon its recommended daily allowance (ICMR, 2009). The crystallinevitamin D2 fortified milk samples were homogenised (Crepaco,AVP Company, Chicago, USA) for uniform mixing, whereas homog-enisation was not essential for encapsulated vitamin D2. Theencapsulated vitamin D2 solution was first mixed in an aliquot oftoned milk and then mixed to the bulk milk. The contents werethen agitated for complete mixing.

2.4. Processing treatments

Milk samples were pasteurised at 63 �C for 30 min in tempera-ture controlled waterbath (PolyScience, USA) in air tight glass bot-tles. The samples were immediately cooled to 4 �C. After 2 h ofstorage at 4 �C, milk samples were analysed for vitamin D2 contentand % recovery of vitamin D2 was calculated.

2.5. Sample preparation

2.5.1. SaponificationSaponification of milk samples for analysis of vitamin D2 was

performed essentially according to the method of Kazmi, Vieth,

and Rousseau, (2007), with several modifications. 3 ml milk waspoured in 15 ml centrifuge tube. 1 ml of 2.5% NH4OH (aqueous)was added and vortexed followed by sonification for 10 min.1 ml of decinormal solution of HCl was then added and vortexedand further sonicated for 10 min. 1.5 ml of 60% aqueous KOH and100 ll of 30% ethanolic pyrogallol (Wehr & Frank, 2004) wereadded and again vortexed. Sample tubes were flushed with nitro-gen, capped and placed in a temperature controlled waterbath at70 ± 1 �C. After 5 min, contents were vortexed and placed back inwaterbath for 30 min. Samples were then cooled in ice chilledwaterbath for 10 min.

2.5.2. ExtractionAbove cooled samples were vortexed and 2 ml of extraction sol-

vent (hexane) was added and again vortexed. 200 ll of ethanol wasadded to prevent emulsion formation. Sample tubes were centri-fuged at 448g (2000 rpm) for 10 min (Fixed Rotor, R8C, Remi Mo-tors, Mumbai, India). Supernatant was separated and thisextraction step was repeated twice. Collected supernatant wasdried by nitrogen flushing. Volume was made up to 1 ml withmethanol and contents were transferred into a vial. The reconsti-tuted sample was centrifuged at 960g (3500 rpm) for 15 min at0 �C (Rotor –RA-53F, Kubota, South Korea) for removal of impuri-ties. The supernatant was then filtered through 0.22 lm filterand transferred to a clean vial for HPLC analysis.

2.6. HPLC conditions

2.6.1. InstrumentationWaters 515 HPLC system was used for estimation of vitamin D2

from vitamin D2 fortified milk samples. It consisted of a high pres-sure gradient binary pump system, manual injector, temperaturecontrolled column chamber and 2998 Photodiode Array Detector.The Waters 2998 Photodiode Array Detector was used for detec-tion and quantification of vitamin D2. Data were collected and ana-lysed using the Empower2 software.

2.6.2. Chromatographic conditionsThe analytical column employed for analysis of vitamin D2 was

C18 (5 lm, 4.5 � 250 mm, 100 Å, Phenomenex, Torrance, CA, USA)with guard column C18 (5 lm, 100 Å, Waters, Ireland). The mobilephase (acetonitrile:methanol:chloroform 88:8:4) flow was iso-cratic at flow rate of 1.0 ml/min. The column temperature wasmaintained at 40 �C in column heater chamber.

2.7. Standards

Vitamin D2 stock solution: 5.0 mg vitamin D2 standard wasweighed and dissolved in methanol and transferred to a 10 mllow actinic volumetric flask and volume was made up with meth-anol. Vitamin D2 stock solution was diluted to prepare standardshaving concentration of 1, 2, 3, 4 and 5 ng per 100 ll injection.

2.8. Linearity of calibration curve

Calibration curves were generated by plotting the peak area ofstandard vitamin D2 versus the theoretical concentration. The cal-ibration curves were run in triplicate and the linearity of the cali-bration curve was evaluated by a linear regression analysis.

2.9. Precision and accuracy

The precision and accuracy were determined using the methodof Sudsakorn, Phatarphekar, O’’ Shea, and Liu (2011). The assayaccuracies were evaluated by the deviation of the mean concentra-tion measurement of the replicates versus the theoretical

R. Kaushik et al. / Food Chemistry 151 (2014) 225–230 227

concentration value expressed as a percentage (% Bias). The assayprecisions were evaluated from the relative standard deviation(RSD) of the concentration measurements and expressed as thepercent coefficient of variation (% CV) from the mean concentrationof the replicates. Inter- and intra-day assays accuracies and preci-sions at standard concentrations of less than or equal to ±15% weredeemed to be acceptable.

2.10. Recovery

% Recovery of vitamin D2 using above developed method fromvitamin D2 fortified milk samples (600 IU/L) was estimated. The% recovery of vitamin D2 from fortified milk samples was calcu-lated against the standardized chromatographic conditions. %Recovery was calculated as

% recovery ¼ XY� 100

where,X = vitamin D2 recovered after analysis.Y = vitamin D2 added to milk.

2.11. Vitamin D2

Encapsulated vitamin D2 was preferred over crystalline vitaminD2 for fortification and for further stability studies, as it was watersoluble and dispersed easily in comparison to crystalline vitaminD2 which needed homogenization for proper mixing of vitaminD2 during fortification in milk.

2.11.1. Storage stability of vitamin D2

The degradation of vitamin D2 in fortified toned milk duringstorage was determined using the above method. Milk sampleswere fortified with encapsulated vitamin D2, pasteurized andstored at 4 �C for seven days. Vitamin D2 content was analysed at0 day and after 3rd day, 5th day and 7th day of storage at 4 �C.

2.11.2. Effect of light on vitamin D2

The effect of light on degradation of vitamin D2 in fortified milkwas determined using the above method. Milk samples were forti-fied with encapsulated vitamin D2, pasteurized and stored atrefrigeration temperature (4 �C) under light intensity 1485 lux.The deterioration of vitamin D2 under light was estimated after2, 4, 8, 16 and 32 h exposure of light.

2.12. Statistical analysis

Means, standard deviation, RSD, linear regression analysis and95% confidence intervals were calculated using Microsoft Excel2007 (Microsoft Corp., Redmond, WA). Data was subjected to a sin-gle way analysis of variance (ANOVA).

3. Results and discussion

3.1. Optimization of the chromatographic conditions and detectorparameters

Three different mobile phases were chosen for estimation ofvitamin D2 viz. acetonitrile:methanol:ethyl acetate (88:8:4), aceto-nitrile:methanol:chloroform (88:8:4) and acetonitrile:methanol(80:20) (Wehr & Frank, 2004). Acetonitrile:methanol:chloroform(88:8:4) gave best resolution in comparison to other two mobilephases. Three flow rates (0.8, 1.0 and 1.2 ml/min) of mobile phasewere assayed for estimation of vitamin D2. Flow rate of 0.8 ml/mingave retention time of 16.7 min, whereas in 1.2 ml/min someshouldering arose in peak. 1.0 ml/min gave good resolution with

no shouldering; therefore, 1 ml/min was used for further study.The retention time of vitamin D2 was 12.30 ± 0.50 min at 1 ml/min flow rate of solvent system acetonitrile:methanol:chloroform(88:8:4) and total run time per sample was 15 min (Fig. 1).

Three injection volumes (20, 100 and 200 ll) of saponified, ex-tracted and filtered sample of milk were used for vitamin D2 anal-ysis. As the concentration of vitamin D2 in fortified milk was less,peak area of vitamin D2 was very less for 20 ll injection. 100 and200 ll sample injection gave sufficient area but lower volumeyielded better peak definition than the large, hence 100 ll waschosen for further study. Three kmax were assayed for vitamin D2

analysis (228, 254 and 265 nm). The kmax for vitamin D2 was254 nm, as it gave maximum peak area for vitamin D2 during HPLCanalysis.

3.2. Optimization of the sample preparation method

3.2.1. Sample sizeThree sample volumes viz. 1, 2 and 3 ml of vitamin D2 (600 IU/L)

fortified milk were analysed for vitamin D2�. 3 ml sample volumewas finally chosen, as (fortified) milk contained very less concen-tration of vitamin D2 (15 ng/ml) and there was difficulty in quanti-fication of low sample volume due to interfering componentspresent in milk. The vitamin D2 peak area and peak height was lessin case of 1 and 2 ml sample volume for quantification; therefore,3 ml sample volume which produced sufficient peak area and peakheight for detection and quantification of vitamin D2 was selectedfor further experimental analysis.

3.2.2. Antioxidant amountThompson, Hatina, Maxwell, and Duval (1982), Faulkner,

Hussein, Foran, and Szijarto (2000) and Wher, and Frank (2004)used pyrogallol as antioxidant for analysis of vitamin D as it didnot interfere with vitamin D2 detection and the vitamin D2 recov-ery was also good. It indicated that pyrogalol effectively protectsthe vitamin D2 during sample preparation further analysis. Theprotecting effect of ethanolic pyrogallol (30% w/v) at differentadditions 50, 100 and 150 ll was assessed. 100 ll of pyrogalloladdition was selected as it gave a higher peak area than 50 lland no change was observed in case of 150 ll.

3.2.3. Aqueous KOHAqueous KOH (60% w/v) was used at 0.5 ml, 0.75 ml and 1.0 ml/

ml for saponification of fortified milk. However, an increase in KOHconcentration did not improve the vitamin D2 recovery; therefore,lesser (0.5 ml/ml of milk) volume was finally selected.

3.2.4. SaponificationCold saponification and hot saponification were both assessed

for vitamin D2 analysis. Cold saponification was time consumingand resulted in low vitamin D2 recovery. Hot saponification takeslesser time and gave higher vitamin D2 recovery in comparisonto cold saponification. Therefore, hot saponification was selectedfor saponification of fortified milk samples.

Three saponification durations (15, 30 and 45 min) were as-sayed. Maximum recovery of vitamin D2 was observed after30 min saponification, therefore, saponification for 30 min dura-tion was selected for further saponification of vitamin D2 fortifiedmilk samples.

3.2.5. Extraction solventChloroform and methanol (4:1) (Kazmi et al., 2007), petroleum

ether (Byrdwell, 2009) and hexane (Grace & Bernhard, 1984) wereused for extraction of vitamin D2 from saponified milk samples.The recovery of vitamin D2 was 83.7%, 89.1% and 99.6% for chloro-form and methanol (4:1), petroleum ether and hexane, respec-

Fig. 1. HPLC chromatogram of vitamin D2.

Table 1Summary of the back-calculated vitamin D2 calibration standards (n = 5) in milk.

Nominal concentration(ng/100 ll)

Concentration found(ng/100 ll)

Accuracy (%Bias)

Precision(% CV)

1.0 0.98 97.94 0.992.0 1.98 99.03 2.363.0 2.99 99.56 1.384.0 4.00 100.11 0.815.0 4.95 99.02 0.51

228 R. Kaushik et al. / Food Chemistry 151 (2014) 225–230

tively. Hexane was finally selected for extraction of vitamin D2

from fortified milk as it gave the highest recovery.

Table 3Storage stability of vitamin D2 in fortified toned milk.

3.2.6. Ethanol (de-emulsifying agent)Ethanol was used to prevent emulsion formation between

hydrophobic and hydrophilic phase. The de-emulsifying agentwas necessary as milk has both hydrophobic as well as hydrophilicphase and also emulsifying agents (phospholipids, amphoteric pro-teins etc.). Ethanol prevented emulsion formation by which vita-min D2 was effectively separated in hydrophobic phase and lesswater came with extraction solvent, which made the followingevaporation step easier. 100, 150 and 200 ll ethanol were addedto extraction tubes. 200 ll ethanol gave clear separation betweenthe hydrophobic and non hydrophilic layers in comparison to100 and 150 ll ethanol.

It was also observed that when hexane and ethanol were bothadded at the same time and then vortexed, there was no properseparation of hydrophobic and hydrophilic phase. Therefore, etha-nol was added after the tube containing milk and hexane was vor-texed and centrifuged thereafter.

Storagestability

0 day 3rd day 5th day 7th day

% Recovery 99.97 ± 0.93a 99.73 ± 0.72a 99.47 ± 0.64a 99.16 ± 0.46a

Data are presented as mean ± sd (n = 3). means with same superscript in row do notvary significantly (p < 0.05) from each other.

3.2.7. Reconstitution solventMethanol and mobile phase (acetonitrile:methanol:chloroform

88:8:4) were used for making up the volume of nitrogen dried milksamples. Methanol reconstitution gave higher area of vitamin D2

Table 2Recovery of vitamin D2 from fortified milk samples.

Level of vitamin D2 fortified milk (IU/L) Encapsulated Vitamin D2

Peak area

400 6913.89 ± 23.96500 8644.73 ± 22.47600 10310.00 ± 32.45

Data are presented as mean ± sd (n = 3).

under HPLC in comparison to mobile phase. Therefore, methanolwas used for volume reconstitution of samples.

The developed vitamin D2 analysis method was used for chro-matographic analysis of vitamin D2 in fortified milk samples.

3.3. Standard curve

Five point calibration curve was prepared using standard vita-min D2. 1, 2, 3, 4 and 5 ng/100 ll solutions were prepared in meth-anol. 100 ll of standards were injected with the mobile phaseacetonitrile: methanol:chloroform (88:8:4), at flow rate of 1.0 ml/min with kmax at 254 nm.

3.4. Analytical parameters of the RP-HPLC method

3.4.1. Linearity of the calibration curveLinearity of standard curve was tested by analysing three sets of

vitamin D2 standards prepared in methanol (1, 2, 3, 4 and 5 ng/100 ll). Calibration curve were plotted with concentration of stan-dard versus peak area of vitamin D2. The resulting simple linearregression and correlation coefficients of the calibration curvewere y = 2076.5x + 1064.7 and R2 = 0.995, respectively. The calibra-tion curves were linear over the range 1–5 ng/100 ll.

3.4.2. Accuracy and precisionThe accuracy of the developed method was assessed by analys-

ing the standard vitamin D2 as reference material at five concentra-tions (1, 2, 3, 4 and 5 ng/100 ll). Three replicates at each level were

Crystalline vitamin D2

% Recovery Peak area % Recovery

99.62 ± 0.34 6785.67 ± 42.60 97.78 ± 0.6299.66 ± 0.26 8519.02 ± 30.07 98.21 ± 0.3599.05 ± 0.003 10041.52 ± 50.21 96.46 ± 0.005

Table 4Effect of light on storage stability of vitamin D2 in fortified toned milk.

Light intensity duration (1485 lux) 0 h 2 h 4 h 8 h 16 h 32 h

% Recovery 99.99 ± 0.76a 99.69 ± 0.90a 99.47 ± 0.75a 99.28 ± 0.71a 99.12 ± 0.83a 98.98 ± 0.70a

Data are presented as mean ± sd (n = 3). means with same superscript in row do not vary significantly (p < 0.05) from each other.

R. Kaushik et al. / Food Chemistry 151 (2014) 225–230 229

analysed on three different days to assess inter day accuracy andprecision with respect to standard curve. The accuracy and preci-sion of HPLC method were within 5% for both intra-day and in-ter-day experiments as shown in Table 1.

3.4.3. Detection and quantification limitsA detection limit of 1.0 pg and a quantification limit of 10 pg in

assay were calculated based on the signal-to-noise ratio of 3 and10. Adaptation of the method to a complex matrix (fortified milk)includes optimization of the sample treatment and chromato-graphic conditions. Its reliability is assured, and the method is pro-posed for determination of vitamin D2 in the routine analysis ofvitamin D2 fortified milk.

3.5. Comparison of developed method with standard methods

AOAC (2005) and also the method developed by Wher andFrank (2004) were tried for analysis of vitamin D2. The methodswere suitable for estimation of vitamin D2 when crystalline vita-min D2 was used for preparation of fortified milk but were not suit-able when encapsulated vitamin D2 was used for fortification as itresulted in lower recovery of the vitamin. Secondly both methodsrequire greater quantity of chemicals and were more time consum-ing and laborious.

European Standard EN 12821, (2009) provided the base for theanalytical methods (HPLC) used for estimation of vitamin D2 andD3 in fortified and non-fortified foodstuffs. It serves as a framefor analytical protocols in accordance to the standard procedure.The principle and sample preparation steps of EN 12821 standardis similar to the developed method. Several chemicals, antioxi-dants, extraction solvents, mobile phase have been suggested forvitamin D analysis. The quantification method required internalstandards which may provide it with better precision. However,the standard does not refer to the estimation of encapsulated vita-min D2 which otherwise would have involved some additionalsteps. It also required greater quantity of chemicals, was more timeconsuming and laborious.

Kazmi et al. (2007) developed a method for estimation of vita-min D3 in milk and dairy products. The % recovery of microencap-sulated as well as of crystalline vitamin D2 using Kazmi et al.(2007) was less, therefore, several modifications were incorporatedfor vitamin D2 analysis.

The main differences between the method developed by Kazmiet al. (2007) and the developed method are as under:

(1) Method was standardized for analysis of vitamin D3 and notfor vitamin D2.

(2) Sonification step with 0.1 N HCl and 2.5% NH3OH was intro-duced to break the microencapsule layer.

(3) The developed method had higher sample size as milk wasfortified with only 600 IU/L of vitamin D2.

(4) Hexane was used for extraction instead of methanol:Chloro-form (2:1) in the present method as recovery was found tobe higher. Extraction was performed three times to improvethe extraction rate.

(5) Acetonitrile:methanol:chloroform (88:8:4) was used asmobile phase as it gave better resolution.

The present developed vitamin D2 analysis method was equallysuitable for analysis of both the crystalline vitamin D2 and alsoencapsulated vitamin D2 in fortified milk.

3.6. Recovery

Pasteurised fortified milk samples containing 400, 500 and600 IU/L vitamin D2 were analysed for vitamin D2 recovery (Ta-ble 2). The percent recoveries were 99.62 ± 0.34%, 99.66 ± 0.26%and 99.05 ± 0.003% for 400, 500 and 600 IU/L for encapsulated vita-min D2 fortified milk and 97.78 ± 0.62%, 98.21 ± 0.35% and96.46 ± 0.005% for crystalline vitamin D2 fortified milk, respec-tively. The % recovery of encapsulated vitamin D2 was higher thancrystalline vitamin D2 due to protecting effect of encapsulatedmaterial on encapsulated vitamin D2. No peak of vitamin D2 wasobserved in control (unfortified milk).

3.7. Storage stability

The percent recovery on 3rd, 5th and 7th day was calculated incomparison to 0 day reading. Non significant degradation of vita-min D2 in fortified milk was observed during storage (Table 3)for seven days at refrigerated temperature (4 �C). The observationswere in accordance with the results obtained by Holick, Shao, Liu,and Chen (1992).

3.8. Effect of light

Light exposure to vitamin D2 fortified milk for 2, 4, 8, 16, and32 h resulted in non significant reduction (Table 4) in vitamin D2

content at refrigerated temperature (4 �C).

4. Conclusion

A simple and rapid protocol has been developed for the estima-tion of vitamin D2 in fortified toned milk. The analytical parame-ters of the optimized RP-HPLC method with PDA detection offerlow detection level and quantification limits and good reproduc-ibility for vitamin D2 determination in fortified toned milk. Thedeveloped method is economical as it saves chemicals and time.

Acknowledgement

This study is part of the DBT-project financially supported bythe Department of Biotechnology (Delhi, India).

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