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Journal of Pharmaceutical and Biomedical Analysis 91 (2014) 24 31

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

jou rn al hom epage: www.elsev ier .com/ locate / jpba

The an byheart- ato

Jing Ma, g HSchool of Medi

a r t i c l

Article history:Received 13 SeReceived in reAccepted 2 NoAvailable onlin

Keywords:Two-dimensioMilk powderCarbohydratesRefractive ind

-dimnts inP-C4drateess wy of s in

was ion, aed thtable

n Co

1. Introduction

Lactose is the main carbohydrate present in milk powder [1].Monosacchadded to morder to imare importaity control dparametersvery impor

To detemethods [3most classiical methodcommerciatedious samised repro

To deal interest in traphy (2DLdemonstratother compuids [18,1

CorresponE-mail add

The aim of this work was to develop a new two-dimensionalliquid chromatography coupled with a refractive index detector(LCLC-RI) for the simultaneous determination of glucose, galac-

0731-7085/$ http://dx.doi.oaride such as fructose, glucose, and galactose are alsoilk powder products by some dairy manufacturers inprove the avor of milk powder. Actually, all of themnt nutrients in the human diet [2]. Therefore, the qual-uring manufacturing process and testing of qualitative

of carbohydrate contents in milk powder products istant.rmine carbohydrate contents in milk powder, many9] were developed in the past few years and the

c and practical one was liquid chromatography analyt-s. Although they were applied to determine series of

l samples successfully, these methods still suffer fromple pretreatment, long analysis time, and even unsat-ducibility.with complex samples, there has been a great deal ofhe development of two-dimensional liquid chromatog-C) [1014]. In the last decade, numerous groups haveed the utility of 2DLC in the separation of a variety oflex mixtures, including plant extracts [1517], bodily9], and beverages [2022].

ding author. Tel.: +86 29 82655392; fax: +86 29 82655451.ress: [email protected] (L. He).

tose, fructose, saccharose and lactose in milk powder. In thismethod, the sample is rst fractionated into intermediate frac-tions on the rst column, and the fraction containing the soluteof interest is collected and injected online into the second columnby a two-position valve to achieve the qualitatively and quan-titatively determination. A conventional one dimensional liquidchromatography (LC-RI) method was also proposed in this paper.After comparison of two methods, the benets of LCLC-RI werediscussed in detail.

2. Experimental

2.1. Chemicals, samples and solvents

The standards of glucose, galactose, fructose, saccharose and lac-tose were purchased from Carl Roth GmbH & Co. KG (Karlsruhe,Germany). HPLC-grade acetonitrile was obtained from Tedia Com-pany Inc. (Faireld, OH, USA). Ultrapure water was prepared using aMillipore Milli-Q purication system (Millipore, Bedford, MA, USA).Other reagents were all of analytical grade, except where noted.Mobile phases used for HPLC were ltered (0.45 m) and ultrason-ically degassed before use. All solutions were prepared daily. Themethod was tested on various commercial milk powders (n = 20),which were all purchased from a local supermarket (Xian, China).

see front matter. Crown Copyright 2013 Published by Elsevier B.V. All rights reserved.rg/10.1016/j.jpba.2013.11.006alysis of carbohydrates in milk powder cutting two-dimensional liquid chrom

Xiaofang Hou, Bing Zhang, Yunan Wang, Langchoncine, Xian Jiaotong University, 76#, Yanta West Road, Xian 710061, Shaanxi, China

e i n f o

ptember 2013vised form 31 October 2013vember 2013e 15 November 2013

nal liquid chromatography

ex detector

a b s t r a c t

In this study, a newheart-cutting twodetermination of carbohydrate contechromatography system, a Venusil XBseparation column, a ZORBAX carbohynal-analysis column. The whole procpreparation procedure. The capabilitin the determination of carbohydratedimensional chromatography methodin terms of linearity, limits of detectobtained with the two methods showchromatography method is more suiinvolved.

Crow a newgraphy method

e

ensional liquid chromatography method for the simultaneous milk powder was presented. In this two dimensional liquid

analysis column was used in the rst dimension (1D) as a pre-s analysis column was used in the second dimension (2D) as aas completed in less than 35 min without a particular samplethe new two dimensional HPLC method was demonstratedvarious brands of milk powder samples. A conventional onealso proposed. The two proposed methods were both validatedccuracy and precision. The comparison between the resultsat the new and completely automated two dimensional liquidfor milk powder sample because of its online cleanup effect

pyright 2013 Published by Elsevier B.V. All rights reserved.

J. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 91 (2014) 24 31 25

2.2. Preparation of standard solutions

Standard stock solutions of glucose, galactose, fructose, sac-charose and lactose were prepared approximately at 20 mg/mL indeionized wfor 1 monthappropriateacetonitrileuse.

2.3. Instrum

Carbohygraphic uniJapan), whicFCV nano an SIL-20A differentialto a compaKyoto, Japa

A VenusAgela, Chin5 m, Agel(4.6 mm 2umn (4.6 m

2.4. One-di

2.4.1. Samp2.0 g of m

4.0 mL of dultra-sonicasium ferrocand 1 mL acand then coat room tem10 min. Thehydrates wv/v) and lInternation

2.4.2. HPLCThe sepa

lactose was(4.6 mm 2system 70:The columnThe injectioarea was uswas injecte

2.5. Two-di

2.5.1. Samp2.0 g of p

10 mL buffeAfter 10 mipletely tra8000 rpm/mto 100 mL b0.45 m nyEngland).

2.5.2. HPLCThe LC

conditions

xperimCLC-tting nrichm

detec

, a Vrd c

d the 0.5ichmed towas lumnraturrd mtion,

ults and discussion

timization of sample preparation procedures

sample preparation method employed in the one dimen-liquid chromatography method (LC-RI) is identical to thatusly published [3,5,79]. The sample preparation methodn the two dimensional liquid chromatography method-RI) only consists of sample dissolution and centrifugation,is totally different from that used before. After summarizingratures [23,24], solution I (6 M carbamide, 0.5% n-Octyl--opyranoside) and solution II (6 M carbamide, 0.1 M BisTris,M dl-Dithiothreitol) were tested as the dissolving buffertively in order to dissolve milk powder completely. The

showed there was no signicant difference. Considering thenmental and economic costs, solution I was employed in our

udy of the chromatographic conditions

objective of this work was to develop a two dimensionalchromatography method for analysis of carbohydrates inowder. Prior to LCLC separation, the two dimensions werezed independently.ater. All these solutions were stored at 4 C in glass vials. Mixed standard solutions were prepared by diluting

volumes of the standard stock solution with water: (1:1, v/v). These solutions were prepared at the day of

entation

drate separation was carried out using a chromato-t from Shimadzu Technologies Inc. (Shimadzu, Kyoto,h consisted of two LC-20AB binary gradient pumps, twoow channel selection valves, two DGU-20A3 degassers,auto sampler, an CTO-20A column oven and a RID-10A

refractive index detector. The apparatus was interfacedtible computer using LC solution software (Shimadzu,n).il XBP-C4 analysis column (4.6 mm 100 mm, 5 m,a) with a 1-cm C4 guard column (4.6 mm 10 mm,a, China), a ZORBAX carbohydrate analysis column50 mm, 5 m, Agilent, USA), a Shim-Pack CLC-NH2 col-m 150 mm, 5 m, Shimadzu, Japan) were used.

mensional chromatography (LC-RI)

le preparationilk powder was accurately weighed, then mixed with

eionized water (approximately 60 C). After 10 min oftion, 2.0 mL Carrez I solution (500 mM aqueous potas-yanide), 2.0 mL Carrez II (500 mM aqueous zinc acetate)etonitrile were added. The contents were gently mixedmpletely transferred to a centrifuge tube kept for 1 hperature. Then it was centrifuged at 8000 rpm/min for

resulting supernatant containing the extracted carbo-as then diluted to 100 mL by water: acetonitrile (3:7,tered through a 0.45 m nylon membrane (Whatmanal Ltd., Maidstone, England).

separationration of glucose, galactose, fructose, saccharose and

carried out by a ZORBAX carbohydrate analysis column50 mm, 5 m, Agilent, USA), using an isocratic solvent30 (v/v) acetonitrile-water at a ow rate of 1 mL/min.

and the RID-cell temperature were maintained at 40 C.n volume of standard mix solutions were 10 L. Peaked for signals evaluation, and each sample or standardd in triplicate.

mensional liquid chromatography (LCLC-RI)

le preparationowdered milk were accurately weighed and mixed withr (6 M carbamide, 0.5% n-Octyl--d-Glucopyranoside).n of ultra-sonication, the resulting solution was com-nsferred to a centrifuge tube, and centrifuged atin for 10 min. The supernatant obtained was dilutedy water: acetonitrile (2:1, v/v), then ltered through alon membrane (Whatman International Ltd., Maidstone,

separationLC-RI system used is shown in Fig. 1. The elutionused are shown in Table 1. In the two-dimensional

Fig. 1. Egraph (Lheart-cuon the ecolumn,

systemC4 gua1D, anrate ofan enrwas usphase The cotempestandaevalua

3. Res

3.1. Op

Thesional previoused i(LCLCwhich the lited-Gluc19.5 mrespecresultsenvirostudy.

3.2. St

Theliquid milk poptimiental procedures of heart-cutting two-dimensional liquid chromato-RI): (a) 1D separations on the pre-separation column (a C4 column),of fraction containing carbohydrate; (b) stored and pre-concentratedent column (a NH2 column); (c) 2D separations on the nal analysis

tion of carbohydrate.

enusil XBP-C4 analysis column coupled with a 1-cmolumn were used as the pre-separation column in

mobile phase was water:acetonitrile (1:1, v/v), ow mL/min. A Shim-Pack CLC-NH2 column was used asent column. A ZORBAX carbohydrate analysis column

separate the carbohydrate completely, and the mobilewater:acetonitrile (1:2, v/v), ow rate of 0.5 mL/min.

temperature was maintained at 30 C and the RID-celle was maintained at 40 C. The injection volume ofix solutions were 10 L. Peak area was used for signals

and each sample or standard was injected in triplicate.

26 J. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 91 (2014) 24 31

Table 1Elution conditions used in the LCLC-RI method.

Time (min) Valve position Mobile phasea Comments

1D 2D

0.002.80 0 Water: acetonitrile (1:1, v/v) Water: acetonitrile (1:2, v/v)

2.814.00 1 Water: acetonitrile (1:1, v/v) Water acetonitrile (1:2, v/v)

4.0140.00 0 Water: acetonitrile (1:1, v/v) Water: acetonitrile (1:2, v/v)

a The ow rate of mobile phase applied in 1D and 2D is 0.5 mL/min.

3.2.1. Choice of columns with different packing materialsFor the experiment, the differential refractive index detector

(RID-10A) was connected to the outlet of the rst column to investi-gate the chromatographic behavior of each carbohydrate standardrstly. Since the milk powder matrix was complex, the primary sep-aration must be performed in a polar medium to separate all thecarbohydrate contents from the other contaminants in milk powderas soon as possible. We tested on three different columns: a VenusilXBP-C4 analysis column (4.6 mm 100 mm, 5 m, Agela, China), aVenusil XBP-C8 analysis column (4.6 mm 100 mm, 5 m, Agela,China), and a Venusil HILIC analysis column (2.1 mm 100 mm,5 m, Agelathe LCLC-Roffered the the optimizof transferetation in mproblems wchromatogrcolumn. A SShimadzu, bohydrate phigher enricstandard so

3.2.2. ChoicOwing t

select the aseparation ing the signand the moprotein-preconsequentorganic lev

Fig. 2. Chromseparation (C4conditions: ac0.5 mL/min, dwere maintainlactose)

column, themended inof 1:1 (v/v)all carbohyd(from1D tothat the sat

In 2D aZORBAX caacetonitrileglucose, galwhile, a hig

ninghe re 2:1e ve

(R

Choic tranansf

carb, thent traimila

min

Impa temn thid pln anahasinalyanalymenper

he oe frainan

colto 2Dluterboh, China). In order to shorten the whole analysis time ofI method, we choose the C4 column in 1D analysis, as itretention time of each carbohydrate within 4 min withed mobile phase. Meanwhile, thanks to the short coursence, other contaminants interfered the nal quanti-ilk powder could not be transferred to 2D. Therefore,ith matrix effect can be avoided. Fig. 2A shows theams of each carbohydrate standard solution on the C4him-Pack CLC-NH2 column (4.6 mm 150 mm, 5 m,Japan) was recommended for the enrichment of car-rior to the second dimensional analysis, as it offeredhment ratios. The chromatograms of each carbohydratelution on the NH2 column are illustrated in Fig. 2B.

e of mobile phaseo the complexity of milk powder, it is a challenge toppropriate 1D mobile phase to achieve the preliminaryof carbohydrates from other contaminants. Consider-icant amounts of protein contained in milk powder

bile phase with higher initial organic level may result incipitation, the water/acetonitrile (1:1, v/v) was testedly. As the use of the mobile phase with higher initialel can shorten the analytical time on a reserve phase

broademeet tlast, thand tholution

3.2.3. The

lytes trof eachFig. 2Adifferetimes s2.84.0

3.2.4. The

used imethoshortebe empdrate aof all requirethe tem

In ttem, thcontamsecondtion inwere eBAX caatograms of each individual carbohydrate standard on the pre-) column (A) and the enrichment (NH2) column (B). Chromatographyetonitrile:water in a 50:50 ratio as mobile phase at a ow rate ofetected by a RI detector. The column and the RID-cell temperatureed at 40 C. (1, fructose; 2, glucose; 3, galactose; 4, saccharose; 5,

chromatogrLCLC-RI m

3.3. Method

Both thetion of carbdetection (LSeparate carbohydrates from the other contaminants in milkpowder sample. The ow path was illustrated in Fig. 1(a) (red line).Collection of the carbohydrates separated from milk powdersample. The ow path was illustrated in Fig. 1(b) (red line).Wash the carbohydrates loaded on the NH2 column, separate themcompletely. The ow path was illustrated in Fig. 1(c) (red line).

1:1 (v/v) acetonitrilewater mobile phase was recom-1D analysis. Moreover, as shown in Fig. 2A, with the use

acetonitrilewater mobile phase, the retention time ofrates in 1D was about 3 min. Since the transference time

2D) starting from 2.8 min to 4.0 min was so concentratedised results of recovery were obtained.nalysis, when the separation was carried out by arbohydrate analysis column using the 70:30 (v/v)water mobile phase, the retention time of fructose,actose and saccharose were all more than 25 min. Mean-her organic level mobile phase in 2D may yield peak, and the poor resolution of related substances cannotequirement of denite quantitative determination. At

(v/v) acetonitrilewater mobile phase was employed analytes could baseline separated and had a good res-

2.4) under the chromatographic conditions.

e of transference time from 1D to 2Dsference time obviously controls the amounts of ana-erred from 1D to 2D. As the chromatographic behaviorsohydrate on the pre-separation column (C4) showed in

retention time of all analytes in 1D were about 3 min,nsference times starting from 2.8 min and ending withr to peak width were tested respectively. Consequently,

was used in our study.

ct of temperatureperature of the ZORBAX carbohydrate analysis columns two-dimensional liquid chromatography (LCLC-RI)ayed an important role: a higher temperature canlysis time of this two dimensional LC system. It shouldzed that when the temperature of the ZORBAX carbohy-sis column maintained higher than 35 C, the resolutiontes was less than 1.0. Obviously, it cannot meet thet of qualitative and quantitative research. As a result,ature of 30 C was chosen.ptimized heart-cutting two dimensional HPLC sys-ction containing carbohydrate separated from the otherts on 1D (a C4 column) were pre-concentrated on the

umn (a NH2 column) prior to subsequent introduc-. After the collection of the carbohydrate, all analytes

d by 2D mobile phase, and then separated on a ZOR-ydrate analysis column. Fig. 3 shows the representative

ams of the mixed standard solution obtained by theethod.

validation

two methods used for the detection and quantica-ohydrate were validated in terms of linearity, limits ofOD), intra- and inter-day precision, accuracy.


Table 2Calibration curve and LOD of sugars (n = 3).

Compounds Analysis methods Matrix Regression equationa (Y = ax b) Regression coefcient Linear range (g/mL) LODb

Fructose LC-RI Standard Y = 315.13x + 21 0.9999 879800 15Milk powder Y = 309.81x 28 0.9989 8910060 1.7

LCLC-RI Standard Y = 310.62x 17 0.9994 879800 25Milk powder Y = 298.86x + 31 0.9991 8910060 2.1

Glucose LC-RI Standard Y = 236.36x 39 0.9999 889900 29Milk powder Y = 223.24x + 36 0.9998 899960 1.6


Galactose LC-RI Standard Y = 241.69x + 59 1.0000 9210300 43Milk powder Y = 219.06x 77 0.9988 9010100 2.9

LCLC-RI Standard Y = 210.91x 63 0.9999 9210300 69Milk powder Y = 193.44x 93 0.9989 9010100 4.1

Saccharose LC-RI Standard Y = 340.03x + 16 0.9999 9510700 22Milk powder Y = 339.27x 25 0.9980 889940 1.7


Lactose LC-RI Standard Y = 311.92x + 55 0.9997 859600 30Milk powder Y = 300.37x + 76 0.9990 889840 2.0


a Y is the average peak area of each analyte (n = 3), x is the mass concentration of the analyte in g/mL.b LOD in standard solutions (in g/mL), in milk powder (in mg/g).

3.3.1. Linearity, limits of detection and quantitationFor linearity, the calibration functions were calculated via

least-square regression. Two sets of calibration curves called,respectively, standard and matrix matched were performed.Standard solutions were prepared by dilution of the workingstandard stose were 987 g/mL; f880 g/mL 6867 g/mLwere 10,7095 g/mL; f853 g/mL

Matrix the mixed

matrices were prepared by mixing and homogenization of 2.0 gof each individual sample completely. The spiked concentrationfor fructose was 503 mg/g; for glucose was 498 mg/g, for galactosewas 505 mg/g, for saccharose was 497 mg/g and for lactose was492 mg/g. Further pretreatment of the spiked pooled samples

arrie samn, there 1mL; f/mL g/m9940mL; f/mL

Fig. 3. The chperformed on olutions. The ve levels of concentration for fruc-800 g/mL, 6533 g/mL, 4356 g/mL, 871 g/mL andor glucose were 9900 g/mL, 6600 g/mL, 4400 g/mL,and 88 g/mL; for galactose were 10,300 g/mL,, 4578 g/mL, 916 g/mL and 92 g/mL; for sacchrose0 g/mL, 7133 g/mL, 4756 g/mL, 951 g/mL andor lactose were 9600 g/mL, 6400 g/mL, 4267 g/mL,and 85 g/mL.matched solutions were prepared by addition ofstandards to pooled powdered milk. The pooled

were cpart ofdilutiotose w89 g/885 g6733 were 88 g/875 gromatograms of ve targeted analytes obtained by theheart-cutting two-dimensionthe C4 column (A); after the transference of ve carbohydrate contents between 2.8 andd out according to the procedures described in the eachple preparation section respectively. After a series ofe ve levels of concentration of spiked samples for fruc-0,060 g/mL, 6707 g/mL, 4471 g/mL, 894 g/mL andor glucose were 9960 g/mL, 6640 g/mL, 4427 g/mL,and 89 g/mL; for galactose were 10,100 g/mL,

L, 4489 g/mL, 898 g/mL and 90 g/mL; for sacchrose g/mL, 6627 g/mL, 4418 g/mL, 884 g/mL andor lactose were 9840 g/mL, 6560 g/mL, 4373 g/mL,and 88 g/mL. The analyte peak area versus analyteal liquid chromatography method (LCLC-RI). The 1D analysis was 4.0 min, they were separated in the 2D analysis section (B).


Fig. 4. Chromatograms of (A) mixed standard solution, (B) the pooled milk pow-der sample, and (C) the spiked pooled blank milk powder sample (20 mg/g) on thetwo-dimensional system (LCLC-RI). Chromatography conditions: see Table 1. Thecolumn temperature was maintained at 30 C and the RID-cell temperature wasmaintained at 40 C. (1, fructose; 2, glucose; 3, galactose; 4, saccharose; 5, lactose)

concentratithe calibrapooled samof the analymentioned rose in the regression eLODs are su

3.3.2. AccurAccuracy

from the spregression were addedprecision wthe three ccision was econsecutive

Fig. 5. Chromatograms of (A) mixed standard solution, (B) the pooled milk powdersample, and (C) the spiked pooled blank milk powder sample (20 mg/g) on the one-dimensional system (LC-RI). Chromatography conditions: a ZORBAX carbohydrateanalysis column (4.6 mm 250 mm, 5 m, Agilent, USA) with acetonitrile:water ina 70:30 ratio as mobile phase at a ow rate of 1 mL/min, detected by a RI detector.The column and the RID-cell temperature was maintained at 40 C. (1, fructose; 2,glucose; 3, galactose; 4, saccharose; 5, lactose)

mparison of the LC-RI and LCLC-RI methods

4 sh stand

undraphblanions

are e allits o

cy ofRI mere 2se, 2

nvenak w

the103.7n th

Table 3Accuracy of th

Compounds

Fructose

Glucose

Galactose

Saccharose

Lactose on in spiked samples were plotted in order to generatetion curves. Detection limits were calculated in theple matrices using progressively lower concentrationstes at a signal/noise ratio of 3/1 (S/N = 3). It should bethat there exist known amounts of lactose and saccha-pooled milk powder matrix. The results including thequations, the linear ranges, regression coefcients andmmarized in Table 2.

acy and precision of the method was expressed in terms of carbohydrates recoveryiked pooled samples calculated by the matrix-matchedlines. The standards at low, medium, and high levels

to the pooled milk powder matrices. The intra-dayas assessed by performing six repeated injections of

oncentration levels during the day. The inter-day pre-stablished by analyzing the pooled sample during ve

days. The results obtained are shown in Table 3.

3.4. Co

Fig.mixedmatrixmatogof the condit(LC-RI)

Oncity, limaccuraLCLC-that wgalactothe cothe pethat of(94.5ter thae LC-RI and LCLC-RI methods.

Added concentration (mg/g) LC-RI

Intra-day (n = 6) Inter-day (n = 5)

Recovery% RSD% Recovery%

21 93.6 4.2 90.1 206 88.5 1.7 87.7 503 94.2 1.5 91.4

20 87.7 3.6 92.3 209 90.4 4.9 91.5 498 89.0 1.4 93.9

19 84.8 4.9 82.0 198 89.7 3.1 87.5 505 90.4 2.2 91.8

22 105.5 3.1 105.4 200 92.9 2.7 94.3 497 104.6 2.0 104.9

23 87.7 3.5 88.0 205 94.4 2.2 91.9 492 107.4 2.7 100.8 ows the representative chromatograms obtained for theards solution, the blank and spiked pooled milk powderer the conditions of the two dimensional liquid chro-y method (LCLC-RI). Representative chromatogramsk and a spiked pooled milk powder matrix under theof the one dimensional liquid chromatography methodillustrated in Fig. 5.

operational conditions have been optimized, linear-f detection (LOD), intra-day and inter-day precision,

the two methods were determined and compared. Theethod has a little higher LOD (in the milk powder matrix,.1 mg/g for fructose, 2.6 mg/g for glucose, 4.1 mg/g for.3 mg/g for saccharose and 3.6 mg/g for lactose) thantional one dimensional method. The main reason wasidth obtained by the LCLC-RI method is broader than

LC-RI method. The recovery of the LCLC-RI method% for intra-day and 92.9103.9% for inter-day) was bet-e LC-RI method mainly because of the simple sampleLCLC-RI

Intra-day (n = 6) Inter-day (n = 5)

RSD% Recovery% RSD% Recovery% RSD%

3.9 95.2 4.8 94.9 4.92.3 96.5 4.0 95.0 4.42.0 98.3 3.2 97.9 4.0

3.7 101.9 3.9 99.3 3.53.6 101.0 3.3 102.4 4.12.2 99.7 2.9 102.8 3.3

4.4 103.7 3.4 99.0 4.74.8 97.5 3.1 92.9 3.35.2 98.3 3.1 97.2 3.8

4.8 96.6 4.8 94.7 4.14.7 102.7 2.6 103.9 3.23.9 100.4 2.0 102.5 3.5

5.7 94.5 3.9 94.5 4.35.0 97.0 2.7 96.0 3.94.4 101.9 2.3 99.8 3.3


Fig. 6. Chromsystem (LCLCgalactose; 4, s

preparationRI method.

3.5. Analysi

The twoRI) was appatograms of (A) whole milk powder 6, (B) skimmed milk powder 3, (C) infant formula -RI). Chromatography conditions: see Table 1. The column temperature was maintaineaccharose; 5, lactose)

steps without sample precipitation used in the LCLC-

s of real samples

dimensional liquid chromatography method (LCLC-lied to a variety of real milk powder samples, including

seven wholformula milocal storescommerciachromatogrin Fig. 6. Insaccharose milk powder 4, and (D) soy milk powder 3 on the two-dimensionald at 30 C and the RID-cell temperature was maintained at 40 C. (3,

e milk powders, ve skimmed milk powders, ve infantlk powders and three soy milk powders available from. Table 4 summarizes the content of carbohydrates inl milk powders discussed above. The representativeams of four types of commercial samples are showed

the various kinds of milk powders, glucose, galactose,and lactose were detected, respectively. The RSDs


Table 4Contents of carbohydrates determined in commercial milk powder by LCLC-RI(n = 3).

Commercial milk powder Carbohydrates Concentrationa RSD (%)

Whole milk Whole milk

Whole milk

Whole milk

Whole milk

Whole milk

Whole milk

Skimmed mSkimmed m

Skimmed m

Skimmed m

Skimmed m

Infant formuInfant formu

Infant formu

Infant formu

Infant formu

Soy milk pow

Soy milk pow

Soy milk pow

a Concentrameans of tripl

of the quatriplicate mpowder samthat the mproducts.

4. Conclus

An efcneously dedeveloped matographyprocess comous. First omilk powdsecondly, itthe loss ofcomplicatedsis protoco35 min, so

faster and more accurate. Especially when the sample is complex,the new LCLC-RI method is much more convenient and practi-cal.

wled

s wo of Cch Fu

nces

uyama deri758. Fulgocult t

he Nat(2011. Indytograp580. Cata

deteperomChvpowder 1 Lactose 498 1.9powder 2 Saccharose 165 2.3

Lactose 350 2.4

powder 3 Saccharose 211 2.9Lactose 308 3.3

powder 4 Glucose 56 4.1Galactose 119 4.0

powder 5 Glucose 23 4.9Lactose 392 2.5

powder 6 Galactose 71 4.4Saccharose 240 3.6Lactose 133 3.8

powder 7 Galactose 25 4.7Saccharose 131 3.1Lactose 232 3.0

ilk powder 1 Lactose 337 1.8ilk powder 2 Glucose 146 4.2

ilk powder 3 Saccharose 141 4.1Lactose 259 3.8

ilk powder 4 Saccharose 162 3.5

Ackno

ThidationResear

Refere

[1] K. Sure585

[2] V.Ldifof t31

[3] H.Ema575

[4] T.Rtiveam

[5] J.L.

Lactose 183 3.6

ilk powder 5 Galactose 37 4.7Saccharose 197 3.8Lactose 366 2.2

la milk powder 1 Lactose 298 3.5la milk powder 2 Galactose 115 3.4

la milk powder 3 Saccharose 66 4.5Lactose 404 2.3

la milk powder 4 Galactose 23 4.7Lactose 59 4.2

la milk powder 5 Saccharose 110 3.3Galactose 47 3.1Lactose 483 2.0

der 1 Saccharose 148 2.5Lactose 316 1.8



tion unit for milk powder: mg/g. The values of the concentration areicate determinations.

ntitative results were in the range of 1.84.9% witheasurements. It was rather strange that three milkple contained some glucose. The possible reason was

anufacturer intentionally added these sugars to their

ions

ient and simple analytical method for the simulta-termination of carbohydrates in milk powder wasin this paper. The new two dimensional liquid chro-

method (LCLC-RI) was a thoroughly automatedpleted in less than 35 min. Its advantages are numer-f all, this method only required the dissolution ofer samples in buffer, which was easy and efcient;

offered better recovery of each carbohydrate since analytes can be avoided without the tedious and

sample preparation procedures; thirdly, the analy-l was performed on an automated instrument withinthe analysis rate is high. Therefore, it is simpler,

disaccharmatograp211215

[6] W. Xinmcosaminechromato

[7] R. SharmHPLC andJ. Dairy T

[8] R. Schustphy methlactulose

[9] P. Manzi,Food Biop

[10] P.W. Carrsive mul(Eds.), Apress.

[11] K.M. Kalioff-line aphy reveTheoretic

[12] E. DavydodifferentA 1271 (2

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The analysis of carbohydrates in milk powder by a new heart-cutting two-dimensional liquid chromatography method1 Introduction2 Experimental2.1 Chemicals, samples and solvents2.2 Preparation of standard solutions2.3 Instrumentation2.4 One-dimensional chromatography (LC-RI)2.4.1 Sample preparation2.4.2 HPLC separation

2.5 Two-dimensional liquid chromatography (LCLC-RI)2.5.1 Sample preparation2.5.2 HPLC separation

3 Results and discussion3.1 Optimization of sample preparation procedures3.2 Study of the chromatographic conditions3.2.1 Choice of columns with different packing materials3.2.2 Choice of mobile phase3.2.3 Choice of transference time from 1D to 2D3.2.4 Impact of temperature

3.3 Method validation3.3.1 Linearity, limits of detection and quantitation3.3.2 Accuracy and precision of the method

3.4 Comparison of the LC-RI and LCLC-RI methods3.5 Analysis of real samples

4 ConclusionsAcknowledgementsReferences

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