(396366426) Ciulu 2011 RP-HPLC Determination of Water-soluble Vitamins in Honey

8/10/2019 (396366426) Ciulu 2011 RP-HPLC Determination of Water-soluble Vitamins in Honey

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.$ Ciulu et al$ % Talanta 83 (2011) 924929.$ Ciulu et al$ % Talanta 83 (2011) 924929

Table 1

2$ G@erimental

2$1$ Samles

The 28 hone- samles use+ in this stu+- #ere o, the ,ollo#

in! botanical t-es" ; ,rom eucal-tus (Gucal-tus calma+ulensis

Denhn)/ 3 ,rom citrus (Citrus s$)/ 3 ,rom asho+el (3sho+elusmicrocarus Salm$)/ 3 ,rom sulla (e+-sarium coronarium *$)/ 3

,rom thistle (5alactites tomentosa .oench)/ 3 ,rom stra#berr-tree

(3rbutus une+o *$)/ 2 ,rom laven+er (*avan+ula s$)/ 2 ,rom acacia

(&obinia seu+acacia *$)/ an+ the last ,our ,rom lin+en (Tilia s$)/

heather (Grica arborea *$)/ rosemar- (&osmarinus o,Acinalis *$) an+

multiBoral/ resectivel-$ G@cet ,or lin+en an+ acacia hone-s (all

,rom northern tal-)/ the remainin! samles #ere obtaine+ ,rom

local beeEeeers (Sar+inia/ tal-)$ The attribution o, the botanical

ori!in ,or uniBoral hone- #as conArme+ b- melissoalinolo!ical

anal-sis L14M$ ll samles #ere store+ at 4 C ,rom the timeimme

+iatel- ,ollo#in! their e@traction ,rom the hone-comb to the time

o, their anal-sis$ 'rior to each anal-tical +etermination/ each

samle #as homo!enie+ ,or 1; min #ith an 6ltraturra@ mi@er

mo+$ T18 (=/ Stau,en/ 5erman-)$

2$2$ Chemicals an+ rea!ents

ll rea!ents #ere anal-tical !ra+e$ &iboBavin/ +antothenic

aci+ hemicalcium salt/ ,olic aci+/ nicotinic aci+ an+ triBuoroacetic

aci+ #ere urchase+ ,rom Si!ma l+rich (.ilan/ tal-)$ l3scorbic

aci+ #as obtaine+ ,rom *ancaster (Gast!ate >hite *un+/ .ore

cambe/ Gn!lan+)/ so+ium +ih-+ro!en hoshate an+ so+ium

h-+ro@i+e #ere obtaine+ ,rom Carlo Grba (.ilan/ tal-)$ cetoni

trile ()'*C !ra+e) #as urchase+ ,rom &ie+el +e aen (.ilan/

0tal-)$ orEstation So,t#are

('erEinGlmer/ .ilan/ 0tal-)$ Gach samle #as reare+ an+ injecte+

in trilicate$

2$4$ Stan+ar+ rearation

The stocE stan+ar+ solution #as reare+ b- #ei!hin! in a

100 m* volumetric BasE/ 10$0 m! o, vitamin ?2C 2;$0 m! o, vita

min ?; C 10$0 m! o, vitamin ?9 an+ b- a++in! 40 m* o, #ater$

Then/

4 m* o, a


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.$ Ciulu et al$ % Talanta 83 (2011) 924929.$ Ciulu et al$ % Talanta 83 (2011) 924929

Table 1

mo+el N922 Alter ,rom 3lltech/ Se+riano/ tal-$ lso/ the hone- solution #as store+ in the +arE at 4 Cuntil injection$

2$N$ &')'*C anal-sis

Chromato!rahic searation #as accomlishe+b- otimiin!

(in terms o,both ro!ram o, elution !ra+ient an+ sensitivit-) the

metho+ +escribe+ b- eu+i et al$ L1;M reviousl- roose+ ,or

mea sures in ol-vitamin remi@es$ urther +etails on theassessment an+ e@erimental con+itions o, the chromato!rahic

metho+ are reorte+ in Section 3$1 (otimiation o,

chromato!rahic metho+)$

3$ &esults an+ +iscussion

3$1$ ?otanical ori!in o, hone- samles

The ualitative melissoalinolo!ical anal-sis #as alie+ to

+etermine the botanical ori!in o, hone- samles$ n +etail/ the

; samles o, eucal-tus hone- #ere characterie+ b- overre

resente+ ollen/ reachin! more than 90Q ,or Gucal-tus t-e/

accomanie+ b- other rare ollens such as Gchium/ &ubus/

Tri,olium retense/ Tri,olium reens/ Cistus/ e+-sarum an+Cruci,erae$The 3 samles o, citrus hone- sho#e+ a relativel- lo#

an+ variable er centa!e o, Citrus ollen/ #ith the most

si!niAcant contaminants bein! Car+uus/ Gchium an+ Tri,olium$

sho+el hone- samles #ere also characterie+ b-

un+erreresente+ ollen/ havin! lar!e/ o,ten isolate+/ !rains an+

a sectrum mainl- comose+ o, srin! Bo#er in! secies"

?rassica/ Cistus/ Grica/ *avan+ula/ 'isticia/ 'runus/ 3cacia/ 5alactites

an+ Tri,olium$ The sectra o, sulla samles sho#e+ more than ;0Q

e+-sarum ollen/ #ith other contaminant ollens such as

Gucal-tus/ Cistus/ Tri,olium/ Gchium/ Cruci,erae$ 5$ tomentosa is the

Arst source o, thistle hone-s #ith aollen content var-in! ,rom ;

to

2;Qollen sectra inclu+e+ various other secies such as 3cacia/

?ora!o/ ?rassica/ Cistus/ Citrus/ Gchium/ Gucal-tus/ e+-sarum/ *otus/Tri,olium$ Stra#berr-tree hone-s #ere characterie+ b- un+er

reresente+ ollen o, rbutus (,rom about 8 to 20Q) moreover/ in

a++ition to ,all or #inter Bo#erin! lant secies (e$!$ Smila@/ e+

era/ 3sara!us/ nula/ &osmarinus an+ &hamnus)/ ollen ,rom

srin! an+ summer Bo#ers #as ,oun+Iarticularl- Citrus an+

Gucal-tus $ The 2 laven+er samles sho#e+ un+erreresente+

ollen o, *avan +ula in their se+iment #ith ercenta!es ,rom N to

10Q/ as reorte+ in literature L1NM$ Gven ,or rosemar-/ heather/

acacia an+ lin+en hone- samles the ollen sectra correson+ to

those reorte+ in the sche+ules o, talian monoBoral hone-s L1NM/

#hile Grica ollen is +ominant in heather hone-s (more than

4;Q)$ cacia an+ lin+en samles also correson+ to the stan+ar+s

reorte+ in the sche+ ules o, talian monoBoral hone-s L1NM/

#hile the multiBoral one is a t-ical srin! Sar+inian ro+uct

comose+ o, various melli,erous secies as *avan+ula/ 3sho+elus/

Gchium/ Tri,olium/ Car+uus/ Cistus an+ &osmarinus L1M$

3$2$


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Time (min) Solvent (T aueous

solution/ 0$02;Q/ v%v) (Q)

Solvent ?

(acetonitrile) (Q)

0a 100 0

;a 100 0

11a

; 2;

11b

; 2;

19b ;; 4;

20b

N0 40

22b 100 0

Glution ro!ram in the &''*C !ra+ient elution o, #atersoluble vitamins inhone-$

lo# rate" 1$0 m* min1

$

aS (i$e$ vitamin ?1 / thiamine

vitamin ?N / -ri+o@ine vitamin ?8 / biotine vitamin ?12 /

c-anocobalamine)" ,or this reason our e,,orts #ere +irecte+ to the

otimiation o, ana l-tical resonses o, the remainin! >S (i$e$

vitamin ?2 / riboBavin vitamin ?3 / nicotinic aci+/ vitamin ?; /

antothenic aci+/ vitamin ?9 / ,olic aci+ an+ vitamin C/ ascorbic

aci+)$

The best sensitivit- comromise ,or all anal-tes #as reache+

usin! t#o +i,,erent oerative #avelen!ths (2;4 nm ,or ?3 an+ C

vitamins/ an+ 210 nm ,or ?2/ ?; an+ ?9 vitamins)$ The chromato

!rahic searation #as er,orme+ at a Bo# rate o, 1$0 m* min1$

The metho+ roose+ is rai+" all anal-tes #ere comletel-

elute+ #ithin 1 min an+ the #hole chromato!rahic run #as

comlete+ in 22 min$ i!$ 1 sho#s the chromato!rams obtaine+

b- anal-in! a mi@ture o, stan+ar+s (i!$ 1a)/ an+ an authentic

hone- samle (i!$ 1b)$

3$3$ >atersoluble vitamins in hone-

The samle anal-tes #ere i+entiAe+ b- comarison to the

reten tion times o, >S solutions an+ the attribution #as

conArme+ b- siEin! the eaE in the real samle #ith a

stan+ar+ solution containin! Eno#n amounts o, ure vitamin$

To +etermine the retention times the re,erence stan+ar+s #ere

injecte+ both in+i vi+uall- an+ as a mi@ture$ RuantiAcation

#as er,orme+ b- the e@ternal stan+ar+ metho+ base+ on eaE

areas o, the elute+ >S8$ or each vitamin/ ,our concentration

levels insi+e the relevant lin earit- interval #ere chosen$

Table 2 reorts the amount o, the >S in all hone- samles

anal-e+/ an+ Table 3 reorts the avera!e >S concentration ,or

hone-s o, +i,,erent botanical ori!in$

urther/ our +ata conArme+ that hone- is not a vitaminrich

,oo+" the hi!hest avera!e value o, the sum o, the concentrationso,

the Ave anal-tes is less than 3N m! E!1 (observe+ ,or Citrus hon

e-s)/ an+ the total amount o, >S ,oun+ in the onl- samle o,

&osemar- hone- anal-e+ is even less than 4$8 m! E!1 $

Concernin! the abun+ance o, each anal-te/ vitamin C #as

uantiAe+ in almost all hone-s onl- in t#o samles (o, lin+en

an+ multiBoral ori!in) its concentration #as lo#er than the *oD$

Desite its ervasiveness/ vitamin C seems not to be abun+ant

in hone-" the hi!hest concentration observe+ in our stu+- #as

;$8 0$; m! E!1 in a eucal-tus hone-/ an+ the avera!e concen

tration ,or uniBoral hone-s ran!es ,rom 1$3 0$8 m! E!1 ,or sullahone- to 3$2 0$ m! E!

1 ,or eucal-tus hone-$

The resence o, the vitamin ?3 #as +etecte+ in all the sam

les anal-e+/ even i, in Ave o, them (t#o stra#berr- tree

hone-s/ an+ one each o, eucal-tus/ laven+er an+ rosemar-) the

concentra tion #as un+er its *oR$ 5enerall-/ the concentration o,

vitamin ?3

i!$ 1$ (a) Chromato!ram o, a stan+ar+ mi@ture o, vitamin C (1)/ vitamin ?3 (2)/vitamin ?; (3)/ vitamin ?9 (4) an+ vitamin ?2 (;)/ each at a concentration o, ;0 m!*

1C

(b) chromato!ram o, an eucal-tus hone-/ (1) vitamin C (2) vitamin ?3 C (3)

vitamin

?; C (4) vitamin ?9 C (;) vitamin ?2$

in hone- is hi!her than those measure+ ,or vitamin C$ n articu

lar/ the ran!e o, the concentrations measure+ ,or vitamin ?3 sans

,rom 2$80 0$04 m! E!1

to 2$1 0$3 m! E!1

,or samles o, citrus

an+ eucal-tus ori!ins/ resectivel-$ This observation su!!eststhat

the botanical ori!in o, the hone- inBuences its content o, vitamin

?3 $ lthou!h the lo# number o, available samles ,or the

uniBoral hone-s Eet us ,rom reachin! conclusions o, !eneral

si!niAcance re!ar+in! this issue/ the evi+ent +i,,erences in the

avera!e concen tration o, vitamin ?3 ,or each botanical ori!in

seems to suort this h-othesis$

itamin ?; seems tobe much less common than vitamin C an+?3$ t #as uantiAe+ onl- in 12 hone- samles an+ #as ualita

tivel- i+entiAe+ in a ,urther nine samles$ Ss) in

articular this haene+ ,or the three samles o, asho+el hone-

(avera!e vitamin

?; concentration o, 1N N m! E!1 )$

urthermore/ vitamin ?9 an+ vitamin ?2 #ere uantiAe+ in

about hal, o, the samles/ an+ their resence #as observe+ in

at least ;Q o, the samles ,or the ,ormer an+ N0Q ,or the lat

ter$ or both vitamins the hi!hest concentrations observe+ are

un+er 10 m! E!1 $ Desite this/ vitamin ?9 is the most abun+ant

>S in eucal-tus hone- (Ave samles/ avera!e concentration o,

;$N 0$4 m! E!1 ) #hile meanin!,ul concentrations o, riboBavin

are al#a-s +etectable in asho+el an+ citrus hone-s (amon!three samles/ avera!e concentration o, 3$ 0$3 m! E!1 an+2$2 0$2 m! E!1 / resectivel-)$


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Table 2

>atersoluble vitamins in Sar+inian hone- samles$

Samle >atersoluble vitamin concentration in hone- (m! E!1 SD

a)


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.$ Ciulu et al$ % Talanta 83 (2011) 924929

Table 4


ali+ation +ata$

itamin Sensitivit- *inearit- &eeatabilit- &ero+ucibilit- ?ias

*oD (m! E!1) *oR (m! E!1) Concentration ran!e (m! *1) &2 CQe@/ra

or&atrb

CQe@/&c

or&ate@/&+ &ecover- (QSDe )

?2 0$2; 0$; 0$01100 1 2$N 0$32 19$8 0$98 100 10

?3 0$2; 0$; 0$0;;00 0$9929 N$9 0$N 18 1$0N 99

?; 0$;8 1$; 0$;1000 0$9993 $N 0$91 $N 0$;; 103

?9 0$1; 0$;0 0$0;100 0$9999 2$9 0$3 12$8 1$00 98 8

C 0$10 0$30 0$0;;00 0$9989 $3 0$N 3$3 0$20 104 3

aG@erimental coe,Acient o, variation o, reeatabilit-$

bThe ratio bet#een CQe@/r an+ the theoretical reeatabilit- +ata (C8Q/r) accor+in! to or#itHs theor-$

cG@erimental coe,Acient o, variation o, rero+ucibilit-$

+The ratio bet#een CQe@/& an+ the theoretical rero+ucibilit- +ata (C8Q)/& ) accor+in! to or#itHs theor-$

eStan+ar+ +eviation$

4$3$ 'recision

'recision #as +etermine+ b- means o, reeatabilit- an+ rero

+ucibilit- measures$

&eeatabilit- #as evaluate+ throu!h Ave consecutive reeti

tions o, the anal-sis o, the same hone- samle/ an+ e@resse+ in

terms o, e@erimental variation (C8Qe@/r)$ vera!e reeatabilit-

ran!e+ ,rom 2$NQ (vitamin ?2) to $NQ (vitamin ?;)$

&ero+ucibilit- #as veriAe+ throu!h Ave reetitions o, theanal

-sis o, the same hone- samle/ er,orme+ over +i,,erent

anal-tical sessions/ #ithin 1 month$ t #as e@resse+ b- the

e@erimental variation/ C8Qe@/& $ &ero+ucibilit- values ran!e+

,rom 3$3Q (vita min C) to 18Q (vitamin ?3 )$ 'recision +ata are

reorte+ in Table 4$n or+er to eliminate an- uncertaint- re!ar+in! theaccetabilit-

o, the level o, recision o, the +ata/ a Atness,orurose

evaluation base+ on or#itHs theor- L19M #as use+$ ccor+in! to

this theor-/ it is ossible to calculate a theoretical C8Q (C8Q )

value on the basis o, the concentration level e@ecte+ an+ to

comare it #ith the e@erimental C8Q (C8Qe@

)$ , the ratio

bet#een e@erimental an+ theoretical C8Q (or&at ratio) is less

than 1$; the recision level o, the e@erimental +ata can be

consi+ere+ accetable$ Table 4 sho#s that or&at ratio values ,or

reeatabilit- an+ rero+ucibilit- are al#a-s lo#er than 1$; ,or all

>Ss$ There,ore/ the rero+ucibilit- arameters can be

consi+ere+ accetable$

4$4$ ?ias

Due to the lacE o, certiAe+ re,erence materials an+ in+een

+ent anal-tical metho+s/ recover- tests #ere er,orme+ to checE

the absence o, bias in the roose+ metho+$ ,ter homo!enia

tion/ 40 ! o, a hone- samle #ere +ivi+e+ into ,our 10 ! aliuots$

liuots 2/ 3/ an+ 4 #ere treate+ #ith a suitable an+ increasin!

vol

ume o, a ,reshl- reare+ bulE solution containin! 100 m! *1 o,

#atersoluble vitamins/ #hile no a++ition #as ma+e to aliuot 1$

Gach aliuot un+er#ent all the anal-tical roce+ures reviousl-

+escribe+ an+ #as anal-e+ in trilicate$ The roce+ure al#a-s

ro+uce+ uantitative avera!e recoveries (criteria" t#otail ttest/

P 0$9;)/ as reorte+ in Table 4$


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Table 4


#as success,ull- alie+ to 2; Sar+inian hone- samles o, 10 +i,

,erent botanical ori!ins/ in a++ition to three monoBoral hone-s

(acacia an+ lin+en) ,rom northern tal-$ The amount o, >S ,oun+

in hone- is uite lo#" the overall concentration o, the Ave

anal-tes

never e@cee+e+ 40 m! E!1 an+ o,ten aeare+ to be si!niAcantl-

+een+ent on the botanical ori!in o, the matri@$ itamin C an+

vitamin ?3 #ere resent in almost all the hone- samles$

nterest in!l-/ the concentration o, vitamin C #as !enerall- lo#

(the hi!hest

concentration +etecte+ #as sli!htl- over ; m! E!1 ) an+ invariant

#ith resect the ori!in o, the hone- (robabl- +ue to the a,ore

mentione+ interactions bet#een ascorbic aci+/ !lucose o@i+ase

an+ catalase in hone-)$ n contrast/ the concentration o, vitamin

?3 #as

observe+ to be as hi!h as ca$ 28 m! E!1 an+ it seeme+ to bestron!l-

+een+ent on the botanical ori!in o, the samles$ The uantiA

cation o, vitamins ?;/ ?2 an+ ?9 #as success,ul onl- in ca$ ;0Q

o, the samles anal-e+" #hile vitamin ?; can reach concentra

tions o, some tens o, m! E!1 in hone-s o, selecte+ botanicalori!in

(i$e$ asho+el hone-)/ the concentrations o, vitamins ?2 an+ ?9 areal#a-s belo# 10 m! E!1 $ >e can resume an inBuence o, the ori!in o, the samles on the concentration o, these anal-tes as

#ell/but the lo# number o, samles anal-e+ Eees us ,rom

+ra#in! such a conclusion at this time$ n conclusion/ althou!h

the con centration o, >S in hone- ma- be too lo# to

!enerate interest in the Ael+ o, nutrition/ its otential

correlation to the botanical ori!in o, the samles ma- rove

use,ul to +etermine the ori!in o, hone-s$

&e,erences

L1M G$ Crane/ one-/ Comrehensive Surve-/ >illiam einemann G+$ *t+$/*on+on/

19;$

L2M G$ *esUEov7/ :$ =ubiEov7/ G$ =ov7cUiEov7/ .$ =osUicE7/ :$ 'orubsE7/ =$

olcUiEov7/ :$oo+ Comos$ nal$ 19 (200N) 2;22N$

L3M 5$ =ites/ $ Schuette/ $ Glvehjem/ :$utr$ (1943) 2412;0$

L4M &$$ Castro/ *$C$ ere+o/ .$$$ ere+o/ C$S$T$ +e Samaio/ :$ *i$Chromato!r$

&elate+ Technol$ 24 (2001) 101;1020$

L;M $ 5hel+o,/ V$ >an!/ $:$ Gn!eseth/ :$ !ric$ oo+ Chem$ ;0 (2002)

;80;8$

LNM $ .atei/ S$ ?ir!hila/ S$ Dobrinas/ '$ Caota/ cta Chim$ Slov$ ;1 (2004)

1N91;$

LM '$ inas/ $ ?alsalobre/ C$ *oeGrro/ .$ ernan+eCor+oba/ :$ !ric$ oo+

Chem$ ;2 (2004) 189194$

L8M 5$ Sanna/ .$$ 'ilo/ '$C$ 'iu/ $ Taaro/ &$ Seeber/ nal$ Chim$ cta 1; (2000)

1N;13$

L9M $ Sano/ *$ Casula/ $ 'ananelli/ .$$ 'ilo/ '$C$ 'iu/ &$ Scanu/ $ Taaro/ 5$

Sanna/ Talanta N8 (200N) 1390139;$

L10M &$ Scanu/ $ Sano/ $ 'ananelli/ .$$ 'ilo/ '$C$ 'iu/ 5$ Sanna/ $ Taaro/ :$

Chromato!r$ 1090 (200;) N80$

L11M $ Sano/ $ 'iras/ .$ Ciulu/ $ loris/ $ 'ananelli/ .$$ 'ilo/ '$C$ 'iu/ 5$ Sanna/ :$

orl+ ;9 (198) 1391;$

L1;M


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L1NM *$ 'ersano

(396366426) Ciulu 2011 RP-HPLC Determination of Water-soluble Vitamins in Honey

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