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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
.$ Ciulu et al$ % Talanta 83 (2011) 924929
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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.$ Ciulu et al$ % Talanta 83 (2011) 924929
Table 4
.$ Ciulu et al$ % Talanta 83 (2011) 924929
#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
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