Chemical characterization of aliphatic and indole glucosinolates in leaves of

Brassica napus by near-infrared spectroscopy.

Rafael Font
1*
, Mercedes del Ro-Celestino
1
, Elena Cartea
2
and Antonio de Haro-Bailn
1
1
Department of Aronom! and "lant Breedin# $nstit%te of &%staina'le Aric%lt%re
(C&$C)# Alameda del *'ispo s+n# 1,-.- Crdo'a# &pain#
2
Misin Biolica de /alicia (C&$C)# Apartado 2., E-01-.- "onte2edra, &pain#
*Correspondin a%t3or4
e-mail4 font5cica#es
6l#4 70, 89: ,88211; fa<4 70, 89: ,88292
+2ar+===+apps+con2ersion+tmp+scratc3>0+220:101,0#doc
1
Abstract
63e potential of near-infrared spectroscop! (?$R&) for screenin t3e total l%cosinolate
(t-/&@) content, and also, t3e alip3atic l%cosinolates l%conapin (/?A),
l%co'rassicanapin (/B?), prooitrin ("R*), l%coal!ssin (/A@), and t3e indole
l%co'rassicin (/B&) in t3e leaf rape (Brassica napus @# 2ar# pabularia), =as assessed#
63is crop is ro=n for edi'le lea2es for 'ot3 fodder and 3%man cons%mption# $n /alicia
(nort3=estern &pain) is 3i3l! appreciated for 3%man n%trition and 3a2e t3e common
name of Ana'icolB# A collection of 01 local pop%lations of na'icol =as anal!sed '!
?$R& for l%cosinolate composition# 63e reference 2al%es for l%cosinolates, as t3e!
=ere o'tained '! 3i3 performance liC%id c3romatorap3! on t3e leaf samples, =ere
reressed aainst different spectral transformations '! modified partial least-sC%ares
(M"@&) reression# 63e coefficients of determination in cross-2alidation (r
2
) s3o=n '!
t3e eC%ations for t-/&@, /?A, /B?, "R*, /A@ and /B& =ere, respecti2el!, -#..,
-#:0, -#.1, -#:., -#0: and -#,1# 63e standard de2iation to standard error of cross-
2alidation ratio, =ere for t3ese constit%ents, as follo=s4 t-/&@, 2#81; /?A4 1#8,; /B?4
2#01; "R*4 2#11; /A@4 1#2:, and /B&4 1#28# 63ese res%lts s3o= t3at t3e eC%ations
de2eloped for total l%cosinolates, as =ell as t3ose for l%conapin, l%co'rassicanapin
and prooitrin, can 'e %sed for screenin t3ese compo%nds in t3e lea2es of t3is species#
$n addition, t3e l%coal!ssin and l%co'rassicin eC%ations o'tained, can 'e %sed to
identif! t3ose samples =it3 lo= and 3i3 contents# From t3e st%d! of t3e M"@&
loadins of t3e first t3ree terms of t3e different eC%ations, it can 'e concl%ded t3at some
maDor cell components as protein and cell%lose, 3i3l! participated in modellin t3e
eC%ations for l%cosinolates#
2
Chemical Abstract
63e near-infrared spectr%m of rapeseed na'icol leaf contains information of t3e
c3emical composition of t3is part of t3e plant# C3emometrics are %sed in t3is =orE to
de2elop predicti2e mat3ematical models to meas%re l%cosinolates in lea2es in an
acc%rate, rapid and cost-effecti2e manner#
Keyword Index
Brassica nap%s; Brasicaceae; Rapeseed na'icol leaf; ?ear-infrared spectroscop!
(?$R&); /l%cosinolates
0
1. Introduction
*2er t3e past t3ree decades, Brassica prod%ction 3as increased to 'ecome one of t3e
most important so%rces of oil and protein of plant oriin for 3%man and animal
n%trition, respecti2el!# $n addition, some species of t3e en%s are 3i3l! cons%med as
reen leaf! 2eeta'les all o2er t3e =orld# $n t3e $'erian "enins%la, t3e 3i3
cons%mption of Brassica crops is reflected '! a lare %se of flo=er '%ds and lea2es of
se2eral of t3ese species# Ho=e2er, t3e information a2aila'le on t3e l%cosinolate (F-
t3iol%coside-?-3!dro<!s%lp3ates) (Fi%re 1) composition of t3ese prod%cts is scarce
(Rosa, 188:)#
63e str%ct%ral di2ersit! of l%cosinolates is d%e almost entirel! to t3e different
s%'stit%ents possi'le at t3e side-c3ain position R, =3ic3 can 'e 2er! 2aria'le (Rosa et
al#, 188:)# 63e 3!drol!sis prod%cts of l%cosinolates are of reat concern 'eca%se t3e!
are t3e compo%nds responsi'le for man! of t3e 'eneficial and 3armf%l properties of
l%cosinolate containin plants# Amon t3e 'eneficial %ses of l%cosinolates are t3eir
anti'acterial and antif%nal properties applied to 'iof%miation (An%s et al#, 188,;
Fa3e! et al#, 2--1), and as cancer-c3emopre2ention aents (Rosa et al#, 188:; &3apiro et
al#, 2--1)# $n addition to t3e a'o2e mentioned attri'%tes, l%cosinolates are also t3e
molec%les responsi'le for t3e p%nent and 3ot fla2ors c3aracteristics of t3e seed of
some Brassica crops, and also of t3e odo%rs of t3e fres3 tiss%es t3at act as stim%lants or
deterrents to insects and 3er'i2ores (@o%da and Mole, 1881)#
63e to<ic and anti-n%triti2e effects of l%cosinolates 3a2e limited t3e %se of Brassica
species for 3%man and animal feed (&orensen, 188-)# Amon t3ese neati2e aspects of
l%cosinolates-containin plants are t3e oitroenic effects in animals (Fen=icE et al#,
18.0a)# 63e neati2e effects of l%cosinolates 3a2e 'een t3e 'asis for researc3 taretin
,
lo= l%cosinolate contents in t3e seed of some Brassica crops (Do=ne! and RG''elen,
18.8)#
Brassica crops are =idel! c%lti2ated in /alicia (nort3=estern &pain), in partic%lar t3ose
correspondin to t3e species B. napus @#, B. oleracea @# and B. rapa @# Crops of t3ese
species are %s%all! cons%med as reen leaf! 2eeta'les '! local 3%man pop%lations, '%t
sometimes t3e! are man%fact%red and commercialised, as occ%rs in t3e case of a 2ariet!
of B. rapa Eno=n as HrelosI#
63e form of B. napus c%lti2ated in /alicia correspond to B. napus 2ar# pabularia
(Cartea et al#, 2--,), termed Hna'icolI, =3ose lea2es are cons%med locall! for 3%man
n%trition# 63e lacE of commercial interest for t3is crop t3ro%3 !ears, 3as allo=ed t3e
conser2ation of local pop%lations of t3is form of B. napus# 63is fact is of reat concern,
as t3e! co%ld ser2e as a 'ase ermplasm for f%t%re researc3 in plant 'reedin
prorammes (Rodr%eJ et al#, 2--0), to enlare t3e enetic 2aria'ilit! of t3is species#
C%rrentl!, t3ose st%dies c3aracterisin t3e different 2arieties of Hna'icolI in /alicia are
minimal# 63ere are pre2io%s st%dies on na'icol pop%lations concernin t3eir
morp3oloical and aronomic attri'%tes (Rodr%eJ et al#, 2--0), and t3eir enetic
di2ersit! (Cartea et al#, 2--,)# Ho=e2er, t3is crop 3as not 'een st%died for n%triti2e
2al%e and t3ere is no information a'o%t t3e leaf composition# 63is is especiall! 2alid for
t3e l%cosinolate composition of t3e lea2es, to =3ic3 t3ese compo%nds i2e a
c3aracteristic p%nent taste =3ic3 is 3i3l! appreciated#
$n recent !ears, man! a%t3ors 3a2e foc%sed researc3 in determinin t3e l%cosinolate
composition of seeds, and to a lesser e<tent, of t3e lea2es of Brassica species (Fa3e! et
al#, 2--1)# Ho=e2er, t3e determination of t3e l%cosinolate content '! t3e standard
met3ods is e<pensi2e and time-cons%min, and in addition, specialised personal is
needed# 63e 3i3 cost and la'o%r inp%t reC%ired for o'tainin t3e l%cosinolate content
9
in samples '! 3i3 performance liC%id c3romatorap3! (H"@C) are serio%s 3andicaps
to anal!se lare sets of samples, =3ic3 is %s%all! necessar! to identif! t3e taret
enot!pes in screenin prorams#
$n contrast, t3e %se of fast anal!tical tec3niC%es s%c3 as near-infrared spectroscop!
(?$R&) res%lts in man! ad2antaes, since anal!sis can 'e carried o%t =it3 a considera'le
sa2in of time, at a lo= cost and =it3o%t %sin 3aJardo%s c3emicals (/ottlie' et al#,
2--,)# ?$R& 3as 'een =idel! %sed for decades for C%alitati2e and C%antitati2e anal!sis
in t3e en2ironmental (ClarE et al#, 18.8; Font et al#, 2--,a), and aric%lt%re and food
researc3, in =3ic3 man! a%t3ors 3a2e %sed t3is tec3niC%e for determinin t3e
l%cosinolate content in t3e seed of Brassica species (Biston et al#, 18..; Da%n et al#,
188,; Kelasco and BecEer, 188.; Font et al#, 2--,')# Ho=e2er, till date no st%dies 3a2e
'een reported on t3e %se of t3e ?$R& tec3niC%e to screen l%cosinolates in plant leaf,
=3ere t3ese compo%nds are present in sinificant lo=er concentrations t3an t3ose
%s%all! fo%nd in t3e seed# 63e p%rpose of t3is =orE =as to test t3e potential of ?$R& for
predictin t3e total l%cosinolate (t-/&@) content, as =ell as t3ose maDor l%cosinolates
fo%nd in t3e lea2es of B. napus 2ar# pabularia (6a'le 1)# $n addition, =e pro2ide some
Eno=lede a'o%t t3e mec3anism %sed '! ?$R& for determinin l%cosinolates
s%ccessf%ll! in t3e lea2es of t3is species#
. !esults
2#1# Reference c3emistr! anal!sis of total and indi2id%al l%cosinolates in t3e samples
FreC%enc! distri'%tions of t3e different l%cosinolates in t3e samples %sed in t3is =orE
(Fi%re 2) s3o=ed t3at t3e t-/&@ (Fi%re 2a) and also, t3e indi2id%al alip3atic
l%cosinolates /?A (Fi%re 2'), /B? (Fi%re 2c) and "R* (Fi%re 2d) e<3i'ited
normal distri'%tions in t3eir inter2als# $n contrast, /A@ and t3e indole /B& s3o=ed
sEe=ed distri'%tions =it3 a reater n%m'er of samples =it3 contents close to -#
1
63e ranes, means and standard de2iations of t3e total and indi2id%al l%cosinolates
%sed in t3is st%d! are s%mmarised in 6a'le 2# $ndi2id%al plants e<3i'ited t-/&@
concentrations t3at raned from 1#-1 to ,8#1. Lmol
-1
d=, and a mean 2al%e of 18#.9
Lmol
-1
d=# 63ese concentrations are similar to t3ose contents pre2io%sl! reported in
B. napus (Fen=icE et al#, 18.0a; Fen=icE et al#, 18.0')# /B? =as t3e l%cosinolate t3at
s3o=ed t3e 3i3er mean content of all t3em, representin t3e ,-M of t3e t-/&@,
follo=ed '! "R* and /?A# 63e alip3atic /A@ and t3e indole /B& l%cosinolates
s3o=ed ranes from - to , Lmol
-1
d=, =it3 mean contents aro%nd 1 Lmol
-1
d=#
$t is important to en3ance t3at some samples s3o=ed t-/&@ 2al%es t3at =ere o2er t3e
recommended intaEe le2els for %se in animal feeds (N 0- Lmol
-1
d=) (Bell, 1889)#
2#2 &pectral data pre-treatments and eC%ation performances
63e application of t3e second deri2ati2e and standard normal 2ariate and de-trendin
alorit3ms to t3e ra= spectra (@o 1+R) (Fi%re 0), res%lted in s%'stantial correction
(Fi%re ,) of t3e 'aseline s3ift ca%sed '! differences in particle siJe and pat3 lent3#
"eaEs and tro%3s in Fi%re , correspond to t3e points of ma<im%m c%r2at%re in t3e
ra= spectr%m, and it 3as a tro%3 correspondin to eac3 peaE in t3e oriinal# 63e
increase in t3e comple<it! of t3e deri2ati2e spectra res%lted in a clear separation
'et=een peaEs =3ic3 o2erlap in t3e ra= spectra#
63e %se of t3e mat3ematical approac3 descri'ed a'o2e !ielded t3e eC%ations =it3 t3e
3i3est prediction a'ilities =3en it =as applied o2er t3e near-infrared sement (11---
29-- nm)# 63e 2isi'le sement of t3e spectr%m =as left o%t of t3e cali'rations as it onl!
contri'%ted =it3 noise to t3e M"@& models for l%cosinolate concentrations, a
p3enomenon t3at 3as 'een pre2io%sl! reported for t3e estimation of ot3er components
(/isl%m et al#, 2--,)#
:
Total glucosinolates#- 63e t-/&@ eC%ation s3o=ed a 3i3 coefficient of determination
in t3e cali'ration (R
2
O -#8:) and lo= standard error of cali'ration (&ECO 1#.. Pmol
-1
d=) (6a'le 2)# ?ine terms =ere selected in cross-2alidation as t3e optim%m n%m'er to
model t3e eC%ation# 63e final model for t-/&@ s3o=ed t3e 3i3est r
2
(-#..) and also
&#D# &ECK
-1
ratio (2#81) (Fi# 9a) s3o=n '! an! of t3e eC%ations for indi2id%al
l%cosinolates#
Gluconapin#- 63e eC%ation for /?A s3o=ed a lo=er &EC (-#11 Pmol
-1
d=) and
3i3er R
2
(-#.,) in cali'ration t3an ra= data or first deri2ati2e# $n cross-2alidation, t3e
second deri2ati2e eC%ation =as modelled =it3 9 terms, and also s3o=ed t3e 3i3est r
2
(-#:0) (Fi%re 9') and &#D# &ECK
-1
(1#8,) ratio of t3ose s3o=n '! t3e ot3er
mat3ematical transformations#
Glucobrassicanapin#- 63e second deri2ati2e eC%ation res%lted in a 3i3 R
2
(-#81) and
lo= &EC (1#,: Pmol
-1
d=) in t3e cali'ration for /B? (6a'le 2)# &e2en terms =ere
selected in cross-2alidation as t3e optim%m n%m'er to fit t3e model# 63e r
2
(-#.1) (Fi#
9c) and &#D# &ECK
-1
(2#01) 2al%es o'tained for t3is l%cosinolate, =ere 3i3er t3an
t3ose s3o=n '! t3e eC%ation for /?A#
Progoitrin#- &imilar prediction a'ilit! to t3at s3o=n '! /B? =as e<3i'ited '! t3e
second deri2ati2e eC%ation for "R* (6a'le 2)# As pre2io%sl! occ%rred for t3e ot3er
l%cosinolates, t3e second deri2ati2e eC%ation (2, 9, 9, 2; &?K7D6) performed o2er t3e
infrared sement e<3i'ited t3e 3i3est prediction a'ilit! of t3e different eC%ations for
t3is l%cosinolate# 63is eC%ation s3o=ed coefficients of determination in cali'ration and
cross-2alidation of -#81 and -#:., respecti2el! (Fi%re 9d), and a &#D# &ECK
-1
ratio of
2#11, =3ic3 =as close to t3ose e<3i'ited '! t3e /?A and /B? eC%ations# 63e eC%ation
for "R* =as modelled =it3 ei3t terms#
.
Glucoalyssin and Glucobrassicin.- EC%ations for /A@ and /B& s3o=ed similar
prediction a'ilities, as it can 'e concl%ded from t3e data reported in 6a'le 2# Bot3
eC%ations s3o=ed R
2
2al%es of -#9-, and also similar &#D &ECK
-1
ratios, =3ic3 =ere
close to 1#0-# 63ese ratios =ere t3e lo=est of t3e different l%cosinolates anal!sed, and
also t3e r
2
2al%es, =3ic3 =ere close to -#, (Fi%re 9e and 9f)# Bot3 eC%ations =ere
modelled =it3 0 terms, as 'ein t3e optim%m n%m'er selected in cross-2alidation#
2#0# &econd deri2ati2e spectra of leaf and modified partial least sC%are loadins
Qa2elent3s noted on Fi%re , as 'ein t3ose of most rele2ance in t3e spectra,
correspond to t3ose =a2elent3s of ma<im%m a'sor'ance s3o=n '! t3e a2erae
spectr%m of t3e freeJe-dried leaf# 63e conspic%o%s 'and in t3e 2isi'le reion at 1:, nm
is d%e to electronic transitions in t3e red and 3as 'een assined to a'sorption '!
c3lorop3!ll (6Eac3%E and R%Jina, 18.2)# $n t3e ?$R sement of t3e spectr%m, t3e main
a'sorption 'ands =ere displa!ed at 1822 nm, =3ic3 3as 'een attri'%ted to *-H stretc3
pl%s *-H deformation; 2-91 nm related to ?-H stretc3 of amides; 22:- nm =3ic3 3as
'een assined to *-H pl%s C-C stretc3 ro%ps (*s'orne et al#, 1880) of cell%lose, and at
201- and 20,. nm related to C-H stretc3in and com'ination 'ands of t3e met3!lene
ro%ps (M%rra! and Qilliams, 18.:)# *t3er minor a'sorptions =ere d%e to t3e first
o2ertone of *-H stretc3in (1,0, nm), &-H stretc3 first o2ertone or C-H stretc3 first
o2ertone of CH
0
ro%ps (118, nm), and C-H stretc3in '! met3!lene ro%ps (1:2. and
1:1, nm)#
63ose =a2elent3s correspondin to a'sorptions '! CH
2
stretc3in and com'ination, ?-
H stretc3in '! amides and *-H stretc3+*H deformation 3!dro<!l (M%rra! and
Qilliams, 18.:) infl%enced 3i3l! t3e first t=o factors of t3e t-/&@ eC%ation (Fi%re 1),
=3ic3 in addition =ere t3e factors most correlated to t3e total content# 63e t3ird factor
8
=as mainl! modelled =it3 t3ose =a2elent3s pre2io%sl! %sed in t3e ot3er factors, '%t
=it3 an increase in t3e participation of 3!dro<!l ro%ps#
Hi3 similarities =ere fo%nd amon t3e first t3ree M"@& loadins of t3e different
l%cosinolate eC%ations and t3at for t-/&@ eC%ation# Qa2elent3s %sed in modellin t3e
first t3ree terms of t3e t-/&@ eC%ation =ere s!stematicall! %sed in modellin t3e
different factors for alip3atic, aromatic and indole l%cosinolate eC%ations#
". #iscussion
63e 2alidit! of cross-2alidation to e2al%ate t3e performance of an ?$R eC%ation 3as
'een s%pported '! different researc3ers (&3enE and Qester3a%s, 1881; Qilliams and
&o'erin, 1881), 3a2in 'een applied s%ccessf%ll! '! t3e a%t3ors of t3is =orE to t3e
anal!sis of l%cosinolates in a pre2io%s report (Font et al#, 2--,')# Follo=in t3e
considerations reported '! &3enE and Qester3a%s (1881) a'o%t t3e estimation of t3e
acc%rac! of a cali'ration eC%ation from cross-2alidation, t3e r
2
o'tained for t-/&@, and
also for t3e alip3atic /?A, /B? and "R* l%cosinolates, =as indicati2e of eC%ations
=it3 ood C%antitati2e information# For t3ese components, t3e mat3ematical models
de2eloped e<plained from -#:0 to -#.. M of t3e 2ariance contained in t3e c3emistr!
data (6a'le 2)# $n contrast, /A@ and t3e indole l%cosinolate /B& s3o=ed coefficients
of determination t3at =ere c3aracteristic of eC%ations %sef%l for ood separation of
samples into 3i3 and lo= ro%ps#
63e n%m'er of M"@& terms of t3e different eC%ations selected as optim%m in cross-
2alidation =as for all t3em, in t3e limits recommended to a2oid o2erfittin, i#e#, one
term '! eac3 ten samples in t3e cali'ration file (&3enE and Qester3a%s, 1889)# From
data s3o=n in 6a'le 2, it can 'e concl%ded t3at t3e 2ariance in t3e reference c3emistr!
2al%es e<plained '! t3e different eC%ations, =as related to t3e mean concentration of
eac3 l%cosinolate in t3e samples# 63e acc%rac! of t3e H"@C anal!sis is 3i3l!
1-
dependent on t3e concentration of t3e component in t3e sample, and acc%rac! in t3e
reference anal!sis is essential to settin %p efficient ?$R cali'rations (Qilliams, 18.:)#
63e e<tremel! lo= concentrations e<3i'ited '! /B& and /A@ in t3e leaf samples
=o%ld maEe t3em more prone to errors in o'tainin t3e c3emistr! 2al%es, t3%s
decreasin t3e correlation =it3 t3e spectral information#
Cross-2alidation res%lted in &#D# &ECK
-1
ratios t3at raned from 1#2: (/A@) to 2#81 (t-
/&@) (6a'le 2)# 63e differences s3o=n '! t3ese ratios for t3e different l%cosinolates,
are e<plained '! t3e fact t3at t3e &ECK 2al%e is limited '! t3e deree of correlation
'et=een c3emistr! reference data and ?$R predictions (Qilliams, 18.:)# 63e 3i3er r
2
s3o=n '! t3e t-/&@ eC%ation =it3 respect to t3ose displa!ed '! t3e indi2id%al
l%cosinolates, =o%ld lead to a lo=er &ECK, t3%s increasin t3e 2al%e of t3e ratio# $n
contrast, t3e lo= coefficients of determination displa!ed '! t3ose minor l%cosinolates,
lead to 3i3er &ECKs# $n addition, =3en t3e rane, and t3erefore, t3e 2ariance in
reference data are lo=, t3e 2al%es for r
2
and also t3e &#D# &ECK
-1
ratio, can not 'e 2er!
3i3, =3ic3 is t3e case for t3e /A@ and /B& l%cosinolates in t3e leaf of na'icol#
63ose =a2elent3s 3i3l! participatin in modellin t3e first t3ree terms of t3e
eC%ations for l%cosinolates (Fi%re 1), =ere, to some e<tent, similar to t3ose pre2io%sl!
reported for determinin l%cosinolates in Brassica juncea seed (Font et al#, 2--,')#
D%e to t3e fact t3at l%cosinolates deri2e from nat%ral aminoacids (Ettliner and RDaer,
181.), it is loical to t3inE t3at t3ose feat%res in t3e spectral 'ands related to protein
a'sorption 'e %sed in t3e eC%ations for l%cosinolates# 63is is t3e case of t3e 'and at
2-92 nm, =3ic3 participated mainl! in t3e constr%ction of t3e first and t3ird loadins of
all t3e l%cosinolates eC%ations# *t3er in3erent correlations 'et=een l%cosinolates and
maDor cell components as it is cell%lose infl%enced s%c3 cali'rations, as it is concl%ded
from t3e 'ands at 20-. and 20,. nm# Ho=e2er, p%re l%cosinolates a'sor' also at t3ese
11
=a2elent3s (Font et al#, 2--,'), and t3%s, it 3as not to 'een discarded t3at, to some
e<tent, a specific HfinerprintI for t3e molec%le of l%cosinolate 'e represented in t3e
spectr%m of t3e na'icol leaf#
From t3e data reported in t3is =orE it is concl%ded t3at t-/&@, and also t3ose maDor
l%cosinolates in t3e lea2es of na'icol, as it is /?A, /B? and "R* can 'e predicted
=it3 s%fficient acc%rac! for screenin p%rposes# /A@ and /B& eC%ations can 'e %sed
for a correct separation of t3e samples into lo= and 3i3 ro%ps# After ?$R& screenin,
more acc%rate anal!ses can 'e carried o%t '! H"@C of t3ose samples of interest# 63%s, a
considera'le sa2in of t3e la'o%r inp%t, time and cost of anal!sis for t3ese compo%nds
is ac3ie2ed# 63e de2elopment of t3ese cali'rations =ill allo= researc3ers leadin =it3
plant 'reedin, 'iof%miation or medical applications, to identif! C%icEl! t3ose
indi2id%als of interest in t3e lea2es of na'icol =it3o%t t3e need of doin H"@C anal!sis#
$. %xperimental
,#1 "lant material and crop manaement
63is =orE is 'ased on 119 indi2id%al plants 'elonin to 01 different accessions of t3e
species Brassica napus 2ar# pabularia, =3ic3 are a part of t3e ermplasm 'anE of
Brassica at t3e Misin Biolica of /alicia (MB/) (C&$C, &pain)# 63ese accessions
represent t3e =3ole 2aria'ilit! of t3is 2ariet! of B. napus in its nat%ral croppin area#
63e plant material =as ro=n in t3e !ears 2--2 and 2--0 in "onte2edra (&pain)# &eeds
of eac3 accession =ere so=n in reen3o%se on A%%st, in small pots containin sterile
commercial pottin mi<t%re, %nder controlled conditions of temperat%re, li3t and
irriation# 63irt! da!s after so=in, t3e seedlins =ere transplanted to field# Mineral
fertilisers =ere %sed in all cases (1-9 S of nitroen; 1-9 S of p3osp3or%s; 1-9 S of
potassi%m)#
12
From 0 to 9 mat%re indi2id%al plants of eac3 one of t3e 01 accessions %sed in t3is st%d!
=ere collected for anal!sis d%rin plant ro=t3 at t3e optimal cons%mption stae# For
eac3 plant, 0 or , lea2es =ere froJen Hin sit%I in liC%id ?
2
, to a2oid l%cosinolate
enJ!matic 3!drol!sis '! t3e enJ!me m!rosinase (t3iol%coside l%co3!drolase, E#C#
0#2#0#1) contained in t3e cell 2ac%oles# "lant samples =ere t3en transported to t3e
la'orator!# 63e reen material =ere ro%nd to a po=der in liC%id ?
2
, Eept at -.- TC and
freeJe-dried in a 6elstar freeJe-drier mod# @ioalfa-1, %ntil anal!sis#
,#2# H"@C anal!sis
@ea2es =ere anal!sed '! H"@C at t3e Department of Aronom! and "lant Breedin
(DA"B) at t3e $nstit%te of &%staina'le Aric%lt%re ($A&, C&$C, &pain)# A'o%t 1-- m
dr! =ei3t (d=) of lea2es =as ro%nd in a UanEe and R%nEel, Model A1- mill ($RA-
@a'ortec3niE) for a'o%t 2- s and a t=o-step l%cosinolate e<traction =as carried o%t in a
=ater'at3 at :9 TC to inacti2ate mirosinase# $n t3e first step t3e sample =as 3eated for
19 min in 2#9 m@ :-M aC%eo%s met3anol and 2-- @ 1- mM sinirin (2-propen!l
l%cosinolate) as an internal standard# A second e<traction =as applied after
centrif%ation (9 min, 9<1-
0
) '! %sin 2 m@ of :-M aC%eo%s met3anol# *ne m@ of
t3e com'ined l%cosinolate e<tracts =as pipetted onto t3e top of an ion-e<c3ane
col%mn containin 1 m@ &ep3ade< DEAE-A29 in t3e formate form# Des%lp3ation =as
carried o%t '! t3e addition of :9@ of p%rified s%lp3atase (E#C# 0#1#1#1, t!pe H-1 from
Heli< pomatia) (&ima) sol%tion# Des%lp3ated l%cosinolates =ere el%ted =it3 2#9 m@
(-#9 m@ < 9) Milli-V (Millipore) %ltra-p%re =ater and anal!sed =it3 Model 1-- H"@C
instr%ment (Qaters) eC%ipped =it3 a Model ,.1 SK t%na'le a'sor'ance detector
(Qaters) at a =a2elent3 of 228 nm# &eparation =as carried o%t '! %sin a @ic3rosp3er
1-- R"-1. in @ic3rocart 129-, col%mn, 9 m particle siJe (MercE)# 63e amo%nt of eac3
10
indi2id%al l%cosinolate present in t3e sample =as calc%lated '! mean of t3e internal
standard, and e<pressed as mol
-1
of d=# 63e total l%cosinolate content =as
comp%ted as t3e s%m of all t3e indi2id%al l%cosinolates present in t3e sample#
,#0# ?$R& proced%re4 recordin of spectra and processin of data
All t3e samples =3ic3 =ere pre2io%sl! anal!sed '! H"@C, =ere t3en anal!sed '!
?$R&# ?ear-infrared spectra =ere recorded on an ?$R& spectrometer model 19-- (Foss-
?$R&!stems, $nc#, &il2er &prin, MD, S&A) in reflectance mode eC%ipped =it3 a
transport mod%le# &amples =ere placed in a 0 cm diameter ro%nd cell sample 3older,
and t3eir spectra =ere reistered as an indi2id%al file, in t3e rane from ,-- to 29-- nm,
at 2 nm inter2als#
63e monoc3romator 19-- consists of a t%nsten '%l' and a rapid scannin 3olorap3ic
ratin =it3 detectors positioned for transmission or reflectance meas%rements# 6o
prod%ce a reflectance spectr%m, a ceramic standard is placed in t3e radiant 'eam, and
t3e diff%sel! reflected ener! is meas%red at eac3 =a2elent3# 63e act%al a'sor'ance of
t3e ceramic is 2er! consistent across =a2elent3s# $n t3is =orE, eac3 spectr%m =as
recorded once from eac3 sample, and =as o'tained as an a2erae of 02 scans o2er t3e
sample, pl%s 11 scans o2er t3e standard ceramic 'efore and after scannin t3e sample#
63e ceramic and t3e sample spectra =ere %sed to enerate t3e final @o (1+R) spectr%m#
63e =3ole time of anal!sis tooE a'o%t 2 min per sample, appro<imatel!#
$n t3e second step, t3e cali'ration file =as formed '! addin t3e reference c3emistr!
2al%es for all l%cosinolates, as t3e! =ere o'tained '! H"@C, to t3e file of spectra, t3%s
formin a ne= file, eac3 spectr%m 3a2in an associated 2al%e for eac3 l%cosinolate#
1,
,#,# De2elopin cali'ration eC%ations
Ssin t3e application /@*BA@ 2# 1#9- (Q$?$&$ $$, $nfrasoft $nternational, @@C, "ort
Matilda, "A, S&A), different cali'ration eC%ations for t-/&@, /?A, "R*, /B?, /A@,
and /B& =ere de2eloped on t3e =3ole set (nO 119)# Cali'ration eC%ations =ere
comp%ted %sin t3e ra= optical data (lo 1+R, =3ere R is reflectance), or first or second
deri2ati2es of t3e lo 1+R data, =it3 se2eral com'inations of sement (smoot3in) and
deri2ati2e (ap) siJes Wi#e#, (-, -, 1, 1; deri2ati2e order, sement of t3e deri2ati2e, first
smoot3, second smoot3); (1, ,, ,, 1); (1, 1-, 1-, 1); (2, 9, 9, 2); (2, 2-, 2-, 2)X# 6o
correlate t3e spectral information (ra= optical data or deri2ati2e spectra) and t3e
c3emistr! 2al%es of t3e different l%cosinolates, as t3e! =ere determined '! t3e
reference met3od, modified partial least sC%ares (M"@&) =as %sed as a reression
met3od, '! %sin =a2elent3s from ,-- to 29-- nm e2er! . nm# $n addition, t3e
alorit3ms termed standard normal 2ariate (&?K) and de-trendin (D6) (Barnes et al#,
18.8) =ere %sed to correct 'aseline offset d%e to scatterin effects prod%ced '!
differences in particle siJe and pat3 lent3 2ariation amon samples#
,#9# Kalidation of t3e eC%ations
63e performances of t3e different cali'ration eC%ations o'tained in t3e cali'ration =ere
determined from cross-2alidation# Cross-2alidation is an internal 2alidation met3od

t3at
liEe t3e e<ternal 2alidation approac3 seeEs to 2alidate t3e cali'ration model on
independent test data, '%t it does not =aste data for testin onl!, as occ%rs in e<ternal
2alidation# 63is proced%re is %sef%l 'eca%se all a2aila'le c3emical anal!ses for all
indi2id%als can 'e %sed to determine t3e cali'ration model =it3o%t t3e need to maintain
separate 2alidation and cali'ration sets# 63e met3od is carried o%t '! splittin t3e
cali'ration set into M sements and t3en cali'ratin M times, eac3 time testin a'o%t a
19
(1+M) part of t3e cali'ration set (Martens and ?aes, 18.8)# $n t3is =orE, t3e different
cali'ration eC%ations =ere 2alidated =it3 9 cross-2alidation sements (ro%ps), as t3is
=as t3e optim%m n%m'er of terms a%tomaticall! selected '! t3e soft=are as a f%nction
of t3e n%m'er of samples emplo!ed#
63e prediction a'ilit! of t3e eC%ations o'tained =as determined on t3e 'asis of t3eir
coefficient of determination in t3e cross-2alidation (r
2
) (&3enE and Qester3a%s, 1881)
(eC# 1) and standard de2iation (&#D#) to standard error of cross-2alidation (&ECK) ratio
(Qilliams and &o'erin, 1881) (eC# 2)#
r
2
O
1
1
2
1
2
) ( ) Y (

,
_

,
_

n
i
i
n
i
y y y y eC# 1
=3ere4
yY
O ?$R meas%red 2al%e;
y
O mean H!I 2al%e for all samples;
i
y
O la' reference
2al%e for t3e it3 sample#
&#D# &ECK
-1
O ( )
1
2 + 1
1
1
2
1 ) Y ( # #

1
]
1

,
_

K N y y D S
n
i
i i
eC# 2
=3ere4
i
y
O la' reference 2al%e for t3e it3 sample;
yY
O ?$R meas%red 2al%e; ?O
n%m'er of samples, RO n%m'er of =a2elent3s %sed in an eC%ation; &#D#O standard
de2iation#
63e statistics s3o=n in eC# 1 and eC# 2 i2e a more realistic estimate of t3e applica'ilit!
of ?$R& to t3e anal!sis t3an t3ose of t3e e<ternal 2alidation, as cross-2alidation a2oids
t3e 'ias prod%ced =3en a lo= n%m'er of samples representin t3e f%ll rane are
selected as 2alidation set (&3enE and Qester3a%s, 1881; Qilliams and &o'erin, 1881)#
11
63e &ECK met3od is 'ased on an iterati2e alorit3m =3ic3 selects samples from a
sample set pop%lation to de2elop t3e cali'ration eC%ation and t3en predicts on t3e
remainin %nselected samples# 63is statistic indicates an estimate of t3e standard error
of prediction (&E") t3at ma! 3a2e 'een fo%nd in an e<ternal 2alidation (QorEman,
1882), and as occ%rred =it3 &E" is calc%lated as t3e sC%are root of t3e mean sC%are of
t3e resid%als for ?-1 derees of freedom, =3ere t3e resid%al eC%als t3e act%al min%s t3e
predicted 2al%e#
,#1# &pectr%m of B. napus leaf and modified partial least sC%are reression loadins
63e M"@& loadin plots of t3e first t3ree factors enerated from t3e M"@& reression
(2, 9, 9, 2; &?K7D6) for t-/&@ and also t3e indi2id%al l%cosinolates, =ere o'tained#
M"@& reression constr%cts its factors capt%rin as m%c3 of t3e 2ariation in t3e spectral
data as possi'le '! %sin t3e reference 2al%es acti2el! d%rin t3e decomposition of t3e
spectral data# 63e loadin plots s3o= t3e reression coefficients of eac3 =a2elent3 to
t3e parameter 'ein cali'rated for eac3 factor of t3e eC%ation# Qa2elent3s represented
in t3e loadin plots as more 3i3l! participatin in t3e de2elopment of eac3 factor, are
t3ose of more 2ariation and 'etter correlated to t3e compo%nd in t3e cali'ration set#
63e deri2ati2e spectr%m of t3e B. napus leaf %sed in t3is =orE =as o'tained '!
transformin t3e oriinal a'sor'ance 2al%es of all samples at eac3 =a2elent3 (ra=
optical data from ,-- to 29-- nm, e2er! 2 nm) to t3eir second deri2ati2e# $n addition,
t3e &?K pl%s D6 alorit3ms =ere applied# $n t3e second step, t3e a2erae spectr%m =as
calc%lated# 63e second order deri2ati2e transformation of t3e oriinal spectr%m res%lted
in a spectral pattern displa! of a'sorption peaEs pointin do=n=ard#
63e information i2en '! t3e second deri2ati2e of t3e spectr%m, toet3er =it3 t3at
information s3o=n '! t3e loadins for t3e factors of t3e different eC%ations, =ere %sed
1:
to identif! some of t3e a'sor'ers emplo!ed in modellin t3ese components# $n t3is =orE
=e %sed 'and assinments from literat%re, to relate some maDor a'sorption 'ands in t3e
spectr%m of na'icol lea2es =it3 t3e main =a2elent3s %sed '! M"@& to constr%ct t3e
first t3ree M"@& terms of t3e l%cosinolate eC%ations#
Ac&nowledgements
Qe t3anE /loria FernZndeJ Marn ($A&, C&$C, Crdo'a, &pain) for t3e performance of
t3e H"@C anal!ses# 63is =orE 3as 'een s%pported '! t3e "roDect MC[6 nT A/@ 2--0-
-1011 of t3e &panis3 /o2ernment#
1.
!eferences
An%s, U#F#, /ardner, "#A#, RirEeaard, U#A#, Desmarc3elier, U#M#, 188,#
Biof%miation4 isot3ioc!anates released from Brassica roots in3i'it ro=t3 of t3e taEe-
all f%n%s# "lant &oil 112, 1-:-112#
Barnes, R#U#, D3anoa, M#&#, @ister, &#U#, 18.8# &tandard normal 2ariate transformation
and de-trendin of near-infrared diff%se reflectance spectra# Appl# &pectrosc# ,0, ::2-
:::#
Bell, U#M#, 1889# Meal and '!-prod%cts %tiliJation in animal n%trition# $n4 Rim'er, D#&#,
Mc/reor, D#$#, (Eds#), Brassica *ilseeds# "rod%ction and StiliJation, CAB
$nternational, Qallinford, pp# 0-1-00:
Biston, R#, Dardenne, "#, C=iEo=sEi, M#, Marlier, M#, &e2erin, M#, Qat3elet, U#"#,
18..# Fast anal!sis of rapeseed l%cosinolates '! near infrared reflectance spectroscop!#
U# Am# *il# C3em# &oc# 19, 1988-11--#
Cartea, M#E#, &oenas, "#, "icoaa, A#, *rdZs, A#, 2--, Relations3ips amon Brassica
napus ermplasm from &pain and /reat Britain as determined '! RA"D MarEers# /en#
Res# Crop E2ol%tion Hin pressI#
ClarE, D#H#, Car!, E#E#, Ma!land, H#F#, 18.8# Anal!sis of trace elements in foraes '!
near infrared reflectance spectroscop!# Aron# U# .1, 81-89#
Da%n, U#R#, Clear, R#M#, Qilliams, "#, 188,# Comparison of t3ree =3ole seed near-
infrared anal!Jers for meas%rin C%alit! components of canola seed# U# Am# *il# C3em#
&oc# :1, 1-10-11-.#
18
Do=ne!, R#R#, RG''elen, /#, 18.8# Brassica species# $n4 RG''elen /#, Do=ne!#, R#R#,
As3ri, A#, (Eds#), *il Crops of t3e Qorld, Mc/ra=-Hill, ?e= [orE, pp# 008-
012#
Ettiner, M#/#, RDaer, A#, 181.# &%lp3%r compo%nds in plants# $n4 Ma'r!, 6#U#, Alston,
R#E#, R%necEles, K#C# (Eds#), Recent Ad2ances in "3!toc3emistr!, Kol# 1# Appleton-
Cent%r!-Crofts, ?e= [orE, pp# .8-1,,#
Fa3e!, U#Q#, \alcmann, A#6#, 6alala!, "#, 2--1# 63e c3emical di2ersit! and distri'%tion
of l%cosinolates and isot3ioc!anates amon plants# "3!toc3emistr! 91, 9-91#
Fen=icE, R#/#, Heane!, R#R#, M%llin, Q#U#, 18.0a# /l%cosinolates and t3eir 'reaEdo=n
prod%cts in food and food plants# C#R#C# Critical Re2ie=s in Food &cience and
?%trition 1., 120-2-1#
Fen=icE, R#/#, /riffit3s, ?#M#, Heane!, R#R#, 18.0'# Bitterness in Br%ssels spro%ts
(Brassica oleracea @# 2ar# gemmifera)4 t3e role of l%cosinolates and t3eir 'reaEdo=n
prod%cts# U# &ci# Food Ar# 0,, :0-.-#
Font, R#, Del Ro-Celestino, M#, K]leJ, D#, De Haro-Bailn, A#, Montoro, R#, 2--,a#
Kisi'le and near-infrared spectroscop! as a tec3niC%e for screenin t3e inoranic
arsenic content in t3e red cra!fis3 (Procambarus clarkii /irard)# Anal# C3em# :1, 0.80-
0.8.#
Font, R#, Del Ro, M#, FernZndeJ-MartneJ, U#M#, De Haro, A#, 2--,'# Sse of near-
infrared spectroscop! for screenin t3e indi2id%al and total l%cosinolate content in
$ndian m%stard seed (Brassica juncea @# CJern# ^ Coss#)# U# Ari# Food C3em# 92,
0910-0918#
/isl%m, R#, MicElander, E#, ?ielsen, U#"#, 2--,# V%antification of nitroen
concentration in perennial r!erass and red fesc%e %sin near-infrared reflectance
spectroscop! (?$R&) and c3emometrics# Field Crops Researc3 Hin pressI
2-
/ottlie', D#M#, &c3%ltJ, U#, Br%%n, &#Q#, Uaco'sen, &#, &_nderaard, $#, 2--,#
M%lti2ariate approac3es in plant science# "3!toc3emistr! Hin pressI
@o%da, &#, Mole, &#, 1881# /l%cosinolates4 c3emistr! and ecolo!# $n4 Rosent3al, /#A#,
Berem'a%m, M#R#, (Eds#), Her'i2ores4 63eir $nteractions =it3 &econdar! "lant
Meta'olites, Kol# 1# Academic "ress, ?e= [orE, pp# 120-11,#
Martens, H#, ?aes, 6#, 18.8# M%lti2ariate cali'ration# Uo3n Qile! ^ &ons, ?e= [orE#
M%rra!, $#, Qilliams, "#C#, 18.:# C3emical principles of near-infrared tec3nolo!# $n4
Qilliams, "#, ?orris, R#, (Eds#), ?ear-$nfrared 6ec3nolo! in t3e Aric%lt%ral and Food
$nd%stries, American Association of Cereal C3emists, $nc#, &t# "a%l, pp# 1:-0,#
*s'orne, B#/#, Fearn, 6#, Hindle, "#H#, 1880# "ractical ?$R spectroscop! =it3
applications in food and 'e2erae anal!sis# @onman &cientific ^ 6ec3nical, Esse<#
Rodr%eJ, K#M#, "adilla, /#, Cartea, M#E#, *rdZs, A#, 2--0# E2al%acin de 2ariedades
alleas de na'icol (B. napus 2ar# pabularia) en siem'ra precoJ# Actas de Hortic%lt%ra
08, 12--121#
Rosa, E#, 188:# /l%cosinolates from flo=er '%ds of "ort%%ese Brassica crops#
"3!toc3emistr! ,,, 1,19-1,18#
Rosa, E#A#&#, Heanc!, R#R#, Fen=icE, /#R#, "ortas, C#A#M#, 188:# /l%cosinolates in
crop plants# Hort# Re2# 18, 88-219#
&3apiro, 6#A#, Fa3e!, U#Q#, Qade, R#@#, &tep3enson, R#R#, 6alala!, "#, 2--1#
C3emoprotecti2e l%cosinolates and isot3ioc!anates of Broccoli spro%ts4 meta'olism
and e<cretion in 3%mans# Cancer Epidemiolo!, BiomarEers ^ "re2ention 1-, 9-1-9-.#
&3enE, U#&#, Qester3a%s, M#*#, 1889# Anal!sis of aric%lt%ral and food prod%cts '! near
infrared reflectance spectroscop!# $nfrasoft $nternational, "ort Matilda#
21
&3enE, U#&#, Qester3a%s, M#*#, 1881# Cali'ration t3e $&$ =a!# $n4 Da2ies, A#M#C#,
Qilliams, "#C#, (Eds#), ?ear $nfrared &pectroscop!4 63e F%t%re Qa2es, ?ir
"%'lications, C3ic3ester, pp# 18.-2-2#
&orensen, H#, 188-# /l%cosinolates4 str%ct%re, properties, f%nction# $n4 &3a3idi, F#,
(Ed#), Canola and Rapeseed# "rod%ction, c3emistr!, n%trition and processin
tec3nolo!, Kan ?ostrand Rein3old, ?e= [orE, pp# 1,8-1:2#
6Eac3%E, R#, R%Jina, F#D#, 18.2# C3lorop3!ll anal!sis of =3ole rapeseed Eernels '!
near infrared reflectance# Can# U# "lant &ci# 12, .:9-..,#
Kelasco, @#, BecEer, H#C#, 188.# Anal!sis of total l%cosinolate content and indi2id%al
l%cosinolates in Brassica spp# '! near-infrared reflectance spectroscop!# "lant
Breedin 11:, 8:-1-2#
Qilliams, "#, 18.:# Karia'les affectin near-infrared reflectance spectroscop! anal!sis#
$n4 Qilliams, "#, ?orris, R# (Eds#), ?ear-$nfrared 6ec3nolo! in t3e Aric%lt%ral and
Food $nd%stries, American Association of Cereal C3emists, $nc#, &t# "a%l, pp# 1,0-11:#
Qilliams, "#C#, &o'erin, D#C#, 1881# Ho= do =e do it4 a 'rief s%mmar! of t3e met3ods
=e %se in de2elopin near infrared cali'rations# $n4 Da2ies, A#M#C#, Qilliams, "#C#,
(Eds#), ?ear $nfrared &pectroscop!4 63e F%t%re Qa2es, ?ir "%'lications, C3ic3ester,
pp# 1.9-1..#
QorEman, Ur# U#U#, 1882# ?ir spectroscop! cali'ration 'asics# $n4 B%rns, D#A#, Ci%rcJaE,
E#Q#, (Eds#), Hand'ooE of ?ear-$nfrared Anal!sis, DeEEer $nc#, ?e= [orE, pp# 2,:-
2.-#
22
'itae
#r. !. (ont) Researc3er of t3e $nstit%te of &%staina'le Aric%lt%re (C&$C, Crdo'a,
&pain)# His =orE deals =it3 t3e %se of t3e ?ear-$nfrared &pectroscop! and its
application to different fields, incl%din plant 'reedin and en2ironmental researc3#
#r. *. del !+o-Celestino) Researc3er of t3e $nstit%te of &%staina'le Aric%lt%re (C&$C,
Crdo'a, &pain)# &3e 3as participated in n%mero%s researc3 proDects dealin =it3 plant
'reedin concernin Brassicas and also, s3e is an e<pertise in p3!toremediation#
#r. %lena Cartea) Researc3er of t3e Misin Biolica of /alicia (C&$C, "onte2edra,
&pain)# Her =orE deals =it3 plant 'reedin of Brassicas#
#r. A. de ,aro--ail.n). Head of t3e Aronom! and "lant Breedin Department of t3e
$nstit%te for &%staina'le Aric%lt%re (C&$C, Crdo'a, &pain)# His =orE 3as dealt for
!ears in t3e impro2ement of t3e C%alit! of different Brassica species and ot3er crops,
leadin different national and international researc3 proDects# &ince 188., 3e is also
leader of different proDects concernin t3e %se of plants for decontaminatin poll%ted
en2ironments '! trace elements#
20
(igure captions
(igure 1. Basic str%ct%re of l%cosinolates#
(igure . FreC%enc! distri'%tion plots of total l%cosinolates (a), l%conapin ('),
l%co'rassicanapin (c), prooitrin (d), l%co'rassicin (e) and l%coal!ssin (f) for t3e
samples %sed in t3is =orE (nO 119) (Lmol
-1
d=)#
(igure ". Ra= spectra (@o 1+R) of t3e leaf samples of Brassica napus 2ar# pabularia
%sed in t3is =orE (nO 119), in t3e rane from ,-- to 29-- nm#
(igure $. &econd deri2ati2e spectra (2, 9, 9, 2; &?K7D6) of t3e ra= optical data in t3e
rane from ,-- to 29-- nm#
(igure /. Cross-2alidation scatter plots of la'orator! 2s# predicted 2al%es '! ?$R& for
total l%cosinolates (a), l%conapin ('), l%co'rassicanapin (c), prooitrin (d),
l%coal!ssin (e) and l%co'rassicin (f) (nO 119) (Lmol
-1
d=)#
(igure 0. M"@& loadin spectra for total l%cosinolates in t3e second deri2ati2e (2, 9,
9, 2; &?K7D6) transformation# From t3e top to 'ottom, panels represent loadins for
factors 1, 2 and 0, respecti2el!#
2,
1ables
1able 1. A''re2iations, tri2ial names and side-c3ain str%ct%res of t3e l%cosinolates
considered in t3is =orE#
Abbreviation 1rivial name Chemical structure
Alip3atic
0-B%ten /l%conapin (/?A) CH
2
OCH-CH
2
-CH
2
-
,-"enten /l%co'rassicanapin
(/B?)
CH
2
OCH-CH
2
-CH
2
-CH
2
-
2-*H-0-B%ten "rooitrin ("R*) CH
2
OCH-CH(*H)-CH
2
-
9-M&" /l%coal!ssin (/@A) CH
0
-&*-CH
2
-CH
2
-CH
2
-CH
2
-CH
2
-
$ndole
0-$M /l%co'rassicin (/B&)
1able . Cali'ration and cross-2alidation statistics for t3e different eC%ations
de2eloped for l%cosinolates (nO 119) (Pmol
-1
d=)#
29
calibration cross-validation
glucosinolate range mean 2.#.
g
2%C
h
!
i
2.#. 2%C'
-13
r
&
nt
l
t-/&@
a
1#-1-
,8#1.
18#.9 11#-
0
1#.. -#8
:
2#81 -#.
.
8
/?A
'
--1#.. 2#:1 1#9, -#11 -#.
,
1#8, -#:
0
9
/B?
c
-#01-
21#-:
.#-0 9#1 1#,: -#8
1
2#01 -#.
1
:
"R*
d
-#1--
10#-0
9#11 0#2. -#8. -#8
1
2#11 -#:
.
.
/A@
e
--,#-- 1#-1 1#19 -#.- -#9
-
1#2: -#0
:
0
/B&
f
-#-1-0#,9 1#-1 -#.0 -#98 -#9
-
1#28 -#,
1
0
a
total l%cosinolates#
'
l%conapin#
c
l%co'rassicanapin#
d
prooitrin#
e
l%coal!ssin#
f
l%co'rassicin#

standard de2iation of t3e reference c3emistr! data#

3
standard error of cali'ration#
i
coefficient of determination of t3e cali'ration#
D
ratio of t3e standard de2iation of t3e reference c3emistr! data to t3e
standard error of cross-2alidation#
E
coefficient of determination of t3e cross-2alidation#
l
n%m'er of terms of t3e eC%ation selected in cross-2alidation#
21
(igure 1.
2:
(igure .
2.
- 1- 2- 0- ,- 9- 1-
-
9
1-
19
2-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
a
meanO 18#.9
&#D#O 11#-0
- 2 , 1 .
-
9
1-
19
2-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanO 2#:1
&#D#O 1#90
'
- , . 12 11 2- 2,
-
9
1-
19
2-
29
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanO :#8
&#D#O 9#-
c
- 2 , 1 . 1- 12 1,
-
9
1-
19
concentration
n
%
m
'
e
r

o
f

s
m
p
l
e
s
meanO 9#11
&#D#O 0#2.
d
28
- 1 2 0 ,
-
1-
2-
0-
,-
9-
1-
:-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanO 1#-1
&#D#O -#.0
e
- 1 2 0 ,
-
1-
2-
0-
,-
9-
1-
:-
concentration
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
meanO 1#-1
&#D#O 1#19
f
(igure ".
0-
(igure $.
01
(igure /.
-10 0 10 20 30 40 50 60
-10
0
10
20
30
40
50
60
a
r2O -#..
&ECKO 0#:2
t
o
t
a
l

g
l
u
c
o
s
i
n
o
l
a
t
e
s

(
p
r
e
d
i
c
t
e
d
)
total glucosinolates (laboratory)
-1 0 1 2 3 4 5 6 7 8
-1
0
1
2
3
4
5
6
7
8
'
r2O -#:0
&ECKO -#:8
g
l
u
c
o
n
a
p
i
n

(
p
r
e
d
i
c
t
e
d
)
gluconapin (laboratory)
-5 0 5 10 15 20 25
-5
0
5
10
15
20
25
c
r2O -#.1
&ECKO 2#2-
g
l
u
c
o
b
r
a
s
s
i
c
a
n
a
p
i
n

(
p
r
e
d
i
c
t
e
d
)
glucobrassicanapin (laboratory)
-2 0 2 4 6 8 10 12 14 16
-2
0
2
4
6
8
10
12
14
16
d
r2O -#:.
&ECKO 1#99
p
r
o
g
o
i
t
r
i
n

(
p
r
e
d
i
c
t
e
d
)
progoitrin (laboratory)
02
-1 0 1 2 3 4 5 6
-1
0
1
2
3
4
5
6
e
r2O -#0:
&ECKO -#8-
g
l
u
c
o
a
l
y
s
s
i
n

(
p
r
e
d
i
c
t
e
d
)
glucoalyssin (laboratory)
-1 0 1 2 3 4
-1
0
1
2
3
f
r2O -#,1
&ECKO -#1,
g
l
u
c
o
b
r
a
s
s
i
c
i
n

(
p
r
e
d
i
c
t
e
d
)
glucobrasicin (laboratory)
(igure 0.
00
0,