Application of Near-Infrared Spectroscopy to the Analysis of Total and Individual

Glucosinolates in Brassica napus Leaves.

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&$C)# Apartado 2., E-01-.- "onte2edra, &pain#
*Correspondin a%t3or4
e-mail4 font5cica#es
6l#4 70, 89: ,88211; fa<4 70, 89: ,88292
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Key words
=ear-infrared spectroscop! (=$R&), /l%cosinolates, Rapeseed na'icol leaf, H%man
feed, Cancer-c3emopre2ention, Biof%miation#
Abstract
63e potential of near-infrared spectroscop! (=$R&) for screenin t3e total l%cosinolate
(t-/&>) content, and also, t3e indi2id%al l%cosinolates l%conapin (/=A),
l%co'rassicanapin (/B=), prooitrin ("R*), l%coal!ssin (/A>) and l%co'rassicin
(/B&) in t3e leaf rape (Brassica nap%s ># 2ar# pa'%laria), ?as assessed# 63is crop is
ro?n for edi'le lea2es for 'ot3 fodder and 3%man cons%mption# $n /alicia
(nort3?estern &pain) is 3i3l! appreciated for 3%man n%trition and 3a2e t3e common
name of @na'icolA# A collection of 01 local pop%lations of na'icol ?as anal!sed '!
=$R& for l%cosinolate composition# 63e reference 2al%es for l%cosinolates, as t3e!
?ere o'tained '! 3i3 performance liB%id c3romatorap3! on t3e leaf samples, ?ere
reressed aainst different spectral transformations '! modified partial least-sB%ares
(M">&) reression# 63e coefficients of determination in cross-2alidation (r
2
) s3o?n '!
t3e eB%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 eB%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 eB%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 eB%ations, it can 'e concl%ded t3at some
maCor cell components as protein and cell%lose, 3i3l! participated in modellin t3e
eB%ations for l%cosinolates#
2
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00
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01
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08
,-
,1
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,,
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. 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 (D-
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 RF''elen,
18.8)#
Brassica crops are ?idel! c%lti2ated in /alicia (nort3?estern &pain), in partic%lar t3ose
correspondin to t3e species B# nap%s >#, 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 GrelosH#
63e form of B# nap%s c%lti2ated in /alicia correspond to B# nap%s 2ar# pa'%laria (Cartea
et al#, 2--,), termed Gna'icolH, ?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
0
10
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conser2ation of local pop%lations of t3is form of B# nap%s# 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%eI et al#, 2--0), to enlare t3e enetic 2aria'ilit! of t3is species#
C%rrentl!, t3ose st%dies c3aracterisin t3e different 2arieties of Gna'icolH in /alicia are
minimal# 63ere are pre2io%s st%dies on na'icol pop%lations concernin t3eir
morp3oloical and aronomic attri'%tes (Rodr%eI 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 reB%ired for o'tainin t3e l%cosinolate content
in samples '! 3i3 performance liB%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#
$n contrast, t3e %se of fast anal!tical tec3niB%es s%c3 as near-infrared spectroscop!
(=$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 3aIardo%s c3emicals# =$R& 3as 'een
?idel! %sed for decades for B%alitati2e and B%antitati2e anal!sis in aric%lt%re and food
researc3, and man! a%t3ors 3a2e %sed t3is tec3niB%e for determinin t3e l%cosinolate
content in t3e seed of Brassica species (Biston et al#, 18..; Da%n et al#, 188,; Jelasco
and BecEer, 188.; Font et al#, 2--,)# Ho?e2er, till date no st%dies 3a2e 'een reported
on t3e %se of t3e =$R& tec3niB%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 maCor l%cosinolates fo%nd in t3e
lea2es of B# nap%s 2ar# pa'%laria (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#
,
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8.
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1--
1-1
1-2
1-0
1-,
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1-.
1-8
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,
!. "aterials and #ethods
2#1# "lant material and crop manaement
63is ?orE is 'ased on 119 indi2id%al plants 'elonin to 01 different accessions of t3e
species Brassica nap%s ># 2ar# pa'%laria, ?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# nap%s 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 K of nitroen; 1-9 K of p3osp3or%s; 1-9 K of
potassi%m)#
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 froIen Gin sit%H in liB%id =
2
, to a2oid l%cosinolate
enI!matic 3!drol!sis '! t3e enI!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 liB%id =
2
, Eept at -.- LC and
freeIe-dried in a 6elstar freeIe-drier mod# >ioalfa-1, %ntil anal!sis#
2#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&$C, &pain)# A'o%t 1-- m
dr! ?ei3t (d?) of lea2es ?as ro%nd in a ManEe and N%nEel, Model A1- mill ($NA-
>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 LC to inacti2ate mirosinase# $n t3e first step t3e sample ?as 3eated for
19 min in 2#9 m> :-O aB%eo%s met3anol and 2-- > 1- mM sinirin (2-propen!l
l%cosinolate) as an internal standard# A second e<traction ?as applied after
centrif%ation (9 min, 9<1-
0
) '! %sin 2 m> of :-O aB%eo%s met3anol# *ne m> of
t3e com'ined l%cosinolate e<tracts ?as pipetted onto t3e top of an ion-e<c3ane
col%mn containin 1 m> &ep3ade< DEAE-A29 in t3e formate form# Des%lp3ation ?as
carried o%t '! t3e addition of :9> of p%rified s%lp3atase (E#C# 0#1#1#1, t!pe H-1 from
Heli< pomatia) (&ima) sol%tion# Des%lp3ated l%cosinolates ?ere el%ted ?it3 2#9 m>
9
10-
101
102
100
10,
109
101
10:
10.
108
1,-
1,1
1,2
1,0
1,,
1,9
1,1
1,:
1,.
1,8
19-
191
192
190
19,
199
191
19:
19.
198
11-
111
9
(-#9 m> < 9) Milli-P (Millipore) %ltra-p%re ?ater and anal!sed ?it3 Model 1-- H">C
instr%ment (Qaters) eB%ipped ?it3 a Model ,.1 KJ t%na'le a'sor'ance detector
(Qaters) at a ?a2elent3 of 228 nm# &eparation ?as carried o%t '! %sin a >ic3rosp3er
1-- R"-1. in >ic3rocart 129-, col%mn, 9 m particle siIe (MercE)# 63e amo%nt of eac3
indi2id%al l%cosinolate present in t3e sample ?as calc%lated '! mean of t3e internal
standard, and e<pressed as mol
-1
of d?# 63e total l%cosinolate content ?as
comp%ted as t3e s%m of all t3e indi2id%al l%cosinolates present in t3e sample#
2#0# =$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, K&A) in reflectance mode eB%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!#
$n t3e second step, t3e cali'ration file ?as formed '! addin t3e reference c3emistr!
2al%es for all l%cosinolates, as t3e! ?ere o'tained '! H">C, to t3e file of spectra, t3%s
formin a ne? file, eac3 spectr%m 3a2in an associated 2al%e for eac3 l%cosinolate#
2#,# De2elopin cali'ration eB%ations
Ksin t3e application />*BA> 2# 1#9- (Q$=$&$ $$, $nfrasoft $nternational, >>C, "ort
Matilda, "A, K&A), different cali'ration eB%ations for t-/&>, /=A, "R*, /B=, /A>,
and /B& ?ere de2eloped on t3e ?3ole set (nR 119)# Cali'ration eB%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) siIes Si#e#, (-, -, 1, 1; deri2ati2e order, sement of t3e deri2ati2e, first
1
112
110
11,
119
111
11:
11.
118
1:-
1:1
1:2
1:0
1:,
1:9
1:1
1::
1:.
1:8
1.-
1.1
1.2
1.0
1.,
1.9
1.1
1.:
1..
1.8
18-
181
182
180
18,
1
smoot3, second smoot3); (1, ,, ,, 1); (1, 1-, 1-, 1); (2, 9, 9, 2); (2, 2-, 2-, 2)T# 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 sB%ares (M">&) ?as %sed as a reression
met3od, '! %sin ?a2elent3s from ,-- to 29-- nm e2er! . nm# $n addition, t3e
alorit3ms termed standard normal 2ariate (&=J) 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 siIe and pat3lent3 2ariation amon samples#
2#9# Jalidation of t3e eB%ations
63e performances of t3e different cali'ration eB%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
(1+M) part of t3e cali'ration set (Martens and =aes, 18.8)# $n t3is ?orE, t3e different
cali'ration eB%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 eB%ations o'tained ?as determined on t3e 'asis of t3eir
coefficient of determination in t3e cross-2alidation (r
2
) (&3enE and Qester3a%s, 1881)
(eB# 1) and standard de2iation (&#D#) to standard error of cross-2alidation (&ECJ) ratio
(Qilliams and &o'erin, 1881) (eB# 2)#
r
2
R
1
1
2
1
2
) ( ) U (

,
_

,
_

n
i
i
n
i
y y y y (eB# 1)
?3ere4
yU
R =$R meas%red 2al%e;
y
R mean G!H 2al%e for all samples;
i
y
R la' reference
2al%e for t3e it3 sample#
:
189
181
18:
18.
188
2--
2-1
2-2
2-0
2-,
2-9
2-1
2-:
2-.
2-8
21-
211
212
210
21,
219
211
21:
21.
218
22-
221
222
220
22,
229
:
&#D# &ECJ
-1
R ( )
1
2 + 1
1
1
2
1 ) U ( # #

1
]
1

,
_

K N y y D S
n
i
i i
(eB# 2)
?3ere4
i
y
R la' reference 2al%e for t3e it3 sample;
yU
R =$R meas%red 2al%e; =R
n%m'er of samples, NR n%m'er of ?a2elent3s %sed in an eB%ation; &#D#R standard
de2iation#
63e statistics s3o?n in eB# 1 and eB# 2 i2e a more realistic estimate of t3e applica'ilit!
of =$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)#
63e &ECJ met3od is 'ased on an iterati2e alorit3m ?3ic3 selects samples from a
sample set pop%lation to de2elop t3e cali'ration eB%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 sB%are root of t3e mean sB%are of
t3e resid%als for =-1 derees of freedom, ?3ere t3e resid%al eB%als t3e act%al min%s t3e
predicted 2al%e#
2#1# &pectr%m of B# nap%s leaf and modified partial least sB%are reression loadins
63e M">& loadin plots of t3e first t3ree factors enerated from t3e M">& reression
(2, 9, 9, 2; &=J7D6) 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 eB%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# nap%s 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 &=J pl%s D6 alorit3ms ?ere applied# $n t3e second step, t3e a2erae spectr%m ?as
.
221
22:
22.
228
20-
201
202
200
20,
209
201
20:
20.
208
2,-
2,1
2,2
2,0
2,,
2,9
2,1
2,:
2,.
2,8
29-
291
292
290
29,
299
.
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 eB%ations, ?ere %sed
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 maCor 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 eB%ations#
$. %esults
0#1# Reference c3emistr! anal!sis of total and indi2id%al l%cosinolates in t3e samples
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# Frec%enc! distri'%tions of t3e different
l%cosinolates in t3e samples %sed in t3is ?orE (Fi%re 2) s3o?ed t3at t3e t-/&> and
also, indi2id%al alip3atic l%cosinolates e<3i'ited normal distri'%tions in t3eir inter2als#
$ndi2id%al plants e<3i'ited t-/&> concentrations t3at raned from 1#-1 to ,8#1. Vmol
-
1
d?, and a mean 2al%e of 18#.9 Vmol
-1
d?# 63ese concentrations are similar to t3ose
reported pre2io%sl! in B# nap%s (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
,-O of t3e t-/&>, follo?ed '! "R* and /=A# 63e alip3atic /A> and t3e indole /B&
l%cosinolates s3o?ed ranes from - to , Vmol
-1
d?, '%t mean contents aro%nd 1
Vmol
-1
d?, d%e to t3e 3i3 'iases displa!ed '! t3eir frec%enc! distri'%tions#
$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 (W 0- Vmol
-1
d?) (Bell, 1889)#
0#2# &pectral data pre-treatments and eB%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 siIe 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#
8
291
29:
29.
298
21-
211
212
210
21,
219
211
21:
21.
218
2:-
2:1
2:2
2:0
2:,
2:9
2:1
2::
2:.
2:8
2.-
2.1
2.2
2.0
2.,
2.9
2.1
2.:
8
63e %se of t3e mat3ematical approac3 descri'ed a'o2e !ielded t3e eB%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--,)#
6otal l%cosinolates#- 63e t-/&> eB%ation s3o?ed a 3i3 coefficient of determination in
t3e cali'ration (R
2
R -#8:) and lo? standard error of cali'ration (&ECR 1#.. Xmol
-1

d?) (6a'le 2)# =ine terms ?ere selected in cross-2alidation as t3e optim%m n%m'er to
model t3e eB%ation# 63e final model for t-/&> s3o?ed t3e 3i3est r
2
(-#..) and also
&#D# &ECJ
-1
ratio (2#81) (Fi# 9) s3o?n '! an! of t3e eB%ations for indi2id%al
l%cosinolates#
/l%conapin#- 63e eB%ation for /=A s3o?ed a lo?er &EC (-#11 Xmol
-1
d?) and
3i3er R
2
(-#.,) in cali'ration t3an ra? data or first deri2ati2e# $n cross-2alidation, t3e
second deri2ati2e eB%ation ?as modelled ?it3 9 terms, and also s3o?ed t3e 3i3est r
2

(-#:0) (Fi%re 9) and &#D# &ECJ
-1
(1#8,) ratio of t3ose s3o?n '! t3e ot3er mat3ematical
transformations#
/l%co'rassicanapin#- 63e second deri2ati2e eB%ation res%lted in a 3i3 R
2
(-#81) and
lo? &EC (1#,: Xmol
-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#
9) and &#D# &ECJ
-1
(2#01) 2al%es o'tained for t3is l%cosinolate, ?ere 3i3er t3an t3ose
s3o?n '! t3e eB%ation for /=A#
"rooitrin#- &imilar prediction a'ilit! to t3at s3o?n '! /B= ?as e<3i'ited '! t3e
second deri2ati2e eB%ation for "R* (6a'le 2)# As pre2io%sl! occ%rred for t3e ot3er
l%cosinolates, t3e second deri2ati2e eB%ation (2, 9, 9, 2; &=J7D6) performed o2er t3e
infrared sement e<3i'ited t3e 3i3est prediction a'ilit! of t3e different eB%ations for
t3is l%cosinolate# 63is eB%ation s3o?ed coefficients of determination in cali'ration and
cross-2alidation of -#81 and -#:., respecti2el! (Fi%re 9), and a &#D# &ECJ
-1
ratio of
2#11, ?3ic3 ?as close to t3ose e<3i'ited '! t3e /=A and /B= eB%ations# 63e eB%ation
for "R* ?as modelled ?it3 ei3t terms#
/l%coal!ssin and /l%co'rassicin.- EB%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
eB%ations s3o?ed R
2
2al%es of -#9-, and also similar &#D &ECJ
-1
ratios, ?3ic3 ?ere
close to 1#0-# 63ese ratios ?ere t3e lo?est of t3e different l%cosinolates anal!sed, and
1-
2..
2.8
28-
281
282
280
28,
289
281
28:
28.
288
0--
0-1
0-2
0-0
0-,
0-9
0-1
0-:
0-.
0-8
01-
011
012
010
01,
019
011
01:
01.
018
02-
021
1-
also t3e r
2
2al%es, ?3ic3 ?ere close to -#,# Bot3 eB%ations ?ere modelled ?it3 0 terms,
as 'ein t3e optim%m n%m'er selected in cross-2alidation#
0#0# &econd deri2ati2e spectra of leaf and modified partial least sB%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 freeIe-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 N%Iina, 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-/&> eB%ation (Fi%re 1),
?3ic3 in addition ?ere t3e factors most correlated to t3e total content# 63e t3ird factor
?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 eB%ations and t3at for t-/&> eB%ation# Qa2elent3s %sed in modellin t3e
first t3ree terms of t3e t-/&> eB%ation ?ere s!stematicall! %sed in modellin t3e
different factors for alip3atic, aromatic and indole l%cosinolate eB%ations#
&. 'iscussion
63e 2alidit! of cross-2alidation to e2al%ate t3e performance of an =$R eB%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 eB%ation from cross-2alidation, t3e r
2
o'tained for t-/&>, and
11
022
020
02,
029
021
02:
02.
028
00-
001
002
000
00,
009
001
00:
00.
008
0,-
0,1
0,2
0,0
0,,
0,9
0,1
0,:
0,.
0,8
09-
091
092
090
09,
099
11
also for t3e alip3atic /=A, /B= and "R* l%cosinolates, ?as indicati2e of eB%ations
?it3 ood B%antitati2e information# For t3ese components, t3e mat3ematical models
de2eloped e<plained from -#:0 to -#.. O of t3e 2ariance contained in t3e c3emistr!
data (6a'le 2)# $n contrast, /A> and t3e indole l%cosinolate /B& s3o?ed coefficients
of determination t3at ?ere c3aracteristic of eB%ations %sef%l for ood separation of
samples into 3i3 and lo? ro%ps#
63e n%m'er of M">& terms of t3e different eB%ations selected as optim%m in cross-
2alidation ?as for all t3em, in t3e limits recommended to a2oid o2erfittin, i#e#, one
term '! eac3 ten samples in t3e cali'ration file (&3enE and Qester3a%s, 1889)# From
data s3o?n in 6a'le 2, it can 'e concl%ded t3at t3e 2ariance in t3e reference c3emistr!
2al%es e<plained '! t3e different eB%ations, ?as related to t3e mean concentration of
eac3 l%cosinolate in t3e samples# 63e acc%rac! of t3e H">C anal!sis is 3i3l!
dependent on t3e concentration of t3e component in t3e sample, and acc%rac! in t3e
reference anal!sis is essential to settin %p efficient =$R cali'rations (Qilliams, 18.:)#
63e e<tremel! lo? concentrations e<3i'ited '! /B& and /A> in t3e leaf samples
?o%ld made 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# &ECJ
-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 &ECJ 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-/&> eB%ation ?it3 respect to t3ose displa!ed '! t3e indi2id%al
l%cosinolates, ?o%ld lead to a lo?er &ECJ, 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 &ECJs# $n addition, ?3en t3e rane, and t3erefore, t3e 2ariance in
reference data are lo?, t3e 2al%es for r
2
and also t3e &#D# &ECJ
-1
ratio, can not 'e 2er!
3i3, ?3ic3 is t3e case for t3e /A> and /B& l%cosinolates in t3e leaf of na'icol#
63ose ?a2elent3s 3i3l! participatin in modellin t3e first t3ree terms of t3e
eB%ations for l%cosinolates (Fi%re 1), ?ere, to some e<tent, similar to t3ose pre2io%sl!
reported for determinin l%cosinolates in Brassica C%ncea seed (Font et al#, 2--,)# D%e
to t3e fact t3at l%cosinolates deri2e from nat%ral aminoacids (Ettliner and NCaer,
181.), it is loical to t3inE t3at t3ose feat%res in t3e spectral 'ands related to protein
a'sorption 'e %sed in t3e eB%ations for l%cosinolates# 63is is t3e case of t3e 'and at
2-92 nm, ?3ic3 participated mainl! in t3e constr%ction of t3e first and t3ird loadins of
12
091
09:
09.
098
01-
011
012
010
01,
019
011
01:
01.
018
0:-
0:1
0:2
0:0
0:,
0:9
0:1
0::
0:.
0:8
0.-
0.1
0.2
0.0
0.,
0.9
0.1
0.:
0..
0.8
12
all t3e l%cosinolates eB%ations# *t3er in3erent correlations 'et?een l%cosinolates and
maCor cell components as it is cell%lose infl%enced s%c3 cali'rations, as it is concl%ded
from t3e 'ands at 20-. and 20,. nm# Ho?e2er, p%re l%cosinolates a'sor' also at t3ese
?a2elent3s (Font et al#, 2--,), and t3%s, it 3as not to 'een discarded t3at, to some
e<tent, a specific GfinerprintH for t3e molec%le of l%cosinolate 'e represented in t3e
spectr%m of t3e na'icol leaf#
(. )onclusion
From t3e data reported in t3is ?orE it is concl%ded t3at t-/&>, and also t3ose maCor
l%cosinolates in t3e lea2es of na'icol, as it is /=A, /B= and "R* can 'e predicted
?it3 s%fficient acc%rac! for screenin p%rposes# /A> and /B& eB%ations can 'e %sed
for a correct separation of t3e samples into lo? and 3i3 ro%ps# After =$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! B%icEl! t3ose
indi2id%als of interest in t3e lea2es of na'icol ?it3o%t t3e need of doin H">C anal!sis#
Ac*nowled+e#ents
Qe t3anE /loria FernYndeI Marn ($A&, C&$C, Crdo'a, &pain) for t3e performance of
t3e H">C anal!ses# 63is ?orE 3as 'een s%pported '! t3e "roCect MCZ6 nL A/> 2--0-
-1011 of t3e &panis3 /o2ernment#
10
08-
081
082
080
08,
089
081
08:
08.
088
,--
,-1
,-2
,-0
,-,
,-9
,-1
,-:
,-.
,-8
,1-
,11
,12
,10
,1,
,19
,11
,1:
,1.
,18
,2-
,21
,22
,20
10
%eferences
An%s, M#F#, /ardner, "#A#, NirEeaard, M#A#, Desmarc3elier, M#M#, 188,#
Biof%miation4 isot3ioc!anates released from Brassica roots in3i'it ro?t3 of t3e taEe-
all f%n%s# "lant and &oil 112, 1-:-112#
Barnes, R#M#, D3anoa, M#&#, >ister, &#M#, 18.8# &tandard normal 2ariate transformation
and de-trendin of near-infrared diff%se reflectance spectra# Appl# &pectrosc# ,0, ::2-
:::#
Bell, M#M#, 1889# Meal and '!-prod%cts %tiliIation in animal n%trition# $n4 Nim'er, D#&#,
Mc/reor, D#$# (Eds#), Brassica *ilseeds# "rod%ction and KtiliIation# CAB
$nternational, Qallinford, pp# 0-1-00:#
Biston, R#, Dardenne, "#, C?iEo?sEi, M#, Marlier, M#, &e2erin, M#, Qat3elet, M#"#,
18..# Fast anal!sis of rapeseed l%cosinolates '! near infrared reflectance spectroscop!#
M# Am# *il# C3em# &oc# 19, 1988-11--#
Cartea, M#E#, &oenas, "#, "icoaa, A#, *rdYs, A#, 2--,# Relations3ips amon Brassica
nap%s ermplasm from &pain and /reat Britain as determined '! RA"D MarEers# /en#
Res# Crop E2ol%tion Gin pressH#
Da%n, M#N#, Clear, N#M#, Qilliams, "#, 188,# Comparison of t3ree ?3ole seed near-
infrared anal!Iers for meas%rin B%alit! components of canola seed# M# Am# *il# C3em#
&oc# :1, 1-10-11-.#
Do?ne!, R#N#, RF''elen, /#, 18.8# Brassica species# $n4 RF''elen, /#, Do?ne!, R#N#,
As3ri, A# (Eds#), *il Crops of t3e Qorld# Mc/ra?-Hill, =e? ZorE, pp# 008-012#
Ettiner, M#/#, NCaer, A#, 181.# &%lp3%r compo%nds in plants# $n4 Ma'r!, 6#M#, Alston,
R#E#, R%necEles, J#C# (Eds#), Recent Ad2ances in "3!toc3emistr!, Jol# $# Appleton-
Cent%r!-Crofts, =e? ZorE, pp# .8-1,,#
Fa3e!, M#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#N#, M%llin, Q#M#, 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#
1,
,2,
,29
,21
,2:
,2.
,28
,0-
,01
,02
,00
,0,
,09
,01
,0:
,0.
,08
,,-
,,1
,,2
,,0
,,,
,,9
,,1
,,:
,,.
,,8
,9-
,91
,92
,90
,9,
,99
1,
Fen?icE, R#/#, /riffit3s, =#M#, Heane!, R#N#, 18.0'# Bitterness in Br%ssels spro%ts
(Brassica oleracea ># 2ar# emmifera)4 t3e role of l%cosinolates and t3eir 'reaEdo?n
prod%cts# M# &ci# Food Ar# 0,, :0-.-#
Font, R#, Del Ro, M#, Domn%eI, M#, FernYndeI-MartneI, M#M#, De Haro, A#, 2--,#
Kse of near-infrared spectroscop! for screenin t3e indi2id%al and total l%cosinolate
content in $ndian m%stard seed (Brassica C%ncea ># CIern# \ Coss#)# M# Ari# Food
C3em# 924 0910-0918#
/isl%m, R#, MicElander, E#, =ielsen, M#"#, 2--,# P%antification of nitroen
concentration in perennial r!erass and red fesc%e %sin near-infrared reflectance
spectroscop! (=$R&) and c3emometrics# Field Crops Researc3 Gin pressH#
>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, Jol# $, 63e C3emical "articipants# Academic "ress, =e? ZorE, pp# 120-
11,#
Martens, H#, =aes, 6#, 18.8# M%lti2ariate cali'ration# Mo3n Qile! \ &ons, =e? ZorE#
M%rra!, $#, Qilliams, "#C, 18.:# C3emical principles of near-infrared tec3nolo!# $n4
Qilliams, "#, =orris, N# (Eds#), =ear-$nfrared 6ec3nolo! in t3e Aric%lt%ral and Food
$nd%stries# American Association of Cereal C3emists, $nc#, &t# "a%l, M=, pp# 1:-0,#
*s'orne, B#/#, Fearn, 6#, Hindle, "#H#, 1880# "ractical =$R spectroscop! ?it3
applications in food and 'e2erae anal!sis# >onman &cientific \ 6ec3nical, Esse<,
Enland#
Rodr%eI, J#M#, "adilla, /#, Cartea, M#E#, *rdYs, A#, 2--0# E2al%acin de 2ariedades
alleas de na'icol (B# nap%s 2ar# pa'%laria) en siem'ra precoI# 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#N#, Fen?icE, /#R#, "ortas, C#A#M#, 188:# /l%cosinolates in
crop plants# Hort# Re2# 18, 88-219#
&3apiro, 6#A#, Fa3e!, M#Q#, Qade, N#>#, &tep3enson, N#N#, 6alala!, "#, 2--1#
C3emoprotecti2e l%cosinolates and $sot3ioc!anates of Broccoli spro%ts4 meta'olism
and e<cretion in 3%mans# Cancer Epidemiolo!, BiomarEers \ "re2ention 1-, 9-1-9-.#
19
,91
,9:
,9.
,98
,1-
,11
,12
,10
,1,
,19
,11
,1:
,1.
,18
,:-
,:1
,:2
,:0
,:,
,:9
,:1
,::
,:.
,:8
,.-
,.1
,.2
,.0
,.,
,.9
,.1
19
&3enE, M#&#, Qester3a%s, M#*# 1889# Anal!sis of aric%lt%ral and food prod%cts '! near
infrared reflectance spectroscop!# Monorap3# $nfrasoft $nternational, "ort Matilda, "A,
pp# 21, ,,-91#
&3enE, M#&#, 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!# Jan
=ostrand Rein3old, =e? ZorE, pp# 1,8-1:2#
6Eac3%E, R#, N%Iina, F#D#, 18.2# C3lorop3!ll anal!sis of ?3ole rapeseed Eernels '!
near infrared reflectance# Can# M# "lant &ci# 12, .:9-..,#
Jelasco, >#, 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.:# Jaria'les affectin near-infrared reflectance spectroscop! anal!sis# $n4
Qilliams, "#, =orris, N# (Eds#), =ear-$nfrared 6ec3nolo! in t3e Aric%lt%ral and Food
$nd%stries# American Association of Cereal C3emists, $nc#, &t# "a%l, M=, 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, Mr#, M#M#, 1882# =ir spectroscop! cali'ration 'asics# $n4 B%rns, D#A#, Ci%rcIaE,
E#Q#(Eds#), Hand'ooE of =ear-$nfrared Anal!sis# DeEEer $nc#, =e? ZorE, pp# 2,:-2.-#
11
,.:
,..
,.8
,8-
,81
,82
,80
,8,
,89
,81
,8:
,8.
,88
9--
9-1
9-2
9-0
9-,
9-9
9-1
9-:
9-.
9-8
91-
911
912
910
91,
919
911
91:
91.
918
11
Table . A''re2iations, tri2ial names and side-c3ain str%ct%res of t3e l%cosinolates
considered in t3is ?orE#
Abbreviation Trivial na#e )he#ical structure
Alip3atic
0-B%ten /l%conapin (/=A) CH
2
RCH-CH
2
-CH
2
-
,-"enten /l%co'rassicanapin
(/B=)
CH
2
RCH-CH
2
-CH
2
-CH
2
-
2-*H-0-B%ten "rooitrin ("R*) CH
2
RCH-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&)
1:
92-
921
922
920
92,
929
921
92:
92.
928
90-
901
902
900
90,
909
901
90:
90.
908
9,-
9,1
9,2
9,0
1:
Table !. Cali'ration and cross-2alidation statistics for t3e different eB%ations
de2eloped for l%cosinolates (nR 119) (Xmol
-1
d?)#
calibration cross-validation
+lucosinolate ran+e #ean S.'. S,) %
!
S.'. S,)-
-
r
!
nt
t-/&> 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= -#01-
21#-:
.#-0 9#1 1#,: -#8
1
2#01 -#.
1
:
"R* -#1--
10#-0
9#11 0#2. -#8. -#8
1
2#11 -#:
.
.
/A> --,#-- 1#-1 1#19 -#.- -#9
-
1#2: -#0
:
0
/B& -#-1-0#,9 1#-1 -#.0 -#98 -#9
-
1#28 -#,
1
0
t-/&>4 total l%cosinolates#
/=A4 l%conapin#
/B=4 l%co'rassicanapin#
"R*4 prooitrin#
/A>4 l%coal!ssin#
/B&4 l%co'rassicin#
&#D#4 standard de2iation of t3e reference c3emistr! data as t3e! ?ere
o'tained '! H">C#
&EC4 standard error of cali'ration#
R
2
4 coefficient of determination of t3e cali'ration#
&#D# &ECJ
-1
4 ratio of t3e standard de2iation of t3e reference c3emistr! data
to t3e standard error of cross-2alidation#
r
2
4 coefficient of determination of t3e cross-2alidation#
nt4 n%m'er of terms of t3e eB%ation selected in cross-2alidation#
1.
9,,
9,9
9,1
9,:
9,.
9,8
99-
991
992
990
99,
999
991
99:
99.
998
91-
911
912
910
91,
919
1.
.i+ure captions
.i+ure . Basic str%ct%re of l%cosinolates#
.i+ure !. FreB%enc! distri'%tion plots of total l%cosinolates (t-/&>), l%conapin
(/=A), l%co'rassicanapin (/B=), prooitrin ("R*), l%co'rassicin (/B&) and
l%coal!ssin (/A>) for t3e samples %sed in t3is ?orE (nR 119) (Vmol
-1
d?)#
.i+ure $. Ra? spectra (>o 1+R) of t3e leaf samples of Brassica nap%s 2ar# pa'%laria
%sed in t3is ?orE (nR 119), in t3e rane from ,-- to 29-- nm#
.i+ure &. &econd deri2ati2e spectra (2, 9, 9, 2; &=J7D6) of t3e ra? optical data in t3e
rane from ,-- to 29-- nm#
.i+ure (. Cross-2alidation scatter plots of la'orator! 2s# predicted 2al%es '! =$R& for
total l%cosinolates, l%conapin, l%co'rassicanapin, prooitrin, l%coal!ssin and
l%co'rassicin (nR 119) (Vmol
-1
d?)#
.i+ure /. M">& loadin spectra for total l%cosinolates in t3e second deri2ati2e (2, 9,
9, 2; &=J7D6) transformation# From t3e top to 'ottom, panels represent loadins for
factors 1, 2 and 0, respecti2el!#
18
911
91:
91.
918
9:-
9:1
9:2
9:0
9:,
9:9
9:1
9::
9:.
9:8
9.-
9.1
9.2
9.0
9.,
9.9
9.1
9.:
9..
9.8
98-
981
982
980
98,
989
981
98:
98.
988
18
.i+ure .
2-
1--
1-1
1-2
1-0
1-,
1-9
1-1
1-:
1-.
1-8
11-
111
112
110
11,
119
111
11:
11.
118
12-
121
122
120
12,
129
121
12:
12.
2-
.i+ure !.
21
128
10-
101
102
100
10,
109
101
21
- 1- 2- 0- ,- 9- 1-
-
9
1-
19
2-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
t-/&>
meanR 18#.9
sdR 11#-0
- 2 , 1 .
-
9
1-
19
2-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanR 2#:1
sdR 1#90
/=A
- , . 12 11 2- 2,
-
9
1-
19
2-
29
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanR :#8
sdR 9#-
/B=
- 2 , 1 . 1- 12 1,
-
9
1-
19
concentration
n
%
m
'
e
r

o
f

s
m
p
l
e
s
meanR 9#11
sdR 0#2.
"R*
22
10:
10.
22
- 1 2 0 ,
-
1-
2-
0-
,-
9-
1-
:-
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
concentration
meanR 1#-1
sdR -#.0
/B&
- 1 2 0 ,
-
1-
2-
0-
,-
9-
1-
:-
concentration
n
%
m
'
e
r

o
f

s
a
m
p
l
e
s
meanR 1#-1
sdR 1#19
/A>
.i+ure $.
20
108
1,-
1,1
1,2
1,0
1,,
1,9
1,1
1,:
1,.
1,8
19-
191
192
190
19,
199
191
20
.i+ure &.
2,
19:
19.
198
11-
111
112
110
11,
119
111
11:
11.
118
1:-
1:1
1:2
1:0
1:,
1:9
1:1
1::
1:.
1:8
1.-
1.1
1.2
1.0
2,
.i+ure (.
-10 0 10 20 30 40 50 60
-10
0
10
20
30
40
50
60
r2R -#..
&ECJR 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
r2R -#:0
&ECJR -#: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
r2R -#.1
&ECJR 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
r2R -#:.
&ECJR 1#99
p
r
o
g
o
i
t
r
i
n

(
p
r
e
d
i
c
t
e
d
)
progoitrin (laboratory)
29
1.,
1.9
1.1
1.:
1..
1.8
18-
181
182
180
18,
189
181
18:
18.
188
29
-1 0 1 2 3 4 5 6
-1
0
1
2
3
4
5
6
r2R -#0:
&ECJR -#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
r2R -#,1
&ECJR -#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)
.i+ure /.
21
:--
:-1
:-2
:-0
:-,
:-9
:-1
:-:
:-.
:-8
:1-
:11
:12
21
2:
:10
:1,
:19
:11
:1:
:1.
:18
:2-
:21
2:
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