Sepa a ion and cha ac e iza ion o p oduc s om acidic 1
me hanolysis o plasmalogenic lipids by wo-dimensional gas 2
ch oma og aphy wi h online educ ion. 3
4
5
6
Pie luigi Delmon e1*, Xabie Belaunza an2, Cla k Ridge1, Noelia Aldai2 and John 7
K.G. K ame 3 8
9
10
11
12
1 O ice o Regula o y Science, Cen e o Food Sa e y and Applied Nu i ion, Food and D ug 13
Adminis a ion, College Pa k, MD, USA14
2 Lac ike Resea ch G oup, Depa men o Pha macy & Food Sciences, Uni e si y o he Basque 15
Coun y (UPV/EHU), 01006, Vi o ia-Gas eiz, Spain16
3Guelph Food Resea ch Cen e, Ag icul u e & Ag i-Food Canada, Guelph, ON, Canada ( e i ed) 17
18
19
20
21
22
23
24
25
∗ Co esponding au ho a : HFS-717, US Food and D ug Adminis a ion, 5001 Campus D i e, 26
College Pa k, MD 20740, USA. Tel.: +1 240 402 1779; ax: +1 240 402 2622. 27
E-mail add ess: Pie luigi.delmon e@ da.hhs.go 28
29
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Jou nal o Ch oma og aphy A 1619 : (2020) // A icle ID 460955,
which has been published in inal o m a h ps://doi.o g/10.1016/j.ch oma.2020.460955. © 2020 Else ie unde CC BY-NC-ND license (h p://
c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
1. Abs ac 30
31
The complexi y o de e mining he composi ion o animal issue lipids is g ea ly inc eased by 32
he p esence o plasmalogens in which he alkyl chain is linked o glyce ol by a inyl e he bond 33
ins ead o being es e i ied. Acidic me hanolysis o animal issue lipids p o ides he simul aneous 34
scission o acyl and alkenyl e he moie ies, bu he complexi y o he p oduc s o eac ion poses a 35
g ea challenge in hei gas ch oma og aphic analysis. Two-dimensional gas ch oma og aphy 36
wi h online educ ion (GC-OR×GC) p o ided he esolu ion o all componen s con ained in acid 37
me hanolyzed animal lipids, aking ad an age o he selec i e hyd ogena ion o alkenyl e he 38
me hanolysis p oduc s p io o he second-dimension sepa a ion (2D). In his s udy, we also 39
s udied he chemical ans o ma ions occu ing du ing he acidic me hanolysis o animal lipids 40
and he subsequen gas ch oma og aphic analysis. In pa icula , we obse ed ha using 41
me hanolysis eagen s con amina ed wi h wa e esul ed in he undesi ed o ma ion o a y 42
aldehydes, and we made ecommenda ions on how o a oid hese side eac ions using p ope 43
me hanolysis condi ions. P oduc s o acidic me hanolysis we e s udied by GC-OR×GC, GC-MS, 44
NMR spec oscopy, and GC wi h lame ioniza ion de ec ion (GC-FID). We de ined he GC-FID 45
elu ion o de o animal lipid acidic me hanolysis p oduc s using 100 m x 0. 25 mm 100% 46
bis(cyanop opyl)siloxane columns and wo di e en se o elu ion condi ions: iso he mal elu ion 47
a 180°C, and a empe a u e p og am op imized o dai y a s. A simple p ocedu e o isola ing 48
DMAs p io o GC analysis is also desc ibed. 49
50
Keywo ds: Dime hyl ace als; plasmalogens; a y acids; GCxGC; animal lipids; wo dimensional 51
gas ch oma og aphy. 52
53
2. In oduc ion 54
55
Mos common lipids in animals consis p ima ily o long-chain alipha ic moie ies linked o a 56
glyce ol backbone by an acyl, alkenyl o alkyl linkage, and wi h (pola lipids) o wi hou (neu al 57
lipids) a pola head g oup on he sn-3 posi ion [1, 2]. Plan s con ain mainly acyl lipids, while 58
ma ine animals, insec s and phyla con ain signi ican p opo ions o alkenyl and alkyl e he lipids 59
[3]. These h ee unc ional g oups show di e ences in hei eac i i y o acid and base ca alys s, 60
which has been exploi ed in hei analysis. While es e bonds a e easily hyd olyzed using an 61
acidic o basic ca alys s, inyl e he linkages a e s able unde basic condi ions bu can be clea ed 62
by s ong acids such as HI [4]. The alipha ic chains a ached o hese h ee unc ional g oups a e 63
gene ally simila in leng h bu di e in unsa u a ion. The gene al me hod o de e mine he alkyl 64
chain composi ion o animal lipids is scission om glyce ol by acid o base ca alyzed 65
me hanolysis, ollowed by GC analysis. As ea ly as 1944, Klenk disco e ed ha acid-ca alyzed 66
me hanolysis o acyl lipids yields a y acid me hyl es e s (FAMEs), while o inyl e he lipids 67
yields dime hylace als (DMAs); e he lipids a e no lyzed om he glyce ol backbone [5]. 68
A ailable ch oma og aphic echniques, p ima ily GC wi h packed columns, we e no capable o 69
sepa a ing he complex animal lipid me hanolysis p oduc s. E en mode n long highly pola 70
capilla y columns ailed o p o ide comple e esolu ion o hese analy es [6, 7, 8, 9]. Despi e 71
hese limi a ions, some au ho s quan i a ed only he main DMAs and FAMEs in me hanolyzed 72
lipids by GC [10, 11] o LC-MS/MS [12] and concluded ha ce ain neu odegene a i e diseases 73
we e associa ed wi h a change in he alkenyl o acyl lipid a io. 74
The need o mo e p ecise composi ional in o ma ion led o he isola ion o he acyl, inyl 75
and alkyl e he me hanolysis p oduc s by hin-laye ch oma og aphy (TLC) p io o GC analysis, 76
o he applica ion o selec i e chemical eac ions. Alkenyl e he s p oduced a y aldehydes when 77
exposed o HCl umes [13, 14, 15], aqueous HCl [16, 17] o 90% ace ic acid [18], which we e 78
analyzed di ec ly o con e ed o alcohol and ace a e de i a i es. Alkenyl e he s we e also 79
con e ed o hyd azones by eac ion wi h 2,4-dini ophenylhyd azine in an acidic en i onmen 80
and analyzed by HPLC [19]. Alkyl e he s we e educed wi h LiAlH4 o hei alkyl glyce ol e he s 81
and analyzed as hei isop opylidene de i a i es [14, 17]. Added o his di icul y o sepa a ing 82
hese complex me hanolyzed samples was he epo ha DMAs decompose o alk-1-enyl me hyl 83
e he s (AME) when exposed o 200-300°C, as in he GC injec ion po [20, 21, 22]. In addi ion, 84
some au ho s ha e specula ed ha DMAs may be decomposed o a y aldehydes [20], which 85
was la e ques ioned by o he esea che s [22]. 86
The e is a need o accu a ely cha ac e ize he eac ion p oduc s o med du ing acid-ca alyzed 87
me hanolysis o p oduc s con aining plasmalogenic lipids. Igno ing he plasmalogenic con en o 88
animal issues is no an op ion, since hey can con ibu e up o 15% o he o al lipids in some 89
issues [6]. In his s udy, we in es iga ed he chemical compounds o igina ing om he acidic 90
me hanolysis o animal issue lipids using GC-FID and ou ecen ly de eloped wo-dimensional 91
gas ch oma og aphy wi h online educ ion (GC-OR×GC). The chemical hyd ogena ion applied 92
be ween 1D and 2D o he GC-OR×GC pe mi ed he sepa a ion in he wo dimensional space o 93
complex samples as FAMEs p epa ed om ma ine oils [23], DHA biohyd ogena ion p oduc s 94
[24], and seed oils [25]. GC/quad upole ime-o - ligh (GC/Q-TOF) mass spec ome y and 95
nuclea magne ic esonance (NMR) spec oscopy we e addi ionally used o con i m which 96
compounds we e p oduced by he acidic me hanolysis, and he eac ions aking place in he GC 97
injec ion po . we p oposed a simple p ocedu e o isola e DMAs om FAMEs p io o GC 98
analysis o simpli y hei ou ine quan i ica ion and in es iga e he o ma ion o a y aldehydes 99
du ing sample p epa a ion. 100
101
3. Ma e ials and Me hods 102
103
All chemicals and sol en s we e o analy ical o ACS g ade. Anhyd ous me hanol was 104
p epa ed by adding Molecula Sie e 5A (Sigma Ald ich S . Louis, MO, USA) o me hanol, and 105
le he molecula sie e o se le o e nigh . Sul u ic acid (98%) and oc adecane we e pu chased 106
om Sigma Ald ich. Pu e FAMEs and GLC 463 e e ence mix u e we e pu chased om Nu 107
Chek P ep Inc. (Elysian, MN). Cis and ans mono-unsa u a ed a y acids we e syn hesized in a 108
p e ious s udy along wi h o he e e ence solu ions [26]. Fa y aldehydes 16:0 and 18:0 we e 109
pu chased om TCI Ame ica (Bos on, MA). Bee hea samples we e pu chased in e ail s o es 110
in College Pa k (MD, USA) and ho se mea samples we e collec ed du ing s udy conduc ed in 111
Spain [27]. 112
113
3.1 P epa a ion o lipid ex ac s. Samples o esh mea (5 g) we e cu in o na ow slices o 114
which 60 mL o chlo o o m/me hanol (1:1) was added, and hen homogenized (IKA, T25 digi al 115
ULTRA TURRAX, A kinson, NH, USA) o one min a 1500 pm. Ten millig ams o 116
oc adecane and glyce ol i idecanoa e (1 mL o 250 mg each in 25 mL e -bu yl me hyl e he ) 117
we e added as in e nal s anda ds, ollowed by 27 mL o aqueous KCl (0.88% by weigh ) and 4 118
d ops o 6N HCl o achie e he inal sol en a io o chlo o o m:me hanol:wa e o 1:1:0.9 [28]. 119
The mix u e was shaken and sepa a ed in o wo laye s by cen i uga ion a 1000 pm o 5 min. 120
The bo om laye was collec ed and he ex ac ion was epea ed by addi ion o ano he 30 mL o 121
chlo o o m [6, 29]. The combined chlo o o m ex ac s we e passed h ough 1 g o anhyd ous 122
Na2SO4 in an SPE ube, and he sol en was emo ed unde a mild s eam o a gon a he bo om 123
o a p e-weighed 20 mL sc ew cap glass con aine . The nea lipids we e weighed and s o ed a -124
20°C dilu ed in 15 mL chlo o o m. 125
3.2 Acid-ca alyzed me hanolysis o animal lipids. App oxima ely 40 mg o animal lipids 126
we e d ied unde a s eam o a gon a he bo om o a 15 mL sc ew-capped es ube. Lipids we e 127
econs i u ed in 1 mL o oluene, ollowed by 2 mL o anhyd ous me hanol con aining 2% H2SO4 128
( / ) and pu ged wi h a gon. The ube was mixed by o ex and hea ed a 80°C o 1 hou wi h 129
occasional mixing. The ube was chilled o oom empe a u e, and 2 mL o hexane we e added 130
ollowed by hand mixing. The H2SO4 was neu alized by illing he es ube (~10 mL) wi h 131
aqueous bu e solu ion (150 g/L sodium hyd ogen ci a e and 100 g/L NaCl) and mixed 132
ca e ully. The wo laye s we e sepa a ed by cen i uga ion, and he uppe o ganic phase was 133
ans e ed in o a second 15 mL sc ew-caped es ube a e passing he con en h ough 1 g o 134
anhyd ous Na2SO4. The hexane ex ac ion was epea ed, and he ex ac s combined. Hal o he 135
me hanolysis p oduc s we e placed in o a 2 mL au o-sample ial and analyzed by GC. 136
3.3 Isola ion o DMAs om FAMEs. The second pa o he me hanolysis p oduc s s ill in 137
he 15 mL sc ew-capped es ube we e aken o d yness, and 2.85 mL o e hanol we e added 138
ollowed by 150 µL o a 15% (w/w) aqueous KOH solu ion. The sample was pu ged wi h 139
ni ogen, mixed and kep 48 hou s in he da k a oom empe a u e. Hyd olysis was e mina ed 140
by addi ion o 2 mL o hexane and 10 mL o wa e . The ube was mixed by o ex and 141
cen i uged a 1500 pm o 5 min o achie e wo disc e e laye s. The uppe o ganic laye 142
(con aining DMAs) was mo ed o a sepa a e es ube, and he lowe laye was ex ac ed again 143
wi h 2 mL o hexane. The combined ex ac s we e washed wi h 10 mL deionized wa e and a 144
ew d ops o he 15% KOH solu ion, and passed h ough 1 g o anhyd ous Na2SO4 p io o GC 145
analysis. 146
147
3.4 P epa a ion o aldehydes om he DMAs. A e GC analysis, he isola ed DMAs 148
we e aken o d yness unde a gen le s eam o ni ogen a he bo om o he GC ial. Toluene 149
(500 μL) and wa e (100 μL) we e added in sequence, ollowed by mixing and addi ion o 40 μL 150
o 37% HCl. The ial was hen hea ed o 2 h a 100ºC. The eac ion mix u e was ex ac ed wi h 151
1 mL o pen ane. Aldehydes we e d ied a he bo om o a second ial unde a s eam o a gon 152
and econs i u ed in 1 mL o isooc ane o GC analysis. 153
154
3.5 GC analysis o FAMEs and DMAs. Analyses we e made wi h an Agilen 6890N gas 155
ch oma og aph (Wilming on, DE, USA) equipped wi h a FID and a Supelco SP-2560 capilla y 156
column (100 m × 0.25 mm i.d. × 0.20 μm ilm hickness; Belle on e, PA). Two di e en elu ion 157
empe a u e condi ions we e used: 1)180°C iso he mal; 2) a empe a u e p og am op imized o 158
dai y a s (45°C o 4 min, amped a 13°C/min o 175°C, main ained a 27 min, amped 4°C/min 159
o 215°C, and main ained 45 min) [30, 31]. The injec o and de ec o empe a u es we e se a 160
250°C. Hyd ogen was used as ca ie gas a a low a e o 1 mL/min, and 1 μL o sample was 161
injec ed wi h a spli a io o 100:1. 162
163
3.6 GC-OR × GC. Two-dimensional sepa a ions we e ca ied ou as p e iously desc ibed 164
[23] wi h modi ica ions. An Agilen 7890A GC wi h a FID and a spli /spli less injec ion po was 165
equipped wi h a ZX2 dual s age he mal modula o (Zoex, Hous on, TX). The column se was 166
sequen ially composed o a SP-2560 capilla y column (100 m × 0.25 mm i.d. × 0.25 μm ilm 167
hickness), a capilla y ube coa ed wi h palladium (0.20 m × 0.18 mm i.d.), a deac i a ed 168
uncoa ed capilla y ube (2 m × 0.10 mm, modula o loop), and a SLB-IL111 capilla y column 169
(2.5 m × 0.10 mm i.d.). The modula ion spo s we e se a 0.05 m a e he beginning o he 170
modula ion loop and 0.05 m om he beginning o 2D. The modula ion ime was se o 3 s. The 171
o en was main ained a he cons an empe a u e o 180°C, and he injec o po a 300°C. 172
Hyd ogen was used as ca ie gas a a cons an p essu e o 55 psi, and he injec ion olume was 1 173
μL. The acquisi ion a e was se o 200 Hz, and da a we e p ocessed wi h he GC Image GC × 174
GC so wa e (Ve sion 2.1, GC Image, LLC, Lincoln, NE). All capilla y connec ions we e made 175
wi h Mic o Unions (SGE Analy ical Science, Aus alia). 176
177
3.7 GC/MS sepa a ions wi h so posi i e chemical ioniza ion (CI). An Agilen 7200A 178
GC/Q-TOF was equipped wi h a SP-2560 capilla y column (100 m × 0.25 mm, 0.20 μm ilm 179
hickness) ollowed by a 2 m × 0.10 mm deac i a ed e en ion gap. The injec ion olume was 1 180
μL, he spli a io was se o 100:1, and He was used as ca ie gas. The o en empe a u e was 181
main ained a 180°C, he injec ion po and ans e line a 250°C, he ion sou ce a 300°C. The 182
mass spec ome e was ope a ed in CI+ mode wi h isobu ane as chemical ioniza ion eagen wi h 183
he ilamen cu en se o 100 μA. 184
185
3.8 NMR analyses. NMR spec a we e collec ed wi h a 600 MHz B uke A ance III 186
spec ome e wi h a s anda d 5mm BBO p obe. Samples we e p epa ed in deu e a ed chlo o o m 187
(“100%” CDCl3, Camb idge Iso ope Labo a o ies, Ando e , MA). S anda d sequences we e used 188
o ob ain 1H, 13C, COSY, HSQC and HMBC spec a. All spec a we e e e enced o he 189
chlo o o m signals: 7.26 ppm o 1H and 77.23 o 13C. Spec al in eg a ion and o he da a 190
p ocessing we e pe o med wi h he Mes ReNo a 10.0 so wa e. 191
192
4. Resul s 193
194
4.1 Analysis o animal issue lipids by GC-FID. 195
In his s udy animal issue lipids we e ex ac ed using he me hod o Bligh and Dye [28], 196
and successi ely me hanolized wi h 2% H2SO4 in anhyd ous me hanol. Bee hea lipids we e 197
selec ed as e e ence because o hei high con en in plasmalogens. Me hanolyzed bee hea 198
lipids we e sepa a ed by GC-FID applying he empe a u e p og am op imized o dai y lipids 199
(Fig. 1A). While DMAs we e elu ed on he GC capilla y column as AMEs, we ha e chosen o 200
label hem as DMAs in Figu e 1 and elsewhe e in he manusc ip , since hey we e in oduced in 201
his o m in o he GC injec ion po . Two in e nal s anda ds we e added p io o lipid ex ac ion: 202
glyce ol i idecanoa e o he comp ehensi e di ec analysis o he me hanolysis p oduc s, and 203
oc adecane o quan i a e only he DMAs a e hei isola ion. Hal o he me hanolysis p oduc 204
was used o di ec GC-FID analysis (Fig. 1A), he emaining po ion was aken o d yness and 205
saponi ied wi h 95:5 ( / ) e hanol/aqueous KOH. A e hyd olysis, he neu al componen s 206
including DMAs we e ex ac ed wi h hexane, while he K+ sal s o a y acids (FAs) epa i ioned 207
in he aqueous phase. The isola ed DMAs we e analyzed by GC (Fig. 1C) and he compa ison 208
wi h he o iginal me hanolyzed sample (Fig. 1A) showed ha FAMEs we e quan i a i ely 209
emo ed. 210
211
The e e ence solu ion o a y aldehydes was p epa ed by eac ing he isola ed DMAs wi h 212
wa e in p esence o concen a ed HCl, and was analyzed applying he same elu ion condi ions 213
(Fig. 1D). FAMEs om bee hea lipids wi h no DMAs we e p epa ed by alkaline ans-214
es e i ica ion wi h 0.5M NaOH in me hanol, and also analyzed applying he same elu ion 215
condi ions (Fig. 1B). The componen s o he acid me hanolyzed bo ine hea lipids (Fig. 1A) 216
we e iden i ied by compa ison o he solu ions con aining only FAMEs (Fig. 1B), only DMAs 217
(Fig. 1C), and only a y aldehydes (Fig. 1D). FAMEs we e iden i ied using a ailable s anda ds 218
and p e iously syn he ized e e ence solu ions [26]. Since indi idual DMA e e ence ma e ials 219
we e no a ailable, iden i ica ion o geome ic/posi ional isome s o unsa u a ed DMAs was 220
made by compa ison wi h he elu ion p o ile o FAMEs. In addi ion, acidic me hanolysis o 221
bo ine hea lipids was epea ed by spiking he me hanolysis eagen (2% H2SO4 in anhyd ous 222
me hanol) wi h 100 L o wa e (Fig. 1E). The compa ison be ween he acidic me hyla ion 223
p oduc s p epa ed wi h and wi hou spiked wa e (Fig. 1E, 1A) showed ha he addi ion o wa e 224
caused he o ma ion o 16:0 and 18:0 a y aldehydes, iden i ied by compa ison wi h he a y 225
aldehydes e e ence solu ion (Fig. 1D). Iden i ica ion o 16:0 and 18:0 a y aldehydes was also 226
con i med by using e e ence s anda ds. The addi ion o wa e also esul ed in a highe con en o 227
c/ -18:2 FAMEs e idenced by compa ing Figu e 1E wi h 1A and 1B. 228
229
Sepa a ions s acked in Figu e 1 show ha mos FAMEs, DMAs and a y aldehydes 230
occu ing in acid me hanolyzed bee hea lipids could be esol ed u ilizing a long highly pola 231
capilla y column (100 m x 0.25 mm, SP2560) and a empe a u e p og am op imized o he 232
sho -medium chain FAMEs o me hanolyzed dai y a s [30]. The DMAs (as AMEs) showed 233
e en ion imes close o hei equi alen FAME one me hylene g oup sho e . Sa u a ed DMAs 234
elu ed sligh ly a e he FAME wi h one less me hylene, and we e all baseline esol ed. 235
Howe e , he mono-unsa u a ed 18:1 DMAs we e elu ed wi h he 17:1 FAMEs, and he 18:2n-6 236
DMA was elu ed among he 18:1 FAMEs. Fa y aldehydes elu ed sligh ly a e hei equi alen 237
FAME. The 18:1 a y aldehydes showed sligh ly highe e en ion ime compa ed o 18:1 238
FAMEs, bu i p esen in he same sample hey would no be esol ed om hem. Sa u a ed a y 239
aldehydes p o ided simila e en ion o DMAs wi h one mo e me hylene g oup, o example 240
15:0 a y aldehyde p o ided same e en ion ime o 16:0 DMA (Fig 1C, 1D). 241
242
4.2 Analysis by GC-OR × GC. 243
GC-OR × GC sepa a ions we e achie ed u ilizing a p e iously desc ibed ins umen al 244
con igu a ion consis ing o a 100% bis(cyanop opyl siloxane) capilla y column o 1D and a 245
sho SLB-IL111 o 2D, bo h main ained a 180°C. The capilla y ube coa ed wi h Pd ( educe ) 246
was pu in on o he c yogenic modula o [24]. The sepa a ion o bee hea lipids is p esen ed 247
di ided in ou 1D e en ion ime in e als: om 12:0 o 16:0 (Fig. 2); om 16:0 o 18:0 (Fig. 3); 248
om 18:0 o 18:3n-3 (Fig. 4); om 18:3n-3 o 22:6n-3 (Fig. 5). Me hanolized animal lipids we e 249
also analyzed by GC-FID wi h he same 1D column main ained a 180°C (Fig. 3-5, lowe panel 250
in each igu e). As p e iously epo ed [23, 24] he FAMEs elu ed on s aigh lines pa allel o he 251
1D ime axis, each de ined by he 2D e en ion ime o a ully sa u a ed FAME. Sa u a ed DMAs 252
elu ed on hei own s aigh line bisec ing he plane, cha ac e ized by a lowe angula coe icien 253
wi h he x-axis compa ed o he one o sa u a ed FAMEs. Simila ly o FAMEs, DMAs di e ing 254
o he numbe /posi ion o double bonds we e elu ed on he same s aigh line pa allel o he 1D 255
ime axis, de ined by he 2D e en ion ime o he sa u a ed o m. Sa u a ed DMAs elu ed a 256
signi ican ly lowe 2D e en ion ime ela i e o he sa u a ed FAME diagonal line a hei 1D 257
e en ion ime, indica ing ha he lowe 2D e en ion was caused by chemical educ ion o he 258
analy e unc ional g oup p io o 2D. FAMEs and DMAs elu ed as wo independen se s o 259
analy es wi h no co-elu ions, and DMAs elu ed below he sepa a ion space cha ac e is ic o 260
FAMEs. 261
262
Fa y aldehydes elu ed in he sepa a ion space below DMAs, and sligh ly abo e he line 263
de ining he 2D dead olume. The 18:0 a y aldehyde elu ed a he same 2D e en ion ime as he 264
in e nal s anda d oc adecane. In some sepa a ions ob ained wi h a educe ha was aged, bu s ill 265
p o iding ull educ ion o FAMEs and DMAs, bo h educed and un educed 16:0 and 18:0 a y 266
aldehydes we e de ec ed (Fig. 3, Fig. 4). The 16:0 and 18:0, in ace amoun s, we e he only a y 267
aldehydes de ec ed in he samples me hanolyzed wi h H2SO4 in anhyd ous me hanol. Applying 268
he 180°C elu ion empe a u e, un educed a y aldehydes elu ed sligh ly a e hei equi alen 269
FAME in 1D and sligh ly be o e o i in 2D, indica ing di e en in e ac ions wi h he wo liquid 270
phases. Fo each alkyl s uc u e, 2D elu ed in sequence: he educed a y aldehyde (an alkane), 271
he DMA (an alkyl e he ), he un educed a y aldehyde, and inally he FAME. 272
273
lipids a e saponi ied o ans-es e i ied wi h alkali, hen eac ed (i.e. es e i ied) wi h a B øns ed-460
Low y acid in me hanol wi hou a lipid ex ac ion and dehyd a ion s ep in be ween. The use o a 461
Lewis acid such as BF3 ins ead o a mine al acid o me hyla e he saponi ied a y acids may 462
esul in he o ma ion o addi ional side p oduc s [4]. In his s udy, he addi ion o wa e o he 463
acid me hanolysis eagen also esul ed in he o ma ion o c/ -18:2 ans a y acids (Fig. 1E), 464
which we e p esen only in ace amoun s in he same sample me hanolyzed wi h he anhyd ous 465
eagen (Fig. 1A), and absen when me hyla ion was ca ied ou by alkaline ans-es e i ica ion 466
(Fig. 1B). 467
468
Rega dless o which eagen is used o he acidic me hanolysis, ei he HCl o H2SO4 in 469
me hanol, i is s ic ly necessa y o analyze he acid me hanolysis p oduc s (o isola ed DMAs) in 470
he sho es possible ime. We obse ed ha he nea DMAs s o ed in he eeze while wai ing 471
o he NMR analysis pa ly con e ed o a y aldehydes, which was con i med by e-analyzing 472
he isola ed DMAs by GC a e he NMR spec um acquisi ion. Also, a la ge po ion o DMAs 473
had con e ed o a y aldehydes in he me hanolyzed ho se mea samples anspo ed om 474
Spain. The abso p ion o small amoun s o a mosphe ic mois u e is ha d o a oid, and can 475
p o ide he o ma ion o a y aldehydes i esidual aces o acid a e p esen and me hanol is 476
absen . 477
478
5.3 GC-OR × GC sepa a ion o me hanolyzed animal lipids. 479
The GC-OR × GC me hodology we de eloped o he analysis o FAMEs p epa ed om 480
complex samples such as ish oils and ege able oils p o ided he sepa a ion o all componen s 481
con ained in he acid me hanolyzed bo ine hea lipids, and c i ical in o ma ion o hei 482
iden i ica ion. The educe placed in on o he GC × GC modula o played a cen al ole o 483
achie ing hese sepa a ions. The esolu ion o FAMEs, DMAs (as AMEs) and a y aldehydes in 484
he 2D plane was achie ed by aking ad an age o he di e en eac i i y o hese compounds 485
owa d ca aly ic hyd ogena ion: AMEs (R-HC=CH-O-R’) we e educed o hei alkyl e he (R-486
CH2-CH2-O-R’) homologs, a y aldehydes o hei alkane homologs, while FAMEs we e le 487
unal e ed. The compounds o iginally in oduced in he GC injec ion po as DMAs we e elu ed 488
by 1D as AMEs, and by 2D as alkyl me hyl e he s. The hyd ogena ion o he AME inyl e he 489
double bond educed he dipole-induced dipole in e ac ions wi h he 2D phase, p o iding he 490
d op in 2D e en ion ime esponsible o he esolu ion o DMAs om FAMEs. Simila ly, he 491
educ ion o he aldehyde unc ional g oup o alkanes elimina ed all he a y aldehydes dipole-492
induced dipole in e ac ions wi h he 2D phase, esul ing in hei elu ion sligh ly abo e he 2D 493
dead olume. These chemical ans o ma ions esul ed in he elu ion o FAMEs, DMAs and a y 494
aldehydes in 3 di e en dis inc a eas o he sepa a ion space wi h no o e laps. DMAs (as 495
AMEs) showed he same elu ion o de as FAMEs using he SP2560 capilla y column, bu 496
occu ed in di e en ela i e amoun s. Subs i u ion o he me hyl es e g oup o FAMEs wi h he 497
me hyl inyl e he o DMAs did no al e he in e ac ions o he analy e ca bon chain wi h he 498
bis(cyanop opyl)siloxane phase, p o iding he same elu ion pa e n o he wo g oups o 499
de i a i es. 500
501
The ex ension o GC-OR × GC (o iginally de eloped o FAMEs) o he analysis o DMAs 502
and a y aldehydes equi ed s udying he hyd ogena ion e iciency o he capilla y educe o 503
he new analy es. In he case o samples consis ing only o FAMEs, he wide di e ence in Pd 504
eac i i y o he hyd ogena ion o ole ins and es e g oups allowed o use educe s o a ious 505
e iciencies, and o p olonged ime [23]. Enol e he s equi e mo e ene gy han ole ins o hei 506
comple e hyd ogena ion, and aldehydes a e only sligh ly mo e eac i e han es e s. To s udy he 507
e ec o empe a u e on he educ ion e iciency, we placed he capilla y educe in an auxilia y 508
o en and es ed hyd ogena ion empe a u es om 140 o 230°C (Figu e 6). Isola ed double 509
bonds and he double bond o inyl e he s we e al eady quan i a i ely educed a 140°C, while 510
he comple e educ ion o aldehydes (excep when p esen in ace amoun s) equi ed a leas 511
210°C. Main aining he educe a 210°C also esul ed in a mino educ ion o es e s (FAME) o 512
alkanes. We p e e ed o ade he ull educ ion o a y aldehydes wi h he simpli ica ion o he 513
sys em by main aining he en i e column se (including he educe ) in he main GC o en a 514
180ºC. Each capilla y educe p o ided he desi ed educ ion e iciency o only 5-10 days p io 515
o showing incomple e educ ion o DMAs. 516
517
5.4 GC-FID analysis o me hanolyzed animal lipids. 518
In his s udy, GC-FID analyses we e ca ied ou applying wo di e en elu ion condi ions 519
widely applied in hese ypes o esea ch s udies and ou ine analyses [30, 31, 45, 46]. The i s 520
se o elu ion condi ions, based on he 180°C iso he mal elu ion (Fig. 2-5, lowe panel) was 521
de eloped o de e mine he a y acid composi ion o ege able oils and was op imized o he 522
sepa a ion o unsa u a ed C18 FAME geome ic isome s [45, 46]. This me hod was la e 523
ex ended o he analysis o gene ic ood lipid ex ac s by inc easing he elu ion empe a u e a e 524
he elu ion o 18:3n-3 [47]. The second me hod was op imized o he analysis o FAMEs 525
p epa ed om dai y lipids, and is based on a empe a u e p og am (Fig. 1) ha ocused on he 526
sepa a ion o sho -chain and mid-chain FAMEs, while s ill p o iding he sepa a ion o mos C18 527
FAME geome ic isome s [26, 30, 31, 38]. 528
529
The applica ion o he iso he mal 180°C me hod (Fig. 2-5, lowe panel) o he analysis o 530
acid me hanolyzed bee hea lipids p o ided he expec ed sepa a ion o C18 FAME geome ic 531
isome s as epo ed in li e a u e [26, 46], bu un a o able esolu ion o DMAs om mid chain 532
FAMEs (Fig. 2-5). All he linea and he iso- mid chain sa u a ed DMAs we e elu ed wi h he 533
FAMEs wi h one less ca bon (i.e., 16:0 DMA wi h 15:0 and c9-14:1 FAMEs), while he iso- and 534
linea 18:0 DMA we e elu ed alone. Rega ding unsa u a ed DMAs, c9-16:1 DMA was esol ed 535
om FAMEs and c11-18:1 DMA was elu ed wi h c9-17:1 FAME. The lack o sepa a ion 536
be ween DMAs and FAMEs, in pa icula he sa u a ed ones, also a ec s he accu a e 537
quan i ica ion o a y acids. The exclusion o he FAME-DMA mixed peaks om he a y acid 538
quan i ica ion esul s in an unde -es ima ion o he con en o o al a as well as o al unsa u a ed 539
a y acids, and hei inclusion p o ides he opposi e esul (o e -es ima ion). While a y 540
aldehydes we e elu ed a e hei homolog FAMEs and DMAs (i.e., 16:0 a y aldehyde in Fig. 3, 541
and 18:0 a y aldehydes in Fig. 4) and esol ed om hem, hey may be mis-iden i ied as e hyl 542
es e s i iden i ica ions a e based uniquely on e en ion ime compa isons. Based on hese esul s, 543
GC-FID me hod ha elies on an iso he mal 180°C elu ion and a 100% bis(cyanop opyl)siloxane 544
capilla y column [45, 47] should no be used o he analysis o samples con aining acid 545
me hanolyzed animal lipids, unless DMAs a e isola ed om FAMEs p io o GC analysis. I only 546
he quan i ica ion o acyl lipids is desi ed, samples may be p epa ed o GC-FID analysis by 547
applying alkaline ans-es e i ica ion wi h no p io acid diges ion. 548
549
The sepa a ions o acid me hanolyzed bee hea lipids, he isola ed DMAs, and FAMEs om 550
alkaline ans-es e i ica ion applying he dai y empe a u e p og am [30, 31], a e shown in Figu e 551
1A, 1C and 1B, espec i ely. The sepa a ion o a y aldehydes p epa ed om bee hea DMAs 552
(Fig. 1D) was included o allow o he iden i ica ion o hese side p oduc s when plasmalogens 553
a e imp ope ly me hanolyzed. The magni ude o he a y aldehydes o ma ion ins ead o DMAs 554
may be moni o ed by quan i ying he 18:0 a y aldehydes, since he co-elu ing 19:0 DMA was 555
no de ec ed in any sample. While DMAs a e cha ac e ized by e y close e en ion o FAMEs 556
wi h one less ca bon, all he sa u a ed DMAs we e baseline esol ed om FAMEs using his 557
empe a u e p og am. Howe e , he 18:1 DMAs co-elu ed wi h he c9-17:1 FAME. 558
Un o una ely, a y aldehydes we e elu ed wi h sa u a ed DMAs wi h mo e ca bon, an 559
incon enience may lead o inco ec DMA quan i ica ion i samples a e imp ope ly 560
me hanolyzed o a e no imely analyzed. 561
562
5.5 De elopmen o a simple p ocedu e o isola e DMAs. 563
We de eloped a simple p ocedu e o isola e DMAs om FAMEs using well known eac ions 564
and pa i ioning p inciples. The DMAs we e p epa ed along wi h FAMEs by acid ca alyzed 565
me hanolysis. FAMEs we e subsequen ly hyd olyzed o ee a y acids unde alkaline condi ions 566
and epa i ioned as K+ sal s in a wa e laye , while DMAs emained in he o ganic phase. 567
FAMEs may be quan i ied wi hou DMAs ollowing wo simple app oaches: 1) neu alize he 568
aqueous solu ion con aining he K+ sal s o he a y acids, eco e he ee FAs wi h o ganic 569
sol en (i.e. 50:50 die hyl e he /pe oleum e he ) and e-p epa e FAMEs wi h using 2% H2SO4 o 570
BF3 in anhyd ous me hanol; o 2) ans-es e i y ano he po ion o animal lipids by alkaline 571
me hanolysis, which con e s acyl lipids o FAMEs bu does no a ec alkenyl e he s [6, 7]. 572
Ano he bene i o he milde alkaline p ocess is ha i does no al e he con en o conjuga ed 573
FAs such as conjuga ed linoleic acid [38], bu i does no me hanolyze N-acyl lipids such as 574
sphingomyelin [6, 38, 39]. The isola ion o DMAs by selec i e emo al o FAMEs is less ime 575
consuming han sepa a ing FAMEs and DMAs by hin-laye ch oma og aphy [6, 7, 33, 39, 48, 576
49], o conduc ing sepa a e analyses o FAMEs and DMAs [14, 15, 22, 19]. In his s udy, he 577
isola ed DMAs we e quan i ied using he oc adecane in e nal s anda d. A p ac ical app oach is o 578
analyze he acid me hanolyzed sample be o e and a e he DMA isola ion, quan i y all DMAs in 579
he isola ed DMA solu ion ela i e o 16:0 DMA, and use he 16:0 DMA concen a ion in he 580
o iginal sample o calcula e he con en in he o he DMAs. 581
582
6. Conclusions 583
584
The GC-OR × GC me hod p o ided he esolu ion o all componen s con ained in he acid 585
me hanolyzed animal lipids. Based on hese esul s, and o he e idences collec ed by GC-QTOF-586
MS and NMR, we concluded ha acidic me hanolysis o he plasmalogens con ained in animal 587
lipids yields DMAs i no wa e is p esen in he eac ion solu ion. The p esence o wa e causes 588
he o ma ion o a y aldehydes. Ou esul s also suppo ha he DMAs a e quan i a i ely 589
py olyzed o AMEs in he GC injec ion po a 250°C, and a e elu ed in his o m. The GC-FID 590
sepa a ion using bis(cyanop opyl)siloxane columns and 180°C elu ion empe a u e p o ided 591
highly un a o able sepa a ion o DMAs om FAMEs. The empe a u e p og am op imized o 592
dai y a analysis p o ided he sepa a ion o almos all DMAs wi h only ew mino coelu ions o 593
DMAs and FAMEs. Pu e DMAs can be p epa ed by saponi ica ion o he acid me hanolyzed 594
ex ac ollowed by emo al o hyd olyzed a y acids. FAMEs ( om only acyl lipids) wi h no 595
DMAs may be p epa ed by base-ca alyzed ans-es e i ica ion o he o iginal lipid ex ac . 596
Hyd ochlo ic acid in me hanol should no be used o acidic me hanolysis o plasmalogens 597
unless i is p epa ed onsi e sho ly p io o use, and in his s udy, we eplaced i wi h 2% H2SO4 598
in anhyd ous me hanol. Sequen ial acidic diges ion, saponi ica ion and acid-ca alyzed 599
es e i ica ion o samples con aining animal lipids should be a oided because he acid-base 600
neu aliza ion eac ions p oduce wa e . Rega dless o wha eagen is used o acid me hanolysis, 601
samples mus be analyzed quickly o a oid deg ada ion o DMAs o a y aldehydes du ing 602
s o age. 603
604
7. Acknowledgemen s 605
606
D . X. Belaunza an hanks he Depa men o Economic De elopmen & Compe i i eness o he 607
Basque Go e nmen (Spain) o inancing his esea ch, and he US Food and D ug 608
Adminis a ion o his in e nship a he Cen e o Food Sa e y and Applied Nu i ion. 609
610
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764
41) Y. Kishimo o, N.S. Radin, A eac ion ube o me hanolysis; ins abili y o hyd ogen chlo ide 765
in me hanol, J. Lipid Res. 6 (1965) 435-436. 766
9 10 11 12 13 14 15 16
Min.
0.00
0.15
0.30
0.45
Sec.
0.60
14:0
15:0
i-
15:0
ai-
15:0
i-
16:0
16:0
13:0
i-
14:0
14:0
15:0
i-
15:0 ai-
15:0
i-
16:0 16:0
c9-
14:1 17:0
ai-
17:0
i-
17:0
12:0
i-
13:0
i-14:1 16:1
pA
1.5
2.5
3.5
4.5
5.5
Min.
9 10 11 12 13 14
16:0
14:0 15:0
i-15:0
ai-
15:0 i-16:0
13:0
i-14:0
12:0 i-13:0
c9-14:1
(17:0)
ai-
17:0
i-
17:0
16:0
(15:0)
(i-15:0)
ai-
15:0 (i-16:0)
i-14:1 14:0 c9-
16:1
DMAs
FAMEs
Figu e 2
Figu e 2
15.6 16.6 17.6 18.6 19.6 20.6 Min.
0.00
0.20
0.40
0.60
Sec.
0.80
1.00
16:0
18:0
17:0
ai-
17:0
i-
17:0
i-
18:0
17:0
18:1
18:0 c9
ans c11
17:1
c9 c11
16:1
c9
c7 c11
ans
c12 c13 c14 c15
16:0 ald.
16:0
(17:0)
18:0
17:1
c9
c11
18:1
18:0
c13
c9
ans
c11 c13
i-
18:0
17:0
ai-
17:0
i-
17:0
16:1
c9
c7
c11
c12
ans c14
16:0 ald.
Min.
13.5 14.0 14.5 15.0 15.5 16.0 16.5 17.0 17.5
pA
2
4
6
8
c12
c13
c12
c10
DMAs
FAMEs
Figu e 3
17:1
c9 c11
Figu e 3
0.0
0.4
0.8
1.2
1.6
Sec.
20 22 24 26 28 30 32 34 36
Min.
18:0
18:1
18:2
c/ c9,c12
17:1
19:0
19:1
c9
c11
c12
c13
6-11 12
c9 c11
20:0
20:1
c9 c11
c7
18:3n-6 18:3n-3
19:2 c9, 11
18:2
c15
c14, 16
c13 c15
18:1
19:0 20:0
18:2
18:0
ald.
13- 14
13
14
18:1 18:2
c/
c9,c12
19:0
19:1
c9 c11
c13
6-11
(c9)
c11 20:0
20:1
18:3n-6
18:3n-3
c9, 11
18:2
c11 c13
c,c
18:0
c9
c6-c8
c15
c14,
16
20:0
18:0
ald. 12
c7
Min.
18 20 22 24 26 28
pA
1.5
2.5
3.5
4.5
5.5
18:1
Figu e 4
Figu e 4
0.00
0.60
1.20
1.80
Sec.
2.40
3.00
35 50 65 80 95 110 min 125
22:1
22:0
24:0
18:3n-3
c9, 11-18:2
23:0
24:1
pA
1.4
1.8
2.2
2.6
3.0
min
30 40 50 60 70 80 90 100
20:5
n-3
20:4
n-6
20:3
n-6
22:5n-3
22:4n-6 22:6n-3
22:5n-6
n-9 n-3
22:5n-3
22:4n-6
23:0 24:0
20:5
n-3
20:4
n-6
20:3
n-6
n-9
n-3
c9, 11-
18:2
22:6n-3
22:5n-6
(24:1)
22:0
20:2
c13-
22:1
20:2
Figu e 5
Figu e 5
Figu e 6
40 45 50 55 60
Min. 35
0.00
1.00
2.00
Sec.
1.50
0.50
40 45 50 55 60
Min. 35
0.00
1.00
2.00
Sec.
1.50
0.50
170°C
230°C
16:0
DMA
16:0 FAME
9 c9
16:1 FAME
16:0
DMA
16:0 FAME
9 c9
16:1 FAME
40 45 50 55 60
Min. 35
0.00
1.00
2.00
Sec.
1.50
0.50
140°C
16:0
DMA
16:0 FAME
9 c9
16:1 FAME
40 45 50 55 60
Min. 35
0.00
1.00
2.00
Sec.
1.50
0.50
210°C
16:0
DMA
16:0 FAME
9 c9
16:1 FAME
16:0 Ald. 16:0 Ald.
16:0 Ald. 16:0 Ald.
16:0 Ald.
Red.
16:0 Ald.
Red.
16:0 Ald.
Red.
16:0 Ald.
Red.
Figu e 6
4
x10
0
1 +CI EIC(283.3056)
4
x10
4
+CI EIC(285.3215)
0
4
x10
0
1
+CI EIC(269.2889)
5
x10
0
2
+CI EIC(271.3058)
4
x10
0
1
+CI EIC(299.3390)
3
x10
0
2
+CI EIC(267.2682)
4
x10
0
1
2
+CI BPC(150.0000-450.0000)
Coun s s. Acquisi ion Time (min.)
32 33 34 35 36 37 38 39 40 41 42 43 44
3
x10
0
5
+CI EIC(281.3264)
18:0
16:0
16:0
18:0
i-18:0
17:0
i-17:0 ai-17:0
17:0
18:0
18:1 c9 c11
c12 c13 c15
c14
11
16:1
c9
c7 c11
c10 c12 c13
17:1
c9
c11
17:1
c9
c11
ans
(18:0)
i-18:0
17:0
17:0
i-17:0 ai-17:0
c9
(c7)
c11
c10
ans
16:1
18:0 18:1 c9
c11
(c15)
17:1
c9
c11
c12
11
c13
(c13)
(c12)
Figu e 7
Figu e 7
Figu e 8
Figu e 8
ΔT
GC injec ion
po
phospha idyle hanolamine
plasmalogen
H+
H2O
+
H+
CH3OH
H+
CH3OH
H2O
H2O
CH3OH
CH3OH
CH3OH
H+
H+
H2O
H+
CH3OH
H2O
H+
H2O
Figu e 9
Figu e 9
FAME
&
DMAs
oge h
e
mg/100g mea
i-14:0
14:0
i-15:0
15:0
i-16:0
16:0
i-17:0
ai-17:0
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
1
0.61
0.61
0.78
0.38
0.38
-0.66
-
0.39
-
0.64
0.65
-1.0
1.44
1.45
-1.0
33.82
34.62
-2.3
0.42
0.45
-7.0
1.75
1.78
-1.5
2
-
0.25
-
0.40
0.41
-2.4
-
0.00
-
0.40
0.42
-4.6
0.62
0.63
-2.2
19.92
20.45
-2.6
-
0.00
-
0.51
0.53
-3.0
3
-
0.18
-
0.25
0.26
-4.0
-
0.00
-
0.52
0.53
-1.8
1.01
1.05
-3.0
26.44
27.40
-3.6
0.21
0.22
-5.4
1.05
1.10
-4.3
4
-
0.52
-
0.56
0.57
-1.8
-
0.00
-
0.67
0.66
0.95
1.20
1.22
-0.92
35.02
35.71
-1.9
-
0.00
-
1.18
1.28
-8.2
5
-
0.25
-
0.39
0.40
-3.1
-
0.25
-
0.54
0.57
-5.4
0.81
0.83
-2.0
24.69
25.45
-3.1
-
0.00
-
0.66
0.67
-1.6
6
-
-
-
0.51
0.54
-5.8
-
0.00
-
0.50
0.54
-7.7
0.69
0.69
-0.90
21.74
22.53
-3.5
-
0.00
-
0.78
0.81
-2.6
7
0.20
0.22
-11.4
0.43
0.46
-7.1
-
0.00
-
0.40
0.43
-6.2
0.87
0.89
-3.1
26.31
27.14
-3.1
0.22
0.22
4.1
0.98
1.02
-4.4
8
-
-
-
0.39
0.41
-3.7
-
0.00
-
0.45
0.46
-2.2
0.98
1.04
-5.6
24.85
25.70
-3.4
0.20
0.21
-4.1
1.03
1.08
-5.4
9
-
-
-
0.32
0.34
-4.4
-
0.00
-
0.33
0.35
-3.7
0.41
0.42
-3.3
17.10
17.53
-2.5
0.16
0.15
4.8
0.68
0.70
-2.6
10
-
-
-
0.51
0.54
-7.0
-
0.00
-
0.48
0.50
-4.5
0.82
0.81
1.5
33.23
34.12
-2.6
0.00
0.00
-
0.75
0.73
3.2
11
-
-
-
0.57
0.59
-3.3
-
0.00
-
0.42
0.45
-7.0
0.67
0.67
0.12
23.13
24.00
-3.7
0.24
0.24
0.70
0.98
1.01
-3.4
12
-
-
-
0.49
0.51
-3.2
-
0.15
-
0.38
0.38
0.68
0.62
0.65
-3.4
26.19
27.13
-3.5
0.21
0.23
-7.4
0.16
0.17
-4.2
13
-
-
-
0.48
0.51
-5.2
-
0.00
-
0.51
0.52
-0.59
0.87
0.91
-4.4
25.33
25.97
-2.5
0.28
0.32
-13
1.17
1.23
-4.7
14
-
0.10
-
0.54
0.55
-1.4
-
0.00
-
0.32
0.35
-8.0
0.75
0.78
-3.9
27.70
28.57
-3.1
0.20
0.20
-0.26
0.89
0.93
-4.6
15
0.14
0.14
-4.7
0.33
0.35
-4.3
-
0.00
-
0.24
0.25
-4.5
0.56
0.60
-7.6
17.30
17.79
-2.8
0.16
0.17
-1.0
0.82
0.85
-3.7
FAME
&
DMAs
oge h
e
mg/100g mea
17:0
i-18:0
18:0
c9-18:1
c11-18:1
18:2n-6
18:3n-3
To al
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
Di .
Isol.
% Di .
1
1.49
1.55
-3.6
1.38
0.65
72
11.53
11.93
-3.4
13.25
13.08
1.3
0.99
1.05
-5.9
2.73
1.34
68
-
0.92
-
70.43
70.85
-0.58
2
0.81
0.84
-3.8
0.45
0.23
65
10.75
11.19
-4.0
9.83
9.62
2.2
0.66
0.69
-5.1
1.02
0.91
11
-
0.62
-
45.37
46.79
-3.1
3
1.09
1.15
-4.9
0.73
0.56
26
10.00
10.38
-3.7
6.47
6.26
3.3
0.53
0.56
-5.8
0.79
0.44
57
-
0.68
-
49.09
50.75
-3.3
4
1.60
1.65
-2.8
1.22
-
-
16.76
16.98
-1.3
15.57
15.06
3.3
0.85
0.93
-9.1
2.89
1.46
66
-
1.56
-
77.52
77.59
-0.09
5
1.03
1.08
-4.9
0.70
0.31
77
11.63
12.04
-3.4
11.47
11.01
4.1
0.54
0.56
-2.3
1.87
1.11
51
-
1.01
-
54.34
55.53
-2.2
6
0.74
0.79
-6.1
0.74
0.28
92
9.07
9.44
-4.0
5.23
5.08
3.0
0.60
0.62
-1.9
0.80
0.34
81
-
0.60
-
41.41
42.23
-2.0
7
0.79
0.83
-5.1
0.54
0.34
44
12.34
12.77
-3.5
9.31
9.12
2.1
0.81
0.86
-5.7
1.34
0.72
60
-
0.95
-
54.52
55.96
-2.6
8
1.12
1.20
-7.0
0.75
0.46
48
13.29
13.79
-3.7
8.77
8.72
0.6
0.61
0.64
-3.8
1.13
0.89
23
-
1.31
-
53.57
55.91
-4.3
9
0.87
0.68
26
0.41
0.27
42
7.80
8.05
-3.2
5.49
5.16
6.1
0.59
0.62
-5.2
0.78
0.53
38
-
0.69
-
34.95
35.50
-1.6
10
0.98
1.01
-2.9
0.74
0.47
44
13.46
13.84
-2.8
9.94
9.77
1.7
0.95
0.98
-3.1
1.01
0.80
23
-
0.99
-
62.87
64.57
-2.7
11
0.96
0.97
-1.4
0.47
0.37
24
11.09
11.50
-3.6
7.50
7.34
2.1
0.81
0.88
-8.4
0.60
0.53
12
-
0.96
-
47.44
49.52
-4.3
12
0.79
0.80
-1.1
0.45
0.39
14
10.28
10.70
-3.9
9.26
9.04
2.4
0.90
0.92
-2.1
0.68
0.55
21
-
0.82
-
50.42
52.42
-3.9
13
0.92
0.97
-5.1
0.73
0.54
30
12.77
13.27
-3.8
7.55
7.28
3.8
1.01
1.02
-1.4
0.93
0.57
47
-
1.09
-
52.56
54.17
-3.0
14
0.80
0.84
-4.0
0.46
0.46
-0.36
11.05
11.47
-3.7
6.81
6.80
0.20
0.74
0.78
-4.3
0.69
0.58
17
-
1.05
-
50.96
53.45
-4.8
15
0.71
0.75
-5.2
0.43
0.38
14
8.38
8.71
-3.8
6.10
5.83
4.6
0.66
0.68
-3.8
0.45
0.37
21
-
0.56
-
36.29
37.42
-3.1
Table 1
Table 1
