V m
`
`% HOKKAIDO UNIVERSITY
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`
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`
`Prediction of Relative Retention Value of the Individual
`
`Title
`
`Molecular Species of Diacyl Glycerolipid on High
`Performance Liquid Chromatography
`
`Author(s)
`
`TAKAHASHI, Koretaro; HIRANO, Tsugihiko
`
`
`Citation
`
`
`Jtifiifijfimfi;tiitiafiiflifé‘t’éiii = BULLETIN or TIIE
`FACULTY OF FISHERIES HOKKAIDO UNIVERSITY,
`38(4): 398-404
`
`information
`
`
`issue Date
`
`1987—11
`
`Doc URL
`
`http://hdl.handle.net/2115/23976
`
`Right
`
`Type
`
`bulletin
`
`Additional
`
`
`
`
`
`H.
`
`Instructions for use
`
`Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
`
`000001
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1023
`
`000001
`
`

`

`Bull. Fae. Fish. Hokkaido Univ.
`38(4), 398—404. 1987.
`
`Prediction of Relative Retention Value of the Individual
`
`Molecular Species of Diacyl Glycerolipid on
`
`High Performance Liquid Chromatography
`
`Koretaro TAKAnAsnI‘ and Tsugihiko HIRANO"
`
`Abstract
`
`The relative retention value of the individual molecular species of amtyldiglyceride
`derived from phosphatidylcholine on reverse phase high performance liquid chromatography
`was considered to be dependant principally on the addition theorem of chemical potentials of
`the two fatty acid residues.
`It was demonstrated that the chemical potential of each fatty acid residue is equivalent
`to the relative retention potential of each fatty acid residue; and that the addition of the
`relative retention potentials of the two fatty acid residues denoted the logarithm of the
`relative retention value, the relative retention time of each individual molecular species of
`acetyldiglyceride.
`
`Martin formulated the formulae“:
`
`AflR/R'T2AflA/R'T+A#X/R'T
`logidn/(ZA) : Alix/R ' T
`
`[ll
`[2]
`
`Where A and B are members of a homologous series differing by the functional group
`X ; [1px is the difference in chemical potential of the group X in the chromatogra-
`phic system. R is the gas constant; and T is the absolute temperature.
`If we
`consider am as the partition coefficient of the standard molecular species and as as
`the partition coefficient of each molecular species,
`(Is/0A will correspond to the
`relative retention value, the relative retention time (RR T) of each molecular species.
`So from formula [2],
`
`10% (aB/aA)Zlog RRT — Aflx/R' T
`
`should hold. Under most of the chromatographic system, T is constant. Therefore,
`1/ R- .T will also be constant.
`
`From the aforementioned aspect, formula [1] can be rewritten as follows:
`
`log RRT oc Alix
`
`This formula [4] can be expanded to cover
`
`Afla=élflA i Alix
`
`Aflnc 7 AIR/11+ Allis/12
`
`[3]
`
`[4]
`
`[5]
`
`where DG- is the diacylglycerolipid and FAl and FA2 are the fatty acid residues of
`DG. From formulae [3], [4] and [5],
`
`
`
`* Laboratory of Food Chemistry 1, Faculty of Fishen'cs, Home; University
`(itifiifiigflfififiefififidhitiifiҤigl
`“ Hitachi Hokkac' Semiconductor Ltd.
`
`(unkfitsnvflaaamra)
`
`- 398 -—
`
`000002
`
`000002
`
`

`

`TAKAHASHI & HIRANO'. Prediction of glycerolipid on HPLC
`
`[6]
`log RRTbczlog RRTFAfirlog RRTFA,
`So by calculating the relative retention potential of the individ—
`can be obtained.
`ual fatty acid residue, it is possible to predict the relative retention value (a RRT )
`of the individual molecular species of DG.
`This idea was demonstrated by comparing the theoretically determined relative
`retention potential of the individual molecular species of D0 and also the empiri—
`cally determined one.
`
`Methods
`
`The RRT data of acetyldiglyceridez’”, obtained by acetylation subsequent to
`phospholipase C hydrolysis of soybean phosphatidylcholine (PC), egg yolk PC, and
`PC from five kinds of fish muscle were reviewed. The relative retention potential
`of each fatty acid residue of the acetyldiglyceride, i.e., the 00, was calculated as
`follows:
`
`From the identified monoacid DG molecular species,
`
`log RRTFALOOg RRTDG)/2
`
`and from the identified diacid DG molecular species,
`log RRTFA1::log RRTnawr-log RRTFAZ
`(Unknown)
`(Known)
`
`or,
`
`[7]
`
`[8]
`
`log RRTFAzzlog RRTDG—log RRTMl
`("UnknOWn)
`(Known)
`The theoretically determined relative retention potential of the individual DG
`molecular species that denotes log RRTDG=10g RRTM1+log RRTFAZ (each term is
`calculated from formulae [7],
`[8] and [9]) was compared with the empirically
`determined one.
`
`[9]
`
`The “Q” value examination proposed by Dean and Dixon" was employed in
`order to minimize the error of BET.
`
`Results
`
`Table 1 shows the theoretically determined relative retention potential of each
`fatty acid residue of the DGs that have been reviewed“). For example, from the
`RRT datazva’ of (22 : 6)(22 : 6), that is, 44.1, 44.2, 44.3, 44.6, 46.2, all of these could
`be employed for
`the mean value calculation according to the “Q” value
`examination“.
`In this case, the mean value of RR T would become 44.6 and the
`relative retention potential of 22 : 6 fatty acid residue was calculated as (log 44.6) /
`220.8247 (See formula [7]).
`In the same way, the relative retention potential of
`20 : 5 fatty acid residue was calculated as (log 37.3) /2 :0.7859, therefore, the relative
`retention potential of (20 : 5)(22 : 6) can be predicted as 0.8247 +0.7859:1.611.
`The actual relative retention potential of (20 : 5)(22 : 6) was 1.613, as shown in Table
`2. The relative error of the retention potential will be (1.613—1.611)/1.61120.12%
`in case of (20: 5)(22 : 6). A retention potential of 16 :0 was calculated as follows.
`The mean value of the RRT of (16 : 0)(20 : 5) was 92.7743); and, the relative reten—
`tion potential of this molecular species was log 92.7: 1.967. Accordingly, from
`__, 399 »»
`
`000003
`
`000003
`
`

`

`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`Table 1. Relative retention potential of the fatty acid residues
`of acetyldiglyceride on reverse phase high performance
`liquid chromatography.‘
`
`Fatty acid
`ruldue
`16 : 2
`17:2
`
`18:2 (06
`
`$222:
`potential
`0.8670
`0.9850
`0.9757
`
`”“111 :3 (03
`"“20:4 (1)6
`22 : 4
`
`0.8580
`0.9—030—“
`1.027
`
`20:5 «)3
`22:5
`
`0.7959
`0.9261
`
`
`
`12:13:22:
`potential
`1.023
`1.102
`1.181
`
`1.229
`1.347
`1.484
`
`0.9985
`1.081
`
`Fatty acid
`reSIdue
`14 :0
`15 :0
`16:0
`
`17:0
`18:0
`20:0
`
`16:1 (09
`17: 1
`
`18:1 (09
`20:1
`24:1
`
`22:6 (03
`
`1.142
`1.313
`1.638
`
`0.8247“
`
`' Relative retention time of (16:0)(22 : 6) is regarded as 100.
`Equipment, Hitachi 638-450; Shodex RI monitor; column,
`LiChrosorb RPJS (250X8mm; tandem); solvent,
`isoe
`propanol/acetone/methanol/acetonitrile
`(1 :
`l
`: 3 : 4, v/v) ;
`flow, 1.5 nil/min; column temperature, ambient.
`
`formula [8], the relative retention potential of 16:0 can be calculated as 1.967 —
`0.7859 2 1.181.
`Incidentally, the relative retention potential of (16 : 0)(22 : 6) can be
`predicted as 1.181 10.8247 : 2.006 and the actual relative retention potential of (16 :
`0)(22 : 6) was 2.000 (See Table 2). All of the relative retention potentials of the
`fatty acid residues in Table 1 were calculated in the same manner. From the
`calculated retention potential of the fatty acid residues shown in Table‘l,
`the
`relative retention potential of the individual molecular species of DG was theoreti-
`cally predicted and it was compared with the empirically determined one as shown
`in Table 2. Though there are small errors between the theoretically determined
`retention potential and the empirically determined one, the validity of this idea is
`well demonstrated.
`
`Discussion
`
`As it is obvious from the theory of Martin”, formulae [1] and [2] should hold
`for all of the partition chromatographic systems.
`It then follows that formulae [6],
`[7], [8] and [9] should also hold for all of the partition chromatographic systems of
`DG lipid molecular species regardless of the analytical condition employed for the
`chromatography. By introducing the RR T data of the dinitrobenzoyl derivatives
`from PC presented by Takamura et al.“ and Kito et alfi’, instead of the acetyl
`derivatives that have been discussed in this study, it was also demonstrated that the
`theoretically determined relative retention potential of this derivative coincides well
`with the empirically determined one as shown in Table 3. This shows that not only
`—400~
`
`000004
`
`000004
`
`

`

`TAKAHASIII & HIRANO: Prediction of glycerolipid on HPLC
`
`Table 2. Comparison of the predicted and the empirically determined relative retention
`' . 1....._.___._—.__—_—.v.4 _ ._._—_._— 7W . V
`potential of the individual molecular species of mtyldiglyceride.’
`
`-0
`
`Molecular specios
`of acetyldlglyceride
`
`Empirically determined
`Predioted relatiye
`relative retention
`retentnon potential
`potential
`
`
`Relative
`error ("/0)
`
`(20 : 5)(20 : 5)
`(20: 5)(22: 6)
`(22 : 6)(22 : 6)
`(18 : 3)(22 : 6)
`(20 : 4)(20 : 5)
`(22 : 5)(20 2 5)
`(20 : 4)(22 : 6)
`(22 : 5)(22 : 6)
`(18 : 2)(20 : 5)
`(16 : 1)(20 : 5)
`(18 : 2)(22 : 6)
`(14 : 0)(20: 5)
`(16: 1)(22 : 6)
`(18 : 2)(18 : 3)
`(20 : 4)(22 : 5)
`(14 : 0)(22 : 6)
`(22 : 4)(22 : 6)
`(22 : 5)(22 : 5)
`(18 : 2)(20 : 4)
`(15 : 0)(20 : 5)
`(17 : 2)(20 : 4)
`(18 : 2)(22 : 5)
`(17: 1)(22 : 6)
`(16: 1)(20 : 4)
`(17 : 2)(22 : 5)
`(16: 1)(22 : 5)
`(15 : 0)(22 : 6)
`(18: 1)(20 : 5)
`(14 : 0)(22 :5)
`(18 : 2)(18 : 2)
`(18: 1)(22 :6)
`(16 : 0)(20 : 5)
`(16: 1)(18 :2)
`(18:1)(18:3)
`(14 : 0)(18 : 2)
`(16 : 0)(22 : 6)
`(16 : 0)(18 :3)
`(16 2 0)(16 : 2)
`(18: 1)(20 2 4)
`(17 : 0)(22 : 6)
`
`1.572
`1.611
`1.649
`1.683
`1.695
`1.712
`1.734
`1.751
`1.762
`1.784
`1.800
`1.809
`1.823
`1.834
`1.835
`1.848
`1.852
`1.852
`1.885
`1.888
`1.894
`1.906
`1.906
`1.908
`1.911
`1.925
`1.927
`1.928
`1.949
`1.951
`1.966
`1.967
`1.974
`1.999
`1.999
`2.006
`2.039
`2.048
`2.051
`2.053
`
`1.572
`1.613
`1.649
`1.688
`1.719
`1.712
`1.722
`1.746
`1.788
`1.784
`1.825
`1.809
`1.824
`1.848
`1.869
`1.843
`1.852
`1.894
`1.917
`1.891
`1.894
`1.900
`1.906
`1.918
`1.895
`1.900
`1927
`1.945
`1.915
`1.981
`1.966
`1.967
`2.000
`1.999
`2.000
`2.000
`2.030
`2.048
`2.064
`2.067
`
`**
`0.12
`**
`0.30
`1.42
`**
`0.69
`0.29
`1.48
`**
`1.39
`an:
`0.01
`0.76
`1.85
`0.27
`#21:
`2.27
`1.70
`0.16
`an:
`0.05
`#21:
`0.52
`0.84
`1.30
`aka:
`0.88
`1.74
`1.54
`**
`*4:
`1.32
`*4:
`0.05
`0.29
`0.88
`**
`0.29
`0.68
`
`— 401,
`
`000005
`
`000005
`
`

`

`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`
`
`Table 2
`
`(continued).
`
`
`
`Molecular species
`of acetyldiglyceride
`
`Predicted relative
`retention potential
`
`5)
`:1)(22:
`(18
`(16
`4)
`:0)(20:
`5)
`1)(20:
`(20 ;
`5)
`:0)(22:
`(16
`2)
`1)(18:
`(18:
`(18:
`5)
`O)(20:
`6)
`1)(22:
`(20:
`.1)
`(1.8:
`1)(16:
`2)
`:0)(18:
`(16
`:0)(18:
`(14
`1)
`6)
`(18
`:0)(22:
`1)
`:0)(16:
`(16
`3)
`:0)(18:
`(18
`2)
`:0)(18:
`(17
`4)
`(16
`:0)(22:
`:0)(18:
`1)
`(15
`4)
`:0)(20:
`(18
`1)
`:0)(17:
`(16
`1)
`(18
`:1)(18:
`1)
`(18
`:2)(20:
`1)
`O)(18:
`(16:
`1)
`:0)(20:
`(14
`O)
`:0)(16:
`(16
`O)(22:
`(18:
`4)
`(24
`:1)(20:
`5)
`:0)
`(18:
`2)(20
`1)
`(18
`:0)(18:
`
`:0)(20:(16
`1)
`
`Empirically determined
`relative retention
`Relatigre
`potential
`error ( /o)
`
`2.067
`0.05
`2.090
`**
`2-098
`0.05
`2-080
`1.28
`2.137
`(194
`2.137
`0.19
`2.1.38
`**
`2.137
`0_ 14
`2-143
`0.42
`2.155
`0.46
`2.172
`**
`2.172
`0,32
`2.184
`095
`2.204
`**
`2.126
`3.71
`2.245
`(104
`2.254
`0.09
`2.245
`0.75
`2.281
`0.09
`2.274
`066
`2.321
`009
`
`2.068
`2.090
`2.099
`2.107
`2.117
`2.133
`2.138
`2.140
`2.157
`2.165
`2.172
`2.179
`2.205
`2.204
`2.208
`2.244
`2.256
`2.262
`2.283
`2.289
`2.323
`2.336
`.10
`2.335
`1.4 4
`2.328
`2.362
`333
`2.283
`2.374
`2.424
`* *
`2.424
`2.460
`* *
`2.460
`Q36
`2.480
`2.489
`2.494
`0.56
`2.480
`
`‘ Relative retention time of (16: 0)(22 :6) is regarded as 100.
`Analytical conditions are the same as in Table 1.
`" Relative retention potentials of the fatty acid residues shown in Table 1 are calculated
`from the log RR’I‘s of these molecular species as explained in the text.
`
`chromatographic conditions such as the column, the solvents, the analytical tempera—
`ture (as long as it is constant), but also the type of derivative does not matter for the
`proposed idea, while the value itself of the relative retention potential of each fatty
`acid residue has to be calculated each time for each chromatographic condition.
`The idea proposed in this study can be expanded to cover the partition
`chromatographic system of triglyceride. This will be reported at a later date.
`
`402 1.
`
`000006
`
`000006
`
`

`

`TAKAHASHI & HmANo : Prediction of glycerolipid on HPLC
`
`Table 3. Comparison of the predicted and the empirically determined relative retention potential
`of the individual molecular species of dinitrobenzoyl derivative of diacylglycerolipid i.e.
`
`diglyceride (DG).'
`
`Molecular species of
`dinitrobenzoyl
`
`derivative of DG
`
`Predicted relative
`retention potential
`
`lmpirieally determind
`relative retention
`
`potential
`
`Relatisre
`
`error (/°)
`
`—l
`
`A
`
`.l
`
`B
`
`C
`
`—l~
`
`
`
`(16 : 1)(22 2 6)
`(18: I)(22 : 6)
`(16 : l)(16: 1)
`(16 : 0)(22 : 6)
`(18: I)(22 : 4)
`(16 : 0)(22 : 4)
`(18: l)(18: 1)
`(16 : 0)(18: 1)
`
`(16 : 0)(16 : 0)
`
`(.16: l)(20 : 5)
`
`(18 : 2)(20 : 5)
`(16: I)(22 : 6)
`
`0.49
`2.090
`2.080
`*1:
`2.225
`2.225
`*1:
`2.238
`2.238
`1:1:
`2.243
`2.243
`0.37
`2.405
`2.414
`*1:
`2.431
`2.431
`*1:
`2.529
`2.529
`2.547
`2.555
`0.33
`
`2.564
`2.583
`0.73
`
`2.190
`
`2.190
`
`*1:
`
`2.210
`2.241
`
`2.223
`2.230
`
`0.60
`0.47
`
`*1:
`2.260
`2.260
`(18 : 2)(22 : 6)
`*1:
`2.305
`2.305
`(16 : 0)(20 : 5)
`*1:
`2.305
`2.305
`(18: l)(20 : 5)
`(16 : 0)(22 : 6)
`2.356
`2.356
`'.O
`
`(16 : 0)(16 : 0)
`2.534
`2.534
`1:1:
`
`(18 : 2)(18 : 2)
`(16 : 0)(20 : 4)
`(18: l)(18 : 2)
`
`2.243
`2.332
`2.394
`
`2.243
`2.332
`2.389
`
`2.490
`(18 : 0)(20 2 4)
`2.544
`(18:1)(18: 1)
`2.564
`(16 : 0)(18: 1)
`2.571
`(18 : 0)(18 : 2)
`
`(16 : 0)(16 : 0)
`2.583
`“n
`. .‘._.._ .
`
`2.490
`2.544
`2.566
`2.571
`2.583
`——.____.__._ i..—
`
`*1:
`*1:
`0.18
`
`r':..0
`*1:
`0.09
`*1:
`*1:
`
`W #_L__
`
`‘ Relative retention time of (12 : 0)(12 : 0) is regarded as 100.
`“ Relative retention potentials of the fatty acid residues are calculated from the log RRTs of
`these molecular species.
`A: Calculated from the data. of Takamura at
`
`(11.5)
`
`Equipment, Hitachi 655— 15 ; Hitachi 6387-41 UV monitor; column, Ifltrasphere ODS (250x
`4.6 mm) ; solvent, acetonitrile/isopropanol (4: 1, v/v) ; flow, 1.0 ml/min; column tempera
`ture, 25'C.
`B: Same condition as A except that the solvent system used is methanol/isopropanol (95 :5,
`v/v).
`C : Calculated from the data of Kite at al.“
`Same condition as A though the analyzed date is dilIerent.
`
`-
`
`~- 402»-
`
`000007
`
`000007
`
`

`

`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`Acknowledgment
`
`Thanks are due to Professors M. I'Iatano, T. Takagi and K. Takama. for their
`advices.
`
`References
`
`1)
`
`3)
`
`4)
`
`5)
`
`6)
`
`Martin, A.J.I’. (1950). Some theoretical aspects of partition chromatography. Biochem. Soc.
`Symposia, (Cambridge, England). 3, 4—20.
`Takahashi, K., Hirano, T., Takama, K. and Zama, K. (1982). Molecular species of fish muscle
`lecithin. Bull. Japan. Soc. Sci. Fish., 48, 1803—1814.
`Takahashi, K.
`(1985). A novel approach for the identification of lipid molecular species.
`Application of high performance liquid chromatography on fish muscle lecithin molecular
`species analysis. Mom. Fae. Fish. Hokkaido Univ., 32, 245—330.
`Dean, RB. and Dixon, W.J. (1951). Simplified statistics for small numbers of observations.
`Anal. Chem, 23, 636-638.
`Takamura, H., Narita, H., Urade, R. and Kite, M. (1986). Quantitative analysis of polyenoic
`phospholipid molecular species by high performance liquid chromatography. Lipids, 21,
`356—361.
`
`Kito, M., Takamura, H., Narita, II. and Urade, R. (1985). A sensitive method for quantita—
`tive analysis of phospholipid molecular species by high performance liquid chromatography.
`J. Biochem, 98, 3277-331.
`
`3404—
`
`000008
`
`000008
`
`