HOKKAIDO UNIVERSITY
`
`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
`
`JtifiiEj@7l<Ia%t§‘i%Bifli5%?%iel1 = I3UI,LEI‘IN or THE
`FACULTY OF FISHERIES HOKKAIDO UNIVERSITY,
`38(4): 398-404
`
`lssue Date
`
`1987-11
`
`Doc URL
`
`http://hdl.handle.IIet/2115/23976
`
`Right
`
`Type
`
`bulletin
`
`Additional
`Information
`
`Instructions for use
`
`Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP
`
`°°°°°1
`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`
`ENZYMOTEC - 1023
`
`000001
`
`

`
`Bull. Fac. 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 TAKAHASHI‘ and Tsugihiko HIRANO"
`
`Abstract
`
`The relative retention value of the individual molecular species of acetyldiglyceride
`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":
`
`Alla/R'T"—‘A#A/R'T+A#X/R'T
`10g(au/am) = A/ix/R ' T
`
`[2]
`
`where A and B are members of a homologous series differing by the functional group
`X ; Apx 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 an, as the partition coeflicient of the standard molecular species and (1,, as
`the partition coefficient of each molecular species, at,/a/A will correspond to the
`relative retention value, the relative retention time (RR T) of each molecular species.
`So from formula [2],
`
`log (as/a/A)=10S RRT—Al1X/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 AM
`
`This formula [4] can be expanded to cover
`
`H/13-_-A/1A i A/ix
`
`Alina 7 A,UFA1'i— Afilr/12
`
`[3]
`
`[4]
`
`[5]
`
`where DG is the diacylglycerolipid and FA, and I/‘A2 are the fatty acid residues of
`DG. From formulae [3], [4] and [5],
`
`= Laborat;-qt, of Food C'h—e.niistry 1, Faculty of Fisheries, Hokkaido rlmmmy
`(S1ti‘§iE7C$7J<F;5i'?‘r‘:'iifI6EiE’:?5?r?“§§E)
`" Hitachi Holclcai Semicmiductor Ltd.
`
`(unkfit5nv!aamML%)
`
`- 398 --
`
`000002
`
`000002
`
`

`
`TAKAHASHI & HIRANOZ Prediction of glycerolipid on HPLC
`
`[6]
`log RRTDG:log .RRTpA,+log RRT“,
`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 DG and also the empiri—
`cally determined one.
`
`Methods
`
`The RRT data of acetyldiglycerideu’, 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 DG, was calculated as
`follows:
`
`From the identified monoacid DG molecular species,
`
`log RRTF,,;(log RRTDG)/2
`
`and from the identified diacid DG molecular species,
`log RRT,.A,::log RRTDG»-log RRT,-A2
`(Unknown)
`(Known)
`
`or,
`
`[8]
`
`log RRTFA,,:log RRTDG—log RRTMI
`(Unknown)
`(Known)
`The theoretically determined relative retention potential of the individual DG
`molecular species that denotes log RRT,,G=log RRT,.,,,+log RRT,-AZ (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 RRT.
`
`Results
`
`Table 1 shows the theoretically determined relative retention potential of each
`fatty acid residue of the DGs that have been rcviewedzi”. For example, from the
`RRT data“) of (22 : 6)(22 1 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 =O.7859, therefore, the relative
`retention potential of (20 : 5)(22 : 6) can be predicted as 0.8247 +0.7859:1.6l1.
`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—1.6l1)/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 92.723’; and, the relative reten-
`tion potential of this molecular species was log 92.7: 1.967. Accordingly, from
`—-2 399 H
`
`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.‘
`
`F
`
`_i
`iiitaiid
`
`Relative
`:.:::f::.°;1
`
`14 :0
`15 :0
`1620
`
`17:0
`18:0
`20:0
`
`16:1 :09
`17: 1
`
`18:1 (29
`20:1
`24:1
`
`1.023
`1.102
`1.181
`
`1.229
`1.347
`1.484
`
`0.9935
`1.081
`
`1.142
`1.313
`1.638
`
`Fa“ acid
`residue
`
`16 : 2
`17: 2
`18:2 (/26
`
`Relative
`;:ii:::::;:
`
`0.867()
`0.9850
`0.9757
`
`0.3590
`'"”'i'§ :3 (123
`"“ %%_‘"
`22 : 4
`1.027
`
`20:5 m3
`22:5
`
`22:0 m3
`
`0.7959
`0.9261
`
`0.§2‘4”7"i
`
`' Relative retention time of (16:O)(22 : 6) is regarded as 100.
`Equipment, Hitachi 638-~50; Shodex RI monitor; column,
`LiChroso1-b
`RP»—18
`(250><8mm; tandem); solvent,
`iso—
`propanol/acetone/methanol/acetonitrile
`(l :
`l
`: 3 : 4, v/v) ;
`flow, 1.5 ml/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‘1,
`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
`
`and [2] should hold
`As it is obvious from the theory of Martin", formulae
`for all of the partition chromatographic systems.
`It then follows that formulae [6],
`[7],
`and
`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 elf”, 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-
`
`0000004
`
`000004
`
`

`
`TAKAIIASIII & HIRAN02 Prediction of glycerolipid on HPLC
`
`Table 2. C0mpa.ris0n of the predicted and the empirically determined relative retention
`_ .V._..._. .V.- __ ——— . V
`potential of the individual molecular species of a.cetyldiglyceri(le.'
`
`_...
`
`Molecular specigs
`of acetyldlglyceride
`
`Predigted relatixfe
`retention potential
`
`Wlflmpirically debermined
`relative retention
`Potential
`
`Relative
`error ("/0)
`
`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
`1.927
`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
`
`=I=*
`0.12
`:I==I<
`0.30
`1.42
`=I==I=
`0.69
`0.29
`1.48
`*=I=
`1.39
`*1:
`0.01
`0.76
`1.85
`0.27
`#21:
`2.27
`1.70
`0.16
`*2:
`0.05
`*1:
`0.52
`0.84
`1.30
`alwk
`0.88
`1.74
`1.54
`**
`**
`1.32
`*1:
`0.05
`0.29
`0.88
`>I==I=
`0.29
`0.68
`
`(20 2 5)(20 : 5)
`(20 : 5)(22 : 6)
`(22 2 6)(22 2 6)
`(18 2 3)(22 2 6)
`(20 2 4)(20 : 5)
`(22 : 5)(20 2 5)
`(20 -. 4)(22 : 6)
`(22 : 5)(22 : 6)
`(18 2 2)(2O 2 5)
`(16 : 1)(20 : 5)
`(18 -. 2)(22 : 6)
`(14 : 0)(20: 5)
`(162 1)(22 2 6)
`(18 2 2)(18 2 3)
`(20 2 4)(22 : 5)
`(14 2 0)(22 2 6)
`(22:11-)(22:6)
`(22 2 5)(22 : 5)
`(18 : 2)(20 : 4)
`(15 2 O)(20 2 5)
`(17 ; 2)(20 : 4)
`(18 2 2)(‘22 2 5)
`(172 1)(22 2 6)
`(16: l)(20 2 4)
`(17 2 2)(22 : 5)
`(162 1)(22 : 5)
`(15 2 O)(22 : 6)
`(18: l)(2O 2 5)
`(14 2 0)(22 :5)
`(18 2 2)(18 : 2)
`(18: 1)(22 2 6)
`(16 2 0)(20 : 5)
`(16: 1)(l8 2 2)
`(182 1)(18 2 3)
`(14 2 O)(18 2 2)
`(16 2 0)(22 : 6)
`(16 : O)(18 2 3)
`(16 2 0)(16 2 2)
`(18: 1)(20 2 4)
`(17 2 0)(22 : 6)
`
`1.572
`1.611
`1.649
`1.683
`1.695
`1.712
`1.734
`1.751
`1.762
`1.784
`1.8()0
`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
`
`— 401
`
`000005
`
`000005
`
`

`
`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`Table 2
`
`(continued).
`
`Molecular species
`of wcetyldiglyceride
`
`Predicted relative
`retention potential
`
`5)
`:1)(22:
`(18
`(16
`4)
`:0)(20:
`5)
`1)(20:
`(20 :
`5)
`:O)(22:
`(16
`2)
`1)(18:
`(18:
`(18:
`5)
`0)(20:
`6)
`1)(22:
`(20:
`.1)
`(1.8:
`1)(16:
`2)
`:O)(18:
`(16
`:O)(18:
`(14
`1)
`6)
`(18
`:0)(22:
`1)
`:0)(16:
`(16
`3)
`:O)(18:
`(18
`2)
`:O)(18:
`(17
`4)
`(16
`:0)(22:
`:O)(18:
`1)
`(15
`4)
`:O)(20:
`(18
`1)
`:0)(l7:
`(16
`1)
`(18
`:1)(18:
`1)
`(18
`:2)(20:
`1)
`O)(18:
`(16:
`1)
`:0)(20:
`(14
`0)
`:0)(16:
`(16
`O)(22:
`(13:
`4)
`(24
`:1)(20:
`11)
`:0)
`(18:
`2)(2o
`1)
`(13
`:O)(18:
`:0)(20:
`(16
`1)
`
`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
`2.362
`2.374
`2.424
`2.460
`2.489
`2.404
`
`Empirically detennined '
`relative retention
`potential
`2.067
`2.090
`2.098
`2.080
`2.137
`2.137
`2.138
`2.137
`2.148
`2.155
`2.172
`2.1.72
`2.184
`2.204
`2.126
`2.245
`2.254
`2.245
`2.281
`2.274
`2.321
`
`2.335
`2.328
`2.283
`2.424
`2.460
`2.480
`2.480
`
`Re1‘“i,§’“
`error ( /°)
`0.05
`*1:
`0.05
`.l.28
`0.94
`0.19
`*1:
`0.14
`0.42
`0.46
`*1!
`0.32
`0.95
`**
`3.71
`0.04
`0.09
`0.75
`0.09
`0.66
`0.09
`
`‘:0
`1.44
`3.83
`* *
`2|: 1|:
`0.36
`0.56
`
`‘ Relative retention time of (16: 0)(22 :6) is regarded as
`Analytical conditions are the same as in Table l.
`" 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 ~-
`
`000006
`
`000006
`
`

`
`TAKAIIASHI & HIRANO 2 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
`
`Prcdiiflted relatiyc
`rewntmn Potential
`
`lmpirieally determind
`relative retention
`potential
`
`Rglatisre
`error (/°)
`
`:17
`
`A
`
`:i
`
`B
`
`—l*
`
`C
`
`(16 2 1)(22 2 6)
`(182 1)(22 : 6)
`(16 : 1)(l6: 1)
`(16 : 0)(22 2 6)
`(18: I)(22 2 4)
`(16 : 0)(22 : 4)
`(18: 1)(18: 1)
`(16 : 0)(18: 1)
`(16 2 0)(16 2 0)
`
`(.16: l)(20 2 5)
`
`(18 : 2)(20 : 5)
`(16: 1)(22 2 6)
`
`(18 : 2)(22 : 6)
`(16 : 0)(20 2 5)
`(18: l)(20 : 5)
`(16 2 O)(22 : 6)
`(16 2 0)(16 : 0)
`
`(18 : 2)(18 ; 2)
`(16 : 0)(20 : 4)
`(18: 1)(18 : 2)
`
`(18 : 0)(20 2 4)
`(18: l)(18: 1)
`(16 : 0)(18: 1)
`(18 2 0)(18 2 2)
`(16 : 0)(16 2 0)
`
`2.080
`2.225
`2.238
`2.243
`2.414
`2.431
`2.529
`2.547
`2.564
`
`2.190
`
`2.210
`2.241
`
`2.260
`2.305
`2.305
`2.356
`2.534
`
`2.243
`2.332
`2.394
`
`2.490
`2.544
`2.564
`2.571
`2.583
`
`2.090
`2.225
`2.238
`2.243
`2.405
`2.431
`2.529
`2.555
`2.583
`
`2.190
`
`2.223
`2.230
`
`2.260
`2.305
`2.305
`2.356
`2.534
`
`2.243
`2.332
`2.389
`
`2.490
`2.544
`2.566
`2.571
`2.583
`
`0.49
`*1:
`ink
`=I==I=
`0.37
`*1:
`*1:
`0.33
`0.73
`
`alnk
`
`0.60
`0.47
`
`akalz
`*2):
`**
`'..O
`*1:
`
`*1:
`=I==|=
`0.18
`
`r':.-0
`*1:
`0.09
`*1:
`=I<aI=
`
`‘ Relative retention time of (12 2 0)(12 2 0) is regarded as 100.
`“ Relative retention potentials of the fatty acid residues are calculated from the log RR'l‘s of
`these molecular species.
`A: Calculated from the data. of Takamura at al.“
`
`Equipment, Hitachi 655-15 ; Hitachi 638--41 UV monitor; column, Ultrasphere ODS (250><
`4.6 mm) ; solvent, acetonitrile/isopropanol (42 1, v/v) ; flow, 1.0 ml/min; column tempera:
`tnre, 25'C.
`B2 Same condition as A except that the solvent system used is methanol/isopropanol (95 2 5,
`v/v).
`C 2 Calculated from the data of Kite et al."
`Same condition as A though the analyzed date is dillerent.
`
`-
`
`-- 403.»-
`
`000007
`
`000007
`
`

`
`Bull. Fac. Fish. Hokkaido Univ. 38(4), 1987.
`
`Acknowledgment
`
`Thanks are due to Professors M. Hatano, T. Takagi and K. Takama for their
`advices.
`
`References
`
`1)
`
`3)
`
`4)
`
`5)
`
`5)
`
`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).
`lecithin. Bull. Japan. Soc. Sci. Fish., 48,
`l803—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. Mam. Fae. Fish. Hokkaido Univ., 32, 245—330.
`Dean, R.B. and Dixon, W.J. (1951). Simplified statistics for small numbers of observations.
`Anal. C'hem., 23, 636--638.
`Takamura, H., Narita, H., Urade, R. and Kito, M. (1986). Quantitative analysis of polyenoic
`phospholipid molecular species by high performance liquid chromatography. Lipids, 21,
`356-361.
`
`Molecular species of fish muscle
`
`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, 327-331.
`
`7404-
`
`000008
`
`000008