513
Characterization and identification of the chemical compositions in a Traditional
514
Uighur medicine prescription Yizhihao granule by LC-ESI-QTOF-MS
515
Dongyu Gu1,2, Yi Yang1, Ba Hang1, Qiaoying Lv1, Haji Akber Aisa1,*
516
517
1
518
Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences,
519
Urumqi 830011, China
520
2
521
Dalian 116023, China
Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization,
School of Marine Science and Environment Engineering, Dalian Ocean University,
522
523
Correspondence:
524
Prof. Dr. Haji Akber Aisa, E-mail: haji@ms.xjb.ac.cn, Tel: +86 991 3835679, Fax:
525
+86 991 3835679.
526
527
528
529
530
531
532
533
534
535
536
Supplementary Material
537
538
539
The identification of other compounds except for the compounds reported in text
540
Characterization of amino acids (Compounds 1, 3, 4, 6, 9, 10, 12 and 14)
541
Compound 1 (tR=1.12 min) displayed a [M+H]+ ion at m/z 175.1198
542
(C6H15N4O2), and its MS2 spectrum gave an ion at m/z 70 as base peak and a weak ion
543
at m/z 116 at the collision energy of 30 eV. The product ion at m/z 116 originated
544
from the loss of guanidinium group, and the ion at m/z 70 was formed by successive
545
loss of CH2O2. It was identified as arginine by comparison with the reference
546
standard.
547
Compounds 4 (tR=1.25 min), 6 (tR=1.32 min), 10 (tR=1.91 min) and 12 (tR=2.11
548
min) displayed [M+H]+ ions at m/z 116.0711 (C5H10NO2), 118.0870 (C5H12NO2),
549
132.1027 (C6H14NO2) and 132.1022 (C6H14NO2), respectively. The characteristic
550
fragment ions of these compounds were the ions at m/z 70 and 53, the ions at m/z 72
551
and 55, the ions at m/z 86 and 69 and the ions at m/z 86 and 69, separately. For all the
552
compounds, the MS2 fragmentation of protonated molecular ion led to the formation
553
of the ions [M+H-CH2O2]+ and [M+H-CH2O2-NH3]+, this fragmentation mechanism
554
was consistent with the literature which reported the fragments of free amino acid
555
with reference standards. Based on TOF-MS data, diagnostic fragment ions and
556
relevant literature, compounds 4, 6, 10 and 12 were identified as proline, valine,
557
isoleucine and leucine, respectively. Among them, valine, isoleucine and leucine were
558
undoubtedly identified by comparison with the reference standards.
559
Compound 9 (tR=1.62 min) was detected in negative-ion mode, and gave
560
molecular ion [M-H]- at m/z 128.0342 (C5H6NO3). The molecular composition was
561
retrieved in Combined Chemical Dictionary Database (Chapman & Hall/CRC,
562
London, UK), and L-pyroglutamic acid was the most possible candidates which from
563
Folium isatidis. Its MS2 spectrum showed weak fragment ions [M-CO]- at m/z 101
564
and [M-CO2]- at m/z 85. Thus, compound 9 was identified as L-pyroglutamic acid
565
which from the leaf of Isatis indigotica.
566
Compound 14 (tR=3.22min) was detected both in positive-ion mode and
567
negative-ion mode, and gave [M+H]+ ion at m/z 166.0866 (C9H12NO2) and [M-H]- at
568
m/z 164.0718 (C9H10NO2), respectively. In the MS2 spectrum of positive-ion mode,
569
the fragment ions [M+H-CH2O2]+ at m/z 120 and [M+H-CH2O2-NH3]+ at m/z 103
570
were observed, suggesting that compound 14 belonged to amino acid. The MS2 data
571
in negative-ion mode confirmed the deduce, which showed the diagnostic fragment
572
ions [M-H-NH3]- at m/z 147 and [M-H-NH3-CO2]- at m/z 103. So compound 14 was
573
identified as phenylalanine by comparison with reference standard.
574
575
Charactization of organinc acids
576
Aliphatic acids (compounds 5 and 7)
577
Two aliphatic acids (compounds 5 and 7) were detected in YG. Compound 5
578
(tR=1.31min) showed a [M-H]- ion at m/z 133.0137 (C4H5O5) and its MS2 spectrum
579
gave fragment ions [M-H-H2O]- at m/z 115 and [M-H-H2O-CO2]- at m/z 71.
580
Compound 7 (tR=1.56min) displayed a [M-H]- ion at m/z 191.0189 (C6H7O7) and its
581
MS2 spectrum gave fragment ions [M-H-H2O]- at m/z 173 and [M-H-2H2O-CO2]- at
582
m/z 111. Their molecular compositions indicated that malic acid and citric acid were
583
the most probable compounds, and the fragmentation mechanisms were consistent
584
with the literature. So, compounds 5 and 7 were plausibly identified as malic acid and
585
citric acid, respectively.
586
Benzoic acid derivatives (compounds 15, 16, 17, 21, 23, 24, 30, 36, 41 and 60)
587
Compounds 15 (tR=3.98min) and 17 (tR=5.78min) all displayed [M-H]- ions at
588
m/z 315.07 (C13H15O9) in negative-ion mode. The MS2 spectra of both compounds
589
were identical. Compound 15 gave a [M-H-163]- ion at m/z 152 (base peak) and a
590
[M-H-163-44]- ion at m/z 108 (ca. 93% of base peak), and compound 17 showed a
591
[M-H-162]- ion at m/z 153 (base peak) and a [M-H-162-44]- ion at m/z 109 (ca. 53%
592
of base peak). The loss of 162 Da suggested the presence of glucosyl group and
593
dihydroxybenzoic acid glucoside was the most probable compound according to
594
TOF-MS data and fragmentation mechanisms. The fragments difference between
595
compounds 15 and 17 may result from the different substituent positions. However,
596
the substitute positions of hydroxyl group and glucosyl group cannot be identified
597
without standard reference. Therefore, compounds 15 and 17 were tentatively
598
characterized as dihydroxybenzoic acid glucoside isomers.
599
Compounds 16 (tR=4.75min), 24 (tR=8.48min), 30 (tR=10.75min) and 41
600
(tR=13.49min) all exhibited [M-H]- ions at m/z 153.01 (C7H5O4) and their MS2 spectra
601
all gave a fragment ion [M-H-CO2]- at m/z 109 as base peak. The most possible
602
compound was dihydroxybenzoic acid according to the TOF-MS data, and the
603
fragment ions supported this deduction. The dihydroxybenzoic acid can be 2,3-, 2,4-,
604
2,5-, 2,6-, 3,4-, 3,5,- dihydroxybenzoic acid, and it was reported that 3,4-, 3,5,-, 2,3-,
605
2,5- and 2,4-dihydroxybenzoic acid was eluted out successively in reversed-phase
606
column. In addition, the MS2 spectrum of compound 41 showed a fragment ion
607
[M-H-18]- at m/z 135 with high abundance (ca. 64% of base peak) which was
608
different from the others. However, the substitute position still can’t be identified
609
based on the above information. Thus, compounds 16, 24, 30 and 41 were tentatively
610
characteristic as dihydroxybenzoic acid isomers.
611
Compounds 21 (tR=7.69min), 23 (tR=8.28min) and 60 (tR=17.46min) all
612
displayed the [M-H]- ions at m/z 137.02 (C7H5O3). Hydroxybenzoic acids were the
613
most probable compounds which from Isatis indigotica [21]. According to the
614
molecular composition, and the fragment ion [M-H-CO2]- at m/z 93 was observed to
615
be base peak in their MS2 spectra, which confirmed the conclusion. The three
616
compounds can be further distinguished by their chromatographic behavior which
617
p-hydroxybenzoic acid, m-hydroxybenzoic acid and o-hydroxybenzoic acid will be
618
eluted out in turn on reversed-phase column. However, some position isomers were
619
still difficult to be identified only by mass spectrometric data.
620
Compound 36 (tR=12.48min) showed [M-H]- ion at m/z 181.0137 (C8H5O5), and
621
its MS2 spectrum displayed ions [M-H-CO2]- at m/z 137 and [M-H-2CO2]- at m/z 93,
622
indicating the presence of two carboxyl group. Based on the molecular composition
623
and
fragmentation
mechanism,
compound
36
were
identified
624
2-hydroxy-1,4-benzenedicarboxylic acid which from Radix isatidis.
as
625
626
Chlorogenic acids(compounds 18, 29, 31, 32, 37, 42, 57, 59 and 64)
627
Compounds 18 (tR=7.69min), 29 (tR=10.46min), 32 (tR=11.25min) and 37
628
(tR=12.50min) showed a [M-H]- ion at m/z 353.08 (C16H17O9) in negative-ion mode,
629
which were considered to be the four isomers of caffeoylquinic acid (CQA). The base
630
peaks in their MS2 spectra were the ions at m/z 191, 191, 173 and 191, respectively,
631
and compound 18 exhibited the ion at m/z 179 with high abundance except 191. Thus,
632
compound 18 and 32 could be identified as 3-O-caffeoylquinic acid and
633
4-O-caffeoylquinic
634
5-O-caffeoylquinic acid or 1-O-caffeoylquinic acid. It is reported that 5-CQA has
635
greater hydrophobicity and will be eluted out first on reversed-phase column, so
636
compound 29 and 37 can be differentiated by the retention time. Compounds 31
637
(tR=11.07min) and 42 (tR=13.67min) both exhibited a [M–H]- ion at m/z 367.10
638
(C17H19O9) indicating that the two compounds were feruloylquinic acid isomers. If the
639
parent ion at m/z 367 leads to a product ion at m/z 193, 173 and 191, the linkage
640
position of feruloyl group should be assigned to the 3-OH, 4-OH or 5-OH position,
641
respectively. According to the rules above, compounds 31 and 42 were identified as
642
3-O-feruloylquinic acid and 4-O-feruloylquinic acid, separately. Their MS2 spectral
643
data are shown in Table 1.
644
acid,
compound
29
and
37
could
be
identified
as
Compounds 57 (tR=17.11min), 59 (tR=17.46min) and 64 (tR=18.32min) all gave
645
the [M-H]- ions at m/z 515.12 (C25H23O12), indicating that they were dicaffeoylquinic
646
acid isomers. These isomers could be distinguished by the intensity of product ions.
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
Figure S1. Negative QTOF-MS/MS spectra for isomeric dicaffeoylquinic acids
668
identified
669
4,5-O-dicaffeoylquinic acid and (C) 3,5-O-dicaffeoylquinic acid.
670
Compounds 57 and 64 all gave the ions at m/z 173 as base peak, so they were 4-acyl
671
dicaffeoylquinic acids. The two compounds can be further discriminated according to
672
the intensity of the ion at m/z 335. The MS2 spectrum of compound 57 exhibited an
673
ion at m/z 335, hence it was identified as 3,4-O-dicaffeoylquinic acid. Since the ion at
674
m/z 335 was not detected in the MS2 spectrum of compound 64, it was identified as
675
4,5-O-dicaffeoylquinic acid. Compound 59 showed the fragment ion at m/z 191 as
676
base peak, and the ion at m/z 335 was undetectable, it was identified as
677
3,5-O-dicaffeoylquinic acid. The MS2 spectra of the three compounds were shown in
678
Fig S1.
679
Charactization of alkaloids
680
Indole type alkaloids（compounds 13, 19, 20, 25, 26, 27, 39, 47 and 54）
in
Yizhihao
granule
(A)
3,4-O-dicaffeoylquinic
acid,
(B)
681
Compound 19 (tR=7.13min) gave a protonated ion at m/z 188.0703 (C11H10NO2)
682
and its MS2 spectrum contained a fragment ion [M+H-42]+ at m/z 146 corresponding
683
to loss of acetyl group and a fragment ion [M+H-42-28]+ at m/z 118 which produced
684
from the consecutive loss of formyl group. Besides, the fragment at m/z 91 was also
685
observed. Based on the molecular composition and fragment mechanism, compound
686
19 was characterized as acetyl-indolecarboxaldehyde.
687
Compound 25 (tR=9.65min) gave a molecular ion [M+H]+ at m/z 327.1346
688
(C18H19N2O4) and its MS2 spectrum showed a fragment ion at m/z 201. The molecular
689
composition suggested us that isaindigodione was the most probable compound. So
690
compound 25 was plausibly identified as isaindigodione which from Radix isatidis.
691
Compounds 26 (tR=9.7min) and 39 (tR=13.46min) both exhibited the [M+H]+
692
ions at m/z 134.06 (C8H8NO) and the characteristic fragment ions of indole alkaloid at
693
m/z 116, 91 and 77 were observed in its MS2 spectrum, suggesting that they were
694
hydroxylindole isomers. Beside the fragment ions above, a fragment ion [M+H-CO]+
695
at m/z 106 was also observed, indicating the substituent position of hydroxyl group
696
should be 2 and 3 in the indole structure, for the transformation between hydroxyl and
697
ketone group. 2-hydroxylindole should be more hydrophobic than 3-hydroxylindole
698
for the intra molecular hydrogen bond effect. Therefore, compounds 26 and 39 were
699
identified as 3-hydroxylindole and 2-hydroxylindole, respectively.
700
Compound 27 (tR=9.74min) yielded a [M-H]- ion at m/z 294.0966 (C14H16NO6)
701
in negative-ion mode. The molecular composition was retrieved in Combined
702
Chemical Dictionary Database (Chapman & Hall/CRC, London, UK), and indican
703
was the most possible candidates which from Isatis indigotica Fort. The MS2
704
spectrum of this ion exhibited a fragment ion at m/z 131 which produced from loss of
705
163 Da, suggesting the presence of glucoside moiety. Thus, compound 27 was
706
plausibly identified as indican (indoxyl-O-β-D-glucoside).
707
Compound 47 (tR=14.97min) gave a [M+H]+ ion at m/z 148.0395 (C8H6NO2) in
708
positive-ion mode and the characteristic fragment ion in its MS2 spectrum were the
709
ions at m/z 120, 92, 77 and 65, indicating that it is a indole type alkaloid. The most
710
possible compound was isatin which was from Folium isatidis. The product ion at m/z
711
120 was produced from the loss of CO. Therefore, compound 47 was identified as
712
isatin (2,3-dioxoindole).
713
Charactization of flavonoids
714
C-glycosidic flavonoids and C,O-glycosidic flavonoids （compounds 34, 35, 38, 43,
715
44, 45, 51 and 56）
716
Compounds 34 (tR=12.22min), 38 (tR=13.21min) and 43 (tR=13.85min)
717
displayed the same [M-H]- ion at m/z 593. The fragment ions [0,3Xo]- at m/z 341 and
718
[0,2Xo]- at m/z 311 were the characteristic ions for the compounds 34 and 43,
719
indicating that apigenin was their common aglycone (MW=270). And the fact
720
together with the molecular mass implied that compounds 34 and 43 most probably
721
contained two hexose. Compound 34 showed the fragment ions at m/z 503 ([M-H-90]-)
722
and 473 ([M-H-120]-) except 341 and 311, suggesting O-glycosidic hexose unit was
723
attached
724
(apigenin-7-O-glucoside-6-C-glucoside). The MS2 spectrum of compound 43 only
725
exhibited the fragment ions at m/z 341 and 311, signaled that O-glycosidic hexose
726
unit was attached to another sugar residue. Thus, it was identified as
727
isovitexin-6’’-O-glucopyranoside (apigenin-6’’-O-hexosyl-6-C-hexoside). The MS2
728
spectra of compound 38 displayed the characteristic fragment ions [0,2Xi0,2Xj]- at m/z
729
353 and [0,2Xi0,3Xj]- at m/z 383, also suggesting that apigenin was their aglycone.
730
According to the molecular mass, compound 38 was considered to contain two hexose
731
moieties. Thus, it were characterized as apigenin-6,8-di-C-hexoside. Their MS2
732
spectra were shown in Fig S2.
733
to
the
aglycone.
Thus,
it
was
identidied
as
saponarin
Compounds 35 (tR=12.34min) gave deprotonated molecular ion [M-H]- at m/z
734
755.2024. The characteristic fragment ions in its MS2 spectra were the ion [0,3Xo]- at
735
m/z 341 and [0,2Xo]- at m/z 311, indicating that apigenin (MW=270) was its common
736
aglycone. By calculating the difference between molecular mass and aglycone mass,
737
compound 35 was considered to contain three hexose units. A fragment ion at m/z 431
738
was also observed due to loss of 324 Da, suggesting that two hexose units connected
739
to the C-glycosidic sugar residue. Based on the above information, compound 35 was
740
characterized
741
(apigenin-6’’-O-dihexosyl-6-C-hexoside).
as
isovitexin-6’’-O-hexosylhexoside
742
Compounds 44 (tR=14.37min) and 45 (tR=14.38min) gave [M-H]- ion at m/z 447
743
and 563, respectively. The MS2 spectrum of compound 44 showed the fragment ion at
744
m/z 357 and 327, and the MS2 spectrum of compound 45 displayed the fragment ion
745
at m/z 503, 473, 443, 383 and 353. The fragment ions were similar to that of
746
luteolin-6-C-hexoside and apigenin-C-hexoside-C-pentoside reported in our previous
747
study. So, compounds 44 and 45 were characterized as luteolin-6-C-hexoside and
748
apigenin-C-hexoside-C-pentoside separately.
749
Compound 56 (tR=16.35min) gave a [M-H]- ion at m/z 461, and its MS2
750
spectrum displayed the characteristic fragment ion [0,3Xo]- at m/z 371 and [0,2Xo]- at
751
m/z 341, suggesting its aglycone molecular mass was 300 and the presence of a
752
hexose
753
(methylkaempferol-6-O-glucoside) which was found in Folium isatidis. In addition,
754
the fragment ion [M-H-163]- at m/z 298 and [M-H-162-14]- at m/z 285 further
755
confirmed the conclusion.
group.
The
most
probable
compound
was
isoscoparin
756
O-glycosidic flavonoids(compounds 50, 53, 55, 58, 62, 65, 66, 68 and 69)
757
Compounds 50 and 53 originated from quercetin. Compounds 50 (tR=15.68min)
758
and 53 (tR=16.21min) gave deprotonated ions at m/z 609 and 463, respectively. In
759
their MS2 spectrum, both displayed high abundance [Y0-H]-. ion at m/z 300 and the
760
product ions at m/z 271, 255 and 151, which were the diagnostic fragments of
761
quercetin. It is reported that flavonols substituted at the 3-OH position should yield a
762
high intensity radical aglycone ion [Y0-H]-, sometimes higher than the aglycone ion
763
[Y0]-, so the glycosylation site of theses two compounds should be 3-OH. By
764
comparing their product ion spectra and retention times with reference standards,
765
compounds 50 and 53 were identified as rutin (quercetin-3-O-rutinoside) and
766
hyperoside (quercetin-3-O-glucoside), respectively.
767
Compounds 55 (tR=16.3min) and 58 (tR=17.46min) both exhibited [M-H]- ions at
768
m/z 447, and their MS2 spectra showed product ion at m/z 285 and 284 as the base
769
peak, respectively. The aglycone fragment ion at m/z 285 can be attributed to luteolin
770
or kaempferol, however, luteolin and kaempferol could be distinguished according to
771
the characteristic fragment ions. The characteristic product ions at m/z 133, 151, 175
772
and 199 led to the aglycone identification as luteolin, whereas, m/z 271, 255, 227, 151
773
were the diagnostic fragment ions for kaempferol. As mentioned before, the cleavage
774
of a glucosyl group at the 3-OH position will produce a higher intensity of [Y0-H]-
775
than [Y0]-, so the glycosylation site of compound 58 should be 3-OH. By comparing
776
the retention times and mass data with the reference standards, compounds 55 and 58
777
were
unambiguously
attributed
to
luteolin-7-O-glucoside
and
astragalin
778
(kaempferol-3-O-glucoside), respectively, the mass spectral data are shown in Table
779
1.
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
Figure S2. Negative QTOF-MS/MS for isomeric flavonoid-C-glycosides identified in
801
Yizhihao
802
apigenin-6’’-O-hexosyl-6-C-hexoside and (C) apigenin-6,8-di-C-hexoside.
granule
(A)
apigenin-7-O-glucoside-6-C-glucoside,
(B)
803
Compounds 62, 65 and 66 originated from methoxylated flavonoids. Compounds
804
62 (tR=17.96min) and 66 (tR=18.46min) gave [M-H]- ions at m/z 607 and 475,
805
respectively. Their MS2 spectra contained a base peak ion [Y0]- at m/z 299 and a
806
[Y0-15]- ion at m/z 284, which was the characteristic fragmentation for the
807
monomethoxylated flavonoids. However, any of the hydroxyl groups of the flavonoid
808
aglycone can be methoxylated, it is difficult to give the definitive assignment of the
809
methoxy group, so the aglycone of these compounds was tentatively characterized as
810
methylkaempferol.
811
methylkaempferol-O-deoxyhexosylhexoside and methylkaempferol-O-glucuronide
812
due to loss of 308 (162+146) Da and 176 Da.
Compounds
62
and
66
were
characterized
as
813
Compound 68 (tR=21.23min) gave a formate adduct ion [M-H+46]- at m/z 637
814
and its MS2 spectrum showed a [Y0]- ion at m/z 283 and a [Y0-15]- ion at m/z 268.
815
This compound was identified as linarin (acacetin-7-O-rutinoside) by comparison of
816
retention time and mass data with the reference standard.
817
Compound 77 (tR=29.12min) exhibited [M-H]- ions at m/z 343, its MS2 spectrum
818
showed the fragment ions at m/z 328, 313, 298 and 270, indicating that it contained
819
three methoxy groups, and the fragments at m/z 270 corresponded to the loss of CO.
820
Based on the above information, compound 77 was plausibly characterized as
821
eupatorin which from Radix isatidis.
822