Fragmentation and Ionization Efficiency of Positional and Functional Isomers of Paeoniflorin Derivatives in Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Paeoniflorin and albiflorin, which are functional isomers, are the major constituents of an herbal medicine derived from Paeonia lactiflora. Those functional isomers and their galloylated derivatives, which are positional isomers, were studied by matrix-assisted laser desorption/ionization–tandem mass spectrometry (MALDI-MS/MS). The resulting mass spectra are discussed based on the fragmentation patterns of the sodium adducts. The product ion spectra of 4-O-galloylalbiflorin and 4′-O-galloylpaeoniflorin differed, even though they were positional isomers. The fragmentations of the ester parts were influenced by the neighboring hydroxyl groups. The ionization efficiency of the sodium adduct of albiflorin was higher than that for paeoniflorin. These results indicate that the carboxylic ester group has a higher affinity for sodium ions than the acetal group, which can be attributed to the carbonyl oxygen being negatively polarized, allowing it to function as a Lewis base.


INTRODUCTION
e roots of Paeonia lacti ora are used in traditional herbal medicine for the treatment of female hormonerelated issues, including menopause, menstruation, blood problems, and general body pain. [1][2][3][4] Paeoni orin and albi orin, terpene functional isomers that contain D-L-βglucopyranosyl and benzoyl groups, are the major constituents of the roots of Paeonia lacti ora. Two new galloylated monoterpene glycosides (4-O-galloylalbi orin and 4′-Ogalloylpaeoni orin) ( Fig. 1) were recently isolated from the roots of Paeonia lacti ora that was obtained in the Nara Prefecture of Japan. 4) ese compounds were galloylated positional isomers and were reported to exhibit androgenmodulating and receptor-binding activities. 4,5) e chemical constituents of Paeonia lacti ora roots, including paeoniorin, albi orin, and their derivatives, were investigated via high-performance liquid chromatography, 6) electrospray ionization mass spectrometry (ESI-MS), and tandem mass spectrometry (MS/MS). [7][8][9][10] Molecular imaging of the metabolites in these plant tissues using an imaging MS technique was recently used to examine their functions. [11][12][13] e imaging MS was used to determine where the metabolites were localized in the plant tissues, since it is di cult prepare antibodies to such small molecules. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is one of the best methods for characterizing small molecules. erefore, the plant metabolites should be studied well in their ionization and fragmentations in MALDI-MS.
We initially attempted to distinguish between the positional isomers of 4′-O-galloylpaeoni orin and 4-Ogalloylalbi orin using their MALDI-MS/MS spectra, and then evaluated the ionization e ciency of the functional isomers of paeoni orin and albi orin based on their sodium adduct ions.

4-O-galloylalbi orin and 4′
-O-galloylpaeoni orin were isolated from the roots of Paeonia lacti ora (Paeoniae Radix), which were collected in the Nara Prefecture of Japan. Rutin was used as an internal standard for estimating the ionization e ciency of paeoni orin and albi orin in MALDI-MS. A 1 mg/mL stock solution of rutin was prepared in methanol. A 10-mg/mL solution of DHBA in 50% ethanol was prepared as a MALDI matrix.

Mass spectrometry
e MS and MS/MS product ion spectra (in the positive re ectron mode) were obtained using an Ultra ex III MALDI-TOF/TOF MS instrument from Bruker Daltonics GmbH (Bremen, Germany). e YAG-laser wavelength used was 355 nm. For the MS experiments, the accelerating voltage was 25 kV. For the MS/MS experiments, the initial accelerating voltage was 18 kV, which was later increased to 19 kV. e product ions were generated by post-source decay.

Estimation of ionization e ciency of paeoni orin and albi orin
e MALDI-MS ionization e ciencies of the analytes as a function of their concentration were estimated from their peak intensities, which were normalized against that of rutin or the internal standard. e concentrations of each analyte studied were 5, 10, 20, 40, 80, 100, and 150 µM. e concentrations of rutin and NaTFA (sodium dopant) in the samples were 100 µM and 1 µg/mL, respectively. e matrix solution was comprised of 10-mg/mL DHB and 0.1% formic acid in 70% methanol. Each MALDI-MS spectrum was generated from 500 scans. Peaks corresponding to paeoni orin and albi orin were both observed at m/z 503. ey were detected as the sodium adduct ions, [M pae +Na] + and [M alb +Na] + , respectively. eir peak intensities were normalized against the rutin peak [rutin+ Na] + at m/z 633. e peak intensities for paeoni orin, albi orin, and rutin were expressed as I pae , I alb , and I rutin , respectively. e ratios of I pae /I rutin and I alb /I rutin were plotted against each analyte concentration. e data presented are the averages of three measurements and the included error bars represent the standard deviations. In the spectrum of 4′-GP, product ion peaks at m/z 501 and 533 were observed, indicating that the galloyl (=154 u) and benzoyl (=122 u) groups had been removed from the molecule (Figs. 2B and 3). However, these product ions were not observed for 4-GA ( Fig. 2A). During the fragmentation of 4′-GP, the galloyl group located next to a hydroxyl group in the glucose unit was removed (Figs. 2B and 3). Meanwhile, the benzoyl group located next to a hydroxyl group in the paeoni orin aglycon was removed. e glycosyl bond of the glucose unit was cleaved with the aid of the C-2 glucosyl hydroxyl group (Figs. 1 and 3). is fragmentation has been previously reported in sugar chain fragmentation in lowenergy collision-induced dissociation ESI-MS/MS. 14,15) In 4-GA, no hydroxyl groups are located close to the benzoate or galloyl groups (Figs. 1 and 4). erefore, the glycosyl bond was cleaved rst due to the neighboring hydroxyl group (C-2) in the glucose unit. is resulted in an easier access to the new neighboring hydroxyl group in the glucose unit, further leading to the removal of the benzoyl group (Fig. 4). However, the galloyl group was not removed due to the absence of a neighboring hydroxyl group. In summary, the fragmentation of esters at the galloyl, benzoyl, and glucosyl groups of the analytes were induced by a neighboring hydroxyl group. ese ndings are critical for understanding the MALDI-MS/MS spectra of paeoni orin and albi orin derivatives, which contain the same structural units.

Ionization e ciency of albi orin and paeoni orin in MALDI
e sodium adduct ion [M+ Na] + dominated the MALDI-MS spectra of paeoni orin and albi orin. Figure  5 shows the ionization e ciencies of albi orin and paeoniorin as a function of concentration. e ionization eciencies were estimated from the intensities of the [M+ Na] + ions, which were normalized using the intensity of the internal standard or rutin (Fig. 5). Interestingly, the relative ion intensity of albi orin was higher than that of paeoni orin for all concentrations that were studied, indicating that the ionization e ciency of albi orin was higher than that of paeoni orin (Fig. 5). is nding suggests that the carboxylic ester of albi orin has a higher a nity for the sodium cation than the acetal group of paeoni orin. e sodium ions were able to more easily attach themselves to the oxygen atom due to the stronger polarization in the carboxylic ester than that in the acetal group. A similar situation was observed with the acetamide group. 16) Li et al. reported on the results of density functional theory calculations (DFT) on the negative charges of the oxygen atoms of paeoni orin and albi orin 10) eir results indicated that the oxygen atoms of the sugar hydroxyl groups and benzoyl esters had lower negative charges than those of the aglycon moieties. is indicates that the sodium cation has a high a nity for the sugar moiety and/or benzoyl esters, as re ected by DFT. However, the di erence in the ionization e ciency between albi orin and paeoni orin cannot be explained by the location of the sodium cation in  the molecule. e observed di erence can be attributed to di erences between the structures of albi orin and paeoniorin. erefore, the a nity of the carboxylic ester in albiorin appears to be stronger than that of the acetal group in paeoni orin for the sodium ion. It was recently reported that the acetamide groups had a much higher a nity for sodium ions compared with the hydroxyl and amino groups in sugars. is caused shi s in the charge center and di erences in the product ion spectra and fragmentation patterns of the sugar chains. 16) e acetamide group can be considered to be a key structure for the attachment of an alkali metal ion in the ion source. e carbonyl oxygen atom in the acetamide group was most likely polarized negatively, and showed Lewis basicity.
is carbonyl oxygen had a strong a nity for sodium ions. e MS/MS product ion spectra of paeoni orin and albi orin are presented in Fig. 6. e concentration of paeoni orin was four times greater than that of albi orin, as shown in Fig. 6. However, the intensities of the product ions of albi orin are stronger than that for paeoni orin (Fig.  6). Since paeoni orin and albi orin only di er in terms of an acetal or a carboxylic ester group, the higher ionization e ciency of albi orin can be attributed to the carboxylic ester group.
For both analytes, the product ions at m/z 341 and 381 were produced by the loss of glucosyl and benzoyl groups, respectively. In the MALDI-MS/MS spectrum of albi orin, the product ion at m/z 341 was the most dominant. is is due to the stability of the sodium adduct that was formed, which was facilitated by the carboxylic ester formed a er the cleavage of the glycosyl bond. Meanwhile, the paeoniorin spectrum contained a product ion at m/z 219 with a relative intensity that was greater than those of the product ions at m/z 341 and 381. is is because the product ions were labile due to the absence of the carboxylic ester group in paeoni orin. e aforementioned results con rm that  I pae and I alb indicate the peak intensity of paeoni orin (pae) and albi orin (alb) at m/z 503 as a sodium adduct ions. eir peak intensities were normalized to the internal standard rutin peak at m/z 633 (I rutin ). e ratios of I pae /I rutin (blue line) and I alb /I rutin (red line) were plotted against each concentration for the estimation of their ion e ciency. ese data represent the average of three measurements and the error bar indicates the standard deviation.
the carboxylic ester group has a higher a nity for a sodium cation than the ester group during ionization.

CONCLUSIONS
e neighboring hydroxyl groups, which are generally polarized, were found to mediate the ester fragmentation in 4′-galloyl paeoni orin and 4-galloyl albi orin. A proton is required during the ester fragmentation, and one is readily available from the neighboring hydroxyl group of the glucose moiety. us, the neighboring hydroxyl groups play a key role in the fragmentation of esters.
Comparing the ionization e ciencies of the functional isomers of albi orin and paeoni orin, the carboxylic ester group had a higher a nity for sodium compared with the acetal group in sodium adducts. e carbonyl oxygen is negatively polarized, thus having Lewis basicity characteristics.
e sodium adduct at the carboxylic ester is more stable than that for the acetal derivative.