Analysis of Hydroxy Fatty Acids from the Pollen of Brassica campestris L. var. oleifera DC. by UPLC-MS/MS

Ultraperformance liquid chromatography coupled with negative electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) was used to determine 7 hydroxy fatty acids in the pollen of Brassica campestris L. var. oleifera DC. All the investigated hydroxy fatty acids showed strong deprotonated molecular ions [M–H]−, which underwent two major fragment pathways of the allyl scission and the β-fission of the alcoholic hydroxyl group. By comparison of their molecular ions and abundant fragment ions with those of reference compounds, they were tentatively assigned as 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (1), 10,11,12-trihydroxy-(7Z,14Z)-heptadecadienoic acid (2), 7,15,16-trihydroxy-9Z,12Z-octadecadienoic acid (3), 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (4), 15-hydroxy-6Z,9Z,12Z-octadecatrienoic acid (5), 15-hydroxy-9Z,12Z- octadecadienoic acid (6), and 15-hydroxy-12Z-octadecaenoic acid (7), respectively. Compounds 3, 5, and 7 are reported for the first time.


Introduction
Pollen contains many kinds of nutrient which are necessary to human body. It is well known as a natural nutrition, health food, and "perfect food. " e pollen preparation (Qian Lie Kang Tablets) made of Brassica campestris L. var. oleifera DC. has also been widely used in China as a good treatment for benign prostatic hyperplasia (BPH), and a chemical study has found plenty of fatty acids in the pollen [1]. Reports have shown promising therapeutic effects of fatty acids and their derivatives in treatment of BPH [2][3][4][5][6]. Hydroxy fatty acids contained in pollen are important bioactive substances. Various kinds of hydroxy fatty acids exhibit many pharmacological activities such as antitumor, antifungal, and prostaglandin E-like [7][8][9]. Although the pollen preparation has been used as phytotherapy for BPH for a long time, its active components and mechanism of action remain unclear. e effects of the supercritical �uid extract and its residue of the plant were screened so as to clarify its active constituents, and its supercritical �uid extract decreased the size of the prostate of androgen-induced prostatic rats by in vivo experiments ( and also demonstrated remarkable inhibitory effects on 5 -reductase and aromatase through in vitro experiments. e chemical investigation of its supercritical �uid extract has led to the isolation of some fatty acids and fatty acid derivatives [10], and the activity test experiments showed that fatty acids possessed strong inhibitory activity on 5 -reductase, which were consistent with literature reports [2][3][4]. e activity test experiments also displayed that fatty acid derivatives possessed strong inhibitory activity on aromatase [10]. 5reductase and aromatase are two important effect targets on prostate hyperplasia [11], so fatty acids and fatty acid derivatives play a coreaction role in treatment of BPH. UPLC-MS combine the efficient separation capability of UPLC and the great power in structural characterization of MS and provide new powerful approach to identify the constituents in plant extracts rapidly and accurately. In this paper, we investigated the fragmentation behaviors of hydroxy fatty acids in a Micromass Q/TOF Mass Spectrometer and emphasized on the structural determination of hydroxy fatty acids 1-7 ( Figure 1) from the supercritical �uid extract by UPLC-ESI-MS/MS.   ionizing source was electrospray ionization (negative ion mode); drying gas (N 2 ) �ow rate was 10.0 L/min; drying gas temperature was 320 ∘ C; capillary voltage was set to 3000 V; fragmentation voltage was set to 120 V; the full-scan second order mass spectra of the investigated compounds from m/z50-350 Da were measured using 500 ms for collection time and three microscans were summed.

Mass Spectrometry Analysis of Reference Compounds 1-2.
At �rst, the two reference compounds 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (1), and 10,11,12-trihydroxy-(7Z, 14Z)-heptadecadienoic acid (2), were analyzed by UPLC-ESI-MS. Both hydroxy fatty acids showed strong [M-H] − ions, which is similar to the hydroxy fatty acids reported in Tydemania expeditionis [12]. e full MS/MS product ion spectrum of compound 1 was shown in Figure 3. Six primary product ions are observed and it is proposed that they are formed in two major fragment pathways (Figure 4). Pathway I involves the -�ssion of alcoholic hydroxyl group along with the neutral loss of propionaldehyde and 2-hydroxy-butyraldehyde or 1hydroxy-2-butanone, which results in the formation of m/z 253 and m/z 223 as the major peaks. Pathway II involves the allyl scission leading to the formation of m/z 183 as the main peak. Some other pathways indicated neutral loss of H 2 O or CO or H 2 from the deprotonated molecular and fragments. e allyl scission of the molecular ion [M-H] − at m/z 313 of 2 leads to the formation of m/z 213, and the -�ssion of OH group of 2 lost 1,3-pentadiene and 2,3-dihydoxy-octa-5-enal or 1,3-dihydoxy-octa-5-en-2-one to produce the major peaks of m/z 245 and m/z 155. min, and compound 7 at 12.71 min, it was found that they showed almost the same fragment pattern with compounds 1-2.

Structural Analysis of Hydroxy Fatty Acids in the Fin
e molecular ion of compound 3 indicated an excess of 16 Da in comparison with that of 1, which suggested a surplus hydroxyl group in compound 2. e full MS/MS product ion spectrum (Figure 3) of compound 3 showed the characteristic product ions of m/z 239, 183, 269, 115, and 89, which were formed from the allyl scission and the -�ssion of OH group respectively (Figure 4). e fragment m/z 115 resulted from a -�ssion of the OH group, which showed a hydroxyl group on C-7. Some other pathways also displayed loss of H 2 O or CO from the deprotonated molecular and fragments. e con�guration of the ole�nic bonds was deduced from the biosynthetic pathway, and the natural sources of unsaturated fatty acids and their derivatives are rich in the cis isomer, so the ole�nic bonds of compound 3 were Z geometry. us, compound 3 was tentatively assigned as 7,15,16-trihydroxy-9Z,12Z-octadecadienoic acid, and it is reported for the �rst time.
e full MS/MS product ion spectrum ( Figure 3) of compound 4 showed the characteristic product ions of m/z 221, and 223, which were formed from the allyl scission, and another major peak of m/z 251 resulted from the -�ssion of the OH group ( Figure 4). ese characteristic product ions suggested an additional ole�nic bond at C-6 in compound 4 in comparison with 1. Some other pathways displayed loss of 18 Da or 30 Da from the deprotonated molecular and fragments. e con�guration of the ole�nic bonds was also determined from the biosynthetic pathway, and compound 4 was tentatively assigned as 15,16-dihydroxy-9Z,12Z-octadecadienoic acid, and 15S,16S-4 was reported in the literature [13].
e molecular ion of compound 5 indicated a lack of 16 Da in comparison with that of 4. e full MS/MS product ion spectrum (Figure 3) of compound 5 showed the characteristic peaks of m/z 73, 207, 221 and 251. e fragment at m/z 251 resulted from the -�ssion of OH group (Figure 4), which showed a hydroxyl group on C-15. e con�guration of the ole�nic bonds was also determined from the biosynthetic pathway, and compound 5 was tentatively assigned as 15hydroxy-6Z,9Z,12Z-octadecatrienoic acid, and it is reported for the �rst time.
e molecular ion of compound 6 indicated a lack of 16 Da in comparison with that of 1. e full MS/MS product ion spectrum (Figure 3) of compound 6 showed the characteristic peaks of m/z 73, 167, 223, and 253. e fragment at m/z 253 resulted from the -�ssion of OH group (Figure 4), which showed a hydroxyl group on C-15. e con�guration of the ole�nic bonds was also determined from the biosynthetic pathway, and compound 6 was tentatively assigned as 15-hydroxy-9Z,12Z-octadecadienoic acid, and 15R-6 was reported in the literature [14].
e molecular ion of compound 7 indicated an excess of 2 Da in comparison with that of 6. e full MS/MS product ion spectrum (Figure 3) of compound 7 showed the characteristic peaks of m/z 73, 127, 225, and 255. e fragment at m/z 255 resulted from the -�ssion of OH group (Figure 4), which showed a hydroxyl group on C-15. e con�guration of the ole�nic bonds was also determined from the biosynthetic pathway, and compound 7 was tentatively assigned as 15hydroxy-12Z-octadecaenoic acid, and it is reported for the �rst time.