Identification and Characterization of Two New Forced Degradation Products of Saikosaponin a under the Stress of Acid Hydrolytic Condition

Saikosaponin (SS) a is a compound with various pharmacological properties and easily degraded into SSb1 and SSg in acid condition. In present work another two new degradation products of SSa formed under acid hydrolytic condition were detected and isolated by analytical and semi-preparative liquid chromatography technology, furthermore their structures were characterized as hydroxyl-saikosaponin a and SSb2 by spectral analysis, which is not only essential in stability-indicating method development and validation but also could be used as a worst case to assess the analytical method performance of SSa. Moreover their structural transformation pathways were also proposed.


Figure 1. Structural formula of SSa During drug product development the International Conference on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines mention the necessity of stress testing to elucidate the inherent stability of the drug substance and the characterization of all degradation products formed in drug products.The information obtained may help to the establishment of the degradation patterns and a better quality control method [7,8].
Previous research indicated that among all the stress conditions mentioned in the ICH guidelines including heating in water, dry heating, oxidation, exposure of alkali solutions and solid power to light, SSa was relatively stable except for the stress of acid hydrolytic condition, in which the H + serves as an effective accelerator in the degradation process.A thorough literature survey revealed that the compounds such as SSb1 and SSg had been well documented as the degradation products of SSa under the acid condition [9][10][11][12][13].However in our detailed studies on the forced degradation of SSa in acid hydrolytic condition another two new degradation products were found, which needed to be characterized in accordance with ICH regulatory guidelines.
The present manuscript describes the structure characterization of the two new degradation products of SSa in acid hydrolytic condition and its structural transformation pathways.

Identification of new acid degradation products
The HPLC method was employed to achieve the best separation of the acid hydrolysis sample of SSa.During the optimization process, preliminary studies were carried out on Hypersil C18 column (4.6 mm×250 mm, 5 mm) using methanol: H2O (70:30, v/v) as a mobile phase [14].However the peaks corresponding to degradation products were not resolved completely.The peak 1 (Figure 2) was mixed with the solvent peaks under 210 nm, and the peak 3 and 4 (Figure 2) were badly overlapped under 254 nm.To get acceptable separation between SSa and its degradation products, the flow rate and composition of the mobile phase were systematically varied to optimize the method [15].Finally, adequate separation of peaks with good resolution was obtained using methanol (A) and H2O (B) in a gradient mode.The solvent program was set as follows: (Tmin/A:B T0/ 60:40; T8.0/60:40; T10/50:50; T20/60:40; T26/60:40).The flow rate of 1.0 mL•min -1 , column temperature at 30 o C, injection volume of 20 μL and wavelength of 210 and 254 nm were found to be suitable to achieve the separation (Figure 2).Under the optimized HPLC conditions, four degradation compound peaks were clearly presented (Figure 2).However the literature search revealed that under acid condition there were only two degradation products reported for SSa, which were SSb1 and SSg [9][10][11][12][13].Therefore there were another two new degradation products of SSa, which had not been clarified.By retention time comparation experiment with the authentic substances the peaks with the retention time of 21.50 min and 16.00 min were identified as SSb1 and SSg respectively.Then the peaks with the retention time of 4.50 min and 14.75 min were recognized as other two new unknown degradation products of SSa (Table 1 ).

Characterization of the two new degradation compounds
The two new degradation compounds, arising from SSa under the tress of acid hydrolyses process, led to the interest of its structure evaluation, which not only would be useful to the stability-indicating method establishment for SSa as a potential drug candidate but also could give more clues for the new understanding of the structure transformation mechanism.The isolation was performed on a semi-preparative HPLC system, and the NMR spectral analyses were used for the structure evaluation.

Degradation compound of peak 1
Peak 1 was isolated as white amorphous powder, positive Molish reaction, which molecular formula was determined to be C42H70O14 by the pseudomolecular ion peak at m/z 821.4546 [M+ Na] + in the positive HRESI-MS experiment.In the UV spectrum, there is no absorption peak, except a slope curve started from 220 nm to 200 nm (Supplementary Materials Figure S2), which indicates the absence of conjugated double bonds.Its 13 C NMR spectrum was similar to that of SSb3 (Table 2).However, the signal of a methoxyl group was detected in SSb3, but not in peak 1.By comparing the 13 C NMR chemical shifts of peak 1 with those of SSb3, the C-11 signal of the aglycone shifted upfield by 9.1 ppm and neighbouring C-9 and C-12 signals shifted downfield by 1.7 and 5.6 ppm (Table 2), respectively.These spectral data suggested that peak 1 possesses a hydroxyl group at C-11 of the aglycone moiety, but not a methoxyl group in SSb3.So its structure was determined as hydroxyl-saikosaponin a (Figure 3).Its NMR data were in agreement with the reported literature [16].

Degradation compound of peak 3
Peak 3 was obtained as white amorphous powder, positive Molish reaction, and exhibited a pseudomolecular ion peak in its positive-ion HRESI-MS at m/z 803.4559 [M+Na] + , corresponding to the molecular formula of C42H68O13.The UV spectrum shows characteristic heteroannular diene absorption (λmax 242, 254, 263 nm) (Supplementary Materials Figure S6).The 13 C NMR spectroscopic data of peak 3 showed close resemblance to those of SSb1 (Table 2).Their main difference was the signal at C16, which shifted upfield by 8.6 ppm in peak 3. The spectral data indicated that an α-OH was located at C16 of the aglycone in peak 3, while a β-OH was linked at C16 in SSb1.Then the structure of peak 3 was decided as SSb2 (Figure 3).The NMR data were identical to the reported literature [16].hydrolysis process.Figure 4 portrayed the proposed structure transformation route of hydroxyl-saikosaponin a. Firstly a proton transfers from hydronium ion (H3O + ) to the O atom of the 13β-28 epoxy ring in SSa forming an alkyloxonium ion, of which a dissociation is involved giving an allylic carbocation at C13.Because a vinyl group is an extremely effective electron-releasing substituent, a resonance interaction of the type shown permits the π electrons of the double bond at C11and C12 to be delocalized dispersing the positive charge, which means that the positive charge is shared by the two end carbons in the allylic unit.The positive charge on carbon and the vacant p orbital combine to make carbocation strongly electrophilic, which could be readily reacted with H2O which serves as a nucleophile.And because of the steric hindrance H2O would more likely react with the carbonation at C11 than C13.The bonds to the positively charged carbon are coplanar and define a plane of symmetry in the carbocation, which is achiral.Theoretically the H2O would attack the carbocation at its two mirror-image faces yielding two alkyloxonium ions with different configuration.But taking the effect of steric hindrance into consideration the alkyloxonium ion with α relative configuration at C11 predominates.At last another H2O acts as a base to remove a proton from the alkyloxonium ion to give the product of hydroxyl-saikosaponin a.

Structure transformation mechanism of SSb2
SSb2 had been known as the degradation product of SSd, an isomer of SSa with the only difference at the configuration of OH at C16.It was firstly detected in the acid hydrolysis system of SSa in our study.Based on the in-depth observation of the structure relationship SSb2 was proposed to be derived from the compound of SSb1.Being similar as hydroxyl-saikosaponin a, the formation of SSb2 was described in Figure 5. Protonation of O atom of the hydroxyl group at C16 firstly takes place mediated by the hydronium ion (H3O+) forming an alkyloxonium ion, which was followed by a dissociation giving a molecule of H2O and a carbocation at C16.Then another H2O acts as a nucleophile to capture the carbocation from the direction with a low level of steric hindrance, which permits the alkyloxonium ion at C16 with α relative congfigration to predominate.Then deprotonation of the alkyloxonium ion yields the product of SSb2, during which H2O acts as a Brønsted base.

Structure transformation path of SSa in acid condition
In the acid condition SSa could not only decomposed to the known degradation compounds SSb1 and SSg but also to SSb2 and hydrol-saikosaponin a which were firstly detected as another two new degradation compounds.It is clear that SSb2 SSb1 and SSg are the isomers of SSa and hydrol-saikosaponin a is derived from the hydration reaction of SSa with H2O.The structure transformation path of SSa in acid condition was displayed in Figure 6.
Moreover, in acidic condition SSa could also be hydrolyzed into prosaikogenin F A H and saikogenin F A H, during which the hydrolysis reaction takes place on the glycosidic bond of the sugar chain.But in our study the above mentioned aglycones and monofucoside of SSa were not detected, which could be explained that it is difficult for the hydrolyses reaction to take place on the sugar side chain under the reaction conditons used in this research.

Chemicals and Reagents
SSa SSb1 and SSg was supplied by Yuanye Bio-Technology Co., Ltd.(Shanghai, China).Methanol (HPLC grade) were purchased from Dikma (Tianjin, China) and used without purification.Analytical reagent grade hydrochloric acid and sodium hydroxide pellets were purchased from Wulian Chemical Factory (Shanghai, China).Ultrapure water was obtained from Milli-Q water (18M) (Millipore, USA).All other chemicals were of AR grade.

Sample preparation
SSa was dissolved in water under the aid of heat and sonication.The stock was diluted 50:50 (v/v) with 0.2N HCl, which was heated at 80 o C for 10 h.After cooling, the solution was neutralized by NaOH solution and applied onto a glass column (20 mm × 300 mm, i.d.) packed with AB-8 macroporous resin (Chemical Plant of Nankai University, Tianjin, China).Deioned water was used to remove the NaCl and 70% aqueous ethanol was used to elute the target compouds.After eluting, solvent was removed using a rotary evaporator under vacuum, and the residue was lyophilized.Three milligrams of the lyophilized sample was redissolved in 10 ml of methanol and filtered through 0.45 μm membrane filters for HPLC analysis.And 100 mg sample was used for the further purification of the degradation compounds on a semi-preparative HPLC.

NMR spectra
NMR spectra were recorded on a Bruker AV-400 spectrometers using C5D5N as solvent.Tetramethylsilane (TMS) was used as internal standard.The Chemical shifts (δ) are in ppm and the coupling constants ( J ) are in Hz.The sample for NMR analysis was obtained by semi-preparative HPLC.

Concluding remarks
According to the ICH guideline on impurities, it is required for the identification of the degradation products formed under a variety of stress conditions, and outlining of degradation pathways and mechanisms.The two new degradation products formed during forced degradation study on SSa under the stress of acid hydrolysis condition were identified as SSb2 and hydrol-saikosaponin a, which is not only essential in stability-indicating method development and validation but also could be used as a worst case to assess the analytical method performance for SSa.Moreover it can also help identify conditions under which additional controls should be employed in manufacturing and storage and help to understand the reactive chemistry of SSa, which is very valuable information with respect to the QbD knowledge space and the establishment of mass balance at early stage during forced degradation studies prevents surprises later during formal stability studies.

Supplementary Materials:
The following are available online at www.mdpi.com/link.

Figure 4 .
Figure 4. Proposed structure transformation pathway of hydroxyl-saikosaponin a

Figure 6 .
Figure 6.Structure transformation map of saikosaponin a under the stress of aicd hydrolytic condition

Table 1
Degradation products of saikosaponin a formed in acid forced degradation experiment No.