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Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla
Accepted Manuscript Title: Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla Author: Wenzhi Yang Yibei Zhang Huiqin Pan Changliang Yao Jinjun Hou Shuai Yao Luying Cai Ruihong Feng Wanying Wu Dean Guo PII: DOI: Reference:
S0731-7085(16)30956-6 http://dx.doi.org/doi:10.1016/j.jpba.2016.10.021 PBA 10905
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Accepted date:
6-6-2016 25-10-2016
Please cite this article as: Wenzhi Yang, Yibei Zhang, Huiqin Pan, Changliang Yao, Jinjun Hou, Shuai Yao, Luying Cai, Ruihong Feng, Wanying Wu, Dean Guo, Supercritical fluid chromatography for separation and preparation of tautomeric 7epimeric spiro oxindole alkaloids from Uncaria macrophylla, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2016.10.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla Wenzhi Yang a,1, Yibei Zhang a,1, Huiqin Pan
a,b,1
, Changliang Yao
a,b
, Jinjun Hou a,
Shuai Yao a, Luying Cai a, Ruihong Feng a, Wanying Wu a,*, Dean Guo a
a
Shanghai Research Center for Modernization of Traditional Chinese Medicine,
National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China b
University of Chinese Academy of Sciences，No.19A Yuquan Road, Beijing 100049,
China
1
These authors contributed equally to this work.
Corresponding authors: Tel.: +86 21 20231000x2221; Fax: +86 21 50272789. E-mail address: [email protected] (W.-y. Wu).
1
Graphical abstract
Highlights
SFC was used to separate and isolate two pairs of 7-epimers of SOAs.
Acetonitrile stabilized two pairs of epimers and was used as modifier in SFC.
Two achiral UPC2 methods were established on the Torus 1-AA and Diol columns.
Preparative SFC enabled isolation of four SOA compounds with the purity >95%.
SFC provides a solution to preparation of high-purity reference standards.
Abstract Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can
2
easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of
ultra-high
performance SFC
(ultra-performance
convergence
chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.
Keywords:
Spiro
oxindole
alkaloid,
Uncaria
macrophylla,
Isomerization,
Ultra-performance convergence chromatography, Preparative scale supercritical fluid chromatography
3
1. Introduction Preparation of high-purity reference standards is a key segment for quality control of traditional Chinese medicine (TCM). However, due to rapid configurational change in polar or aqueous solvent or thermal instability [1], some natural compounds fail to be obtained in high purity and could not be used as the reference standards. On the other hand, the conventional separation and isolation techniques are always laborious and time-consuming, during which some unstable structures might suffer from transformation [2]. Thereby to develop new, efficient analytical and isolation approaches for preparation of high-purity reference standards is of vital significance for more scientific quality control of TCM. Spiro oxindole alkaloids (SOAs) are the bioactive components in the medicinal Uncaria species (Rubiaceae) [3-5], showing a broad spectrum of pharmacological properties (such as antihypertensive, sedative, antiarrhythmic, anticonvulsive, neuroprotective, immunostimulant, and depressed locomotive activities, etc). Moreover, SOAs from U. rhynchophylla have shown the potential to treat neurodegenerative diseases [6−9]. Despite the significant bioactivity and the vital role in quality control of Uncariae Ramulus cum Uncis (Gou-Teng) [10,11], SOAs are frequently reported to interconvert (isomerism) between 7-epimers in aqueous solution via retro-Mannich reaction, and the composition of equilibrium mixture depends on pH, temperature, and solvent polarity as well [12]. For pentacyclic SOAs (i.e. mitraphylline and isomitraphylline, formosanine and isoformosanine), four 3,7-isomers and two 7-isomers were formed for the cis and trans D/E ring junctions, 4
respectively.
In
contrast,
tetracyclic
SOAs
(i.e.
rhynchophylline
and
isorhynchophylline, corynoxeine and isocorynoxeine), were prone to produce two 7-isomers [13,14]. As a typical
case, refluxing of rhynchophylline (or
isorhynchophylline) in pyridine could yield a mixture of rhynchophylline and isorhynchophylline at the ratio of 3:7 [15]. The commonly-used purification technique by reversed-phase HPLC (RP-HPLC) fails to obtain high-purity SOA compounds in laboratories at ambient environment, due to the containing of water in the mobile phase (Fig. A.1). Therefore, new methods should be developed, aiming to capacitate efficient analysis and preparation of tautomeric SOA compounds. Supercritical fluid chromatography (SFC) that uses a mobile phase consisting of pressurized, heated CO2 and a proportion (less than 40%) of miscible polar organic solvents (modifier), is a powerful separation technique complementary to conventional LC and GC [16,17]. The low viscosity and good diffusivity (close to gas), high density and good solvating power (similar to liquid), of the supercritical or subcritical mobile phase enable a fast and high-resolution analysis by SFC. Addition of organic modifier, such as alcohols (methanol, ethanol, isopropanol, and trifluorethanol), dioxane, tetrahydrofuran, and acetonitrile, etc, facilitates the elution of polar and high molecular weight compounds [18]. On the other hand, addition of low concentrations of additives (acid or base) in the organic modifier is beneficial to peak shape and selectivity in SFC [19]. UPC2 integrates SFC and sub-2 µm particles, providing significantly improved repeatability, stability, and reliability, compared to conventional SFC instruments. Those problematic components, such as the lipophilic, 5
hydrophilic, and basic ingredients, could be well resolved by optimized UPC2 conditions [18].
Very recently, UPC2 has been employed in separation, isolation, or
quantitation of TCM components, such as curcuminoids [20], alkaloids [21,22], anthraquinones [23], and steroid saponins [24,25], which exhibited the superiority of higher efficiency, higher selectivity, and consumption of less organic solvents over RP-UHPLC.
In
addition,
hybridation
of
SFC
×
RP-UHPLC
in
offline
two-dimensional liquid chromatography achieved highly orthogonal separation of amide alkaloids from Piper longum [26]. Rhynchophylline (R; 7R/20R) and isorhynchophylline (IR; 7S/20R), corynoxine (C; 7S/20S) and corynoxine B (CB; 7R/20S) (Fig. 1), are present as two pairs of 7-epimers rich in U. macrophylla [27]. The aim of this study is to establish UPC2 methods targeting the rapid analysis of two pairs of 7-epimers, and a convertible preparative SFC approach for their isolation. Initially, the long-term stability of R, IR, C, and CB as a mixture in versatile solvents was tested to seek a solvent that can stabilize the epimers and is suitable for sample dissolving and also as the modifier in SFC. Subsequently, key parameters (i.e. stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate) were compared to optimize the UPC2 conditions. Ultimately, preparative scale SFC was used to purify four alkaloids based on the optimization results. As a result, two different achiral UPC 2 methods were established enabling the rapid analysis of four SOA components in U. macrophylla extract, and high-purity (> 95%) SOA reference standards were obtained by preparative SFC isolation on a Viridis Prep Silica 2-EP OBD column. To our 6
knowledge, it is the first report that utilizes SFC to separate and isolate SOAs from the Uncaria genus.
2. Experimental 2.1. Materials and reagents HPLC-grade methanol (MeOH), acetonitrile (MeCN), ethanol (EtOH) (Merck KGaA, Darmstadt, Germany), isopropanol (iPOH) (Burdick & Jackson, Honeywell International, Inc., USA), and ultra-pure water (18.2 MΩ·cm at 25°C) in-house prepared by a Millipore Alpha-Q water purification system (Millipore, Bedford, MA), were examined as the dissolving solvent or as the modifier for SFC. Diethylamine (TCI, Tokyo, Japan; DEA), diethanolamine (Aladdin, shanghai, China; DEOA), ammonium hydroxide (J&K, Beijing, China; AH), di-n-butylamine (Acros, New Jersey, USA; DBA), and triethylamine (J&K, Beijing, China; TEA), of HPLC grade were applied as the candidate additives in modifier. High-purity CO2 (99.999%) was purchased from Shanghai Yizhi Industry Gases Co., Ltd. (Shanghai, China). Analytical grade petroleum ether (60-90ºC), chloroform, ethyl acetate, methanol, ethanol, isopropanol, and acetonitrile used for extraction and column chromatography of U. macrophylla, were supplied by Shanghai Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Japan) was used to isolate and prepare SOAs-enriched fractions. The stems with hooks of U. macrophylla were collected from Guangxi Province of China in September, 2014, and were authenticated according to the morphological features as recorded in Flora of China. 7
2.2. Extraction and enrichment of alkaloids by MCI column chromatography Air-dried and pulverized crude sample of U. macrophylla (12 kg) was extracted under reflux with 95% ethanol for 2 h twice and subsequently with 60% ethanol for another time. The pooled extracts were evaporated to dryness under reduced pressure, yielding a dry residue (1.4 kg). After being suspended with water, the residue was successively extracted with petroleum ether, chloroform, and ethyl acetate, producing three fractions. An aliquot of 40 g of the ethyl acetate fraction was further separated on an MCI gel column eluted with gradient aqueous ethanol to yield five fractions (Fr. 1 to Fr. 5). Fr. 3 (0.8 g) and Fr. 4 (1 g), which severally contained rich IR/R and C/CB, were further purified by preparative SFC.
2.3. Column screening for UPC2 Seven achiral UPCC® columns from Waters Corporation (Milford, MA, USA) encompassing different functional groups were tested to optimize the UPC2 conditions, which involved BEH (3.0 × 100 mm, 1.7 μm), BEH 2-EP (3.0 × 100 mm, 1.7 μm), Torus DEA (3.0 × 100 mm, 1.7 μm), Torus Diol (3.0 × 100 mm, 1.7 μm), Torus 1-AA (3.0 × 100 mm, 1.7 μm), Torus 2-PIC (3.0 × 100 mm, 1.7 μm), and CSH FP (3.0 × 100 mm, 1.7 μm).
2.4. UPC2 Ultra-high performance SFC separation was performed on a Waters ACQUITY UPC2 system controlled by MassLynx V4.1 software, which involved a binary solvent manager, a sample manager (with a 10-μL loop), a column manager, an automatic 8
back pressure regulator (ABPR), and a PDA detector. Two achiral UPC2 chromatographic conditions were optimized in this study on two different columns: (1) on the Torus 1-AA column using 22% acetonitrile-0.1% DEA in CO2 as the mobile phase, a flow rate of 1.2 mL/min, column temperature of 45ºC, and ABPR pressure of 2000 Psi; (2) on the Torus Diol column using 21% acetonitrile-0.1% AH in CO2 as the mobile phase, a flow rate of 1.2 mL/min, column temperature of 30ºC, and ABPR pressure of 1800 Psi. The PDA detector was set at 245 nm for monitoring of four SOAs. An injection volume of 1 µL was used.
2.5. Preparative scale SFC Preparative isolation of four SOA isomers from the U. macrophylla extract was conducted on a Waters Prep 100q SFC System equipped with 2545 Quaternary Gradient Module, 2676 Sample Manager, the Column Oven, 2998 Photodiode Array Detector, the Injection Module, SFC Flow Splitter, Automatic Back Pressure Regulator, and a Heat Exchanger. A Viridis Prep Silica 2-EP OBD column (30 × 250 mm, 5 μm; Waters, MA, USA) was used. The mobile phase composed of 12% MeCN containing 0.2% DEA in CO2 and 8% MeCN containing 0.2% DEA were employed to isolate the mixtures of C/CB and R/IR obtained by MCI gel column chromatography, respectively. ABPR pressure was set at 150 bar and the flow rate of 80 mL/min was employed. The injection volumes were 1200 μL for isolation of C and CB (equivalent to 33.3 mg/mL of the drug material), and 500 μL for isolation of R and IR (40 mg/mL of the drug material), respectively. A makeup solvent of MeCN at 25 mL/min was used. 9
2.6. Identification of R, IR, C, and CB by high-resolution MS and NMR analyses Structural elucidation of R, IR, C, and CB, was performed by combined analyses of the high-resolution MS data acquired on a Waters Xevo® G2-S QTOF mass spectrometer (Waters Corporation, Milford, MA, USA), and 1H (500 MHz), 13
C-NMR (125 MHz) data (see Electronic Supplementary Information) obtained on a
Bruker AM-500 spectrometer (Karlsruhe, Germany). Structures of R, IR, C, and CB, were confirmed by comparing the 1H and
13
C-NMR data with those reported in
literature [28].
3. Results and discussion 3.1. Isomerization of R, IR, C, and CB in different solvents Given that polar protic solvents enable the interconversion of 7-epimeric SOAs [12-15], stability of R, IR, C, and CB in a mixture in different solvents was examined for nine days at 25ºC, aiming to find a suitable solvent for sample dissolving and as the modifier for SFC. Different protic/aprotic organic solvents (MeOH, EtOH, iPOH, and MeCN), aqueous organic solvents (each containing 20% H2O, v/v), and different ratios of mixtures containing two organic solvents (iPOH:MeCN 1:3, 1:1, and 3:1), were involved. A rapid UPC2 method (on a BEH column eluted by 7% methanol containing 0.1% DEA in CO2 at a flow rate of 1.8 mL/min was used to determine the dynamic isomerization of R/IR and C/CB. The relative content of each compound was expressed as the peak area percentage to the total of each pair of epimers. As a result, remarkable conversion from CB to C and from R to IR was observed, representing the transformation from 7R to 7S 10
configuration. 3.1.1. Isomerization between C and CB, R and IR, as a mixture The degree and rate of isomerization from CB to C (Fig. 2A1) and from R to IR (Fig. 2A2) maintained in different organic solvents displayed remarkable differentiation. Apparently, less than 5% of either CB or R was transformed in MeCN and iPOH within nine days, and, in contrast, the isomerization proportions of CB and R in MeOH were 26.75% and 16.97%, and 16.73% and 4.75% in EtOH. In particular, MeOH most favored the interconversion between 7-epimers. Rapid transformation occurred in the first four days, and the isomerization rate in different organic solvents was MeOH > EtOH > MeCN or iPOH. The addition of 20% H2O in four organic solvents further enhanced the conversion of both CB and R. Particularly, a large proportion of CB and R were transformed into their 7S-epimers in aqueous MeCN and MeOH (the order of transformation degree: aqueous MeCN > aqueous MeOH > aqueous EtOH > aqueous iPOH). The observation can be relevant to the fact that semi-preparative RP-HPLC that uses aqueous MeCN or MeOH failed to obtain high-purity CB and R in practice. Since extremely rare isomerization of R/IR and C/CB occurs in MeCN and iPOH, whether their combination at different ratios (MeCN:iPOH=3:1, 1:1, and 1:3) could better stabilize these epimers needs further investigation. However, actually the mixture of MeCN and iPOH at three different ratios all accelerated the isomerization, compared to pure MeCN or iPOH for both CB (Fig. 2B1) and R (Fig. 2B2). Previous studies have proposed a retro-Mannich ring opening, rotation and 11
Mannich ring closure reaction for the underlying isomerization mechanism [15]. As Gerhard Laus proposed, on one hand, more polar solvents stabilize the zwitterionic intermediate to reduce the energy barrier of reaction [13], which could be used to interpret the differential isomerization rate in different solvents (MeOH > EtOH > MeCN or iPOH), and on the other hand, apolar solvents stabilize the more lipophilic isomer in the equilibrium [14], which could be associated with the differentiated isomerization degree. Overall, MeCN has the potential to be a suitable solvent that can be used to dissolve samples and as the modifier in preparative SFC to isolate four target SOA compounds in high purity. 3.1.2. Isomerization of pure C, CB, R, and IR In order to validate the observed isomerization of R/IR and C/CB, the transformation of four later obtained pure compounds dissolved in MeOH and MeCN was further compared at 25ºC within 10 days. Despite an equilibrium ratio for R and IR had not been achieved up to the 10th day, the isomerization degree of four pure compounds in MeOH basically accorded with that obtained in a mixture, that is, the protic MeOH favored isomerization compared with the aprotic MeCN (Fig. A.2). When dissolved in MeOH, CB easily converted and C became predominant in equilibrium composition, while IR was more abundant estimated by the conversion tendency even if an equilibrium state had not reached. In detail, 31.7% of CB and 6.5% of R isomerized into the corresponding epimers in MeOH within 24 hours, which rendered it rather difficult to obtain pure SOA compounds, especially for CB. In contrast, these two pairs of epimers in MeCN 12
were relatively stable. Remarkable interconversion of epimers was detected from the 4th day, and the transformed proportions of C, IR, CB, and R at the 10th day were 6.47%, 3.97%, 0, and 1.46%, respectively. This evidence testifies that MeCN is a satisfactory solvent for sample dissolving and as the modifier in SFC preparation of pure SOA compounds at room temperature.
3.2. Optimization of UPC2 condition 3.2.1. Stationary phase The first concern for optimization of UPC2 condition is to select the proper stationary phase. In this study, seven achiral UPC2 customized columns were compared by gradient elution linearly increasing from 18% to 32% of MeCN containing 0.1% DEA in CO2. These columns, with the same dimension and particle size, possess various bonded functional groups, involving 2-ethyl pyridine (BEH 2-EP), 1-aminoanthracene (Torus 1-AA), diethylamine (Torus DEA), 2-picolylamine (Torus 2-PIC), and high-density diol (Torus Diol) on the framework of ethylene bridged hybrid particle, fluoro-phenyl (CSH FP) based on charged surface hybrid technology, and no bonded ethylene bridged hybrid particle (BEH) (Fig. 3). Significant selectivity difference in separation of four SOAs for seven stationary phases was witnessed (Fig. 3). Stereochemistry of two chiral centers at C-7 and C-20 gives rise to the structural difference among R, IR, C, and CB (Fig. 1). Generally, in addition to Torus DEA, well resolution between 7-epimers, that is, IR/R and C/CB, was achieved on the other six columns. However, resolution of IR and CB was poor on Torus 2-PIC, BEH, and BEH 2-EP columns. With regard to the CSH FP column, in 13
spite of its good resolution capacity, severe peak tailing occurred for all four SOAs, which correlates to its specific charged surface hybrid technology. Comparatively, better separation was obtained on Torus 1-AA and Torus Diol columns, as the former enabled baseline separation of all four analytes, whereas the latter exhibited the highest column efficiency (with the narrowest peaks). Therefore, these two columns (Torus 1-AA and Torus Diol) were selected for further optimization. 3.2.2. Additives in the modifier Basic components are prone to interact with silanols on the stationary phase (due to incomplete bonding reaction), thus displaying distorted peak shapes and stronger retention [29,30]. This situation can be improved by the addition of aliphatic amines, such as isopropylamine, DEA, ethyldimethylamine, or TEA, in organic modifier [18]. Herein, five basic additives, involving 0.1% DEA, 0.1% AH, 0.1% TEA, 0.1% DBA, and 5 mM DEOA, added in MeCN (modifier) were tested on Torus 1-AA and Torus Diol columns, respectively, to compare the influence on peak shape and selectivity (Fig. 4). Addition of TEA in MeCN as the modifier caused baseline fluctuation at the initial elution stage for both two columns. In the case of the Torus 1-AA column, the retention was enhanced and resolution was improved when DEOA or AH was added in the modifier, whilst little influence on retention capacity was observed by addition of DEA or DBA. In light of resolution and peak shape, the use of DEA as the additive facilitated the best chromatography performance. With respect to the Torus Diol column, beneficial effects on resolution obtained by addition of DEOA and AH were 14
significant, however, resolution of IR/CB sharply decreased when DEA or DBA was used as the additive. Given the low solubility in MeCN and low volatility (boiling point: 269ºC), DEOA may be a candidate additive for UPC2 analysis of four SOAs from U. macrophylla, but not suitable for preparative SFC isolation of pure SOA compounds. Therefore, AH was the best choice of additive on the Torus Diol column. These results were consistent with Aranyi’s report in UPC2 enantioseparation of amino-naphthol analogues, in which DEOA and AH had a beneficial influence on resolution [30]. The collaborative or competitive interactions between additives and stationary phase/basic analytes by two active sites contribute to the improvement in selectivity. Notably, use of AH as the additive on the Torus Diol column can be developed into a UPC2 method by MS detection. Subsequently, three different concentrations of each additive were compared, including 0.05%, 0.1%, and 0.2%, of DEA and AH in MeCN on Torus 1-AA and Torus Diol columns, respectively (Fig. A.3). Clearly, the influence arising from different concentrations of additives on both two columns was not remarkable. For the Torus 1-AA column, a tendency was discerned that increasing concentration of DEA weakened the retention capacity of four SOAs. However, for the Torus Diol column, less use of AH otherwise could weaken the retention, and negligible difference in both resolution and retention was observed between 0.1% and 0.2% of AH as the additive. In addition, four SOAs in a mixture dissolved in MeCN separately containing 0.05%, 0.1%, 0.2% DEA and AH could keep stable within five days at 25ºC (Fig. A.4). It suggests that use of MeCN containing 0.1% DEA as the modifier on the Torus 1-AA 15
column and MeCN containing 0.1% AH on the Torus Diol column is suitable for the establishment of UPC2 methods for SOAs analysis. 3.2.3. Column temperature In addition to the composition of modifier, temperature and pressure are another two vital factors influencing the density and solubility of the mobile phase, and thus determining the retention of analytes in SFC [18,31]. Here, the influence of different column temperature (15ºC, 30ºC, 45ºC, and 60ºC) on two columns was compared in terms of retention (tR), plate number (N), and resolution (Rs). First, the increase of column temperature weakened the retention of four SOAs on both two columns (Fig. 5). This observation disagrees with the common sense of SFC that decreased density of mobile phase at higher column temperature can enhance the retention of analytes, but accords with that in RP separation [30]. Second, plate number of four analytes dramatically increased when column temperature elevated from 15ºC to 60ºC on the Torus 1-AA column. Different results were obtained on the Torus Diol column that, despite the same increase displayed when column temperature increased from 15ºC to 45ºC, slight decrease was observed when temperature further elevated from 45ºC to 60ºC (R as an exception). Third, resolution of IR and CB showed different alternating trends on two columns. Better resolution for both IR and CB was obtained at 45ºC on the Torus 1-AA column and at 30ºC on the Torus Diol column. Taken together, the column temperature of 45ºC and 30ºC was set for Torus 1-AA and Torus Diol columns, respectively. 3.2.4. ABPR pressure and flow rate 16
Analogous to column temperature, ABPR pressure and flow rate also are vital parameters affecting SFC separation, given they can influence the density and solvent power of mobile phase [31]. ABPR pressure varying among 1600 Psi, 1800 Psi, 2000 Psi, 2200 Psi, and 2400 Psi, on both columns were examined. Negligible effect on resolution and small influence on retention were detected by altering ABPR pressure on UPC2 separation of four SOAs on two columns (Fig. A.5). Increasement of ABPR pressure enhanced the density and solvent power of mobile phase, and thus rendered decreased retention. However, the retention variations were not remarkable (from 4.57 min to 4.04 min for R on Torus 1-AA; from 6.46 min to 5.98 min for R on Torus Diol), which could be attributed to the less compressibility of the mobile phase due to higher ratio of organic solvent modifier (22% MeCN on Torus 1-AA and 21% MeCN on Torus Diol) [32]. In addition, the resolution of IR and CB remained in general in spite of the changes in ABPR pressure. Ultimately, ABPR pressure for the Torus 1-AA and Torus Diol columns were set at 2000 Psi and 1800 Psi, respectively. Influence of flow rate (0.8, 1.0, 1.2, 1.5, and 1.8 mL/min on Torus 1-AA; 1.0, 1.2, 1.5, 1.8, and 2.0 mL/min on Torus Diol) was assessed, given the differentiated optimal ABPR pressure of two columns (Fig. A.6). With the increase of flow rate, the retention of four SOA analytes weakened, giving rise to higher analytical efficiency on both two columns. It is ascribed to the stronger solvent power at higher flow rate. Slight decrease on resolution was distinguished when higher flow rate was used. In contrast, remarkable decrease occurred to plate number when flow rate elevated, 17
Hence, taking into account both analytical efficiency and resolution, an ideal flow rate at 1.2 mL/min was set for UPC2 separation on both two columns. Ultimately, after the systematic optimization of chromatographic conditions and instrument parameters, two rapid UPC2 analytical methods by isocratic elution were established separately on the Torus 1-AA column (Method I: 22% MeCN containing 0.1% DEA in CO2; flow rate, 1.2 mL/min; column temperature, 45ºC; ABPR pressure, 2000 Psi) and the Torus Diol column (Method II: 21% MeCN containing 0.1% AH in CO2; flow rate, 1.2 mL/min; column temperature, 30ºC; ABPR pressure, 1800 Psi). These two methods were further applied to analyze the total extract of U. macrophylla (Fig. 6). The first UPC2 approach, enabling 5-min rapid separation of four SOA analytes, is especially suitable for UV detection, whilst the second one is compatible to mass spectrometry, and thus can be used to probe into new minor SOAs from U. macrophylla and other Uncaria species as well.
3.3. Isolation of four SOA compounds by preparative scale SFC Since extremely fast interconversion occurs between SOA epimers when kept in aqueous organic solvents, it is rather difficult to isolate four SOA compounds from U. macrophylla in high purity by the conventional semi-preparative RP-HPLC. Taking advantage of the optimization results performed on UPC2, a preparative scale SFC approach was established by use of a commercially available Viridis Prep Silica 2-EP OBD column (30 × 250 mm, 5 μm). MeCN was used to dissolve samples and as the modifier, given isomerization between epimers can be inhibited, and DEA was added in MeCN as the additive to obtain symmetric peak shape. Moreover, MeCN was 18
selected as the makeup solution. Simultaneous baseline separation of four SOAs on the preparative Silica 2-EP OBD column was difficult with discounted analytical efficiency. Therefore, column chromatography was initially employed to fractionate them into different samples. Interestingly, MCI gel enabled the separation of four target SOAs by the difference in 20-configuration, and two pooled fractions containing mixed R/IR (R:IR 3:7) and mixed C/CB (C:CB 4:1) were obtained. Given the BEH 2-EP column was potent of separating 7-epimers as discussed above, a preparative column involving the same bonded functional group but on Viridis Hybrid particles with reduced surface silanol activity (Viridis Prep Silica 2-EP OBD) should have the potential to isolate these two pairs of 7-epimers. As a result, good separation of C/CB and R/IR was achieved within 15 min and 25 min, respectively (Fig. 7). 500 mg of C and 44 mg of CB (by duplicate preparation), 254 mg of IR and 61 mg of R (by duplicate preparation), were isolated from Fr. 3 and Fr. 4, respectively. The purity of four isolated compounds was all higher than 95% determined by the UPC2 method. In contrast to RP-HPLC, the established preparative SFC methods use water-free mobile phase (aprotic modifier and inert CO2), and allow efficient preparation and purification of SOAs.
4. Conclusion Preparation of high-purity reference standards is a vital segment in quality control of TCM. However, increasing difficulties resulting from chirality and configurational changes render is rather difficult to obtain high-purity reference 19
standards. This study used SFC to solve this issue by use of SOAs as an example. A proper solvent that could stabilize SOA epimers was screened in the first step. MeCN was able to suppress the epimeric interconversion of four target analytes both in a mixture and as pure compounds within three days, and thereby was employed to dissolve samples and as the modifier in SFC. Subsequently, the Torus 1-AA and Torus Diol columns showed good selectivity, and DEA, AH were separately used as the additives on these two columns to improve resolution. Two isocratic achiral UPC2 analytical methods were established, which suited UV and MS detection, respectively. Combined with MCI gel column chromatography, efficient preparative isolation of high-purity R, IR, C, and CB, was achieved on a Viridis Prep Silica 2-EP OBD column eluted by MeCN containing 0.2% DEA in CO2, which would lay a foundation for better quality control of Uncaria-derived herbal medicines and the future bioactivity screening of natural molecules against Alzheimer’s disease. SFC, by use of inert CO2 and aprotic modifier as the mobile phase, is proven as a feasible solution to rapid analysis and preparation of high-purity SOA reference standards, which is also recommended when other chiral or configurational issues are encountered in quality control of TCM.
Acknowledgements We gratefully acknowledge the financial support from the National Science and Technology Major Project for Major Drug Development (2013ZX09508104 and 2014ZX09304-307-001-007). We also appreciate Dr. Lirui Qiao and Yongwei Xu from Waters Corporation for the technical assistance. 20
Appendix A. Supplementary data Supplementary data associated with this article is available in the online version.
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spectrometry,
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Figure Caption Fig. 1 Structures of rhynchophylline (R), isorhynchophylline (IR), corynoxine (C), and corynoxine B (CB), four major spiro oxindole alkaloids in Uncaria macrophylla.
26
Fig. 2 Conversion of corynoxine B to corynoxine and rhynchophylline to isorhynchophylline dissolved in different pure organic and aqueous organic solvents (A) and different ratios of isopropanol and acetonitrile (B) within nine days at 25℃. R: rhynchophylline, IR: isorhynchophylline, C: corynoxine, CB: corynoxine B.
27
Fig. 3 Comparison of seven achiral chromatographic columns to the separation of two pairs of SOA epimers.
28
Fig. 4 Comparison of five different base additives in modifier (0.1% AH, TEA, DEA, DBA, and 5 mM DEOA) to the separation of two pairs of SOA epimers on the Torus 1-AA and Torus Diol columns.
29
Fig. 5 Comparison of different column temperature (15ºC, 30ºC, 45ºC, and 60ºC) to the separation of two pairs of SOA epimers on the Torus 1-AA and Torus Diol columns, evaluated by retention (tR), plate number (N), and resolution of IR and CB (Rs).
30
Fig. 6 The UV spectra (245 nm) of the total extract of U. macrophylla obtained on the Torus 1-AA (A) and Torus Diol columns (B). The BPI spectrum (C) was recorded on a Waters UPLC/QTOF MS instrument. (A: Torus 1-AA: 78% CO2-22% MeCN containing 0.1% DEA; flow rate, 1.2 mL/min; ABPR pressure, 2000 Psi; column temperature, 45ºC; B: Torus Diol: 79% CO2-21% MeCN containing 0.1% AH; flow rate, 1.2 mL/min; ABPR pressure, 1800 psi; column temperature, 30ºC; C: Torus Diol: 0-7 min, 21% MeCN containing 0.1% AH, 7-8 min, 21-40% MeCN containing 0.1% AH, 8-10 min, 40% MeCN containing 0.1% AH; flow rate, 1.2 mL/min; ABPR pressure, 1800 Psi; column temperature, 30ºC).
31
Fig. 7 Illustration of the preparative SFC isolation (A) and purity check by UPC 2 (B) of four SOA compounds from U. macrophylla. Mobile phase: 12% acetonitrile containing 0.2% DEA for the isolation of C and CB; 8% acetonitrile containing 0.2% DEA for the isolation of IR and R; ABPR pressure, 150 bar; flow rate, 80 mL/min; the make-up solution, 25 mL/min of MeCN.
32
S0731-7085(16)30956-6 http://dx.doi.org/doi:10.1016/j.jpba.2016.10.021 PBA 10905
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Accepted date:
6-6-2016 25-10-2016
Please cite this article as: Wenzhi Yang, Yibei Zhang, Huiqin Pan, Changliang Yao, Jinjun Hou, Shuai Yao, Luying Cai, Ruihong Feng, Wanying Wu, Dean Guo, Supercritical fluid chromatography for separation and preparation of tautomeric 7epimeric spiro oxindole alkaloids from Uncaria macrophylla, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2016.10.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla Wenzhi Yang a,1, Yibei Zhang a,1, Huiqin Pan
a,b,1
, Changliang Yao
a,b
, Jinjun Hou a,
Shuai Yao a, Luying Cai a, Ruihong Feng a, Wanying Wu a,*, Dean Guo a
a
Shanghai Research Center for Modernization of Traditional Chinese Medicine,
National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Haike Road 501, Shanghai 201203, China b
University of Chinese Academy of Sciences，No.19A Yuquan Road, Beijing 100049,
China
1
These authors contributed equally to this work.
Corresponding authors: Tel.: +86 21 20231000x2221; Fax: +86 21 50272789. E-mail address: [email protected] (W.-y. Wu).
1
Graphical abstract
Highlights
SFC was used to separate and isolate two pairs of 7-epimers of SOAs.
Acetonitrile stabilized two pairs of epimers and was used as modifier in SFC.
Two achiral UPC2 methods were established on the Torus 1-AA and Diol columns.
Preparative SFC enabled isolation of four SOA compounds with the purity >95%.
SFC provides a solution to preparation of high-purity reference standards.
Abstract Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can
2
easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of
ultra-high
performance SFC
(ultra-performance
convergence
chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.
Keywords:
Spiro
oxindole
alkaloid,
Uncaria
macrophylla,
Isomerization,
Ultra-performance convergence chromatography, Preparative scale supercritical fluid chromatography
3
1. Introduction Preparation of high-purity reference standards is a key segment for quality control of traditional Chinese medicine (TCM). However, due to rapid configurational change in polar or aqueous solvent or thermal instability [1], some natural compounds fail to be obtained in high purity and could not be used as the reference standards. On the other hand, the conventional separation and isolation techniques are always laborious and time-consuming, during which some unstable structures might suffer from transformation [2]. Thereby to develop new, efficient analytical and isolation approaches for preparation of high-purity reference standards is of vital significance for more scientific quality control of TCM. Spiro oxindole alkaloids (SOAs) are the bioactive components in the medicinal Uncaria species (Rubiaceae) [3-5], showing a broad spectrum of pharmacological properties (such as antihypertensive, sedative, antiarrhythmic, anticonvulsive, neuroprotective, immunostimulant, and depressed locomotive activities, etc). Moreover, SOAs from U. rhynchophylla have shown the potential to treat neurodegenerative diseases [6−9]. Despite the significant bioactivity and the vital role in quality control of Uncariae Ramulus cum Uncis (Gou-Teng) [10,11], SOAs are frequently reported to interconvert (isomerism) between 7-epimers in aqueous solution via retro-Mannich reaction, and the composition of equilibrium mixture depends on pH, temperature, and solvent polarity as well [12]. For pentacyclic SOAs (i.e. mitraphylline and isomitraphylline, formosanine and isoformosanine), four 3,7-isomers and two 7-isomers were formed for the cis and trans D/E ring junctions, 4
respectively.
In
contrast,
tetracyclic
SOAs
(i.e.
rhynchophylline
and
isorhynchophylline, corynoxeine and isocorynoxeine), were prone to produce two 7-isomers [13,14]. As a typical
case, refluxing of rhynchophylline (or
isorhynchophylline) in pyridine could yield a mixture of rhynchophylline and isorhynchophylline at the ratio of 3:7 [15]. The commonly-used purification technique by reversed-phase HPLC (RP-HPLC) fails to obtain high-purity SOA compounds in laboratories at ambient environment, due to the containing of water in the mobile phase (Fig. A.1). Therefore, new methods should be developed, aiming to capacitate efficient analysis and preparation of tautomeric SOA compounds. Supercritical fluid chromatography (SFC) that uses a mobile phase consisting of pressurized, heated CO2 and a proportion (less than 40%) of miscible polar organic solvents (modifier), is a powerful separation technique complementary to conventional LC and GC [16,17]. The low viscosity and good diffusivity (close to gas), high density and good solvating power (similar to liquid), of the supercritical or subcritical mobile phase enable a fast and high-resolution analysis by SFC. Addition of organic modifier, such as alcohols (methanol, ethanol, isopropanol, and trifluorethanol), dioxane, tetrahydrofuran, and acetonitrile, etc, facilitates the elution of polar and high molecular weight compounds [18]. On the other hand, addition of low concentrations of additives (acid or base) in the organic modifier is beneficial to peak shape and selectivity in SFC [19]. UPC2 integrates SFC and sub-2 µm particles, providing significantly improved repeatability, stability, and reliability, compared to conventional SFC instruments. Those problematic components, such as the lipophilic, 5
hydrophilic, and basic ingredients, could be well resolved by optimized UPC2 conditions [18].
Very recently, UPC2 has been employed in separation, isolation, or
quantitation of TCM components, such as curcuminoids [20], alkaloids [21,22], anthraquinones [23], and steroid saponins [24,25], which exhibited the superiority of higher efficiency, higher selectivity, and consumption of less organic solvents over RP-UHPLC.
In
addition,
hybridation
of
SFC
×
RP-UHPLC
in
offline
two-dimensional liquid chromatography achieved highly orthogonal separation of amide alkaloids from Piper longum [26]. Rhynchophylline (R; 7R/20R) and isorhynchophylline (IR; 7S/20R), corynoxine (C; 7S/20S) and corynoxine B (CB; 7R/20S) (Fig. 1), are present as two pairs of 7-epimers rich in U. macrophylla [27]. The aim of this study is to establish UPC2 methods targeting the rapid analysis of two pairs of 7-epimers, and a convertible preparative SFC approach for their isolation. Initially, the long-term stability of R, IR, C, and CB as a mixture in versatile solvents was tested to seek a solvent that can stabilize the epimers and is suitable for sample dissolving and also as the modifier in SFC. Subsequently, key parameters (i.e. stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate) were compared to optimize the UPC2 conditions. Ultimately, preparative scale SFC was used to purify four alkaloids based on the optimization results. As a result, two different achiral UPC 2 methods were established enabling the rapid analysis of four SOA components in U. macrophylla extract, and high-purity (> 95%) SOA reference standards were obtained by preparative SFC isolation on a Viridis Prep Silica 2-EP OBD column. To our 6
knowledge, it is the first report that utilizes SFC to separate and isolate SOAs from the Uncaria genus.
2. Experimental 2.1. Materials and reagents HPLC-grade methanol (MeOH), acetonitrile (MeCN), ethanol (EtOH) (Merck KGaA, Darmstadt, Germany), isopropanol (iPOH) (Burdick & Jackson, Honeywell International, Inc., USA), and ultra-pure water (18.2 MΩ·cm at 25°C) in-house prepared by a Millipore Alpha-Q water purification system (Millipore, Bedford, MA), were examined as the dissolving solvent or as the modifier for SFC. Diethylamine (TCI, Tokyo, Japan; DEA), diethanolamine (Aladdin, shanghai, China; DEOA), ammonium hydroxide (J&K, Beijing, China; AH), di-n-butylamine (Acros, New Jersey, USA; DBA), and triethylamine (J&K, Beijing, China; TEA), of HPLC grade were applied as the candidate additives in modifier. High-purity CO2 (99.999%) was purchased from Shanghai Yizhi Industry Gases Co., Ltd. (Shanghai, China). Analytical grade petroleum ether (60-90ºC), chloroform, ethyl acetate, methanol, ethanol, isopropanol, and acetonitrile used for extraction and column chromatography of U. macrophylla, were supplied by Shanghai Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). MCI gel (75-150 μm, Mitsubishi Chemical Corporation, Japan) was used to isolate and prepare SOAs-enriched fractions. The stems with hooks of U. macrophylla were collected from Guangxi Province of China in September, 2014, and were authenticated according to the morphological features as recorded in Flora of China. 7
2.2. Extraction and enrichment of alkaloids by MCI column chromatography Air-dried and pulverized crude sample of U. macrophylla (12 kg) was extracted under reflux with 95% ethanol for 2 h twice and subsequently with 60% ethanol for another time. The pooled extracts were evaporated to dryness under reduced pressure, yielding a dry residue (1.4 kg). After being suspended with water, the residue was successively extracted with petroleum ether, chloroform, and ethyl acetate, producing three fractions. An aliquot of 40 g of the ethyl acetate fraction was further separated on an MCI gel column eluted with gradient aqueous ethanol to yield five fractions (Fr. 1 to Fr. 5). Fr. 3 (0.8 g) and Fr. 4 (1 g), which severally contained rich IR/R and C/CB, were further purified by preparative SFC.
2.3. Column screening for UPC2 Seven achiral UPCC® columns from Waters Corporation (Milford, MA, USA) encompassing different functional groups were tested to optimize the UPC2 conditions, which involved BEH (3.0 × 100 mm, 1.7 μm), BEH 2-EP (3.0 × 100 mm, 1.7 μm), Torus DEA (3.0 × 100 mm, 1.7 μm), Torus Diol (3.0 × 100 mm, 1.7 μm), Torus 1-AA (3.0 × 100 mm, 1.7 μm), Torus 2-PIC (3.0 × 100 mm, 1.7 μm), and CSH FP (3.0 × 100 mm, 1.7 μm).
2.4. UPC2 Ultra-high performance SFC separation was performed on a Waters ACQUITY UPC2 system controlled by MassLynx V4.1 software, which involved a binary solvent manager, a sample manager (with a 10-μL loop), a column manager, an automatic 8
back pressure regulator (ABPR), and a PDA detector. Two achiral UPC2 chromatographic conditions were optimized in this study on two different columns: (1) on the Torus 1-AA column using 22% acetonitrile-0.1% DEA in CO2 as the mobile phase, a flow rate of 1.2 mL/min, column temperature of 45ºC, and ABPR pressure of 2000 Psi; (2) on the Torus Diol column using 21% acetonitrile-0.1% AH in CO2 as the mobile phase, a flow rate of 1.2 mL/min, column temperature of 30ºC, and ABPR pressure of 1800 Psi. The PDA detector was set at 245 nm for monitoring of four SOAs. An injection volume of 1 µL was used.
2.5. Preparative scale SFC Preparative isolation of four SOA isomers from the U. macrophylla extract was conducted on a Waters Prep 100q SFC System equipped with 2545 Quaternary Gradient Module, 2676 Sample Manager, the Column Oven, 2998 Photodiode Array Detector, the Injection Module, SFC Flow Splitter, Automatic Back Pressure Regulator, and a Heat Exchanger. A Viridis Prep Silica 2-EP OBD column (30 × 250 mm, 5 μm; Waters, MA, USA) was used. The mobile phase composed of 12% MeCN containing 0.2% DEA in CO2 and 8% MeCN containing 0.2% DEA were employed to isolate the mixtures of C/CB and R/IR obtained by MCI gel column chromatography, respectively. ABPR pressure was set at 150 bar and the flow rate of 80 mL/min was employed. The injection volumes were 1200 μL for isolation of C and CB (equivalent to 33.3 mg/mL of the drug material), and 500 μL for isolation of R and IR (40 mg/mL of the drug material), respectively. A makeup solvent of MeCN at 25 mL/min was used. 9
2.6. Identification of R, IR, C, and CB by high-resolution MS and NMR analyses Structural elucidation of R, IR, C, and CB, was performed by combined analyses of the high-resolution MS data acquired on a Waters Xevo® G2-S QTOF mass spectrometer (Waters Corporation, Milford, MA, USA), and 1H (500 MHz), 13
C-NMR (125 MHz) data (see Electronic Supplementary Information) obtained on a
Bruker AM-500 spectrometer (Karlsruhe, Germany). Structures of R, IR, C, and CB, were confirmed by comparing the 1H and
13
C-NMR data with those reported in
literature [28].
3. Results and discussion 3.1. Isomerization of R, IR, C, and CB in different solvents Given that polar protic solvents enable the interconversion of 7-epimeric SOAs [12-15], stability of R, IR, C, and CB in a mixture in different solvents was examined for nine days at 25ºC, aiming to find a suitable solvent for sample dissolving and as the modifier for SFC. Different protic/aprotic organic solvents (MeOH, EtOH, iPOH, and MeCN), aqueous organic solvents (each containing 20% H2O, v/v), and different ratios of mixtures containing two organic solvents (iPOH:MeCN 1:3, 1:1, and 3:1), were involved. A rapid UPC2 method (on a BEH column eluted by 7% methanol containing 0.1% DEA in CO2 at a flow rate of 1.8 mL/min was used to determine the dynamic isomerization of R/IR and C/CB. The relative content of each compound was expressed as the peak area percentage to the total of each pair of epimers. As a result, remarkable conversion from CB to C and from R to IR was observed, representing the transformation from 7R to 7S 10
configuration. 3.1.1. Isomerization between C and CB, R and IR, as a mixture The degree and rate of isomerization from CB to C (Fig. 2A1) and from R to IR (Fig. 2A2) maintained in different organic solvents displayed remarkable differentiation. Apparently, less than 5% of either CB or R was transformed in MeCN and iPOH within nine days, and, in contrast, the isomerization proportions of CB and R in MeOH were 26.75% and 16.97%, and 16.73% and 4.75% in EtOH. In particular, MeOH most favored the interconversion between 7-epimers. Rapid transformation occurred in the first four days, and the isomerization rate in different organic solvents was MeOH > EtOH > MeCN or iPOH. The addition of 20% H2O in four organic solvents further enhanced the conversion of both CB and R. Particularly, a large proportion of CB and R were transformed into their 7S-epimers in aqueous MeCN and MeOH (the order of transformation degree: aqueous MeCN > aqueous MeOH > aqueous EtOH > aqueous iPOH). The observation can be relevant to the fact that semi-preparative RP-HPLC that uses aqueous MeCN or MeOH failed to obtain high-purity CB and R in practice. Since extremely rare isomerization of R/IR and C/CB occurs in MeCN and iPOH, whether their combination at different ratios (MeCN:iPOH=3:1, 1:1, and 1:3) could better stabilize these epimers needs further investigation. However, actually the mixture of MeCN and iPOH at three different ratios all accelerated the isomerization, compared to pure MeCN or iPOH for both CB (Fig. 2B1) and R (Fig. 2B2). Previous studies have proposed a retro-Mannich ring opening, rotation and 11
Mannich ring closure reaction for the underlying isomerization mechanism [15]. As Gerhard Laus proposed, on one hand, more polar solvents stabilize the zwitterionic intermediate to reduce the energy barrier of reaction [13], which could be used to interpret the differential isomerization rate in different solvents (MeOH > EtOH > MeCN or iPOH), and on the other hand, apolar solvents stabilize the more lipophilic isomer in the equilibrium [14], which could be associated with the differentiated isomerization degree. Overall, MeCN has the potential to be a suitable solvent that can be used to dissolve samples and as the modifier in preparative SFC to isolate four target SOA compounds in high purity. 3.1.2. Isomerization of pure C, CB, R, and IR In order to validate the observed isomerization of R/IR and C/CB, the transformation of four later obtained pure compounds dissolved in MeOH and MeCN was further compared at 25ºC within 10 days. Despite an equilibrium ratio for R and IR had not been achieved up to the 10th day, the isomerization degree of four pure compounds in MeOH basically accorded with that obtained in a mixture, that is, the protic MeOH favored isomerization compared with the aprotic MeCN (Fig. A.2). When dissolved in MeOH, CB easily converted and C became predominant in equilibrium composition, while IR was more abundant estimated by the conversion tendency even if an equilibrium state had not reached. In detail, 31.7% of CB and 6.5% of R isomerized into the corresponding epimers in MeOH within 24 hours, which rendered it rather difficult to obtain pure SOA compounds, especially for CB. In contrast, these two pairs of epimers in MeCN 12
were relatively stable. Remarkable interconversion of epimers was detected from the 4th day, and the transformed proportions of C, IR, CB, and R at the 10th day were 6.47%, 3.97%, 0, and 1.46%, respectively. This evidence testifies that MeCN is a satisfactory solvent for sample dissolving and as the modifier in SFC preparation of pure SOA compounds at room temperature.
3.2. Optimization of UPC2 condition 3.2.1. Stationary phase The first concern for optimization of UPC2 condition is to select the proper stationary phase. In this study, seven achiral UPC2 customized columns were compared by gradient elution linearly increasing from 18% to 32% of MeCN containing 0.1% DEA in CO2. These columns, with the same dimension and particle size, possess various bonded functional groups, involving 2-ethyl pyridine (BEH 2-EP), 1-aminoanthracene (Torus 1-AA), diethylamine (Torus DEA), 2-picolylamine (Torus 2-PIC), and high-density diol (Torus Diol) on the framework of ethylene bridged hybrid particle, fluoro-phenyl (CSH FP) based on charged surface hybrid technology, and no bonded ethylene bridged hybrid particle (BEH) (Fig. 3). Significant selectivity difference in separation of four SOAs for seven stationary phases was witnessed (Fig. 3). Stereochemistry of two chiral centers at C-7 and C-20 gives rise to the structural difference among R, IR, C, and CB (Fig. 1). Generally, in addition to Torus DEA, well resolution between 7-epimers, that is, IR/R and C/CB, was achieved on the other six columns. However, resolution of IR and CB was poor on Torus 2-PIC, BEH, and BEH 2-EP columns. With regard to the CSH FP column, in 13
spite of its good resolution capacity, severe peak tailing occurred for all four SOAs, which correlates to its specific charged surface hybrid technology. Comparatively, better separation was obtained on Torus 1-AA and Torus Diol columns, as the former enabled baseline separation of all four analytes, whereas the latter exhibited the highest column efficiency (with the narrowest peaks). Therefore, these two columns (Torus 1-AA and Torus Diol) were selected for further optimization. 3.2.2. Additives in the modifier Basic components are prone to interact with silanols on the stationary phase (due to incomplete bonding reaction), thus displaying distorted peak shapes and stronger retention [29,30]. This situation can be improved by the addition of aliphatic amines, such as isopropylamine, DEA, ethyldimethylamine, or TEA, in organic modifier [18]. Herein, five basic additives, involving 0.1% DEA, 0.1% AH, 0.1% TEA, 0.1% DBA, and 5 mM DEOA, added in MeCN (modifier) were tested on Torus 1-AA and Torus Diol columns, respectively, to compare the influence on peak shape and selectivity (Fig. 4). Addition of TEA in MeCN as the modifier caused baseline fluctuation at the initial elution stage for both two columns. In the case of the Torus 1-AA column, the retention was enhanced and resolution was improved when DEOA or AH was added in the modifier, whilst little influence on retention capacity was observed by addition of DEA or DBA. In light of resolution and peak shape, the use of DEA as the additive facilitated the best chromatography performance. With respect to the Torus Diol column, beneficial effects on resolution obtained by addition of DEOA and AH were 14
significant, however, resolution of IR/CB sharply decreased when DEA or DBA was used as the additive. Given the low solubility in MeCN and low volatility (boiling point: 269ºC), DEOA may be a candidate additive for UPC2 analysis of four SOAs from U. macrophylla, but not suitable for preparative SFC isolation of pure SOA compounds. Therefore, AH was the best choice of additive on the Torus Diol column. These results were consistent with Aranyi’s report in UPC2 enantioseparation of amino-naphthol analogues, in which DEOA and AH had a beneficial influence on resolution [30]. The collaborative or competitive interactions between additives and stationary phase/basic analytes by two active sites contribute to the improvement in selectivity. Notably, use of AH as the additive on the Torus Diol column can be developed into a UPC2 method by MS detection. Subsequently, three different concentrations of each additive were compared, including 0.05%, 0.1%, and 0.2%, of DEA and AH in MeCN on Torus 1-AA and Torus Diol columns, respectively (Fig. A.3). Clearly, the influence arising from different concentrations of additives on both two columns was not remarkable. For the Torus 1-AA column, a tendency was discerned that increasing concentration of DEA weakened the retention capacity of four SOAs. However, for the Torus Diol column, less use of AH otherwise could weaken the retention, and negligible difference in both resolution and retention was observed between 0.1% and 0.2% of AH as the additive. In addition, four SOAs in a mixture dissolved in MeCN separately containing 0.05%, 0.1%, 0.2% DEA and AH could keep stable within five days at 25ºC (Fig. A.4). It suggests that use of MeCN containing 0.1% DEA as the modifier on the Torus 1-AA 15
column and MeCN containing 0.1% AH on the Torus Diol column is suitable for the establishment of UPC2 methods for SOAs analysis. 3.2.3. Column temperature In addition to the composition of modifier, temperature and pressure are another two vital factors influencing the density and solubility of the mobile phase, and thus determining the retention of analytes in SFC [18,31]. Here, the influence of different column temperature (15ºC, 30ºC, 45ºC, and 60ºC) on two columns was compared in terms of retention (tR), plate number (N), and resolution (Rs). First, the increase of column temperature weakened the retention of four SOAs on both two columns (Fig. 5). This observation disagrees with the common sense of SFC that decreased density of mobile phase at higher column temperature can enhance the retention of analytes, but accords with that in RP separation [30]. Second, plate number of four analytes dramatically increased when column temperature elevated from 15ºC to 60ºC on the Torus 1-AA column. Different results were obtained on the Torus Diol column that, despite the same increase displayed when column temperature increased from 15ºC to 45ºC, slight decrease was observed when temperature further elevated from 45ºC to 60ºC (R as an exception). Third, resolution of IR and CB showed different alternating trends on two columns. Better resolution for both IR and CB was obtained at 45ºC on the Torus 1-AA column and at 30ºC on the Torus Diol column. Taken together, the column temperature of 45ºC and 30ºC was set for Torus 1-AA and Torus Diol columns, respectively. 3.2.4. ABPR pressure and flow rate 16
Analogous to column temperature, ABPR pressure and flow rate also are vital parameters affecting SFC separation, given they can influence the density and solvent power of mobile phase [31]. ABPR pressure varying among 1600 Psi, 1800 Psi, 2000 Psi, 2200 Psi, and 2400 Psi, on both columns were examined. Negligible effect on resolution and small influence on retention were detected by altering ABPR pressure on UPC2 separation of four SOAs on two columns (Fig. A.5). Increasement of ABPR pressure enhanced the density and solvent power of mobile phase, and thus rendered decreased retention. However, the retention variations were not remarkable (from 4.57 min to 4.04 min for R on Torus 1-AA; from 6.46 min to 5.98 min for R on Torus Diol), which could be attributed to the less compressibility of the mobile phase due to higher ratio of organic solvent modifier (22% MeCN on Torus 1-AA and 21% MeCN on Torus Diol) [32]. In addition, the resolution of IR and CB remained in general in spite of the changes in ABPR pressure. Ultimately, ABPR pressure for the Torus 1-AA and Torus Diol columns were set at 2000 Psi and 1800 Psi, respectively. Influence of flow rate (0.8, 1.0, 1.2, 1.5, and 1.8 mL/min on Torus 1-AA; 1.0, 1.2, 1.5, 1.8, and 2.0 mL/min on Torus Diol) was assessed, given the differentiated optimal ABPR pressure of two columns (Fig. A.6). With the increase of flow rate, the retention of four SOA analytes weakened, giving rise to higher analytical efficiency on both two columns. It is ascribed to the stronger solvent power at higher flow rate. Slight decrease on resolution was distinguished when higher flow rate was used. In contrast, remarkable decrease occurred to plate number when flow rate elevated, 17
Hence, taking into account both analytical efficiency and resolution, an ideal flow rate at 1.2 mL/min was set for UPC2 separation on both two columns. Ultimately, after the systematic optimization of chromatographic conditions and instrument parameters, two rapid UPC2 analytical methods by isocratic elution were established separately on the Torus 1-AA column (Method I: 22% MeCN containing 0.1% DEA in CO2; flow rate, 1.2 mL/min; column temperature, 45ºC; ABPR pressure, 2000 Psi) and the Torus Diol column (Method II: 21% MeCN containing 0.1% AH in CO2; flow rate, 1.2 mL/min; column temperature, 30ºC; ABPR pressure, 1800 Psi). These two methods were further applied to analyze the total extract of U. macrophylla (Fig. 6). The first UPC2 approach, enabling 5-min rapid separation of four SOA analytes, is especially suitable for UV detection, whilst the second one is compatible to mass spectrometry, and thus can be used to probe into new minor SOAs from U. macrophylla and other Uncaria species as well.
3.3. Isolation of four SOA compounds by preparative scale SFC Since extremely fast interconversion occurs between SOA epimers when kept in aqueous organic solvents, it is rather difficult to isolate four SOA compounds from U. macrophylla in high purity by the conventional semi-preparative RP-HPLC. Taking advantage of the optimization results performed on UPC2, a preparative scale SFC approach was established by use of a commercially available Viridis Prep Silica 2-EP OBD column (30 × 250 mm, 5 μm). MeCN was used to dissolve samples and as the modifier, given isomerization between epimers can be inhibited, and DEA was added in MeCN as the additive to obtain symmetric peak shape. Moreover, MeCN was 18
selected as the makeup solution. Simultaneous baseline separation of four SOAs on the preparative Silica 2-EP OBD column was difficult with discounted analytical efficiency. Therefore, column chromatography was initially employed to fractionate them into different samples. Interestingly, MCI gel enabled the separation of four target SOAs by the difference in 20-configuration, and two pooled fractions containing mixed R/IR (R:IR 3:7) and mixed C/CB (C:CB 4:1) were obtained. Given the BEH 2-EP column was potent of separating 7-epimers as discussed above, a preparative column involving the same bonded functional group but on Viridis Hybrid particles with reduced surface silanol activity (Viridis Prep Silica 2-EP OBD) should have the potential to isolate these two pairs of 7-epimers. As a result, good separation of C/CB and R/IR was achieved within 15 min and 25 min, respectively (Fig. 7). 500 mg of C and 44 mg of CB (by duplicate preparation), 254 mg of IR and 61 mg of R (by duplicate preparation), were isolated from Fr. 3 and Fr. 4, respectively. The purity of four isolated compounds was all higher than 95% determined by the UPC2 method. In contrast to RP-HPLC, the established preparative SFC methods use water-free mobile phase (aprotic modifier and inert CO2), and allow efficient preparation and purification of SOAs.
4. Conclusion Preparation of high-purity reference standards is a vital segment in quality control of TCM. However, increasing difficulties resulting from chirality and configurational changes render is rather difficult to obtain high-purity reference 19
standards. This study used SFC to solve this issue by use of SOAs as an example. A proper solvent that could stabilize SOA epimers was screened in the first step. MeCN was able to suppress the epimeric interconversion of four target analytes both in a mixture and as pure compounds within three days, and thereby was employed to dissolve samples and as the modifier in SFC. Subsequently, the Torus 1-AA and Torus Diol columns showed good selectivity, and DEA, AH were separately used as the additives on these two columns to improve resolution. Two isocratic achiral UPC2 analytical methods were established, which suited UV and MS detection, respectively. Combined with MCI gel column chromatography, efficient preparative isolation of high-purity R, IR, C, and CB, was achieved on a Viridis Prep Silica 2-EP OBD column eluted by MeCN containing 0.2% DEA in CO2, which would lay a foundation for better quality control of Uncaria-derived herbal medicines and the future bioactivity screening of natural molecules against Alzheimer’s disease. SFC, by use of inert CO2 and aprotic modifier as the mobile phase, is proven as a feasible solution to rapid analysis and preparation of high-purity SOA reference standards, which is also recommended when other chiral or configurational issues are encountered in quality control of TCM.
Acknowledgements We gratefully acknowledge the financial support from the National Science and Technology Major Project for Major Drug Development (2013ZX09508104 and 2014ZX09304-307-001-007). We also appreciate Dr. Lirui Qiao and Yongwei Xu from Waters Corporation for the technical assistance. 20
Appendix A. Supplementary data Supplementary data associated with this article is available in the online version.
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Figure Caption Fig. 1 Structures of rhynchophylline (R), isorhynchophylline (IR), corynoxine (C), and corynoxine B (CB), four major spiro oxindole alkaloids in Uncaria macrophylla.
26
Fig. 2 Conversion of corynoxine B to corynoxine and rhynchophylline to isorhynchophylline dissolved in different pure organic and aqueous organic solvents (A) and different ratios of isopropanol and acetonitrile (B) within nine days at 25℃. R: rhynchophylline, IR: isorhynchophylline, C: corynoxine, CB: corynoxine B.
27
Fig. 3 Comparison of seven achiral chromatographic columns to the separation of two pairs of SOA epimers.
28
Fig. 4 Comparison of five different base additives in modifier (0.1% AH, TEA, DEA, DBA, and 5 mM DEOA) to the separation of two pairs of SOA epimers on the Torus 1-AA and Torus Diol columns.
29
Fig. 5 Comparison of different column temperature (15ºC, 30ºC, 45ºC, and 60ºC) to the separation of two pairs of SOA epimers on the Torus 1-AA and Torus Diol columns, evaluated by retention (tR), plate number (N), and resolution of IR and CB (Rs).
30
Fig. 6 The UV spectra (245 nm) of the total extract of U. macrophylla obtained on the Torus 1-AA (A) and Torus Diol columns (B). The BPI spectrum (C) was recorded on a Waters UPLC/QTOF MS instrument. (A: Torus 1-AA: 78% CO2-22% MeCN containing 0.1% DEA; flow rate, 1.2 mL/min; ABPR pressure, 2000 Psi; column temperature, 45ºC; B: Torus Diol: 79% CO2-21% MeCN containing 0.1% AH; flow rate, 1.2 mL/min; ABPR pressure, 1800 psi; column temperature, 30ºC; C: Torus Diol: 0-7 min, 21% MeCN containing 0.1% AH, 7-8 min, 21-40% MeCN containing 0.1% AH, 8-10 min, 40% MeCN containing 0.1% AH; flow rate, 1.2 mL/min; ABPR pressure, 1800 Psi; column temperature, 30ºC).
31
Fig. 7 Illustration of the preparative SFC isolation (A) and purity check by UPC 2 (B) of four SOA compounds from U. macrophylla. Mobile phase: 12% acetonitrile containing 0.2% DEA for the isolation of C and CB; 8% acetonitrile containing 0.2% DEA for the isolation of IR and R; ABPR pressure, 150 bar; flow rate, 80 mL/min; the make-up solution, 25 mL/min of MeCN.
32