Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

Research Article Open Access

Ndorbor et al., J Chromat Separation Techniq 2013, 4:3http://dx.doi.org/10.4172/2157-7064.1000176

Research Article Open Access

Chromatography Separation Techniques

Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition MechanismTheophilus Ndorbor1, Yong Wang2, Ding Huaijing2, Ding Zhizhang2, Jacob A Kolawole3 and He Hua1,4*1China Pharmaceutical University, Nanjing 210009, China2 Zhenjiang Xiangju Pharmaceutical Company, Taizhou, China3School of Pharmacy, University of Liberia, Monrovia, Liberia4Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China

*Corresponding author: He Hua, China Pharmaceutical University, Nanjing 210009, China, E-mail: hanfazou@dicp.ac.cn

Received March 11, 2013; Accepted March 25, 2013; Published March 28, 2013

Copyright: © 2013 Ndorbor T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

AbstractA baseline separation was achieved for the direct HPLC separation of atracurium besylate stereoisomers;

atracurium trans-trans, atracurium trans-cis, and atracurium cis-cis, on a Cellulose tri- 3, 5-dimethylphenycarbamate (CDMPC) column. Acetonotrile (ANC) and Potassium hexaflourophosphate (KPF6) were used as mobile phase. The effect of organic modifier, pH, buffer concentration, temperature, and flow rate on retention time and enantioselectivity, was investigated. Binding energy differences, mode of interaction as determined by computer simulation method, were used to elucidate chiral recognition mechanism and explain the effect of organic modifier on enantioselectivity. The result suggests that the isomers of atracurium besylate could be well resolved on a CDMPC column by a 50:50 ANC: KPF6 (0.1 M, pH 3.0-3.5) mobile phase in the temperature range of 30-38°C, at a flow rate between 0.5-1, and wavelength of 280 mm. It was further observed that both ANC and KPF6 did influence enantioselectivity. From computer simulation, π-π interaction, Hydrogen bonding and Vander Waal force were noted to be responsible for chiral recognition. Results from this research are useful in designing chromatography method for separating atracurium besylate and related substances on CDMPC column and other chiral selectors.

Keywords: Atracurium besylate; Cisatracurium besylate; Cellulose tri- 3, 5-dimethylphenycarbamate; Chiral separation; Recognition mechanism

Introduction The study of charility has become a subject of interest in many

disciplines including agriculture, pharmacy and its related disciplines

[1-3]. Chiral substances are found to be widely applied in medical sciences, the food and petroleum industries [1-5]. In drugs development research, the chirality of a drugs substance add a difference challenge to principles of its development, especially when the chiral drugs substance shows enantioselective pharmacokinetics (PK) and/or pharmacodynamics (PD). In certain cases one enantiomer could be therapeutically active while the other may be less active or even having toxic effect. It is therefore essential to recognize and separate both enantiomers separately to ensure the safety and efficacy of the drugs substance under consideration [5-9].

Cisatracurium besylate, also known as NIMBEX, is a nondepolarizing neuromuscular blocking agent, currently used as single enantiomer for anesthetic purpose. It is one of ten isomers of the chiral drug Atracurium besylate (Figure 1a) that relaxes the skeleton muscle during surgery [10-13]. The three most important isomers present in substantial amount are the cis-cis, cis-trans, and trans-trans all being positional isomers and hard to separate [14]. The single enantiomer, the R-cis, R’-cis isomer of atracurium besylate (Cisatracurium besylate), is approximately 3-fold more potent than the mixture of isomers that constitute the parent drug. It has also been

(a)

N+

MeO

MeO

OMe

MeO

Me

OO

N+

O O Me

OMe

OMe

OMe

OMe

(b)

X

XX

OCO4

OO

OCO

OCO

n

X = CH3, n = CH3

Figure 1: Structure of (a) Atracurium besylate, (b) cellulose 3,5-dimethyltriphenylcarbamate.

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

reported that the chances of Cisatracurium besylate releasing histamine and other side effects are lower compared to its parent compound [1-13,15]. This has therefore increased the need to seek for a suitable method of separation of Cisatracurium besylate as a pure enationmer from its parent compound. Several methods in the form of patents have been advanced for the preparation and separation of atracurium besylate isomers. Stealake et al. [16], suggested a method preparation of quaternary ammonium compounds such as Atracurium besylate, Atracurium mesylate, R-atracurium besylate but the method does not cover the aspect of getting the cisatracurium besylate. The US Patent No: 5,453,510 [17], suggests an HPLC separation method of the Isomers of Atracurium besylate but in the presence of a strong acid. Such acids might have a damaging effect on HPLC columns over a period of time since they are usually made of stainless steel [18]. However, in 2008, Oded et al. [19], put forward a chromatographic method for separating the isomers of (IR, l’R) atracurium besylate by HPLC without the use of strong acid, using silica gel, HPLC column and eluting the compound containing an polar aprotic co solvent and a week acid. Industrially this method may be expensive. Several other methods have been formulated for the separation of the atracurium besylate isomers [20-22]. However, up to present, very little has been done to elucidate the recognition mechanism of chiral RCis-RCis and its positional isomers on a CSP(s). Chiral separation and recognition mechanism go together. To effectively develop a chiral separation method, using a particular CSP, it is important to first understand and interpret the interaction between the enantiiomer and the chiral selector [23]. Most of the methods developed for the separation of atracurium besylate isomers are either expensive or use solvent posing problem to the column and CSP [16-20]. Unlike other chiral compounds, the chiral recognition and separation of atracurium besylate is cumbersome and achievable on relatively fewer CSPs and mobile phases that are pH, concentration and temperature sensitive [16-22]. The choice of column, appropriate mobile phase and specific HPLC conditions do affect its recognition [16-22].

Considering its structural makeup; with phenyl moiety for π-π interaction and sites for H-bond formation (acceptor), the stereoisomers of atracurium besilate could better be recognized on a Polysaccharides column such as cellulose or amylase. Phenylcarbamate of polysaccharide cellulose base CSP; Cellulose tri- 3, 5-dimethylphenycarbamate (CDMPC (Figure 1b) could forms hydrogen bond with the atracurium besilate isomers and π-π interaction via the phenyl groups of both the Polysaccharides and the stereoisomers of atracurium besilate. Phenylcarbamate of polysaccharide cellulose base CSP has been shown to be powerful in the resolution of a wide range of racemates and nearly 90% of chiral compound could be resolved on it. It is very selective, and can associate individual chiral carbohydrate monomer in a long range helical secondary structure. This association has proven to be very effective for HPLC resolution [23,24]. Nevertheless, it major drawbacks is the limited amount of solvent it can tolerate. Polar solvent such as acetone, present even in minute proportion may damage the column [25]. Combination of suitable organic modifier such as actonitrile, ethanol, and methanol, with ionic liquids therefore presented a suitable mobile phase for the chiral resolution of many compounds on the CDMPC column including compounds hard to separate.

The analyte, CSP, and mobile phase, are all essential for developing a chiral separation method. The key, therefore, to a successful chiral resolution of a particular class of enantiomer on a given CSP is the understanding of the possible chiral recognition mechanism. Computer simulation method is one of the best tools of understanding interactions at the molecular level. The application of Computer simulation in chiral

drugs separation has become a source of information about CDMPC–guest interaction [26]. Binding energy difference, host-guest structural distinction resulting form CDMPC -Guest interaction, can be elucidated by computer simulation methods. Molecular Docking could also be used to predict the strength of association or binding affinity between two molecules using  scoring functions. Prediction from molecular docking method reduces time and increases accuracy and precision in chiral recognition. It allows researcher to quickly screen large database for potential CSPs or drugs which would otherwise require tedious and prolong work in the laboratory [26-29]. In this paper we therefore apply combine HPLC method and computer simulation to study the resolution of atracurium besilate stereoisomers on a CDMPC column and it recognition mechanism.

ExperimentChemicals and reagents

Atracurium besylate racemate and Ciatracurium besylate pure enanatiomer were donated by Zhejaing Xianju pharmaceutical Company (Xiangju, China), Acetronitrile, Methanol, and Phosphoric acid (HPLC grade) were purchased from TEDIA Company INC, (Fairfield, Ohio USA), Ethanol was purchased from Nanjing Chemical Reagent Company (Nanjing, China), Potassium hexaflourophosphate and potassium dihydrogen phosphate all analytical grades were purchased from Aladdin Industrial Corporation, Shanghai, China.

Equipment

The HPLC experiment was conducted on a Waters 2695 Alliance Separations Module HPLC System, consisting of 2996 photodiode array detector, Chromatography data were acquired and processed by the Empower software. The Column used was Cellulose tri- 3, 5-dimethylphenycarbamate (Chiralcel OD-RH) from CHIRALCEL® (Europe). PH meter used to measure PH (PHs 3c) was purchased from Shanghai Jing Ke Instrument CO., Ltd, China. The solvents were degassed by ultrasonic bath purchased from Shanghai Ke Dao Ultrasonic Instrument Company, China.

Chromatographic conditions

The mobile phase system were Acetonitrile:Potassium hexafluoride phosphate (0.05 M, 0.1 M, pH 2-4), Methanol:Potassium hexafluoride phosphate (0.05 M, 0.1 M, pH 2-4), and Ethanol:Potassium hexafluoride phosphate (0.05 M, 0.1 M, pH 2-4) each at a ratio of 20/80, 40/60, 50/50, 60/40, and 80/20. The sample was dissolved in the mobile phase. The solvents were filtered using a Millipore 0.45-um filter and degassed by ultrasonic bath before used. The flow rate was set between 0.5 to 1.0 ml/min-1 and column temperature varying from between 30 to 38°C. UV detection was at 280 nm and 20 μl was injected in each run. The following perimeters were calculated:

During the run, the following parameters were calculated: Separation factor (α1, α2) using α1=K2 / K1, α2 =K2 / K3 with α1 as the selective factor between first and second eluting isomers, α2 the selective factor between the second and third eluting enantiomers isomers, K1, K2 and K3 were capacity factors of the first , second and third eluting enantiomers, respectively. The resolutions factors (Rs1 and Rs2) were calculated as Rs1= 2(t2 – t1)/w1+w2, Rs2= 2(t3 – t2)/w2+w3), where Rs1 measures the resolution between the first and second eluting isomers, while Rs2 measures the resolution the second and the third eluting isomers, w is the baseline bandwidth obtained by drawing tangents to the inflexion points of the chromatographic peak.

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

Molecular molding

The structures of atracurium besylate isomers and CDMP were prepared using MOE (Molecular Operating Environment), then minimized using force field parameter Merck Molecular Force field (MMFF94X) implemented in MOE at gradient 0.01 with partial charges based on bond-charge increments. Discovery Studio (DS) then was used for Geometric optimization and visualizing docking results. Atoms, bond types and charges were inspected using sibyl 6.0.

Molecular docking simulation

The molecular docking program GOLD (version 3.0.1, Cambridge Crystallographic Data Center), which uses a powerful genetic algorithm (GA) method for ligand conformational and docking searches, was employed to generate an ensemble of docked conformations. It has implemented in it a scoring function called Goldscore, for ranking ligand binding poses [30,31]. For each complex, 20 independent docking runs were performed. All docking runs were carried out using standard default settings with a population size of 100, a maximum number of 100,000 operations, and a mutation and crossover rate of 95. The best generated 10 solutions of each ligand were ranked according to their fitness scores calculated by the GOLD Score In each group, the binding energy with the highest % frequency was selected as the group representative.

Result and DiscussionChromatographic parameters, capacity (k), separation factor

(α) and resolution factor (Rs) for resolving the positional isomers of Atracurium besylate on a cellulose tri-3,5-dimethylphenylcarbamate (Chiralcel OD-RH) column using ANC : KPF6, MeOH: KPF6, EtOH : KPF6 (20:80, 40:60, 50:50 , 60:40, 80:20 v/v) are given in tables 1-4. The values of separation factor of resolved isomers were in the range of A 1.1 to 1.4. The typical chromatograms of resolved and unresolved isomers

ANC:KPF6 (0.05 M), pH 3.0, 30°C, Flow rate: 1 ml/minConcentration K1 K2 K3 R1 R2 α α

80:20 1.69 1.94 2.28 - 0.99 1.14 1.1760:40 3.15 3.69 4.58 1.19 1.57 1.17 1.250:50 4.13 5.00 6.61 1.37 1.57 1.2 1.2140:60 - - - - - - -20:80 - - - - - - -

Table 1: The ANC: KPF6 Mobile phase concentration effect on retention factor and enantioselectivity of Atracurium besylate isomers at pH 3.0.

ANC:KPF6 (0.05 M), pH 2.0, 3°C, Flow rate: 1 ml/minConcentration K1 K2 K3 R1 R2 α1 α2

80:20 - - - - - - -60:40 2.07 2.33 2.79 0.76 1.13 1.12 1.1950:50 5.70 6.758 8.66 1.13 1.61 1.16 1.2540:60 - - - - - - -

20:80 - - - - - -

Table 2: The ANC: KPF6 Mobile phase concentration effect on retention factor and enantioselectivity of Atracurium besylate isomers at pH 2.0.

50:50 ANC:KPF6 (0.05 M and 0.1 M), pH 3, 30°C, Flow rate: 1 ml/minCon K1 K2 K3 R1 R2 α1 α2 plate Plate Plate

0.05 M 5.6 6.6 8.5 1.13 1.61 1.1 1.3 1111 860 8760.1 M 7.5 9.1 12.3 1.78 2.24 1.2 1.4 1935 1480 1136

Table 3: The effect of KPF6 concentration on retention factor, theoretical plate and enantioselectivity of Atracurium besylate isomers at pH 3.0.

50:50 ANC:KPF6 (0.1 M), pH 3, 30°C, Flow rate: 1 ml/minpH K1 K2 K3 R1 R2 α α2.02.53.0 7.5 9.1 12.3 1.7 2.2 1.2 1.43.2 7.6 9.5 13.0 1.7 2.1 1.2 1.43.3 4.1 5.0 6.6 1.5 1.7 1.1 1.34.0 4.73 5.75 7.7 1.37 1.7 1.2 1.3

Table 4: The effect of 50:50 ANC:KPF6 (0.1 M) on retention factor and enantioselectivity of Atracurium besylate isomers at between pH 2.0 – 4.0.

of Atracurium besylate using different mobile phases are shown in figure 2. It was observed that the trans-trans isomer eluted first followed by the trans-cis and cis-cis isomers respectively. Chromatography parameters such as pH, flow rate and temperature were varied to obtain the best resolution. Several organic modifiers and aqueous solutions proportionally adjusted were also investigated.

Considering the nature of the cellulose carbamate column (Chiralcel OD-RH), the choice of mobile was noted to be very important. Many of the solvents commonly used as HPLC eluents including acetone, chloroform, dimethylformamide, dimethylsulfoxide, ethylacetone, mrthylene chloride, and Tetrahydrofuran could destroy the chiral stationery phase if they are present even in residual quantities, in the system [25,32]. Choice of mobile organic modifier was thus limited to methanol (MeOH), ethanol (EtOH) and Acetonitrile (ANC). The Atracurium besylate isomers are most stable in acidic environment. Potassium hexaflourophaosphate (KPF6) or potassium dihydrogen phosphates (KH2P) were therefore considered as choice of aqueous mobile phase. Phosphoric acid was used for pH adjustment.

Organic modifier selection

Three solvent systems were prepared and investigated for mobile phase. Mobile phase I consisted of EtOH : KPF6, Mobile phase II consisted of MeOH : KPF6, and mobile phase III consisted of ANC: KPF6. Ethanol and methanol as organic modifier failed to give meaningful resolution of the Atracurium besylate isomers (Figures 2b and 2c). The isocratic mobile phase consisted of KPF6 (0.05 M) and Methanol in ratios of 20:80, 40:60, 50:50, 60:40, and 80:20, with flow rate and temperature were held at 1 ml/min and 30°C respectively. The pH was considered at 2.0, 2.5, 3.0, and 3.5. The same condition was repeated for ethanol and ANC separately. The result shows that both ethanol and methanol were poor organic modifier. The methanol/ethanol: KPF6 compositions at between pH 3.0 - 3.5, give an unrealistic signal. This could be due to interaction between methanol/ethanol and KPF6 which was confirmed by computer simulation. Hydrogen bonds were form between the KPF6 and the alcohols (Figures 3a and 3b). This is in agreement with recent report by Xueying Zhu and group, where it was suggested that interaction could occur between the H of the CH3 or OH group of methanol and the F ions of KPF6 via hydrogen bond in a two point interaction [33]. Similar report on ionic liquid and their interaction with alcohol has been published [34]. However, when ANC is used as organic modifier under similar condition, peaks of trans-trans, cis-trans, and, cis-cis are obtained, indicating that ANC is the best organic modifier. Further studies by computer simulation show no interaction between ANC and KPF6 (Figure 3c).

Aqueous phase selection

Two coordination salts, Potassium hexaflurophosphate and potassium dihydrogenphosphate at concentration of 0.05 M and 0.1 M, were evaluated to determine their compatibility with ANC. Their

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

composition with ANC was set at 50:50, 40:60, and 60:40. The pH was considered at 3.0, and 4.0. Result shows that KH2P pairing with ANC showed only one peak and not three (Figure 3d). So, kpF6 combination with ANC was still the choice of aqueous phase.

ANC: KPF6 Mobile phase composition

To determine the requisite condition for the separation of Atracurium besylate isomers on a CDMPC column, ANC: KPF6 Mobile phase composition and its resolution ability were studied in different proportion: 80:20, 60: 40, 50:50, 40:60, 20:80, temperature 30°C, pH 2.0, - 4.0, flow rate 1 μl/min. The results from tables 1 and 2 suggest that Atracurium besylate isomers could not be resolved completely by mobile phase 80:20, 60:40, 40:60, 80:20 between pH 2.0 and 3.0 (Figures 2e-2h). Retention time and enatioselectivity were greatly influenced. Increasing ANC concentration and lowering that of KPF6, reduces retention time and enantioselectivity(α). On the other hand, lowering the concentration of ANC and raising that of KPF6 resulted in extension of retention time. However, chiral recognition was also not achievable. As such 50:50 ratio was realized to be the best option remain for achieving baseline separation of Atracurium besylate isomers on a CDMPC column.

Effect of buffer concentration and pH

Table 2 shows that the resolution factors Rs1 and Rs2 of 50:50, at

A

B

C

Figure 3: The stable geometries of (a) ethanol-anion complexes (E-Aˉ, PF6ˉ), (b) methanol-anion complexes (M-Aˉ, PF6ˉ), (c) acetronitrile-anion complexes (ANC-Aˉ, PF6ˉ).

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 2: Chromatogram of (a) ANC: KPF6 (0.1 M, pH.3.2) (b) MeOH:KPF6 (0.1 M, pH.3.2) (c) EtOH:KPF6 (0.1 M, pH.3.2) (d) ANC:KH2P (0.1 M, pH.3.2) （e）ANC:KPF6 (0.5 M, pH.3.0, 60:40), (f) ANC: KPF6 (0.1 M, pH.3.0, 80:20), (g) ANC: KPF6 (0.1 M, pH.3.0, 40:60), (h) ANC: KPF6 (0.1 M, pH.3.0, 20:80),

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

pH 3.0, temperature 30°C, flow rate 1 ml/min, with UV detector set at 280 mm, is 1.37 and 1.61 respectively while the selective factors (α1, α2) were 1.1 and 1.3 respectively. In accordance with ICH guideline, baseline resolution was only achieved between the trans-cis and cis-cis isomers. Since the resolution factor between the trans-cis and trans –trans isomers is less than 1.5 per ICH standard, baseline resolution was not achieved. However, when the concentration of KPF6 was doubled, baseline separation was achieved (Table 3). Resolution factor (Rs1) between the trans –cis and the trans-trans isomer increased from 1.13 to 1.78. The degree of chiral recognition, as shown by the magnitude of the separation factors (α1), increased from 1.16 to 1.2. Increased enantioselectiveity was also enhanced between the trans-cis and cisi-cis isomers. The Rs2 increased from 1.78 to 2.24 while α2 increased from 1.3 to 1.4. Thus, enantioselectivety was very sensitive to the concentration of KPF6. Best separations were achieved by doubling the concentration of KPF6. However, retention time corresponding to trans-trans, cis-trans, cis-cis (K1, K2 and K3) was increase from 5.6 to 7.5 for K1, from 6.6 to 9.1 for K2, and 8.5 to 12.3 for K3. This result suggest a liquid –solid adsorption process on the surface of the packing in which the cis-cis isomer exhibits a more stable equilibrium with the surface than the trans-cis and the trans-trans. This is agreement with computer simulation study. Also from computer simulation study, the process is predicted to involves hydrogen-bonding, π-π, vander waal interaction.

The effect of pH on resolution was also considered (Table 4). Result shows that Atracurium besylate isomers can best be resolved on a CDMPC column in the pH interval of 3.0 to 3.35. When the pH was increased to 4 the resolution between the trans-cis and trans-trans isomers could not be achieved. However, baseline separation was still achieved between trans-cis and trans-cis isomers. At pH below 3.0 no resolution was achieved.

Effect of Flow rate and temperature (Table 5)

The flow rate and temperature mainly affected the column performance. At lower temperature, retention time was increased while low theoretical plates were observed. However, when the temperature was increased, the retention decreased and plate increased considerably. Notwithstanding, increase in temperature led to decrease resolution. To control the resolution and keep retention time with in acceptable range while at the same time increasing the plate value, the flow rate was decreased. So, by controlling both the flow rate and the temperature a better theoretical plate was achieved, retention time kept within acceptable range and baseline separation maintained. At 30°C, flow rate 1.0, pH 3.32, R1 and R2 are 1.7, and 2.1, with retention time ((K1, K2 and K3) 7.5, 9.5, 13.0, and selective factors of 1.2 and 1.4. The R1 drop by 0.1 factor and remain the same for R2 when temperature increased by 5°C, and flowrate reduced by 0.15 ml/min. Column performance which is a function of theoretical plate was increased and K2 and K3 slightly increased. When the change in temperature was further increase by 2°C (37°C), and flow rate also further reduced by 0.2, K1, K2 , K3 again slightly increased, while R1 remain the same and R2 increased by 0.2 while plate

rate reduced by 0.15 (0.5), retention time continue to slightly increase and R1 remain the same with R2 increasing by 0.1. This suggest that separation of Atracurium besylate isomers on a CMPD column using ANC and KPF6 (0.1 M) in a 50:50 ratio mobile phase, at increasing temperature and reducing flow rate, column performance is enhanced and enantioselectivity is maintained (Figure 2a).

Computer simulation and chiral recognition mechanism

Result from molecular docking was evaluated on the bases of binding energy difference of diatereomeric complexes, hydrogen bonds formation, and geometrics. Table 6 shows that the binding energy difference between the trans-trans and the trans-cis is -0.04. With such a small binding energy difference (G) chiral recognition is considerably difficult, thus giving reason for the poor resolution. The binding energy difference is far better between trans-cis and cis-cis thus it was easy to achieve satisfactory baseline separation. The magnitude of elution is therefore in the order trans-trans > trans-cis > cis-cis Computer simulation shows that the cis-cis isomers can interact with the CSP through π-π interaction. These π-π interactions mainly occur between the phenyl group of the Atracurium besylate isomers and the phenyl group of the CSP. π-π interaction involving the phenyl rings has been noted to be one factor responsible for chiral recognition on a cellulose column [35]. The substituents on the cis-cis phenyl group may also interact with substituents on the glucose ring of the CSP. However, fitness score from molecular docking (S (hbex) = 0.0) and geometrics show no evidence of hydrogen bond. This could be due to the effect of π-π interaction between the phenyl groups mostly involving the aromatic rings. Hydrogen bonding could be insulated in the environment mostly of aromatic interaction where the π-acceptor and π-denotes dominate [36]. Unlike the cis-cis, molecular docking also shows that the trans-cis isomer can interact with the CSP via hydrogen bonding (S (hbex) = 1.04). The hydrogen bond occurs between the isomer carbonyl group (O) and the amide group (H) of the CSP. It can be noted that the polar carbamate on the CSP is the most important site of chiral recognition [35]. Interaction between the trans-trans isomer and the CSP is somewhat similar to that of the trans-trans, in that both can form bond with the NH of the CSP. On the contrary, the trans-trans form a very week and unstable hydrogen bond with the CSP. Vander Waal interaction was also observed to be involved with the formation of complexes (Figure 4).

These observations show that Atracurium besylate isomers can be recognition on the CDMPC column. First, chiral recognition resulted when diastereomeric complexes were formed from the CDMPC-isomer interaction, and their difference in free energy of formation. Increased G value leads to better enantiomeric separation. Since G determines enantioselectivity, it is important to maximize G while minimizing the adsorption energies, GR and GS [37,38]. The results from Gold docking (Table 6) show that binding energy difference (G) increased with increased discrimination of isomers. Second, the stability of their

50:50 ANC:KPF6 ( 0.1 M), pH 3.2 T FR K1 K2 K3 R 1 R2 α1 α2 P1 P2 P330 1.0 7.5 9.5 13.0 1.7 2.1 1.2 1.4 1564 1191 97035 0.85 7.5 9.6 12.4 1.6 2.1 1.2 1.3 2278 1920 153637 0.65 9.1 10.6 13.5 1.6 2.3 1.2 1.3 2531 2134 177838 0.5 11.7 13.5 17.0 1.6 2.4 1.2 1.3 2911 2437 2082

Table 5: The effect of Temperature and flow rate on retention factor, theoretical plate and enantioselectivity of Atracurium besylate isomers at pH 3.2.

a. Binding free energies derived from Gold 3.0.1b. Binding free energy difference between the (R) - and (S)-enantiomes complexes, where △△G= △GR –△GS, negative △△G = S>R while positive △△G= R>S.Table 6: Binding energy and binding energy difference obtained from Gold docking between CDMPC and Atracurium besylate isomers.

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

Receptor Anayte △Ga S(hb_ex) S(vdw_ext) S(hb_int) S(int) △△Gb

CDMPC SS 16.85 0.00 46.55 0.00 -47.12-0.04

CDMPC RS 16.89 1.04 53.79 0.00 -58.15CDMPC RR -17.06 0.00 26.13 0.00 -53.62 -33.0

continue to increase. By increasing the temperature to 38°C, and flow

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

complexes is a requirement for chiral recognition. The trans-trans isomer is least stable and elutes first, followed by trans-cis. Thus cis-cis isomer forms a more stable complex with the CSP and elutes last. Third, hydrogen bonds, π-π interaction and Vander Waal forces are believed to be the primary forces responsible for chiral recognition.

Furthermore, these results are in agreement with the three points rule for chiral recognition on a CDMPC column. The three isomers interact with the CSP differently which give rise to difference in binding energy and complex stability.

Our observation from molecular docking aimed at explaining the chiral recognition mechanism between Atracurium besylate isomers and the CDMPC CSP column shows that chiral recognition between Atracurium besylate isomers depends on selection of CSP and solvents (with control concentration) that allows both hydrogen bonding and π-π interaction coexisting.

Effect of solvent on chiral recognition

In chiral recognition mechanism, the formation of diastereomeric complexes marks the beginning of chiral recognition. Thus, successful chiral recognition is a function of the interaction between the enantiomer and the CSP in chromatography separation as well as in capillary electrophoresis. However, this interaction could be influenced by the mobile phase. As such, chiral recognition on a CSP depends

A B C

Figure 4: Stable complex and interactions between (a) cis-cis and CDMPC （b）cis-trans and CDMPC, (c) trans-trans and CDMPC: Hydrogen bond, Vander waals interactions and π-π interaction are represented by green dotted lines, pink dotted lines, green arrow and brown arrow.

.

ΔEa

0

2

4

6

8

10

12

14

16

18

20

1:01 1:02 1:03 1:04 1:05 1:06 1:07

系列1

ANC: CDMPC ratioFigure 5: Interaction energy between ANC and CDMPC obtained from MOE and schiochemetry ratio. a. ΔE is the interaction energy calculated using equation: ΔE = | Ecomplex - Ehost - Eguest |

on the interaction occurring among the CSP, analyte, and the mobile phase. The following equations could be used to demonstrate the chiral recognition process involving CSP, analyte, and mobile phase.

The effect of acetonitrile on π-π interaction and subsequent influence on recognition mechanism is: Molecular docking results show that acetonitrile can interact with the cellulose CSP and thus did influence the interaction between Atracurium besylate isomers and CDMPC CSP. Figure 5 shows that ANC: CDMPC interact at a ratio of 5:1 with an interaction energy difference of -17.671. This interaction energy difference result from the interaction of ANC with the CDMPC CSP at binding sites also sought for by the Atracurium besylate isomers. This competition for binding sites did influence chiral recognition. The N atom of ANC could form H-bond with H of the amide on the CSP (Figure 5), thus competing with the analyte for hydrogen bond formation. It could also interaction with the phenyl group of the CSP, thus hindering pi-pi interaction between the analyte and the CSP (Figure 5). This is in agreement with our chromatography result. At higher concentration of ANC; 80% and 60%, retention time significantly reduced and chiral resolution failed to occur (Figures 2e and 2f). This is because π-π interaction, a major factor for recognition, as suggested by molecular docking result is hindered and less stable CSP- isomers complexes are formed. Thus, retention time and enatio selectivity of the CSP with regards to the isomers decreases with decreasing π-π

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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Volume 4 • Issue 3 • 1000176J Chromat Separation TechniqISSN:2157-7064 JCGST, an open access journal

interaction resulting from increasing ANC concentration. On the other hand when the concentration was decrease to 40%, 20%, retention time was greatly increased but chiral recognition was also not achievable. First, retention time was increased because as CSP- isomers complexes interaction became strong and stronger, more stable complexes were formed. Second, from molecular docking it was noticed that H-bond is also a key factor in accounting for the recognition of isomers on the CDMPC column. As such, by reducing the ANC concentration, π-π interaction is increased. These increase π-π interactions insulate H_B [36]. Thus chiral recognition is not achieve in the absent on H-bond. This further support our elucidation of the three point rule that is, for chiral recognition of all three isomers on the CSP column, the isomer must interaction differently with the CSP (one of the three point of

interaction should be stereochemical) [23,24]. From these observations it is tempting to say ANC was not the best organic modifier for the recognition of Atracurium besylate isomers on the CDMPC column. However, further observation shows that at 50% concentration baseline separation was achieved. At this concentration, π-π interaction was effectively control such that it could tolerate H-B. It can therefore be suggested that the best organic modifier for the separation of Atracurium besylate isomers on a CDMPC column should be able to control π-π interaction such that it remain effective but not completely insulating H-B (Figures 6 and 7).

Role of potassium hexaflourophasphate in the recognition mechanism: Regarding the effect of potassium hexaflourophasphate on chiral recognition of Atracurium besylate isomers on the column, is being considered. However, our initial investigation shows that KPF6 could interact with the CSP via hydrogen bond (Figure 7). We are of the opinion that other than hydrogen bonding, KPf6 and the CSP could interact in some other ways. Investigation continues. We strongly assume that the recognition mechanism was influence by KPF6, especially at 50:50 ratios. Expectation was that the increasing concentration of ANC should continue to vary directly with retention time and resolution. However, it was observed that above 50% ANC, concentration varies directly with retention time but inversely with resolution. We assumed that at 50% the effect of pi-pi on the recognition process was equally balanced. KPF6 (pH 3-3.5, 0.1 M) is assumed to have been involved in establishing this balance. However, this assumption could not be substantiated by the current simulation parameters used. To measure the effect of KPF6 ions and other ionic liquid on enantioselectivity using computer simulation is a challenging and requires long computational time. In our next study we intend to define both the experimental and theoretical parameters require to clearly measure this effect. The exact measurement of π-π interaction will also be considered.

ConclusionHPLC and computer simulation methods were used to study the

Chiral recognition and mechanism of Atracurium besylate isomers on a CDMPC column. Several mobile phase solvents and HPLC conditions were considered. The result obtained suggest that the best solvents and chromatography condition for the separation of Atracurium besylate isomers on a CDMPC column is ANC: KPF6, (0.1 M, pH 3.0-3.35), flow rate 0.5-1.0, 30-38°C 280 mm. It was observed that pH, temperature, and buffer concentration influenced enantioselectivety as well as retention time. Molecular docking aimed at explaining the chiral recognition mechanism between Atracurium besylate isomers and the CPMD CSP column shows that chiral recognition between Atracurium besylate isomers and CSP depended on hydrogen bonding, π-π interaction, and vander waal force. As such the choice of mobile phase should take into consideration solvents effect on π-π interaction as well as Hydrogen bonding.Acknowledgements

Let God Almighty take all the glory for this work. We would like to however acknowledge the support of Xiangju Pharmaceutical Company, China. Special thanks go to the staffs of the Quality Research Department, Xiangju Pharmaceutical Company.

References

1. Wu T, Li X, Huang H, Zhang S (2011) Enantioselective oxidative damage of chiral pesticide dichlorprop to maize. J Agric Food Chem 59: 4315-4320.

2. Sekhon BS (2010) Enantioseparation of chiral drugs – An overview. Int J ChemTech Res 2: 1584-1594.

3. Li B, Haynie DT (2006) Chiral Drug Separation. In Encyclopedia of Chemical Processing. Taylor & Francis: 449-458.

Figure 6: Stable complex and interactions between ANC and CDMPC (a) Hydrogen bond (green dotted line) between ANC (N atom) and CDMPC (amide H atom) (b) π-π interaction (pink dotted line) between ANC and the phenyl group of CDMPC.

2.41919

Figure 6: Stable complex and interactions between ANC and CDMPC (a) Hydrogen bond (green dotted line) between ANC (N atom) and CDMPC (amide H atom) (b) π-π interaction (pink dotted line) between ANC and the phenyl group of CDMPC.

Equation 1. Demonstration of the chiral recognition mechanism process, Where R, RS and S are the analytes, A is the mobile phase and B is the CSP

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

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4. Kim B.I, Hanson J, Turner M, Reeder L (2012) Influence of solvent on the chiral resolution of organic molecules on Au(111): EC-STM study of biphenyl dicarboxylic acid on Au(111) in an aqueous environment. Surf Sci 606: 1340-1344.

5. Leffingwell JC (2003) Chirality and Bioactivity I.: Pharmacology. Leffingwell Report. 3: 1-27.

6. Ariëns EJ (1984) Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics and clinical pharmacology. Eur J Clin Pharmacol 26: 663-668.

7. Ward TJ, Ward KD (2010) Chiral separations: fundamental review 2010. Anal Chem 82: 4712-4722.

8. Fosse TK, Toutain PL, Spadavecchia C, Haga HA, Horsberg TE, et al. (2011) Ketoprofen in piglets: enantioselective pharmacokinetics, pharmacodynamics and PK/PD modelling. J Vet Pharmacol Ther 34: 338-349.

9. Bertera F, Di Verniero CA, Mayer MA, Chiappetta D, Buontempo F, et al. (2012) Pharmacokinetic and pharmacodynamic properties of carvedilol in fructose hypertensive rats. Xenobiotica 42: 206-219.

10. Chuang CT, Fan SZ, Shieh JS (2009) Rule extraction by fuzzy modeling algorithm for fuzzy logic control of cisatracurium as a neuromuscular block. Eng Appl Artif Intel 22: 129-140.

11. Chuang CT, Fan SZ, Shieh JS (2006) Muscle relaxation controlled by automated administration of cisatracurium. Biomed Eng-app Bas c 18: 284-295.

12. Kisor DF, Schmith VD (1999) Clinical pharmacokinetics of cisatracurium besilate. Clin Pharmacokinet 36: 27-40.

13. Torres RF, Mochón MC, Sánchez JC, Bello López MA, Pérez AG (2001) Electrochemical oxidation of cisatracurium on carbon paste electrode and its analytical applications. Talanta 53: 1179-1185.

14. Nehmer U (1988) Separation of cis-cis, cis-trans and trans-trans isomers of (±)-atracurium besylate and cis and trans isomers of its major quaternary decomposition products and related impurity by reversed-phase.

15. Hillier K (2007) Cisatracurium. xPharm: The Comprehensive Pharmacology Reference, Elsevier, New York .

16. Stenlake JB, Waigh RD, Dewar GH, Urwin J, Dhar NC (1979) US patent 4,179,507. Burroughs Wellcome Co.

17. Hill DA, Turner GL (1995) US Patent 5,453,510. Burroughs Wellcome Co.

18. Loto CA, Popoola API, Fayomi OS, Loto RT (2012) Corrosion Polarization Behaviour of Type 316 Stainless Steel in Strong Acids and Acid Chlorides. Int J Electrochem Sci 7: 3787-3797.

19. ARAD O (2008) 1R,1’R)-ATRACURIUM SALTS SEPARATION PROCESS: WIPO Patent Application WO/2008/107887.OSTROVSKY , Elena (9/12 Horovitz Street, Rishon Le-zion, 75233, IL).

20. Venkatesan SC, Santhiyanarayanan S, Rao BV, Nadamuni G (2010) Novel Process For The Preparation Of Cisatracurium Besylate: WIPO Patent Application WO/2010/128518.

21. Zhang H, Wang P, Bartlett MG, Stewart JT (1998) HPLC determination of cisatracurium besylate and propofol mixtures with LC-MS identification of degradation products. J Pharm Biomed Anal 16: 1241-1249.

22. Liu C, Wang Y, Zhou T (1994) Determination of R-S-isomer and S-S-isomer of Cisatracurium Besilate by High Performance Liquid Chromatography. Acta Pharmaceutica Sinica 01.

23. Berthod A (2011) Chiral Recognition in Separation Methods. Mechanisms and applications. Anal Bioanal Chem 400: 5-6.

24. Gübitz G, Schmid MG (2004) Chiral Separation Methods and protocols. In: Methods in Molecular Biology, Volume 243, Humana Press Totowa, New Jersey.

25. Chiral Technologies Europe (2003) Instruction Manual For Chiralcel® Od-H Columns.

26. Yan C, Xiu Z, Li X, Hao C (2007) Molecular modeling study of beta-cyclodextrin complexes with (+)-catechin and (-)-epicatechin. J Mol Graph Model 26: 420-428.

27. Zhu JL, He J, He H, Tan SH, He XM, et al. (2011) Study on the interaction between ketoprofen and bovine serum albumin by molecular simulation and spectroscopic method. Spectroscopy 26: 337-348.

28. Lengauer T, Rarey M (1996) Computational methods for biomolecular docking. Curr Opin Struct Biol 6: 402-406.

29. Li W, Liu C, Tan G, Zhang X, Zhu Z, et al. (2012) Molecular Modeling Study of Chiral Separation and Recognition Mechanism of β-Adrenergic Antagonists by Capillary Electrophoresis. Int J Mol Sci 13: 710-725.

30. Warren GL, Andrews CW, Capelli AM, Clarke B, LaLonde J, et al. (2005) A critical assessment of docking programs and scoring functions. J Med Chem 49: 5912-5931.

31. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD (2003) Improved protein-ligand docking using GOLD. Proteins 52: 609-623.

32. Dong X, Wu R, Dong J, Wu M, Zhu Y, et al. (2008) The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography. J Chromatogr B Analyt Technol Biomed Life Sci 875: 317-322.

33. Zhu X, Sun H, Zhang D, Liu C (2011) Theoretical study on the interactions between methanol and imidazolium-based ionic liquids. J Mol Model 17: 1997-2004.

34. Gao T, Andino JM, Alvarez-Idaboy JR (2010) Computational and experimental study of the interactions between ionic liquids and volatile organic compounds. Phys Chem Chem Phys 12: 9830-9838.

35. Li W, Zhang W, Wang X, Dou J, Chen L, et al. (2003) Chiral separation of novel chiral tetrahedrone-type clusters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase. Anal Chim Acta 495: 77-83.

36. Narasimhamurthy T, Benny JC, Pandiarajan K, Rathore RS (2003) Herring-bone pi-pi interactions in trans-2,6-diphenyl-2,3,5,6-tetrahydrothiapyran-4-one. Acta Crystallogr C 59: o620-621.

37. Pirkle W, Pochapsky T (1989) Considerations of Chiral Recognition Relevant to the Liquid Chromatographic Separation of Enantiomers. Chem Rev 89: 347-362.

38. Li W, Tan G, Zhao L, Chen X, Zhang X, et al. (2012) Computer-aided molecular modeling study of enantioseparation of iodiconazole and structurally related triadimenol analogues by capillary electrophoresis: chiral recognition mechanism and mathematical model for predicting chiral separation. Anal Chim Acta 718: 138-147.

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Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176

Citation: Ndorbor T, Wang Y, Huaijing D, Zhizhang D, Kolawole JA, et al. (2013) Chromatographic and Molecular Simulation Study on the Chiral Recognition of Atracurium Besylate Positional Isomers on Cellulose Tri- 3, 5-Dimethylphenycarbamate (CDMPC) Column and its Recognition Mechanism. J Chromat Separation Techniq 4: 176. doi:10.4172/2157-7064.1000176