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JSSC jssc201200275 Dispatch: April 28, 2012 CE:
Journal MSP No. No. of pages: 6 PE: XXXXX
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UNCORRECTEDPROOF
J. Sep. Sci. 2012, 00, 1–6 1
Francisco Garcıa SanchezAurora Navas DıazAlfonso AguilarIgnacio Medina LamaManuel Algarra
Department of AnalyticalChemistry, Faculty of Science,University of Malaga, Malaga,Spain
Received March 14, 2012Revised March 30, 2012Accepted April 3, 2012
Research Article
HPLC enantioseparation of alkaloidmalacitanine using fluorimetric/polarimetricdetection
This work reports two methods developed for the separation and determination of the enan-tiomers of the new alkaloid malacitanine (MLC) and the determination of the enantiomericpurity in mixtures. First, the isomers were separated using a Chirex 3020 (250 mm × 4.6mm, 5 �m) chiral column with a mobile phase of cyclohexane–1,2-dichloroethane–ethanol–trifluoroacetic acid (64:30:6:0.6, v/v) at a flow rate of 1 mL/min and fluorimetric detection.Obtained retention times were 12.4 and 15.9 min (+ and −) with a resolution Rs of 1.13.Relative standard deviations (RSDs) were 2.5 and 2.4% at the 0.5-�g level (four determi-nations). Second, a nonenantioselective procedure for the determination of enantiomericpurity of MLC using a Lichrospher R© Si-60 (250 mm × 5 mm, 5 �m) normal phase witha mobile phase of 100% ethanol at a flow rate of 0.9 mL/min coupled to two detectors inseries, fluorimetric and polarimetric. RSD of 3.3% was obtained. Calculated enantiomericpurity by chiral chromatography gave 48.6% (−)-MLC in the near racemic product. Usingpolarimetric signal of the nonseparated enantiomers and comparing the slopes of the cal-ibration curves (enantiomers) from the racemic product gave 47.8% (−)-MLC content. Astudy of accuracy of (−)-MLC gave recoveries from 98.3 to 100.7%.
Keywords: Alkaloids / Ceratocapnos heterocarpa / Chiral separation / Fluorimetric/ Malacitanine / Polarimetric detectionDOI 10.1002/jssc.201200275
1 Introduction
Botanicals represent a formidable source of potential pharma-ceutical and nutraceutical compounds because of the molec-ular diversity found in nature. Two main challenges can befaced in the analysis of phytochemicals: the lack of suitablestandards, and the fact that many bioactive compounds arefound at low levels in complex mixtures or plant extracts mak-ing detection and quantification very difficult. Frequently,natural products analysis suffers an additional difficulty; dueto its inherent optical activity and since it is well known thata pair of enantiomers can display quite different activity andtherapeutic and toxicological profiles, the analytical chemistryof these compounds is a promising research field strongly de-manded by pharmaceutical companies.
Protoberberines represent a structural class of organiccations that have been mainly distributed in several plantsand widely used in western natural medicine and traditionalChinese herbal medicine [1, 2]. Protoberberine alkaloids arereported to have anticancer [3–5], anti-infective [6, 7], antidi-
Correspondence: Professor Francisco Garcıa Sanchez, Departa-mento de Quımica Analıtica, Facultad de Ciencias, Universidadde Malaga, Malaga 29071, SpainE-mail: [email protected]: +34 952131884
Abbreviations: CD, circular dichroism; MLC, malacitanine;OR, optical rotation; RIA, refractive index artefact
abetic [8], immunomodulatory [9, 10], and antimalarial ac-tivities [11, 12]. Most published papers have focused on theseparation of chiral pharmaceutical products, obtained by or-ganic synthesis, because the production and commercializa-tion of enantiomerically pure drugs is a warranty of goodpharmaceutical practices. Although, in several cases, signifi-cant differences between the pharmacology and toxicology ofthe individual enantiomers and the racemate have not beenclearly demonstrated; in other instances, serious differencesbetween them have been found. For example, it has been re-ported that l-tetrahydropalmatine has analgesic activity witha more potent tranquilizing effect than the d-isomer [13]. Re-cently, the antimalarial activity and structure–activity relation-ship of 22 protoberberines has been studied and discussed[7,12]. No references to malacitanine (MLC) have been founduntil now.
(−)-MLC is an isoquinolinic alkaloid that can be extractedfrom Ceratocapnos herocarpa, endemic plant found in southof Spain and north of Africa [14]. Together with heterocarpineis the unique tetrahydroprotoberberine containing a hydrox-ymethyl group at C-8.
MLC molecule has two chiral centers, C-8 and C-14, thuspresenting two enantiomeric pairs. In this work, we usedpure 8S,14S-(−)-MLC (Fig. 1) and a nonracemic mixture ofboth enantiomers. The other enantiomeric pair (epimalac-itanine) has not been used. Total synthesis has been per-formed by proton and C13 NMR. 8S,14S-(−)-MLC has a spe-cific rotation of [�]D = −87.3� (c = 0.05, methanol). MLC is
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UNCORRECTEDPROOF
2 F. G. Sanchez et al. J. Sep. Sci. 2012, 00, 1–6
Figure 1. Structure of 8S,14S-(−)-MLC (A)and 8R, 14R-(+)-MLC (B).
soluble in methanol and slightly soluble in ethanol. Theethanolic solution of MLC shows absorption spectrum max-ima at �max (log ε) 205 (4.75) and 286 (3.56) nm and a shoul-der at 228 (406) nm. This ethanolic solution shows intensefluorescence emission centered at 318 nm when excited at285 nm. Figure 2 shows absorption (A), excitation (B), andemission (C) spectra of MLC.
The enantiomeric form found in the nature is (−)-MLC in several papaveraceas such as C. heterocarpa. Biologi-cal/pharmacological effects have not been studied until now,however, it is probable that these effects are similar as inother protoberberines (antioxidant, anticolinergic, antisecre-tive, astringent, and antimicrobial agents).
In the present paper, we proposed a chromatographicmethod for the separation and determination of the enan-tiomers of MLC based on the use of a chiral col-umn Chirex 3020 (250 mm × 4.6 mm, 5 �m) fromPhenomenex (Torrance, CA, USA). Mobile phase wascyclohexane–1,2-dichloroethane–ethanol–trifluoroacetic acid(TFA) (64:30:6:0.6, v/v) at a flow rate of 1 mL/min. No previ-ous analytical methods have been found for the determinationof MLC and its enantiomers.
In addition, a procedure for the determination of enan-tiomeric purity using a conventional chromatographic col-umn Si-60 (250 mm × 4 mm, 5 �m) from Merck (Darmstadt,Germany) in normal phase (100% ethanol) at a flow rateof 0.9 mL/min coupled to a polarimetric detector (AppliedChromatographic Systems Limited, Macclestfield, England)placed in series with the fluorimetric detector gives additionalinformation to determine enantiomeric purity, because theabsorbance or fluorescence signal is proportional to the totalamount of stereoisomer present, while the polarimetric de-tector signal is dependent on the ratio of enantiomers present[15–17]. This methodology, based on the use of a combinedsignal from a generic detector (UV) and a chiroptical detec-tor (O.R.) has been used previously in the enantiomeric ratiodetermination of several compounds [18–21].Q1
2 Experimental
2.1 Chemicals and reagents
(S)- (−)-MLC and racemic (±)-MLC were obtained from theQ2Department of Organic Chemistry, in University of Malaga,Suau et al. [14, 22].
(S)-(−)-MLC was isolated from the plant (C. hetero-carpa) by extraction with methanol and subsequently sep-arated by liquid chromatography column and preparativethin-layer chromatography [14]. The racemic was synthesized Q3[22] (joined with (±)-epimalacitanine) of which was separatedby preparative thin-layer chromatography reacting glycolalde-hyde norcrasilofoline with 2.5 M hydrochloric acid medium.
2.2 Instrumentation
Measurements were performed with a Merck-Hitachi (Darm-stadt, Germany) liquid chromatograph consisting of an L-6200 pump, an AS-4000 autosampler, an L-4250 UV-visibledetector, an F-1080 fluorescence detector, and a D-6000 in-terface. Instrument parameters were controlled by Hitachi-Merck HM software.
A ChiraMonitor 2000 optical rotation (OR) detector(Applied Chromatography Systems Limited) placed in seriesafter above-mentioned detectors. This detector was equippedwith a collimated laser diode providing up to 30 mW of lightat 830 nm, and a flow cell of 0.48 dm path length, 73 �Lvolume.
Data acquisition and transformation were accomplishedby the Pico ADC-142 (Picotechnology Ltd., Cambridge, UK),which is an analog-to-digital converter with two input ranges.The instrumental parameters were controlled by Picolog soft-ware and the calculation of the areas (negative and positivepeaks) with Microsoft Origin 7.5.
2.3 Chromatographic conditions
Enantiomeric separation was accomplished using a chiralchromatographic column (Chirex 3020, Phenomenex) con-sisting of (S)-tert-leucine and R-1-(�-naphtyl)ethylamine innormal-phase conditions. Several percentages of tertiary mix-ture were assayed and Rs values calculated from chromato-graphic data. The obtained data show that a percentage of64% n-hexane, 30% dichloroethane, 6% ethanol, and 0.6%TFA gives the best resolution at a flow rate of 1 mL/min, with1-�L injection.
Nonenantioselective chromatographic conditions wereselected to obtain a good separation between injection andanalyte peaks. A Si-60 (250 mm × 4 mm, 5 �m) column wasused as stationary phase, 100% ethanol as mobile phase, and0.9 mL/min flow rate.
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J. Sep. Sci. 2012, 00, 1–6 Liquid Chromatography 3
Figure 2. Absorption (A), excitation (B), and emission (C) spectraof (−)-MLC. [MLC] = 35 �g/mL in ethanol.
2.4 Standard solutions
Stock standard solutions of (S)-(−)-MLC (1600 mg/L) and(±)-MLC (1600 mg/L) were prepared by dissolving the com-pounds in ethanol. Dilutions were in the appropriate mo-biles phase. The solvents used as mobile phase were gradi-
ent grade from Lichrosolv Merck (methanol, ethanol, 1,2-dichloroethane, TFA, and cyclohexane). All these solventswere filtered through 0.2-�m nylon membrane and then de-gassed for 1 h in ultrasonic bath before using.
3 Results and discussion
3.1 Enantioselective HPLC
3.1.1 Mobile-phase effect on retention
and stereoselectivity
As expected, large contents on hexane promote higher reten-tion time and polar alcohols give short retention time. How-ever, the solubility of the analyte in cyclohexane is higherand this solvent was preferred as a mobile phase in spite oftheir high viscosity. The use of ethanol as a modifier appearedto be better to obtain short retention times than 2-propanol.To improve selectivity, the addition of 1,2-dichloroethane hasproved to be effective, and TFA gives best chromatographicprofiles. Finally, the mobile phase giving best separationof both enantiomers was cyclohexane–1,2-dichloroethane–ethanol–TFA (32:15:3:0.3; v/v) at a flow rate of 1 mL/min.
Preliminary experiences show that neither chiral station-ary phase Chiradex, Merck (�-cyclodextrine inclusion mech-anism) nor Chiraspher, Merck (Pirkle type) gave good sepa-ration of MLC enantiomers.
Using a chiral column Chirex 3020 and the mobile phaseabove cited, separation at baseline of both enantiomers canbe obtained in a short total time. Figure 3A shows the fluo-rimetric chromatogram of the near racemic mixture of bothenantiomers and Fig. 3B shows the chromatographic profileof the enantiomer (−)-MLC. As can be seen, retention time of12.4 and 15.9 min was obtained for (+)-MLC and (−)-MLC,respectively. From these data and using the equation Rs =2�t/w1 + w2, a resolution Rs value of 1.13 and a value of �
= 1.4 were obtained. Once separated the enantiomers, themethod validation was established. Linearity was assessedwith four series at six concentrations levels and the correla-tion coefficients were calculated in order to prove the linearityof the calibration curves. To evaluate the intraday precision,three control samples at the 500 ng/mL level (fluorescencedetector) (150 �g/mL in O.R.) were injected. Statistical testswere performed at a level of confidence of 95% (P = 0.05).The limits of detection and quantitation were considered tobe the concentrations that produced signal-to-noise ratios of3 and 10, respectively. In Table 1, the obtained results areordered. The enantiomeric purity determination was carried Q4out using the expression:
%S( − ) − MLC = [mS/mS+mR]100
where mS and mR are the values of the slopes of the cali-bration curve for each enantiomer [23, 24]. From these data,the enantiomeric purity of the sample used was found to be48.60% (−)-MLC.
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4 F. G. Sanchez et al. J. Sep. Sci. 2012, 00, 1–6
Figure 3. (A) Mixture of (+)-MLC and (−)-MLC chromatogram(flow rate 1 mL/min, fluorimetric detection, and sample concen-tration 16.00 mg/L, therefore, injection mass 0.160 �g). Retentiontimes 12.4 and 15.9 min for (+)-MLC and (−)-MLC, respectively.(B). (−)-MLC chromatogram (injection mass 0.40 �g).
3.2 Nonenantioselective HPLC: polarimetric
detection
The best option to establish a method for the determina-tion of the enantiomers of a mixture, or proportion in themixture (enantiomeric purity), is the enantioseparation bya suitable chromatographic procedure and comparison withstandard pure enantiomers. However, sometimes there is no
methodology or appropriate chiral column that separates theenantiomers, and in these cases the use of chiral detectionby circular dichroism (CD) or OR), whose response is spe-cific for compounds exhibiting optical activity, can solve theproblem. In this case, we used a column Si-60 (250 mm × Q55 mm, 5 �m) and 100% ethanol mobile phase at a flow rateof 0.9 mL/min. The choice of the mobile phase was advisedby the better solubility in ethanol. The Si-60 column givesresults with a narrow elution peak than with a reverse-phasecolumn.
Several samples containing 160 �g in an injection vol-ume of 100 �L were injected in the chromatograph. Sampleswere prepared from standard R-(−)-MLC (1600 mg/L) andstandard racemic (±)-MLC (1600 mg/L). Each sample wasprepared by mixing different aliquots in the volume of bothsolutions. The result is a mixture containing a fix concen-tration of 1600 mg/L of MLC with different enantiomericproportion. All measurements were performed by triplicate.Figure 4 shows the polarimetric profile of a blank, 100, 93.3,87.1, 81.3, and 72.8% (−)-MLC (from upper to lower). Ascan be seen, a false-positive signal appears before the truepolarimetric signal (negative) corresponding to the excessof (−)-MLC. This false-positive signal is due to the so-called
Figure 4. Polarimetric chromatograms. Polarimetric profile ofseveral mixtures of MLC containing a fix total concentration anddifferent percentages of enantiomers: blank, 100, 93.3, 87.1, 81.3,and 72.8% (−)-MLC (from upper to lower). Injected volume 100�L, total injected mass 160 �g.
Table 1. Analytical performances
Detector Enantiomer Range RSD LD LQ R2 Slope Intercept(ng) % (ng) (ng)
S(−) 217–1000 2.9 65.38 217.9 0.9993 0.0515 4.48FL
R(+) 61–1000 2.5 18.41 61.3 0.9992 0.0572 2.10S(−)
OR (115–160) × 103 3.3 35 × 10−3 115 × 10−3 0.9956 −0.0297 1.40R(+)
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J. Sep. Sci. 2012, 00, 1–6 Liquid Chromatography 5
Table 2. Recovery assay of MLC
MLC S(−) MLC R(+) MLC S(−) MLC R(+) % recovery % recoveryadded (�g) added (�g) found (�g) found (�g) MLC S(−) MLC R(+)calculated calculated
145.7 14.3144 16 143.8 16.2 100.74 ± 0.76 92.08 ± 8.15
146.3 13.7134.7 25.3
136 24 132.5 27.5 98.30 ± 0.81 109.58 ± 4.64133.9 26.1126.9 33.1
128 32 125.4 34.6 99.18 ± 0.31 105.29 ± 2.48126.6 33.4
refractive index artefact (RIA) [25], because no separation wasassociated with the polarimetric signal. Obtained polarimet-ric signals are noisiest because sensitivity of polarimetry, asdispersive phenomena, is reduced. However, this methodcan be useful when no separation can be obtained and anindicative report is needed.
From these data, the plot of polarimetric sig-nal/fluorimetric signal (peak area) against% (−)-MLC [26]gives a linear fit of P = 1.4 – 0.03x (x = % (−)-MLC)with a correlation coefficient of −0.998. Figure 5 shows thatthe crossing zero abscissa axis occurs at 47.8% (−)-MLC,in good concordance with that obtained by the fluorimet-ric/chromatographic method. If a true racemic mixture ofMLC was used as a standard, crossing of fittest curve withabscissa axis would be at 0.0%.
4 Recovery assay
To determine the accuracy of the developed methods, a recov-ery assay at 80, 85, and 90% levels o (−)-MLC was performed.
Figure 5. Optical rotation/RFI versus % (−)-MLC. Injected volume100 �L.Q6
For the analytical recovery assay study, three different quanti-ties (�g) of (−)-MLC and (±)-MLC were injected in the chro-matograph and submitted to the chiral method. The sum of(−)-MLC and (±)-MLC was 160 �g in a total volume of 100mL. Each sample was prepared by mixing different aliquotsof both solutions to obtain a final percentage of 80, 90, and95% of (−)-MLC. The spiked samples were analyzed in tripli-cate and the obtained results are ordered in Table 2. Recoveryassay shows that the accuracy of the method is excellent be-cause recoveries are within 109–92% with standard deviationbelow 5%.
5 Conclusions
The new isoquinolinic alkaloid MLC was analyzed, previouschiral HPLC separation with a resolution of 1.13, and enan-tiomeric excess of mixtures of both enantiomers determined.A second nonenantioselective method with polarimetric de-tection, for the determination of enantiomeric excess of mix-tures, was proposed.
The enantioselective chromatographic method of thisnew alkaloid is fast (10 min), selective (Rs 1.13), precise (RSD2.5%), and exact (recovery values between 98 and 101%. Thus,it is advisable for phytochemical enantiomeric analysis and toassess enantiomeric excess in enantioselective synthesis. Thenonenantioselective method shows good analytical specifica-tions and in addition is very simple, therefore, can be usefulas first analysis results in organic synthesis.
Results obtained show that the methods developed in thisstudy might be applied for the wide application in separationand determination of enantiomeric excess of the componentsof mixtures of enantiomers.
The authors have declared no conflict of interest.
6 References
[1] Gao, W. H., Lin, S. Y., Jia, L., Guo, X. K., Chen, X. G., Hu,Z. D., J. Sep. Sci. 2005, 28, 92–97.
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