774
A. Nardi ef al.
Annalisa Nardi' Salvatore Fanali' FrantiSek Foret2 'Istituto di Cromatografiadel C.N.R., Monterotondo Scalo (Roma) 21nstituteof Analytical Chemistry, Czechoslovak Academy of Sciences, Brno
Electrophoresis 1990, 11. 774-716
Capillary zone electrophoretic separation of cyclodextrins with indirect UV photometric detection A method for the separation of cyclodextrins by capillary zone electrophoresis with indirect detection is described. It is based on the formation of inclusion complexes with benzoate which simultaneously forms a UV absorbing constituent of the background electrolyte used for the separation.
1 Introduction Cyclodextrins (CD) are neutral polymers of glucose with a shape of a truncated cone and a cavity able to form inclusion complexes with several kinds of chemical compounds. Due to their properties C D s are frequently applied in different fields, e.g. in analytical chemistry for the resolution of structural, positional and enantiomeric isomers [ 1-61. In pharmaceutical applications C D s are used as complexing agent for drugs in order to improve their solubility [71. In view ofthe importance of C D s in different pharmaceutical areas, e.g. control ofdrugs, pharmacokinetic studies in plasma,etc.,it isimportant to have an analytical method for their determination. Methods used so far for the determination of C D s are thin-layer chromatography (TLC) and high performance liquid chromatography i8,91. Capillary zone electrophoresis (CZE) is a relatively new analytical technique for the separation of ionogenic compounds [ 101. The advantages of C Z E arise from its high separation efficiency, good reproducibility, low consumption of either electrolytes and/or samples and speed of analysis. The separated zones are detected by a suitable on-line detector. UV absorbance, fluorescence, conductivity and electrochemical detectors are common in C Z E analysis [ 101. Indirect detection modes can also be used in C Z E for the determination of compounds which do not provide sufficient signal in direct detection [ I I, 121. This paper deals with the resolution of a, p and y-CD by C Z E with indirect UV photometric detection. Since C D s are neutral compounds, their electromigration is assured by the formation of inclusion complexes with benzoic acid, which also serves as a UV absorbing constituent of the background electrolyte (BGE). Indirect UV detection of C D is possible probably due to the shift ofthe absorbance spectraofthecomplexed species [71.
2 Materials and method 2.1 Apparatus Electrophoretic experiments were performed in a fused silica capillary tube 50 cm x 0.075 mm i.d. (S.G.E:., Ringwood, Vic-
Correspondence: Dr. Salvatore Fanali, Istituto di Cromatografia del C.N.R., AreadellaRicercadiRoma, C.P. 10,1-00016 Monterotondo Scalo (Roma), Italy Abbreviations: BGE, background electrolyte: CD, cyclodextrin; CZE, capillary zone electrophoresis
0VCH Verlagsgesellschaft mbH. D-6940 Weinheim. I990
toria, Australia). The distance between the anode end of the capillary and the on-column detection cell was 36 cm. A Varian 2550 UV detector (Walnut Creek, CA, USA) was used for indirect UV detection at a wavelength of 254 nm. The detector cell and the refilling block were described previously [ 13 I. The operator security was guaranteed by a plexiglass enclosure that was interlocked. Chromatopac C-RSA (Shimadzu, Kyoto, Japan) was used to record the electropherograms. A Glassman LH60R high voltage power supply (Witehouse Station, NJ, USA) was used at aconstantvoltagemode. Sample loading was performed by siphoning.
2.2 Chemicals All chemicals used were of reagent grade. P-Cyclodextrin (0-CD) and y-cyclodextrin (y-CD) were purchased from Sigma (St. Louis, MO, USA). L-histidine and a-cyclodextrin (a-CD) were from Fluka (Buchs, Switzerland). Benzoic acid and tris(hydroxymethy1)aminomethane (Tris) were obtained from Carlo Erba (Milan, Italy). Doubly distilled water was used to prepare the solutions. Cicladol (Master Pharma, Parma, Italy), declared to contain piroxicam, complexed with P-cyclodextrin, was used as a real sample.
3 Results and discussion C D s are neutral polymers of glucose. Their shapeis afunction of the number of glucose units; the most common ones used are a, and y-CD, with six, seven and eight units, respectively. Being uncharged compounds, they are difficult to analyze by CZE. Uncharged compounds have been separated in CZE, e.g. by using micellar electrokinetic chromatography [ 141. In the proposed analytical method CDs are moved toward the detector by electroosmotic flow and the selectivity of the separation is obtained by using inclusion complexing equilibria between C D s and the anion of the BGE. As C D s show virtually no absorbance in the UV range, the BGE was selected, considering the possibility to perform indirect UV detection. We selected benzoate as anion in the BGE that provided either complexing properties towards CDs and UV absorbtion. Figure 1 shows the mechanism of the movement ofCD. In this mechanism we suppose that the BGE is pumped towards the cathode by the electroosmotic flow. During this process C D s are in contact with benzoate, which is a UV absorbing constituent of the BGE and forms inclusion complexes. The inclusion complexes CD-benzoate are negatively charged and migrate as anions. The selectivity of the separation of a, p and y-CD is a result of the combination of the electroosmotic flow and the inclusion complexation. Of course, the two parameters should be controlled in order to 0173-0835/90/0909-0774 $3.50+.25/0
Eleclrophoresis 1990.11, 714-776
CZE of cyclodextrins
optimize the analysis. Several experiments were performed by changing the concentration of benzoate in the range of 10-80 mMin order to find the optimum operationalconditions. By increasing the concentration of benzoate, the migration time of CDs increased. The detection order was always y, a a n d p. The analysis of p-CD was performed easily, even at a low concentration of benzoate and high electroosmotic flow. However, the peaks of a and y-CD were too close to the solvent (H,O) peak and thus the analysis of a and y-CD was not possible. This is due to different association constants between benzoate and CDs. It was shown in capillary isotachophoresis that the mobility of benzoate is influenced by the type and the amount of CD [ 15, 161. Optimum separation conditions were obtained by using as BGE a buffer solution of 30 mM benzoic acid titrated to p H 6.2 with Tris. The separation of a, p and yCD could be achieved in less than 7 min. In order to test thereproducibility of the migration times of CDs, a model mixture was injected seven times and the standard deviation found was 1.2, 1.8 and 1.3 for a, p and y-CD, respectively. The capillary was rinsed with the BGE after each run. Figure 2 shows the effect of the concentration of benzoate in the BGE on the effective mobilities (UCDB-) of a, p and y-CD inclusion complexes. The order of effective mobilities of CDBcomplexes was always GPCDB-> G i a c ~ ~G,CDB- > and since the sample zones were moving towards the cathode the order of migration times was ~PCDE-> t a c ~ r C>~ C D B - Of . course this is due to different association constants between CDs and benzoate. The strongest interaction was observed for benzoate
775
and [I-CD. It seems that at the concentration of 80 mM of benzoate in the BGE, p-CD is fully complexed with benzoate and at this concentration we can estimate the electrophoretic mobility of the 0-CDB- complex to be -8 x lo-' c m Z V 's-I. The formation of a and y-CD-benzoate complexes is much weaker than with p-CD. From the dependencies shown in Fig. 2 and from the definition of the effective mobility we can estimate the molar fraction of CD present in the complexed form i.e. CDB-. For the dependence of the effective mobility UCDB- of a zone of CD moving in the BGE with different concentrations of benzoate, Eq. (1) 1171 applies: :CDB-
= XCD UCD
(1)
-t XCDB- UCL)B-
wherexcDand XCDBare the molar fractions of non-complexed and complexed CD and U C D and U C D B - their electrophoretic mobilities, respectively. It is clear that the mobility of noncomplexed CD, U C D , is zero and that the molar fraction of complexed CD at a given concentration of benzoate in BGE can be calculated as
where [CDB-1and CCD are the equilibrium and analytical concentrations of cyclodextrin in a moving zorie. Thus, e.g. at the concentration of 20 mM of benzoatein the BGE, approximately 50 % of P-cyclodextrin is complexed with benzoate. Two times and four times lower complexation is observed for a and y-CD, respectively. Figure 3 demonstrates a separation record of a model mixture of a, p and y-CD, obtained in a BGE consisting of 30 mM benzoic acid/Tris, pH 6.2. The first peak, detected at about 5 min, corresponds to the peak of water originating from the sample solution. This peak can be used to follow the speed of electro-
I CD
H,O +
B--
CDB-
Figure I . Scheme of the separation mechanism of C D s in CZE.
-
U
*
10
254
8 /------
Y 6 .
'
4 [
2
;
A
./.i /
x
li
/
,
.
-
-x- -x,x
-
-
c
0
20
40
60
80
mM
Figure 2. The dependence of effective mobilities (in I 0 - b n Z / V s) of C D benzoate inclusion complexes on the concentration of benzoate in BGE.
I
1
I
I
I
0
2
4
6
8 min
Figure3.Theseparationofamodel mixtureofa,p andy-CD(103~ e a c h ) . Separation voltage: 20 kV, 7 yA. A,,,-absorbance at 254 nm. Sample loading by siphoning for 5 s, 10 cm (30 nL).
776
Electrophoresis 1990,11, 114-116
A. Nardi et al.
osmotic flow. Figure 4 shows the analysis ofapharmaceutical sample: we can determine both the UV non-absorbing zone of p-CD and the UV absorbing zone of the active component piroxicam (4-hydroxy-2-methyl-N-2-pyridinyl-2H1Jbenzothiazine-3-carboxamide 1,1 -dioxide). Here 10 mM of benzoate in the BGE was used in order to perform fast analysis with sufficient resolution.
From the presented results it is clear that CZE is a powerful tool for a rapid and efficient separation of both ionic and nonionic components in pharmaceutical chemistry.
Thanks are due to Mr. G. Caponecchi andMr. M . Cristallifor technical assistance. Received April 23, 1990
-I2
0
4 References
3
I11 Hinze, W. L., Pharr, D. Y., Fu, Z. S. and Burkert, W. G.,Anal. Chem.
Q nr 0
8 p-CD
‘1
PIR
Figure 4. Determination of p-CD and piroxicam (PIR) in a Cicladol tablet. One tablet containing 191.2 mg of p-CD and 20 mg of piroxicam was dissolved in 50 mL of water and injected by siphoning (5 s, 10 cm) into the separation capillary. Voltage: 25 kV, 10 PA. BGE: 10 mM benzoic acid/ Tris, pH 6.2.
1989,61,422-428. 121 Hinze, W. L., Sep. Purif: Methods 1981,10, 159-237. [31 Jelinek, I., Snopek, J. and Smolkova-Keulemannsova, E., J. Chromatogr. 1987,405, 379-384. 141 Ward, T. W. and Armstrong, D. W., J. Liq. Chromatogr. 1986, 9, 407-423. [51 Sybilska, D., Debowski, J., Juczak, J. and Zukowski, J., J. Chromatogr. 1984,286, 163-170. 161 Fanali, S., J . Chrornatogr. 1989,474,441-446. (71 Szejtli, J., Cyclodextrins and their Inclusion Complexes, Akademia Kiado, Budapest 1982, pp. 204-232. 181 Ibid., pp. 53-54. [91 Tacheuchi, T., Murayama, M. and Ishii, D., J. High Resol. Chromatogr. 1990,13,69-70. I101 Foret, F. and BoEek, P.,Adv. Electrophoresis 1989,3,273-342. [ I 11 Kuhr, W. G. andYeung, E. S., Anal. Chem. 1988,60, 1832-1834. I121 Foret, F., Fanali, S., Ossicini, L. and BoEek, P.,J.Chrornatogr. 1989, 470,299-308. [ 131 Fanali, S., Ossicini, L., Foret, F. and BoEek, P.,J. Microcolumn Sep. 1989,1, 192-134. 1141 Terabe, S., Trends Anal. Chem. 1989,8, 129-134. [151 Snopek, J., Jelinek, I. and Smolkova-Keulemansova, E., J . Chromatrogr. 1987,411, 153-159. [ 161 Fanali, S . and Sinibaldi, M., J. Chromatogr. 1988,442, 371-377. I171 Tiselius,A.,NovaActaReg.Soc.Sci. Uppsaliensis 1930,7,1-107.
A. Nardi ef al.
Annalisa Nardi' Salvatore Fanali' FrantiSek Foret2 'Istituto di Cromatografiadel C.N.R., Monterotondo Scalo (Roma) 21nstituteof Analytical Chemistry, Czechoslovak Academy of Sciences, Brno
Electrophoresis 1990, 11. 774-716
Capillary zone electrophoretic separation of cyclodextrins with indirect UV photometric detection A method for the separation of cyclodextrins by capillary zone electrophoresis with indirect detection is described. It is based on the formation of inclusion complexes with benzoate which simultaneously forms a UV absorbing constituent of the background electrolyte used for the separation.
1 Introduction Cyclodextrins (CD) are neutral polymers of glucose with a shape of a truncated cone and a cavity able to form inclusion complexes with several kinds of chemical compounds. Due to their properties C D s are frequently applied in different fields, e.g. in analytical chemistry for the resolution of structural, positional and enantiomeric isomers [ 1-61. In pharmaceutical applications C D s are used as complexing agent for drugs in order to improve their solubility [71. In view ofthe importance of C D s in different pharmaceutical areas, e.g. control ofdrugs, pharmacokinetic studies in plasma,etc.,it isimportant to have an analytical method for their determination. Methods used so far for the determination of C D s are thin-layer chromatography (TLC) and high performance liquid chromatography i8,91. Capillary zone electrophoresis (CZE) is a relatively new analytical technique for the separation of ionogenic compounds [ 101. The advantages of C Z E arise from its high separation efficiency, good reproducibility, low consumption of either electrolytes and/or samples and speed of analysis. The separated zones are detected by a suitable on-line detector. UV absorbance, fluorescence, conductivity and electrochemical detectors are common in C Z E analysis [ 101. Indirect detection modes can also be used in C Z E for the determination of compounds which do not provide sufficient signal in direct detection [ I I, 121. This paper deals with the resolution of a, p and y-CD by C Z E with indirect UV photometric detection. Since C D s are neutral compounds, their electromigration is assured by the formation of inclusion complexes with benzoic acid, which also serves as a UV absorbing constituent of the background electrolyte (BGE). Indirect UV detection of C D is possible probably due to the shift ofthe absorbance spectraofthecomplexed species [71.
2 Materials and method 2.1 Apparatus Electrophoretic experiments were performed in a fused silica capillary tube 50 cm x 0.075 mm i.d. (S.G.E:., Ringwood, Vic-
Correspondence: Dr. Salvatore Fanali, Istituto di Cromatografia del C.N.R., AreadellaRicercadiRoma, C.P. 10,1-00016 Monterotondo Scalo (Roma), Italy Abbreviations: BGE, background electrolyte: CD, cyclodextrin; CZE, capillary zone electrophoresis
0VCH Verlagsgesellschaft mbH. D-6940 Weinheim. I990
toria, Australia). The distance between the anode end of the capillary and the on-column detection cell was 36 cm. A Varian 2550 UV detector (Walnut Creek, CA, USA) was used for indirect UV detection at a wavelength of 254 nm. The detector cell and the refilling block were described previously [ 13 I. The operator security was guaranteed by a plexiglass enclosure that was interlocked. Chromatopac C-RSA (Shimadzu, Kyoto, Japan) was used to record the electropherograms. A Glassman LH60R high voltage power supply (Witehouse Station, NJ, USA) was used at aconstantvoltagemode. Sample loading was performed by siphoning.
2.2 Chemicals All chemicals used were of reagent grade. P-Cyclodextrin (0-CD) and y-cyclodextrin (y-CD) were purchased from Sigma (St. Louis, MO, USA). L-histidine and a-cyclodextrin (a-CD) were from Fluka (Buchs, Switzerland). Benzoic acid and tris(hydroxymethy1)aminomethane (Tris) were obtained from Carlo Erba (Milan, Italy). Doubly distilled water was used to prepare the solutions. Cicladol (Master Pharma, Parma, Italy), declared to contain piroxicam, complexed with P-cyclodextrin, was used as a real sample.
3 Results and discussion C D s are neutral polymers of glucose. Their shapeis afunction of the number of glucose units; the most common ones used are a, and y-CD, with six, seven and eight units, respectively. Being uncharged compounds, they are difficult to analyze by CZE. Uncharged compounds have been separated in CZE, e.g. by using micellar electrokinetic chromatography [ 141. In the proposed analytical method CDs are moved toward the detector by electroosmotic flow and the selectivity of the separation is obtained by using inclusion complexing equilibria between C D s and the anion of the BGE. As C D s show virtually no absorbance in the UV range, the BGE was selected, considering the possibility to perform indirect UV detection. We selected benzoate as anion in the BGE that provided either complexing properties towards CDs and UV absorbtion. Figure 1 shows the mechanism of the movement ofCD. In this mechanism we suppose that the BGE is pumped towards the cathode by the electroosmotic flow. During this process C D s are in contact with benzoate, which is a UV absorbing constituent of the BGE and forms inclusion complexes. The inclusion complexes CD-benzoate are negatively charged and migrate as anions. The selectivity of the separation of a, p and y-CD is a result of the combination of the electroosmotic flow and the inclusion complexation. Of course, the two parameters should be controlled in order to 0173-0835/90/0909-0774 $3.50+.25/0
Eleclrophoresis 1990.11, 714-776
CZE of cyclodextrins
optimize the analysis. Several experiments were performed by changing the concentration of benzoate in the range of 10-80 mMin order to find the optimum operationalconditions. By increasing the concentration of benzoate, the migration time of CDs increased. The detection order was always y, a a n d p. The analysis of p-CD was performed easily, even at a low concentration of benzoate and high electroosmotic flow. However, the peaks of a and y-CD were too close to the solvent (H,O) peak and thus the analysis of a and y-CD was not possible. This is due to different association constants between benzoate and CDs. It was shown in capillary isotachophoresis that the mobility of benzoate is influenced by the type and the amount of CD [ 15, 161. Optimum separation conditions were obtained by using as BGE a buffer solution of 30 mM benzoic acid titrated to p H 6.2 with Tris. The separation of a, p and yCD could be achieved in less than 7 min. In order to test thereproducibility of the migration times of CDs, a model mixture was injected seven times and the standard deviation found was 1.2, 1.8 and 1.3 for a, p and y-CD, respectively. The capillary was rinsed with the BGE after each run. Figure 2 shows the effect of the concentration of benzoate in the BGE on the effective mobilities (UCDB-) of a, p and y-CD inclusion complexes. The order of effective mobilities of CDBcomplexes was always GPCDB-> G i a c ~ ~G,CDB- > and since the sample zones were moving towards the cathode the order of migration times was ~PCDE-> t a c ~ r C>~ C D B - Of . course this is due to different association constants between CDs and benzoate. The strongest interaction was observed for benzoate
775
and [I-CD. It seems that at the concentration of 80 mM of benzoate in the BGE, p-CD is fully complexed with benzoate and at this concentration we can estimate the electrophoretic mobility of the 0-CDB- complex to be -8 x lo-' c m Z V 's-I. The formation of a and y-CD-benzoate complexes is much weaker than with p-CD. From the dependencies shown in Fig. 2 and from the definition of the effective mobility we can estimate the molar fraction of CD present in the complexed form i.e. CDB-. For the dependence of the effective mobility UCDB- of a zone of CD moving in the BGE with different concentrations of benzoate, Eq. (1) 1171 applies: :CDB-
= XCD UCD
(1)
-t XCDB- UCL)B-
wherexcDand XCDBare the molar fractions of non-complexed and complexed CD and U C D and U C D B - their electrophoretic mobilities, respectively. It is clear that the mobility of noncomplexed CD, U C D , is zero and that the molar fraction of complexed CD at a given concentration of benzoate in BGE can be calculated as
where [CDB-1and CCD are the equilibrium and analytical concentrations of cyclodextrin in a moving zorie. Thus, e.g. at the concentration of 20 mM of benzoatein the BGE, approximately 50 % of P-cyclodextrin is complexed with benzoate. Two times and four times lower complexation is observed for a and y-CD, respectively. Figure 3 demonstrates a separation record of a model mixture of a, p and y-CD, obtained in a BGE consisting of 30 mM benzoic acid/Tris, pH 6.2. The first peak, detected at about 5 min, corresponds to the peak of water originating from the sample solution. This peak can be used to follow the speed of electro-
I CD
H,O +
B--
CDB-
Figure I . Scheme of the separation mechanism of C D s in CZE.
-
U
*
10
254
8 /------
Y 6 .
'
4 [
2
;
A
./.i /
x
li
/
,
.
-
-x- -x,x
-
-
c
0
20
40
60
80
mM
Figure 2. The dependence of effective mobilities (in I 0 - b n Z / V s) of C D benzoate inclusion complexes on the concentration of benzoate in BGE.
I
1
I
I
I
0
2
4
6
8 min
Figure3.Theseparationofamodel mixtureofa,p andy-CD(103~ e a c h ) . Separation voltage: 20 kV, 7 yA. A,,,-absorbance at 254 nm. Sample loading by siphoning for 5 s, 10 cm (30 nL).
776
Electrophoresis 1990,11, 114-116
A. Nardi et al.
osmotic flow. Figure 4 shows the analysis ofapharmaceutical sample: we can determine both the UV non-absorbing zone of p-CD and the UV absorbing zone of the active component piroxicam (4-hydroxy-2-methyl-N-2-pyridinyl-2H1Jbenzothiazine-3-carboxamide 1,1 -dioxide). Here 10 mM of benzoate in the BGE was used in order to perform fast analysis with sufficient resolution.
From the presented results it is clear that CZE is a powerful tool for a rapid and efficient separation of both ionic and nonionic components in pharmaceutical chemistry.
Thanks are due to Mr. G. Caponecchi andMr. M . Cristallifor technical assistance. Received April 23, 1990
-I2
0
4 References
3
I11 Hinze, W. L., Pharr, D. Y., Fu, Z. S. and Burkert, W. G.,Anal. Chem.
Q nr 0
8 p-CD
‘1
PIR
Figure 4. Determination of p-CD and piroxicam (PIR) in a Cicladol tablet. One tablet containing 191.2 mg of p-CD and 20 mg of piroxicam was dissolved in 50 mL of water and injected by siphoning (5 s, 10 cm) into the separation capillary. Voltage: 25 kV, 10 PA. BGE: 10 mM benzoic acid/ Tris, pH 6.2.
1989,61,422-428. 121 Hinze, W. L., Sep. Purif: Methods 1981,10, 159-237. [31 Jelinek, I., Snopek, J. and Smolkova-Keulemannsova, E., J. Chromatogr. 1987,405, 379-384. 141 Ward, T. W. and Armstrong, D. W., J. Liq. Chromatogr. 1986, 9, 407-423. [51 Sybilska, D., Debowski, J., Juczak, J. and Zukowski, J., J. Chromatogr. 1984,286, 163-170. 161 Fanali, S., J . Chrornatogr. 1989,474,441-446. (71 Szejtli, J., Cyclodextrins and their Inclusion Complexes, Akademia Kiado, Budapest 1982, pp. 204-232. 181 Ibid., pp. 53-54. [91 Tacheuchi, T., Murayama, M. and Ishii, D., J. High Resol. Chromatogr. 1990,13,69-70. I101 Foret, F. and BoEek, P.,Adv. Electrophoresis 1989,3,273-342. [ I 11 Kuhr, W. G. andYeung, E. S., Anal. Chem. 1988,60, 1832-1834. I121 Foret, F., Fanali, S., Ossicini, L. and BoEek, P.,J.Chrornatogr. 1989, 470,299-308. [ 131 Fanali, S., Ossicini, L., Foret, F. and BoEek, P.,J. Microcolumn Sep. 1989,1, 192-134. 1141 Terabe, S., Trends Anal. Chem. 1989,8, 129-134. [151 Snopek, J., Jelinek, I. and Smolkova-Keulemansova, E., J . Chromatrogr. 1987,411, 153-159. [ 161 Fanali, S . and Sinibaldi, M., J. Chromatogr. 1988,442, 371-377. I171 Tiselius,A.,NovaActaReg.Soc.Sci. Uppsaliensis 1930,7,1-107.
