130 M Jung, I<. 0. Bornsen and E. Francotte E/ecrrophore.\is 1996, 17, 130-136

Martin Jung K. Olaf Bornsen Eric Francotte

Ciba-Geigy, Basel, Switzerland

Dextrin sulfopropyl ether: A novel anionic chiral buffer additive for enantiomer separation by electrokinetic chromatography

Dextrin 10 sulfopropyl ether (DSPE) was prepared and characterized by matrix-assisted laser-desorption ionization-mass spectrometry (MALDI-MS) using a recently developed matrix. The anionic compound was used as a novel chiral buffer additive for enantiomer separation by capillary electrophoresis, thereby changing into the elektrokinetic chromatography (EKC) mode. DSPE was systematically evaluated as a chiral selector and was compared to the respective nonderivatized maltodextrin. DSPE showed an increased separation power for cationic racemic solutes. Although not quite as versatile and powerful as cyclodextrins, the inexpensive dextrin 10 and its derivative DSPE showed remarkable

1 Introduction

In recent years, cyclodextrins and some of their deriva- tives have been introduced and extensively used as powerful chiral buffer additives for efficient and easy enantiomer separations by CE [I]. The cyclodextrin deriv- atives are usually neutral and can be used for the enan- tiomer separation of charged analytes by capillary zone electrophoresis [(CZE). Enantiomer separation of neutral analytes can be accomplished by the addition of a micelle agent, e.g. sodium dodecyl sulfate, thereby changing into the micellar electrokinetic chromatography (MEKC) mode. In comparison to HPLC, important advantages of CE for enantiomer analysis consist in sim- plicity, efficiency, low operating cost, and versatility (easy switching from one chiral selector to another). P-Cyclo- dextrin sulfobutyl ether (p-CD-SBE) and sulfopropyl ether (P-CD-SPE) have recently been found to be partic- ularly powerful chiral selectors for CE even at very low concentrations, down into the DM range [2-71. Because of their sulfonic acid functions, they are negatively charged above pH 2 and thus have their own electropho- retic mobility. Hence, the separation mode is electroki- netic chromatography (EKC). As a consequence, the enantioselectivity towards cationic analytes was often found to be better than with uncharged cyclodextrins, and the enantiomer separation of neutral analytes became feasible without addition of a micelle agent. In addition to cyclodextrins, some commercially available, linear, negatively charged polysaccharides have also been used as chiral additives for EKC [8-101.

A few reports [ 11-14] describe the use of linear, electri- cally neutral carbohydrates as chiral selectors for the sep- aration of anionic analytes, such as several profen drugs and warfarin, as well as the cationic analytes verapamil

Correspondence: Dr. E. Francotte, Pharmaceutical Research. K-122.P.25, Ciba-Geigy AG, 4002 Basel, Switzerland

Nonstandard abbreviations: P-CD-SBE, p-cyclodextrin sulfobutyl ether; DHB, 2,s-dihydroxybenzoic acid; DSPE, dextrin sulfopropyl ether; HIC, 1-hydroxyisochinolin

Keywords: Maltodextrins / Enantiomer separation / Electrokinetic chromatography / Chiral capillary electrophoresis / Matrix-assisted laser-desorption ionization mass spectrometry

enantioselectivity in some cases.

and norverapamil, by CZE. Among a series of oligosac- charides, maltodextrins with a degree of polymerization beyond ca. 8 were found most useful for this purpose [12, 131 and display excellent water solubility. Dextrin 10 is commercially available at very low cost; “10” refers to a glucose equivalent of 10 (reducing power equivalent to 10% w/w glucose monomer content), i.e. the average degree of polymerization is also approximately 10. In this work, dextrin 10 sulfopropyl ether (DSPE) is pre- pared, characterized and systematically evaluated as a chiral selector for CE in comparison to native dextrin 10.

2 Materials and methods

2.1 Chemicals

Dextrins, cyclodextrins, 3-hydroxy-4-aminobenzoic acid and 1-hydroxyisochinolin were obtained from Fluka (Buchs, Switzerland). Tris, 2,5-dihydroxybenzoic acid and 1,3-propane sultone were obtained from Sigma (Buchs, Switzerland), and 1,4-butane sultone was purchased from Aldrich (Steinheim, Germany). Racemic samples were commercial products (Sigma). 1,l‘-Binaphthyl-2,2’- diylphosphate was kindly provided by Prof. V. Schurig (Tubingen, Germany).

2.2 Preparation of DSPE

In a round-bottom flask equipped with a dropping funnel, log dextrin 10 was dissolved in 50 mL water.

%og\ H n

H x “A-” Figure I . Struclure of dextrin sulfopropyl ether (DSPE). Remark: DSPE is a mixture of products with varying values for m and n, refering to G,S,, c[ Fig. 2. Moreover, it is not excluded that substitu- tion also occurs to a small extent in the 2- and 3-positions of the glu- cose moiety.

0 VCH Verlagsgesellschaft mhH, 6945 I Weinheim, 1996 0173-0835/96/0101-0130 $5.00+.25/0

Electrophoresis 1996, 17. 130-136

I

~ I

7 500

Dextrin 10

N

reaction mixture

Dextrin sulfopropyl ether 13 1

0 cule The respective adduct contains ( n + l \ Na+ ions. For exoerimental details

mlz

With vigorous stirring, 8g of 25% wlw aqueous sodium hydroxide were added dropwise during 2.5 h at room temperature, and 5 g solid propane sultone were simulta- neously added in three portions. Then 10 mL water were added at room temperature, and the solution was neutra- lized to pH 6 with 4 M hydrochloric acid. Inorganic salt was removed by threefold membrane filtration in a stirred cell (Spec, Basel, Switzerland; exclusion size: 1000 Da; initial volume of the solution, 150 mL; final volume, 20 mL; cell pressure, 4 bar). After concentration in a rotary evaporator, drying overnight at 0.1 torr160"C yielded 5g of white powder. The product was character- ized by matrix-assisted laser desorption ionization-MS (MALDI-MS; see Section 2.4).

2.3 Preparation of 6-CD-SBE

P-CD-SBE was prepared in a similar way as previously described [2, 3, 15-18]. In a round-bottom flask equiped

Figure 2. MALDI-MS spectra of (a) dex- trin 10; (b) dextrin 10 sulfopropyl ether directly from the reaction mixture; (c) dextrin 10 sulfopropyl ether after desalting by membrane filtration in a stirred cell. The spectra show the respec- tive [M+Na]+ adduct for every molecular mass. The counterions of the polyanions are also Na'. Nomenclature: G,S, stands for a molecule consisting of m glu- cose units (G,) and bearing a total of n sulfopropylether groups (S,) per mole-

~,

see Section 2.

with a reflux condenser, 1 mmol fbcyclodextrin mono- hydrate was dissolved with vigorous stirring in 2.4 mL of 25 Yo wlw aqueous sodium hydroxide. To the solution, 10 mmol butane sultone were added, and stirring was continued for 18 h at 50°C. Neutralization, membrane fil- tration and drying were performed as described for P-CD-SBE (yield: 800 mg).

2.4 MALDI-MS

Measurements were performed on a linear time-of-flight mass spectrometer (prototype of Linear Scientific, Reno, NV, USA) [19]. All spectra represent an accumulation of 30-50 shots. A vacuum in the flight tube of approxi- mately 2E-6 to IE-6 torr was observed. The length of the tube was 1.7m. The intensity of the nitrogen laser (337 nm) pulses varied between 3 and 6 pJ. The DHBI HIC matrix [20] was prepared as a 3:l mixture of 2,5-dihydroxybenzoic acid (DHB) and l-hydroxyisochi-

132 M. Jung. K. 0. Bornsen and E. Francotte Electrophoresis 1996, 17, 130-136

1 2 3 4

p-CD-SBE 946.9

L\,

46.9

“c,

6 7 8 9 10-SBE

I, I I I I I I

1000 1200 14100 1600 1800 2000 2200 2400 2600 2800 3000 3200

mlz

Table 1. Enantiomer separations by EKC using DSPE as a buffer additivea’

Solute Bufferb) Migration times Resolution Efficiency t l [min] tz [min] R, N / m

Figure 3. Positive ion MALDI-MS spec- trum of B-cyclodextrin sulfobutyl ether after desalting by membrane filtration in a stirred cell. The spectrum shows the respective [M+Na]+ adduct for every molecular mass. The counterions of the polyanions are also Na+. For experi- mental details see Section 2.4.

1 1,1 ’-Binaphthyl-2,2’- 2 % DSPE in P6 4.69 4.72 0.3 561000

2 l,l’-Binaphthyl-2,2‘-diol 3 % DSPE in P6 2.86 9000 3 Ibuprofen 6% DSPE in P I 3.90 340000 4 Hexobarbital 6% DSPE in P7 2.40 472000 5 Warfarin 2% DSPE in P6 3.18 700000

6% DSPE in P7 3.73 755000 6 Dansyl-leucin 2% DSPE in P6 8.37 8.43 0.1 98000 7 Verapamil 4% DSPE in TP3 11.61 11.95 1.3 117000 8 Ephedrin 6 % DSPE in TP3 5.46 380000 9 Norephedrin 6% DSPE in TP3 5.41 330000

10 Pseudoephedrin 6 % DSPE in TP3 5.52 5.54 0.1 348000 11 Troger’s base 4% DSPE in TP3 11.91 12.07 0.8 249000 12 Trasicor (Oxprenolol) 4 % DSPE in TP3 7.75 376000 13 Salbutamol 4% DSPE in TP3 7.74 375000 14 Clenbuterol 4 % DSPE in TP3 9.83 9.93 0.8 249000 15 Terbutalin 4 % DSPE in TP3 7.47 275000 16 Formoterol 4% DSPE in TP3 12.45 12.61 0.6 111000 17 Mianserin 4% DSPE in TP3 12.24 13.37 10.0 149000

a) Conditions: 47 cm X 50 pm capillary, 40 cm injector to detector; voltage, 30 kV. b) Buffers: P6, phosphate buffer (pH 6, 40 mM); P7, phosphate buffer (pH 7, 35 mM); TP3, Tris-phos-

phate buffer (ptI 3, 80 mM Tris adjusted to the respective pH with 1 M phosphoric acid). The weight percentage (% w/w) of DSPE is indicated.

diylhydrogenphosphate

nolin (HIC) by mixing equal volumes ( 5 or 10 pL each) of 0.2 M DHB, 0.6 M HIC (both dissolved in water/ acetonitrile 5050 v/v, 30 mM sodium chloride (in water), and aqueous sample solution (approximately 1 mg/mL). For analysis of reaction mixtures, a drop of the solution was desalted prior to preparation of the matrix by placing it for 30 min on the hydrophobic side of a circular cellulose membrane filter (pore size 0.025 pm Millipore, Volketswil, Switzerland) floating on distilled water in a bowl [21, 221.

2.5 Capillary electrophoresis

All CE experiments were performed on a Beckman P/ACE 5000 series capillary electrophoresis instrument

with diode-array UV detection (214 nm, 4 nm band width). An untreated 50 pm ID X 47 cm (40 cm from injector to detector) fused silica capillary was used. The voltage was 30 kV. The capillary was rinsed for 2 min with buffer before every run and was regenerated for 2-5 min with 0.1 M NaOH and for 2 rnin with water after every run. Phosphate and borate buffers were ob- tained from the in-house central service department and were diluted to the desired concentration with distilled water. If necessary, the pH was adjusted with phosphoric acid in the same concentration. Tris-phosphate buffer (80 mM) was prepared by adjusting the pH of 80 mM Tris with 1 M phosphoric acid. Running buffer was prepared by dissolving the additive in the respective buffer in 4.5 mL vials or 500 pL minivials and sonicating briefly.

Electrophoresis 1996, 17, 130-136 Dextrin sulfopropyl ether 133

1 0 11 12

Figure 4. Molecular structures of the racemic solutes used in this work.

3 Results and discussion

3.1 Preparation and characterization of DSPE

Before attempting the synthesis of DSPE (cf: Fig. l), P-CD-SBE was prepared as previously described [2, 3, 15-18]. Amount and concentration of the aqueous sodium hydroxide used as solvent were found to be crit- ical to obtain a homogeneous, but not too viscous solu- tion. When applying the same synthetic procedure to the preparation of DSPE, it was found that dextrin 10 quickly turned black when dissolved in sodium hydrox- ide at 50°C. As a consequence, the procedure was modi- fied to obtain milder conditions. The described synthetic procedure yields a complex mixture of products hereafter refered to as G,S,, i.e. molecules consisting of m glucose residues (G,) and bearing n sulfopropyl ether groups (S,,). DSPE was chosen rather than the butyl homologue DSBE because the SBE group (Na salt) and an additional glucose unit are so similar in molecular mass that an unambiguous characterization of that prod- uct by MS would be difficult. In other words, e.g., G,S, and G,S, would be almost indistinguishable.

3.1.1 Characterization by MALDI-MS

MALDI-MS allows the convenient, easy, fast and unam- biguous characterization of carbohydrate mixtures by their molecular mass distribution. The recently devel- oped DHB/HIC matrix [20] optimizes performance and signal-to-noise ratio for carbohydrates and was preferred over 3-amino-4-hydroxybenzoic acid, which also proved to be useful. The appearance of multiple, split or broad peaks, caused by formation of different adducts, was often a problem. Single, narrow peaks for the respective

[M-tNa]' adducts can, however, be obtained when working at an optimized, well-defined sodium concen- tration in the matrix (e.g. , Fig. 2a). The analysis of reac- tion mixtures with their extreme salt content is facili- tated by desalting a drop of solution on a membrane filter as described in Section 2.4. In comparison to elec- trospray MS [23] of identical samples (V. Schurig, unpu- blished results), MALDI-MS showed similar results with respect to molecular mass distribution. Electrospray MS produces somewhat narrower peaks, but involves consi- derably higher efforts with respect to cost, simplicity, robustness, and ease of operation and sample prepara- tion.

3.1.2 Removal of residual inorganic salt

The reaction mixtures contain high concentrations of sodium chloride, which has to be removed prior to using the product as a buffer additive in CE. Membrane filtra- tion in a stirred cell was found to be a suitable method for the workup of batches as large as log per run during about two days, in spite of the high viscosity of the con- centrated solutions. However, since the smallest avail- able pore size has an exclusion limit of 1000 Da, exces- sive treatment should be avoided because the smaller molecules within the product mixture are not fully retained. Consequently, the product composition slowly and gradually changes. This is revealed by the respective MALDI-MS spectra of DSPE recorded before (Fig. 2b) and after desalting in a stirred cell (Fig. 2c). For mole- cules with similar molecular mass (e.g., G,S, and G,S,), a comparison of Fig. lb and c indicates that those mole- cules with more glucose units and fewer sulfopropyl ether groups are somewhat more effectively retained. In Fig. 3, the molecular mass distribution for P-CD-SBE is

134 M Jung, K. 0. Bomsen and E Francotte

a ) Mianscrin b) Vcrapamil c) Clenbukrol d) Formolcrol c) Trogcr's Base

4% 4% 8%

DSPE

- - 10 12 10 12 12 14

4% 4% 8%

D

3 4 3 4 4

20% 20% 20%

----I- - 14 16 16 i a

6 % 6% I

D high conc.

5 2 4 6 6 7

20% 20%

Electrophoresis 1996, 17, 130-136

f) 1,I'-Binaph- g) l.I'-Binaph- thyl-2,2'-dioI thyl-2,2'diyl-

hydr. phosphat

8 6 7 8 6

Time [min]

3 Yo

I 7

4 5 6

3%

i 3

20%

J- 8

4

L

7 --

Figure 5. Comparison of Dextrin 10 sulfopropyl ether ("DSPE) and underivatized Dextrin 10 ("D") as chiral buffer additives for several racemic solutes in CE. Upper row: DSPE added to the buffer in suitable weight percentages as indicated; center row: same weight percentages D added; bottom row: D used in a higher concentration, i.e. 20% w/w. Running buffers: (a)-(e) 80 mM Tris-phosphate, pH 4 (cationic analytes); (0 40 mM phosphate for DSPE (uncharged analyte) and 50 mM borate, pH 10, for D (anionic analyte); (8) 40 mM phosphate, pH 6 (uncharged analyte). Other conditions: 47 cm x 50 vm capillary, 40 cm from injector to detector; voltage, 30 kV.

shifted a little towards higher masses, as compared to 3.2 DSPE as a novel buffer additive in CE the respective MS spectrum recorded prior to desalting in a stirred cell (data not shown). Our 6-CD-SBE mole- A selection of successful and attempted enantiomer sep- cules bear more sulfoalkyl substituents (3-8) than the arations using DSPE is listed in Table 1 (for structures, 6-CD-SPE used by Schurig and co-workers [3], who see Fig. 4). The comparison of underivatized 0-CD and employed electrospray MS for characterization. The P-CD-SBE as buffer additives suggests that DSPE B-CD-SBE product used in [4-71 was characterized by should be advantageous for cationic and neutral racemic CE with indirect UV detection [18]. analytes, as compared to native dextrin. Indeed, several

Electrophoresis 1996, 17. 130-136 Dextrin sulfopropyl ether 135

150 I Phosphate pH>

’?€US-Phosphate pH 3 “ T 2

- 0 2 4

% DSPE (w/w)

6 8 0 2 4

Yo DS P E (w/w)

6 8

Figure ti Current vs. weight percentage of DSPE in the running Figure 8. Enantiomer resolution R, of mianserine and clenbuterol vs. buffer: (a) 35 mM phosphate buffer, pH 7; b) 80 mM Tris-phosphate weight percentage of DSPE in the running buffer, i.e., 80 mM Tris- buffer, pH 3 . phosphate buffer, pH 3.

.- = 20 E L--.l

E“ b = 10

LLI

0

6

2

0

Clenbu terol

0 2 4 6 2 ‘ 4 6 i

‘30 DS PE (w/w) PH Figure 7. Migration time of dimethylformamide (electroosmotic flow) vs. weight percentage of DSPE in the running buffer: (a) 50 mM phos- phate buffer, pH 3; (b) 40 mM phosphate buffer, pH 6 ; (c) 40 mM ace- tate buffer, pH 4.65; (d) 40 mM phosphate buffer, pH 7.

Figure 9. pH dependence of the enantiomer resolution R,: (a) mianse- rine and (b) clenbuterol: 6% w/w DSPE in an 80 mM Tris-phosphate buffer; (c) l,l’-binaphthyl-2,2‘-diylhydrogenphosphate: 3% w/w DSPE in 40 mM phosphate and acetate buffers.

examples for this behavior were found. A systematic comparison of DSPE and native dextrin 10 as buffer additives under otherwise identical conditions is shown in Fig. 5 (top and center rows); in addition, the bottom row shows chromatograms obtained with dextrin at a higher concentration, i.e. 20% w/w. For the cationic ana- lytes (chromatograms a to e), it can be seen that DSPE produces a considerably higher enantioselectivity and also longer migration times than dextrin. Clenbuterol and formoterol can only be separated with DSPE, but not with dextrin. For mianserin, verapamil and Troger’s base, a higher dextrin concentration can be used to compensate the lower enantioselectivity, and similar enantiomer resolution can thus be achieved as with DSPE. Binaphthol (f, top), which is neutral at pH 6, mi- grates more slowly than the electroosmotic flow (EOF) when DSPE is used, which indicates an interaction, but enantioselectivity is not sufficient for a separation. This racemate can, however, be resolved as an anion at pH 10 with dextrin (f, center). The anionic l,l’-binaphthyl-2,2’- diylhydrogenphosphate (g) interacts so strongly that it can be partially separated with DSPE in spite of the electrostatic repulsion. However, the separation is much better with unmodified dextrin. The remarkably good enantiomer separations of the trycylic base mianserin (a) and of l,l’-binaphthyl-2,2’-diylhydrogenphosphate (g)

with dextrin have not previously been reported. Previous reports on the use of dextrin focused on anionic ana- lytes such as profen drugs or warfarin. None of those molecules could be separated with DSPE.

The analytical conditions were systematically optimized, as depicted in Fig. 6-9. The charge of the DSPE mole- cules contributes to the current through the capillary, as displayed in Fig. 6. The use of up to approximately 10% w/w DSPE in a SO pm (ID) capillary is still possible without excessive Joule heating caused by the current. This is fortunate since the much lower enantioselectivity of dextrins, as compared to cyclodextrins, requires the use of higher concentrations (e.g., typically 10 to SO mM, corresponding to (ca. 1 to 6% w/w) for cyclodextrins, but 10-20°/0 w/w for dextrin 10). As shown in Fig. 7, the addition of DSPE reduces the EOF. Whereas the effect is usually not great, it was reproducibly found to be extreme in phosphate buffer pH 6 above 3% w/w DSPE as well as in phosphate buffer pH 3. We have verified that the buffer pH was not affected by the addition of DSPE. Fortunately, the application of DSPE focuses on cationic analytes that have their own electrophoretic mobility and do not require the EOF in order to reach the detector. The influence of the DSPE percentage on the enantiomer resolution is shown in Fig. 8 for mianse-

136 M Jung, K. 0. Bornsen and E Francotte EIerrmphuresis 1996, /7, 130-136

rine and clenbuterol. The curves suggest that a maximum in resolution has not yet been reached. Higher DSPE percentages are, however, not possible because of excessive current and reduced baseline sta- bility. As shown in Fig. 9, the enantiomer resolution of the cationic analytes mianserine and clenbuterol remains almost constant over a wide pH range, whereas for the binaphthyl phosphate there is a maximum at pH 6. The cationic racements could not be resolved in the basic pH range. This observations is in contrast to the report of Terabe and co-workers [6], who claimed that a basic pH should give optimum resolution for ephedrine alkaloids with P-CD-SBE.

3.3 Peak shape

Although the separation efficiency with DSPE in usually high (cJ Table I), somewhat asymmetric, tailing peaks were observed for verapamil. This phenomenon was de- scribed before [6, 141, and switching to other buffer sys- tems with different mobilities (Tris-phosphate, acetate, citrate, phosphate), addition of 10 mM tetrabutylam- monium bromide, or the use of a polyacrylamide-coated capillary (Beckman “Neutralcap”) did not bring any improvement.

4 Concluding remarks

The described procedure is suitable for the easy prepara- tion and desalting of DSPE in the range of 10-2Og. MALDI-MS allows convenient and unambiguous cha- racterization. Although versatility and range of applica- tion of the novel DSPE and of dextrins in general are more limited than observed with cyclodextrins, remark- ably high enantioselectivity was found in some cases. As expected, DSPE showed increased separation power for cationic analytes, as compared to unmodified dextrin.

5 References

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The recording qf the MALDI-MS spectra by H. Miiller is gratejailfy acknowledged.

Received August 18, 1995