JOURNAL OF MASS SPECTROMETRY, VOL. 31, 1364-1370 (1996)

Multicomponent Non-covalent Associations of fl-Cyclodextrin @-CD)-Drug Inclusion Complexes with Diethanolamine (DEA). Detection and Characterization of Gaseous Protonated 1 : 1 : 1 p- CD-Drug-DEA Adducts by Ionspray Ionization and Tandem Mass Spectrometry

~ ~~

Antonio %ha* C.N.R.--Centro di Studio sulle Sostanze Organiche Naturali, Diparthento di Chimica del Politecnico di Milano, Via Mancinelli, 7,I-20131 Milano, Italy

Enrico Redeati, Paolo Ventura and Marghenta ZanoI Chemical and Biopharmaceutical Department, Chiesi Farmaceutici SPA, Parma, Italy

Bruno Casetta Perkin-Elmer, European Life Science Center, Applied Biosystems Division, Langen, Germany

The multicomponent non-covalent associations of fbcyclodextrin (p-CD)-drug host-guest complexes with appropri- ate molecules are of current interest to the pharmaceutical industry, as they can increase dramatically the solubility in water of scarcely soluble guest drugs. The present study by ionspray and tandem mass spectrometry (MS) of the multicomponent associations with diethanolamine (DEA) of the &CD host-guest complexes of two drugs, namely glybenclamide and furosemide, allowed the detection and characterization of the gaseous protonated 1 : 1 : 1 drug-fl- CD-DEA adducts. Their dissociation patterns upon collision activation provided interesting information on the relative strength of the interactions binding the components of such charged non-covalent associations in the gas phase. In particular, from the results above it clearly emerged that the stability of the 1 : 1 drug+CD inclusion complexes within such gaseous ternary associations was much weaker than that shown previously for the analogous 1 : 1 terfenadine (TFN)+CD complex originating by the tandem MS dissociation of the 1 : 1 : 1 TFN+CD-HA (HA = tartaric or citric acid) protonated adducts, generated by ionspray MS. Also, we noted that these findings appear consistent with the relative stability of the corresponding 1 : 1 drug-&CD host-guest complexes in solution.

KEYWORDS : /?-cyclodextrin; host-guest complexes; supramolecular associations; ionspray-MS and MS/MS; glybenclamide ; furosemide

INTRODUCTION

Host-guest (or inclusion) complexes are supramolecular associations of current scientific and technological inter- est, whose existence has been demonstrated either in solution or in the solid state by spectroscopic and other physico-chemical methods. Mass spectrometry (MS) was applied to the study of host-guest chemistry in the gas phase’ and almost all of the so-called ‘soft’ ioniza- tion MS methods have been exploited on various types of host-guest systems for: (i) the characterization of the complexes; (ii) the study of association/dissociation equilibria in solution; (iii) the evaluation of molecular and/or chiral recognition capability and (iv) the study of the complex formation processes in the gas phase. For the hydrophobic host-guest associations, however, it was argued that the presence of a charge, which is

Author to whom correspondence should be addressed.

essential for any MS processes, could dramatically affect the weak van der Waals interactions binding a neutral guest within the non-polar cavity of various macrocyclic hosts, and could eventually preclude the surviving of the charged inclusion complex in the gas phase.’ This should apply, for instance, to the host-guest complexes of cyclodextrins’ (CDs), which are mostly used as hosts for the complexation of hydrophobic or barely water soluble guest molecules of interest in many fields, e.g. pharmaceutics, cosmetics, agriculture, food and bever- ages.

We performed a few MS studies on CDs inclusion complexes, actually existing in solution, by ionspray3 (IS), fast atom bombardment4 (FAB) and electro~pray~ (ESI) ionizing methods, which showed the possibility to detect protonated or cationated 1 : 1 CD-guest adducts. These findings suggested obviously that such weak host-guest complexes did survive as charged species in the gas phase. Since two of the guest molecules of the complexes studied by US,^'^ namely terfenadine (TFN) and piroxicam, beside the appropriate lipophilic moi- eties for the inclusion, have also suitable basic sites for

CCC 1076-5174/96/1213W-07 0 1996 by John Wiley & Sons, Ltd.

Received 3 June 1996 Accepted 8 August 1996

/9-CYCLODEXTRIN NON-COVALENT MULTICOMPONENT ASSOCIATIONS 1365

protonation or cationation, we did also consider that their protonated or cationated 1 : 1 CD-guest gaseous species might even be just electrostatic ion-molecule adducts. However, pertinent experiments provided indirect evidence,4s6 which did not support such an alternative possibility. Accordingly, Ramanathan and Prokai' have most recently observed that host-guest complexes of CDs with amino acids are stable in the conditions for the ESI MS analysis, by which it is possible to determine the stoichiometry and the relative abundance of the complexes formed in solution.

Conversely, it had been just before aftirmed by other authors' that only relatively strong electrostatic bonds, such as those between CDs and molecules bearing protonated amino groups, could be able to survive the rather hard desolvation processes taking place within an ESI source. These authors concluded that, consider- ing the high affinity of saccharides toward protonated amines, all of the previously detected protonated or cationated gaseous species generated from hydrophobic inclusion complexes, actually existing in solution between CDs and guest amino-compounds, were just electrostatic adducts formed during the ESI process.'

Interestingly, it has been recently found that CDs inclusion complexes can associate with other suitable molecules, yielding non-covalent multicomponent adducts of increasing scientific and technological rele- vance for their physical, chemical and/or biological proper tie^.^.' O In the field of pharmaceutical prep- arations, for instance, multicomponent associations of CDs-drug host-guest complexes with appropriate mol- ecules can dramatically increase the solubility in water of hydrophobic or sparingly soluble guest drugs."." We are presently engaged with the characterization of such multicomponent supramolecular associations, a really challenging problem indeed, which would poss- ibly demand contributions by different analytical approaches. On this topic we have already exploited the capability of MS, showing the first interesting results obtained with the multicomponent non-covalent adducts of the organic hydroxy acids (HAS) salts of the TFN-#I-CD inclusion complex,13 from which the gaseous protonated 1 : 1 : 1 HA-TFN-B-CD ternary adducts (HA = tartaric or citric acid) were detected by IS MS and characterized by tandem-MS.

On these preliminary results we would prospect that the investigation of such multicomponent associations by MS methods, besides its intrinsic interest, could also represent a useful experimental approach for gaining a deeper insight into the surviving aptitude of CDs host- guest complexes as gaseous charged species. With such a perspective, the present paper will deal with a study by IS and tandem MS of the multicomponent associ- ations with diethanolamine (DEA) of the 8-CD inclu- sion complexes of two guest drugs: glybenclamide (l), namely 5-chloro-N-[2-[4-[[[(cyclohexylamino)arbon- yl]amino]sulfonyl]phenyl]ethyl]-2-methoxybenzamide (CAS number 10238-21-81, a second generation sulfo- nylurea derivative for the oral treatment of type I1 dia- betes; and furosemide (2), namely 4-chloro-N-(2- furylmethy1)sulfamoylanthranilic acid [CAS number 54-31-91, a diuretic agent for the oral treatment of renal hypertension.

f'

2

EXPERIMENTAL

Mass spectrometry

A bench-top Perkin Elmer-Sciex API 300 triple quad- rupole mass spectrometer, equipped with a standard API-IonSprayTM ionization source, operated in positive ion mode with a cluster breaking orifice voltage of 60 V, was employed for the MS and tandem-MS experiments, which were performed with a resolution of 0.8 m u (measured at 1/2 height) for both the resolving quadru- poles. The acquired data spectra were processed with Multiview 1.2 proprietary software package.

CAD tandem-MS experiments were made through the closed-design 4 2 collision cell operating with a col- lision energy (Elab) of 30 eV, using nitrogen as a colli- sion gas at a pressure of 8 mtorr (1 Torr = 133.3 Pa).

The samples of the multicomponent complexes 1-8- CD-DEA or 2-B-CD-DEA were dissolved in water- acetonitrile 50/50 (v/v) containing 2 mM of ammonium acetate, diluted to concentrations of 1000 or 100 ppm and then introduced into the ion source by an infusion pump in continuous flow mode with a flow rate of 2 pl/min.

Preparation of the multicomponent complexes

The multicomponent complex 1-B-CD-DEA was pre- pared by lyophilizing a water solution containing the components in molar ratios of 1:2:3. The multi- component complex 2-B-CD-DEA was prepared by lyophilizing a water solution containing the com- ponents in molar ratios of 1:2:2. Both the samples were prepared by Cyclolab, Budapest, Hungary.

RESULTS AND DISCUSSION

The IS MS spectra of both the multicomponent associ- ations l-#?-CD-DEA [Fig. l(a) and (b)] and 2-B-CD- DEA [Fig. 2(a) and (b)] show the peaks of the corresponding 1 : 1 : 1 protonated drug-B-CD-DEA

1366 A. SELVA ET AL.

3.9e7

3.6e7

3.3e7

3.0e7

2.7e7

vI 2.4e7

i e 2. le7

P

5 4- -

1 .Be7

1 S e 7

1.2e7

9.0e6

6.0eE

3.0eE

2.4eE

2.2eE

2.0e6

1.8e6

1 h e 6

1.4e6 B

1 .Oe6

B.Oe5

6.0eS

4.0ed

2.0eS

3f 06.3

0

4:

5

zoo 400

5s

9

.1

L E

1733.7

2

1 1

1004.4

987.4

3

1240.6

1645.6

1628.6 I I

1733.7 J 860 1 do0 li00 1 i o o 1 600 1800

m/z, amu

1734.7

I I , I

1728 1731 1734 1737 1740 1743 1746 1749 m/z, amu

Figure 1. (a) lonspray MS spectrum of the 1-b-CO-DEA multicomponent complex (at 1000 ppm sample concentration); (b) the expand- ed region of the protonated 1 : 1 : 1 ternary adduct (at 1000 or 100 ppm sample concentrations).

200 400 600 800 1000 1200 l i00 1600 1 so0 m/z, amu

p-CYCLODEXTRIN NON-COVALENT MULTICOMPONENT ASSOCIATIONS

106.0

1367

(a)

11 52.4

436.0 I

1240.5

I I 1570.5

1.35e6

1.26e6

1.17e6

1.08e6

9.90e5

9.OOeS

8.1 Oe5

.- 5 7.20e5

6.3Oe5

5.40e5

4.50e5.

3.60e5'

2.70e5.

1.80.S.

2

!A C V

b

9.0Oe4

m/z, amu

Figure 2. (a) lonspray MS spectrum of the 2-p-CD-DEA multicomponent complex (at 1000 ppm sample concentration); (b) the expand- ed region of the protonated 1 : 1 : 1 ternary adduct (at 1000 or 100 ppm sample concentrations).

1368 A. SELVA ET AL.

ternary adducts at m/z 1733.7 and 1570.5, respectively, while the relative abundance of the related natural iso- topic peaks is in good agreement with the values calcu- lated for the respective elemental compositions. These spectra show some evident analogies. In particular we can observe the peaks corresponding to: (i) drug-p-CD host-guest complexes, i.e. the protonated and the ammonium cationated species of 1-fl-CD at m/z 1628.6 and 1645.6, respectively, and the ammonium cationated species of 2-p-CD at m/z 1482.5; (ii) fl-CD-DEA proto- nated adducts at m/z 1240.5; (iii) 1 or ZDEA proto- nated adducts at m/z 599.2 and 436.0, respectively; (iv) protonated DEA (m/z 106.3) and ammonium cationated p-CD (m/z 11 52.5).

In addition the IS MS spectrum -of 1-j-CD-DEA multicomponent association [Fig. l(a)] shows other intense signals due to the protonated species of the free drug (m/z 493.9) and of its dimer (m/z 987.4), the latter observed also as ammonium adduct (m/z 1004.4), while the peaks at m/z 169.0 and 369.0 could be reasonably attributed to the fragmentation of 1, as reported in the Scheme 1.

The tandem MS spectra of the collision activated (CA) protonated species of 1-j?-CD-DEA (Fig. 3) and Zp-CD-DEA (Fig. 4) 1 : 1 : l ternary adducts at m/z 1733.7 and 1570.5, respectively, show a similar and straightforward fragmentation pattern, both giving pre- dominant protonated 1 : l p-CD-DEA at m/z 1240.5 and the less abundant 1 or 2-DEA protonated adducts at m/z 599.2 and 436.0, respectively, while only negligi-

1.12e6

1.05e6

9.8oe5

9.1oe5

8.40e5

7.7oe5

7.0Oe5

6.3Oe5

t 5.6Oe5 4.9oe5

4.2Oe5

3.50e5

2.8oeE

2.1oe:

1.4oe:

a I .-

Y C -

R - N H ~ + a mlr 369.0

R-NHEO + 0

02- H,C'

m h 189.0 mh 395.0

Scheme 1

ble or minor protonated DEA at m/z 106 are formed. It is remarkable that in both these tandem MS spectra no significant signals of the protonated species of the 1 : 1 drug-8-CD host-guest complexes are observed at m/z 1628.3 and 1465.5, respectively.

A strikingly different behaviour was previously shown13 by the CA protonated 1 : 1 : 1 HA-TFN-P-CD parents fragmentation, which yielded the most abun- dant protonated 1 : 1 TFN-p-CD adduct (m/z 1606.7), by the loss of HA as a neutral fragment, and the proto- nated drug molecule as the only product ions, while the abundance of the protonated HA species was absolutely negligible.

Such relevant difference between the fragmentation patterns of the CA gaseous protonated 1 : 1 : 1 ternary adducts of 1 or 2 in respect to those of TFNl3 under comparable operative conditions appears worthy of comment for the important information that can be gained on the relative strength of the non-covalent

124 3.5

1733.3 I 599.3

105.7 L 1 1 I I

. -- _ - - --" m/z, amu

Figure 3. Tandem-MS spectrum of the protonated 1 : 1 : 1 ternary adduct generated by ionspray MS from the 1-b-CD-DEA multi- component complex.

j-CYCLODEXTRIN NON-COVALENT MULTICOMPONENT ASSOCIATIONS

4 . 0 ~ 4 - b

1369

436.0 81 1.5 I t

1

121

m/z, arnu

Figure 4. Tandem-MS spectrum of the protonated 1 : 1 : 1 ternary adduct generated by ionspray MS from the 2-j-CD-DEA multi- component complex.

binding interactions among the components of such charged supramolecular associations and in particular between drug and P-CD, which could cast some light on the gas phase stability of the corresponding protonated or neutral gaseous 1 : 1 inclusion complexes.

In particular, the formation of a rather strong proto- nated B-CD-DEA adduct by loss of the neutral drug molecule 1 or 2, respectively, supports that the non- covalent interactions between DEA and B-CD are stronger than those binding the drug-B-CD host-guest partners within the related parent ions. On the contrary, for the CA parent ions of TFN ternary ad duct^'^ the predominant formation of protonated 1 : 1 drug-B-CD species takes place by the loss of the HA molecule, which therefore should reasonably be the most weakly bonded component within these charged gaseous associations. In conclusion the present study at first shows the possibility to obtain the gaseous protonated 1 or 2-fi-CD-DEA 1 : l : l ternary adducts from the related non-covalent multicomponent associations in water solution by IS MS, as we did previously ob~erve '~ with another multicomponent system.

Second, the comparison of the CA dissociation patterns of such ternary adducts provides interesting evidence on the relative strength of the binding interactions among their components and in particular on the relative stability of the gaseous charged 1 : l drug-#I-CD inclusion complexes, which could form as a protonated or neutral fragmentation product.

Eventually, we would just mention that the tandem- MS observations made in the present work are consis- tent with the much lower stability of 1-B-CD than that of TFN-j?-CD 1 : 1 host-guest complexes in water solu- tion, according to their association constants, i.e. K , , = 2121 M-' for the formeri4 (at pH 7.9, deter- mined from the straight line portion of the isothermal phase solubility curve, and 20269 2976 M-' for the HCl salt of the latter, measured by microcalorimetry.'5 However, these few data available do not allow us pre- sently to assess if any correlations could be established between the stability of the hydrophobic host-guest complexes in solution and that of the corresponding gaseous protonated or neutral species originating from multicomponent ternary parent ions by tandem MS.

REFERENCES

1. M. Vincenti, J. Mass Spectrom. 30,925 (1 995). 2. (a) J. Szejtli, Cyclodextrins and their Inclusion Complexes.

Akadbmiai Kiadd, Budapest (1 982); (b) J. Szejtli, Cyclo- dextrin Technology. Kluwer Academic, Dordrecht (1 988) ; (c)

K.-H. Fromming and J. Szejtli, Cyclodextrins in Pharmacy. Kluwer Academic, Dordrecht (1 994).

3. A. Selva. E. Redenti, M. Zanol, P. Ventura and B. Casetta, Org. Mass Spectrom. 28,983 (1 993).

1370 A. SELVA ET AL.

4. A. Mele and A. Selva, J. Mass Spectrom. 30,645 (1 995). 5. A. Selva, A. Mele and G. Vago, Eur. Mass Spectrom. 1, 215

6. A. Selva, E. Redenti, M. Zanol, P. Ventura and B. Casetta, fur.

7. R . Ramanathan and L. Prokai, J. Am. SOC. Mass Spectrom. 6,

8. J. B. Cunniff and P. Vouros, J. Am. SOC. Mass Spectrom. 6.

9. M. T. Esclura Diaz, M. B. Perez Marcos, J. L. Vila Lato and J. J. Torres Labandeira. in 8th International Cyclodextrins Sym- posium, Budapest, March 30-April 2, Book of Abstracts 3, p. 33 (1 996).

10. E. Redenti, M. Pasini, L. Carima, M. Zanol, A. Bacchi, M.

(1 995).

MassSpectrom. 1,105,330 (1995).

866 (1 995).

437 (1 995).

Vikmon, J. Szejtli and P. Ventura, in Proceedings of the 1st World Meeting on Pharmaceutics, Biopharmaceutics, Phar- maceutical Technology, APGI/APV, Budapest, p. 601 (1 995).

11. P. Chiesi, P. Ventura, M. Pasini, E. Redenti, J. Szejtli and M. Vikmon, WO 94/16733 (1 994).

12. P. Chiesi, P. Ventura, M. Delcanale, E. Redenti, D. Acerbi, M. Pasini, J. Szejtli, M. Vikmon and E. Fenyvesi, W095/28965

13. A. Selva, E. Redenti, M. Pasini, P. Ventura and B. Casetta, J . Mass Spectrom. 30,219 (1 995).

14. M. Vikmon and J. Szejtli, Personal communication. 15. W.-Q. Tong, J. L. Lach, T.-F. Chin and J. K. Guillory, Pharm.

(1995).

Res. 8, 951 (1991).