Two-Dimensional and Deuterium-Induced, Differential-lsotopeShift Nuclear Magnetic Resonance Characterization of Potassium Sucrose Octasulfate and Sucralfate GARYL. SILVEY Received April 9, 1991. from Marion Menell Dow Inc., Park A, Marion Park Drive, Kansas City, MO 64137.

June 29, 1991.

Abrtrrct 0 Two-dimensional and deuterium-induced, differentiaiisotope-shift NMR measurements have made it possible to assign completely all of the 'H and I3C resonances of potassium sucrose octasulfate (K,SOS) and thereby prove complete sulfation of the sugar moiety during synthesis. In addition, a comparison of the I3C NMR chemical shifts obtained for K,SOS and sucralfate made it possible to demonstrate that the sugar moiety in sucralfate is sucrose octasulfate (SOSB-).Finally, deuterium-induced, differential-isotope-shiftand 13C NMR techniques were shown to be useful tools for detecting and characterizing carbon-containing impurities and hydrolysis products in solutions containing SOSB-.

Sucrose octasulfate anion (SOSs-) effectively protects esophageal mucosa against acid-induced injury.14 It is also believed to be an active component6 of sucralfate, an amorphous complex of aluminum hydroxide with SOSs- used in the treatment of duodenal ulcers. Evidence supporting the structure of SOS8- came from single-crystal X-rays results on potassium sucrose octasulfate (K,SOS). However, singlecrystal X-ray results are not necessarily representative of a bulk sample, with measurements o h n being made on crystals isolated after recrystallization. This situation is especially true for K,SOS, which is produced as a fine powder and must be recrystallized, with some difficulty, to obtain crystals suitable for X-ray analysis. More recently, Baker obtained FAB MS (fast-atombombardment mass spectrometry) results on sucralfate7 and proved conclusively that SOS8- exists in sucralfate. However, the FAB MS results indicated that large amounts of sucrose heptasulfate (SHS7-) and sucrose hexasulfate (SXS6-) were also present in sucralfate, although the relative abundance8 of SHS7- and SXS6- detected did not represent the actual amounts of these species in sucralfate. Baker7 suggested that the smaller masses of SHS7- and SXS6-, compared with SOS8-, might result in more efficient sputtering from the thioglycerol matrix to give the misleading relative abundances. Alternatively, SOSs- may degrade in the thioglycerol matrix to form increased amounts of SHS7- and SXS6-. NMR spectrometry, in contrast to X-ray and MS techG6 CH2OR

I

H

F1

OR

OR

R = SO;

sosoOO22-3549/92/0500-O471$02.50/0 Q 1992, American Pharmaceutical Association

H

Accepted for publication

niques, can provide an overall picture of sample identity and purity. In the present study, two-dimensional (2-D) and deuterium-induced, differential-isotope-shift (DIS) NMR techniques were used to demonstrate complete sulfation of SOP- in K,SOS and sucralfate. In addition, the use of 13C and DIS NMR techniques to detect and identify partially sulfated sucrose species in KsSOS and sucralfate was examined.

Experimental Section Materiala43amples of potassium sucrose octasulfate (K,SOS) and sucralfate (vendor A) used in the characterization of SOS8- and SOS8- hydrolysis products were obtained internally from Marion Merrell Dow Inc. Sucralfate samples labeled vendor B, C, and D in Figure 4 were obtained from three other suppliers through the purchasing department of Marion Merrell Dow. NMR spectra were collected at ambient temperature on a Bruker AC-250 spectrometer operating a t 250.13 and 62.9 MHz for 'H and "C NMR measurements, respectively. Both 5-mm 'W13C dual and 10-mm broad-band NMR probes were used in the study. 2-D NMR-A sample of K8SOS of unknown weight was dissolved in D20 that w a spiked ~ with a small amount of TSP (34trimethylsily1)propionic acid, sodium salt) and filtered through Whatman 44 filter paper into a 5-mm NMR tube for analysis. The 'H, 13C, COSY (homonuclear 2-D correlation spectroscopy), DEFT (distortionless enhancement by polarization transfer), and XHCORR (heteronuclear 2-D correlation spectroscopy) NMR spectra were referenced internally to TSP. DIS NMR-Two samples of K8SOS, weighing 50-100 mg, were dissolved separately in H 2 0 and D20 and placed in 10-nun NMR tubes for analysis. To ensure that both sets of data were referenced to the same chemical shift point, a coaxial NMR tube insert was filled with 5% methanol (vlv) in D20 and placed inside each 10-mm NMR tube prior to analysis. For comparison, samples of free sucrose, also weighing 50-100 mg, were dissolved separately in H 2 0 and D20 and analyzed in a manner identical to that used for the KBSOSsolutions. The 13C NMR spectra were referenced to the methanol contained in the coaxial tube insert. DIS NMR Study of Acid Hydrolysi+Two samples of K,SOS, weighing 86 and 84 mg, were dissolved in 1.0-mL quantities of H,O and D20, respectively. Then, l d r o p quantities of H3P04 and DsP04 were added to the H 2 0 and D20 solutions, respectively. The solutions were then brought to a quick boil on a hot plate, immediately removed, and allowed to air cool. Portions of the two solutions were transferred to separate 5-mm NMR tubes, into which a coaxial tube insert containing dimethyl sulfoxide-d, (DMSO-d,) in D20 would be placed prior to NhfR analysis. The 13C NMR spectra were referenced to the DMSO-d, contained in the coaxial tube insert. NMR Analysis of Sucralfate-Excess amounts of sucralfate samples were stirred for several minutes with -3-mL quantities of a 50 :50 mixture of concentrated ammonium hydroxide (28%ND40D in D20) and 2 M NaOH, after which several drops of DMSO-$ were added. The resulting solutions were filtered through 0.45-/~m Gelman Acrodiec disposable filters into 5-mm NMR tubes for analysis. The 13C NMR spectra were referenced internally to DMSO-d6. Journal of Pharmaceutical Sciences I 471 Vol. 81, No. 5, May 1992

G6 G2

Results and Discussion K,SOS Characterization-K,SOS was chosen over sucralfate for the 2-D and DIS NMR measurements because of its significantly greater solubility in aqueous media. A sample of K,SOS was dissolved in D,O at a concentration of -100 mg/mL and examined with a combination of NMR techniques to arrive at the chemical shift assignments (Table I). The COSY (Figure 1)and XHCORR (Figure 2) contour maps made it possible to assign completely all the 'H and 13C resonances of K,SOS with respect to their positions in the sucrose backbone of SOS8-. However, it was not possible to prove, on the basis of the 2-D NMR measurements alone, that the sucrose moiety was fully sulfated. StudiesB-11 on the effects of sulfation on monosaccharide 13Cchemical shifts indicate that sulfation generally results in a downfield shift of 6 1 0 ppm for directly bonded carbons, whereas a-carbons are shifted upfield by 1-2.5 ppm. One might therefore predict that 13C resonances of K,SOS corresponding to G2, G4, F3, and F4 would shift 3.5-9 ppm downfield; those corresponding to G6,F1, and F6 would shift 6-10 ppm downfield, those corresponding to G5, F2, and F5 would shift 2-5 ppm upfield; that corresponding to G3 would shift 1-8 ppm downfield; and that corresponding to G1 would shift 1-2.5 ppm upfield relative to free sucrose. The observed differences in the chemical shifts of K,SOS and free sucrose are in good agreement with these predictions (Table 11)and support the conclusion that the sucrose moiety of KBSOSis fully sulfated. Additional support for complete sulfation of K,SOS is provided by DIS 13C NMR measurements. This technique involves the detection of subtle differences induced in the chemical shifts of carbons directly bonded to hydroxyls on substitution of deuterium for the exchangeable hydroxyl protons. The DIS technique was first applied to mono- and disaccharide5 by Pfeffer et a1.,12 who observed that carbons directly bonded to hydroxyls exhibit DIS shifts of 0.13-0.24 ppm, whereas carbons not directly bonded to hydroxyls exhibit DIS shifts of 0.07 ppm or less. Later, Archbald et al.6 applied DIS techniques to a series of monosulfated D-glucose and &galactose derivatives. They found that carbons directly bonded to hydroxyls exhibit DIS shifts of 0.12-0.23 ppm, whereas carbons directly bonded to sulfates exhibit DIS shifts of 0.07 ppm or less. Notable exceptions are D-galactose 2-sulfate, D-glucose 2-sulfate, and &glucose 3-sulfate, which exhibit DIS shifts as high as 0.13 ppm for carbons directly bonded to sulfates. However, in every case, DIS shifts obtained for carbons directly bonded to sulfates were significantly smaller than DIS shifts obtained prior to sulfation; this result is consistent with the findings of Pfeffer et a1.12 In Table 11, DIS 13CNMR results for K,SOS are presented.

F5 G5

HDO G1

- 4.2

- 4.4 - 4.6

a

8

- 4.0 - 5.0 z2

B

d)

I

I

5.0

5.6

-

I

5.4

~

52

I

'

5.0

I

'

4.0

I

4.6

~

4.4

G1 G2 G3 G4 G5 G6 F1 F2 F3 F4 F5 F6

Atom Type

13C Shift,

CH CH CH CH CH CH2 CH2 C CH CH CH CH2

915 9 75.47 76.84 74.99 70.76 67.55 69.61 104.30 80.49 80.22 80.49 67.74

PPm

'H Shift, PPm 5.80 4.45 4.79 4.49 4.39 4.41 4.2w.52

-

5.17 4.87 4.52 4.30

Integrated as a group. Not determined. 472 I Journal of Pharmaceutical Sciences Vol. 81, No. 5, May 1992

1

under HDO 10' 10' 1oa 10'

-

b

1 1

1oa 1oa

~

I

-

42

NMR contour map for K,SOS. F3 F5

- 4.2 - 4.4 - 4.6 - 4.8

- 5.0

I a a

- 5.2 - 5.4 - 5.6 - 5.0

'H Integration 10'

I

PPM

Flgure 1-COSY

Table CNMR Data for K,SOS Position

- 5.2 - 5.4 - 5.6 - 5.0

I 1 I I ~ 1 1 1 1 ~ 1 1 1 1 , 1 1 1 1 ~ 1 1 1 1 ~ 1 1 1 1 ~ l I I I ~ I I ~

100

Flgure 2-XHCORR

95

90

85 PPM

80

75

70

NMR contour map for K,SOS.

Shifts of 0.07 ppm or less were obtained for all 12 carbons of K,SOS, whereas shifts of 0.13-0.20 ppm were obtained for the eight carbons directly bonded to hydroxyls in free sucrose. These results are in complete agreement with earlier findings by Archbald e t al.8 and Pfeffer et a1.12 and prove conclusively that no free hydroxyls remain on the sucrose moiety. When

I

Table iiCNMR Data for Predomlnant Hydroiyak Product of KISOS

Table iC--Comparleon of NMR R08ub for K,SOS and Free

sucro~

13CShift (ppm) for':

Position G1 62 G3 G4 G5 G6 F1 F2 F3 F4 F5 F6 a

13C Shift (ppm) in:

A$

kSOS

Sucrose

90.30(0.02) 74.19(0.03) 75.56(0.02) 73.75(0.03) 69.50(0.02) 66.28(0.03) 68.39(0.07) 102.97(0.06) 79.19(0.05) 78.94(0.02) 79.19(0.05) 66.49(0.05)

92.25(0.04) 71.13(0.15) 72.62(0.20) 69.26(0.14) 72.46(0.06) 60.1 q0.15) 62.44(0.15) 103.75(0.02) 76.40(0.15) 74.03(0.15) 81A(0.07) 61.36(0.13)

- 1.95 3.06 2.94 4.49 -2.96 6.13 5.95 -0.78 2.79 4.91 -2.25 5.13

Internal standard was tetramethylsilane. Values in parentheses are

DIS shifts.bA8 = chemical shift for GSOS - chemical shift for sucrose.

combined with chemical shift arguments made previously, these findings prove that K,SOS is fully sulfated. Hydrolysis Products of K,SOSSuccess in application of DIS methodologyto K,SOS led to attempts to characterize the acid hydrolysis products of SOS8-. The 13C NMR spectrum obtained for hydrolyzed K,SOS in D,O (Figure 3) demonstrates that a number of hydrolysis producta are formed, none of which correspond to desulfation a t G6, F1, or F6. Desulfation a t primary sulfate positions should result in the appearance of 13C resonances upfield of 66 ppm. The absence of resonances upfield of 66 ppm indicates that SOS8- hydrolyzes more readily at secondary, rather than primary, sulfate positions. Although primary sulfate positions should be more accessible to attack by H', greater steric crowding at secondary sulfate positions must result in weaker, more labile 0430, bonds. One hydrolysis product appears to be formed preferentially over the others by a factor of at least 2 to 1. Chemical shifts and corresponding DIS values (Table III) indicate that this hydrolysis product is desulfated at only one position, corresponding to a 13C resonance of 66.74ppm. On the basis of a reversal of the arguments used to prove complete sulfation of K,SOS, the 66.74-ppm resonance must be upfield and the a-carbon resonances must be downfield of the corresponding SOS8- resonances. This argument would indicate that the hydrolysis product cannot be desulfated at G6,F1, and F6, which are all upfield of the 66.74-ppm resonance. In addition, one can also rule out F3 and F4, which are too far downfield;

DIS, ppm

HZO

DZO

65.14 65.70 66.86 67.09 69.59 73.37

65.12 65.68 66.74 67.04 69.58 73.39 77.42 77.48 77.61

77.40 77.52 77.58 77.77 89.46 101.59

0.02 0.02 0.128 0.05 0.01 -0.02 -0.02 0.04 -0.03 -0.03 -0.01 -0.04

77.80 89.47 101.63

'Position of desulfation. therefore, only G2, G3, and G4 are possible sites of desulfation. In Table IV, SOS8- chemical shies and shift differences for these three possibilities are presented. Desulfation at G3 or G2 requires downfield shifts of 4.22and 0.17 ppm for G2 and G1,respectively. These shifts are significantly outside the anticipated range of 1-2.5 ppm for carbons a to a desulfation site and lead one to conclude that the predominant hydrolysis product is not desulfated at either G2 or G3.This conclusion leaves G4 as the only remaining possibility. Desulfation at G4 results in the most reasonable chemical shift differences from SOS8-. In addition, one might expect that G4 would be more accessible to H' attack than either G2 or G3. Sucralfate Characterization-Sucralfate can be dissociated in either acidic or basic media prior to NMR analysis. However, as demonstrated by DIS measurements on K,SOS, SOS8- hydrolyzes readily under acidic conditions to form desulfated sucrose species. Fortunately, SOS8- is stable in basic media. Apparently, the eight negative charges distributed about the SOS8- anion reduce the accessibility of the 0430, bonds to OH- hydrolysis by charge repulsion. In Table V, the "C N M R chemical shifts obtained for sucralfate in aqueous base are compared with those obtained previously for K,SOS in neat water. The good agreement observed between the two sets of data demonstrates that sucralfate contains SOS8-. In addition, no SHS7- or SXSgspecies were detected in sucralfate by NMR, a result supporting Baker's conclusion7 that relative abundance8 of SHS7and SXS6- obtained by FAB MS analysis do not represent actual amounts of these species in sucralfate. Table IV-Predicted Chemical ShHt8 for G2,G3, and G4 SHS7Models and Chamlcal ShHt Difference8 Relative to SOSO-

Position Position of Desullation

I

I

(

I

I

I

100

I

(

I

I

I

I

J

80

90

I

I I

1

1

70

'

1

I

I

I 60

PPM

Flgure3-l3C NMR spectrum of D,PO.-hydroiyred K,SOS in D,O. Key: *,

hydrolysis product.

G1 G2 G3 G4 G5 G6 F1 F2 F3 F4 F5 F6

13CShift (ppm) fors:

soso-

G2

G3

64

89.30 73.20 74.56 72.73 68.48 65.29 67.34 102.00 78.20 77.93 78.20 65.48

89.47 (0.17) 66.74(-6.46) 77.42 (2.86) 73.39 (0.66) 69.58 (1.10) 65.12( -0.17) 67.04(-0.30) 101.63(-0.37) 77.80(-0.40) 77.48(-0.45) 77.61(-0.59) 65.68 (0.20)

89.47 (0.17) 77.42 (4.22) 66.74(-7.82) 73.39 (0.66) 69.58 (1.10) 65.12(-0.17) 67.04( -0.30) 101.63(-0.37) 77.80(-0.40) 77.48(-0.45) 77.61(-0.59) 65.68 (0.20)

89.47 (0.17) 73.39 (0.19) 77.42 (2.86) 66.74(-5.99) 69.58 (1.10) 65.12(-0.17) 67.04(-0.30) 101.63(-0.37) 77.80(-0.40) 77.48(-0.45) 77.61 (-0.59) 65.68 (0.20)

a Values in parentheses are chemical shift differences:A6 = chemical shift of G2 (or G3 or G4) - chemical shift of SOSe-.

Journal of Pharmaceutical Sciences I 473 Vol. 81, No. 5, May 1992

Table V-Comparlson of NMR Results for K.SOS and Sucralfate

so$-

I.

13CShift (ppm) for:

Label G1 G2 G3 G4 G5 G6 F1 F2 F3 F4 F5 F6

K,SOS

Sucralfate

91.59 75.47 76.84 74.99 70.76 67.55 69.61 104.30 80.49 80.22 80.49 67.74

91.21 75.57 77.14 75.35 70.84 67.64 70.32 103.38 79.97 79.73 79.97 68.07

Vendor

D

The use of 13C NMR to detect lower sulfated species in SOS8- solutions was demonstrated for hydrolyzed K8SOS, but the effectiveness of this technique can also be demonstrated by an examination of the 13C NMR spectra, expanded about the F2 region, for sucralfate from four vendors. As shown in Figure 4, additional carbon-containing species are present in three of the four sucralfate samples. These additional species appear to be sucrose related and may be the result of incomplete sulfation.

Conclusions This study proves that the sugar moieties in K,SOS and sucralfate are fully sulfated and correspond to SOS8-. In addition, 13C NMR is an effective tool for detection of sucroserelated impurities and decomposition products. The DIS 13C NMR measurements on KsSOS indicated that SOSs- hydrolyzes in the presence of acid to form lower sulfated, sucrosebased species such a s SHS7- and SXS6- and that secondary sulfate positions hydrolyze preferentially over primary sulfate positions. Under the hydrolysis conditions used, the formation of one particular SHS7- species, possibly desulfated at G4, was favored over other possible hydrolysis products.

References and Notes 1. Szabo,S.; Brown, A. Proc. Soc. Exp. Bio. Med. 1987,185,493.

2 . Orlando, R. C.; Tujman, N. A.; Tobey, N. A.; Schreiner, V. J.; Powell, D. W. Gastroenterology 1987,93, 352. 3. Nagashima, R.; Hirano, T. Anneim.-Forsch. 1980,30, 80. 4. Nagashima, R.; Yoshida, N. Anneim.-Forsch. 1979,29, 1668. 5. Nagashima, R.; Hinohara, Y.; Hirano, T.;Tohira, Y.; Kamiyama, H. Arzneim.-Forsch. 1980,30, 84. 6. Nawata, Y.; Ochi, K.; Shiba, M.; Morita, K. Acta Crystullogr. 1981, B37,246.

474 I Journal of Pharmaceutical Sciences Vol. 81, No. 5, May 1992

105

104

103

PPM

Figure 6%NMR spectra, expanded about the F2 region, for sucralfatesamples obtainedfrom four vendors. Key: *, carbon-containing impurity.

I. Baker, R. Org. Mass Spec. 1989,24, 895. 8. Archbald, P. J.;Fenn, M. D.; Roy, A. B. Carbohydr.Res. 1981,93, 117. 9. Ragan, M. A. Can. J . Chem. 1978,56,2681. 10. Usov, A. I.; Yarotakii, S. V.; Takiura, K. Bioorg. Khim. 1975, 1, 1583. 11. Honda, S.; Yuki, H.; Takiura, K. Carbohydr. Res. 1973,28, 150. 12. Heffer, P. E.; Valentine, K. M.; Parrish, F. W. J . Am. Chem. Soc. 1979,101, 1265.