December 1992 Volume 81, Number 12

JOURNAL OF PHARMACEUTICAL SCIENCES A publication of the American Pharmaceutical Assodation

A RTlCLES

Utility of Fourier Transform-Raman and Fourier Transform-Infrared Diffuse Reflectance Spectroscopy for Differentiation of Polymorphic Spironolactone Samples G.A. NEVILLE*’, H. D. BECKSTEAD*, AND H. F. SHURVELL* Received November 20, 1991. from the ‘Bureau of Drug Research, Health Protection Branch, Health and Welfare Canada, Ottawa, ON, KlA,OL?, Canada, and the *Department of Chemistry, Queen’s University, Kingston, ON, K7L 3N6,Canada. Accepted for publication April 15, 1992.

spironolactone by crystallization from acetonitrile, ethyl acetate, and ethanol, respectively; the forms obtained differed in their rate of dissolution in water:methanol mixtures. Salole and Al-Sarraj,8.9 using the same crystallization approach, identified five solvated forms of spironolactone and characterized three polymorphs and five solvated forms with IR spectrometry, thermal analysis, and X-ray diffraction. Unfortunately, these X-ray diffraction patterns were not indexed; hence, it is not possible with this technique to identify each of the spironolactone forms prepared by the latter workers. Just recently, Agafonov et a l . l O J 1 reported the preparation of two polymorphic and four solvated crystalline forms of spironolactone, each of which (except the form obtained from methanol) they characterized by X-ray crystallographic procedures. In this paper, we report the results of examination of 13 bulk pharmaceutical preparations (from five different suppliers) of spironolactone for residual solvents (including the hydrolysis product, thioacetic acid), evidence for the lack of enolic tautomeric forms, and the utility of Fourier transform @“)-Raman spectroscopy and diffuse reflectance infrared Spironolactone [3-(3-oxo-7a-acetylthio-l7~-hydroxy-4androsten-l7a-y1)propionicacid y-lactone11 is a diuretic ateroidal aldosterone known to show variable and incomplete 0 oral behavior because of poor water solubility and dissolution rate.2 Different crystallographic varieties (polymorphs)of the same molecule can result in differences in bioavailability of the polporphs.3 Mesley and Johnson4 distinguished two crystalline forms of spironoladone in 1965 with IR spectrometry (with liquid parailin mulls) and X-ray diffradometry. A crystal structure for spironoladone was first reported in 1972 I by Dideberg and Dupont,5 and a new crystalline modification 0 of spironolactone was reported in 1989 by Agafonov et al.6 In Splronolectone the interim, El-Dalsh et al.7 prepared three different forms of

Thirteen bulk pharmaceuticalpreparations of spironolactone were examined by Fourier transform (FT)-Raman spectroscopy and by diffuse reflectance infrared Fourier transfom spectroscopy (DRIFTS) for residual solvents (includingthe hydrolysis product, thioacetic acid),the presence of enolic tautomericforms, and evidence for different polymorphic forms. One sample (L) only was found to contain solvent residue (benzene).No evidence for the possible existence of enolic tautomers in the d i d state was found. From these specimens, four different repre sentative polymorphic samples (A, 6 , C, and D) were selected on the basis oftheir DRIFTS patterns in the 36004200cm-’ region. Samples K and L were considered to represent mixtures of two or more of the above representative types. Similar differentiation of the samples was made on the basis of their Raman spectra over the frequency range 1800-400an-’.The various fundamental stretching frequencies for the C = 0 and C = C bonds have been assigned,and these assignments, in turn. were used to account for all the bands in the 360&32OO-an-’ region as overtone and combination frequencies of the fundamentals. The Raman lines at 637 and 655 an-’were assigned to the two CS stretching modes of the thioacetyl moiety. Abrtract

4

This artide is not subject to U S . copyright. Published 1992, American Pharmaceutjcal Association

Journal of Pharmaceutical Sciences I 1141

HO Flgure 2-Possible enolic forms of spironolactone eliminated by deuterium exchange studies (where R represents the remainder of the spironolactone structure and R' is the thioacetyl moiety).

a Lo

3a

Ln

i

P

-

O N + f.

Y

Ln

F-

Is

fC9 3 N

0

Y

Z 3 Tf.

L

8

I

.

aY

-I

W

9 0 Y ' 3LtQQ 3200 WRVENUMBER (Cm-0 CRYSTRLS FROM METHFINOL

3600

50

3850

WAVENUMBER

3950

3.50

3350

3250

3150

(ad)

f.

Flgure 1-Expanded DR spectra (in Kabelka-Munk units) in the 332Wcm-' region of four representative polymorphic spironolactone samples (A, B, C, and D) and suspected mixtures of polymorphic forms (Samples K and L).

Fourier transform spectroscopy (DRIFTS)for differentiating solid state forms of spironolactone.

kc

5x1

Experimental Section DRIFTS spectra were recorded from 4000 to 650 cm-' with a Nicolet model 6OSX FT-IR spectrometer. Macro diffuse reflectance cups (13-mm diameter) and a DRIFTS accessory made by SpectraTech, Inc, were used. DRIFTS samples were prepared by mixing 60 mg of the bulk drug with 450 mg of spectral-grade potassium chloride in a dental amalgamator (Wig-L-Bug) for 10 8. After filling of the macro cup with a samplf+KCl blend, e x a m material was removed by placement of a microscope slide (with frosted face towards the powder) against the open cup in a rotary motion to leave a level but roughened surface of the sample to minimize the specular reflectance compon e n P in the diffuse reflectance (DR) spectrum. DR spectra were obtained without the uae of a blocker. For each sample 500 8~8118were collected employing a mercury-cadmium-telluride detector. Conventional fused KBr disk spectra were recorded with a deuterated triglyceride sulfate detector from samples prepared by compressing a 0.3% mixture of bulk spironolactone with spectral grade potassium bromide. Residual solvent analysis was conducted by gently heating -100 mg of each bulk spironolactone material just to the molten state in a 1-mL vial sealed with a Teflon-lined crimped cap (Hypo-vial, Pierce Chemical Company) followed by gas chromatography FT-IR analysis (a Nicolet 60SX interferometer with an MCT-A detector coupled to a Hewlett-Packard 5890 gas chromatograph) of the head space vapor. Injections of 1000 pL of headspace vapor were made onto a 60-m (i.d., 0.75-mm) SPB-1 (Supelco) column (5-pm film) whose initial temper-

Ill

I\ I

3600 3;QO 3200 LJFIVENUMBER(cm-') CRYSTFILS FROM METHFINOL-Dl

Flgure 3-DR spectra of a sample of spironolactone recrystallized (2x) from methanol (top) and from methanold, (bottom).

ature (100 "C) was held for 3 min before being raised at the rate of 25 "C/min to a final temperature of 280 'C. Helium was used as the carrier gas, with a flow velocity of 20 c d s (5 mumin). The temperature of both the iqjjector and detector was 280 "C and that of the transfer line and light pipe was 255 "C. Samplee for FT-Raman spectroscopy were prepared in the form of disks 3 mm in diameter with a KBr backing. The disks were formed in a hand-held minipress. FT-Raman spectra were recorded at the Thornton Research Centre, Shell Research Ltd. (Chester, Great Britain). Details of the experimental arrangement have been given in two recent publications.1sJ4 The excitation murce is a continuouswave Nd:YAG laser operating at 1064.1 nm (9397.6 cm-'). The laser beam is focused to a 1-mm spot at the sample, which is placed at one focus of an ellipsoidal mirror. The scattered radiation is collected by

Table CSummary of Fundamental Stretchlng Frequencles for C=O and C=C Bonds In Splronolactone Samples' Vibrational Mode

Stretching Frequency (cm-') for Samples: A

B

C

D

E

F

G

H

I

J

K

L

M

u ~ (ylactone)b = ~ uc=o (thioacetyl)b uc,o (a,punsat.)"

1768 1691 1675

1768 1691 1675

1769 1691 1675

1768 1691 1675

1768 1691 1675

1768 1691 1675

1769 1691 1675

1769 1691 1675

1769 1691 1675

1617

1771 1696 1678 (1688) 1619

1778 1701 1678

~ , - . , ~ (&%unsat.)d a,

1776 1700 1678 (16w 1619

1619

1617

1618

1617

1617

1617

1618

1618

1618

1776 1701 1678 (1688) 1619

a

Numbers in parentheses indicate minor differentiating peaks. Maximum Au, 10. " Maximum Au, 3. Maximum Au, 2.

1142 I Journal of Pharmaceutical Sciences

Table ICSummary of Overtone and Combindon Frequencler Arlslng from Fundamental vcs0and v

~Frequencies. , ~ ~

Frequency (cm-') for Samples:

Vibrational mode

A

B

C

D

E

F

G

H

I

J

1 st overtone vcs0 (rlactone)

3516

3537

3537 (3518)

3538

3516

3517

3515

3516

3515

3518

(3536) (3537) 3516 3516 3523 (3381) (3382) (3382)

1 st overtone

3362

3362

3361

3362

3362

3362

3362

3362

3362

3362

3362

3362

-

Combination from vc,o (thioacetyl) and vc,o (a, punsat.)

3362

3362

3361 3362 3362 (3374) (3354) (3354) (3351)

3362

3362

3362

3362

3362

3362

3362

-

1st overtone vc,o (a, punsat.)

3324

3332

3332

3332

3324

3323

3323

3325

3324

3324

3327

3326

(3353) 3332

Combination from vc,o (thioacetyl) and vc,c (a,punsat.)

3297

3296

3301

3296

3297

3297

3298

3297

3297

3297

3298

3297

3298

Cornbination from vcm0 (a,punsat.) and vc,c (a,punsat.)

3287

-

3267

-

3286

3267

3286

3286

3286

3287

3287

3285

-

K

L

M

vcx0 (thioacetyl)

a

Numbers in parentheses indicate additional peaks.

this mirror and directed into the Jacquinot stop of a Perkin-Elmer model 1760 near-IR spectrometer. Laser powers of 100-200 mW at the sample were used, and 100-200 scans at a nominal resolution of 4 cm-' were collected. The nitrogen-cooled germanium detector covere the spectral range 9400-6200 cm-', which is equivalent to a Raman shift range of 0-3200 cm-'. The WOO-cm-' region is obscured by the filter needed to remove the intense Rayleigh scattering and any unscattered laser radiation. The detector response is not linear and is very low in the CH stretching region of the Raman spectrum near 3000 cm-'. The Raman spectra were not corrected for

FREQUENCY ASSIGNMENTS (SAMPLE A) Fundamentals: uCd %o

(thioacetyl) ucd (steroidal)

1766 m' I691 an-' 1675 W'

Uc-c (steroidal)

1617 cm-'

Oldactone)

Overtone and Combination Frequencies:

-

3516 an" (overtone) from (2x 1766)= 3536 a m 1 3362 an" (overtone) from (2 x 1691) 3382 cml 3362 an" (combination) from (1691 + 1675)= 3368 am1 3324 m' (overtone) from (2 x 1675) = 3350 W' 3297 an-' (combination) from (1691 1617) = 3308 an-' 3287 an" (Comwnation) from (1675 1617)= 3292 an-'

detedor response and are presented only for qualitative comparison.

Results and Discussion Thirteen different lots of spironolactone from four different pharmaceutical sources [I (A-D), I1 (HA), 111 (K, L), IV (E-G)], and one chemical supplier, Sigma (MI,were examined in this study. Each of the samples (A-M) showed the same chemical identity by conventional FT-IR examination as KBr disks; this is not a surprising finding because one might expect any individual polymorphic differences t o be destroyed by the compression required to produce fused KBr disks that would result in a common solid state form. GC-FT-IR head space analysis of vapor sampled over the molten specimens failed to show any evidence for residual solvents of crystal-

-3-

in

E

+ +

Sample A

.

c

).)

Flgure -and assignments for the DR spectrum of sample A showing how the fundamental C = 0 and C = C stretching frequencies of the 1 ~ 1 6 0 0 - c m - 'region can account for the major overtone and combination frequencies observed in the 360&3200-cm-' region.

1600

1000 WaVMUlllbWD

100

(em-')

Flgure !+Comparison of IR-DR and Raman spectra of spironolactone sample A over the spectral region 1800-1100 cm-'. Journal of Pharmaceutical Sciences I 1143

I

A

I

Wavenumbers (cm-')

1boo

1000

Wavenumbers (cm-')

Wavenumbers (cm-')

Wavenumbers (cm-')

Wavenumbers (cm-')

500

Wavenumbers (cm-')

Figure 6-Comparison of FT-Raman spectra for spironolactone samples A, B, C, D, K, and L over the range 1800-400cm-'.

lization. One sample (L), however, was found by FT-Raman spectroscopy to contain benzene (see discussion below), but the residual benzene was below the level of detection by the GC-FT-IR head space vapor analysis technique.15 No evidence was found for thermal instability of the thioacetyl moiety in any of the spironolactone samples during head space analysis, although sample L clearly contained some free thioacetic acid whose presence was readily detected by smell (see discussion below). Interesting, subtle differentiation of the 13 spironolactone samples was obtained with DRIFTS, especially in the 36003200 cm-' region (Figure 1).From these specimens, four different representative samples (A, B, C, and D) wert selected on the basis of their patterns in the 3600-3200 cmregion. In addition, samples K and L were considered to represent mixtures of two or more of the above representative types. The other samples (E, F, G, H, I, and J)were found by DRIFTS to be identical with sample A, and sample M matched sample B. Although there is much similarity between the IR spectra from B and D, B shows a pronounced shoulder on the low frequency side of the band near 3500 cm-' (Figure 1)as well as an additional band in the carbonyl stretching region at 1688 cm-', which is also seen in samples C and M (Table I). Neither of these features are shown by sample D. Given the nature of the IR bands between 3600 and 3200-1 in Figure 1, we were concerned whether possible enolic tautomeric forms (Figure 2) of spironolactone might co-exist with the generally preferred keto form. To determine if there might be contributing enolic uOpH bands in 3600-3200-cm-' region, a sample of spironolactone was recrystallized twice from methanol-d, and no shift or alteration in the DRIFTS pattern for the 3600-3200-cm-' region was noted (Figure 3). All the bands of the 3600-3200-cm-' region, however, could be accounted for on the basis of overtone and combination frequencies arising from four fundamental frequencies, viz. vC=, (y-lactone) 1768 cm-', v,=, (thioacetyl) 1691 cm-', (steroidal with a,/?-unsaturation), and vc=, (steroidal with conjugation to a-keto) 1617 cm- (Table I1 and Figure 4). The IR-DR and Raman scattering of Sample A were compared over the frequency range 400-1800 cm-' (Figure 1144 I Journal of Pharmaceutical Sciences

5). Both the IR and Raman spectra are rich i n detail. Of major interest are the vC=, and vcZc bands as well as those for vCmq of the thioacetyl moiety. In particular, the strong IR vC=, bands at 1768 and 1691 cm-' for the y-lactone and thioacetyl keto groups, respectively, are matched by weak to moderately strong Raman lines at 1764 and 1689 cm-', respectively, for these groups. In contrast to the strong IR steroidal vc=, band at 1675 cm-', a very strong band is seen in the Raman at 1667 cm-' that is presumably due to enhanced polarization of the keto group through conjugation with the adjacent double bond. Likewise, this polarizability of the double bond must contribute to the greater Raman intensity of the steroidal vcx7 line at 1616 cm-' compared with its IR bands at 1617 cm- . The Raman lines at 637 and 655 cm-' are assigned to vcs of the thioacetyl moiety and, by anology with the polarization argument given above for the carbonyl Raman enhancement, the Raman line at 655 cm-' is assigned to vs-c=o (i.e., to the S-C bond conjugated to the thioacetyl carbonyl group), whereas the weaker line at 637 cm-' can be attributed to vcs remote from the carbonyl group. A band in the IR is observed at 657 cm-' to correspond with the 655 cm-' Raman line for vS-,;, of the thioacetyl group. Raman spectra of spironolactone samples A, B, C, D, K, and L (Figure 6) over the frequency range 1800400 cm-l indicate many subtle differences between the representative sample spectra A, B, C, and D in addition to the differences afforded by the spectra of samples K and L that are regarded as mixtures of two or more of the other types. In particular, note the various relative intensities of the thioacetyl vC=, line near 1690 cm- ' and its appearance as a shoulder in spectrum C. Considerably more variation is seen in the region of,,v near 650 cm-' where there is both variation in relative intensity and multiplicity of lines. Other differences can be seen between the spectra of samples A, B, C,and D, as well as with L, in the interim 700-1500-cm-' regions (Figure 6); in particular, although the Raman spectra for H and D are very similar, that of D shows two new lines near 1500 cm-' and one new line a t -1360 cm-' as well as other more subtle differences from the spectrum of sample B. One striking

t

N 0111- 1

UAVENUWBER

Figure I-Chmparison of FT-Raman spectra of spironolactone sample L as originally recorded (top trace) and as recorded 5 months later (bottom trace), showing marked reduction in the intensity of the line at loo0 cm-' due to a ring-breathingvibrational mode of benzene unique to the Raman effect.

B

"""*~""....j~"...... L-.............-

h

so

0

XU?

ctr

a

(WN) 192

00

161

5.50

1 6

4.00

15,

O W # POINTS

Figure &(A) Vapor-phase IR spectrum of benzene determined by GC-FT-IR head space analysis of spironolactone recrystallized from C,H,). (6)CorrespondingGramSchmidt reconbenzene (C,,H,,O.S struction chromatograph.

-

0.01

o.oO0ol

lo00

1800

800

em-1

Figure &Comparison of FT-Raman spectra of spironolactone recrystallized from benzene (top trace) and of spironolactone sample L (bottom

trace) containing benzene residue. feature, however, of the Raman spectrum of sample L is the sharp, intense line at 1000 cm-'. This was proven (as d i d below) to arise from benzene residue, but in too low concentration for detection by the GC-FT-IR headspace analysis. When, however, the Raman spectrum of sample L was recorded again 6 months after the original recording, the line at 1000 cm-' had nearly completely disappeared (bottom trace of Figure 7), presumably from slow release of entrapped benzene. To verify that the Raman line at 1000cm-' did indeed arise from adsorbed benzene, the spectrum of a sample of spironolactone, recrystallized from benzene, was compared with that recorded originally from sample L. Interestingly, a small frequency shift of 10 cm-' (Figure 8)was found between the line at 990 cm-' of the spironolactone recrystallized from benzene and that at 1000 cm-' from Sample L. The same scattering line occurs at 992 cm-' in free benzene due to a ring-breathing vibrational mode which is unique to the Raman effect and not found in the IR spectrum.16 The spironolactone material recrystallized from benzene waa also examined by GC-FT-IR head space analysis (Figure 9).The major vapor peak of the GramSchmidt reconstruction chromatograph (RT = 2.15 min) is due to benzene whose vapor-phase spectrum is seen above showing vCH near 3030

cm-' as the strongest band. Elemental microanalysis of this material gave excellent duplicate determinations for C, H, and S for the monobenzene solvate of spironolactone.

Conclusions All 13 spironolactone samples showed the same chemical identity by conventional FT-IR examination as KBr disks. Only one sample (L) was found to contain solvent residue (benzene). No evidence wa8 found for thermal instability of the thioacetyl moiety in any of the spironolactone samples, although one sample (L) clearly contained some free thioacetic acid. No evidence for the possible existence of enolic tautomeric forms was found. Both the DRIFTS technique and FT Raman spectroscopy provide a rapid and convenient means for differentiating solid state forms of spironoladone. In this study, four of the 13 samples were designated as different representative samples (A, B, C, and D),whereas samples K and L were considered to represent mixtures of two or more of the representative types. Samples E, F, G, H, I, and J were identical to sample A, and sample M matched sample B.

References and Notes 1. The Merck Index, 11th Ed.; Merck Rahway, NJ, 1989;p 8721. 2. Seo, H.; Tsuruoka,M.; Hashimoto, T.;Fujinaga, T.;Otarigi, M.; Uekama, K. Chem. Phurm. Bull. 1983,31,286291. 3. Haleblain, J. K.; Koda, R. T.;Biles, J. A. J.Pharm. Sci.1971,60, 1485-1489. 4. Mesley, R.J.; Johnson, C. A. J. Pharm. Phanacol. 1965, 17, 329-340. Journal of Pharmaceutical Sciences I 1145

5. Dideberg, 0.; Dupont, L.Acta Crystallogr. 1972,B28,3014-3022. 6 . Agafonov, V.; Legendre, B.; Rodier, N. Acta Crystallogr. 1989, C45, 1661-1663. 7 . El-Dalsh, S.S.; El-Sayed, A. A; Badawi, A. A.; Khattab, F. I.;

Fouli, A. Drug Dev. Ind. Pharm. 1983,9,877-894. 8. Salole, E. G.; Al-Sarraj,F. A. Drug Dev. Ind. Pharm. 1985,11,

855-864. 9. Salole, E. G.; Al-Sarrqj, F. A. Drug Dev. Ind. Pharm. 1985,1 1 ,

2061-2070. 10. Agafonov, V.;Le endre, B.; Rodier, N.; Wouessidjewe, D.; Cense, J.-M. J. Phurm. f5ci. 1991, 80, 181-185. 11. Agafonov, V.; Legendre, B.; Rodier, N. Ada Crystallogr. 1991, c47. 36.5-369. --- - - I

12. Messerschmidt, R.G.Appl. Spectrosc. 1985,39,737-739.

1 140 I Journal of Pharmaceutical Sciences

13. Bergin, F. J.;Shurvell, H. F. Appl. Spectrosc. 1989,43,516622. 14. Shurvell, H.F.; Bergin, F. J. J. Raman Spectrosc. 1989, 20, 163-168. 15. Beckstead, H.D.;Neville, G. A. Can. Soc. Forensic. Sci.J. 1987, 20,71-76. 16. Dollish, F. R.;Fatele , W.G.; Bentley, F. F. Characteristic Raman Frequencies of drrganic Molecules; Wiley: New York, 1974; p 163.

Acknowledgments Presented a t the 36th Canadian Spectroscopy Conference, 1 3 August 1990, at Brock University, St. Catharines, ON, Canada.