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Forced Degradation of Betamethasone Sodium PhosphateUnder Solid State: Formation, Characterization, andMechanistic Study of All Four 17,20-Diastereomers ofBetamethasone 17-Deoxy-20-Hydroxy-21-Oic Acid

MIN LI,1 XIN WANG,1 BIN CHEN,1 TZE-MING CHAN,2 ABU RUSTUM1

1Global Quality Services - Analytical Sciences, Schering-Plough Corporation, 1011 Morris Avenue, Union,New Jersey 07083

2Structural Chemistry, Schering-Plough Research Institute, Kenilworth, New Jersey 07033

Received 14 November 2007; revised 3 February 2008; accepted 12 May 2008

Published online 11 July 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21477

Corresponde908-284-2171; E

Journal of Pharm

� 2008 Wiley-Liss

894 JOURNA

ABSTRACT: Four diastereomers of betamethasone 17-deoxy-20-hydroxy-21-oic acidwere found to be degradants of betamethasone sodium phosphate when the latterwas stressed by heat under the solid state. The structure elucidation of these fourdiastereomers was achieved by a combination of LC–MSn (n¼ 1–3), various 1D and 2DNMR experiments, and mechanistic consideration of potential degradation pathways. Amechanism for the formation of the four degradants has been proposed, which involveshydration of a key intermediary degradant, betamethasone enol aldehyde, followed byintramolecular Cannizzaro reaction. The proposed mechanism is supported by a modelreaction which converted betamethasone enol aldehyde into the four diastereomericdegradants in good yields, albeit in solution chemistry. � 2008 Wiley-Liss, Inc. and the

American Pharmacists Association J Pharm Sci 98:894–904, 2009

Keywords: corticosteroids; degradation

; betamethasone enol aldehyde; betametha-sone 17-deoxy-20-hydroxy-21-oic acid; LC–MS

INTRODUCTION

Corticosteroids such as betamethasone, dexa-methasone, and prednisolone are anti-inflam-matory drugs; they are often available as thecorresponding 21-phosphoric esters that are for-mulated into various dosage forms due to theirmuch enhanced solubility in an aqueous environ-ment. Yet, the introduction of the phosphoric

nce to: Min Li (Telephone: 908-820-3391; Fax:-mail: min.li@spcorp.com)

aceutical Sciences, Vol. 98, 894–904 (2009)

, Inc. and the American Pharmacists Association

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group at the 21-hydroxyl of the steroid corestructures might impart additional degradationpathways to the resulting phosphonosteroids.Although these drugs have been on the marketfor many decades, not much information isavailable in the literature regarding their degra-dation behaviors. In our laboratory, we have beenprobing their degradation mechanisms throughvarious forced degradation studies; the degra-dants formed are usually analyzed by LC-PDA/UV-MSn (n is typically 1–3) first and, if necessary,by 1D and 2D NMR spectroscopy followingisolation by preparative HPLC.1 During one ofour recent forced degradation studies of the drugsubstance, betamethasone sodium phosphate (1),

H 2009

Scheme 1. Formation of the four diasteoremers of betamethasone 17-deoxy-20-hydroxy-21-oic acid from betamethasone sodium phosphate by heat stress in the solidstate.

FORCED DEGRADATION OF BETAMETHASONE SODIUM PHOSPHATE 895

it became evident that additional degradationpathways due to the phosphoric moiety did indeedexist. In this paper, we present the evidence for anovel degradation pathway of 1 under solid statewhich leads to the formation of four degradantsthat are 17,20-diastereomers of betamethasone17-deoxy-20-hydroxy-21-oic acid (2a, 2b and3a, 3b, Scheme 1). These four degradants havesince been observed in the drug substancelots that were stored under the ICH long termstorage condition (308C/60% RH) and in productsformulated with 1, during related analyticalmethod development activities in our laboratory.At least one of such analogous isomers wasfound to be among many acidic metabolites ofcortisol.2,3

EXPERIMENTAL

Chemicals

Betamethasone sodium phosphate (1) and beta-methasone enol aldehyde (both E- and Z-isomers)were in-house materials. All other chemicals andreagents are either reagent grade or HPLC gradeand were obtained from Sigma-Aldrich (St. Louis,MO, USA) or Fisher Scientific (Pittsburgh, PA,USA).

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Heat Stress of Betamethasone Sodium Phosphate (1)in Solid State

A crystalline sample of 1 was heated at 105 8C forup to 3 weeks. The heat-stressed sample wasanalyzed by LC-PDA/UV-MSn.

LC-PDA/UV-MSn Experiments

A Thermo Electron Surveyor HPLC systemcoupled with a PDA detector and a linear iontrap mass spectrometer (LTQ) was used in the LC-PDA/UV-MSn studies. The mobile phase A of theHPLC separation contained 10% tetrahydrofuran,10% acetonitrile, 80% 10 mM ammonium acetatebuffer at pH 4.5. The mobile phase B contained15% tetrahydrofuran, 30% acetonitrile, 55%10 mM ammonium acetate buffer at pH 4.5. Thegradient started from 0 to 80% B in 25 min andfollowed by a 10 min equilibration at 0% B witha flow rate of 1.5 mL/min. The HPLC flow wassplit at a ratio of 10:1 after the PDA detector;approximately 150 mL/min of the HPLC flow wasdirected into the ion source of the MS detector. Forpositive electrospray ionization, a spray voltage of4.5 kV was used, while for negative electrosprayionization, a spray voltage of �4.5 kV was used.The capillary temperature was 3008C and thesheath gas flow rate was 40 units. A full MS scanwas obtained in the mass range of 200–1000 Da.The MS/MS experiments were performed using

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helium as the collision gas, the parent ionisolation width was 2.8 Da, and the normalizedcollision energy was 28.

Preparative HPLC Separation

An Agilent preparative HPLC system coupledwith a PDA detector and a MSD mass spectro-meter was used to isolate the four degradantpeaks of interest. A Supelcosil LC8 preparativecolumn (250 mm� 21.1 mm i.d.) was used duringthe first isolation. The mobile phase A contained10% acetonitrile, 90% 10 mM ammonium acetatebuffer at pH 4.5. The mobile phase B contained50% acetonitrile, 50% 10 mM ammonium acetatebuffer at pH 4.5. The gradient started at 40% B for30 min and increased to 100% B in 2 min at a flowrate of 25 mL/min. Six fractions were collectedbased on the retention times and mass responses.The collected fractions were then analyzed usingthe analytical LC–MS method outlined above.Only four of the collected fractions were foundto contain the degradant peaks of interest. Asecondary purification for the four collectedfractions were performed using the same systembut with a modified HPLC method that did notcontain buffer in the mobile phases. The modifiedmobile phase A contained 95% water, 5% acet-onitrile with 0.1% trifluoroacetic acid and themobile phase B contained 100% acetonitrile with0.1% trifluoroacetic acid. An isocratic run with35% B was used at a flow rate of 25 mL/min. Thepurified fractions were evaporated to dryness andeach dried fraction was confirmed by the analy-tical LC–MS to contain the degradant peaks ofinterest, respectively, prior to NMR analysis.

NMR Experiments

NMR spectra (Proton, Carbon, Carbon APT,HSQC, HSQCTOXY, HMBC, COSY and ROESYspectra) were taken at 258C on a Varian 500 MHzspectrometer with either a 3 mm dual probe or a3 mm indirect detection probe. The samples weredissolved in either DMSO-d6 or CD3OD.

Base-Catalyzed Conversion of Betamethasone EnolAldehyde Into the Four Diastereomers, 2a, 2b,3a, and 3b (Model Reaction)

Betamethasone enol aldehyde was dissolved inmethanol/water (1:1, v/v) at a concentration of

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�1 mg/mL. To �1 mL of this solution was added�50 mL of 1 N NaOH solution. The resultingsolution was kept at room temperature andmonitored continuously by LC–MS on a ThermoElectron Surveyor HPLC system equipped with aPDA detector and an MSQ Plus MS detector. Theflow was directed into the MS source withoutsplitting after the PDA detector. The MS wasoperated in electrospray positive ion mode, withthe cone voltage at 75 V and the probe tempera-ture at 6008C. Full MS scans were obtained in themass range of 150–1000 Da. The LC parametersare identical to those described in the section forLC-PDA/UV-MSn experiments.

RESULTS AND DISCUSSION

Initial LC-PDA/UV-MS Analysis of Heat-StressedBetamethasone Sodium Phosphate (1) at 105-C

The heat stressed sample was analyzed by LC–MSand four degradant peaks with the same mole-cular weight of 392 (m/z 393) were observed atretention times of 11.5, 12.8, 13.3, and 14.6 min,respectively, as shown in Figure 1. The molecularweights of the four degradants (392 Da) werefound to be the same as that of betamethasone, thestarting material as well as a potential hydrolyticdegradant of 1. The yields of these four unknownspecies were between �2% and �5% in the heat-stressed sample which allowed enough quantitiesof them to be isolated for further structuralcharacterization using a preparative LC–MSsystem.

LC–MSn (n U 2, 3) Analysis of the FourIsolated Degradants

The identity and purity of each isolated fractionwas confirmed by LC–MSn. All the fractionsdisplayed the molecular ions at m/z 393, each ofwhich gave rise to essentially identical MS2

fragmentation patterns (results not shown) upondissociation by collision in the gas phase. Themajor daughter ions at m/z 355 in all four casesabove were further fragmented, respectively, andthe resulting MS3 spectra now showed similar butdistinguishable fragmentation patterns (Fig. 2).In our laboratory, we have utilized MSn fragmen-tation pattern (or MSn fingerprinting) as a keyanalytical tool for tracing the unknown peaks fortheir formation in the stress studies and through-

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Figure 1. Upper panel: Extracted ion chromatogram at m/z 393 of the heat stressedbetamethasone sodium phosphate sample. Peaks eluting at 11.5, 12.8, 13.3, and 14.6 mincorrespond to the four degradants of 2a, 3b, 2b, and 3a. The peak at 15.8 minis betamethasone. Lower panel: Representative mass spectrum corresponding to thepeak at �13.3 min.

FORCED DEGRADATION OF BETAMETHASONE SODIUM PHOSPHATE 897

out their isolation process. Frequently, fragmen-tation patterns revealed by MS2 spectra providereliable molecular fingerprinting, which has beenshown to be reproducible under identical LC–MSn

conditions (on the same type of MS spectrometer)in our laboratory and can be used to distinguishmolecules with similar structural features. For

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instance, the MS2 spectra of the two regioisomers,the Z- and E-isomers of betamethasone enol alde-hyde, display different fragmentation patterns.4

In the case of betamethasone versus dexametha-sone, which are a pair of epimers with the onlydifference lying in the orientation of the 16-methylgroup, their MS2 spectra are still distinguishable

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Figure 2. MS3 spectra of the four diastereomers, 2a (A), 2b (B), 3a (C), and 3b(D): fragmentation patterns of the m/z 355 daughter ions (m/z 393!m/z 355!). Theabsolute stereochemistry at the 20-position could not be determined due to the fact thatthe 1H NMR signal of 20-OH could not be observed.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 3, MARCH 2009 DOI 10.1002/jps

FORCED DEGRADATION OF BETAMETHASONE SODIUM PHOSPHATE 899

although the majority of the fragment peaks andtheir intensities are essentially identical.1,5 Inthe present case, the MS2 spectra of the four m/z393 isomers showed no substantial differenceamong themselves; nevertheless, the MS3 frag-mentation of their common daughter ion, m/z 355,displayed similar but distinguishable patterns(Fig. 2). These results suggested that the four m/z393 species should be structurally very similar(since MS2 displayed no difference) and yet withsubtle differences (since MS3 displayed smalldifferences).

Mechanistic Consideration Based on the Results ofLC–MSn and Subsequent 1D and 2D NMR Analysis

The collected fractions of the four degradants fromthe preparative HPLC were dried through eva-poration and lyophilization; the residues wereanalyzed by various 1D and 2D NMR techniques.Similar to the situation of the LC–MSn analysis,

Scheme 2. Proposed mechanism for the fbetamethasone 17-deoxy-20-hydroxy-21-oic acby heat stress in the solid state.

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the 1D and 2D NMR spectra of these fourbetamethasone isomers showed similar peaksand/or patterns with subtle differences. Althoughthe spectra were still distinguishable, the similarresults made the elucidation of the four structuresperplexing. At the meantime, certain physico-chemical properties of these four isomers startedto reveal themselves during the sample prepara-tion. For example, these four isomers seemedquite polar as they could be relatively easilyextracted into aqueous solutions; this behaviorhad been observed in our laboratory for some ofthe known acidic degradants of betamethasone.Based on these results and observation, itoccurred to us that betamethasone enol aldehyde(5), which can be considered as a dehydrateddegradant of betamethasone, could be the inter-mediary degradant from which these four iso-meric degradants would be formed through re-hydration. A degradation mechanism was thusproposed (Scheme 2) which led to the predication

ormation of the four diastereomers ofid from betamethasone sodium phosphate

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900 LI ET AL.

of the four degradant structures (2a, 2b, 3a, and3b)6 which are isomeric to betamethasone. Areview of all the 1D and 2D NMR results indicatedthat they are consistent with the four degradantstructures predicated by the proposed mechan-ism. Due to the similarity among the fourdegradant structures and their NMR data, onlythe 1D 1H and 13C NMR results of 2a are shown inTables 1 and 2 for simplicity.

Table 1. 1H and 13C NMR Signal Assignme17, 20-Diastereomers of Betamethasone 17-D

13C NMR

1a 153.0 d2 128.9 d3 185.3 s4 124.0 d5 167.3 s6 30.4 t 2.29 d7 27.7 t8 33.8 d 2JCF¼ 19.5 Hz9 101.5 s 1JCF¼ 175.2 Hz

10 48.1 s 2JCF¼ 22.7 Hz11 71.1 d 2JCF¼ 37.1 Hz12 38.1 t13 42.3 s14 45.9 d15 35.1 t16 30.3 d17 58.5 d18 27.7 q19 22.9 q 3JCF¼ 5.7 Hz20 69.4 d21 176.1 s22 23.1 q

�NMR spectra (Proton, Carbon, Carbon APT, HSspectra) were taken at 258C on a Varian 500 MHz sp3 mm indirect detection probe. The sample was dis

aThe numbering of the steroid rings follows the

.

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Summary of the Key 2D NMR Results

For 2a, its protonated carbon resonances (Tabs. 1and 2) were assigned by HSQC and its protoncoupling patterns were established by COSY andHSQCTOXY. The nonprotonated carbon reso-nances were assigned by HMBC, which was alsoused to establish the proton–carbon network.Important HMBC cross peaks observed were: H22

nt for 2a (in DMSO-d6), One of the Foureoxy-20-Hydroxy-21-Oic Acid�

1H NMR

7.29 d 10 Hz6.20 dd 1.6, 10 Hz

5.98 br s

d 3.8, 13.5 Hz; 2.60 dt 5.5, 13.5, 13.5 Hz1.30 m; 1.79 m

2.30 m

4.09 br d 3JHF¼ 10 Hz1.55 br d 13 Hz; 1.99 br d 13 Hz

1.72 m0.97 m; 1.71 m

2.07 m1.49 br s

1.05 s1.48 s

3.95 br s

0.96 d 6.5 Hz

QC, HSQCTOXY, HMBC, COSY and ROESYectrometer with either a 3 mm dual probe or asolved in about 200 mL of DMSO-d6.usual convention as shown below for 2a.

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Table 2. 1H and 13C NMR Signal Assignment for2a (in CD3OD), One of the Four Diastereomers ofBetamethasone 17-Deoxy-20-Hydroxy-21-Oic Acid�

13C NMR 1H NMR

1 156.4 d 7.45 d 10.2 Hz2 129.5 d 6.27 dd 1.9, 10.2 Hz3 189.1 s4 124.8 d 6.06 br s5 171.5 s6 32.3 t 2.37 m; 2.70 dt 6.2, 13.5, 13.5 Hz7 29.1 t 1.50 m; 1.90 m8 35.8 d 2.41 m9 102.6 s

10 50.4 s11 73.5 d 4.22 br d 3JHF¼ 11 Hz12 40.2 t 1.67 br d 13.8 Hz; 2.14 br d 13 Hz13 43.7 s14 47.5 d 1.89 m15 36.7 t 1.17 m; 1.79 m16 31.0 d 2.03 m17 60.8 d 1.71 dd 1.7, 5.0 Hz18 24.6 q 1.15 s19 23.5 q 1.59 s20 72.5 d 3.94 d 1.7 Hz21 181.1 s22 23.6 q 1.05 d 7.1 Hz

�13C NMR spectrum was not recorded due to the smallquantity of sample available. Carbon chemical shifts wereextracted from the HSQC and HMBC spectra (For chemicalshift reference, the carbon chemical shift of the solvent was setat 49.15 ppm). The carbon-fluorine coupling constants were notobtained; however, a large carbon-fluorine coupling in theorder of 175 Hz was observed for C9 in the HMBC spectrum.NMR spectra were taken at 25 C on a Varian 600 MHzspectrometer with a 3 mm indirect detection probe. The samplewas dissolved in about 100 mL of CD3OD and transferred to a3 mm shigemi tube. Proton, HSQC, HMBC, COSY and NOESYspectra were taken.

FORCED DEGRADATION OF BETAMETHASONE SODIUM PHOSPHATE 901

and H18 to C17; H20 to C17, C16 and C21. TheseHMBC correlations are consistent with thestructure of 2a. From the HSQC spectrum, thecoupling constant between H16 and H17 wasdetermined to be about 4–5 Hz, which isconsistent with the trans relationship of thetwo protons. NOE signals between protonswere obtained by a 2D ROESY experiment.NOE signals were observed between H17 andthe methyl groups H18 and H22, which isconsistent with the stereochemistry at C17 (17bdeoxy). Only a small NOE signal was observedbetween H16 and H17, which is also consistentwith the trans relationship of the two protons. Forthe NMR results of 2b, the HSQC and HMBCspectra are very similar to those of 2a, indicatingsimilar structures. From the HSQC spectrum,

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the coupling constant between H16 and H17was determined to be about 4–5 Hz, which isconsistent with the trans relationship of the twoprotons. Similar coupling constants had beenobserved for this type of 17b deoxy compounds asdiscussed above. In the ROESY spectrum, NOEsignals were observed between H17 and bothmethyl group H18 and H22, which are consistentwith the 17b deoxy stereochemistry. Degradants2a and 2b have different stereochemistry at C20;during the course of this study, their absoluteconfigurations with regard to whether 2a has Rand 2b has S at the C20 position or vice versacould not be determined due to the fact that theproton signal of 20-OH could not be observed. Forthe NMR results of 3a, the HSQC and HMBCspectra are very similar to those of 2a and 2b,indicating similar structures. From the HSQCspectrum, the coupling constant between H16 andH17 was determined to be about 10 Hz, which isconsistent with the cis relationship of the twoprotons. In the ROESY spectrum, no NOE wasobserved between H17 and both methyl groupH18 and H22; NOE was observed between H16and H17. These two results are consistent withthe 17a deoxy stereochemistry. For the NMRresults of 3b, the HSQC and HMBC spectra arevery similar to 2b, 3a, and 3b, indicating similarstructures. From the HSQC spectrum, the cou-pling constant between H16 and H17 wasdetermined to be about 10 Hz, which is consistentwith the cis relationship of the two protons.Similar coupling constants had been observed forthis type of 17a deoxy compounds. In the ROESYspectrum, no NOE was observed between H17 andboth methyl group H18 and H22; NOE wasobserved between H16 and H17. These two resultsare again consistent with the 17a deoxy stereo-chemistry. Degradants 3a and 3b have differentstereochemistry at C20 and their absolute config-urations at that position could not be determinedfor the same reason discussed above.

Verification of the Proposed Mechanism Via aModel Reaction (Based-Catalyzed Reaction ofBetamethasone Enol Aldehyde)

In the proposed mechanism (Scheme 2), beta-methasone enol aldehyde (5a) could be formeddirectly from betamethasone sodium phosphate(1). This direct pathway apparently would gothrough the betamethasone sodium phosphateenol form (4), followed by a concerted rearrange-

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Scheme 3. Formation of betamethasone enol aldehyde from betamethasone 17,21-dipropionate via the variation of Mattox rearrangement under alkaline condition.4

902 LI ET AL.

ment of the virtual six member ring (involving17-OH, 17-C, 20-C, 21-C, and one P–O bond in thephosphate) which could be triggered by 17-OHattacking the phosphate group. This proposedconcerted rearrangement in which 1 directlydegrades to the enol aldehyde (5a) somewhatresembles a variation of the classic Mattoxrearrangement recently reported by our group.4

In the variation of the Mattox rearrangement, the17,21-diester of betamethasone (and relatedcompounds) has been found to degrade directlyto the enol aldehyde (5a) under an alkalinecondition (Scheme 3); the degradation, pre-sumably via a corresponding enol form (9), isapparently triggered by the nucleophilic attack onthe 21-acyl group. In the current case, once theenol aldehyde (5a) would be formed, its re-hydration could occur by incorporating a watermolecule into the aldehyde group of the a-ketotautomer (5b, which is a glyoxal) of the enolaldehyde (Scheme 2, Pathway a). The a-ketoaldehyde group should be readily susceptible tohydration due to the activation of the aldehydegroup by the neighboring keto group. Alternately,the hydration of 5b could also be mediated by thephosphate group (Scheme 2, Pathway b). The

Scheme 4. Intramolecular Cannizzaro reacnan-21-al (10) catalyzed by NaOH in aqueous

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hydrated and/or the phosphonated species (6 and/or 7) would then undergo an intramolecularCannizzaro reaction to yield the four diastereo-mers of betamethasone 17-deoxy-20-hydroxy-21-oic acid (17S, 20S; 17S, 20R; 17R, 20S; 17R, 20R).A search of the literature indicated that the samekind of intramolecular Cannizzaro transforma-tion occurs quite efficiently for steroidal glyoxals(20-keto-21-aldehyde) such as 3a-hydroxy-11,20-dioxo-5b-pregnan-21-al (10) when catalyzed byNaOH in aqueous solutions (Scheme 4).7 In orderto verify that betamethasone enol aldehyde (5a),which is a tautomer of the glyoxal form (5b),would also undergo the intramolecular Canniz-zaro reaction, a solution of 5a8 in a mixture ofmethanol and water was treated with a smallaliquot of 1 N NaOH solution at room temperatureand the resulting solution was monitored by LC-PDA/UV-MS. Approximately 5 min after theaddition of NaOH, two partially coeluting, iso-meric species at retention times of 12.3 and13.1 min and both with molecular weights of392 Da (and thus are isomers to betamethasone aswell as 2 and 3) were found to have formed insignificant yields, while trace amounts of 2a, 2b,3a, and 3b were also observed (Fig. 3).9 With

tion of 3a-hydroxy-11,20-dioxo-5b-preg-solutions.7

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Figure 3. Base-catalyzed conversion of betamethasone enol aldehyde (E-isomer):initial formation of the two enol aldehyde hydrates followed by intramolecular Canniz-zaro reaction. Reaction condition: Betamethasone enol aldehyde was dissolved inmethanol/water (1:1, v/v) at a concentration of �1 mg/mL. To �1 mL of this solutionwas added �50 mL of 1 N NaOH solution. The solution was kept at room temperatureand monitored continuously by LC-PDA/UV-MS analysis. Identities of chromatograms(A–D, UV chromatograms at 240 nm; E: Extracted ion chromatogram at m/z 393):(A) Betamethasone enol aldehyde, before addition of NaOH; (B) 30 min after additionof NaOH; (C) 90 min after addition of NaOH; (D) 16 h after addition of NaOH; (E) 16 hafter addition of NaOH. Identity of peaks: (a) Betamethasone enol aldehyde, E-isomer;(b) Betamethasone enol aldehyde, Z-isomer; (c) Two betamethasone enol aldehydehydrates; (d) Peaks 2a, 3b, 2b, and 3a.

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elapse of time (20 min through overnight), 2a, 2b,3a, and 3b started to increase and become majorproducts, which coincided with diminishing of thetwo initial isomers at 12.3 and 13.1 min. Based onthe proposed mechanism (Scheme 2) and theknown intramolecular Cannizzaro reaction(Scheme 4), the two initial isomers at 12.3 and13.1 min seem to be the two epimers of theenol aldehyde hydrate (6) with epimerizationoccurring at the 17-position. It appears that thesetwo epimers of the hydrate (6) readily converted tothe four diastereomers of betamethasone 17-deoxy-20-hydroxy-21-oic acid (2a,b and 3a,b)apparently through the intramolecular Canniz-zaro reaction. This facile model reaction, althoughoccurring in the solution phase, demonstrates thefeasibility for the same transformation frombetamethasone enol aldehyde to the four diaster-eomers of betamethasone 17-deoxy-20-hydroxy-21-oic acid (2a,b and 3a,b) to happen in thesolid phase. It is interesting to note that thedistributions of the four degradants are quitesimilar between the solid state degradation andthe model reaction in solution (Fig. 1, upper vs.Fig. 3E).

CONCLUSION

In summary, we have shown the formation ofthe four diastereomers of betamethasone 17-deoxy-20-hydroxy-21-oic acid from betametha-sone sodium phosphate by heat stress in the solidstate. A mechanism for the formation of the fourdegradants has been proposed which is supportedby a model reaction involving initial hydration ofthe key intermediary degradant, betamethasoneenol aldehyde, followed by an intramolecularCannizzaro rearrangement. The critical struc-tural and mechanistic results were obtained viaLC-PDA/UV-MSn and 1D and 2D NMR analyses.The elucidation of this degradation pathwayshould facilitate the understanding of the stabilitybehavior of betamethasone sodium phosphate and

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pharmaceutical products formulated with beta-methasone sodium phosphate and similar phos-phonosteroid drug substances.

REFERENCES

1. Li M, Chen B, Lin M, Rustum A. (by invitation)Application of LC-MSn in Conjunction with Mechan-ism-based Stress Studies in the Elucidation of DrugImpurity/Degradation Product Structures and Degra-dation Pathways. Am Pharm Rev 2008. 1–2:98–103.

2. Weiss G, Monder C, Bradlow L. 1976. 17-Deoxygena-tion: a new pathway of Cotisol metabolism isolationof 17-deoxycortolonic acids. J Clin Endocrinol Metab43:696–699.

3. Singer CJ, Iohan F, Monder C. 1986. 11ß, 20-Dihydroxy-3-oxopregna-4, 17(20)-dien-21-al: anintermediate in the biological 17-dehydroxylationof Cortisol. Endocrinology 119:1356–1361.

4. Li M, Chen B, Lin M, Chan T-M, Fu X, Rustum A.2007. A variation of Mattox rearrangement mechan-ism under alkaline condition. Tetrahedron Lett 48:3901–3905.

5. Arthur KE, Wolff J-C, Carrier DJ. 2004. Analysis ofbetamethasone, dexamethasone and related com-pounds by liquid chromatography/electrospray massspectrometry. Rapid Commun. Mass Spectrom18:678–684.

6. The four degradants 2a, 2b, 3a, and 3b correspond tothe peaks eluting at retention times of 11.5, 13.3,14.6, and 12.8 minutes (Fig. 1), respectively.

7. Lewbart ML, Mattox VR. 1963. Conversion of Ster-oid-17-al Glyoxals to Epimeric Glycolic Esters. J OrgChem 28:1779–1786.

8. Both the E- and Z-isomers of betamethasone enolaldehyde were found to undergo the intramolecularCannizzaro reaction although the distribution of thefour diastereomers were slightly different. Only thereaction of the E-isomer is shown.

9. The retention times of the four diastereomers, 2a,2b, 3a, and 3b, were somewhat different from thosein the previous LC-PDA/UV-MSn studies, whichappeared to be caused partly by different instru-ments used and by the presence of THF in the mobilephase. THF is a strong organic modifier and anysmall variation in its concentration tends to givelarge impact to retention times.

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