J. Soc.Co,•met.Chem., 25, 495-506 (September1974)

Kineticsof Degradation of the Parabens SEYMOUR M. BLAUG, Ph.D.,* and DONALD E. GRANT, Ph.D.t

Synopsis-The effect of pH and TEMPERATURE on the HYDROLYSIS of methyl, ethyl, propyl, and n-butyl PARABEN was studiedat 70øC in 0.1M phosphatebuffer so]utions at ionic strength0.3 from pH 2.75 to 9.16 and at 40ø and 50øC at pH 9.16. The REACTION xvasfirst order with respectto paraben. ENERGIES OF ACTIVATION

were

determined from Arrhenius plots. RATE CONSTANTS and HALF-LIVES of each paraben at 25øC were obtainedby extrapolationof the Arrheniusplots. The half-life of each parabenat 70øC and pH 8.24 was essentiallyindependentof the initial concentrationof paraben. Increasingionic strengthresulted in a slight increasein the rate of hydrolysisof each paraben.Increasingthe phosphateconcentrationin buffer solutionsat pH 8.24 at 70øC producedan increasein the rate of hydrolysisof each paraben. This indicated that the parabensundergo general base catalysisand that hydroxyl ion is not the only speciesthat can catalyzetheir hydrolysis.

INTRODUCTION

The parabens havebeenreportedto be stable,and to showno hydrolysis under conditionsof heat steril/zat'on(2 hours at 100øC or 0.5 hour at

120øC)at a pH rangeof 3-8 (1). Pekkarinen andTommila(2) studiedthe alkalinehydrolysisof ethyl estersof para and meta hydroxybenzoates in potassiumhydroxidesolution,and in variousorganicsolvents.They conc]udedthat the hydrolysis takesplacesolelyas a reactionbetweenthe ester ion andthe hydroxideion. Otherauthorsfoundthat the methylesterof p-

hydroxybenzoic acidis hydrolyzed at hightemperature andin strongly acid solution(3). RavelandParrott(4) studiedthehydrolysis of methylparaben in aqueous solutions at temperatures of 70-85øCin a pH rangeof 6-9. * Universityof North Carolina,Schoolof Pharmacy.ChapelHill, N.C. Investigationwas conductedat University of Iowa, Iowa City, Iowa 52242.

'• Beecham-Massengill Pharmaceuticals, Bristol,Tenn. 495

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Methyl,ethyl,propyl,andbutyl parabenarewidelyusedin manytypesof pharmaceutical preparationssuch as ophthalmic,oral, and topicaldosage formsandin cosmeticcreamsandlotions.They are usedsinglyor in combination with one another.Mixturesof the parabenshavebeen shownto be more effectiveaspreservatives thanthe individualparabens(5, 6). Their hydrolysis yieldsp-hydroxybenzoic acid, which possesses little preservativeactivity.It was the purposeof this investigationto study the stability of the estersof

p-hydroxybenzoic acidoverpH rangeof 3-9 andvarioustemperatures. EXPERIMENTAL

Reagentsand Apparatus Reagentsused were p-hydroxybenzoicacid (mp 213-214øC), methyl paraben (top 126-128øC), ethyl paraben (mp 116-117øC), propyl paraben (mp 95-97øC), and n-butyl paraben (mp 73øC), all recrystallized from hydroalcoholicsolution;also,sodiumhydroxide,monobasicpotassium phosphate, potassium chloride,phosphoric acid,andchloroform, all AR grade. All pH's were measuredusing a Beckmanmodel H-2 pH meter* and spectrawere obtainedwith a BeckmanDU spectrophotometer* with 1-cm quartz cells. A constanttemperaturebath with a Haake thermoregulator, series ED,t with a thermometer calibrated to 0.1øC was used for all the studies. Procedure

Preparationo[ Buffers

The buffersusedin this investigationwere preparedfrom monobasic potassiumphosphatesolutionsof desiredmolaritywhich were adjustedto the desiredpH with sodiumhydroxidepelletsand phosphoricacid. Bufferswere adjustedto constant ionicstrengthof either0.3,0.6,0.9,or 1.2by the addition of potassiumchloride.All bufferswere preparedat 25øC. Since the studies were conductedat elevatedtemperatures,the pH of the buffers at those temperatureswould be differentfrom the pH determinedat 25øC. The pH valuesof the bufferswere calculatedusingthe ion productof water at the various temperaturesof the study, 40ø, 50ø, and 70øC. For example, a buffer of pH 9 at 25øC would have a calculatedpH of 8.24 at 70øC. The pKwof water at 70øC is 12.82 (7, 8 ). CalibrationCurvesfor the Paraber• For each of the parabensstudied,calibrationcurveswere preparedusing chloroformas the solvent.The optical densityof eachparabendilutionwas --

*Beckman Instruments, Fullerton, Calif. ?Gebriider Haake, Berlin, Germany.

KINETICS

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497

determinedat 253 nm, the wavelengthof maximumabsorbance, usingchloroform as the blank. A Beeifslaw relationshipwas obtained for methyl, ethyl, propyl, and n-butyl parabenin chloroformat 253 nm. Method of Analysis

The followinggeneralprocedurewas developedand usedfor stud,vingthe effectsof variousfactors,i.e., pH, temperature,ionic strength,on the rate of hydrolysisof the parabens. An accuratelyweighedquantityof parabenwasaddedto a 100-mlvolumetfie flaskand dissoIvedin a smallvolume of appropriatebuffer solutionwith the aid of heat. The solution was diluted to volume with the buffer solution

whichhad beenpreheatedto the temperatureat whichthe particularanalysis was made.The flaskwas placedin a constanttemperatureeirculatorbath that had been previouslyadjustedto the desiredtemperature(-0.1øC). The solutionwas allowedto thermallyequilibratefor 10 min, then a 5-ml aliquot,representing the zerohoursample,waswithdrawnand transferredto a separatoryfunnel which containedg0 ml of chloroform.At pH 9.0, phydroxybenzoie acid (pK• 4.48) existsas a salt which remained in the aqueousphasewhen the solutionwas extractedwith chloroform.Complete extractionof the unclecomposed parabenwasaccomplished using3 x 20 ml of chloroform extractions. The combined chloroform extracts were filtered into a

100-mlvolumetricflaskthroughfilter paper that had beenpreviouslywetted with chloroform.The solutionwas diluted to volume with chloroform, and the

absorbance wasdetermined at 253nmusingchloroform astheblank.Samples wereremovedat definitetime intervalsand analyzedby thisprocedure.

Sincep-hydroxybenzoie acidis themajordegradation productof hydrolysis of theparabens (1, 9), mixtures containing knownconcentrations of methyl, ethyl,propyl,andn-butylparaben andp-hydroxybenzoic acidwereprepared. The esterconcentrationin eachmixture was determinedusingthe extraction

procedure previously described. Theper centrecovery of theparabens alone and in combination with varyingconcentrations of p-hydroxybenzoic acid is shownin TabIe I. The initial concentration of eachparabenin all subsequentstudieswas5 rag/1.

pH andTemperatureEffects

In ordertostudytheeffectofpH andtemperature ontherateof hydrolysis of methyl,ethyl,propyl,andn-butylparaben, thereactions werecarriedout at threetemperatures in buffersolution asfollows:70øC-pH2.75,3.66,4.58, 5.49,6.41,7.33,8.24,and 9.16;50øC-pH 9.16;40øC-pH 9.16.Reactions were carriedout for a maximumof 160hours,and all aliquotswere analyzedusing the extractionproceduredescribed above.

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Table I

Recoveryof Parabenfrom a Mixture of Parabenand p-HydroxybenzoicAcid Composition of Mixture

(mg/l.) Paraben

Amount ofParaben Acid

Found (mg/1.)

% Recovery

Methyl Paraben 503

0.00

4.95

4.02 3.02 2.01

1.03 2.06 2.58

4.05 3.00 2.03

98.4 100.7 99.3 100.9

1.01

5.16

0.98

97.0

0.00 1.03 2.06 2.58 5.16

4.70 3.70 2.77 1.85 0.95

100.9 99.2 98.9 99.5 102.2

5.31 4.25

0.00 1.03

101.7 99.3

3.19

2.06

2.12 1.06

2.58 5.16

5.40 4.22 3.20 2.10 1.07

0.00 1.03 2.06 2.58 5.16

5.30 4.12 3.17 2.05 1.10

100.2 97.4 100.0 96.7 103.8

Ethyl Paraben 4.66 3.73 2.80 1.86 0.93

Propyl Paraben

100.3

99.1 100.9

n-Butyl Paraben 5.29 *$.23 3 17 2.12 1.06

Effect of ParabenConcentration

Solutionsof methyl, ethyl, propyl, and n-butyl parabenwere preparedat at 70øC in 0.0005-0.20M phosphatebuffer at pH 8.24 and constantionic strengthof 0.3, and the hydrolysiswas followed at 70øC in the manner previouslydescribed. Effect of Ionic Strength

The effectof ionic strengthon the hydrolysisof the parabenswas investigated by studyingthe hydrolyricrate, at 70øC,of parabensoluti.ons in 0.1M phosphatebuffer at pH 8.24. The bufferswere adiustedto ionic strengthsof 0.3, 0.6, 0.9, and 1.2by the additionof potassiumchloride.The effectof buffer concentration on hydrolyticrate was alsostudiedby followingthe hydrolysis at 70øC in 0.005-0.20M phosphatebuffer at pH 8.24 and constantionic strengthof 0.6.

KINETICS

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499

RESULTS AND DtSCUSS•ON

All of the rate constants •vcrccalculatedfrom the first-orderrate equation. The slopesof the lineswere calculatedby regressionanalysisusingan electronic calculator.*

The effect of pH and temperatureon the hydrolysisof the parabenswas studiedat 3 temperaturesand at pH valuesrangingfrom 2.75-9.16. The calculatedvaluesof the rate constantsand half-livesat differentpH valuesand temperaturesare summarizedin TablesII and III. Typical plotsshowingthe hydrolysisof ethyl paraben at 70øC and various pH values and at various temperaturesat pH 9.16 are shownin Figs. 1 and 2. Theseplotsindicatethat the reactionwas first order with respectto ethyl paraben sincestraightlines were obtainedwhen the log of concentration was plotted againsttime, which •vas true for each parabenstudied.The reactionoverall would be pseudofirst order sincethe concentrationof water can be regardedasconstant. As shownin TablesII and III and in Figs. 1 and 2, as the pH and temperature increase,the rate of hydrolysisof the parabensincreases.One would expect n-butyl parabento have a lower rate constantthan propyl paraben,yet the data shown in Tables II and III

show an increase in the rate constants for

*Wang model 360K/362K, Wang Laboratories,Inc., Tewksbury, Mass.

Table

II

Specific Rate Constantsand Half-life Periods for the Hydrolysis of Parabensat 70øC in 0.1M PhosphateBuffer, Ionic Strength0.3, at VariouspH Values

Paraben

pH

k x 10a (hours-•)

Methyl

9.16

30.63

22.6

8.24

17.80

38.9

7.33

10.24

Ethyl

Propyl

n-Butyl

t'/• (hours)

67.6

6.41

2.710

255.0 1673.0

5.49

0.414

9.16

8.877

78.1

8.24

7.667

90.4

7.33 6.41

6.540 1.203

106.0 576.0

9.16

7.163

8.24

5.982

7.33

4.238

164.0

6.41

0.568

1218.0

9.16

9.391

8.24

6.387

109.0

7.83 6.41

4.580

15]..0 601.0

1.153

96.7 120.0

73.8

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2.75

4.59

pH 3.66

•.

0.6

pH6.41

z

r•

0.4 pHZ55 0.2

q

o

i

i0

4'0 TIME

IN

i

I

140

160

HOURS

Figure 1. Log of concentration of ethylparabenagainsttime at 70øC in 0.1M phosphate buffer, ionic strength0.3, at variouspH values

Table III

SpecificRate Constants and Half-life Periodsfor the Hydrolysisof Parabensat Various Temperatures in 0.1M Phosphate Buffer,pH 9.16, and Ionic Strength0.3

Paraben

Temperature (øC)

kx-i0 •

- -t•a

(hours-')

M;thyl

40

1.941

Ethyl Propyl n-Butyl Methyl Ethyl Propyl n-Butyl

40 40 40 50 50 50 50

0.735 0.529 0.536 5.776 1.752 1.205 1.347

Methyl Ethyl Propyl n-Butyl

70 70 70 70

30.63 8.877 7.163 9.391

(hours)

943.0 1310.0 1292.0 120.0 396.0 575.0 515.0 22.6 78.1 96.7 73.8

n-butylparaben,at the pH's and temperatures usedin this study,when comparedto the valuesobtainedfor propylparaben.At 70øCin 0.1M phosphate buffer at pH 9.16, n-butyl parabenhydrolyzedfaster than ethyl and propyl parabens.This phenomenon is difiieultto explainsincethere is little differeneebetweenthe activationenergies.ofthe parabensat pH 9.16, asshownin

KINETICS

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DEGRADATION

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501

PARABENS

0.8

400 C

0.6

0.4

'

OG - 0 '-ø"---- soøc

70ø C

0.2

o

2b

do

6b TIME

,oo IN

HOURS

Figure 2. Log of concentrationof ethyl paraben againsttime at various temperatin'esin 0.1M phosphatebuffer,pH 9.16, and ionicstrength0.3 Table

IV

Energies of Activation of the Parabensas Determined from the Slopesof the Arrhenius Plots at pH 9.16 in 0.1M PhosphateBuffer and Ionic Strength 0.3 Paraben

Energy of Activation (kcal/mole)

Methyl Ethyl Propyl n-Bury1

20.8 18.7 19.3 21.0

Table IV. It is difficultto rationalizea changein the mechanism of hydrolysis for a simpleesterof p-hydroxybenzoic acid. The effectof temperatureon the reactionrate can be expressedusingthe Arrheniusequation(10). Plotsof log k versus1/T yielded a straight-line relationshipwith a negativeslopefor eachof the parabensstudied,as shown in Fig. 3 for ethyl paraben.This indicatesthat the mechanismfor the degradationof the parabensdoesnot changewith temperatureat pH 9.16.Energies of activationfor eachparabenare shownin Table IV. Thesevaluesare in goodagreementwith literaturevaluesfor the hydrolysisof simplesesterssuch as methyl acetate and ethyl benzoateas reported by Newling and Hinshelwood(11). By extrapolating the Arrheniusplotsof eachparabento 23øC,rate constants and half-liveswere predicted,asshownin Table V. It is valid to fol-

502

JOURNAL OF THE SOCIETY OF COSMETIC

CHEMISTS

2.œ

1.2

0.4

I/Tx

IO$

Figure3. Arrheniusplot showingtemperaturedependence of the ethyl parabenhydrolysis at pH 9.16 in 0.1M phosphatebuffer and ionic strength0.3 Table

V

SpecificRate Constantsand Half-life Periodsfor the Hydrolysisof Parabensat 25øC in 0.1M PhosphateBuffer, pH 9.16 and Ionic Strength0.3, as Calcu',ated from Arrhenius Plots

Paraben

Methyl Ethyl Propyl n-Butyl

k x 10•

t 1/•

(hours-•)

(hours)

4.015 1.514 1.122 1.096

1726 4577 6176 6323

low the extrapolatedvalue for the rate constantif the experimentalactivation energyfalls within a rangeof 10-30 keal/mole ( 12, 13). Althoughdata alreadyobtainedshowedthat the reactionof the parabensat constanthydrogenion concentrationwas first order with respectto paraben, this first-orderdependencywas further verified by a study of the effect of initial paraben concentrationon the hydrolysisrate. The resultsshownin Table VI indicate that for each parabenstudiedbetweenconcentrations of 4-7 rag/l, the rate constantswere essentiallyindependent of the initial concentrationof paraben.

KINETICS

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OF PARABENS

503

Table VI

Effect of Paraben Concentrationon the Specific Rate Constantsat 70øC in 0.1M PhosphateBuffer,pH 8.24, and Ionic Strength0.3 Paraben

Initial Concentration

Rate x 105

(mg/1.)

(hours-•)

6.90

16.86

Methyl

Ethyl

Propyl

n-Butyl

5.00

17.81

3.62

17.48

7.00

7.946

5.00

7.667

4.10

8.096

5.95

6.063

5.00

5.982

4.00

6.229

6.96

6.449

5.00 3.77

6.387 6.295

METHYL

ETHYL

PROPYL

N-BUTY•

o, o.•

0.8

ø'7o

!

!

0.6

o.9

,h

Figure 4. Effect of ionic strengthon the hydrolysisof the parabensat 70øC in 0.1M phosphatebuffer, pit 8.24

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table VII

Effect of PhosphateConcentration on the SpecificRate Constants for the Hydrolysisat 70øC, pH 8.24, and Ionic Strength0.6 Paraben Methyl

Ethyl

Moles of Phosphate

k x 10:• (hours-•)

t v2 (hours)

0.02

11.46

60.3

0.04

12.84

53.9

0.08

16.42

42.2

0.10 0.20

19.05 26.87

36.4 25.8

0.02

5.175

133.0

0.08

6.920

100.0

0.10

7.862

0.20

Propy]

n-Butyl

10.45

88.1 66.3

0.005

2.638

262.0

0.02

3.470

199.0

0.04

4.747

146.0

0.10

6.145

113.0

0.20

7.947

0.005

2.486

87.2

278.0

0.02

3.836

181.0

0.04

4.759

146.0

0.08

5.634 6.418 8.008

108.0 86.5

0.10 0.20

123.0

Figure 4 showsthe effect of ionic strengthon the hydrolysisrate of the parabens.A positiveslopewas obtainedfor each paraben,which indicatesa reactionof ionsof like sign,i.e., a reactionbetweenthe hydroxylion and the negativelychargedp-hydroxybenzoate. Also, the rate of hydrolysisof each parabenwas influencedslightly by the presenceof potassiumchlorideas notedby an increasein the rate constants with an increasein ionicstrength. The effect of buffer concentrationon hydrolytic rate is summarizedin Table VII. The rate constantswere obtained from log concentrationversus' time plotsfor the parabensat eachbufferconcentration. Each plot yieldeda straight-linerelationshipindicatingthat the reactionwas first order with respectto parabenat all of the phosphate concentrations. Figure5 showsthe relationshipbetweenphosphateconcentrations and rate constants for ethyl paraben.A linear relationshipwas alsoobtainedfor methyl parabenbut the propyl and butyl estersproduceda distinctbreak in the curvewhich may havebeendue to the catalyticeffectof otherspeciesin the phosphate buffer (mainly monohydrogen phosphateion at pH 8.24). As shownin Fig. 5 and Table VII, the rate constantsand the half-lives decreasedwith increasing

phosphate concentration for all of the parabens. Thiswouldindicatethat the

KINETICS

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DEGRADATION

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PARABENS

5O5

1 1.0

10.0

9.0

8.0 7.0

6.0

5.0

.....

I 0.04

I 0.08

I 0.12

I 0.16

I 0.20

TOTAL PHOSPHATE, moles/liter Figu"e 5. Effect of phosphateconcentrationon the rate of hydrolysisof ethyl parabenat 70 øC, pH 8.24, and ionic strength 0.6

parabensunderg•o generalbasecatalysis, andthat hydroxylion is not the only specieswhichcan catalyzetheir hydrolysis. ( ReceivedOctober25, 1973) REFERENCES

(1) Aalto, T. R., Fi•Tnan,M. C., and Rigler, N. E., Uses, antibacterialand antifungal studies, properties and determination, J. Amer. Pharm. Ass., Sci. Ed., 42, 449-57 (1953).

(2) Pekkarinen,L., and Tomrolla, E., Hydrolysisof alkyl salicy]atein alkaline solutions, Acta Chem.Scand.,13, 1019-30 (1959). (3) Shou,A. S., and Frauch, P., Untersuchungen iiber die stabilitStvon p-aminobenzoes•iure-methylesterbei kiinstlicher alterung, Pharm. Acta Helv., 34, 37-52 (1959). (4) Ravel, N. N., and Parrott, E. L., Hydrolysis of methyparaben,1. Pharm. Sci., 56, 274-5 (1967). (5) Littlejohn,O. M., and Husa, W. J., The potentizingeffect of antimoldingagentsin syrups,]. Amer. Pharm. Ass., Sci. Ed., 44, 305-8 (1955).

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JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS

(6) SchimmeI, J., and Husa, W. J., The effect of various preservationson microorganismsisolated from deterioratedsy•nps,Ibid., 45, 204-8 (1956). (7) Harned, H. S., and Robinson,R. A., Temperature variation of the ionization constants of weak electrolytes, Trans. Faraday Soc., 36, 973--8 (1940). (8) Martin, A. N., Swarbick, J., and Cammarata, A., Physical Pharmactt, 2nd Ed., Lea and Febiger, Philadelphia,Pa., 1969, p. 196. (9) SokolskiW. T., Chidester,C. G., and Honeywell,G. E., The hydrolysisof methyl phydroxybenzoateby Cladosporiumresinae, Develop. Ind. Microbiol., 3, 179-197 (1962). bei der inversionyon rohrzucker (10) Arrhenius,S., tYber die Reaktionsgeschwidnidkeit durch s•iuren,Z. Physik. Chest., 4, 226-48 (1889). (11) Newling, W. B. S., and Hinshelwood,C. N., The kineticsof the acid and the alkaline hydrolysisof esters,J. Chest. Soc., 1357-61 (1936). (12) Garrett, Predictionof stability in pharmaceuticalpreparations.III. Comparisonof vitamin stabilitiesin different multivitamin preparations,J. Aster. Pharst. Ass., Sci. Ed., 45, 470-3 (1956). (13) Martin, A. N., et al., op cit., p. 397.