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1/7

Analyst, M a y ,

1980,

Vol.

105, p p

455-467

455

Quality Control

of

Prednisolone Sodium Phosphate

N. Stroud, N. E. Richardson, D. J. G. Davies and D.

A.

Norton

Centre or Drug Formulat ion Studies , School o f Pharmac y and Pharmac ol ogy , Uni v e r s i t y

of

Ba t h , Cl av e rt on

Down Bath, BA2 7 A Y

Prednisolone sodium phosphate is believed to undergo hydrolysis of the

phosphate ester group as its primary degradation pathway . Most published

assay methods do not determine the phosphate ester directly, and therefore

a high-performance liquid chromatographic method has been developed for

prednisolone sodium phosphate in the presence of its breakdown products,

which has been val idated in th e presence of excipients used in ophtha lmic

solutions. Stability data are presented tha t are comparable to those obtained

for related steroid phosphate esters. The

stability data indicate that a

simpler ultraviolet spectrophotometric assay method can be used for routine

stability testing.

Ke y wo r d s : P v e d n i s o lo n e s o d iu m p l zo s pl ia t e d e t e r m in a t i o n ; h ig h - p e r fo r m a n c e

l iq u id chvomatograp lay

;

p r e d n i s o lo n e s o d i u m p la os p ha t e s t a b i l i ty

The efficiency of corticosteroids such as prednisolone for the treatment of ocular inflammatory

conditions is now well established. Prednisolone has a low solubility in water and for

aqueous formulations the more water-soluble phosphate ester is used, which can be formulated

for both parenteral and topical administration. Several workers have reported that cortico-

steroids such as prednisolone undergo thermal degradation in aqueous solution, involving

the 17-dihydroxyacetone ~ide-cha in. l-~Transformations and eliminations have been shown

to occur in both the presence and absence of air. In the presence of air under alkaline

conditions, the predominant reaction appears to involve cleavage of the C17 side-chain to

yield the corresponding etianic acid. In the absence of air, two reactions predominate,

yielding the 17-keto steroid and the hydroxy acid. Degradation of the A ring has also been

shown to occur in a related steroid, hydrocortisone, formulated in a polyethylene glycol

base.5 However, the A ring is an inherently stable structure and the rate of degradation

was much slower than that for the C,, side-chain. The degradation of steroid phosphate

esters has not been studied as extensively, although Marcus6 has reported that the degrada-

tion of hydrocortisone phosphate in aqueous solution involved hydrolysis as the only signifi-

cant degradative pathway and was dependent on the hydrogen-ion concentration. It would

appear, therefore, that the thermal degradation of prednisolone sodium phosphate in aqueous

solution would involve the pathways illustrated in Fig.

1

and that hydrolysis of the phosphate

group on the C17 side-chain would be predominant.

Both

prednisolone sodium phosphate and the parent prednisolone possess the 3-keto group and

related conjugated system and have similar absorption spectra in the ultraviolet region.

Kaplan and Levine7 have developed a column chromatographic method for separating the

two compounds using ion-pair formation between the ester and trihexylammonium chloride.

However, the method is tedious and lengthy for routine analysis. The C,, side-chain of

prednisolone has been determined by complexation with tetrazolium blue followed by

spectrophotometric determination of the coloured complex.* This method, however, is

specific for the

C,,

side-chain and prednisolone sodium phosphate would require preliminary

hydrolysis to the parent alcohol, which is difficult to achieve quantitatively. Other methods

reported are the determination of the inorganic phosphate produced as the ester hydrolysese

and gas - liquid chromatography. Upton

et aL9

have reported

a

high-performance liquid

chromatographic (HPLC) method for steroid phosphates using a reversed-phase column.

However, preliminary work in our laboratories indicated that prednisolone sodium phosphate

was eluted immediately after a non-retained compound (potassium dichromate) on

a

Spherisorb

S5

ODS reversed-phase column. It is essential that any assay method distin-

guishes between the parent compound and its degradation products and

we

have therefore

developed an HPLC assay for prednisolone sodium phosphate using an anion-exchange

column, as the phosphate ester is present in an anionic form in aqueous solution.

Most published assay methods do not determine the phosphate ester directly.

Publishedon01January

1980.DownloadedbyUNIVERSIT

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456 STROUD et

d

QUALITY CONTROL Amzlyst,

Vol.

105

Experimental

Apparatus

Chromatograms were determined routinely using

a

Pye LC20 system, which has a fixed-

wavelength detector set

at

254nm. Injections were made on-column with a Pye Unicam

fixed-volume 10-pl loop valve. All measurements were made at ambient temperature in

replicate.

Spectrophotometric determinations were made using

a

Pye Unicam SP1800 spectrophoto-

meter.

pH determinations were performed using either a Pye Unicam 291 pH meter

or a

Radio-

meter Type 27

pH

meter fitted with

a

PHA 630P scale expander. Both pH meters were

used in conjunction with Pye-Ingold combined glass - silver electrodes. All pH measure-

ments were carried out on solutions equilibrated

to 25

0.1 C; meters were standardised

with two appropriate standard buffers.

CH,OPO, Na2

Prednisolonesodium

& phosphate

0

CH20H

Predn solone

I

Prednisolone

COOH

Fig. 1 . Thermal degradation pathways of prednisolone

sodium phosphate.

Mat eri a1s

Prednisolone sodium phosphate was a gift from Smith and Nephew Ltd. and was used as

received. All buffer salts were of analytical-reagent grade and other reagents were of

at

least laboratory-reagent grade. Potassium hydrogen phthalate was of an NPL certificated

grade supplied by BDH Chemicals Ltd. Solvent:; were of analytical-reagent grade. Water

was freshly distilled from an all-glass still.

Poly(viny1 alcohol) (Gohsenol N300, Nippon

Goshei) was supplied by British Traders and Shippers Ltd. Trihexylammonium chloride

was prepared from trihexylamine (Eastman Kodak Co.) according to the method

of

Kaplan

and L e ~ i n e . ~



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M a y ,

1980 O F PREDNISOLONE SODIUM PHOSPHATE 457

The stationary phase was Partisil 10 SAX (Whatman), an anion-exchange material , packed

into either

250

x

4.6

mm or 100 x

4.6

mm stainless-steel columns. The mobile phase

consisted of a 1

9 V / V

mixture of methanol and one-fifth strength McIlvaines citrate -

phosphate buffer (pH 5.2), and was de-gassed before use. The actual pH of the mobile

phase

was 5.6 .

Chromatography

of

Prednisolone Sodium Phosphate in Aqueous Solution

Aqueous solutions are prepared to contain 0.004-0.024~0

m/

V prednisolone sodium

phosphate and 0.12

m/V

potassium hydrogen phthalate

as

internal standard and

10

pi

are injected on to the column with a mobile phase flow-rate of 1.5 ml min-l. The chromato-

gram is recorded at a detector wavelength of 254 nm. The concentration of prednisolone

sodium phosphate is then determined by comparing the peak-height ratio of drug to internal

standard with that obtained with a standard solution containing

0.02 m/V

of prednisolone

sodium phosphate and 0.12

m/V

of potassium hydrogen phthalate.

Chromatography

of

Prednisolone Sodium Phosphate in the Presence of a Viscoliser

Place 2 ml of prednisolone sodium phosphate solution (concentration range

0.

1-0.5y0

m/V)

n

a

10-ml glass centrifuge tube containing 3

ml

of double-strength Sprrensens phosphate

buffer (pH 5 . 0 ) ,mix and add 5 ml of a 5

V/V

solution of trihexylammonium chloride in

dichloromethane. Stopper the tube, shake it vigorously for

30s,

then centrifuge it

at

4000 rev min-l for

15

min. Remove the aqueous phase, transfer 3 ml of the organic phase

into a fresh centrifuge tube containing ml of

0.1 M

sodium hydroxide solution, shake for

30

s and then centrifuge at 4000 rev min-l for

5

min. Pipette 3 ml of the aqueous phase

into a 25-ml calibrated flask containing 3 ml of

0.1

M hydrochloric acid and 3 ml of 1

m/V

potassium hydrogen phthalate solution and dilute to volume with water. The solution

in the flask is then chromatographed as described above.

Thermal Degradation of Prednisolone Sodium Phosphate

priate buffer were sealed into glass ampoules and heated in an oil-bath.

removed after known periods and assayed for residual drug.

Aliquots of 10 ml of a 0.5

m/V

solution of prednisolone sodium phosphate in an appro-

The ampoules were

Results and Discussion

Prednisolone sodium phosphate forms an anion in aqueous solution and should therefore

be retained by an anion-exchange column to an extent dependent on the pH of the mobile

phase, which would be reflected in longer retention times with increase in pH. Fig. 2 shows

the capacity factors for 0.02yo

m/V of

drug using benzyl alcohol as the non-retained com-

1 1

3

4

5

6

Influence of pH of mobile

phase on capacity factors of: A, pred-

nisolone sodium phosphate; and B,

potassium hydrogen phthalate.

PH

Fig.

2.



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458 STROUD e t al.

: QUALITY

CONTROL Analyst,

Vol. 105

pound, over the mobile phase pH range 2.95-6.10.

It

is apparent that the capacity factor

decreases with increase in pH, and this is probably due to competition by the buffer com-

ponents dominating the extent of the interactions between column and solute. Knox and

VasvarilO have shown tha t the capacity factor

of

phthalic acid on an anion-exchange column

can be selected by manipulation of the mobile phase pH. Fig. 2 also shows the capacity

factors for the more water-soluble potassium hydrogen phthalate over the mobile phase pH

range 3.50-6.10, and again it is observed that an increase in pH decreases the retention time.

However, it is apparent tha t over this pH range good resolution is obtained between predniso-

lone sodium phosphate and potassium hydrogen phthalate, and the latter was therefore

selected as the internal standard using a mobile phase pH of 5.2. The addition of 10

V/V of methanol as organic modifier was found to reduce the analysis time and improve

peak symmetry, although the final pH of the mobile phase increased slightly to 5.6.

Initially, chromatograms were obtained using; the 250-mm column with a mobile phase

flow-rate of

1.5

ml min-l. Subsequently, a 100-mm column with a mobile phase flow-rate

of 1.2 ml min-l was shown to give improved pleak symmetry and

a

reduction in analysis

time.

A typical chromatogram using this system is shown in Fig. 3 ( a ) .

10 5 0

L

10

5 0

Time/min

C )

i

1

C

10

5

Fig. 3. Chromatograms

of

prednisolone

sodium phosphate and its degradation pro-

ducts.

a)

Prednisolone :sodium phospha te,

0.02'70

m / V ;

( b )

prednisolone sodium phos-

phate, 0.02 m/V prednisolone, 0.018 6

m V ;

nd (c) prednisolone sodium phosphate,

0.02

m/V in pH 8 buffer heated a t 110 C

for 24 h. Conditions: ambient temperature;

flow-rate 1.2 ml min-I; stationary phase 100

x 4.6 mm of Part isil 10 SAX (10

p m ;

mobile

phase

10 V / V

methanol in McIlvaines

citrate

-

phosphate pH

5.2

buffer and ionic

strength

0.1 M

(final pH of mobile phase

was

5.6); detector, ultraviolet a t

254

nm; sensi-

tivity 0.16 a.u.f.s.

1 ,

Prednisolone sodium

phosphate ; 2, potassium hydrogen phthalate

(internal standard)

;

3, prednisolone and 4

degradation products.

The principal degradation product of prednisolone sodium phosphate in aqueous solution

is

probably the parent steroid, prednisolone, which may break down further to give the

corresponding etianic acid, hydroxy acid or 17-keto steroid. Prednisolone is un-ionised in



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M a y , 1980 O F PREDNISOLONE SODIUM PHOSPHATE 459

aqueous solution and should be non-retained by an anion-exchange column. Fig.

3 (b )

shows a chromatogram of

0.02y0 m/V

prednisolone sodium phosphate in the presence

of

0.0186~0m/V of prednisolone, and it is apparent tha t the peak due to the latter is non-

retained. A 0.018670

m/V

solution of prednisolone buffered at pH 7.4 was heated at 100

C

for 48 h and the chromatogram again showed only one non-retained peak, indicating that

any further degradation products would not interfere in the assay for prednisolone sodium

phosphate. Finally,

a

0.5

m/V

solution of prednisolone sodium phosphate buffered at

pH 8.0 was heated at 100 C for 20 h and, after appropriate dilution, the chromatogram

[Fig.

3(c)3

shows that adequate resolution is obtained between drug, internal standard and

degradation products.

The linearity of the response was checked by injecting solutions of prednisolone sodium

phosphate over the concentration range 0.004-0.024~0

m/V

in the presence of 0.12

m/V

of potassium hydrogen phthalate and calculating the peak-height ratios. Replicate cali-

bration graphs constructed on two consecutive days were linear, with slopes

of

63.08 [standard

deviation (s.d.) 0.181 and 63.8 (s.d. 0.22) and intercepts of -0.014 (s.d. 0.01) and -0.039

(s.d. 0.18), respectively. Comparison by

a

Student's t distribution showed them to be not

significantly different tt;:: 0.42; tinter epta,c. 1.20; ttabulated 2.45;

n

10,

p

0.05).

Simple aqueous formulations of prednisolone sodium phosphate containing only the drug,

buffer and

a

preservative such as benzalkonium chloride can be chromatographed directly

after appropriate dilution and the addition of an internal standard. The peak-height ratios

of drug to internal standard can then be compared between the sample and

a

standard

solution

of

prednisolone sodium phosphate. In the presence of formulatory excipients such

as

polymeric viscolisers, pre-extraction of the drug is necessary.

Preliminary extraction

experiments were monitored by determining the absorbance of prednisolone sodium phosphate

in the aqueous phase

at

248 nm. An aqueous phase consisting of double-strength McIlvaines

citrate phosphate buffer (pH

5.0)

and an organic phase consisting of a

5 V / V

solution of

trihexylammonium chloride in methylene chloride was found to transfer 98.0 (s.d.

O.lyo,

n

3)

of the drug to the organic phase. The extent of subsequent re-extraction into 0.1 M

sodium hydroxide solution was

1 0 l . l ~ o

s.d. 0.86y0,

n

3) .

Formulations of 0.1

m/V

and

0.5 m/V

prednisolone sodium phosphate containing 4.25

m/V

of Gohsenol

N300

as

viscoliser, O.Olyo m/V of benzalkonium chloride and 0.01

yo

m/V of EDTA, disodium sal t,

buffered

at

pH

8

were extracted and assayed by HPLC

as

described. Recoveries were

100.8 (s.d. 1.7 ,

n

3) and 98.6 (s.d. 0.62y0, 12 3) compared to injection of the

standard aqueous prednisolone sodium phosphate solutions, which was considered to be

satisfactory.

Applicability of the Assay to Stability Studies

Stability studies were carried out as described above and residual prednisolone sodium

phosphate was determined using the simple non-extraction HPLC assay procedure.

Degradation was generally followed to below

50 .

Preliminary experiments showed that

in Smensens phosphate buffer (pH 6.1 and 8.2) at 90 C the data for the degradation of

prednisnlone sodium phosphate could be fitted to first-order rate plots, leading to values for

the rate constants of 4.7

x

and 6.52

x

10-3h-1, respectively, which are close to the

values

of

about x h-I determined by Marcus6 for the hydrolysis

of

hydrocortisone phosphate at pH 6 and 7.5 and 91

C.

However, it was observed that

prednisolone

sodium

phosphate underwent an initial rapid degradation of about

5y0,

and

this was overcome by the addition of O.Olyom/V of EDTA disodium salt. The influence of

temperature on the degradation of prednisolone sodium phosphate was determined over the

range 80-110

C

in Smensens phosphate buffer (pH 8) containing 0.01

m/V

of EDTA,

disodium salt . When the da ta (Table I) were plotted according

t o

the Arrhenius relationship,

a value for the activation energy

of

126.2 kJ mol-l was obtained, which compares with a

value at pH 7.5 of

113

kJ

mol-l for methylprednisolone phosphate reported b y Flynn and

Lambll and 71

kJ mol-I

determined by Marcus6

at

the same pH for hydrocortisone phosphate.

The low value

of

71 kJ mo1-I for hydrocortisone phosphate reported by Marcus has been

attributed to the reaction system being of a significant micellar character a t the

drug

con-

centration studied and that the micellar fraction changes with temperature.ll

and 8 x



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460 STROUD et

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:

QUALITY

CONTROL Analyst, Vol. 105

Applicability

of

the Assay to other Dosage Forms and Related Drugs

Most liquid formulations of prednisolone sodium phosphate are simple aqueous solutions,

which may contain a buffer (pH 5-8), a preservative such as benzalkonium chloride, EDTA,

disodium salt, or

a

viscoliser, and the drug can be determined by the procedures described.

The method should also be applicable to solid dosage forms utilising the extraction procedure

described.

Related steroid phosphate esters can also be determined directly by the HPLC procedure.

Fig. 4 shows the chromatogram of 0.02

m/V

dexamethasone sodium phosphate, which is

similar to th at obtained for prednisolone scldium phosphate. The retention time for

prednisolone sodium phosphate on this column it; the same as that for dexamethasone sodium

phosphate. Burgess,12however, has reported good resolution of related steroidal esters using

an elevated temperature and reversed-phase ZIPLC. Whether the method reported here

can also be used for this purpose must await further work, which is proceeding in our

laboratories.

FIRST-ORDER

ATE CONSTANTS FOR THE

DEGRADATION OF PREDNISOLONE

SODIUM PHOSPHATE AT pH 8.0

AT

DIFFERENT

TEMPERATURES

IFirst-order ra te

Temperaturel'C constaat/h-

80 1 13 x

90

4.145 x

100 1 271 x

110 3 323 x

Routine Quality Control of Aqueous Prednisolone Sodium Phosphate Formulations

During the stabili ty studies it was observed thad if the degraded drug solution was extracted

prior to HPLC assay, the chromatographic peak due to the degradation products was negligible

down to about 60 of residual drug.

It

was thought possible, therefore, that the residual

drug could be determined by UV spectroscopy of the extraction solution.

A solution con-

taining 0.5:/,

m/V

of drug, O.Olyo

m/V

of benzalkonium chloride and

O.Olyo

m/V of EDTA,

disodium salt, in Sorensens phosphate buffer (pl3 7.4) was prepared and the degradation of

the prednisolone sodium phosphate a t 100

C

was determined using the following assay

procedures

:

Solutions were appropriately diluted with water, internal standard was added and

the mixture was subsequently assayed by HPLC.

Solutions were extracted and assayed by

IIPLC

as described above.

Solutions were extracted and 2 ml of the aqueous 0.1 M sodium hydroxide phase were

added to

a

100-ml calibrated flask containing

2

ml of 0.1

M

hydrochloric acid, diluted

to volume with water and the

UV

absorbance of a

1

cm layer of this solution was

determined at 248 nm against an appropriate blank.

The percentage residual drug concentrations were calculated relative to the values of the

drug to internal standard peak-height ratios or absorbance at 248nm determined at zero

time. Fig. 5 shows graphs of the percentage residual concentration on a logarithmic scale

against time for the three assay procedures. Both the direct HPLC and extraction

-

HPLC

methods gave straight lines, leading to rate constant values of 1.87 x and 1.88 x

10-2

h-l, respectively. With the extraction

-

1JV method the percentage residual con-

centration

of

drug is in agreement with that determined by the

HPLC

techniques down to

about 60 of residual drug. I t is apparent, t'herefore, th at until 20-30 of t he drug is

degraded, insufficient degradation products are transferred in the extraction procedure to

interfere significantly in the

UV

determination, and this method can be used for routine

quality control purposes.

1

2

3.



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1

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al

.

.

OF

PREDNISOLONE SODIUM PHOSPHATE

461

aJ

t

6

t

10 5 0

Time/min

Fig.

4.

Chromatogram

of

0.02 m/V

dexameth-

asone sodium phosphate.

Conditions as for Fig.

3;

detector, ultraviolet a t

254 nm, sensitivity 0.16

a.u.f.s. 1 , Dexameth-

asone sodium phosphate ;

and 2, potassium hydro-

gen phthalate (internal

standard).

20 40

Time/m in

Fig. 5. Comparison

of

assay techniques

to determine the degradation of 0 . 5

prednisolone sodium phosphate a t pH 7.4

and 100

C .

0 Direct HPLC; 0

extraction HPLC; and

4

extraction

-

ultraviolet.

Conclusions

It

has been shown that an anion-exchange column can be used for the

HPLC

assay

of

prednisolone sodium phosphate in the presence of its degradation products and th at the

assay is suitable for stability studies. Using

a

simple extraction procedure, prednisolone

sodium phosphate can be separated from interfering formulatory excipients such

as

viscolisers.

The extraction -

UV

assay procedure described is particularly useful for the routine analysis

of prednisolone sodium phosphate in quali ty control laboratories.

References

1 .

2.

3.

4.

5.

6.

7.

I).

9.

10.

1 1 .

12.

Mason, H. L.,

J Biol.

Chem. , 1938, 124 475.

Hertzig,

P.

T., and Ehrenstein,

M., J

Org. Chem. , 1951, 16 1050.

Wendler,

N .

L., and Graber,

R.

P., Chem.

Ind. London),

1956, 549.

Guttmann, D. E., and Meister, P. D., J . A m . P h a rm . Assoc., Sci.Ed., 1958, 47 773

Allen, A.

E.,

and Das Gupta, V.,

J P h a r m .

Sci . ,

1974,

63,

107.

Marcus, A. D.,

J

Am.

Pharm. As s oc . ,

Sci.

E d . , 1960, 49, 383.

Kaplan,

G.

B., and Levine,

J . ,

J

Assoc .

Ofl.

nal. Chem. , 1973, 57 735.

Mader, W. J., and Buck, R. R., Anal.

C h e m . ,

1952, 24 666.

Upton, L.

M. ,

Townley,

E.

R. , and Sancilio, F.

P., J .

P h a r m .

Sci., 1978, 67 913.

Knox, J .

H.,

and Vasvari, G.,

J .

Chromatogr. ScZ.

1974,

12,

449.

Flynn, G. L., and Lamb, D. J., J . Phavm. Sci.,

1970, 59, 1433.

Burgess, C.,

J

Chromatogr. , 1978, 149 233.

Received September loth, 1979

Accepted December 12th, 1979



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