Analyst, 1980,105, 455-461
Transcript of Analyst, 1980,105, 455-461
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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.
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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
al.
:
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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M a y , 1,980
1
J
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.,
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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.,
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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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