Solid forms of pharmaceuticals: Polymorphs, salts and cocrystals

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E L S E V I E R

Journal of Pharm aceutical and Biomedical Analysis

13 (1995) 1321 1329

J O U R N A L O F

PHARMACEUTICAL

AN D BIOMEDICAL

A N A LY S I S

A n a l y s i s o f p h a r m a c e u t i c a l c r e a m s : a u s e f u l a p p r o a c h

b a s e d o n s o l i d - p h a s e e x t r a c t i o n ( S P E ) a n d U V

s p e c t r o p h o t o m e t r y

D . B o n a z z i , V . A n d r i s a n o , R , G a t t i , V . C a v r i n i *

Dip art im ento di Scie nze Farmaceut iche. Unicers itgl di Bologna, via Belm e/oro 6, 401 26 Bologna, l tu lv

Received for review 11 Nove mb er 1994: revised man uscript receixed 20 Februa ry 1905

Abstract

Solid-phase extract ion (SPE) using C-18, diol and ion-exchange sorbents fol lowed by UV spectrophoto-

metric (conv ent ional and de rivat ive mo de) assay was appl ied to the analysis of basic, acidic and neutral

drugs c omm ercial ly avai lable in creams.

A rep resentat ive set of drugs (p rom ethazine, chlorhexidine, benzyd amine , ketop rofen, ibuprofe n, fenti -

azac, pi roxicam, f luorouraci l , crotamiton and hydrocort isone acetate) was selected, and for each drug the

appropriate SPE condi t ions (adsorpt ion, washing and elut ion) were invest igated to obtain a pract ical and

reliable sample clean-up. I t was shown that the deve loped SPE proc edures were capab le of removing

interfering cream c om pon ents (excipients including preservatives) al lowing accu rate spectro photo me tric

analyses to be perform ed. In some appl icat ions, derivat ive spec troph otom etry was advantag eous ove r the

convent ional absorpt ion mode with respect to higher select ivi ty and versat i l i ty .

K e y w o r ~ b : Pharmaceut ical creams; Sample clean-up; Sol id-phase extract ion: Drug analysis : Derivat ive

s p ec t ro p h o t o me t ry

1 . I n t r o d u c t i o n

A n u m b e r o f d r u g s t h a t p o s se s s a v a r ie t y o f

p h a r m a c o l o g i c a l a c t i v i t i e s ( e . g . a n t i - i n f l a m m a -

t o r y , a n t i m y c o t i c ) a r e c u r r e n t l y u s e d a s c r e a m s

f o r t o p i c a l t r e a t m e n t [ 1 , 2 ] . T h e a n a l y s i s o f

t h e s e c o m p l e x f o r m u l a t i o n s c a n s o m e t i m e s b e

p e r f o r m e d b y s i m p l e , n o n - s p e c if i c t i t r im e t r i c

m e t h o d s i n n o n - a q u e o u s s o l v e n t s [ 1 - 3 ] o r i n

h e t e ro g en eo u s s y s t ems [2 ,4 ,5 ], b u t s p ec i f i c d e -

t e r m i n a t i o n s ( s p e c t r o p h o t o m e t r i c a n d c h r o -

m a t o g r a p h i c m e t h o d s ) a r e u s u a l l y r e q u i r e d .

S p e c t r o p h o t o m e t r i c a n a l y s e s a r e g e n e r a l l y

b a s e d o n c h r o m o g e n i c r e a c t i o n s i n o r d e r t o

p e r f o r m a b s o r b a n c e m e a s u r e m e n t s i n t h e v i s i -

b l e r e g i o n , s o a v o i d i n g i n t e r f e r e n c e f r o m t h e

e x c i p i e n t s . D i r e c t U V a s s a y s a r e n o t a l w a y s

* Corresponding author.

0731-7085/95/$09.50 © 1995 Elsevier Scie nce B.V. All rights

S S D I I)731-7085(95)01536-1

s u i t ab l e w i t h o u t p r i o r s amp l e c l ean -u p [1 , 2 , 5 ] .

G a s c h r o m a t o g r a p h y [ 1 , 2 , 5 , 6 ] , h i g h - p e r f o r -

m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C )

[ 1 , 2 , 4 , 5 , 7 ] a n d t h i n - l a y e r c h r o m a t o g r a p h y

( H P T L C ) [ 8 - 1 0 ] a r e w i d e l y a p p l i e d t o t h e

a n a l y s i s o f p h a r m a c e u t i c a l c r e a m s ; t h e m e t h -

o d s u s u a l l y i n v o l v e t i m e - c o n s u m i n g a n d l a b o r i -

o u s s a m p l e c l e a n - u p s t e p s , s u c h a s

l i q u id l i q u id e x t r a c t i o n a n d e x c i p i e n t p r e c i p i -

t a t io n b y c o o l in g o f a n a p p r o p r i a t e s a m p l e

s o l u t i o n . W h e n t h i s p r e l i m i n a r y c l e a n - u p i s

o m i t t e d a n d t h e s a m p l e s o l u t i o n s a r e d i r e c t l y

s u b j e c t e d t o H P L C a n a l y s i s , i n t e r m e d i a t e a n a -

l y t i c a l c o l u m n w a s h i n g s w i t h a n o r g a n i c s o l -

v e n t a r e r e c o m m e n d e d [ 7 , 1 1 ] t o r e m o v e t h e

l ip h o p h i li c c o m p o n e n t s o f t h e c r e a m b a se .

R e c e n t l y , i t h a s b e e n o b s e r v e d t h a t s o l i d -

p h a s e e x t r a c t i o n ( S P E ) c a n o f f e r t h e o p p o r t u -

n i t y o f s im p l e a n d p r a c t i c a l p r o c e d u r e s f o r

reserved



1322

D . B o n a z z i e t a l . / J . P h a r m . B i o m e d . A n a l . 1 3 1 9 9 5 ) 1 3 2 1 - 1 3 2 9

sample clean-up in the analysis of imidazole

antimycotic drugs in creams [12,13]. The role

of SPE technique in drug analysis is well

known [14-18], but systematic investigations

on its application to the analysis of creams

have not been reported; some specific applica-

tions have been developed only in the HPLC

analysis of steroid topical dosage forms using

conventional SPE [19-21] and on-line column

switching procedures [22-24].

Thus, the aim of this study was to provide

convenient and reliable SPE procedures as

useful guidelines for sample clean-up in the

analysis of pharmaceutical creams. A repre-

sentative set of basic, acidic, and neutral

drugs, belonging to different structural groups

(promethazine, chlorhexidine, benzydamine,

ketoprofen, ibuprofen, fentiazac, piroxicam,

fluorouracil, crotamiton and hydrocortisone

acetate) and commercially available as creams

were selected. For each drug, the appropriate

SPE conditions (sorbent, adsorption, washing,

elution) were investigated using UV spec-

troscopy (conventional and derivative mode) to

monitor the development of the SPE method.

In this application, UV spectroscopy proved to

be a suitable technique to evaluate the perfor-

mance of the different SPE steps; moreover,

with the derivative mode, the residual non-spe-

cific matrix absorption was suppressed to allow

accurate analyses of commercial creams to be

performed.

2. Experimental

2. I. Materials

Promethazine hydrochloride, ketoprofen,

piroxicam, chlorhexidine, ibuprofen were ob-

tained from Sigma Chimica (Italy), fluorouracil

and hydrocortisone acetate were from Fluka

(Switzerland) and benzydamine hydrochloride

was from Aldrich (Italy). Crotamiton was

kindly supplied by Ciba-Geigy (Italy) and fen-

tiazac was from LPB Istituto Farmaceutico

(Italy).

All other chemicals were obtained from Far-

mitalia C. Erba (Italy). Phosphate buffer solu-

tions (pH 4.5; 7 .4; and 8.0) were prepared

according to s tandard methods. Solid-phase ex-

tractions were performed on Bond Elut car-

tridges (500mg; 2.8ml) from Analytichem

(Varian, USA), using a Baker-10 SPE vacuum

manifold connected to a water aspirator.

2.2. Apparatus

All the spectrophotometric analyses were

performed on a Jasco Uvidec-610 double-beam

spectrophotometer using 1-cm quartz cells with

a slit width of 2 nm. Suitable settings were:

scan speed 100 nmmi n-~, chart speed

20 nm cm -1, absorbance scale 0-2.0. For the

derivative mode, the A2 value and the ab-

sorbance scale were selected in accordance with

the nature and concentration of the analyte.

2.3. Calibration graphs

Standard solutions of appropriate concentra-

tions in suitable solvents (Table 1) were pre-

pared for each drug. The absorbance at 2max

(zero-order spectrum) as well as the selected

amplitudes ~D and 2D (first- and second-order

derivative spectrum, respectively) were plotted

against the corresponding concentration to ob-

tain the calibration graphs.

2.4. Solid phase extraction SPE)

Before use, the SPE columns were properly

conditioned as follows: C-18 sorbent by rinsing

with 6 ml of methanol; strong anion exchange

(SAX) sorbent by rinsing with 6ml of

methanol and then with 3 ml o f methanol

buffer solution (pH 8) (1:1, v/v); strong cation

exchange (SCX) propylsulphonate (PRS)

(propylsulphonic acid) and SCX (benzenesul-

phonic acid) sorbents by rinsing with 6 ml of

methanol and then with 3 ml o f buffer solution

(pH 4.5); diol sorbent by rinsing with 6ml

of dichloromethane and then with l ml of n-

hexane.

A 3.0-ml aliquot of the sample solution was

applied to the appropriate SPE column as de-

scribed below for each drug.

2.5. Sample preparation

Fluorouracil cream (Efudix)

A sample was dissolved in water-methanol

(80:20, v/v) to give a final concentration of

about 20 ~tg ml 1 of the drug. A 3.0-ml aliquot

was applied to a conditioned C-18 SPE column

and the column was then washed with 3.0 ml of

water. The filtrate and the washings were com-

bined and subjected to UV analysis by com-

parison with a s tandard solution (20 ~tg ml -l ).



D. B ona zz i e t a l . / J . Phar m. B iom ed . Ana l . 13 19 9 5 ) 13 21 13 29

Table 1

Data for the calibration graphs (n = 6)

1323

Drug Method Slope Intercept Correlation Concentration

coefficient range (lag ml ~)

F l u o r o u r a c i l A 2 6 6

0.0503 -0.0137 0.9997 5- 30

2D292~ 26v 2 0.0102 0.0175 0.9980

Promethazine A2492 0.0878 - 0.0017 0.9994 8.05 20.25

2D~,~ - 2st.2 0.0211 0.0059 0.9988

Piroxicam A ~2~ 6 0.0649 0.0012 0.9953 10.25 20.5

2Dw~ 2 ~26 0.0149 0.0117 0.9990

Ketoprofen A26~, 0.0658 0.110 0.9995 10.64- 35.47

2D2~2 2ssg 0.0196 0.0046 (I.9999

Crotamiton i D2~4 4 0.0045 - (I.0027 0.9998 I 1.60 46.70

20261 6 0.0042 (I.0019 0.9999

2D261 6 2472 0.0082 -- (I.0072 0.9996

Benzydamine A~o56 0.0150 - (I.0071 0.9976 14.4 43.4

Hydrocortisone A248 0.0473 --0.0194 0.9956 12 30

a c e t a t e 2D271 6 248.8 0.0090 0.0042 0.9971

Chlorhexidine A256 s 0.0518 -0.0133 0.9995 7.3 36.5

I D272

2484 0.0089 0.0005 0.9997

2D2592

24(} 0.0062 -0.0003 (I.9998

2D240 229.6 0.0062 0.0021 (}.9996

Ibuprofen A2716 0.0151 I).0059 0.9998 6 42

A26~ ~, 0.0197 I).0071 (I.9997

Fentiazac A3oos 0.0186 -0.0094 0.9998 5.1 25.5

I D~27 6 0.0256 0.0038 {).9999

i D2544 0.0344 0.0030 0.9999

P r o m e t h a z i n e c r e a n l (Prometazina)

A sample, equiva lent to about 1.34 mg of the

drug, was dissolved in 100ml of methanol-

0.01 M buffer solution (pH 4.5) (1:4, v/v) and a

3.0-ml aliquot of the sample solution was ap-

plied to a C-18 SPE column. The column was

washed with two l-ml portions of the same

solvent mixture and the retained drug was

eluted with 3 ml of met hanol. The eluate was

subjected to UV analysis by comparison with a

sta nda rd solu tion (15.0 lag ml ~) in metha nol .

P i r o x i c a m c r e a m (Feldene)

A sample, equivalent to a bout 0.143 mg of

the drug, was dissolved in 10ml of

dic hlor ome tha ne-n -hex ane (2:3 , v/v). A 3.0-ml

aliquot was applied to a diol SPE column,

which was washed with two 1-ml porti ons of

the same solvent mixture and the drug was

eluted with 3 .0ml of d ichloromethane. UV

analysis was performed using a stan dard

solu tion of 14.0 lag ml ~ in dich lor ome than e.

K e t o p r o J e n c r e a n l (Ketofen)

A sample, equivalent to about 0.3 mg of the

drug, was dissolved in 20 ml of me than ol

0.3 M buffer s olu tio n (pH 7.4) (1:9, v/v). A

3.0-ml volume of the solution was applied to a

SAX column. The column was washed with

two 1-ml portions of the same solvent system

and the drug was eluted with three 1-ml por-

tions of methanol-0.01 M buffer solution (pH

4.5) (9:1, v/v). The standard solution contained

14,0 lag ml ~ of t he dru g.

C r o t a m i t o n c r e a m (Eurax)

A sample, equ ivalent to abo ut 4.0 mg of the

drug, was dissolved in 100ml of methanol

buffer solution (pH 4.5) (3:7, v/v) and a 3.0-ml

volume of this solution was applied to a C-18

SPE column. The retained drug was then

eluted with 3 ml of meth anol -bu ffe r solut ion

(pH 4.5) (6:4, v/v) and the eluate was analysed

by comparison with a crotamiton s tandard so-

lu ti on (40 lag ml ~).



1324

D. Bonazzi et al./ J. Pharm . Biomed. Anal. 13 1995) 1321-1329

I b u p r o f e n c r e a m

(Brufen)

A sample, equivalent to about 3.0 mg of the

drug, was dissolved in 100ml of methanol-

phosphate buffer solution (pH 8.0 ) (1:1, v/v)

and a 3.0-ml aliquot was applied to a SAX

SPE column. The column was washed with

2 ml o f methanol -buffe r solution (pH 8.0) (1:1,

v/v) and then the drug was eluted with 3 ml of

methanol-phosphate buffer solution (pH 4.5)

(9:1, v/v). The eluate was analysed by compari-

son with a standard solution of ibuprofen in

the same solvent system (27 tag ml 1).

tional and derivative mode) before and after

the SPE step. The absorbances (zero-order

spectrum) and the amplitudes ~D and

2 D

(first-

and second-order derivative) at the selected

wavelengths were compared with the corre-

sponding values obtained from an appropriate

standard solution of the drug to calculate the

drug content in each sample.

3 . R e s u l t s a n d d i s c u s s i o n

C h l o r h e x i d i n e c r e a m

(Trust)

A sample equivalent to about 3.0 mg of the

drug was dissolved in 100ml of methanol-

phosphate buffer solution (pH 4.5) (1:9, v/v)

and a 3.0-ml aliquot was applied to a PRS SPE

column. The column was washed with 2 ml of

the buffer solution (pH 4.5) and then the drug

was eluted with two 1.5-ml portions of buffer

solution (pH 7.4). The combined eluates were

subjected to spectrophotometric analysis using

a standard solution (26 lag ml ~) in the same

solvent.

B e n z y d a m i n e h y d r o c h l o r i d e c r e a m

(Tantum)

The sample solution was prepared as de-

scribed for chlorhexidine but a SCX cartridge

was used for the SPE.

In creams, the active components (basic,

acidic and neutral drugs of different polarity)

are dispersed in complex emulsions containing

hydrophobic and hydrophilic compounds,

emulsifying agents and preservatives. Depend-

ing on the drug and formulation nature, differ-

ent SPE processes have been developed in this

study. The performance of the SPE methods

was first evaluated on standard solutions of

each drug and then the suitability of the

methods was verified on solutions of commer-

cial samples. To this end, UV spectra (conven-

tional and derivative mode) obtained from the

sample solution before and after the SPE step

were compared with the spectrum of a stan-

dard solution of the drug in the same solvent

system.

H y d r o c o r t i s o n e a c e t a t e c r e a m

(Lenirit)

A sample equivalent to 0.5 mg of steroid was

dissolved in 20ml of n-hexane-dichlorome-

thane (7:3, v/v) and 3.0ml of the resulting

solution was applied to a diol SPE column.

The column was washed with 2ml of di-

chloromethane and the drug was eluted with

two 1.5-ml portions of methanol. UV analyses

were performed by comparison with a drug

standard solution (25 lag m1-1) in methanol .

F e n t i a z a c c r e a m

(Norvedan)

A sample equivalent to about 5.0 mg of the

drug was dissolved in 100 ml o f n-h exane-

dichloromethane (7:3, v/v) and a 3.0-ml aliquot

was applied to a diol SPE column. The column

was washed with two 1-ml portions o f n-hex-

ane-dichlorometh ane (7:3, v/v) and the re-

tained drug was eluted with two 1.5-ml

portions of methanol.

2 . 6 . A s s a y p r o c e d u r e

The sample solution of each drug was sub-

jected to spectrophotometric analysis (conven-

3 .1 . S o l i d - p h a s e e x t r a c t i o n S P E )

The SPE strategy generally comprises the

isolation (and concentration) of the analytes

from a complex matrix by adsorption onto an

appropriate sorbent, the removal of interfering

impurities by washing with a suitable solvent

system and then the selective recovery of the

retained analytes with a modifed solvent sys-

tem of suitable elution strength. If necessary,

this process can be modified by selection of

sorbent and solvent systems, so that the inter-

fering components are retained by a sorbent

and the an~dytes are then recovered in the

filtrate-eluate.

The last approach was found to be effective

for the sample clean-up of commercial fluorou-

racil creams which also contained methyl- and

propyl-p-hydroxybenzoate. These preservatives

exhibit UV spectral properties which can inter-

fere with the spectrophotometric assay of the

drug. Thus, using a C-18 sorbent and dissolv-

ing the cream sample in aqueous medium (20%

v/v methanol), the hydrophobic parabens were



D . B o n a z z i e t a l . / J . P h a r m . B i o m e d . A n a l . 1 3 1 9 9 5 ) 1 3 2 1 - 1 3 2 9 1325

completely retained by the C-18 sorbent while

fluorouracil passed through.

When the usual, general SPE process was

adopted, the conditions were adjusted to in-

duce the interactions matrix -analyt e, analyte

sorbent and analyte-eluent, increasing in that

order. Thus, for the analysis of the basic, hy-

drophobic drug promethazine a C-18 sorbent

was used to isolate the analyte from a 20%

v/v methanol sample solution; after washing

with the same solvent system to remove the

excipients, the drug was recovered with

methanol. A similar procedure was applied to

the clean-up of crotamiton creams. When ba-

sic, comparatively hydrophilic drugs such as

chlorhexidine and benzydamine were analysed,

a SCX packing material was preferred. At

pH 4.5 (aqueous medium), the protonated

chlorhexidine was retained by a PRS sorbent,

while the uncharged excipients passed through

the column; subsequent elution with a solvent

(pH 7.4) provided quantitative drug recovery.

The same procedure was applied to the analy-

sis of benzydamine hydrochloride cream, but

an arylsulphonic sorbent (SCX) was used. The

last cation exchange material was found to be

unsuitable for the extraction of chlorhexidine;

the different size and nature of the drugs are

probably responsible for different secondary

interactions with the aliphatic-aromatic moiety

of the SPE sorbent.

Ion-exchange methodology also proved to

be suitable for the clean-up of cream samples

containing hydrophobic, acidic drugs such

as Ketoprofen (pK~=5.9) and Ibuprofen

(pK~,= 5.2). The drugs, in the carboxylate

form in a basic solvent system, were retained

by a SAX sorbent; after appropriate washing

to remove the excipients, the drugs were re-

covered by eluting with an acidic solvent sys-

tem. This procedure allows elimination of

neutral components such as methyl- and

propyl-parabens which could interfere with the

spectrophotometric determination.

A diol sorbent, previously applied to the

sample clean-up of imidazole antimycotic

creams [12,13], was found to be suitable for

the analysis of formulations containing neu-

tral or acidic drugs of different polarity, such

as hydrocortisone acetate, fentiazac and

piroxicam. In these applications, the cream

sample was dissolved in an appropriate di-

chloromethane-n-hexane mixture, where the

dichloromethane n-hexane ratio was suitably

adjusted to give weak matrix-analyte interac-

tions and to favour the sorbent analyte inter-

actions. Under these conditions, the drug was

adsorbed onto the diol sorbent while most of

the excipients were eliminated; the drug was

then recovered by eluting with methanol or

dichloromethane.

On the basis of this and previous experience

[12,13], the use of a diol sorbent as the first

approach for the clean-up of a formulated

cream can be suggested. The hydrophobic and

acidic-basic properties of the drug as well as

the nature of the excipients constitute critical

elements for determining the choice of a re-

versed-phase or ion-exchange methodology for

optimisation of the SPE.

3 .2 . U V s p e c t r o s c o p y

Conventional (zero-order) and derivative

(first- and second-order) UV spectroscopy was

applied to the analysis of the representative,

selected drugs and to monitor the develop-

ment of the appropriate SPE method. For

each drug, linear relationships between the ab-

sorption maximum absorbance (zero-order) or

the selected amplitudes ~D and 2D (first- and

second-derivative), and the corresponding

drug concentration were found (Table 1).

When commercial creams were analysed, the

applicability of the different UV methods un-

der different conditions (with and without

SPE step) were evaluated. The results are

given in Table 2.

Direct conventional spectrophotometric

analysis of the sample solutions was not found

to be applicable: in each case, inflated assay

results were obtained, the extent depending on

the excipient structure and the measurement

wavelength. This effect was marked for formu-

lations containing parabens (fluorouracil, ke-

toprofen, ibuprofen, benzydamine) or

butylhydroxyanisole (chlorhexidine). Minor

effects were observed when the cream base

components were of an aliphatic nature; inter-

ference with the assay of piroxicam from an

aromatic compound (phenylethyl alcohol) was

prevented by using high wavelengths for mea-

surements.

Derivative UV spectrophotometric analysis

of the sample solution, without a preliminary

SPE clean-up did not offer significant im-

provements in accuracy.

When the commercial cream samples were

subjected to the SPE procedures prior to the



1326 D . B o n a z z i e t a l . / J . P h a r m . B i o m e d . A n a l . 1 3 1 9 9 5 ) 1 3 2 1 - 1 3 2 9

T a b l e 2

A s s a y r e s u l t s ~ f o r t h e s p e c t r o p h o t o m e t r i c ( c o n v e n t i o n a l a n d d e r i v a t iv e m o d e ) a n a l y s i s o f p h a r m a c e u t i c a l c r e a m s b e f o r e

a n d a f t e r S P E c l e a n - u p s t e p

D r u g M e t h o d B e f or e S P E A f t e r S P E

( S P E )

% f o u n d R S D % % f o u n d R S D %

C r o t a m i t o n ~D 106.22 2.5 99.41 1.24

254.4

(C - 18) 2D26t 6 104.13 2.7 98. I 1 0.73

2D261.6 247.2

107.86 2.3 98.9 0 1.71

I bu pr o f en A 27~ .6 > 150 . 20 b 98 . 21 0 . 93

( SA X ) A2636 > 150 . 15 b 98 . 29 1 .01

B e n z y d a m i n e ( S C X ) A 3 o5 6 1 3 5 . 70 2 . 9 9 8 . 6 6 1 . 20

C h l o r h e x i d i n e A 2 5 6 . 8 146 . 46 2 . 2 102 . 95 0 . 38

(PR S) ID272 248.4 140.93 3.0 98.32 0.16

2D259.2 240 142.31 2.9 98. 30 0.2 9

2D24o 229.9 145.25 2.1 97.83 0.25

H y d r o c o r t i s o n e A 24 8 1 1 0 .0 7 2 . 3 9 9 . 7 3 0 . 6 5

ace t a t e ( 2 - O H) 2D271 6 2488 90 . 82 2 . 6 99 . 71 0 . 65

F l u o r o u r a c i l A266 125 . 35 3 . 2 102 . 62 0 . 27

( C- I 8 )

2D292.8 267.2 89.1 0 3.1 99 .80 1.19

P r o m e t h a z i n e A 2 4 9 .2 140.74 2.3 100.52 1.58

(C-18 ) 20261.2 251.2 107.04 2.8 99.7 0 2.66

P i r o x ic am A 325 .6 103 . 89 2 . 5 98 . 09 0 . 79

( 2 - OH) 20371 . 2 326 102 . 96 3 . 0 98 . 80 0 . 70

K e t o p r o f e n A 2 6o 127.63 2.4 97.71 0.35

(SA X ) 2D232_258.8 113 . 76 3 .1 98 . 58 0 . 88

Fe n t i a zac A3oo4 112. 81 2 . 6 98 . 05 0 . 89

( 2 - O H) 10327 . 6 86 . 20 2 . 9 98 . 35 0 . 75

1D 88.41 2.8 97.50 0.90

254.4

T h e r e s u lt s , e x p r e s s e d a s a p e r c e n t a g e o f t h e c l a i m e d c o n t e n t , a r e

d e t e r m i n a t i o n s .

b ( _ ) : n o t d o n e .

t h e m e a n s o f fi v e ( a f te r S P E ) a n d t h r e e ( b e f o re S P E )

spectrophotometric assay, interference was

eliminated and accurate analyses were per-

formed. In general, no significant differences

were observed between the conventional and

the derivative mode of analysis; slight inter-

ference observed in the conventional determi-

nation of fluorouracil and chlorhexidine was

completely suppressed using the derivative

mode. It should be emphasized that crotami-

ton was analysed only by derivative spec-

trophotometry. Crotamiton is a typical

compound with a UV spectrum characterized

by slight shoulders in the usual analytical re-

gion 220-300 nm (Fig. 1); using the derivative

mode, the shoulders are converted to intense,

sharp peaks whose amplitudes can be used for

quantitative applications. Moreover it was

shown that the SPE method could provide an

effective clean-up of the crotamiton sample so-

lution; the derivative spectrum after SPE over

the 250-350 nm region was essentially identi-

cal to that of an equimolar standard solution.

The performance of the adopted SPE proce-

dures can be further illustrated by the exam-

ples reported in Fig. 2 (benzydamine

hydrochloride), Fig. 3 (hydrocortisone ac-

etate), Fig. 4 (ketoprofen) and Fig. 5 (ibupro-

fen). Generally, after the SPE step, the UV

spectra appear to recover their correct profiles

allowing accurate analysis to be.performed. It

should be pointed out that the method of

sample dissolution was chosen to be suitable

for the subsequent SPE step; a different

method of sample preparation could probably

be devised to provide more accurate direct

spectrophotometric analyses. The accuracy of

the proposed methods was determined by

analysing commercial samples spiked with a

known quantity of drug; the recoveries were

98.00-100.64%. The precision of the method

(SPE and UV assay) was good, as indicated

by the RSD (Table 2).



D. B ona zz i e t a l . / J . Phar m. B iom ed . Ana l . 13 19 9 5 ) 13 21 13 29

1327

3 0 ~ 7 - ~ - ~

1 5

0

i

' , .

\

\

'\

\

\ ,

/ \

2s,l.~

. ~..

26•.6

B D C 2 D

~ . . . . . . . o

I I

i

200 300 200 300 200 300

n m

Fig . I . Ze ro -order (A) , f i r s t -o rder (B) and se cond-order (C) de r iva t ive UV spec t ra o f c ro tami ton sample so lu t ion

(45.6 pg ml ~), before (- . -) and after ( ) the SPE (C-18 sorbent) clean-up step.

B ecause of t he gene ra l l y h i gh recove r i e s and

t he go od prec i s ion ( R S D < 1 .5% , wi t h t he ex-

cep t i on of p rome t haz i ne ) whi ch can be a tt a i ned

f r o m e x p e r i e n c e w i t h t h e m e t h o d , S P E - U V

s p e c t r o p h o t o m e t r y c a n b e c o n s i d e r e d t o b e a

use fu l combi na t i on for t he rou t i ne qua l i t y con-

t ro l o f pha rm aceut i ca l c reams .

4 . C o n c l u s i o n s

Sol i d-phase ex t rac t i on (SPE) proved t o be an

e f fec t ive t oo l fo r pe r form i ng adequ a t e sam pl e

c l ean-up for t he spec t rophot ome t r i c ana l ys i s o f

drugs i n c reams . The SPE condi t i ons can be

opt i mi zed by se l ec t i ng t he appropr i a t e sorben t

and e l uen t sys t ems accord i ng t o t he prope r t i e s

of t he drug and exc i p ien t s ( a c i d ba se ,

l ipophi l ic i ty) . Afte r the SPE s teps , both con-

vent i ona l ( z e ro-orde r ) and de r i va t i ve spec -

t roscopy can be gene ra l l y app l i ed . The

de r i va t i ve mode , howeve r , o f fe r s a more cha r -

ac ter i s t i c spec t ra l prof i le tha t i s useful for drug

i den t i f i c a t i on and for i mprovi ng t he accuracy

of t he me t hod , by us i ng sa t e ll it e peaks a t

higher wavelengths and suppress ing the res id-

ua l non-spec i f i c ma t r i x absorp t i on . The pro-

l

A

200 300 n

Fig . 2 . Zer o-order UV spec t ra o f benzydamine sample

so lu t ion (30 pg ml ~), be fore ( ) and a f te r ( - . . ) the

SPE (SCX sorben t ) c lean-up s tep .

0.75

~ . ~

o , ~ . . . _

200 300

r im

I I I

200 300

2 D

B

Fig . 3 . Zero-order (A) and second-order (B) de r iva t ive UV

spec t ra o f hydrocor t i sone ace ta te sample so lu t ion

(25 pg ml- ~) , be fore ~ ) and a f te r ( . . . ) the SPE (d io l

sorbent) clean-up step.



1328 D . B o n a z z i e t a l . / J . P h a r m . B i o m e d . A n a l . 1 3 1 9 9 5 ) 1 3 2 1 - 1 3 2 9

2.0

1 0

/ \

:

°

220 260

L_

i ° • I

300 340

r i m

2D

Fig . 4 . Ze ro-order UV spec trum of ke toprofen sample

solutio n (14.2 pg m l 1; pH 7.4) before ( ) the SPE

(SAX sorben t) c lean-up s tep . Zero-order ( . . . ) and second-

order ( - - - ) UV spec tra o f the so lu tion (pH 4 .5) a f te r the

SPE step.

1.0

0 S

A

l i

I i \

250 3S0

N m

2 D

0

Fig . 5 . Ze ro-order UV spec trum of ibuprofen sample so lu-

t ion (27 lag m l- t ; pH 8 .0) be fore ( ) the SPE (SAX

sorbent) c lean-up s tep . Zero-order ( . . . ) and second-order

( . • ) UV spec tra o f the so lu t ion (pH 4 .5) a f te r the SPE

step.

p o s e d S P E p r o c e d u r e s m a y a l s o be a d o p t e d i n

c h r o m a t o g r a p h i c ( H P L C ) a n a l y s e s t o a v o i d

o v e r l o a d i n g a n d d e l e t e r io u s e f f ec t s u p o n t h e

a n a l y t i c a l c o l u m n p e r f o r m a n c e a n d l i fe t im e .

Acknowledgements

T h a n k s a r e d u e t o M i s s C e c i l ia L i m i n a a n d

M i s s M a u r a B o t t u r a f o r th e i r v a l u a b l e t e c hn i -

c a l a s s i s ta n c e . T h i s w o r k w a s s u p p o r t e d b y t h e

M U R S T ( R o m e , I t al y ).

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Solid forms of pharmaceuticals: Polymorphs, salts and cocrystals

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