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Automated colorimetric assay of Norgestrel
217
AUTOMATED COLORIMETRIC ASSAY OF. NORGESTREL.
by P. M. Short, School of Pharmacy, Portsmouth Polytechnic, Portsmouth P01 2DZ, England. and C. T. Rhodes, F a c u l t y of Pharmaceutical Sciences, U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Received:
June I0, .1970 ABSTRACT
A r e l a t i v e l y s e n s i t i v e assay f o r the contraceptive s t e r o i d n o r g e s t r e l , in pharmaceutica] p r e p a r a t i o n s , is d e s c r i b e d . The procedure i n v o ] v e s the b]ue t e t r a z o l i u m r e a c t i o n . Optimum c o n d i t i o n s f o r c o l o r development were e s t a b ] i s h e d . The r e l a t i v e standard d e v i a t i o n f o r the assay, which has a sample r a t e of 30 h r - ] , were of the o r d e r of 0.9% at a c o n c e n t r a t i o n o f 5 ug ml - I n o r g e s t r e ] . The s e n s i t i v i t y of the method is such t h a t 500 ng ml-] can e a s i ] y be e s t a b ] i s h e d and w i t h more e ] a b o r a t e equipment ]ower c o n c e n t r a t i o n s cou]d be determined. The method is u n ] i k e ] y to be o f use in assaying the drug in b i o ] o g i c a | systems. INTRODUCTION Norgestrel formulated identifying
is a p o t e n t progesterone type c o n t r a c e p t i v e which may be
in t a b l e t s or suspensions ( l ) . t h i s drug,
13
-ethyl-]7
3-one, have p r e v i o u s l y been d e s c r i b e d . spectrophotometry, thin-]ayer
P o s s i b l e methods of
-ethiny]-]7
hydroxygon-4-ene-
These inc]ude u n t r a v i o ] e t
chromatography and f l u o r i m e t r y
The assay of c o r t i c o s t e r o i d s
utilising
zo]ium has been automated by Beyer (3).
the r e a c t i o n w i t h b]ue t e t r a -
The r e d u c t i o n o f blue t e t r a -
zo]ium ( 3 : 3 ' - D i a n i s o ] o n e - b i s - 4 : 4 ' - ( 3 : 5 - d i p h e n y l ) - t e t r a z o l i u m in the presence of a s t r o n g o r g a n i c base, by n o r g e s t r e ] , diformazan (4).
Using t h i s
(2).
ch]oride) gives the red
r e a c t i o n , which depends on the presence in
n o r g e s t r e ] of the ketone group at carbon t h r e e , s u i t a b ] e c o n d i t i o n s
2 18
S T E R O I D S
16:2
have been found which provide a reproducible and sensitive colorimetric assay of norgestrel.
Further, the technique has been automated. EXPERIMENTAL
Equipment. - Automatic sampler II, proportioning pump, "solvaflex" tubing, glass fittings, colorimeter and recorder supplied by Technicon Ltd., and a heating bath. The filter interference assembly used in the colorimeter was o? the type 522-18-28. The ~ max, wavelength o? maximum absorbance, of the norgestrel solutions was found by a manual technique to be 524 nm. The flowcell used in the colorimeter was of the tubular type with a path length of 0.6 inches, 15 mm. Reagents. - Absolute ethanol, blue tetrazolium, (B.T.) tetramethylammonium hydroxide (T.M.A.) supplied by British Drug Houses Ltd. Blue tetrazolium solutions were made in 90% ethanol and tetramethylammonium hydroxide in absolute ethanol. Standards. - Accurately weighed quantities of norgestrel were dissolved in absolute alcohol; the material used has been fully characterized (2). Solutions containing l, 5, 8, 12 and 15 yg ml -l norgestrel were used as standards. Procedure. - The flow diagram (Fig. l) shows the relative positions of the instruments. Samples were mixed initially with the B.T. solution and then with the T.M.A. solution. The solutions were heated in a time delay coil (length 40.7 feet, 12.2 m, internal diameter 0.064 inches 1.6 mm, wall thickness 0.04 inches l.O mm) for 5.25 mins. before passing to the debubbler and colorimeter. Absorbancies of the solutions were calculated making allowances for the absorbance of the solvent. Using this system the optimum conditions for the assay were developed. The following were some of the tests performed. Effect of Temperature. - The absorbance was measured for different concentrations of norgestrel after the heating bath was set at various temperatures. B.T. 0.05% w/v and T.M.A., 0.5% v/v solutions were used. Effect of B.T. Concentration. - At a temperature of 55°C and a T.M.A. concentration of 5% v/v the absorbance for various solutions of norgestrel were measured. Effect of T.M.A. Concentration. - At a temperature of 55oc and a B.T. concentration of O.O15% w/v the absorbances for various solutions of norgestrel were measured. ?recision. - Concentrations of l, 5, 8, 12 and 15}~g ml -I norgestrel in absolute alcohoi were assayed using B.T. 0.015% WlV, T.M.A. 5% v/v and a temperature of 55oc. A latin square was used to present the samples in a random order. The standard deviations, means, ranges and relative standard deviations were calculated.
Aug. 1970
ST ER O I D S
219
TUBE I.D. (inches)
SAMPLER II
A g SO LUTE"lc~'ee'~, A L C O H O L ~ (7))
0.090 ~ 3 ~ SAMPLES o.o9o ~ P E R HOUR
m-4..~
0
0.056
BLUE TETRAZOLIUM
0.045 0.056
AIR T - - ~ M E TH YL A M M O N I U M HYDROXIDE
0.090
WA5TE
4"
PROPORTIONING IME ELAY COIL WASTE,
COLORIMETER Fig.
I
PUMP
RECORDER
Flow diagram f o r automated c o ] o r i m e t r i c
assay o f . n o r g e s t r e l .
220
ST ER O I D S
16:2
RESULTS AND DISCUSSION Fig. 2 shows the effect of temperature on the assay. The absorbancies of the solutions increased with increasing
temperature.
However, although the absorbancies at 65oc are greater than at 55°C, the assay loses s e n s i t i v i t y at the higher temperature• This is due to two factors, a reduction absorbance.
in peak height at 65° and a high background
The increese in absorbance with temperature is probably due
to increase in the reaction rate to produce more diformazan. Above a certain temperature the decrease in s e n s i t i v i t y is probably due to a slower increase in the color development of the samples as compared to the color development of the solvent.
o.1 O4 Z 0 0.2'
0
5
10
15
NORGESTREL CONCENTRATION pg ml. "1
Fig. 2
Effect of temperature on the colorimetric assay of norgestrel. Key: triangles, 55o; open circles, 65o; squares, 46o; solid circles, 35 o.
Aug. 1970
Fig.
STEROIDS
3 shows a s i m i l a r
concentration
is considered.
221
r e l a t i o n s h i p when the e f f e c t of the B.T. Although the absorbancies
increase up to
a B.T. c o n c e n t r a t i o n of 0.025~ w/v and then decreased, the optimum conc e n t r a t i o n was found to be 0.015~ w/v.
The decrease in s e n s i t i v i t y
q u i t e considerable when the B.T. c o n c e n t r a t i o n w/v to 0.025~ w/v.
The reason f o r t h i s
discussed above in c o n j u n c t i o n with
O
is
is increased from 0.015~
is probably that which was
the e f f e c t
of temperature.
0.4
m
0,2
I
I
I
$
10
15
NOIIGE$TilEL COHCENI"RAIIOM
Fig. 3
,IJII m l . "I
E f f e c t of blue t e t r a z o l i u m c o n c e n t r a t i o n on the assay of n o r g e s t r e l . Key: ~ w/v B.T.; s o l i d c i r c l e s , 0.004; t r i a n g l e s , 0 . 0 ] 5 ; open c i r c l e s , 0.025; squares, 0. I00.
222
STE
ROIDS
Both absorbancies and the sensitivity T.M.A. concentration
16:2
increased with increase in
up to 5~ v/v and then decreased
(Fig. 4).
The
decrease is probably due to the very high background absorbance of the solvent.
Z
."
o4,
02'
i~0
1~5
NORGESTREL CONCENTRATION pg ml -I
Fig. 4
Effect of tetramethylammonium hydroxide concentration on the assay of norgestrel. Key: % w/v T.M.A.; triangles, 7.5; open circles, 5.0; squares, 2.0; solid circles, 0.5. TABLE l
REPRODUCIBILITY OF THE COLORIMETRIC ASSAY OF NORGESTREL USING A LATIN SQUARE SEQUENCE, A TYPICAL SET OF RESULTS.
Norgestrel g ml -l I 5 8 12 15
Mean Absorbance of 5 readings 0.067 0.281 0.437 0.624 0.751
Range
Relative Standard Deviation
0.006 0.005 O.O14 0.005 0.007
3.9 0.9 l.l 0.5 0.5
Aug. 1970
S T ER O I D S
22 3
Using the optimum conditions of 0.015~ w/v B.T., 5% v/v T.M.A. and a time delay coil at a temperature of 55°C the flow characteristics the system are indicated by the recorded curve shown in Fig. 5.
of
Also,
using these optimum conditions the precision was checked using a latin square sequence. conditions,
A number of runs have been performed under these
all giving similar results.
shown in Table I.
Results of a typical
run are
The low relative standard deviations emphasize the
precision of the technique.
The method
is sensitive enough to detect
less than 500 ng ml -l and with the incorporation of a scale expander quantities below 200 ng ml -l could be investigated. ml -l was detected
200 ng
in the instrumentation described above but the
absorbance was considered
to be too low for dependable results.
found that using smaller reagent tubes, 0.02 inches,
Norgestrel
It was
(internal diameter 0.04 and
l.O and 0.05 mm) the sensitivity was further
at the expense of excluding the upper concentration
0,8
increased but
range from the
15ug/ml
ml f'
0_6
r O~ug/ml
0.4
r
Z ~C o
~
0.2
l~ug/ml
\ Fig.
5
N o r g e s t r e l assay, recorded curve o b t a i n e d using the developed optimum c o n d i t i o n s ,
224
ST E R O I D S Beer-Lambert Law r e l a t i o n s h i p . project,
f o r which t h i s
thus the c o n d i t i o n s
For the b i o p h a r m a c e u t i c a l
assay was d e v e l o p e d ,
outlined
Using the c o n d i t i o n s
were o b t a i n e d
described
in t h i s
The scope of t h i s limited
to s o l u t i o n s .
in t h i s
technique with
tablets
The l a c k o f s p e c i f i c i t y
assay i n d i c a t e s
that
paper the Beer-Lambert studied.
this
Highly
law
reproducible
region.
the assay c o u l d not be r e a d i l y alone.
t h i s was u n d e s i r a b l e and
the above a p p a r a t u s
However, using a s i m i l a r
used f o r h y d r o c o r t i s o n e
research
above were u t i l i z e d .
was obeyed over the range o f c o n c e n t r a t i o n s results
16:2
is o b v i o u s l y
t e c h n i q u e which Beyer
(3)
t h e r e does not seem to be any reason why adapted f o r
tablets
o f the c o l o r i m e t r i c
assay is u n l i k e l y
containing
norgestrel
reaction
used in t h i s
to be o f use f o r b i o l o g i c a l
systems (5). ACKNOWLEDGEMENTS We thank Dr. M.H. Briggs of Schering Chemicals for the generous supply of norgestrel. C.T.R. thanks the Medical Research Council of Canada for the V i s i t i n g Scientist award made for the academic year 1969-70. REFERENCES (1) L. Fotherby, BRIT. MED. J., 2._33, 489 (1968). (2) P.M. Short, E.T. Abbs and C.T. Rhodes, CANAD. J. PHARM. SCI., ~, 8 (1969). (3) W.F. Beyer, J. PHARM. SCI., 55, 200 (1966). (4) C.R. NoIIer, TEXTBOOK OF ORGANIC CHEMISTRY, 3rd ed., W.B. Saunders Company, London, p. 524 (1966). (5) A.S. Meyer and M.C. Lindberg, ANAL. CHEM., 2.J_7, 813 (1955).
AUTOMATED COLORIMETRIC ASSAY OF. NORGESTREL.
by P. M. Short, School of Pharmacy, Portsmouth Polytechnic, Portsmouth P01 2DZ, England. and C. T. Rhodes, F a c u l t y of Pharmaceutical Sciences, U n i v e r s i t y o f B r i t i s h Columbia, Vancouver 8, Canada. Received:
June I0, .1970 ABSTRACT
A r e l a t i v e l y s e n s i t i v e assay f o r the contraceptive s t e r o i d n o r g e s t r e l , in pharmaceutica] p r e p a r a t i o n s , is d e s c r i b e d . The procedure i n v o ] v e s the b]ue t e t r a z o l i u m r e a c t i o n . Optimum c o n d i t i o n s f o r c o l o r development were e s t a b ] i s h e d . The r e l a t i v e standard d e v i a t i o n f o r the assay, which has a sample r a t e of 30 h r - ] , were of the o r d e r of 0.9% at a c o n c e n t r a t i o n o f 5 ug ml - I n o r g e s t r e ] . The s e n s i t i v i t y of the method is such t h a t 500 ng ml-] can e a s i ] y be e s t a b ] i s h e d and w i t h more e ] a b o r a t e equipment ]ower c o n c e n t r a t i o n s cou]d be determined. The method is u n ] i k e ] y to be o f use in assaying the drug in b i o ] o g i c a | systems. INTRODUCTION Norgestrel formulated identifying
is a p o t e n t progesterone type c o n t r a c e p t i v e which may be
in t a b l e t s or suspensions ( l ) . t h i s drug,
13
-ethyl-]7
3-one, have p r e v i o u s l y been d e s c r i b e d . spectrophotometry, thin-]ayer
P o s s i b l e methods of
-ethiny]-]7
hydroxygon-4-ene-
These inc]ude u n t r a v i o ] e t
chromatography and f l u o r i m e t r y
The assay of c o r t i c o s t e r o i d s
utilising
zo]ium has been automated by Beyer (3).
the r e a c t i o n w i t h b]ue t e t r a -
The r e d u c t i o n o f blue t e t r a -
zo]ium ( 3 : 3 ' - D i a n i s o ] o n e - b i s - 4 : 4 ' - ( 3 : 5 - d i p h e n y l ) - t e t r a z o l i u m in the presence of a s t r o n g o r g a n i c base, by n o r g e s t r e ] , diformazan (4).
Using t h i s
(2).
ch]oride) gives the red
r e a c t i o n , which depends on the presence in
n o r g e s t r e ] of the ketone group at carbon t h r e e , s u i t a b ] e c o n d i t i o n s
2 18
S T E R O I D S
16:2
have been found which provide a reproducible and sensitive colorimetric assay of norgestrel.
Further, the technique has been automated. EXPERIMENTAL
Equipment. - Automatic sampler II, proportioning pump, "solvaflex" tubing, glass fittings, colorimeter and recorder supplied by Technicon Ltd., and a heating bath. The filter interference assembly used in the colorimeter was o? the type 522-18-28. The ~ max, wavelength o? maximum absorbance, of the norgestrel solutions was found by a manual technique to be 524 nm. The flowcell used in the colorimeter was of the tubular type with a path length of 0.6 inches, 15 mm. Reagents. - Absolute ethanol, blue tetrazolium, (B.T.) tetramethylammonium hydroxide (T.M.A.) supplied by British Drug Houses Ltd. Blue tetrazolium solutions were made in 90% ethanol and tetramethylammonium hydroxide in absolute ethanol. Standards. - Accurately weighed quantities of norgestrel were dissolved in absolute alcohol; the material used has been fully characterized (2). Solutions containing l, 5, 8, 12 and 15 yg ml -l norgestrel were used as standards. Procedure. - The flow diagram (Fig. l) shows the relative positions of the instruments. Samples were mixed initially with the B.T. solution and then with the T.M.A. solution. The solutions were heated in a time delay coil (length 40.7 feet, 12.2 m, internal diameter 0.064 inches 1.6 mm, wall thickness 0.04 inches l.O mm) for 5.25 mins. before passing to the debubbler and colorimeter. Absorbancies of the solutions were calculated making allowances for the absorbance of the solvent. Using this system the optimum conditions for the assay were developed. The following were some of the tests performed. Effect of Temperature. - The absorbance was measured for different concentrations of norgestrel after the heating bath was set at various temperatures. B.T. 0.05% w/v and T.M.A., 0.5% v/v solutions were used. Effect of B.T. Concentration. - At a temperature of 55°C and a T.M.A. concentration of 5% v/v the absorbance for various solutions of norgestrel were measured. Effect of T.M.A. Concentration. - At a temperature of 55oc and a B.T. concentration of O.O15% w/v the absorbances for various solutions of norgestrel were measured. ?recision. - Concentrations of l, 5, 8, 12 and 15}~g ml -I norgestrel in absolute alcohoi were assayed using B.T. 0.015% WlV, T.M.A. 5% v/v and a temperature of 55oc. A latin square was used to present the samples in a random order. The standard deviations, means, ranges and relative standard deviations were calculated.
Aug. 1970
ST ER O I D S
219
TUBE I.D. (inches)
SAMPLER II
A g SO LUTE"lc~'ee'~, A L C O H O L ~ (7))
0.090 ~ 3 ~ SAMPLES o.o9o ~ P E R HOUR
m-4..~
0
0.056
BLUE TETRAZOLIUM
0.045 0.056
AIR T - - ~ M E TH YL A M M O N I U M HYDROXIDE
0.090
WA5TE
4"
PROPORTIONING IME ELAY COIL WASTE,
COLORIMETER Fig.
I
PUMP
RECORDER
Flow diagram f o r automated c o ] o r i m e t r i c
assay o f . n o r g e s t r e l .
220
ST ER O I D S
16:2
RESULTS AND DISCUSSION Fig. 2 shows the effect of temperature on the assay. The absorbancies of the solutions increased with increasing
temperature.
However, although the absorbancies at 65oc are greater than at 55°C, the assay loses s e n s i t i v i t y at the higher temperature• This is due to two factors, a reduction absorbance.
in peak height at 65° and a high background
The increese in absorbance with temperature is probably due
to increase in the reaction rate to produce more diformazan. Above a certain temperature the decrease in s e n s i t i v i t y is probably due to a slower increase in the color development of the samples as compared to the color development of the solvent.
o.1 O4 Z 0 0.2'
0
5
10
15
NORGESTREL CONCENTRATION pg ml. "1
Fig. 2
Effect of temperature on the colorimetric assay of norgestrel. Key: triangles, 55o; open circles, 65o; squares, 46o; solid circles, 35 o.
Aug. 1970
Fig.
STEROIDS
3 shows a s i m i l a r
concentration
is considered.
221
r e l a t i o n s h i p when the e f f e c t of the B.T. Although the absorbancies
increase up to
a B.T. c o n c e n t r a t i o n of 0.025~ w/v and then decreased, the optimum conc e n t r a t i o n was found to be 0.015~ w/v.
The decrease in s e n s i t i v i t y
q u i t e considerable when the B.T. c o n c e n t r a t i o n w/v to 0.025~ w/v.
The reason f o r t h i s
discussed above in c o n j u n c t i o n with
O
is
is increased from 0.015~
is probably that which was
the e f f e c t
of temperature.
0.4
m
0,2
I
I
I
$
10
15
NOIIGE$TilEL COHCENI"RAIIOM
Fig. 3
,IJII m l . "I
E f f e c t of blue t e t r a z o l i u m c o n c e n t r a t i o n on the assay of n o r g e s t r e l . Key: ~ w/v B.T.; s o l i d c i r c l e s , 0.004; t r i a n g l e s , 0 . 0 ] 5 ; open c i r c l e s , 0.025; squares, 0. I00.
222
STE
ROIDS
Both absorbancies and the sensitivity T.M.A. concentration
16:2
increased with increase in
up to 5~ v/v and then decreased
(Fig. 4).
The
decrease is probably due to the very high background absorbance of the solvent.
Z
."
o4,
02'
i~0
1~5
NORGESTREL CONCENTRATION pg ml -I
Fig. 4
Effect of tetramethylammonium hydroxide concentration on the assay of norgestrel. Key: % w/v T.M.A.; triangles, 7.5; open circles, 5.0; squares, 2.0; solid circles, 0.5. TABLE l
REPRODUCIBILITY OF THE COLORIMETRIC ASSAY OF NORGESTREL USING A LATIN SQUARE SEQUENCE, A TYPICAL SET OF RESULTS.
Norgestrel g ml -l I 5 8 12 15
Mean Absorbance of 5 readings 0.067 0.281 0.437 0.624 0.751
Range
Relative Standard Deviation
0.006 0.005 O.O14 0.005 0.007
3.9 0.9 l.l 0.5 0.5
Aug. 1970
S T ER O I D S
22 3
Using the optimum conditions of 0.015~ w/v B.T., 5% v/v T.M.A. and a time delay coil at a temperature of 55°C the flow characteristics the system are indicated by the recorded curve shown in Fig. 5.
of
Also,
using these optimum conditions the precision was checked using a latin square sequence. conditions,
A number of runs have been performed under these
all giving similar results.
shown in Table I.
Results of a typical
run are
The low relative standard deviations emphasize the
precision of the technique.
The method
is sensitive enough to detect
less than 500 ng ml -l and with the incorporation of a scale expander quantities below 200 ng ml -l could be investigated. ml -l was detected
200 ng
in the instrumentation described above but the
absorbance was considered
to be too low for dependable results.
found that using smaller reagent tubes, 0.02 inches,
Norgestrel
It was
(internal diameter 0.04 and
l.O and 0.05 mm) the sensitivity was further
at the expense of excluding the upper concentration
0,8
increased but
range from the
15ug/ml
ml f'
0_6
r O~ug/ml
0.4
r
Z ~C o
~
0.2
l~ug/ml
\ Fig.
5
N o r g e s t r e l assay, recorded curve o b t a i n e d using the developed optimum c o n d i t i o n s ,
224
ST E R O I D S Beer-Lambert Law r e l a t i o n s h i p . project,
f o r which t h i s
thus the c o n d i t i o n s
For the b i o p h a r m a c e u t i c a l
assay was d e v e l o p e d ,
outlined
Using the c o n d i t i o n s
were o b t a i n e d
described
in t h i s
The scope of t h i s limited
to s o l u t i o n s .
in t h i s
technique with
tablets
The l a c k o f s p e c i f i c i t y
assay i n d i c a t e s
that
paper the Beer-Lambert studied.
this
Highly
law
reproducible
region.
the assay c o u l d not be r e a d i l y alone.
t h i s was u n d e s i r a b l e and
the above a p p a r a t u s
However, using a s i m i l a r
used f o r h y d r o c o r t i s o n e
research
above were u t i l i z e d .
was obeyed over the range o f c o n c e n t r a t i o n s results
16:2
is o b v i o u s l y
t e c h n i q u e which Beyer
(3)
t h e r e does not seem to be any reason why adapted f o r
tablets
o f the c o l o r i m e t r i c
assay is u n l i k e l y
containing
norgestrel
reaction
used in t h i s
to be o f use f o r b i o l o g i c a l
systems (5). ACKNOWLEDGEMENTS We thank Dr. M.H. Briggs of Schering Chemicals for the generous supply of norgestrel. C.T.R. thanks the Medical Research Council of Canada for the V i s i t i n g Scientist award made for the academic year 1969-70. REFERENCES (1) L. Fotherby, BRIT. MED. J., 2._33, 489 (1968). (2) P.M. Short, E.T. Abbs and C.T. Rhodes, CANAD. J. PHARM. SCI., ~, 8 (1969). (3) W.F. Beyer, J. PHARM. SCI., 55, 200 (1966). (4) C.R. NoIIer, TEXTBOOK OF ORGANIC CHEMISTRY, 3rd ed., W.B. Saunders Company, London, p. 524 (1966). (5) A.S. Meyer and M.C. Lindberg, ANAL. CHEM., 2.J_7, 813 (1955).