Quantitative Colorimetric Determination of Residual 9-alpha-Fluoroprednisolone and 9-alpha-Fluorohydrocortisone in Triamcinolone Samples CHARLES J. SIH,
S. C.
PAN, and
R. E. BENNETT
Squibb Institute for Medical Research, New Brunswick, N.
J.
A colorimetric method for the simultaneous quantitative estimation of 9afluoroprednisolone and 9a-fluorohydrocortisone in the presence of large quantities of triamcinolone depends upon the formation of a blue chromogen from 9a-fluoroprednisolone (Amax 625 mp) and a reddish purple chromogen from 9a-fluorohydrocortisone (Am.= 548 mp) in the presence of sulfuric acid, fructose, and cysteine. Within the range of 20 to 100 y of 9a-fluorohydrocortisone and/or 9a-fluoroprednisolone, these steroids can be determined accurately in triamcinolone samples with a precision to f3.0%. The advantage of this method over the conventional one employing partition chromatography is discussed.
EXPERIMENTAL
I
microbiological and chemical production of triamcinolone, 9afluoro-16a-hydroxypredniso1one(1), the amount of residual Sa-fluoroprednisolone and Sa-fluorohydrocortisone must be kept to a minimum, as they may give undesirable side effects such as salt retention (3). Thus, a rapid method for the quantitative estimation of Safluoroprednisolone and Sa-fluorohydrocortisone in the presence of a large excess of their 16-hydroxylated analogs is desirable. Because the side chain of Sa-fluoroprednisolone and Sa-fluorohydrocortisone might be considered as a triose and that of its 16a-hydroxylated derivatives as a tetrose, the method of Dische and Dische (8) for the estimation of tetroses was chosen to test this assumption. Although the results did not conform to this hypothesis, the color reactions were interesting. When Sa-fluoroprednisolone and Sa-fluorohydrocortisone were heated in the presence of fructose and concentrated sulfuric acid, the former produced a blue chromogen and the latter a reddish purple chromogen, whereas its 16-hydroxylated analogs gave none. This report deals with a quantitative colorimetric method for the determination of Sa-fluoroprednisolone and 9a-fluorohydrocortisone in the presence of their 16-hydroxylated analogs based on this observation. N THE
Reagents and Steroid Samples. Sulfuric acid reagent, 6 parts of concentrated sulfuric acid mixed with 1 part of distilled water. Fructose solution, 0.01% C.P. fructose (Pfanstiehl) in water. Cysteine hydrochloride solution, 3.0% L-cysteine hydrochloride (Merck) in water. All steroids used are of Squibb stock collection, twice recrystallized. Steroids are dissolved in methanol to give a concentration of 1 mg. per ml. For solutions of higher concentrations, a solution of 100 mg. of the steroid in 1ml. of dimethylformamide is prepared and diluted to the desired concentration with methanol. Procedure. To each tefit tube containing 10 to 100 y of Sa-fluoroprednisolone or Sa-fluorohydrocortisone dissolved in 0.1 ml. of methanol are added in order 0.4 ml. of water, 0.5 ml. of the fructose solution, and 4.5 ml. of the sulfuric acid reagent. The test tubes are immersed in an ice water bath during these additions. Five minutes after the additions are completed, the tubes are shaken while still immersed to ensure thorough mixing and cooled for 5 more minutes. The tubes are first warmed to room temperature by immersion in tap water for approximately 10 minutes and then placed for exactly 5 minutes in a water bath held a t 60" C. A blank containing no steroid is run simultaneously. The tubes are immediately cooled in tap water and 0.1 ml. of the cysteine hydrochloride solution is added. The reaction mixture is left a t room temperature for approximately 15 hours (overnight) to obtain a maximum color development, which is then terminated by the dilution with 1.2 ml. of distilled water. The chromogens formed showed an absorption maximum a t 625 mp for 9a-fluoroprednisolone and 548 mp for Sa-fluorohydrocortisone and both appeared to be stable for a t least 3 days. Prolonged incubation intensified the color only slightly. The absorbance a t 625 or a t 548 mp is read in a Beckman Model DU spectrophotometer against a blank containing no steroid, or against a blank with triamcinolone. Effect of Temperature on Chromogen Formation. Direct application of the originally given procedure by
Dische and Dische (9) to the estimation of Sa-fluoroprednisolone or Sa-fluorohydrocortisone was unsatisfactory, especially in the presence of their 16hydroxylated analog, where a brown color developed after heating a t 100' C. for 3 minutes. To determine optimum conditions for chromogen formation, the rate of chromogen development a t different temperatures was studied. Figure 1 shows that with heating a t about 60" C., the chromogens from Safluoroprednisolone or Sa-fluorohydrocortisone disappeared rapidly. At 100" C. after 3 minutes, considerable quantities of chromogens have already been destroyed, while with heating a t 60" C., the chromogens reached their maximum smoothly in 5 minutes and no brown color was observed with triamcinolone. Sa-Fluoroprednisolone and Sa-fluorocortisone behaved identically in this experiment. Although the color development was more rapid a t 80" C., heating a t 60" C. for 5 minutes was chosen for subsequent determinations because it gave more reproducible results and eliminated the ebullition of the organic solvent. Relationship between Steroid Concentration and Absorbance. With the procedure thus developed, the absorption spectra of the chromogens from Sa-fluoroprednisolone, Sa-fluorohydrocortisone, 9 a-fluoro- 16ahydroxyhydrocortisone, and triamcinolone were determined. Figure 2 shows t h a t Sa-fluoroprednisolone has an absorption maximum a t 625 mp and 9a-fluorohydrocortisone has a peak a t 548 mp, whereas its 16-hydroxylated analogs show no significant absorption in the visible region. The absorption of the chromogens from 901fluoroprednisolone a t 625 mp and 9afluorohydrocortisone a t 548 mp obeys Beer's law within the range of 10 to 100 y per tube (6.7 ml.). To determine whether Sa-fluoroprednisolone and 9afluorohydrocortisone behave independently of each other in the reaction, a mixture containing equal parts of the two compounds ranging from 20 to 100 y was tested (Table I). 9wFluorohydrocortisone shows no absorbance at 625 mp and therefore its presence will not VOL. 32, NO, 6, MAY 1960
669
50 ' C
10 oc
70 C '
8 0 *c
O
* 400
I 450
M 500
. 550
600
;
Y
650
700
Wive length in m,u
0
4 6 TIME IN MINUTES
2
0
Figure 2.
IO
Absorption spectrum of chromogen produced
I.
Figure 1. Effect of temperature and time on color development
la.
II. Ill.
125 y 100 y 100 y 100 y
Pa-fluoroprednirolone 9a-fluorohydrocortirone 9a-fluoro- 1 6a-hydroxyhydrocortirone triamcinolone
100 y of 9a4uoroprednirolone or 100 y of 9a-Auorohydrocortirone per tube
interfere in the determination of Safluoroprcdrisolone (Figure 2). SaFluoroprednisolone, on the other hand, contributes significant absorption a t 548 mp. The absorbance a t 548 mp is equal to the sum of the absorbances con= tributed by both compounds (C, A u ~ BLd8).To obtain the absorbance at 548 mp contributed to Sa-fluorohydrocortisone alone (Bars),the absorbance a t 548 mp due to Sa-fluoroprednisolone (.4ars) must be subtracted from the total absorbance (Csd*), Table I shows that a mixture of Sa-fluorohydrocortisone and Sa-fluoroprednisolone can be analyzed with an accuracy to *3.0%,. To determine the effect of triamcinolone on the color reaction, tests were run first with Sa-fluoroprednisolone and 9afluorohydrocortisone in the presence of triamcinolone. The data presented in Table I1 show that triamcinolone exerts no detcctahle interference up to 1 mg. per tube. In the presence of 5 mg. per tube, another straight line with a lower slope than that of the original is obtained. However, a t higher levels of the minor components in bulk quantities
+
of triamcinolone, recovery becomes considerably poorer. The results (Table I) show that the percentage deviation from theory for Sa-fluorohydrocortisone and Sa-fluoroprednisolone changes from
Table 11. Relaiionship of Steroid Concentration and Absorbance
Sa-Fluorohydrocortisone, y
Absorbance a t
Sa-Fluoropredniaolone, y 20 40 60 80 100
Analysis
548 M p
B
A 20 40 60 80 100
C
0.090 0.090 0.180 0.270 0.350 0.460
0.085 0.170 0.250 0.320 0.380
0.180 0.265 0.355 0.450
Absorbance a t 0.055 0.110 0.155 0.210 0.265
625 Mp 0.055 0.105 0.150 0.205 0.280
0.050
0.093 0.125 0.155 0.210
A. KO addition. B. 1 m of triamcinolone added to each test tu%e. C. 5 mg. of triamcinolone per tube added.
Table I. Recovery of Mixtures of 9a-FIuorohydrocortisone and 9a-Fluoroprednisolone
Steroid
Sa-Fluorohydrocortisone %Da B %D
Added, y 20 40 60 80
A
100
100
a
20
0.00
40
0.00
60 80
0.00 0.00 0.00
19 39 55 69 83
-
-
5.0 2.5 8.0
-14.0 -17.0
Sa-Fluoroprednisolone A 20 41 62 80
98
%D
B
%D
0.0 $2.5 +3.0 0.0 -2.0
16 33
-20.0
49
59 75
-17.5 -18.0 -26.0 -25.0
yo deviation from theory.
A . Equal arts of Sa-fluorohydrocortisone and Sa-fluoroprednisolone.
B. EquaP parts of 9a-fluorohydrocortisone and Sa-fluoroprednisolone in 5 mg. of triamcinolone. 670
ANALYTICAL CHEMISTRY
3 to 8% and 18 to 20% for the lower level (20 to 60 y) to 14 to 17% and 25% for the higher level (80 to 100 y), respectively. Obviously, triamcinolone, which itself is rather inert in this color reaction, interferes with the chromogen formation from Sa-fluorohydrocortisone and with that from Sa-fluoroprednisolone when it is present to an extent of 50 to 500 times the amount of these compounds.
of Triamcinolone Samples.
Because Sa-fluoroprednisolone and Sa-fluorohydrocortisone are as a rule present in only small percentages in commercial triamcinolone samples, the interference described above must be taken into account in formulating the analytical procedure. The following procedure is therefore followed when t h e method is applied t o t h e analysis of commercial triamcinolone samples. 1. Use 1 mg. of sample per test tube. 2. Carry out the color reaction with the procedure described above. 3, Read absorbance against a blank run simultaneously with 1 mg. of pure triamcinolone a t 548 and 625 mp. 4. Prepare a standard curve with each group of samples, using 10 to 100 y of Sa-fluorohydrocortisone plus 1 mg. of triamcinolone per tube. Read the absorbance a t 548 mp. 5, Prepare two standard curves with each group of samples, using 10 t o 100 y of Sa-fluoroprednisolone plus 1 mg. of triamcinolone per tube. Construct one standard curve with absorbance at 548 mp and the other with absorbances a t 625 mp. 6. Calculate the content of Safluoroprednisolone by using the standard curve tit 625 mp. 7, Find from the standard curve at 548 mp for Sa-fluoroprednisolone the absorbance corresponding to the content of Sa-fluoroprednisolone calculated
0.5
x
r
Table 111. Comparison of Results Obtained by Partition Chromatography and Colorimetric Method
0.4 -
Partition Sam- Chroma- Coloriple tography, metric, No. % %
Y
z u
2
0.3
-
0.2
-
0.1
-
U In 0
m 4
1 2 3 4 5 6
420
440
460
480
NAVE Figure 3.
Absorption spectra
500 LENGTH IN
of
520
560
540
580
600
(my)
chromogens from different steroids
25 y A'-androstenedione 50 y Reich's Compound S 3. 25 7 Pa-fluoro-11 &hydroxyandrostenedione 4. 50 y 9a-fluorohydrocortisone 1. 2.
in 6. Subtract this from the total absorbance a t 548 mp. Calculate the content of Sa-fluorohydrocortisone by using this difference and the standard curve a t 548 mp for Sa-fluorohydrocortisone as constructed in 4.
If the analytical result indicates that the samples contain 1% or less of either or both of these steroids, the analysis is repeated using a 5-mg. sample. In this case, the blank and the standard curves must be prepared with 5 mg. of pure triamcinolone per tube in place of 1 mg. per tube. Results obtained by this method are compared (Table 111) with those determined by partition chromatography (6) and agree well with a maximum deviation of 0.35% for the determination of 9wfluoroprednisolone. Specificity of Reaction. To gain further information concerning the formation of this chromogen a t 548 mp, a series of steroids was tested (Table IV). Besides Sa-fluorohydrocortisone, Reichstein's Compound S (cortexolone) A'-cortexolone, prednisolone, deoxycorticosterone, testosterone, A4-androstenedione, and 9a-fluoro-118-hydroxyandrostenedione all gave considerable chromogen a t 548 mp. As shown in Figure 3, the chromogen from Su-fluorohydrocortisone has the same absorption spectrum as Sa-fluoro-1lg-hydroxyandrostenedione with an absorption maximum a t 548 mp; and cortexolone has an absorption spectrum identical to that of A4-androstenedione with an absorption maximum a t 523 mp. It therefore seems that Sa-fluorohydrocortisone and cortexolone might be first converted to their respective C19 steroid analogs or that they aye further converted to common intermediates, which then react with hydroxymethylfurfural[(hydroxymethylmethyl) - 2 - furaldehyde] to give the red chromogen a t 548 mp. As can be seen from Table IV, the formation of the blue chromogen a t 625 mp is specific and only prednisolone besides 9a-fluoroprednisolone gives con-
siderable color. Possibly a double bond a t the 1- position is a prerequisite for the formation of the blue chromogen a t 625 mp. The requirement of cysteine is rather nonspecific, as i t can be replaced by other sulfhydryl reducing agents such as mercaptoacetic acid or glutathione. The addition of cysteine not only raises the extinct absorptivity coefficient but also shifts the absorption to a longer wave length-e.g., from 500 to 625 mp or from 534 to 548 mpthis indicates that the two steroids form chromogens when heated with sulfuric acid alone. DISCUSSION
These data show that the method is applicable only to samples containing no other steroids which produce a color in the reaction. This is true for the finished product of triamcinolone. In this instance, a determination of the residual Sa-fluoroprednisolone and 9afluorohydrocortisone is of mein concern and the method presented here is satisfactory. The advantage of the present method over that of partition chromatography (6) is twofold. It is rapid and as many as 100 samples per day can be analyzed, as compared to only one per day. Also, this method is more sensitive; small quantities of Qa-fluorohydrocortisone have been determined in certain triamcinolone samples where partition chromatography fails to show its presence. During the course of this work, a paper by Walser and Shlunke (6) came to our attention. These investigators applied the Porter-Silber reaction (4) to 9a-fluorohydrocortisone and triamcinolone. The former gave a chromogen with an absorption maximum a t 410 mp, whereas the latter had insignificant absorption in that region. These results are similar to ours for Qafluorohydrocortisone and perhaps the reaction can also be adapted to the
0.85 0.50
0.00
0.08 0.70 1.10 1.20
0.42
0.000 -0.07 +0.02
+0.15 $0.35 -0.08
0.46 $0.03 1.04 +0.33 0.52 $0.02 5 Per cent Sa-fluoroprednisolone in triamcinolone samples. 7
400
0.00" 0.15 0.68 0.95
Deviation, %
8 9
0.43 0.71 0.50
~
Table IV. Chromogenic Values of Several Steroids
Amount Used Per Tube,
Kara" K82bb Compound y A'-Androstene0.0 4.0 25 dione
[email protected] 19.2 25 stenedione 0.0 3.8 50 Testosterone 0.0 0.2 50 A'-Testolactone 0.0 0.0 50 Progesterone 11a-Hydroxypro0.0 0.0 50 gesterone 17a-Hydroxypro0.2 0.6 50 gesterone Reichstein's Com0.0 2.4 50 pound S 0.0 0.0 50 Cortisone 0.8 0.0 50 Hydrocortisone Deoxycorticoster0.6 5.6 50 one 9a-Fluorohydro0.0 4.6 50 cortisone9a-Fluoroprednis3.0 1.3 50 olone 9 a-Fluoro-l6ahydroxyhydro0.0 0.0 50 cortisone 0.0 0.0 50 Triamcinolone 0.6 2.6 50 AI-Cortexolone 1.6 3.4 50 Prednisolone 0 Absorbance at 548 m# per mg. steroid reacting. * Absorbance at 626 mp per mg. steroid reacting.
determination of Sa-fluorohydrocortisone in triamcinolone samples. LITERATURE CITED
(1) Bernstein, S., Lenhard, R. H., Allen,
W. S., Heller, M., Littell, R., Stolar, S. H., Feldman, L. I., Blank, R. H., J . Am. Chem. So.: 78,5693 (1956): (2) Dische, Z., Dische, M. R., Bzochim. et Biophys. Acta 27,184 (1958). (3) Fried, J., Borman, A., Vitamins and Hormones 15,303 (1958). (4) Silber, R. H., Porter, C. C., J. Biol. Chem. 210, 923 (1954). (5) Smith, L. L., Foell, T., DeMaio, R., Halwer, M., J . Am. Pharm. Assoc. 45, KO.9, 528 (1959). (6) Walser, A., Schlunke, H. P., Experientza 15, 71 (1959). RECEIVED for review November 4, 1959. Accepted February 4,1960. VOL. 32, NO. 6, MAY 1960
671
