LITERATURE CITED
(1) Handbook of Chemistry and Physics,
45th Ed., D-78. The Chemical Rubber Co., Cleveland, Ohio, 1964. (2) Harned, H. S., Owen, B. B., “The Physical Chemistry of Electrolytic Solutions,” 3rd Ed., pp. 310-16. Reinhold, New York, 1958.
(3) Katchalsky, A., Eisenberg, H., Lifson, S., J. Am. Chem. SOC.73,5889 (1951). (4) MacInnes, D, A., Ibid., 48, 2068 (1926). (5) McDonald, H. J., “IonograPhy- Electrophoresis in Stabilized Media,” pp. 57-8.
The Year Book Publishers, Inc., Chicago, Ill., 1955. (6) Onsager, L., PhY5ik. 2 . 2 8 ~ 2 7 7(1927). (7) Strickland, R. D., Ann. N. Y . Acad. Sci. 137, 139 (1966). RECEIVEDfor review March 18, 1966. Accepted April 29, 1966.
Comparison of Four Methods of Determining Urinary Pregnanediol R. D. BARRY, MICHAEL GUARNIERI, P. K. BESCH, WILLIAM RING, and N. F. BESCH Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, Columbus, Ohio
4321 0
and Medical Research Consultants, Inc., Columbus, Ohio
b Four methods for determining urinary pregnanediol are compared. The procedures involve several hydrolysis, extraction, and purification methods, and utilization of gas chromatography and spectrophotometry for quantitation. Enzymatic hydrolysis of pregnanediol glucosiduronate, preliminary purification using silica gel column chromatography, and quantitation b y means of gas chromatography was found to be the preferred method.
T
HE DEVELOPMENT of methods for determining urinary pregnanediol (5p-pregnane-3a,20a-diol) originated with Venning (16, 17) and involved the gravimetric assay of pregnanediol glucosiduronate. In this procedure sodium pregnanediol glucosiduronate was extracted with butanol and purified by an acetone precipitation technique. Among the disadvantages of this method were the occurrence of emulsions during the butanol extraction and the difficulty of purifying the extract (14). Astwood and Jones (1) attempted to overcome the difficulties of the Venning method by hydrolyzing the conjugate with mineral acid in the presence of toluene. This method has gained wide acceptance, although many modifications have been reported. The advent of gas chromatography of steroids prompted many investigators to apply this technique to the determination of pregnanediol in biological extracts. In most cases the toluene-acid hydrolysis procedure has been utilized (2, 3, 7 , I S , 16, 18) and the extract submitted to analysis either directly or following prepurification and acetylation. Enzyme hydrolysis has been reported by some investigators and the extract is either purified by means of thin layer or column chromatography and acetylated, converted to the trimethylsilyl ether, or analyzed directly
(9-12). Unfortunately, very little information has been given concerning the reliability of these methods. The present investigation was undertaken to evaluate these possible methods for determining urinary pregnanediol using gas chromatography. The methods were : toluene-acid hydrolysis and extraction ( I S , 15); extraction of the conjugate with ether-ethanol (8) and hydrolysis with perchloric acid in anhydrous tetrahydrofuran (6); and enzyme hydrolysis, chloroform extraction, and silica gel column purification. These methods were compared with the spectrophotometric method of Goldzieher and Nakamura (4) used in these laboratories for 2 years. The methods were evaluated by study ing the recovery of free pregnanediol added t o urine and by analyzing a serially diluted pregnancy urine pool. EXPERIMENTAL
Apparatus. Model 15 and Series 5000 Barber-Colman gas chromatography units equipped with strontium-90 detectors (A-4150) and a Packard Model 7503 dual column gas chromatograph equipped with tritium detectors were used in this study. The stationary phase consisted of a mixture of 1% XE-60 and lyOSE-30 coated by the filtration technique (6) on 100-1 10 mesh Anakroxy ABS (Analabs, Inc., Hamden Conn.) previously washed with methanol and methylene chloride. Glass columns 1.8 meters X 6 mm. were used, with a column temperature of 205’-215’ C. and a gas flow rate of 50-150 ml./ minute, the temperature and flow rate being appropriately adjusted to give a retention time of 15-30 minutes for pregnanediol. Quantitation was performed by the peak height method, following injection of the samples dissolved in methylene chloride (injection of 1-8 pl. was sufficient). Samples of 1-5 pl. of standards containing 1.0 and 2.0 mg. of pregnanediol per ml. of
methylene chloride were used for relating the peak height to the amount injected. The detectors were calibrated once each day using the standards. Method 1. h 100-ml. aliquot of urine was mixed with 20 ml. of toluene, heated to boiling, and 10 ml. of concentrated hydrochloric acid was added. The mixture was boiled under reflux for 15 minutes, cooled, and the toluene layer removed. The aqueous layer was extracted with t w o 10-ml. portions of toluene, the extracts were combined, washed once with an equal volume of water, dried over sodium sulfate, and evaporated in v,zcuo. The residue was transferred to a 1-ml. volumetric flask and diluted to volume with methylene chloride. Method 2. A 50-ml. aliquot of urine was shaken with 24 grams of ammonium sulfate (enough to ensure saturation with ammonium sulfate), and extracted three times with a 25-ml. 3 : 1 mixture of ether and ethanol. The extracts were combined, dried over sodium sulfate, and evaporated in vacuo. The residue was treated with 50 ml. of 0.1N perchloric acid in anhydrous tetrahydrofuran (prepared by adding the required amount of 70y0 perchloric acid to tetrahydrofuran) and kept a t 50’ C. for 3 hours. The mixture was neutralized with 50 ml. of 0.3N sodium hydroxide and the tetrahydrofuran was removed in vacuo. The remaining aqueous mixture was extracted with three 50-ml. portions of methylene chloride. The extracts were combined, dried over sodium sulfate, and evaporated in vacuo. The residue was transferred. to a 1-ml. volumetric flask and diluted to volume with methylene chloride. Method 3. A 50-nil. aliquot of urine was adjusted to p H 4.5-4.8, and treated with 3 ml. of Ketodase (Warner-Chilcott form of beef liver p-glucuronidase) and 5 ml. of acetate buffer (prepared from three parts of 1.66X sodium acetate solution and two parts of 1 M acetic acid). The mixture was incubated a t 37’ C. for 24 hours, extracted with two 50-ml. VOL. 38, NO. 8, JULY 1966
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Table 1.
Urine Dilution Study
Comparison of Methods Method (pregnanediol, mg./l.)
Dilution group
Urine v o l . ~(ml.)
Relative concn.b
1
2
3
I I1 I11 IV
650.0 325.0 162.5 81.2 40.2
16.0 8.0 4.0 2.0 1.0
22.1 f 4 . 8 10.6 f 2 . 9 4 . 2 f 0.5 1.5 f0.6 0.5 f 0 . 3
15.9 f 0 . 9 9.7 f0.9 5.6 f 1.6 4.1 f 1 . 7 1.9 f0.3
13.9 f 0 . 9 7.4 f 0 . 4 4.1 f 0.1 2.1 k 0.1 1.1 f 0.1
v
4 16.1 f 4 Oc 7.7 f1.0 3 . 9 f 0.4 1.8 f0.2 1.0 f0.1
2 3 . 7 f 8.2d 12.1 f 0.8 5.5 f0.4 2.2 f0.3 1.5 f0.4
Each sample brought to a volume of 650 ml. by addition of mrater. bBased on the most dilute urine as 1.0, arbitrarily assigned to permit a basis for comparing the results for the several dilutions, c Values obtained using the same urine pool as for Methods 1 and 3. d Values obtained using the same urine pool as for Xethod 2. a
portions of chloroform, and the extracts were combined and washed with 50 ml. of O.1N sodium hydroxide, followed by 150 ml. of mater, and dried over sodium sulfate. The chloroform was removed in vacuo and the residue was dissolved in 5 ml. of benzene. This solution was chromatographed on a silica gel column made from 1-cm. 0.d. glass tubing as previously described (4). Air bubbles were dislodged by tapping after introducing 2-3 ml. of benzene, and a glass wool plug 5 mm. deep was pushed to the bottom of the column. A mixture of 1.0 gram of silica gel (Davidson Chemical Co., Grade 923, Code KO. 923-08-08-226, 100-120 mesh) and 3 ml. of benzene was introduced portionwise into the column using a pipet. The sealed tip of the column was immediately broken off after introduction of the first portion of silica gel, and the remainder of the silica gel mixture was added, followed by Peveral rinses of the container with benzene to ensure complete transfer of the silica gel. The 5-ml. solution of the sample in benzene was quantitatively transferred to the column and the benzene eluate was discarded. The column was de-
Table ll. Recovery of Pregnanediol Added to 100 ml. of Male Urine using Method 2
Pregnanediol Added (mg.) Recovered (mean) 1.0 2.0 4.0
5.0
1.0 1.6 3.6
4.0
Table 111. Recovery of Pregnanediol from Silica Gel Column Using Method 3 (1 .O mg. added ta 50-ml. aliquots)
Fraction
Pregnanediol recovered (mg.)"
0.025 0.008 0.032 0.045 0.565 3 0,555 0.060 4 0.146 Total (70) 75.4 68.2 a Values are for three replicates. 1 2
0.013 0.034 0.584
0.153 78.3
Fraction 1-original tube containing the chloroform extract 2-25y0 ethyl acetate-benzene 3-50% ethyl acetate-benzene 4-ethyl acetate
984
0
ANALYTICAL CHEMISTRY
veloped with 10 ml. of 25% ethyl acetate in benzene, and the pregnanediol fraction was eluted with 10 ml. of 50% ethyl acetate in benzene. The eluate was evaporated under a stream of nitrogen, transferred to a 1-ml. volumetric flask, diluted to volume with methylene chloride, and submitted to gas chromatographic analysis. Method 4. The method described by Goldzieher and Nakamura was used (4). RESULTS
Method 1. Recovery using the toluene-hydrochloric acid method was determined by analyzing male urine specimens containing 1 mg./100 ml. of added free pregnanediol. An average recovery of 75% was obtained from several determinations. I n order to gain information about the recovery of pregnanediol glucosiduronate, a third trimester pregnancy urine pool high in conjugated pregnanediol was collected. The pool was successively diluted with water and the individual diluted specimens rvere analyzed by each method. The relative recovery of each method at various levels of conjugated pregnanediol was determined in this manner. The dilution method is outlined in Table I, where there were five groups indicated by Roman numerals. The dilution was such that each succeeding group was one half of the concentration of the previous group. It should be emphasized that the exact value for each individual group in this series was not known. However, the relative concentration of pregnanediol a t each dilution was known, as the values obtained from analyzing the samples of each group should reflect the dilution pattern. The results for each method were calculated as mg./liter, and plotted as the log of the analyzed amount us. the log of the relative concentration based on the dilution. Theoretically, such a plot should yield a straight line with slope 1.00. Using Method I, five samples from each dilution group were analyzed and the results are given Table I. A plot of the log value us. the log of the dilution is shown in Figure 1. The slope was 1.46
calculated by the method of least squares. Method 2. The recovery of free pregnanediol from male urine was determined by adding various amounts of the steroid to 100-ml. urine aliquots. The results are given in Table 11, and the overall average recovery was 87%. The urine dilution study gave the values in Table I, and a plot of the d a t a is shown in Figure 1 where the slope was 0.80. Method 3. The recovery of free pregnanediol was 83y0 at a level of 0.03 mg./100 ml., but a t higher concentrations (2.0 mg./100 ml.) the recovery was less as shown in Table 111. The data in Table I11 emphasize the loss of pregnanediol during silica gel Chromatography and based on fraction 3 (eluate used for analysis), the mean recovery is 57%. At the higher levels difficulty is encountered in eluting all of the pregnanediol. An evaluation of this method using the urine dilution sequence gave the results shown in Table I, and the data are plotted in Figure 1 where the slope was 0.92. Method 4. This procedure has been in these laboratories during t h e past two years, and the recovery of pregnanediol was found t o be 7 5 4 0 % at levels of 0.03-0.3 mg./100 ml. of urins. The urine dilution study was applied to this procedure t o serve as a basis for comparing the other three methods. Two dilution studies were made using Method 4,one on the same pool as Methods 1 and 3 and the other with Method 2 given in Table I. A plot of the results is shown in Figure 2 where the slope was 1.01 for (c) in Table I and 1.04 for ( d ) in Table I. DISCUSSION
Accuracy. RECOVERY OF FREE PREGNANEDIOL. The original Astwood-Jones toluene-acid hydrolysis technique has been reported t o give a n average recovery of 68yo ( I ) and the gas chromatography modification 6095y0 (13,16). The average recovery of free pregnanediol using this procedure (Method 1) was 75y0 a t a level of 1.0
2.3
2.I 1.9
J
1.7
.-ti c
J \
&
1.5
E
Y
J
0
n w
1.3
2 U
5
1.1
w
a II
g
0.9 0.0
0.9
I
I
0.3
0.6
I
as
1.2
LOG DILUTION
J
Figure 2. Recovery of pregnanediol at various urine concentrations using Method 4
0.7
0 .t #O
1. Slope 1.04 2. Slope 1.01
0.3
0.6
0.9
LOG DILUTION Figure 1. tions
1.2
Recovery of pregnanediol at various urine concentra1. Method 1 2. Method 2 3. Method 3
mg./100 ml. of urine in our hands, and a better recovery could not be realized. The recovery of added free pregnanediol using Method 2 averaged 87% over a range of 1-5 mg./100 ml. of urine. On this basis Method 2 gave a better recovery of pregnanediol indicating the acid hydrolysis procedure in anhydrous tetrahydrofuran does not destroy as much of the free steroid as hydrolysis by Xethod 1. However, Method 2 depends on extraction of the conjugate before hydrolysis, and addition of free pregnanediol to urine to evaluate the recovery of Method 2 is not reliable since extraction of the free steroid is more readily accomplished than that of the conjugate which is highly water soluble. Method 3 gave a recovery of 83y0 at a level of 0.03 mg./100 ml. of urine and 57y0 a t 2.0 mg./100 ml. Indeed, in this instance the recovery depended on the level of pregnanediol and the low recovery a t higher levels is believed due to losses on the silica gel column used for preliminary purification (Table 111). Because of this fact, pregnancy urines which are high in pregnanediol (20-40 mg./24 hours) must be diluted 1 : l O before analysis by Method 3. Method 4 gave recoveries of i’5-80y0 for 0.03-0.3 mg./ 100 ml. of added free pregnanediol.
RECOVERY OF CONJUGATED PREGThe recovery of free pregnanediol added to urine does not give a true evaluation of a method since pregnanediol occurs largely as a conjugate with glucosiduronic acid. This limitation on using the addition of free steroid to urine to assess an analytical method must be considered when evaluating any steroid procedure since most steroids are present in urine as a conjugate. In this instance the addition of sodium pregnanediol glucosiduronate would be required in order to permit a more realistic evaluation of the methods in question. Because pure samples of sodium pregnanediol glucosiduronate could not be obtained, evaluation of the recovery of added conjugate was not possible. As an alternate approach, a urine dilution study was developed where a pregnancy urine pool high in conjugated pregnanediol was divided into five groups and diluted such that each group was one-half the concentration of the succeeding one. The analysis of samples from each group using the methods under scrutiny should permit the evaluation of each method over a range of concentrations of pregnanediol. Inspection of the results in Table I does not permit an evaluation of the methods,
NANEDIOL.
but by plotting the data as log values us. the log of the dilution (arbitrarily set a t 1 to 16) a straight line with slope of 1.00 should be obtained. This is based on the log separation of 0.3010 for units for dilution in increments of one half. Therefore, if the pregnanediol levels for each group determined by the analytical method in question are accurate, a slope of 1.00 should be obtained. The urine dilution procedure permits an evaluation of the overall accuracy of a method, although the exact value for an individual group is not known. In order to evaluate the methods using this procedure two separate urine pools were obtained. To one pool RIethods 1, 3, and 4 were applied, and for the second pool Methods 2 and 4. Method 4 was used as the reference method and gave slopes of 1.Q1 and 1.04 for the two separate studies in close agreement with the theoretical slope. Separation between the two slope lines (Figure 2 ) is attributed to different pools which would be expected to vary in total pregnanediol content. The close agreement between slope values obtained for Nethod 4 using different urine pools attest to the reliability of this method. The slope obtained for Method 1 was greater than theory (1.46), indicating a poor overall accuracy. Similarly, Method 2 gave a poor result with a slope of 0.80, perhaps because of the inability to extract all of the conjugate a t elevated levels. Method 3, with a slope of 0.92, approached that of theory, and in view of the low recovery of added free VOL. 38, NO. 8, JULY 1966
985
pregnanediol a t levels above 20 mg./ liter, the low slope is probably caused by losses a t the high levels. Methods 3 and 4 showed the best agreement with predicted slope and are considered to be the most accurate, accuracy in this instance being based on the slope values since the exact amount of pregnanediol is not known. Using the urine dilution procedure, it is assumed that dilution of the urine does not affect: hydrolysis of the conjugate; extraction of free pregnanediol or the conjugate; and the purity of the extract. The urine pool was diluted with water rather than urine to eliminate the introduction of other urine products. The first assumption is important when considering Methods 3 and 4 since an enzyme method is used, whereas acid hydrolysis (Methods 1 and 2) should not be influenced. Inhibitors in urine are known to interfere with the enzyme activity of p-glucuronidase and dilution of the urine would serve to dilute the inhibitors. However, a large excess of enzyme is used in each case and this effect should be negligible. hlethod 2 relies on the extraction of conjugated steroid from urine prior to hydrolysis, and dilution of the urine can be expected to exert an influence on the extraction. Because the urine is sa& urated with ammonium sulfate before the extraction this factor also is minimized. For the other methods the extraction of free pregnanediol should be little affected by dilution of the urine. The purity of the extract is affected by dilution of the urine but the ratio of impurities to the pregnanediol should be relatively the same a t each dilution. Methods 3 and 4 utilize a column purification step but because Methods 1 and 2 do not require this step, changes in the purity of the extract will influence these methods. However, this factor also is considered to be minimal. Specificity. The gas chromatographic columns used in this work were 1% XE-60 and 1% SE-30, a biphasic mixture which does not adequately separate all of the metabolites of progesterone, especially pregnanediol and pregnanolone (5p-pregnan-3a-ol-20-one). Pregnanolone is the only other major metabolite of progesterone, but pregnanediol occurs a t 3 to 16 times the level of pregnanolone. Methods 1 and 2 do not utilize prepurification before gas chromatography, and therefore, interference by pregnanolone can occur. On the other hand, Methods 3 and 4 utilize a silica gel column purification step which removes nearly all of the pregnanolone and interference with the pregnanediol analysis is minimal (4). Therefore, analysis of urinary pregnanediol using Methods 1 and 2 furnishes results which include pregnanolone. Other substances presumably also can interfere with the 986
0
ANALYTICAL CHEMISTRY
analysis of pregnanediol, even when gas chromatography is used, but no information is available to ascertain whether this occurs. Such a problem would have to be resolved by collecting the gas chromatographic fraction of pregnanediol from several typical analyses, and perform an analysis by other methods. However, preliminary column purification using thin layer or column chromatography serves to increase the specificity of the determination as well as to extend the life of the gas chromatography column and decrease detector contamination. Acetylation before gas chromatography is not necessary and merely imparts an additional step in the procedure. Method 4 in which final quantitation is accomplished with a nonspecific colorimetric technique requires two silica gel column purifications and acetylation to establish a high specificity. Goldzieher and Nakamura carried out extensive studies of the specificity of this method, and as long as the columns are used properly, reasonable specificity is obtained. The specificity of Methods 1 and 2 using the XE-60 and SE-30 column is not as good as that of Method 3 but by using a more polar phase such as QF-1, selectivity of the gas chromatography column can be improved, thus increasing the specificity of the methods. However, in our experience the QF-l column has a short life and frequent detector cleaning is necessary. Precision. Method 4 has been shown to have a high precision (4). From this study Method 3 had the best precision in terms of the deviation (Table I). As expected, the precision was better for most of the methods as the level of pregnanediol decreased. Sensitivity. The smallest amount of pregnanediol which can be determined in a sample submitted for analysis was found to be 0.1 mg./24 hour urine. However, Method 1 requires 100 ml. of urine and Methods 2, 3, and 4 require 50 ml. Sensitivity was ascertained by stipulating that the gas chromatography peak must be 1 cm. high, a height found to be reproducible from repeated injections. Convenience. The most rapid procedure for analyzing pregnanediol is Method 1 and analyses can be completed in one day. hlethods 2 and 3 require two days for completion and Method 4 can require up to 3 days, depending on the laboratory organization. Although Method 1 can be performed in one day, a large amount of technician time is required, whereas Method 3 requires little time during 24hour enzyme hydrolysis. In terms of running a large number of analyses, Method 3 readily lends itself to this type of operation, whereas Method 1 limits a technician to six analyses a t one time.
Choice of Method. I n terms of the overall accuracy, specificity, precision, sensitivity, and convenience, Method 3 is preferred. For high levels of pregnanediol the urine specimen must be diluted 1:10 with water before analysis (such as pregnancy wines), Method 3 has been in use in this laboratory for more than one year and has been proven to be reliable. Care must be used in performing the silica gel column chromatography step to obtain good results. If a quick pregnanediol analysis is required, Method 1 can be used, but Method 4, although good, is no longer utilized due to the inconvenience of analysis. Use of Method 1 will give a result which more appropriately should be called “progesterone metabolites” rather than pregnanediol since the various isomers of pregnanediol and pregnanolone are not separated adequately by the gas chromatography column. ACKNOWLEDGMENT
The authors thank J. W. Goldzieher for helpful comments. LITERATURE CITED
(1) Astwood, E. B., Seegar Jones, G. E., J. Biol. Chem. 137, 397 (1941). (2) Chamberlain, J., Knights, B. A., Thomas, G. H., J . Endocrinol. 28, 235 (1964). (3) Cox, R. I., J. Chromatog. 12, 242 (1963). (4) Goldzieher, J. W., Nakamura, Y., Acta Endocrinol. 41 , 371 (1962). (5) Horning, E. C., Mascatelli, E. A.,
Sweeley, C. C., Chem. Ind. (London)
1959, p. 751. (6) Jacobsohn, G. &I.,Lieberman, S., Bzol. Chem. 237, 1469 (1962).
J.
(. 7,) Jansen. A. P.. Clin. Chim. Acta 8 ,
785 (1963). (8) Kellie. A. E.. Wade. A. P.. Acta ~~, Endoci-&ol. 23. 357 (1965). (gjKirschner, M. A.,‘ Lipsett, M. B., Steroids 3 , 277 (1964). (10) Marmorstan, J., Crawley, L. G.,
Myers, S. M., Stern, E., Hopkins, C. E.. Am. J . Obstet. Gunecol. 92. 447 (1965 j. (11) Patti, A. A,, Bonanno, P., Frawley, T. F., Stein, A. A., Obstet. Gynecol. 21, 302 (1963).
(12).Raman, P. B., Avraman, R., hIcNiven, N. L., Dorfman, R. I., Steroids 6 , 177‘ (1965): (13) Seegar Jones, G. E., Turner, D.,
Sarlos, I. J., Barnes, A. C., Cohen, R., Fertility Sterility 13, 544 (1962). (14) Sinowara, G. Y., Reinhart, H. L., Am. J . Clin. Pathol. S u p p l . 4, 77
(1940). (15) Turner, D., Seegar Jones, G. E.,
Sarlos, I. J., Barnes, A. C., Cohen, R.,
Anal. Biochem. 5 , 99 (1963). (16) Venning, E. H., J. Biol. Chem. 119, 473 (1937). (17) Venning, E. H., Ibid., 126, 595 (1938). (18) Wotiz, H. H., Biochim. Biophys. Acta 69, 415 (1963).
RECEIVED for review January 24, 1966. Accepted May 4, 1966. Work supported by a N.I.H. Institution General Research Support Grant and by the J. A. Hartford Foundation, Inc.
