New derivatives for electron capture—Gas chromatography of steroids: A simplified procedure for measuring plasma testosterone

ANALYTICAL

30, 346-357

BIOCHEMISTRY

New A Simplified

MARVIN Endocrinology

(1969)

Derivatives for Electron Capture-Gas Chromatography of Steroids: Procedure

for Measuring

A. KIRSCHNER

Plasma

JOANNA

AND

Brunch, National Cancer Institute, National Bethesda, Maryland i?OOl4 Received

December

Testosterone

P. TAYLOR Institutes

of Health,

13, 1968

Since steroids have little affinity for electrons, they must first be converted to appropriate derivatives in order to be measured by electron captur+gas liquid chromatography. Derivatives currently being used that confer electron-capturing properties include chloroacetates (l-3), heptafluorobutyrbtes (49)) and pentafluorophenylhydrazones (10). We have evaluated two new derivatives formed from polyfluorinated acyl chlorides and hydroxyl-containing steroids. These derivatives, the hexadecafluoronanoates (HFN) and eicosafluoroundecanoates (EFU) (Fig. 1) have great%r electron-capturing properties and longer retention times compared to heptafluorobutyrates (HFB). Using the HFN, it

9-H-HEXADECAfLUORONANOATE

-

-

11.H-EICOSAFLUOROUNDECANOATE -

FIG.

1. Formulas

of testosterone 346

(HFN)

(EFU)

derivatives.

NEW

DERIVATIVES

has been possible to simplify plasma.

FOR

ELECTRON

the measurement

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347

of testosterone in human

MATERIALS

Solvents were obtained from Burdick and Jackson Laboratories, Muskegon, Michigan, and used without further purification. Tetrahydrofuran was purified as previously described (9). Reagents: Heptafluorobutyric anhydride, perfluorooctanoyl chloride, 9-H-hexadecafluoronanoyl chloride, and 11-H-eicosafluoroundecanoyl chloride were obtained from Peninsular ChemBesearch, Gainesville, Florida (courtesy of Mr. L. Williams). The anhydride was redistilled over P,05 as previously described (9)) and other reagents were used without further purification. Gas-liquid chromatography: The GLC system consisted of an F & M model 400 containing a siliconized glass U-tube 4 ft X 3.4 mm i.d. packed with 2.0% XE-60 on Diatoport S 60-80 mesh, or 2.0% QF-1 on Diatoport S 6&80 mesh. Temperatures used were 190 to 220’. GLC phases were purchased from F & M Scientific Co., Avondale, Pa. Carrier gas, argon/methane (95/5), was used at a flow rate of 100 ml/min. No purge flow was used. The detector consisted of a tritium foil operating at a pulse interval of 150 ,psec, a pulse width of 0.75 psec, and a pulse amplitude of 30 V. The electrometer gain was 10 with attenuation of 16, resulting in a full-scale deflection of 6 X lo-l1 A. A second unit, Glowall ,#310 containing a 6 ft x 3.4 mm i.d. column packed with 3.0% SE-30 Gas Chrom Q 80-100 mesh at 220” with hydrogen flame detection, was used to obtain retention time data. METHODS

Testosterone

Derivatives

were prepared in micro amounts of Wotiz and Clark (4, 9). Crystalline testosterone was prepared and characterized previously (9). Testosterone HFN was prepared in bulk adding 2.0 ml of a 5% solution of HFN acid chloride in benzene and 0.1 ml pyridine to approximately 20 mg testosterone. After incubating at 56°C for 30 min, reagents were dried under nitrogen, and the product was dried, purified by sublimation, and crystallized from acetone-hexane. The crystalline material had a melting point of 9g99”C. The infrared spectrum was virtually identical with that seen for testosterone 17-mono-heptafluorobutyrate, with major bands at 2950, 1773, and 1675 and a.minor band at 1615 cm-l. These bands indicate an ester carbonyl group, and an Testosterone

.heptafluorobutyrates

after the method heptafluorobutyrate

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KIRSCHNER

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intact A4-3-ketone. For the preparation of micro quantitites of testosterone HFN, 0.1 ml of a 5% solution of the acid chloride in benzene was added to the dried steroid. Pyridine was omitted. Following incubation at 56’C for 30 min, reagents were dried under nitrogen and a single partition between 5 ml hexane and 1 ml 70% methanol was used to remove unreacted testosterone. The reaction was usually quantitative, with less than 2% of testosterone remaining in the 70% methanol. Testosterone EFU was prepared in a similar manner, except that the acid chloride (solid) is made up to a 2% solution in benzene; 5 ml of this solution and 0.1 ml pyridine were added to the pure testosterone. Following incubation for 30 min, partition, sublimation, and crystallization yielded a slightly yellow rhomboid-shaped crystal melting sharply at 93-95’C. This slightly yellow color per&ted through several crystallizations. Infrared spectrum again revealed major bands at 2950, 1773, and 1675 and a minor band at 1615 cm-l. For the preparation of micro quantities of testosterone EFU, 0.2 ml of the 2% solution of the acid chloride in benzene was used, and pyridine was omitted. Following the reaction, excess reagents and unreacted testosterone were removed by the hexane/70% methanol (5/l) partition. Again reactions were usually quantitative. Testosterone perfluorooctanoate (PFO) was prepared from a 5% solution of the acid chloride in benzene, with pyridine as above. The melting point of 88’C agreed well with that reported by Nakagawa et al. (11). An infrared spectrum was not obtained owing to laboratory accident. Estradiol

Derivatives

The derivatives of estradiol were prepared in micro quantities using the method described above. Unreacted estradiol was separated from the 17-mono- and 3,17-di-derivatives either by partition between 70% methanol and hexane or by use of a small column (Pasteur pipet) containing Sephadex LH-20 swelled in benzene. In the first system, unreacted estradiol remained in the methanol. In the latter, the unreacted estrogen remained in the column, whereas the derivative was all eluted within a volume of benzene equal to the pore volume of the column. Formation ,of 17-monoderivatives: To 10 pg estradiol was added 25,000 dpm 3H-estradiol (ca. 0.5 ng). The 3,17-di-derivatives were formed as above. Following their formation, the products were spotted and developed in the thin-layer system, benzene/ethyl acetate, 80/20, using silica gel GF-254. Under these conditions, the phenolic hydroxyl group is cleaved, to the 3-hydroxy-17-mono-derivative, which is simul-

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349

CAPTURGGLC

taneously chromatographed. After eluting with benzene, an aliquot was counted to establish recovery. Benzene was then added to make a solution containing 1 ng/pl. Separation of 17-mono- from 3,17-di-derivatives: In some instances, derivatization was incomplete (especially with PFO derivatives). Separation of the 17-mono- from the 3,17-di-derivatives was accomplished by using reversed-phase thin-layer chromatography. Acetonitrile/water, 3/l, was used as the mobile phase, vs. 10% decane in isooctane impregnated on siliconized silica gel G (Reversil-3, Applied Science Laboratories, State College, Pennsylvania). In this system, estradiol, 3,17diPF0, as well as the diHFB, diHFN, and diEFU remain on the origin, whereas their respective 17-mono-derivatives travel with the solvent front. RESULTS

Retention

Time Characteristics

Retention times of the various electron-capturing derivatives of testosterone and estradiol are listed in Table 1. Retention times obtained using the phase SE-30 generally vary with molecular weight. The shorter Relative

Retention

TABLE Times

1 of Steroid

SE-30a

Derivatives XE-Gob

Testosterone 17-mono-HFB I‘ -PFO “ -HFN “ -EFU ‘L chloroacetate

1.00 1.61 2.80 3.46 2.86

Estrodiol 17-mono-HFB ‘I -PFO ‘I -HFN ‘L -EFU “ chloroacetate

1.04 0.88 1.68 2.29 2.86 2.64

1.20 4.50 5.10 -

0.32 0.27 0.68 3.40 4.40 1.25

3,17-di-HFB ‘I -PFO L‘ -HFN I‘ -EFU

0.95 1.77 2.64 3.32

0.20 0.31 2.80 3.20

0.33 1.16 5.40 8.15

0 3.0% * 2.0% c 2.0y0

1.10

(6.0 min)

2.50 1.00 1.12 4.30 Ti.10 2.50

QF-1~

(4.2 min)

3.50 1.10

SE-30 on Gas Chrom Q 80-100 mesh, 220°C. XE-60 on Diatoport S 60-80 mesh, 200°C. QF-1 on Diatoport S 60-80 mesh, 21O”C, flow

100 ml/min.

0.80 1.00 1.70 3.50 4.60 3.00

(4.5 min)

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retention times of the HFB derivatives compared to their parent alcohols have been described previously (6, 12). The retention times of the HFN derivative are approximately double those of the PFO. These differences in retention times are equally striking on QF-1 and XE-60 phases. The HFN and EFU derivatives of testosterone have long retention times, as do those of the chloroacetate. Derivatives of estradiol have differing retention times on the polar phases &F-l and XE-60 depending on whether one or two functional groups are derivatized. On XE-60, polarity appears to be the major factor, since the 3-hydroxy-17-mono-derivative in each case has a longer retention time compared to its 3,17-“di”-derivative. On &F-l, however, the increased molecular weight of the 3,17-di-derivatives results in longer retention times compared to the more polar 3-hydroxy-17-monoderivatives. Relative

Area Responses

The relative electron-capturing properties of the testosterone and estrogen derivatives are compared in Table 2. As previously reported, testosterone PFO provides a greater response when compared with equimolar amounts of the chloroacetate or HFB (6, 11). The HFN and EFU derivatives even exceed the PFO in this respect and give more than twice the area response of testosterone HFB. A comparison of retention times and relative area responses for equimolar amounts of the various testosterone derivatives is seen in Figure 2. There is a marked discrepancy between the electron-capturing propTABLE 2 Relative Area Responsesa Derivative

Testosterone (17-mono)b

Estradiol (17-mono)c

Estradiol (3,17-di)

Chloroacetate HFB PFO HFN EFU

0.22 1.00 1.82 2.30 2.55

0.03 0.05 0.08 0.07

2.65~ 2.740 3.34* 3.00*

0 Derivatives were purified through one chromatographic step. The concentration was then adjusted to 1 ng/pl based on recovery of added radioactivity. Area response was measured as the product of retention time and peak height. 2 ng testosterone-17-monoHFB = 6700 mm2. h 2.60/n &F-l in Diatoport S 60-80 mesh, 220”, flow 100 ml/min. c Column temperature 210”, flow 100 ml/mm. * Column temperature 225”, flow 100 ml/min.

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PFO

PIG. 2. Area responses of 2 ng testosterone HFB, PFO, HFN, tively. Conditions: 2% &F-l, 210°C; for GLC settings see text.

and EFU, respec-

erties of 3,17-di-derivatives of estradiol compared to those of the 3-hydroxy-17-mono-derivative. Virtually all of this affiity is associated with derivative formation at the phenolic position. The 3,17di-HFN and di-EFU have only slightly greater area responses compared to those of the di-HFB and di-PFO. Development

of a Simplified Procedure for Measuring in Human Plasma

Testosterone

Rf values of the various HFN derivatives of steroids known or thought to be present in human plasma are shown in Table 3. From these data, it is apparent that most HFN derivatives are separated from testosterone HFN by thin-layer chromatography. Of the possible remaining steroidal contaminants, the HFN derivatives of 20/3- and 20ahydroxyprogesterone and epitestosterone are separated from testosterone HFN by GLC on the phase &F-l. Thus, by using the HFN derivative instead of the heptafluorobutyrate it was possible to eliminate the preliminary thin-layer chromatography step of the earlier method (9). In addition, the increased sensitivity of testosterone HFN compared to the HFB makes it possible to use smaller aliquots of plasma for the determination. By eliminating an acetic acid wash (Q), troublesome oils are

352

XIRSCHNER

AND

TABLE 3 Thin Layer RI Values and GLC Retention HFN derivative

of

TAYLOR

Times on HFN Derivatives Rel. retention

W

Androsterone Etiocholanolone Dehydroepiandrosterone Testosterone Epitestosterone 5@-Dihydrotestosterone 5a-Dihydrotestosterone 20@-Hydroxyprogesterone 20a-Hydroxyprogesterone 17a-Hydroxyprogesterone Hydrocortisone 11-Desoxycortisol Cortisone

0.55 0.56 0.56 0.43 0.43 0.54 0.54 0.43 0.43 0.13 0.13 0.15 0.10

1.00 0.75 1.33 1.52 -

= Benzene/ethyl acetate, 80/20. Similar RI’s are obtained using benzene/ethyl 95/5-sandwich system. b 2y0 &F-l on Diatoport S 60-80 mesh, 220°C flow 100 ml/min.

no longer a problem, and a “prerun” 100% hexane was also eliminated. Method

timeb

acetate,

of the plasma extract in TLC using in Detail

In men, l-4 ml plasma is diluted to 10 ml with distilled water. In women, 5-10 ml plasma are used. With each set of 13-24 samples, two water blanks of 10 ml are also processed. Following the addition of 4000 cpm (0.4 ng) 3H-testosterone to monitor methodologic losses, 0.5 ml N NaOH is added, and the plasma is extracted twice with 3 vol ether. The ether extract is washed twice with l/l0 vol water, and allowed to dry in a hood overnight. The dried ether extract is transferred to a 13 ml ground-glass stoppered centrifuge tube and HFN derivatives are made by addition of 0.1 ml of a 5% solution of HFN acid chloride in benzene. The tubes are shaken and heated in a sand bath at 56°C for 30 min. The reagents are then evaporated under nitrogen and the products partitioned between 5 ml hexane and 1 ml 70% methanol. The hexane layer is transferred to a new tube, dried under nitrogen, and spotted with benzene on precoated silica gel GF-254 plates (Brinkmann Instruments, Great Neck, New York), and developed in a sandwich system using 95/5 benzene/ethyl acetate (9). The testosterone-containing region is located opposite testosterone EFU used as a marker. The silica gel is sucked into Pasteur pipets packed with glass wool, and eluted with 3 ml benzene. After the benzene is dried, appropriate

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353

amounts of a solution containing 0.2 ml testosterone EFU in benzene is added as an internal standard. Ten microliters of the sample is taken for counting and 10 ~1 is injected into the GLC system. A standard curve is established with each set of determinations as follows: 0.5 ng standard testosterone HFN is mixed in a syringe with 2 ng testosterone EFU. This mixture is chromatographed. The area responses of both compounds are measured and expressed as a ratio. Similar ratios are obtained relating area response of 1.0 and 2.0 ng testosterone-HFN to that of 2 ng internal standard. The ratios are plotted on the ordinate vs. ng testosterone on the abscissa. From the chromatograms of a given plasma sample or water blank, the area of testosterone HFN and the internal standard are calculated, and their ratios obtained. By referring this ratio to the standard curve, the amount of testosterone present in the final 10 ~1 aliquot is obtained. Since an equal aliquot is counted: cpm 3H-testosterone added to sample cpm 3H in final sample X ng teetost,erone in final sample - water blank value in ng/lO m

ng in original sample =

Measurement ‘of A4-Andros tenedione in Plasma

Measurement of A4-androstenedione in plasma is performed using the HFN derivative; 8 ml plasma is required. Following extraction of the plasma with ether as above, thin-layer chromatography is performed using the system benzene/ethyl acetate 64/40, in order to separate A’androstenedione from testosterone. The fraction containing A4-androstenedione is eluted with 4 ml ethyl acetate, dried, and converted to testosterone (9). The HFN is then prepared, and the sample is thereafter handled in the same manner as testosterone. Evaluation

of the Method

Sensitivity: The minimum amount of testosterone readily quantified is 0.1 ng and full-scale deflection is 2.0 ng (Fig. 3). Recovery through the procedure ranges 25 to 40%. This relatively poor recovery probably reflects the difficulty with which testosterone HFN is eluted from the TLC plates. This has been improved recently by using an eluting mixture of benzene/methanol 10/l. Since l/3 of the final sample can be injected into the GLC system, a minimum of 0.7 to 1.2 ng per sample can be seen and quantified. Water blanks, and plasma blanks (plasma from patients with endocrine glands surgically removed) generally show a peak in the testosterone region of the GLC chromatogram. This “blank” value is independent of the amount of plasma used, and equals about 0.1 ng in the final chromatogram. The coefficient of variation for estimates below 80 ng/lOO ml is 30%; thus, when using 8 ml of plasma from women, a sample containing 23

354

AND

( 1

TAYLOR

2.0 nonogroms TESTO-HFN

\

FIG. 3. Left, area response of 0.1 ng testosterone HFN. This represents the lowest quantifiable peak area for the conditions used. Right, 2.0 ng testosterone HFN. Shaded area represents testosterone EFU used BS internal standard.

ng/lOO ml of testosterone can be differentiated from zero, with p < 0.05. From men with 3 ml of plasma, a minimum of 63 ng/lOO ml, and with 1 ml of plasma, 190 ng/lOO ml can be differentiated from zero, with p < 0.05. Specificity: As noted in Table 3, most of the steroids that are now known to be present as contaminants are separated from testosterone HFN by the thin-layer and GLC. Since this method eliminates one thinlayer purification step from that previously described by this laboratory, it was necessary to compare results obtained by the longer “heptafluorobutyrate method” vs. the shorter “HFN method.” The results of samples analyzed by both procedures are shown in Figure 4. There is adequate correspondence of values between the methods. Thus, specificity does not seem to suffer by elimination of the preliminary thin-layer step. Precision: The adequacy of precision is attested to by the excellent agreement between the HFB and HFN methods. Based on this comparison, coefficients of variation in the normal female range are 30%. Elevated levels, 90-160 ng/lOO ml are measured with a coefficient of variation of 137%. In men, using 3 ml aliquots, values less than 600 ng/lOO ml have coeffcients of variation of 13% and values greater than 600 ng/ 100 ml have a 4% coefficient of variation. Representative GLC tracings from a normal man and woman are presented in Figure 5.

NEW

FIG. 4. Comparison (long method).

DERIVATIVES

of values

FOR

obtained

ELECTRON

by

HFN

355

CAPTUREGLC

method

(short

method)

vs. HFB

DISCUSSION

In considering which derivative to use for of the factors to be considered are: (1) will ficient sensitivity and (2) will the derivative separated from the background impurities? chloroacetates and heptafluorobutyrates fulfill

electron capt.ure GLC, two the derivative provide sufenable the compound to be As previously reported, the these criteria for the mea-

MALE

FIG. 5. Left, testosterorw Right, testosterone content represents testosterone EFU

PLASMA

content in 8 ml extract of plasma from a normal woman. in 4 ml extract from a normal male. Shaded area used as internal standard.

356

KIRSCHNER

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surement of testosterone, as well as for 20~ and SO@hydroxyprogesterones in human plasma. They are not adequate, however, for the measurement of other steroids in biological fluids. More sensitive derivatives are needed. Although heptafluorobutyrates are generally considered as “sensitive” derivatives for electron-captur+GLC, it seems clear, from the studies of Nakagawa et al. (11) and those reported here, that the perfluorooctanoates of testosterone offer twice the sensitivity of the HFB using the conditions described herein. Sensitivity can be augmented even further by preparing the HFN or EFU. An additional advantage of the PFO and HFN derivative is the longer retention times on polar and nonpolar phases, which offer a better chance to free the steroid from contaminant peaks associated with solvents, silica gel, etc. The EFU derivative of testosterone provides an even longer retention time requiring lower loaded phases or higher column temperatures in order to elute at a reasonable retention time. Since tritium foil detectors cannot be used above 225OC, testosterone EFU is not an ideal derivative. Such temperature limitations do not exist for 63Ni detectors however, and thus the EFU may be better suited for use in such systems. The relative area responses of the 17-mono-derivatives of estradiol, are in agreement with the studies of Exley and Chamberlain (12) and van der Molen, Maas, and Groen (6) using estradiol monoHFB. Estradiol derivatives at the 17-position are virtually inert-all of the electron capture properties reside in the phenolic derivative. It is of interest that all four derivatives tested gave approximately the same molar response. By using the HFN derivative, it has been possible to simplify and shorten the time required for the measurement of testosterone in human plasma, without losing accuracy. The simplified method described here requires extraction, derivatization, one thin-layer chromatography, and then GLC. It would appear that measurement of testosterone by the HFN derivative and electron captureGLC is as simple, rapid, and accurate as the newer competitive protein-binding methods (13,14). If the C, and C,, derivatives described here represent an advance in electron-capturing derivatives, it should be possible to find other derivatives, possibly of higher chain length, that would be even better. Furthermore, by condensing chlorinated alcohols with polyfluorinated alcohols, it might be possible to make derivatives with far more sensitivity than are now available. SUMMARY

Two new derivatives are described that properties to hydroxyl-containing steroids.

confer electron-capturing The 9-H-hexadecafluoro-

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nanoate (HE’N) and ll-H-eicosafluoroundecanoate (EFU) derivative of testosterone have 2.30 and 2.55 times the area response of equimolar amounts of testosterone heptafluorobutyrate, as well as longer retention times on both polar and nonpolar phases. Thin-layer and GLC properties of the 17-mono-derivatives and the 3,17-di-derivatives of estradiol are presented, Using the HFN derivative, a simplified method is presented for the measurement of testosterone in human plasma. ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Dr. Hildegarde Wilson and Mortimer B. Lipsett in revising the manuscript. The assistance of Dr. Robert Highet in preparing sublimates, and the useful discussions of Dr. Henry Fales, are appreciated. REFERENCES C. BROWNIE, H. J. VAN DER MOLEN, E. E. NISHIZAW.~, AND K. B. EIK-NES, J. Clin. Endocrinol. 24, 1091 (1964). 2. H. J. VAN DER MOLEN, AND D. GROEN, J. Clin. Endocrinol. 25,1625 (1965). 3. H. J. VAN DER MOLEN, D. GROEN, AND J. H. VAN DER MAAS, Steroids 6, 195 (1965). 4. S. J. CLARK, AND H. H. WOTIZ, Steroids 2, 535 (1963). 5. D. EXLEY, in “Androgens in Normal and Pathological Conditions” (D. Exley, 1. A.

6. 7. 8.

9. 10. 11. 12. 13.

14.

ed.), p. 11. Excerpta Medica International Congress Series #lOl, Amsterdam, 1966. H. J. VAN DER MOLEN, J. H. MAAS, AND D. GROEN, European J. Steroids 2, 119 (1967). H. H. WOTIZ, G. CHARRANSOL, AND I. N. SMITH, Steroids 10, 127 (1967). D. EXLEY, B&hem. J. 107,285 (1968). M. A. KIRSCHNER, AND G. D. COFFMAN, J. Clin. Endowi&. 28,1347 (1968). J. ATTAL, S. M. HENDELES, AND K. B. EIK-NES, AnaE. Biochem. 20, 394 (1967). K. NAKAG.~WA, N. L. MCNIVEN, E. FORCHIELLI, A. VERMEULEN, AND R. I. DORFMAN, Steroids 7, 329 (1966). D. EXLEY, AND J. CHAMBERLAIN, Steroids 10,509 (1967). R. HORTON, T. KATO, AND R. SHERINS, Steroids 10,257 (1967). D. MAYES, AND C. A. NUGENT, J. Clin. Endocrinol. 28, 1169 (1963).