Gas-L iquid Chromatographic Separation of Thyroid Hormones SIR: A study has been undertaken to develop a method for quantitation of the physiologically active components of thyroid drug preparations by gas-liquid chromatography. A satisfactory method must be capable of determining the active components 3,5,3 ’ 3‘-tetraiodothyronine (thyroxin, T4) and 3,5,3 ’-triiodothyronine (T3) and must also be capable of detecting and determining nonphysiologically active components such as 3,3 ’,5 ’-triiodothyronine (T3’), 3,5-diiodothyronine (T2), and 3,5-diiodotyrosine (DIT) (I). At present, a variety of procedures are being used for quantitation of thyroid preparations based on the non-specific cericarsenite color reaction first described by Sandell and Kolthoff (2) or upon the U.S.P. thiosulfate titration procedure (3). Column, paper, and thin-layer chromatographic methods ( 4 ) may give more specific information as to composition but few of these have been applied to thyroid preparations. Gasliquid chromatographic methods ( 5 , 6 )have been proposed for thyroid substances in blood or serum but the authors have not included T3’ in their standards and have used two-step volatile derivative preparation procedures. Recently Klebe, Finkbeiner, and White (7) proposed bis(trimethylsilyl) acetamide (BSA) as a reagent for obtaining volatile trimethylsilyl derivatives of amino acids in one step. We have found that thyronine, tyrosine, and their halogen analogs form the tris(trimethylsily1) derivatives because the reagent silylates the carboxyl, amino, and phenol groups. These derivatives are formed rapidly and are very stable. Gas-liquid chromatography of these derivatives on a temperature programmed short column with flame ionization detection allows separation and quantitation of iodinated amino acids in submicrogram quantities. EXPERIMENTAL
Apparatus. A Barber-Colman Model 5000 was equipped with both flame ionization and electron capture (A-4072 at 25 Volts) detectors, two 1-mV recorders and a splitting device. A 2 ft x 3 mm i.d. glass column packed with 2 z SE-33 on 100/120 mesh Chrom Q (Applied Science Laboratories, Inc.) was used with a nitrogen flow rate of 50 ml/min at 18 p.s.i. The splitting device was used to channel 9 5 x of column effluent to the flame detector and 5 to the electron capture detector. Operating temperatures were: injector, 270 “C; detector, 275 “C; column programmed from 150 to 280 “C at 10 “C/min. (1) S. B. Barker, “Methods in Hormone Research,” Vol. I, R. I. Dorfman, Ed., Academic Press, New York, 1962, p 3531. (2) E. B. Sandell and I. M. Kolthoff, J. Am. Chem. SOC.,56, 1426, (1934). (3) “Pharmacopeia of the United States of America,” Rev. XVI, Mack Publishing Co., Easton, Pa., 1960, p 759. (4) C. A. Johnson and R. E. A. Drey, Anulyst, 92, 328, (1967). (5) P. 1. Jaakonmaki and J. E. Stouffer, J. Gus Chromatogr. 5, 303, (1967). (6) A. H. Richards and W. B. Mason, ANAL. CHEM.,38, 1751 (1966). (7) J. F. Klebe, H. Finkbeiner, and D. M. White, J. Am. Chem. SOC.,88:14,3390 (1966).
1.
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TIME, MINUTES 11’0
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COLUMN
250
TEMPERATURE
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264
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Figure 1. Chromatograms of thyroid hormone standard mixture with flame detector See text for conditions and abbreviations
A Perkin-Elmer Model 337, Grating infrared spectrometer with cardboard micro disk forms (Limit Research Corp., Darien, Conn.) was used. Reagents used were BSA (Perco Supplies, P. 0. Box 201, San Gabriel, Calif. 91778); chlorotrimethylsilane (CTMS) (Eastman); KBr, infra red quality, (Harshaw Chemical Co., Cleveland, Ohio); SE-33 (Barber-Colman); squalene (Barber-Colman); tetrahydrofuran, methanol, ammonium hydroxide-reagent grade; amino acids, tyrosine (Ty), DIT, T2,T3,and T4(Nutritional Biochemicals), T3’(Research sample from Division of Pharmaceutical Chemistry, Food and Drug Administration, Washington, D. C.). Solutions. For the reagent mixture, mix 5.00 ml of THF, 2.00 ml of BSA, and 5 drops of CTMS in a 2-dram vial with a foil lined screw cap. The internal standard was 25.0 mg of squalene per 50.0 ml in methanol. Ammoniacal methanol used was 5% vjv. Amino acid standards were 6.00 microVOL. 40, NO. 10, AUGUST 1968
* 1587
moles per 5.00 ml in ammoniacal methanol. Standard mixtures were made by mixing equal volumes of squalene and each amino acid solution. and dilute to desired volume with ammoniacal methanol. Procedure. Evaporate an appropriate aliquot of a mixed standard to dryness in a 2-dram vial. Add 100 pl of reagent mixture. Seal vial with a foil lined screw cap and warm on front edge of a water bath under a hood for 1 minute. Remove and allow to cool to room temperature. This derivative mixture is stable for several weeks if the vial is wrapped in foil and stored in a cool place. Suitable aliquots are taken for injection in the gas chromatograph.
RESULTS AND DISCUSSION
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Figure 2. Chromatogram of thyroid hormone standard mixture with electron capture detector
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(8) D. M. Oaks, H. Hartrnann, and K. P. Dimick, ANAL.CHEM., 36,1560(1964).
See text for conditions and abbreviations
.7
Figure 1 shows the chromatograms obtained from two standard mixtures using a flame ionization detector. Curve A represents a solution containing 4.50 X micromole of each amino acid and 1.50 micrograms of squalene and curve B was obtained from a solution containing 7.50 x 10-5 micromole of each amino acid. Curve A represents approximately 3 micrograms of T3 and curve B approximately 50 nanograms of Ts. The peaks correspond to the retention times obtained with the individual components and are symetrical. Figure 2 illustrates the chromatogram obtained from an aliquot containing approximately 50 nanograms of T3 using the electron capture detector in a low sensitivity mode to minimize base-line drift caused by the temperature program and subsequent changes in flow rate. Similar results are obtained using the splitting device and larger aliquots. Comparison of the peak areas for the identical quantities shown in Figure 2 and curve B, Figure 1 substantiates that EC/FL ratios are large for compounds containing iodine and small for non-iodinated compounds as suggested by Oaks, Hartmann, and Dimick (8). Such 4 values can be obtained readily using the splitter.
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3 4 5 ti MOLE R A T I O = MOLE BSA/MOLE
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.AM I NO A C I D
Figure 3. Moles of reagent (BSA) required for derivatization of amino acids 1588
ANALYTICAL CHEMISTRY
IO
3500
3000
2500
2000
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Figure 4. Portions of infrared spectra of free amino acids (-) (....)
Results of three replicate determinations on each of three consecutive days of 4.5 x loda micromole aliquots of a standard mixture showed standard deviations [calculated from the range (9) on each set of three] of from 0.17 to 2.08 % of the amounts present. The average standard deviation for all compounds was 1.14%. A study of the BSA derivatives was conducted using phenylalanine, tyrosine, and diiodotyrosine. Phenylalanine should give a bis derivative while the tyrosines should give tris derivatives. The completeness of reaction between various mole-ratios of BSA and the individual amino acids was determined using l , 2, 3,4, and 10 moles of BSA to l mole of the acid. Each reaction vial was purged with nitrogen and closed before heating for 15 minutes. Aliquots were then assayed with the results shown in Figure 3. The reaction with phenylalanine is complete with slightly more than two moles of BSA per mole of acid while the tyrosines require slightly more than three moles of BSA to react completely. Figure 4 shows portions of the infrared spectra obtained for phenylalanine and the tyrosines. The solid line spectra are of the free amino acids. The dotted line spectra are the corresponding silyl derivatives obtained by trapping approximately 60 pg from the GLC column on KBr. The silylation of the phenolic groups is shown by the disappearance of the strong phenolic absorbance at approximately 3250 cm-1 and by the appearance of the Si-0-AR stretching vibration band at 930 cm-’ (10). It should be noted that these bands are not present in the spectra of the phenylalanine.
1200
1100
IO00
900
800
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and corresponding tris-trimethylsilyl derivatives
The silylation of the nitrogen is shown by the disappearance of the weak nitrogen overtone band at 2100 cm-’ which is distinctive for amino acids and which is absent if the nitrogen atom is substituted (10). Infrared spectra of T2,Ts,and Td show the appearance of the Si-0-AR band at 920 cm-’ but the nitrogen overtone band at 2100 cm-I is too weak to observe for the quantities of materials used. The method is now being used to evaluate the effects of variations in conditions for hydrolysis of thyroid preparations and to study methods of separation of the iodinated amino acids from the hydrolysate. ACKNOWLEDGMENT
The author is grateful to Joseph Levine, Division of Pharmaceutical Chemistry, Food and Drug Administration, Washington, D. C., for the sample of 3,3 ‘,5 ‘-triiodothyronine and to C. T. Kenner, Science Advisor to the Dallas District, FDA, for technical assistance. LYDELL B. HANSEN Dallas District Food and Drug Administration 3032 Bryan St., Dallas, Texas 75204 RECEIVED for review August 7,1967. Accepted May 31,1968. (10) R. M. Silverstein and G. C. Bassler, “Spectrometric Identification of Organic Compounds,” 2nd Ed., Wiley, New York,
(9) R. B. Dean and W. J. Dixon, ANAL.CHEM.,23,636, (1951).
1967, pp 97, 102.
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