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Gas chromatographic determination of iodinated compounds
ANALYTICAL
BIOCHEMISTRY
Gas
27,150-161
(1969)
Chromatographic
Determination
lodinated KAZUHISA National National
Compounds
FUNAKOSHP Institutes Institutes
AND
of Arthritis
of Health, Received
of
May
H. J. CAHNMANN
and Metabolic Diseases, Bethesda, Maryland 20014 23, 1968
The classical methods for the detection and quantitative determination of iodoamino acids and related iodinated compounds are paper and thinlayer chromatography. Only in recent years has gas chromatography been applied to the major iodoamino acids (1, 2). After it had been discovered that BSA2 is a powerful reagent for the conversion of nonvolatile or insufficiently volatile compounds to their volatile TMS derivatives (3)) we began to test the suitability of this method of derivatization for the gas chromatographic analysis of iodoamino acids as well as of a large number of other iodinated compounds. The method is now being used routinely in our laboratory for purity checks, for the identification of unknown compounds by comparison with known standard samples, and for the resolution and quantitative evaluation of complex mixtures of iodinated compounds. A few applications of the method have been reported by us in a preliminary fashion (4). Subsequently Alexander and Seheig described the gas chromatographic determination of the major iodoamino acids using a similar method of derivatization (5). The present paper gives the details of our procedure for the determination of a large variety of iodinated compounds and discusses various characteristics of the TMS derivatives of the iodoamino acids. The compounds tested comprise not only the known iodinated tyrosines and thyronines, but also their deamino, position, and side-chain analogs as well as various intermediates in the synthesis of the thyroid hormones. The ‘Visiting Scientist from the Faculty of Pharmaceutical Sciences, Kiushu University, Japan. ’ Abbreviations : EC, electron capture; BSA, N,O-bis(trimethylsilyl)acetamide; TMS, trimethylsilyl; TYR, tyrosine ; MIT, 3-iodotyrosine ; DIT, 3,54iodotyrosine, T, thyronine ; T,, 3-iodothyronine ; Tz, 3,6diiodothyronine; T,‘, 3,3’-diiodothyronine Ta, 3,5,3’-triiodothyronine ; T31, 3,3’5’-triiodothyronine ; T,, thyroxine. 150
;
GAS
CHROMATOGRAPHY
OF
IODO
COMPOTXDS
l.=ll
amounts determined were usually a few micrograms, but when the normally used hydrogen flame ionization detector was replaced with an EC detector samples in the nanogram and picogram range could be analyzed as well. EXPERIMENTAL
Starting Materials and Equipment
4-Hydroxy-3-iodobenzaldehyde was synthesized by the controlled iodination of p-hydroxybenzaldehyde (6). The method is a modification of that described by Barnes et al. (7). 4-Hydroxy-3,5-diiodobenzaldehyde and most of the other unbranched deamino analogs of DIT were synthesized as described previously (8). The a,/?-dihydroxypropionic acid analog of DIT was synthesized by borohydride reduction of a T,-precursor described by Nishinaga et al. (4). 4- (4-Acetoxy-3,5-diiodobenzal) -2-methyl-5-oxazolone, an intermediate in the synthesis of the keto acid analog of DIT, was synthesized as described for the 1311-labeled compound (9). The keto acid itself, 4-hydroxy-3,5-diiodophenylpyruvic acid, was purchased from the Osaka Laboratory of Synthetic Organic Chemicals, Nishinomiya, Japan, or from J. T. Baker Chemical Co., Phillipsburg, N. J., and once recrystallized from glacial acetic acid. The corresponding 3-monoiodinated oxazolone and keto acid were prepared in analogous manner (6). 4- (p-Hydroxyphenoxy) -3,5-diiodobenzylmalonic acid, thr a-carboxypropionic acid analog of DIT, was synthesized according to HSfer and Cahnmann (10). T, and T, in the form of the free acids were a gift of Dr. J. F. Kerwin (Smith Kline and French Laboratories). The other thyronines were commercial products or gifts from various sources. Most of the side-chain analogs of the iodothyronines were a gift of Dr. R. I. Meltzer (Warner Lambert Research Institute). The keto acid analog of T, was generously supplied by Dr. R. Pitt-Rivers. All acids were used in the free form since their salts react only sluggishly with BSA. The meta analogs of T, were synthesized as described previously (11). All other starting materials were commercial products. As BSA is hydrolyzed by moisture and decolorized on long standing in rubber-sealed vials, it was purchased in 1 ml glass-sealed ampoules (Pierce Chemical Co.). Opened ampoules could be kept for up to a few weeks in Teflonlined screw-cap vials which contained a powerful drying agent (calcium hydride) under a wad of glass wool. All solvents were reagent grade. A Chromalab (Glowall Corp.) gas chromatograph or a Microtek MT220 gas chromatograph (Mikrotek Instruments Corp.) was used in conjunction with a hydrogen flame ionization detector. Only the latter instrument was employed with an EC detector (63Ni) which could be
152
FUNAKOSHI
AND
CAHNMANN
supplied with either direct or pulsed current. Direct current (45 V) was used in most instances. Glass columns were filled with either lo/O OV-1 (a methylsiloxane polymer) or 1% OV-17 (a phenylmethylsiloxane polymer) on S&100 mesh Chromosorb W HP (Supelco). The length of the columns ranged from 2 to 6 ft and their i.d. from 1.7 to 3.3 mm. The carrier and scavenger gas (frequently omitted) was nitrogen (ultra high purity, Southern Oxygen) or argon (Matheson), except when a pulsed power supply was used, in which case nitrogen was replaced with argon10% methane (Matheson). A LKB gas chromatograph-mass spectrometer type 9000 was used for mass spectrometric analyses. Analytical
Procedures
Glass vials of NO-250 ~1 capacity equipped with a Teflon-lined silicon rubber septum (Kontes K-774150-0500) and an aluminum screw-cap assembly were used to prepare the TMS derivatives. A weighed amount of the compound to be analyzed (e.g., 1.0 mg) was placed in the vial. Then 50 or 100 ~1 of BSA (for quantitative determinations and for the detection of picogram and nanogram amounts) or of BSA containing 10% acetonitrile (for qualitative purposes) was added by injection through the rubber septum. Heating at 80°, if required, was carried out by inserting a series of up to twelve vials into wells in an aluminum heating block (Temp-Blok module heater, Lab-Line Instruments). The contents of the vial were mixed at the beginning of the heating period by means of a vibrator (Lab-Line Instruments) in order to facilitate dissolution. The stock solution of the TMS derivative, prepared either at room temperature or at SO”, was then diluted with BSA in a second or in a second and third vial. The final concentration was such that the amount injected (l-2 ~1) contained 500 ng to 10 pg and in the case of EC detection about 200 pg to 20 ng. Columns were heated either at constant or programmed temperatures depending on how much the compounds in the mixture to be analyzed differed in molecular weight. Temperatures ranged from about 130-285’. Carrier gas flow rates usually ranged from 4&60 ml/min, even when narrow columns were used in conjunction with EC detection. In that case the scavenger gas flow rate was about 50-60 ml/min. Higher carrier gas flow rates (120-150 ml/min) and no scavenger gas were used for the determination of picogram and nanogram amounts of the iodothyronines. In order to remove nonvolatile residues from the column after a series of injections, Silyl-8 (Pierce Chemical Co.), a mixture of several silylating reagents, was injected from time to time.
GAS
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
153
RESULTS
A few of the iodinated compounds tested (compounds l-4 and 17-18) 3 were sufficiently volatile to be chromatographed without derivatization*. All other compounds were converted to their TMS derivatives, either at room temperature or at 80”. The only compounds which required heating for rapid derivatization were the amino acids and diiodooctopamine hydrochloride (compound 22). When BSA containing 10% acetonitrile was used for the conversion to the TMS derivatives, heating for 5-7 min at 80’ was sufficient for maximal conversion. In the presence of 50% acetonitrile the reaction was faster (l-2 min), but in this case the stock solutions of the TMS derivatives of T, and T, (10 pg/pl) separated into two layers upon cooling to room temperature. In the absence of acetonitrile much longer heating (from 210 min for MIT to 235 min for T,) was required. BSA containing 10% acetonitrile was frequently used for the qualitative evaluation of microgram amounts. For all others purposes BSA alone was used in spite of the longer reaction times because the TMS derivatives are considerably less stable in the presence of acetonitrile. For the same reason concentrated stock solutions of the TMS derivatives were diluted with BSA alone. Figure 1 illustrates the greatei stability in the absence of acetonitrile (compare A with B and C wit,h D). Figure 1 also shows that very dilute solutions are less stable than more concentrated ones (compare A with C and B with D) . Tables 1 and 2 list most compounds test.ed, together with the retention times observed under typical conditions which were selected arbitrarily out of a large number of runs. It can be seen that all TMS derivatives, even those of compounds of very high molecular weight, are quite volatile at reasonably low column temperatures. Figure 2 illustrates the great power of resolution of the gas chromatographic method. A mixture of ten tyrosines and thyronines was completely resolved into single, nearly symmetrical peaks. Figure 3 showsthe resolution of three iodothyronines in a mixture containing picogram or nanogram amounts of each. In order to determine how well other structurally closely related iodinated compounds can be resolved by gas chromatography, a large number of mixtures of compounds was tested. A few mixtures could not be resolved on OV-1. The binary mixtures of compounds 6 + 8 or 33 + 36 and the ternary mixtures of compounds 7 + 10 + 13 or 37 + 38 + 44, as well as a few other binary or ternary mixtures emerged from an OV-1 column as single peaks. The higher ‘Compoundsare designatedby numbers,which are explainedin Tables1 and 2. ‘Alcohols shouldnot be usedaa solvents for the aldehydes3 and 4. which are slowly convertedby alcoholsto the muchmore volatile acetals.
154
FUNAKOSHI
Typical
AND
CAHNMANN
TABLE 1 Data for ledinated Compounds
Chromotogrophic
Containing
Chromatographic
No. 1 2 3 4 5 6 7
a Q 10 11 12 13 14 15 16 17
R,
R,
R3
R4
R5
0 OH CHO I,
1 ,
CH2C00~ (CH,),COOH (CH,),COOH CHZC~(CH3)C00H CH(OH)COOH tH,c~(0H)C00H CH(OH)CH(OH)COOH CH&H(NH$OOH I, CH = CHCOOH CH,CH(COOH), CH=C-C=O I I
ia 19 20 21 22 Column:
COCOOH CH2COC00H II CH(OH)CHZNHz.HCI 3.3 mm x 6 ft. 1% OV-1;
inlet and detector
temperatures
Data
Column Temp. (“C)
Flow Rate (ml/min.)
132 160 132 160 197 197 197 197 202 202 202 208 208 204 204 212 213
50 50 50
50 50 50 50 50 54 54 54 50 50 53 54 50 50
2’32” 4’11” 1’53” 2’46” 1’57” 2’46” 3’44” 2’44” 1’58” 3’22” 4’14” 1’23” 3’10” 2’55” 4’15” 3’54” 1’25”
213 199 199 199 200
50 53 53 53 50
3’05” 1’42” 2’29” 6’31” 2’07”
were lo-2Q”above
column
Retention
Time
temper&we.
power of resolution of OV-17 is shown by the fact that each of these mixtures was completely resolved on OV-17. While OV-17 is superior to OV-1 as far as resolution is concerned, it requires higher column temperatures. In order to counteract some decomposition of the TMS derivatives of the iodothyronines on the column at elevated temperat,ures (see below), we have. therefore used exclusively short and narrow columns (1.7 mm X 2 ft) of OV-1 for the determination of picogram and nanogram amounts of T, and T,. In both the microgram and the nanogram range the dose-response curve was found to be linear within appropriate dose limits for several compounds tested. Two examples are given in Figures 4 and 5. A decrease of the peak areas with increasing amount of column packing was
GAS
CHROMATOGFtAPHY
OF TABLE
Typocal
Chromotogruph,c
Doto for lod,nated
IODO
1%
COMPOUNDS
2
Compounds
Contalnlng
Two Benzene
Chromatographic
No
R,
R>
Rx
R5
R4 II ,.
26 2, 28 29 30 3, 32 33 34 35 36 37 38 39 40 4, 42 43 44 45 46 47 48 Column
,’ 11 I ” ‘. H I ” ‘( H ‘I I ” ” .’ ” H I ”
” ”
1, / ,,
ii ” ” ” 1 ” H ” I ” H ” ” ” I ” ” H ” I
n I n I H I ” ‘( ” ” H / H I H I ‘. H I ”
Column Temp. t C)
Flow Raw ‘ml ‘mn.)
275 227 227 227 275 275 237 228 237 275 275 275 237 275 275 274 250 250 250 276 276 276 285 275 275 282
56 52 52 53 Sb 56 53 50 53 56 56 56 50 56 56 56 50 50 50 56 56 56 55 55 55 55
COOH CH?COOH I.
., .,
.,
/
.I I
., ,, ,,
CHICH(OH)COOH CHzCH(NH$OOH/ ,,
,,
I
.a
,,
>,
.I
II
CH>CH(NH$OOH I.
3 3 m m x 6 ft 1% OV-1,
inlet
CH&OCOOH I /,
and detector
temperatures
were IO-20
oboe
Rings
Doto
Retention
T,me
2’26” 1’57” 3’50” 4’39” 1’25” 2’44” 1’49” 4’45” 3’52” 1’57” 2’24” 3’37” 4’06” 2’16” 4’25” 4’57” 1’49” 2’55” 3’36” 2’14” 2’57” 4’30” 2’03” 0’52” 2’33” 3’32”
colu,nn temperature.
observed with picogram and nanogram amounts of the TMS derivatives of the iodothyronines, particularly with that of T,. This is indicative of some destruction of these derivatives in the course of their passage through the column. Dilutions made from stock solutions (10 ,Ag/$) of the TMS derivatives in BSA either immediately after their preparation or 18 hr later gave the same chromatographic response. More di1ut.e solutions (0.5-l pg/pl), however, showed a diminished response after they had been kept at room temperature in hermetically closed vials for the same length of time. The peak areas obtained after 18 hr with the TMS derivatives of the iodoamino acids and their deamino analogs (500 ng/pl) varied between 69 and 82% of the original values. The changes which the TMS derivatives undergo in dilute solutions upon standing are partly but not completely
156
FUNAKOSHI
AND
IlM*
CAHNMANN
60 MINUTES
Fro. 1. Inffuence of solvent and concentration on stability of trimethylsilyl (TMS) derivatives of thyroxine (TJ. A stock solution (10 ag/&l) of TM&T, in N,O-bis (trimethylsilyl)acetamide (BSA) was diluted either with BSA (solutions A and C) or with acetonitrile containing 10% BSA (solutions B and D). Aliquots of 1 pl were chromatographed immediately and up to 1 hr later. The peak areas obtained are expressed as per cent recorder response whereby the response in the absence of acetonitrile is designated as 100%. Solutions A and B: 500 ng/,ul; hydrogen flame ionization; 3.3 mm X 6 ft column of OV-I at 285” ; inlet, 290” ; detector, 300”. Solutions C and D: 1 ng/gl; electron capture; 1.7 mm X 2 ft column of OV-1 at 210”; inlet, 26.5” ; detector, 270”.
0 I 135
4 I 155
8 I 175
12 I 195
16 I 215
20 I 235
24 mm I 2559
Fra. 2. Resolution of eight iodoamino acids and their deiodo analogs (hydrogen flame ionization). Abbreviations as given in footnote 2 ; 5 nmoles of each amino acid injected; 1.7 mm X 6 ft column of OV-1; temperature programmed as shown; inlet, 280” ; detector, 295” ; maximum recorder response 3.2 X IO-’ A.
GAS
CHROMATOGRAPHY
0~
10~0
COAIP~UNDS
157
FIG. 3. Resolution of three iodothyronines (electron capture). Abbreviations ah given in footnote 2; 300 pg 3,5-diiodothyronine, 300 pg 3,5,3’4riiodothyronine, 1.5 ng thyroxine; 1.7 mm X 2 ft column of OV-1; temperature programmed from 180 to 225”, then kept constant; inlet, 270’ ; detector, 269” ; maximum recorder response 8.0 X 10-l” A.
reversible. Upon heat,ing at 80’ for 30 min the response was restored to about 90% of the original value. The mass spectra of the TMS derivatives of DIT and of T,, obtained either from unfractionated, freshly prepared solutions in BSA or by injecting only the chromatographic effluents (peak areas) into the mass spectrometer, showed no higher mass peaks than those corresponding to the triply trimethylsilylated derivatives with molecular weights of 649 and 993, respectively. DISCUSSION
Gas chromatography has a number of advantages over the more widely used methods of paper or thin-layer chromatography. In particular, the detection of small amounts of impurities and the quantitation of unlabeled compounds is in general much easier. In the case of iodinecontaining substances, gas chromatography has the added advantage of extremely high sensitivity. The detection of as little as 500 pg of T, and 4 ng of T, by means of an EC detector has been reported (12) _ The derivatives used in that case were the iV,O-dipivalyl methyl esters,
158
FUNAKOSHI
AX-D
CAHNMANN
FIQ. 4. Dose-response curve for 3,5,3’-triiodothyronine from 500 ng to 4 pg (hydrogen flame ionization). 1.7 mm X 4 ft column of OV-1 at 250” ; inlet, 21%” ; detector, 275”. Range of four determinations is shown by vertical lines. Peak areas were transposed onto heavy paper, then cut out and weighed.
In our own investigations we used the TMS derivatives, which can be prepared with great ease in a single operation. TMS derivatives are easily hydrolyzed by moisture, but this offers no difficulty if they are prepared in sealed vials and in the presence of an excess of BSA. The vials and the heating device used by us were found to be convenient for the simultaneous derivatization of many samples. The absence of higher mass peaks than those corresponding to the triply trimethylsilylated derivatives from the mass spectra of the TMS derivatives of DIT and T4 clearly show that the gas chromatographically determined TMS derivatives of the iodoamino acids contain three and not four TMS groups. This proves unequivocally what others had already suspected (5). The interesting phenomenon of the reversible reaction slowly taking place in moderately dilute solutions (0.5-l pg/pl) has not been investigated in detail. It is therefore difficult at present to speculate on its chemical or physical basis. The destruction of the TMS derivatives on standing is never. completely reversible. The degree of reversibility is concentration-dependent. With increasing dilution the destruction be-
GAS
120
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
159
r
FIG. 5. Dose-response curve for thyroxine from 500 pg to 5 ng (electron capture). 1.7 nun X 2 ft column of OV-1 at 215” ; inlet, 265’ ; detector, 270”. Range of three determinations is shown by vertical lines. Peak areas were transposed onto heavy paper, then cut out and weighed.
comes less and less reversible. Microgram quantities of the iodoamino acids can therefore be determined with a good to excellent degree of accuracy even after the derivative solutions have been kept overnight, provided they are reheated before being chromatographed. On the other hand, picogram and nanogram quantities should always be determined immediately after derivatization. Destruction of the TMS derivatives of the iodothyronines during passage through heated columns is negligible in the case of microgram quantities. Thus, in the example shown in Figure 2 where 3.88 pg of TMS-T, was injected into a 6 ft column (retention time 23.6 min), the peak area was not significantly smaller than that obtained in a similar run with a shorter (4 ft) column. In the quantitative evaluation of a few nanograms or less, difficulties arising from partial destruction have been largely overcome by the use of narrow and short columns, relatively low temperatures and high flow rates, and alternating injections of the test compounds with known amounts of reference samples. The gas chromatographic analyses carried out in our laboratory on a large number of iodinated compounds belonging to different chemical
166
FUNAKOSHI
AND
CAHNMANN
categories show that trimethylsilylation with BSA is a convenient method of derivatization of those iodinated compounds which are not sufficiently volatile per se. Gas chromatography of TMS derivatives permits the qualitative and quantitative evaluation of iodinated compounds in picogram to microgram amounts. The EC detector, although much more sensitive than the hydrogen flame ionization detector, gives a lesser degree of reproducibility. This is due not only to the various factors discussed above but also to those inherent in the EC method which have been previously discussed by Lovelock (13). Considerably more work will be necessary before the method can be applied to the routine determination of T, and T, in serum or other body fluids. In preliminary experiments which were based on the passage of serum through an anion-exchange resin (14)) followed by a series of solvent extractions, a satisfactory quantitation of T, and T, was not possible on account of the high background caused by contaminants. Future work must therefore be mainly directed toward the elimination of these contaminants. Note added in pro@. In a recent paper [Anal. Biochem. 24,281 (1968) ] which appeared after the present paper had been submitted for publication, F. Shahrokhi and C. Gehrke report the synthesis of TMS derivatives of various iodoamino acids by means of a mixture of hexamethyldisilazane, trimethylchlorosilane, and pyridine. These authors believe that the derivatives they prepared from iodotyrosines contain three TMS groups, but that those prepared from iodothyronines contain only two (one at the carboxyl group and one at the amino group). Mass spectrometric determinations carried out in our laboratory by injecting the effluent from gas chromatographic columns directly into the mass spectrometer clearly show that BSA converts both, iodotyrosines and iodothyronines, to the triply trimethylsilylated derivatives. SUMMARY
The qualitative and quantitative gas chromatographic evaluation of a large variety of iodinated substances (iodoamino acids and their side chain and position analogs, intermediates in the synthesis or degradation of the thyroid hormones, and related compounds) is described. As little as a few hundred picograms can he determined accurately in most cases by means of an electron capture detector. Nonvolatile compounds have been converted to their volatile trimethylsilyl derivatives by treatment with N,O-bis(trimethylsilyl)acetamide at room temperature or at 80”. Various characteristics of these derivatives as well as precautions which must be taken for the determination of picogram and nanogram amounts
GAS
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
161
are described. Mass spectrometry shows that the gas chromatographically determined derivatives of the iodoamino acids contain three trimethylsilyl groups. ACKNOWLEDGMENT We wish to thank Mr. R. C. Pittman mination of the mass spectra.
for his most valuable help with the deter-
REFERENCES I.
2. 3. 4.
5. 6.
7. 8. 9. 10. 11.
12. 13. 14.
STOUFFER,J. E., JAAKONM~KI, P. I., AND WENGER, T. J., Biochim. Biophys. Actrr 127,261 (1966). RICEARDS, A. H., AND MASON, W. B., Anal. Chem. 38, 1751 (1966). KLEFIE, J. F., FINKBEINER, H., AND WHITE, D. M., J. Am. Chem. Sot. 88, 3399 (1966). NISHINAGA, H., CAHNMANN, H. J., KON, H., AND MATSUURA, T., Biochemistry 7, 388 (196%. ALEXANDER, N., AND &LHEIO, R., Anal. Biochem. 22, 187 (1968). FUNAKOSHI, K., AND CAHNMANN, H. J., in preparation. BARNES, J. H., BORROWS,E. T., ELKS, J., HEMS, B. A., AND LONG, A. G., J. Chem. sot. 1950, 2824. MATSUUBA, T., AND CAHNMANN, H. J., J. Am. Chem. Sot. 81, 871 (1959). SHIBA, T., AND CAHNMANN, H. J., J. Org. Chem. 27, 1773 (1962). HSFER, A., AND CARNMANN, H. J., J. Med. Chem. 7,326 (1964). SHIBA, T., H~FER, A., AND CAHNMANN, H. J., J. Org. Chem. 29, 3171 (1964). JAAKONMB~I, P. I. AND STOUFFER,J. E., in “Advances in Gas Chromatography” (A. Zlatkis, ed.), p. 149. Preston Technical Abstracts Co., Evanston, Illinois, 1967. .L~wEL~~K, J. E., Anal. Chem. 35, 474 (1963). LEWALLEN, C. G., in “Evaluation of Thyroid and Parathyroid Functions” (F. W. Sunderman and F. W. Sunderman, Jr., eds.), p. 37. Lippincott, Philadelphia, Pa., 1963.
BIOCHEMISTRY
Gas
27,150-161
(1969)
Chromatographic
Determination
lodinated KAZUHISA National National
Compounds
FUNAKOSHP Institutes Institutes
AND
of Arthritis
of Health, Received
of
May
H. J. CAHNMANN
and Metabolic Diseases, Bethesda, Maryland 20014 23, 1968
The classical methods for the detection and quantitative determination of iodoamino acids and related iodinated compounds are paper and thinlayer chromatography. Only in recent years has gas chromatography been applied to the major iodoamino acids (1, 2). After it had been discovered that BSA2 is a powerful reagent for the conversion of nonvolatile or insufficiently volatile compounds to their volatile TMS derivatives (3)) we began to test the suitability of this method of derivatization for the gas chromatographic analysis of iodoamino acids as well as of a large number of other iodinated compounds. The method is now being used routinely in our laboratory for purity checks, for the identification of unknown compounds by comparison with known standard samples, and for the resolution and quantitative evaluation of complex mixtures of iodinated compounds. A few applications of the method have been reported by us in a preliminary fashion (4). Subsequently Alexander and Seheig described the gas chromatographic determination of the major iodoamino acids using a similar method of derivatization (5). The present paper gives the details of our procedure for the determination of a large variety of iodinated compounds and discusses various characteristics of the TMS derivatives of the iodoamino acids. The compounds tested comprise not only the known iodinated tyrosines and thyronines, but also their deamino, position, and side-chain analogs as well as various intermediates in the synthesis of the thyroid hormones. The ‘Visiting Scientist from the Faculty of Pharmaceutical Sciences, Kiushu University, Japan. ’ Abbreviations : EC, electron capture; BSA, N,O-bis(trimethylsilyl)acetamide; TMS, trimethylsilyl; TYR, tyrosine ; MIT, 3-iodotyrosine ; DIT, 3,54iodotyrosine, T, thyronine ; T,, 3-iodothyronine ; Tz, 3,6diiodothyronine; T,‘, 3,3’-diiodothyronine Ta, 3,5,3’-triiodothyronine ; T31, 3,3’5’-triiodothyronine ; T,, thyroxine. 150
;
GAS
CHROMATOGRAPHY
OF
IODO
COMPOTXDS
l.=ll
amounts determined were usually a few micrograms, but when the normally used hydrogen flame ionization detector was replaced with an EC detector samples in the nanogram and picogram range could be analyzed as well. EXPERIMENTAL
Starting Materials and Equipment
4-Hydroxy-3-iodobenzaldehyde was synthesized by the controlled iodination of p-hydroxybenzaldehyde (6). The method is a modification of that described by Barnes et al. (7). 4-Hydroxy-3,5-diiodobenzaldehyde and most of the other unbranched deamino analogs of DIT were synthesized as described previously (8). The a,/?-dihydroxypropionic acid analog of DIT was synthesized by borohydride reduction of a T,-precursor described by Nishinaga et al. (4). 4- (4-Acetoxy-3,5-diiodobenzal) -2-methyl-5-oxazolone, an intermediate in the synthesis of the keto acid analog of DIT, was synthesized as described for the 1311-labeled compound (9). The keto acid itself, 4-hydroxy-3,5-diiodophenylpyruvic acid, was purchased from the Osaka Laboratory of Synthetic Organic Chemicals, Nishinomiya, Japan, or from J. T. Baker Chemical Co., Phillipsburg, N. J., and once recrystallized from glacial acetic acid. The corresponding 3-monoiodinated oxazolone and keto acid were prepared in analogous manner (6). 4- (p-Hydroxyphenoxy) -3,5-diiodobenzylmalonic acid, thr a-carboxypropionic acid analog of DIT, was synthesized according to HSfer and Cahnmann (10). T, and T, in the form of the free acids were a gift of Dr. J. F. Kerwin (Smith Kline and French Laboratories). The other thyronines were commercial products or gifts from various sources. Most of the side-chain analogs of the iodothyronines were a gift of Dr. R. I. Meltzer (Warner Lambert Research Institute). The keto acid analog of T, was generously supplied by Dr. R. Pitt-Rivers. All acids were used in the free form since their salts react only sluggishly with BSA. The meta analogs of T, were synthesized as described previously (11). All other starting materials were commercial products. As BSA is hydrolyzed by moisture and decolorized on long standing in rubber-sealed vials, it was purchased in 1 ml glass-sealed ampoules (Pierce Chemical Co.). Opened ampoules could be kept for up to a few weeks in Teflonlined screw-cap vials which contained a powerful drying agent (calcium hydride) under a wad of glass wool. All solvents were reagent grade. A Chromalab (Glowall Corp.) gas chromatograph or a Microtek MT220 gas chromatograph (Mikrotek Instruments Corp.) was used in conjunction with a hydrogen flame ionization detector. Only the latter instrument was employed with an EC detector (63Ni) which could be
152
FUNAKOSHI
AND
CAHNMANN
supplied with either direct or pulsed current. Direct current (45 V) was used in most instances. Glass columns were filled with either lo/O OV-1 (a methylsiloxane polymer) or 1% OV-17 (a phenylmethylsiloxane polymer) on S&100 mesh Chromosorb W HP (Supelco). The length of the columns ranged from 2 to 6 ft and their i.d. from 1.7 to 3.3 mm. The carrier and scavenger gas (frequently omitted) was nitrogen (ultra high purity, Southern Oxygen) or argon (Matheson), except when a pulsed power supply was used, in which case nitrogen was replaced with argon10% methane (Matheson). A LKB gas chromatograph-mass spectrometer type 9000 was used for mass spectrometric analyses. Analytical
Procedures
Glass vials of NO-250 ~1 capacity equipped with a Teflon-lined silicon rubber septum (Kontes K-774150-0500) and an aluminum screw-cap assembly were used to prepare the TMS derivatives. A weighed amount of the compound to be analyzed (e.g., 1.0 mg) was placed in the vial. Then 50 or 100 ~1 of BSA (for quantitative determinations and for the detection of picogram and nanogram amounts) or of BSA containing 10% acetonitrile (for qualitative purposes) was added by injection through the rubber septum. Heating at 80°, if required, was carried out by inserting a series of up to twelve vials into wells in an aluminum heating block (Temp-Blok module heater, Lab-Line Instruments). The contents of the vial were mixed at the beginning of the heating period by means of a vibrator (Lab-Line Instruments) in order to facilitate dissolution. The stock solution of the TMS derivative, prepared either at room temperature or at SO”, was then diluted with BSA in a second or in a second and third vial. The final concentration was such that the amount injected (l-2 ~1) contained 500 ng to 10 pg and in the case of EC detection about 200 pg to 20 ng. Columns were heated either at constant or programmed temperatures depending on how much the compounds in the mixture to be analyzed differed in molecular weight. Temperatures ranged from about 130-285’. Carrier gas flow rates usually ranged from 4&60 ml/min, even when narrow columns were used in conjunction with EC detection. In that case the scavenger gas flow rate was about 50-60 ml/min. Higher carrier gas flow rates (120-150 ml/min) and no scavenger gas were used for the determination of picogram and nanogram amounts of the iodothyronines. In order to remove nonvolatile residues from the column after a series of injections, Silyl-8 (Pierce Chemical Co.), a mixture of several silylating reagents, was injected from time to time.
GAS
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
153
RESULTS
A few of the iodinated compounds tested (compounds l-4 and 17-18) 3 were sufficiently volatile to be chromatographed without derivatization*. All other compounds were converted to their TMS derivatives, either at room temperature or at 80”. The only compounds which required heating for rapid derivatization were the amino acids and diiodooctopamine hydrochloride (compound 22). When BSA containing 10% acetonitrile was used for the conversion to the TMS derivatives, heating for 5-7 min at 80’ was sufficient for maximal conversion. In the presence of 50% acetonitrile the reaction was faster (l-2 min), but in this case the stock solutions of the TMS derivatives of T, and T, (10 pg/pl) separated into two layers upon cooling to room temperature. In the absence of acetonitrile much longer heating (from 210 min for MIT to 235 min for T,) was required. BSA containing 10% acetonitrile was frequently used for the qualitative evaluation of microgram amounts. For all others purposes BSA alone was used in spite of the longer reaction times because the TMS derivatives are considerably less stable in the presence of acetonitrile. For the same reason concentrated stock solutions of the TMS derivatives were diluted with BSA alone. Figure 1 illustrates the greatei stability in the absence of acetonitrile (compare A with B and C wit,h D). Figure 1 also shows that very dilute solutions are less stable than more concentrated ones (compare A with C and B with D) . Tables 1 and 2 list most compounds test.ed, together with the retention times observed under typical conditions which were selected arbitrarily out of a large number of runs. It can be seen that all TMS derivatives, even those of compounds of very high molecular weight, are quite volatile at reasonably low column temperatures. Figure 2 illustrates the great power of resolution of the gas chromatographic method. A mixture of ten tyrosines and thyronines was completely resolved into single, nearly symmetrical peaks. Figure 3 showsthe resolution of three iodothyronines in a mixture containing picogram or nanogram amounts of each. In order to determine how well other structurally closely related iodinated compounds can be resolved by gas chromatography, a large number of mixtures of compounds was tested. A few mixtures could not be resolved on OV-1. The binary mixtures of compounds 6 + 8 or 33 + 36 and the ternary mixtures of compounds 7 + 10 + 13 or 37 + 38 + 44, as well as a few other binary or ternary mixtures emerged from an OV-1 column as single peaks. The higher ‘Compoundsare designatedby numbers,which are explainedin Tables1 and 2. ‘Alcohols shouldnot be usedaa solvents for the aldehydes3 and 4. which are slowly convertedby alcoholsto the muchmore volatile acetals.
154
FUNAKOSHI
Typical
AND
CAHNMANN
TABLE 1 Data for ledinated Compounds
Chromotogrophic
Containing
Chromatographic
No. 1 2 3 4 5 6 7
a Q 10 11 12 13 14 15 16 17
R,
R,
R3
R4
R5
0 OH CHO I,
1 ,
CH2C00~ (CH,),COOH (CH,),COOH CHZC~(CH3)C00H CH(OH)COOH tH,c~(0H)C00H CH(OH)CH(OH)COOH CH&H(NH$OOH I, CH = CHCOOH CH,CH(COOH), CH=C-C=O I I
ia 19 20 21 22 Column:
COCOOH CH2COC00H II CH(OH)CHZNHz.HCI 3.3 mm x 6 ft. 1% OV-1;
inlet and detector
temperatures
Data
Column Temp. (“C)
Flow Rate (ml/min.)
132 160 132 160 197 197 197 197 202 202 202 208 208 204 204 212 213
50 50 50
50 50 50 50 50 54 54 54 50 50 53 54 50 50
2’32” 4’11” 1’53” 2’46” 1’57” 2’46” 3’44” 2’44” 1’58” 3’22” 4’14” 1’23” 3’10” 2’55” 4’15” 3’54” 1’25”
213 199 199 199 200
50 53 53 53 50
3’05” 1’42” 2’29” 6’31” 2’07”
were lo-2Q”above
column
Retention
Time
temper&we.
power of resolution of OV-17 is shown by the fact that each of these mixtures was completely resolved on OV-17. While OV-17 is superior to OV-1 as far as resolution is concerned, it requires higher column temperatures. In order to counteract some decomposition of the TMS derivatives of the iodothyronines on the column at elevated temperat,ures (see below), we have. therefore used exclusively short and narrow columns (1.7 mm X 2 ft) of OV-1 for the determination of picogram and nanogram amounts of T, and T,. In both the microgram and the nanogram range the dose-response curve was found to be linear within appropriate dose limits for several compounds tested. Two examples are given in Figures 4 and 5. A decrease of the peak areas with increasing amount of column packing was
GAS
CHROMATOGFtAPHY
OF TABLE
Typocal
Chromotogruph,c
Doto for lod,nated
IODO
1%
COMPOUNDS
2
Compounds
Contalnlng
Two Benzene
Chromatographic
No
R,
R>
Rx
R5
R4 II ,.
26 2, 28 29 30 3, 32 33 34 35 36 37 38 39 40 4, 42 43 44 45 46 47 48 Column
,’ 11 I ” ‘. H I ” ‘( H ‘I I ” ” .’ ” H I ”
” ”
1, / ,,
ii ” ” ” 1 ” H ” I ” H ” ” ” I ” ” H ” I
n I n I H I ” ‘( ” ” H / H I H I ‘. H I ”
Column Temp. t C)
Flow Raw ‘ml ‘mn.)
275 227 227 227 275 275 237 228 237 275 275 275 237 275 275 274 250 250 250 276 276 276 285 275 275 282
56 52 52 53 Sb 56 53 50 53 56 56 56 50 56 56 56 50 50 50 56 56 56 55 55 55 55
COOH CH?COOH I.
., .,
.,
/
.I I
., ,, ,,
CHICH(OH)COOH CHzCH(NH$OOH/ ,,
,,
I
.a
,,
>,
.I
II
CH>CH(NH$OOH I.
3 3 m m x 6 ft 1% OV-1,
inlet
CH&OCOOH I /,
and detector
temperatures
were IO-20
oboe
Rings
Doto
Retention
T,me
2’26” 1’57” 3’50” 4’39” 1’25” 2’44” 1’49” 4’45” 3’52” 1’57” 2’24” 3’37” 4’06” 2’16” 4’25” 4’57” 1’49” 2’55” 3’36” 2’14” 2’57” 4’30” 2’03” 0’52” 2’33” 3’32”
colu,nn temperature.
observed with picogram and nanogram amounts of the TMS derivatives of the iodothyronines, particularly with that of T,. This is indicative of some destruction of these derivatives in the course of their passage through the column. Dilutions made from stock solutions (10 ,Ag/$) of the TMS derivatives in BSA either immediately after their preparation or 18 hr later gave the same chromatographic response. More di1ut.e solutions (0.5-l pg/pl), however, showed a diminished response after they had been kept at room temperature in hermetically closed vials for the same length of time. The peak areas obtained after 18 hr with the TMS derivatives of the iodoamino acids and their deamino analogs (500 ng/pl) varied between 69 and 82% of the original values. The changes which the TMS derivatives undergo in dilute solutions upon standing are partly but not completely
156
FUNAKOSHI
AND
IlM*
CAHNMANN
60 MINUTES
Fro. 1. Inffuence of solvent and concentration on stability of trimethylsilyl (TMS) derivatives of thyroxine (TJ. A stock solution (10 ag/&l) of TM&T, in N,O-bis (trimethylsilyl)acetamide (BSA) was diluted either with BSA (solutions A and C) or with acetonitrile containing 10% BSA (solutions B and D). Aliquots of 1 pl were chromatographed immediately and up to 1 hr later. The peak areas obtained are expressed as per cent recorder response whereby the response in the absence of acetonitrile is designated as 100%. Solutions A and B: 500 ng/,ul; hydrogen flame ionization; 3.3 mm X 6 ft column of OV-I at 285” ; inlet, 290” ; detector, 300”. Solutions C and D: 1 ng/gl; electron capture; 1.7 mm X 2 ft column of OV-1 at 210”; inlet, 26.5” ; detector, 270”.
0 I 135
4 I 155
8 I 175
12 I 195
16 I 215
20 I 235
24 mm I 2559
Fra. 2. Resolution of eight iodoamino acids and their deiodo analogs (hydrogen flame ionization). Abbreviations as given in footnote 2 ; 5 nmoles of each amino acid injected; 1.7 mm X 6 ft column of OV-1; temperature programmed as shown; inlet, 280” ; detector, 295” ; maximum recorder response 3.2 X IO-’ A.
GAS
CHROMATOGRAPHY
0~
10~0
COAIP~UNDS
157
FIG. 3. Resolution of three iodothyronines (electron capture). Abbreviations ah given in footnote 2; 300 pg 3,5-diiodothyronine, 300 pg 3,5,3’4riiodothyronine, 1.5 ng thyroxine; 1.7 mm X 2 ft column of OV-1; temperature programmed from 180 to 225”, then kept constant; inlet, 270’ ; detector, 269” ; maximum recorder response 8.0 X 10-l” A.
reversible. Upon heat,ing at 80’ for 30 min the response was restored to about 90% of the original value. The mass spectra of the TMS derivatives of DIT and of T,, obtained either from unfractionated, freshly prepared solutions in BSA or by injecting only the chromatographic effluents (peak areas) into the mass spectrometer, showed no higher mass peaks than those corresponding to the triply trimethylsilylated derivatives with molecular weights of 649 and 993, respectively. DISCUSSION
Gas chromatography has a number of advantages over the more widely used methods of paper or thin-layer chromatography. In particular, the detection of small amounts of impurities and the quantitation of unlabeled compounds is in general much easier. In the case of iodinecontaining substances, gas chromatography has the added advantage of extremely high sensitivity. The detection of as little as 500 pg of T, and 4 ng of T, by means of an EC detector has been reported (12) _ The derivatives used in that case were the iV,O-dipivalyl methyl esters,
158
FUNAKOSHI
AX-D
CAHNMANN
FIQ. 4. Dose-response curve for 3,5,3’-triiodothyronine from 500 ng to 4 pg (hydrogen flame ionization). 1.7 mm X 4 ft column of OV-1 at 250” ; inlet, 21%” ; detector, 275”. Range of four determinations is shown by vertical lines. Peak areas were transposed onto heavy paper, then cut out and weighed.
In our own investigations we used the TMS derivatives, which can be prepared with great ease in a single operation. TMS derivatives are easily hydrolyzed by moisture, but this offers no difficulty if they are prepared in sealed vials and in the presence of an excess of BSA. The vials and the heating device used by us were found to be convenient for the simultaneous derivatization of many samples. The absence of higher mass peaks than those corresponding to the triply trimethylsilylated derivatives from the mass spectra of the TMS derivatives of DIT and T4 clearly show that the gas chromatographically determined TMS derivatives of the iodoamino acids contain three and not four TMS groups. This proves unequivocally what others had already suspected (5). The interesting phenomenon of the reversible reaction slowly taking place in moderately dilute solutions (0.5-l pg/pl) has not been investigated in detail. It is therefore difficult at present to speculate on its chemical or physical basis. The destruction of the TMS derivatives on standing is never. completely reversible. The degree of reversibility is concentration-dependent. With increasing dilution the destruction be-
GAS
120
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
159
r
FIG. 5. Dose-response curve for thyroxine from 500 pg to 5 ng (electron capture). 1.7 nun X 2 ft column of OV-1 at 215” ; inlet, 265’ ; detector, 270”. Range of three determinations is shown by vertical lines. Peak areas were transposed onto heavy paper, then cut out and weighed.
comes less and less reversible. Microgram quantities of the iodoamino acids can therefore be determined with a good to excellent degree of accuracy even after the derivative solutions have been kept overnight, provided they are reheated before being chromatographed. On the other hand, picogram and nanogram quantities should always be determined immediately after derivatization. Destruction of the TMS derivatives of the iodothyronines during passage through heated columns is negligible in the case of microgram quantities. Thus, in the example shown in Figure 2 where 3.88 pg of TMS-T, was injected into a 6 ft column (retention time 23.6 min), the peak area was not significantly smaller than that obtained in a similar run with a shorter (4 ft) column. In the quantitative evaluation of a few nanograms or less, difficulties arising from partial destruction have been largely overcome by the use of narrow and short columns, relatively low temperatures and high flow rates, and alternating injections of the test compounds with known amounts of reference samples. The gas chromatographic analyses carried out in our laboratory on a large number of iodinated compounds belonging to different chemical
166
FUNAKOSHI
AND
CAHNMANN
categories show that trimethylsilylation with BSA is a convenient method of derivatization of those iodinated compounds which are not sufficiently volatile per se. Gas chromatography of TMS derivatives permits the qualitative and quantitative evaluation of iodinated compounds in picogram to microgram amounts. The EC detector, although much more sensitive than the hydrogen flame ionization detector, gives a lesser degree of reproducibility. This is due not only to the various factors discussed above but also to those inherent in the EC method which have been previously discussed by Lovelock (13). Considerably more work will be necessary before the method can be applied to the routine determination of T, and T, in serum or other body fluids. In preliminary experiments which were based on the passage of serum through an anion-exchange resin (14)) followed by a series of solvent extractions, a satisfactory quantitation of T, and T, was not possible on account of the high background caused by contaminants. Future work must therefore be mainly directed toward the elimination of these contaminants. Note added in pro@. In a recent paper [Anal. Biochem. 24,281 (1968) ] which appeared after the present paper had been submitted for publication, F. Shahrokhi and C. Gehrke report the synthesis of TMS derivatives of various iodoamino acids by means of a mixture of hexamethyldisilazane, trimethylchlorosilane, and pyridine. These authors believe that the derivatives they prepared from iodotyrosines contain three TMS groups, but that those prepared from iodothyronines contain only two (one at the carboxyl group and one at the amino group). Mass spectrometric determinations carried out in our laboratory by injecting the effluent from gas chromatographic columns directly into the mass spectrometer clearly show that BSA converts both, iodotyrosines and iodothyronines, to the triply trimethylsilylated derivatives. SUMMARY
The qualitative and quantitative gas chromatographic evaluation of a large variety of iodinated substances (iodoamino acids and their side chain and position analogs, intermediates in the synthesis or degradation of the thyroid hormones, and related compounds) is described. As little as a few hundred picograms can he determined accurately in most cases by means of an electron capture detector. Nonvolatile compounds have been converted to their volatile trimethylsilyl derivatives by treatment with N,O-bis(trimethylsilyl)acetamide at room temperature or at 80”. Various characteristics of these derivatives as well as precautions which must be taken for the determination of picogram and nanogram amounts
GAS
CHROMATOGRAPHY
OF
IODO
COMPOUNDS
161
are described. Mass spectrometry shows that the gas chromatographically determined derivatives of the iodoamino acids contain three trimethylsilyl groups. ACKNOWLEDGMENT We wish to thank Mr. R. C. Pittman mination of the mass spectra.
for his most valuable help with the deter-
REFERENCES I.
2. 3. 4.
5. 6.
7. 8. 9. 10. 11.
12. 13. 14.
STOUFFER,J. E., JAAKONM~KI, P. I., AND WENGER, T. J., Biochim. Biophys. Actrr 127,261 (1966). RICEARDS, A. H., AND MASON, W. B., Anal. Chem. 38, 1751 (1966). KLEFIE, J. F., FINKBEINER, H., AND WHITE, D. M., J. Am. Chem. Sot. 88, 3399 (1966). NISHINAGA, H., CAHNMANN, H. J., KON, H., AND MATSUURA, T., Biochemistry 7, 388 (196%. ALEXANDER, N., AND &LHEIO, R., Anal. Biochem. 22, 187 (1968). FUNAKOSHI, K., AND CAHNMANN, H. J., in preparation. BARNES, J. H., BORROWS,E. T., ELKS, J., HEMS, B. A., AND LONG, A. G., J. Chem. sot. 1950, 2824. MATSUUBA, T., AND CAHNMANN, H. J., J. Am. Chem. Sot. 81, 871 (1959). SHIBA, T., AND CAHNMANN, H. J., J. Org. Chem. 27, 1773 (1962). HSFER, A., AND CARNMANN, H. J., J. Med. Chem. 7,326 (1964). SHIBA, T., H~FER, A., AND CAHNMANN, H. J., J. Org. Chem. 29, 3171 (1964). JAAKONMB~I, P. I. AND STOUFFER,J. E., in “Advances in Gas Chromatography” (A. Zlatkis, ed.), p. 149. Preston Technical Abstracts Co., Evanston, Illinois, 1967. .L~wEL~~K, J. E., Anal. Chem. 35, 474 (1963). LEWALLEN, C. G., in “Evaluation of Thyroid and Parathyroid Functions” (F. W. Sunderman and F. W. Sunderman, Jr., eds.), p. 37. Lippincott, Philadelphia, Pa., 1963.