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Fused Silica Capillary Column Gas Chromatography of Derivatized Thyroid Hormone Standards with Electron Capture Detection Jeffrey A. Corklll and Roger W. Glese" Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions and Institute of Chemical Analysis, Northeastern University, Boston, Massachusetts 02 1 15
Standards of some lodiothyronlnes as their N,O-dlheptafluorobutyryl methyl estler derivatives are analyzed at the plcogram level by electron capture gas chromatography. Injectlon by dlrect sampling Is employed onto fused sillca caplllary columns as shairt as 7.5 m. The recovery of these compounds depends not only on the various temperatures In the gas chromatograph (Injector, column, and detector) but also on the flow rate of carrier gas In the Injector and the column and on the column sectlon and length. Most Important Is to use a hlgh flow rate (above 6 cm3 mlm-'), which not only Increases the recovery but also mlnlmlzes any dlfferences In recovery between alternate sectlons from a given column. A hlgh flow rate further Improves the performance of the Internal standard. In splte of the high molecular welghts (In the range of 900-1200), lablltty (lodllne atoms) and trace amounts of the derlvatlzed lodothyronlnes In this analysis, temperature programming can be used (2-8 mln hold at 200 O C , program up to 275 "C). Thls approach allows a llnear working range of 0.4-700 pg, and a detectlon llmlt of 30 fg.
The thyroid hormones and their metabolites are challenging solutes for analysis by gas chromatography for three reasons. First of all, they contaiin three types of functional groups (phenol, carboxyl, and amino) requiring derivatization prior to sample injection. Secondly, their molecular weights after a typical derivatization procedure fall into the range of 900-1200,which is high for GC solutes, particularly polar ones. Finally, the high tempeiratures required to volatilize these substances also tend to dlestroy them due to their content of' labile iodine atoms. In spite of these problem, GC has been found to be a useful method for analyzing the iodothyronines. For example, we previously used this technique to measure the dialyzable fractions (involving picogram amounts) of these hormones from serum samples (I). A regular packed GC column was employed along with an electron capture detector (ECD). This and earlier GC work on the iodothyronines has been reviewed (2). I n this article, the andysis of thyronine standards is investigated by GC-ECD on a fused silica capillary column. The major aspects are the roles of temperature, flow rate, and column section and length. Optimization of these parameters defies the nature of some of the losses in the system, increases t h e recovery, improves the performance of the internal standard, and allows temperature programming by GC-ECD at the trace level.
EXPERIMENTAL SECTION Materials and Methods. The thyronines (Tz,T3,and T4)were obtained as free amino acids from Sigma Chemical Co. and stored in a desiccator over Drierite at -4 "C. Acetonitrile (Burdick and Jackson) was distilled from calcium hydride (e.g., 10 g of CaH, for 250 mL of acetonitrile) and stored under a nitrogen atmosphere
at room temperature in the dark in a glass flask with a Teflon-lined lid. The toluene and methanol (Burdick and Jackson) were used as received. The silanizing compounds, trimethylchlorosilane (TMCS) and hexamethyldisilazane (HMDS) were obtained from Pierce Chemical Co. and were "Specially Purified Grade". The glassware, including the 0.1,1.0, and 5.0 cma Reacti-vials, matching caps (open-topped), Mininert valve caps, "Tuf-bond Teflon silicone disks and "Silli-vap" nitrogen evaporator were acquired from Pierce Chemical Co. The Teflon-coated silicone disks of the open-topped screw caps were allowed to stand for 30 min in hot methanol, rinsed with methanol, dried overnight at 60 OC, and stored in a desiccator over Drierite. All glassware used in this work was cleaned in a solution of concentrated nitric acid at 50-60 "C overnight. The glassware was then rinsed twice with water and heated in boiling water for 1 h. After being rinsed with water and methanol, it was dried at 200 O C in a vacuum oven ( P < 0.1 torr) overnight. The surfaces were silanized by HMDS in vacuo at 200 "C similar to a method described previously (3). The glassware was stored sealed at room temperature. All the reagents used in this cleaning and silanizing procedure were stored either in glass bottles with Teflon-lined caps or in glass/Teflon Oxford Pipettors (VWR Scientific). The N,O-diheptafluorobutyryl methyl ester derivatives of the iodothyronineswere synthesized as described (4). The derivatives obtained were purified by recrystallination twice from toluene and stored in the dark at -4 "C. No decomposition of these derivatives was detected by GC-ECD over a period of 9 months. Stock solutions of derivatized thyronines were stored in 4-mL screw cap borosilicate septum vials with the 'Tuf-bond Teflon-silicon disks (Pierce). Solutions in the 10410-3M range in toluene were stable if stored at 4 "C for 3-4 weeks. These stock solutions were diluted to 10-8-10-e M concentrations and used within a several hour period. In toluene these solutions were stable for 12 h whereas in acetonitrile only 4-6 h. Instability was evidenced by decreased recoveries and the appearance of extraneous peaks by GC-ECD. All solutions were stored in the dark. Injections into the gas chromatograph were made with silanized 1O-hL syringes, either type 701N or 170RN (Hamilton Co.), fitted with a type 26s needle. A simpler cleaning procedure than reported previously (2) was found to be effective. By means of a Tuf-bond disk mounted on a screw-thread connector (Wheaton) with an open top screw cap at the end of a water aspirator vacuum line, 20 volumes of each of the following solutions at 60 "C in an ultrasonic bath were drawn through the syringes from the barrel end (1)20% (v/v) nitric acid in water, (2) water, (3) acetonitrile, and (4) methanol. The plungers were immersed in the same sequence of solutions. The unassembled syringes were then dried at 60 "C overnight. The syringes were silanized by filling with 5% trimethylchlorosilaneand 5% HMDS in toluene and allowing them to stand for 30 min at 50-60 "C. Toluene and methanol then were drawn through the syringes (60 "C, 20 volumes each), and they were dried at 60 "C overnight and stored as above. This cleaning and silanization process was repeated whenever the syringes became contaminated as evidenced by injecting solvent blank into the gas chromatograph or when the tailing of the solvent peak increased to the extent observed with a nonsilanized syringe (e.g., after 20-30 injections). Immediately after each injection into the gas chromatograph, the syringe is treated as follows: (1)remove plunger and place the lower two-thirds into hot toluene (85 "C), (2) pull 10 volumes of hot toluene and 10 s of air through the syringe from the barrel
0003-2700/81/0353-1867$01.25/00 1981 American Chemical Soclety
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end, (3) wipe plunger dry and insert into syringe still attached to vacuum, (4) pump once and pull 10 s more air, (5) remove syringe from vacuum and fill to 10 pL with 1% HMDS in toluene, (6)store at 40 "C (temperature on top of the gas chromatograph) until reuse (but not longer than 30 min), (7) expel HMDS solution, (8) repeat steps (1)-(4), (9) flush the syringe once with 8 pL of the next solution to be injected, (10) pump out the air with needle immersed in the solution to be injected, (11)f i i to a certain volume (e.g., 0.5 pL), draw up 1pL of nitrogen into the syringe barrel, and (12) inject. With the injection technique usually employed (rapid needle withdrawal after a cold needle injection) the residual solution in the syringe occupied about half of the needle volume. When the syringe is not to be used for a period longer than 30 min between injections,the HMDS solution is expelled after 10-20 min and the syringe is cleaned with toluene as above and then stored in a plastic bag. Instrumentation. A Varian Model 3740 gas chromatograph equipped with a constant current, pulse modulated 63Ni(8 mCi) electron capture detector was utilized. The electrometer response was monitored with a Varian Model 9176 recorder (full scale response time
