Anal. Chem. 1987, 59, 2899-2703
the double modulation scheme drives the Faradaic process fairly hard, with the possibility of electrode fouling. The current densities will typically be comparable to microdisk electrodes rather than the usual millimeter scale solid electrodes. The advantages of double modulation voltammetry are several and will be particularly important when background current is severe. First, Figure 9 demonstrates outstanding rejection of background current compared to conventional voltammetry. Compared to HMV, the SHMACV technique has the advantage of a peak rather than wave response, leading to improved detection limits and resolution. As has been amply demonstrated with differential pulse and square wave voltammetry compared to dc polarography, the peak response resulting from the differential nature of these methods is more analytically useful. Finally, the rejection of background extends the potential range of the electrode/electrolyte combination farther into the solvent breakdown region, permitting significant (0.2-0.3 V) extension of the useful potential range accessible to solid electrodes.
ACKNOWLEDGMENT The authors thank Dale Karweik, director of the Chemical Instrumentation support group, for fabrication of the triple function generator and for discussions on signal processing. Registry No. BHMF, 62524-59-8; ferrocene, 102-54-5.
LITERATURE CITED Wang, J. Talanta 1981, 2 8 , 369-376. Miller, B.; Bruckenstein. S. J. Electrochem. SOC. 1974, 121, 1558-1562. Miller, B.; Bruckenstein, S. Anal. Chem. 1974, 46, 2026-2033. Tokuda, K.: Bruckenstein, S.; Miller, B. J. Electrochem. SOC. 1975, 122, 1316-1322. Tokuda, K.; Bruckenstein, S.; Miller, B. J. Electrochem. SOC. 1979, 126, 431-436. Kanzaki, Y.; Bruckenstein, S. J. Nectrochem. SOC. 1979, 126, 437-441.
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(7) Blaedel, W. J.; Engstrom, R. C. Anal. Chem. 1978, 5 0 , 476-479. (8) Blaedel, W. J.; Boyer, S. L. Anal. Chem. 1971. 43, 1538-1540. (9) Blaedel, W. J.; Iverson, D. G. Anal. Chem. 1977, 49, 1563-1566. (10) Blaedel, W. J.; Wang. J. Anal. Chern. 1979, 5 1 , 799-602. (11) Blaedel, W. J.; Yim, Z. Anal. Chem. 1980, 5 2 , 564-566. (12) Blaedel, W. J.; Wang, J. Anal. Chlm. Acta 1980. 116, 315-322. (13) Blaedel, W. J.; Wang, J. Anal. Chem. 1980, 5 2 , 1697-1700. (14) Wang, J.; Dewald, H. D. Anal. Chim. Acta 1982. 136, 77-84. (15) Wang, J.; Freiha, B. A. Analyst(Lond0n) 1883, 108, 685-690. (16) Pratt, K. W., Jr. Ph.D. Thesis, Iowa State University, 1081. (17) Pratt, K. W., Jr.; Johnson, D. C. Electrochlm. Acta 1082, 2 7 , 1013- 1021. (18) Schuette, S. A.; McCreery, R. L. Anal. Chem. 1988, 5 8 , 1776-1762. (19) Bard, A. J.; Faulkner, L. R. ~lectrochem/calMethods; Wiley: New York, 1960; p 28. (20) Lindsey, A. J. J. W y s . Chem. 1952, 56, 439-442. (21) Harris, E. D.; Lindsey, A. J. Analyst (London) 1951. 76, 647-649. (22) Moorhead, E. D.; Bhat, G. A.; Stephens, M. M. J. Chem. Technol. Blotechnol. 1981, 3 1 , 259-272. (23) Moorhead, E. D.; Stephens, M. M.; Bhat. G. A. Anal. Left. 1981, 14(A4), 219-240. (24) Weber, S . G. J. Electroanal. Chem. 1983, 145, 1-7. (25) Hanekamp, H. 8.; deJong, H. G. Anal. Chim. Acta 1982, 135, 351-354. (26) Hanekamp, H. 8.; Van Nleuwkerk, H. J. Anal. Chim. Acta 1980, 121, 13-22. (27) Smith, D. E. I n .€lecfroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1966; Vol. 1, Chapter 1. (28) Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1977, 8 2 , 157-171. (29) Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1978, 9 0 , 149-163. (30) Compton, R. G.; Sealy, G. R. J. Nectroanal. Chem. 1983, 145, 35-41. (31) Jolly, W. J. The Synthesis and Characterizatlon of Inorganic Compounds; Prentlce Hall: Englewood Cliffs: NJ, 1970, p 486. (32) Schuette, S. A. Ph.D. Thesis, The Ohio State University, 1987. (33) Schuette, S. A.; McCreery, R. L. J. €/echoanal. Chem. 1985, 191, 329-342. (34) Pratt, K. W. Anal. Chem. 1984, 5 6 , 1967-1970. (35) O'Dea, J. J.; Osteryoung, J.; Osteryoung, R. A. Anal. Chem. 1981, 5 3 , 695-701. (36) O'Dea, J. J.; Osteryoung, J.; Osteryoung, R. A. J. Phys. Chem. 1983, 8 7 , 3911-3916.
RECEIVED for review March 2,1987. Accepted July 3, 1987. The work was supported by the Chemical Analysis Division of the National Science Foundation.
Liquid Chromatography-Photolysis-Electrochemical Detection for Organoiodides. 1. Optimization and Application Carl M. Selavka and Ira 5.Krull* Barnett Institute of Chemical Analysis and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115
An improved high-performance liquid chromatographic detection method has been applied for the trace determination of iodinated organic compounds. The method, which incorporates postcolumn, on-line UV irradiatlon prior to oxidative electrochemical (EC) detection, exploits the facile photochemical dissociation of the GI bond to form anionic iodide and a number of soivoiyzed products. Following bond cleavage, iodide is readily detected amperometricaily at moderate oxidative potentials, allowing for the determination of a number of OrganolOdMes at the 25-75 pg level. Fdlowing optimization of experimental parameters, the detectlon approach is linear over 8 orders of magnitude, and enhanced selectivity is demonstrated through the utilization of chromatographic retention times, dual electrode response ratios, and qualitative lamp onloff responses for anaiyte identification. The method is validated in a slngie-biind study and is successfully applied to the determination of llothyronlne (T,) in tablets.
Halogenated organic compounds are widely employed as solvents, reagents in organic syntheses, additives in manufactured products, argicultural fumigants, flame retardants, and pharmaceuticals (1,2). Unfortunately, many of these compounds are toxic, mutagenic, or carcinogenic. Typically, gas chromatographic (GC) methods are employed for the determination of organohalogens, due to the volatile nature of most of these compounds, with selective and sensitive detection obtained by using the electron capture detector (ECD) or mass spectrometry. Additionally, halogen-selective detection in GC may be obtained using near-infrared emission in an inductively coupled plasma (3) or a microwave induced plasma ( 4 ) . There is a need for alternative methods of analysis for those organohalogens which are insufficiently volatile for routine GC-ECD analysis, or for laboratories which require an analytical method to confirm GC determinations (5). In addition, time-consuming sample cleanup may be necessary prior to GC
0003-2700/87/0359-2699$01.50/00 1987 Amerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
analysis, to avoid column overload with matrix materials or to remove potential interferents prior to the separation. To circumvent GC deficiencies, or to provide the alternative method needed, high-performance liquid chromatography (LC) methods may be applied for the trace determination of organohalogens. It has been acknowledged that LC methods generally allow for less complex extraction and sample cleanup procedures (6). However, the detectors available in LC are less sensitive and far less selective for organohalogens than those used in GC. Derivatization can often be used to improve the selectivity and sensitivity of detection for an analyte during LC assay. An on-line, postcolumn derivatization method has been reported for some chlorinated phenols (7), and several off-line, precolumn derivatization approaches have been used to improve the detectability of organohalogens in LC assays (8-10). Unfortunately, precolumn derivatizations generally add complexity and time to an analysis procedure and introduce sources of contamination and error, while the reported POstcolumn derivatization method was only applicable to a very select class of halogenated compounds. For the past several years, we have been involved with the development of derivatization schemes for improved electrochemical (EC) detection in LC (LCEC). One of the methods produced involves the incorporation of postcolumn irradiation prior to oxidative EC detection. This approach, known as LC-photolysis-EC (LC-hv-EC), employs continuous, on-line, UV photolysis a t 254 nm to generate relatively stable electrophores from nonelectroactive analytes, following their separation on a reversed-phase LC column. LC-hv-EC has been successfully utilized for the trace determination of explosives and related nitro compounds in postblast residues (11,12),thiophosphate pesticides in wheat extracts (13),plactam antibiotics in commercial formulations (14),barbiturates, benzodiazepines, and other controlled substances in biological fluids (15,16), and cocaine in simulated illicit preparations (17). The lack of a suitable detection method for the trace determination of organohalogens (especially aliphatic species) has now led to the consideration of LChu-EC as a possible approach. T o date, LCEC has found limited usefulness for the determination of organo halogens, due to the poor electroactivity of these compounds within the potential range (+1.2 to -1.1 V) commonly incorporated for EC detection. However, a number of workers have demonstrated that under the proper pH and solvent conditions, and using the proper electrode material, halogen anions may be electrochemically oxidized (1419). In addition, it is generally understood that organo halogens, when irradiated with light of the proper wavelength, may undergo photodissociation to release halogen ions or radicals (20). Organoiodides, in particular, are susceptible to facile dehalogenation upon irradiation a t wavelengths below 300 nm, and a number of methods used for the determination of “organic” iodide in natural waters are based upon photochemical release of the halide from its matrix forms (21). The thyroid hormones thyroxine (T4)and liothyronine (T,), and the structurally related diiodothyronine (TJ, are important organoiodides, in terms of the need for improved analytical methods. While precolumn, off-line derivatization approaches enable capillary GC-ECD analysis, the extraordinary (subpicogram) limits of detection are obtained at considerable cost, especially with regard to sample cleanup, analysis time, convenience, and precision. Polarographic methods have been developed for T4and T, (22), but quam titation at the low levels found in complex biological matrices necessitates the use of chromatographic separation, which makes the application of these reductive EC methods rather difficult. For this reason, other LC detection methods have
exploited the oxidative electroactivity of the phenolic moiety within the structure of the iodinated hormones to advantage (23). Although it has not been demonstrated in actual Samples, these oxidative approaches would probably allow for but would be insufroutine quantitation of total T4 and TS, ficiently sensitive for the determination of free levels in plasma. I t was our desire to possibly improve upon these methods through the use of LC-hu-EC.
EXPERIMENTAL SECTION Reagents and Materials. Standards of the test analytes chosen for study, including 1-iodopentane,3-iodo-1-propene (allyl iodide), 1,2-diiodoethane (ethylene diiodide), iodobenzene, and 1,4-diiodobenzene, as well as inorganics, were obtained in the highest purity available from Aldrich (Milwaukee, WI) except iodopentane, which was obtained from Chem Services (West Chester, PA). Reagent grade HC1, H3P04, and NHIOH were obtained from J. T. Baker (Phillipsburg,NJ), iodinated thyronine standards from Sigma (St. Louis, MO), and water and methanol (MeOH) were obtained from EM Science (Cherry Hill, NJ) as the Omnisolv grade. Cytomel (50 pg of liothyronine) tablets were a gift from Smith, Kline & French (S,K&F, Philadelphia, PA). Apparatus. The construction of the LC-hV-EC apparatus has been described in detail elsewhere (16);basically, its components include a conventional reversed-phase LC system, an on-line, postcolumn irradiator,and a thin-layer amperometric EC detector. LC separations were obtained by using either a CN (10 pm) 100 X 8 mm, or CI8 (5 pm) 100 X 5 mm Radial-PAK cartridge in a radial compression module (Waters Chromatography Division, Millipore Corp., Milford, MA). The irradiator was constructed with a low-pressure Hg discharge lamp (Model 816 UV batch irradiator, Photronix Corp., Medway, MA) and a knitted open tubular (KOT) reactor composed of 0.5 mm I.D. X 1.6 mm 0.d. Teflon tubing. The geometry of the KOT was developed in our laboratory, and its construction and optimization have been reported (24). The amperometric EC detector incorporated dual LC-4B controllers, as dual glassy carbon working electrode, an Ag/AgCl reference electrode, and a stainless steel auxiliary electrode (BioanalyticalSystems, Inc. (BAS),West Lafayette,IN). A t times, tandem UV detection was incorporated (LC-UV-huEC), using either a Laboratory Data Control (Riviera Beach, FL) Model UV-I11 or a Waters Model 440 detector fixed at 254 nm. Procedure. Optimization and Characterization of LC-hv-EC. In the experiments described in this paper, unless otherwise noted, the mobile phase consisted of mixtures of MeOH and 0.2 M NaCl (adjusted to pH 3 with HC1). For maximum response from the organoiodides, the method required optimization of the period of photolysis, the EC potential used, the separation conditions, and solution pH. A temporal conversion study was performed by using a flow injection analysis (F1A)-hv-EC method. In this experiment, the flow rate of the mobile phase (50:50 MeOH:0.2 M NaC1) was altered in order to produce residence times of 1.0-3.5 min (at 0.5-min increments) for analytes in the KOT reactor. At each residence time, four replicate 50-fiL injections were made for the test organoiodides, as well as I-, at the 5 ppm level. Peak areas were collected while monitoring at +1.0 V and were normalized to the response for I-. FIA-hu-EC was also used to generate hydrodynamic voltammograms (HDVs) for 10 ppm solutions of iodide and the five test analytes, using four replicate 20-pL injections at 50-mV incrementa between 0.0 and +1.2 V. A “series HDV” was constructed for I- by first positioning the upstream (generator) electrode potential at +LO5 V and changing the downstream (collector) electrode potential to maximize response and then repeating the procedure by using a collector electrode potential of 0.0 V and varying the generator potential. Two analytical columns (CISand CN) were evaluated for the retention and resolution of test organoiodides. Once the optimal chromatographic conditions were selected, a KOT reactor was constructed from Teflon tubing having sufficient length to deliver optimum residence (irradiation) times, at the chromatographic flow rates chosen for optimum separations. The linearity of detection for the test analytes was determined by using five replicate 200-pL injections of each analyte at concentrations between 5 ppm and 5 ppb (ng/mL). In this experiment, tandem
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
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Table I. Summary of LC-I, u-EC Analytical Performance Characteristics analyte allyl iodide
iodopentane diiodoethane iodobenzene diiodobenzene
RR (+l.OV/+O.85V)"
slopeb
intrcptb
LOD-EC,' ppb/pg
LOD-UV,' ppb/ng
2.15 f 0.17 2.80 f 0.32 2.30 f 0.17 2.19 f 0.07 2.00 f 0.10
10.10 f 0.02 6.20 f 0.05 19.50 f 0.50 12.60 f 0.10 10.40 0.01
0.79 f 0.55 -0.68 f 1.09 2.40 f 2.97 0.45 f 0.39 0.29 f 0.25
0.2150 0.4175 0.1125 0.2140 0.2150
912 611 3016 80116 1012
*
OMean standard deviation (n = 5). bCalculatedusing linear regression, slope values in (nA/ppb) X 100, intercept values in nA; values are mean f standard deviation (n = 5). Calculated using the method of Foley and Dorsey (25): NFP= 0.04 nA, r = 5, 200-pL injections. UV detection at 254 nm. UV and hv-EC were incorporated to facilitate comparison of the two detection methods. These linearity plots were then employed for calculation of the limits of detection (LODs) for both UV and hv-EC detection (25). Similar studies were performed for dual series EC detection, and the single blind validation was performed in the usual manner. LC-hv-EC for Iodinated Thyronines. By use of conditions from the literature (26), separations of T4, T3, and T2 were achieved by using a mobile phase composition of 6535 MeOHH20 (the water was adjusted, before mixing, to pH 3 with H3P04),at a flow rate of 0.85 mL/min, on a CIScolumn. Dual parallel EC responses were monitored at +1.0 and +0.85 V, and calibration plots were generated by using four replicate 200-pL injections of standards at five concentrations between 10 ppm and 10 ppb. Stock solutions of the iodoamino acids were prepared at the 1 ppth level by using ammoniacal methanol (1:99 NH40HMeOH) and were diluted as needed with mobile phase on the day they were to be used. Assay of Cytomel tableb for liothyronine incorporated a mobile phase of 60:40 MeOH:O.l% H3P04at 1 mL/min, on CIS. An extraction solvent was prepared by adding 20 mL of concentrated HCl to 1-L of water in a 2-L volumetric flask and then diluting to volume with MeOH. The sampling and extraction were performed by accurately weighing and then crushing 10 pills. About 520-mg (accurately weighed) of the fine powder was transferred to a 100-mL volumetric flask, about 70 mL of extraction solvent wm added to the flask, and the sample was ultrasonicated for 10 min. The sample was taken to volume with extraction solvent and agitated to mix and then about 10 mL of the sample solution was centrifuged at 3000 rpm for 5 min. Four replicate 50-pL aliquota of the clarified solution were injected onto the chromatograph, and T3 was quantitated by the external standard method using both UV (254 nm) and EC (+1.0 V) responses. These analytically determined values were compared with each other, and with the stated (label) value, using standard statistical methods (27). RESULTS AND DISCUSSION Optimization and Characterization of LC-h r E C . The results of the FIA-hv-EC conversion study for 1,4-diiodobenzene and iodopentane are provided in Figure 1. 1,4-Diiodobenzene (and iodobenzene, not shown) exhibited the parabolic conversion relationship that had been previously observed for a number of other analytes (7, 11-17) during optimization experiments. This parabolic form indicates that initial photolytic generation of electroactive species from nonelectroactive precursors is followed by a competing photolytic or solvolytic process that destroys one or more of the generated analyte(s). Therefore, there is an optimum residence time for 1,l-diiodobenzene (and iodobenzene), in order to obtain the maximum yield of electroactive product while minimizing concomitant degradation. It was noted that the plateaus for aryl organoiodides were very broad, and attenuation of responses with longer residence times was much smaller than in previous experiments. In contrast with the results for the aromatic compounds, the conversion plots for allyl iodide, 1,2-diiodoethane, and iodopentane were nearly linear from 1- to 3.5-min residence times. These results suggested that maximum hv-EC responses for allyl iodide, 1,2-diiodoethane, and iodopentane
20
1
4 x
a
m IS
5 =
0-IP A DIB
-
II
-E& IO I
2
3
RESIDENCE TIME (Min)
Figure 1. Results of conversion study for 1,Cdiiodobenzeneand iodopentane.
could be achieved by using the longest feasible residence time. Conversion results led to the incorporation of a KOT having a volume of approximately 1.8 mL, which allowed for residence times between 1.1and 2.3 min when using chromatographic flow rates between 1.6 and 0.8 mL/min. The tubing length needed for this volume KOT (9.14 m) was also the longest which, following the 7:l reduction in length during knitting, would cover the window portion of the photolysis lamp with only one layer of the KOT. Single electrode HDVs for all the organoiodides were identical with that for iodide, exhibiting no response a t potentials below approximately +650 mV, a steeply rising portion from +725 to about +900 mV (Eqz = +810 mV), and diffusion limited response at E 2 +935 mV. Photolytic cyclic voltammograms (PCVs) exhibited points of distinction (28,29),while the HDVs were similar for all species. This contrasting behavior was probably due to the minor generation of phenolic products from low concentrations of iodobenzene and 1,4diiodobenzene in the HDV experiment, with respect to the larger generation of iodide. The CV experiments were performed at much higher analyte concentrations (1ppth vs. 10 ppm) than in the HDV studies. When dual parallel working electrode potentials of +1.0 V and +OB5 V were incorporated in LC-hv-EC, response ratios (Rh)were found to be quite similar for all of the test compounds except, inexplicably, iodopentane (Table I). Although it had been demonstrated that aromatic and aliphatic organoiodide precursors could be differentiated by using response ratios at +1.0 and +0.75 V (28))the linearity of the RRs a t these potentials was poor. This finding can probably be attributed to nonlinearity in the amplification of the small currents generated at +0.75, when concentrations of analyte below 1 ppm were injected. Therefore, dual parallel electrode potentials of +LO and +OB5 V were used in the remainder of the LC-hv-EC applications. The last FIA experiment involved the optimization of generator and collector electrode potentials in dual series EC detection of I-. As demonstrated in the "dual HDV" depicted in Figure 2, it was determined that reductive response at the collector electrode was maximized by using upstream and downstream potentials of +1.05 and 0.00 V, respectively. At these potentials, the collection efficiency for series detection
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987
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Table 11. Results of Single Blind Study-MeOH Spiked with 1,2-Diiodoethane
determined level,” ppb
spiked level, ppb
difference,
sample 1 2 3 4 5
990 f 15 11 f 0.5 50 f 1.1 0 509 f 8
1000 10 50 blank 500
-1.0 +10 0 0 +1.8
W, .Abscissa
wp0.ov Monitor W2 W,. + 1.05V W2=Abscirra
%
“Mean f standard deviation (n = 9).
Monitor Wz
Table 111. Figures of Merit for Iodinated Thyronines Using
LC-h V-EC -3 - 2
-I
00
I
2
3
4
5
6
7
8
9
IO
II
12
APPLIED P O T E N T I A L ( V o l t s v o Ag/AgCI)
Figure 2. Dual hydrodynamic voltammogram: W, upstream (pnerator) electrode, W, downstream (collector) electrode.
.o
.I
I
analyte
Tz LC-hv-EC
FOR
ORGANOIODIDES
T3
T4
85V
k’
lamp on RR(+1.0/ LOD,b +0.85)O ppb/ng
1.79 1.45 f 0.02 2.74 1.75 f 0.17 4.24 1.28 k 0.01
0.6/0.1 0.8/0.2 1.4/0.3
lamp off RR(+1.0/ LOD,b +0.85)” ppb/ng 9.67 f 0.03 8.05 f 0.31 2.69 f 0.01
1/0.2 2/0.4 5/1.0
“Mean f standard deviation (n = 4). bCalculated using the method of Foley and Dorsey (25): Nvp = 0.1 nA, r = 5.
I~
y. Y
I - 200ppb AL-l 2- 6Oppb ED1 3- 1OOppb I8 4- I8Oppb I-IP 8- IOOppb D I 8
L
f
oov
(MINUTES)
Figure 3. LC-h v-EC separation and lamp onloff detection of organoiodides.
(reductive current/oxidative current) was 0.36 f 0.02 ( n = 10). However, when the dual series EC approach was attempted for organo iodides in LC-hv-EC, reductive current responses at the downstream electrode were only linear over approximately 1order of magnitude (from 10 to 1ppm). At lower concentrations, severe peak tailing and reduced responses were observed for the test analytes and iodide. One possible explanation for this effect was nonlinearity in the iodine absorption process on the electrode during oxidation of iodide, perhaps as a function of the character of the electrode surface and the concentration of the absorbing species. Even with electrochemical pretreatment (preanodization at +1.5 V for 30 min) of the electrodes (30),or plug injections of high concentrations of iodide before collection of analytical
measurements (19),the nonlinearity of response could not be rectified. For these reasons, the dual series approach for organoiodides was not competitive with the dual parallel detection method in terms of reproducibility, linearity, and sensitivity. As demonstrated in Figure 3, adequate separation of the test analytes could be achieved by using the CN column, with a mobile phase consisting of 40:60 Me0H:O.Z M NaC1. This figure also demonstrates the unusual selectivity available in LC-hv-EC, in that the identity of a compound in a sample mixture is based on the capacity factor, dual electrode response ratio, and qualitative lamp on/off behavior. In other words, misassignment of a peak identity to interfering compounds in a sample would require that the interferents possess similar chromatographic behavior, EC behavior, and photolytic behavior. These three modes of selectivity are unique to LChv-EC, making this method highly specific when compared to similarly priced LC detection methods. Table I provides a summary of the linearity of detection, LODs, and RRs for the test analytes. Injections of organo iodides at the levels of the calculated LODs revealed that the precision (% RSD at +LO V) at these concentrations was k23% ( n = 25). A t levels >3 X the LODs, the precision was better than * 5 % RSD. The RRs were linear for concentrations between 5 ppm and about 10 ppb, below which the values tended toward unity. It was noted the RRs were reproducible within-day but could change by as much as 20% between days, depending on the condition of the electrode and the mobile phase composition. Therefore, daily generation of dual electrode calibration plots is suggested before using LC-hwEC in qualitative or quantitative experiments. The results from the single blind study, wherein 1,2-diiodoethane was spiked into MeOH, are shown in Table 11. These data indicate that LC-hv-EC is, indeed, a valid quantitative method for the trace determination of organoiodides. LC-h V-EC for Iodinated Thyronines. Even though T4, T3,and T2 could be oxidized in their native forms, the selectivity of detection was enhanced through the use of changes in RRs under photolytic and nonphotolytic conditions. As noted in Table 111, identification of these compounds in a sample would be facilitated through the use of RRs, owing to the large differences in lamp on/off values. In addition, the sensitivity of detection was enhanced under photolytic
ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 L c - h". E c
+ LOOV
LC-EC
LC-UV
-I-
0-0
I-
Figure 4. Chromatograms of LC-UV-h u-EC assay for liothyronine in Cytomel tablet extracts: Left trace: lamp on; center trace; lamp off; right trace, UV (254 nm).
conditions, although not nearly to the extent that had been expected or desired when the application of LC-hv-EC to these compounds was undertaken. It is possible that through the use of longer residence times, or more intense light sources, the photolysis-EC method could significantly lower the LODs for these iodoamino acids. The determination of liotliyronine in Cytomel tablets, using tandem UV and dual parallel hv-EC detection, gave quantitative values (in pg of T3per tablet) of 52.3 f 3.1 and 53.1 f 2.3 (n = 27 for both assays), respectively. Statistical comparison of these means with the stated value (50 f 5, as allowed by the USP), using the Student's t test at the (P = 0.01) level, revealed that the LC assay results were not significantly different from the stated value. As shown in Figure 4, neither UV, EC, or hv-EC detection traces exhibited any extraneous peaks that could be confused for that corresponding to the analyte of interest. However, this figure shows that the selectivity of detection for T3was improved through the use of the dual electrode response ratio differences, in both the lamp-on and lamp-off mode. The use of postcolumn photolysis enhanced the specificity and sensitivity of detection over that available with conventional oxidative methods, as well as UV detection, and should allow for determination of total T4and T3levels in biological fluids (23). However, at this time, quantitation of free levels of these thyroid hormones in biological matrices would still not be possible, even with postcolumn photolysis. As a final note, it should be emphasized that this study represents the first step in a three-part research program to design an improved LC detection method for organohalogens. The results obtained in work with organoiodides were important in designing similar approaches for brominated and chlorinated compounds. While the number of analytically important organoiodides is admittedly rather limited, the ultimate generation of LC-hv-EC approaches for brominated and chlorinated organics will fill a void in the applicability of LC methods for routine determinations of such compounds in regulatory, toxicological, pharmaceutical, or research laboratories.
ACKNOWLEDGMENT The EC detectors used were donated by Bioanalytical Systems,Inc., and we are grateful to P. Kissinger and R. Shoup
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for their continued technical assistance and support. We also extend our appreciation to J. Paul of Smith, Kline & French for coordinating donation of the Cytomel samples and E. Rogers of Northeastern University for providing standards of the thyroid hormones and helpful discussions. The work was facilitated by the assistance of W. Lacourse, S. Colgan, J. Burton, L-R. Chen, and R. Nelson at N.U. and M. Lookabaugh at the Boston district office of the FDA. Registry No. TZ, 1041-01-6;T3,6893-02-3;T4,51-48-9;cytomel, 55-06-1; allyl iodide, 556-56-9; 1-iodopentane,628-17-1; 1,2-diiodoethane, 624-73-7; iodobenzene, 591-50-4;1,4-diiodobenzene, 624-38-4.
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RECEIVED for review December 9, 1986. Accepted June 25, 1987. Funding was provided by a Phase 1 NIH-SBIR (No. 1R43ES04057-01) grant to Northeastern University and Cambridge Analytical Associates (Boston, MA), an NIH Biomedical Research Grant to Northeastern University (No. RR07143), and an unrestricted grant from Pfizer, Inc. (Groton, CT). Support for C.M.S. was provided by a National Institute of Justice, U S . Department of Justice, Graduate Research Fellowship (No. 86-IJ-CX-0058), and a scholarship from the Northeastern Association of Forensic Scientists. Points of view or opinions stated in this document are those of the authors and do not necessarily represent the official position or policies of the US. Department of Justice. This is contribution number 326 from the Barnett Institute of Chemical Analysis and Materials Science at Northeastern University.
