700
Anal. Chem. 1980, 52, 700-704
(31) A. Craggs, G. J. Moody, and J. D. R. Thomas, J . Chem. fduc,51, 541 (1974). (32) T. Treasure, Intens. Care Med., 4, 83 (1978). (33) N. A. J. Platzer, "Plasticization and Plasticizer Processes", Advances in Chemistry Series, Vol. 48, American Chemical Society, Washington, D.C., 1965. (34) J. L. Hill, L. S. Gettes, M. R . Lynch, and N. C. Hebert, Am. J. Physiol. 235, H455 (1978). (35) D. Ammann, R . Bissig, 2. Cimerman, U. Fiedler, M. Guggi, W. E. Morf, M. Oehme, H. Osswald, E. Pretsch, and W. Simon, in "Proceedings of the International Workshop on Ion Selective Electrodes and on Enzyme Electrodes in Biology and Medicine", M. Kessler et al., Eds., Urban & Schwarzenberg, Munich, 1976, p 22. (36) W. Simon, D. Ammann, M. Oehme. and W. E. Morf, Ann. N . Y . Acad. Sci., 307, 52 (1978). (37) M. Guggi, Ph.D. Thesis, ETH 5866, Zurich, 1977.
(38) J. A. Riddick and W. B. Bunger, "Organic Solvents", Techniques of Chemistry, Vol. 11, Wiley-Interscience, New York, 1970. (39) W. D. Stein, "The Movement of Molecules across Cell-Membranes", Academic Press, New York, 1967. (40) A. Leo, C. Hansch. and D. Elkins, Chem. Rev., 71,525 (1971). (41) J. Petrgnek and 0. Ryba, Anal. Chim. Acta, 72, 375 (1974). (42) A. M. Y. Jaber, G. J. Moody, and J. D. R. Thomas, Analyst(London) 101, 179 (1976). (43) U. Fiedler, Anal. Chim. Acta, 89, 101 (1977). (44) J. W. Ross, Orion Research Inc., Cambridge, Mass., private communication.
RECEIVEDfor review October 30, 1979. Accepted December
26, 1979.
Reversed-Phase Liquid Chromatography of Basic Drugs and Pesticides with a Fluorigenic Ion-Pair Extraction Detector Carmen van Buuren, J. F. Lawrence,' U. A. Th. Brinkman, I. L. Honigberg,2 and R. W. Frei" Department of Analytical Chemistry, Free University, D e Boelelaan 1083, 108 7 HV Amsterdam, The Netherlands
fluorescence detection of some basic drugs, pesticides, and their metabolites is described. The addition of the ion-pairing reagent dimethoxyanthracene sulfonate (DAS) prior to the column permits a drastic simplification of the detector design. The Influence of the DAS concentration, pH, buffer concentration, and organic polarity modifier (methanol) on the separation of the above compounds carried out in the reversed-
DAS
--a
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7
-
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g
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-
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1
f l u o r o r e s c o n c o detuctor
B -
l r o m HPLC
DAS AIR
The use of post-column reaction detectors ( 1 , Z ) to expand the usefulness of existing detectors in liquid chromatography (LC) has gained widespread acceptance. Different types of reactor designs ranging from tubular ( 3 )and bed ( 4 ) reactors with nonsegmented streams to segmented-flowprinciples have been proposed ( Z ) , the latter being particularly useful for longer reaction times on the order of 5 min and longer ( 4 ) . Recently it has been shown (5,6)that segmentation techniques can also be used to advantage for relatively fast reactions, mainly in cases where the signal of the excess reagent interferes with the signal of the product. In these cases, a dynamic micro-extraction procedure has been adopted to separate the excess reagent from the less polar product. The feasibility of such an approach has been tested with tertiary amines of pharmaceutical and agricultural importance,
'On a transfer o f work 1978 79 f r o m the Food Directorate, Health Protection Branch, Ottawa, danada. 2 0 n sabbatical leave 1Y78 from the University of Georgia, School of Pharmacy, Athens, Ga.
QRG
+ I
waste
k
g g
extraction coils
Y-
Figure 1. Schematics for ion-pair extraction and fluorescence detection. A = with air segmentation. B = without air segmentation (solvent segmentation)
and dimethoxyanthracene sulfonate (DAS) proposed earlier by Westerlund and Borg (7) as a fluorescent counterion for ion-pair formation. The schematic of the first detector construction coupled to reversed-phase separation and based on a three-phase segmentation system (5,6) is shown in Figure 1A. The reagent is added to the column effluent right after the column, air segmentation being used to efficiently reduce band broadening.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
H2
H2
/CH3
C -- C--hl
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701
C PA
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ChloropPeniromine
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Bromopheniramina BPA
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Diphenhydramine
\ CH3
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Atropine
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SAN
,SAN
HA
Figure 2. Compounds investigated
The high-density organic extractant is then added and, after extraction of the ion pair into the organic phase, the layers are separated in a conventional phase-separator. Fluorescence detection of the ion pair then takes place in the organic solvent. In a further step the air bubble was dropped and the water-immiscible plug of the extracting solvent now was also
used for controlling band broadening (see Figure 1B). The possibilities and advantages of this solvent-segmentation approach have been described (8). I t was the purpose of this study to further investigate the potential of the extraction-detector system for the determination of basic drugs, pesticides, and their metabolites and
702 * ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
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-
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+ DASON
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Schematic for ion-Dair extraction and detection: usina t h e solvent segmentation principie, when mobile phase contains bAS counterion. Figure 3.
to use pre-column technology for partial sample cleanup and trace enrichment (6, 9, 10). Attention was chiefly devoted to (i) the addition of the ion-pairing agent to the mobile phase prior to the column, in order to further simplify the detection system, and (ii) the chromatographic behavior of the said types of compounds on a series of reversed-phase columns of widely different polarity. Finally an application of this technique to urine analysis was attempted.
AtrOP
’ SCOP I SAN
EXPERIMENTAL Reagents. The tertiary amine compounds investigated are listed in Figure 2 with structures, names and abbreviations used in this study. H T and HA are the major metabolites of the herbicides terbutylazine and atrazine and have been obtained through CibaGeigy Ltd. Basle, Switzerland; chloro- and bromopheniramines and diphenhydramine have been provided by the laboratory of one of the authors, I. L. Honigberg. SAN is a pesticide manufactured by Sandoz Ltd. and has been donated along with the tropa alkaloids and the dimethoxyanthracene sulfonate (DAS)by the same company (Sandoz,Basle, Switzerland). The compounds in Figure 2 were dissolved in distilled water in concentrations of 1 mg/mL except HA and HT, for which 0.1 mg was dissolved in 1 mL methanol. DAS was dissolved in its sodium-salt form in doubly distilled water at a concentration of 10-4 M. All organic solvents and chemicals were of analytical-grade quality (J. T. Baker, Deventer, The Netherlands). Chromatography and Apparatus. Reversed-phase LC of the basic compounds was carried out on home-packed columns (10 cm X 3 mm i.d.) with experimental and commercial batches of chemically bonded 10-pm silica gels of the diol- and CN-type of LiChrosorb RP-2, RP-8, and RP-18 (Merck,Darmstadt, G.F.R.). For some applications, a Varian (Walnut Creek, Calif.) Micro-Pack CN-10 column (30 cm X 4 mm i.d.) has been used. The mobile phases are described in the following text and in the legends to the figures. A Perkin-Elmer Series 2 high-pressure pump and a six-port injection valve were employed. Detection System. The schematic of the arrangement when DAS was added after the column is represented in Figure 1B. Figure 3 shows the simplified arrangement with pre-column reagent addition. A simple two-channel peristaltic pump can be used for delivering the organic solvent (dichloroethane or tetrachloroethane) and for drawing the organic phase through the phase-separator and detector to waste. The pump, pump tubing, glass extraction spirals, and phaseseparators were conventional AutoAnalyzer components (Technicon, Tarrytown, N.Y.). A modification of the phase-separator to reduce the dead volume has been described earlier (11). An Aminco Fluoromonitor (Aminco, Silver Springs, Md.) equipped with a Corning-16 primary filter and a Wratten 2-E emission filter was used for the application. All other measure-
0’
’ 10-4
5 10-4
10-
Pconcentration D A S ( M )
Figure 4. Effect of DAS concentration on k’values using an RP-2 column and a mobile phase consisting of 25% MeOHlO.1 N NaH,PO,, pH 3.5. Flow = 1.0 mL/min. No DAS added at point 0
ments were done with a Perkin-Elmer fluorescence detector model 204A. Excitation and emission maxima of the extracted ion-pairs were at 386 and 446 nm, respectively. In some studies a variable-wavelength UV detector (LC 55, Perkin-Elmer) was placed between the column and the extraction detector (Perkin-Elmer, Norwalk, Conn.). Sample Preparation. Samples of up t o 10 mL of undiluted urine were concentrated on a pre-column containing a 2-mm thick (4-mm i.d.) plug of 5-pm LiChrosorb RP-18, at a flow-rate of approximately 3 mL/min. The compounds were then transferred with the mobile phase on-line to the analytical column and separated. The pre-column construction and performance have been described in detail elsewhere (10). For details, see legend in Figure 8.
RESULTS AND DISCUSSION Chromatography. The studies on the dependence of retention on DAS-concentration (Figure 4) were done with LiChrosorb RP-2 material which has intermediate properties between the RP-8 and RP-18 phases on one side and the polar diol and nitrile phases on the other and permitted us to obtain measurable values of the capacity ratios, i.e., u p to k ’ 2 0 for all compounds studied. With DAS concentrations increasing from 0 to approximately M, all model compounds show about a 1.5-fold increase in k’value (see Figures 4 and 5). Influence of t h e Stationary Phase. Different stationary phases ranging from the polar diol and nitrile materials to the reversed phase supports RP-2, RP-8, and RP-18 were investigated. In order to facilitate the comparison of the retention behavior of the five stationary phases investigated, Figure 5 shows k’ values of all model compounds using a single mobile-phase composition, viz. 25% methanol in water (0.1 N NaH2P04;p H 3.5) without (Figure 5 A) and with (Figure 5B) DAS added.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
20 -
A
703
B
A
HT
Figure 5. Variation in k’values with different stationary phases for the compounds studied. Mobile phase as in Figure 4. A = without DAS in mobile phase. B = with lo-‘ M DAS in the mobile phase. (See Figure 2 for abbreviations)
ttime
-
Figure 6. Chromatograms for a urine sample after pre-concentration.
A = UV trace at 225 nm; arrow indicates position of HA. B = fluorescence trace. I = blank urine, I1 = urine spiked with 0.5 ppm HA. 10 mL urine preconcentrated each time. Chromatographic conditions as in Figure 4
Retention invariably reaches a maximum with the RP-8 support. Very little influence is exerted when adding DAS under the prevailing conditions. The largest difference was observed for the Diph with an increase of k’from 9 to 14 or a k ’ ~ * ~ / k ’ value of 1.56. The somewhat anomalous behavior of the RP-8 material in comparison to the other reversed-phase supports (Figure 5, A and B) has been found consistently with a home-
packed column as well as with two commercial columns of different origin. Detection. From the previous results it can be concluded that pie-column addition of the ion-pairing reagent is feasible without necessitating drastic changes in the mobile phase. The resulting simplification of the detector is evident from Figure 3. Since the reagent is already homogeneously mixed at the column outlet, the extraction equilibrium state can be reached more efficiently and less reactor volume is needed. A similar simplified design with only two pump channels has previously been used for the coupling of normal-phase chromatography with this DAS ion-pair extraction principle; there the column effluent (CHC13+ methanol) acted as the nonmiscible organic segment and an aqueous reagent solution was added a t the column outlet ( 2 1 ) . The quantitative analytical potential of these extraction detectors is good. The reproducibility for multiple injection ( N = 4) of the same sample solution is 3% RSD for all designs discussed here. A linear dynamic range has been observed over two orders of magnitude (6, 8). Detection limits as recorded with the Perkin-Elmer Model 204A fluorescence detector were in the lower nanogram range except for SAN (400 ng). Application to Urine Samples. An argument which is as important as sensitivity is the enhanced selectivity of this detection mode, which permits trace analysis in complex matrices with a minimum of sample preparation. The potential of this detection technique for handling urine samples has been demonstrated earlier (6). In this study a method has been developed with potential for routine screening of the metabolite HA in urine samples. The determination of HA, which is relatively polar and present in very low concentrations in the matrix has posed considerable
704
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
t
. D A S ( ~ O - ~DMA S )
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L
.:
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Figure 7. Effect of sample volume on pre-concentration of 1 .O ppm HA from water. I = direct pre-concentration on RP-18 5 - ~ m pre-column; length 2 mm, i.d. 4 . 6 mm. I1 = pre-concentration in the ion-pair form with addition of M DAS
problems in routine analysis via conventional extraction and detection techniques. Pre-column technology had again to be used for pre-concentration of the urine samples, for partial cleanup and for protection of the analysis column (6). In Figure 6, one can see chromatograms obtained for 10-mL urine samples spiked with 0.5 ppm HA. The 10-mL urine sample was pre-concentrated on the pre-column without further sample treatment and the compounds were transferred on-line with the mobile phase to the separation column. The advantage of the ion-pair extraction detector mode as compared to UV detection is obvious from this figure. Pre-concentration of 10-mL samples of HA on 2-mm LiChrosorb RP-18 layers can already be quite critical with regard to recoveries due to breakthrough of the HA (see Figure 7). The reason for this probably is not an overloading phenomenon. HA and many other metabolites are often significantly more polar than their parent compounds and would have a higher mobility (lower k 9. With large sample volumes, this mobility will permit them to move through the entire pre-column during the injection step and eventually this results in a loss of the component. The polarity can be reduced by forming an ion pair and pre-concentrating the compound as an ion pair. This was done in the case of HA by adding M solution. As can be seen in Figure 7, DAS to give a the breakthrough point is thus shifted to a significantly higher volume. The effect of this phenomenon on an actual chromatogram is shown in Figure 8 with a more than 50% gain in recovery when injecting the HA in the DAS ion-pair form.
CONCLUSIONS The influence of pH, buffer concentration, and counterion concentration of the mobile phase is relatively small for the separation of the basic model compounds on chemically bonded stationary phases of divergent polarity. However, as is to be expected, retention is highly dependent on the proportion of methanol present in the mobile phase. Fortunately, experience has shown us that the extraction detector can tolerate up to 20-30% of methanol without unreasonable
tlrnQ
-
Figure 8. Resulting chromatograms for the pre-concentration and analysis of 25 mL of urine. (---) = direct pre-concentration; (-) = pre-concentration after the addition of M DAS. HA spiked at 0.1 ppm. chromatography conditions as described in Figure 4. Column RP-2 10 k m , length 10 cm, i.d. 4 . 6 mm
increase in background fluorescence. In summary, there is good flexibility in adjusting the mobile phase composition to the best conditions needed for the detection system; the availability of different types of stationary phases enhances this flexibility (8). The study of the influence of ion-pair formation on separation behavior reveals only small effects when a 25% MeOH in water mobile phase is used. The possibility of adding the ion pairing reagent t o the mobile phase prior to the column permits considerable simplification of the extraction, detector design and eliminates one source of contribution to band broadening.
ACKNOWLEDGMENT We thank F. Eisenbeiss of Merck, Darmstadt, G. F. R., for providing us with different chemically bonded HPLC materials. The gift of a CN-bonded column by R. Majors of Varian is also acknowledged. LITERATURE CITED Lawrence, J. F.; Frei, R. W. "Chemical Derivatization in Liquid Chromatography", Elsevier: Amsterdam, 1976; Chapter 4. Frei, R. W.; Scholten. A. H. M. T. J. Chromatogr. Sci. 1979, 17, 152. Frei, R. W.; Michel, L.; Santi, W. J . Chromatogr. 1977, 142, 251. Deelder. R. S.; Kroll, M. G. F.; Beeren, A . J. B.; van den Berg, J. H. M. J . Chromatogr. 1970, 149. 669. Gfeller, J . C.;Frey, G.; Huen, J. M.; Thevenin, J. P., J. High Res. Chromatogr. Chromatogr. Commun. 1970, 4 1 , 213. Frei, R. W.; Lawrence, J. F.; Brinkman, U. A. Th.; Honigberg. I. J . High Res. Chromatogr. Chromatogr. Commun. 1979, 2, 1 1 . Westerlund, D.; Borg. K . 0. Anal. Chim. Acta 1976, 67, 89. Lawrence, J. F.; Brinkman. U. A. Th.; Frei, R. W. J. Chromatogr. 1979, 171, 73. Frei, R. W. Int. J. Environ. Anal. Chem. 1970, 5, 143. van Vliet, H. P. M.; Bootsman, Th. C.; Frei, R. W.; Brinkman, U. A . Th. J. Chromatogr. 1979, 185. 483. Lawrence, J . F.; Brinkman, U. A. Th.; Frei, R. W. J. Chromatogr. 1979, 185, 473.
RECEIVED for review August 6, 1979. Accepted January 14, 1980. Partial support has been given for this study by Sandoz Ltd., Basle, Switzerland.
