Anal. Chem. 1987, 59, 2127-2130
LITERATURE CITED Van Bladeren, P. J. J. Am. Coll. Toxlcol. 1983, 2 , 73-83. Borman, S. Anal. Chem. 1984, 56, 573A. US EPA Report, Fed. Reg. 1983. 48(No. 197), 46229-46230. Isaacson, P. J.; Hankln, L.; Frlnk, C. R. Sdence 1984, 225. 672. Casanova, J.; Rogers, H. R. J . Org. Chem. 1974, 39, 2408-2410. Hawley, M. D. I n Encyclopedic of Electrochemistry of the Elemsnts; Bard, A. J., Lund, H., Eds.; Dekker: New York, 1980; Vol. XIV, pp 1-136. (7) Fry, A. J. Synthetic Organic Electrochemishy; Harper and Row: New York, 1972; pp 170-187. (8) Tokoro, R.; Bllewicz, R.; Osteryoung, . - J. Anal. Chem. 1986, 58, 1964- 1969. HIII, H. A. 0.; hatt, J. M.; O'Riordan, M. P.; Williams, F. R.; Williams, R. J. P. J. Chem. SOC.A 1971, 1859-1862. Connors, T. F.; Arena, J. A,; Rusllng, J. F.. unpublished results, 1987. Sharma, M. K.; Shah, D. 0. Introduction to Macro and Mlcroemulslons: Shah, D. 0.Ed.; ACS Symp. Ser. No. 272; American Chemical Society, Washington, DC, 1985; pp 1-18. Franklin, T. C.; Iwunze, M. Anal. Chem. 1980, 52, 973-976. Berthod, A.; Georges, J. Anal. Chim. Acta 1983, 147, 41-51. Georges, J.; Berthod, A. J. Electroanal. Chem. Interfaclel Electrochem. 1984, 175, 143-152. Fendler, J. H.; Nome, F.; Van Woert, H. C. J. Am. Chem. SOC.1974, 96, 6745-6753. Osteryoung, J.; O'Dea, J. J. I n Electroanalytical Chemlsfty; Bard, A. J., Ed.; Marcel Dekker: N. Y. 1986; Vol. 14, pp 209-308. Kamau, G. N.; Wlllls, W. S.; Rusllng, J. F. Anal. Chem. 1985, 57, 545-55 1.
(1) (2) (3) (4) (5) (6)
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(18) Nicholson, R. S.; Shain. I. Anal. Chem. 1964, 36, 709-723. (19) Mato, F.; Hernandez, J. L. An. Qulm. 1969, 65, 9-18. (20) Connors, T. F. PhD. Thesis, University of Connecticut, $torrs, CT, 1986. (21) Lexa, D.; Saveant, J. M. Acc. Chem. Res. 1983, 76,235-243. (22) Nicholson, R. S. Anal. Chem. 1985, 37, 1351-1355. (23) Andrleux, C. P.; Blocman, C.; Dumas-Bouchiat, J. M.; M'Halla, F.; Saveant, J. M. J. Electroanal. chem. Interfacial Electrochem. 1980, 113, 19-40. (24) Arena, J. A.; Rush'ng, J. F. Anal. Chem. 1986, 58, 1481-1488. (25) Winters, L. J.; Grunwald, E. J . Am. Chern. SOC. 1965, 87, 4608-461 1. (26) Rothbart, H. L.; Snook, M. E.; Rusling, J. F.; Scott, W. E. J. Am. Oil. Chem. SOC.1969, 46, 327-331. (27) Hoag, G. E.; Marly, M. C.; Brull, C. J. Proceedings of the Conference on Oil and Fresh Water: Chemical and Biological Technology; Edmonton, Alberta, Canada, Oct 1984. (28) Colgan, S.T.; Krull, I. S.; Dorschel, C.; Bidlingmeyer, B. Anal. Chem. 1988, 58, 2366-2372.
RECEIVED for review January 5,1987. Accepted May 26,1987. This work was supported by US PHS Grant ES03154 awarded by the National Institute of Environmental Health Sciences and, partly, by the donors of the Petroleum Research Fund, administered by the American Chemical Society. This paper is part 4 in the series Electrocatalytic Reactions in Organized Assemblies.
Anodic Behavior of the Antineoplastic Agent Amethopterin at a Mercury Electrode and Its Determination in Body Fluids by Liquid Chromatography with Indirect Anodic Polarographic Detection Antonio Guerrieri and Francesco Palmisano* Laboratorio di Chimica Analitica, Dipartimento di Chimica dell' Universitd, Via G . Amendola, 173, 70126 Bari, Italy
Amethopterln has been found to be anodlcaHy electroactlve on mercury due to a depolarlratlon effect arlslng from the formation of a sparingly soluble mercury complex. Hlghpetformame HquM chromatography wlth anodlc pdarographk (sampled dc) detectlon at +0.19 V vs. Ag/AgCI has been employed for the determlnatlon of the drug In body flulds. Detector response was found to be Unear In the range 5-1000 ng on column. A detectlon llmlt of 1.5 FM amethopterln in serum was achieved. The wlthlnday and day-today coefflclent of varlatlon at the 10 bg/ml level were 1.5 and 3.7% respectively.
Amethopterin (Methotrexate, MTX or L-(+)-N-(~-[ ((2,4diamino-6-pteridinyl)methyl)methylamino] benzoy1)glutamic acid) is a competitive inhibitor of dihydrofolate reductase currently used in the treatment of several human cancers including acute lymphocytic leukemia, osteosarcoma, nonHodgkin's lymphoma, breast carcinoma, and choriocarcinoma. The effectiveness of MTX increases with high dosage regimens but the risk of haematologic and renal toxicity also increases. Citrovorum factor (Leucovorin) rescue is used to protect patients from overdosage toxicity effects associated with high concentrations of MTX in plasma and/or with a delayed MTX elimination. However, a 6% incidence of drug-related 0003-2700/87/0359-2127$01.50/0
deaths has been reported (I). Such a noticeable mortality rate accounts for the stringent need of clinical and pharmacokinetic monitoring of high-dose MTX treatments. Several protocols (2)are presently used for monitoring high-dose MTX therapy, all of them requiring measurement of serum or plasma drug concentration. Fluorometry (3),radioimmunoassay (4-6), enzyme immunoassay (7), and high-performance liquid chromatography (HPLC) with UV detection (8-12)are the most often used techniques. A significant amount of work in the field of liquid chromatography/electrochemical detection (LC/EC) methodology for the determination of pteridine derivatives, other than MTX, has been accomplished by Lunte and Kissinger (13, 14), Picomole amounts of several oxidized and reduced pteridines were simultaneously determined in biological samples by LC/EC with a dual electrode (glassy carbon) amperometric detector. Recently (15) some preliminary data concerning the anodic electroadivity of MTX on glassy carbon have been reported, as well as a LC/EC method for MTX determination in body fluids. In this paper the peculiar anodic behavior of MTX at a mercury electrode is described. An anodic wave caused by the formation of an insoluble film has been observed. Although the ability of purine and pyrimidine derivatives to yield anodic polarographic currents arising from formation of sparingly soluble mercury compounds has been extensively 0 1987 American Chemical Society
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reported (16, 17), this is, as far as we know, the first report about the anodic activity of pteridine-related compounds at a mercury electrode. The anodic electroactivity of mercury suggests a possibility of considerable analytical interest, e.g., the determination of analytes I,”-, forming a mercury complex according to electrode processes of the kind
2Hg
0.5pA
+ 2L”- + Hg2L22(1-n)+ + 2e-
(2) by liquid chromatography coupled to anodic polarographic detection (LC-APD). Such electrode processes involve oxidation of the mercury in the presence of a mercury complexing agent and occur at potentials less positive than for oxidation of mercury itself. Electrochemical detection methods based on such processes would, in theory, be highly selective because of both the low value of the applied potential and the selectivity of the electrode reaction. The usefulness of anodic detection on mercury for the determination of some biologically important thiols has been widely recognized (78-22). The general applicability of this principle of detection to non-sulfur-containing compounds of biological significance has been recently demonstrated in the authors’ laboratory. LC-APD has been successfully used for the determination of purine and pyrimidine derivatives (23-25) in serum and urine. The applicability of this detection mode to MTX, taken as a representative member of pteridine derivatives, enhances further the potential of the method.
EXPERIMENTAL SECTION Instrumentation. A PAR Model 174A polarographic analyzer (EG&G Princenton Applied Research Co.) coupled to a PAR Model 303 stationary mercury drop electrode (SMDE) was used to perform polarographic measurements. Cyclic and stripping voltammetric experiments were done with the SMDE operating in the “hanging mercury drop” mode. A Perkin-Elmer Model 3B pump module coupled to a Rheodyne 7125 injection valve and a reversed-phase column (Brownlee RP18, 5-gm, 250 X 4.6 mm i.d.) was used a.. the chromatographic system. A Brownlee RP-GU guard cartridge (30 X 4.6 mm i.d.) fitted into a Brownlee MPLC holder was used to protect the analytical column unless otherwise specified. The polarographic detector was the above cited SMDE converted (23) in a wall-jet flow cell and operated in the dropping mode with a drop time of 1s and a “medium”drop size (estimated area 0.016 cm?. The receiving solution was the same as the mobile phase. PTFE tubing (15 cm X 0.25 mm i.d.) was used to connect the detector flow cell to the column. A pulse dampener, consisting of a Bourdon tube and an empty chromatographic column both in a tee configuration, was placed between the pump outlet and the injector in order t o ensure a relatively pulseless delivery of the column effluent to the flow-sensitive electrochemical detector. Chemicals. MTX, folic acid, folinic acid, and 5-bromouracil were obtained from Sigma (St. Louis, MO). All other reagents (Carlo Erba, Milan, Italy) were analytical grade. Solvents used were HPLC grade (J. T. Baker, Deventer, Holland). Buffers used in the mobile phase were filtered through a 0.45-gm membrane (Gelman Sciences, Ann arbor, MI). Strong anion exchange disposable microcolumns (Baker 7091-3 Quaternary Amine) used for sample treatment were obtained from J. T. Baker. Chromatographic Conditions. Unless otherwise specified the following conditions were used: mobile phase, 0.05 M phosphate buffer pH 6.4-methanol-acetonitrile (92:44 v/v); flow rate, 1mL/min; injection volume, 20 gL; temperature, ambient. Sample Treatment. Serum. To 250 pL of serum were added 10 p L of the internal standard (5-bromouracil)methanolic solution and 250 g L of 0.8 M trichloroacetic acid (TCA). The sample was vortex-mixed for 1 min and then centrifuged at ca. l8OOg for 5 min. Twenty microliters of the supernatant was directly injected. When a preconcentration step WRS required, the microcolumn extraction procedure described in ref 16 was followed. Urine. T o 1 mL of urine was added 1 mL of 0.5 M Na2HP0, and the resulting solution was loaded on a Raker microcolumn
L
P
O
T
E
N
T
I
A
L
Figure 1. Typical cyclic voltammograms of MTX recorded at a hanging mercury drop electrode in a 0.05 M borate buffer pH 9.2: [MTX] = 2.0 X M; scan rate, 50 mV s-‘. The cathodic part of the cyclic voltammogram B has been obtained after a 20-s preconcentration time at 4-0.1 V before scan reversal. Potential are referred to an AgIAgCI, (3 M CI-) reference electrode.
previously treated as follows. Three milliliters of each of the solvents CH,OH, 9 M “,OH, and H20was flushed through the column and the column briefly air-dried between each solvent addition. Three milliliters of 0.2 N HC1 was then flushed without allowing the column to dry at this stage. After sample addition the column was washed with 6 mL of water followed by 6 mL of methanol and eluted with two 3-mL aliquots of CH3COOH/ CH,OH (1:3v/v). The eluate was evaporated to dryness and the residue reconstituted with 0.1 mL of mobile phase. Quantitation. The MTX concentration in serum can be calculated by the peak-height ratio of analyte to the internal standard and comparison with a standard curve. For urine an internal standard with MTX comparable extractability and electrochemical characteristics has not yet been found. An external standard quantitation method was thus adopted. A working standard was prepared (by spiking drug-free urine with a known amount of MTX) and processed according to the described procedure. The MTX concentration in the unknown sample could then be obtained by direct comparison of peak heights in the sample and in the working standard.
RESULTS AND DISCUSSION Polarographic Behavior. Typical cyclic voltammograms of MTX at a concentration of 20 yM in a 0.05 M borate buffer pH 9.2 are presented in Figure 1. A well-defined anodic peak is evident as well as a cathodic one whose height increased as the accumulation time before scan reversal increased. The concentration dependence (at fixed deposition time) of the stripping peak current is presented in Figure 2; a linear region is followed by a flat region where, evidently, saturation of the surface occurs. The electrqde charge at full coverage, determined by integration of the background-corrected currentlpotential curve under saturation conditions was around 60 pC/cm2. A similar electrochemical behavior was observed for other pteridines such as folic and folinic acid. All these features strongly indicate the formation, as for some purine and pyrimidine derivatives, of a sparingly soluble Hg-MTX compound in the course of the anodic reaction. The value of the electrode charge at full coverage suggests that the anodically formed film can grow up to a monolayer and could indicate a flat orientation of the pteridine ring of the MTX molecule on the electrode surface. Ac polarography and electrocapillary studies have shown (27)that xanthopterin (2-amino-4,6-dioxypteridine) is adsorbed at a mercury electrode with the plane of the ring parallel to the electrode surface and that the area occupied by a molecule is 55 A2. If we take this value as a close estimation of the area occupied by the MTX pteridine ring, then the calculated electrode charge at
ANALYTICAL CHEMISTRY, VOL. 59, NO. 17, SEPTEMBER 1, 1987
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300
200 0
0
5
15 I
I
[ M T X ] I O ' hf
Flgure 2. Plot of MTX cathodic stripping peak current vs. MTX concentration in a 0.05 M borate buffer, pH 9.2: deposition potential, +0.16 V vs. Ag/AgCi (3 M Cl-) reference electrode: deposition time,
2 min from unstlrred solution.
i
L
C
D
Flgwe 4. Chromatograms of blank serum (A) and serum spiked with 10 pglmL of MTX (6): sample treatment, TCA deproteinization; polarographic detection in sampled dc mode at +O 19 V vs AgIAgCI; drop time, 1 s; chromatographic conditions, see Experimental Section.
C and D represent typical chromatograms obtained on serum samples spiked with MTX (1.2and 0.2pg/mL. respectively) and treated according to the microcolumn extraction procedure described in ref 15: column, RP C18 (250X 4.6 mm) 10-prn packing: mobile phase, 0.025 M phosphate buffet pH 6.5/acetonltrile/methanoi(87:6.5~6.5 (vlv)); flow rate, 1.5 mL/min; polarographic detection as above.
POTENTIAL
/ v vs.Ag/AgCI
Flgure 3. Hydrodynamic voftammogram of MTX dissolved in mobile phase: injectbn volume, 20 pL; injected quantity, 250 ng. For chromatographic conditions, see Experimental Section.
full coverage would be 57 pC/cm2, which compares well with the experimental value and could support the above hypothesis. The M T X anodic peak shifted toward more positive potentials with decreasing pH values, suggesting the involvement of an ionized species in the electrode process. The pK, values (27) of the drug indicate MTX" (e.g., MTX molecules having the pteridine moiety and the glutamic acid residue in a neutral and ionized form, respectively) as the species which predominates a t basic p H values or around neutrality. The welldefined anodic waves observed at the above pH values could then be ascribed to the MTX2- electroactive species. E1[PLC-Polarographic Detection. The peculiar behavior of MTX at a mercury electrode is of considerable interest from an analytical point of view since it makes possible the use of a HPLC polarographic detector operating in the oxidative mode. In this way the unique advantage of a polarographic detector, e.g., the continuously renewed electrode surface, can be coupled to the main advantage of the oxidative operating mode, e.g., no requirement for mobile phase deoxygenation. T h e current-potential profile of M T X obtained at a dropping mercury electrode under hydrodynamic conditions is shown in Figure 3. The wave remains practically unshifted along the potential axis on changing the concentration of the MTX solution injected. A working potential of +0.19 V vs. Ag/AgCl was chosen for HPLC detection purposes.
The limit of detection for MTX dissolved in the mobile phase a t a signal-to-noise ratio of 3 (faradaic current to background ratio, peak to peak, at the time of elution) was 3 ng (6.6 pmol). Calibration curves of MTX dissolved in mobile phase were linear (correlation coefficient typically better than 0.999 and intercept not significantly different from zero at the 95% confidence level) in the range 5-1000 ng injected. The within-day relative standard deviation (RSD) for repetitive injections of 50 ng of MTX dissolved in mobile phase was around 1% (n = 10) and the within-day RSD around 2.5%. Typical chromatograms obtained on a drug-free serum and on a serum sample spiked with MTX (10 pg/mL) are shown in Figure 4. The excellent specificity of the detector is clearly evidenced by the blank chromatogram; a nearly flat base line is observed at the retention time of MTX so that interferences from endogenous serum components can be excluded notwithstanding the isocratic and short chromatographic run. Detection limits were substantially similar to those reported in mobile phase; the lowest MTX detectable concentration was 1.5 pM (a larger injection volume would accordingly lower this limit). A linear calibration curve could be obtained up to 150 pM MTX in serum ( r > 0.998, typical slope 0.25 nA/ng). Recovery data are those previously (15) published (e.g., 95.3 f 2.570, N = 3 a t the 8 pM level); the within-day and the day to day coefficient of variation ( N = 5 ) at 10 kg/mL were 1.5% and 3.770,respectively. Detection limits reported above can be lowered by about 1 order of magnitude using the microcolumn extraction procedure already described (15) which provides a 10-fold preconcentration factor and recovery of 93.6 f 3.5 70 ( N = 5) at the 0.1 pM level. Typical chromatograms obtained on serum samples spiked with MTX and treated according to the cited procedure are shown in Figure 4. Note that (28) MTX serum levels persisting above 1 p M for 48 h, and above 0.1 pM for 7 2 h, are indicative of impending severe toxicity which can be averted by continuing the rescue treatment a t an increased dosage level. LC-APD then possesses the sensitivity required for monitoring serum drug levels in high-dose MTX therapy.
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by detection limits which compare well with those offered by
UV detection but are generally higher than those offered by solid-electrode-based detectors. The possibility of using an amalgamated gold electrode solid-state detector coupled to pulsed techniques (which could reduce electrode fouling by electrochemical cleaning of the electrode surface) is now under investigation.
ACKNOWLEDGMENT We thank P. G. Zambonin for helpful discussions. Registry No. MTX, 59-05-2.
LITERATURE CITED
F w e 5. (Left) Chromatogram relevant to an urine sample (direct injection wlthout prior treatment) spiked with MTX (50 gg/ml): moblle phase, 0.05 M phosphate buffer pH 6.4/methanol(85: 15 (vlv)); other conditlons as for chromatograms C and D in Figure 4. (Right) Chromatogram relevant to an urine sample spiked with MTX (1.5 phi) and extracted as described In the Experimental Section. Other conditions are as for chromatograms A and B in Figure 4.
Figure 5 shows representative chromatograms relevant to an untreated urine sample (left) and to an urine extract (right). The microcolumn extraction procedure described in the Experimental Section gave an extensive cleanup and an average recovery of 85.1 f 3.2 % (n = 5 ) at the 7 pM level. Interferences. A certain number of substances that could be coadministered with MTX have been tested for interferences effects. Adriamycine (anticancer drug), paracetamol, diazepam, caffeine, and morphine do not interfere because of their eledroinactivity. &Fluorouracil (anticancer drug) and folinic acid (MTX antidote), which are anodically electroactive on mercury, do not interfere because, under the given chromatographic conditions, they are practically unretained. A combined electrochemical/chromatographic study of 7-hydroxy-MTX and 4-deoxy-4-amino-N(10)-methylpteroic acid, the major metabolites of MTX in humans, has not yet undertaken because of the lack of suitable standards.
CONCLUSION The method presented in this paper represents the first LC-APD approach for a selective and rapid MTX determination in body fluids. The unique characteristics of LC-APD should be acknowledged even if they are counterbalanced by practical difficulties in operating such an awkward device and
Von Hoff, 0. D.; Penta, J. S.; Helmann, L. J.; Slavik, M. Cancer Treat. Reo. 1977. 61. 745-748. Evans, W. E. I~Ap~lledPhermec~lneflcs; Evans, W. E., Schentag, J. J., Jusko, W. J., Eds.; Applied Therapeutics, Inc.: Sen Francisco, CA, 1980 p 516. Kinkade, M. M.; Vogler, W. R.; Dayton, P. G. Biochem. Med. 1974. IO. 337. Raso, V.; Scherelber, R. Cancer Res. 1975, 35, 1407. Bohoun, C.; Dobrey, F.; Bouden, C. Clln. Chem. (Wlnsfon-Salem, N. C . ) 1974, 5 7 , 283. Hendel, J.; Sarek, L. J.; HuMberg, E. F. Clln. Chim. Acta 1978, 2 2 , 813. Gashaw, J. B.; Miler, J. 0. Clln. Chem. (Wlnston-Salem, N.C.)1978, 2 4 , 1032. Lawson, G. J.; Dlxon, P. F.; Aherne, G. W. J. Chromafogr. 1981, 223, 225. Buice, R. J.; Sidhu, P. J. Pham. Scl. 1982, 71, 74. Calrnes, D. A.; Evans, w. E. J . Chromatogr. 1982, 231, 103. El Yazigl, A.; Cazemiro, R. M. J. Ll9. Chromatogr. 1984, 7 , 1579. So, N.; Chandra, D. P.; Alexander, I. S.; Webster, W. J.; OGormenHughes, D. W. J. Chromafogr. 1985, 337, 81. Lunte, C. E.; Kissinger, P. T. Anal. Chem. 1983. 55, 1458. Lunte, C. E.; Kissinger, P. T. Anal. Blochem. 1983, 129, 377. PalmisanO, F.; Cataldl, T. R. 1.; Zambonin, P. G. J. Chromatogr. 1985, 344, 249. Paiecek, E.; Jelen. F.; Hung, M. A,; Lasovsky, J. Bioelechochem. Blmnerg. 1981, 6 . 621. Palecek, E.; Osteryoung, J.; Osteryoung, R. A. Anal. Chem. 1982, 54, 1389. Bond, A. M.; Thomson, S. 6.; Tucker, D. J.; Briggs, M. H. Anal. Chlm. Acta 1984, 156. 33. Thomson, S. 6.; Tucker, D. J.; Briggs, M. H. J. Chromatogr. 1985, 336,201. Saetre, R.; Rabenstein, D. L. Anal. Chem. 1978, 5 0 , 276. Eggli, R.; Asper, R. Anal. Chlm. Acta 1978, 101, 253. Allison. L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8 . Paimisano, F.; Cataldi, T. R. I.; Zambonin, P. G. Ann. Chlm. (Rome) 1984, 7 4 , 187. Paimisano, F.; Zambonin, P. 0. Ann. Chlm (Rome) 1984, 74, 833. Palmisano, F.; Desimoni. E.; Zambonin, P. 0. J. Chromatogr. 1984, 306. 205. AsahiYY. Yakugaku Zaasskl 1959, 79, 1559. Chem. Abstr. 1980, 5 4 , 10592. Hansen, J.; Kreilgard, B.; Nielsen, 0.; Veje, J. Int. J. Pharm. 1983, 16, 141. Sadee, W.; El Sayed, Y. M. I n Liquid Chromatography In Cllnlcal Analysis; Kabra, P. M.. Marton, L. J.. Eds.; The Humana Press: Clifton, NJ, 1981; Chapter 9. pp 212-218.
RECEIVEDfor review December 2,1986. Accepted May 1,1987. Work carried out with financial assistance from “Minister0 della Pubblica Istruzione”. This work is part of the PhD thesis of A. Guerrieri still in progress in the authors’ laboratory.
