Quantitation of Pyrimethamine and Related Diaminopyrimidines In Situ by Enhancement of Fluorescence after Thin Layer Chromatography William S. Simmons and Richard L. DeAngelis The Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, N. C. 27709
Of the many 2,4-diaminopyrimidines synthesized and tested for antimalarial activity ( I ) , pyrimethamine has been widely used for many years. The 2,4-diaminopyrimidines exert their cytotoxic action by strongly binding to and inhibiting the enzyme dihydrofolate reductase ( 2 ) (EC 1.5.1.4). Increased interest in this class of compounds has been stimulated by the recent report of Geils et al. ( 3 ) , who observed that pyrimethamine given in rather high doses achieved remission of meningeal leukemia of six-months duration in an adult patient suffering from acute myeloblastic leukemia. The increased interest in pyrimethamine has led to the development in this laboratory of a new method of analysis for several 2,4-diaminopyrimidines needed for pharmacokinetic studies and clinical trials of these compounds. A previous method of determining the level of pyrimethamine in urine or plasma was based on extraction into an organic solvent and direct spectrophotometric measurement of the extract a t 270 nm. This method, used by Schmidt et al. ( 4 ) , lacks specificity as well as sensitivity, Golinsky et al. ( 5 ) have recently reported a method for measuring levels of pyrimethamine and several related 2,4-diaminopyrimidines by selective desorption from Sephadex G-25 and G-10 columns. The concentration was calculated from spectrophotometric measurements made a t 270 nm. Although this method has the required specificity, it lacks sensitivity and is very time consuming. Jones et al. (6) attempted to measure pyrimethamine levels in urine utilizing the weak natural fluorescence of the compound. These investigators chromatographed an extract of urine on a TLC plate and scanned it in a specially modified spectrophotofluorometer. This method of analysis is attractive because of the convenient and versatile power of separation of thin layer chromatography and the potential sensitivity of fluorescence as a means of quantitation. Jones et al. (6) reported a lower limit of measurement of 0.1 Fg/ml for pyrimethamine in urine, which is inadequate sensitivity for supporting clinical trials where urine or cerebrospinal fluid levels may be considerably lower a t several days after administering the drug. In this communication, a spectrophotofluorometric method is described which may be used to measure very low levels of pyrimethamine in biological fluids. Evidence is also presented which indicates that the method may be satisfactorily applied to the measurement of many other compounds in this class. It should be noted that Sigel and Grace (7) of Wellcome Research Laboratories have developed a similar method (1) E. A . Falco, L. G . Goodwin, G . H . Hitchings, I . M . Roilo, and P. B. Russel, Brit. J. Pharrnacoi., 6 , 185 (1951). (2) G . H. Hitchings and J. J . Burchall, Advan. Enzymoi., 27, 417 (1965). (3) G. F. .Geils, C. W. Scott, Jr., C. M . Gaugh, and C . E. Butterworth, Jr., Blood, 38, 131 (1971). (4) L. H. Schmidt, H . B. Hughes, and I . G . Schmidt, J . Pharmacoi. Exp. Ther., 107,92 (1953). (5) G. F. Golinsky and V . S. Repin, Byuii. Eksp. Bioi. Med.. 71 ( 6 ) , 122 (1971). (6) C. R . Jonesand L. A. King, Eiochem. Med., 2, 251 (1968)
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
for assaying trimethoprim (2,4-diamino-5-[3’4’5’trimethoxybenzyl]pyrimidine) and its metabolites. This method utilizes the strong natural fluorescence of these compounds, which arises upon standing for a short period of time on a TLC plate (7).
EXPERIMENTAL Apparatus. The measurement of fluorescence was made with a Schoeffel SD3000-1 Spectrodensitometer equipped with a reflection mode assembly especially designed for spectrofluorescence determinations. The fluorescence intensity, resulting from a n excitation at 300 nm and total nonselective emission, was recorded as a peak on a strip chart recorder. The area under the peak was directly proportional to the mass of compound present. The areas were manually determined with a Hewlett-Packard calculator, integrator trace combination. Procedure. Extraction from Plasma and Urine The extraction of pyrimethamine from plasma or urine was done in the following manner, which is a modification of that used by Jones et al. (6). Plasma or urine ( 2 ml) containing pyrimethamine was made alkaline (pH >12) by addition of 0.5 ml of 2.ON NaOH. Ethylene dichloride (6.0 ml) was added to the sample in a 15-ml glass centrifuge tube, and the mixture was shaken a t room temperature for 15 minutes. The tubes were then centrifuged briefly, and a 4-mi aliquot of the organic phase was evaporated to dryness. The residue was dissolved in 0.1 ml of CHC13 and a suitable aliquot (1050 pl) was spotted on a prescored TLC plate using an electrically driven, modified Hamilton-Terasaki multichanhel microliter syringe. Standard samples ranging from 50 ng to 300 ng were run on each plate. Thin La3er Chromatograph3 Solutions (10-50 pl) containing the test compounds were spotted on a prescored Brinkmann nonfluorescent silica gel G TLC plate using a Hamilton microliter syringe. The plate was then developed in chloroform: methanol (75325) to a height of 11 cm. This solvent system was satisfactory for the compounds tested; the R f for most of the compounds was around 0.5. After the plate was developed, it was allowed to air dry for 10-15 minutes before it was sprayed with 2.OM XH4HS04 (aqueous). The plate was sprayed until the surface was saturated. After spraying, the plate was allowed to air dry for approximately one hour, after which time it was analyzed quantitatively.
RESULTS AND DISCUSSION While attempting to develop a sensitive direct assay for pyrimethamine, we observed that acids, particularly HC104 and HzS04, (lM), enhanced the fluorescence of pyrimethamine on silica gel TLC plates. This observation led us to investigate several other reagents, of which NHdHS04 has proved to be most satisfactory. The fluorophor generated will not fluoresce in either aqueous or nonaqueous systems, but only in the solid state, such as on a TLC plate or clean glass surface. Once generated, the fluorescent product is stable for several weeks. Attempts to identify the fluorophor by mass spectrometry, after TLC development, scrapping, and elution, show only the parent compound plus several oxides of sulfur. (7) C. Sigel and M. Grace,J. Unrornatogr., 80, 111 (1973)
Table I. Enhancement of the Fluorescence of Several 2,4-Diaminopyrimidines and Related Compounds M , = Slope of fluorescence/mass curve before spraying. M 2 = Slope of fluorescence/mass curve after spraying. M 2 / M 1 = Relative enhancement
B
of fluorescence.
"i
H2
X2/!ll
,355
13.75
38.7
,225
3.35
14.9
,215
5.36
2-.9
?JN W C I
rzN
A
..
3 ,
.5
._,i
^ ^
2.3
Figure 1. Chromatogram of pyrimethamine before spraying ( A ) and after spraying ( B ) with 2.OM NH4HS04.F = Solvent front, D = Pyrimethamine fluorescence (400 ng) 3.8:
12.86
3.3
6
O
I I
2
3
4
EISULTAIE M O L A R I T Y
Figure 2. Graph showing increase of fluorescence intensity of 200 ng of pyrimethamine with increasing concentration of .. ,. .\ .
2.67
6.7
A chromatogram of the fluorescence of 400 ng of pyrimethamine, on a developed plate, before and after spraying with 2.OM NH4HS04, is presented in Figure 1. The instrument settings were the same for both scans. In a n attempt to determine the optimum concentration of N H 4 H S 0 4 to use, several 200-ng spots of pyrimetha-
NH4HS04 mine were spotted on a TLC plate and overspotted with 10 pl of varying concentrations of NH4HS04 (Figure 2). The fluorescence intensity increases with increasing KH4HS04 concentration in a nearly linear manner u p to a concentration of 4 moles per liter. Above this concentration, the reagent has a n adverse effect on the surface of the TLC plate. However, we have found t h a t for most compounds tested 2 M NH4HS04 gave sufficient sensitivity. Inherent to the technique of spraying TLC plates is the uneven distribution of spray reagent, which often results in inconsistent activation of a given plate. T o determine whether or not this was a problem in our method, we ANALYTICAL CHEMISTRY, VOL. 45, NO. 8, JULY 1973
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Table II. Data Showing Efficiency of Quantitation of Pyrimethamine in Urine Amount of pyrimethamine added to 5 ml of urine, ng
Theoretical amount on plate, ng
50 100 150 200 250
Actual amount measured, ng
20
20.65 40.1 5 58.70 74.7 103.3
40 60
80 100
Recovery, %' 103.3 100.4 97.8 93.4 103.3
aAverage recovery = 99.65 f 2.73%.
Table Ill. Data Showing Efficiency of Quantitation of Pyrimethamine in Plasma over a Wide Range of Drug Concentration 4------&-----Ll-
0
1
2
3
4
MICROGRAMS Graph showing Increased slope of standard curve c Figure pyrimethamine after spraying with 2.OM NH4HS04. A = Before spraying. = After spraying
Spiked, wg/ml
Measured, wg/ml
0.20 0.25 0.30 0.35 0.40 9.50 0.60 0.75 0.80 0.90 1.oo 1.20 1.25
0.184 0.227 0.277 0.334 0.393 0.491 0.622
0.800 0.752 0.941 1.000 1.225 1.180
Recovery, %" 97.6
90.8 92.3 95.4 95.6 98.2 103.7 106.7 94.0 104.6 100.0 102.1 94.4
'Average recovery = 98.1 1 = 4 . 7i 9%
NANOGRAMS Figure 4. Graph showing sensitivity of the fluorescence assay for pyrimethamine. As little as 5 ng can be measured on a TLC
date spotted eight 200-ng samples of pyrimethamine on a TLC plate and measured the relative fluorescence resulting from spraying with 1.OM NH4HS04. The samples gave a mean of 0.676 area unit, with a standard deviation of f0.016 unit or 2.4%. These data are very consistent, and we have concluded that the precision of the method is not limited by the spraying procedure. Figure 3 is a comparison of the fluorescence of several concentrations of pyrimethamine before and after spraying. The gain setting was the same for both curves,
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ANALYTICAL CHEMISTRY, VOL. 45, N O . 8, JULY 1973
but the 0.d. setting had to be adjusted to compensate for the great difference in fluorescence between the sprayed and unsprayed sample. The peak areas have been normalized for the purpose of comparison. The curves are linear and extrapolate through zero. The slopes of the two curves may be taken as a measure of the degree to which fluorescence has been enhanced by the NH4HS04 spray reagent. The slope of the sprayed material ( M z ) is approximately 40 times greater than the slope before spraying ( M I ) . We have similarly studied several 2,4-diamino pyrimidines and related compounds, and these data are summarized in Table I. It is obvious that a wide variety of amino pyrimidines can be made to fluoresce more strongly when treated with ammonium bisulfate. The data presented in Table I1 show the efficiency of measuring nanogram quantities of Pyrimethamine in urine. For drug concentrations of 0.3 to 5.0 Fg per ml of urine, a recovery of 99.42 f 7.5% ( n = 28) was obtained. For pyrimethamine in plasma (Table 111), the efficiency of quantitation from 0.2 to 1.25 pg/ml was 98.11 f 4.7970 ( n = 13). The sensitivity of the method is illustrated in Figure 4, which shows a standard curve of pyrimethamine with quantities of drug ranging from 10 ng to 100 ng. Received for review December 7, 1972. Accepted February 7, 1973.
