A modified fluorimetric determination of chloroquine in biological samples

Atwlyrica i’

Chimicrt

Elscvier Scicntik

SHORT

College of JOHN

70 ( 1974)

229-232

Publisl~VingCompany.

Amstcrdum

- Prinkd

in The Nctherlunds

229

COMMUNICATION

A modified

STEPHEN

Acta.

fluorimetric

determination

of chloroyuine

in biological

samples

G. SCHULMAN Plrarrwcy.

UuiL’crsi!y

o/’ i;‘loritkc.

Gtriwsrillc.

I~loritla

32610

( U.S.A.)

F. YOUNG

The antimalarial chloroquine ha; enjoyed recent popularity as an agent in the treatment of rheumatoid arthritis. However. in the large doses employed in the treatment of arthritis. toxic side effects have been noted’, notably deformation of the lens of the eye. Consequently, the determination of chloroquine in biological samples is of current interest. A fluorimetric procedure for the determination of chloroquine in biological fluids has been reported by Rubin et trl. *. In this procedure, the sample was diluted tenfold with ethanoi, centrifuged. adjusted to pH 9.5 with borate buffer. and extracted with methylene chloride: the methylene chloride extract was then shaken with pH 7.85 phosphate-borate buffer. The aqueous phosphate-borate buffer extract was diluted with an equal volume of 0.1 M sodium hydroxide in 5Oo/0 ethanol, and the fluorescence of this solution was measured to determine the metabolites of chloroquine. The unmetabolized chloroquine was then determined by extracting a portion of the methylene chloride extract with 0.1 bf hydrochloric acid. adjusting the extract to pH 13 with 0.2 A4 sodium hydroxide in 50% ethanol, and comparing fluorimetrically with an appropriate standard. In the present study, the variations of the absorption and fluorescence spectra of chloroquine were investigated throughout the pH range and in concentrated sulfuric acid media; it was found that while the pH conditions specified by Rubin et al.’ are probably ideal for the separation of chloroquine from its metabolites. these pH conditions do not provide maximal sensitivity in the fluorimetric analysis. Experintert trill A pure sample of chloroquine phosphate was donated by Dr. S. Archer of the Sterling-Winthrop Institute, Rensselaer, N.Y. Analytical reagent-grade sulfuric acid and sodium hydroxide (Mallinckrodt Chemical Works, St. Louis. MO.) were used. Sulfuric acid solutions were prepared by dilution with distilled deionized water; the corrected Hammett acidity scale of Jorgenson and Hartter2 was employed to calibrate the sulfuric acid solutions. Solutions in the pH region l-3 were prepared by dilution of sulfuric acid. Acetate, phosphate and borate buffers

SHORT

230

COMMUNICATION

4-10. Dilute sodium were employed to prepare solutions in the pH region hydroxide solutions were employed for solutions of pH 11-14. A lOO-/tl aliquot of a 1. lows M stock solution of chloroquine phosphate in ethanol was delivered to a lO.OO-ml volumetric flask filled with buffer or acid solution, and mixed by inversion, to prepare each solution for spectromctric measurement. Each solution was prepared immediately before spectra were taken, in order to minimize decomposition errors. Fluorescence spectra were taken on a Perkin-Elmer MPF-2A fluorescence spectrophotometer. the monochromators of which were calibrated against the xenon line emission spectrum; output was corrected for wavelength variable response of lamp. monochromators and phototube by means of a rhodamine B quantum counter. Absorption spectra were taken on a Beckman DB-GT spectrophotometer. pH measurements were made on an Orion model 801 digital pH meter with a Corning combination pH electrode. Results

ant1 tliscwssiort Chloroquine has three basic nitrogen atoms and is therefore capable of demonstrating three protolytic equilibria involving four distinct species. These species along with their long wavelength absorption (&,) and fluorescence (&) maxima and the pK,, values corresponding to their interconversions are depicted in scheme 1. Scheme

1

H

4H3

H I-I tH3 Id 631 H-N-CH(CH2)3N(C2H5)2

H+CHKH,,3&,H,), -H+

pK,

Cl (T)

= - 9.1

A, =32Fanm A,

=411

h,=344nm 330nm

“In

h,=365nrn 357nm y3

y%

T@

H ,,,,,CH

H,N,CHKH2)3NK2H,),

(M)

X, h,

=331 d306nm

nm

(N)

A,=

331

(CH2)3 N (C2H,),

“In

ht=3B6rim

The triply charged cation (T), singly charged cation (M) and neutral molecule (N) ali demonstrate structureless fluorescence bands and long wavelength absorption bands with blurred vibrational structure. The spectral features of these species given in scheme 1 correspond to the band maxima. The doubly charged cation (D), however, exhibits well defined vibrational line structure in absorption and fluorescence, and in this case the two most distinct and intense vibrational features of the absorption and fluorescence bands are presented. The doubly charged cation (D) is represented as a protonated cyclic vinyl-type amidine rather than as a

SHORT

COMMUNICATION

231

protonated aminoquinoline. The basis for this structural assignment has been well established for 4-aminoquinoline3 and is apparently applicable here as well. The conversion from (T) to (D) produces substantial changes in the absorpthe variations of both types of spectra yielding tion and fluorescence spectra, This is in contrast to 4-aminoquinoline, in which the corresponding P&I = -9.1. pK,, value obtained by fluorimetry is some two orders of magnitude more basic than that determined by absorptiometry, a result attributed to ionization of the latter compound in the excited state from which fluorescence arises. That the same phenomenon is not observed in chloroquine indicates that the life-times of the excited states of the tri-cation and di-cation derived from chloroquine are too short for excited-state dissociation to compete with fluorescence4. The conversion also yields the same pK,, value whether deterfrom (D) to (M) in chloroquine mined absorptiometrically or fluorimetrically. This is analogous to the behavior of 4-aminoquinoline with respect to dissociation from the heterocyclic ring nitrogen atom3. Whereas the dissociations of (T) to (D) and that of (D) to (M) both involve dissociation from a site intimately coupled to the aromatic system, and therefore perturb the absorption and fluorescence spectra with respect to both wavelength and intensity, the dissociation of (M) to (N) arises from a site far removed from the aromatic part of chloroquine. Consequently, the dissociation of (M) to (N) does not at all affect the absorption or lluorescence spectra. The pK, for this equilibrium was determined potentiometrically. No changes in the absorption or fluorescence spectra above pH 10 would thus be expected,. and in fact, no changes in the absorption spectra were observed with increasing pH, above pH 10. However, at pH IQ the fluorescence of chloroquine was quenched, vanishing completely at pH 14. The midpoint of this quenching occurred at pH 11.7. A similar phenomenon was observed in 4-aminoquinoline3 and was attributed to proton abstraction in the fluorescent state, from the amino group. .Presumably. this is also occurring in chloroquine as represented in scheme 2. Scheme

2 y3

H.

,c(CH~)~N(C&)~ N

CL (N)

(A)

The inflection point in the quenching curve of chloroquine*fluorescence with increasing pH may be assigned to the dissociation constant for the excited state equilibrium represent%d in scheme 2 and has the value 11.7. The corresponding dissociation cannot be observed in the absorption spectra, because the anion (A) is much too basic in the ground state to exist in water. That (A) can be generated in the excited state in the accessible pH range indicates that in the excitation of (N) from the ground state to the excited state, the amino group of(N) loses electronic charge to the aromatic ring, rendering (N) a stronger acid (or conversely, A a weaker base) in the excited state.

232

SHORT

COMMUNICATION

The relative quantum yields of fluorescence of each species derived from chloroquine were estimated from the integrated area under the fluorescence peak divided by the molar absorptivity at the wavelength of excitation. Comparison of these relative quantum yields permits the identification of the most intensely fluorescing species and therefore the species most desirable for the most sensitive fluorimetric analysis. The relative quantum yield of(T) was measured at Hammett acidity H 0 - 10 (96% H2S05). that of (D) at pH 1.0 and that of (M) or (N) at pH 9.8. The relative quantum yields of (T). (D) and (M) or (N) were found to be in the ratio 1: 1: 100 indicating that the mono-cation or neutral species ax-e certainly the most desirable for fluorimetry. All of this leads to the original point of this puper. Because of the excitedstate ionization which quenches the fluorescence of (N) at high pH and which could not bc detected by ordinary chemical means. the fluorimetric assay of chloroquine. carried out by monitoring the fluorescence of the neutral species (N) at pH 13. is about 14 times less sensitive than if the solution which is measured fluorimetrically for chloroquine is adjusted to pH 9.8-10.0 and the fluorescence then monitored. Therefore. it is suggested that the procedure of Rubin et al.’ be modified by neutralizing the 0.1 M hydrochloric acid extract of the methylene chloride extract with an equal volume of 6.1 M sodium hydroxide and then adjusting to pH 9.8-10.0 carefully with phosphate or borate buffer before fluorescence is measured. The standard solution should also. of course, bc measured at the same pH. Since the metabolites of chloroquine are simply dealkylated derivatives’. the same pH considerations are presumably also applicable to their analysis. REFERENCES I M. Rubin. N. Zwillcr. H. N. Bcrnstcin and A. Mansour. Proc,. flff. fJlItrrr~?ac~o/. Alcrrirrg. Pcrgamon Press. Oxford. 1965. p. 467: and rcfcrcnccs contained thcroin. 2 M. J. Jorgenson and D. R. Harttcr. J. .Arrwr. Chw. SW.. 85 ( 1963) 87% 3 P. J. Kovi, A. C. Cupomacchia and S. G. Schulman, rlrwl. C’/IO~I.. 44 ( 1972) 161 I. 4 S. G. Schulmim. kw. Arm/. C/W~~I.. I ( lY71) 85.

AId.