Penicillin-Enhanced Chemiluminescence of the Luminol-H202-Co2+System S. CHEN,G. YAN, M. A. SCHWARTZ, J. H. PERRIN, AND S. G. SCHULMAN' Received November 6, 1990, from the College of Pharmacy, University of Florida, Gainesville, FL 32670. January 16, 1991. Abstract The Iu~~~oI-H,O,-CO~+ system has been widely used in chemical and biological analysis. We report here an investigation of the observation that penicillins have the ability to prolong and enhance the intensity of chemiluminescence from luminol. The basis of this phenomenon appears, as revealed by differencespectroscopy, to be the formation of a complex between the p-lactam and the superoxide ion. The latter is the oxidizing species responsible for the oxidation of luminol in alkaline solution and has a mean lifetime, in solution, of milliseconds. The stabilization of the superoxide ion by penicillin complexation extends the effective lifetime of the superoxide ion by a few orders of magnitude and thereby allows for more efficient oxidation of the p-lactam. Several penicillins were determined by their enhancement of luminol chemiluminescence. A detection limit of 100 ng mL was obtained for penicillin G with a less-than-ideal detection system.

The chemiluminescence system luminol-H202-Co2+(LHC) has been widely used in chemical and biological systems and has been applied to pharmaceutical analysis.'" The origin of luminescence in this system can be briefly described as the interaction of luminol, under alkaline conditions, with oxygen to form an adduct which decomposes into N, and excited aminophthalate. The latter species fluoresces. The Co2+is a catalyst which accelerates the decomposition of H,O, to supply the reactive form of oxygen which is needed to interact with luminol. There are two very reactive types of oxygen which are able to exist in solution, one5is singlet O,, the other is *Oi, the superoxide iom6 Currently, there is considerable experimental evidence to show that singlet oxygen is not predominant in strong alkali ~ o l u t i o nThe . ~ current experiments support this conclusion. The only active oxygen species which plays an important role under alkaline conditions is -0;; its lifetime in alkali is only several milliseconds.' The peroxide adduct formed between it and luminol can exist for -4 s. This is the reason why the sensitivity of chemiluminescence is low, and the duration short. It is proposed that if one compound (in this case penicillin) can interact with -0,to form a peroxide adduct and the adduct can then interact with luminol (thereby freeing the compound), then the sensitivity and duration of luminescence will be promoted. The structure of penicillin has some similarities to that of luminol, in particular a highly strained heterocyclic ring. Consequently, it seems possible that penicillin can form peroxide adducts with -0;. The current investigations show that the sensitivity and duration of luminescence are enhanced by adding penicillin to the LHC system. This phenomenon should be useful for the determination of penicillins at picomolar concentrations or lower, as might be desirable for checking for contamination by penicillins in "clean" rooms.

Experimental Section R e a g e n t e T h e H,Oz (30%) was supplied by Fisher Scientific Company. Penicillin G and penicillin V were purchased from Sigma Chemical Company, St. Louis, MO.Piperacillin-Na was from AmerOO22-3549/91/1100- 1 01 7$02.50/0 0 799 1, American Pharmaceutical Association

Accepted for publication

ican Cyanamid, Pearl River, NY. 3-Aminophthalhydrazide (luminol), CoC1, hydrate (99.99%), and 1,4-diazabicyclo(2,2,2)octane(DABCO) were purchased from Aldrich Chemical Company Inc., Milwaukee, WI. All reagents were used as supplied. Instruments-Absorption and fluorescence spectra were measured on a double-beam UV-vis spectrophotometer (model Lamda-3B) and fluorescence spectrometer (model LS-5, Perkin-Elmer, Norwalk, CT), respectively. All spectroscopic measurements were blank-corrected. Absorbance measurements were carried out in matched reference and sample cells. Chemiluminescence was measured by a spectrofluorimeter (model MK-1; Farrand Optical Company, Valhalla, NY) and recorded on a Fisher Recordall series 5000 strip chart recorder (Fisher Scientific, Pittsburg, PA). Procedure for Measuring Detection Limits of Penicillin and Co2+-A 0.001 M luminol solution was prepared in 0.1 M NaOH. A 0.001 M aqueous solution of H,O, was used throughout the detection limit measurements. The CoCl, and penicillin aqueous solutions were prepared at concentrations appropriate for a given experiment. The ratio of solution volumes for luminol: H,Oz: Co2+:penicillin was 0.8:0.7:0.03:0.03throughout. The luminol, CoCl,, and penicillin solutions were placed in the cuvette that was positioned in the cell holder of the fluorimeter. The excitation slit was closed and the emission slit was opened as wide as possible. The emission monochromator was set at 420 nm, the wavelength maximum of luminol fluorescence. The cover of the cell compartment was replaced in part by the phosphorescence accessory which allowed the addition of the H,Oz solution without exposure to light. The dependence of chemiluminescence on time was then recorded. The peak height and area under the decay curve were used to measure the sensitivity. Both gave consistent results. When the detection limit of Co2+ was determined, the concentration of penicillin was kept constant a t -1 x M. When the detection limit of penicillin was measured, the Co2+concentration was kept constant at M. All of the measurements were made at room -1.9 x temperature. For each sample, at least three measurements were made with good reproducibility.

Results and Discussion Chemiluminescence Promoted by Penicillin-The chemiluminescence of luminol-H,0,-Co2+ and luminolH,02-Co2+-penicillin were measured under the same conditions. As shown in Figure 1, the behaviors of these two systems are quite different. For the luminol-H,0,-Co2+ system, only a brief pulse of light was observed, but on the addition of penicillin, the sensitivity is much improved and time of luminescence lengthened. The dependence of chemiluminescence intensity on the concentrations of luminol, Co2+, H,02, and penicillin has been studied. The linear relationship between them allows chemiluminescence to be used for quantitative measurements of Co2+ and penicillin. Measurement of Detection Limits of Co2+ and Penicillin-The concentration of luminol, H,O,, and penicillin were kept constant and the Co2+ concentration varied. Chemiluminescence intensity-time curves were obtained, and at a range of lo-' to g/mL, a straight line was obtained with a 0.99 linear correlation coefficient. Reproducibility was very good. At this concentration, the noise was very small so the Journal of Pharmaceutical Sciences I 1017 Vol. SO, No. 11, November 1991

Time (min)

Flgure1-Dependence of chemiluminescenceon time. Key:(A) luminolH202-Co2+;(B) luminol-H202-Co2+-penicillin.The concentrations were M; Co2', 1.9 x M; H202, 4.4 x as follows: penicillin, 9.4 x M in 0.05 M NaOH solution. M; and luminol, 5.0 x

detection limit was 6 x lo-" g/mL or lower. This is in spite of the fact that the optical system was not ideal because the cuvette is at least 60 cm from the photomultiplier as multiple reflections are used in the emission optics of the Farrand spectrofluorimeter. Usual chemiluminescence instruments have a sample-to-detector distance of only -2 cm. The concentration ratios of the reactants probably was not optimal, giving further opportunity for increasing the sensitivity. Using constant concentrations of luminol, H202, and Co2+, the detection limit of penicillin G was found to be 100 ng/mL. Effect of Different Penicillins on the Catalytic ActivityPenicillin G, Penicillin V, and pipericillin promoted the chemiluminescence to varying extents. Penicillin G and pipericillin had almost the same effect, while that of penicillin V was a little smaller. Reaction Mechanism-The cobalt ion (Co2+)is a catalyst for the luminol-H,O, system. However, penicillin alone is not a catalyst because in an alkaline solution of luminol-H,O,penicillin without Co2+,chemiluminescence is not observed. With the concentrations of luminol, H,02, and penicillin fixed and the concentration of Co2+ varied, two types of luminescence curves were observed. When the ratio Co2+:penicillinwas high, the intensity of chemiluminescence increased rapidly to a maximum, then it decayed following a single-decay exponential. On the other hand, when the ratio was low, the chemiluminescence intensity increased rapidly to a maximum, then it decreased rapidly subsequently, increasing slowly to a second maximum before decreasing again to zero, as shown in Figure 2. These experimental results indicate that the reaction should be divided into two parts. The rapid increase and decrease is contributed by the luminol-H,0,-Co2+ system and the slower part is derived from the role of penicillin added to the solution. As is well known, the chemiluminescence reaction mechanisms can be briefly expressed by the following reaction:

+ hv

(1)

In solution, there are two forms of oxygen which may play an important role in reacting with luminol. One is singlet oxygen and the other is the superoxide anion C Oi). The very effective singlet oxygen scavenger DABC06*' was added to the luminol-H20~+-penicillin system, and no observable change in the chemiluminescence was found. Previous work has shown that singlet oxygen is not stable in alkaline it is concluded that the role of singlet s ~ l u t i o nTherefore, .~ oxygen can be excluded and the superoxide anion is probably the only species involved in the oxidation of luminol to aminophthalate. 1018 I Journal of Pharmaceutical Sciences Vol. 80, No. 11, November 7991

Figure 2-Dependence of chemiluminescence on time. The concentrations were the same as in Figure 1.

The lifetime of the superoxide anion is only several milliseconds: even in alkaline solution. Addition of penicillin to the luminol-H,O,-Co2+ system extends the duration of chemiluminescence to several minutes. This indicates that penicillin can combine with the superoxide anion to form a peroxide adduct which prevents the decomposition of the superoxide. The luminescent species is aminophthalate with or without penicillin. This means that the penicillin peroxide adduct can react with luminol to exchange superoxide anions. The reaction mechanism can be expressed as follows:

luminol

+ - Oi-*luminol-peroxide adduct

luminol peroxide adduct-*aminophthalate -+

aminophthalate

+ - Oi-tpenicillin-peroxide adduct penicillin-peroxide adduct + luminol+luminolpenicillin

peroxide adduct

(2)

+ hv

+ penicillin

luminol-peroxide adduct-aminophthalate

Luminol + [Oxygen+Peroxide AdductAminophthalate-*Aminophthalate

Time (min)

(3)

(4)

+ hv

(5)

A critical aspect of the above proposal is to find evidence of the existence of a penicillin-peroxide adduct. As discussed above, the lifetime of the superoxide anion is very short, but the penicillin-peroxide adduct should have a relatively long lifetime. An ordinary UV-vis spectrophotometer was used to measure the absorption spectrum of the H,02.-Co2 +-penicillin alkaline solution from 240 to 300 nm by using a penicillinCo2+ alkaline solution in the reference cell. At these wavelengths, in the absence of interaction in the sample cell, the absorptions of penicillin in the reference and sample cells should cancel. The superoxide anion has a very short lifetime, and cannot be detected. If light is absorbed at these wavelengths, it should result from the penicillin-superoxide adduct. The experimental result is shown in Figure 3. This

quenchers of 3-aminophthalate fluorescence (at concentrations high enough to cause diffusional quenching: i.e. > 1 x mol L-') which include ions derived from elements of high atomic number and many aromatic molecules as well as Lewis bases which are coordinated by Co2+ may interfere, decreasing the analytical sensitivity. Consequently, the system probably ought to be adapted to becoming the detector ancillary to an efficient separation process such as HPLC or flow injection analysis.

-

B

01-

/

References and Notes 2w

220

240

260

280

3w

Wavelength (nm)

Absorption spectrum of penicillin-peroxide adduct. The concentrations were as follows: penicillin G, 9.16 x 1 0-4 M;Co2', 1.83 x M; NaOH, 4.6 x M; and H,02, 4.76 x M. (B) M penicillin G. Absorption spectrum of 6.7 x Figure +(A)

result confirms the existence of a penicillin-peroxide adduct. The enhanced luminescence apparent from the penicillinLHC system is certainly of analytical value. However,

1. Tsai, T. S.,Clin. Chem. 1985,31,1248. 2. Leupold, C.;Volkl,A.;Fahimi, H. D.AnaZ.Biochem. 1985,151,63. 3. Nieman, T. A. Abstracts of 3rd International Symposium on

uantitative LumineacenceSpectrometry in Biomedical Sciences, 2Milbrawth, hent, Belgium, D.

1989,p 34. S: Pmeedings o N t h International Bwluminescence and Chemrluminescence ymposaum. 1986;pp 515-518. 5. Selinger, H . H.Photochem. Photobiol. 1975,73,335. 6. Rabani, J.;Niesen, S. 0. J . Phys. Chem. 1969,73,3736. 7. Ware, W. R.;Richter, M. P. J . Chem. Phys. 1968,48,1595. 8. Natl. Standard Ref. Data Series; Ross, F; Ross, A. B. Eds.; Natl. Bur. Stand., 1977;p 59. 4.

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Journal of Pharmaceutical Sciences I 1019 Vol. SO, No. 7 7 , November 7997

Penicillin-enhanced chemiluminescence of the luminol-H2O2-Co2+ system.

The luminol-H2O2-Co2+ system has been widely used in chemical and biological analysis. We report here an investigation of the observation that penicil...
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