7/18/2019 Penicillin-Enhanced Chemiluminescence of The http://slidepdf.com/reader/full/penicillin-enhanced-chemiluminescence-of-the 1/3 Penicillin-Enhanced Chemiluminescence of the Luminol-H202-Co2+System S. CHEN, . YAN, M. A. SCHWARTZ, . H. PERRIN, ND S. G. SCHULMAN' Received November 6, 1990, from the College of Pharmacy, University o Florida, Gainesville, L 32670. January 16, 1991. Accepted for publication Abstract The Iu~~~oI-H,O,-CO~+ ystem 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 phenom- enon appears, as revealed by difference spectroscopy, 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, n 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 ofluminol chemilu- minescence. A detection limit of 100 ng mL was obtained for penicillin G with a less-than-idealdetection system. The chemiluminescence system luminol-H202-Co2+ LHC) has been widely used in chemical and biological systems and has been applied to pharm aceutical analysis.' The origin of luminescence in this system can be briefly described as the interaction of luminol, under al kal ine conditions, with oxy- gen to form an adduct which decomposes into N, and excited aminophthalate. The latte r species fluoresces. The Co2+ s a catalyst which accelerates the decomposition of H,O, to supply the reactiv e form of oxygen which is needed to interact with luminol. There are two very reactive types of oxygen which ar e able to exist in solution, one5 s singlet O,, the other is *Oi, he superoxide iom6 Currently, there is considerable experimental evidence to show that singlet oxygen is not predominant in strong alkali ~olution.~he current experi- ments support this conclusion. The only active oxygen species which plays an important role under alkaline conditions is 0;; ts 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 chemilumi- nescence 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 sen- sitivity and duration of luminescence will be promoted. The structure of penicillin has some similarities to th at of luminol, in particular a highly strained heterocyclic ring. Conse- quently, 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 ReagenteThe H,Oz (30%) was supplied by Fisher Scientific Company. Penicillin G and penicillin V were purchased from Sigma Chemical Company, St. Louis, MO. iperacillin-Na was from Amer- ican Cyanam id, 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 spectrofluorim- eter (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 0.8:0.7:0.03:0.03 hroughout. 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 pa rt 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 penicil- lin was kept constant at -1 x M. When the detection limit of penicillin was measured, the Co2+ concentration was kept constant at -1.9 x M. ll of the measurements were made at room 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 luminol- H,02-Co2+-penicillin were measured under the same condi- tions. As shown in Figure 1, the behaviors of these two systems are quite different. For th e luminol-H,0,-Co2+ sys- tem, only a brief pulse of light was observed, but on the addition of penicillin, th e sensitivity is much improved and time of luminescence lengthened. The dependence of chemilu- minescence intensity on the concentrations of luminol, Co2+, H 02 nd 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 Penicil- lin-The concentratio n of lum ino l, H,O,, and penicillin were kept constant and the Co2+ concentration varied. Chemilu- minescence intensity-time curves were obtained, and at a range of lo-' to g/mL, a str aig ht line was obtained with a 0.99 line ar correlation coefficient.Reproducib ility was very good. At this concentration, th e noise was very small so the OO22-3549/9 1100 7 02.50/0 0 799 1 American Pharmaceutical Association Journal of Pharmaceutical Sciences 1017 Vol. SO, No. 11, November 1991