penicillin-enhanced chemiluminescence of the
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Penicil l in-Enh anced Chemilum inescence of the
Luminol-H202-Co2+System
S. CHEN, .
YAN ,
M.
A.
SCHWARTZ,
.
H.
P E R R IN ,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 I u ~ ~ ~ o I - H , O , - C O ~ +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
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 oxidationof 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 of luminol chemilu-
minescence. 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 oxy-
gen to form an adduct which decomposes into N, and excited
aminophthalate. The la tte r species fluoresces. The Co2+ s a
catalyst which accelerates the decomposition of H,O, t o
supply the reactive 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 i t and luminol can exist for
-4
s. This is the reason why the sensitivity of chemilumi-
nescence is low, and the duration short. I t 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 that 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 tha t 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 an d penicillin V were purchased from Sigma
Chemical Company, St. Louis, MO. iperacillin-Na was from Amer-
ican Cyanam id, Pea rl River, NY. 3-Aminophthalhydrazide (luminol),
CoC1, hydr ate (99.99 ), an d 1,4-diazabicyclo 2,2,2)octaneDABCO)
were purchased from Aldrich Chemical Company Inc., Milwaukee,
WI. All reagents were used as supplied.
Instruments-Absorption an d fluorescence spectra were measured
on a double-beam U V-vis spectrophotometer (model Lamda-3B) and
fluorescence spectro meter (model LS-5,Perkin -Elm er, Norwalk, CT),
respectively. All spectroscopic measur emen ts w ere blank-corrected.
Absorbance meas urem ents were carried out in matched reference and
sample cells. Chemiluminescence was m easure d 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, Pittsbu rg, PA).
Procedure for Measuring Detect ion Limits of Penicillin and
Co2+-A
0.001 M luminol solution was prepared in
0.1
M NaOH. A
0.001 M aqu eous solution of H,O, was used thro ughout the detection
limit measu reme nts. The CoCl, an d penicillin aqueous solutions
were prepared at concentrations ap prop riate for a given experiment.
The ra tio of solution volumes for luminol: H,Oz: Co2+:penicillin was
0.8:0.7:0.03:0.03hroughout.
The luminol, CoCl,, an d penicillin solutions were placed in the
cuve tte tha t was positioned i n the cell holder of the fluorimeter. The
excitation slit was closed and th e emission slit was opened
as
wide
as
possible. The emission monochromator was set
at
420 nm, the
wavelength max imum of luminol fluorescence. The cover of the cell
comp artme nt was replaced in pa rt by the phosphorescence accessory
which allowed th e additio n of th e H,Oz solution without exposure to
light. The dependence of chemiluminescence on time was then
recorded. The peak heigh t a nd a rea u nder th e decay curve were used
to measure t he sensitivity. Both gave consistent results. When the
detection limit of Co2+ was dete rmin ed, the co ncentration of penicil-
l in was kept constant a t
-1
x M. When th e detection limit of
penicillin w as 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 b y 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, the 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 concentration of luminol, 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 aight line was obtained with
a 0.99 linear correlation coefficient. Reproducibility was very
good. At this concentration, the noise was very small
so
the
OO22-3549/9 1100 7 02.50/0
0
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American Pharmaceutical Association
Journal of Pharmaceutical Sciences 1017
Vol. SO,No.
11,
November 1991
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Time min)
Flgure1-Dependence of chemiluminescenceon time. Key:
(A)
luminol-
H202-Co2+;B) luminol-H202-Co2+-penicillin.
The concentrations were
as follows: penicillin,
9.4 x
M;
H20 2, 4.4 x
M; and luminol, 5.0 x
M;
Co2 , 1.9 x
M in
0.05 M
NaOH solution.
detection limit was 6
x lo-
g/mL or lower. This is in spite
of
the fact tha t the optical system was not ideal because the
cuvette is a t 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 opti-
mal, 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 Activity-
Penicillin 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+) s 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 i t decayed following a
single-decay exponential. On the other hand, when the ratio
was low, the chemiluminescence intensity increased rapidly
t o
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 mecha-
nisms can be briefly expressed by the following reaction:
Luminol
+
[Oxygen+Peroxide Adduct-
Aminophthalate-*Aminophthalate
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 th e superoxide anion C O i ) . The very
effective singlet oxygen scavenger DABC06*' was added t o
the luminol-H20~+-penicillinystem, and no observable
change in the chemiluminescence was found. Previous work
has shown that singlet oxygen is not stable in alkaline
s ~ l u t i o n . ~herefore, it is concluded that the role of singlet
oxygen can be excluded and the superoxide anion is probably
the only species involved in the oxidation of luminol to
aminophthalate.
Time (min)
Figure 2-Dependence of chemiluminescenceon time.
The
concentra-
tions were the same as in Figure 1.
The lifetime of the superoxide anion is only several milli-
seconds: 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 t o 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
(2)
-+ aminophthalate + hv
penicillin Oi-tpenicillin-peroxide adduct (3)
penicillin-peroxide adduct
+
luminol+luminol-
peroxide adduct penicillin (4)
luminol-peroxide adduct-aminophthalate 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 penicillin-
Co2+ alkaline solution in the reference cell. At these wave-
lengths, 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 wave-
lengths,
it
should result from the penicillin-superoxide ad-
duct. The experimental result is shown in Figure
3.
This
101
8 Journal
of Pharmaceutical Sciences
Vol. 80,
No.
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B
0 1
2w 220 24 260
280 3w
Wavelength nm)
Figure +(A) Absorption spectrum of penicillin-peroxide adduct. The
concentrations were as follows: penicillin
G, 9.1
6 x 0 4 M;
o2 ,
1.83
x
M; NaOH,
4 6 x
M; and H,02,
4 76
x M. (B)
Absorption spectrum of 6 7
x
M penicillin G.
result confirms the existence of a penicillin-peroxide adduct.
The enhanced luminescence apparent from the penicillin-
LHC system is certainly of analytical value. However,
quenchers of 3-aminophthalate fluorescence (at concentra-
tions 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 ar e coordinated by Co2+ may interfere,
decreasing the analytical sensitivity. Consequently, the sys-
tem probably ought to be adapted to becoming the detector
ancillary to an efficient separation process such as HPLC or
f low injection analysis.
References and No tes
1.
Tsai, T. S., l in. Chem. 1985,31, 248.
2. Leupold,
C.;
olkl,A.;Fahimi, H. D.AnaZ.Biochem.1985,151,63.
3.
Nieman, T. A. Abstracts
of 3rd
International Symposium on
uantitative Lumineacence Spectrometry in Biomedical Sciences,
4.
Milbrawth, D.
S:
P m e e d i n g s o N t h International Bwlumines
5. Selinger, H H. hotochem. Photobiol. 1975, 73, 35.
6.
Rabani,
J.;
Niesen,
S.
0 J . Phys . Chem.
1969, 73, 736.
7. Ware, W. R.;Richter,
M.
P.
J Chem. Phys.
1968,48, 595.
8 .
Natl .
Stan dard Re f. Data Series; Ross F; Ross A.
B.
Eds.; Natl.
ent, Belgium, 1989, 34.
cence
and
ChemrluminescenceBmposaum. 1986; p 515-518.
Bur. Stand., 1977; 59.
Journal of Pharmaceutical Sciences
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Vol.
SO,
No. 7 7 November 7997