A&&a Chzmrca Acta, 255 (1991) 383-385 Elsevler Science Pubhshers B V , Amsterdam

Determination of cephalothin sodium by a chemiluminescence method

S G Schuhnan *, J H Pemn, Guo Fan Yan and Shangxlan Chen CoNege of Pharmacy, Uruvemty of Fiorda, Gamesvdle, FL 32610 (USA)

(Recetved 13th March 1991, rewed manuscript received 1st July 1991)

Absbgct

The cephalosporm antlblotlc cephalothm has the ability to prolong and mtenstfy the chennlummescence derived from the cobalt(U)-lummol-hydrogen peroxide system Thus IS beheved to result from the formatlon of a peroxide complex of cephalothm which IS longer hved than the superoxlde ion m aqueous solution It can be used as the baals for the deternunatlon of cephalothm m the range 0 4-400 cg ml- Keywords Chenulummescence, Antlblotlcs, Cephalosporms, Cephalothm

The hunmol-hydrogen peroxide-metal Ion system has been widely used m the analysts of chemical and blologcal samples, and more than 30 years ago it was used to determine metal Ions [1,21 Smce then, many papers have dlscussed the reaction mechanism of the system [3-61 To date, it 1s still an Interesting problem although the mechanism is not well understood [7,8] Rarely has this system been used to study pharmaceutl- cal compounds [9], but recently [lo] it was shown that it could be used to determine pemcllhn A possible mechanism was suggested which mvolved the formation of a complex between pemcdhn and the superoxlde ion This pemallm-peroxide adduct mteracts wth lummol to form the luml- nol-peroxide adduct, which decomposes to form excited ammophthalate which emlts lummes- cence In this paper, the use of this system to determme the cephalosporm cephalothm 1s re- ported

EXPERIMENTAL

Reagents Cephalothm [7-(2-thlenylacetarmdo) cephalo-

sporamc acid] was supplied by Eh Lilly (Indl-

anapohs, IN), hydrogen peroxide (30%) by Fisher Sclentdic (Pittsburgh, PA) and cobalt@) chloride hydrate (99 99%) and 3-ammophthalhydrazlde (lummol) by Aldrich (Milwaukee, WI), and all were used as received

Instrurnentatzon Chemllummescence was measured by a Far-

rand Optical (New York) Model MK-1 spec- trofluornneter, with a phosphorescence accessory wlthout the chopper The output was recorded on a Fisher Saentlfic Recordall Series 5000 stnp- chart recorder

Procedure for measunng the concentratwn and detectwn hmrt of cephalothm

A 0 001 M solution of lummol m 0 1 M NaOH solution and 10m3 M aqueous solutions of hydro- gen peroxide and cobalt were used throughout for the measurements Cephalothm aqueous solu- tlons were prepared at concentrations appropn- ate for a given experunent Ratios of solution volumes of lummol.hydrogen peroxide cobah cephalothm of 08 0.7 003 003 were used throughout

0003-2670/91/$03 50 0 1991 - Elsevler Science Pubhshers B V All nghts reserved

S G SCHULMAN ET AL

The lummol, cobah chlonde and cephalo- thm solution were placed m the cuvette and posltroned m the cell holder of the fluoruneter The excltatlon slit was closed and the ermsslon sht was opened fully (7 x 20 mm21 The emlsslon monochromator was set at 420 nm, the wave- length maxnnum of hmunol fluorescence The cover of the cell compartment was replaced, m part, by the phosphorescence accessory, which allowed the addition of hydrogen peroxide solu- tion without exposure to light The peroxide solu- tton was plpetted mto the cuvette under pressure to ensure total mlxlng The dependence of cheml- lummescence on tnne was recorded The peak height and area under the decay curve were used to measure the sensltlvlty

RFJSULTS AND DISCUSSION

It has already been reported that addmg pem- dim to the lummol-hydrogen peroxide-cobalt system will mcrease the chemAnnmescence m- tens@ and prolong the duration of the chemllu- mmescence The same phenomenon was ob- served m the lummol-hydrogen peroxlde- cobah(cephalothm system, as shown m Fig 1

when the concentrations of l-01, hydrogen peroxide and cobalt011 were kept constant, a concentration of cephalothm of 0 4 pg ml- gave a photocurrent of near 1 mA The plot of the logarrthm of the chemdummescence signal versus the logarithm of cephalothm concentration 1s lm- ear over at least three orders of magnitude above

Time (mln)

Rg 1 Chenulummescence of the Co(H)-lummol-pcrondc system m (A) the presence and (B) the absence of cephalothm (A) Concentrations lummol, 4 5 X 10e4 M, HzOz, 5 1 X 10m4 M, C&II), 19 X 10m6 M, cephalothm sodmm, 9 6 x 10m4 M (B) Concenhatlons as m (A) except no cephalothm sodmm.

DETERMINATION OF CEPHAJLWHIN SODIUM BY CHEMIJJMINESCENCE 385

this concentration Peak-height or peak-area measurements gave sumlar results The optlcal system used 1s far from Ideal because the cuvette was at least 60 cm from the photomultlpher, as multiple reflections are used If a detector of short optical path is used the senntWy wrll be increased by several orders of magmtude, makmg the system an ideal detector for hqmd chro- matography

A possible mechanism for the reactlon 1s [lOI

lummol + superoxlde ion --f

lummol-peroxtde adduct +

excited ammophthalate

cephalothm + superoxlde ion --f

cephalothm-perorade adduct

cephalothm-peroxide adduct + lummol +

lummol-peroxide adduct + cephalothm

As shown m Fig 1, the pattern of the chemdu- mmescence of the lummol-hydrogen peroxlde- cobalt(II)-cephalothm system 1s sundar to that of the lummol-hydrogen peroxlde-cobalt(II)-pem- alhn system, so the same mechamsm 1s probably involved m the cephalothm system There are several functional groups m both cephalothm and penmllm, including the carboxyl group and two amldo groups One of the latter 1s m the p-lactam rmg and the other 1s exocychc Schwartz [ll] reported that pemcdlm and cephalosporm showed different behaviors m forming complexes, be- cause the carboxyl groups were m different posl- tions on the p-lactam rmg However, here pem-

c&n and cephalothm have sundar behaviors, so the role, if any, of the carboxyl group 1s not clear In unpublished work, glycylglycme has been found to quench the chemdummescence of this cobalt(II)-lummol-peroxide system, gwmg a con- fusing picture of whether or not the amide group- mg 1s essential for adduct formation Much work IS necessary to elucidate the structure necessary to enhance the lummescence of the cobalt(II)- lummol-peroxide system

REFERENCES

1 2

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