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Clinica Chimica Acta, 78 (1977) 203-207 @ Elsevier/North-Holland Biomedical Press

CCA 8541

THE SIMULTANEOUS QUANTITATIVE DETERMINATION OF CEPHALOTHIN AND CEFAZOLIN IN SERUM BY HIGH PRESSURE LIQUID CHROMATOGRAPHY

J.S. WOLD and S.A. TURNIPSEED Toxicology Division, Lilly Research Laboratories, 46140 (U.S.A.) (Received

December

29th,

Eli Lilly and Company,

Greenfield,

Ind.

1976)

Summary Conventional microbiological assay procedures for cephalosporins in serum do not allow the determination of serum concentrations if more than one cephalosporin is present in a single sample. An HPLC procedure has been developed which permits the simultaneous quantitative determination of cefazolin sodium and cephalothin sodium in serum. Reverse phase chromatography using methanol in 0.2 M ammonium acetate as the mobile phase was employed to separate and quantitate the two cephalosporins in a trichloroacetic acid supematant solution prepared from serum.

Introduction Antibiotics in general, including the cephalosporins, have conventionally been determined by microbiological assay. Cephalosporins in serum are usually determined by the cylinder plate method [ 11 or the agar well diffusion method

[a. The microbiological assay methods obviously cannot differentiate between two cephalosporins present in the same serum sample. Separation of the cephalosporins by paper chromatography followed by bioautography [3,4] permits only a general indication of the relative amount of each antibiotic present. The administration of more than one parenteral cephalosporin to an individual patient is not a common practice. However, the combination of cefazolin (Kefzola) and cephalothin (Keflin@) has been used in cardiopulmonary surgery procedures involving the insertion of prosthetic materials (Csicsko, J., personal communication). Cephalothin was administered intravenously during and after surgery because of the wide experience with the use of this antibiotic in surgical prophylaxis [5] while cefazolin was selected for preoperative administration

204

because of the relatively low pain associated with intramuscular use of this newer cephalosporin [ 61. The use of cardiopulmonary bypass and hypothermia may induce alterations in the pharmacokinetics of the antibiotics which would make the dosage regimens that were developed to provide adequate serum levels for use in prophylaxis in conventional procedures unreliable for use in these specialized procedures. It was desirable, therefore, to determine antibiotic concentrations in serum of patients receiving intramuscular cefazolin preoperatively and intravenous cephalothin during and after surgery involving cardiopulmonary bypass. This report describes the development of a high performance liquid chromatography (HPLC) assay to permit the simultaneous determination of cephalothin and cefazolin in serum. Methods

and materials

Serum samples were prepared for HPLC by addition of an equal volume of 0.36 M aqueous trichloroacetic acid. The samples were cooled, centrifuged and kept at 0°C until analysis. Chromatography was carried out on a Waters model ALC 202 liquid chromatograph (Waters Associates, Milford, Mass.). A 90 cm X 2 mm stainless steel column pack with Phenyl Corasil @, 37/50 pm reverse phase packing was used (Waters Associates, Milford, Mass.). The mobile phase consisted of lo13% methanol in 0.2 M aqueous ammonium acetate. The concentration of methanol in the mobile phase was adjusted daily for optimum separation and peak shape. Flow rate was maintained at 2 ml/min at 1500-2000 p.s.i. Absorption was monitored at 254 nm recorded at 0.02 absorbance units full scale. Standard solutions were prepared by adding weighed quantities of cefazolin sodium and cephalothin sodium to human serum. 20+1 aliquots of the trichloroacetic acid supernatant solution prepared from standard or experimental serum samples were injected onto the chromatographic column. Standard curves were constructed daily relating peak area (determined by triangulation) to cefazolin sodium and cephalothin sodium concentrations. Results Representative chromatographic tracings resulting from the injection of aliquots of solutions prepared from blank human serum and serum containing 75 E.cg/ml cefazolin sodium and 75 pg/ml cephalothin sodium are presented in Fig. 1. Blank human serum contained no ultraviolet-absorbing components that would significantly interfere with the peaks corresponding to cefazolin and cephalothin. The chromatographic method provided adequate resolution of cefazolin and cephalothin as well as separation of the cephalosporins from other ultraviolet-absorbing components of serum. The retention times were 2.5 and 5.0 min for cefazolin and cephalothin, respectively. The linearity and reproducibility of the standard curve is shown in Fig. 2 which presents a standard curve produced from triplicate analyses of serum containing combinations of cefazolin sodium, 10-100 pg/ml and cephalothin sodium, 10-100 pg/ml.

205

4

I

0.002 A

“E v B 3 $ ii B

CEFAZOLIN

2

1 CEPHALOTHIN

50

-

w/ml

.

Fig. 1. Chromatography of blank human serum (left) and human serum containing cefazolin sodium. 75 pg/ml, and cephalothin sodium, 75 pglml (right). on 90 cm X 2 mm i.d. Phenyl Cons@‘; mobile phase 13% methanol in 0.2 M ammonium acetate, 2.0 ml/min. Retention times: cefazolin, 2.5 min; cephalothin, 5.0 min. and cephalothin Fig. 2. Standard curves for simultaneous determination of cefazolin sodium ( -_) sodium (- - - - - -) in human serum. Regression lines are from triplicate analyses. Vertical bars represent range of peak areas at each concentration. Cefazolin sodium: slope, 5.0818 X 10e2; intercept, 0.181463; correlation coefficient, 0.999. Cephalothin sodium: slope, 4.05497 X 10m2: intercept, 0.166585; correlation coefficient, 0.997.

The reproducibility of replicate analyses was examined by the determination of cefazolin and cephalothin in 10 aliquots of human serum containing known amounts of each antibiotic by comparison against a single standard curve. The results are presented in Table I. The assay procedure was applied to samples obtained from a pediatric patient who had received 350 mg intramuscular cefazolin sodium preoperatively and 1 g intravenous cephalothin sodium during surgery. The concentra-

TABLE

I

REPRODUCIBILITY IN SERUM

OF SIMULTANEOUS

DETERMINATION .__~~__

-___. Actual

___

Cefazolin sodium

Cephalothin sodium

40.0 38.6 31.742.4 3.18 8.2

60.0 57.9 47.8-64.0 4.96 8.6

concentration

@g/ml) MeaIl Range Standard deviation Coefficient of variation

(4%)

OF CEFAZOLIN ._

AND CEPHALOTHIN

206 TABLE THE

II HPLC

DETERMINATION

OF

CEFAZOLIN

AND

CEPHALOTHIN

IN

Sk:RUhI

OF

:\ SURGICAL

PATIENT Hours

after:

Cefazolin

Cephalothin

(350

(1 g i.v.)

mg i.m.)

Ccfuolin

Cephalothin

sodium

sodium

c.e/mI)

Wa/mI)

4.5

1.0

“4.9

67.6

6.17

3.5

22.5

13.1

tions are presented in Table II. Thus, the HPLC assay is capable of determining cephalosporin serum concentrations over the range of concentrations encountered in the surgical procedures of interest. A detailed account of the cephalosporin concentrations in serum of surgical patients will be reported elsewhere (Csicsko, J, to be published). Discussion The limit of sensitivity for the simultaneous determination of cefazolin and cephalothin was approximately 10 lug/ml of either antibiotic. In an HPLC assay for cefazolin in serum, described in a previous report from this laboratory [ 71, concentrations of 1.6 pg/ml were readily determined. The difference in sensitivity of the two assays is due to the maximum volume of the trichloroacetic acid supernatant solution that can be injected onto the chromatographic column. Due to the presence of ammonium acetate in the mobile phase of this assay system which is necessary to effect separation of the two antibiotics, injection of volumes of acid supernatant solution greater than 20 ~1 caused a disruption of the chromatography. In the assay for cefazolin alone, the mobile phase was acidic (lo-15% methanol in 0.16 M aqueous acetic acid) which permitted injection of at least 90 ~1 of the trichloroacetic acid supernatant solutions without alteration of peak shape or retention time. The simultaneous assay was also somewhat less reproducible than the assay for cefazolin alone (coefficient of variation of 8.2--8.6% vs. 4.9%) [ 71. The deficiencies of this simultaneous assay (sensitivity and reproducibility) do not, however, seriously affect the utility of the assay. The sensitivity of the assay permitted the determination of concentrations of cefazolin and cephalothin in serum from a subject undergoing cardiopulmonary bypass surgery and the precision of the assay is sufficient to allow valid judgments about pharmacokinetics under these conditions. Metabolism of the cephalosporins did not interfere with the HPLC assay. Cefazolin is not metabolized by man [8] although cephalothin is metabolized in part to deacetylcephalothin [9]. Cooper et al. [lo] have described the determination of cephalothin and deacetylcephalothin in serum by HPLC after ion-pair extraction and have shown that serum levels of deacetylcephalothin are less than 8% of serum cephalothin concentrations. Deacetylcephalothin chromatographed with a retention time of 1.6 min in the system used in this assay when injected in aqueous solution. When deacetyl-

207

cephalothin was added to serum and treated within trichloroacetic acid prior to chromatography it was converted to cephalothin lactone (lactone of 7(thiophene-2-acetamido)-cephalosporanic acid) as would be expected [ 111. Cephalothin lactone had a broad peak with a retention time of 10.5 min. Thus, deacetylcephalothin does not interfere with this assay procedure. However, this procedure does not permit the quantitation of deacetylcephalothin because the product, cephalothin lactone, chromatographs as a broad peak that cannot be quantified in the range of concentrations expected (less than 8% of cephalothin concentrations). The use of HPLC for the determination of a single cephalosporin in serum offers the advantage of greatly increased speed with slightly greater accuracy compared to conventional microbiological assay as demonstrated previously [ 71. These advantages are only relative, however, while the simultaneous determination of two cephalosporins in serum as described here using HPLC, is not possible by conventional microbiological assays and represents a unique accomplishment of HPLC. Although the procedures described apply to cephalothin and cefazolin, the general approach should be applicable to other combinations of cephalosporins. Acknowledgements The authors wish to express their appreciation to Dr. J. Csicsko, Department of Surgery, Indiana University Medical Center, for his collaboration. References 1

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The simultaneous quantitative determination of cephalothin and cefazolin in serum by high pressure liquid chromatography.

203 Clinica Chimica Acta, 78 (1977) 203-207 @ Elsevier/North-Holland Biomedical Press CCA 8541 THE SIMULTANEOUS QUANTITATIVE DETERMINATION OF CEPHA...
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