development high liquid chromatographic assay measuring serum

Infectious Diseases in Obstetrics and Gynecology 1:71-75 (1993)(C) 1993 Wiley-Liss, Inc.

Development of a High Performance LiquidChromatographic Assay for Measuring Mezlocillin in

Serum or Tissue

Sandra Mya, Ron E. Gain, and Bryan LarsenDepartments of Obstetrics and Gynecology (S.M.), Biological Science (R.E.G.), and Microbiology and

Obstetrics and Gynecology (B.L.), Marshall University School of Medicine, Huntington, WV

ABSTRACT

Objective: This study evaluated the blood and uterine tissue concentration of mezlocillin, a broad-spectrum penicillin.

Methods: We adapted a liquid chromatographic method to measure mezlocillin in serum andtissue. Mezlocillin reference standard was diluted in water, chromatographed on a reversed phaseC18 column eluted at 1.5 ml/min with acetonitrile and phosphate buffer (1:3 v:v), and detectedspectrophotometrically at 210 nm. Mezlocillin was administered to 14 premenopausal women sched-uled to undergo vaginal hysterectomy. Each patient received a 4 g IV infusion of the drug 30 to 60min prior to surgery. During surgery, tissue was removed from the uterine cervix and blood wasobtained for assay of mezlocillin content.

Results: Chromatography of the mezlocillin standard furnished a discrete peak with a retentiontime of 2.4 min. The sensitivity of the assay was 0.1 pg/ml with a linear response up to 100 g/ml.The correlation coefficient for the standard curve was 0.9997. When reference standard was dilutedin pooled human serum, the assay was complicated by interfering compounds. These were removedby ether extraction. The sensitivity of the assay performed in serum was 3 Cg/ml. Serum samplescontained from 81.2 to 358 pg of mezlocillin/ml with an average serum concentration of207.5 g/ml.When serum containing a known amount of mezlocillin was homogenized for a period of timesimilar to that required to homogenize tissue samples, a detectable loss of drug was observed andwas applied as a correction factor to the measured tissue levels. After correction, the average tissuelevel was 117.2 g/ml and ranged from 27% to 98% of the serum levels.

Conclusions: The serum concentration ofmezlocillin after IV infusion of4 g was greater than thatrequired to inhibit the majority of the most significant organisms responsible for post-hysterectomysepsis. Although tissue levels appeared to be consistently lower than serum levels, they could beexpected to provide an inhibitory effect against many of the bacterial strains that contaminate thesurgical site. (C) 1993 Wiley-Liss, Inc.

KEY WORDS

Antibiotics, pharmacodynamics, ureidopenicillin

otent broad-spectrum congeners of penicillinare finding increasing use in a variety of clinical

settings. The rational use ofsuch antibacterial agentsrequires knowledge of the pharmacokinetics andpharmacodynamics of these drugs. Sensitive meth-ods for assaying drugs in serum or tissue are needed

for developing such information, and high perfor-mance liquid chromatography (HPLC) has becomea useful technique for obtaining such information.

The present study adapted an HPLC method foranalysis of serum and tissue samples from patientstreated with rnezlocillin. HPLC methods for de-

Address correspondence/reprint requests to Dr. Bryan Larsen, Department of Microbiology, Marshall University School ofMedicine, Huntington, WV 25701.

Clinical StudyReceived December 23, 1992

Accepted August 12, 1993

MEZLOCILLIN IN TISSUE MYA ET AL.

termination of the related compounds mezlocillinand azlocillin in plasma have been reported, butthese methods have not been tailored for use withtissue. The methods available for measuring plasmalevels were evaluated and adapted for use with se-

rum and tissue specimens.

MATERIALS AND METHODSMezlocillin as the sodium salt with a potency of920 Ixg/mg was provided by Miles Pharmaceuticals(New Haven, CT). The drug was reconstitutedwith distilled water to make a stock solution of100.28 txg/ml. Stock solutions were stored at-74C.The 14 patients studied were scheduled to un-

dergo vaginal hysterectomy and were given preop-erative prophylaxis consisting of 4 g mezlocillindissolved in 500 ml of normal saline by IV infu-sion. The antibiotic infusion was started withinhr of the time the surgical procedure began.

During the surgical procedure, when the uterus

was being removed, a blood sample was drawnfrom the antecubital vein, and the blood was al-lowed to clot. A section was cut from the uterinecervix, and both blood and tissue were transportedto the laboratory, where the serum was removedfrom the clot and serum and tissue stored at -74Cuntil analysis.

The method for tissue and serum analysis wasestablished experimentally (see Results). Prior to

assay, tissue and blood samples were thawed at room

temperature. Approximately 0.25 g was cut fromthe tissue sample, weighed to the nearest 0.1 mg,minced, and placed in a tube containing 3 ml ofwater. These samples were kept on ice until thetissue was liquified with a Tekmar tissue homoge-nizer (Tekmar, Cincinnati, OH). The homoge-nate was deproteinized by the addition of 5 mlacetonitrile, followed by centrifugation for 15 minat 2,000g. The supernatant fluid was removed andextracted 3 times with 3 ml of diethyl ether. Theaqueous phase was then injected onto the chromato-graph.

The serum samples were deproteinized withequal volumes of acetonitrile, centrifuged at 2,000gfor 15 min, and extracted 3 times with 2.5 volumesof diethyl ether. The aqueous phase was chromato-graphed.

Samples were analyzed by I--IPLC on a 4.6

220 mm octadecyl silyl, 5 Im particle size columnwith a guard column of identical composition. Sam-ples of 20 Ixl were injected and eluted with 1:3acetonitrile:0.01 M potassium dihydrogen phos-phate buffer, pH 7, at a flow rate of 1.5 ml/min.The column effluent was monitored spectrophoto-metrically at 210 nm. Except where specificallynoted, all measurements were made by triplicatedeterminations on each sample, and the identity ofmezlocillin was based on retention time while quan-titation was based on peak height.

Dilutions of mezlocillin standard were made inwater or pooled human serum to determine linear-ity, repeatability, and sensitivity of the assays. Stan-dard curves made in serum were used to establishdrug levels in the patient specimens.A control to determine whether drug may have

been lost during the process of tissue disruption wasprepared. Serum containing a known amount ofmezlocillin was homogenized for a period of timeequal to the time required to disrupt the tissuesamples. This control was then subjected to depro-teinization and ether extraction as for other sam-

ples. The peak height obtained from this samplewas compared with the result obtained from a se-rum sample with an identical concentration of me-zlocillin that had not undergone homogenization.This provided a correction factor that was appliedto tissue concentrations to correct for drug loss dueto tissue homogenization.

RESULTSFigure shows that the mezlocillin standard di-luted in water produced a symmetrical peak with a

retention time of 2.4 min. Thus, the chromato-graphic method used was deemed acceptable fordetecting the pure compound.A series of dilutions of mezlocillin standard from

0.1 Ixg/ml to 100.28 Ixg/ml were made in water toallow determination of the sensitivity and linearityof the assay method. The smallest concentrationdetected was 0.1 Ig/ml, which furnished an aver-

age peak height of 0.00083 absorbance units at 210nm. The assay was linear over the full range ofconcentrations and provided a correlation coeffi-cient (r2) Of 0.9997.

Ten replicate chromatograms of a standard con-

taining 100.28 Ixg mezlocillin standard/ml wereevaluated to determine the repeatability of peak

72 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY


lpgMEZ=0.0079

TIME

Fig. I. Mezlocillin (MEZ) reference standard diluted in wa-ter chromatographed on a reversed phase column underconditions described in the Materials and Methods section. Asingle symmetrical peak representing the pure compoundwas observed. Chromatography of a larger concentrationrevealed a linear relationship between peak height and con-centration.

height and retention time. The average retentiontime was 2.40 + 0.024 rain (SD). The averagepeak height was 0.774 + 0.0118 absorbance units(SD). The coefficient of variation (relative SD) forretention time was 1% and for peak height was

1.5%. The procedure was therefore considered to

be highly repeatable.The methods for preparation of serum samples

for chromatographic determination of mezlocillinwere evaluated. A known concentration of mezlocil-lin was added to a pool of human serum, and vari-ous methods were used to prepare aliquots of thatserum for analysis. The first method involved ad-dition of ml of 5% perchloric acid to ml of theserum sample followed by centrifugation. This pro-vided a protein-free sample for chromatography.As illustrated in Figure 2, this method of prepara-tion was unsuitable because exposure to perchloricacid appeared to generate at least two new peaks,which may have been degradation products of me-zlocillin. Furthermore, it was not possible to iden-tify the mezlocillin peak unequivocally in the acid-treated serum.

The second method of sample preparation em-

ployed the addition of ml of acetonitrile to ml ofserum to precipitate the protein from the sample. Itwas believed that the acetonitrile would be less likely

to cause degradation of the mezlocillin than wouldperchloric acid. However, the protein-free super-natant fluid from acetonitrile-treated serum yieldeda chromatogram similar to that obtained when se-

rum without mezlocillin was treated with acetoni-trile. This finding suggested that the mezlocillin wasbeing masked by some component of the serum.

It was subsequently found that diethyl ether ex-

traction removed the interfering compounds fromthe acetonitrile-precipitated samples, as shown inFigure 3. In addition, the acetonitrile entered theether phase, which eliminated the dilution of thesample due to the deproteinization step. Consequently,the acetonitrile precipitation followed by ether extrac-tion was adopted for sample preparation.When standards prepared in water, treated with

acetonitrile, and extracted with ether were com-

pared with standards diluted in water, no apprecia-ble loss of drug due to the extraction procedure was

observed. Recoveries ranged from 97.7% to 106%for the concentrations of mezlocillin observed inthe patient samples. Based on these findings, stan-dard curves for mezlocillin assay were prepared inpooled serum, and patient samples were comparedwith these standards.

Tissue samples were also compared with a stan-

dard curve prepared in serum. However, it was

determined that the tissue homogenization was ex-

pected to have reduced the amount of mezlocillindetectable by 37.5% based on homogenizing serum

samples with added mezlocillin. As a consequenceof this finding, the observed tissue values wereincreased by a factor of 0.375.

The serum and tissue levels observed in the 14patients studied are summarized in Table 1. Thelevels observed in serum were consistently higherthan those found in tissue. The highest serum levelstended to be associated with the highest tissue lev-els, but the relationship of serum to tissue levelsrevealed an r2 of 0.58.

This study was not designed to be a pharmaco-kinetic study, but rather an evaluation of the serumand tissue levels obtained simultaneously after IVinfusion. Therefore, the time after infusion that thetissue sample was removed and the serum samplewas taken was not identical in each case and variedfrom 5 rain to 130 min. In the limited number ofsamples available in the study, no linear correlationbetween the time after infusion and tissue or serumlevels was apparent.

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 73


E

MEZ

MEZ

$

SC

A B

2.5 min 2.5 min 2.$ min

Fig. 2. Evaluation by chromatograms of perchloric acid precipitation for preparation of serumsamples. A: Mezlocillin (MEZ) standard (6.25 Ig/ml, final concentration) diluted in water with aretention time of approximately 2.4 min. B: Mezlocillin, 6.25 Ig/ml, with 2.5% perchloric acid. Theheight of the mezlocillin peak was diminished (compared with A), and multiple new peaks appeared,suggesting degradation products’ (2: Mezlocillin, 6.25 Ig/ml (final concentration), in serum deprotein-ized with perchloric acid, showing that mezlocilin is obscured by serum components. S: Start of eachchromatographic run.

DISCUSSIONThe use of antimicrobial prophylaxis in such surgi-cal procedures as vaginal or abdominal hysterec-tomy is so common that one may be tempted to

consider studies involving prophylaxis as trivial.Despite the frequent application of prophylaxis, thefundamental reasons why this type of drug use iseffective is still a matter of conjecture. It is assumedthat antibiotics administered preoperatively affectmicroorganisms that contaminate the operative site,but it is not clear whether any of the organisms are

killed, prevented from replicating, or simply pre-vented from producing virulence factors. It is like-wise not clear what relevance in vitro minimal in-hibitory concentrations have for these tissues. Thelength of time and concentration of drug requiredto be present in the tissue for effective prophylaxis

TIME

Fig. 3. A: Pattern obtained when ether-extracted serum is

chromatographed. B: Results when the serum is spiked withmezlocillin (M) standard and extracted with ether. The me-zlocillin produces a resolved and quantifiable peak.

74 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY


TABLE I. Observed concentrations of mezlocillin inserum and uterine cervix from women given 4 g ofmezlocillin prior to vaginal hysterectomy

Concentration(lg/g) Ratio

Tissue Serum (tissue/serum)

Mean 117.2 207.5 0.567Standard 66.9 93.0 0.233

deviationRange 27.5-228. 81.2-358.0 0.268-0.982

are also open to question. Analysis of tissue levels ofvarious antimicrobials as well as serum levels rep-resents a necessary tool for enhancing our under-standing of the effects of prophylaxis at the opera-tive site.

The work presented here adapted a chromato-graphic procedure for use in determining the se-rum and tissue levels of mezlocillin. An attemptwas made to take into account the possibility thatinteraction of the drug with tissue or serum compo-nents may reduce the amount of drug apparent inour assays. It cannot be claimed that subjectingmezlocillin-spiked serum to the homogenizationstep provided a true .control for possible loss intissue samples, although the data suggest that suchan observation is not irrelevant. An ideal studywould have added drug to tissue from a patient not

treated with antibiotic and processed the tissue as

described; however, obtaining tissue samples frompatients who had not received the drug was outsidethe bounds of the protocol approved by the IRB,leaving the serum homogenization test as the sub-optimal alternative. Certainly, any future applica-tion of this method could be improved by includinga control using tissue from a patient not exposed toantibiotic.

The question of why the homogenizing proce-dure made a portion of the drug unrecoverable isnot explained by the studies presented. Oxidativedamage to the drug or some irreversible reactionwith protein or other constituents of the biologicalmatrix are potential explanations. The possibilitythat drug was incorporated into micelles during thehomogenization process may be less likely, since

the solvent extraction steps should destroy suchstructures.

In view of the above considerations, it may bestated that in all cases, some mezlocillin was presentin uterine tissue at the time of incision, and thetissue concentration was at least one quarter thatpresent in serum if one accepts the correction fac-tor. A more conservative estimate (without the cor-rection factor) of tissue concentrations ranges from17.2 to 142.5 txg/g and would average 35% of thesimultaneous tissue values.

For therapy of established infections, it is desir-able to achieve serum and tissue concentrationsgreater (generally by 2-4 times) than the minimalinhibitory concentrations of the etiologic agents.Somewhat less certain are the serum or tissue levelsrequired to achieve prophylaxis against infection ofthe operative site. It is not clear whether it is neces-

sary to achieve minimal inhibitory concentrationsagainst microorganisms at the surgical site. Thelevels of mezlocillin observed in serum and tissueare well above the minimal inhibitory concentra-tions reported for some ofthe microorganisms com-monly involved in infections after hysterectomy. 2

For example, 90% of Bacteroidesfragilis strains areinhibited by 32 Ig of mezlocillin/ml and 90% ofEscherichia coli strains are inhibited by 64 Ig/ml.It should be noted, however, that minimal inhibi-tory concentrations are determined in vitro with a

constant concentration of drug and with the test

organism exposed to that drug for many hours. Inthe patient, the concentration of the drug is con-

stantly changing. The present study, not being a

pharmacokinetic study, did not sample tissue andserum at identical times after infusion. Thus, whilesignificant levels of antibiotic were detected in thepatients studied, future work will need to elucidatethe relevance of brief exposures of microorganismsto these concentrations of mezlocillin. This chro-matographic technique has proved an appropriatetool in establishing the concentration of mezlocillinto be studied in future investigations.

REFERENCES

1. Rouan MC: Antibiotic monitoring in body fluids. J Chro-matogr 340:361-400, 1985.

2. Parry MF, Pancoast SJ: Antipseudomonal penicillins. InCunha B, Ristuccia A (eds): Antimicrobial Therapy. New.York: Raven Press, pp 197-208, 1984.

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