BIOMEDICAL CHROMATOGRAPHY, VOL. 7,204-207 (1993)

Simple Analysis of Amoxycillin in Plasma by High Performance Liquid Chromatography with Internal Standardization and Ultraviolet Detection

Bruce Charles* and Suvatna Chulavatnatol Department of Pharmacy, The University of Queensland, Steele Building, Brisbane, Oueensland 4072, Australia

A simple high performance liquid chromatographic method with ultraviolet detection at 229 nm is described for quantitation of amoxycillin in plasma. After deproteination of plasma samples with perchloric acid and adjustment of the pH to 4.9, the supernatant was injected onto a reversed phase C18 column, using acetonitrile: phosphate buffer (0.01 M, pH 7.4) (1 : 25 v/v) as the mobile phase. Amoxycillin and the internal standard, cefadroxil, were eluted at 23 min and 12 min, respectively, without interference from endogenous substances. Processed samples were stable for at least 24 h at room temperature which permitted automated batch processing overnight. Calibration plots of the amoxycillin to cefadroxil peak-height ratio vs. amoxycillin concentration were linear (P<O.OOol; r z 0.995) from 0.25 mg/L to at least 16.0 mg/L. Between-day and within-day imprecision (CV) ranged between 3.7% and 17.7%. Absolute recovery for amoxycillin and cefadroxil exceeded 82%. The application was demonstrated by the analysis of amoxycillin in human plasma after a single oral dose of amoxycillin (250 mg) suspen.rion.

INTRODUCTION

Amoxycillin, an amino-substituted p-lactam penicillin, enjoys widespread clinical use not only for its broad antibacterial spectrum but also because its rapid and extensive oral absorption is unhindered by food and it is well tolerated by most patients (Brogden et al., 1975).

Early analytical methods based on bioassay (Bennett et al., 1966) or chemical derivatization and spectropho- tometry (Davidson, 1976) were time-consuming and non-specific. Reversed phase high performance liquid chromatography (HPLC) has been used in more recent times; however, this approach presents significant diffi- culties due to the amphoteric structure of amoxycillin which causes it to elute among other polar, endogenous substances contained in biological fluids. The drug’s high polarity also precludes the use of standard liquid extraction steps in sample clean-up and it is unstable when exposed to media of high or low pH. Some HPLC methods involve direct column injection of plasma supernates following deproteination with strong acid, with or without the addition of an organic solvent (Vree et al., 1978; Carlqvist and Westerlund, 1979; Mascher and Kikuta, 1990). While rapid and convenient, this practice often culminates in rapid column deterioration (Carlqvist and Westerlund, 1979). Further, perchloric acid treated plasma samples containing amoxycillin must be chromatographed immediately to avoid excess- ive degradation (Lee and Brooks, 1984; Mascher and Kikuta, 1990). Ultrafiltration (Foulstone and Reading, 1982), and solid phase or ion pair column extraction (Lee and Brooks, 1984; Jonkman et al., 1985; Nelis el

* Author to whom correspondence should be addressed.

al., 1992) have been applied to avoid this drawback. To enhance performance, pre- or post-column denvatiza- tion reactions have been developed (Carlqvist and Westerlund, 1979; Miyazaki et al., 1983; Haginaka and Wakai, 1985; Carlqvist and Westerlund, 1985; Mascher and Kikuta, 1990) using ultraviolet (UV) detection (Carlqvist and Westerlund, 1979; Haginaka and Wakai, 1985) or fluorometry (Miyazaki et al., 1983; Carlqvist and Westerlund, 1985; Mascher and Kikuta, 1990). Ion pairing principles or column switching have also been applied to increase selectivity and specificity (Jonkman et al., 1985; Haginaka and Wakai, 1985; Carlqvist and Westerlund, 1985). While some of these techniques claim detection limits around 10 pg/L (Miyazaki et al., 1983; Carlqvist and Westerlund, 1985) they are fre- quently laborious and require complex instrumen- tation. Almost all published methods have neglected to employ an internal standard (IS) to control variation despite extensive sample preparation in some cases. A recent paper (Nelis et al., 1992) described a procedure in which cefadroxil was used as the IS for analysis of amoxycillin in bovine plasma. However, its usefulness was compromised by a significant endogenous peak which coeluted with amoxycillin, and the need for complex sample clean-up involving successive extrac- tions on two types of solid phase columns.

We presently describe a reversed phase HPLC method suitable for assaying amoxycillin in plasma. Cefadroxil was used as the IS to control experimental variation, while simple pH adjustment of samples prior to injection provides for adequate stability of amoxycil- lin. The assay is simple, specific and precise. Minimal sample preparation is involved and standard UV detection provides adequate sensitivity for application of the method in pharmacokinetic and bioavailability studies.

0269-3879/93/040204-04 $07.00 0 1993 by John Wiley PC Sons, Ltd.

Received 30 December 1992 Accepted (rerjised) 30 Murch 1993

HPLC OF AMOXYCILLIN IN PLASMA 205

EXPERIMENTAL

Materials. Amoxycillin trihydrate BP (Batch No. M0313) was supplied by Alphapharm Pty Ltd., Brisbane, Australia. Cefadroxil monohydrate was from Sigma Chemical Co., St. Louis, MO, USA. An amoxycillin metabolite, amoxycillin (2K)-piperazine-2',5'-dione was kindly donated by Dr J. Haginaka, Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Japan. The penicilloic acid metabolite was prepared by alkaline hydrolysis of amoxycillin as pre- viously described (Masada el af., 1980). Acetonitrile (Mal- linkrodt, Clayton, Australia) was of HPLC grade. Water was freshly distilled, filtered through a 0.45 pm membrane and degassed under vacuum before use. Anhydrous disodium hydrogen orthophosphate, perchloric acid, phosphoric acid, citric acid and sodium hydroxide were AR grade. Citrate/phosphate buffer (pH 5.4), and a perchloric acid protein-precipitation reagent and pH-adjustment solution (pH 6.9) were prepared as described previously (Carlqvist and Westerlund, 1985).

Instrumentation. The HPLC system was from Millipore-Waters (Milford. MA, USA) and comprised a Model 501 pump, a Model 712 WISP automatic sampler and a Model 484 tunable absorbance detector. Separations were performed on a reversed phase column (Ultrasphere CI8, 5 pm, 250 mm X 4.6 mm i.d.; Beckman Instruments Inc., San Ramon, CA, USA). Chromatograms were recorded on an OmniScribe chart recorder (Model EB 5517-5, Houston Instruments, Austin, TX, USA).

Standards and controls. A master stock solution of amoxycil- lin trihydrate was freshly prepared on each assay day by dissolving amoxycillin trihydrate 0.04591 g (equivalent to 0.04 g amoxycillin base) in 50 mL of citratelphosphate buffer, pH 5.4. Plasma standards were freshly prepared by adding 0.02 mL of amoxycillin master stock solution to 0.980 mL of drug-free human plasma, and then performing 1 mL doubling dilutions in drug-free plasma to give concentrations of 16, 8, 4, 2, 1, 0.5 and 0.25mg/L amoxycillin base, respectively. Seeded controls containing amoxycillin base concentrations of 15, 6, 1.5 and 0.3 mglL were prepared in drug-free plasma and stored at -70°C. A master IS stock solution was pre- pared by dissolving 52.7 mg of cefadroxil monohydrate (equi- valent to 50.2mg of cefadroxil base) in 100ml of citratel phosphate buffer (pH5.4). A working IS solution was pre- pared as a 1 in 4 dilution in buffer of the master solution, which when added to plasma gave a final concentration equivalent to 6 mg/L of cefadroxil base.

Preparation of samples. 20pL of working IS solution were added into labelled centrifuge tubes, and also into each of four seeded controls which had been thawed at room temper- ature shortly before analysis. Plasma standards and unknown samples were pipetted (400 pL) into the appropriate tube to each of which was added 200 pL of perchloric acid protein- precipitation reagent. The contents were mixed with a vortex- ing action for 1 min. The pH-adjustment solution (400pL) was added, the contents agitated with a vortexing action for a few seconds and the tubes centrifuged at approximately 12,OOOxg for 5 min. The supernates were filtered through 25mm diameter filter units (0.45pm pore size; Lida Manufacturing Corporation, Bensenville, IL, USA) into 1 mL HPLC autosampler vials. Preliminary experiments indi- cated that amoxycillin and cefadroxil were not retained by the filtration units.

Chromatography. HPLC analyses were performed at 20 +. 2 "C. The mobile phase consisting of acetonitrile (100 mL), 0.01 M phosphate buffer pH 7.4 (2500 mL) was filtered (0.45 pm pore size) and degassed under negative pressure before use. The flow rate was 1 mL/min which generated a system back-pressure averaging about loo0 p.s.i. The injec- tion volume was 15 pL. Peaks were detected at 229nm at a sensitivity of 0.003 absorbance units full scale and recorded at a chart speed of 2.5 mm/min. The run-time was 24 min.

Quantitation. Standard calibration plots were constructed by linear regression analysis of the peak-height ratios of amoxy- cillin to IS vs. amoxycillin concentration. Unknown concen- trations of amoxycillin were calculated by inverse prediction.

Imprecision. Assay imprecision (CV) was assessed by expressing the standard deviation as a percentage of the mean value. Within-day imprecision was estimated by analysing the four seeded controls 18 times over one day. Between-day imprecision was estimated from the analysis of seeded controls over 18 assay days as part of a clinical study.

Recovery. Drug-free plasma from two volunteers was supple- mented with amoxycillin to give concentrations of 16 ,8 ,1 and 0.5 mg/L. Absolute recoveries were estimated by expressing the peak-heights of both amoxycillin and IS in plasma as a percentage of the heights measured following injection of appropriate amounts of the respective amoxycillin control solutions in citratelphosphate buffer (pH 5.4).

Stability. Two concentrations (16 mg/L and 4 mg/L) of amoxycillin in plasma were analysed to determine the stabi- lity of plasma specimens stored at -70 "C for four months and of processed specimens during overnight storage in the HPLC autosampler carousel.

Specificity. Assay specificity was examined in relation to interferences from endogenous plasma constituents in the drug-free plasma of 18 healthy volunteers. Potential interfer- ence from two known amoxycillin metabolites, amoxycilloic acid and amoxycillin (2R)-piperazine-2' ,5'-dione was assessed by chromatography of aqueous samples of these compounds and comparison of their retention times with that of amoxycillin.

Application. Amoxycillin concentrations were measured in the plasma of 18 volunteers, each of whom had taken by mouth 250mg of amoxycillin in a sugar-free suspension (Imacillin; Astra Pharmaceuticals, Sodertalje, Sweden) fol- lowing an overnight fast.

RESULTS AND DISCUSSION

Amoxycillin (1) and the IS, cefadroxil (2), were eluted as sharp, symmetrical peaks after 23 and 12 min, respectively. The results in Fig. 1 were from the analy- sis of drug-free plasma from a healthy volunteer and from plasma containing amoxycillin concentrations of 16 mg/L and 1 mg/L. Amoxycilloic acid and amoxycil- lin (2R)-piperazine-2',5'-dione did not interfere. Amoxycilloic acid, which is far more polar than the parent compound, was eluted 3.6 min after injection. A solution of the dione derivative in the mobile phase

206 B. CHARLES AND S. CHULAVATNATOL

0.001 I h_s 1 3 0

J I ) L

0 30 15 0 30 15 0 m in

(4 @) (c)

Figure 1. Chromatograms of (a) drug-free human plasma, (b) plasma containing 16 mg/L of amoxycillin and (c) plasma containing 1 mglL of amoxycillin. 1 = Amoxycillin, 2 = cefadroxil; detector response in absorbance units is indicated by the scale bar.

H H

C o o H

Formula 1

Formula 2

gave two peaks at retention times of 29.6 and 38.6 min, although this metabolite was undetectable in the plasma of subjects following oral administration of a single 250 mg dose of amoxycillin. The retention time of amoxycillin on the reversed phase column could be altered significantly by adjustment of the pH of buffer in the mobile phase in the pH range 7.2-7.4. The pK, of the primary amino group on the amoxycillin side- chain is approximately 7.2 and reduction in the proto- nation of this group makes the molecule more lipophilic and, therefore, more retentive on the reversed phase column. Minor adjustments in acetonitrile concentra- tion of the mobile phase (range: 3.5-3.8% v/v) were required occasionally to fine-tune the separation of amoxycillin from small endogenous peaks in some sam- ples. Slight broadening of the amoxycillin peak

occurred after about 60 injections, presumably due to accumulation of endogenous substances on the column. Column life was extended to ca. 500 injections by washing it with 30 mL of methanol, then injecting 1 mL of dimethyl sulphoxide, followed by 30 mL of 50% v/v methanol in methylene chloride. In some cases, perfor- mance could be restored by replacing the top few millimetres of the column packing with fresh material.

The sample clean-up procedure was simple and involved a one-step precipitation of plasma proteins with 10% perchloric acid in citrate/phosphate buffer (pH 5.4). Since the p-lactam ring of amoxycillin cannot tolerate extended exposure to the acidic and oxidative environment produced by perchloric acid, adjustment of the supernate pH to 4.9 prior to HPLC injection was essential for optimal stability (Carlqvist and Westerlund, 1985; Erdmann et al., 1990). We checked that amoxycillin was stable at this pH by storing pro- cessed samples (4mg/L and 16mg/L) in the auto- sampler at room temperature and reinjecting periodi- cally up to 24 h. No degradation was observed in any sample. It was found previously that plasma samples containing amoxycillin can be kept stable for at least one year at -70°C (Carlqvist and Westerlund, 1985). Analysis up to four months storage confirmed adequate amoxycillin stability at this temperature (Table 1).

The regression equation from pooled calibration data from 18 replicate standard curves of peak-height ratio (Y) versus plasma amoxycillin concentration (X) was Y=0,1193(X) - 0.0072. It showed highly significant linearity ( P < 0.0001; r 2 0.995) with statistically insig- nificant ( P > 0.05) non-linear elements in the residual sum of squares, as determined by analysis of variance.

The within-day and between-day imprecision data presented in Table 1 shows that acceptable imprecision was obtained over the entire assay range. The mini-

HPLC OF AMOXYCILLIN IN PLASMA 207

Table 1. Within-day and between-day imprecision. Coefficients of variation are in parenthesis

Target

Within-day 15.0 6.0 1.5 0.3

Between-day 15.0 6.0 1.5 0.3

concentration (mglL1 Found

(Mean f SDI 14.2 f 0.77 (5.42) 5.5 k 0.26 (4.73) 1.39 k0.06 (4.32) 0.37 f 0.03 (8.1 1) 15.0 & 0.55 (3.67) 5.8+ 0.28 (4.83) 1.49 k 0.09 (6.04) 0.34+0.06 (17.7)

Table 2. Recovery of amoxycillin and IS. Results of two repli- cate determinations are given in parenthesis

Amoxycillin concentration Mean recovery (XI

(mglL1 Arnoxycillin IS

16.0 87.6 (90.9.84.2) 91.5 (89.7, 93.3) 8.0 88.6 (88.2.88.9) 90.7 (88.0, 93.4) 1 .o 85.7 (85.7,85.7) 84.3 (83.6,84.9) 0.5 82.4 (82.4,82.4) 88.2 (88.7,87.7)

.- 4

I I I 1 I I

0 1 2 3 4 5 6 Time (hr)

Figure 2. Mean (SE) plasma amoxycillin concentrations after administration of a single oral dose (250 mg) of arnoxycillin suspension.

mum quantifiable concentration (MQC) was 0.3 mg/L, the lowest concentration of amoxycillin in plasma at which maximum acceptable imprecision (between-day CV: 17.7%) was obtained. Recoveries of amoxycillin and IS were comparable and greater than 82% in all cases (Table 2).

Cefadroxil, the cephalosporin counterpart of amoxy- cillin, was chosen as the IS because both molecules possess a p-hydroxyphenyl and an a-amino side-chain substituent and have similar solubilities, UV spectra and stabilities. Interestingly, almost all previously pub- lished HPLC assays of amoxycillin neglected to employ internal standardization, while compounds that were used as such standards differed markedly in structure from amoxycillin (Jonkman et al., 1985; Erdmann ef

af., 1990). Interfering endogenous peaks in plasma specimens precluded the use of an IS in some published methods (Lee and Brooks, 1984; Foulstone and Reading, 1982).

The data in Fig. 2 demonstrate the practical appli- cation of the method in an amoxycillin pharmacokinetic study. Plasma amoxycillin concentrations arising from a normal clinical dose (250 mg) could be measured for at least four half-lives (mean half-life: 56 min) after peak concentrations were observed (median time: 55 min). The mean area under the curve extrapolated from the last sampling time to infinity was only 5.2%. The relatively small plasma volume required makes the method ethically acceptable for kinetic studies in which large numbers of samples are to be drawn, and in studies involving paediatric subjects.

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