Assay of Amoxicillin and Clavulanic Acid, the Components of Augmentin, in Biological Fluids with High-Performance Liquid
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 1982, p. 753-762 Vol. 22, No. 5 0066-4804/82/110753-10$02.00/0 Copyright © 1982, American Society for Microbiology Assay of Amoxicillin and Clavulanic Acid, the Components of Augmentin, in Biological Fluids with High-Performance Liquid Chromatography MARK FOULSTONE AND CHRISTOPHER READING* Beecham Pharmaceuticals-Research Division, Brockham Park, Betchworth, Surrey RH3 7AJ, England Received 12 April 1982/Accepted 9 August 1982 Augmentin is a new antibacterial formulation comprised of amoxicillin and the ,B-lactamase inhibitor clavulanic acid. In the present paper, the use of high- performance liquid chromatography (HPLC) to provide a rapid assay of the components of Augmentin in body fluids is described. Clavulanic acid was assayed by reacting the sample with imidazole, which readily produces a derivative absorbing at 311 nm. This derivative chromatographs on reverse-phase HPLC columns clear of interfering components in both human serum and urine. Downloaded from aac.asm.org by on June 29, 2010 Concentrations of clavulanic acid as low as 0.1 ,ug/ml were readily detectable in human serum with this procedure. There was no interference from amoxicillin, amoxicillin penicilloic acid, or the acid and alkali degradation products of clavulanic acid when this assay system was used. Amoxicillin in body fluids was assayed directly by HPLC without derivatization. The same chromatographic conditions were employed for the assay of amoxicillin and the clavulanic acid derivative, simplifying the methodology. Amoxicillin, however, was determined by monitoring at 227 nm, and the limits of detection in human serum were 0.5 jig of the antibiotic per ml. An alkali blanking procedure for amoxicillin and clavulanic acid is also described which allows the detection of any underlying peaks which may cochromatograph. The use of ultrafiltration to remove protein from serum samples before HPLC was successfully applied to the assay of clavulanic acid and amoxicillin. Ultrafiltration is not an essential procedure for these assays, but it prolongs column life and reduces interference in the amoxicillin assay. Results obtained by HPLC were compared with those obtained by using, microbiological assays. Clavulanic acid is a novel P-lactam compound chromatography (HPLC) to rapidly determine which was isolated from the culture fluid of low concentrations of clavulanic acid in biologi- Streptomyces clavuligerus (8). The compound is cal fluids is described, and the assay of amoxicil- a potent inhibitor of a large number of ,-lacta- lin by using the same chromatographic system is mase enzymes which are responsible for the also reported. These methods may be of use in resistance of many bacteria to ,-lactam antibiot- the study of the pharmacokinetics of formula- ics. In the presence of clavulanic acid, P-lacta- tions of penicillins and clavulanic acid, such as mase-labile penicillins are protected from degra- Augmentin, and for rapidly determining body dation by cell-free P-lactamase preparations (8) fluid concentrations of the components of these and by whole bacterial cultures (7). One such formulations during clinical use. penicillin-clavulanic acid formulation is Aug- mentin, which is comprised of amoxicillin and MATERIALS AND METHODS clavulanic acid (Fig. 1) and which has been HPLC equipment. A Waters M6000A pump was reported (1, 5) to give good results in clinical used, and injections were made with a Hamilton 25-pI use. syringe via a U6K injector (Waters Associates, North- Clavulanic acid may be detected by using a wich, England). The column eluant was monitored by special microbiological assay method (2) which using a Cecil 2012 variable-wavelength UV detector with a 1-cm 8-,il flow cell (Cecil Instruments Ltd., has been adapted for routine assay purposes (1). Cambridge, England). The HPLC column (25 cm; It may also be determined biochemically by inner diameter, 4.6 mm) contained a C18 ,u-Bondapak using its enzyme inhibitory properties against a support prepacked by Waters Associates. A guard suitable P-lactamase preparation (9). In the pres- column (Whatman column survival kit) was used ent paper, the use of high-performance liquid throughout these studies and was frequently repacked 753
754 FOULSTONE AND READING ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. HPLC on C18 ,u-Bondapak reverse-phase columns Compounds to be assayed Solvent system Detection wavelength Clavulanic acid Buffer' 220-227 Amoxicillin in serum or pure solution Buffer + 6% methanol 227 Amoxicillin in urine Buffer + 4% methanol 227 Amoxicillin penicilloate in pure solution Buffer + 4% methanol 227 Derivatized clavulanic acid in serum Buffer + 6% methanol 311 Derivatized clavulanic acid in urine (>2 ,ug/ml) Buffer + 6% methanol 311 Low concentrations (
VOL. 22, 1982 ASSAY OF AUGMENTIN COMPONENTS 755 HO f .CH-CO-NH z I Hr NH2 COO H20j~ amox icillin trihydrate *OHV 101L potassium clavlanate (BRL 14151) C02,4imidazol (room temperature) N3-C-CH C-N-CFrC H -CFPH20H B 1-(8-hydroxy-6-oxo-4-azaoct-2-enoyl)-imidazole FIG. 1. Structures of amoxicillin and clavulanic acid and the reaction of clavulanic acid with imidazole. Downloaded from aac.asm.org by on June 29, 2010 was added to neutralize the sample, and 25 ,ul was then applied to the HPLC column. Amoxicillin standards in 15 10 5 diluted control urine or buffer were treated identically Time (min) to the samples to provide test and blanks when neces- t inject sary. For the routine assay of clavulanic acid in urine, the FIG. 2. HPLC of amoxicillin (Amox.) and clavu- samples were diluted 1/10 into the phosphate buffer, lanic acid (underivatized) in human serum. The col- and samples were then derivatized and applied to the umn was eluted with 0.1 M potassium phosphate HPLC column. Blanking procedures were not normal- buffer (pH 3.2) at 2.5 ml/min and monitored at 227 nm ly required for clavulanic acid assay. However, when and 0.005 AUFS. Samples (25 ,ul) contained both concentrations of clavulanic acid of less than 2 jig/ml amoxicillin (5 ,ug/ml) and clavulanic acid (2.5 ,ug/ml). in neat urine were required to be assayed, test and (A) Human serum. (B) 0.05 M potassium phosphate blanked samples were prepared. The sample in neat buffer (pH 7.3). urine (0.6 ml) was diluted with 0.2 ml of phosphate buffer and then derivatized by adding 0.2 ml of the imidazole reagent. After 10 min at room temperature, 25 pR1 of the reaction mixture was applied to the HPLC ole. The buffer eluants shown in Table 1 were used for column. The blanked sample was prepared by adding amoxicillin assay, and the eluant was monitored at 227 0.1-ml portions of alkali and acid to 0.6 ml of the nm; the earlier assays of the clavulanate-imidazole sample, as described for the amoxicillin blanks. This derivative were monitored at 311 nm under the condi- was then derivatized by the addition of 0.2 ml of tions given in Table 1. imidazole reagent, and after 10 min at room tempera- Microbiological assay methods. Amoxicillin was as- ture, 25 ,u1 was applied to the HPLC column. sayed by a hole-in-plate method with Sarcina lutea as HPLC conditions. Table 1 summarizes the chro- the test organism. Clavulanic acid was determined by matographic and detection conditions used for the using the special synergistic assay system devised in determination of clavulanic acid, amoxicillin, and these laboratories (2) and adapted for the assay of amoxicillin penicilloate without reaction with the imid- clavulanic acid in the presence of amoxicillin (R. azole reagent, as well as for the imidazole reaction Horton, unpublished work) as described by Ball et al. products of clavulanic acid and amoxicillin penicil- (1). This method used agar which contained benzyl loate. penicillin and Klebsiella pneumoniae ATCC 29665 as Assay of a sample for both clavulanic acid and the test organism. Clavulanic acid in the samples amoxicillin. Where assays of both components in a inhibited the 1-lactamase produced by this organism sample were required, the clavulanic acid content was and so allowed the penicillin in the agar to produce a determined first. This order was used because clavu- zone of inhibition. lanic acid was less stable than amoxicillin when stored in solution in body fluids. After removal of sample for RESULTS derivatization and clavulanic acid assay, serum, ultra- HPLC of underivatized clavulanic acid and filtered serum, or urine samples were stored in closed amoxicillin. Clavulanic acid is poorly retained on vessels at 5°C if amoxicillin assays were to be per- formed on the same day; they were stored at -40 to C18 reverse-phase columns. Retention times can -70°C if the assays were to be performed at a later be lengthened by using buffer eluants at acid pH, time. Amoxicillin assays were carried out on the but even under these conditions it was not stored samples that had not been reacted with imidaz- possible to obtain clavulanic acid peaks distinct
756 FOULSTONE AND READING ANTIMICROB. AGENTS CHEMOTHER. I C Swumn A UlNtred D seu L-r. Downloaded from aac.asm.org by on June 29, 2010 Ultrflteod B 4~~~ Pi I lb 5 10 5 0 lime (min) FIG. 3. Amoxicillin (Amox.) in human serum. Samples were chromatographed in 0.1 M potassium phosphate (pH 3.2)-6% (vol/vol) methanol, and the eluant was monitored at 227 nm and 0.01 AUFS. (A) I 15 lb I 5 I I 0 Serum (25 1I). (B) Serum containing 2.5 Fg of amoxi- cillin per ml (25 ,ul injected [Inj.]). (C) Serum diluted lime (min) 1/2 into 0.1 M sodium phosphate buffer (pH 7.0) and FIG. 4. Amoxicillin (Amox.) in urine. Samples ultrafiltered (50 p.1 injected). (D) Serum containing 2.5 were chromatographed in 0.1 M potassium phosphate ,ug of amoxicillin per ml diluted and ultrafiltered (50 ,ul (pH 3.2)4%o (vol/vol) methanol. The eluant was moni- injected). tored at 227 nm and 0.01 AUFS. Amoxicillin (200 p.g/ml) in urine was diluted 1/10 into 0.1 M sodium phosphate buffer (pH 7.0). (A) 0.6 ml of diluted urine plus 0.2 ml buffer (25 p.1 injected [Inj.]). (B) 0.6 ml of from interfering components in human serum or diluted urine blanked with 0.1 ml 1 M NaOH (30 min at plasma samples (Fig. 2). Clavulanic acid, like room temperature) neutralized with 0.1 ml of 1 M HCI the penicillins, has no specific chromophore, (25 g.d injected). and to obtain reasonable detection limits, eluants must be monitored at low wavelengths (
VOL. 22, 1982 ASSAY OF AUGMENTIN COMPONENTS 757 A E -C .6a -Cdco B a.0) Downloaded from aac.asm.org by on June 29, 2010 Time (min) V1bI FIG. 6. Effect of reagent concentration on the rate of derivatization of clavulanic acid. Clavulanic acid (100 ,Lg/ml) in phosphate buffer (pH 7.3) was added 4 3 2 1 0 (0.5 ml) to (0)0.4 ml, (0)0.2 ml, (U) 0.1 ml, and (0) (min) 0.05 ml of imidazole reagent. Reaction volumes were Time t made up to 1 ml with 0.05 M potassium phosphate inject buffer (pH 7.3). Formation of the clavulanic acid FIG. 5. HPLC of the imidazole-clavulanic acid re- derivative at room temperature was followed with time action product in human serum. The column was by loading 25-,ul samples onto the HPLC column eluted with 0.1 M potassium phosphate buffer (pH under the conditions described in the legend to Fig. 4. 3.2)-6% (vol/vol) methanol at 2.5 ml/min and moni- Peak height (millimeters) was measured at 0.5 AUFS. tored at 311 nm and 0.01 AUFS. Samples (25 ,ul) were On the basis of these data, a 1:4 addition of reagent to (A) serum plus imidazole reagent after 10 min at room sample (0) was chosen for routine use with a 10-min temperature and (B) 2 ,ug of clavulanic acid per ml in reaction period. human serum plus imidazole reagent after 10 min at room temperature. greatly decrease the sensitivity of the assay by leaving the majority of the antibiotic behind in units full scale (AUFS). The ultrafiltration of the protein-rich retentate. The dilution of the serum samples and standards containing amoxi- serum sample with 0.1 M sodium phosphate cillin was not absolutely essential. This proce- buffer (pH 7.0) before filtration served two pur- dure did, however, reduce the level of high- poses. The percentage binding was decreased by molecular-weight material that was applied and dilution, but the main reason for diluting with so greatly extended the efficient working life of buffer was to maintain the pH of the filtrate at the column. Ultrafiltration also removed inter- neutrality. It was found that the pH of the serum fering peaks on the HPLC trace, as illustrated in after ultrafiltration was in excess of 8.0 when Fig. 3, making measurements of peak height unbuffered serum was used. This actually had easier and more accurate. This procedure can be little effect on the stability of amoxicillin, but used for both amoxicillin and clavulanic acid clavulanic acid was relatively unstable at alkali because both these P-lactams are not highly pH. Since these ultrafiltered samples were to be serum bound (4); thus, ultrafiltration does not assayed for both components of Augmentin, the
758 FOULSTONE AND READING ANTIMICROB. AGENTS CHEMOTHER. I lin in serum, a range of standard concentrations A Serum C serum in serum was used, and standard lines of peak Ukraf lered height versus concentration were plotted from which the concentration of amoxicillin in the serum sample was determined. If the serum samples were to be assayed after ultrafiltration, then the standards in serum were similarly dilut- ed and ultrafiltered. With ultrafiltered samples and standards, the injection volume was in- creased from 25 to 50 iLl to allow for the dilution step. Standard lines for amoxicillin in serum and ultrafiltered serum were linear up to at least 20 ,ug/ml. When amoxicillin in urine was assayed, the samples were initially diluted 1/10 into phos- phate buffer to reduce the level of extraneous material that was loaded onto the column. In urine containing low concentrations of amoxicil- lin, interference from urine components can occur. For this reason, such urine samples must Downloaded from aac.asm.org by on June 29, 2010 be blanked by the addition of alkali followed by neutralization, as described in Materials and Methods. In this way, the absorbance of minor i D D Ul Serum ie litere peaks which cochromatograph with amoxicilUin may be subtracted from the peak heights ob- tained for the untreated urine samples contain- ing amoxicillin. The alkali treatment generates the faster-running penicilloic acid well separated from the original amoxicillin position (Fig. 4). The addition to urine of alkali followed by HCl does not appear to affect the chromatographic results for other urine components. Even with the use of blanked samples, it proved difficult to assay amoxicillin in concentrated urine at less than 100 ptg/ml. After oral administration of amoxicillin, urine concentrations of the antibiot- ic can, however, be very high. For assays of amoxicillin in urine, the methanol content of the eluant was 4% (vol/vol). Standard lines for amoxicillin in urine were linear up to at least 1 mg/ml. Assay of derivatized davulanic acid. The gen- eration of a more suitable chromophore by de- 5 0 5 0 rivatizing clavulanic acid before chromatogra- lime (min) phy was considered as a means of overcoming FIG. 7. Clavulanic acid (Clav.) (derivatized) in hu- the interference problems discussed earlier. The man serum. Samples were chromatographed with 0.1 reaction of imidazole with penicillins in the M potassium phosphate (pH 3.2)-6% (vol/vol) metha- presence of mercuric chloride to form penicil- nol, and the eluant was monitored at 311 nm and 0.005 lenic acid mercuric mercaptides was first de- AUFS. (A) Serum plus imidazole (25 p.l). (B) Clavu- scribed by Bundgaard and Ilver (3). The reaction lanic acid (1.25 Pg/ml) in serum, derivatized (25 Fal of imidazole with clavulanic acid in the absence injected [IIU.]). (C) Serum diluted 1/2 into 0.1 M of mercuric chloride occurs readily at room sodium phosphate buffer (pH 7.0) and ultrafiltered. temperature to form a relatively stable product Imidazole was added, and 50 was injected. (D) Serum containing 1.25 ,ug of clavulanic acid per ml, (Fig. 1) absorbing at 311 nm (A. E. Bird, J. M. diluted by 1/2 and ultrafiltered. The ultrafiltrate was Bellis, and B. C. Gasson, The Analyst, in derivatized and 50 Fl was injected. press). HPLC examination of this product by using C18 reverse-phase columns revealed a single peak when chromatographed in the phos- buffer dilution stage was routinely adopted when phate buffer (pH 3.2) containing 6% methanol ultrafiltration was used. For assays of amoxicil- (Fig. 5).
VOL. 22, 1982 ASSAY OF AUGMENTIN COMPONENTS 759 Materials and Methods). The sample (0.4 ml) was added to 0.1 ml of the reagent, and after 10 A min at room temperature, it was injected (25 pl) onto the column. The rate of formation of the clavulanate-imidazole product was limited by reagent concentration (Fig. 6). A 1:4 addition of reagent to sample and a 10-min reaction at ambient temperature were selected as routine procedure, although shorter reaction times may be used if a more concentrated reagent is em- ployed or if larger reagent volumes are added. As can be seen in Fig. 6, the reaction product was stable over a period of at least 2 h. With the routine procedure, linearity was obtained for plots of peak height versus concentration up to at least 200 p,g/ml of clavulanic acid. With a 25- pl injection volume, the detection limit was 0.1 jig/ml at 311 nm and 0.005 AUFS. In serum samples, clavulanic acid was readily derivatized, and there was no interference, even Downloaded from aac.asm.org by on June 29, 2010 at 0.005 AUFS, during HPLC when the buffer (pH 3.2) plus 6% methanol was used (Fig. 7). Ultrafiltration of serum samples for the assay of derivatized clavulanate had no benefit in terms of reducing interference from serum compo- B nents because at 311 nm very few of these other components were detectable (Fig. 7). The ultra- filtration step in this instance therefore served merely to prolong column life by reducing the amount of extraneous material that was loaded onto the column. If only a few samples are to be assayed on an occasional basis, then ultrafiltra- tion can probably be avoided. As for the assay of amoxicillin in serum, standards of clavulanate were prepared in serum (see Materials and Methods), and a standard line of peak height versus concentration was used to determine clavulanate concentration in the serum samples. For the assay of clavulanic acid in urine after derivatization, the methanol level in the buffer eluant was normally 6% (vol/vol). However, the selection of suitable methanol concentrations and the correct buffer pH can be used to opti- mize separation conditions on a particular col- umn, as these appear to vary from column to lb 6 0 column and individual columns also become less Time (min) efficient with use. In the assay of urine samples after derivatization (Fig. 8), problems only oc- FIG. 8. Clavulanic acid (Clav.) (derivatized) in curred with minor cochromatographing peaks urine. Samples were chromatographed with 0.1 M when assays of clavulanic acid at concentrations potassium phosphate (pH 3.2)-6% (vol/vol) methanol, and the eluant was monitored at 311 nm and 0.1 of less than 2 p,g/ml in concentrated urine at 0.02 AUFS. (A) Neat urine plus imidazole (25 ,ul injected AUFS were attempted. In this situation, the [Inj.]). (B) Clavulanic acid (10 pLg/ml) in neat urine was alkali blanking procedure described in Materials derivatized and 25 ,ul was injected. and Methods was used. For the assay of low concentrations of clavulanic acid (
760 FOULSTONE AND READING ANTIMICROB. AGENTS CHEMOTHER. examination when monitored at 311 nm. The presence of 1 mg of penicilloic acid per ml had no effect when standard solutions of clavulanic acid were derivatized and chromatographed. If, however, the reagent included mercuric chlo- ride, the penicilloic acid did indeed produce a reaction product after the 10-min reaction at room temperature. Figure 9 shows that penicil- loic acid produces one main component, distin- guishable from the clavulanate derivative. This penicilloate product was not produced when penicilloic acid was reacted with mercuric chlo- ride alone. The absorbance at 311 nm for the major product of the penicilloic acid-imidazole-mercu- ric chloride reaction was significantly lower than that generated by clavulanic acid with either reagent. As shown in Fig. 9, even after optimiz- ing the reaction with penicilloic acid by using a more concentrated reagent (see Materials and Downloaded from aac.asm.org by on June 29, 2010 Methods) and reacting for 20 min at room tem- perature, the assay of the penicilloic acid deriva- tive was 100-fold less sensitive than that achieved with clavulanic acid. Although the two derivatives were readily separated on fresh C18 ,u Bondapak columns, it was noted that column Time (min) t performance rapidly decreased in terms of re- taining the penicilloic acid derivative. This oc- inject cuffed even though retention times for deriva- FIG. 9. HPLC separation of the reaction products tized clavulanic acid remained fairly constant. of clavulanic acid and amoxicillin penicilloic acid with This problem, combined with poor sensitivity, imidazole. The column was eluted with 0.1 M potassi- did not allow the use of the imidazole reaction um phosphate buffer (pH 3.2) at 2.5 ml/min and for penicilloic acid determinations. For the rou- monitored at 311 nm and 0.005 AUFS. The sample (25 tine determination of clavulanic acid, therefore, ,ul) was from a reaction of 0.4 ml of a solution a mercuric chloride-free reagent was used, total- containing clavulanic acid (1 ,g/ml) and penicilloic ly eliminating the possibility of interference from acid (100 Rg/ml) with 0.1 ml of imidazole-mercuric chloride reagent after 20 min at room temperature. amoxicillin penicilloate in assay samples. Comparison of HPLC and microbiological as- says for Augmentin. Human serum and urine with the imidazole reagent either with or without were spiked with mixtures of amoxicillin and mercuric chloride at room temperature (3). potassium clavulanate and were assayed by us- When clavulanic acid was derivatized in the ing both HPLC and microbiological assay meth- presence of 1 mg of amoxicillin per ml with the ods. The results of this comparison are shown in routine procedure, the standard line for clavu- Tables 2 and 3. lanic acid was unaffected. Although amoxicillin Samples from healthy human volunteers who disappears from the reaction mixture under had been given Augmentin were also assayed by these conditions and hence must interact with using both methods. A typical result for serum the imidazole reagent, the product did not ab- concentrations obtained from a single male vol- sorb at 311 nm. unteer given 500 mg of amoxicillin and 125 mg of Degradation products of clavulanic acid were potassium clavulanate is shown in Fig. 10. prepared by both mild acid and alkaline hydroly- Assay of amoxicfllin and davulanate in uremic sis of the compound (1 mg/ml). Neither of the serum. Sera from uremic patients may contain hydrolyzed preparations produced detectable high concentrations of various components not peaks on HPLC after derivatization with the present in normal serum. The direct HPLC imidazole reagent and did not affect the reaction assay of amoxicillin in such sera proved difficult, with fresh clavulanic acid. even after ultrafiltration of the samples. Under Using the imidazole reagent (no mercuric these circumstances, the alkali blanking proce- chloride), the penicilloic acid of amoxicillin at dure described for the assay of amoxicillin in approximately 1 mg/ml produced no detectable urine can be used to reveal interfering compo- peaks on C18 reverse-phase chromatographic nents, and the methanol concentration in the
VOL. 22, 1982 ASSAY OF AUGMENTIN COMPONENTS 761 TABLE 2. Comparison of microbiological and HPLC assays for spiked samples of human serum containing both amoxicillin and clavulanic acid Amoxicillin (,ug/ml) Clavulanic acid (tg/ml) Sample Actual Microbiological HPLC Actual Microbiological HPLC concn assay assay concn assay assay 1 2.80 3.10 3.00 1.30 1.35 1.35 2 11.30 13.00 11.20 10.20 11.20 10.40 3 5.20 5.30 5.50 2.80 2.30 2.90 4 15.00 13.80 14.60 3.80 3.10 3.80 5 0.47 0.30 0.85 0.23 0.19 0.35 6 3.10 3.70 3.10 1.60 1.40 1.70 7 0.90 0.86 0.85 0.7 0.64 0.75 eluant may be adjusted to provide better separa- may be achieved by using larger sample vol- tions. Some interference from a component in umes. The HPLC assay is highly specific for uremic serum also occurred when low concen- clavulanic acid, and under the routine conditions trations of clavulanate were assayed with the used for derivatization, penicillins, penicilloic precolumn derivitization assay. This was over- acids, and clavulanic acid degradation products Downloaded from aac.asm.org by on June 29, 2010 come by reducing the methanol level in the do not interfere with the assay. Although HPLC eluant to 4% (vol/vol), thus improving the sepa- assays provide rapid results, in contrast to bio- ration of the derivative from the interfering logical methods, for which overnight incubation peak. is required, there is often a limitation on sample throughput. With serum samples for clavulanic acid assay, the short retention time for the DISCUSSION derivative peak allows one assay every 5 to 6 The use of HPLC and precolumn derivatiza- min, providing reagent addition is timed in se- tion for the determination of clavulanic acid, quence to allow a 10-min reaction period before described in the present paper, provides a sim- injection. With urine samples which contain ple rapid assay method. The optional use of slower-running components, the assay time is ultrafiltration, combined with the addition of a somewhat longer. The assay of clavulanic acid single reagent and a short reaction period before in urine by using HPLC has been previously HPLC, is in contrast to many procedures for the described by Haginaka et al. (6). This ion pair HPLC assay of clinical samples, for which com- HPLC method had a detection limit of 5 ,ug/ml plex extraction and precipitation procedures are for clavulanic acid and was not suitable for the required. The reaction of the imidazole reagent assay of clavulanate in serum. with clavulanic acid and the generation of a Amoxicillin determination by direct UV moni- product absorbing at 311 nm considerably re- toring of HPLC column eluants has been de- duces interference from UV-absorbing compo- scribed previously (10). In the present study, nents in human serum and urine. The high amoxicillin was assayed in column eluants at 227 sensitivity obtained for serum samples, with 0.1 nm with chromatographic conditions similar to p,g of clavulanic acid per ml being readily detect- those used for the clavulanic acid-imidazole able with a 25-,ual sample, is similar to that derivative. Ultrafiltration of serum samples has obtained with special microbiological assays for been introduced for amoxicillin assay, which this compound. Further increases in sensitivity markedly reduces interference from high-molec- TABLE 3. Comparison of microbiological and HPLC assays of human urine spiked with both amoxicillin and potassium clavulanate Amoxicillin (>g/ml) Clavulanic acid (,ug/ml) Sample Actual Microbiological HPLC Actual Microbiological HPLC concn assay assay concn assay assay 1 262.0 258.0 260.0 62.5 54.0 60.5 2 103.0 120.0 90.0 28.1 27.0 30.0 3 513.0 500.0 525.0 263.0 280.0 255.0 4 56.0 67.5 40.0 15.6 15.6 18.0 5 250.0 220.0 240.0 256.0 280.0 257.0 6 350.0 320.0 350.0 150.0 140.0 153.0 7 38.0 24.0 20.0 9.4 7.5 10.6
762 FOULSTONE AND READING ANTIMICROB. AGENTS CHEMOTHER. It is hoped that the HPLC assay of clavulanic E acid and amoxicillin will provide a useful meth- od for pharmacokinetic studies on Augmentin, 6 as well as providing the clinical laboratory with a rapid and reliable assay of body fluid concentra- Cu tions in patients under treatment. 0= ACKNOWLEDGMENTS We thank M. Cole and R. Sutherland for their encourage- ment and advice, D. Jackson for human volunteer studies, and 0 R. Horton and his staff for their cooperation in supplying 1! 25 samples and microbiological assay expertise. ~ ~ ~ ~ LITERATURE CITED E 1. Ball, A. P., P. G. Davey, A. M. Geddes, I. D. Farrel, and G. Brookes. 1980. Clavulanic acid and amoxycillin: a 0 ~ ~ ~ ~ ....... .... clinical, bacteriological and pharmacological study. Lan- cet 1:620-623. 2. Brown, A. G., D. Butterworth, M. Cole, G. Hanscomb, Time (hours) J. D. Hood, C. Reading, and G. N. Rolinson. 1976. Natu- FIG. 10. Concentrations of amoxicillin and clavu- rally occurring 3-lactamase inhibitors with antibacterial activity. J. Antibiot. 29:668-669. lanic acid in human serum as determined by microbio- 3. Bundgaard, H., and K. Ilver. 1972. A new spectrophoto- Downloaded from aac.asm.org by on June 29, 2010 logical and HPLC assay. A healthy male volunteer metric assay for the determination of penicillins. J. was given amoxicillin (500 mg) plus potassium clavu- Pharm. Pharmacol. 24:790-794. lanate (125 mg) orally. Microbiological assays were 4. Comber, K. R., R. Horton, S. J. Layte, A. R. White, and carried out for amoxicillin (0) and clavulanate (O), R. Sutherland. 1980. Augmentin: antibacterial activity in and HPLC assays were carried out for amoxicillin (0) vitro and in vivo, p. 11-38. In G. N. Rolinson and A. and clavulanate (U). Watson (ed.), Augmentin: Proceedings of the First Sym- posium, July 1980. Excerpta Medica, Amsterdam. 5. Goldstein, F. W., M. D. Kitzis, and J. F. Acar. 1979. Effect of clavulanic acid and amoxycillin formulation ular-weight serum compounds and prolongs col- against P-lactamase producing gram-negative bacteria in umn life. Amoxicillin concentrations down to urinary tract infections. J. Antimicrob. Chemother. 5:705- 0.5 ,ug/ml were assayed by using these proce- 709. dures. The use of alkali treatment to provide 6. Haginaka, J., T. Nakagawa, Y. Nishino, and T. Uno. 1981. High performance liquid chromatographic determination blanks when assaying urine containing amoxicil- of clavulanic acid in human urine. J. Antibiot. 34:1189- lin has been used, and this improves the reliabil- 1194. ity of HPLC determinations by revealing any 7. Hunter, P. A., K. Coleman, J. Fisher, D. Taylor, and E. components which may cochromatograph with Taylor. 1979. Clavulanic acid, a novel ,-lactam with broad spectrum 3-lactamase inhibitory properties. Synergistic amoxicillin in these complex and variable sam- activity with ampicillin and amoxycillin. Drugs Exp. Clin. ples. Res. 5:1-6. Comparison of HPLC and microbiological as- 8. Reading, C., and M. Cole. 1977. Clavulanic acid: a beta- says for serum and urine samples which were lactamase-inhibiting beta-lactam from Streptomyces cla- vuligerus. Antimicrob. Agents Chemother. 11:852-857. spiked with amoxicillin plus clavulanic acid 9. Reading, C., and P. Hepburn. 1979. The inhibition of showed that both methods gave reliable results. staphylococcal P-lactamase by clavulanic acid. Biochem. Good correlation was also obtained when micro- J. 179:67-76. 10. Vree, J. B., Y. A. Hekster, A. M. Baars, and E. Van Der biological and HPLC systems were compared Klerjn. 1978. Rapid determination of amoxycillin and for samples from human volunteers given amox- ampicillin in body fluids by means of high performance icillin plus clavulanic acid. liquid chromatography. J. Chromatogr. 145:496-501.
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