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.
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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
753754 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.
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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 distinct756 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, the758 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
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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
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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 theVOL. 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 10.6762 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
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