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Enzyme and Microbial Technology 46 (2010) 292–296

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Enzyme and Microbial Technology

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simple, sensitive and green bienzymatic UV-spectrophotometric assay ofmoxicillin formulations

heerasak Rojanarataa,∗, Praneet Opanasopita, Tanasait Ngawhirunpata, Choedchai Saehuanb,uthep Wiyakruttac, Vithaya Meevootisomc

Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, ThailandFaculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, ThailandFaculty of Science, Mahidol University, Bangkok 10400, Thailand

r t i c l e i n f o

rticle history:eceived 11 September 2009eceived in revised form2 November 2009ccepted 22 November 2009

eywords:nzymatic method

a b s t r a c t

A simple, fast, sensitive and inexpensive UV-spectrophotometric method for the determination of amox-icillin in pharmaceutical preparations has been developed based on two enzymatic reactions. In thismethod, d-4-hydroxyphenylglycine side chain of amoxicillin was selectively cleaved off by penicillinacylase. Subsequently, it was reacted with 2-oxoglutarate, by the catalysis of d-phenylglycine amino-transferase, to yield the product with high UV absorption namely 4-hydroxybenzoylformate. The amountof amoxicillin was then determined as a change in absorbance at 335 nm. In this work, the assay con-ditions were studied and optimized and the method was validated. The calibration curve presented an

2

moxicillinpectrophotometry

excellent linearity with r of 0.9998 (0–100 �M amoxicillin). Detection and quantitation limits were 0.77and 2.55 �M, respectively. Good accuracy and precision were obtained when the method was tested withamoxicillin capsules and powder for oral suspension. No interference from common excipients in the for-mulations or degradation products was observed. Finally, since all procedures were performed withoutthe use of any organic solvents or hazardous chemicals which were detrimental to the environment and

f reaamox

had a low consumption ofor the quality control of

. Introduction

Amoxicillin is a semi-synthetic �-lactam antibiotic belongingo the group of penicillins. The chemical structure of amoxi-illin consists of d-4-hydroxyphenylglycine side chain attachedo 6-aminopenicillanic acid (6-APA) moiety. Because of its broadpectrum of bactericidal activity and therefore widespread use inedicines, various preparations of this drug alone including cap-

ules, tablets, powder for oral suspension, and injections as well asn combination with other ingredients, e.g. amoxicillin/clavulanateablets are commercially available [1]. Currently, several analyti-al methods for the quantitation of amoxicillin in pharmaceuticalormulations have been reported. Examples of these methods areodometric titration [2], fluorometry [3], chemiluminescence [4,5],

oltammetry [6], spectrophotometry [7–9] and high-performanceiquid chromatography (HPLC) [2,10,11]. Though the latter methods specified in the pharmacopoeias, it requires instruments withigh cost compared to the spectrophotometric assay. In addition,

∗ Corresponding author. Tel.: +66 34 255800; fax: +66 34 255801.E-mail addresses: theerasak@email.pharm.su.ac.th, rtheerasak@yahoo.com

T. Rojanarata).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.11.011

gents, this proposed assay was an ideal green analytical method suitableicillin in pharmaceuticals.

© 2009 Elsevier Inc. All rights reserved.

some chromatographic systems use organic solvents as mobilephases which are on the issues of environmental concern or aworldwide shortage crisis such as acetonitrile.

Enzymatic assay is an alternative for the drug analysis. Thehigh specificity of enzymes can be used to determine preciselyconcentrations of compounds even in a complex matrix by employ-ing techniques, e.g. spectrophotometry to monitor the course ofenzyme-catalyzed reaction upon the compound is converted tothe measurable product [12]. Furthermore, enzymatic methodsare ideally suited to green chemistry because most enzymes workefficiently under mild conditions and often use no undesirablechemicals or solvents.

In the present paper, we report the development and validationof a new UV-spectrophotometric method for the determination ofamoxicillin in capsules and powder for oral suspension by the useof two enzymes. One enzyme is penicillin acylase (PA; EC 3.5.1.11)from Escherichia coli which is commercially available. The enzymehas been used in pharmaceutical industry for the hydrolytic cleav-

age of the side chains of penicillin G or V to obtain 6-APA, a precursorof various semi-synthetic penicillins [13]. The other enzyme is d-phenylglycine aminotransferase (d-PhgAT; EC 2.6.1.72) purifiedfrom recombinant E. coli expressing the cloned gene encodingthe d-PhgAT from Pseudomonas stutzeri ST-201. It catalyzes a

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eversible stereo-inverting transamination of d-phenylglycine or-4-hydroxyphenylglycine and 2-oxoglutarate to yield benzoylfor-ate or 4-hydroxybenzoylformate and l-glutamate [14]. Currently,

-PhgAT has been applied for the synthesis of optically pure d-henylglycine [15] and the analysis of l-glutamate in foods [16,17].ue to a high substrate specificity of the enzymes, an intenseV absorption property of formed product as well as simple andasy measuring procedures, this proposed method is very sensi-ive, rapid and convenient and does not require any complicated orxpensive instruments and environmentally detrimental reagents.

. Experimental

.1. Instrumentation

The spectra and absorbance at 335 nm were measured by an Agilent G1103Aodel UV–visible spectrophotometer (Agilent, USA) using a semi-micro quartz

uvette type (1 cm path length). The HPLC system consisted of an Agilent 1100Seriend a diode array detector (Agilent, USA). The column used was reversed phaseertiSepTM GES ODS column, 5 �m, 150 mm × 4.5 mm (Verical Chromatography,angkok, Thailand).

.2. Reagents and chemicals

Reference standard amoxicillin trihydrate was purchased from Laboratory ofr. Ehrenstofer Company (Augsburg, Germany) with the purity of 99.6%, calculatedn the anhydrous basis, determined by HPLC method. The enzyme PA from E. colias obtained as a commercial reagent from Sigma Aldrich (St. Louis, MO). It was

upplied as suspension in 3 M ammonium sulfate and phosphate buffer. d-PhgATas purified from a recombinant E. coli expressing the cloned gene encoding the-PhgAT from P. stutzeri ST201 as previously described [18] and kept in phosphateuffer containing 25% glycerol for a long term use. Both enzymes were freshly dilutedith water to desired activity before use. Double distilled water was used to prepare

ll solutions throughout the experiments. All other chemicals were of the analyticalrade from Sigma Aldrich (St. Louis, MO) The commercial capsules and powder forral suspension were prepared from Amoxicillin trihydrate USP by manufacturers inhailand. Their expiration dates were at least 30 months beyond the date of analysisn this study.

.3. Assay of amoxicillin by bienzymatic UV-spectrohotometric method

.3.1. Analytical procedureIn order to minimize pipetting steps and increase both precision and accuracy,

he reaction cocktail was prepared containing 135 mM Tris buffer (pH 9.0), 135 �Myridoxal-5′-phosphate, 270 �M 2-oxoglutarate, 0.27 U mL−1 d-PhgAT enzyme andater. The assay, conducted in a total volume of 1 mL, was started with the pre-

ncubation of 250 �L standard or sample solution and 745 �L reaction cocktail at7 ◦C so thatd-4-hydroxyphenylglycine impurity which may be present with amox-

cillin was converted to 4-hydroxybenzoylformate. After 5 min, the absorbance at35 nm of the solution was read, designated as “A”. Subsequently, 5 �L of the enzymeA was added into the reaction at the final concentration of 1 U mL−1. The reactionolution containing both enzymes was further incubated at 37 ◦C for 15 min andeasured again for the absorbance, as “B”. The difference in absorbance values (�

bsorbance = B − A) was calculated. The concentration of amoxicillin in the sampleas determined by comparing � absorbance which was obtained from the sampleeasurement with a calibration curve.

.3.2. Construction of calibration curveThe reference standard amoxicillin trihydrate of 33.6 mg was accurately

eighed, transferred into a 100-mL volumetric flask and diluted with water to pre-are 800 �M amoxicillin solution. Further dilutions were made by pipetting 1, 2, 3,and 5 mL of 800 �M solution into 10-mL volumetric flask and diluted with water

o the marks to obtain five stock standard solutions containing 80, 160, 240, 320nd 400 �M amoxicillin. The calibration curve was constructed by precisely pipet-ing stock solution in the volume of 250 �L into the 1-mL reaction and assays wereerformed as above. Then the results of � absorbance were plotted against themoxicillin concentration (20, 40, 60, 80 and 100 �M) and used to determine a lin-ar regression equation. The standard solution was freshly prepared and used withinh.

.3.3. Sample preparation for capsules and powder for oral suspension

The contents of 20 capsules were weighed and a quantity of capsule content

r powder for oral suspension was transferred into a volumetric flask, dissolvedn water, sonicated if necessary to ensure the complete dissolution and adjustedo volume with water. Further dilutions were made to the concentration of about00 �M and the solution was filtered through 0.45 �M-membrane filter, discardinghe first portion of the filtrate. In the analysis, 250 �L of filtered sample solution was

ial Technology 46 (2010) 292–296 293

used in a 1-mL reaction, so the final concentration of amoxicillin was about 50 �M,which was in the middle of the calibration curve.

2.4. Assay of amoxicillin by official HPLC method [2]

The United State Pharmacopoeia HPLC method was used as a reference methodto determine the quantity of amoxicillin in commercial samples in comparison withthe proposed method. Briefly, the aliquot of combined contents from 20 capsuleswas accurately weighed and diluted to the concentration of about 1 mg amoxicillinper mL.

After filtered through 0.45 �M-membrane filter, the sample solution wasinjected into a C18 column, using mobile phase consisting of monobasic potassiumphosphate solution, pH 5.0 and acetonitrile (96:4). Flow rate was 0.8 mL per min.The detector was set at 230 nm. For oral suspension, powder was reconstituted asdirected on the label and aliquot was diluted to the concentration of about 1 mgamoxicillin per mL. After filtered, the sample solution was analyzed using the sameprocedures as for capsules. The quantity of amoxicillin in the sample was calculatedby comparing the peak area obtained from the sample to that obtained from the ref-erence standard solution which was prepared at the concentration of about 1.2 mgamoxicillin per mL.

2.5. Method validation

2.5.1. LinearityThe linearity of the developed method was investigated by replicate analysis

(n = 6) at six concentration levels, i.e. 0, 20, 40, 60, 80 and 100 �M of reference stan-dard amoxicillin. The � absorbance obtained at concentration was plotted againstthe concentration of amoxicillin and the linear regression equation was evaluatedby statistical treatment of calibration data. The other regression characteristics werecalculated using Microsoft Excel 2003 software.

2.5.2. Limit of detection and quantitationLimit of detection (LOD) and limit of quantitation (LOQ) were calculated from

the residual standard deviation of regression line (�) of the calibration curve and itsslope (S) in accordance with the equation LOD = 3(�/S) and LOQ = 10(�/S).

2.5.3. PrecisionThe method repeatability was evaluated by spiking the sample solution pre-

pared from capsules or powder for oral suspension with lower, middle and higherconcentrations of the analytical curve (25, 50 and 75 �M amoxicillin). The addedconcentrations were analyzed in three independent series on the same day to estab-lish the intra-day precision. For intermediate or inter-day precision, samples wereprepared as above and analyzed in 3 consecutive days from triplicate measurementsof every sample in each series.

2.5.4. AccuracyAccuracy was calculated as the percentage recovery of a known amount of stan-

dard added to the sample according to standard addition procedures. Amoxicillinstandard (99.6% purity) solution was therefore added to sample solutions whichwere prepared from capsules or powder for oral suspension at three concentrationlevels 25, 50 and 75 �M and analyzed by the proposed method in triplicate.

2.5.5. SpecificityThe specificity of the method was evaluated by determining the concentration

of amoxicillin in the presence of pharmaceutical excipients, i.e. lactose, sucrose,starch, magnesium stearate and sodium benzoate as well as structurally relateddegradation products, i.e. 6-APA and d-penicillamine.

3. Results and discussion

3.1. Principle of the assay

The assay consisted of two enzymatic reactions (Scheme 1).Firstly, amoxicillin was hydrolyzed by PA to yield d-4-hydroxyphenylglycine and then d-PhgAT catalyzed thetransamination of d-4-hydroxyphenylglycine and 2-oxoglutarateto form l-glutamate and 4-hydroxybenzoylformate which stronglyabsorbed UV light at 335 nm (ε335 = 2.2 × 104 L mol−1 cm−1). Sinced-PhgAT did not accept d-4-hydroxyphenylglycine which attachedto 6-APA as a substrate, in a preliminary investigation, a chemicalhydrolysis was tested in the hydrolytic cleavage reaction in order

to save the cost of PA enzyme by treating amoxicillin with sodiumhydroxide. However, we found that the chemical hydrolysis ofamoxicillin did not only unreliably cleave the amide bond torelease d-4-hydroxyphenylglycine from 6-APA moiety but alsoopened the �-lactam ring yielding undesired products [13]. Hence,

294 T. Rojanarata et al. / Enzyme and Microbial Technology 46 (2010) 292–296

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Fig. 2. The effect of pH on the enzymatic reactions. The absorbance at 335 nm were

ig. 1. Absorption spectra of the assay solutions conducting at pH 9.0, before startingdash line) and after finishing (solid line) the reactions.

he greater selective enzymatic hydrolysis mediated by PA wasmployed for this purpose. Based on the end-point method, themount of amoxicillin was evaluated as a change in absorbance at35 nm after the reactions were complete. Although the hydrolysisf amoxicillin in the first step was not completely irreversible,e found that the equilibrium state was constant at about 80%

ubstrate conversion at the end of the reaction. From this definedquilibrium state, an appropriate factor may be used to derivectual initial substrate concentration. In another way, as performedy our study, a calibration curve for known standards which wasssayed under the same condition as samples could be employed.ig. 1 represents the spectra of the assay solutions before startingnd after finishing the reactions. It can be seen that while thenzymes and starting substrate components (e.g. amoxicillin and-oxoglutarate) show a very low background in the measuredavelength range, 4-hydroxybenzoylformate product has a high

bsorbance at the wavelengths which discriminate from those athich the initial reaction solution absorbs.

.2. Optimization of method variables

.2.1. Order of mixingSince the working conditions, e.g. pH and temperature for

oth PA and d-PhgAT enzymes are compatible, the two reactionsnvolved are not necessary to conduct stepwise and thus all assayomponents can be added together. However, in order to diminishhe possible interference from d-4-hydroxyphenylglycine impu-ity which may be formerly present in the samples [10], the assay

Scheme 1. Principle of the determination o

followed periodically from the assay reactions conducted at pH (�) 7.5, (�) 8.0, (�)8.5, (�) 9.0 and (×) 9.5 in 100 mM Tris buffer at 37 ◦C. The concentrations of PA andd-PhgAT used were 1 and 0.2 U mL−1, respectively.

was started by incubating the sample and d-PhgAT for 5 min inabsence of PA enzyme. The increase in absorbance at 335 nm in thisstep was a result of the contaminating d-4-hydroxyphenylglycinein the sample and this increment was used to subtract from theabsorbance obtained from the end of the reactions consisting ofboth enzymes. The subtraction of the initial absorbance before theaddition of second enzyme offered the further advantage because iteliminated the background absorption from the starting reactions.For PA, at the low amount used in the assay, it can be considered asnon-absorbing specie in the working wavelength range.

3.2.2. pHFrom the literatures, the activity of PA and d-PhgAT are favored

by alkaline pH [13,14]. Therefore, the assay reactions in 100 mMTris buffers of different pH (7.5–9.5) were tested and followed. Thetime-course of the reactions is shown in Fig. 2. The results revealedthat the higher rate of product formation was obtained when thepH raised, and the assays conducted at the pH above 9.0 reachedthe end point within 15 min. In addition, since the UV absorption of4-hydroxybenzoylformate at 335 nm increased with pH (data notshown), the measurement of its absorbance under alkaline condi-tions could enhance the sensitivity of the method. Therefore, we

deemed the optimal pH for this assay to be 9.0. Excessive alka-line condition may adversely affect the stability of enzymes [15,19]and/or cause the degradation of amoxicillin via undesirable sidereactions [20].

f amoxicillin by bienzymatic method.

T. Rojanarata et al. / Enzyme and Microbial Technology 46 (2010) 292–296 295

Fig. 3. The effect of 2-oxolglutarate concentration on the enzymatic reactions. Thetime-courses of reactions were followed from the assays conducted at differentc20

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Table 1Method validation.

Linearity (n = 3; 0–100 �M)Slope ± S.D. = 0.0185 ± 0.0001Intercept ± S.D. = 0.0131 ± 0.0047r2 = 0.9998

Limit of detectionLOD = 0.77 �M

Limit of quantitationLOQ = 2.55 �M

Precision (L = 25, M = 50, H = 75 �M)Intra-day (n = 3)

R.S.D. tested in capsules = 2.60% (L), 1.13% (M), 1.08 (H)R.S.D. tested in oral suspension = 2.53% (L), 1.24% (M), 0.82 (H)

Inter-day (n = 3)R.S.D. tested in capsules = 3.30% (L), 2.13% (M), 1.14 (H)R.S.D. tested in oral suspension = 1.58% (L), 1.47% (M), 0.85 (H)

Accuracy (L = 25, M = 50, H = 75 �M, n = 6)Tested in capsules

24.52 ± 0.58 (% recovery = 98.07 ± 2.32%) (L)49.51 ± 0.92 (% recovery = 99.07 ± 1.85%) (M)74.65 ± 1.09 (% recovery = 99.53 ± 1.46%) (H)

Tested in oral suspension24.78 ± 0.93 (% recovery = 99.12 ± 3.73%) (L)

oncentrations of amoxicillin (25, 75, 100 �M) and 2-oxoglutarate; (—–) 100, (– –)00, (— ·) 300 �M at 37 ◦C. The concentrations of PA and d-PhgAT used were 1 and.2 U mL−1, respectively.

.2.3. Concentration of 2-oxoglutarateThe 2-oxoglutarate acts as a co-substrate in the d-PhgAT-

atalyzed reaction. So, it should be added in an excess concentrationo push the equilibrium. However, it must not result in a substrate-nhibition towards the enzyme activity. When we tested the effectf the 2-oxoglutarate concentration, it was found that 200 �M-oxoglutarate was sufficient for the reaction containing initial00 �M amoxicillin and did not cause any deteriorate effects on thenzyme activity (Fig. 3). Higher concentration of 2-oxoglutarate didot significantly help drive the equilibrium of the transaminationowards 4-hydroxybenzoylformate and was therefore consideredasteful.

.2.4. Concentration of enzymesThe optimal concentrations of the two enzymes for the assay

ere investigated by conducting a 1-mL reaction mixture con-aining 100 �M amoxicillin and 200 �M 2-oxoglutarate. Under thisondition, using PA and d-PhgAT at the concentrations of 1 and.2 U mL−1, respectively, resulted in a satisfactory reaction rate, andhe end point of the assay could be obtained within 15 min (dataot shown).

.3. Analytical method validation

Validation of an analytical procedure is the prerequisite processy which it is established, by laboratory studies, that the perfor-ance characteristics of the method meet the requirements for the

ntended analytical application.

.3.1. Linearity, LOD and LOQIn developed enzymatic method, a calibration curve was

btained using six different concentrations of amoxicillin. Eachoncentration was analyzed six times. The results, summarizedn Table 1, showed an excellent linearity (r2 = 0.9998) between �bsorbance (y) and initial amoxicillin concentration in �M (x) over

he concentration range of 0–100 �M. LOD and LOQ were 0.77 and.55 �M, respectively. Compared with the USP chromatographicethods in which the sample solution was prepared at the con-

entration of 2.75 mM for the analysis, the proposed showed muchigher sensitivity. In addition, it was more sensitive than former

49.39 ± 0.77 (% recovery = 99.77 ± 1.55%) (M)74.73 ± 0.95 (% recovery = 99.64 ± 1.27%) (H)

S.D. = standard deviation; R.S.D. = relative standard deviation; L, M, H = low, middle,high level of spiked amoxicillin standard.

spectrophotometric methods which were based on chemical reac-tion with amoxicillin [8,9].

3.3.2. PrecisionThree different concentrations of amoxicillin (25, 50 and 75 �M)

in capsule and oral suspension samples were analyzed in threeindependent series on the same days (intra-day precision) and 3consecutive days (inter-day precision) from triplicate measure-ments of every sample in each series. As shown in Table 1, the%R.S.D. values varied from 0.82 to 2.60 for intra-day and from 0.85to 3.30 for inter-day precision. Intra-day precision was better thaninter-day precision as expressed in the lower %R.S.D. values. Inoverall, the precision parameters were considered satisfactory.

3.3.3. AccuracyThe accuracy of the proposed method was also examined by

performing recovery experiments through the standard additionmethod. The sample solutions were spiked with three levels of ref-erence standard amoxicillin to the final added concentration of 25,50 and 75 �M. Table 1 illustrates the method accuracy with recov-eries from 98.07 to 99.53% for capsules and 99.12–99.77% for oralsuspensions. The low bias values (S.D.) and high recovery percent-ages indicated that the method was highly accurate and relativelyindependent on other excipients present in the samples.

3.3.4. SpecificityOne prominent advantage of enzymatic assay is its high

specificity and the analysis may be done by skipping tedious pre-separation steps of the analyte from the sample matrix. Particularly,in this case, the enzyme d-PhgAT has been reported to have ahigh selectivity ford-4-hydroxyphenylglycine andd-phenylglycinesubstrates which are not usually found in the nature [14]. In theinterference study performed by adding pharmaceutical excipi-ents and diluents that are commonly formulated in dosage forms,

i.e. lactose, sucrose, starch, magnesium stearate and sodium ben-zoate, no interference was observed. Also, other structurally relateddegradation products, 6-APA and d-penicillamine, did not interferewith the proposed method. For the possible interference from d-4-hydroxyphenylglycine, this could be eliminated by subtracting

296 T. Rojanarata et al. / Enzyme and Microbial Technology 46 (2010) 292–296

Table 2Amoxicillin determination in pharmaceutical preparations by proposed and official methods.

Preparation Brand Claimed (mg)a Proposed method Official method

% LA S.D. % LA S.D.

Capsules Brand 1 250 97.26 1.51 96.80 0.24Brand 2 250 101.25 1.44 100.18 0.15Brand 3 500 101.36 1.46 101.11 0.10Brand 4 500 101.59 0.93 101.20 0.05

Powder for oral suspension Brand 5 125 98.01 0.97 98.86 0.20

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LA: percentage labeled amount (expressed as mean) and S.D.: standard deviationa Claimed amount is labeled amount in mg of amoxicillin per capsule or mg of am

he absorbance obtained after incubating the sample and d-PhgATor 10 min in absence of the PA from the absorbance obtained athe end of the reactions consisting of both enzymes. However,hroughout our study we found that average background due to D--hydroxyphenylglycine was as minimal as 0.03 absorbance scale.

.4. Assay of commercial pharmaceutical preparations

The applicability of the method to the real samples were evalu-ted by determining the percentage labeled amount of amoxicillinn commercial dosage forms including capsules and powder for oraluspension available in Thailand. The results of the analysis by thenzymatic method were in good agreement with those obtainedy the pharmacopoeial HPLC method (Table 2). This reveals thathe proposed method can be applied for the assay of amoxicillinreparations without interference.

. Conclusion

In this study, a bienzymatic UV-spectrophotometric method forhe determination of amoxicillin in pharmaceutical preparationsas been developed, validated and evaluated with commercial sam-les in comparison with the official method. We found that ourroposed method is simpler and more sensitive than the officialhromatographic method and requires no expensive instruments,xcept spectrophotometers which are commonly available in allaboratories. The considerably low limit of detection also convincess to further develop the assay with an improved sensitivity whichay be applied for the determination of drug in biological speci-ens or residues in food samples. However, the specificity of theethod needs to be verified with such complex samples. Never-

heless, the assay was proven in this work to be free from thenterference such as common pharmaceutical excipients and most

ajor related degradation products. Since the total proceduresncluding the incubation take less than 30 min, this method is par-icularly appropriate for rapid assay of large number of samplesnd may be applicable to multi-assay using autoanalyzer. Finally,ince few reagents are required and the procedures are entirely per-ormed without the use of any organic solvents or harsh chemicalshich are employed in the other methods, this enzymatic assay is

n ideal green analytical method suitable for the quality control ofmoxicillin in pharmaceuticals.

cknowledgements

The authors wish to thank Commission of Higher EducationThailand) and the Thailand Research Funds for the financial sup-ort through the grant MRG5080333.

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