Enzyme and Microbial Technology 46 (2010) 292296

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Enzyme and Microbial Technology

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A simple, sensitive and green bienzymatic UV-spectrophotometric assay of amoxicillin formulations

Theerasak Rojanarata a, , Praneet Opanasopit a , Tanasait Ngawhirunpat a , Choedchai Saehuan b , Suthep Wiyakrutta c , Vithaya Meevootisom c
a b c

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

a r t i c l e

i n f o

a b s t r a c t
A simple, fast, sensitive and inexpensive UV-spectrophotometric method for the determination of amoxicillin in pharmaceutical preparations has been developed based on two enzymatic reactions. In this method, d-4-hydroxyphenylglycine side chain of amoxicillin was selectively cleaved off by penicillin acylase. Subsequently, it was reacted with 2-oxoglutarate, by the catalysis of d-phenylglycine aminotransferase, to yield the product with high UV absorption namely 4-hydroxybenzoylformate. The amount of amoxicillin was then determined as a change in absorbance at 335 nm. In this work, the assay conditions were studied and optimized and the method was validated. The calibration curve presented an excellent linearity with r2 of 0.9998 (0100 M amoxicillin). Detection and quantitation limits were 0.77 and 2.55 M, respectively. Good accuracy and precision were obtained when the method was tested with amoxicillin capsules and powder for oral suspension. No interference from common excipients in the formulations or degradation products was observed. Finally, since all procedures were performed without the use of any organic solvents or hazardous chemicals which were detrimental to the environment and had a low consumption of reagents, this proposed assay was an ideal green analytical method suitable for the quality control of amoxicillin in pharmaceuticals. 2009 Elsevier Inc. All rights reserved.

Article history: Received 11 September 2009 Received in revised form 12 November 2009 Accepted 22 November 2009 Keywords: Enzymatic method Amoxicillin Spectrophotometry

1. Introduction Amoxicillin is a semi-synthetic -lactam antibiotic belonging to the group of penicillins. The chemical structure of amoxicillin consists of d-4-hydroxyphenylglycine side chain attached to 6-aminopenicillanic acid (6-APA) moiety. Because of its broad spectrum of bactericidal activity and therefore widespread use in medicines, various preparations of this drug alone including capsules, tablets, powder for oral suspension, and injections as well as in combination with other ingredients, e.g. amoxicillin/clavulanate tablets are commercially available [1]. Currently, several analytical methods for the quantitation of amoxicillin in pharmaceutical formulations have been reported. Examples of these methods are iodometric titration [2], uorometry [3], chemiluminescence [4,5], voltammetry [6], spectrophotometry [79] and high-performance liquid chromatography (HPLC) [2,10,11]. Though the latter method is specied in the pharmacopoeias, it requires instruments with high 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). 0141-0229/$ see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2009.11.011

some chromatographic systems use organic solvents as mobile phases which are on the issues of environmental concern or a worldwide shortage crisis such as acetonitrile. Enzymatic assay is an alternative for the drug analysis. The high specicity of enzymes can be used to determine precisely concentrations of compounds even in a complex matrix by employing techniques, e.g. spectrophotometry to monitor the course of enzyme-catalyzed reaction upon the compound is converted to the measurable product [12]. Furthermore, enzymatic methods are ideally suited to green chemistry because most enzymes work efciently under mild conditions and often use no undesirable chemicals or solvents. In the present paper, we report the development and validation of a new UV-spectrophotometric method for the determination of amoxicillin in capsules and powder for oral suspension by the use of two enzymes. One enzyme is penicillin acylase (PA; EC 3.5.1.11) from Escherichia coli which is commercially available. The enzyme has been used in pharmaceutical industry for the hydrolytic cleavage of the side chains of penicillin G or V to obtain 6-APA, a precursor of various semi-synthetic penicillins [13]. The other enzyme is dphenylglycine aminotransferase (d-PhgAT; EC 2.6.1.72) puried from recombinant E. coli expressing the cloned gene encoding the d-PhgAT from Pseudomonas stutzeri ST-201. It catalyzes a

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reversible stereo-inverting transamination of d-phenylglycine or d-4-hydroxyphenylglycine and 2-oxoglutarate to yield benzoylformate or 4-hydroxybenzoylformate and l-glutamate [14]. Currently, d-PhgAT has been applied for the synthesis of optically pure dphenylglycine [15] and the analysis of l-glutamate in foods [16,17]. Due to a high substrate specicity of the enzymes, an intense UV absorption property of formed product as well as simple and easy measuring procedures, this proposed method is very sensitive, rapid and convenient and does not require any complicated or expensive instruments and environmentally detrimental reagents.
2. Experimental 2.1. Instrumentation The spectra and absorbance at 335 nm were measured by an Agilent G1103A model UVvisible spectrophotometer (Agilent, USA) using a semi-micro quartz cuvette type (1 cm path length). The HPLC system consisted of an Agilent 1100Serie and a diode array detector (Agilent, USA). The column used was reversed phase VertiSepTM GES ODS column, 5 m, 150 mm 4.5 mm (Verical Chromatography, Bangkok, Thailand). 2.2. Reagents and chemicals Reference standard amoxicillin trihydrate was purchased from Laboratory of Dr. Ehrenstofer Company (Augsburg, Germany) with the purity of 99.6%, calculated on the anhydrous basis, determined by HPLC method. The enzyme PA from E. coli was obtained as a commercial reagent from Sigma Aldrich (St. Louis, MO). It was supplied as suspension in 3 M ammonium sulfate and phosphate buffer. d-PhgAT was puried from a recombinant E. coli expressing the cloned gene encoding the d-PhgAT from P. stutzeri ST201 as previously described [18] and kept in phosphate buffer containing 25% glycerol for a long term use. Both enzymes were freshly diluted with water to desired activity before use. Double distilled water was used to prepare all solutions throughout the experiments. All other chemicals were of the analytical grade from Sigma Aldrich (St. Louis, MO) The commercial capsules and powder for oral suspension were prepared from Amoxicillin trihydrate USP by manufacturers in Thailand. Their expiration dates were at least 30 months beyond the date of analysis in this study. 2.3. Assay of amoxicillin by bienzymatic UV-spectrohotometric method 2.3.1. Analytical procedure In order to minimize pipetting steps and increase both precision and accuracy, the reaction cocktail was prepared containing 135 mM Tris buffer (pH 9.0), 135 M pyridoxal-5 -phosphate, 270 M 2-oxoglutarate, 0.27 U mL1 d-PhgAT enzyme and water. The assay, conducted in a total volume of 1 mL, was started with the preincubation of 250 L standard or sample solution and 745 L reaction cocktail at 37 C so that d-4-hydroxyphenylglycine impurity which may be present with amoxicillin was converted to 4-hydroxybenzoylformate. After 5 min, the absorbance at 335 nm of the solution was read, designated as A. Subsequently, 5 L of the enzyme PA was added into the reaction at the nal concentration of 1 U mL1 . The reaction solution containing both enzymes was further incubated at 37 C for 15 min and measured again for the absorbance, as B. The difference in absorbance values ( absorbance = B A) was calculated. The concentration of amoxicillin in the sample was determined by comparing absorbance which was obtained from the sample measurement with a calibration curve. 2.3.2. Construction of calibration curve The reference standard amoxicillin trihydrate of 33.6 mg was accurately weighed, transferred into a 100-mL volumetric ask and diluted with water to prepare 800 M amoxicillin solution. Further dilutions were made by pipetting 1, 2, 3, 4 and 5 mL of 800 M solution into 10-mL volumetric ask and diluted with water to the marks to obtain ve stock standard solutions containing 80, 160, 240, 320 and 400 M amoxicillin. The calibration curve was constructed by precisely pipetting stock solution in the volume of 250 L into the 1-mL reaction and assays were performed as above. Then the results of absorbance were plotted against the amoxicillin concentration (20, 40, 60, 80 and 100 M) and used to determine a linear regression equation. The standard solution was freshly prepared and used within 6 h. 2.3.3. Sample preparation for capsules and powder for oral suspension The contents of 20 capsules were weighed and a quantity of capsule content or powder for oral suspension was transferred into a volumetric ask, dissolved in water, sonicated if necessary to ensure the complete dissolution and adjusted to volume with water. Further dilutions were made to the concentration of about 200 M and the solution was ltered through 0.45 M-membrane lter, discarding the rst portion of the ltrate. In the analysis, 250 L of ltered sample solution was

used in a 1-mL reaction, so the nal concentration of amoxicillin was about 50 M, which was in the middle of the calibration curve. 2.4. Assay of amoxicillin by ofcial HPLC method [2] The United State Pharmacopoeia HPLC method was used as a reference method to determine the quantity of amoxicillin in commercial samples in comparison with the proposed method. Briey, the aliquot of combined contents from 20 capsules was accurately weighed and diluted to the concentration of about 1 mg amoxicillin per mL. After ltered through 0.45 M-membrane lter, the sample solution was injected into a C18 column, using mobile phase consisting of monobasic potassium phosphate 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 as directed on the label and aliquot was diluted to the concentration of about 1 mg amoxicillin per mL. After ltered, the sample solution was analyzed using the same procedures as for capsules. The quantity of amoxicillin in the sample was calculated by comparing the peak area obtained from the sample to that obtained from the reference standard solution which was prepared at the concentration of about 1.2 mg amoxicillin per mL. 2.5. Method validation 2.5.1. Linearity The 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 standard amoxicillin. The absorbance obtained at concentration was plotted against the concentration of amoxicillin and the linear regression equation was evaluated by statistical treatment of calibration data. The other regression characteristics were calculated using Microsoft Excel 2003 software. 2.5.2. Limit of detection and quantitation Limit of detection (LOD) and limit of quantitation (LOQ) were calculated from the residual standard deviation of regression line ( ) of the calibration curve and its slope (S) in accordance with the equation LOD = 3( /S) and LOQ = 10( /S). 2.5.3. Precision The method repeatability was evaluated by spiking the sample solution prepared from capsules or powder for oral suspension with lower, middle and higher concentrations of the analytical curve (25, 50 and 75 M amoxicillin). The added concentrations were analyzed in three independent series on the same day to establish the intra-day precision. For intermediate or inter-day precision, samples were prepared as above and analyzed in 3 consecutive days from triplicate measurements of every sample in each series. 2.5.4. Accuracy Accuracy was calculated as the percentage recovery of a known amount of standard added to the sample according to standard addition procedures. Amoxicillin standard (99.6% purity) solution was therefore added to sample solutions which were prepared from capsules or powder for oral suspension at three concentration levels 25, 50 and 75 M and analyzed by the proposed method in triplicate. 2.5.5. Specicity The specicity 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 related degradation 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-4hydroxyphenylglycine and then d-PhgAT catalyzed the transamination of d-4-hydroxyphenylglycine and 2-oxoglutarate to form l-glutamate and 4-hydroxybenzoylformate which strongly absorbed UV light at 335 nm (335 = 2.2 104 L mol1 cm1 ). Since d-PhgAT did not accept d-4-hydroxyphenylglycine which attached to 6-APA as a substrate, in a preliminary investigation, a chemical hydrolysis was tested in the hydrolytic cleavage reaction in order to save the cost of PA enzyme by treating amoxicillin with sodium hydroxide. However, we found that the chemical hydrolysis of amoxicillin did not only unreliably cleave the amide bond to release d-4-hydroxyphenylglycine from 6-APA moiety but also opened the -lactam ring yielding undesired products [13]. Hence,

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Fig. 1. Absorption spectra of the assay solutions conducting at pH 9.0, before starting (dash line) and after nishing (solid line) the reactions.

Fig. 2. The effect of pH on the enzymatic reactions. The absorbance at 335 nm were 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 and d-PhgAT used were 1 and 0.2 U mL1 , respectively.

the greater selective enzymatic hydrolysis mediated by PA was employed for this purpose. Based on the end-point method, the amount of amoxicillin was evaluated as a change in absorbance at 335 nm after the reactions were complete. Although the hydrolysis of amoxicillin in the rst step was not completely irreversible, we found that the equilibrium state was constant at about 80% substrate conversion at the end of the reaction. From this dened equilibrium state, an appropriate factor may be used to derive actual initial substrate concentration. In another way, as performed by our study, a calibration curve for known standards which was assayed under the same condition as samples could be employed. Fig. 1 represents the spectra of the assay solutions before starting and after nishing the reactions. It can be seen that while the enzymes and starting substrate components (e.g. amoxicillin and 2-oxoglutarate) show a very low background in the measured wavelength range, 4-hydroxybenzoylformate product has a high absorbance at the wavelengths which discriminate from those at which the initial reaction solution absorbs. 3.2. Optimization of method variables 3.2.1. Order of mixing Since the working conditions, e.g. pH and temperature for both PA and d-PhgAT enzymes are compatible, the two reactions involved are not necessary to conduct stepwise and thus all assay components can be added together. However, in order to diminish the possible interference from d-4-hydroxyphenylglycine impurity which may be formerly present in the samples [10], the assay

was started by incubating the sample and d-PhgAT for 5 min in absence of PA enzyme. The increase in absorbance at 335 nm in this step was a result of the contaminating d-4-hydroxyphenylglycine in the sample and this increment was used to subtract from the absorbance obtained from the end of the reactions consisting of both enzymes. The subtraction of the initial absorbance before the addition of second enzyme offered the further advantage because it eliminated the background absorption from the starting reactions. For PA, at the low amount used in the assay, it can be considered as non-absorbing specie in the working wavelength range. 3.2.2. pH From the literatures, the activity of PA and d-PhgAT are favored by alkaline pH [13,14]. Therefore, the assay reactions in 100 mM Tris buffers of different pH (7.59.5) were tested and followed. The time-course of the reactions is shown in Fig. 2. The results revealed that the higher rate of product formation was obtained when the pH raised, and the assays conducted at the pH above 9.0 reached the end point within 15 min. In addition, since the UV absorption of 4-hydroxybenzoylformate at 335 nm increased with pH (data not shown), the measurement of its absorbance under alkaline conditions could enhance the sensitivity of the method. Therefore, we deemed the optimal pH for this assay to be 9.0. Excessive alkaline condition may adversely affect the stability of enzymes [15,19] and/or cause the degradation of amoxicillin via undesirable side reactions [20].

Scheme 1. Principle of the determination of amoxicillin by bienzymatic method.

T. Rojanarata et al. / Enzyme and Microbial Technology 46 (2010) 292296 Table 1 Method validation. Linearity (n = 3; 0100 M) Slope S.D. = 0.0185 0.0001 Intercept S.D. = 0.0131 0.0047 r2 = 0.9998 Limit of detection LOD = 0.77 M Limit of quantitation LOQ = 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 suspension 24.78 0.93 (% recovery = 99.12 3.73%) (L) 49.39 0.77 (% recovery = 99.77 1.55%) (M) 74.73 0.95 (% recovery = 99.64 1.27%) (H)

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Fig. 3. The effect of 2-oxolglutarate concentration on the enzymatic reactions. The time-courses of reactions were followed from the assays conducted at different concentrations of amoxicillin (25, 75, 100 M) and 2-oxoglutarate; () 100, ( ) 200, ( ) 300 M at 37 C. The concentrations of PA and d-PhgAT used were 1 and 0.2 U mL1 , respectively.

3.2.3. Concentration of 2-oxoglutarate The 2-oxoglutarate acts as a co-substrate in the d-PhgATcatalyzed reaction. So, it should be added in an excess concentration to push the equilibrium. However, it must not result in a substrateinhibition towards the enzyme activity. When we tested the effect of the 2-oxoglutarate concentration, it was found that 200 M 2-oxoglutarate was sufcient for the reaction containing initial 100 M amoxicillin and did not cause any deteriorate effects on the enzyme activity (Fig. 3). Higher concentration of 2-oxoglutarate did not signicantly help drive the equilibrium of the transamination towards 4-hydroxybenzoylformate and was therefore considered wasteful. 3.2.4. Concentration of enzymes The optimal concentrations of the two enzymes for the assay were investigated by conducting a 1-mL reaction mixture containing 100 M amoxicillin and 200 M 2-oxoglutarate. Under this condition, using PA and d-PhgAT at the concentrations of 1 and 0.2 U mL1 , respectively, resulted in a satisfactory reaction rate, and the end point of the assay could be obtained within 15 min (data not shown). 3.3. Analytical method validation Validation of an analytical procedure is the prerequisite process by which it is established, by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application. 3.3.1. Linearity, LOD and LOQ In developed enzymatic method, a calibration curve was obtained using six different concentrations of amoxicillin. Each concentration was analyzed six times. The results, summarized in Table 1, showed an excellent linearity (r2 = 0.9998) between absorbance (y) and initial amoxicillin concentration in M (x) over the concentration range of 0100 M. LOD and LOQ were 0.77 and 2.55 M, respectively. Compared with the USP chromatographic methods in which the sample solution was prepared at the concentration of 2.75 mM for the analysis, the proposed showed much higher sensitivity. In addition, it was more sensitive than former

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 reaction with amoxicillin [8,9]. 3.3.2. Precision Three different concentrations of amoxicillin (25, 50 and 75 M) in capsule and oral suspension samples were analyzed in three independent series on the same days (intra-day precision) and 3 consecutive days (inter-day precision) from triplicate measurements 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 0.85 to 3.30 for inter-day precision. Intra-day precision was better than inter-day precision as expressed in the lower %R.S.D. values. In overall, the precision parameters were considered satisfactory. 3.3.3. Accuracy The accuracy of the proposed method was also examined by performing recovery experiments through the standard addition method. The sample solutions were spiked with three levels of reference standard amoxicillin to the nal added concentration of 25, 50 and 75 M. Table 1 illustrates the method accuracy with recoveries from 98.07 to 99.53% for capsules and 99.1299.77% for oral suspensions. The low bias values (S.D.) and high recovery percentages indicated that the method was highly accurate and relatively independent on other excipients present in the samples. 3.3.4. Specicity One prominent advantage of enzymatic assay is its high specicity and the analysis may be done by skipping tedious preseparation steps of the analyte from the sample matrix. Particularly, in this case, the enzyme d-PhgAT has been reported to have a high selectivity for d-4-hydroxyphenylglycine and d-phenylglycine substrates which are not usually found in the nature [14]. In the interference study performed by adding pharmaceutical excipients and diluents that are commonly formulated in dosage forms, i.e. lactose, sucrose, starch, magnesium stearate and sodium benzoate, no interference was observed. Also, other structurally related degradation products, 6-APA and d-penicillamine, did not interfere with the proposed method. For the possible interference from d4-hydroxyphenylglycine, this could be eliminated by subtracting

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Table 2 Amoxicillin determination in pharmaceutical preparations by proposed and ofcial methods. Preparation Brand Claimed (mg)a Proposed method % LA Capsules Brand 1 Brand 2 Brand 3 Brand 4 Brand 5 Brand 6 250 250 500 500 125 250 97.26 101.25 101.36 101.59 98.01 99.12 S.D. 1.51 1.44 1.46 0.93 0.97 0.72 Ofcial method % LA 96.80 100.18 101.11 101.20 98.86 101.19 S.D. 0.24 0.15 0.10 0.05 0.20 0.15

Powder for oral suspension

% LA: percentage labeled amount (expressed as mean) and S.D.: standard deviation calculated from six replication. a Claimed amount is labeled amount in mg of amoxicillin per capsule or mg of amoxicillin per 5 mL of reconstituted oral suspension.

the absorbance obtained after incubating the sample and d-PhgAT for 10 min in absence of the PA from the absorbance obtained at the end of the reactions consisting of both enzymes. However, throughout our study we found that average background due to D4-hydroxyphenylglycine was as minimal as 0.03 absorbance scale. 3.4. Assay of commercial pharmaceutical preparations The applicability of the method to the real samples were evaluated by determining the percentage labeled amount of amoxicillin in commercial dosage forms including capsules and powder for oral suspension available in Thailand. The results of the analysis by the enzymatic method were in good agreement with those obtained by the pharmacopoeial HPLC method (Table 2). This reveals that the proposed method can be applied for the assay of amoxicillin preparations without interference. 4. Conclusion In this study, a bienzymatic UV-spectrophotometric method for the determination of amoxicillin in pharmaceutical preparations has been developed, validated and evaluated with commercial samples in comparison with the ofcial method. We found that our proposed method is simpler and more sensitive than the ofcial chromatographic method and requires no expensive instruments, except spectrophotometers which are commonly available in all laboratories. The considerably low limit of detection also convinces us to further develop the assay with an improved sensitivity which may be applied for the determination of drug in biological specimens or residues in food samples. However, the specicity of the method needs to be veried with such complex samples. Nevertheless, the assay was proven in this work to be free from the interference such as common pharmaceutical excipients and most major related degradation products. Since the total procedures including the incubation take less than 30 min, this method is particularly appropriate for rapid assay of large number of samples and may be applicable to multi-assay using autoanalyzer. Finally, since few reagents are required and the procedures are entirely performed without the use of any organic solvents or harsh chemicals which are employed in the other methods, this enzymatic assay is an ideal green analytical method suitable for the quality control of amoxicillin in pharmaceuticals. Acknowledgements The authors wish to thank Commission of Higher Education (Thailand) and the Thailand Research Funds for the nancial support through the grant MRG5080333.

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