Analytical Methods PAPER

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1 Analytical Methods PAPER Cite this: Anal. Methods, 2014,6, 1523 Direct determination of ampicillin and amoxicillin residues in food samples after aqueous SDS extraction by micellar liquid chromatography with UV detection Fawzia A. Ibrahim and Jenny Jeehan M. Nasr* Received 12th November 2013 Accepted 24th December 2013 DOI: /c3ay42011f A simple, sensitive and rapid micellar HPLC method was optimized and validated for the analysis of amoxicillin and ampicillin residues in food samples. Analytical separation was performed in less than 7 min using a RP C 18 column with UV detection at 220 nm and a micellar solution of 0.05 M sodium dodecyl sulphate, 5% 1-propanol and 0.3% triethylamine in 0.02 M phosphoric acid buffered at ph 5 as the mobile phase. The flow rate was 1 ml min 1 and the effluent was monitored at 220 nm. The micellar method was successfully applied to quantitatively determine amoxicillin and ampicillin residues in spiked chicken muscles, chicken liver, bovine muscles, liver, kidney and eggs. The method was fully validated in accordance with ICH guidelines. Linearity was in the range mg ml 1 for each drug and the percentage recoveries of both drugs ranged from 95.5 to 102.3% for amoxicillin and 95.6 to 101.7% for ampicillin. High extraction efficiency of amoxicillin and ampicillin was obtained without matrix interference in the extraction process and in the subsequent chromatographic determination. An aqueous solution of SDS surfactant only was used in extraction. No organic solvent was used during the pretreatment step. Hence, it is considered an interesting technique for green chemistry. 1. Introduction Ampicillin ((2S,5R,6R)-6-{[(2R)-2-amino-2-phenylacetyl]amino}- 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid) (AMP) and amoxicillin ((2S,5R,6R)-6-{[(2R)-2-amino-2-(4- hydroxyphenyl)-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid) (AMX) 1,2 are two broadspectrum b-lactam antibiotics (Fig. 1). They are used in veterinary medicine for the treatment and prevention of primary respiratory, gastrointestinal, urogenital, and skin bacterial infections in food-producing animals. 3 They are widely used because of their broad spectrum and low cost. 4 This widespread use can result in the presence of residues in food that can lead to health problems for individuals who are hypersensitive to penicillins. 3 Even more important, low-level doses of antibiotics in foodstuff for long periods may lead to the emergence of penicillin-resistant bacterial strains. 4 To ensure human food safety, the European Union (EU) has set maximal residue limits (MRLs) for penicillins in tissues and eggs. The EU has established a maximum residue limit of 50 mg kg 1 of AMP and AMX in animal tissues and 4 mg kg 1 in milk. 5 The determination of antimicrobial drugs is mainly carried out using microbiological techniques. 6,7 These techniques are very sensitive but not speci c because they cannot distinguish among different b-lactam antibiotics. Also, the relatively long time required for analysis by such techniques is a disadvantage. 8 A number of analytical methods in addition to microbiological assay have been reported for the determination of these compounds in food samples which include HPLC with uorescence detection using precolumn derivatization, 4,9 HPLC with UV detection, 10,11 and HPLC with photodiode array detection a er mixed micelle-cloud point extraction 12 or liquid chromatography-mass spectrometry (LC-MS). 3 Ampicillin was determined also in powder for injection by Fourier-transform infrared spectroscopy (FT-IR) 13 and by turbidimetry. 14 Micellar liquid chromatography (MLC) allows complex matrices to be analyzed without the aid of extraction and with direct injection of samples. 15 Micelles tend to bind proteins competitively, thereby releasing protein-bound drugs and Faculty of Pharmacy, Department of Analytical Chemistry, University of Mansoura, Mansoura 35516, Egypt. nasrjj@yahoo.com; Fax: ; Tel: Fig. 1 Chemical structures of the drugs investigated. This journal is The Royal Society of Chemistry 2014 Anal. Methods, 2014,6,

2 Analytical Methods proteins, rather than precipitating into the column. Proteins are solubilized and washed harmlessly away, eluting with the solvent front. This means that costs and analysis times are cut considerably. 16 Micellar mobile phases usually need less quantity of organic modi er and generate less amount of toxic waste in comparison with aqueous-organic solvents, so that they are less toxic, non- ammable, biodegradable and relatively inexpensive. 17 Because of these advantages, MLC is considered an interesting technique for green chemistry that copes with current concern about the environment. 18 It proved to be a useful technique in the determination of diverse groups of compounds in several matrices, including food samples. 22,23 MLC was used for the determination of some penicillins in pharmaceutical formulations 8 and in urine samples. 24 The present study describes a rapid, simple, selective and green MLC method with UV detection for the simultaneous determination of AMP and AMX residues in chicken muscles, chicken liver, bovine meat, liver, kidney and eggs. The proposed procedure bene ts from the two main advantages associated with the use of micellar mobile phases, namely, the use of environment-friendly eluents and fast and easy sample preparation. 2. Experimental 2.1. Apparatus Chromatographic analyses were carried out using a Shimadzu Prominence HPLC system (Shimadzu Corporation, Japan) with a LC-20 AD pump, DGU-20 A5 degasser, CBM-20A interface, and SPD-20A UV-VIS detector with a 20 ml injection loop. The columns used were Supelco Discovery HS C18 column (250 mm 4.6 mm i.d., 5 mm particle size), Supelco, Pennsylvania, USA and Nucleodur MN-C18 column (150 mm 4.6 mm i.d., 5 mm particle size), Macherey-Nagel, Düren, Germany. Centrifugation was carried out using a TDL-60B Centrifuge (Anke, Taiwan). A BHA-180T Sonicator (Abbotta Corporation, USA) was used. Tissue homogenization was carried out using a Tissue Master- 125 Homogenizer (Omni International, Georgia, USA) Reagents and materials All reagents and solvents used were of HPLC grade. AMP and AMX raw materials were kindly provided by the Arab Company for Gelatin and Pharmaceutical Products (Alexandria, Egypt), their purities were 99.7%. Tinidazole (TNZ) was kindly provided by Sigma Pharmaceutical Company, Cairo, Egypt, its purity was %. Methanol, 1-propanol, acetonitrile and sodium dodecyl sulphate (SDS) were from Sigma-Aldrich (Seelze, Germany). Triethylamine and phosphoric acid were from RiedeldeHaën (Seelze, Germany). Regenerated cellulose membrane lters and syringe lters (Minisart RC25) with pore size 0.45 mm were from Sartorius-Stedim (Goettingen, Germany). Chicken muscles, chicken liver, bovine meat, liver, kidney samples and eggs were purchased from the local market Preparation of stock and standard solutions Stock solutions of 0.4 mg ml 1 of each AMX and AMP were prepared by dissolving in water. These stock solutions were freshly prepared each time and stored below 4 C and protected from light. Working solutions were prepared by diluting the stock solutions with the mobile phase just before performing the analysis General recommended procedures Construction of calibration curves. Working solutions containing mg ml 1 of each of AMP and AMX were prepared by serial dilutions of aliquots of the stock solutions together with an aliquot of internal standard solution containing 10 mg ml 1 of TNZ. 20 ml aliquots were injected (triplicate) and eluted with the mobile phase under the reported chromatographic conditions. The average peak area ratios between the internal standard and each drug were plotted versus the concentrations of each drug in mg ml 1. Alternatively, the corresponding regression equations were derived Analysis of the bulk substance. The method mentioned under the previous section was applied to the determination of the purity of AMX and AMP raw materials. The percentage recoveries were calculated by referring to the calibration graphs previously prepared or by applying the regression equations Sample preparation. 2.5 g of each sample of chicken muscles, chicken liver, bovine muscles, bovine liver, bovine kidney and eggs were accurately weighed and spiked with aliquots of AMX, AMP and TNZ solutions. The spiked samples were mixed with 25 ml of 0.05 M SDS solution of ph 5. The solid samples were then homogenized at 5000 rpm for 5 min, then, the homogenate was sonicated for 15 min, and then centrifuged at 3000 rpm for 5 min. The egg samples were not homogenized, but they were only sonicated for 2 min without centrifugation. The supernatant of all samples was ltered through 0.45 mm membrane lters using a vacuum pump and aliquots of 20 ml were injected (triplicate) and eluted with the mobile phase under the reported chromatographic conditions. 3. Results and discussion Paper The proposed method permits the quantitation of AMX and AMP in bulk drug materials, in chicken muscles, chicken liver, bovine meat, liver, kidney and eggs. Fig. 2A shows a chromatogram indicating good resolution of AMX (t R ¼ 3.2 min) and AMP (t R ¼ 6.7 min). The proposed method offers high sensitivity: as low as 0.05 mg ml 1 of AMX and 0.13 mg ml 1 of AMP could be determined Selection and optimization of chromatographic conditions To achieve the best chromatographic conditions, the mobile phase composition was optimized to provide sufficient selectivity and sensitivity in a short separation time. The different chromatographic conditions affecting the separation and resolution of AMX and AMP were carefully studied and optimized. The results of the optimization study are summarized in Table Anal. Methods, 2014, 6, This journal is The Royal Society of Chemistry 2014

3 Paper Analytical Methods Fig. 2 Chromatograms showing (a) solvent front, (b) 5 mgml 1 AMX, (c) 10 mgml 1 TN, and (d) 5 mgml 1 AMP in (A) AMX and AMP standards, (B) chicken liver, (C) bovine kidney, and (D) eggs. This journal is The Royal Society of Chemistry 2014 Anal. Methods, 2014,6,

4 Analytical Methods Table 1 Optimization of experimental factors affecting the chromatographic performance of the proposed method Parameter Number of theoretical plates (N) Tailing factor Resolution (R s ) AMX AMP AMX AMP AMX/AMP Organic modi er nature 1-Propanol Acetonitrile Methanol Propanol concentration (%) SDS concentration (M) ph Choice of appropriate detection wavelength. The UV detector responses of AMX and AMP were carefully studied and the best wavelength for detection was found to be 220 nm showing the highest sensitivity Choice of column. Two different columns were used for performance investigation, including Supelco Discovery HS C18 column and Nucleodur MN-C18 column. The experimental studies revealed that the rst column was more suitable, since it produced well-resolved peaks with a very high sensitivity and free from tailing Choice of ow rate. The effect of ow rate on the formation and separation of peaks of the studied compounds was studied in the range ml. A minimum ow rate as well as minimum run time gives the maximum saving on the usage of solvents. From the experiments, it was found that a ow rate of 1 ml min 1 was ideal for successful elution of the analytes in a reasonable time Mobile phase composition. Several modi cations in the micellar mobile phase composition were performed in order to study the possibilities of changing the selectivity of the chromatographic system. These modi cations included the change of the concentration and type of organic modi er, the surfactant concentration, the ph, and the ow rate. The mobile phase was prepared using 0.3% triethylamine and 0.02 M phosphoric acid. The effect of changing the type of organic modi er on the selectivity and retention times of AMX and AMP was investigated using mobile phases containing 10% 1-propanol, acetonitrile or methanol. 1-Propanol was found to be the best one, giving well-resolved peaks and the highest number of theoretical plates. The effect of changing the concentration of the organic modi er on the selectivity and retention times of AMX and AMP was investigated using mobile phases containing concentrations of 4 7% 1-propanol and containing 0.05 M SDS and buffered at ph 5. Well-resolved peaks and the highest number of theoretical plates were obtained using 5% 1-propanol. So, a small amount of 1-propanol is added to accelerate and control the elution of the drugs. The effect of changing the concentration of surfactant on the selectivity and retention times of AMX and AMP was investigated using mobile phases containing SDS concentrations in the range M and containing 5% 1-propanol and buffered at ph 5. SDS with concentration 0.05 M was the best, giving well-resolved peaks and the highest number of theoretical plates. Also, the effect of changing the ph of the mobile phase on the selectivity and retention times of AMX and AMP was investigated using mobile phases of ph ranging from A ph of 5.0 was the most appropriate, giving well-resolved peaks and the highest number of theoretical plates (Table 1). Values of ph higher than 5.5 resulted in a very low number of theoretical plates. A er these experimental investigations, the assay was carried out using a Supelco Discovery HS C18 column with the mobile phase consisting of 0.05 M sodium dodecyl sulphate 5% 1-propanol 0.3% triethylamine 0.02 M phosphoric acid at ph 5.0 and UV detection at 220 nm with a ow rate of 1.0 ml min System suitability test parameters To ascertain the reproducibility of the MLC method, system suitability tests were performed using the working standard solutions of AMX and AMP. Resolution (R s ), theoretical plate number (N) and tailing factor (T) were measured as the criteria for system suitability testing. These results are satisfactory compared to the minimum values necessary for an acceptable method as depicted in Table Method validation The validity of the proposed MLC method was tested in terms of linearity, ranges, limits of detection, limits of quanti cation, accuracy and precision Linearity and range Under the above-described experimental conditions, linear relationships were established by plotting AMX and AMP concentrations against peak area ratios for AMX and AMP to the internal standard. The concentration ranges were found to be mg ml 1 for each of AMX and AMP, respectively. Linear regression analysis of the data gave the following equations: AMX: P ¼ C (r ¼ ) AMP: P ¼ C (r ¼ ) Paper 1526 Anal. Methods, 2014, 6, This journal is The Royal Society of Chemistry 2014

5 Paper Table 2 Statistical analysis of the results obtained by the proposed and reference methods for pure samples of amoxicillin and ampicillin Parameter Table 3 Accuracy and precision data from determination of amoxicillin and ampicillin using the proposed method a Analyte Concentration (mg ml 1 ) AMX Intra-day b Recovery (mean S.D.) Er (%) AMP Proposed Reference Proposed Reference % Recovery Mean (X) S.D Variance Student's t-value a Variance ratio F-value a a Tabulated t- and F-values at p ¼ 0.05 are 2.26 and 4.74, respectively. Inter-day c Recovery (mean S.D.) Er (%) AMX AMP a N.B.: each result is the average of three separate determinations. b Intra-day: within the day. c Inter-day: three consecutive days. where P is the peak area ratio and C is the concentration of drug in mg ml 1 and r is the correlation coefficient. The high values of the correlation coefficients (r-values > 0.999) indicate good linearity of the calibration graphs. Statistical analysis of the data gave small values of the standard deviation of the residuals, (S y/x ) and ,of slope, (S b ) and and of intercept, (S a ) and and the % relative error, (% Er) 0.33% and 0.35% for both AMX and AMP, respectively Limit of quantitation (LOQ) and limit of detection (LOD) The limit of quantitation (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B recommendations 26 below which the calibration graph is non-linear and was found to be 1.7 mg g 1 and 4.5 mg g 1 for AMX and AMP, respectively. The limit of detection (LOD) was determined by establishing the minimum level at which the analyte can be reliably detected (S/N ¼ 3); it was found to be 0.5 mgg 1 ( M) and 1.3 mgg 1 ( M) for AMX and AMP, respectively Accuracy The accuracy of the analytical method is de ned as the agreement of the results obtained by this method with the true values. To test the validity of the proposed method, it was applied to the determination of pure samples of AMX and AMP over the range of mg ml 1. The proposed method was favorably compared with the comparison HPLC method. 4 Using Student's t-test and the variance ratio F-test revealed no significant difference between the performance of the two methods regarding the accuracy and precision, respectively (Table 2). 25 The proposed procedure offers additional advantages over the comparison procedure in that the former is simpler, avoiding lengthy derivatization procedures with no use of organic solvents in extraction maintaining good accuracy and precision. The comparison method depends on the reversed phase liquid chromatographic determination of AMX and AMP, in eggs, with uorescence detection using precolumn derivatization. The derivatization depends on heating the drugs with salicylaldehyde and trichloroacetic acid in a boiling water bath for 60 min. The chromatographic procedure uses a C18 column and a mobile phase composed of 0.01 M potassium dihydrogen phosphate, adjusted to ph 5.5 and acetonitrile. Detection was carried out at 345 nm for excitation and 445 nm for emission using liquid liquid extraction with acetonitrile and dichloromethane for sample clean-up Precision The intra-day precision was evaluated through replicate analysis of different concentrations of the pure forms of two drugs within the speci c working concentration ranges. Each sample was analyzed three successive times. Similarly, the inter-day precision was evaluated through replicate analysis of the three different concentrations on three successive days. The results obtained are summarized in Table 3. The data presented in Table 3 indicate high precision of the developed method. Good values of the average percentage recoveries and the small values of standard deviations indicate the high accuracy and precision, respectively. 5. Applications Analytical Methods The applicability of the developed procedure to determine AMX and AMP was tested by analyzing it in spiked chicken muscles, liver, bovine meat, liver, kidney, and eggs. All samples were collected from the local market. Table 4 shows the results of the analysis of AMX and AMP determined in all samples a er homogenization with micellar solution, sonication, centrifugation and ltration. Samples were spiked at the following concentration levels: 5, 10 and 15 ppm for each of AMX and AMP. Three replicates of each concentration were injected into the chromatograph. The data obtained (Table 4) show satisfactory recoveries of AMX and AMP in all samples, and the This journal is The Royal Society of Chemistry 2014 Anal. Methods, 2014, 6,

6 Analytical Methods Paper Table 4 Assay of amoxicillin and ampicillin in food samples using the proposed and reference methods AMX AMP AMX JM Method Prop. Ref. Prop. Ref. Prop. Ref. Prop. Ref. Sample type Chicken muscle Chicken liver Mean recovery (X) a S.D Variance Student's t-value b Variance ratio F-value b Sample type Bovine muscle Bovine liver Mean recovery (X) a S.D Variance Student's t-value b Variance ratio F-value b Sample type Bovine kidney Eggs Mean recovery (X) a S.D Variance Student's t-value b Variance ratio F-value b a Number of experiments ¼ 3. b Tabulated t- and F-values at p ¼ 0.05 are 2.78 and 19.00, respectively. results fall in the range of % and % for AMX and AMP, respectively. Fig. 2 depicts the chromatograms obtained from different spiked samples of AMX and AMP analyzed with the optimum mobile phase. These chromatograms reveal how a surveillance programme for AMX and AMP residues can be performed under the proposed chromatographic conditions. The low quanti cation limits of the proposed method are useful for the determination of any traces of AMX and AMP residues. 6. Conclusion The proposed procedure is useful for food quality testing and control areas to determine the content of AMX and AMP in chickenmuscles,liver,bovinemuscles,liver,kidneyandegg samples. One advantage of this procedure is the possibility of injecting the samples directly into the chromatographic system with no previous treatment other than homogenization, dilution and ltration, thus avoiding tedious extractions from matrices. Validation according to ICH regulation provides satisfactory results in terms of sensitivity, linearity, accuracy and recoveries and at the ppm level. The results fall in the range of % and % for AMX and AMP, respectively. It is noteworthy that the use of micellar mobile phases endows the procedure with advantages such as non- ammability, biodegradability and low cost. The current concern about the environment also reveals MLC as an interesting technique for green chemistry because it uses mobile phases containing low amounts of organic solvents. These micellar mobile phases have a low toxicity and are not producing hazardous wastes. Conflict of interest Fawzia Ibrahim declares that she has no con ict of interest. Jenny Jeehan Nasr declares that she has no con ict of interest. Compliance with ethics requirements This article does not contain any studies with human or animal subjects. References 1 The United States Pharmacopeia 37, the National Formulary 32, US Pharmacopeial Convention, Rockville, MD, Electronic version, The British Pharmacopeia, Her Majesty's Stationery Office, London, Electronic version, 2013, vol. I. 3 S. Bogialli, V. Capitolino, R. Curini, A. Di Corcia, M. Nazzari and M. Sergi, J. Agric. Food Chem., 2004, 52, K. Xie, L. Jia, D. Xu, et al., J. Chromatogr. Sci., 2012, 50, The European Agency for the Evaluation of Medicinal Products, Veterinary Medicines and Inspections, Reg. 675/ 92, A. Strasser, E. Usleber, E. Schneider, R. Dietrich, C. Bürk and E. Märtlbauer, Food Agric. Immunol., 2003, 15, S. N. Jiao, P. Wang, G. X. Zhao, H. C. Zhang, J. Liu and J. P. Wang, J. Environ. Sci. Health, Part B, 2013, 48, P. Srisom, B. Liawruangrath and S. Liawruangrath, Chromatographia, 2007, 65, Anal. Methods, 2014, 6, This journal is The Royal Society of Chemistry 2014

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