(c). Scheme. Structures of Lora (a), Ceft (b) and Cefa (c). Development and Validation of Spectrophotometric Methods for Determination of Some Cephalosporin ...
ISSN 10619348, Journal of Analytical Chemistry, 2012, Vol. 67, No. 2, pp. 144–150. © Pleiades Publishing, Ltd., 2012.
ARTICLES
Development and Validation of Spectrophotometric Methods for Determination of Some Cephalosporin Group Antibiotic Drugs1 Derya Tarinc and Aysegul Golcu University of Kahramanmaras Sütçü Imam, Faculty of Science and Arts, Department of Chemistry, Campuse of Avsar, Kahramanmaras, 46100 Turkey Received October 6, 2010; in final form April 5, 2011
Abstract—A simple and direct spectrophotometric method is developed for the determination of some ceph alosporin group antibiotic drugs such as Loracarbef (Lora), Ceftazidime (Ceft), and Cefaclor (Cefa) in bulk and pharmaceutical formulations. The optimum conditions for the analysis of aqueous solutions of drugs are studied. Under the optimum conditions, the three drugs could be assayed in the concentration range 2–9 × 10–5, 2–6 × 10–5, and 3–9 × 10–5 M for Lora, Ceft and Cefa, respectively. Detection and quantification limits are calculated. The obtained results showed good recoveries of 100.4, 107.4, and 100.7% for Lora, Ceft, and Cefa, respectively. The results obtained are compared favorably with those given by literature methods. Keywords: spectrophotometric determination, antibiotic drugs, validation. DOI: 10.1134/S1061934812020128
The cephalosporins are a class of βlactam anti biotics originally derived from Acremonium, which was previously known as Cephalosporium. Cepha losporins are sometimes grouped into generations by their antimicrobial properties. The first cepha losporins were designated first generation, whereas later, more extended spectrum cephalosporins were HO
classified as secondgeneration cephalosporins. Lora and Cefa are second generation, Ceft is a third generation cephalosporins [1]. Different methods for determination of the Lora, Ceft and Cefa were reviewed. The structural formulas of these drugs are illustrated in Scheme.
O O
Cl
N
O N H
(a)
H2N O OH
N O N H2N
H
H N
S
S
O
N+
N
O (b)
O−
O
NH2 H N O
H N
O
S Cl
(c) O OH Scheme. Structures of Lora (a), Ceft (b) and Cefa (c). 1
The article is published in the original.
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DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHODS
Reviewing the literature revealed that several meth ods have reported for the analysis of this cephalospor ins group in pharmaceutical preparations or biological fluids. Among these methods are spectrophotometry [2–5], chromatography [6, 7] and electrochemical methods [8, 9]. These methods, particularly the chro matographic techniques are time consuming, costly and require expertise. A simple and precise UVspec trophotometric method can be highly applicable for routine analysis of bulk, formulations and dissolution samples. The aim of the present study is to improve a simple, precise, accurate and.economic analytical method with a better detection range for the estima tion of Lora, Ceft and Cefa in bulk and pharmaceuti cal formulations. No extraction, derealization or evaporation step, no complexation agent and no harmful chemicals are involved in the suggested method, in that connection decreasing time and the error in quantitation. To our awareness, there is no direct UV spectro photometric method for determination of these drugs in pharmaceutical compounds for water media in lit erature. Hence, the objective of the present study is the development and sensitive analytical methods for the determination of Lora, Ceft and Cefa in pharmaceuti cal preparations. The method was validated to analyze the dosage forms. The obtained results from spectro photometeric and literature methods [10–12] in water media were statistically compared. EXPERIMENTAL Apparatus. PerkinElmer Lambda 45 UV–vis dou ble beam spectrophotometer with a slit width of 2 nm. The absorbance values were measured using 1 cm quartz cells. Reagents. Lora and dosage forms (Lorabid) cap sule (400 mg), Cefa and dosage forms (Losefar) cap sule (250 mg), Ceft and dosage forms (Zidim) flakon (1 flakon + 10 mL solvent ampule) were kindly pro vided by Lilly Pharmaceutical Co. (Istanbul, Turkey), Eczacibasi Pharmaceutical Co. (Istanbul, Turkey), and TümEkip Pharmaceutical Co. (Istanbul, Tur key), respectively. Preparation of standard and quality control solu tions. An aqueous primary stock solution of 1 × 10–3 M of Lora (0.546 mg/mL), Ceft (0.340 mg/mL) and Cefa (0.380 mg/mL) were prepared in water. All measure ments were performed at 25°C. The standard solutions were prepared by the suitable dilutions of the primary stock solution with ultra pure water to obtain working standards in the concentration range of 2–9 × 10–5, 2– 6 × 10–5 and 3–9 × 10–5 M for Lora, Ceft and Cefa, respectively. The absorbances of these solutions were then fitted in the calibration curve to calculate the accuracy and precision of the method. Method validation. Linearity. The method was val idated according to International Conference on Har JOURNAL OF ANALYTICAL CHEMISTRY
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monization Q2B guidelines [13] for validation of ana lytical procedures in order to determine the linearity, sensitivity, precision and accuracy for each analyte [14–17]. Precision and accuracy. The precision of the assay was determined by repeatability (intraday) and inter mediate precision (interday) and reported as RSD, % for a statistically significant number of replicate mea surements [18]. The intermediate precision was stud ied by comparing the assays on three different days and the results are documented as standard deviation and RSD, %. Accuracy is the percent of analyte recovered by assay from a known added amount. Data from nine determinations over three concentration levels cover ing the specified range were obtained. The repeatabil ity of the method was determined by assaying six sam ple solutions of the highest test concentration [19]. LOD and LOQ. In this study, LOD and LOQ were based on the standard deviation of response and the slope of the corresponding curve using following equa tions [18]; LOD = 3 s/m; LOQ = 10 s/m, where s, the noise estimate, is the standard deviation of the absorbance of the blank, m is the slope of the related calibration graphs. The limit of quantification (LOQ) is defined as the lowest concentration that can be measured with acceptable accuracy, precision and variability [18]. The values of LOD and LOQ are given in Table 1. Extraction of active ingredient. Five capsules were accurately weighed and emptied as so completely as possible. The emty capsules were weighed again, and the differences were given as the total amount of the five capsules contents. The combined contents of the capsules were thoroughly ground to a fine powder. A sufficient amount of this powder for preparing a stock solution of 1 × 10–3 M accurately weighed and trans ferred into a 25 mL calibrated flask and volume was completed with distilled water for Lora and Cefa. Then, sufficient amount of equivalent injection powder (contains only 1 g ceft) was weighed and volume was completed to 25 mL with distilled water for 1 × 10–3 M Ceft and it was stirred 30 min with magnetic stirrer. Appropriate dilutions were made and the samples were subjected to UV analysis. Recovery experiment from dosage forms. Recovery of the analytes of interest from a given matrix can be used as a measure of the accuracy or the bias of the method. The same range of concentrations as employed in the linearity studies was used. To study the accuracy, precision and reproducibility of the pro posed methods and to check the interference from the excipients used in the dosage forms, recovery experi ments were carried out using the standard addition method. These studies were performed by addition of known amounts of pure Lora, Ceft and Cefa to the preanalysed dosage forms and the mixtures were No. 2
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Table 1. Regression data of the calibration lines for quantitative determination of Lora, Ceft and Cefa by UV method Compounds Lora Measured wavelength (λmax), nm Linearity range, M Slope Absorbance range ε* Intercept Correlation coefficient LOD, M LOQ, M Repeatability of absorbance (RSD %) Repeatability of wavelength (RSD %) Reproducibility of absorbance (RSD %) Reproducibility of wavelength (RSD %)
Ceft
261 2×
Cefa
256
× 1.08 × 104 0.201–0.968 11700 –0.0271 0.998 1.5 × 10–6 5 × 10–6 2.62 0.03 0.75 0.02 10–5–9
10–5
2×
264
× 1.43 × 104 0.248–0.880 14340 –0.004 0.999 1.6 × 10–6 5.5 × 10–6 2.75 0.01 1.84 0.07 10–5–6
3×
× 10–5 8.3 × 103 0.220–0.727 8040 –0.0258 0.999 1.7 × 10–6 6 × 10–6 2.79 0.03 1.53 0.03
10–5
10–5–9
* Molar absorption coefficients have been calculated for 5 × 10–5 M.
analysed by the proposed techniques. After parallel analyses, the recovery results were calculated using the related calibration equations. RESULTS AND DISCUSSION Analytical parameters of developed method. The development of simple, rapid, sensitive and accurate analytical method for routine quantitative determina tion of samples will reduce unnecessary tedious sam
1.6
A 1.2 1.0 0.8 0.6 0.4 0.2
1.4
0
A 2.2 2.0 1.8
1.2
ple preparations and cost of materials and labor. Lora, Ceft and Cefa are UV absorbing molecules with spe cific chromophores in their structures that absorb at a particular wavelength and this fact was successfully employed for their quantitative determinations by UV spectrophotometric method. The absorption spectra and calibration graphs of Lora, Ceft and Cefa in aque ous solutions are illustrated in Figs. 1–3. Calibration curve data were constructed in the range of expected
y = 10802x – 0.0271 R2 = 0.9957
0.00006 0.00010 0.00002 0.00008 c, M 0.00004
1.0 0.8 0.6 0.4 0.2 0 200
220
240
260
280
300 λ, nm
Fig. 1. UV spectrum and calibration graph of Lora (5 × 10–5 M) in aqueous solution. JOURNAL OF ANALYTICAL CHEMISTRY
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DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHODS A 2.30 2.25 2.20 2.15 2.10 2.05
A 3.0 2.5 2.0 1.5 1.0 0.5
147
y = 14271x – 0.0044 R2 = 0.9995
0.00005 0.00010 0.00015 0.00020 0.00025 c, M
0
2.00 1.95 1.90 1.85 1.80 1.75 1.70 1.65 230
250
240
260
270
280 λ, nm
Fig. 2. UV spectrum and calibration graph of Ceft (4 × 10–5 M) in aqueous solution.
A 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 230
A 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
240
250
y = 8389.3x – 0.0258 R2 = 0.9994
0.00002 0.00004 0.00006 0.00008 0.00010 c, M
260
270
280 λ, nm
Fig. 3. UV spectrum and calibration graph of Cefa (3 × 10–5 M) in aqueous solution.
concentrations of 2–9 × 10–5, 2–6 × 10–5 and 3–9 × 10–5 M for Lora, Ceft, and Cefa respectively. Beer’s law was obeyed over this concentration range. The regression equations were found. y = 10800x ⎯ 0.0271 for Lora (r2 = 0.9960), y = 15000x ⎯ 0.0586 for Ceft (r2 = 0.9980) and y = 8300x ⎯ 0.0258 for Cefa (r2 = 0.9980). The stock solutions and working standards were made in aqueous media. The λmax of the drugs for JOURNAL OF ANALYTICAL CHEMISTRY
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analysis were determined by taking scans of the drug sample solutions in the entire UV region (256.37– 264.8 nm). The characteristics of the calibration plots are sum marized in Table 1 and the analytical characteristics and necessary validation parameters for UV tech niques for three drugs are presented. Performing replicate analyses of the standard solu tions was used to assess the accuracy, precision and No. 2
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Table 2. Assay results from Lora, Ceft and Cefa dosage forms and mean recoveries in spiked dosage forms Lora
Ceft
Cefa
Lorabid
Zidim
Losefar
Labeled claim, mg
400
1000
250
Amount found, mga
401.5
1148.9
251.7
RSD, %
0.48
0.37
1.56
Bias, %
–0.4
–14.9
–0.7
ttest (ttheoretical = 2.31)
0.40
0.39
0.51
Ftest (Ftheoretical = 2.60)
0.32
0.59
0.73
Added, mg
40
250
25
Found, mga
40.5
232.6
25.174
Recovery, %
100.4
107.4
100.7
RSD, % of recovery
1.20
2.0
0.33
–0.36
–7.4
–0.7
Product
Bias, % a
Each value is the mean of 5 experiments.
reproducibility of the proposed methods. The selected concentration within the calibration range was pre pared in water and analyzed with the relevant calibra tion curves to determine intraday and interday vari ability. The intra and interday precision were deter mined as the RSD %. The precision, accuracy and reproducibility results given in Table 1 demonstrate a good precision, accuracy and reproducibility. The commercial dosage forms showed 100.4, 107.4, and 100.7% recovery by this method for Lora, Ceft and Cefa respectively, which were within the specified limits of content uniformity. Moreover, the UV method offers a cost effective and time saving alternative to other methods for example colorimetric, complexometric and chromatographic. The proposed methods can be successfully applied for Lora, Ceft and Cefa assay in dosage forms without any interference. The assay showed the drug content of this product to be in accordance with the labeled claim (Table 2). The recovery of the analyte of interest from a given matrix can be used as a measure of the accuracy of the method. In order to check the accuracy and pre cision of the developed method and to prove the absence of interferences by excipients, recovery stud ies were carried out using the standard addition tech nique. Recovery studies were carried out after the addition of known amounts of the pure drug to various preanalyzed formulations of all drugs. The applica tion of this procedure is explained in Experimental Section. The obtained results demonstrate the validity
and accuracy of the proposed method for the determi nation of all drugs in tablets (Table 2). These results reveal that all methods have adequate precision and accuracy, and consequently, can be applied to the determination of all drugs forms in pharmaceuticals without any interference from the excipients. Comparison between literature method and devel oped method. The results of spectrophotometric and literature methods (electroanalytical methods) [18– 20] were compared with each other in Table 2. The amounts of drugs are fairly close to the labeled amounts for all techniques. Student’s t and Ftests were carried out on the data and statistically examined the validity of the obtained results by spectrophoto metric and literature methods. At the 95% confidence level, the values of t and Ftests (calculated from the experiments) were less than those of theoretical t and Fvalues showing that there are no significant differ ences between the spectrophotometric and literature methods with regard to accuracy and precision. How ever, the proposed spectrophotometric methods could be successfully applied for Lora, Cefa and Ceft assay in capsule dosage form without any interference. According to Student’s ttest, the calculated T value did not exceed the theoretical value for significance level of 0.05. There was no significant difference between the performance of all techniques with regards to applicability and simplicity. To prove the absence of interferences by excipients, recovery stud ies were also carried out for all techniques. The accu
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DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHODS Wavelength 265 264 263 262 261 260 259 258 257 256 255 0 1
149
(A)
Lora Ceft Cefa
2
3
4
5
6
7
8 Time, h
(B)
Absorbance 1.2 1.0
Lora Ceft Cefa
0.8 0.6 0.4 0.2 0
1
2
3
4
5
6
7
8 Time, h
Fig. 4. Wavelength (A) and absorbance (B) curves of Lora, Ceft and Cefa aqueous solutions stability stored at 25°C.
racy of the analysis of all methods was determined by calculating the percentage relative error (Bias %) between the measured and actual concentrations (Table 2) [21, 22]. Stability. The stability of three drugs in aqueous solution was evaluated to verify that any spontaneous degradation occur when the samples were prepared. Fig. 4A and Fig. 4B show the stability profile that belong to absorbance and wavelength at 25°C for 1, 2, 3, 4, 5, 6 and 7 h. In addition, as soon as the solutions are prepared, they have been measured and results have been recorded. The results were expressed as per centage of drugs remaining. The obtained data showed that sample solutions were stable during 7 h when stored at 25°C with a degradation less than 6%. That is, the absorbances and wavelengths belonging to the spectral maximum of these drugs have not changed seriously during this time. *** The UV method for the determination of Loracar bef, Cefaclor and Ceftazidime in powder is simple, rapid, precise, accurate and sensitive. In summary, the JOURNAL OF ANALYTICAL CHEMISTRY
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proposed method can be used for the drug analysis in routine quality control. ACKNOWLEDGMENTS This research was supported by a grant TUBITAK. (Grand no. 105T371) for Assoc. Prof. Dr. Aysegul Golcu. REFERENCES 1. en.wikipedia.org/wiki/Cephalosporin 2. Hiremath, B., Mathada, B.H., and Mruthyunja yaswamy, Acta Pharm., 2008, vol. 58, no. 3, p. 275. 3. Moreno, A.D. and Salgado, H.R.N., Anal. Lett., 2008, vol. 41, no. 12, p. 2143. 4. AlMomani, I.F., J. Pharm. Biomed. Anal., 2001, vol. 25, nos. 5–6, p. 751. 5. Ivama, V.M., Rodrigues, L.N.C., and Guaratini, C.C.L., Zanoni, M.V.B., Quimica Nova, 1999, vol. 22, no. 2, p. 201. 6. Alangary, A.A., Anal. Lett., 1995, vol. 28, no. 10, p. 1819. 7. Klancke, J.W., J. Chromatogr., A, 1993, vol. 637, no. 1, p. 63. No. 2
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(
(
(
8. Moreno, A.D. and Salgado, H.R.N., AOAC Int., 2008, vol. 91, no. 4, p. 739. 9. Prasad, B.B. and Arora, B., Electroanalysis, 2003, vol. 15, no. 14, p. 1212. 10. Dogan, B., Golcu, A., and Ozkan, S.A., Anal. Lett., 2009, vol. 42, p. 689. 11. United States Pharmacopoeia, 2000. 12. Golcu, A., Dogan, B., and Ozkan, S.A., Talanta, 2005, vol. 67, no. 4, p. 703. 13. Golcu, A., Dogan, B., and Ozkan, S.A., Anal. Lett., 2005, vol. 38, p. 1913. 14. Golcu, A. and Ozkan, S.A., Die Pharmazie, 2006, vol. 61, p. 760. 15. Golcu, A., Anal. Chem., 2008, vol. 63, no. 6, p. 538. 16. Laviron, E., Rouillier, L., and Degrand, C., J. Electro anal. Chem., 1980, vol. 112, no. 1, p. 11.
17. Development and Validation of Analytical Methods, Riley, C.M. and Rosanske,T.W., Eds., New York: Elsevier, 1996. 18. Golcu, A., Dogan, B., and Ozkan, S.A., Anal. Lett., 2009, vol. 42, p. 689. (
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19. Muslu, H., Golcu, A., and Ozkan, S.A., Curr. Anal. Chem., 2010, vol. 6, p. 299. 20. Tarinc, D., Topal, B., Dolaz, M., Golcu, A., and Ozkan, S.A., Curr. Anal. Chem., 2010, vol. 6, p. 316. 21. Method Validation in Pharmaceutical Analysis, Ermer, J. and Miller, J.H. McB., Eds., Germany: Wiley, 2005. 22. Analytical Method Development and Validation, Swartz, M.E. and Krull, I.S., Eds., New York: Marcel Dekker, 1997.
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