Revista Brasileira de Ciências Farmacêuticas Brazilian Journal of Pharmaceutical Sciences vol. 37, n. 2, maio/ago., 2001
Performance characteristics of bioassay, UV-spectrophotometry and high performance liquid chromatographic determination of sparfloxacin in tablets Hérida Regina Nunes Marona1,2*, Elfrides Eva Scherman Schapoval2 1
Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, SP, 2 Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul
*Correspondence: H. R. N. Marona Fac. de Ciências Farmacêuticas UNESP Rod. Araraquara-Jaú, km 1 14801-902 – Araraquara - SP - Brasil
The microbiological bioassay, the UV-spectrophotometry and the high performance liquid chromatography (HPLC) methods for assaying sparfloxacin in tablets were compared. The accuracy, repeatability, and precision of each method was assessed and proved to be satisfactory. All methods were reliable within acceptable limits for antibiotic pharmaceutical preparations being accurate and precise. The microbiological bioassay and HPLC are more specific than UV-spectrophotometric analysis. However, the microbiological bioassay requires 20 h to get results, and HPLC is the most expensive analysis. The application of each method as a routine analysis should be investigated considering cost, simplicity, equipment, solvents, speed, and application to large or small workloads.
Uniterms: • Bioassay • Fluoroquinolone • HPLC • Pharmaceutical analysis • Sparfloxacin • UV-spectrophotometry
E-mail:
[email protected]
INTRODUCTION Sparfloxacin (Figure 1), a difluoroquinolone derivative, is a potent antibacterial agent active against a wide range of Gram-positive and Gram-negative organisms including Mycobacteria (Goa et al., 1997; Shimada et al., 1993). A drawback of fluoroquinolones is their photoreactivity (Engler et al., 1998; Phillips et al., 1990). Many factors can influence its toxicity. Sparfloxacin has been studied in terms of therapeutic activities (Goa et al., 1997; Shimada et al., 1993). However, few reports about analytical methods of sparfloxacin are available in the literature (Marona, Schapoval, 1999; Marona et al., 1999). Because the half-life is approximately 20 hours and some factors may influence toxicity and therapeutic dosis, careful and frequent monitoring of the drug’s concentrations has been studied, especially in the case of patients with hepatic and renal insuffiency.
FIGURE 1 - Chemical structure of sparfloxacin. In this paper, the results of three methods are compared with respect to accuracy, precision and speed. They included the microbiological bioassay (BA), the spectrophotomethic analysis (SPA) and the high performance liquid chromatographic determination
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(HPLC). The aim of this work was to determine the performance characteristics of these methods considering simplicity, cost, time and sensitivity to quantify sparfloxacin and determine the presence of any impurities and photodegradation products.
MATERIAL AND METHODS Material Sparfloxacin powder (purity 99.5%) was supplied by Dainippon Pharmaceutical Co., Osaka, Japan and Rhône-Poulenc Rorer, U.S.A. The sparfloxacin tablets were obtained commercially. The tablets were claimed to contain 200 mg of active drug. All other chemicals used were of analytical grade. The sparfloxacin powder and tablets were stored protected from light. Methods The bioassay, the spectrophotometry and the HPLC can be considered routine methods to analyse both raw materials and pharmaceutical preparations. Bioassay The bioassay was performed with antibiotic media Grove-Randall no 1 and no 11, the indicator organism was Staphylococcus epidermidis ATCC 12228 at 35 o C overnight diluted in nutrient broth 2:100 inoculated to the surface of each plate. Six 8 mm diameter cilinders were used and 200 μL of each sample was added to each cilinder. The standard curve was linear over the range 4.0 to 25.0 μg/mL. The bioassay plates were incubated at 35 oC aerobically for 18 h. The zone sizes were carefully measured with calipers. A standard curve was prepared by measuring the zone diameters of three concentrations (4.0; 10.0 and 25.0 μg/mL) of sparfloxacin (Marona, Schapoval, 1998). Spectrophotometric analysis Solutions of sparfloxacin powder in methanol (1 mg/mL) were prepared by accurately weighing 10.0 mg of sparfloxacin to a 50 mL volumetric flask, adding 10.0 mL methanol, followed by sterile distilled water up to volume. The sparfloxacin standard solution was diluted in 50 mL volumetric flasks with sterile distilled water to give a range of solutions with final concentrations of 2-12 mg/L. The absorbance value of each solution at 292 nm was determined in a 10 mm quartz cell in a UV-VIS
H. R. N. Marona, E. E. S. Schapoval
spectrophotometer (Shimadzu, Japan). To analyse the concentratrion of sparfloxacin in tablets each tablet was weighed to obtain the average tablet weight. The tablets were ground up and 960.0 mg (representing three times the average weight of each tablet) were transferred to a 1 000 mL volumetric flask; 200 mL methanol was added followed by sterile distilled water to volume. Aliquots of 10 mL of this solution were transferred to a 100 mL volumetric flask and sterile distilled water added to volume to give an estimated concentration of 60.0 mg/L. After rotation in a centrifuge flask, the solution was diluted 1:10 to give a final estimated concentration of 6.0 mg/L. This solution was prepared six times and the absorbance of each determined at 292 nm. HPLC HPLC analysis was performed on a Waters SCL-6A chromatograph equipped with a model LC-10AS pump; SPD-10A variable-wavelength detector (set at 292 nm); SCL-10A system controller; C-R6A integrator and Rheodine injection valve with a 20 μL loop. A Shim-pack CLC-ODS column (250 mm x 4.6 mm I.D., 5 mm particle size, 100 Å pore diameter) was used with aqueous acetic acid 5%:methanol:acetonitrile (70:15:15, v/v/v) isocratic as mobile phase at a flow-rate of 1.0-mL/min; the sensitivity was 0.5 AUFS and the chart speed was 0.5 cm/min. The HPLC system was operated at ambient temperature (20 ± 1 oC). The mobile phase was filtered by membrane filter (Supelco) 0.45 μm x 47 mm and degassed with helium sparge for 15 minutes. The analysis required less than ten minutes. All determinations were carried out in triplicate. Recovery Test The recoveries were determined by adding known amounts of sparfloxacin reference substance to the samples at the beginning of the process (USP, 1999). A recovery exercise was then performed. Accuracy and Precision Accuracy and precision of the assay were determined intra-day and inter-day on three different days. Precision was expressed as the percent coefficient of variation of each curve. The statistical analysis employed mean, standard deviation, coefficient of variation and for performance of least-squares linear regression and analysis of variance (ANOVA).
Performance characteristics of bioassay, UV-spectrophotometry...
RESULTS
TABLE III - Analysis of variance of spectrophotometric results of sparfloxacin
The bioassay developed is a microbiological procedure for the analysis of sparfloxacin in raw material and tablets. The linear regression for this fluoroquinolone was calculated to be y = 13867 + 2.5374 ln(x) with correlation coefficient of 0.9977. The statistical data were calculated by ANOVA (Table I). TABLE I Analysis of variance of microbiological results of sparfloxacin by agar diffusion Source of variation
d.f.
SS
Preparation Regression Parallelism Square Difference Doses Within dishes Residual
1 1 1 1 1 5 5 25
1.206 128.390 0.004 0.001 0.169 129.770 0.596 1.476
Total
35
131.842
MS
F
1.206 20.439* 128.390 2175.28 0.004 0.059 0.001 0.010 0.169 2.866 25.954 439.731* 0.119 2.021 0.059 -
Source of variation
d.f.
SS
Preparation Regression Parallelism Residual
5 1 4 12
1.102796 1.102510 0.000286 0.000313
Total
17
1.1033109
The recoveries were determined by adding known amounts of the sparfloxacin reference substance (10.0, 20.0 and 30.0 µg/mL) to the samples at the beginning of the process (Table II). TABLE II - Experimental values obtained in the recovery test for sparfloxacin tablets by bioassay
MS
F
0.220559 8450.54* 1.102510 42241.76* 0.0000715 2.74 0.0000261 -
-
* P < 0.05 The recoveries were determined by adding known amounts of the sparfloxacin reference substance (2.0, 4.0 and 6.0 µg/mL) to the samples at the beginning of the process (Table IV). TABLE IV - Experimental values obtained in the recovery test for sparfloxacin tablets by spectrophotometric method Amount of standard (µg/mL)
-
* P < 0.05
Added (µg/mL)
Recovered (µg/mL)
Recovery (%)
2.0 4.0 6.0
1.96 4.09 5.99
98.00 100.21 99.83
R1 R2 R3
The calibration curves for sparfloxacin were constructed by plotting the peak area versus concentration. It was found to be linear with a correlation coefficient of 0.9997. The representative linear regression was 0.9994. The statistical results was calculated by ANOVA (Table V). TABLE V - Analysis of variance of sparfloxacin by HPLC
Amount of standard (µg/mL)
R1 R2 R3
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Added (µg/mL)
Recovered (µg/mL)
Recovery (%)
10.0 20.0 30.0
9.79 19.82 29.41
97.90 99.10 98.03
The spectrophotometric method gave rise to linear data in the range 2-12 µg/mL. The linear regression for sparfloxacin was calculated to be y = 0.0725x + 0.0008 with a correlation coefficient of 0.9998. The statistical data were analysed by ANOVA and linear simple regression by the least-squares method (Table III).
Source of variation
d.f.
SS
Preparation Regression Parallelism Residual
5 1 4 12
Total
17 3.422 × 1012
MS
3.41 × 1012 6.83 × 1011 3.41 × 1012 3.41 × 1012 4.8 × 108 1.9 × 109 9 7.2 × 10 6.0 × 108 -
F 1136.20* 5677.81* 0.80 -
* P < 0.05 The recoveries were determined by adding known amounts of the sparfloxacin reference substance (15.0,
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H. R. N. Marona, E. E. S. Schapoval
25.0 and 40.0 µg/mL) to the samples at the beginning of the process (Table VI). TABLE VI - Experimental values obtained in the recovery test for sparfloxacin tablets by HPLC Amount of standard (µg/mL)
R1 R2 R3
Added (µg/mL)
Recovered (µg/mL)
Recovery (%)
15.0 25.0 40.0
14.95 24.90 39.38
99.67 99.60 98.45
The precision of each method was determined by evaluation of the intra- and inter-day variation. The percent coefficient of variation (CV) on the basis of each assay for three replicate determinations were found to be between 0.98 and 2.42%. These methods developed in our laboratory had also been used to quantify sparfloxacin in tablets. The results obtained, the precision data and recovery test are shown in Table VII. TABLE VII - Interassay precision and accuracy of three methods proposed of sparfloxacin Assay
Mean* (mg)
CV %
Recovery (%)
BA SPA HPLC
186.82 194.16 188.22
2.42 1.07 0.98
98.34 100.04 99.24
*Each preparation assayed at least in triplicate in 3 different days. BA = bioassay; SPA = spectrophometric analysis; HPLC = high performance liquid chromatography Estimates of the slope of the line of the best fit and of the proportional error illustrating the best criteria for evaluating the linearity are presented in Table VIII. TABLE VIII - Between-day linear regression analysis comparing three sparfloxacin analytical methods Assay
r2
Equation for the line
SPA HPLC BA
0.9998 0.9994 0.9978
y = 0.0008 + 0.0725x y = 895145 + 41290x y = 13867 + 2.5374ln(x)
BA = bioassay; SPA = spectrophometric analysis; HPLC = high performance liquid chromatography
The data obtained by bioassay, spectrophotometric analysis and HPLC were statistically comparable by ANOVA test. The mean percent levels determined by the method proposed did not differ significantly at the p < 0.01 (Table IX). TABLE IX - Analysis of variance of sparfloxacin tablets determination by bioassay, spectrophotometry and HPLC Source of variation
d.f.
SS
MS
F
Preparation Error Total
2 9 11
99.20 88.77 187.97
49.6 9.86 -
5.03 -
* P < 0.01
DISCUSSION We had developed three methods of analysis of sparfloxacin both raw material and tablets (Marona, Schapoval, 1999; Marona et al., 1999). This paper compared these methods on the basis of precision, accuracy, repeatability, cost, and ease of handling. All methods proposed yielded coefficients of variation less than 2.5%. These results can indicate a good precision. The reported methods for the determination of sparfloxacin in pharmaceutical forms give accurate and precise results. It is evident that HPLC was the most expensive method. This procedure requires specialized equipment and expensive solvents as well. The spectrophotometric analysis also requires specialized equipment but cost of spectrophotometer is less than an HPLC apparatus. The bioassay is clearly the least expensive method studied, and requires no specialized equipment. Ease of handling, as measured by technician time required for performance of a single assay can be another determination factor to select a laboratory routine method. Spectrophotometry and HPLC both provide results in 2 hours. Bioassay is limited by incubation and counting time resulting 20 h. The data obtained by bioassay, spectrophotometric analysis and HPLC were statistically comparable by ANOVA test. The mean percent levels determined by the method proposed did not differ significantly at the p < 0.01. However, UV-spectrophotometry method can quantify impurities and degradation products which present quite similar structure but they do not have any antibacterial activity (Marona, 2000). Because the photodegradation
Performance characteristics of bioassay, UV-spectrophotometry...
products of sparfloxacin showed similar profile of this pure fluoroquinolone and some of them present absorption maximum in the UV spectrum at 292 nm, careful analysis has been studied (Marona et al., 1999; Marona, 2000). On the other hand, HPLC technique can show interfering peaks and bioassay can quantify antibacterial activity. Our study leads to conclude that the bioassay and HPLC have clearly displayed advantages.
ACKNOWLEDGMENTS The authors are grateful to Dr. Sayuri Kitada (Dainippon Co., Japan) and Dr. Michael Pease (RhônePoulenc Rorer, E.U.A.) for providing the sparfloxacin reference substance and Helder Teixeira for providing the tablets. This work was supported by CAPES/PICDT program (Brasília - Brazil).
RESUMO Comparação dos parâmetros dos métodos desenvolvidos para a determinação de esparfloxacino em comprimidos Este trabalho compara, por análise estatística, parâmetros de validação de três metodologias desenvolvidas para a determinação de esparfloxacino em comprimidos. Os parâmetros, tais como exatidão e precisão, determinados para cada método, apresentaram valores satisfatórios. Os métodos microbiológico e cromatográfico (CLAE) demonstraram maior especificidade do que o método espectrofotométrico na região de ultravioleta. Entretanto, o ensaio microbiológico exigiu 20 horas para sua execução e a CLAE apresentou maior custo. Desta forma, a aplicação de cada um dos métodos como rotina deve ser estudada, considerando diferentes fatores, tais como custo, simplicidade, equipamentos e solventes, bem como o volume de análises a serem realizadas. UNITERMOS: CLAE. Análise Esparfloxacino. UV-espectroscopia.
farmacêutica.
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GOA, K. L., BRYSON, H. M., MARKHAM, A. Sparfloxacin: a review of its antibacterial activity, pharmacokinetic properties, clinical efficacy and tolerability in lower respiratory tract infections. Drugs, Auckland, v.53, p.700-725, 1997. MARONA, H. R. N., SCHAPOVAL, E. E. S. Desarrollo de análisis microbiológico para la determinación de esparfloxacino en polvo y en comprimidos de 200 mg. Información Tecnológica, La Serena, v.9, p.251-4, 1998. MARONA, H. R. N., SCHAPOVAL, E. E. S. A highperformance liquid chromatographic assay of sparfloxacin. J. Pharm. Biomed. Anal., Amsterdam, v.20, p.413-7, 1999. MARONA, H. R. N., SCHAPOVAL, E. E. S. Spectrophotometric determination of sparfloxacin in tablets. J. Antimicrob. Chemother., Oxford, v.44, p.1367, 1999. MARONA, H. R. N., ZUANAZZI, J. A. S., SCHAPOVAL, E. E. S. High-performance liquid chromatographic assay of sparfloxacin and its degradation products. J. Antimicrob. Chemother., Oxford, v.44, p.301-2, 1999. MARONA, H. R. N. Esparfloxacino: Estudo QuímicoFarmacêutico e Caracterização de Mutantes Resistentes. Porto Alegre, 2000. 456 p. [Tese de Doutorado em Ciências Farmacêuticas – Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul]. PHILLIPS, G., JOHNSON, B. F., FERGUSON, J. The loss of antibiotic activity of ciprofloxacin by photodegradation. J. Antimicrob. Chemother., Oxford, v.26, p.783-9, 1990. SHIMADA, J., NOGITA, J., ISHIBASHI, Y. Clinical pharmacokinetics of sparfloxacin. Clin. Pharmacokin., Auckland, v.25, p.358-69, 1993 UNITED STATES PHARMACOPOEIA. 24.ed. Rockville: United States Pharmacopeial Convention, 1999. p. 214952. Recebido para publicação em 26 de outubro de 2000.
