Indirect Spectrophotometric Determination of Gentamicin and Vancomycin Antibiotics Based on Their Oxidation by Potassium Permanganate

DOI: 10.2478/s11532-006-0035-z Research article CEJC 4(4) 2006 708–722

Indirect spectrophotometric determination of gentamicin and vancomycin antibiotics based on their oxidation by potassium permanganate

Akram M. El-Didamony1∗, Alaa S. Amin2, Ahmed K. Ghoneim1, Ayman M. Telebany1

1 Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt 2 Chemistry Department, Faculty of Science, Banha University, Banha, Egypt

Received 3 May 2006; accepted 11 July 2006

Abstract: Four simple, accurate, sensitive and economical procedures (A - D) for the estimation of gentamicin sulphate and vancomycin hydrochloride, both in pure form and in pharmaceutical formulations have been developed. The methods are based on the oxidation of the studied drugs by a known excess of potassium permanganate in sulphuric acid medium and subsequent determination of unreacted oxidant by reacting it with amaranth dye (method A), acid orange II (method B), indigocarmine (method C) and methylene blue (method D), in the same acid medium at a suitable λmax=521, 485, 610 and 664 nm, respectively. The reacted oxidant corresponds to the drug content. Regression analysis of Beer-Lambert plots showed good correlations in the concentration ranges 4-8, 3-8, 4-9 and 5-9 µg ml−1 with gentamicin and 4-8, 1.5-4, 1.5-4 and 3.5-5.5 µg ml−1with vancomycin for methods A, B, C, and D, respectively. The molar absorptivity, sandell sensitivity, detection and quantification limits were calculated. The stoichiometric ratios for the cited drugs were studied. The optimum reaction conditions and other analytical parameters were evaluated. The influence of the substance commonly employed as excipients with these drugs were studied. The proposed methods were applied to the determination of these drugs in pharmaceutical formulations. The results have demonstrated that the methods are equally accurate and reproducible as the official methods. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

Keywords: Oxidation reaction, potassium permanganate, gentamicin; vancomycin, dosage forms, indirect spectrophotometry

∗ E-mail: ak [email protected] A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 709

1 Introduction

Gentamicin is a broad spectrum aminoglycoside antibiotic, which belongs to the class of medicinal compounds capable of inhibiting the growth of Gram-positive and Gram- negative bacteria. Gentamicin is one of the most effective drugs used in the treatment of serious suppurative and septic processes, especially those that are caused by Gram-negative microorganisms. The advantages of gentamicin over other aminoglycosides (kanamycin, neomycin) are its activity towards Pseudomonas aeruginosa and microorganisms of the Serratia–Klebsiella –Enterobacter group, a faster bactericidal effect, and the rare development of gentamicin resistant strains [1]. It can be quantified using colorimetric technique [2], amperometric enzyme-immunosensors [3], flow injection chemiluminescence [4], spectrofluorometry [5–7], capillary electrophoresis with UV detection [8], liquid chromatography (LC) [9–14] and high performance liquid chromatography (HPLC) [15–23]. Vancomycin is an amphoteric glycopeptide antimicrobial substance produced by the growth of certain strains of Nocardia orientalis. It is bactericidal against many gram- positive organisms [24, 25]. Vancomycin is not chemically related to any of the presently used antimicrobial agents. Vancomycin hydrochloride has been reported to be effective alone or in combination with an aminoglycoside for treatment of endocarditis caused by S. viridans or S. bovis. For endocarditis caused by enterococci (e.g., Enterococcus faecalis), vancomycin hydrochloride has been reported to be effective only in combination with an aminoglycoside. Vancomycin can be determined by several analytical methods, such as voltametric [26], spectrophotometric [27], radioimmunoassay and fluorescence polarization immunoassay [28], LC [29], and HPLC [30–37]. There are a few spectrophotometric methods reported for the assay of the cited drugs, and most of the analytical techniques used for their determination are radioimmunoassay, LC and HPLC. However, all these methods are expensive, tedious, time consuming and prior separation of the drugs is required. The present work describes four visual spectrophotometric methods (A-D) for assaying the cited drugs in bulk form and in commercial pharmaceutical formulations. Methods A-D are indirect procedures, involving the addition of an excess of KMnO4 and the determination of unreacted oxidant by the decrease in absorbance of the different dyes.

2 Experimental

2.1 Apparatus

All the absorption spectral measurements were made using a Biotech (UV-VIS) spec- trophotometer (Biotech Engineering Ltd. (UK) world wide distributor sole agent Sedico Ltd. Cyprus), with a scanning speed 400 nm/min and band width of 2.0 nm, equipped with 10 mm matched quartz cells. 710 A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722

2.2 Materials and reagents

All of the chemicals used were of analytical grade and all of the solutions were freshly prepared in double distilled water. Pure gentamicin and vancomycin were obtained from the Egyptian International Phar- maceutical Industries Company (EIPICO). Stock solutions of the drugs studied were freshly prepared daily by dissolving 20 mg of the drug in distilled water and then, made upto 100 ml in a calibrated flask. The stock solutions were further diluted stepwise with water to achieve the working standard solutions. An aqueous solution of amaranth (AM) (Merck; 5.0×10−4 M), acid orange II (AO) (Merck; 5.0×10−4 M), indigocarmine (Indigo) (Aldrich; 5.0×10−4 M) and methylene blue (MB) (Merck; 1.0×10−4 M) were prepared by dissolving the appropriate weight of the dye in a very small volume of water and then made upto 100 ml in a calibrated flask. The stock solutions of dyes were allowed to stand at room temperature for a few weeks without any significant decay. −3 A stock solution of 5.0×10 M KMnO4 (Aldrich) was prepared by dissolving the appropriate weight in 10 ml of warm distilled water, made upto 100 ml in a calibrated flask. This solution was then standardized using sodium oxalate and stored in a dark −4 bottle [38]. A 5.0×10 M solution of KMnO4was prepared by diluting the stock solution.

2.0 M H2SO4 was also prepared.

2.3 General procedure and calibration

1.0 ml aliquots of the examined drug solutions were pipetted (concentration range as indicated in Table 1, 2) into 10 ml calibrated ﬂasks in a series, followed by acidiﬁcation −4 by adding 0.5 ml of 2.0 M H2SO4. 2.0 ml and 1.7 ml of 5.0×10 M KMnO4 solution was added to gentamicin and vancomycin, respectively, and heated in a boiling water bath for 25 and 15 min. The mixture was cooled and 1.3 ml of 5.0×10−4 M AM was used for method A, 1.7 ml of 5.0×10−4 M of AO for method B, 1.6 ml of 5.0×10−4 M Indigo for method C, and 2.5 ml of 5.0×10−4 M MB for method D, for both gentamicin and vancomycin. The volume was made up to 10 ml with water. The decrease in color intensities in A, B, C and D were measured spectrophotometrically at their corresponding maximum wavelengths (Table 1, 2). The concentration of each drug was determined from a calibration graph constructed under the same conditions.

2.4 Procedure for pharmaceutical formulations: 2.4.1 Procedure for injections

The contents of ten garamycin ampoules were pooled and from this an accurately measured volume equivalent to 20 mg of drug was added to a calibrated ﬂask and diluted to 100 ml with distilled water. These solutions were further diluted to get the working A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 711 concentrations and analyzed as described above.

2.4.2 Procedure for eye drops The contents of ten bottles of gentamytrex eye drops were mixed and the average volume of one bottle was determined. An accurate measured volume equivalent to 20 mg of gentamicin was diluted to 100 ml with with water in a calibrated ﬂask. Accurate volumes of this solution were analyzed as described above.

2.4.3 Procedure for vials The contents of ﬁve vancocin vials were pooled and from this an accurate amount of the powder equivalent to 20 mg of drug was diluted to 100 ml with water in a calibrated ﬂask. The above described procedure was used to determine the drug concentration.

3 Results and discussion

The optimum conditions for color development in each method were established by varying the parameters one at a time, keeping the others ﬁxed and observing the eﬀect produced on the absorbance of the colored species.

3.1 Absorption spectra

The spectrophotometric method for the determination of gentamicin and vancomycin is based on their oxidation with known excess of KMnO4 in acidic medium and subsequent determination of residual oxidant by reacting it with ﬁxed amount of AM, AO, indigo and MB. The absorption spectra of the reaction product between KMnO4 and AM, AO, indigo and MB in methods A, B, C and D shows characteristic λmax value (Table 1, 2).

3.2 Eﬀect of heating time

In order to obtain the highest and most stable absorbance, the eﬀect of heating time on the oxidation reaction was studied. The reactions were performed in a water bath at 100 ± 2 oC for the periods ranging from 10 – 30 min. Maximum and constant absorbance was obtained after 25 min for gentamicin, and after 15 min for vancomycin (Fig. 1).

3.3 Eﬀect of oxidant concentration

When a study on the eﬀect of KMnO4 on color development was performed, it was observed that in both cases the absorbance increased with increase in the volume of −4 5.0×10 M KMnO4. It reached maximum when 2.0 ml and 1.7 ml of KMnO4 solution was added to a total volume of 10 ml for gentamicin and vancomycin, respectively. The

color intensity decreased above the upper limits. Therefore, 2.0 ml and 1.7 ml of KMnO4 were used for all measurements (Fig. 2). 712 A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722

1.5 (b)

1.2 (a)

0.9

0.6 Absorbance Absorbance

0.3

0 5 10 15 20 25 30 Time (min)

Fig. 1 Eﬀect of heating time on the oxidation of: (a) gentamicin-MB and (b) vancomycin- AO.

1.4 (b) (a)

1.12

0.84

0.56 Absorbance

0.28

0 0.5 1.1 1.7 2.3 2.9 3.5

V KMnO4

−4 Fig. 2 Eﬀect of volume of 5.0×10 M KMnO4 on the development of the reaction product: (a) 9.0 µg ml−1 gentamicin with MB and (b) 4.0 µg ml−1vancomycin with AO.

3.4 Eﬀect of acidity

Studies on the variation of sulphuric acid concentration indicated that a constant absorbance is obtained at 0.5 ml of 2.0 M H2SO4. Above this volume, the absorbance decreased for gentamicin, where as for vancomycin the absorbance remained constant.

Therefore, a volume of 0.5 ml of 2.0 M H2SO4, was used for all measurements. A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 713

3.5 Eﬀect of dye concentration

In order to ascertain the linear relationship between the volume of added KMnO4 and the decrease in absorbance of AM, AO, Indigo and MB, experiments were performed using

0.5 ml of 2.0 M H2SO4 with varying volumes of KMnO4. The decrease in absorbance −4 was found to be linear up to 2.0 ml and 1.7 ml of 5.0×10 M KMnO4 with 1.3 ml of AM, 1.7 ml of AO, 1.6 ml of indigo and 2.5 ml of MB for gentamicin and vancomycin, respectively. The color was found to be stable up to 24 h.

3.6 Stoichiometry

The stoichiometric ratio between gentamicin and oxidant was checked by Job’s method of continuous variation [39]. The Job’s plot (Fig. 3) reached a maximum value at mole fraction of 4.0, which conﬁrmed that molar ratio between gentamicin and oxidant is 1 : 4 (Table 1).

1.5 c a d 1.2 b

0.9

0.6 Absorbance

0.3

0 0.1 1.88 3.66 5.44 7.22 9 [Oxid] / [Drug]

Fig. 3 Continuous variations graph for the reaction between 5.0×10−4 M gentamicin and −4 5.0×10 M KMnO4 with: (a) AM (b) AO (c) Indigo and (d) MB.

The stoichiometry of vancomycin and oxidant was determined by continuous variation of potassium permanganate concentration with the concentration of vancomycin being constant. The plot obtained by the molar ratio method indicated that the reaction proceeded by molar ratio of 1 : 16 (Fig. 4). Additionally, the stoichiometric ratio between drugs and dyes, and dyes and oxidant were examined as shown in Tables 1, 2. 714 A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722

0.5

d 0.4 a c b 0.3

0.2 Absorbance

0.1

0 5 8 11 14 17 20 [Oxid] / [Drug]

Fig. 4 Molar ratio plot for the reaction of 1.0×10−4 M vancomycin and 1.0×10−4 M

KMnO4 with: (a) AM (b) AO (c) Indigo and (d) MB.

Table 1 Analytical parameters and optical characteristics of the proposed methods with gentamicin sulphate.

Parameter A B C D

λmax 521 485 610 664 Beer Lambert’s law limits (µg ml−1) 4.0–8.0 3.0-8.0 4.0–9.0 5.0–9.0 Molar absorptivity (l mol−1cm−1) 7.19×104 6.12× 104 5.86×104 7.07×104 Sandell sensitivity (µg ml−1) 6.64 7.80 8.15 6.75 Detection limits (µg ml−1) 9.24×10−3 5.19×10−3 5.1×10−3 6.69×10−3 Quantiﬁcation limits (µg ml−1) 0.030 0.016 0.017 0.022 Regression equation∗ Slope (b) 0.2388 0.1998 0.1917 0.2744 Intercept (a) -0.7018 -0.4019 -0.6550 -1.04

S y/x 0.4360 0.4180 0.4041 0.5018 SD of slope (Sb) 0.1378 0.0999 0.0965 0.1586 SD of intercept (Sa) 1.8497 1.3461 1.5380 2.483 Correlation coeﬃcient (r) 0.9996 0.9998 0.9955 0.9989 Stoichiometric ratio [Drug] : [Oxid.] 1:4 1:4 1:4 1:4 [Drug] : [Dye] 1:4 1:4 1:4 1:4 [Oxid.] : [Dye] 1:1 1:1 1:1 1:1

∗A = a + b C, where C is the concentration in µg ml−1. A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 715

3.7 Quantiﬁcation

Beer Lambert’s law limits, molar absorptivity, Sandell’s sensitivity, regression equations and correlation coefficients obtained by linear square treatment of the results are given in Table 1, 2. The standard deviation of the absorbance measurements was obtained from a series of 13 blank solutions. The detection (k = 3) and quantification (K = 10) limits of the method were established according to the IUPAC definitions (C 1= K S 0/ s) where

C 1 is the detection limit, S 0 is the standard error of blank determination, s is the slope of the standard curve, and K is the constant related to the confidence interval [40]. In order to study the accuracy and precision of the proposed methods, three concentration levels of the drugs studied within the linearity range were selected and analyzed in five replicates. The measured standard deviation (SD), relative standard deviation (RSD) and confidence limits are summarized in Table 3, 4 and can be considered satisfactory, at least for the concentrations examined.

Table 2 Analytical parameters and optical characteristics of the proposed methods with vancomycin hydrochloride.

Parameter A B C D

λmax 521 485 610 664 Beer Lambert’s law limits (µg ml−1) 4.0–8.0 1.5-4.0 1.5-4.0 3.5–5.5 Molar absorptivity (l mol−1cm−1) 4.96×105 4.46×105 4.11×105 3.23×105 Sandell sensitivity (µg ml−1) 2.99 3.33 3.61 4.6 Detection limits (µg ml−1) 5.19×10−3 6.36×10−3 5.19×10−3 0.0108 Quantiﬁcation limits (µg ml−1) 0.017 0.021 0.017 0.036 Regression equation∗ Slope (b) 0.2573 0.0386 0.3483 0.0260 Intercept (a) -0.7188 -0.3685 0.3038 -0.2100

S y/x 0.4702 0.4046 0.3653 0.2387 SD of slope (Sb) 0.1486 0.0193 0.1747 0.0150 SD of intercept (Sa) 1.993 1.3030 1.176 1.518 Correlation coeﬃcient (r) 0.9992 0.9994 0.9973 0.9984 Stoichiometric ratio [Drug] : [Oxid.] 1:16 1:16 1:16 1:16 [Drug] : [Dye] 1:16 1:16 1:16 1:16 [Oxid.] : [Dye] 1:1 1:1 1:1 1:1

∗A = a + b C, where C is the concentration in µg ml−1. 716 A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722

Table 3 Evaluation of accuracy and precision of the proposed methods with gentamicin sulphate.

Method Taken Recovery RSDa REb Conﬁdence limitsc µg ml−1 % % %

A 5.0 100.12 0.74 0.86 0.435 ± 3.72×10−3 7.0 100.04 0.42 0.48 0.986 ± 4.74×10−3 9.0 100.02 0.34 0.39 1.332 ± 5.26×10−3 B 6.0 99.98 0.49 0.56 0.808 ± 4.55×10−3 7.0 99.97 0.54 0.62 1.016 ± 6.32×10−3 8.0 99.96 0.35 0.41 1.150 ± 4.66×10−3 C 7.0 99.96 0.44 0.51 0.768 ± 3.88×10−3 8.0 100.04 0.44 0.50 0.915 ± 4.59×10−3 9.0 100.02 0.42 0.48 1.060 ± 5.13×10−3 D 6.0 100.04 0.78 0.90 0.698 ± 6.26×10−3 7.0 99.92 0.87 1.00 1.097 ± 0.0109 8.0 100.02 0.33 0.38 1.183 ± 4.48×10−3 a Relative standard deviation for five determinations. b Relative Error. c 95% confidence limits and five degrees of freedom.

Table 4 Evaluation of accuracy and precision of the proposed methods with vancomycin hydrochloride.

Method Taken Recovery RSDa REb Conﬁdence limitsc µg ml−1 % % %

A 5.0 100.06 0.57 0.66 0.596±3.93×10−3 6.0 100.02 0.38 0.44 0.856±3.75×10−3 7.0 99.94 0.50 0.58 1.061±6.12×10−3 B 3.0 99.98 0.58 0.66 0.800±5.20×10−3 3.5 100.02 0.96 1.10 0.903±9.97×10−3 4.0 99.98 0.56 0.64 1.172±7.52×10−3 C 3.0 100.06 0.88 1.01 0.703±7.12×10−3 3.5 99.98 0.64 0.74 1.141±8.42×10−3 4.0 100.00 0.77 0.89 1.242±0.0109 D 3.5 100.04 0.98 1.13 0.702±7.91×10−3 4.0 99.94 0.66 0.75 0.856±6.39×10−3 4.5 100.02 0.41 0.47 1.156±5.44×10−3 a Relative standard deviation for five determinations. b Relative Error. c 95% confidence limits and five degrees of freedom. A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 717

4 Interferences

In pharmaceutical analysis, it is important to test the selectivity towards excipients added to the pharmaceutical preparations. It is clear from the results obtained for the pharmaceutical preparations that the commonly encountered excipients such as starch, talc, glucose, alginate and stearate did not interfere (Table 5, 6).

5 Analytical Application

The proposed methods were successfully applied to determine the drugs studied in tablets and injections. The results obtained were compared statistically by student’s t- test (for accuracy) and variance ratio F- test (for precision) with official methods [41, 42] at 95% confidence level with five degrees of freedom (Table 5, 6). The results showed that the t- and F- values were less than the critical value [43] indicating that there was no significant difference between the proposed and official methods. Because the proposed methods were more reproducible with high recoveries they can be recommended for routine analysis in majority of drug quality control laboratories.

Table 5 Determination of gentamicin sulphate in garamycin pharmaceutical preparation using the proposed methods.

Preparation Taken Proposed methods Oﬃcial and Supplier µg ml−1 Recovery ± SD (%)a method A B C D

4.0 100.15±0.62 100.21±0.51 99.86±0.64 99.98±0.73 Fb = 1.38 F = 2.04 F = 1.30 t = 0.53 t = 0.49 t = 0.26 5.0 100.13±0.68 99.90±1.05 100.03±0.75 100.02±0.87 99.93±1.12 F = 2.71 F = 1.13 F = 2.23 F = 1.65 t = 0.38 t = 0.54 t = 0.63 t = 0.49 6.0 100.02±0.59 99.94±0.95 99.96±0.67 100.05±0.73 99.89±0.81 F = 1.88 F = 0.72 F = 1.46 F = 1.23 t = 0.47 t = 0.70 t = 0.59 t = 0.68 7.0 100.03±0.56 100.08±0.74 100.08±0.50 100.16±0.59 100.04±0.57 F = 1.03 F = 0.59 F = 1.29 F = 0.93 t = 0.98 t = 0.47 t = 0.38 t = 0.79

Garamycin 40 mg/ml 8.0 100.06±0.52 99.94 ± 0.58 100.05±0.44 100.05±0.62 99.96±0.56 Memphis Co. for Pharm. F = 1.15 F = 0.93 F = 1.61 F = 0.81 t = 0.44 t = 0.37 t = 0.78 t = 0.46 9.0 99.98 ± 0.68 99.92±0.71 F = 1.09 t = 1.18 a The average of six determinations. b Theoretical values for t- and F-values for ﬁve degrees of freedom and 95% conﬁdence limits are 2.57 and 5.05, respectively. 718 A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722

Table 6 Determination of gentamicin sulphate in gentamytrex pharmaceutical preparation using the proposed methods.

Preparation Taken Proposed methods Oﬃcial and Supplier µg ml−1 Recovery ± SD (%)a method A B C D

4.0 99.78±0.50 99.94±0.55 100.58±0.53 99.91 ± 0.57 Fb= 1.29 F = 1.07 F = 1.15 t = 0.78 t = 0.57 t = 1.42 5.0 99.88±0.43 100.1±0.54 99.86±0.48 99.96±0.63 98.95 ± 0.59 F = 1.88 F = 1.19 F = 1.51 F = 0.87 t = 0.51 t = 1.19 t = 0.70 t = 0.85 6.0 99.96±0.61 100.07±0.89 100.05±0.67 100.08±0.56 98.95 ± 0.79 F = 1.67 F = 0.78 F = 1.39 F = 1.99 t = 0.62 t = 0.65 t = 0.86 t = 0.66 7.0 99.96±0.32 99.96±0.38 100.1±0.51 100.27±0.44 100.14 ± 0.41

Minapharm F = 1.64 F = 1.16 F = 0.64 F = 0.86 t = 1.25 t = 1.09 t = 0.62 t = 0.96 8.0 100.03±0.73 100.1±0.48 99.94±0.83 99.96±0.65 99.95±0.63 Gentamytrex 5.0 mg/ml F = 0.74 F = 1.72 F = 0.57 F = 0.93 t = 0.84 t = 0.95 t = 0.53 t = 1.34 9.0 99.98±0.41 99.91±0.46 F = 1.25 t = 0.91 a The average of six determinations. b Theoretical values for t- and F-values for ﬁve degrees of freedom and 95% conﬁdence limits are 2.57 and 5.05, respectively.

6 Conclusion

The order of λmax values among the proposed methods for the determination of the cited drugs is D > C > A > B. The higher λmax of the visible spectrophotometric methods over reported UV and visible spectrophotometric methods is decisive and advantageous since the interference from the excipients should be far less at higher wavelengths. The proposed methods are accurate and precise as indicated by good recoveries of the drugs and low RSD values. The proposed methods could be applied for routine analysis and in quality control laboratories for quantitative determination of the cited drugs both in the pure and dosage forms. A.M. El-Didamony et al. / Central European Journal of Chemistry 4(4) 2006 708–722 719

Table 7 Determination of vancomycin hydrochloride in vancocin pharmaceutical preparation using the proposed methods.

Preparation Taken Proposed methods Oﬃcial and Supplier Recovery ± SD (%)a method A B C D

2.0 100.12±0.58 99.98±0.63 99.96±1.56 99.89±0.79 Fb = 1.85 F = 1.57 F = 0.26 t = 0.67 t = 0.94 t = 0.83 2.5 100.02±0.84 99.94±0.60 99.74±0.75 99.80±0.72 99.95±0.82 F = 0.95 F = 1.86 F = 1.19 F = 1.29 t = 1.28 t = 0.68 t = 0.47 t = 1.35 3.0 99.96±0.98 99.98±1.20 100.04±0.80 100.12±0.73 99.97±0.99 F = 1.02 F = 0.68 F = 1.53 F = 1.83 t = 0.94 t = 0.52 t = 0.68 t = 0.96 3.5 99.96±0.96 99.98±0.68 100.021.07±0.88 100.06±0.91 98.94±0.87 F = 0.82 F = 1.63 F= 0.97 F = 0.91 t = 0.57 t = 0.35 t = 1.07 t = 0.49 Vancocin 500mg/ml 4.0 99.94 ± 0.66 100.03±0.52 99.98±0.42 100.06±0.68 99.70±0.54 Lilly Pharma Fertigung F = 0.66 F = 1.07 F = 1.65 F = 0.63 t = 0.64 t = 1.15 t = 0.87 t = 0.88 4.5 99.99±0.74 100.06±0.69 100.05±0.86 99.98±0.68 99.97±0.76 F = 1.05 F = 1.21 F = 0.78 F = 1.24 t = 0.49 t = 0.56 t = 0.72 t = 1.47 a The average of six determinations. b Theoretical values for t- and F-values for ﬁve degrees of freedom and 95% conﬁdence limits are 2.57 and 5.05, respectively.

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