Azitromicina Metanol PDF
Azitromicina Metanol PDF
Ramallah, Palestine
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Journal of Chromatographic Science, Vol. 48, February 2010
Time (min) Table I. Chromatographic Parameters for the Separated Peaks in Figure 2
Figure 2. Chromatogram of azithromycin and its related compounds. Parameter Desosaminylazithromycin N-Demethylazithromycin Azithromycin
Analytes: 1, Desosaminylazithromycin; 2, N-demethylazithromycin; and 3,
Azithromycin. Mobile phase: methanol–phosphate buffer, pH 7.5 (80:20, Resolution – 2.1 5.3
v/v), flow rate 2.0 mL/min, injection volume 20 mL. Column: reversed phase Capacity factor 2.8 3.3 7.7
C18, 5 mm, 25 cm length, 4.6 mm inner diameter, column temperature: Asymmetry 1.09 1.18 1.32
50°C. UV detection: 210 nm. Selectivity – 1.18 2.33
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Journal of Chromatographic Science, Vol. 48, February 2010
optimum percentage. Temperature was increased to facilitate from 80–120% of the target concentration. For content unifor-
mass exchange with the corresponding decrease of peak broad- mity testing, the minimum range is from 70–130% of the test or
ening and increase in sensitivity; 50°C was a good selection. We target concentration (14).
have selected low wavelength (210 nm) to be used for UV detec- Acceptance criteria for linearity are that the correlation coeffi-
tion due to the lack of chromophores other than the ester group cient (R2) is not less than 0.990 for the least squares method of
(Figure 1). In the current study, the elution is simplified by using analysis of the line. Additionally, the relative standard deviation
isocratic elution (80:20, methanol–buffer) with a flow rate of (RSD) will not be greater than 5.0% at all standard concentra-
2.0 mL/min, compared with gradient elution employed by tions (14).
Miguel et. al for the separation of azithromycin and its related Standard solutions covering the range between 30–120% of
substances (4). the nominal standard concentration (1.0 mg/mL azithromycin)
After this optimization, this method has been used for the sep- have been prepared by diluting specific volume of the stock stan-
aration of azithromycin from its related compounds (e.g., des- dard to get several concentrations (0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.20,
osaminylazithromycin and N-demethylazithromycin) (Figure 2) 1.60, and 2.0 mg/mL). Then, these standards have been chro-
as well as separation from azaerythromycin A (Figure 3). Good matographed using UV detector at 210 nm. Three runs have been
separation with adequate resolution has been obtained (Figures performed for every concentration. The peak responses (e.g.,
2–3). Chromatographic parameters of the separated peaks peak area) have been recorded and plotted versus standard con-
(desosaminylazithromycin, N-demethylazithromycin, and centrations. Results have shown that the method is linear over
azithromycin) (Table I). the specified range with R2 of 0.9999, insignificant y-intercept
(6977), and a slope of 3 × 1006 has also been obtained. Standard
Method validation
After method development, the validation of the current test
method for azithromycin has been performed in accordance with
USP requirements for assay determination (Category I:
Analytical methods for quantitation of active ingredients in fin-
ished pharmaceutical products), which include accuracy, preci-
sion, specificity, linearity, and range (13).
AU
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Journal of Chromatographic Science, Vol. 48, February 2010
deviation of the slope and y-intercept is 145,921 and 15,761, have shown that the mean recovery of the assay for both drug
respectively. Standard error was 23,798. These findings demon- suspension and capsules as well as for azithromycin raw material
strate linearity of this method over the specified range. is within 100 ± 2.0% at each concentration, and the RSD is lower
The obtained R2 value for the current method (0.9999) is com- than 1.0% (Table II). Furthermore, results have shown that
parable to the value obtained by Zubata et. al (9) for the LC recovery data obtained was within the 99.9–101.3% range for
method for azithromycin analysis in raw material and in capsule formulation (mean = 100.4%) and 99.5–100.0% range
azithromycin tablets (0.9994), and better than the value obtained for dry suspension (mean = 99.8%), and the mean recovery for
by Miguel et. al (4) for the LC method for azithromycin analysis raw material at the nominal concentration (1.0 mg/mL) is 99.6%
in azithromcin tablets (0.996). (Table II). Zubata et al. has obtained comparable recovery data for
azithromycin in raw material and in azithromycin tablets
Accuracy (99.8–100.0% with a mean of 99.4%) (9).
The accuracy of an analytical procedure measures the close-
ness of agreement between the value, which is accepted either as Precision
a conventional true value or an accepted reference value and Precision is the measure of the degree of repeatability of an
value found (i.e., accuracy is a measure of exactness of an analyt- analytical method under normal operation and is normally
ical method). Accuracy is measured as the percent of analyte expressed as the RSD for a statistically significant number of
recovered by assay after spiking samples in a blind study (15). For samples. Precision is performed at one level (repeatability).
the assay determination of azithromycin in drug formulations Repeatability is the result of the method operating over a short
(capsules and dry suspension), accuracy is evaluated by ana- time interval under the same conditions (injection precision or
lyzing synthetic mixtures spiked with known quantities of instrument precision). It is determined from a minimum of nine
azithromycin. determinations covering the specified range of the procedure
To document accuracy, a minimum of nine determinations (for example, three levels, three repetitions each), or from a min-
over a minimum of three concentration levels covering the spec- imum of six determinations, at 100% of the test or target con-
ified range (for example, three concentrations, three replicates for centration. RSD for replicate injections should not be greater
each) were collected. It is performed at 80, 100, and 120% levels than 1.5% (16).
of label claim. At each level studied, replicate samples are evalu- The RSD of the peak areas for the recovery data analyzed in
ated. The RSD of the replicates provides the analysis variation and accuracy study (see the Method validation section) for each level
gives an indication of the precision of the test method. Moreover, (80%, 100%, and 120% of the nominal concentration) has been
the mean of the replicates, expressed as % of label claim, indicates calculated, and it has been found to be less than 1.0% for each
the accuracy of the test method. The mean recovery of the assay level (Table III). The RSD of the peak areas of six replicate injec-
should be within 100 ± 2.0% at each concentration over the range tions for the nominal standard concentration (100%) has also
of 80–120% of nominal concentration (15). been calculated to be 0.2%. These results show that the current
To prepare accuracy standard solutions, placebo of the drug method for azithromycin analysis is repeatable.
formulation (e.g., capsule or drug suspension) has to be prepared
according to the formulation procedure. To the required quan- Specificity (stability-indicating evaluation)
tity of placebo, a known quantity of azithromycin with the same Specificity is the ability to assess unequivocally the analyte in
proportion as in the drug formulation has been added to get the presence of components that may be expected to be present,
three concentrations [0.8, 1.0 (nominal concentration), and 1.2 such as impurities, degradation products, and matrix compo-
mg/mL of azithromycin]. These standards, then, have been chro- nents (17). It is a measure of the degree of interferences from
matographed. Three runs have been performed for every con- such components, ensuring that a peak response is due to a single
centration, and then peak area has been recorded. The average component only. Specificity is measured and documented in a
recovery and the RSD for each level have been calculated. Results separation by the resolution, plate count (efficiency), and tailing
AU
AU
Time (min)
Time (min) Figure 6. Chromatogram of 1, azithromycin in a drug formulation product
Figure 5. Chromatogram of 1, azithromycin (1.0 mg/mL azithromycin, added to (capsule). Other experimental conditions are the same as in Figure 2.
it 10% of 2M hydrochloric acid). Other experimental conditions are the same Resolution, capacity factor, and peak asymmetry for azithromycin peak are
as in Figure 2. 1: azithromycin is no more present as it is completely degraded. 4.8, 4.4, and 1.34, respectively.
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Journal of Chromatographic Science, Vol. 48, February 2010
10 σ/S
13. United States Pharmacopeia (2007), National Formulary, Validation of
Compendial Methods <1225>, Rockville, MD, 549.
14. International Conference on Harmonization (ICH), “Validation of Analytical
Procedures-PA/PH/OMCL (05) 47 DEF”, elaborated by OMCL Network/EDQM
Results showed that the detection and quantitation limits for of the Council of Europe, June 2005.
azithromycin using this method are 0.0005 and 0.0008 mg/mL, 15. M. Green, a Practical Guide to Analytical Method Validation; Analytical
Chemistry News and Features, May 1, 1996, P. 309A.
respectively. 16. L. Huber. Validation of Analytical Methods, in “Validation and Qualification in
After successful development and validation of this method, the Analytical Laboratories” 1998, Interpharm Press, Buffalo Grove, IL, 107.
17. A WHO Guide to Good Manufacturing Practice (GMP) Requirements, Part 2:
we have employed it for the analysis of azithromycin in two drug Validation, World Health Organization, Geneva, 1997.
formulations (capsules and drug suspension) as well as in raw Manuscript received March 2, 2008;
material (Figure 6). revision received May 30, 2008.
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