Spectrophotometric and HPLC Methods for the determination of AMIKACIN
Amikacin is chemically known as 2(S)-4-amino-N-[(2S,3S,4R,5S)-5amino-2-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl) oxan-2-yl]oxy-4-[(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxyoxan-2-yl]oxy-3-hydroxy-cyclohexyl]-2-hydroxy-butanamide Amikacin is an aminoglycoside antibiotic used to treat different types of bacterial infections. Amikacin works by binding to the bacterial ribosomal subunit, causing misreading of mRNA and leaving the bacterium unable to synthesize proteins vital to its growth. Amikacin is most often used for treating severe, hospital-acquired infections with multidrug resistant gram negative bacteria such as Pseudomonas aeruginosa, Acinetobacter, and Enterobacter. Amikacin may be combined with a beta-lactam antibiotic for empiric therapy for people with neutropenia and fever. Side effects of amikacin are similar to other aminoglycosides. Kidney damage and hearing loss are the most important side effects.
Fig.1.5.1 Structure of amikacin
Systematic (IUPAC) Name 2(S)-4-amino-N-[(2S,3S,4R,5S)-5-amino-2-[(2S,3R,4S,5S,6R)-4-amino-3,5dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4-[(2R,3R,4S,5R,6R)-6(aminomethyl)-3,4,5-trihydroxy-oxan-2-yl]oxy-3-hydroxy-cyclohexyl]-2hydroxy-butanamide. Formula
Table-I.5.1 : List of important brand names of amikacin formulations.
Brand Name AMISTAR
Cadila Pharmaceuticals Limited, Mahemdabad Road,Ahmedabad.
Ind-Swift Ltd 102-103, ChambersW.E Highway, Service Road Mumbai.
Lupin laboratory Bandra Kurla complex, Mumbai.
Very few spectrophotometric methods for the determination of amikacin have been reported ( Chapter-I). Literature survey revealed that few analytical methods were reported for the determination of amikacin.1-13 In this chapter catechol-NaIO4 and p-amino acetophenone ( AAP-NaIO4) have been used as coupling agents for the determination of amikacin. A detailed review of literature for catechol ( or AAP) and sodium meta per iodate was reported in chapter-I.
SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF AMIKACIN USING CATECHOL AND SODIUM METAPERIODATE
Results and Discussion
EXPERIMENTAL Preparation of Solutions Catechol: 0.1% solution was prepared by dissolving 0.1 g of catechol sample (A.R.grade: SDFCL Mumbai) in 100 ml of distilled water. Oxidising Agent. Sodium meta periodata, NaIO4 : 2.1392 g of NaIO4 (A.R grade: Hi Media laboratories Mumbai-66) was dissolved in distilled water and the total volume was brought to 1 Lt (0.01M). Standard solution of Amikacin. Standard solution of amikacin was prepared by dissolving 100 mg of drug sample [ ALFAKIM-Ranbaxy ] in 100mL of distilled water. Working solutions of drug sample (100 g / mL) were prepared by diluting aliquots of the stock solutions with distilled water. Instrumentation Spectral measurements and absorbance readings were made on Elico SL 177 double beam Spectrophotometer. pH measurements were carried out using Elico pH meter model LI 615. Absorbance curves
species formed on mixing amikacin with suitable reagents in appropriate pH medium exhibiting maximum absorbance, the absorption spectra were scanned on a spectrophotometer in the range 400 – 550 nm against the
In order to ascertain the optimum wave lengths ( λmax) of the colored
reagent blank using the proposed procedure under experimental conditions (Table – II.5.1) and the results are graphically presented in Fig-.2.5.1.
A b s o r b a n c e
0.6 0.4 0.2 0 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 Concentration µg
Fig.2.5.1—Standard Curve of Amikacin
Establishment of Optimum Conditions Concentration of Reagents: The optimum conditions were established in each case basing on the
in Table – II.5.1. Among the various oxidizing agents tried, IO4- is the best one, followed by H2O2. The other oxidizing agents such as IO3-, Fe(III), MnO4-, clo-, Fe(CN)63-, are inferior. The efficiency of the oxidizing agent
development of maximum color and its stability and results are presented
depends upon its relative reactive tendency towards reactants, (drug, catechol) products (indo-dyes) and also on the behavior of its reduced form.
The formation of colored species of same λmax in the case of
amikacin with each pair of reagents, (Catechol – IO4- and AAP-IO4-) suggests that the indo dye formed with both compounds is the same. However for operational feasibilities only catechol-IO4- related results are presented although experiments were conducted with AAP-IO4- reagent also. Order of addition of reagents. The author has carried out series of experiments to test whether variation in the order of addition of reactants ( amikacin, oxidizing agent and catechol) effect the absorbance. The suitable order of addition of reactants for getting maximum absorbance and stability has been found to be, amikacin, catechol oxidizing agent.
The order of addition of reactants influences color
development. Any delay in adding catechol to oxidant causes considerable decrease in absorbance depending upon the nature of oxidant. These studies reveal that the oxidant is capable of oxidizing catechol under
chosen experimental conditions.
Effect of temperature : All experiments and absorbance measurements were carried out at laboratory temperature (280 + 30 C). At low temperature (< 200c) the stability of the colored species is less. Effect of solvent: A mixture of, requisite concentrations of amikacin, catechol and oxidizing agent were placed in a separating funnel and was diluted to 25mL with distilled water. After keeping it for some time, for allowing the reaction to complete, 10mL of chloroform or n-butanol (if insoluble in chloroform) was added to the separating funnel and the contents were shaken well for 2 min. and left 10 min to get clear separation of two phases. It was noticed that the colored species formed in the case of amikacin with the reagent (Catechol- IO4-) is extractable in butanol but not into chloroform. The absorbance of the organic phase was measured at appropriate wave length against reagent blank. As solvent extraction did not give any additional advantage, it was excluded in further investigations. The studies on the influence of other water miscible (polar) solvents such as acetonitrile, methanol, t-butyl alcohol, or acetone instead of water
revealed that the aqueous medium was the best one for maximum color
Effect of Buffer: Aqueous medium without usage of any buffer (or Potassium acid phthalate buffer,3.4-4.0) was found suitable in the determination of amikacin with catechol-per iodate. Stability of Color: The influence of time for maximum color development and stability of the colored species formed between amikacin, catechol, per iodate was found to be 5 min. and the results are incorporated in Table-II.5.1 Table-II.5.1 Experimental conditions
Optimum conditions Catechol
Time for max. Color in min
Stability of Color in min
Optical Characteristics. Adherence to Beer’s Law: In order to test whether the amikacin-catechol-per iodate system
varying amounts of amikacin, specified concentrations of catechol and sodium meta per iodate (Table-II.5.1) were measured against reagent blank on a spectrophotometer. The linearity of the plot between absorbance and the concentration range specified in Table-II.5.1 shows that the color
adheres to Beer’s, the absorbance at λmax of a set of solutions containing
system obeys Beer’s law, Fig-3.5.1. Beer’s law limits, molar absorptivity, optimum photometric range, and Sandell’s Sensitivity values were calculated and the results are incorporated in Table-III.5.1
0.1 0.05 0 0
Fig-3.5.1 : Beer’s Law Plot for Amikacin
Table – III.5.1 : Optical Characteristics
Molar Absorptivity Lt/mol/cm
Sandell’s Sensitivity µg/cm2/0.001 absorbance units
Optimum Photometric Range µg/25 ml
4.65 X 103
56 - 316
Beer’s Law Range µg/25 ml
In view of all the observations it is felt that the following procedure for the spectrophotometric assay of amikacin using catechol, (or AAP) and oxidizing agent will be highly suitable for routine analysis.
Assay procedure: For Amikacin-using Catechol-IO4- : Aliquots ranging from 1-4 mL of the working standard solution of Amikacin along with 1 mL of catechol solution and 1 mL of sodium per iodate solution were added to a series of 10 mL graduated test tubes and the tubes were kept aside at room temperature for 5 min. Appropriate quantities of distilled water was added to each tube to make the volume. The absorbance of red colored complex formed was measured at 460 nm against reagent blank, prepared in a similar manner. The amount of amikacin was read from calibration curve prepared with its standard solution under identical conditions.
Precision and accuracy: The precision and accuracy of the methods in the determination of
containing a final concentration value, approximately ¾ of Beer’s range. The % relative standard deviations and confidence limits (0.05 and 0.01 levels) are presented in Table-IV.5.1.
amikacin was tested by measuring the absorbance of six replicates each
Table.IV.5.1.Precesion and Accuracy. Amikacin
Amount of Drug * Taken Found mg mg
% Range of Error 95% 99% Confidence Confidence Limit Limit
*Average of six replicates
The accuracy of the methods was determined by taking known different amounts (within Beer’s law range) of amikacin and estimating these amounts with the proposed methods. The results are incorporated in TableIV.5.1. The accuracy of the method was further tested in injections with proposed and reported methods. The results of these estimations are incorporated in Table-V.5.1 Table-V.5.1 Analysis of Formulations-Recovery Experiments.
Mean of % amount found Reported Proposed method method
% Recovery Experiments Amount %Recovey added
Amikacn injection Amikacn injection
Labeled Amount mg
RESULTS AND DISCUSSION As mentioned on page 5.7, AAP-IO4- is behaving in a similar manner like catechol-IO4- in case of λmax, of course with a negligible improvement in color intensity. However the mechanism of color formation is different in case of catechol-IO4- and AAP-IO4- reagent systems. Based on the results furnished in Tables II.5.1—V.5.1 reveal that the method proposed for the spectrophotometric determination of amikacin is simple, rapid, sensitive and specific with reasonable precision and accuracy. The proposed method appears to be superior to many of the reported methods and so it can be employed in routine determinations.
Catechol undergoes oxidation in the presence of mild oxidizing agent per iodate to form o-benzo quinone. This o-benzo quinone undergoes coupling with the – amino group of amikacin to form red colored chromogen. The oxidative coupling reaction can be represented as follows.
Red colored indo-dye As AAP contains electron withdrawing group,- CO-CH3 in para position to aromatic amine, AAP-IO4- can successfully be used for the estimation of amikacin. The failure of resorcinol (or pyrogellol) to develop color with all the proposed pairs of reagents may be due to the less reactive nature of its oxidative product, m-benzo quinone, and so it does not
undergo coupling reaction giving indo dye.
Conclusion. Hence the author concludes that the proposed spectrophotometric method is sensitive and reproducible for the analysis of Amikacin in pharmaceutical dosage forms with short analysis time.
DEVELOPMENT AND VALIDATION OF AMIKACIN BY RP-HPLC METHOD
Experimental Results and Discussion
Materials and Methods. Instrumentation. The author attempted to develop a liquid chromatographic method for the quantitative estimation of Amikacin. A Scimadzu HPLC equipped with a Luna C18 column (250 nm X 4.6nm,5µ) an LC 20 AD pump and a SPD 20 AD UV- Visible detector was employed in this study. Chromatographic analysis and data acquision was monitored by using Spinchrome software. A 20 µL Hamilton syringe was used for sample injection. Degassing of the mobile phase was done by using a spectra lab.DGA 20A3 Ultra sonic bath sonicator. A Shimadzu electronic balance was used for weighing the materials. The reference samples of Amikacin was supplied by Venus Remedies Limited, India
and the branded
formulations of Amikacin (AMISTAR and AMLTOP) were used.
Chemicals and Solvents. Methanol – HPLC grade (Merck Ltd, Worli ,Mumbai), Acetonitrile-HPLC
Fine Chemicals, Chennai) were used.
grade(Merck Ltd,Worli,Mumbai) ortho-phosphoric acid HPLC grade (SD
Preparation of mobile phase and stock solutions. A mobile phase is a mixture of acetonitrile, methyl alcohol, and ophosphoric acid, OPA, (0.1%) in 50:35:15 (v/v) which was prepared by mixing 500 mL of acetonitrile, 350 mL of methyl alcohol, and 150 mL of 0.1% o-phosphoric acid in one Liter flask. This mixture was used as a diluent for preparing working standard solutions of the drug.
About 100mg of Amikacin was weighed accurately and transferred into a 100 mL volumetric flask containing 20 mL of mobile phase. The solution was sonicated for 20 min. and then the volume was made up with a further quantity of mobile phase to get 1 mg/mL solution. This solution was suitably diluted with mobile phase to get a working standard solution of 100µg/mL of Amikacin.
Optimization of Chromatographic Conditions. Method Development: A systematic study was followed for developing the method for optimization of chromatographic conditions. This was carried out by varying one parameter keeping the other conditions constant
Column: A non polar C18, 250 nm x 4.6 nm column was chosen as the stationary phase for this study.
at an instant of time.
Mobile Phase: In order to get sharp peak and base line separation of the components, the author has carried out a number of experiments by varying the commonly used solvents with different compositions and its flow rates. In order to establish ideal separation of the drug under isocratic conditions mixtures of commonly used solvents like water, methanol, acetonitrile, o-phosphoric acid with or without different buffers, in different combinations were tested as mobile phases on a C18 stationary phase.
A mixture of acetonitrile, methyl alcohol, and o-phosphoric acid, OPA, (0.1%) in 50:35:15 (v/v) was proved to be the most suitable of all the combinations since the chromatographic peaks obtained were well defined, resolved and free from tailing. A flow rate of 1.0 mL/min mobile phase was found to be suitable in the studied range of 0.5—1.5 mL/min.
Wave length: The spectra of diluted solutions of Amikacin in methanol were record on UV spectrophotometer. The peaks of maximum absorbance wavelengths were observed. The spectra of Amikacin showed a balanced wavelength at 272 nm.
min was obtained for Amikacin. A typical model chromatogram showing the separation of Amikacin is presented in Fig.1.5.2.
Retention Time: Under the above optimized conditions a retention of 9.6
After a thorough study of the various parameters the following optimized conditions mentioned in Table-I.5.2 were followed for the determination of Amikacin in bulk samples and pharmaceutical formulations.
Fig-.1.5.2 Chromatogram of Amikacin
HPLC Report A.P.IAMIKACIN
• CONCENTRATION-25µg/ml • RETENTION TIME (R.T)-9.6 min • AREA--945655.8 • THEORETICAL PLATES-46721.5 • WAVE LENGTH--272nm • MOBILE PHASE-MeOH 35%:ACN,50%: (0.1%)OPA,15% • COLUMN--C18 • FLOW RATE-1.0 ml/min • RUN TIME--12 min • PH-- 4.6 • LINEARITY RANGE--1.05.0µg/ml
Table.1.5.2. HPLC Report of amikacin
Linearity and Construction of Calibration Curve The quantitative determination of the drug was accomplished by the external standard method. The mobile phase was filtered through a 0.45µ membrane filter before use. The flow rate of the mobile phase was adjusted to 1.0 ml/min. The column was equilibrated with mobile phase for at least 30 min. prior to injection of the drug solution. The column temperature was maintained at 25±10C throughout the study. Linearity of the peak area response was determined by taking six replicates at seven concentration points. Working solutions of Amikacin (range 100µg/ml) were prepared by diluting 10ml volumetric flasks with mobile phase. 20 microliters of the dilution was injected six times into the column. The drug in the eluents was monitored at 272 nm and corresponding chromatograms were obtained. The mean peak areas were noted from the chromatograms and a plot of concentrations over the peak areas was constructed. The regression of the plot was computed by least square method. The linearity was found to be in the range of 1—5 µg/mL between the concentration of Amikacin and peak area response. This regression equation was later used to estimate the amount of Amikacin in pharmaceutical dosage forms. The linearity was
linearity plot are reported in Tables II.5.2 and III.5.2
shown in Fig.2.5.2 and the linearity data and statistical parameters for
200000 150000 100000 50000 0 0
Fig 3.5.2 Linearity Graph of amikacin Table – II.5.2: Linearity of Amikacin by the proposed HPLC method. CONCENTRATION µg/mL
Table III.5.2:Regression Characteristics of the linearity plot of Amikacin VALUE
VALIDATION OF THE PROPOSED METHOD The method was validated in compliance with guidelines of International Conference on Harmonization (ICH). The following parameters were determined for validation. Specificity: The specificity of the method was assessed by comparing
chromatograms obtained from the drug with the most commonly used excipients mixture with those obtained from the blank solution. The blank solution was prepared by mixing the excipients in the mobile phase without the drug. The drug to excipient ratio used was similar to that in the commercial formulations. The commonly used excipients in formulations like lactose, microcrystalline cellulose, ethyl cellulose, hydroxyl propyl methyl cellulose, magnesium stearate and colloidal silicon di oxide were used for the study. The mixtures were filtered through 0.45µ membrane filter before injection. An observation of chromatograms indicates absence of excipients peaks near the drug peak in the study runtime. This indicates that the method is specific. Precision:
normal operational conditions. The precision of the method was studied in terms of repeatability in intra-day assay and inter-day assay (intermediate precision). Method repeatability was studied by repeating the assay three
Precision is the degree of repeatability of an analytical method under
times in the same day for intra-day precision, and intermediate precision was studied by repeating the assay on three different days, three times each day(inter day precision). The intra day and inter day variation for determination of Amikacin was carried out at four different concentrations. %RSD values are presented in the Table-IV.5.2 shows that the method provides acceptable (<2) intra day and inter day variation.
Table-IV.5.2 Intra and Inter-Day Precision Intra-Day Precision
Concentration Mean % Mean % of Amikacin amount amount %RSD amount amount %RSD µg/mL found found found found 40
Accuracy: Accuracy of the method is evaluated by standard addition method. An amount of the pure drug at three different concentrations in its solution has
sample solutions were analyzed in triplicate at each level as per the proposed method. The percent individual recovery and %RSD for recovery at each level are calculated. The results are tabulated (Table-V.5.2). A
been added to the pre analyzed working standard solution of the drug. The
mean recovery ranged from 99.53 – 99.90 has been obtained by the method indicates its accuracy. Table-V.5.2 Accuracy Data Amount taken mg 1.0
Robustness: A study was conducted to determine the effect of deliberate variations in the optimized chromatographic condition of the mobile phase, flow rate, and the pH of the mobile phase. The effect of these changes on the system suitability parameters like tailing factors, the number of theoretical plates, and on assay was studied. A single condition was carried at a time keeping
allowed limits indicate that the method is robust.
Variation in composition of mobile phase: The effect of variation in percent organic content in mobile phase was evaluated by changing the
all other parameters constant. The results were found to be within the
composition of organic component in the mobile phase. The tailing factor and the number of theoretical plates showed a little change with change in mobile phase composition. The values are presented in Table-VI.5.2.
Variations in flow rates—A study was conducted to determine the effect of variation in flow rate. The system suitability parameters were evaluated at 0.9 ml/min and 1.1 ml/min. The results were within the acceptance criteria. Hence the allowable variation in flow rate is 1.0 mL/min. Table - VI.5.2 Results of Robustness Study Variation of Mobile Phase
Tailing factor 1.83
Theoretical plates 46650
Stability of the analytical solution: A study to establish bench to top stability of the drug solution was performed. A freshly prepared working standard solution (25µg/mL of the drug) was analyzed immediately at different time intervals. The tailing factor theoretical plates, and the
the results are given in Table-VII.5.2. A maximum difference of 0.361% in the assay at the end of 24 hours was observed. The difference in percent assay meets the acceptance standard. The above study concludes that the standard drug solution is stable for twenty four hours on bench top.
difference in percent assay at different time intervals were calculated and
Table—VII.5.2 Stability of Standard solution. AMIKACIN Time in hours
Limit of Detection and Limit of Quantification. Limit of detection (LOD) is defined as the lowest concentration analyte that gives a measurable response. LOD is determined based on signal to noise ratio (S/N) of three times typically for HPLC methods.
LOD = 3.3X S.D of y intercept÷ Slope of Calibration curve
The limit of quantification (LOQ) is defined as the lowest concentration that can be quantified reliably with a specified level of accuracy and precision. It is the lowest concentration at which the precision expressed by RSD of less than 2%.
In this study the analyte response is 10 times greater than the noise response. For this study six replicates of the analyte at lowest concentration in the calibration range were measured and quantified. The LOD and LOQ 5.28
LOQ = 10X S.D of y intercept÷ Slope of Calibration Curve
of Amikacin obtained by the proposed method were 20 and 65 µg/mL respectively. (Table-VIII.5.1)
Table-VIII.5.2 LOD and LOQ of Amikacin Parameter
System Precision and System Suitability: System precision and system suitability studies were carried out by injecting six replicates of the working standard solution. The % RSD for the peak areas obtained was calculated. The data presented in Table VIII.5.2 reveals that %RSD is <1 and establishes reproducible performance of the instrument. The system suitability parameters are presented in Table-IX.5.2. Table- IX.5.2. System Precision
Theoretical Plates 46413
Estimation of the drug from dosage forms. In view of the satisfactory results obtained with the method development for the assay of Amikacin, the author has attempted its applicability for the estimation of the drug in its formulations. Ten tablets of Amikacin were weighed and powered into uniform size in a mortar. An average weight of a tablet was calculated from this powder. An accurately weighed portion from this powder equivalent to 100mg of Amikacin was transferred to 100mL volumetric flask containing 20 mL of mobile phase. The contents of the flask were sonicated for about 20 min. for complete solubility of the drug and the volume was made up to 100 mL with water. Then the mixture was filtered through 0.45µ membrane filter 4mL of above solution was taken into a separate 100 mL volumetric flask and made up to the volume with mobile phase and mixed well. The above solution (20µL) was then injected six times into the column. The mean peak area of the drug was calculated and the drug content in the formulation was calculated by the regression equation of the method. The results of the recovery are tabulated. The percent recovery was reported in Table-X.5.2.
Labeled Amount Amount found %Recovery
Table-X.5.2.Analysis of formulations & Recovery experiments
RESULTS AND DISCUSSION The present study was a
humble presentation of the author in
developing a sensitive, precise and accurate HPLC method for the analysis of Amikacin in bulk drug and pharmaceutical dosage forms. In order to affect analysis of the component peaks, mixtures of acetonitrile with phosphate buffer in different combinations were tested as mobile phase on a C18 stationary phase. A mixture of acetonitrile, methyl alcohol, and ophosphoric acid ,OPA, (0.1%) in 50:35:15 (v/v) was proved to be the most suitable of all combinations since the chromatographic peaks were better defined and resolved and almost free from tailing. The retention time obtained for Amikacin was 12 min.
Each of the samples was injected six times and the same retention times were obtained in all cases. The peak areas of Amikacin were reproducible as indicated by low coefficient of variation. A good linear relationship (r = 0.995) was observed between the concentration of Amikacin and the respective peak areas. The regression curve was
was Y=38820.9X-0.03 where Y gives peak area and X is the concentration of the drug. The regression characteristics are given in Table II.5.2. When Amikacin solutions containing 40,80,120,160 µg/mL was analyzed by the proposed method for finding out intra and inter day variations, low % RSD
constructed by linear regression fitting and its mathematical expression
was observed. High recovery values obtained from the dosage form by the proposed method indicates the method is accurate. The absence of additional peaks indicates non interference of common excipients used in the tablets. The drug content in tablets was quantified using the proposed analytical method. The tablets were found to contain an average of
the labeled amount of the drug. The deliberate changes in the method have not much affected the peak tailing theoretical plates and percent assay. This indicates that the present method is robust. The lowest values of LOD and LOQ as obtained by the proposed method indicate the method is sensitive. The standard solution of the drug was stable up to 24 hours as the difference in percent assay is within acceptable limit. System suitability parameters were studied with six replicates standard solution of the drug and the calculated parameters are within the acceptance criteria. The tailing factor and the number of theoretical plates are in the acceptable limits.
Hence the author concludes that the proposed HPLC method is sensitive and reproducible for the analysis of Amikacin in pharmaceutical dosage forms with short analysis time.
REFERENCES 1. Juan Manuel Serrano and Manuel Silva, Journal of Chromatography A (2006), 1117, # 2,176-183. 2. Lorena Baietto, Analytical and Bioanalytical Chemistry,396, #2, 791798. 3. Gallon, O; Guillet-Caruba, C.; Lamy, B.; Laurent, F.; Doucet-Populaire, F.; Decousser, European Journal of Clinical Microbiology & Infectious
Diseases,( 2009), 28, 10, 1209-1215. 4. Baietto L, D'Avolio A, De Rosa FG, Garazzino S, Michelazzo M, Ventimiglia G, Siccardi M, Simiele M, Sciandra M, Di Perri G. Anal Bioanal Chem.( 2010),396(2):791-8. E.pub (2009) Nov 9. 5. Yokoyama yoko HPLC Annals of Gunma University School of Health Sciences,. (2005) 25;183-189. 6. Grazyna Ginalska, Dorota Kowalczuk,Monika Osińska
Journal of Pharmaceutics (2007); 339(1-2):39-46. 7. Sa Nicoli; Patrizia Santi (pp.). Journal of Pharmaceutical and Biomedical Analysis .41, #3, 994-997. 8. Gordana Brajansoki J. Chromatogr. B, (2008), 867 149-152.
Waraska and Bruce Corona® ultra Detection ,Doinex online
9. Christopher Crafts, Marc Plante, Ian Acworth, Paul Gamache, John
10. A. Papp Catherine A. Knuppa and Rashmi H. Barbhaiya Journal of Chromatography B: Biomedical Sciences and Applications, (1992) 574, # 1, 93-99. 11. Burkhard Wichert, Hans Schreier and Hartmut Derendorf Journal of Pharmaceutical and BiomedicalAnalysis , (1991)Volume 9, Issue 3, 251. 12. Juan Manuel Serranoa and Manuel Silva Journal of Chromatography B, (2006), 843, # 1, 20-24. 13. C. H. Feng, S. J. Lin, H. L. Wu and S. H. Chen Chemistry and Materials Science Chromatographia 53, Supplement 1.