Sie sind auf Seite 1von 21
1
1

PONTIFÍCIA UNIVERSIDADE CATÓLICA DO PARANÁ CAMPUS TOLEDO CURSO DE FARMÁCIA

BRUNNA RICCI FALCÃO

DESENVOLVIMENTO E VALIDAÇÃO DE METODOLOGIA DE DISSOLUÇÃO DE COMPRIMIDOS REVESTIDOS DE DESLORATADINA POR ESPECTROFOTOMETRIA NO ULTRAVIOLETA

TOLEDO

2016

1
1

BRUNNA RICCI FALCÃO

DESENVOLVIMENTO E VALIDAÇÃO DE METODOLOGIA DE DISSOLUÇÃO DE COMPRIMIDOS REVESTIDOS DE DESLORATADINA POR ESPECTROFOTOMETRIA NO ULTRAVIOLETA

Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia da Pontifica Universidade Católica do Paraná, como requisito parcial à obtenção do titulo de bacharel em farmácia.

Orientador: Prof. Msc. Tiago Rafael Sausen.

TOLEDO

2016

2
2

BRUNNA RICCI FALCÃO

DESENVOLVIMENTO E VALIDAÇÃO DE METODOLOGIA DE DISSOLUÇÃO DE COMPRIMIDOS REVESTIDOS DE DESLORATADINA POR ESPECTROFOTOMETRIA NO ULTRAVIOLETA

Trabalho de Conclusão de Curso apresentado ao Curso de Graduação em Farmácia da Pontifícia Universidade Católica do Paraná, como requisito parcial a obtenção do título de bacharel em Farmácia.

COMISSÃO EXAMINADORA

_____________________________________ Msc. Tiago Rafael Sausen Pontifícia Universidade Católica do Paraná

_____________________________________ Dr. Inara Staub Prochnau Pontifícia Universidade Católica do Paraná

_____________________________________ Dr. Liberato Brum Júnior Prati – Donaduzzi

Toledo, Outubro de 2016.

Artigo:

3

Artigo: 3 Development and validation of a dissolution UV spectrophotometric method for desloratadine coated tablets Artigo

Development and validation of a dissolution UV spectrophotometric method for desloratadine coated tablets

Artigo submetido ao Latin American Journal of Pharmacy

4

4 Development and validation of a dissolution UV spectrophotometric method for desloratadine coated tablets Brunna R.

Development and validation of a dissolution UV spectrophotometric method for desloratadine coated tablets

Brunna R. FALCÃO 1 , Letícia M. TEIXEIRA 1 , Francine Z. PHILIPPSEN 1 , Tiago R. SAUSEN 1 *

  • 1 Curso de Farmácia, Pontifícia Universidade Católica do Paraná, campus Toledo, Brasil.

*Correspondence: Tiago Rafael Sausen, Pontifícia Universidade Católica do Paraná – PUCPR, Av. da União, 500, 85902–532, Toledo/PR, Brasil. Tel: +55 (45) 3277 8600. E-mail: tiago.sausen@pucpr.br

ABSTRACT.

5

ABSTRACT. 5 The aim of this work was the development and validation of a dissolution methodology

The aim of this work was the development and validation of a dissolution methodology for desloratadine-coated tablets by spectrophotometry on ultraviolet (UV). Based on the physiological conditions of the body, 0.1 M hydrochloric acid (HCl), pH 4.5-citrate buffer and pH 6.8 phosphate buffer were tested as dissolution medium. In addition, influences of apparatus, and rotation speed were evaluated. After an UV scan spectrum from 500 to 200 nm, in order to determine the maximum wavelength absorbance, samples were analyzed by UV visible spectrophotometric method. The better dissolution profile obtained for desloratadine tablets was 0.1 M HCl as dissolution medium, using USP type II (paddles) as apparatus is a speed of 100 rpm, with analysis at wavelength of 280 nm, resulting in a discriminating dissolution method successfully developed. Samples were analyzed by UV spectrophotometric method and validated as per ICH guidelines, showing specificity, linearity, precision and accuracy.

Key words: desloratadine, dissolution, validation.

INTRODUCTION

6

INTRODUCTION 6 The dissolution tests allow to obtain information regarding the drug biological availability, being recognized

The dissolution tests allow to obtain information regarding the drug biological

availability, being recognized also as an important tool to pharmaceutical companies

on development and quality control, because helps on the research and development

to guide the best formulation and ensure the lot-to-lot quality of pharmaceutical

dosage forms or for justifying post-approval product changes such as in the

formulation and the manufacturing process 1, 2, 3, 4, 5. 6, 7, 8, 9 .

Dissolution is the only test that addresses product performance, therefore

when developing a dissolution methodology, the knowledge related to solubility,

permeability, and pharmacokinetics of the drug should be considered and the test must

be capable to display the maximum discriminative profile and also to allow to detect

any deviation in quality standards initially proposed. The results from dissolution

assays provides important information, including to establish in vitro/in vivo

correlation 2, 7, 10, 11, 12, 13, 14

.

Methodology

validation

enables

to

know

limitations

and

reliability

of

measurements performed. Validation process is essential to ensure that the analytical

method is suitable for the intended purpose, demonstrating reliable information

through parameters such as specificity, linearity, accuracy and precision suitable for

analysis 2, 15, 16, 17, 18, 19, 20, 21

.

Desloratadine (8Chloro-11-piperidin-4-ylidene-6,11-diydro-5H-benzo

[5,6]cyclohepta[1,2-b]pyridine) an antihistamine drug used for the relief of symptoms

of seasonal allergic rhinitis, perennial (non-seasonal) allergic rhinitis by blocking the

action of histamine, is commercially available in Brazil by the brand name of

Desalex ® as coated tablets with a labeled amount of 5 mg in boxes with 20 tablets

7

7 each, and also available as 0.5 mg/mL syrup. Desloratadine is classified, according to Biopharmaceutical Classification

each, and also available as 0.5 mg/mL syrup. Desloratadine is classified, according to

Biopharmaceutical Classification System (BCS) as a Class I drug 21, 22, 23 .

Therefore, this paper describes the development and validation of a dissolution

test for desloratadine coated-tablets using a simple, fast and inexpensive ultraviolet

method. The development and validation were carried out in compliance with the

International Conference on Harmonization 17 .

EXPERIMENTAL

Materials

All chemicals and reagents were of analytical reagent grade. Desloratadine

reference substance (99.72 %) was kindly donated by the pharmaceutical company

Prati–Donaduzzi (Toledo, Brazil). The reference drug product (Desalex ® ), labelled as

containing 5 mg of desloratadine and the following excipients (dibasic calcium

phosphate, microcrystalline cellulose, starch, talc, hypromellose, titanium dioxide,

polyethylene glycol, white wax and carnauba wax) were obtained commercially.

Instrumentation

Equipment and instruments used in the present study were analytical scale

(Gehaka, AG–200 model), dissolution test apparatus (Nova Ética, 301-6 AUT model),

ultrasonic bath (Químis, Q355D model) and UV spectrophotometer (UV-1600 Pró–

Análise).

Methodology

Maximum wavelength absorption determination

8

Methodology Maximum wavelength absorption determination 8 It was prepared three different standard solutions with 5.56 μg/mL

It was prepared three different standard solutions with 5.56 μg/mL of

desloratadine, and each solution were prepared in the following medium: 0.1 M

hydrochloric acid (HCl), pH 4.5-acetate buffer and pH 6.8 phosphate buffer. The

samples were submitted to an UV scan spectrum between 500 to 200 nm in order to

determinate desloratadine maximum wavelength absorption.

Dissolution test conditions

Dissolution testing of tablets was perfomed with the reference drug product

(Desalex ® 5 mg), to define the method conditions. Initially, in order to determinate the

more discrimating dissolution medium, using paddles (USP apparatus II) at a stirring

speed of 100 rpm, a volume of 900 mL of the following dissolution media, pre-heated

to 37 °C ±0.5°C, were tested: 0.1 M HCl; pH 4.5-citrate buffer and pH 6.8–phosphate

buffer. Manual sampling aliquots of 20.0 mL were withdrawn at 5, 10, 15, 30 and 60

minutes, filtered in a Millex ® filter and analyzed on a UV/VIS Spectrophotometer

(280 nm). There was no sample dilution nor medium replacement after the sampling.

The desloratadine standard solution was prepared in order to obtain a final

concentration of 5.56 μg/mL.

With the results of the dissolution medium, tests to determinate the apparatus

(paddles and baskets) and rotation speed (50 and 100 rpm for paddles; 75 and 100

rpm for baskets) were performed using as dissolution medium 0,1 M HCl maintained

at 37 ± 0.5ºC. Again, samples of 20.0 mL were withdrawn manually at 5, 10, 15, 30,

45 and 60 minutes, filtered (Millex ® filter) and analyzed on a UV/VIS

Spectrophotometer (280 nm), along with a desloratine standard solution in 5.56

μg/mL final concentration.

9

9 To perform the filter evaluation, tests were performed in the mentioned dissolution mediums and the

To perform the filter evaluation, tests were performed in the mentioned

dissolution mediums and the samples withdrawn were not filtered. Filtration of

dissolution samples is usually necessary to prevent undissolved drug particles from

entering the analytical sample and removes insoluble excipients that otherwise cause

high background or turbidity. The filter evaluation is necessary to verify whether it

can be used in the dissolution test without adsorption of the drug into the filter 24 .

Validation

In order to demonstrate the method’s suitability for use as a dissolution test, it

was validated based on specificity, linearity, precision and accuracy parameter 16, 25 .

Specificity

Placebo samples of desloratadine, a mix of the excipients from the

desloratadine reference drug tablets (Desalex ® ) were prepared in their usual

compositions, according to literature 22, 26 . The placebo samples were transferred to

different vessels (n=6) with 900 mL of 0.1 M HCl as dissolution medium at 37 °C ±

0.5 °C and stirred for 1 h at 100 rpm using a paddle (USP apparatus II). Aliquots of

these solutions were filtered through a Millex ® filter and analyzed by the UV method

(280 nm).

Linearity

Desloratadine stock solutions at 55.55 mg/mL, using 0.1 M HCl as solvent,

were prepared. Aliquots of this solution were transferred to volumetric flasks to obtain

final concentrations of 1.11, 2.78, 4.17, 5.56, 6.94 and 8.33 µg/mL. Each solution was

prepared in triplicate. The linearity was evaluated by linear regression analysis, which

was calculated by the least squares regression method and analysis of variance

(ANOVA).

Precision

10

Precision 10 Precision was evaluated on two levels, repeatability and intermediate precision. The evaluation of intermediate

Precision was evaluated on two levels, repeatability and intermediate

precision. The evaluation of intermediate precision (inter-day precision) of the

dissolution test was performed on three different days. The repeatability was

evaluated on the same day for intra-day precision in six vessels used for the

dissolution test, in a concentration of 5,56 µg/mL. The relative standard deviation

(RSD) from the results was calculated.

Accuracy

Accuracy was determinate by the recovery percentage of a known amount of

desloratadine reference substance added to a placebo solution. A recovery study was

conducted by adding known amounts of the desloratadine stock solution to the

dissolution vessels containing the placebo solution at 50 % (2,78 μg/mL), 100 % (5,55

μg/mL) and 150 % (8,25 μg/mL) of the nominal assay (5 mg). Each concentration

was prepared in triplicate and analyzed by the UV method at 280 nm.

RESULTS AND DISCUSSION

Dissolution method development

In order to determine which wavelength desloratadine shows UV absorption,

an UV scan spectrum between 500–200 nm was performed. The results demonstrated

that desloratadine showed a maximum absorption at 280 nm (Figure 1).

11

11 Figure 1. UV spectrum of desloratadine solution at 5.56 µg/mL in 0.1 M HCl During
11 Figure 1. UV spectrum of desloratadine solution at 5.56 µg/mL in 0.1 M HCl During

Figure 1. UV spectrum of desloratadine solution at 5.56 µg/mL in 0.1 M HCl

During the development of a dissolution methodology, different parameters

were evaluated. As dissolution method development for immediate release drugs must

be tested in dissolution mediums ranging physiological pH (1.2 to 6.8), the dissolution

medium selected 0.1 M HCl, pH 4.5-citrate buffer and pH 6.8–phosphate buffer were

evaluated in order to determinate which medium showed better discriminative

dissolution profile 27 . The results (Figure 2) demonstrated that pH 4.5-citrate buffer

and pH 6.8–phosphate buffer as dissolution medium showed dissolution profiles very

similar, with an initial dissolution around 70 % (71.64 % and 14.72 %, respectively).

However, both medium doesn’t allowed total desloratadine dissolution, since the final

drug dissolution was 74,76 % for pH 4.5-citrate buffer and 80,79 % for pH 6.8-

phosphate buffer.

12

12 Figure 2: Dissolution profiles of desloratadine tablets in 0.1 M HCl, pH 4.5-acetate buffer 4.5
12 Figure 2: Dissolution profiles of desloratadine tablets in 0.1 M HCl, pH 4.5-acetate buffer 4.5

Figure 2: Dissolution profiles of desloratadine tablets in 0.1 M HCl, pH 4.5-acetate buffer 4.5 and pH 6.8-phosphate buffer as dissolution mediums.

The dissolution medium where desloratadine showed complete dissolution,

was 0.1 M HCl. Also, the dissolution profile at five points (5, 10, 15, 30 and 60

minutes) showed that desloratadine dissolved more than 85 % in 15 minutes (Figure

2). As desloratadine is an antihistamine drug used to relieve allergy symptoms, it must

be ready to act in the body around 30 minutes after the drug intake 22 , a rapidly drug

dissolution is required for immediate-release dosage forms 16,26 . This results,

corroborates with the fact that desloratadine is classified as a Class I drug (high

solubility and high permeability) in Biopharmaceutical Classification System (BCS) 16 .

So, the choice of 0.1 M HCl as dissolution medium was due to its good solubility,

accessibility, low cost and the fact that it is a typical dissolution medium 18 .

13

13 Figure 3: Dissolution profile of desloratadine tablets in paddles (50 and 100 rpm) and baskets
13 Figure 3: Dissolution profile of desloratadine tablets in paddles (50 and 100 rpm) and baskets

Figure 3: Dissolution profile of desloratadine tablets in paddles (50 and 100 rpm) and baskets (75 and 100 rpm) using 0.1 M HCl as dissolution medium.

After the dissolution medium was determinate, apparatus type and speed were

tested. The most common dissolution conditions are basket (USP apparatus I) at 75 or

100 rpm as rotation speed and paddles (USP apparatus II) in a rotation of 50 or 100

rpm 13 . According with Figure 3, the use of baskets as apparatus doesn’t allowed a

complete desloratadine dissolution, since the drug dissolved at the end of the test was

92.32 % to baskets as 75 rpm and 96.15 % to baskets as 100 rpm. Meanwhile, paddles

apparatus, from both speeds (50 rpm and 100 rpm), showed dissolution profiles very

similar, with a fast dissolution at the first sampling point (around 90 % of drug

dissolver). At the end of the test, paddles at 50 rpm showed 98.08 % of drug

dissolution and paddles at 100 rpm, 99.90 % of dissolution. Thus, the conditions

selected were paddles (USP apparatus II) in a rotation of 100 rpm.

The results between the solutions (filtered or centrifuged) must be very

similar for acceptance of the filter. The evaluation of the filter demonstrated that there

14

14 was no significant drug adsorption, as the difference in values between the solutions was less

was no significant drug adsorption, as the difference in values between the solutions

was less than 2 % 27, 29 .

Based on the results, is possible to determinate that a good dissolution method

for desloratadine-coated tablets is using 900 mL 0.1 M HCl as dissolution medium

with paddles at 100 rpm, samples withdraw at 5, 10, 15, 30 and 60 minutes, filtered

and analyzed at spectrophotometer at 280 nm.

Dissolution method validation

Validation of an analytical method is defined as the process that establish that

performance characteristics of the procedure meets the requirements for the intended

analytical applications 16 . In order for the developed dissolution method meets the

requirements of the analytical applications, ensuring results reliability, it should be

validated.

The determinate specificity, which is the ability to assess unequivocally the

analyte in the presence of components that may be expected to be present 16 , analysis

of a desloratadine placebo solutions showed that the UV method suffer no

interference from the formulation of the tablet evaluated, demonstrating to be specific

(Figure 4). Since there was no interference from the excipients with the selected

wavelength (280 nm), UV can be used in order to quantify desloratadine. Analysis by

UV are usually used in quality control of pharmaceuticals because most of drugs

absorbed energy in UV region, and is a method, which does not require a complex or

expensive equipment, and there is no need for toxic solvents 19 . In addition, by using

UV method, results can be obtained faster, analysis is simpler and fewer solvents are

used, making this valuable in routine analysis 16, 20, 26, 30 .

15

15 Figure 4. UV spectrum of desloratadine and excipients solutions for specificity. Linearity of an analytical
15 Figure 4. UV spectrum of desloratadine and excipients solutions for specificity. Linearity of an analytical

Figure 4. UV spectrum of desloratadine and excipients solutions for specificity.

Linearity of an analytical method is its ability to elicit test results that are

directly proportional to the analyte concentration in samples within a given range 26 .

In order to assess linearity, three calibration curves of desloratadine were constructing

and plotted graphically as concentration (µg/mL) versus absorbance. The results

showed a good correlation coefficient (R 2 : 0.9994) in the studied concentration range

(1.11, 2.78, 4.17, 5.56, 6.94 and 8.33 µg/mL). Also, the representative linear equation

was y = 0,0432x + 0,0019 and the data were validated by means of the analysis of

variance (ANOVA), which demonstrated significant linear regression and no

significant linearity deviation (p < 0.01). According to the results, linearity was

proved because an appropriate linear correlation was found since the obtained

correlation coefficients showed values higher that 0.99 16, 24, 31, 32 .

The precision of an analytical procedure is the degree of agreement among

individual test results when the procedure is applied repeatedly to multiple samplings

of a homogeneous sample 15 . Precision of the method was determined by measuring

the repeatability and intermediate precision on the concentration of 5.56 µg/mL. The

16

16 0.71 and 1.58 % from each of the three days and an RSD of 1.04

0.71 and 1.58 % from each of the three days and an RSD of 1.04 % for inter-day

precision. As RSD values were lower than 5 %, the results indicated the good

precision of this method 16, 18, 25, 32 .

The accuracy of the analytical procedure, the accordance between the accepted

value and the value found 15 , was demonstrated by the recovery of known amounts of

desloratadine in the dissolution vessels. In the present study, three concentrations

were evaluated (2.78, 5.55 and 8.25 µg/mL) and each concentration was measured

three times. The recovery percentage found ranged from 100.16 to 102.04 %,

resulting in an average recovery of 101.37 % with a RSD of 0.90 %, indicating

method’s accuracy. As the measured recovery is typically 95–105 % 26 , the results

indicated good accuracy of the method. The recovery percentage was calculated in

triplicate and the mean value was considered 16, 31 .

CONCLUSIONS

A discriminative dissolution method to evaluate desloratadine tablets was

successfully developed and demonstrate to be an easy, fast and simple method. The

conditions allowing dissolution determination were 900 mL of 0.1 M HCl as

dissolution medium at 37.0 ± 0.5 ºC, using USP type II apparatus (paddles) at 100

rpm and analysis by spectrophotometric detection in a wavelength of 280 nm. The

spectrophotometric method was validated and showed to be specific, linear, precise

and accurate. The developed method is suitable for its purpose and could be applied in

routine quality control of desloratadine tablets since there is no official monograph

using spectrophotometric method for this drug in the pharmacopoeias.

Acknowledgements

17

Acknowledgements 17 The authors would like to thanks Fundação Araucária for the research’s scholarship to the

The authors would like to thanks Fundação Araucária for the research’s

scholarship to the first author.

REFERENCES

18

REFERENCES 18 1. Grady, L. T. (1996) Drug. Inf. J. Philadelphia. 30: 1063-70. 2. Marcolongo, R
  • 1. Grady, L. T. (1996) Drug. Inf. J. Philadelphia. 30: 1063-70.

  • 2. Marcolongo, R (2003). Dissolução de medicamentos: fundamentos, aplicações,

aspectos regulatórios e perspectivas na área farmacêutica. São Paulo: USP/

Faculdade de Ciências Farmacêuticas.

  • 3. Sausen T.R., C. Pavei, A.P.C. Silva, S. Fialho & P. Mayorga (2010) Lat. Am. J.

Pharm. 29: 719-24.

  • 4. Kulkarni A.P., S. Mohd, Z. Zahi, & M.H.G. Deghan (2012) Dissol Tech. 19: 36-47.

  • 5. Garcia, C.V., C.S. Paim, M. Steppe & E.E.S. Schapoval (2006) J. Pharm Biomed

Anal. 41: 833-837.

  • 6. Martins, M.T., C.S. Paim & M. Steppe (2010) Braz. J. Pharm. Scien. 46: 179-186.

  • 7. Dressman, J (2014) Dissol Tech. 21: 6-10.

  • 8. Fortunato, D (2005) Dissol Tech. 12: 12-14.

  • 9. Marroum, P.J (2014) Dissol Tech. 21: 11-16.

    • 10. Rossi, R.C., C.L. Dias, L. Bajerski, A.M. Bergold & P.E. Froehlich (2011) J.

Pharm, Biomed. Anal. 54: 429-444.

11. Shah,

R.,

S.

Patel, H. Patel,

S.

Pandey,

S.

Shah &

D. Shah.

(2011).

Braz. J.

Pharm. Sci. 47: 899-906.

  • 12. Martin G.P. & V.A Gray (2011). J. Valid. Tech. 17: 8-11.

  • 13. FDA. (1997) Guidance for Industry. Dissolution testing of immediate release solid

oral dosage forms.

  • 14. Manadas R., M.E. Pina & F. Veiga (2002) Rev. Bras. Ciên. Farm. 38: 375–399

  • 15. Brito N.M., O.P.A. Junior, L. Polese & M.L. Ribeiro (2003) Rev. Ecol. Amb. 13:

129-156.

  • 16. Brasil. (2003) Ministério da Saúde, Agência Nacional de Vigilância Sanitária.

Resolução Específica nº 899 de 29 de maio de 2003. Determina a publicação do

"Guia para validação de métodos analíticos e bioanalíticos".

19

19 17. ICH. (2005) International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals forSilva W.M, F.R. Santos & J .P. Batistuti (2013) Braz. J. Food. Nutr. 24: 275–282 31. ICH. (1995) International Conference on Harmonization of Technical Requirements for Registration of Pharmaceutical for Human use. Validation of Analytical Procedures: Methodology: Q2B. ICH Harmonized Tripartite Guideline. " id="pdf-obj-19-4" src="pdf-obj-19-4.jpg">

17.

ICH. (2005) International Conference on Harmonization of Technical

Requirements for Registration of Pharmaceuticals for Human Use: Guideline on

Validation of Analytical Procedures Q2 (R1).

 

18.

FDA. (2015) Analytical Procedures and Methods Validation for Drugs and

Biologics. Guidance for Industry.

 

19.

FIP. (1996) Guidelines for dissolution testing of solid oral products (Final draft,

1995). Drug Inf. J, Philadelphia, 30, 1071–84.

 

20.

Fonseca, L. B. (2007) Desenvolvimento e validação de método de dissolução

aplicado a suspensões orais de nimesulida. Rio de Janeiro: UFRJ / Faculdade de

Farmácia.

 

21.

USP. (2013) The United States Pharmacopoeia. Rockville, USA: United States

Pharmacopoeial Convention. 37 th Ed.

 

22.

Goodman, A. (2008) The Pharmacological Basis of Therapeutics. Elmsford, Ny:

McGraw-Hill.

 

23.

Amidon G. L., H. Lennernas, V.P. Shah & J.R. Crison (1995) Pharm. Res. 12:

413420.

 

24.

USP. (2014) The United States Pharmacopeia and National Formulary USP 37

NF 32; The United States Pharmacopeial Convention, Inc.: Rockville, MD.

 

25.

ICH.

(1996)

International

Conference

on

Harmonization

of

Technical

Requirements for Registration of Pharmaceutical for Human Use. Validation of

Analytical Procedures: Methodology: ICH4. ICH Harmonized Tripartite Guideline.

26.

Kibbe, A. Handbook of Pharmaceutical Excipients. 6th ed. Washington, DC:

American Pharmaceutical Association, 2009.

 

27.

Gibaldi, M. (1991) Biopharmaceutics and Clinical Pharmacokinetics. 4th ed.

Philadelphia: Lea & Febiger.

 

28.

Brazilian Pharmacopoeia. (2010) Brazilian Health Surveillance Agency: Brasília,

BR: Brasil. 5th ed.

 

29.

Priya M.B.V & T.E.G.K Murthy. (2012) Dissol. Tech.19: 38-42.

 

30.

Silva W.M, F.R. Santos & J.P. Batistuti (2013) Braz. J. Food. Nutr. 24: 275–282

31.

ICH.

(1995)

International

Conference

on

Harmonization

of

Technical

Requirements for Registration of Pharmaceutical for Human use. Validation of

Analytical Procedures: Methodology: Q2B. ICH Harmonized Tripartite Guideline.

20

20 32. Swartz M.E. & I.S Krull (1998) Pharm Tech . 12: 12-20.

32. Swartz M.E. & I.S Krull (1998) Pharm Tech. 12: 12-20.