Date Coming into Force: 02-Jun-2003 Link to IDRAC Amended Version

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RESOLUÇÃO-RE Nº 899, DE 29 DE MAIO DE 2003

O Adjunto da Diretoria Colegiada da Agência Nacional de Vigilância Sanitária, no uso

da atribuição, que lhe confere a Portaria n.º 238, de 31 de março de 2003,
considerando o disposto no art.111, inciso II, alínea “a” § 3º do Regimento Interno
aprovado pela Portaria nº 593, de 25 de agosto de 2000, republicada no DOU de 22 de
dezembro de 2000, considerando que a matéria foi submetida à apreciação da Diretoria
Colegiada, que a aprovou em reunião realizada em 6 de março de 2003, resolve:

Art. 1º Determinar a publicação do "Guia para validação de métodos analíticos e

bioanalíticos" anexo

Art. 2º Fica revogada a Resolução RE n o 475, de 19 de março de 2002.

Art. 3º Esta Resolução entra em vigor na data de sua publicação.

DAVI RUMEL
Date Coming into Force: 02-Jun-2003

ANEXO
GUIA PARA VALIDAÇÃO DE MÉTODOS ANALÍTICOS E BIOANALÍTICOS
MÉTODOS ANALÍTICOS

1. Considerações gerais
1.1. As informações contidas nesse Anexo apresentam as características a serem
consideradas durante a validação de procedimentos analíticos. O objetivo de uma
validação é demonstrar que o método é apropriado para a finalidade pretendida, ou seja,
a de-terminação qualitativa, semi-quantitativa e/ou quantitativa de fármacos e outras
substâncias em produtos farmacêuticos.
1.2. Essas informações aplicam-se a:
1.2.1. técnicas analíticas que façam uso de métodos de cromatografia gasosa (CG) ou
cromatografia líquida de alta eficiência (CLAE);
1.2.2. métodos nãocromatográficos, desde que estes ofereçam uma seletividade aceitável
(por ex. titulometria, espectrofotometria UV-VIS);
1.2.3. testes imunológicos ou microbiológicos, desde que observado o grau de
variabilidade usualmente associado a estas técnicas.
1.3. A validação deve garantir, por meio de estudos experimentais, que o método atenda
às exigências das aplicações analíticas, assegurando a confiabilidade dos resultados.
Para tanto, deve apresentar especificidade, linearidade, intervalo, precisão,
sensibilidade, limite de quantificação, exatidão, adequados à análise.

1.4. Deve-se utilizar substâncias de referência oficializadas pela Farmacopéia Brasileira

ou, na ausência destas, por outros códigos autorizados pela legislação vigente. No caso
da inexistência dessas substâncias, será admitido o uso de padrões de trabalho, desde
que a identidade e o teor sejam devidamente comprovados.
1.5. Para efeito desse guia, considera-se corrida analítica as medições sucessivas de um
mesmo analito, efetuadas nas mesmas condições: método, analista, instrumentação,
local, condições de utilização e em intervalo de tempo curto entre as medições.
1.6. No caso de metodologia analítica descrita em farmacopéias ou formulários oficiais,
devidamente reconhecidos pela ANVISA, a metodologia será considerada validada.
1.7. No caso de metodologia analítica não descrita em farmacopéias ou formulários
oficiais, devidamente reconhecidos pela ANVISA, a metodologia será considerada
validada, desde que sejam avaliados os parâmetros relacionados a seguir, conforme
especificado nas Tabelas 1 e 2.
1.7.1. Especificidade e Seletividade
1.7.2. Linearidade
1.7.3. Intervalo
1.7.4. Precisão
1.7.5. Limite de detecção (sensibilidade)
1.7.6. Limite de quantificação
1.7.7. Exatidão
1.7.8. Robustez
1.8. No caso da transferência de metodologias da matriz para suas subsidiárias no Brasil
e/ou das empresas nacionais para os centro de estudos de equivalência farmacêutica, a
metodologia será considerada validada, desde que sejam avaliados os parâmetros de pre-
cisão, especificidade e linearidade. Cópia de toda a documentação original da validação
da metodologia deverá ser anexada, como prova de que a metodologia foi originalmente
validada e deverá conter, no mínimo, todos os parâmetros relacionados no item 1.7.
Date Coming into Force: 02-Jun-2003

1.9. Para a garantia da qualidade analítica dos resultados, todos os equipamentos

utilizados na validação devem estar devidamente calibrados e os analistas devem ser
qualificados e adequadamente treinados.
1.10. Os testes são classificados em 4 categorias, conforme a Tabela 1.
Tabela 1. Classificação dos testes, segundo sua finalidade:

1.11. Para cada categoria será exigido um conjunto de testes, relacionados na Tabela 2.
Tabela 2. Ensaios necessários para a validação do método analítico, segundo sua
finalidade:

* pode ser necessário, dependendo da natureza do teste específico.

** se houver comprovação da reprodutibilidade não é necessária a comprovação da
Precisão
Intermediária.
1.12. metodologia analítica deverá ser revalidada nas seguintes circunstâncias:
1.12.1. mudanças na síntese da substância ativa;
1.12.2. mudanças na composição do produto acabado;
1.12.3. mudanças no procedimento analítico.
Determinadas outras mudanças podem requerer validação também, dependendo da
natureza das mudanças.
2. Metodologia
2.1. Especificidade e Seletividade
É a capacidade que o método possui de medir exatamente um composto em presença de
outros componentes tais como impurezas, produtos de degradação e componentes da
matriz.
2.1.1. Para análise qualitativa (teste de identificação) é necessário demonstrar a
capacidade de seleção do método entre compostos com estruturas relacionadas que
podem estar presentes. Isto deve ser confirmado pela obtenção de resultados positivos
(preferivelmente em relação ao material de referência conhecido) em amostras contendo
o fármaco, comparativamente com resultados negativos obtidos com amostras que não
contém o fármaco, mas compostos estruturalmente semelhantes.
2.1.2. Para análise quantitativa (teor) e análise de impurezas, a especificidade pode ser
de-terminada pela comparação dos resultados obtidos de amostras (fármaco ou
medicamento) contaminadas com quantidades apropriadas de impurezas ou excipientes
e amostras não contaminadas, para demonstrar que o resultado do teste não é afetado
Date Coming into Force: 02-Jun-2003

por esses materiais. Quando a impureza ou o padrão do produto de degradação não

estiverem disponíveis, pode-se comparar os resultados do teste das amostras contendo
impurezas ou produtos de degradação com os resultados de um segundo procedimento
bem caracterizado (por exemplo metodologia farmacopéica ou outro procedimento
validado). Estas comparações devem incluir amostras armazenadas sob condições de
estresse (por ex. luz, calor umidade, hidrólise ácida/básica, oxidação).
2.1.3. Em métodos cromatográficos, deve-se tomar as precauções necessárias para
garantir a pureza dos picos cromatográficos. A utilização de testes de pureza de pico
(por exemplo, com auxilio de detector de arranjo de fotodiodos ou espectrometria de
massas) são interessantes para demonstrar que o pico cromatográfico é atribuído a um
só componente.
2.2. Linearidade
É a capacidade de uma metodologia analítica de demonstrar que os resultados obtidos
são diretamente proporcionais à concentração do analito na amostra, dentro de um
intervalo especificado.
2.2.1. Recomenda-se que a linearidade seja determinada pela análise de, no mínimo, 5
concentrações diferentes. Estas concentrações devem seguir os intervalos da Tabela 3.
2.2.2. Se houver relação linear aparente após exame visual do gráfico, os resultados dos
testes deverão ser tratados por métodos estatísticos apropriados para determinação do
coeficiente de correlação, intersecção com o eixo Y, coeficiente angular, soma residual
dos quadrados mínimos da regressão linear e desvio padrão relativo. Se não houver
relação linear, realizar transformação matemática.
2.2.3. O critério mínimo aceitável do coeficiente de correlação (r) deve ser = 0,99.
2.2.4. Deve-se apresentar as curvas obtidas (experimental e a resultante do tratamento
matemático).
2.3. Intervalo
O intervalo especificado é a faixa entre os limites de quantificação superior e inferior de
um método analítico. Normalmente é derivado do estudo de linearidade e depende da
aplicação pretendida do método (Tabela 3). É estabelecido pela confirmação de que o
método apresenta exatidão, precisão e linearidade adequados quando aplicados a
amostras contendo quantidades de substâncias dentro do intervalo especificado.
Tabela 3. Limites porcentuais do teor do analito que devem estar contidos no intervalo
de linearidade para alguns métodos analiticos.

2.4. Precisão
Date Coming into Force: 02-Jun-2003

A precisão é a avaliação da proximidade dos resultados obtidos em uma série de

medidas de uma amostragem múltipla de uma mesma amostra. Esta é considerada em
três níveis.
2.4.1. Repetibilidade (precisão intra-corrida): concordância entre os resultados dentro de
um curto período de tempo com o mesmo analista e mesma instrumentação.
A repetibilidade do método é verificada por, no mínimo, 9 (nove) determinações,
contemplando o intervalo linear do método, ou seja, 3 (três) concentrações, baixa, média
e alta, com 3 (três) réplicas cada ou mínimo de 6 determinações a 100% da concentração
do teste;
2.4.2. Precisão intermediária (precisão inter-corridas): concordância entre os resultados
do mesmo laboratório, mas obtidos em dias diferentes, com analistas diferentes e/ou
equipamentos diferentes.
Para a determinação da precisão intermediária recomendase um mínimo de 2 dias
diferentes com analistas diferentes.
2.4.3. Reprodutibilidade (precisão inter-laboratorial): concordância entre os resultados
obtidos em laboratórios diferentes como em estudos colaborativos, geralmente aplicados
à padronização de metodologia analítica, por exemplo, para inclusão de metodologia em
farmacopéias. Estes dados não precisam ser apresentados para a concessão de registro.
A precisão de um método analítico pode ser expressa como o desvio padrão ou desvio
padrão relativo (coeficiente de variação) de uma série de medidas.
A precisão pode ser expressa como desvio padrão relativo (DPR) ou coeficiente de
variação (CV%), segundo a fórmula, em que, DP é o desvio padrão e CMD, a
concentração média determinada.
O valor máximo aceitável deve ser definido de acordo com a metodologia empregada, a
concentração do analito na amostra, o tipo de matriz e a finalidade do método, não se
admitindo valores superiores a 5%.
2.5. Limite de Detecção
Limite de detecção é a menor quantidade do analito presente em uma amostra que pode
ser detectado, porém não necessariamente quantificado, sob as condições experimentais
estabelecidas.
2.5.1. O limite de detecção é estabelecido por meio da análise de soluções de
concentrações conhecidas e decrescentes do analito, até o menor nível detectável;
2.5.2. No caso de métodos não instrumentais (CCD, titulação, comparação de cor), esta
determinação pode ser feita visualmente, onde o limite de detecção é o menor valor de
concentração capaz de produzir o efeito esperado (mudança de cor, turvação, etc).
2.5.3. No caso de métodos instrumentais (CLAE, CG, absorção atômica), a estimativa
do limite de detecção pode ser feita com base na relação de 3 vezes o ruído da linha de
base. Pode ser determinado pela equação, em que: DPa é o desvio padrão do intercepto
com o eixo do Y de, no mínimo, 3 curvas de calibração construídas contendo
concentrações do fármaco próximas ao suposto limite de quantificação.
Este desvio padrão pode ainda ser obtido a partir da curva de calibração proveniente da
análise de um número apropriado de amostras do branco; IC é a inclinação da curva de
calibração.
2.6. Limite de Quantificação
É a menor quantidade do analito em uma amostra que pode ser determinada com
precisão e exatidão aceitáveis sob as condições experimentais estabelecidas.
O limite de quantificação é um parâmetro determinado, principalmente, para ensaios
quantitativos de impurezas, produtos de degradação em fármacos e produtos de
Date Coming into Force: 02-Jun-2003

degradação em formas farmacêuticas e é expresso como concentração do analito (por

exemplo, porcentagem p/p ou p/V, partes por milhão) na amostra.
2.6.1. O limite de quantificação é estabelecido por meio da análise de soluções contendo
concentrações decrescentes do fármaco até o menor nível determinável com precisão e
exatidão aceitáveis.
Pode ser expresso pela equação, em que: DPa é o desvio padrão do intercepto com o
eixo do Y de, no mínimo, 3 curvas de calibração construídas contendo concentrações do
fármaco próximas ao suposto limite de quantificação.
Este desvio padrão pode ainda ser obtido a partir da curva de calibração proveniente da
análise de um apropriado número de amostras do branco; IC é a inclinação da curva de
calibração.
2.6.2. Também pode ser determinado por meio do ruído.
Neste caso, determina-se o ruído da linha de base e considerase como limite de
quantificação aquela concentração que produza relação sinal-ruído superior a 10:1.
2.7. Exatidão
A exatidão de um método analítico é a proximidade dos resultados obtidos pelo método
em estudo em relação ao valor verdadeiro.
Várias metodologias para a determinação da exatidão estão disponíveis:
2.7.1. Fármaco
2.7.1.1. aplicando-se a metodologia analítica proposta na análise de uma substância de
pureza conhecida (padrão de referência);
2.7.1.2. comparação dos resultados obtidos com aqueles resultantes de uma segunda
metodologia bem caracterizada, cuja exatidão tenha sido estabelecida;
2.7.2. Forma Farmacêutica
2.7.2.1. na análise de uma amostra, na qual quantidade conhecida de fármaco foi
adicionada a uma mistura dos componentes do medicamento (placebo contaminado);
2.7.2.2. nos casos em que amostras de todos os componentes do medicamento estão
indisponíveis, aceita-se a análise pelo método de adição de padrão, no qual adiciona-se
quantidades conhecidas do analito (padrão de referência) ao medicamento.
2.7.3. Impurezas
2.7.3.1. análise pelo método de adição de padrão, no qual adiciona-se quantidades
conhecidas de impurezas e/ou produtos de degradação ao medicamento ou ao fármaco;
2.7.3.2. no caso da indisponibilidade de amostras de certas impurezas e/ou produtos de
degradação, aceita-se a comparação dos resultados obtidos com um segundo método
bem caracterizado (metodologia farmacopéica ou outro procedimento analítico
validado).
A exatidão é calculada como porcentagem de recuperação da quantidade conhecida do
analito adicionado à amostra, ou como a diferença porcentual entre as médias e o valor
verdadeiro aceito, acrescida dos intervalos de confiança.
A exatidão do método deve ser determinada após o estabelecimento da linearidade, do
intervalo linear e da especificidade do mesmo, sendo verificada a partir de, no mínimo,
9 (nove) determinações contemplando o intervalo linear do procedimento, ou seja, 3
(três) concentrações, baixa, média e alta, com 3 (três) réplicas cada. A exatidão é
expressa pela relação entre a concentração média determinada experimentalmente e a
concentração teórica correspondente:
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2.8. Robustez
A robustez de um método analítico é a medida de sua capacidade em resistir a pequenas
e deliberadas variações dos parâmetros analíticos. Indica sua confiança durante o uso
normal.
Durante o desenvolvimento da metodologia, deve-se considerar a avaliação da robustez.
Constatando-se a susceptibilidade do método à variações nas condições analíticas, estas
deverão ser controladas e precauções devem ser incluídas no procedimento.
A Tabela 4 relaciona os principais parâmetros que podem resultar em variação na
resposta do método.
Tabela 4. Fatores que devem ser considerados na determinação da robustez do método
analítico.

MÉTODOS BIOANALÍTICOS
1. Definições
Amostra - termo geral que abrange: controles, brancos, amostras processadas e
desconhecidas.
Amostra branco - amostra de uma matriz biológica na qual nenhum analito foi
adicionado, utilizada para avaliar a especificidade do método bioanalítico.
Amostra de Controle de Qualidade (CQ) - amostra de matriz biológica adicionada do
analito, usada para monitorar o desempenho de um método bioanalítico e para avaliar a
integridade e validade dos resultados das amostras desconhecidas analisadas numa
corrida individual.
Amostra processada - extrato final (anterior à análise instrumental) de uma amostra que
foi submetida a várias manipulações (ex.: diluição, extração, concentração).
Amostra desconhecida - amostra biológica que é objeto de análise.
Analito - composto químico específico a ser mensurado, podendo ser o fármaco não-
transformado, biomolécula ou seu derivado, metabólito ou produto de degradação em
uma matriz biológica.
Corrida analítica (ou lote) - conjunto completo de amostras em estudo, com um número
apropriado de padrões e CQs para sua validação e que tem sua análise completa nas
mesmas condições.
Date Coming into Force: 02-Jun-2003

Especificidade - habilidade do método bioanalítico de medir e diferenciar o analito de

componentes que possam estar presentes na amostra, tais como metabólitos, impurezas,
compostos de degradação ou componentes da matriz.
Estabilidade - parâmetro que visa determinar se um analito mantém-se quimicamente
inalterado numa dada matriz sob condições específicas, em determinados intervalos de
tempo.
Exatidão - representa o grau de concordância entre os resultados individuais
encontrados e um valor aceito como referência.
Faixa de quantificação - corresponde a uma faixa de concentração, incluindo o LSQ e o
LIQ, que pode ser confiável e re-produtivelmente quantificada com exatidão e precisão,
por meio da relação concentração-resposta.
Limite de Detecção (LD) - menor concentração de um analito que o procedimento
bioanalítico consegue diferenciar confiavelmente do ruído de fundo.
Limite Inferior de Quantificação (LIQ) - menor quantidade de um analito numa amostra
que pode ser determinada quantitativamente com precisão e exatidão aceitáveis.
Limite Superior de Quantificação (LSQ) - maior quantidade de um analito numa
amostra que pode ser determinada quantitativamente com precisão e exatidão.
Linearidade - corresponde à capacidade do método de fornecer resultados diretamente
proporcionais à concentração da substância em exame (analito).
Matriz biológica - material distinto de origem biológica, que pode ser amostrado e
processado de modo reprodutível.
Resolution - RE n. 899, of May 29, 2003

The Deputy of the Collegiate Board of Directors of the Brazilian Sanitary Surveillance
Agency, in the use of the attribution vested in him by Administrative Order n. 238, of March
31, 2003,

WHEREAS

provided in Article 111, clause II, item “a” of paragraph 3 of the Bylaws approved by
Administrative Order 593, of August 25, 2000, re-published in the Federal Official Journal
of December 22, 2000

that the matter was submitted to the examination of the Collegiate Board of Directors,
which approved the matter in a meeting held on March 6, 2003, decides:

Article 1 - To determine the publication of the "Guide for validation of analytical and
bioanalytical methods”, attached.

Article 2 – Resolution RE n. 475, of March 19, 2002, is hereby revoked.

Article 3 - This Resolution enters into force on the date of its publication.

DAVI RUMEL

ANNEX

GUIDE FOR VALIDATION OF ANALYTICAL AND BIOANALYTICAL METHODS

1. General considerations

1.1. The information contained in this Annex presents the characteristics to be considered
during the validation of analytical procedures. The objective of a validation is to
demonstrate that the method is appropriate for the intended purpose, that is, the
qualitative, semi-quantitative and/or quantitative determination of drugs and other
substances in pharmaceutical products.

1.2. This information is applicable to:

1.2.1. analytical techniques that employ methods of gas chromatography (GC), high
performance liquid chromatography (HPLC);

1.2.2. non-chromatographic methods, as long as they provide acceptable selectivity (e.g.

titlemetry, UV-VIS spectrophotometry);

1.2.3. immunological or microbiological tests, as long as the degree of variability usually

associated to these techniques is observed.

1.3. The validation must guarantee, through experimental studies, that the method meets
the requirements of the analytical applications, ensuring the reliability of the results. For
this, it must present suitable specificity, linearity, range, precision, sensitivity, quantification
limit and accuracy.

1.4. Reference standard substances made official by the Brazilian Pharmacopoeia or, for
lack of it, by any other code authorized by the current legislation must be used. In the
absence of these substances, working standards will be admitted provided their identity
and levels are proved.

1.5. For the purpose of this guide, analytical run is the successive measurements of the
same analyte, carried out under the same conditions: method, analyst, instrumentation,
place, conditions of use and at short time interval between the measurements.

1.6. In the case of analytical methodology described in official pharmacopoeia or official

forms, duly recognized by ANVISA, the methodology will be considered validated.

1.7. In the case of analytical methodology not described in official pharmacopoeia or

official forms, duly recognized by ANVISA, the methodology will be considered validated,
provided the following parameters are evaluated, as specified in Tables 1 and 2.

1.7.1. Specificity and Selectivity

1.7.2. Linearity

1.7.3. Range

1.7.4. Precision

1.7.5. Detection limit (sensitivity)

1.7.6. Quantification limit

1.7.7. Accuracy

1.7.8. Robustness

1.8. In the case of the transference of methodologies from headquarters to subsidiary in

Brazil and/or of the national companies to the center of pharmaceutical equivalence
studies, the methodology will be considered validated, as long as the parameters of
precision, specificity and linearity are evaluated. A copy of all the original documentation
referring to methodology validation must be attached, as proof that the methodology was
originally validated and must contain, at least, all the parameters listed in item 1.7.

1.9. To ensure the analytical quality of the results, all the equipment used in the validation
must be properly calibrated and the analysts must be qualified and adequately trained.

1.10. The tests are classified in 4 categories, according to Table 1,

Table 1. Classification of tests, according to their purpose:

Category Test purpose

Quantitative tests for determination of active principle
I
in pharmaceutical products or raw-materials.
Quantitative tests or limit assay for determination of
II impurities and degradation products in
pharmaceutical products or raw-materials.
Performance tests (e.g. dissolution, release of active
III
principle)
IV Identification tests

1.11. For each category a set of tests, listed in Table 2, will be demanded:

Category II
Category Category
Parameter Category I Limit
Quantitative III IV
assay
Specificity Yes Yes Yes * Yes
Linearity Yes Yes No * No
Interval Yes Yes * * No
Repeatability Yes Yes No Yes No
Precision
Intermediate ** ** No ** No
Detection limit No No Yes * No
Quantification limit No Yes No * No
Accuracy Yes Yes * * No
Robustness Yes Yes Yes No No

* may be necessary, depending on the nature of the specific test.

** if there is proof of reproducibility, the proof of Intermediate Precision is not necessary.

1.12. the analytical methodology must be revalidated in the following circumstances:

1.12.1. changes in the synthesis of the active substance;

1.12.2. changes in the composition of the finished product;

1.12.3. changes in the analytical procedure.

Certain other changes may also require validation, depending on the nature of the
changes.

2. Methodology

2,1. Specificity and Selectivity

It is the capacity of the method to accurately measure a compound in the presence of

other components such as impurities, degradation products and components of the matrix.
2.1.1. For qualitative analysis (identification test) it is necessary to demonstrate the
capacity of selection of the method between compounds with related structures that may
be present. This must be confirmed by obtaining positive results (preferably in relation to
known reference material) in samples containing the drug, comparatively with negative
results obtained with samples that do not contain the drug, but structurally similar
compounds.

2.1.2. For quantitative analysis (content) and analysis of impurities, specificity can be
determined by the comparison of the results obtained from samples (drug or drug product)
contaminated with appropriate amounts of impurities or excipients and samples not
contaminated, to demonstrate that the result of the test is not affected by these materials.
In the absence of impurity or standard of the degradation product, the specificity of the
method can be determined by comparing the results of analysis of samples containing
impurities or degradation products with the results of a second, well characterized
procedure (e.g. pharmacopeial methodology or other validated procedure). These
comparisons must include samples stored under stressful conditions (e.g. light, heat,
humidity, acid/base hydrolysis and oxidation).

2.1.3. In chromatographic methods, the necessary precautions must be taken to

guarantee the purity of the chromatographic peaks. The use of peak purity tests (e.g. with
the aid of photodiode arrangements or mass spectrometry) is interesting to demonstrate
that the chromatographic peak is attributed to a single component.

2.2. Linearity

It is the capacity of an analytical methodology to demonstrate that the results obtained are
directly proportional to the concentration of the analyte in the sample, within a specified
interval.

2.2.1. It is recommended that linearity be determined by the analysis of at least 5 different

concentrations. These concentrations must follow the intervals in Table 3.

2.2.2. If there is apparent linear relation after visual examination of the graph, the results
of the tests must be handled with appropriate statistical methods for determination of the
correlation coefficient, intersection with the Y axis, angular coefficient, residual addition of
the minimum squares of the linear regression and relative standard deviation. If there is
no linear relation, undertake mathematical transformation.

2.2.3. The acceptable minimum criterion of the correlation coefficient (r) must be = 0.99.

2.2.4. The curves obtained must be presented (from the experiment and from the result of
the mathematical treatment).

2.3. Interval

The specified interval is the range between the limits of upper/lower quantification of an
analytical method. Normally it is derived from the linearity study and depends on the
intended application of the method (Table 3). It is established by the confirmation that the
method presents adequate accuracy, precision and linearity when applied to samples
containing amounts of substances within the specified interval.
Table 3. Percentage limits of the analyte content that must be contained in the linearity
interval for some analytical methods.

Assay Scope
Quantitative determination of the analyte 80% to 120% of theoretical concentration
in raw materials or pharmaceutical forms of the test

From expected impurity level to 120% of

maximum expected limit. When significant
toxicological effects or unexpected
Determination of impurities pharmacological effects occur, the
quantification and detection limits must be
adequate in relation to the amount of
impurities to be controlled.

70% to 130% of theoretical concentration of

Uniformity of content
Dissolution assay
the test
Of approx. 20% over value specified for the
interval. If specification for dissolution
involves more than one time, the scope of
the method must include –20% over lower
value and +20% over higher value.

2.4. Precision

Precision is the assessment of the closeness of the results obtained in a series of

measurements of a multiple sampling of the same sample. This is considered at three
levels.

2.4.1. Repeatability (intra-run precision): match between the results within a short period
of time with the same analyst and the same instrumentation.

The repeatability of the method is verified through a minimum of 9 (nine) determinations

contemplating the linear interval of the method, that is, 3 (three) concentrations, low,
medium and high, with 3 (three) replications each, or a minimum of 6 determinations at
100% concentration of the test;

2.4.2. Intermediate precision (inter-run precision): match between the results from the
same laboratory, but obtained in different days, with different analysts and/or different
equipment.

For the determination of intermediate precision, a minimum of 2 different days with

different analysts is recommended.

2.4.3. Reproducibility (inter-laboratory precision): match between the results from different
laboratories as in collaborative studies, generally applied to the standardization of
analytical methodology, for example, for inclusion of methodology in pharmacopoeias.
This data need not be presented for the concession of registration.
The precision of an analytical method can be expressed as the standard deviation or
relative standard deviation (variation coefficient) of a series of measurements.

The precision can be expressed as relative standard deviation (RSD) or coefficient of

variation (CV%), according to the formula,

SD
RSD = ---------------- x 100
ACD

where SD is the standard deviation and ACD is the average concentration determined.

The maximum acceptable value must be defined according to the methodology

employed, the concentration of the analyte in the sample, the type of matrix and the
purpose of the method, values over 5% not accepted.

2.5. Detection limit

Detection limit is the lowest amount of the analyte present in a sample that can be
detected, however not necessarily quantified, under the established experiment conditions.

2.5.1. The detection limit is established by means of analysis of solutions of known and
decreasing concentrations of the analyte, to the lowest detectable level;

2.5.2. In the case of non-instrumental methods (CCD, titling, comparison of color), this
determination can be done visually, where the detection limit is the lowest value of
concentration capable of producing the expected effect (change of color, clouding, etc).

2.5.3. In the case of instrumental methods (HPLC, GC, atomic absorption), the detection
limit can be estimated based on the ratio of 3 times the noise of the base line. It can be
determined by the equation,

SDa x 3
DL = -------------------
CI
where: SDa is the standard deviation of the intercept with the Y axis of at least 3
calibration curves containing concentrations of the drug close to the presumed
quantification limit. The standard deviation can also be obtained from the analysis of the
calibration curve obtained from a suitable number of blank samples; CI is the slope of the
calibration curve.

2.6. Quantification limit

It is the lowest amount of the analyte in a sample that can be determined with acceptable
precision and accuracy under the established experiment conditions.

The quantification limit is a parameter determined, mainly, for quantitative impurity assays,
degradation products in drugs and drug products, and is expressed as concentration of
the analyte (e.g. percentage w/w or w/V, parts per million) in the sample.

2.6.1. The quantification limit is established by means of analysis of solutions containing

decreasing concentrations of the drug to the lowest determinable level with acceptable
precision and accuracy. It can be represented by the equation,

SDa x 10
DL = -----------------
CI

where: SDa is the standard deviation of the intercept with the Y axis of at least 3
calibration curves containing concentrations of the drug close to the presumed
quantification limit. The standard deviation can also be obtained from the analysis of the
analysis of a suitable number of blank samples; CI is the slope of the calibration curve.

2.6.2. It can also be determined through noise. In this case, the noise of the base line is
determined and the concentration that produces a signal-noise over 10:1 is considered the
quantification limit.

2.7. Accuracy

The accuracy of an analytical method is the closeness of the results obtained through the
method under study in relation to the true value.
Several methodologies for determination of accuracy are available:

2.7.1. Drug

2.7.1.1. applying the analytical methodology proposed in the analysis of a substance of

known purity (reference standard);

2.7.1.2. comparison of the results obtained with those resulting from a second well
characterized methodology, whose accuracy has been established;

2.7.2. Dosage Form

2.7.2.1. in the analysis of a sample, in the which a known amount of the drug was added
to a mixture of the components of the drug product (contaminated placebo);

2.7.2.2. in the cases where samples of all the components of the drug product are
unavailable, analysis though the method of standard addition may be accepted, in which
known amounts of the analyte (reference standard) are added to the drug product.

2.7.3. Impurities

2.7.3.1. analysis through the method of standard addition, in which known amounts of
impurities and/or degradation products are added to the drug product or the drug;

2.7.3.2. in the case of unavailability of samples of certain impurities and/or degradation

products, comparison of the results obtained with a second well characterized method may
be accepted (pharmacopeial methodology or another validated analytical procedure).

The accuracy is calculated as percentage of recovery of the known amount of the analyte
added to the sample, or as the percentage difference between the averages and the
accepted true value, with the addition of the reliability intervals.

The accuracy of the method must be determined after the establishment of the linearity,
the linear interval and its specificity, being verified from, at least, 9 (nine) determinations
contemplating the linear interval of the procedure, that is, 3 (three) concentrations, low,
average and high, with 3 (three) replications each. The accuracy is expressed by the
relation between the average concentration determined experimentally and the
corresponding theoretical concentration:

average experimental concentration

Accuracy = ----------------------------------------------------- X 100
theoretical concentration

2.8. Robustness

The robustness of an analytical method is the measurement of its capacity to resist small
and deliberate variations of the analytical parameters. It indicates its reliability during
normal use.

The robustness assessment must be considered during the phase of method

development. If susceptibility to variations is verified in the analytical conditions, they must
be properly controlled or precautions must be included in the procedure.

Table 4 lists the main parameters that can result in variation in the response of the
method.

Table 4. Factors that must be considered in the determination of the robustness of the
analytical method.

Preparation of ·Stability of analytical solutions

samples ·Extraction time
·pH variation of the solution
Spectrophotometry ·Temperature
·Different solvent manufacturers
·pH variation of the mobile phase
·Variation in the composition of the mobile phase
Liquid
·Different batches or column manufacturers
Chromatography
·Temperature
·Flow of mobile phase
·Different batches or column manufacturers
Gas chromatography ·Temperature
·Speed of residual gas

BIOANALYTICAL METHODS
1. Definitions

Sample – general term that includes: controls, blank, processed and unknown samples.

Blank sample – sample of a biological matrix to which no analyte was added, used to
evaluate the specificity of the bioanalytical method.

Quality Control Sample (QC) – sample of added biological matrix of the analyte, used to
monitor the performance of a bioanalytical method and to assess the integrity and validity
of the results of the analyzed unknown samples in an individual run.

Processed sample – final extract (prior to the instrumental analysis) of a sample that has
been submitted to several manipulations (e.g.: dilution, extraction, concentration).

Unknown sample – biological sample that is the object of analysis.

Analyte – specific chemical compound to be measured, may be the untransformed drug,

biomolecule or its derivative, metabolite or degradation product in a biological matrix.

Analytical run (or batch) – complete set of samples under study, with an appropriate
number of standards and QCs for its validation and that undergoes complete analysis in
the same conditions.

Specificity – ability of the bioanalytical method to measure and distinguish the analyte from
components that may be present in the sample, such as metabolites, impurities,
degradation compounds or components of the matrix.

Stability – parameter aimed at determining if an analyte remains chemically unchanged in

a given matrix under specific conditions, at certain time intervals.

Accuracy – represents the degree of match between the individual results found and a
value accepted as reference.

Quantification range– corresponds to a concentration range, including the HLQ and the
LLQ, that can reliable and can be quantified through replication with accuracy and
precision, by means of the concentration-response ratio.

Detection Limit (DL) – lowest concentration of an analyte that the bioanalytical procedure
can distinguish reliably from the background.

Lower limit of quantification (LLQ) – lowest amount of an analyte in a sample that can be
quantitatively determined with acceptable precision and accuracy.

Higher limit of quantification (HLQ) – largest amount of an analyte in a sample that can be
quantitatively determined with precision and accuracy.

Linearity – corresponds to the capacity of the method to provide resulted directly

proportional to the concentration of the substance under examination (analyte).

Biological matrix – distinct material of biological origin, that can be sampled and
processed in a manner that can be reproduced.

Method – understandable description of all the procedures used in analyses of samples.

Calibration standard – calibration matrix to which a known amount of analyte was added.
The calibration standards are used to build the calibration curve, with which the
concentrations of the analyte in the QCs and the unknown samples under study are
determined.

Internal standard (IS) – generally composed of structural characteristics similar to the

analyte, added to the calibration standards and samples in known and constant
concentrations, to facilitate the determination of the analyte.

Precision – represents the degree of repeatability between the results of individual

analyses, when the procedure is applied various times to the same homogeneous sample,
in identical assay conditions.

Recovery – extraction efficiency of an analytical method, expressed as the percentage of

the known amount of an analyte, obtained from the comparison of the analytical results of
blank samples added to standard and submitted to the extraction process, with the
analytical results of standard solutions not extracted.

Reproducibility – precision between two laboratories. It also represents the precision of

the method under the same operational conditions, in a short period of time.

Partial validation – modification in the validated bioanalytical method that does not require
the need for a total revalidation.

Total validation – establishment of all the validation parameters of a bioanalytical method,

applicable to the analysis of the samples.

2. General considerations

2.1. The information contained in this guide is applicable to bioanalytical methods, such
as gas chromatography (GC, high efficiency liquid chromatography (HPLC) and these
combined with mass spectrometry (MS) such as LC-MS, LC-MS-MS, GC-MS, GC-MS-MS,
used for the quantitative drug determination and their metabolites in biological matrixes,
such as blood, serum, plasma or urine. It is also applicable to other analytical techniques,
such as microbiological and immunological methods, or for other biological matrixes,
although in these cases, a high degree of variability can be observed.

2.2. The validation must guarantee, through experimental studies, that the method meets
the requirements of the analytical applications, ensuring the reliability of the results. For
this, it must present precision, accuracy, linearity, detection limit and limit of quantification,
adequate specificity, reproducibility, stability and recovery suitable for the analysis. Thus, it
is important to highlight that all the equipment and materials must be properly calibrated
and the analysts must be qualified and adequately trained.

2.3. Reference chemical substances and/or biological standards made official by the
Brazilian Pharmacopoeia or by any other code authorized by the current legislation must
be used. In the absence of reference chemical substances and/or biological
pharmacopeial standards, studies using secondary standards will be admitted provided
their certification is proved.

2.4. For the relative bioavailability and bioequivalence studies the internal standard must
be used whenever chromatographic methods are used. The impossibility of its use must
be justified.

2.5. Total validation must be carried out before the implementation of a bioanalytical
method for the quantification of a drug and/or metabolites.

2.6. Partial validations must be carried out when modifications take place in the
bioanalytical method already validated. The partial validation assays can involve from a
small determination, such as the determination of intra-assay accuracy and precision, to
close to total validation. The typical changes that require partial validation include,
among others:

2.6.1. change of methods between laboratories and analysts;

2.6.2. changes in the analytical methodology, for example, substitution of the detection
system;

2.6.3. change of anticoagulant in the collection of the samples;

2.6.4. matrix change, for example, from plasma to urine;

2.6.5. change in the sample preparation procedure;

2.6.6. significant changes in the concentration range;

2.6.7. changes of instruments and/or software;

2.6.8. demonstration of selectivity of the analyte in the presence of concomitant

medications;

2.6.9. demonstration of selectivity of the analyte in the presence of specific metabolites.

2.7. The robustness assessment must be considered during the phase of method
development. If susceptibility to variations is found in the analytical conditions, they must
be properly controlled or precautions must be included in the procedure. Variation
examples are:

2.7.1. stability of the analytical solutions.

2.7.2. extraction time.

Typical variations in liquid chromatography are:

2.7.3. influence of the pH variation in the mobile phase.

2.7.4. influence of the variation of the mobile phase composition.

2.7.5. different columns (different batches and/or manufacturers).

2.7.6. temperature.

2.7.7. flow rate.

Typical variations in gas chromatography are:

2.7.8. different columns (different lots and/or manufacturers);

2.7.9. temperature;

2.7.10. flow speed.

3. Pre-study validation

3.1. Specificity

3.1.1. Samples of the biological matrix must be analyzed (blood, plasma, serum, urine, or
other) obtained from six individuals, four normal samples, a lipemic sample and hemolized
sample, under controlled conditions in terms of time, feeding and other important factors
for the study. Each blank sample must be tested using the procedure and the
chromatographic conditions proposed. The results must be compared with those obtained
with aqueous solution of the analyte, in concentration close to the LLQ.

3.1.2. Any blank sample that presents significant interference in the retention time of the
drug, metabolite or internal standard, must be rejected. If one or more of the analyzed
samples presents such interference, new samples of six other individuals must be tested.
If one or more of the samples of this group present significant interference in the retention
time of the drug, the method must be modified with a view to eliminating it.

3.1.3. The interferents may be components of the biological matrix, metabolites,

decomposition products and drug products used concomitantly with the study. The
interference of nicotine, caffeine, products sold without prescription and metabolites must
be considered whenever necessary.

3.1.4. If the method is intended for quantification of more than one drug, each one must
be injected separately to determine the individual retention times and to ensure that the
impurities of a drug do not interfere with the analysis of the other.

3.1.5. The response of interfering peaks in the retention time of the drug must be lower
than 20% of the response of the LLQ. The responses of interfering peaks in the retention
time of the drug and the internal standard must be lower, respectively, than 20% and 5%
of the response in the concentration employed.

3.2. Calibration/linearity curve

3.2.1. The calibration curve represents the ratio between the response of the instrument
and the known analyte concentration. A calibration curve must be built for each drug and
analytical run, which will be used to calculate the concentration of the drug in the samples,
using the same biological matrix proposed for the study. The calibration curve must
include the analysis of the blank sample (biological matrix free from the drug standard and
internal standard), of the zero sample (biological matrix plus the internal standard) and at
least 6 (six) samples containing drug standard and internal standard, contemplating the
expected variation limit, of the LLQ up to 120% of the highest concentration intended to be
analyzed.

3.2.2. For the determination of the calibration curve, samples extracted from the
appropriate matrix must be analyzed, at least 6 (six) different concentrations. Alternative
procedures must be justified, such as in the attainment of a nonlinear correlation, where a
higher number of concentrations of standards will be necessary.

3.2.3. The results must be analyzed by appropriate statistical methods such as, for
example, the calculation of linear regression by the method of the square minimums. The
curves obtained (experimental and those resulting from the mathematical treatment), the
coefficient of linear correlation, the angular coefficient and intercept must be presented.

3.2.4. Acceptance criteria of the calibration curve:

3.2.4.1. deviation less than or equal to 20% (twenty per cent) in relation to the nominal
concentration for the LLQ;

3.2.4.2. deviation less than or equal to 15% (fifteen per cent) in relation to the nominal
concentration for the other concentrations of the calibration curve;

3.2.4.3. at least four of the six concentrations of the calibration curve must comply with the
former criteria, including the LLQ and the largest concentration of the calibration curve;

3.2.4.4. the coefficient of linear correlation must be equal to or higher than 0.98.

3.3. Precision

3.3.1. The repeatability of the method is verified by using, at least, 3 (three)

concentrations (low, medium and high), contemplating the variation range of the
procedure, carrying out, at least, 5 (five) determinations per concentration.

3.3.2. The precision must be determined in the same run (intra-run precision) and in
different runs (inter-run precision).

3.3.3. It may be expressed as relative standard deviation (RSD) or coefficient of variation

(CV%), values greater than 15% not accepted, except for the LLQ, for which values lower
than or equal to 20% are admitted, according to the formula:

SD
RSD = ------------ x 100
ACD

where, SD is the standard deviation and ACD is the average concentration determined.
3,4. Accuracy

3.4.1. The accuracy of the method must be determined using, at least, 3 (three)
concentrations (low, medium and high), contemplating the variation range of the
procedure, carrying out, at least, 5 (five) determinations per concentration.

3.4.2. The accuracy must be determined in the same analytical run (intra-run accuracy)
and in different runs (inter-run accuracy).

3.4.3. The deviation should not exceed 15% (fifteen per cent), except for the quantification
limit for which values lower than or equal to 20% are allowed.

3.4.4. The accuracy is determined by the ratio between the average concentration
determined experimentally and the corresponding theoretical concentration:

average experimental concentration

Accuracy = -------------------------------------------------------- x 100
theoretical concentration

3.5. Lower limit of quantification (LLQ)

3.5.1. Established by means of the analysis of the biological matrix containing decreasing
concentrations of the drug up to the lowest quantifiable level with acceptable precision and
accuracy.

3.5.2. The ratio of 5:1 between the signal and the noise of the base line can also be used,
specifying the method used for the determination of the LLQ.

3.5.3. The LLQ must be, at least, five times higher than any interference of the blank
sample in the retention time of the drug.

3.5.4. The response peak of the drug in the LLQ must be identifiable and reproducible
with 20% (twenty percent) precision of and 80 – 120 % (eighty to one hundred and twenty
percent) accuracy, through the analysis of, at least, 5 (five) samples of standards.

3.6. Detection limit (DL)

Established by means of the analysis of solutions of known and decreasing concentrations

of the drug, up to the lowest detectable level. It is recommended that the DL be 2 to 3
times higher than the noise of the base line.

3.7. Recovery

The recovery measures the efficiency of the extraction procedure of an analytical method
within a variation limit. Recovery percentages near 100% are desirable, nevertheless lower
values are accepted, provided the recovery is precise and accurate.

3.7.1. This test must be done comparing the analytical results of samples extracted from
three concentrations (low, medium and high), contemplating the linearity range of the
method with the results obtained with standard solutions not extracted, presenting 100%
recovery.

3.7.2. The calculation of the recovery must be based on the ratio of the area of the
extracted and not extracted standard, both for the analyte and the internal standard
separately.

3.8. Quality control (QC)

3.8.1. QC of the quantification limit (QC-LLQ): same concentration as the LLQ.

3.8.2. QC of low concentration (QCL): less than or equal 3 x LLQ.

3,8,3. QC of middle concentration (QCM): approximately the average between QCL and
QCH

3,8,4. QC of high concentration (QCH): 75 to 90% of the highest concentration of the

calibration curve.

3.9. Stability study of drugs in biological Liquids:

3.9.1. Relevant specific Considerations

For the stability study the parameters for accuracy, precision, linearity, detection limit, limit
of quantification, specificity, variation and robustness limits previously validated must be
observed.

The drug stability in biological liquids depends on its chemical properties, the biological
matrix and the packing material used. The stability determined for a type of matrix and a
specific packing material can not be extended to others.

The conditions for carrying out the stability assays must reproduce the actual conditions of
handling and analysis of the samples. The stability of the analyte during the collection
and handling of the sample must be assessed, after long term storage (freezing) and
short term storage (room temperature), after cycles of freeze and thaw and under analysis
conditions. Assessment of the stability of the analyte in the standard solutions must also
be included, prepared with appropriate solvent in known concentrations.

The stability determinations must use a set of samples, prepared from a recent solution
reserve of the drug analyzed, added to a biological matrix free from interference.

3.9.2. Stability after freeze and thaw cycles

The drug stability must be tested after three freeze and thaw cycles using a minimum of
three samples of low and high concentrations determined in the validation of the analytical
method, in the following conditions: the samples must be frozen at temperature indicated
for storage and kept for 24 hours, after which they are defrosted at room temperature.
When completely defrosted, the samples must be frozen again at temperature indicated
for storage, for 12 to 24 hours, and so on successively, until the three cycles are complete,
the drugs in the samples being quantified after the third cycle. The results must be
compared to those obtained from the analysis of the recently prepared samples.
3.9.3. Short term stability

For verification of this stability, at least three samples of the low and high concentrations
determined in the validation of the analytical method are used. Each one must remain at
the room temperature for 4 (four) to 24 (twenty and four) hours (based on the time in
which the samples of the study are to be kept at room temperature) and analyzed. The
results must be compared to those obtained from the analysis of the recently prepared
samples.

3.9.4. Long term stability

3.9.4.1. The period of storage for the study of long term stability must exceed the interval
of time comprised between the first sampling and the analysis of the last, according to the
schedule presented in the protocol of the relative bioavailability/bioequivalence study.

3.9.4.2. The temperature used in the assay must reproduce the one recommended for
storage of the samples, usually -20ºC.

3.9.4.3. For verification of this stability at least three samples of the low and high
concentrations determined in the validation of the analytical method are used. The
concentrations of all the stability samples must be compared to the average of the values
previously calculated for the samples on the first day of the test.

3.9.5. Post-processing stability

If equipment is used that employs an autosampler, a study of the drug stability must be
undertaken, in the sample processed in the analysis, including the internal standard, at the
temperature in which the test is to be carried out and for a period of time longer than the
duration of the analytical race. At least, three samples of the low and high concentrations
determined in the validation of the analytical method are used. The results must be
compared to those obtained from the analysis of the recently prepared samples.

3.9.6. Stability of the standard solutions

3.9.6.1. The standard solutions stability of the drug and the internal standard must be
assessed, at room temperature, after a minimum of 6 (six hours) of preparation.

3.9.6.2. In the case of such solutions to be stored under refrigeration or freezing, the
stability must also be assessed, contemplating their temperature and period of storage.

3.9.6.3. The results of this test must be compared to the ones obtained using recent
solutions prepared from the drug and internal standard.

3.9.7. Analysis of the results

The samples will be considered stable when there is no deviation higher than 15% of the
value obtained from the recently-prepared samples, with the exception of the LLQ, for
which a deviation of up to 20% is accepted. Whatever the statistical method used to
assess the results of the stability studies, it must be described clearly in the standard
operational procedure (SOP).
4. Criteria for application of the validated bioanalytical method

4.1. The analysis of all the samples of an analyte in biological matrix must be concluded
within the period of time for which the stability has been determined.

4.2. An analytical run must contain: QC samples, calibration standards and unknown
samples of one or more subjects of the study. It is preferable that all the samples of a
same subject be analyzed in a single run.

4.3. It is not to estimate the concentration of the samples through extrapolation of the
calibration curve below the LLQ or above of the highest standard. Instead, the curve
must be redefined or the higher concentration samples must be diluted and re-analyzed.

4.4. In the routine use of the validated analytical method, its precision and accuracy must
be monitored regularly to ensure continuity of the satisfactory performance. For this, QC
samples must be analyzed together with the other samples, in each analytical run.

4.5. The QC samples must be incorporated at appropriate intervals, depending on the

total number of samples in the run, always in the same number of the replicates of each
concentration (QCL, QCM and QCH).

4.6. The number of QC samples (in multiples of three) to be incorporated in each

analytical run must not be lower than 5% (five percent) of the number of unknown
samples. For analytical runs consisting of up to 120 samples, at least 6 (six) QCs (a
duplicate of each concentration) must be present.

4.7. The results of the QC samples will serve as basis for acceptance or rejection of the
analytical run. At least 67% (four out of six) of the QC samples must be within
approximately 15% of their respective nominal values, except for the LLQ, for which a
deviation lower than or equal to 20% is admitted; 33% (two out of six) QC samples may
be outside these limits, but not for the same concentration.