Location via proxy:   [ UP ]  
[Report a bug]   [Manage cookies]                

Method Validation in Pharmaceutical Analysis From Theory To Practical Optimization PDF

Download as pdf or txt
Download as pdf or txt
You are on page 1of 3

Commentary SCIENCE

Method validation in pharmaceutical analysis: from theory to practical optimization


Jaqueline Kalleian Eserian, BPharm, MS and Márcia Lombardo, BPharm, MS
Centro de Medicamentos, Cosméticos e Saneantes, Instituto Adolfo Lutz, São Paulo, Brazil

Keywords: validation studies, chromatography, quantitative analysis.

Conflicts of Interest: We declare no conflicts of interest or financial interests that the authors or members of their immediate
families have in any product or service discussed in the manuscript, including grants (pending or received), employment, gifts, stock
holdings or options, honoraria, consultancies, expert testimony, patents, and royalties.

Abstract
The validation of analytical methods is required to obtain high-quality data. For the pharmaceutical industry, method validation is
crucial to ensure the product quality as regards both therapeutic efficacy and patient safety. The most critical step in validating a
method is to establish a protocol containing well-defined procedures and criteria. A well planned and organized protocol, such as the
one proposed in this paper, results in a rapid and concise method validation procedure for quantitative high performance liquid
chromatography (HPLC) analysis.

Introduction
Analytical methods can be considered as a complex and High performance liquid chromatography (HPLC) is the most
multi-step issue, ranging from sampling to generating the common analytical technique applied in pharmaceutical
result. It is internationally recognized that the validation of analysis. For the pharmaceutical industry, the demonstration
1
methods is required to obtain high-quality data. that an analytical method is able to quantify the drug and its
related compounds is crucial to ensure the product quality as
2
According to Araujo (2009), the word valid - from the Latin regards both therapeutic efficacy and patient safety.
validus - means strong. To validate means to officially state
that something that has been proven to be true is useful and Although different areas of work have specific characteristics,
of an acceptable standard. the validation of analytical methods might be applied for
different types of analyses, as it does not depend on the
4
The validation of analytical procedures intends to establish matrix of the sample or analytical technology employed.
the performance characteristics of analytical applications
through experimental tests, resulting in a suitable analytical What to be considered in the validation study
3
method for its purpose. Analytical procedures might be validated for investigating the
identity of the analyte in a sample (identification tests);
Considerations about method validation have been obtaining the analyte content (quantitative tests); and
5
highlighted since the late 1940s when mathematics and analyzing impurities (quantitative or limit tests).
statistics were considered to be essential prerequisites for
the development of analytical methods. Its implementation in Aiming the validation of HPLC quantitative methods, the
analytical laboratories was established in the late 1970s, analytical performance characteristics to be considered are
reflecting the recognition of this process as an important way linearity, range, precision, accuracy, specificity and
of obtaining standard methods. Currently, several robustness, according to the International Conference on
5
international organizations are committed to continuous Harmonisation guideline Q2(R1). Although not necessary for
2
processing improvement. quantitative analytical procedures, detection and
quantitation limits (LOD and LOQ) might be included as
complementary tests, in addition to stability and system
Corresponding author: Jaqueline Kalleian Eserian, Scientific suitability.
Researcher, Centro de Medicamentos, Cosméticos e
Saneantes, Instituto Adolfo Lutz; Avenida Doutor Arnaldo, Proposing an optimized validation scheme
355, Prédio BQ, 5º andar, CEP 01246-000, São Paulo, SP, The most critical step in validating a method is to establish a
Brazil. Tel.: +55 11 3068 2930; Fax: +55 11 3068 2926; protocol containing well-defined procedures and criteria.
E-mail: jkeserian@ial.sp.gov.br Testing validation parameters consumes time and resources;
however, a well-planned and organized protocol, such as the
http://z.umn.edu/INNOVATIONS 2015, Vol. 6, No. 1, Article 194 INNOVATIONS in pharmacy 1
Commentary SCIENCE

5
one presented in Figure 1, results in a rapid and concise same analyst and same equipment. It is obtained by
method validation procedure. analyzing standard solutions of analyte at 3 different
concentrations covering the specified range in triplicate. As
Figure 1. Scheme for a rapid quantitative HPLC method shown in the scheme, it can be directly obtained from the
validation linearity test.

2.2. Intermediate precision (IP)


IP expresses within-laboratory variation, considering different
5
days, different analysts or different equipments. In practice,
it is repeatability performed for 3 times. Therefore, the first
determination of IP can be obtained from the repeatability
test; the second and third determinations are obtained by
repeating the test for 2 more consecutive days.

3. Accuracy
Accuracy is the closeness of agreement between test results
5
across the specified range and an accepted reference value.
It is obtained by the standard addition method, in which the
sample is spiked with known quantities of the analyte. In
practice, it can be evaluated by spiking a sample with the
standard solutions of analyte at 3 different concentrations
used in the linearity test in triplicate. It is expressed as the
percentage of analyte recovery, standard deviation and
The following parameters should be considered in order to relative standard deviation.
obtain the proposed scheme:
4. Specificity
1. Linearity Specificity is the ability to assess unequivocally the analyte in
Linearity is the ability of obtaining results that are directly the presence of substances that are expected to be present,
proportional to the analyte concentration in the sample, such as excipients, impurities and components of the mobile
5
across the specified range, i.e., the interval between the phase. It might be assessed by verifying the absence of
5
upper and lower levels of analyte. It might be evaluated interfering peaks at the analyte retention time when spiking a
through a calibration curve with 5 points in triplicate within a sample with appropriate levels of impurities or excipients.
minimum range of 80 to 120% of the test concentration. It is Peak purity determination with a diode array detector can be
estimated by the linearity's curve correlation coefficient, y- performed to demonstrate that the target peak corresponds
intercept, slope of the regression line and residual sum of to a single component.
squares.
5. Robustness
This test should be run first, since its data might be used for Robustness is the capacity of the analytical method to remain
assessing repeatability, intermediate precision (IP), accuracy, unaffected by small variations in its parameters, indicating
5
LOD, LOQ, stability and system suitability. reliability. Concerning liquid chromatography, variations
such as mobile phase composition, mobile phase pH, column
2. Precision temperature, column lots, column suppliers and flow rate
5
Precision is the closeness of agreement between individual might be set. It can be evaluated by the analysis of the
results obtained from a repeatedly applied procedure in a standard solution of analyte at 100% of expected
5
homogeneous sample, comprising repeatability and IP. It is concentration in triplicate for each isolated condition and
expressed as the standard deviation or coefficient of expressed as the standard deviation and relative standard
variation. deviation.

2.1. Repeatability 6. Limits of detection and quantitation


Repeatability is defined as precision over a short interval of Both LOD and LOQ parameters can be directly obtained from
time under the same operating conditions, i.e., same day, the linearity test. The lowest amount of analyte which can be
detected under the stated experimental conditions is the

http://z.umn.edu/INNOVATIONS 2015, Vol. 6, No. 1, Article 194 INNOVATIONS in pharmacy 2


Commentary SCIENCE

5
LOD. It can be estimated based on the signal-to-noise ratio can be applied easily to routine quality control, as well as
of 3:1 or standard deviation of the y-intercepts divided by the scientific research, producing more rigorous results and
slope of the calibration curve obtained in the linearity test, in enabling methodological problems solving.
5
a ratio of 3.3:1.
The main advantage of the proposed scheme is the time
LOQ is the lowest amount of analyte which can be optimization, since all the required data can be obtained in a
quantitatively determined with precision and accuracy under maximum of four days of tests. Additionally, it leads to
5
the stated experimental conditions. This parameter can be solvents and reference standards savings, consequently
evaluated in the same way as for the LOD; however, in a ratio generating less chemical waste. Nevertheless, in practice
5
of 10:1 for both cases. minor adaptations might be required according to the
laboratory routine, difficulty level of sample preparation and
7. Stability total analysis time.
Since chemical decomposition may occur during storage, drug
1
stability should be evaluated by the analysis of sample and Besides composing the first level of quality assurance (QA),
standard solutions during a preselected period of time, validation of methods provides valuable information about
allowing the estimation of the maximum interval between the specific characteristics of method performance and its
6
sample preparation and analysis. critical steps. Given the significance of obtaining reliable
results in pharmaceutical analysis, further research is needed
A short-term storage test might be performed by maintaining to improve the processes related to the validation of
sample solution and standard solution of analyte at 100% at analytical methods.
temperature of interest and testing at time, e.g., 0, 24 and 48
hours in triplicate. It is expressed as the standard deviation References
and relative standard deviation. The standard solution of 1. Taverniers I, De Loose M, Van Bockstaele E. Trends in
analyte at 100% can be obtained from the linearity test. quality in the analytical laboratory. II. Analytical
method validation and quality assurance. Trends
8. System suitability Analyt Chem. 2004; 23(7):480-490.
The system suitability is characterized by a set of tests 2. Araujo P. Key aspects of analytical method validation
applied to verify the reproducibility of the chromatographic and linearity evaluation. J Chromatogr B Analyt
system. It can be obtained by performing 5 injections of the Technol Biomed Life Sci. 2009; 877 (23):2224–2234.
standard solution of analyte at 100% and assessing the 3. United States Pharmacopeia and National Formulary
following parameters: peak tailing, resolution between peaks, (USP 35 NF30), The United States Pharmacopeial
theoretical plates, capacity factor and peak symmetry. It is Convention, Inc., Rockville, MD, 2012.
expressed as the relative standard deviation. As in the 4. Rozet E, Ceccato A, Hubert C, et al. Analysis of recent
stability test, system suitability can be evaluated using the pharmaceutical regulatory documents on analytical
3
standard solution of analyte at 100% from the linearity test. method validation. J Chromatogr A. 2007; 1158(1-
2):111–125.
Final considerations 5. Validation of Analytical Procedures: Text and
Method validation is an essential step of quantitative Methodology Q2(R1). International Conference on
analysis, ensuring the level of measurement and increasing Harmonisation.
the confidence in the results. Reference guidelines present http://www.ich.org/fileadmin/Public_Web_Site/ICH
the definition of the validation parameters and provide _Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1_
recommendations on how to evaluate them; however, these _Guideline.pdf. Accessed on January 10, 2014.
guidelines do not present detailed information on how to test 6. Eurachem Guide: The Fitness for Purpose of
these parameters simultaneously. Analytical Methods – A Laboratory Guide to Method
Validation and Related Topics. 2nd. ed. B.
In this paper, we aimed at introducing an idea of method Magnusson and U. Ornemark (eds.). 2014.
validation optimization; however, different schemes
considering the necessary parameters might be proposed in
order to optimize the validation process. To our knowledge,
this is the first study to address the optimization of method
validation through a rapid and concise procedure, improving
usual and customary processes. This working methodology

http://z.umn.edu/INNOVATIONS 2015, Vol. 6, No. 1, Article 194 INNOVATIONS in pharmacy 3

You might also like