03 Reference Standards

Kuliah VMA 2014 - Reference Standards 5/8/2014

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Kuliah Validasi Metode Analisis

Reference StandardsFlorentinus Dika Octa Riswanto, M.Sc.

Fakultas Farmasi

Universitas Sanata Dharma

2014

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HPLC methods rely on reference standards in order to provide accurate data.

The quality and purity of reference standards are critical and these materials should be highly purified and well characterized.

1. Reference standards from the USP/NF

2. Non-compendial primary reference standard

3. Working reference standards

Reference Standards

Kuliah Validasi Metode Analisis 2014

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Ahuja and Dong, 2005, Handbook of pharmaceutical analysis by HPLC, p. 212.


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"pure" materials by legal definition

unless a purity value other than 100%

has been assigned by the USP,

and no further characterization is

required prior to their use.


Reference standards from the USP/NF

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If USP reference standards are not available,

a non-compendial primary reference standard may be certified for use

(commercially supplied RS obtained from a reputable commercial source)

These should be of the highest purity available and well characterized

to assure the purity, strength, identity, and quality of the material.

Methods using non-compendial reference standards

must incorporate any purity correction factor into the calculations.

It is important that any and all reference materials used

for method validation should be well documented in the validation report.


Non-compendial primary reference standard

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Working reference standards are usually materials that have been characterized

and have had their purity established against a compendial reference standard.

These may be used in cases where it is more cost effective

to certify an in-house lot than to purchase USP reference materials

for routine analysis.


Working reference standards

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1. Quantitation by External Standard

This quantitation technique is the most straightforward.

It involves the preparation of one or a series of standard solutions that approximate the concentration of the analyte.

Chromatograms of the standard solutions are obtained, and peak heights or areas are plotted as a function of concentration of the analyte.

The plot of the data should normally yield a straight line. This is especially true for pharmaceuticals of synthetic origin.

Other forms of mathematical treatment can be used but will need to be justified.


Types of quantitation

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Chan, 2004, Analytical Method Validationand Instrument Performance Verification, p. 13.

1. Quantitation by External Standard

There are some potential instrumental sources of error that could occur using this quantitation technique.

It is critical to have minimal variability between each independent injection, as the quantitation is based on the comparison of the sample and standard areas.

However, the current autosamplers are able to minimize this variability to less than 0.5% relative standard deviation (RSD).



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External Standard

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C1

C4

C3

C2

ConcentrationArea

A1

A2

A3

A4C1 C2 C3 C4

A1

A2

A3

A4

Concentration

Peak a

rea

Calibration curve

2. Quantitation by Internal Standard

Quantitation by internal standard provides the highest precision because uncertainties introduced by sample injection are avoided.

In this quantitation technique, a known quantity of internal standard is introduced into each sample and standard solutions.

As in the external standard quantitation, chromatograms of the standard and sample solutions are integrated to determine peak heights or peak areas.

The ratio of the peak height or area of the analyte to an internal standard is determined. The ratios of the standards are plotted as a function of the concentration of the analyte. A plot of the data should normally yield a straight line.



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2. Quantitation by Internal Standard

Due to the presence of the internal standard, it is critical to ensure that the analyte peak be separated from the internal standard peak.

A minimum of baseline separation (resolution >1.5) of these two peaks is required to give reliable quantitation.

In addition, to quantitate the responses of internal standard accurately, the internal standard should be baseline resolved from any significant related substances and should have a peak height or area similar to that of the standard peak.



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Internal Standard

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C1

C4

C3

C2

Concentration Area

A1

A2

A3

A4C1/CIS C2 /CIS C3 /CIS C4 /CIS

A1/AIS

A2 /AIS

A3 /AIS

A4 /AIS

Concentration of target substance /

Concentration of internal standard

Are

a f

or

targ

et

substa

nce / A

rea for

inte

rnal sta

ndard

Calibration curveTarget

substance

Internal

standard

CIS

CIS

CIS

CIS

AIS

AIS

AIS

AIS


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Advantages of Internal Standard Method

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Not affected by inconsistencies in injection volume.

10 L

injected

9 L

injected

CX / CIS

AX / AIS

XIS

XIS

Same area ratio


Advantages of Internal Standard Method

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Not affected by the pretreatment recovery rate.

100%

recovery

rate

90%

recovery

rate

CX / CIS

AX

/ A

IS

XIS

XIS

Same area ratio


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It is preferable to choose an internal standard which has a similar chemical structure to the analyte in order to mimic its behaviour.

Its peak must appear relatively near that of the target substance.

It must not already be contained in the actual samples.

Its peak must be completely separated from those of other sample components.

It must be chemically stable.

It must give a similar respon to the same detector

Available in high purity


Selection Criteria for Internal Standard

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In many instances in the pharmaceutical industry, drug products may be

manufactured in a variety of strengths (e.g., levothyroxine tablets in strengths of

50, 100, 150, 200, 500, and 750 g). To develop and validate these potency

methods, three strategies may be followed.

1. Single-Point Calibration

2. Multiple-Point Calibration

3. One Standard Calibration for Each Strength


Standard Plots for Quantitation

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A method may be developed and validated using only one standard analyteconcentration.

The standard plot generated is used to assay the complete range of tablet strengths.

This strategy should be adopted wherever possible due to the simplicity of standard preparation and minimal work for quantitation of the sample.

However, this method will require different extraction and dilution schemes of the various drug product strengths to give the same final concentration that is in the proximity of the one standard analyte concentration.


Single-Point Calibration

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Another strategy involves two or more standard concentrations that will bracket the complete range of the drug product strengths.

In this strategy it is critical that the standard plots between the two extreme concentration ranges be linear.

Therefore, this is a valid calibration method as long as the sample solutions of different strengths are prepared within the concentration range of the calibration curve.

Its advantage is that different strengths can utilize different preparation procedures and be more flexible. Its disadvantage is that multiple weighing of standards at different concentrations may give a weighing error.


Multiple-Point Calibration

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The least favored method is to develop and validate using one standard concentration for each product strength.

This situation will arise when the analyte does not exhibit linearity within a reasonable concentration range.


One Standard Calibration for Each Strength

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03 Reference Standards

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