Method Development

(High Performance Liquid Chromatography)

by Alfan Danny Arbianto
OUR GOAL
• Memahami langkah-Langkah Ketika akan
melakukan sebuah analisis sampel

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 Outline

Validation
1 Guidelines
q Kenali Sampel
q ICH Guideline

q Journal

3 4
q Pharmacopeia

Examples

2 Verification

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Guide Line

Start from
q Kenali Sampel
q Pharmacopeia
q Journal

01

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Kenali Sampel

SAMPEL
q Jenis &
karakteristiknya
q Known Sampel
Tak Kenal
q Unknown Sampel maka Tak Sayang
q Polarity
q Solubility

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Pharmacopeia
• Validitas tinggi
• Hanya perlu verifikasi
• Waktu developmentnya lebih singkat
• Mengurangi cost analisa

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 Pharmecopeia
1 Indonesia 3 JP
Pharmacopeia
q Physic Analysis
q Physic Analysis q Chemical Analysis
q Chemical Analysis

2 USP 4 EP
q Physic Analysis q Physic Analysis
q Chemical Analysis q Chemical Analysis

http://www.uspbpep.com/

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European Pharmacopeia 6.0

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 Informasi
Penting
q Solubility
q Konsentrasi sampel &
standard
q Column
q Detector
q Flow
q Run Time

01
Column?

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Column HPLC

https://www.ich.org/page/quality-guidelines

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Journal

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Kondisi HPLC

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Kualitas Peak

Resolusi Peak

Peak tailing

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Preparasi Sampel

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Data Validasi

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Data Validasi

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Verification & Validation

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Verification Process
USP EDQM AOAC
Specificity Specificity Specificity
Precision Precision Precision
Linearity Linearity -
Range - -
- Accuracy Accuracy

Catatan: Karena kompendial bersifat territorial, maka harus dilihat secara

detail dan akan berbeda beda persyaratan serta batas nilai yang di ijinkan

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Validation Process
Based on ICH guideline Protocols

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Validation Process
Based on ICH guideline Protocols

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Specificity
Specificity is the ability to assess unequivocally the analyte in the
presence of components which may be expected to be present.
Typically these might include impurities, degradants, matrix, etc.

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Linearity
The linearity of an analytical procedure is its ability (within a given
range) to obtain test results which are directly proportional to the
concentration (amount) of analyte in the sample.

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Range
The range of an analytical procedure is the interval between the
upper and lower concentration (amounts) of analyte in the
sample (including these concentrations) for which it has been
demonstrated that the analytical procedure has a suitable level of
precision, accuracy and linearity.

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Accuracy
The accuracy of an analytical procedure expresses the closeness
of agreement between the value which is accepted either as a
conventional true value or an accepted reference value and the
value found.

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Precision
The precision of an analytical procedure expresses the closeness of
agreement (degree of scatter) between a series of measurements
obtained from multiple sampling of the same homogeneous sample
under the prescribed conditions. Precision may be considered at three
levels: repeatability, intermediate precision and reproducibility

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LOD & LOQ
Detection Limit, The detection limit of an individual analytical
procedure is the lowest amount of analyte in a sample which can
be detected but not necessarily quantitated as an exact value

Quantitation Limit, The quantitation limit of an individual

analytical procedure is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable precision
and accuracy

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Cara? LOD & LOQ
Standard stock solutions of cefotaxime sodium (6.25, 12.50, 25, 50,
100, and 200 μg/mL were prepared) by diluting the standard stock
solution (1000 ppm) with mobile phase.

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Robustness
The robustness of an analytical procedure is a measure of its
capacity to remain unaffected by small, but deliberate variations
in method parameters and provides an indication of its reliability
during normal usage.

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System Suitability Testing

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 Q&A

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