ANALYTICAL METHOD DEVELOPMENT

OPTIMISATION AND VALIDATION OF

HPLC

Sudheer kumar kamarapu

Assistant professor

Sri Shivani college of pharmacy

INTRODUCTION

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• The drug or drug combination may not be official in any pharmacopoeias.

• A proper analytical procedure for the drug may not be available in the literature due to the patent regulations.

• Analytical procedures may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.

• Analytical methods for the quantisation of the drug in the biological fluids may not be available.

• Analytical methods for a drug in combination with other drugs may not be available.

• The existing analytical procedures may require expensive reagents and solvents.

• The existing analytical procedures involve cumbersome extraction and separation procedures and these may not be reliable.

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• As simple as possible.

• Most specific.

• Most productive economical and convenient.

• As accurate precise as required.

• Multiple source of key components(reagents,columns,TLC plates) should be avoided.

• To be fully optimized before transfer for validation of characteristics such as accuracy,precision,sensitivity, ruggedness etc.

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STEPS IN HPLC METHOD

DEVELOPMENT

Information on sample,

define separation goals.

Need for special HPLC procedure, sample pretreatment..etc..

Choose detector

Choose LC method ; preliminary run; estimate best separation conditions

Optimize separation conditions.

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Check for problems or

requirement for special procedure

Validate method for release to routine

laboratory.

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Important information concerning sample composition and properties:

Number of compounds present.

Chemical structures (functionality) of compounds.

Molecular weights of compounds.

Pka values of compounds.

UV spectra of compounds.

Concentration range of compounds in samples of interest.

Sample solubility.

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SEPARATION GOALS

Is the primary goal quantitative analysis ,the detection of an substance, the characterization of unknown sample components or the isolation of purified material ?

Is it necessary to resolve all sample components?

If quantitative analysis is requested , what levels of accuracy and precision are required

For how many different sample matrices should the method be designed

How many samples will be analyzed at one time

What HPLC equipment and operator skills are present in the laboratory that will use the final method ?

SAMPLE PRE TREATMENT AND DETECTION

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Samples come in various forms: • Solutions ready for injection. • Solutions that require dilution, buffering, addition of an internal standard, or other volumetric manipulation. • Solids that must first be dissolved or extracted. • Samples that require sample pretreatment to remove interferences and/or protect the column or equipment from damage.

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SAMPLE PRETREATMENT • Removal of insoluble material

Filteration Centrifuge

• Control of concentration Dilution

• Extraction

Liquid phase extraction Solid phase extraction

• Derivatization for detection

Selection of detectors • UV-VIS Ultraviolet/ visible detector

• PDA Photodiode Array Detector

• RF Fluorescence detector

• CDD Conductivity detector

• RID Refractive index detector

• ECD Electrochemical detector

• ELSD Evaporative light scattering detector

• MS Mass spectrometer detector

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DEVELOPING THE SEPARATION

13 Ref:Practical HPLC method development 2nd edition by Lioyd R.Snyder Chapter 1

Improving The Separation

14 Ref:Practical HPLC method development 2nd edition by Lioyd R.Snyder Chapter 1

Problem Comment

Low plate numbers Poor choice of column

Column variability Poor choice of column

Short column life Poor choice of column, need for

sample pretreatment

Retention shift Insufficient column equilibrium, need

for sample pretreatment, loss of

bonded phase

Poor quantitative precision Need for better calibration,

identification of sources of error

New interference peaks discovered Initial inadequate or initial samples

not representative.

Checking for problems

15 Ref:Practical HPLC method development 2nd edition by Lioyd R.Snyder Chapter 1

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Optimization of a separation is principally directed by the following goals: • To separate better (higher resolution), • To separate faster (shorter retention time), • To see more (lower detection limit), • To separate at lower cost (economic effort), • To separate more (higher throughput). Optimization of a method can follow either of two general approaches: • Manual . • Computer driven.

METHOD OPTIMIZATION

The various parameters that include to be optimized during method development are :

• Mode of separation .

• Selection of stationary phase .

• Selection of mobile phase.

• Selection of detector .

Mode of separation: For the separation of polar or moderately polar

compounds, the most preferred mode is reverse phase

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Selection of stationary phase/column: The appropriate choice of separation column includes different

approaches:

• The particle size and the nature of the column packing.

• The physical parameters of the column i.e. the length and the diameter.

Selection of mobile phase : The following are the parameters, which shall be taken into

consideration while selecting and optimizing the mobile phase:

• Buffer and its strength: The retention times are depend on the molar strengths of the buffer – Molar strength is increasingly proportional to retention times.

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• pH : It is important to maintain the pH of the mobile

phase in the range of 2.0 to 8.0 as most columns does not withstand to the pH which are outside this

range. • Mobile phase composition:

• Experiments were conducted with mobile phases having buffers with different pH and different organic phases to check for the best separations between the impurities.

• A mobile phase which gives separation of all the impurities and degradants from each other and from analyte peak should be preferred

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Selection of detector: The characteristics that are to be fulfilled by a detector to

be used in HPLC determination are:

• High sensitivity, facilitating trace analysis

• Negligible baseline noise. To facilitate lower detection

• Large linear dynamic range

• Non destructive to sample

• Inexpensive to purchase and operate

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VALIDATION OF HPLC METHOD

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VALIDATION, USP:

“Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications.”

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Basic Parameters for the Validation of Method:

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Method

validation

Specificity

Linearity

Accuracy

Limit of detection

Limit of quantification

Precision

Range

Robustness

System suitability

Validation Characteristics

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Assay Impurities Identificatio

n limit

quantita

tive

+ - + - Accuracy

+ - + - Precision

+ + + + Specificity

- + - - Detection Limit

-

- +

- Quantitation

Limit

+ - + - Linearity

+ - + - Range

+ + + + Robustness

Ref:Quality assurance of pharmaceuticals(A compendium of guidelines and related materials),volume 1 ,Chapter 4 World Health Organization Geneva

Accuracy:

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

According to the ICH, accuracy should be determined using a minimum of nine determinations over a minimum of three concentration levels covering the range .

Obtained value - Expected value %Error = ----------------------------------------- * 100 Expected value

%Error <1% Highly accurate 1 to 5% Moderately accurate >5% Low accurate

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Precision : Definition : The Precision is a measure of the ability of the method to generate reproducible results. The precision of a method is evaluated for repeatability, intermediate precision and reproducibility.

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Limit of Detection (LOD, DL):

The LOD of an analytical procedure is the lowest amount of

analyte in sample which can be detected but not necessarily

quantitated as an exact value. Determination is usually based on

– Signal to noise ratio (~3:1) (baseline noise)

or

– Standard deviation of response (s) and Slope (S)

3.3 s/S

SNR = H/h

Where,

H = height of the peak corresponding to the component.

h = absolute value of the largest noise fluctuation from

the baseline of the chromatogram of a blank solution .

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Limit of Quantitation (LOQ, QL)

– The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.The quantitation limit is used particularly for the determination of impurities and/or degradation products.

Determination is usually based on

– Signal to noise ratio (~10:1) (baseline noise)

or

– Standard deviation of response (s) and Slope (S)

10 s/S

- The Quantitation limit of a method is affected by both the detector sensitivity and the accuracy of sample preparation.

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Noise

LOD Signal to Noise = 3:1

LOQ

Signal to Noise = 10:1

LOD, LOQ and Signal to Noise Ratio (SNR)

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Range:

ICH Definition: The range of an analytical procedure is the interval

between the upper and lower concentrations of analytes in the for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy, and linearity.

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Range For Different Tests:

• Assay

80 to 120% of test concentration.

• Content uniformity

70 to 130% of test concentration

• Dissolution

Q-20% to 120%

• Impurities

Reporting level – 120% of specification limit (with respect to test concentration of API)

• Assay & Impurities

Reporting level to 120% of assay specification

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Linearity:

Definition : Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration of analytes in the sample. •If there is a linear relationship test results should be evaluated by appropriate statistical methods like, •Correlation coefficient •Y-intercept •Slope of regression line •Plot of the Data

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Linearity Ranges and Acceptance Criteria for

Various Pharmaceutical Methods

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Ref:Modern HPLC by Michel Dong.

SYSTEM SUITABILITY TESTING (SST)

System suitability testing (SST) is used to verify resolution,

column efficiency, and repeatability of the analysis system to ensure its adequacy for performing the intended application on a daily basis.

Which Parameters??

•Number of theoretical plates (efficiency) •Capacity factor, •Separation (relative retention) •Resolution, •Tailing factor •Relative Standard Deviation (Precision) 34 34

•Plate number or number of theoretical plates (n) n=L/H, where L is Length of Column H is HETP or height of one theoretical plate •Capacity factor (capacity ratio) k

k= (tr-tm) /tm where tr is retention time

tm is dead time •Separation Factor (relative retention)

α=k1/k2 where k1 is capacity factor of compound a and k2 is capacity factor of compound b

•Tailing factor ,T T=W/2f where W is width at 5% at peak height, f is distance between max and leading edge of the peak

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36 Ref:Modern HPLC by Michel Dong,

Robustness : Definition : Robustness is reliability of an analytical procedure with respect to deliberate variations in method parameters. •If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure.

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PARAMETERS TO BE EVALUATED FOR ROBUSTNESS •Mobile Phase

• pH (±0.1–0.2 units) • Buffer concentration (±5–10mM) • Percentage organic modifier (±1–2% MP)

•Sample •Injection volume • sample concentration •Column temperature (±5°C) •Detector wavelength (±3nm)

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Selectivity and Specificity : •Selectivity is the ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix. •Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipients and/or degradation products and/or impurities.

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REFERENCES

1. Modern HPLC by Michel Dong, Chapter 9.

2. Analytical Method Validation and Instrument Performance Verification by Herman Lam and Y.C. Lec, Chapter 3 and Chapter 11.

3. Practical HPLC method development 2nd edition by Lioyd

R.Snyder Chapter 1

4. Quality assurance of pharmaceuticals(A compendium of guidelines and related materials),volume 1 ,Chapter 4 World Health Organization Geneva

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