ANALYTICAL METHOD

DEVELOPMENT OF BULK DRUGS

Rishabh Nagar

M.Pharma.(Analysis)

Introduction:- The number of drugs introduced into the market is increasing

every year. These drugs may be either new entities or partial structural modification of the existing one. It becomes necessary, therefore to develop newer analytical methods for such drugs.

Analytical Method Development

Analytical method development plays important role in the discovery, development, and manufacturing of pharmaceuticals.

The official analytical methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency, and performance of drug products. (James et al, 1988 )

Basic criteria for new method development of drug analysis :-

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 patent regulations,

Analytical methods 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 quantitation of the drug in 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. It may also involve extraction and separation procedures and these may not be reliable.

There may not be a suitable method for a particular analyte in the specific sample matrix.

Existing methods may be have error, artifact , or contamination prone, or they may be unreliable (have poor accuracy or precision).

Existing methods may not provide adequate sensitivity or analyte selectivity in samples of interest.

Newer instrumentation and techniques may have evolved that provide opportunities for improved methods, including improved analyte identification or detection limits, greater accuracy or precision, or better return on investment.

Analytical Method Development is required for :» Herbal Products» New process and reactions» New molecules » Active ingredients (Macro analysis)» Residues (Microanalysis) » Impurity Profiling » Component of Interest in different matrices

NEEDS:- Affordability and necessity based eg. academic and

personnel Laboratories. Simple, easier, cost effectiveness method is preferred. Need for alternative method of analysis for better accuracy

and precision. Better recovery of bulk drugs. Evaluation and rethought over the already existing methods. New and innovative ideas are encouraged – advances in

analytical field. Broadens the scope of research and exploration of new

properties of drugs those can be exploited for their estimation and quantitative determination.

Availability of solvents/ reagents costs issue.

GOAL’s:- To improve the degree of accuracy and precision For better recovery of drugs. Improvement of sensitivity and/or specifity. Simpler and easier method. Reduce the cost (cost of operation and running cost of HPLC are

high)etc.

Validation of developed Analytical method:-

Validation of analytical procedures is the process of

determining the suitability of a given methodology for

providing useful analytical data.

J. Guerra, Pharm. Tech. March 1986

Method validation is the process of demonstrating that

analytical procedures are suitable for their intended use and

that they support the identity, strength, quality, purity and

potency of the drug substances and drug products.

G. Maldener , Chromatographia , July 1989

Analytical method development requirement :-

Validation of analytical methods is a prerequisite for prequalification of product dossiers○ Non-compendial APIs are tested with methods

developed by the manufacturer○ For compendial APIs the „applicability“ of

pharmacopoeial methods to particular products must be demonstrated (verification)

Analytical methods must be developed and validated according to TRS 823, Annex 5, Validation of analytical procedures used in the examination of pharmaceutical materials ; ICH Q2 (R1)○ To be used within GLP and GMP environments

PHYSICAL PARAMETRS OF BULK DRUGS

Structure Molecular weight Melting point/Boiling point Solubility profile pH and pka value Refractive index Optical rotation Salt formation Partition coefficient Moisture content Method of synthesis of bulk

drugs Impurity profile Related substance

METHOD CYCLE :-

Validation

Development Optimization

Method Development, Optimization, and Validation Approaches:-

Method development usually requires selecting the method requirements and deciding on what type of instrument to utilize and why . In the development stage, decisions regarding choice of column, mobile phase, detector(s), and method of quantization must be addressed. In this way, development considers all the parameters pertaining to any method.

During the Optimization stage, the initial sets of conditions that have evolved from the first stages of development are improved or maximized in terms of resolution, peak shape and area under curve, plate counts, asymmetry, capacity, elution time, detection limits, limit of quantization, and overall ability to quantify the specific analyte of interest.

In the Validation stage, an attempt should be made to demonstrate that the method works with samples of the given analyte, at the expected concentration in the matrix, with a high degree of accuracy and precision.

Method Validation Parameters (ICH guideline)

Various parameters are used to validate the methods:

Linearity Range Accuracy Precision

• Repeatability

• Intermediate Precision

• Reproducibility

Limit of Detection Limit of Quantitation Specificity Ruggedness Robustness

Definition

Linearity :-

Ability of an assay to elicit a direct and proportional response to changes in analyte

concentration.

By Visual Inspection of plot of signals vs. analyte concentration

By Appropriate statistical methods

Linear Regression (y = mx + c)

Correlation Coefficient, y-intercept (c), slope (m)

Acceptance criteria: Linear regression r2 > 0.95 (min 5 concentration require )

Range :-

Acceptable range having linearity, accuracy, precision.

For Drug Substance & Drug product Assay

80 to 120% of test Concentration

For Content Uniformity Assay

70 to 130% of test Concentration

For Dissolution Test Method

+/- 20% over entire Specification Range

For Impurity Assays

From Reporting Level to 120% of Impurity Specification for Impurity Assays

From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods

Accuracy :- Closeness of the test results obtained by the method to the true value.

It Should be established across specified range of analytical procedure.

It Should be assessed using a minimum of 3 concentration levels, each in

triplicate (total of 9 determinations)

Should be reported as:

Percent recovery of known amount added

The difference between the mean assay result and the accepted value

Precision :- Expresses the closeness of agreement between a series of measurements

obtained from multiple sampling of the same homogenous sample Is usually expressed as the standard deviation (S), variance (S2) or coefficient

of variation (RSD) of a series of measurements Precision may be considered at three levels

○ Repeatability (intra-assay precision)○ Intermediate Precision (variability within a laboratory)○ Reproducibility (precision between laboratories)

Repeatability

Express the precision under the same operating conditions over a short interval of

time.

Also referred to as Intra-assay precision

Should be assessed using minimum of 9 determinations

(3 concentrations/ 3 replicates) or Minimum of 6 determinations at the 100% level. Intermediate Precision Express within-laboratory variations. Expressed in terms of standard deviation, relative standard deviation (coefficient of

variation) and confidence interval. Depends on the circumstances under which the procedure is intended to be used. Studies should include varying days, analysts, equipment, etc.

Reproducibility

It is an Ability reproduce data within the predefined precision

Determination: SD, RSD and confidence interval

Repeatability test at two different labs.

LOD

Lowest amount of analyte in a sample that can be detected but not necessarily

quantitated.

Estimated by Signal to Noise Ratio of 3:1.

LOQ

Lowest amount of analyte in a sample that can be quantified with suitable

accuracy and precision.

Estimated by Signal to Noise Ratio of 10:1.

LOQ & LOD Estimated by :-

1. Based in Visual Evaluations

- Used for non-instrumental methods

2. Based on Signal-to Noise-Ratio

- 3:1 for Detection Limit

- 10:1 for Quantitation Limit

3. Based on Standard Deviation of the Response and the Slope

Ybl

LOD LOQ

Statistical estimate of LOD & LOQ

LOD = 3.3 Sbl / b LOQ = 10 Sbl / b

Y = b X + a

Specificity & Selectivity:-

Ability of an analytical method to measure the analyte free from interference due

to other components.

Selectivity describes the ability of an analytical method to differentiate various

substances in a sample

Original term used in USP

Also Preferred by IUPAC

Also used to characterize chromatographic columns

Degree of Bias (Used in USP)

The difference in assay results between the two groups

- the sample containing added impurities, degradation products, related

chemical compounds, placebo ingredients

- the sample without added substances

Robustness :-

Definition: Capacity to remain unaffected by small but deliberate

variations in method parameters

Determination: Comparison results under differing conditions with

precision under normal conditions

Examples of typical variations in LC

Influence of variations of pH in a mobile phase

Influence of variations in mobile phase composition

Different columns (different lots and/or suppliers)

Temperature

Flow rate

Ruggedness :-

Degree of reproducibility of test results under a variety

of conditions

○ Different Laboratories

○ Different Analysts

○ Different Instruments

○ Different Reagents

○ Different Days

Expressed as %RSD

Techniques:- Chromatographic Methods - HPLC, GC, TLC,

GC/MS,LCMS, etc. Spectrophotometric Methods – UV/VIS, IR, NIR, AA,

NMR, XRD,MS Electrochemical techniques:-Potentiometry,

Polarography ,Conductometric titration.

UV-Visible spectrophotometry:-

A molecule which has either n , pie or sigma valance electrons, absorb the characteristics radiations and undergoes transition from ground state to exited state.

Almost all organic compounds absorb in the UV range(180nm-380nm)

Solution in appropriate solvent is prepared in a given concentration range and then scanned in the UV range to obtain. Lambda-max(nm)

Presence of auxochrome and chromophores is identified by examining the structure which is must for absorption in UV-range.

If conjugate double bond is present ,a better peak is obtained. Determination whether and how a chromophore can be

derivatised or auxochrome may be introduced to obtained better peak values.

Introduction To Hplc Methods Of Analysis For Drugs In Combination 

HPLC defined as a separation technique in which pressure is applied to the column forcing the mobile phase through at much higher rate. It is based upon number of theoretical plates available for different component to be separated.

Most of the drugs in multi component dosage forms can be analyzed by HPLC method because of the several advantages like rapidity, specificity, accuracy, precision and ease of automation in this method. HPLC method eliminates tedious extraction and isolation procedures.

Some of the advantages are: Speed (analysis can be accomplished in 20 minutes or less), Greater sensitivity (various detectors can be employed), Improved resolution (wide variety of stationary phases),

Easy sample recovery, handling and maintenance, Instrumentation tends itself to automation and quantitation (less

time and less labour), Precise and reproducible, Suitable for preparative liquid chromatography on a much larger

scale. Reusable columns (expensive columns but can be used for many

analysis), Ideal for the substances of low volatility. There are different modes of separation in HPLC. They are

normal phase mode, reversed phase mode, reverse phase ion pair chromatography, affinity chromatography and size exclusion chromatography.

HPTLC

(High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows a lot of advantages in comparison to other separation techniques.

Layer of Sorbent

100µm Efficiency

High due to smaller particle size generated Separations

3 - 5 cm Analysis Time

Shorter migration distance and the analysis time is greatly reduced Solid support

Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes

Development chamber

New type that require less amount of   mobile phase Sample spotting

Auto sampler Scanning

Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer

Features of HPTLC

Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard

Several analysts work simultaneously Lower analysis time and less cost per analysis Low maintenance cost Simple sample preparation - handle samples of divergent nature No prior treatment for solvents like filtration and degassing Low mobile phase consumption per sample No interference from previous analysis - fresh stationary and

mobile phases for each analysis - no contamination Visual detection possible - open system Non UV absorbing compounds detected by post-chromatographic

derivatization

Parameter’s involved during Analytical method development :-1. Optimization of Mobile Phase .

2. Selection of Buffers

3. Selection of Ion pair reagent

4. pH of buffer/mobile phase

5. Composition of Mobile Phase

6. Selection of Column

7. Optimization of Analyte signal

8. Objectives of Separation( Analysis time, Resolution, k factor (Capacity factor), Peak height, Asymmetry, Theoretical Plates)

9. 9. Flow rate

The parameters that are affected by the changes in chromatographic conditions are:-

Resolution (Rs), Capacity factor (k’), Selectivity (a), Column efficiency (N) and Peak asymmetry factor (As). Peak purity

Resolution- Rs, of two neighboring peaks is defined as the ratio of the distance between maxima of two peak. It is the difference between the retention times of two solutes divided by their average peak width. For baseline separation, the ideal value of Rs is 1.5. It is calculated by using the formula,

where,      Rt1 and Rt2 are the retention times of components 1 and 2 and

W1 and W2 are peak width of components 1 and 2.

Capacity Factor (k’): Capacity factor is the ratio of the reduced retention volume to the dead volume.

Capacity factor, k’, is defined as the ratio of the number of molecules of solute in the stationary phase to the number of molecules of the same in the mobile phase. Capacity factor is a measure of how well the sample molecule is retained by a column during an isocratic separation. The ideal value of k’ ranges from 2-10. Capacity factor can be determined by using the formula,

Where, tR = retention volume at the apex of the peak (solute) and

  t0 = void volume of the system.

Capacity Factor (k') changes are typically due to:

· Variations in mobile phase composition · Changes in column surface chemistry (due to aging) · Changes in operating temperature. In most chromatography modes, capacity factor (k')

changes by 10 percent for a temperature change of 5 C.

Adjusting Capacity Factor (k')

Good isocratic methods usually have a capacity factor (k') in the range of 2 to 10 (typically between 2 and 5).  Lower values may give inadequate resolution.  Higher values are usually associated with excessively brood peaks and unacceptably long run times.

If the analyte fall outside their specified windows run the initial column test protocol to compare the results obtained with a new column.

Capacity Factor (k') values are sensitive to: · solvent strength · composition · purity · temperature · column chemistry · sample

Selectivity(a):

The selectivity (or separation factor), a, is a measure of relative retention of two components in a mixture. Selectivity is the ratio of the capacity factors of both peaks, and the ratio of its adjusted retention times. Selectivity represents the separation power of particular adsorbent to the mixture of these particular components.

This parameter is independent of the column efficiency; it only depends on the nature of the components, eluent type, and eluent composition, and adsorbent surface chemistry. In general, if the selectivity of two components is equal to 1, then there is no way to separate them by improving the column efficiency.

The ideal value of a is 2. It can be calculated by using formula, a= V2 – V1 / V1 – V0     = k1’/ k2’

Where, V0 = the void volume of the column,

         V1 and V2 =the retention volumes of the

second and the first peak respectively.

Column Efficiency/ Band broadening: Efficiency, N, of a column is measured by the number of theoretical plates per meter. It is a measure of band spreading of a peak. Similar the band spread, higher is the number of theoretical plates, indicating good column and system performance. Columns with N ranging from 5,000 to 100,000 plates/meter are ideal for a good system. Efficiency is calculated by using the formula

Where,       tR is the retention time and W is the peak width.

Peak asymmetry factor (Tf):- Peak asymmetry factor, Tf, can be used as a criterion of column performance. The peak half width, b, of a peak at 10% of the peak height, divided by the corresponding front half width, a, gives the asymmetry factor.

For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.

Peak purity:  The null hypothesis “these spectra are identical” can in this

case (purity) with two sided significance.  During the purity test the spectrum taken at the first peak slope is correlated with the spectrum of peak maximum [r(s,m)] and the correlation of the spectra taken at the peak maximum with the one from the down slope or peak end [r(m,e)] which is used as a reference spectra for statistical calculation.  An error probability of 1% only be rejected if the test value is greater than or equal to 2.576

REFERENCES :- Snyder, L.R.; Kirkland, J.J.; Glajch , J.L. “Practical

HPLC Method Development,” 2nd ed. John Wiley & Son: New York, 1997.

WHO guidelines for Analytical Method Development. FDA, “Analytical Procedures and Methods Validation:

Chemistry, Manufacturing, and Controls ,” Federal Register (Notices) 65 (169) 52,776–52,777 (30 August 2000).

An Introduction to Analytical method development of formulation (http://www.pharmainfo.net)

Thank You