Chromatographic Methods

Chromatographic analytical procedures are analysed by two types of method there are

1. External Standard methods

2. Internal Standard methods

External Standard methods:

An external standard method is used when the standard is analyzed on a separate chromatogram from the sample. Quantitation is based on a comparison of the peak area or height (HPLC or GC) or spot intensity (TLC) of the sample to that of a reference standard of the analyte of interest.

The external standard method is more appropriate for samples as follows:
1. Sample with a single target concentration and narrow concentration range, e.g., acceptance and release tests.
2. Simple sample preparation procedure.
3. Increased baseline time for detection of potential extraneous peaks, e.g., impurities test.

Internal Standard Methods:

With an internal standard method, compound of known purity that does not cause interference in the analysis is added to the sample mixture.

Quantitation is based on the response ratio of compound of interest to the internal standard vs the response ratio of a similar preparation of the reference standard (HPLC or GC). 


This technique is rarely used for TLC methods.

The internal standard method is more appropriate for samples as follows:

1. Complex sample preparation procedures, e.g., multiple extractions.
2. Low concentration sample (sensitivity being an issue), e.g., pharmacokinetics studies.
3. Wide range of concentrations expected in the sample for analysis, e.g., pharmacokinetics studies.

HPLC methods for release and stability and TLC methods use external standards; and methods for biological fluids and GC methods use internal standards.

Reference Standards For Analytical Procedures

Types of Standards
Reference standards are of two type’s a. Primary standards and b. secondary standards.
 

Primary Reference standards or primary standards:
Primary standards are the chemical substances which are accepted without reference to other standards.

Primary standard may be obtained from the USP/NF or EP or BP and other official sources or prepared by the manufacturer if there is no official source to obtain material (In-house products).
 

Secondary reference standards or working standards:
A working standard is a standard that is qualified against and used instead of the reference standard. There are generally used for regular analysis of drug substances.
 

Manufacturing of Reference standards:
The manufacturing process of references standards can be different from the final processing of the drug substance.It is most widely acceptable practice in the industry.

Generally purification methods such as distillation, crystallization or extraction are used where as Prepaprative HPLC used only for rare or expensive products.

In all cases, the analytical procedure (purity and assay) used for characterization must be validated.
 

Characterization of Primary or working standards:
The characterization of primary standards or working standards generally involves the elucidation of chemical structure by IR, UV, H'-NMR, C13-NMR, XRD, Mass spectroscopy and elemental Analysis etc.
 

Purity/Potency Determination
The degree of potency is to be established by chromatographic validated method.
If the monograph contains only one method for assay (without purity determination), it is preferable to use a stability indicating method of the drug substance for purity determination.
If no drying is required, the assay and the water determination may be analyzed individually and the assay recorded "as is". The assay refers to the declared amount, and the figure is to be used in calculating the results of the assay.

The standard potency is calculated as follows:
%Potency = %Assay-(%water+% residual solvents)
 

Drying
Working standards are to be dried in accordance with the labeling of the same Pharmacopeial or primary standard.

If a standard requires drying, the required amount must be transferred to a clean weighing bottle for drying. The weighing bottle must be protected from light during use. Dried standards must not be returned to the original bottle in order to avoid any possible contamination of the stock. After drying, place the standard into the desiccator to equilibrate before assaying.
 

Retest or Expiration dating:
The retest or expirations dating for primary reference standards can be obtained from the official source. In case of in-house reference standards retest date can be assigned by the stability and sensitivity of drug substance. 

In case of secondary standards, usually one year is the retest period. This parameter always depends on the stability of the drug substance.

Storage of Standards:
Working standards are stored in the same manner as indicated by the official source or stability of the standard and are stored in amber glass containers, protected from light, heat and moisture.
 

Working standards should be kept at ambient conditions in an amber dissector cabinet, unless otherwise specified by the individual monograph and if the standard that require storage in a "cold" or "dry" place are to be stored in a refrigerator at 2-8°C.

Types of Analytical Procedures

Analytical procedures are used to evaluate characteristics of the drug substance. Analytical procedures are classified into three types. There are

1. Regulatory analytical procedures
2. Alternative Analytical procedures
3. Stability Indicating Methods
 

Regulatory Analytical Procedures or Compendial Methods
The analytical procedures which are defined in the USP/EP/EP/JP/IP etc.. are termed as regulatory analytical procedure for a particular Drug substance. If the manufacturer claims that his/her material is EPgrade, then the manufacturer must analyse the drug substances as per the procedures given in the EP and the material must meet the specifications given by EP. 

Eg: Famotidine, Ciprofloxacin Hydrochloride etc..

Alternative Analytical Procedures or In-House procedures:
There are situations were the regulatory analytical procedures may not be sufficient to analyze the drug substance, in such cases applicant can come up with own analytical methods. These methods are called as alternative analytical procedures. The method should be same or better than the regulatory method and fully validated and also comparative to the regulatory analytical procedures.
 

Stability Indicating Methods(SIM):
A SIM is a quantitative analytical procedure used to detect a decrease in the amount of the active pharmaceutical ingredient (API) present due to degradation.

According to FDA guidelines, a SIM is defined as a validated analytical procedure that accurately and precisely measures active ingredients (drug substance or drug product) free from potential interferences like degradation products, process impurities, excipients, or other potential impurities.

All the assay procedures for stability studies be stability indicating.