HPLC in Industry - Biagini

Question 1

Where is HPLC used?

Answer 1

• Drug analysis for ID and quantification on drugs
• Discovery: Research
• Development: Chemical synthesis and formulation
• Manufacturing: Quality control and API and Dosage Forms

Question 2

What is HPLC method validation?

Answer 2

• HPLC methods used for development of pharmaceuticals and for determination of their quality have to be validated.
• Specificity or selectivity, precision, accuracy or trueness or bias, linearity range, limit of detection, limit of quantification and robustness need to be determined.

Question 3

What are selectivity/specificity?

Answer 3

Selectivity and specificity are used interchangeably.

Selectivity is the ability to measure accurately an analyte in the presence of interference such as synthetic precursors, excipients, enantiomers and known degradation products that might be present in the sample.

Question 4

What is the precision of a method?

Answer 4

• Precision of a method is measured by injecting a series of standards and measuring the variability of the quantitative results.
• Precision can be separated into repeatability, intermediate precision and reproducibility.

Question 5

What is repeatability?

Answer 5

Repeatability is obtained when one operator using one system over a short time span carries out the analysis in one laboratory. At least 5/6 determinations of three different matrices at two or three concentrations should be done and the relative standard deviation should be calculated.

Question 6

What is intermediate precision?

Answer 6

• Intermediate precision is the long-term variability of the measurement and is determined by the results of a method run within a single laboratory over a number of weeks.
• It may reflect discrepencies in results obtained by different operators, from different instruments etc.
• The objective is to verify that in the same laboratory the method will provide the same results once the development phase is over

Question 7

What is reproducibility?

Answer 7

Reproducibility represents the precision obtained between different laboratories.

The objective is to verify that the method will provide the same results in different labs, preparing it for transfer to other sites.

Typical variations affecting reproducibility are room temperature/humidity, operators with different expertise, different equipment, different solvents, reagents and materials etc.

Question 8

What is the accuracy of a method?

Answer 8

Accuracy is the extent to which test results are close to their true value

It is measured from the result of the quantitative determination of a well characterised known sample. The amount measured is compared to the known

Question 9

What is the linearity of a method?

Answer 9

• Linearity is determined by a series of three-six injections of five/more standards whose concentration’s span is 80-120% of the expected concentration range.
• Response should be proportional to the concentrations of analytes, directly or by means of a well-defined mathematical calculation.
• A linear regression equation should have an intercept not significantly different from zero.
• If significant non-zero is obtained, it should be demonstrated there is no affect on accuracy of method.

Question 10

What is the limit of detection and what is the limit of quantification?

Answer 10

• Limit of detection is the lowest concentration of analyte in a sample that can be detected but not necessarily quantified.
• In chromatography, DL is the injected amount that results in a peak height of at least twice/three times as high as baseline.
• Limit of quantification is the minimum injected amount that gives precise measurements
• Typically requires a peak of 10-20x higher than baseline noise at precision of <10-15% RSD between results.

Question 11

What is the identification test of HPLC?

Answer 11

• ID test of HPLC is aimed to confirm the identity of the API inside the samples of either drug substance or drug product.
• Two independent drug tests are needed e.g. one chromatographic and one spectroscopic.
• Thus a chromatographic run with a MS detector will fulfil both parameters, the retention time in the chromatogram and the MS of the eluting peak matched against a known standard.

Question 12

Why is it necessary for substances of abuse to be quantified?

Answer 12

• Under Misuse of Drugs Act 1971, there is a requirement for the amount of drugs to be determined.
• This is related to the severity of the sentence that will be imposed.

Question 13

What is the profiling of substances of abuse?

Answer 13

Profiling of drugs is where the same impurities observed in one batch are compared with another to determine whether they are linked. Four comparisons are:

• Drug identification
• Drug quantification
• ID of the cutting agents
• Identification of the impurities.

Question 14

What are quality control checks?

Answer 14

• Sample runs are followed by blanks
• Checked for no cross-contamination
• Calibrants or real samples are required.
• Internal quality controls
• External quality controls
• Validation of results by another lab.
• Instrumentation must be functional, test sampling must be consistent and data handling must be double-checked.