Development and Validation of a RP-HPLC method

1. DEVELOPMENT AND
VALIDATION OF A RP-
HPLC METHOD
SUBMITTED BY,
Usha Khanal
M.Pharm 1st year
Roll no.-18HMPA06
Pharmaceutical Analysis&QualityAssurance

2.  Method: A method is a set of experimental conditions designed to create a good
analysis of a particular sample.
 Developing a method: Method development encompasses many stages and can take
months to complete, depending on the complexity and goals of the method.
 The process usually includes the following steps:

3. STEPS IN HPLC METHOD
DEVELOPMENT
Information on sample, define separation goal
Need for special HPLC procedure, sample, pretreatment,
etc.
Choose detector and detector settings
Choose LC method; preliminary run; estimate best
separation conditions

4. Optimize separation conditions
Check for problems or requirements for special procedure
Recover purified
material
Quantitative
calibration
Qualitative method
Validate method for release
to routine laboratory

5. NATURE OF THE SAMPLE
 To know the composition and properties of sample; particularly the properties likely to
be affected by the analysis.
 The sample properties we need to know before developing a method are:
Sample property Details
Matrix To know the compounds other than the
analyte are in the sample.
Concentration The amount of the compound is
present in the sample.
The concentration range (low or high).

6. Sample property Details
Quantity The number compounds are present in the
sample.
Chemical/physical properties •pKa values
• molecular size and weight
•electrical charge
• sample solubility
•sample volatility
• stability and toxicity
• hydrophobicity/polarity
• chemical/biological reactivity
•UV spectra

7. METHOD GOALS
Method goal Details
Detect The compound is present or not in the sample.
Quantitate The amount of the compound is present in the
sample
Identify Compound identification
Characterize Properties of the compound
Purify/ isolate To collect the compound for further use, if
required

8. DETERMINE THE ANALYSIS
REQUIREMENT
 These are variables associated with the method goals.
Method goal Analysis requirement
Detection Detection technique that can be used to analyze
the sample
Quantitation Method for quantify (e.g., internal standard,
external standard)
The concentration range or sample amount
(low or high)
Levels of accuracy and precision are required
Identification Process required for identifying the
compound
To determine purity

9. Method goal Analysis requirement
Characterization To determine the properties or property
levels
Purification/isolation To isolate purified material
To recover 100% of your sample
Sample matrix If there is more than one sample matrix
before analysis and either the sample matrix
interfere with analysis
Properties The analysis technique allow you to
determine sample properties

10. CONDUCT RESEARCH
 Conduct research to determine if the analysis has been performed before.
 Previously developed methods with quantitation and sample matrices that are close to the requirements
can form a starting point for the method.
 Resources to consult include:
• Internet
• United States Pharmocopeia (USP)
• FDA requirements
• EPA requirements
• USDA methods
• Colleagues
• Professional/technical journals and meetings

11. SELECT THE ANALYSIS TECHNIQUE
Analysis technique Capabilities
Liquid chromatography (LC) Separates samples in solution based on
physical properties such as polarity, ionic
strength, and molecular size.
Liquid chromatography-mass spectrometry
(LC-MS)
Combines the physical separation
capabilities of LC with the mass analysis
capabilities of MS

12. SAMPLE PRETREATMENT AND
DETECTION
 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

13.  Most HPLC analysis require weighing and/or volumetric dilution before injection.
 Best results are often obtained when the composition of the sample solvent is close to
that of the mobile phase, since this minimizes baseline upset and other problems.
 This means that it is important to know the nature of the sample matrix and the
probable concentrations of various analytes.
 Before the first sample is injected during HPLC method development, we must be
reasonably sure that the detector selected will sense all sample components of interest.
 variable-wavelength ultraviolet (UV) detectors normally are the first choice, because of
their convenience and applicability for most samples.

14.  For this reason, information on the UV spectra can be an important aid tor method
development.
 UV spectra can be found in the literature, estimated from the chemical structures of
sample components of interest, measured directly (if the pure compounds are available),
or obtained during HPLC separation by means of a photodiode-array (PDA) detector.
 When the UV response of the sample is inadequate, other detectors are available
(fluorescence, electrochemical, etc.), or the sample can be derivatized for enhanced
detection.

15. DEVELOPING THE SEPARATION(METHOD)
 Selecting an HPLC Method and Initial Conditions
 Based on a knowledge of sample composition and the goals of separation.
 If HPLC is chosen for the separation, the next step is to classify the sample as regular or
special.
SAMPLE
HPLC
Regular Special

16. Using information about the sample to select conditions for the initial experimental separation.
Regular Special
Neutral Ionic
exploratory run
(reversed-phase)
•Inorganic ions
•Isomers
•Enantiomers
•Biological samples
Peptides
Carbohydrates
Neuclotides
Macromolecules
Proteins
Carbohydrates
Nucleic acids
Synthetic polymers

17.  When the goal of separation is the isolation of purified material, an optimized final
HPLC method will differ from one developed for routine quantitative analysis.
 However, the beginning of the method development proceeds in exactly the same way
for both cases.
 Thus method can be developed using one of the following approaches:
a. stepwise incremental (one-factor-at-a-time) approach based on results from previous
experiment
b. systematic screening protocol, in which we evaluate factors such as stationary
phases, solvents, pH and retention and thereby enhance resolution.

18.  Preferred Experimental Conditions for the Initial HPLC Separation
Separation Variable Preferred Initial Choice
Column
Dimensions 15 x 0.46 cm
Particle size 5µm
Stationary phase C8 or C18
Mobile phase
Solvent A &B Buffer and acetonitrile
% B 80-100%
Buffer (compound, pH, concentration) 25 mM potassium phosphate,
2.0<Ph<3.0
Additives (e.g., amine modifiers, ion- pair reagents) Do not use initially
Flow rate 1.5-2.0 mL/min
Temperature 35-45°C
Sample Size
Volume
Weight
< 25µL
< 100µg

19. GETTING STARTED ON METHOD
DEVELOPMENT
 we assume that the sample is regular unless noted otherwise.
 Although the initial and final conditions required for special samples will differ from those
listed for regular samples, the general strategy and approach to method development is
similar for both regular and special samples.
 Thus the separation of regular samples will therefore prove applicable in many respects to
method development for special samples.
 Start with an isocratic mobile phase composition use water & Methanol or ACN according to
solubility in 50:50 ratio.
 Feed other parameter like wavelength, ambient temperature, 1ml/min Flow rate.

20.  If there are more than 10 components in particular sample it is usually not
recommended to begin method development with an intermediate strength mobile
phase.
 A better alternative is to use a very strong mobile phase ( e.g., 100-80% B), then reduce
the %B as necessary.
 Example: For the separation of mixture of triazine herbicides (6 components) as a
function of mobile phase conditions. Conditions: 25 x 0.46-cm C18 column; water-
methanol mobile phase; ambient temperature; 1.7 mL/min. (a) 50% B; (b) 100% B; (c)
80% B; (d) 60% B; (e)5-100%B in 20 min (gradient) (f) 70%B (isocratic)

25. IMPROVING THE SEPARATION
Goal Comment
Resolution Precise and rugged quantitative analysis requires
that R, be sgreater than 1.5.
Separation time <5-10 min is desirable for routine procedures.
Quantitation ≤2% for assays; ≤5% for less-demanding analyses;
≤15% for trace analyses
Pressure <150 bar is desirable, <200 bar is usually essential.
Peak height Narrow peaks are desirable for large signal/noise
ratios.
Solvent consumption Minimum mobile-phase use per run is desirable.

26. METHOD OPTIMIZATION
 The experimental conditions should be optimized to get desired separations and
sensitivity after getting appropriate separations.
 Stability indicating assay experimental conditions will be achieved through
planned/systemic examination on parameters including pH (if ionic), mobile phase
components and ratio, gradient, flow rate, sample amounts, injection volume and
diluents solvent type.

27. VALIDATION OF METHOD
 Validation of an analytical procedure is the process used to confirm that the analytical
procedure employed for a specific test is suitable for its intended use.
 The methods validation process for analytical procedures begins with the planned and
systematic collection by the applicant of the validation data to support analytical
procedures.
 The validation of analytical methods is done as per ICH guidelines Q2(R1).

28. COMPONENTS OF METHOD VALIDATION
The following are typical analytical performance characteristics which may be tested during methods validation:
 1. System Suitability
 2. Accuracy
 3. Precision
 4. Repeatability
 5. Intermediate precision
 6. Linearity
 7. Detection limit
 8. Quantitation limit
 9. Specificity
 10. Range
 11. Robustness
 12. Forced degradation studies

29. 1. System Suitability
 System suitability testing originally believed by the industry of pharmaceuticals to decide whether a
chromatographic system is being utilized day to day in a routine manner in pharmaceutical laboratories
where quality of results is most important which is suitable for a definite analysis.
 The parameters used in the system suitability tests (SST) report are as follows:
 1. Number of theoretical plates or Efficiency (N).
 2. Capacity factor (K).
 3. Separation or Relative retention (α).
 4. Resolution (Rs).
 5. Tailing factor (T).

30. 1. Number of theoretical plates/Efficiency (N)
 In a specified column, efficiency is defined as the measurement of the degree of peak
dispersion. The efficiency is conveyed in terms of number of theoretical plates.
 The formula of calculation of N is illustrated below in the following

31. 2. Capacity ratio or Capacity factor (k)
 K’=tR-tM/tM
 The above said capacity factor sometimes is called as a retention factor which has no
dimension and independent from flow rate of mobile phase as well as column
dimensions which is the measure of extent of retention relating to an analyte relative to
an un-retained peak.
 Where tR implies retention time of the sample peak and retention time of an un-
retained peak is tM. k' = 0 means no compound is left in the column.
 Generally the value of k' is > 2.

32. 3. Relative retention or separation factor (α)
 α = t2-ta/t1-ta
 α = Relative retention.
 t2= Retention time calculated from point of injection.
 ta= Unretained peak time (Retention time (tR) of an inert component not retained by
the column).
 t1= the retention time from the point of injection of reference peak defined. (Suppose
no reference peak is found, value would be zero).

33. 4. Resolution (Rs)
 Resolution is the capability of the column to separate 2 drugs in 2 individual peaks or
chromatographic zones and it is improved by enhancing column length, reduction of
particle size and rising temperature, altering the eluent or stationary phase.
 By using the following formula resolution is calculated.

34. Determination of resolution between two peaks.
 tR1 and tR2 are the retention times for the two peaks of components.
 tw1 and tw2 = At the baseline lies between tangents drawn to the sides of the peaks.

35. 5.Tailing factor or Asymmetry factor
 Chromatographic peak assumed to have a Gaussian shape under ideal conditions.
 However in practical conditions, there is always a deviation from normal distribution which
indicates non- uniform migration and non-uniform distribution process.
 The asymmetry factor and tailing factor are roughly same and rarely accurate and equal in
most cases.
 Values should normally between 1.0-1.5 and values greater than 2 are unacceptable.
 The peak asymmetry is computed by utilizing the following formula.
As = B/A
Where:
As = peak asymmetry factor.
B = distance from the point at peak midpoint to the trailing edge. (measured at 10 % of peak
height).
A = distance from the leading edge of peak to the midpoint. (measured at 10 % of peak height).

36.  Ideally, peaks should be Gaussian in shape or totally symmetrical.
Determination of tailing and asymmetric factor.

38. 2. 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.
 Lack of specificity of an individual analytical procedure may be compensated by other supporting
analytical procedure(s).
 This definition has the following implications:
 Identification: To ensure the identity of an analyte.
 Purity Tests: Ensure that all the analytical procedures performed allow an accurate statement of the
content of impurities of an analyte, i.e. related substances test, heavy metals, residual solvents content,
etc.
 Assay (content or potency): To provide an exact result which allows an accurate statement on the content
or potency of the analyte in a sample.

39. 3. 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.
 This is sometimes termed trueness.

40. 4. 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.
 Precision should be investigated using homogeneous, authentic samples.
 However, if it is not possible to obtain a homogeneous sample it may be investigated
using artificially prepared samples or a sample solution.

41.  Repeatability
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision .
 Intermediate precision
Intermediate precision expresses within-laboratories variations: different days, different
analysts, different equipment, etc.
 Reproducibility
Reproducibility expresses the precision between laboratories (collaborative studies,
usually applied to standardization of methodology).

42. 5. 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.

43. 6. 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.
 The quantitation limit is a parameter of quantitative assays for low levels of compounds
in sample matrices, and is used particularly for the determination of impurities and/or
degradation products.

44. 7. 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.

45. 8. 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

46. 9. 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.

47. Forced Degradation Studies
 Forced degradation or stress studies are undertaken to deliberately degrade the sample.
 These studies are used to evaluate an analytical method’s ability to measure an active
ingredient and its degradation products, without interference, by generating potential
degradation products.
 During validation of the method, drug substance are exposed to acid, base, heat, light and
oxidizing agent to produce approximately 10% to 30% degradation of active substance.
 The studies can also provide information about the degradation pathways and degradation
products that could form during storage.
 These studies may also help in the formulation development, manufacturing, and packaging
to improve a drug product.

48. REFERENCES
 Lloyd R. Snyder, Joseph J. Kirkland, Joseph L. Glajch. Practical HPLC method
development. 2nd edition,1988,1-15
 Vibha Gupta et al,. Development and validation of HPLC method. International
Research Journal of Pharmaceutical and Applied Sciences. 2(4),2012,17-25.
 Pallavi Nemgonda Patil. HPLC method development. Journal of Pharmaceutical
Research & Education. 1(2),2017,243-260.
 ICH, Validation of Analytical Procedures: Text and Methodology. International
Conference on Harmonisation, EMEA.2006,2-15.