HPLC Method Development and Validation for the Quantification of Drug in 100 mg

Tablet

1. Preparation of Stock Solution (1000 µg/mL)

The first step in the process is the preparation of the stock solution. In HPLC experiment, ppm
(µg/mL) concentration is prepared. For assay method, we prefer to make same ppm as the
label claim. E.g. if our tablet label claim is 100 mg/tablet then we will use 100 ppm
standard. For that we generally prepare stock solution 10 times higher concentration than
required. Means we would prepare 1000 ppm stock and then perform 1/10 th dilution to get
100 ppm standard.

Stock Solution Preparation:

 Weigh accurately 100 mg of the drug substance (tablet powder, or API).

 Transfer the drug into a 100 mL volumetric flask.
 Dissolve the drug in a suitable solvent (commonly, a mixture of water and organic
solvent like acetonitrile or methanol depending on the solubility of the compound).
 Make up the volume to 100 mL with the solvent.
 This gives a stock solution with a concentration of 1000 µg/mL (1 mg/mL).

EXPLANATION OF PPM CALCULATION

2. Preparation of Standard Solution (100 ppm), Which is termed as 100% concentration

level. This will be used for method development.

The next step is the preparation of the standard solution.

Standard Solution Preparation:

 Take an aliquot of the stock solution (for example, 1 mL).

  Dilute the aliquot to 10 mL with the mobile phase to obtain a concentration of 100 ppm
(100 µg/mL).
 The standard solution concentration should be 100 ppm.

EXPLANATION

Objective: Prepare a standard solution with a concentration of 100 ppm from the stock solution.

Dilution: From the 1000 ppm stock solution, take 1 mL and dilute it to 10 mL with the
mobile phase.

C1xV1=C2xV2

Where:

 C1 = initial concentration (1000 ppm)

 V1 = volume taken from the stock (1 mL)
 C2 = final concentration (desired concentration, 100 ppm)
 V2 = final volume (10 mL)

Let’s calculate

1000ppm×1mL=C2×10mL

C2 = 100 ppm

Thus, the concentration of the standard solution is 100 ppm.

IN OTHER WORDS:

10 1
------ X -------- X 1000

100 10

(First step is stock preparation, second step is dilution of stock and 1000 is there for
conversion of mg to mcg)

3. Preparation of Calibration Standards (50, 75, 100, 125, and 150 ppm)

For calibration curve development, prepare a range of (50%-150%) standards from 50 ppm to
150 ppm, representing the linearity range.

Calibration Standards Preparation:

 From the 100 ppm standard solution, prepare five calibration standards by serial dilution.
o 50 ppm: Take 1 mL of the 1000 ppm solution and dilute it to 10 mL with the
mobile phase.
o 75 ppm: Take 0.75 mL of the 1000 ppm solution and dilute it to 10 mL with the
mobile phase.
o 100 ppm: Take 1.00 mL of the 1000 ppm solution and dilute it to 10 mL with the
mobile phase.
o 125 ppm: Take 1.25 mL of the 100 ppm solution and dilute it to 10 mL with the
mobile phase.
o 150 ppm: Take 1.5 mL of the 100 ppm solution and dilute it to 10 mL with the
mobile phase.

4. Method Development

Before Starting:

 Check pump seals and lamp status.

 Ensure no leakages in tubing.
  Clean injector if not used for a while.
 Turn on the system and allow it to equilibrate.

4.1 Mobile phase selection:

For Reverse-Phase Chromatography (most commonly aqueous and organic mixture is used in
a volumetric ratio, e.g. 50:50%v/v, 40:60%v/v, (note this ratio is always 100), etc.) Also
remember when we are doing RP-HPLC, the MOBILE PHASE IS POLAR and stationary
phase is NONPOLAR.

 Aqueous component: Typically water, but often buffered with weak acids (like
phosphoric acid, formic acid) to help maintain pH stability. A buffer also helps minimize
peak tailing and improves resolution.
 Organic component: Common solvents are acetonitrile (ACN) or methanol (MeOH).
These are often chosen because of their high miscibility with water and good solvent
properties.
 Buffer pH: Typically, a pH of around 2-7 is used for reverse-phase methods, with pH
adjustment depending on the analyte's stability and solubility.

Once you have selected a mobile phase or a range of possible mobile phases:

 Run test injections: Start by running standard injections with your mobile phase and
analyzing the retention time, resolution, and peak shape.
 Adjust parameters based on results:
o Peak Shape: If peaks are broad or tailing, you may need to adjust the pH, modify
the solvent composition, or try different flow rates.
o Retention Time: If the analyte is retained for too long, consider increasing the
organic solvent concentration or using a higher flow rate.
o Resolution: If the analytes are not well separated, fine-tune the mobile phase
composition or consider gradient elution.
Practical Example

Let’s say you are developing a method for analyzing a moderately polar compound (polarity
known from logP values) in a tablet using RP-HPLC.

1. Select mobile phase solvents: You know your analyte is moderately polar, so you
choose water and acetonitrile (ACN) or methanol as the solvents.
2. Start with a 50:50 water:methanol %v/v or 70:30 water:ACN %v/v mixture: This is a
typical starting point for reverse-phase chromatography.
3. Test the mixture: Run the HPLC, and if the analyte takes too long to elute, increase the
Methanol/ACN percentage to 40% or 50%.

 If resolution is poor (means peaks are poorly separated, adjust the pH or change the
flow rate.
 If peak shape is poor, tailing or fronting is seen, replace water with appropriate
buffer.

4. Fine-tuning: After optimizing the solvent composition, you notice that the analyte is
eluting too quickly, so you adjust the mobile phase to a 60:40 water:ACN mixture, which
gives a good separation.
5. Buffer: IF peaks are poor use buffer instead of water to stabilize the pH and improve
peak shape.

TWO IMPORTANT PARAMETERS OF DRUG COMPOUNDS NEEDS TO BE

CONSIDERED ARE LogP AND pKa

LogP (the partition coefficient or octanol-water partition coefficient) is an important

physicochemical property that describes the hydrophobicity or lipophilicity of a compound. It is
the logarithm of the ratio of the concentration of a compound in octanol to its concentration in
water. This value gives insight into how a compound interacts with water (hydrophilic) versus
organic solvents (hydrophobic).

In Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), LogP plays a

crucial role in determining the retention behavior of analytes on the C18 (or other reversed-
phase) columns, which are the most commonly used stationary phases in RP-HPLC.

How LogP Affects RP-HPLC

1. Retention Behavior:
o Hydrophobic Interaction: In RP-HPLC, the stationary phase is non-polar (e.g.,
C18, a hydrocarbon chain). The mobile phase is typically a mixture of water (or
buffer) and an organic solvent like acetonitrile or methanol. Compounds with a
higher LogP (more hydrophobic) will have stronger interactions with the
stationary phase and will be retained longer.
 High LogP: The higher the LogP value (meaning the compound is more
hydrophobic), the longer it will typically stay on the column because it has
a stronger affinity for the non-polar stationary phase.
 Low LogP: Compounds with low LogP (more hydrophilic) will interact
less with the non-polar stationary phase and will be eluted faster as they
prefer to stay in the aqueous mobile phase.
2. Elution Order:
o In RP-HPLC, the elution order is generally governed by the LogP value:
 More hydrophobic compounds (with a higher LogP) will be retained
longer and elute later in the chromatogram.
 More hydrophilic compounds (with a lower LogP) will be eluted earlier,
as they prefer to stay in the mobile phase.
3. Optimization of Mobile Phase:
o LogP also influences how the mobile phase should be optimized:
 For highly hydrophobic compounds: A more polar mobile phase (e.g.,
more water or less organic solvent like acetonitrile) might be required to
elute them.
  For hydrophilic compounds: A more non-polar mobile phase (more
acetonitrile or methanol) may be needed to reduce their affinity for the
stationary phase and promote faster elution.
4. Retention Time Prediction:
o A general relationship exists between LogP and retention time in RP-HPLC. As
LogP increases, so does the retention time, meaning that compounds with a
higher LogP value will typically have a longer retention time because of their
stronger interactions with the stationary phase.
o However, other factors such as the size of the molecule, the presence of functional
groups, and the mobile phase composition also play roles in retention, so LogP is
just one factor to consider.
5. Separation Efficiency:
o The differences in LogP between analytes can contribute to good separation in
RP-HPLC, as compounds with significantly different LogP values will have
different retention times, helping to achieve better resolution.
o If LogP values are too similar, it can lead to poor separation, as the compounds
will interact similarly with the stationary phase and elute at nearly the same time.
6. Effect of pH on LogP:
o The LogP value can be influenced by pH, especially if the compound is ionizable
(such as acidic or basic compounds). The ionization state of the compound in the
mobile phase can change depending on the pH, and this will affect its
hydrophobicity and interaction with the stationary phase.
 For instance, if a weak acid is ionized at high pH, it will be more
hydrophilic (lower LogP) and elute faster than its unionized form.
 Conversely, at a lower pH, the acid might remain unionized (higher LogP)
and be more retained.

Practical Example

Consider a scenario where you are analyzing a set of compounds using RP-HPLC:

 Compound A has a LogP of 3 (highly hydrophobic).

  Compound B has a LogP of 1 (moderately hydrophobic).
 Compound C has a LogP of -2 (very hydrophilic).

In this case, you would expect the elution order to be:

1. Compound C (lowest LogP) will elute first because it is hydrophilic and prefers to stay
in the aqueous mobile phase.
2. Compound B (moderately hydrophobic) will elute next, as it has an intermediate affinity
for the stationary phase.
3. Compound A (highest LogP) will elute last because it is the most hydrophobic and
interacts strongly with the non-polar stationary phase.

ROLE OF PKA IN REVERSE-PHASE HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY (RP-HPLC)

The pKa (acid dissociation constant) of a compound is a measure of the compound’s ability to
donate or accept protons in solution, indicating the pH at which the compound is 50% ionized.
This property is especially important for ionizable compounds, as it determines the compound’s
ionization state at different pH values, which directly affects its behavior in reverse-phase
HPLC (RP-HPLC).

How pKa Affects RP-HPLC

1. Ionization State and Interaction with Stationary Phase:

o Ionization of the Compound: Compounds with a pKa in the range of the mobile
phase's pH will be partially or fully ionized, and this will affect their interaction
with the non-polar stationary phase (like C18) in RP-HPLC.
 Ionized species: Ionized compounds tend to be more hydrophilic and
therefore interact less with the non-polar stationary phase. As a result, they
typically elute more quickly.
  Neutral species: The neutral form of a compound tends to be more
hydrophobic and interacts more strongly with the stationary phase, leading
to longer retention times.
o RP-HPLC Column: In RP-HPLC, the stationary phase (C18 or another
hydrophobic phase) interacts more with hydrophobic compounds than with
hydrophilic ones. Thus, whether a compound is ionized or neutral (which depends
on its pKa) significantly affects its retention time.
2. Retention Behavior Based on pKa:
o If a compound has a pKa value close to the pH of the mobile phase, it will exist
in a mix of ionized and neutral forms. The relative amounts of these two forms at
any given pH will determine the compound's retention time:
 At pH < pKa: The compound will be mostly in its protonated (neutral)
form if it’s a weak acid, or in its non-protonated (neutral) form if it’s a
weak base. The neutral form tends to interact more with the hydrophobic
stationary phase, leading to a longer retention time.
 At pH > pKa: The compound will be mostly in its ionized form (if it’s an
acid, it will lose protons; if it’s a base, it will gain protons). The ionized
form tends to be more hydrophilic and interacts less with the hydrophobic
stationary phase, leading to a shorter retention time.
3. Elution Order and Separation:
o The pKa of different compounds can lead to differences in their retention times
and separation in RP-HPLC. Compounds with different pKa values will ionize
differently at the same pH, leading to different extents of interaction with the
stationary phase.
 Compounds with higher pKa (less acidic, more basic) may become more
ionized at higher pH, resulting in quicker elution.
 Compounds with lower pKa (more acidic) may stay neutral at higher pH
and thus elute later.
4. Optimization of pH for Separation:
o The mobile phase pH is one of the key parameters in RP-HPLC, and adjusting it
can enhance separation:
  If compounds in a mixture have similar pKa values, adjusting the pH can
help maximize differences in their ionization states, leading to better
separation.
 If the analytes have a wide range of pKa values, selecting a mobile phase
pH where one or more compounds are predominantly ionized and others
are neutral can aid in optimizing retention times.
5. Effect of pKa on Peak Shape:
o Compounds that undergo ionization/deionization during the chromatographic run
may exhibit peak tailing or broader peaks if the pH is not controlled properly.
The ionization equilibrium can be shifted during the run, leading to inconsistent
interactions with the stationary phase and poor peak shape.
o Careful selection of the mobile phase pH can minimize these issues.

Practical Example

Suppose you are working with a mixture of two compounds, Compound A and Compound B,
in reverse-phase HPLC:

 Compound A is a weak acid with a pKa of 4.

 Compound B is a weak base with a pKa of 9.

Now, consider the following mobile phase conditions:

 Mobile Phase pH 3 (acidic):

o Compound A (pKa 4) will be mostly neutral at this pH, as the pH is lower than
its pKa. Since the neutral form is more hydrophobic, it will be retained more
strongly by the C18 stationary phase, leading to a longer retention time.
o Compound B (pKa 9) will be mostly protonated (ionized) at this pH. The
protonated form is more hydrophilic and interacts less with the stationary phase,
leading to faster elution.
 Mobile Phase pH 7 (neutral):
o Compound A will be partially ionized (since pH 7 is above its pKa of 4) and thus
more hydrophilic. As a result, it will have a shorter retention time.
 o Compound B will be partially protonated and thus exist in a mix of neutral and
ionized forms, potentially giving it a moderate retention time.
 Mobile Phase pH 10 (basic):
o Compound A will be completely ionized at this pH, as it is an acid with a pKa of
4. The ionized form is more hydrophilic and will elute faster.
o Compound B will be mostly neutral (because pH 10 is above its pKa of 9), and
the neutral form will interact more with the hydrophobic stationary phase, leading
to a longer retention time.

4.2 Flow Rate Selection: The flow rate can also affect the separation. If the flow rate is too high,
peaks may not have enough time to separate. If too low, the run time may be excessively long.
Generally, a flow rate of 0.5 to 1.5 mL/min is typical for HPLC, but this depends on the column
size and type.

4.3 Selection of the HPLC Column:

 Flush column with 100% methanol/acetonitrile (10 min)

 Then flush with mobile phase for at least 30 minutes (till baseline stabilizes).
 Select a column suitable for the separation of the active ingredient in the tablet. A typical
column might be a C18 (reverse-phase) column, which is commonly used for drug
analysis.

1. C18 Stationary Phase

 Hydrophobic: C18 is the most commonly used reverse-phase stationary phase, ideal for
retaining hydrophobic compounds. Its long alkyl chains (18 carbon atoms) provide strong
hydrophobic interactions with a wide range of analytes.
 Versatility: Suitable for many small to medium-sized pharmaceutical compounds,
providing reproducible results across various sample types.
2. Column Length (150 or 250 mm)

 150 mm: Provides a good balance between separation and analysis time. Ideal for most
standard separations.
 250 mm: Offers higher resolution for complex mixtures but increases analysis time.
Suitable when you need greater peak separation or when dealing with closely related
compounds.

3. Column Diameter (4.6 mm ID)

 4.6 mm ID is the most common and optimal choice for analytical work. It provides
adequate sample load capacity and ensures a proper resolution without overloading the
system.

4. Particle Size (5 µm)

 5 µm particles provide a good balance between resolution and pressure. It’s the standard
for most analytical applications, offering sharp peaks and good separation efficiency
without causing excessive backpressure.

4.4 Wavelength Selection (Detection):

 Set the UV detector to an appropriate wavelength, Select the wavelength based on the
maximum absorption of the analyte or based on Isosbestic point.

4.5 Column Temperature:

 Set the column temperature to room temperature (about 25°C), or slightly higher (30-
40°C) to improve peak shape and resolution if needed.
4.6 Run Initial Trials:

 Inject between 5-100 µL of the 100 ppm standard solution to check the retention time,
peak shape, and resolution.
 Analyze the chromatogram and optimize the mobile phase composition or flow rate if
needed.

4.7 Determine Retention Time and Peak Shape:

 The retention time (RT) should be stable and reproducible.

 Peaks should be sharp and well-resolved from the baseline. If peaks are not adequately
separated, adjust the mobile phase or flow rate.

4.8 How adjustments are made:

 % Organic is changed → to control retention time

 pH is changed → for better selectivity or peak shape
 Flow rate is changed → to optimize resolution or shorten runtime

Use one variable at a time.

Focus on: (system suitability criteria, also explained separately in next section)

 Retention time (preferably 2–8 min and should remain consistent when runs are
repeated)
 Symmetrical peaks (Tailing factor < 2)
 Good separation (Resolution > 2 for nearby peaks)
 %RSD of the peak area should be less than 2 for a minimum of five runs performed
from a same standard vial.
 Theoretical plates more than 2000
5. Calibration Curve (draw calibration curve in Microsoft Excel)

Once the method is developed, prepare the calibration standards (50, 75, 100, 125, and 150 ppm)
and inject them into the HPLC.

 Plot the concentration (ppm) on the x-axis and the peak area (or height) on the y-axis.
 Ensure that the data points fall on a straight line, which indicates linearity.
 Use linear regression to determine the equation of the calibration curve.

6. Sample Preparation

For sample analysis, dosage form solution need to be appropriately prepared. (here is an
example of tablet dosage form)

Tablet Sample Preparation:

 Weigh an appropriate number of tablets (e.g., 10 tablets) and determine the average
weight.
 Crush the tablets into a fine powder.
 Accurately weigh the powder equivalent to 100 mg of the active ingredient.
 Prepare a solution in a similar manner to the stock solution. (we prefer sample stock will
be prepared of the same concentration as of standard solution)
 Typically, dissolve the powder in the mobile phase or a suitable solvent and then dilute to
the required volume.

SYSTEM SUITABILITY TEST

Why Do We Perform SST?

To confirm:

 The instrument and method are working as expected.

 Accuracy, precision, and consistency of results.
 The system is suitable for the intended analysis.

How to Perform SST in Practice:

1. Prepare Standard Solution (e.g., a known concentration of your analyte).

2. Inject Standard Multiple Times (usually 5–6 injections).
3. Record the Following from the Chromatogram:
o Retention time
o Peak area
o Peak width
o Tailing factor
o Resolution (if multiple peaks)
4. Calculate:
o % RSD for peak area and retention time
o Theoretical plates
o Tailing factor
o Resolution (in case of more than one analytes)
5. Compare the results with the pre-defined acceptance criteria.

Common System Suitability Parameters in HPLC:

Parameter What it Checks Typical Criteria

Retention Time (Rt) Consistency of peak position RSD ≤ 1% (for replicate injections)
Theoretical Plates
Column efficiency N should be high (e.g., >2000)
(N)
Tailing Factor (T) Peak symmetry T≤2
Resolution (Rs) Separation between two peaks Rs ≥ 2
% RSD of Peak Repeatability (precision) of RSD ≤ 2% (typically for 5–6
Area injections injections)

7. Method Validation
7.1 Accuracy:

 To evaluate the accuracy of the method, prepare a spiked sample by adding known
amounts of the API (at concentrations 100 ppm, 50 ppm, and 150 ppm) to the sample.
 Calculate the recovery rate by comparing the measured value to the known added value.
 Acceptable recovery is typically within 97-103% at individual levels and 98-102% at
mean.

Detailed steps

 Prepare Sample: Take a placebo or matrix (blank sample) that does not contain the
analyte.
 Spike Known Amount: Add a known amount of the standard analyte to the sample at
three concentration levels — typically 50%, 100%, and 150% of the target concentration
(which means 50, 100 and 150 µg/mL).
 Analyze the Sample: Process the sample using your analytical method (e.g., HPLC).
 Calculate Amount Recovered: Measure the amount of analyte recovered from the
sample.
 Use the Formula to compute recovery at each level.

AMOUNT ADDED : It known: i.e. 50 ppm for 50%, 100 ppm for 100% and 150ppm for 150
%

AMOUNT RECOVERED/FOUND:

FROM CALIBRATION CURVE (linearity curve) WE ALREADY HAVE THE

EQUATION

Inject standard solutions of known concentrations and plot peak area (Y-axis) vs concentration
(X-axis).

You'll get a linear equation:

Peak Area= m × Concentration + c

Where:
  m = slope
 c = intercept

Peak area - c

Concentration Recovered= -------------------------

m

7.2 Precision (Repeatability and Intermediate Precision):

 Repeatability: Inject the same sample multiple times (usually six times) and calculate
the % RSD (relative standard deviation) for the peak area or height. It should be ≤2%.
 Intermediate Precision: Perform the analysis on different days by different analysts or
using different equipment. Calculate the % RSD for each set.
 Acceptance criteria : %RSD <2

7.3 Specificity:

 Check for interference from excipients or other components in the tablet formulation.
 Run a placebo (without the active ingredient) and ensure no interfering peaks appear at
the same retention time as the analyte.
 Run a diluent and ensure no interfering peaks appear at the same retention time as the
analyte.

7.4 Linearity:

 Verify that the calibration curve is linear over the entire concentration range (50-150
ppm). The correlation coefficient (R²) should be ≥0.998.
7.5 Robustness:

 Evaluate how small variations in experimental conditions

 Slight changes are made in flow rate/temperature/mobile phase composition
 One change made at a time
 Multiple runs are taken from standard solution
 Acceptance criteria: As same as system suitability section

8. Sample Analysis

Once the method is validated, analyze the sample of the 100 mg tablet.

 Inject the prepared sample solution and calculate the concentration of the drug based on
the calibration curve.

Assay Calculation

The concentration of the drug in the sample can be calculated using the equation derived from
the calibration curve (y = mx + b). Where:

 y = peak area
 x = concentration (ppm)
 m = slope of the calibration curve
 b = y-intercept