Senthamizh Selvan International Flavors & Fragrances Inc.

1. First, you shoud know analytes structure, solubility and choose the detector based on
analytes (or your interest) if the analytes are uv active use UV or RID, LSD and MSD
etc.,HPLC method development start with literature survey to get rough idea about
development. start with solubility of the analytes,if it is soluble in polar solvents we can try
with reverse phase if is is not we should use normal phase chromatography. Mostly many
organic drug molecules are polar or mid polar so we usually go for RPC

2. selection of wavelength by PDA (not always wavelength max, based on related

compounds, UV cut off of mobile phase, to avoid matrix intereferencese etc.) . if the
analytes are not UV active then there is dervatisation method.

3. column selection (start with C18 inertsil and short column (100mm or 150 mm) if it is
revesed phase or u can use GL Sciences Inertsil ODS, waters Xbridge, Agilent Zorbax
eclips columns or phenomenex Gemini etc)

4. Mobile Phase: Gradient setting(Always develope the method by using gradient than
isocratic). use buffer or modifier if the analytes are ionisable because ionisable compounds
will show as two peaks or split peak as it is existing in two form (HA and A-); so if you
buffered the mobile phase into basic or acidic the analyte will go to single form. maximum
use acidic pH (2-4) as the mostly column will hydrolyse more in basic condition

HA------------>H+ + A-

0.1% TFA, H3PO3, H2SO4, 1-2 % Acetic acid are the simple modifiers to restrict the pH
into acidic side to improve the peak shape) and well known phasphate buffer (10 mmol
100 mmol phasphate buffer), optimum is 30-50 mmol
start with mobile phase composition Use water & Methanol or ACN according to solubility in
50:50 ratio. feed other parameter like wavelength, ambient Temp, 1ml/min Flow rate. About
sample preparation for assay preparation use conc in between 100-500 ppm as per peak
response.
keep the peak response below 1000mV = 1V or 1000 mAU= 1AU to keep the UV detector
linearity.
If there are more than 10 molecule in particular product / sample go for gardient method for
better resolution & short run time.you can start with simple greadient

Time(min)---------%A(Water or Buffer)-------------%B (Organic Modifier-ACN,MeOH)

0---------------------80---------------------------------------20
25-------------------20---------------------------------------80
35-------------------20---------------------------------------80
40-------------------80---------------------------------------20
50-------------------80---------------------------------------20
while developing any method always follow ICH parameter abt Tailing factor, Capacity
factor, Assymetry, thereotical plates.Change MP composition ,Column.
Once you get better result then for sharpness or to save time adjust flowrate & increase
Temp.

If our desired peak is not retain on water /ACN/Methanol composition MP then go for Buffer.
Phosphate buffer is widly use buffer for its PKa value,easily Available, gives more
Hydrophillic effect, stable or non degradable for 2-3 days.

...............Before starting any method development on

5. HPLC Flush the system with water, check pressure, saturate column with MP, check
callibration status, lamp Hours.

Always keep data about whatever trials you have done during method development.

Method Development is very challenging task so always be logical before any changes in
any parameter.

Mostly For HPLC method Development 10 gm sample is enough but for overall
development max 100 gm sample is sufficient.

Its a tentative quantity.

After method development method validation must be done. use ich Q2(R2) for method
validation

http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html

Regards
Selvan