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[24]
reflux in a water bath for 30 minutes, and cribed in THP. The separation was per-
o
filter. Evaporate the filtrate at 50 C under re- formed on a C18 column (4.6 × 150 mm) pro-
duced pressure to dryness and dissolve the tected by a C18 guard column (4.0 × 3.0 mm).
residue in sufficient methanol. Transfer quan- The mobile phase was a mixture of methanol
titatively to a 100 mL volumetric flask, dilute and water (50:50) at a flow rate of 1 mL/min.
with mobile phase to volume and mix. Filter Detection was monitored at 224 nm. Ac-
through a nylon membrane having a 0.45 μm cording to ICH Q2(R1) Validation of Analyti-
[32]
porosity. The filtrates were then analyzed for cal Procedures: Text and Methodology ,
their andrographolide contents using HPLC. change in sample preparation is considered
1.2 Sonication as modification of validated method which
In order to optimize the ultraso- required partial validation to ensure that the
nication extraction conditions, extracting sol- analytical method maintains its characteris-
vent and time were investigated so as to tics. Therefore, the method was validated for
obtain satisfactory extraction effciency and specificity, linearity, accuracy, and precision.
quantitative results. Different concentrations Specificity is demonstrated by comparing the
of solvent (100% and 50% methanol) were in- representative chromatogram obtained from
vestigated using 10 different lots of samples the sample solution with that of the standard
as a preliminary study. Weigh, finely powder solution. The calibration curves were ge-
and sieve through mesh size No. 180 the con- nerated by plotting the peak area against the
tents of not less than 20 capsules. Transfer concentration of andrographolide. Accuracy
an accurately weighed quantity of the pow- was evaluated using standard addition
der about 400 mg to a 100 mL volumetric method. Three different concentrations (26.2
flask, add 70 mL of extracting solvent and μg/mL, 44.9 μg/mL and 89.7 μg/mL) of stan-
ultrasonicate for the duration of 5, 10, 15, 20 dard were added to a previously analyzed
and 25 minutes at room temperature. Cool to sample solution. Triplicate experiments were
room temperature and dilute with extracting performed at each concentration level. The
solvent to volume, mix, and filter. The sample average recoveries were estimated and should
solutions were then assayed for their andro- be in the range of 97.0 -103.0%. Precision was
grapholide content using HPLC. performed using 6 determinations of the same
2. Method validation sample and should be within 2.0 %RSD.
The content of andrographolide was 3. Comparison of extraction method
determined using HPLC-UV method as des- A total of 30 different lots of Andro-
