Objective:
Development and validation of spectrophotometric an
d RP-HPLC methods for the simultaneous determination
of Hydroquinone (HQ),
Hydrocortisone (HC) and Tretinoin (TRT) ternary combi
nation in pharmaceutical preparation.
Methods:
The proposed spectrophotometric method was able to
determine TRT directly from its absorption spectrum
at 362 nm, however, HQ and
HC from their first derivative spectra at 284 nm and
252 nm, respectively, without any separation step.
The RP-HPLC method was developed using a
C
18
Sunfire
©
waters column with a mobile phase composed of acet
onitrile: phosphate buffer (adjusted to pH 6.1 using
ortho-phosphoric acid) in the
ratio of 30:70 %, v/v, respectively at a flow rate
of 0.8 ml/min. Quantification was based on measurin
g peak areas at 260 nm.
Results:
The spectrophotometric method was able to selective
ly quantify each of HQ, HC and TRT in the ranges of
10-50 μg/ml, 2-10 μg/ml and 0.5-5 μg/ml,
respectively. The RP-HPLC method was able to produc
e well-resolved peaks after 3.0, 8.2 and 20.2 min,
in the ranges of 2-10 μg/ml, 0.1-1 μg/ml and 0.05-2
μg/ml, for HQ, HC and TRT, respectively. The obtain
ed A, D
1
or peak areas values plotted against the concentra
tion of each of the three components showed
linear response in the stated ranges. Both methods
were validated in terms of linearity, LOD, LOQ, pre
cision, accuracy and selectivity.
Conclusion:
Both developed proposed methods were applied for th
e determination of the active ingredients in the ph
armaceutical formulation and
the common excipients did not interfere in the anal
ysis. The RP-HPLC method proved to be more sensitiv
e when compared to the applied
spectrophotometric method. However, the applied spe
ctrophotometric methods, considered as green analyt
ical chemistry, is a simple, time-saving
method that requires minimal use of a hazardous sol
vent.