S.T. Ulu, F.T. Elmali / Spectrochimica Acta Part A 77 (2010) 324–329
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2.5. Determination of molar ratio
Applying the continuous variation method [21] for the reaction
of DDQ reagent with DP and refer to the stoichiometry of the drug
donor (D) to the reagent acceptor (A) of ratio 1:1 (D:A).
2.6. Method validation
Fig. 1. Molecular structure of diphenhydramine hydrochloride.
The method was validated according to International Con-
ference on Harmonization guidelines for validation of analytical
procedures [22,23].
2.2. Apparatus
2.6.1. Linearity
The linearity was evaluated by linear regression analysis, which
was calculated by the least square regression method. The cali-
bration curves were constructed by plotting concentration versus
absorbance, using linear regression analysis.
An UV-160A UV–visible spectrophotometer (Shimadzu, Japan)
was used for the absorbance measurements.
TGA–DSC (thermogravimetric analysis–differential scanning
calorimetry) curves were obtained with a TA SDT Q 600 thermal
analyzer apparatus using flowing nitrogen 100 mL min−1 temper-
,
ature range 25–750 and 25–1200 ◦C, at heating rate of 20 ◦C min−1
.
2.6.2. Limit of detection and limit of quantification
The FTIR spectra were recorded with ATR technique on a
PerkinElmer Spectrum One Bv 5.0 spectrophotometer. 1H NMR
spectra were recorded on a Varian UNITY INOVA 500 MHz spec-
trometer. TMS was used as internal standard in 1H NMR.
The limit of detection (LOD) and limit of quantification (LOQ)
were calculated as 3.3ꢁ/S and 10ꢁ/S, respectively, where S is the
slope of the calibration curve and ꢁ is the standard deviation of
intercept of regression equation.
2.6.3. Precision and accuracy
2.3. Solutions
DP samples (12.5, 75.0 and 150 g/mL) in five replicates were
analyzed on the same day to determine the intra-day precision and
accuracy, and on each of five separate days to determine inter-
day precision and accuracy. Precision was expressed as the relative
standard deviation (RSD %). Accuracy was expressed as the mean
relative error (RME %).
10 mg mL−1 of DPH stock solution was prepared in water. DDQ
solution was prepared 2.0 mg mL−1 in acetonitrile. The DPH stock
and DDQ solutions were stored at 4 ◦C.
2.3.1. Preparation of diphenhydramine base solution
A 1 mL standard stock solution of DPH was transferred into the
12 mL each glass tube. Then, 1 mL of concentrated ammonia solu-
tion was added and shaken for 1 min. The alkaline aqueous layer
was extracted with three portions of chloroform each of 3 mL. The
chloroform phase was filtered through anhydrous sodium sulfate,
into a 10 mL calibrated flask and the volume. It was made up to
10 mL by adding chloroform (1.0 mg mL−1, calculated as diphenhy-
dramine base (DP)). From this solution, 5.0 mL was transferred into
10 mL calibrated flasks and evaporated to dryness in a water bath at
45 ◦C under a stream of nitrogen. Then, the residue completely dis-
solved in acetonitrile using an ultrasonic bath and then completed
to the mark with acetonitrile (0.5 mg mL−1, calculated as free base).
2.6.4. Recovery
The % recovery of the added pure drug was calculated as, %
recovery = [(Ct − Cs)/Ca] × 100, where Ct is the total drug concentra-
tion measured after standard addition, Cs the drug concentration
in the formulation sample and Ca is the drug concentration added
to formulation.
2.6.5. Robustness
The robustness of the spectrophotometric method was deter-
mined by analysis of samples under a variety of conditions such as
small changes in the reagent concentration and reaction time.
3. Results and discussion
2.4. Procedures
3.1. Absorption spectra and mechanism of the reaction
2.4.1. General procedure
The method is based on the formation of intensely red colored
DDQ•− radical anion by interaction of the drug with DDQ in acetoni-
trile at ambient temperature. As can be represented in the following
scheme, the radical anion results from dissociation of the original
charge-transfer complex (CT-complex) formed by interaction of the
drug (n-electron donor, D) with DDQ (-acceptor, A) (Scheme 1).
Interaction of DDQ with DP formed a red color product in ace-
tonitrile with absorption maxima at 460, 541 and 586.5 nm (Fig. 2).
Measurements were carried out at 460 nm, at which higher sensi-
tivity was achieved.
Transfer aliquots of 0.25–3.0 mL of diphenhydramine base solu-
tion (0.5 mg mL−1) into a series of 10 mL calibrated flasks. Bring
the volume to 3.0 mL with acetonitrile, added 1.0 mL DDQ reagent.
The reaction mixture was mixed and the volume was completed
to 10 mL with acetonitrile. The immediately formed dark red color
was measured at ꢀmax = 460 nm against a blank prepared in the
same manner except for an addition of drug and the calibration
graph was obtained.
tubes. The solution was alkalized with 1.0 mL of concentrated
ammonia and then extracted with chloroform as indicated in Sec-
tion 2.3.1. After preparation of diphenhydramine base solution,
proceed as described in Section 2.4.1.
3.2. Optimization of reaction conditions
3.2.1. Effect of reagent concentration
The influence of the concentration of DDQ was examined by
addition of different volumes of 0.2% reagent in the range of