Asian Journal of Applied Sciences (ISSN: 2321 – 0893)

Volume 10 – Issue 4, August 2022

UV –Visible Spectroscopy Method Development and Its

Validation for the Analysis of Marketed Hair Dyes for Amine
Content
K. Bhavyasri1, K. AmukthaMalyada2, M. Sumakanth3
1Department of Pharmaceutical Analysis RBVRR Women’s College of Pharmacy
Hyderabad, INDIA
Email: bhavya.khagga [AT] gmail.com
2Department of Pharmaceutical Analysis , RBVRR Women’s College of Pharmacy
Hyderabad, INDIA
Email: amukthamalyada2910 [AT] gmail.com
3Deparment of Pharmaceutical Chemistry, RBVRR Women’s College of Pharmacy
Hyderabad, INDIA
Email: Suma.mogili [AT] gmail.com

____________________________________________________________________________________________________________
ABSTRACT---- To develop a simple rapid, accurate and spectrophotometric reproducible method were developed for
estimation of PARA - PHENYLENE DIAMINE in different Marketed Hair dyes . The analysis of PPD was performed
using NaOH solution as diluent and using folins reagent at 432nm respectively .The methods were linear in the
concentration range from 0-50µg/ml. The methods were validated with respect to system suitability, linearity, precision,
limit of detection, limit of quantification, accuracy, ruggedness and robustness . The developed method can be used for
routine analysis of PARA PHENYLENE DIAMINE in marketed hair products. The methods were validated in
accordance to the ICH guidelines.

Keywords----ParaPhenylenediamine , folins reagent Spectrophotometric method

_________________________________________________________________________________________________

1. INTRODUCTION
Paraphenylenediamine (PPD) is a permanent hair dye it is chemical substance that is widely used . PPD is used in hair dye
because that gives a natural look, and even after shampooing the hair dyed will not loose its colour .PPD hair dyes leads
to cancer and mutagenicity.Apart from that, PPD also causes skin irritation and many such related allergies . Limit of PPD
in hair dyes is 6%. The initiation of allergic reactions are by oxidation of PPD on the surface and within the skin .

Figure 1 : Chemical structure of Para phenylenediamine

Natural black hair colour is due to melanin clusters dispersed within the colourless keratin-based cortex of the hair.
Melanin is responsible for the colour determination of hair. White hair is due to age as we get older pigment cells in the
hair die. Even though PPD has several disadvantages people are crazy about putting dyes on their hair as beauty has more
significance in day to day life. this is because of the attraction in a high definition way and also it is the practice of changing
hair colours. but before that skin sensitivity tests are important. Hair dyes and colours are substances that contain hundreds
of chemicals that are combined to bring out the desired properties in the product. Many products brands do not contain
PPD or PPD free, ammonia free that doesn’t mean the hair is completely free from chemicals and also still cause allergic
reactions.Ammonia and PPD free hair dyes contain Emollient oils, walnut oils, argan oil so help the texture to remain fine
and the cuticles do not loose their moisture while dyeing .

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

1.1 MECHANISM OF ACTION OF HAIR DYES:

Hair shaft is the outer layer which is covered by Cuticle and Keratin cells , protein for the inner layers of hairs. Cortex
contain more Keratin cells which helps to provide proteins to Melanin. Melanin gives Natural black colour . In a hair dye
it generally consist of developer , primary intermediates , couplers .
Developer : hydrogen peroxide
Primary intermediates : PPD
Couplers : Ammonia
Solution hair dye consist of Ph 11 which will help to expand or swell the hair shaft and thereby it leads to separation of
keratin cells and thereby the color sieves into the cortex . Here the ammonia opens the cuticle for color to enter the cortex
the swollen molecules join together and remain in the cortex making it impossible to escape and thereby oxidation of colour
occurs on the hair and colour is produced

1.2 Structure of PPD :

Molecular formula : C6H4(NH2)2

Molecular weight : 108.4

2. MATERIALS AND METHOD

2.1 Chemicals:Para-Phenylenediamine , NaOH , Distilled water , Folins reagent

2.2 Instruments: ELICO SL 210 double beam UV-Visible Spectrophotometer, glass cuvettes, analytical weighing
balance , Sonicator were used .

3. METHOD DEVELOPMENT

3.1Preparation of standard stock solution:

10ml volumetric flask was taken in that 10mg of Pure PPD was dissolve with diluent NaOH and make up to the mark
which gives the concentration of 1000μg/ml.
3.1.1 Working standard solution preparation:
For preparation of 100μg/ml standard solution, pippete out 1ml from standard stock solution and transfer it to 10ml
volumetric flask and make up to the mark with 0.1N NaOH. From this 100ug/ml pippete out 1ml and transfer into 10ml
volumetric flask .And then pippete out 1ml of FC and 1ml of 0.1N NaOH and makeup the volume upto the mark with
distilled water

3.2 Preparation of 0.1N NaOH:

0.4gms of NaOH is dissolved 100ml of distilled water and and transferred to 100ml volumetric flask

3.3 Preparation of FC Solution:

Take 1ml of FC reagent and transfer it into 10ml volumetric flask and to this add 1ml of 0.1N NaOH and add distilled
water upto the mark .

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

3.4 Determination of wavelength of maximum absorption:

10ppm standard solution was prepared by taking 10ml volumetric flask and pippete 1ml of 100µg/ml of standard solution
to this add 1ml NaOH , 1ml FC reagent and make up the volume upto the mark by distilled water and check the absorbance
under UV Spectroscopy within the range 400-800nm using the diluent as blank. The maximum absorption were found at
432nm.

4. VALIDATION
ICHQ2(R1) guidelines was followed for analytical method validation. The following are the validation parameters
performed for PPD.

4.1 Linearity:

Linearity of analytical procedure is defined as concentration of analyte in sample is directly proportional to obtained test
result.A linear relationship should be developed across the range of analytical procedure. Linear standard solutions were
prepared from the working standard solutions. From the working standard solution ,serial dilutions were made to get 0-
50ppm were prepared and absorbance was measured at 432nm using NaOH as diluent and as blank and the calibration
curve is plotted.

4.2 Precision:
It is determined by keeping the same homogeneous sample for at least six times and noting the absorbance at lambda
max.The consistency of homogenous sample. Then calculating the %RSD.

For performing precision, 50ppm standard solution of PPD was selected. The absorbance of 50ppm solution was checked
at 432nm and this is repeated for 6 times and all 6 absorbance’s were noted. The formula for calculating %RSD was given
below.

4.3 Accuracy:
It is also Known as trueness. Accuracy is done by comparing the obtained test results with that of true value.

The accuracy of the proposed method was tested by recovery studies at 100%, 200%, and 300% by adding a known amount
of pure drug to the pre-analyzed formulation of concentration 10µg/ml. The accuracy was determined by spiking standard
solution to sample solution at three concentrations i.e., 100μg/ml,200μg/ml,300μg/ml. Standard concentrations equal to
100,200.300 percent is added to sample. . 2ml of 200ppm sample was spiked to 2ml of 100ppm standard solution, 2ml of
200ppm of sample was spiked to 200ppm of standard solution, 2ml of 200ppm sample solution was spiked to 2ml of
300ppm of standard solution. At 432nm ,absorbance was checked for three times. The below formula is used to calculate
% Recovery.

4.4 Robustness
Robustness of analytical procedure is minute changes in method are done to see the stability of the method. Robustness is
performed by measuring the absorbance at 431,432,433nm i.e., ±1nm from the lambda max.

4.5 Ruggedness:
The results obtained by analysis of sample under different conditions must be reproducible. Different conditions may be
different analyst, different instrument, different days etc.

In our research we did robustness studies were done by two different analysts.

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4.6 Limit of Detection:

The analyte in sample that can be detected that is too less to quantify but can be detected. The formula for calculating LOD
is given below:

where

σ = standard deviation

S = slope

4.7 Limit of Quantification:

The amount of analyte in the sample that can be just quantified. The formula for calculating LOQ is given below:

where,

σ = standard deviation

S = slope

5. RESULTS AND DISCUSSION

FIGURE:1 λmax of NaOH

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

Table 1: Absorbance values for Calibration curve of PPD

Concentaration Absorbance

0ppm 0

2ppm 0.038

4ppm 0.128

6ppm 0.242

8ppm 0.346

10ppm 0.465

20ppm 0.586

25ppm 0.724

30ppm 0.823

35ppm 0.943

40ppm 1.032

50ppm 1.162

5.1Lineaity :

Figure 2: Calibration curve of PPD

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

5.2 Precision:
Table 2: Results of Precision

Concentration Absorbance(x)

50 1.243
50 1.244

50 1.236

50 1.246

50 1.254

50 1.224

Average
1.241167
Standard deviation 0.010206

RSD% 0.822308

5.3Accuracy:

Table 3: Results of Accuracy

% LEVEL ABSORBANCE % RECOVERY MEAN % RECOVERY

100% 1.004 97%

(100ppm+200ppm) 1.004 97% 97%

1.024 98.9%

200% 1.1508 99.5%

(200ppm+200ppm) 1.1499 99.5% 99.5%

1.1548 99.9%

300% 1.382 99.2%

(300ppm+200ppm) 1.3724 98.5% 98.5%

1.3899 99.7%

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

5.4 Robustness:

Table 4: Results of Robustness

Concentration 431nm 432nm 453nm
50ppm 1.2422 1.2305
1.2553
50ppm 1.2675 1.248 1.2415

50ppm 1.275 1.2542 1.2463

50ppm 1.2798 1.2461 1.2364

50ppm 1.2668 1.2542 1.2358

50ppm 1.2818 1.2242 1.2564

1.244817 1.241483
Mean 1.271033
SD 0.009864 0.011133 0.009881

%RSD 0.77603 0.89385 0.795884

5.5 Ruggedness:

Table 5: Results of Ruggedness

Concentration Analyst-1 Analyst-2
50 1.2422
1.2675
50 1.2553 1.248

50 1.275 1.2542

50 1.2818 1.2461

50 1.2668 1.2242

50 1.2798 1.2542

Average 1.244817
1.271033
Standard deviation 0.009864 0.011133

RSD% 0.77603 0.89385

Table 6: Summary of Results

Parameters PPD
Linearity range 0-50ppm

Slope 0.1141
Standard Deviation 0.010206
%RSD 0.822308
LOD 0.2951µg/ml

LOQ 0.8944µg/ml

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 Asian Journal of Applied Sciences (ISSN: 2321 – 0893)
Volume 10 – Issue 4, August 2022

6. CONCLUSION
A simple method has been developed for estimation of PPD in hair dyes. A method has been developed and validated
according to ICHQ2(R1) guidelines. All the validation parameters have been performed and all the parameters were found
to be within the limits.

7. ACKNOWLEDGEMENT
I want to acknowledge our beloved principal Prof. M. Sumakanth and Faculty of Department of Pharmaceutical Analysis
for giving me the opportunity to perform the research work.

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