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Chemical Derivatization UV Spectrophotometric Method for Detection of P-

Aminophenol and Energy of Activation Approach to Set Degradation Protocol
for Forced Degradation Studies

Article · January 2016

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International Journal of Pharma Research & Review, Feb 2016;5(2):1-12 ISSN: 2278-6074

Research Article

Chemical Derivatization UV Spectrophotometric Method for Detection of P-

Aminophenol and Energy of Activation Approach to Set Degradation Protocol for
Forced Degradation Studies
Gitanjali S. Deokar, Indrayani S. Pagare, Ansari Md Naseem*, Sanjay Kshirsagar, Lavesh Shindhe

Department of Quality Assurance, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon Nashik,
Maharashtra, India.
ABSTRACT
A spectrophotometric method has been developed for the determination of paracetamol in a dosage form
and in Pharmaceutical preparation. The method is based on the reaction of p-aminophenol group of the
drug with ninhydrin in N, N. dimethylformamide (DMF) medium producing a coloured complex which
absorbs maximally at 547 nm. Beers law is obeyed in the concentration range 1-3μg/ml and with set
parameter the method is validated, LOD and LOQ were found to be 0.254, 0.770 and 0.2849, 0.863 by
standard deviation of blank and calibration curve method respectively. As it is having least sum square
error that is 1.448667 × 10 -5. Method is found to be specific for the para amino phenol, data supported
by ANOVA test with at P ≤ 0.05. For accuracy and precision we are 99℅ sure that the results lie between
100.32-99.26℅ and 100.19 to 100.07℅ respectively. From the principle of energy of activation the force
degradation of Paracetamol in tablet dosage form is carried out at 100ᵒc for 3 days, degradation of
Paracetamol was found to be 0.7℅ Hence the method is developed and validated for the detection of p-
amino phenol, a degradation product of paracetamol and by using concept of energy of activation p-amino
phenol from paracetamol drug product is estimated the results were validated statistically.

Keywords: Ninhydrin, paracetamol, pharmaceutical preparation, spectrophotometry

Received 6 Jan 2016 Received in revised form 28 Jan 2016 Accepted 30 Jan 2016

*Address for correspondence:

Ansari Md Naseem,
Department of Quality Assurance, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon Nashik,
Maharashtra, India.
E-mail: naseempharma1990@gmail.com
_________________________________________________________________________________________________________________________
1. INTRODUCTION
Paracetamol, acetaminophen or N-acetyl-p- level of 0.005 % (w/w) i.e.50 ppm in the
aminophenol is commonly used analgesic drug substance by European
and antipyretic drug, present in different pharmacopoeia .The significant limit of p-
pharmaceutical formulations such as aminophenol may vary in different products
tablets, soluble powder syrups and depends on dosage forms and formulations.
suppositories. It is administered to both The monograph of paracetamol tablet in BP
children and adults. [1, 2] The primary allows 0.1 %. For drug product containing
degradation product of paracetamol is 4- paracetamol often less tight limits are
aminophnol or 1-hydroxy-4- applied such as 1000 ppm or 0.1 % (w/w).
aminobenzone.It is formed during the Internally the lower drug substances
synthesis of paracetamol or during the specification limit is applied to product 50
storage condition of pharmaceutical ppm is equivalent to 25g of p-aminophenol
formulation such as heat, pH, temp etc. [3, per 500 mg tablet. [5] The present work
4] Paracetamol degrades slowly forming a describes simple, sensitive, accurate,
mixture of contaminants such as 4- precise, economical visible spectrophoto-
aminophenol and acetic acid. [3, 4] meteric method using ninhydrin reagent for
It is reported that p-aminophenol have estimation of p aminophenol in paracetamol
significant nephrotoxic and tetratogenic dosage form. Ninhydrin is a 2, 2,
effects, therefore its amount should be dihydroyindane 1,3-dione [6] which react
strictly controlled. [5] It is limited to a low with the primary amino group of p-

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aminophenol to yield purple color by the formation of coordination (in which

derivative called Ruhemans purple and both electrons are donated by the ligand)
hydroquinone. [7] and covalent bonds ligands with two or
1.1. Chemical Derivatization more donating groups may share more than
Derivatization is a technique which one pair of electrons with a single metal
transforms a chemical compound into a atom by coordinating to two or more
product of similar chemical structure. It positions. [8]
changes chemical and physical properties. 1.2. Ninhydrin
Chemical derivatives may be used to The chemistry of ninhydrin has been
facilitate analysis. Chemical Derivatization studied extensively. Ninhydrin is a triketo
modifies or converts substances with a low, compound; much of the work has been
UV absorption into highly sensitive directed towards the reaction of amines
products. [8, 19] with ninhydrin. This pigment serves as the
1.1.1. Types of Chemical Derivatization basis of detection and quantitative
Methods estimation of amino acids, the reaction is
1. Diazotisation and coupling of primary usually carried out by heating for short time
aromatic amines in a mixture of water and organic solvents.
The amine is diazotised with aqueous [7, 9, 10]
solution of Nitrous acid, the colorless 1.3. Energy of Activation
diazonium salt is very reactive and when Activation energy is a term introduced in
treated with suitable coupling reagent like 1889 by the Swedish scientist Syante
aromatic amine or phenol undergoes an Arrhenius that is defined as the energy that
electrophilic substitution reaction to must be overcome in order for a chemical
produce an azo derivative. The azo reaction to occur. Activation energy may
derivatives are colored and have absorption also be defined as the minimum energy
maximum in UV visible region at λ max550 required starting a chemical reaction. The
nm. [8] activation energy of a reaction is usually
2. Condensation Reaction denoted by Ea and given in units of
Many Colorimetric procedures are based on kilojoules per mole. [11]
rapid reaction that occurs under suitable
condition between amines and carbonyl [ ]
compounds. The reaction involves the
nucleophilic attack by the amine on the
carbonyl carbon with the elimination of Where
water. [8] Ea is the activation energy of the reaction in
3. Reduction of tetrazolium salts J/mol
Tetrazolium salts are reduced to their R is the ideal gas constant = 1.98 cal / deg·
colored form azan derivatives in the mol
presence of a Steroid with α-ketol (21- T1 and T2 are absolute temperatures
hydroxy-20-keto) side chain group. [8] Convert T1 and T2 in Kelvin
4. The acid-dye method K1 and K2 are the reaction rate constants at
The addition of an amine in its ionized form T1 and T2
to an ionized acidic dye, i.e. methyl orange 2. MATERIALS AND METHODS
or bromocresol purple, yields a salt (ion- 2.1. Apparatus
pair). [8] A UV-visible spectrophotometer (Shimadzu,
5. Oxidation methods model 1800) with 1 cm quartz cells was
Oxidation of side chain of weakly absorbing used for the absorbance measurements.
compounds containing a simple phenyl 2.2. Materials
group produces a carbonyl derivative that Para amino phenol in the form of powder
has a much greater absorptivity than the was provided by Thomas Baker, Mumbai,
parent compound. [8] and DMF in the form of liquid was provided
6. Metal-ligand complexation by LOBA Chemie, India, which was used in
Many organic reagents that are called
ligands forms complexes with metal atoms

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International Journal of Pharma Research & Review, Feb 2016;5(2):1-12 ISSN: 2278-6074

analytical method development and 2.5.3. Method based on Calibration Curve

validation. Prepare 0.001ppm to 2.0 ppm of para
2.3. Determination of wavelength of aminophenol solution from stock solution
maximum absorption respectively. Analysis were carried out
A standard stock solution of Para Amino using 2% w/v ninhydrin reagent; 3 ml were
Phenol (100 μg/ml) was prepared using added and mixed, heated on water bath at
DMF as diluents and 1 to 5 ml of standard 700 C ± 20 C for 30 minutes, cooled at room
solution was then diluted to obtain 10 ml temperature and volume in each were
with DMF to obtain 10 to 50 μg/ml Para adjusted to 10ml with DMF, The absorbance
Amino Phenol. An UV spectroscopic of resulting solution was measured 10 times
scanning (200– 600nm) was carried out to at 547nm.
determine the λ max. In this study, LOD and LOQ were based on
2.4. Method the standard deviation of the response and
2.4.1. Preparation of stock solution the slope of the corresponding curve using
10mg of the pure drug was weighed and the following equations
transferred to a 100ml volumetric flask;
50ml DMF was added to the above flask and
dissolved, the volume was made up with the Where,
DMF. = Standard Deviation
2.4.2. Preparation of ninhydrin reagent S = Slope
2gm of the ninhydrin was weighed and Cut off range was found to be 0.03
transferred to a 100ml volumetric flask,
50ml DMF was added to the above flask and Table 1: Determination LOD and LOQ
dissolved, the volume make up 100ml with based on calibration curve calculations
the DMF. Sr. No Conc. µg/ml Abs at 547nm
2.5. Method validation 1 0.03 0.00209
Analytical method development and 2 0.04 0.0025
validation for detection of p-aminophenol in 3 0.05 0.0035
the paracetamol bulk dosage form by ICH 4 0.06 0.0037
guideline. 5 0.07 0.0045
2.5.1. Limit of Detection (LOD) and Limit 6 0.08 0.00507
of Quantitation (LOQ) 7 0.09 0.0056
2.5.1.1. LOD 8 0.1 0.0061
It is the lowest amount of analyte in a 9 0.2 0.0077
sample that can be detected but not 10 0.3 0.01012
necessarily quantitated under the stated 11 0.4 0.01774
experimental conditions. [12-15] 12 0.5 0.01906
2.5.1.2. LOQ 13 0.6 0.02381
Lowest concentration or amount of analyte 14 0.7 0.02792
that can be detected quantitatively with an 15 0.8 0.03302
acceptable level of repeatability, precision 16 0.9 0.03697
and trueness. [12-15] 17 1.0 0.04062
2.5.2 Method based on Standard Deviation 18 1.1 0.04554
of blank 19 1.2 0.0494
Analysis were carried out using 2% w/v 20 1.3 0.0532
ninhydrin reagent; 3 ml were added and 21 1.4 0.05762
mixed, heated on water bath at 700 C ± 20 C 22 1.5 0.062
for 30 minutes, cooled at room temperature 23 1.6 0.06821
and volume in each were adjusted to 10 ml 24 1.7 0.07252
with DMF, The absorbance of resulting 25 1.8 0.07751
solution was measured 10 times at 547nm. 26 1.9 0.0797
27 2.0 0.0868

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Figure 1: Calibration Curve for p-amino phenol for LOD & LOQ (0.03 to 2µg/ml)

Standard stock solution para amino phenol

(1.0 ml) were transferred to a 10ml of
LOD = 3.3 x 0.00388597 ambered colored volumetric flask, 2% w/v
0.045 ninhydrin reagent; 3.0 ml were added and
mixed. The flask were immediately
LOD = 0.284971 µg/ml immersed in a water bath 700 C ± 20 C for 30
minutes, cool to room temperature and the
volume were adjusted to 10 ml with DMF.
The absorbance of resulting solution was
LOQ = 10 x 0.00388597 measured at 547nm against blank.
0.045 The absorbance of the samples in the range
LOQ = 0.8635 µg/ml of 2–12μ g/ml was linear with a correlation
coefficient (R2) 0.999.
2.5.4. Linearity and Range
2.5.4.1 Linearity Table 2: Data for calibration curve
Linearity indicates the ability to produce
results that are directly proportional to the Sr. Concentration Absorbance
concentration of the analyte in samples. [12, no (μg /ml) at 447nm
13, 15, 16] 1 2 0.0812
2.5.4.2 Range 2 4 0.1801
Range is an expression of the lowest and 3 6 0.2682
highest levels of analyte that have been 4 8 0.3593
demonstrated to be determinable for the 5 10 0.4449
product. [12, 13, 15, 16] 6 12 0.5392

Figure 2: Calibration Curve for p-amino phenol (2 to 12µg/ml)

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2.5.5. Specificity 2.5.6. Accuracy

Specificity (selectivity) is the ability to Accuracy is the degree of agreement of test
measure unequivocally the desired analyte results with the true value, or the closeness
in the presence of components such as of the results obtained by the procedure to
excipients and impurities. [12, 13, 17, 18] the true value. [12, 13, 15, 17, 18]
Standard solution of para amino phenol (0.4 Prepare 20 µg/ml of para aminophenol
ml) from stock solution A. Add 3.0 ml of 2% solution in three sets from stock solution.
w/v ninhydrin reagent and mixed, Spike standard para amino phenol in
immediately heated on water bath 700 C ± concentration of 50, 100,150% respectively.
20 C for 30 minutes, cooled at room Analysis were carried out using 2% w/v
temperature. Now spiking Paracetamol in 3 ninhydrin reagent; 3.0 ml were added and
different levels 50,100,150% respectively mixed, immediately heated on water bath
from stock solution B, make up the volume 700 C ± 20 C for 30 minutes, cooled at room
to 10ml with DMF, The absorbance of temperature and volume adjusted to 10 ml
resulting solution was measured at 547nm with DMF, The absorbance of resulting
against blank. solution was measured at 547nm against
Spiking Paracetamol in 3 different levels blank. Solution stability was also performed
50,100,150% respectively from stock simultaneously for 2 hours highlighted in
solution B to determine the amount of (Table 4).
℅recovery at 547nm.

Table 3: % Recovery studies for specificity

Sr. Conc. of API Add ℅ Spike stock Abs. at Drug found ℅ Recovery
No μg/ml level sol. μg/ml 547nm μg/ml
1 4 50 2 0.1812 4.14 103.5
2 4 100 4 0.1812 4.136 103.4
3 4 150 6 0.1816 4.146 103.7

Table 4: Accuracy Study with Solution Stability

After 1 Hour
Sr. No conc. Of ml of Level of Spike Total conc. Abs. at Drug ℅
API stock add. ℅ API in μg/ml 547 nm found recovery
sol. ml μg/ml
1 20 2 50 1 30 1.334 29.76 99.31
2 20 2 100 2 40 1.797 40.03 100.1
3 20 2 150 3 50 2.242 49.93 99.91
After 2 Hours
1 20 2 50 1 30 1.336 29.8 99.42
2 20 2 100 2 40 1.799 40.66 100.2
3 20 2 150 3 50 2.243 49.86 99.97

2.5.7. Precision mixed, immediately heated on water bath

Precision is the degree of agreement among 700 C ± 20 C for 30 minute, cooled at room
individual results. The complete procedure temperature and volume adjusted to 10 ml
should be applied repeatedly to separate, with DMF, The absorbance of resulting
identical samples drawn from the same solution was measured at 547nm against
homogeneous batch of material. [12, 13, 15, blank. The same complex solution were
17, 18] taken and the absorbance were measured at
Prepare 20µg/ml of para aminophenol 547nm at 4 different time intervals (i.e. at
solution in three sets from stock solution. 30 minutes, 60 minutes, 90 minutes, 120
Spike standard para aminophenol in minutes).
concentration of 50,100,150% respectively. The interday precision and intraday
Analysis were carried out using 2% w/v precision are applied to determine the
ninhydrin reagent; 3.0 ml were added and sample stability. The interday precision and

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intraday precision was found RSD is less

than 2℅.
3. Calculation
3.1. Energy of Activation Ea [11, 20]
3.1.1. Water bath

Table 5: Calibration Curve of Paracetamol in 0.01M H2SO4

Sr. No Concentration µg/ml ml of Stock Solution ml of 0.01M H2SO4 Absorbance
1 2 0.2 9.8 0.2349
2 3 0.3 9.7 0.2383
3 4 0.4 9.6 0.3140
4 5 0.5 9.5 0.4028
5 6 0.6 9.4 0.4761
6 7 0.7 9.3 0.5410
7 8 0.8 9.2 0.6035
8 9 0.9 9.1 0.6835

Figure 3: Calibration curve of Paracetamol in 0.01M H2SO4 ( max-372 nm)

Table 6: Determination of specific reaction rate constant (K1, at room temperature)

Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K1 = 2.303 log a
No Minutes µg/ml t (a-x)
1 Initial 0.3208 4.0112 0.60327 ---
2 30 0.3450 4.3521 0.63869 0.27188 x 10-4
3 60 0.3135 3.9084 0.59199 4.3292 x 10-4
4 90 0.3129 3.90 0.59106 3.12331 x 10-4
5 120 0.3123 3.8915 0.59011 2.5254 x 10-4
6 150 0.2905 3.5845 0.55442 7.49847 x 10-4
7 180 0.2866 3.5295 0.54771 7.106124 x 10-4
8 210 0.2627 3.1929 0.50418 0.108602 x 10-4
9 240 0.2614 3.1746 0.50168 9.748373 x 10-4
Mean = 9.041325 x 10-4/min

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Table 7: Determination of specific reaction rate constant (K2, at 60ºC)

Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K2 = 2.303 log a
No Minutes µg/ml t (a-x)
1 Initial 0.3553 4.4971 0.65293 ---
2 30 0.3265 4.0915 0.61188 3.1509 x 10-3
3 60 0.2932 3.6225 0.5590 3.60503 x 10-3
4 90 0.1584 1.72394 0.23652 0.010651
5 120 0.1527 1.64366 0.21579 8.3883 x 10-3
6 150 0.1580 1.71830 0.23509 6.41384 x 10-3
7 180 0.1350 1.39436 0.14437 6.5044 x 10-3
8 210 0.0611 0.35352 - 0.45158 0.012105
9 240 0.0507 0.20704 - 0.68394 0.012828
Mean = 7.561975 x 10-3/min
Table 8: Determination of specific reaction rate constant (K3, at 800C)
Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K3 = 2.303 log a
No minutes µg/ml t (a-x)
1 Initial 0.3400 4.2816 0.65293 ---
2 30 0.1924 2.2080 0.61188 0.0220769
3 60 0.1326 1.3605 0.5590 0.01910947
4 90 0.0099 - 0.3676 0.23652 5.03874 x 10-3
5 120 0.0071 - 0.4070 0.21579 4.62862 x 10-3
6 150 0.0067 - 0.4126 0.23509 3.79344 x 10-3
7 180 0.0026 - 0.47042 0.14437 3.8893 x 10-3
8 210 - 0.0418 - 1.09577 - 0.45158 6.49056 x 10-3
9 240 - 0.0013 - 0.5253 - 0.68394 3.3778281 x 10-3
Mean = 8.55060725x 10-3/min

3.1.1.1. Calculation for Energy of Activation (Ea): For room temp and 600C
[ ]

Where K1 and K2= Mean of Room temperature and 600C

Convert T1 and T2 in Kelvin, hence add 273
Ea= 2.303 [(20 + 273) (60 + 273) 1.98] X Log 0.007561975
[(20 + 273) – (60 + 273)] 0.0009041325

Ea= 444908.7859 X (-2.121364763) – (-3.043767919)

40
Ea= 11122.71965 X 0.922403156 = 10.25963171 Kcal/mole

3.1.1.2. Energy of Activation (Ea): For room temp and 800C

[ ]

Where K1 and K3 = Mean of Room temperature and 800C

Convert T1 and T3 in Kelvin, hence add 273
Ea= 2.303 [(20 + 273) (80 + 273) 1.98] X Log 0.00855060725
[(20 + 273) – (80 + 273)] 0.0009041325
Ea= 471630.0343 X (-2.068003054) – (-3.043767919)
60
Ea= 7860.500571 X 0.975764865 = 7.670000278 Kcal/mole.

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3.1.2. Hot air oven

Table 9: Calibration Curve of Paracetamol in 0.01M NaOH
Sr. Concentration ml of Stock ml of 0.01M Absorbance
No µg/ml Solution H2SO4
1 2 0.2 9.8 0.3194
2 4 0.3 9.7 0.4103
3 6 0.4 9.6 0.4984
4 8 0.5 9.5 0.5895
5 10 0.6 9.4 0.6738
6 12 0.7 9.3 0.7514

Figure 4: Calibration curve of Paracetamol in 0.1M NaOH ( max-257 nm)

Table 10: Specific reaction rate constant Paracetamol Degradation in Bulk in hot air oven
(K1,at 1200 C)
Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K1 = 2.303 log a
No Minutes µg/ml t (a-x)
1 Initial 0.5995 8.4534 0.9270 ---
2 60 0.5979 8.4162 0.9251 7.25382 x 10-5
3 120 0.5648 7.6460 0.8834 8.36684 x 10-4
4 180 0.5413 7.10 0.8512 9.69482 x 10-4
5 240 0.5281 6.7930 0.8321 9.106414 x 10-4
6 300 0.5211 6.630 0.8215 8.0989185 x 10-4
Mean=7.06790614x 10-4/min
Table 11: Specific reaction rate constant Paracetamol Degradation in Bulk in hot air oven
(K2, at1400 C)
Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K2 = 2.303 log a
No Minutes µg/ml t (a-x)
1 Initial 0.5995 8.4534 0.9270 ---
2 60 0.5990 8.4418 0.9264 2.3028 x 10-5
3 120 0.5460 7.2093 0.8579 1.326029 x 10-3
4 180 0.5152 6.4930 0.8124 1.466192 x 10-3
5 240 0.4739 5.5326 0.7429 1.766592 x 10-3
6 300 0.4337 4.5977 0.6625 2.0304872 x 10-3
Mean = 1.3183206 x 10-3/min

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Table 12: Specific reaction rate constant Paracetamol Degradation in Bulk in hot air oven
(K3, at 1600 C)
Sr. Time Abs (a-x) Conc. (a-x) Log (a-x) K3 = 2.303 log a
No Minutes µg/ml t (a-x)
1 Initial 0.5995 8.4534 0.9270 ---
2 60 0.5619 7.57907 0.8796 1.819212 x 10-3
3 120 0.5266 6.7581 0.82983 1.8646923 x 10-3
4 180 0.4740 5.5349 0.7431 2.35289016 x 10-3
5 240 0.4214 4.31163 0.63464 2.805436859 x 10-3
6 300 0.3688 3.0884 0.48973 3.356790609 x 10-3
Mean =2.439804386 x 10-3/min

3.1.2.1. Calculation for Energy of Activation (Ea): For 1200 C and 1400 C

[ ]

Where K1 and K2 = Mean of 1200 C and 1400 C

Convert T1 and T2 in Kelvin, hence add 273
Ea= 2.303 [(120 + 273) (140 + 273) 1.98] X Log 0.0013183206
[(120 + 273) – (140 + 273)] 0.000706790614
Ea= 740119.3015 X (-2.879978962) – (-3.150709226)
20
Ea= 37005.96507 X 0.270730266 = 10.01863477 Kcal/mole

3.1.2.2. Calculation for Energy of Activation (Ea): For 1400 C and 1600C

[ ]

Where K2 and K3 = Mean of 1200 C and 1400 C

Convert T2 and T3 in Kelvin, hence add 273

Ea= 2.303 [(140 + 273) (160 + 273) 1.98] X Log 0.0024398043

[(160 + 273) – (140 + 273)] 0.0013183206
Ea= 815449.5103 X (-2.612644992) – (-2.879978962)
20
Ea= 40772.47551 X 0.26733397 = 10.89986774 Kcal/mole.

3.1.2.3. Calculation for Energy of Activation (Ea): For 1600 C and 1200 C

[ ]

Where K1 and K3 = Mean of 1200 C and 1600 C

Convert T1 and T3 in Kelvin, hence add 273
Ea= 2.303 [(120 + 273) (160 + 273) 1.98] X Log 0.0024398043
[(160 + 273) – (120 + 273)] 0.0013183206
Ea= 775960.4299 X (-2.612644992) – (-3.150709226)
40
Ea= 19399.01075 X 0.538064234 = 10.43791386 Kcal/mole.

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Energy of Activation for paracetamol was considering the energy of activation to be

found to be 10 Kcal/mol 10 Kcal/mole. The degraded product was
Assuming Energy of 12 Kcal/mol affords estimated by the prepared method. [20]
nearly doubling of reaction rate for every 3.2. Paracetamol Degradation
10ºC rise in temperature. Take 9 tablets of Calpol 500 mg, covered
With this relationship one can calculate the with aluminum foil was kept in hot oven at
amount of time a sample should be stored at 100 0C for 3 days. [5, 10]
a specified temperature to achieve the After heating each tablet was triturated,
energy equivalent of exposure at Weight equivalent to 5 mg of paracetamol,
accelerated stability conditions (40 ºC for 6 5.7 mg of tablet powder was for analysis
months) so to keep at 80 ºC (every 10 ºC using 2% w/v ninhydrin reagent; 3.0 ml
rise 24) were added and mixed, immediately heated
180 days (6 months) / 24 = 11.25 = 12days. on water bath 700 C ± 20 C for 30 minutes,
A sample of bulk drug substance stored at cooled at room temperature and volume
80 ºC would be stored for 12 days is adjusted to 10 ml with DMF, The
kinetically equivalent to 40 ºC for 6 months. absorbance of resulting solution were
Using the same concept paracetamol drug measured at 547nm against blank.
product was kept at 100 ºC for 3 days

Table 13: ̅ Found (For tablet degradation) at 1000C for 3 days

Tab. Powder mg Abs 547 nm Mean ̅
5.7 1.7
5.7 1.8
5.7 1.7
5.7 1.6
5.7 1.5 1.6412
5.7 1.4
5.7 1.5
5.7 1.4
5.7 1.6
Absorbance of degraded paracetamol i.e. P-amino phenol is 1.6412,
Concentration of p-amino phenol =
Y = 0.0454x – 0.0054
1.6412 = 0.0454 x – 0.0054
X= 36.2687 µg/ml

Weight equivalent to 5 mg of paracetamol, In present study by using chemical

5.7 mg of tablet powder was analyzed and Derivatization, UV method is developed for
P- amino phenol was found to be the determination of the Para -Amino
36.2687µg/ml, thus the degradation is 0.7% phenol, which is a degraded product of
from the tablet dosage form. paracetamol. For the preparation of stable
4. RESULTS AND DISCUSSION complex of para amino phenol solvents like
A spectrophotometric method was Water, Methanol, Acetone, Ethanol, DMF are
developed for quantitative determination of used, and DMF showing stable readings
degraded amino group in sample and among all other solvents. Accordingly
reduces unnecessary tedious sample concentration of Ninhydrin 3ml of 2% w/v,
preparation. heating time 30 minutes at 70 ºC decided
The ninhydrin has been known as a reagent for formation of stable complex.
for detection of amino acid and amines. It With set parameter the method is validated,
was suggested that the reaction of LOD and LOQ were found to be 0.254(Table
ninhydrin with amine, amino acid and imino 1, and Fig. 1), 0.770 and 0.2849 (Table 1,
acids all produced by the mechanism to give and Fig. 1), 0.863 by standard deviation of
the Ruhemans purple. blank and calibration curve method

Ansari Md Naseem et.al, IJPRR 2016; 5(2) 10

International Journal of Pharma Research & Review, Feb 2016;5(2):1-12 ISSN: 2278-6074

respectively. Among the entire range 1 to was considered, indicates that the results lie
30 µg/ml Linearity range was found to be 2 between 100.32-99.26% and 100.19 to
to 12 µg/ml (Table 2, and Fig. 2) as it is 100.07% respectively (Table 4). From the
having least sum square error i.e. 1.448667 principle of energy of activation the forced
x 10-5. Method is found to be specific for the degradation of paracetamol in tablet dosage
para amino phenol, data supported by form is carried out at 100ºC for 3 days,
ANNOVA test with at P ≤ 0.05. For accuracy degradation of paracetamol was found to be
and precision confidence interval of 99% 0.7% (Table 13).

Table 14: Result of various validation parameters

Parameters Values
Linearity and range (μ/ml) 2-12 μg/ml
Precision (% RSD) RSD< 2%
Accuracy(% Recovery) 99.86 %
ANOVA P ≤ 0.05
LOD (μg/ml) 0.284971
LOQ (μg/ml) 0.8635
Energy of Activation 10.43791386 Kcal/mole
Degradation of Paracetamol 0.7 %

5. CONCLUSION Institute of Pharmacy, Bhujbal Knowledge

In present study by using chemical City, Nashik for providing necessary
derivatization, UV method is developed for research facilities. Thanks are also extended
the determination of the Para -Amino to S.D FINE CHEM ltd for providing gift
phenol, which is a degraded product of sample of pure Paracetamol and P-
paracetamol for the preparation of stable aminophenol.
complex of Para amino phenol and DMF REFERENCES
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