part-[b] simultaneous reverse phase high performance...
TRANSCRIPT
PART-[B]
Simultaneous reverse phase
high performance liquid
chromatographic [RP-HPLC]
determination of salicylamide,
salicylic acid and deferasirox in
the bulk API dosage forms
SECTION-[II]
HPLC method development and
validation of salicylic acid
Section-[II] Salicylic acid
PART-[B] Page 72
[1.0] Introduction of salicylic acid
Salicylic acid (from Latin Salix, willow tree, from the bark of which the
substance used to be obtained) is chemically 2-hydroxybenzoic acid. This
colorless crystalline organic acid is derived from the metabolism of salicin. Its
molecular formula is C7H6O3 and molecular is weight 138.12 gm/mole. It is
poorly soluble in water (2 g/L at 20 °C) [1, 2]. It has bacteriostatic, fungicidal,
keratolytic actions and widely used in organic synthesis and functions as a
plant hormone. The salts and esters of salicylic acid are known as salicylates.
Salicylate salt is used as analgesics [3-5]. In addition to being an important
active metabolite of aspirin (acetylsalicylic acid), which acts in part as a prodrug
to salicylic acid, it is probably best known for its use as a key ingredient in
topical anti-acne products. Aspirin (acetylsalicylic acid or ASA) can be prepared
by the esterification of the phenolic hydroxyl group of salicylic acid with the
acetyl group from acetic anhydride or acetyl chloride.
Salicylic acid is known for its ability to ease aches and pains and reduce
fevers. These medicinal properties, particularly fever relief, have been known
since ancient times and it is used as an anti-inflammatory drug [6]. For
example, methyl salicylate is used as a liniment to soothe joint and muscle pain
and choline salicylate is used topically to relieve the pain of mouth ulcers.
Cotton pads soaked in salicylic acid can be used to chemically exfoliate skin as
with other hydroxy acids, salicylic acid is a key ingredient in many skin-care
products for the treatment of seborrhoeic dermatitis, acne, psoriasis, calluses,
corns, keratosis pilaris, acanthosis nigricans, ichthyosis and warts. The
standard treatment for all uses is a 6 % aspirin suspension in petroleum jelly,
applied on the callus for one hour and then removed with washing. Salicylic
acid works as a keratolytic, comedolytic and bacteriostatic agent, causing the
cells of the epidermis to shed more readily, opening clogged pores and
neutralizing bacteria within, preventing pores from clogging up again by
constricting pore diameter and allowing room for new cell growth [7, 8]. Use of
concentrated solutions of salicylic acid may cause hyper pigmentation on
unpretreated skin for those with darker skin types, as well as with the lack of
use of a broad spectrum sun block [9, 10]. Bismuth subsalicylate, a salt of
bismuth and salicylic acid, is the active ingredient in stomach relief aids such
as Pepto-Bismol, is the main ingredient of Kaopectate and displays anti-
Section-[II] Salicylic acid
PART-[B] Page 73
inflammatory action (due to salicylic acid) and also acts as an antacid and mild
antibiotic [11-13].
It is derived from a bitter powder that comes from the bark of a willow
tree. Though it does not have any effect on the acne bacteria in the pores or the
excess production of sebum that cause acne, 1.5 % - 2 % concentrations of
salicylic acid are effective at clearing mild to moderate acne effectively without
over drying the skin. In some skin creams for dry or mature skin, salicylic acid
is used in lower concentrations as an exfoliating ingredient which increases the
efficacy of active ingredients. Since salicylic acid can be slightly drying to the
surface of the skin, it is advised not to use it along with topical acne
prescriptions, aggressive cleansers and astringents. For the best results, a
balanced acne regimen with moisturizing and soothing products is
recommended along with salicylic acid products.
The P.acnes bacterium that causes acne cannot live in an oxygen rich
environment which benzoyl peroxide when it comes in contact with the skin so
breakouts are reduced. It is also a keratolytic, providing exfoliating benefits in
the pores to prevent clogging. It is used in 2.5 %, 5 % and 10 % concentrations
depending on the severity of acne and sensitivity level of the skin although
higher concentrations are not always more effective. Benzoyl peroxide can be
found in many over the counter creams, gels and lotions as well as in stronger
prescription products. It can also bleach fabric so it’s best to protect your
clothing and linens when using products with this ingredient. Side effects range
from mild to severe dryness, peeling and swelling so careful selection of other
skin care products should be made so as not to increase the severity of these
effects.
[2.0] Chemical structure, IUPAC and common name of salicylic acid
Section-[II] Salicylic acid
PART-[B] Page 74
Common name
2-Carboxyphenol, 2-hydroxy-benzoic acid, o-carboxyphenol, o-hydroxybenzoic
acid, salicylic acid, SAX, Ionil, Keralyt, Saligel, Salonil, Duoplant, Freezone,
Rutranex, Stri-Dex and verrugon
[3.0] Brief overview of synthetic pathway of salicylic acid
Salicylic acid is biosynthesized from the amino acid phenylalanine. In
arabidopsis thaliana it can also be synthesized via a phenylalanine-independent
pathway. Sodium salicylate is commercially prepared by treating sodium
phenolate with carbon dioxide at high pressure (100 atm) and high temperature
(125ºC), a method known as the Kolbe-Schmitt reaction. Acidification of the
product with sulfuric acid gives salicylic acid. It can also be prepared by the
hydrolysis of aspirin (acetylsalicylic acid or methyl salicylate (oil of wintergreen)
with a strong acid or base.
[4.0] Description
[4.1] Pharmacodynamics
Salicylic acid treats acne by causing skin cells to slough off more readily,
preventing pores from clogging up. This effect on skin cells also makes salicylic
acid an active ingredient in several shampoos meant to treat dandruff.
Subsalicylates in combination with bismuth form the popular stomach relief aid
known commonly as Pepto-Bismol. When combined the two key ingredients
help control diarrhea, nausea, heartburn, and even gas. It is also very mildly
anti-biotic.
[4.2] Mechanism of action
Salicylic acid directly and irreversibly inhibits the activity of both types of
cyclo-oxygenases (COX-1 and COX-2) to decrease the formation of precursors of
prostaglandins and thromboxanes from arachidonic acid. Salicylic acid is a key
ingredient in many skin-care products for the treatment of acne, psoriasis,
Section-[II] Salicylic acid
PART-[B] Page 75
calluses, corns, keratosis pilaris, and warts. Because of its effect on skin cells,
salicylic acid is used in several shampoos used to treat dandruff.
Salicylic acid shows its anti-inflammatory effects via suppressing the
activity of cyclooxygenase (COX), an enzyme that is responsible for the
production of pro-inflammatory mediators such as the prostaglandins. It does
this not by direct inhibition of COX like most other non-steroidal anti-
inflammatory drugs (NSAIDs) but instead by suppression of the expression of
the enzyme [11]. Salicylic acid has also been shown to activate adenosine
monophosphate-activated protein kinase (AMPK), and it is thought that this
action may play a role in the anticancer effects of the compound and its
prodrugs aspirin and salsalate. In addition, the antidiabetic effects of salicylic
acid are likely mediated by AMPK activation primarily through allosteric
conformational change that increases levels of phosphorylation [12]. Salicylic
acid also uncouples oxidative phosphorylation, which leads to increased ADP :
ATP and AMP : ATP ratios in the cell. As a consequence, salicylic acid may alter
AMPK activity and subsequently exert its anti-diabetic properties through
altered energy status of the cell. Even in AMPK knock-out mice, however, there
is an anti-diabetic effect, demonstrating that there is at least one additional,
yet-unidentified action of the compound [13].
Table 1: Physical and chemical properties of salicylic acid
Physical and chemical properties
Property Value
Molecular Weight 138.12 g/mol
Molecular Formula C7H6O3
Density 1.44 g/cm3
Solubility Easily soluble in methanol
CAS number 69-72-7
Physical state colorless to white crystals
Melting point 158-161°C
Boiling point 211°C
Stability Stable, substances to be avoided include oxidizing agents, strong bases, iodine, fluorine, combustible, Sensitive to light.
Categories Antifungal agents, anti-infective agents, keratolytic agents
Section-[II] Salicylic acid
PART-[B] Page 76
Prescription, indications and usage of salicylic acid
Salicylic acid 6 % is a topical aid in the removal of excessive keratin in
hyperkeratotic skin disorders including verrucae, and the various ichthyoses
(vulgaris, sex-linked and lamellar), keratosis palmaris and plantaris keratosis
pilaris, pityriasis rubra pilaris, and psoriasis. Salicylic acid gel is used to treat
scalp itching, flaking, irritation, redness, and scaling due to psoriasis, dandruff,
or seborrhea. It may also be used for other conditions as determined by your
doctor. Salicylic acid gel is a topical salicylate.
Topical salicylic acid comes as a cloth, cream, lotion, liquid, gel,
ointment, shampoo, wipe, pad, and patch, several strengths, including certain
products that are only available with a prescription to apply to the skin or
scalp. It treats other skin conditions by softening and loosening dry, scaly, or
thickened skin so that it falls off or can be removed easily. It is used to help
clear and prevent pimples and skin blemishes in people who have acne. It is
also used to treat skin conditions that involve scaling or overgrowth of skin cells
such as psoriasis, ichthyoses, dandruff, corns, calluses, and warts on the
hands or feet. Salicylic acid is in a class of medications called keratolytic
agents. It may be used as often as several times a day or as infrequently as
several times a week, depending on the condition being treated and the
products are being used.
Table 2: Salicylic acid topical dosage
Topical Dosage
Salicylic acid topical
1 % pad
Cleanse affected area; apply 2 to 3 times daily. If
dryness occurs, reduce to every other or once a day
Salicylic acid topical
16.7 % liquid
Wash and dry area thoroughly. Apply enough to cover
each wart 1 to 2 times daily
Salicylic acid 3 %
topical soap
Apply to affect areas at least twice weekly. Leave lather
on scalp or skin for two minutes then rinse
Salicylic acid topical
6 % cream
Apply to affected area once daily. Hydrate area for 5
minutes prior to application if possible. Occlude the
area at night. Wash off in morning
Salicylic acid topical
6 % lotion
Apply to affected area once daily. Hydrate area for 5
minutes prior to application if possible. Occlude the
area at night. Wash off in morning
Salicylic acid topical
6 % foam
Apply to affected area once daily at bedtime. Hydrate
area for 5 minutes prior to application if possible.
Occlude the area after application. Wash off in morning
Section-[II] Salicylic acid
PART-[B] Page 77
Salicylic acid - clinical pharmacology
Salicylic Acid has been shown to produce desquamation of the horny layer of
skin while not effecting qualitative or quantitative changes in the structure of
the viable epidermis. The mechanism of action has been attributed to
dissolution of intercellular cement substance.
Follow the directions on the package label or your prescription label
carefully, and ask your doctor or pharmacist to explain any part you do not
understand. Use salicylic acid exactly as directed. Do not use more or less of it
or use it more often than directed on the package or prescribed by your doctor.
If you are using topical salicylic acid to treat acne, your skin may become dry or
irritated at the beginning of your treatment. To prevent this, you may apply the
product less often at first, and then gradually begin to apply the product more
often after your skin has adjusted to the medication. If your skin becomes dry
or irritated at any time during your treatment, you may apply the product less
often.
[5.0] Survey of analytical method/literature reviews
The literature reviews regarding salicylic acid suggest that various analytical
methods were reported for its determination in pharmaceutical formulation and
in various biological fluids. As per discussion in the literature reviews UV, LC-
MS, HPLC methods for the determination of salicylic acid in pharmaceutical
dosage forms are reported. The analytical methods including UV, HPLC, HPTLC,
LC-MS/MS, GC and fluorimetric have been reported for the estimation of
salicylic acid alone or simultaneously with other drugs. However no method has
been reported for simultaneous estimation of salicylamide, salicylic acid and
deferasirox. The present work describes simultaneous estimation of
salicylamide, salicylic acid in bulk API pharmaceutical dosage forms [14-31].
The literature reviews for analysis of salicylic acid are as under:
[1] PH. Patel, B. Shrivastava, J. Prajapati, J. Akhtar et al, have developed
and validated a simple, precise and accurate Rp-HPLC method for simultaneous
estimation of niacinamide and salicylic acid in semi solid dosage form. The
separation is done by using column oyster BDS C-18 (4.6 x 250 mm, 5 µm, 110
Å) at 25ºC and 74 : 26 (V/V) water: methanol as mobile phase at flow rate of 1
mL/min and pH adjusted with triethylamine and glacial acetic acid. Detection
is carried out at 280 nm. The method has been validated according to ICH
Section-[II] Salicylic acid
PART-[B] Page 78
guideline in terms of linearity, precision, accuracy, specificity and solution
stability. The linearity of proposed method is investigated in the range of 250-
750 µg/mL (R2 = 0.999 %) for niacinamide and 100-300 µg/mL (R2 = 0.999 %) for
salicylic acid. The percentage recoveries of niacinamide and salicylic acid are
found to be 99.07-100.08 % and 99.57-100.25 % respectively. The proposed
method provides an accurate and precise quality control tool for analysis of
niacinamide and salicylic acid in semisolid dosage forms [32].
[2] JS. Mary, Vimal Kumar et al, have developed and validated a rapid
reversed-phase high-performance liquid chromatographic procedure for the
simultaneous quantitation of aspirin, salicylic acid, and caffeine extracted from
an effervescent tablet. The method uses a Hypersil C18 column (5 μm, 15 cm ×
4.6 mm) for an isocratic elution in a water : methanol : acetic acid mobile phase
at a wavelength of 275 nm. The tablets’ buffering effects and acid neutralizing
capacity require an extraction solvent of methanol : formic acid. The range of
linearity for aspirin is 0.5-1.25 mg/mL, caffeine 0.065-0.195 mg/mL, and
salicylic acid 0.4-6.0 % of aspirin. The overall recovery is 100.2 %, 100.7 %, and
99.2 % for aspirin, caffeine, and salicylic acid, respectively. Under the
conditions of the method, aspirin, caffeine, and salicylic acid are adequately
resolved with proper peak symmetry in less than 7 min. [33].
[3] B. Patel, H. Raj, V. Jain, V. Sutariya, M. Bhatt et al, have developed and
validated a simple, gradient, rapid and accurate reversed phase high
performance liquid chromatography method for simultaneous determination of
Clobetasol propionate and salicylic acid in its pharmaceutical dosage forms.
Unisphere C-18 column (5 μm, 250 mm x 4.6 mm i.d.) at 45 ºC temperature
and UV detector at 240 nm was used. (Methanol: Sodium dihydrogen
phosphate monohydrate (pH 5.5 adjusted with NaOH): acetonitrile (6 : 29 : 65)
was used as mobile phase with 1 mL/min with injection volume 20 μL. The
method has been validated according to ICH guidelines. Calibration graph was
found to be linear in 300-900 μg/mL and 5-15 μg/mL for salicylic acid and
clobetasol propionate respectively. The regression coefficient (R2) obtained was
found to be 0.9999 % for both drugs. The retention time were found to be 2.258
min and 6.083 min respectively [34].
[4] Safeena Sheikh, Suhail Asghar, Showkat Ahmad Patni et al, have
developed and validated a stability indicating high performance liquid
Section-[II] Salicylic acid
PART-[B] Page 79
chromatography (HPLC) method for the quantification of salicylic acid (SA) and
tolnaftate (TF) in combined pharmaceutical ointment base formulations. The
separation was performed on a Merck” C-18 column with the mobile phase
consisting of Acetonitrile : Methanol : Water (50 : 20 : 30 v/v) at flow rate 1.5
mL/min. Both the drugs were resolved successfully with retention time 1.318
min and 8.805 min, when detection was carried out at UV 245 nm. The overall
retention times of analytes were 10.0 minutes. The method was validated with
respect to linearity, precision, accuracy and recovery. The relative standard
deviation for six replicate measurements of SA and TF were 0.259 % and 0.240
% respectively. Total recoveries of analytes were 100.56 %, 100.63 %, 100.58 %
and 100.23 %, 100.73 %, 100.22 % of SA and TF respectively when examine
over the range of 80 %, 100 %, and 120 % of added drugs in placebo. The
linearity of SA and TF were found in the range of 256-384 μg/mL and 32.0
μg/mL to 48.0 μg/mL respectively [35].
[5] M. Shou, WA. Galinada, Yu-ChienWei, Q. Tang, RJ. Markovich, AM.
Rustum et al, developed a reversed-phase high performance liquid
chromatography (RP-HPLC) method for simultaneous determination of salicylic
acid, betamethasone dipropionate, and their related compounds in diprosalic
lotion®. A 150 mm 4.6 mm I.D. YMC J’sphere ODS-H80 column at 35◦C and
UV detection at 240 nm was used. A gradient elution was employed using 0.05
% (v/v) methanesulfonic acid solution and acetonitrile as mobile phases. A total
of thirty three compounds from diprosalic lotion® samples were separated in 38
min. [36].
[6] Pushpa Kumari K, Gowri Sankar, P. Sowjanya, S. Madhubabu et al, have
developed and validated a stability indicating RP-HPLC method for the
determination of salicylic acid in trilisate tablets using Xorabax XBD C18
column (150 4.6 mm, 5 μm) with mobile phase consisting of phosphate buffer
(pH 3.0) : methanol (80 : 20 v/v) with a flow rate of 1.0 mL/min. Detection was
carried out at 230 nm. Retention time was found to be 4.6 min. Linearity was
observed over the concentration range of 5-30 μg/mL (R2 = 0.999 %) with
regression equation y = 30.55x + 5.302. Salicylic acid was subjected to stress
conditions including acidic, alkaline, photolysis and thermal degradation. The
drug is more sensitive towards alkaline degradation. The method was validated
as per ICH guidelines [37].
Section-[II] Salicylic acid
PART-[B] Page 80
[7] FH. Havaldar, DL. Vairal et al, have developed and validated a simple,
specific, accurate and economical isocratic reversed phase liquid
chromatographic (RP-HPLC) method for the determination of paracetamol,
acetyl salicylic acid, mefenamic acid and cetirizine dihydrochloride. Separation
was achieved with a Nucleodur 100 C–18 column having 250 x 4.6 mm i.d. with
5 μm particle size and disodium hydrogen phosphate buffer adjusted to pH 6.5
using diluted orthophosphoric acid and acetonitrile (60 : 40 v/v) as eluent at a
constant flow rate of 1.0 mL per min. UV detection was performed at 220 nm.
The retention time of acetyl salicylic acid, paracetamol, mefenamic acid and
cetirizine dihydrochloride were 2.01 min, 2.92 min, 4.91 min and 10.2 min
respectively. This method is simple, rapid and selective and can be used for
routine analysis of analgesic and antipyretic drugs in pharmaceutical
formulations [38].
[6.0] Aim and scope of present work
The primary objective of the present work was thus to develop and validate a
RP-HPLC method for the assay of salicylic acid from API dosage forms. Hence,
the method is useful for routine quality control analysis and also for
determination of stability. Purpose of the present study was to develop and
validate a RP-HPLC method for determination of salicylic acid in API
pharmaceutical dosage forms. The aim and scopes of the proposed work are as
under:
1. To select suitable mobile phase (solvent buffer ratio)
2. To optimize RP-HPLC conditions
3. To develop suitable HPLC method for salicylamide
4. Perform the validation for the developed method
[7.0] Experimental
[7.1] Materials
Salicylic acid & Orthophosphoric acid were obtained from s d Fine
Chemical Limited. Acetonitrile (fisher Qualigens, HPLC grade) were
obtained from Thermo Fisher Scientific India Pvt. Ltd. and potassium
dihydrogen phosphate was obtained from (Merck Specialties Private
Limited).
Section-[II] Salicylic acid
PART-[B] Page 81
[7.2] Equipment
Equipment Apparatus
HPLC System Dionex ultimate 3000 (Germany) High performance liquid chromatographic system equipped with ultimate 3000 Pump, Auto Sampler, Column Compartment and RS Diode Array Detector
Software Dionex Chromeleon ® 7 (Version 7.1, Simply Intelligent)
Column oven Ambient
Column Waters symmetry C18 (4.6 x 250mm, 5µm, 110 Å)
[7.3] Preparation of stock and sample solutions
[7.3.1] Preparation of buffer
Potassium dihydrogen phosphate (1.36 g) was dissolved in 1000 mL high
purity demonized Milli-Q water [Millipore, Milli-Q, Bedford, MA, USA,
purification system] and pH was adjusted 3.2 with ortho-phosphoric acid
and filtered through 0.22 μ size nylon filter under vacuum.
[7.3.2] Preparation of mobile phase
The mobile phase was prepared by mixing 400 mL phosphate buffer pH
3.2 and 600 mL of acetonitrile [HPLC Grade]. The mixture was sonicated
in Expo-Hi Tech sonicator for 5 minutes.
[7.3.4] Preparation of standard and sample solution
The Standard stock solutions were prepared by accurately weighing
100mg of each salicylic acid in 100 mL volumetric flask (1000 µg/mL) in
acetonitrile. Sample solutions were prepared by appropriate dilution of
the standard solutions with the diluent.
[8] Method development and optimization of chromatographic conditions
(UV graph/chromatograms)
To develop a precise, accurate and suitable RP-HPLC method for the
estimation of salicylic acid different mobile phases, solvent-buffer ratios and pH
were tried to proposed final chromatographic conditions. Deferasirox is soluble
in methanol and acetonitrile-water mixture. The peak shape, resolution and
symmetry of salicylic acid were good with above gradient elution at a 1.0
mL/min flow rate. The method developed was unique in determining the
impurities even at low levels than that of specifications. The developed method
was successfully applied to estimate the amount of deferasirox.
Section-[II] Salicylic acid
PART-[B] Page 82
Optimized chromatographic conditions
Parameter Optimized condition
Flow rate 1.0 mL/min
Mobile phase 40 : 60 v/v (Buffer : ACN)
Buffer pH Potassium phosphate buffer pH 3.2 adjusted by OPA
Wavelength 245 nm
Injection volume 10 µL
Run time 15 min
Column and column
oven temperature
30ºC
Figure 1: Chromatogram and UV calibration curve for salicylic acid
standard (Mobile phase: ACN : buffer, 60 : 40 v/v, pH 3.2 with OPA)
Section-[II] Salicylic acid
PART-[B] Page 83
Figure 2: Chromatogram and UV calibration curve for salicylic acid sample
(Mobile phase: ACN : buffer, 60 : 40 v/v, pH 3.2 with OPA)
Figure 3: Chromatogram and UV calibration curve for salicylic acid sample
(Mobile phase: ACN : buffer, 60 : 40 v/v, pH 3.2 with OPA).
Section-[II] Salicylic acid
PART-[B] Page 84
Figure 4: Chromatogram and UV calibration curve for salicylic acid sample
(Mobile phase: CAN : buffer, 60 : 40 v/v, pH 3.2 with OPA).
Section-[II] Salicylic acid
PART-[B] Page 85
[9] Analytical method validation
The optimized RP-HPLC assay method was validated for specificity,
linearity, accuracy, precision (repeatability and intermediate precision), recovery
and system suitability according to International Conference on Harmonization
(ICH) guidelines for the validation of bioanalytical method [39] and the US Food
and Drug Administration (FDA) [40].
[9.1] System suitability
System suitability was performed by using 100 µg/mL of salicylic acid by
making six replicate injections. Chromatographic parameters calculated from
experimental data, such as Number of theoretical plates, % RSD of peak area
and resolution factors (Rs) are given in table-3. The system was deemed to be
suitable for use if the capacity factors were in the range of 11.77-20 (11.77 <K’
< 20), lower than 2 for tailing factor, more than 2 for resolution (Rs), greater
than 4357 number of theoretical plates (N), resolution between salicylic acid of
at least two and less than 2 % relative standard deviation (% RSD) for peak
area.
Table 3: System suitability parameters
System suitability parameters
Sr. No. Parameters Salicylic acid
1 Linearity range (µg/mL) 10.0-100.0 µg/mL
2 Retention time (Min.) 3.397
3 Theoretical plates (N) 4357
4 Peak Asymmetry (T) 3.38
5 Resolution (Rs) 11.77
6 Accuracy 100.015 %
7 Precision 99.59 %
8 % RSD (For peak area) 0.025 %
[9.2] Precision
The precision of an analytical method is the degree of agreement among
individual test results when the method is applied repeatedly to multiple
samplings of homogenous samples. This is usually expressed as the standard
deviation or the relative standard deviation.
The precision of the assay was studied with respect to both intra-day
(Repeatability) and Inter-day (Intermediated) precisions. Repeatability was
calculated from five replicate injections of three different concentrations of
salicylic acid in the same equipment on the same day. Inter day precision was
Section-[II] Salicylic acid
PART-[B] Page 86
checked with the same concentrations as intra-day assay and the
determination of each compound was repeated day by day during three days.
The method was found to be precise with RSD values within for intra-day and
inter day assay. Evaluation of the intra-day and inter-day precision for the
determination of salicylic acid by the proposed HPLC method according to ICH
guidelines.
Intra Day (Repeatability) precision
Repeatability can be defined as the precision of the procedure when
repeated by same analyst under the same operating conditions over a short
interval of time or same day. It is normally expected that at least six replicates
be carried out and individual result provided from mean, standard deviation
and coefficient of variation should be calculated for set of n value. The RSD
values are important for showing degree of variation expected when the
analytical procedure is repeated several time in a standard situation (RSD
below 2 % for assays in finished product).
Inter day (Intermediate) precision
Repeatability can be defined as the precision of the procedure when
repeated by same analyst under the same operating conditions and the
determination of each compound was repeated day by day during three days or
study repeat three days over a long interval of time.
Table 4: Intra-day and Inter-day precision data for salicylic acid sample
Sample
Con.
(µg/mL)
Intra day Inter day
Area
(mAU*m
in)
Mean
area
% SD %
RSD
Area
(mAU*m
in)
Mean
area
% SD %
RSD
100 16.538 16.554 0.016 0.096 16.539 16.551 0.016 0.095
16.565 16.551
16.574 16.545
16.539 16.578
16.558 16.542
500 78.552 78.505 0.050 0.064 78.542 78.562 0.021 0.027
78.495 78.549
78.433 78.553
78.489 78.593
78.554 78.573
1000 165.275 165.258 0.042 0.025 165.277 165.275 0.017 0.010
165.279 165.285
165.183 165.258
165.278 165.259
165.278 165.297
Section-[II] Salicylic acid
PART-[B] Page 87
Table 5: Intra-day and Inter-day precision data for salicylic acid standard
Standard con. (µg/mL)
Intra day Inter day
Area (mAU*min) Area (mAU*min)
1000 165.283 165.279
165.195 165.252
165.239 165.287
165.279 165.299
165.245 165.245
Average 165.248 165.272
% SD 0.03565 0.02317
% RSD 0.02158 0.01402
Standard potency 99.66 %
[1] % Assay =
P
AT = Average area of obtained in sample preparation
AS = Average area of obtained in standard preparation
W1 = Weight taken of reference standard (mg)
W2 = Weight taken of test sample (mg)
P = Potency of reference standard (%)
[2]
[1] Intra day
1. % Assay =
99.66
= 1.00 0.9993 99.66 = 99.59 %
2.
=
= 0.025 %
[2] Inter day
1. % Assay =
99.66
= 1.00 0.9995 99.66 = 99.61 %
2.
=
= 0.010 %
[9.3] Limit of detection (LOD) and Limit of Quantification (LOQ)
The limit of detection (LOD) is defined as the lowest concentration of an
analyte that can reliably be differentiated from background levels. The standard
solutions of the compounds for LOD were prepared by diluting them
sequentially. Limit of quantification (LOQ) of an individual analytical procedure
is the lowest amount of analyte that can be quantitatively determined with
Section-[II] Salicylic acid
PART-[B] Page 88
suitable precision and accuracy (ICH Guideline Q2B, 2005). LOD and LOQ were
determined calculating the signal-to-noise ratio of each compound by injecting
a series of solution until the S/N ratio 3 for LOD and 10 for LOQ. where S is the
standard deviation of y-intercepts of regression.
[9.4] Specificity
Specificity of method can be absence of any interference at retention
times of samples. The specificity of the method was demonstrated by injection
of standard solution of salicylic acid at concentration of 100 µg/mL. The elution
peaks of salicylic acid presented in representative chromatograms shown in
Figure 4. The representative chromatogram for simultaneous determination of
the studied drugs in API pharmaceutical dosages forms.
Table 6: Specificity study of salicylamide
Con. (µg/mL) Sample Standard
Area (mAU*min) Area (mAU*min)
1000 16.479 16.538
16.577 16.565
16.539 16.574
16.547 16.539
16.499 16.558
Average 16.5282 16.5548
% SD 0.0391306 0.015927963
% RSD 0.2367502 0.096213562
Standard potency 99.66 %
[1] % Assay =
99.66
= 1.00 0.9998 99.66 = 99.64 %
[2]
=
= 0.2367 %
[9.5] Linearity
The linearity of salicylic acid was studied by preparing standard solution at five
different concentrations ranging from 10.0-100.0 µg/mL. Each concentration
was injected in a five replicates and mean value of peak area was taken for
calibration curve.
Construction of the calibration curves
Working solutions containing (10.00-100.00) μg/mL were prepared by
serial dilution of standard solution with the acetonitrile. In all cases, 10 µL
Section-[II] Salicylic acid
PART-[B] Page 89
aliquots were injected (triplicate) and eluted with the mobile phase under the
following chromatographic conditions. The average peak area ratio of each drug
and the internal standard were plotted versus the final concentration of the
drug in μg/mL to get the calibration graph. Alternatively, the corresponding
regression equation was derived.
Sr. No. Concentration (µg/mL) Area (mAU*min)
1 1 0.225
2 10 1.751
3 20 3.603
4 30 5.065
5 40 6.847
6 50 8.662
7 100 16.538
Figure 5: Linearity curve
Section-[II] Salicylic acid
PART-[B] Page 90
Table 7: Summary of linearity data Summary of linearity data
Sr. No. Parameters Salicylic acid
1 Linearity range (µg/mL) 10.0-100.0 µg/mL
2 Slope ± Standard error 0.1872
3 Intercept ± Standard error 0.1649x
4 Linearity equation Y = 0.1649x + 0.1872
5 R2 0.9993
[9.6] Accuracy
Accuracy of the assay method was calculated for salicylic acid by recovery
studies at three concentrations of 50 %, 100 %, 150 % and 200 % levels by
standard addition method. The mean % recovery for salicylamide, salicylic acid
and deferasirox were found is given in the following table.
Table 8: Sample and standard area for salicylic acid
Sample Area (mAU*min)
Standard Area
(mAU*min)
50% 100% 150% 200% 1000%
8.662 16.479 24.742 32.545 165.283
8.659 16.577 24.755 32.569 165.195
8.643 16.539 24.739 32.485 165.239
8.678 16.547 24.688 32.539 165.279
8.654 16.499 24.788 32.583 165.245
Average 8.6592 16.528 24.7424 32.544 165.248
% SD 0.0128 0.0391 0.0361 0.0376 0.0357
% RSD 0.1473 0.2368 0.1459 0.1155 0.0216
Standard potency
99.66 %
[1] Amount added (µg/mL) =
=
= 5.241 µg/mL
[2] Amount found (µg/mL) =
=
= 5.240 µg/mL
[3] % Recovery =
= 100.015 %
Section-[II] Salicylic acid
PART-[B] Page 91
Table 9: Accuracy data for salicylic acid
Sr.
No.
Salicylic
acid
Added
(µg/mL)
Found
(µg/mL)
%
Recovery
% Mean
Recovery
SD % RSD
1 50% 5.241 5.240 100.015 99.94 0.2075 0.2076
5.248 5.24 99.847
5.245 5.23 99.720
5.238 5.25 100.258
5.245 5.24 99.847
2 100% 10.08 9.97 99.524 99.705 0.571 0.572
10.04 10.03 99.916
10.10 10.0 99.095
9.95 10.01 100.637
9.99 9.98 99.944
3 150% 14.97 14.97 100.017 100.059 0.1262 0.1263
14.96 14.98 100.203
14.99 14.97 99.872
14.93 14,94 100.067
14.98 15.00 100.137
4 200% 19.69 19.71 99.922 99.6680 0.442 0.444
19.65 19.70 100.300
19.82 19.66 99.184
19.79 19.69 99.499
19.83 19.72 99.433
[10.0] Summary and Conclusion
An accurate, sensitive, and precise RP-HPLC method with PDA detection was
developed and validated for salicylic acid in their bulk API dosages forms. The
proposed method is very rapid, where the total analytical run time for salicylic
acid (Rt = 3.397 min.) and the internal standard is less than 10 min. The
method can also be readily adapted to routine quality control analysis.
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