part-[b] simultaneous reverse phase high performance...

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-


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


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,


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


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


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


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


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


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


PART-[B] 7

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


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


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].


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).


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.


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)


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)


(Mobile phase: ACN : buffer, 60 : 40 v/v, pH 3.2 with OPA).


PART-[B] Page 84


(Mobile phase: CAN : buffer, 60 : 40 v/v, pH 3.2 with OPA).


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


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


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


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


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


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 %


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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