PART-[E] Reverse phase high performance liquid ...shodhganga.inflibnet.ac.in/bitstream/10603/110204/18/18_part e.pdf · coordinated as chelate ligands to form a complex with a transition

PART-[E]

Reverse phase high

performance liquid

chromatography [RP-HPLC]

determination of 2, 3 and 4-

aminopyridine with pyridine in

the bulk API dosages forms

RP-HPLC determination of aminopyridine with pyridine

PART-[E] Page 224

[1.0] Introduction of pyridine and 2, 3, 4-aminopyridine

Pyridine has molecular formula C5H5N and molecular weight 79.10

gm/mole. Pyridine is a basic heterocyclic organic compound with the chemical

formula C5H5N. It is structurally related to benzene, with one methine group

(=CH-) replaced by a nitrogen atom. The pyridine ring occurs in many important

compounds, including azines and the vitamins niacin and pyridoxal. Pyridine

was discovered in 1849 by the Scottish chemist Thomas Anderson as one of the

constituents of bone oil. Two years later, Anderson isolated pure pyridine

through fractional distillation of the oil. It is a colorless, highly flammable,

weakly alkaline, water-soluble liquid with a distinctive, unpleasant fish-like

odor.

Pyridine is used as a precursor to agrochemicals and pharmaceuticals

and is also an important solvent and reagent. Pyridine is added to ethanol to

make it unsuitable for. It is used in the in-vitro synthesis of DNA [1] in the

synthesis of sulfapyridine, antihistaminic drugs tripelennamine and

mepyramine, as well as water repellents, bactericides, and herbicides. Some

chemical compounds, although not synthesized from pyridine, contain its ring

structure. They include B vitamins niacin and pyridoxal, the anti-tuberculosis

drug isoniazid, nicotine and other nitrogen-containing plant products [2]

historically; pyridine was produced from coal tar and as a by-product of the

coal gasification. However, increased demand for pyridine resulted in the

development of more economical methods of synthesis from acetaldehyde and

ammonia, and more than 20,000 tonnes per year are manufactured worldwide.

2-Aminopyridine is chemically pyridine-2-amine. Its molecular formula

C5H6N2 has molecular weight 94.04 gm/mole. 2-Aminopyridine is an organic

compound; it is one of three isomeric amino pyridines. It is a colourless solid

that is used in the production of the drugs piroxicam, sulfapyridine, tenoxicam,

and tripelennamine. It is produced by the reaction of sodium amide with

pyridine, the chichibabin reaction [3, 4]. 2-Aminopyridine is used in the

synthesis of pharmaceuticals especially for antihistamines, antinflammatories

and other drugs. Example is pyrilamine, an antihistaminic.

3-Aminopyridine is chemically pyridine-3-amine. Its molecular formula

C5H6N2 has molecular weight 94.04 gm/mole. It is used in the synthesis of


PART-[E] Page 225

organic ligand 3-pyridylnicotinamide. Examples of end-products derived from 3-

aminopyridine are piroxicam, tenoxicam, ampiroxicam. Pyridine molecules are

coordinated as chelate ligands to form a complex with a transition metal ion. 3-

Aminopyrodine is also used as an intermediate for agrochemicals,

pharmaceuticals and colorants.

4-Aminopyridine (4-AP, fampridine, dalfampridine) is an organic

compound with the chemical formula C5H4N-NH2 and molecular weight 94.04

gm/mole. It is chemically pyridine-4-amine, used primarily as a research tool

for subtypes of potassium channels. It is use to manage some of the symptoms

of multiple sclerosis [5, 6] and is indicated for symptomatic improvement of

walking in adults with several variations of the disease [7]. It is very effective

avicide and bird repellent and is highly toxic to human; it strongly excites

central nervous system. It was undergoing Phase III clinical trials as of 2008

[8]. Fampridine is also marketed as ampyra (pronounced am-PEER-ah,

according to the maker's website) in the United States by Acorda Therapeutics

[9] and as Fampyra in Europe. In Canada, the medication has been approved

for use by Health Canada since February 10, 2012 [10].

4-Aminopyridine (Dalfampridine) is a non-selective K+ channel blocker,

which can block a wide variety of K+ channels with different state dependences.

Therefore, in search for clues for the structural determinants of K+ channels

that are important for the state dependences of drug-channel interactions, 4-AP

serves as a useful tool. Potassium channel blocker used to help multiple

sclerosis patients walk [11-15]. This is the first drug that was specifically

approved to help with mobility in these patients [16]. Fampridine has been

shown to improve visual function and motor skills and relieve fatigue in

patients with multiple sclerosis (MS). 4-AP is most effective in patients with the

chronic progressive form of MS, in patients who are temperature sensitive, and

in patients who have had MS for longer than three years. Common side effects

include dizziness, nervousness and nausea and the incidence of adverse effects

was shown to be less than 5 % in all studies. Potassium channel blockade

reverses this effect. A study has shown that 4-AP is a potent calcium channel

activator and can improve synaptic and neuromuscular function by directly

acting on the calcium channel beta subunit [17]. MS patients treated with 4-AP

http://en.wikipedia.org/wiki/Organic_compound

http://en.wikipedia.org/wiki/Organic_compound

http://en.wikipedia.org/wiki/Chemical_formula

http://en.wikipedia.org/wiki/Multiple_sclerosis

http://en.wikipedia.org/wiki/4-Aminopyridine#cite_note-austpre-3

http://en.wikipedia.org/wiki/Clinical_trial#Phase_III

http://en.wikipedia.org/wiki/Clinical_trial

http://en.wikipedia.org/wiki/4-Aminopyridine#cite_note-4

http://en.wikipedia.org/wiki/Acorda_Therapeutics


http://en.wikipedia.org/wiki/Health_Canada



PART-[E] Page 226

exhibited a response rate of 29.5 % to 80 %. A long-term study (32 months)

indicated that 80-90 % of patients who initially responded to 4-AP exhibited

long-term benefits. Although improving symptoms, 4-AP does not inhibit

progression of MS. These improvements include sensory, motor and pulmonary

function, with a decrease in spasticity and pain [18]. Dalfampridine completed

Phase 2 clinical trials for parkinson’s disease in July 2014 [19]. Clinical studies

have shown that 4-AP is capable of reversing the effects of tetrodotoxin

poisoning in animals, however, its effectiveness as an antidote in humans has

not yet been determined [20-22]. Case reports have shown that overdoses with

4-AP can lead to paresthesias, seizures [23] and atrial fibrillation [24].

The drug was originally intended, by Acorda Therapeutics, to have the

brand name amaya, however the name was changed to ampyra to avoid

potential confusion with other marketed pharmaceuticals [25]. The drug 4-

aminopyridine (4-AP) is known to block voltage-activated K+ channels expressed

in a variety of cell types including neurons [26-27] and muscle [28]. Cells of the

immune system, such as B and T lymphocytes and macrophages, express

voltage activated K+ channels also sensitive to 4-AP [29-30].

In addition, 4-AP blocks different lymphocytic functions, including

mitogen induced cell proliferation [31, 32] and killing by natural killer (NK) cells

and cytotoxic T cells. However, the concentration of 4-AP required to inhibit cell

functions may be 5-10 fold higher than that required to block K+ channels

during voltage clamp experiments [33-35]. Part of this effect may result from a

decrease in the channel blocking potency of 4-AP in the presence of serum

contained in the culture medium. Alternatively, the discrepancy may be due to

the voltage dependent properties of 4-AP binding, observed in experiments on

nerve and muscle. Previous studies have suggested that 4-AP binds to closed

channels, is released from open channels when the cell is depolarized and

interferes with inactivation [36]. On the basis of experimental data and

computer simulations, we conclude that 4-AP blocks lymphocyte K+ channels

when they are in their open state.

Furthermore, the drug remains trapped in the channels at

hyperpolarized potentials and relief of block only occurs upon cell

depolarization. It has also been shown that 4-AP block K+ channels when


PART-[E] Page 227

applied outside or inside the cell. We found that in isolated patches of

membrane, the K+ channels are blocked only if the drug is present on the

cytoplasmic side. Finally, demonstration of a pH dependence of 4-AP actions led

us to propose that the drug crosses the membrane in its no ionized form, and

that it blocks the channels from the inside once reionized [37].

The FDA granted dalfampridine orphan drug status, which will provide

market exclusivity for the drug for 7 years [38-39]. Dalfampridine is a

potassium channel-blocker that enhances conduction in focally demyelinated

axons, improves synaptic transmission and potentiates muscle contraction [40].

Based on these actions, the clinical use of dalfampridine has been found to

improve walking in patients with multiple sclerosis (MS) as demonstrated by an

increase in walking speed. It has shown efficacy in patients with all four major

types of MS [41-45]. Dalfampridine is mostly unbound to plasma protein (97–99

%) and the apparent volume of distribution is 2.6 L/kg. Studies with human

liver microsomes indicated that CYP2E1 was primarily accountable for the 3-

hydroxylatoin of dalfampridine. The common adverse effects in patients

administered dalfampridine include urinary tract infections, insomnia,

dizziness, headache, nausea, weakness, back pain, ataxia and visual

disturbances. The FDA’s approval of dalfampridine was based on the results of

one phase 2 and two phase 3 randomized, double-blind, placebo-controlled,

parallel-group clinical trials (MS-F202 and MS-F203)[46, 47]. During the study

period, 300 patients were randomly assigned to receive either dalfampridine 10

mg or placebo twice daily. After the 14-week treatment period, patients were

observed for an additional four weeks.

Multiple sclerosis (MS) is a neurological disorder that affects

approximately 400,000 people in the U.S. and 2.5 million people worldwide,

with women twice as likely to be affected as men [48-52]. MS is manifested by

mental and physical symptoms characteristic of the illness [53, 54]. As the

disease progresses, clinical symptoms begin to reveal cognitive deficits and

increased neuropathic discrepancies [55]. In addition to clinical data,

diagnostics such as neuroimaging, evoked potentials, and cerebrospinal fluid

(CSF) are performed in order to definitively identify the presence of CNS lesions

that may vary over time and space [56-58]. Currently, the McDonald criteria


PART-[E] Page 228

constitute the most widely used diagnostic modality because they focus on

clinical, laboratory, and radiologic data of MS lesions and their dissemination in

time and space [59, 60]. More than 90 % of patients with MS report difficulty in

walking [61]. An oral medication, dalfampridine is quickly and completely

absorbed in the gastrointestinal (GI) tract. A slight and clinically insignificant

increase in the Cmax of dalfampridine is noted when it is taken with food;

therefore, this drug may be taken without regard to meals [62-64].

[2.0] Chemical structure of pyridine and 2, 3, 4-aminopyridine

Table 1: IUPAC and common names Name IUPAC Name Common Name

Pyridine pyridine Azine, Azabenzene

2-Aminopyridine Pyridin-2-amine NSC 431, AKOS 91158, AURORA KA-680,

Aminopyridine, 2-pyridinamine, o-

aminopyridine, α-aminopyridine, amino-2-

pyridine, 2-AP, 2-pyridylamine, α-

pyridylamine

3-Aminopyridine Amino-3-Pyridine 3-pyridinamine, pyridin-3-amine, 462-08-8, 3-pyridylamine, pyridin-3-ylamine, m-

aminopyridine, β-aminopyridine, 3-AP, 3-

pyridylamine, m-aminopyridine, Amino-3-

pyridine, β-pyridylamine

4-Aminopyridine Amino-4-Pyridine 4-pyridinamine, 4-pyridylamine, 4-AP, p-

aminopyridine, Amino-4-pyridine, γ-

aminopyridine, Fampridine, Compound 1861, γ-pyridylamine, gamma-

aminopyridine, mi-w-3, Phillips 1861,

phillips1861, EL-970, Ampyra, Avitrol,

Avitrol 200, Pimadin

[3.0] Brief overview of synthetic pathway of pyridine and 2, 3, 4-

aminopyridine salicylic acid

[1] Synthetic pathway of pyridine

In the synthesis of pyridine, in first step, acrolein is formed by

Knoevenagel condensation of the acetaldehyde and formaldehyde. It is then

condensed with acetaldehyde and ammonia into dihydropyridine and then

oxidized with a solid-state catalyst to pyridine. This process is carried out in a

gas phase at 400-450 °C. The product consists of a mixture of pyridine, simple

methylated pyridines (picoline) and lutidine; its composition depends on the

catalyst used and can be adapted to the needs of the manufacturer. The

catalyst is usually a transition metal salt such as cadmium (II) fluoride or


PART-[E] Page 229

manganese (II) fluoride, but cobalt and thallium compounds can also be used

[65].

Formation of acrolein from acetaldehyde and formaldehyde

Condensation of pyridine from acrolein and acetaldehyde

[2] Synthetic pathway of 2-Aminopyridines

The N-oxide is transformed to N-tert-butylamino intermediate and then

deprotected in situ with TFA in a one-pot operation. The procedure works well

with a wide variety of substrates: pyridines, quinolines, isoquinolines. The few

examples of unsymmetrically-substituted pyridine-oxides that had both the 2

and 6 positions free provided a mix of the two ortho amino regioisomers [66].


3-Aminopyridine can be prepared by heating nicotinamide with sodium

hypobromite which is in turn prepared in situ by the reaction of sodium

hydroxide and bromine at 70 °C [67]


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4-Aminopyridine is prepared by the decarbonylation of pyridine-4-carboxamide

using sodium hypochlorite via the hoffmann rearrangement. The pyridine

carboxamide is generated from the corresponding nitrile, which in turn is

obtained from ammoxidation of 4-methylpyridine [68].

[4.0] Description

[4.1] Pyridine

Table 2: Physical and chemical properties

Physical and chemical properties

Property Value

Molecular Weight 79.10 g/mol

Exact Mass 79.042199 g/mol

Molecular Formula C5H5N

Density 0.9819 g/cm3

Solubility Miscible with water at 20 °C

CAS number 110-86-1

Physical state colorless liquid

Melting point -41.6 °C

Boiling point 115.2-115.3 °C

Stability Mixtures with formamide + iodine + sulfur trioxide are

storage hazards, releasing carbon dioxide & sulfuric acid


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[4.2] 2-aminopyridine



Property Value



Molecular Formula C5H6N2

Density 1.065 g/cm3

Solubility soluble in water, alcohol, benzene, and ether

CAS number 504-29-0

Physical state colorless to white solid

Melting point 59 - 60 °C

Boiling point 204 – 210 °C

Stability Stable under normal temperatures and pressures

Categories




Property Value




Density 1.26 g/cm3

Solubility soluble in water, alcohol, and ether

CAS number 462-08-8

Physical state white to light yellow-brown crystals

Melting point 64 °C

Boiling point 251°C

Stability Stable under normal temperatures and pressures

Categories


Table 5: Physical and chemical properties Physical and chemical properties

Property Value




Density 1.26 g/cm3

Solubility soluble in water, slightly soluble in benzene, and ether

CAS number 504-24-5

Physical state off-white to white crystals

Melting point 158.9 °C

Boiling point 273.5°C

Stability Stable under recommended storage conditions, Store at

room temperature.

Categories potassium channel-blocking agent


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Pharmacodynamics

Dalfampridine is a board-spectrum lipophillic potassium channel blocker and

binds favourably to the open state than closed state of the potassium channel

in the CNS. Their pharmacological targets are the potassium channels exposed

in MS patients.

Mechanism of action

In MS, axons are progressively demyelinated which exposes potassium

channels. As a result, there is a leak of potassium ions which results in the

repolarization of cells and a decrease in neuronal excitability. The overall

impact is the impairment of neuromuscular transmission as it is harder to

trigger an action potential. Dalfampridine inhibits voltage-gated potassium

channels in the CNS to maintain the transmembrane potential and prolong

action potential. In other words, dalfampridine works to make sure that the

current available is high enough to stimulate conduction in demyelinated axons

that are exposed in MS patients. Furthermore, it facilitates neuromuscular and

synaptic transmission by relieving conduction blocks in demyelinated axons.

[5.0] Survey of analytical method/literature reviews

The literature reviews regarding pyridine and 2, 3 and 4-aminopyridine

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

pyridine and 2, 3 and 4-aminopyridine in pharmaceutical dosage forms are

reported. Most of the reported methods; either do not include stress

degradation studies or are not completely optimized and validated and they are

cumbersome, time-consuming and expensive. The literature reviews for analysis

of pyridine and 2, 3 and 4-aminopyridine are as under:

[1] D. Choquet, H. Korn et al, have studied the mechanism by which 4-

aminopyridine (4-AP) blocks the delayed rectifier type potassium (K+) channels

present on lipopolysaccharide-activated murine B lymphocytes was investigated

using whole-cell and single channel patch clamp recordings. 4-AP, was super

fused for 3-4 min before applying depolarizing pulses to activate the channel.

During the first pulse after application of 4-AP above 50 M , the current

inactivated faster, as compared with the control, but its peak was only reduced


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at high concentrations of 4-AP (Kd = 3.1 mM). During subsequent pulses, the

peak current was decreased (Kd = 120 mM), but the inactivation rate was slower

than in the control, a feature that could be explained by a slow unblocking

process. After washing out the drug, the current elicited by the first voltage step

was still markedly reduced, as compared with the control one, and displayed

very slow activation and inactivation kinetics; this suggests that the K+

channels move from a blocked to an unblocked state slowly during the

depolarizing pulse [69].

[2] SW. Casteel, BR. Thomas et al, have developed an analytical method to

measure 4-aminopyridine in tissues and urine to determine appropriate

diagnostic samples in acute poisoning cases. Tissues from rats dosed with 4-

aminopyridine were extracted with methylene chloride. Extracts were analyzed

by high performance liquid chromatography using an isocratic solvent system of

acetonitrile and aqueous solution (15 : 85 v/v) consisting of 0.015 M sodium

salt of 1-heptane-sulfonic acid, 0.002 M tetramethylammonium bromide, and

0.01 M sodium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid

[70].

[3] RF. Donnelly et al, have determined the chemical stability of 4-AP capsules

containing 10 mg of active ingredient. Ten-milligram capsules were prepared

from 4-AP obtained from 2 suppliers, with either lactose or microcrystalline.

The hard gelatin capsules were stored in amber snap top prescription vials at

room temperature (20°C to 25°C) with protection from light. Two capsules were

collected from each group on days 0, 14, 28, 62, 96, 125, 180, and 365 and

stored in a rubber stoppered glass test tube containing desiccant material at

70°C. On the day of analysis, solutions were prepared from the contents of the

capsules, which had been accurately weighed and appropriately diluted; the

solutions were assayed, in duplicate, by means of a stability indicating high-

pressure liquid chromatography assay. Ten-milligram capsules of 4-AP,

prepared from material obtained from each supplier and diluted with either

lactose or microcrystalline cellulose, retained at least 94 % of the initial content

for 365 days when stored in plastic prescription vials at room temperature with

protection from light. Extemporaneously prepared 10 mg capsules of 4-AP were


PART-[E] Page 234

considered stable for 365 days when stored in plastic prescription vials at room

temperature with protection from light [71].

[4] LA. Trissel, BS, RY. Zhang, BS Quanyun A. Xu et al, have studied the

chemical stability of 4 aminopyridine 5-mg capsules and 3,4-diaminopyridine 5-

mg capsules under a variety of storage conditions. Each of the two drug

preparations was extemporaneously prepared in hard gelatin capsules; lactose

and micronized silica gel were used as excipients. Samples were stored under

three conditions: refrigeration at 4°C and protected from light for 6 months,

protected from light at room temperature that ranged from 22°C to 24°C for 6

months, and at a temperature of 37°C and protected from light for 1 month.

The hard gelatin capsules remained clear and colorless, and the content

of the capsules remained an off-white powder when viewed under normal

fluorescent room light. Capsule content weight did not change during the study.

Both 4-aminopyridine and 3,4-diaminopyridine exhibited excellent chemical

stability under all study conditions. Little or no loss of drug content occurred in

either product under refrigeration, at room temperature, and even at the

elevated temperature of 37°C. The oral 5 mg capsules of 4-aminopyridine and

3,4-diaminopyridine did not undergo decomposition or other adverse changes

within 6 months at refrigerated or room temperature or within 1 month of

storage at 37°C [72].

[5] S. Goulay-Dufay, B. Do, MD. Le Hoang, JA. Raust, H. Graffard, F. Guyon,

D. Pradeau et al, have developed an analytical method based on high-

performance liquid chromatography with electrochemical detection (HPLC–EC)

whose purpose is to obtain first a sensitive method and second a satisfying

separation between 3,4-DAP and phenylephrine. The analytical method is

accurate, specific, and linear between 10 and 500 g of 3,4-DAP per litre. The

recovery of 3,4-DAP is estimated at 70.8 % with a 95 % confidence interval of

(66.0-75.6 %). Intermediate precision was evaluated on three quality control

samples; the intra-day precision was estimated at 13.5, 9.1, 7.8 % and the

inter-day precision at 17.9 %, 8.4 %, 9.3 %. The limit of quantification of the

method was evaluated at 10 gl−1. First toxico kinetic parameters determined on

dogs plasma samples after one 3,4-DAP oral administration of 1 mg kg−1 were:


PART-[E] Page 235

Cmax = 395.7 gl−1; Tmax = 15 min; t1/2 = 113.6 min; Clearance/F = 16.8 m kg−1

min−1 and Vd/F = 2.7 l kg−1 [73].

[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 pyridine and 2, 3 and 4-aminopyridine 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 pyridine

and 2, 3 and 4-aminopyridine 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 pyridine and 2, 3 and 4-

aminopyridine

4. Perform the validation for the developed method

[7.0] Experimental

[7.1] Materials

Pyridine and 2, 3 and 4-aminopyridine were sigma Aldrich. Orthophosphoric

acid was 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).

[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

1.64 g of sodium acetate was dissolved in 1000 mL high purity

demonized Milli-Q water [Millipore, Milli-Q, Bedford, MA, USA,


PART-[E] Page 236

purification system] and pH was adjusted 4.0 with HPLC grade glacial

acetic 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 450 mL acetate buffer, 350 mL

of methanol and 200 mL of acetonitrile [HPLC Grade]. The mixture was

sonicated in Expo-Hi Tech sonicator for 5 minutes

7.3.3 Preparation of standard and sample solution

The Standard stock solutions were prepared by accurately weighing 100

mg of each pyridine and 2, 3 and 4-aminopyridine in 100 mL volumetric

flask (1000 µg/mL) in methanol. Sample solutions were prepared by

appropriate dilution of the standard solutions with the diluent.

[8.0] Method development and optimization of chromatographic

Conditions/UV graph/chromatograms

To develop a precise, accurate and suitable RP-HPLC method for the

estimation of pyridine and 2, 3 and 4-aminopyridine different mobile phases,

solvent buffer ratios and pH were tried to proposed final chromatographic

conditions. The peak shapes, resolution and symmetry of pyridine and 2, 3 and

4-aminopyridine 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 pyridine and 2, 3 and 4-aminopyridine.

Optimized chromatographic conditions

Parameter Optimized condition

Flow rate 1.0 mL/min

Mobile phase 45 : 35 : 20 v/v (Buffer : MeOH : ACN)

Buffer Sodium acetate buffer pH 4.0 adjusted by Acetic acid

Wavelength 245 nm

Injection volume 5.0 µL

Run time 15 min

Column and column

oven temperature

30ºC


PART-[E] Page 237

[1] Pyridine + 2-Aminopyridine

Figure 1: Chromatogram and UV calibration curve for standard (Mobile

phase: 45 : 35 : 20 v/v, Buffer : MeOH : ACN).

Figure 2: Chromatogram and UV calibration curve for sample (Mobile phase:

45 : 35 : 20 v/v, Buffer : MeOH : ACN).


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Figure 10: Chromatogram and UV calibration curve for sample (Mobile





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[9.0] Analytical method development/validation and its result and

discussion

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 [74] and the US Food

and Drug Administration (FDA) [75].

[9.1] System suitability

System suitability was performed by using 100 µg/mL of pyridine and 2,

3 and 4-aminopyridine 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-6. The

system was deemed to be suitable for use if the capacity factors were in the

range of 2-20 (2 < K’ < 20), lower than 2 for tailing factor, more than 2 for

resolution (Rs), greater than 1650 number of theoretical plates (N), resolution

between pyridine and 2, 3 and 4-aminopyridine of at least two and less than 2

% relative standard deviation (% RSD) for peak area.

Table 6: System suitability parameters [Pyridine + 2-Aminopyridine]

Sr. No. Parameters Pyridine 2-Aminopyridine

1 Linearity range (µg/mL) 10.0-100.0 µg/mL 10.0-100.0 µg/mL

2 Retention time (Min.) 3.237 2.207

3 Theoretical plates (N) 1698 1650

4 Peak Asymmetry (T) 2.95 2.77

5 Resolution (Rs) 2.45 3.89

6 Accuracy 99.997 % 99.998 %

7 Precision 99.86 % 99.90 %

8 % RSD (For peak area) 0.060 % 0.096 %








6 Accuracy 99.995 % 99.997 %

7 Precision 99.90 % 99.91 %

8 % RSD (For peak area) 0.044 % 0.051 %


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6 Accuracy 100.002 % 99.992 %

7 Precision 99.91 % 99.85 %

8 % RSD (For peak area) 0.037 % 0.035 %

[9.2] Precision 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

salicylamide in the same equipment on the same day. Inter day precision was

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 deferasirox 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.


PART-[E] Page 247

Table 9: Intra-day and inter-day precision data for pyridine sample

Sample con. (µg/mL)

Intra day Inter day

Area (mAU*min)

Mean area

% SD % RSD

Area (mAU*min)

Mean area

% SD % RSD

100 28.493 28.477

0.022

0.077

28.492 28.474

0.021

0.073 28.498 28.499

28.457 28.448

28.449 28.457

28.489 28.475

500 39.545 39.562

0.024

0.060

39.543 39.557

0.024

0.060 39.548 39.558

39.598 39.598

39.545 39.535

39.578 39.549

1000 54.824 54.856

0.027

0.050

54.825 54.849

0.031

0.056 54.889 54.824

54.857 54.889

54.879 54.832

54.835 54.879

Table 10: Intra-day and inter-day precision data for 2-aminopyridine sample

Sample Con. (µg/mL)

Intra day Inter day

Area (mAU*min)

Mean area

% SD % RSD

Area (mAU*min)

Mean area

% SD % RSD

100 9.925 9.957

0.024

0.247

9.924 9.954

0.029

0.291 9.945 9.929

9.989 9.958

9.954 9.998

9.972 9.964

500 14.578 14.567

0.022

0.151

14.574 14.566

0.026

0.178 14.589 14.598

14.554 14.545

14.582 14.534

14.535 14.578

1000 18.572 18.568

0.018

0.096

18.571 18.571

0.021

0.113 18.589 18.579

18.559 18.589

18.541 18.535

18.579 18.583

[1] Calculation for Intra and inter day precision for pyridine

[1] % Assay =

P

AT = Average area of obtained in sample preparation

AS = Average area of obtained in standard preparation


PART-[E] Page 248

W1 = Weight taken of reference standard (mg)

W2 = Weight taken of test sample (mg)

P = Potency of reference standard (%)

[2]

Table 11: Intra-day and inter-day precision data for standard

Con. (µg/mL) Intraday [Area (mAU*min)] Inter day [Area (mAU*min)]

Pyridine 2-Aminopyridine Pyridine 2-Aminopyridine

1000 54.825 18.575 54.826 18.574

54.829 18.579 54.857 18.578

54.898 18.594 54.883 18.595

54.874 18.545 54.849 18.542

54.883 18.589 54.874 18.567

Average 54.8618 18.5764 54.858 18.571

% SD 0.03293 0.01913 0.0223 0.0192

% RSD 0.06003 0.10298 0.0406 0.1033

Standard potency 99.95 % 99.99 %

[1] Intra-day precision for pyridine

1 % Assay =

99.95

= 99.98 0.9993 99.95 = 99.86 %

2

=

= 0.060 %

[2] Inter day precision for Pyridine

1 % Assay =

99.90

= 0.9998 0.9996 99.95 = 99.90 %

2

=

= 0.056 %

[2] Calculation for Intra and inter day precision for 2-aminopyridine

[1] Intra-day precision for 2-aminopyridine

1 % Assay =

99.99

= 0.9995 0.9996 99.99 = 99.90 %

2

=

= 0.096 %

[2] Inter day precision for 2-aminopyridine

1 % Assay =

99.99

= 1.00 0.9995 99.99 = 99.94 %

2

=

= 0.0011 %


PART-[E] Page 249


Sample

Con.

(µg/mL)

Intra day Inter day

Area

(mAU*

min)

Mean

area

% SD % RSD Area

(mAU*

min)

Mean

area

% SD % RSD

100 27.33 27.338

0.032

0.119

27.32 27.304

0.037

0.136

27.38 27.35

27.35 27.25

27.29 27.31

27.34 27.29

500 34.548 34.554

0.035

0.102

34.547 34.564

0.036

0.106

34.598 34.599

34.578 34.577

34.505 34.589

34.545 34.508

1000 60.319 60.356

0.027

0.044

60.317 60.358

0.032

0.054

60.345 60.349

60.389 60.398

60.375 60.385

60.354 60.345

Table 13: Intra-day and inter-day precision data for sample and 3-amino pyridine standard Con.

(µg/mL)

Intraday [Area (mAU*min)] Inter day [Area (mAU*min)]


1000 60.318 105.48 60.319 105.47

60.376 105.49 60.354 105.58

60.398 105.58 60.378 105.45

60.358 105.59 60.398 105.55

60.345 105.54 60.345 105.45

Average 60.359 105.536 60.359 105.5

% SD 0.03036 0.0503 0.0304 0.0608

% RSD 0.05031 0.04766 0.0504 0.0577

Standard potency

99.95 % 99.98 %



1 % Assay =

99.95

= 0.9999 0.9996 99.95 = 99.90 %

2

=

= 0.044 %

[2] Inter day precision for pyridine

1 % Assay =

99.95

= 0.9999 0.9992 99.95 = 99.86 %


PART-[E] Page 250

2

=

= 0.053 %


[1] Intra-day precision for 3-aminopyridine

1 % Assay =

99.98

= 1.00 0.9993 99.98 = 99.91 %

2

=

= 0.051 %

[2] Inter day precision for 3-aminopyridine

1 % Assay =

99.98

= 1.00 0.9994 99.98 = 99.92 %

2

=

= 0.052 %



Intra day Inter day

Area (mAU*min)

Mean area

% SD % RSD Area (mAU*min)

Mean area

% SD % RSD

100 55.048 55.067

0.023

0.041

55.045 55.064

0.022

0.039 55.087 55.098

55.045 55.054

55.059 55.078

55.098 55.049

500 64.545 64.565

0.022

0.034

64.543 64.561

0.026

0.040 64.549 64.549

64.599 64.578

64.578 64.598

64.554 64.535

1000 105.46 105.556

0.054

0.051

105.45 105.54

0.055

0.052 105.57 105.54

105.59 105.59

105.57 105.54

105.59 105.58


PART-[E] Page 251



Intra day Inter day

Area (mAU*min)

Mean area

% SD % RSD

Area (mAU*min)

Mean area

% SD % RSD

100 30.643 30.667

0.024

0.078

30.643 30.664

0.023

0.075 30.649 30.648

30.659 30.655

30.698 30.699

30.688 30.678

500 46.595 46.572

0.026

0.055

46.595 46.564

0.026

0.055 46.578 46.545

46.555 46.558

46.535 46.589

46.598 46.535

1000 55.387 55.38

0.021

0.037

55.389 55.367

0.021

0.037 55.399 55.358

55.375 55.345

55.345 55.354

55.394 55.393



Intra day Inter day

Area (mAU*min)

Mean area

% SD % RSD

Area (mAU*min)

Mean area

% SD % RSD

100 47.361 47.366

0.021

0.044

47.359 47.373

0.021

0.044 47.398 47.389

47.378 47.398

47.354 47.374

47.343 47.345

500 64.329 64.357

0.025

0.038

64.328 64.358

0.028

0.043 64.335 64.343

64.389 64.398

64.375 64.345

64.359 64.378

1000 84.917 84.951

0.030

0.035

84.915 84.940

0.026

0.030 84.925 84.919

84.954 84.979

84.989 84.954

84.974 84.934


PART-[E] Page 252

Table 17: Intra-day and inter-day precision data for pyridine 4-

Aminopyridine

Con.

(µg/mL)

Intraday [Area (mAU*min)] Inter day [Area (mAU*min)]


1000 55.387 84.918 55.389 84.917

55.389 84.921 55.398 84.933

55.398 84.943 55.355 84.924

55.357 84.945 55.378 84.954

55.378 84.923 55.384 84.945

Average 55.3818 84.93 55.381 84.935

% SD 0.01558 0.01292 0.0162 0.0151

% RSD 0.02813 0.01521 0.0292 0.0177

Standard

potency

99.95 % 99.99 %



1. % Assay =

99.95

= 0.9999 0.9997 99.95 = 99.91 %

2.

=

= 0.037 %


1. % Assay =

99.95

= 0.9997 0.9990 99.95 = 99.82 %

2.

=

= 0.037 %



1 % Assay =

99.99

= 1.00 0.9986 99.99 = 99.85 %

2

=

= 0.035 %


1 % Assay =

99.99

= 1.00 0.9991 99.99 = 99.90 %

2

=

= 0.030 %


PART-[E] Page 253

[9.3] Limit of quantification and Limit of detection

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

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 pyridine and 2, 3 and 4-aminopyridine at concentration of

100 µg/mL. The elution peaks of pyridine and 2, 3 and 4-aminopyridine

presented in representative chromatograms shown in Figure 4. The

representative chromatogram for simultaneous determination of the studied

drugs in API pharmaceutical dosages forms.

Table 18: sample and standard area for sample and standard

Con.

(µg/mL)

Sample [Area (mAU*min)] Standard [Area (mAU*min)]


100 28.492 9.924 28.493 9.925

28.499 9.929 28.498 9.935

28.448 9.938 28.457 9.932

28.457 9.928 28.479 9.925

28.475 9.914 28.489 9.919

Average 28.4742 9.9266 28.4832 9.9272

% SD 0.021879 0.008706 0.01622 0.00634

% RSD 0.076839 0.087707 0.05696 0.06387

Standard

potency

99.95 % 99.99 %

[1] % Assay =

99.95

= 0.9996 0.9998 99.95 = 99.89 %

[2]

=

= 0.076 %


PART-[E] Page 254

[1] % Assay =

99.99

= 0.9999 0.9995 99.99 = 99.93 %

[2]

=

= 0.087 %

Table 19: sample and standard area

Con. (µg/mL)



100 27.32 55.045 27.33 55.048

27.29 55.098 27.35 55.087

27.25 55.054 27.35 55.045

27.31 55.078 27.29 55.059

27.29 55.049 27.34 55.058

Average 27.292 55.0648 27.332 55.0594

% SD 0.0268328 0.0225544 0.0249 0.016592

% RSD 0.0983175 0.0409597 0.091101 0.030135

Standard potency

99.95 % 99.98 %

[1] % Assay =

99.95

= 0.9985 0.9993 99.95 = 99.73 %

[2]

=

= 0.098 %

[1] % Assay =

99.98

= 1.00 0.9988 99.98 = 99.86 %

[2]

=

= 0.040 %

Table 20: sample and standard area

Con. (µg/mL)



100 30.643 47.359 30.643 47.361

30.648 47.389 30.649 47.398

30.655 47.398 30.659 47.378

30.699 47.374 30.67]8 47.354

30.678 47.345 30.688 47.343

Average 30.6646 47.373 30.6598 47.3668

% SD 0.023437 0.021575 0.01996 0.02158

% RSD 0.076431 0.045544 0.06509 0.04556

Standard potency

99.95 % 99.99 %

[1] % Assay =

99.95

= 1.00 0.9998 99.95 = 99.93 %


PART-[E] Page 255

[2]

=

= 0.076 %

[1] % Assay =

99.99

= 1.00 0.9996 99.99 = 99.95 %

[2]

=

= 0.045 %

[9.5] Linearity

The linearity of pyridine and 2, 3 and 4-aminopyridine were 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 water. In all cases, 10 µL 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.

Sr. No. Concentration (µg/mL) Area (mAU*min)

Pyridine 2-Aminopyridine

1 10 2.878 1.589

2 20 6.897 3.549

3 30 9.786 3.987

4 40 12.456 4.543

5 50 14.234 5.453

6 100 28.492 9.904

Table 21: Summary of linearity data Sr. No. Parameters Pyridine 2-Aminopyridine


2 Slope ± Standard error 0.9396 1.2156

3 Intercept ± Standard error 0.2764x 0.0869x

4 Linearity equation y = 0.2764x + 0.9396 y = 0.0869x + 1.2156

5 r2 0.9957 % 0.9831 %


PART-[E] Page 256

Figure 13: Linearity curve



PART-[E] Page 257



1 10 3.364 5.478

2 20 8.232 12.433

3 30 9.342 17.345

4 40 12.543 23.898

5 50 14.324 27.654

6 100 27.32 55.002

Table 22: Summary of linearity data






5 r2 0.9909 % 0.998 %



PART-[E] Page 258



PART-[E] Page 259



1 10 3.443 5.436

2 20 8.345 11.455

3 30 11.876 16.564

4 40 14.456 20.348

5 50 15.879 25.897

6 100 30.643 47.359

Table 23: Summary of linearity data






5 r2 0.9882 % 0.997 %



PART-[E] Page 260


[9.6] Accuracy

Accuracy of the assay method was calculated for pyridine and 2, 3 and 4-

aminopyridine by recovery studies at three concentrations of 50 %, 100 % 150

% and 200 % levels by standard addition method. The mean % recoveries for

pyridine and 2, 3 and 4-aminopyridine were found are given in below table 22.

Table 24: Accuracy data for pyridine sample and standard Pyridine sample area (mAU*min)

Standard area (mAU*min)

50% 100% 150% 200% 1000%

14.234 28.492 32.578 35.898 54.825

14.259 28.454 32.589 35.857 54.828

14.249 28.499 32.599 35.879 54.899

14.254 28.434 32.554 35.854 54.835

14.255 28.478 32.534 35.869 54.879

Average 14.2502 28.471 32.5708 35.8714 54.853

% SD 0.00973 0.0271 0.0265273 0.017897 0.0336

% RSD 0.06829 0.0951 0.0814452 0.049892 0.0613

Standard

potency

99.95 %


PART-[E] Page 261

Table 25: Accuracy data for pyridine

Sr. No.

Pyridine Added (µg/mL)

Found (µg/mL)

% Recovery

% Mean Recovery

SD % RSD

1 50% 25.945 25.949 100.01 99.996

0.013

0.013 25.999 25.994 99.98

25.976 25.976 100.00

25.989 25.985 99.98

25.989 25.987 99.99

2 100% 51.949 51.942 99.98 99.997

0.0108

0.0108 51.867 51.872 100.01

51.957 51.955 99.99

51.843 51.836 99.98

51.914 51.916 100.00

3 150% 59.399 59.391 99.98 99.994

0.010

0.010 59.414 59.411 99.99

59.435 59.429 99.99

59.341 59.347 100.01

59.319 59.311 99.98

4 200% 65.45 65.443 99.98 99.997

0.013

0.013 65.37 65.369 99.99

65.41 65.409 100.00

65.37 65.363 99.98

65.38 65.390 100.01

[1] Amount added (µg/mL) =

=

= 25.945 µg/mL

[2] Amount found (µg/mL) =

=

= 25.949 µg/mL

[3] % Recovery =

= 100.01 %

Table 26: Accuracy data for 2-Aminopyridine sample standard

2-aminopyridine sample area (mAU*min)


50% 100% 150% 200% 1000%

14.324 27.32 30.453 32.342 60.32

14.329 27.39 30.489 32.349 60.39

14.358 27.98 30.498 32.354 60.4

14.398 27.78 30.456 32.398 60.39

14.374 27.55 30.474 32.358 60.35

Average 14.3566 27.604 30.474 32.3602 60.37

% SD 0.031 0.2746 0.0197864 0.021959 0.033

% RSD 0.21591 0.9949 0.0649287 0.067858 0.055

Standard potency

99.95 %


PART-[E] Page 262

Table 27: Accuracy data for 2-Aminopyridine Sr.

No.

2-Amino

pyridine

Added

(µg/mL)

Found

(µg/mL)

%

Recovery

% Mean

Recovery

SD % RSD

1 50% 29.375 29.370 99.986 99.988

0.016

0.016

29.412 29.403 99.970

29.512 29.505 99.978

29.615 29.618 100.012

29.539 29.537 99.996

100% 53.508 53.501 99.986 99.998

0.010

0.010

53.735 53.732 99.995

53.802 53.802 100.001

53.612 53.619 100.014

53.785 53.781 99.992

150% 61.12 61.090 99.951 99.992

0.024

0.024

61.129 61.127 99.998

61.149 61.154 100.009

61.292 61.289 99.995

61.149 61.154 100.009

200% 69.47 69.465 99.995 100.00 0.013

0.013

69.46 69.476 100.025

69.36 69.363 100.00

69.23 69.234 100.010

69.34 69.331 99.991


=

= 29.375 µg/mL

[2] Amount found (µg/mL)

=

=

= 29.370 µg/mL

[3] % Recovery =

= 99.98 %

Table 28: Accuracy data for pyridine sample standard

Pyridine sample area (mAU*min)


50% 100% 150% 200% 1000%

50% 100% 150% 200% 60.32

14.324 27.32 30.453 32.342 60.39

14.329 27.39 30.489 32.349 60.4

14.358 27.98 30.498 32.354 60.39

14.398 27.78 30.456 32.398 60.35 Average 14.374 27.55 30.474 32.358 60.37 % SD 14.3566 27.604 30.474 32.3602 0.033 % RSD 0.0309968 0.274645 0.01978636 0.02195905 0.055 Standard potency

99.95 %


PART-[E] Page 263


Sr. No.

pyridine Added (µg/mL)

Found (µg/mL)

% Recovery

% Mean Recovery

SD % RSD

1 50% 23.729 23.727 99.994 100.026

0.043

0.043 23.732 23.736 100.017

23.789 23.784 99.979

23.83 23.850 100.085

23.798 23.810 100.053

2 100% 45.259 45.255 99.992 99.995

0.012

0.012 45.377 45.371 99.988

46.354 46.349 99.989

46.01 46.017 100.016

45.259 45.636 99.988

3 150% 50.449 50.445 99.993 99.996

0.025

0.025 50.512 50.505 99.986

50.53 50.520 99.980

50.43 50.450 100.040

50.49 50.480 99.980

4 200% 53.58 53.574 99.992 99.998

0.007

0.007 53.58 53.586 100.006

53.59 53.594 100.006

53.67 53.667 99.991

57.19 57.187 99.997


=

= 23.729 µg/mL


=

= 23.727 µg/mL

[3] % Recovery =

= 99.99 %

Table 30: Accuracy data for 3-aminopyridine sample standard

3-Aminopyridine sample area (mAU*min)


50% 100% 150% 200% 1000%

27.654 55.043 59.567 62.234 105.45

27.659 55.054 59.589 62.258 105.54

27.689 55.089 59.598 62.254 105.59

27.654 55.069 59.554 62.245 105.54

27.678 55.054 59.576 62.245 105.58

Average 27.6668 55.062 59.5768 62.2472 105.54

% SD 0.01635 0.0178 0.017427 0.009311 0.05523

% RSD 0.0591 0.0323 0.0292513 0.014959 0.05233

Standard potency


PART-[E] Page 264

Table 31: Accuracy data for 3-Aminopyridine Sr.

No.

3-Amino

pyridine

Added

(µg/mL)

Found

(µg/mL)

%

Recovery

% Mean

Recovery

SD % RSD

1 50% 26.208 26.202 99.978 99.998

0.021

0.021

26.203 26.207 100.015

26.239 26.235 99.986

26.195 26.202 100.028

26.229 26.225 99.952

2 100% 52.16 52.153 99.989 99.997

0.0108

0.0108

52.16 52.164 100.004

52.19 52.197 100.010

52.18 52.178 99.998

52.17 52.164 99.982

3 150% 56.45 56.440 99.982 99.998

0.011

0.011

56.465 56.461 99.993

56.467 56.469 100.004

56.429 56.427 99.998

56.442 56.448 100.011

4 200% 58.97 58.967 99.996 100.007

0.015

0.015

58.98 58.989 100.013

58.99 58.986 99.995

58.96 58.977 100.031

58.98 58.977 99.999


=

= 26.208 µg/mL


=

= 26.202 µg/mL

[3] % Recovery =

= 99.97 %

Table 32: Accuracy data for pyridine sample standard Pyridine sample area (mAU*min)

Standard area

(mAU*min)

50% 100% 150% 200% 1000%

15.879 30.643 34.678 41.897 55.389

15.859 30.689 34.685 41.899 55.359

15.856 30.654 34.654 41.887 55.345

15.877 30.656 34.678 41.854 55.353

15.845 30.698 34.698 41.834 55.397

Average 15.8632 30.668 34.6786 41.8742 55.3686

% SD 0.0144983 0.02401 0.0159937 0.0288218 0.023

% RSD 0.0913957 0.078291 0.0461199 0.0688296 0.04153

Standard

potency

99.95 %


PART-[E] Page 265


Sr. No.

Pyridine Added (µg/mL)

Found (µg/mL)

% Recovery

% Mean Recovery

SD % RSD

1 50% 28.679 28.678 99.997 100.017

0.046 0.046 28.647 28.642 99.983

28.632 28.636 100.017

28.677 28.674 99.992

28.589 28.616 100.097

2 100% 55.35 55.343 99.989 100.002

0.012 0.012 55.43 55.426 99.998

55.36 55.362 100.010

55.37 55.366 99.995

55.43 55.442 100.020

3 150% 62.61 62.630 100.032 100.003

0.017 0.017 62.65 62.643 99.989

62.589 62.587 99.997

62.584 62.587 100.005

62.669 62.666 99.996

4 200% 75.67 75.668 99.999 100.023

0.040

0.040 75.68 75.672 99.990

75.61 75.650 100.053

75.53 75.590 100.080

75.56 75.554 99.994


=

= 28.679 µg/mL


=

= 28.678 µg/mL

[3] % Recovery =

= 99.99 %

Table 34: Accuracy data for 4-aminopyridine sample standard

4-Aminopyridine sample area (mAU*min)


50% 100% 150% 200% 1000%

25.897 47.359 52.456 57.578 84.917

25.899 47.399 52.459 57.598 84.921

25.854 47.378 52.487 57.556 84.989

25.878 47.349 52.454 57.578 84.954

25.895 47.376 52.498 57.543 84.974

Average 25.8846 47.372 52.4708 57.5706 84.951

% SD 0.01903 0.0192 0.0202657 0.02142 0.0318

% RSD 0.07353 0.0406 0.0386229 0.037206 0.0374

Standard potency


PART-[E] Page 266

Table 35: Accuracy data for 4-aminopyridine

Sr. No.

4-Amino pyridine

Added (µg/mL)

Found (µg/mL)

% Recovery

% Mean Recovery

SD % RSD

1 50% 30.484 30.484 100.002 99.9947

0.016

0.016 30.492 30.497 100.018

30.439 30.434 99.983

30.465 30.462 99.991

30.489 30.482 99.977

2 100% 55.75 55.748 100.006 99.992

0.016

0.016 55.8 55.795 99.9940

55.79 55.770 99.965

55.73 55.736 100.005

55.78 55.768 99.988

3 150% 61.745 61.748 100.005 100.001

0.011

0.011 61.759 61.752 99.988

61.787 61.785 99.996

61.735 61.746 100.018

61.799 61.797 99.998

4 200% 67.779 67.777 99.998 100.000

0.004

0.004 67.805 67.801 99.994

67.749 67.751 100.004

67.779 67.777 99.998

67.732 67.736 100.006


=

= 30.484 µg/mL


=

= 30.484 µg/mL

[3] % Recovery =

= 100.00 %

[10.0] Summary and Conclusion

We have successfully developed a new simple RP-HPLC method for the

simultaneous estimation of pyridine with 2, 3 and 4-aminopyridine in bulk API

dosages forms using simple mobile phase buffer, methanol and acetonitrile. The

developed method was validated as per ICH guidelines and was found to be an

accurate, sensitive, economical and precise. The proposed method is rapid,

where the total analytical run time for all four APIs [pyridine (Rt = 3.237 min.)

and 2-aminopyridine (Rt = 2.207 min.), 3-aminopyridine (Rt = 3.240 min.) and


PART-[E] Page 267

4-aminopyridine (Rt = 2.073 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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PART-[E] Reverse phase high performance liquid ...shodhganga.inflibnet.ac.in/bitstream/10603/110204/18/18_part e.pdf · coordinated as chelate ligands to form a complex with a transition