CHAPTER - III

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DEVELOPMENT AND VALIDATION OF RP - HPLC METHOD FOR THESIMULTANEOUS ESTIMATION OF DOXYLAMINE SUCCINATE,

PYRIDOXINE HYDROCHLORIDE AND FOLIC ACID IN COMBINEDDOSAGE FORM

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76

3. DETERMINATION OF DOXYLAMINE SUCCINATE, PYRIDOXINEHYDROCHLORIDE AND FOLIC ACID

3.1. PROFILE OF THE DRUGS

Doxylamine Succinate: Doxylamine is a member of the ethanolamine class of

antihistamines and has anti-allergy power superior to almost every other

antihistamine with the exception of diphenhydramine. Doxylamine is a Histamine

H1 antagonist [1] with pronounced sedative properties. It can be used by itself as

a short-term sedative and in combination with other drugs to provide night-time

allergy and cold relief. Doxylamine is also used in combination with the

analgesics paracetamol (acetaminophen) and Codeine as an analgesic/calmative

preparation, and is prescribed in combination with vitamin B6 (Pyridoxine) to

prevent morning sickness [2] in pregnant women. In USA, Doxylamine succinate

and Pyridoxine (Vitamin B6) are the ingredients of “Diclegis”, approved by the

FDA in April 2013 as the only drug for morning sickness with a class ‘A’ safety

rating for pregnancy.

NOCH3 N

CH2

CH3HO

OH

O

O

Fig 3.1.1: Structure of Doxylamine succinate

Chemical name : (RS)-N, N-dimethyl-2-(1-phenyl-1-pyridin-2-yl-ethoxy)-ethanamine,butanedioate(1:1).

Molecular formula : C17H22N2O · C4H6O4

Molecular weight : 388.46 g/mol

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77

Categories : Histamine H1 Antagonists, Antiemetics

Trade names : Unisom

Pyridoxine Hydrochloride: Vitamin B6, also called pyridoxine. It occurs in three

active forms-pyridoxol, pyridoxal and pyridoxamine, all of which undergo

reversible transformation in the body. Vitamin B6 assists in the balancing of

sodium and potassium as well as promoting red blood cell production. It takes

part in the nitrogen metabolism of linoleic and linolenic acid, neurotransmitters

(norepinephrine, dopamine, GABA, serotonin, histamine) and also

carbohydrates. It is linked to cardiovascular health by decreasing the formation of

homocysteine. Pyridoxine may help balance hormonal changes in women and

aid the immune system. Lack of pyridoxine may cause anemia, nerve damage

[3], seizures, skin problems, and sores in the mouth. It is highly required for the

production of the monoamine neurotransmitters serotonin, dopamine,

norepinephrine and epinephrine, as it is the precursor to pyridoxal phosphate:

cofactor for the enzyme aromatic amino acid decarboxylase. Pyridoxal

phosphate (PALP) takes part in the synthesis of aminolevulinic acid, the

precursor of heme porphyrin ring. Therefore, vitamin B6 deficiency may result in

anemia, as well as in neuropathy and depression [4]. This enzyme is responsible

for converting the precursors 5-hydroxytryptophan into serotonin and melatonin,

and levodopa into dopamine, noradrenaline and adrenaline. As such it has been

implicated in the treatment of depression and anxiety.

N

HO

HO

HOCH3

HCl

Fig 3.1.2: Structure of Pyridoxine hydrochloride

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78

Chemical name : 4,5-Bis(hydroxymethyl)-2-methylpyridin-3-olhydrochloride

Molecular formula : C8H12ClNO3

Molecular weight : 205.63852 g/mol

Categories : Vitamin B Complex,

Special dietary and nutritional additives

Trade names : Beesix,Hexa-Betalin and Becilan

Folic Acid: Folic acid part of the vitamin B group (vitamin B9) is a water soluble

vitamin. Folate is the general term including folic acid (pteroylglutamate, PteGln)

and poly-y-glutamyl conjugates with the biological activity of folic acid. It is one of

the most important coenzyme of the haemopoietic system that controls the

generation of ferrohaeme. Folic acid biological importance is due to

tetrahydrofolate and other derivatives after its conversion to dihydrofolic acid in

the liver [5]. Vitamin B9 (folic acid and folate) is essential to numerous bodily

functions. Folates are a group of ‘B’ vitamins required for the synthesis of DNA

and RNA. Health benefits of folates including prevention of neural tube defects,

coronary heart diseases and colon cancer [6] received the considerable attention

in recent years. Folates possess a diverse array of compounds that vary by

oxidation state of the pyridine ring structure, one-carbon moieties carried by

specific folate, and the number of conjugated glutamate residues on the folate.

These vitamins cofactors are essential for the synthesis of purines and

pyrimidines and in the production of methionine from homocysteine. Storage of

foods by freezing does not seem to affect the concentration of folate in spinach,

potatoes and broccoli. It is especially important in aiding rapid cell division and

growth, such as in infancy and pregnancy. Children and adults both require folic

acid to produce healthy red blood cells and prevent anemia. In 1996, the United

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79

States Food and Drug Administration published regulations requiring the addition

of folic acid to enriched breads, cereals, flours, corn meals, pastas, rice, and

other grain products.

HN

N N

NNH

NH

CO2H

H2N

O

CO2HO

Fig 3.1.3: Structure of Folic acid

Chemical name : (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-6-yl)

methyl]amino} phenyl)formamido]pentanedioic acid

Molecular formula : C19H19N7O6

Molecular weight : 441.3974 g/mol

Categories : Hematinics, Vitamin B Complex

Trade names : Folicet, Folvite, Folvron, Folacin etc.,

3.2. REVIEW ON ANALYTICAL METHODS OF DOXYLAMINE SUCCINATE,PYRIDOXINE HYDROCHLORIDE AND FOLIC ACID

Various analytical methods were reported in literature for the

determination of Doxylamine succinate, Pyridoxine hydrochloride and Folic acid

in pure drug, pharmaceutical dosage forms and in biological samples using High

performance liquid chromatography [7-31], Spectrophotometry [32-46], Ultra

performance liquid chromatography [47], LC-MS [48-49], Voltametric method

[50], Electrophoresis method [51], High performance thin layer chromatography

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80

[52], Chemometric methods [53-56], Fluorimetry [57] and Densitometric methods

[58-59] either in single or in combined forms.

Anant P. Argekar et al., [9] developed and validated simple, precise, and

rapid ion pair reversed-phase high-performance liquid chromatographic (RP-

HPLC) method for the simultaneous estimation of Pyridoxine hydrochloride

(PYR) and Doxylamine succinate (DOX) in tablets dosage form. The stationary

phase was a Microbondapak C18 column (10 μ, 300 mm x 3.9 mm). The mobile

phase was water:methanol (60: 40) containing 10 mM heptanes sulphonic acid

and 0.25% triethylamine and the pH was adjusted to 2.2 with orthophosphoric

acid buffer. Detection was carried out at 263 nm using an UV detector. The flow

rate was 1.0 mL/min, and retention times were 3.65 min and 7.32 min for PYR

and DOX, respectively. The linearities were in the concentration range

0.5-500 μg/mL for PYR and DOX. Mean percentage recoveries were 100.20%

and 101.20% for PYR and DOX respectively.

AMID@1 et al., [10] developed and validated simple and sensitive

reversed-phase, ion-pair HPLC method for the simultaneous determination of ‘B’

group vitamins, Thiamine chloride hydrochloride (B1), Nicotinamide (B3),

Pyridoxine hydrochloride (B6) and Folic acid in Pentovit coated tablets. The

cyanocobalamine (B12) was determined separately, because of its low

concentration in the multivitamin preparation. RP-HPLC analysis was performed

with methanol 5 mM heptanes sulphonic acid sodium salt and 0.1% triethylamine

TEA (25:75 v/v) at pH 2.8 as the mobile phase. For the determination of B12 a

methanol-water 22: 78 (v/v) as the mobile phase was used. The column effluents

were monitored at 290 nm for B1, B3, B6 and Folic acid, and at 550 nm for B12.

The obtained results and statistical parameters for all investigated vitamins of the

‘B’ group in Pentovit coated tablets were satisfactory and ranged from 90.4 % to

108.5%.

Novi Yantih et al., [13] developed and validated HPLC method for

determination of four vitamins viz., Thiamine hydrochloride, Riboflavin,

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81

Nicotinamide, and Pyridoxine hydrochloride in the multivitamin syrup. The

chromatographic separation was achieved by using a C18 column with dimension

of 3.9x300 mm and particle size of 10 μm. A mixture of methanol and 1% acetic

acid by using 7 mM 1-hexane sulphonic acid sodium salt 20: 80 (v/v) as mobile

phase with flow rate of 1 mL/min. The effluent was monitored at 280 nm. The

separation and quantification was achieved in less than 20 min.

P Jin et al., [14] developed and validated a simple, isocratic, and stability-

indicating high-performance liquid chromatographic method for the rapid

determination of thiamine (VB1), niacinamide (VB3), pyridoxine (VB6), ascorbic

acid (VC), pantothenic acid (VB5), riboflavin (VB2) and folic acid (VB9) in

Vitamins with Minerals Tablets (VMIT). An Alltima C18 column (250 mmx4.6 mm

and 5 μm) was used for the separation at ambient temperature, with 50 mM

ammonium dihydrogen phosphate (adjusting with phosphoric acid to pH 3.0) and

acetonitrile as the mobile phase at the flow rate of 0.5 mlmin. VB1, VB3, VB6, VC

and VB5 were extracted with a solution containing 0.05% phosphoric acid (v/v)

and 0.3% sodium thiosulphate (w/v) and simultaneously analyzed by the mobile

phase of phosphate buffer-acetonitrile 95:5 (v/v), while VB2 and VB9 were

extracted with a solution containing 0.5% ammonium hydroxide solution (v/v),

and were then simultaneously analyzed by using the mobile phase of phosphate

buffer-acetonitrile (85:15,v/v). The detection wavelengths were 275 nm for VB1,

VB3, VB6, VC, 210 nm for VB5, and 282 nm for VB2 and VB9. All the seven water-

soluble vitamins were separated from other ingredients and degradation

products. The developed method was reliable and convenient for the rapid

determination of VB1, VB3, VB6, VC, VB5, VB2 and VB9 in VMT.

Paulo Cesar Pires Rosa et al., [17] developed and validated a simple, fast,

reproducible and sensitive reversed phase HPLC method, using the stationary

phase containing embedded urea polar groups for the simultaneous

determination of Clobutinol hydrochloride (CLO) and Doxylamine succinate

(DOX) in syrups. The estimation was carried out on a C8 column (125 mm x 3.9

mm, 5 μm size). The mobile phase is the mixture of acetonitrile: methanol:

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82

phosphate buffer (pH 2.5) in the gradient mode. The diode array detector

operated at 230 nm for CLO and 262 nm for DOX. This method showed

adequate precision, with the RSD value less than 1%. The excipients did not

interfered in the results of the analysis. The analytical curves were linear (R²=

0.9999 for CLO and R²= 0.9998 for DOX) over the concentration range (2.4-336

μg/mL for CLO and 2.3-63 μg/mL for DOX). The solutions were stable for 72

hours at room temperature.

Kamble Reema et al., [21] developed and validated simple and sensitive

RP-HPLC method for the simultaneous estimation of ‘B’ group vitamins like

Pyridoxine hydrochloride (B6), folic acid (B9), Methylcobalamine (B12) and

Atorvastatin (ATO) in Atherochek Tablets. B12 was determined separately due to

its low concentration. The procedures for the determination of B6, Folic acid and

Atorvastatin carried out with the detection wavelength of 254 nm for Atorvastatin,

vitamin B6, Folic acid and 265 nm for Vitamin B12.

Chatzimichalakis PF et al., [27] developed and validated and HPLC

method for the simultaneous determination of seven water-soluble vitamis

(Thiamine, Riboflavin, Nicotinic acid, Nicotinamide, Pyridoxine, Cyanocobalamin,

and Folic acid) in multivitamin pharmaceutical formulations and biological fluids

blood serum and urine. Separation was performed at ambient temperature.

Gradient elution was started at a 99: 1 of A: B (v/v) composition, where A is 0.05

M CH3COONH4/CH3OH (99/1) and B is H2O/CH3OH (50/50), at a flow rate of 0.8

ml/min. After a 4 min isocratic elution the composition was changed to 100% of B

in 18 min and the elution was continued isocratically for 8 min. Detection was

performed with a photodiode array detector at 280 nm. Detection limits were in

the range of 1.6-3.4 ng per 20 µL injection, while linearity held up to 25 ng/µL.

Sample preparation of biological fluids was performed by SPE on

Supelclean LC-18 cartridges with methanol-water 85: 15 (v/v) as eluent.

Extraction recoveries were from biological matrices ranged from 84.6% - 103.0%.

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83

Suvarna Y et al., [28] developed and validated a simple

spectrophotometric method for simultaneous estimation of Pyridoxine

hydrochloride and Doxylamine succinate in tablet dosage form as per ICH

guidelines; this method is first order derivative spectroscopy. 10 μg/mL of each of

PYR and DOX were scanned in 200-400 nm range. The sampling wavelengths

were 231.8 nm for PYR where DOX showed zero crossing point and 253 nm for

DOX where PYR showed zero crossing point in first order derivative

spectroscopy. For this method, the linearity was observed in the concentration

range of 1-40 μg/mL for PYR and 2.5-80 μg/mL for DOX. LOD of DOX and PYR

were 0.2826 and 0.3571 μg/mL respectively. LOQ of DOX and PYR were 0.4621

and 0.5918 μg/mL respectively.

P. Giriraja et al., [29] developed and validated a Simple, precise, accurate

and economic method for the simultaneous estimation of Doxylamine succinote

and Pyridoxine hydrochloride in pure and pharmaceutical dosage form as per

ICH guidelines. This method measures the absorbance at the wavelengths of

260 nm and 291.2 nm. Calibration curves were found to be linear in the

concentration ranges of 10-100 μg/mL and 10-60 μg/mL with their correlation

coefficient (R²) values 0.9999 and 0.9996 for Doxylamine succinate and

Pyridoxine hydrochloride respectively. The LOD and LOQ of Doxylamine

succinate and Pyridoxine hydrochloride were found to be 0.3888 μg/mL,

0.1229 μg/mL and 1.1782 μg/mL, 0.3724 μg/mL respectively. Precision and

recovery studies were < 2 % of RSD.

Nayak et al., [33] developed and validated a simple, rapid UV

spectrophotometric method for the simultaneous determination of Pyridoxine

hydrochloride (PYR) and Doxylamine succinate (DOX) in bulk and tablet dosage

as per ICH guidelines. PYR and DOX showed absorption maxima at 290 nm and

260 nm respectively. Standard curves of both drugs obeyed Beer-Lambert’s law

in concentration range of 4-20 μg/mL. Simultaneous equations were developed

and validated for accuracy, linearity and precision. Marketed sample of tablets

was analyzed by using this method and yielded accurate results.

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84

Rajput SJ et al., [45] developed and validated three simple, rapid, and

accurate methods, i.e., the derivative ratio spectra-zero-crossing method

(method I), double divisor-ratio spectra derivative method (method II), and

column RP-HPLC method (method III) for the simultaneous determination of

Doxylamine succinate (DOX), Pyridoxine hydrochloride (PYR) and Folic acid

(FA) in their ternary mixtures and in tablets. In methods I and II, the calibration

graphs were linear in the range of 2.5-80, 1.0-40, and 1.0-30 μg/mL for DOX,

PYR and FA respectively. In the HPLC method, the separation of these

compounds was performed using mobile phase consisting of 0.05 M phosphate

buffer (pH 6.3)-methanol-acetonitrile 50: 20: 30, (v/v/v), and UV detection was

performed at 263 nm. Linearity was observed between the concentrations of the

analytes and peak areas [correlation coefficient (R²) = 0.9998] in the

concentration range of 1-200, 4-600, and 4.0-600 μg/mL for DOX, PYR, and FA,

respectively. The standard deviation of retention time in method III was

0.011, 0.015, and 0.016 for DOX, PYR and FA respectively. The precision

studies for all the three methods gave %RSD values of <2%.

Sadhna Rajput et al., [56] developed and validated the Simultaneous

estimation of Doxylamine succinate (DOX), Pyridoxine hydrochloride (PYR) and

Folic acid (FA) was carried out by UV spectrophometric assisted chemometric

methods. Four chemometric methods i.e. classical least square (CLS), inverse

least square (ILS), principal component regression(PCR) and partial least

squares (PLS) were applied to simultaneous assay of DOX,PYR and FA in

tablets without any chemical separation and any graphical treatment of the

overlapping spectra of three drugs. The chemometric calculations performed by

Chemometrics Toolbox 3.02 software (Kramer) along with MATLA B6. Mean

recoveries and the RSD of ILS, CLS, PCR, PLS methods were found to be

98.77/1.76, 100.59/1.53, 97.91/1.50, 97.53/1.73 for DOX; 99.79/1.22,

100.22/0.58, 100.31/1.68 and 99.33/1.10 for PYR; 99.79/1.37, 100.57/1.56 and

98.38/0.96 for FA respectively. These four Chemometric methods developed can

satisfactorily used for the quantitative analysis of multi-component dosage form.

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85

The literature survey revealed that there were some HPLC methods for

the simultaneous estimation of Doxylamine succinate, Pyridoxine hydrochloride

and Folic acid drugs in pharmaceutical formulations and only a few analytical

methods were reported for pharmaceutical preparations. Moreover, most of the

available methods are based on involvement of buffer which was not favourable

for column efficiency for longer usages. Keeping, in view of this an attempt was

made to develop a simple, precise and accurate RP-HPLC method for the

simultaneous estimation of Doxylamine succinate, Pyridoxine hydrochloride and

Folic acid in pharmaceutical dosage forms.

3.3. EXPERIMENTAL AND RESULTS

3.3.1. Materials and Methods

Equipment

The author had developed a liquid chromatographic in bulk samples and

pharmaceutical formulations. In this study PEAK 7000 isocratic HPLC with

rheodine manual sample injector with switch (77251) was employed and the

column used was thermo hypersil BDS C18 (250 mmx4.6 mm, particle size 5 µm)

column, Waters 2695 alliance with binary HPLC pump and a Waters 2998 PDA

detector. Waters Empower 2 software was used for monitoring chromatographic

analysis and data acquisition. Spectra lab DGA 20 A3 ultrasonic bath sonicator

was used for degassing the mobile phase. Electronic balance ELB 300 was used

for weighing the materials. The syringe used for injecting was 20 µL Hamilton

syringe. DIGISUN pH meter was used for all pH measurements.

Drugs:

The working standards of Doxylamine succinate, Pyridoxine hydrochloride

and Folic acid were provided as gift samples from Dr. Reddy’s Laboratories Ltd,

Hyderabad. Marketed formulation of combination was purchased from local

market.

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86

Chemicals and reagents:

Methanol - HPLC grade (Merck)

Sodium acetate

solution

- AR grade (Merck)

Water - Triple distilled water was prepared by

using Borosil Glass Distillation Unit.

Preparation of mobile phase:

The mobile phase composition used for elution was 0.01M sodium acetate

solution and methanol in the ratio of 600: 400 (v/v). It was prepared by diluting

400 mL methanol and 600 mL water (pH 5.2 adjusted with sodium acetate) in

one litre flask. It was filtered through 0.45 µ nylon membrane filter before use.

This mixture was also used as diluents for preparing working standard solutions

of the drug.

Preparation of stock and working standard solutions:

Pure standards of Doxylamine succinate, Pyridoxine hydrochloride and

Folic acid were used as external standards in the analysis. Different

concentrations of the standards were used based on the range required to plot a

suitable calibration curve.

A standard stock solution of 1 mg/mL of Doxylamine succinate, Pyridoxine

hydrochloride and folic acid were prepared separately used methanol as solvent.

In order to get the required ratio (4: 4: 1) of the drugs Doxylamine succinate,

Pyridoxine hydrochloride and Folic acid, appropriate quantities of respective

solutions of each drug were mixed and diluted with the mobile phase. The flask

containing standard solution was sonicated for 10 minutes to degas it. The

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87

standard solution was then filtered with 0.45 µm membrane filter paper. A series

of different dilutions (50-100 µg/mL) were prepared using above stock solution

with selected mobile phase and analyzed using the same chromatographic

conditions as those of the target compounds and a calibration curve was

generated.

Sample preparation:

Accurately weighed Quantity of sample powder equivalent to 10 mg of

Doxylamine succinate, 10 mg of Pyridoxine hydrochloride and 2.5 mg of Folic

acid was transferred into 100 mL of volumetric flask added 50mL of water and

sonicated for 30 mins and make up the volume with mobile phase and filtered

through the 0.45 µm membrane filter paper. 5 mL of the above solution is taken

into 25 mL volumetric flask make up the volume with mobile phase. An aliquot of

this solution was injected into HPLC system.

3.3.2. Method Development and Optimization of Chromatographic………………………………………………………………………………..Conditions

Inorder to develop the method, a study base line was recorded with the

optimized chromatographic conditions set for Doxylamine succinate, Pyridoxine

hydrochloride and Folic acid and stabilized for about 30 minutes. A non-polar

C18 column was chosen as the stationary phase for this study. The following

studies were carried for this purpose.

Mobile Phase:

For getting sharp peak and line separation of the components, successive

aliquots of the sample solution were recorded by the author until the

reproducibility of the peak areas were adequate.

For ideal separation of the drug isocratic conditions, mixtures of commonly

used solvents with or without different buffers in different combinations were

tested as mobile phases on C18 stationary phase. A mixture of 0.01 M sodium

acetate solution and methanol in the ratio of 600: 400 (v/v) was found to be the

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88

most suitable of all the combinations since the chromatographic peaks were

better defined, resolved and showed a low tailing factor of 1.622 for Doxylamine

succinate, 1.180 for Pyridoxine hydrochloride and 1.062 for Folic acid. The

analysis was carried at a flow rate of 1 mL/min. The injecting volume is 20 µL and

the total run time 7 minutes.

Detection Wavelength:

UV-spectrophotometer was used to record the spectra of diluted solution

of Doxylamine succinate, Pyridoxine hydrochloride and Folic acid in methanol.

The peaks of maximum absorbance wavelengths were observed. The spectra of

the Doxylamine succinate, Pyridoxine hydrochloride and Folic acid showed that a

balanced wavelength was found to be 247 nm.

Fig 3.3.2.1: Standard chromatogram for Doxylamine succinate andPyridoxine hydrochloride and Folic acid

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89

Fig 3.3.2.2: Formulation chromatogram for Doxylamine succinate , Pyridoxinehydrochloride and Folic acid

Retention time of Doxylamine succinate, Pyridoxine hydrochloride and Folic acid:

Under the above optimized conditions a retention of 1.469, 2.231 and

4.432 minutes were obtained for Doxylamine succinate, Pyridoxine hydrochloride

and Folic acid. A typical chromatograms showing the separation of Doxylamine

succinate, Pyridoxine hydrochloride and Folic acid was shown in Fig. 3.3.2.1 and

Fig. 3.3.2.2.

The following optimized chromatographic conditions mentioned in Table

3.3.2.1 were followed for the determination of Doxylamine succinate, Pyridoxine

hydrochloride and Folic acid in bulk samples and pharmaceutical formulations

after a detailed study of various parameters.

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90

Table 3.3.2.1: Optimized Chromatographic conditions

S.No Parameter Value

1 Column Inertsil- ODS C18 (250 mmx4.6 mm,particle size 5 µm)

2 Mobile phasewater (pH 5.2 adjusted with sodiumacetate) and methanol in the ratio of

600: 400( v/v)

3 Flow rate 1.0 mL/min

4 Diluent Mobile phase

5 Columntemperature 25°C

6 pH 5.2

7 APIConcentration

Doxylamine succinate- 20 µg/mLPyridoxine hydrochloride- 20 µg/mL

Folic acid- 5 µg/mL

8 Run time 6 min

9 Retention timeDoxylamine succinate-1.4 min,

Pyridoxine hydrochloride-2.2 min,Folic acid-4.4 min.

10 Volume ofinjection 10 µL

11 Detectionwave length 247 nm

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91

3.3.3. Validation of the Developed Method

The developed method was validated interms of different parameters like

linearity, specificity, precision, accuracy, LOD & LOQ in compliance with ICH [29]

guide lines.

Linearity:

External standard method was employed for the quantitative determination

of the drug. The mobile phase was filtered through 0.45 µ nylon membrane filter

before use. The flow rate of the mobile phase was adjusted to1.0 mL/min. Prior

to the injection of the drug solution, the column was equilibrated with the mobile

phase for atleast 30 min. The column temperature was maintained at 25±10c

throughout the study.

Linearity of the peak areas were determined by taking six replicate

measurements. Working dilutions were prepared by mixing standard solutions of

Doxylamine succinate in the range of 10-30 µg/mL, Pyridoxine hydrochloride in

the range of 10-30 µg/mL and Folic acid in the range of 2.5-7.05 µg/mL each in

different 10 mL volumetric flasks and diluted up to the mark column. The eluents

in the drug were monitored at 247 nm to obtain the corresponding

chromatograms. From the chromatograms, linearity plots were drawn individually

by taking concentration on x-axis and area of peaks on y-axis. The regression

values of the plots were computed by least squares method. The plot of peak

area versus the respective concentrations of Doxylamine succinate , Pyridoxine

hydrochloride and Folic acid were found to be linear in the concentration range of

10-30 µg/mL, 10-30 µg/mL and 2.5-7.05 µg/mL respectively. These regression

equations were later used to estimate Doxylamine succinate, Pyridoxine

hydrochloride and Folic acid in pharmaceutical dosage forms. The response of

the drug was found to be linear in the investigation concentration range and the

linear regression equation for Doxylamine succinate was y = 37490 x with

coefficient of correlation (R2) of 0.99(~1.0) (Fig.3.3.3.1), for Pyridoxine

hydrochloride was y = 61968x with coefficient of correlation (R2) of 0.99(~1.0)

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92

(Fig.3.3.3.2) and for Folic acid was y = 35929x with correlation coefficient (R2) of

0.99(~1.0) (Fig.3.3.3.3). Where x is the concentration in µg/mL and y is the peak

area in absorbance unit. The linearity data was reported in Table 3.3.3.1, Table

3.3.3.2 and Table 3.3.3.3, the linearity plots were shown in Fig.3.3.3.1, Fig.

3.3.3.2 and 3.3.3.3. The statistical parameters of the linearity plots were show in

Table 3.3.3.4. The overlay chromatograms of Linearity of Doxylamine succinate,

Pyridoxine hydrochloride and Folic acid shows in Fig 3.3.3.4. The results showed

that an excellent correlation exists between areas and concentration of drugs

within the concentration range indicated above.

Table 3.3.3.1: Linearity data of Doxylamine succinate

S.NoConcentration

(µg/mL) Peak area

1 10 1877189 Slope = 37490

C.C = 0.99(~1.0)

2 15.00 2812563

3 20.00 3747683

4 25 4688354

530 5621489

Table 3.3.3.2: Linearity data of Pyridoxine hydrochloride

S.NoConcentration

(µg/mL) Peak area

1 10 3099184

Slope = 61968

C.C = 0.99(~1.0)

2 15 4642641

3 20 6197340

4 25 7744800

530.00 9298119

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93

Table 3.3.3.3: Linearity data of Folic acid

S.NoConcentration

(µg/mL) Peakarea

1 2.5 1793210Slope = 35929

C.C = 0.99(~1.0)

2 3.75 2694269

3 5.00 3593031

4 6.25 4491038

5 7.5 5390636

Table 3.3.3.4: Regression characteristics of the Linearity plot of Doxylaminesuccinate, Pyridoxine hydrochloride and Folic acid

ParametersDoxylamineSuccinate

Pyridoxinehydrochloride Folic acid

CorrelationCoefficient 0.99 (~1.0) 0.99 (~1.0) 0.99 (~1.0)

RegressionEquation y = 37490 x y = 61968x y = 35929 x

Theoreticalplates 4124 5888 5640

Tailing 1.622 1.180 1.062

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94

Fig.3.3.3.1: Linearity Curve for Doxylamine succinate

Fig.3.3.3.2: Linearity Curve for Pyridoxine hydrochloride

y = 37490xR² = 0.99 (~1)

0

1000000

2000000

3000000

4000000

5000000

6000000

0 50 100 150 200

Series1

Linear (Series1)

y = 61968xR² = 0.99 (~1)

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

0 50 100 150 200

Series1

Linear (Series1)

Chapter-III

95

Fig.3.3.3.3: Linearity Curve for Folic acid

Fig. 3.3.3.4: Overlay chromatograms of Linearity for Doxylamine succinate,Pyridoxine hydrochloride and Folic acid.

y = 35929xR² = 0.99 (~1)

0

1000000

2000000

3000000

4000000

5000000

6000000

0 50 100 150 200

Series1

Linear (Series1)D

OXL

YAM

INE

SUC

CIN

ATE

- 1.4

67

PYR

IDO

XIN

E H

YDR

OC

HLO

RID

E - 2

.248

FOLI

C A

CID

- 4.

447

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

Chapter-III

96

Specificity:

The chromatograms obtained from the drug with most commonly used

interfering materials were compared with those obtained from the solution to

determine the specificity of the method. The interfering materials were mixed in

the mobile phase without the drug to prepare the blank solution. The interfering

materials used for the study were magnesium stearate, colloidal silicon dioxide,

starch, lactose, micro crystalline cellulose, ethyl cellulose and hydroxyl propyl

cellulose, which were commonly used in formulation. 0.45 µ membrane filter was

used to filter the mixtures before injection. In the chromatogram it was observed

that there are some impurity peak, however study runtime of the drugs indicated

the absence of interfering material peaks near the drug peaks. This indicates the

specificity of the proposed method.

Precision:

Precision of an analytical method is the degree of agreement among

individual test results when the method is applied repeatedly to multiple sampling

of homogeneous samples. The precision of the method, as intra-day repeatability

was evaluated by performing six independent assays of the test sample

preparation and the corresponding peak areas were recorded. It is expressed as

the standard deviation or the relative standard deviation. The intermediate

(inter-day) precision of the method was checked by performing same procedure

on different days by another person under the same experimental conditions.

Data obtained from precision experiments are given in Table 3.3.3.5 and

Table 3.3.3.6 for intraday and inter-day precision study respectively for

Doxylamine succinate, Pyridoxine hydrochloride and Folic acid. The %RSD

values for intraday precision study and inter-day precision study were less than

2% for Doxylamine succinate, Pyridoxine hydrochloride and Folic acid. Which

confirm that the developed RP-HPLC method was found to be precise.

Chapter-III

97

Table 3.3.3.5: Intra – day precision of Doxylamine succinate, Pyridoxinehydrochloride and Folic acid

Table 3.3.3.6: Inter – day precision of Doxylamine succinate, Pyridoxinehydrochloride and Folic acid

Accuracy:

Accuracy of the method was determind by performing recovery studies

using regular addition method. The recovery studies were carried out at three

concentration levels (50%, 100%and 150%). Each level was repeated six times.

For all the three drugs, recovery was performed in the same way. The

S.NoSampleWeight

(mg)

Doxylaminesuccinate

Pyridoxinehydrochloride Folic acid

% Assay(Doxylamine

succinate)

% Assay(Pyridoxine

hydrochloride)

%Assay(Folicacid)

1 482.20 3746397 6199675 3595004 99 100 99

2 482.20 3746266 6195760 3592678 99 100 99

3 482.20 3741869 6197389 3590231 99 100 99

4 482.20 3740761 6199444 3591778 99 100 99

5 482.20 3740569 6195273 3599453 99 100 99

6 482.20 3749990 6195000 3593793 99 100 99

AverageAssay: 99 100 99

STD 0.10 0.03 0.09

%RSD 0.10 0.03 0.09

S.NoSampleWeight

(mg)

Doxylaminesuccinate

Pyridoxinehydrochloride

Folicacid

% Assay(Doxylamine

succinate)

% Assay(Pyridoxine

hydrochloride)

%Assay(Folicacid)

1 482.20 3746548 6198328 3595456 99 100 99

2 482.20 3745985 6195284 3594689 99 100 99

3 482.20 3746892 6194983 3594867 99 100 99

4 482.20 3748657 6195749 3598746 99 100 99

5 482.20 3746829 6194862 3598743 99 100 99

6 482.20 3748219 6194168 3598749 99 100 99AverageAssay: 99 100 99

STD 0.03 0.02 0.06

%RSD 0.03 0.02 0.06

Chapter-III

98

percentage recovery and standard deviation of the percentage recovery were

calculated. A recovery of 99.9% which is almost equal to 100% (Table 3.3.3.7)

for Doxylamine succinate, 98.95% which is almost equal to 100% (Table 3.3.3.8)

for Pyridoxine hydrochloride and 99.8% which is almost equal to 100%

(Table 3.3.3.9) for Folic acid have been obtained by this method. The results

indicated good accuracy of the method for the determination of analysed drugs

as revealed by mean recovery data. Chromatograms obtained during accuracy

study were shown in Fig.3.3.3.5, Fig.3.3.3.6 & Fig.3.3.3.7.

Fig.3.3.3.5: Accuracy Chromatograms-50% of Doxylamine succinate, Pyridoxinehydrochloride and Folic acid

Chapter-III

99

Table 3.3.3.7: Accuracy data of Doxylamine succinate

SpikedLevel

SampleWeight(mg)

SampleArea

µg/mLadded

µg/mLfound % recovery mean

50% 241.50 1875005 9.910 9.92 100.10 (~100)

100.04(~100)

50% 241.50 1875013 9.910 9.92 100.10 (~100)

50% 241.50 1879474 9.910 9.94 100.30 (~100)

50% 241.50 1871012 9.910 9.90 99.89 (~100)

50% 241.50 1870158 9.910 9.89 99.79 (~100)

50% 241.50 1876423 9.910 9.92 100.10 (~100)

100% 483.00 3747599 19.820 19.82 100.00 (~100) 99.99(~100)100% 483.00 3745976 19.820 19.81 99.94 (~100)

100% 483.00 3749990 19.820 19.83 100.05 (~100)

150% 725.00 5626444 29.751 29.76 100.03 (~100)

99.99(~100)

150% 725.00 5623774 29.751 29.75 99.99 (~100)

150% 725.00 5620015 29.751 29.73 99.92 (~100)

150% 725.00 5624450 29.751 29.75 99.99 (~100)

150% 725.00 5626008 29.751 29.76 100.03 (~100)

150% 725.00 5626410 29.751 29.76 100.03 (~100)

Fig.3.3.3.6: Accuracy Chromatograms-100% of Doxylamine succinate,Pyridoxine hydrochloride and Folic acid

Chapter-III

100

Table 3.3.3.8: Accuracy data of Pyridoxine hydrochloride

SpikedLevel

SampleWeight(mg)

Sample Area µg/mLadded

µg/mLfound % recovery mean

50% 241.50 3099476 9.970 9.98 100.10 (~100)

100.03(~100)

50% 241.50 3097631 9.970 9.97 100.00(~100)

50% 241.50 3097853 9.970 9.97 100.00(~100)

50% 241.50 3099674 9.970 9.98 100.10 (~100)

50% 241.50 3099739 9.970 9.98 100.10 (~100)

50% 241.50 3092668 9.970 9.96 99.89 (~100)

100% 483.00 6199374.00 19.940 19.96 100.10 (~100)100.03(~100)100% 483.00 6193618.00 19.940 19.94 100.00(~100)

100% 483.00 6195000.00 19.940 19.94 100.00(~100)

150% 725.00 9297069 29.931 29.93 99.99 (~100)

99.99(~100)

150% 725.00 9295944 29.931 29.93 99.99 (~100)

150% 725.00 9293624 29.931 29.92 99.96 (~100)

150% 725.00 9299360 29.931 29.94 100.03 (~100)

150% 725.00 9299492 29.931 29.94 100.03 (~100)

150% 725.00 9293506 29.931 29.92 99.96 (~100)

Fig. 3.3.3.7: Accuracy Chromatograms-150% of Doxylamine succinate,Pyridoxine hydrochloride and Folic acid

Chapter-III

101

Table 3.3.3.9: Accuracy data of Folic acid

SpikedLevel

SampleWeight(mg)

SampleArea

µg/mLadded

µg/mLfound % recovery mean

50% 241.50 1796227 2.478 2.47 99.67 (~100)

99.80(~100)

50% 241.50 1797935 2.478 2.48 100.08 (~100)

50% 241.50 1794166 2.478 2.47 99.67 (~100)

50% 241.50 1798889 2.478 2.48 100.08 (~100)

50% 241.50 1796892 2.478 2.47 99.67 (~100)

50% 241.50 1793796 2.478 2.47 99.67 (~100)

100% 483.00 3595593 4.955 4.95 99.89 (~100) 99.89(~100)100% 483.00 3590965 4.955 4.95 99.89 (~100)

100% 483.00 3593793 4.955 4.95 99.89 (~100)

150% 725.00 5391597 7.438 7.43 99.89 (~100)

99.89(~100)

150% 725.00 5394134 7.438 7.43 99.89 (~100)

150% 725.00 5395568 7.438 7.43 99.89 (~100)

150% 725.00 5395112 7.438 7.43 99.89 (~100)

150% 725.00 5398058 7.438 7.43 99.89 (~100)

150% 725.00 5397639 7.438 7.43 99.89 (~100)

Limit of detection and Limit of Quantification (LOD&LOQ) study:

LOD is the smallest concentration of the analyte which gives a

measurable response. It is calculated by taking the concentration of the peak of

interest divided by three times the signal to noise ratio (s/n). LOQ is the smallest

concentration of the analyte, which gives response that can be absolutely

quantified. It is determined by analyzing samples containing known quantities of

the analyte and determining the lowest level at which acceptable degrees of

accuracy and precision are attainable.

The limit of detection and limit of quantification were evaluated by

serial dilutions of Doxylamine succinate, Pyridoxine hydrochloride and Folic acid

stock solutions by the proposed method in order to obtain signal to noise ratio of

3:1 for LOD and 10:1 for LOQ. The LOD value for Doxylamine succinate,

Pyridoxine hydrochloride and Folic acid were found to be 0.050, 0.0370 and

0.033 respectively and the LOQ value 0.167, 0.1233 and 0.109 respectively.

Chapter-III

102

Chromatograms of LOD and LOQ study were shown in Fig 3.3.3.8 &

Fig 3.3.3.9.

Fig 3.3.3.8: Chromatogram of LOD study of Doxylamine succinate andPyridoxine hydrochloride and Folic acid

Fig 3.3.3.9: Chromatogram of LOQ study of Doxylamine succinate andPyridoxine hydrochloride and Folic acid

Chapter-III

103

Table 3.3.3.10: LOD and LOQ of Doxylamine succinate

LOD 0.050

LOQ 0.167

Table 3.3.3.11: LOD and LOQ of Pyridoxine hydrochloride

LOD 0.0370

LOQ 0.1233

Table 3.3.3.12: LOD and LOQ of Folic acid

LOD 0.033

LOQ 0.109

Ruggedness and system suitability:

Ruggedness and system suitability were studied by injecting seven

replicates of working standard solution at six minutes interval. The %RSD was

calculated for the peak areas. %RSD <2 (data presented in Table 3.3.3.13,

Table 3.3.3.14 and Table 3.3.3.15.) establishes the reproducibility. The system

suitability parameters are show in Table 3.3.3.13, Table 3.3.3.14 and Table

3.3.3.15.

Chapter-III

104

Table 3.3.3.13: Ruggedness of Doxylamine succinate

Table 3.3.3.14: Ruggedness of Pyridoxine hydrochloride

Injection number Peak area

16197213

26196234

36183723

46173210

56163846

66153012

76140371

Mean 6172515.571SD 19955.0915

% RSD 0.32328

Injection number Peak area

13742362

23740123

33739472

43720912

53701634

63692345

73681204

Mean 3716864.5714SD 23370.9432

% RSD 0.62878

Chapter-III

105

Table3.3.3.15: Ruggedness of Folic acid

Injection number Peak area

13599456

23585012

33580835

43575031

53570237

63565023

73560284

Mean 3576554

SD 12272.3103

% RSD 0.343132

Robustness study:

Robustness of the method was studied by varying single condition in the

optimized chromatographic conditions such as mobile phase composition, pH,

column temperature, flow rate and wavelength at a time keeping all other

parameter constant. The effect of the above changes on system suitability

parameters like tailing factor, number of theoretical plates and on peak area were

studied. The results of the above parameter variation of Doxylamine succinate

(Table 3.3.3.16), Pyridoxine hydrochloride (Table 3.3.3.17) and Folic acid

(Table 3.3.3.18) were found to be within the acceptable limits. The result of

robustness study of the developed assay method was established in Table

3.3.3.16, Table 3.3.3.17 and Table 3.3.3.18. The result shown that during all

variance conditions, assay value of the test preparation solution was not affected

and it was in accordance with that of actual. System suitability parameters were

also found satisfactory; hence the analytical method would be concluded as

robust.

Chapter-III

106

Table 3.3.3.16: Robustness data of Doxylamine succinate

Table 3.3.3.17: Robustness data of Pyridoxine hydrochloride

Table 3.3.3.18: Robustness data of Folic acid

SNo

Samplename Change Name RT Area Tailing Plate

count

1 Flow1 0.2 mL/min(1.0-0.2)

Doxylaminesuccinate 1.827 4646829 1.524 2701

2 Flow2 0.2 mL/min(1.0+0.2)

Doxylaminesuccinate 1.294 2973403 1.541 2932

3 Temp1 5oC(25-5)

Doxylaminesuccinate 1.444 3674010 1.586 2788

4 Temp2 5oC(25+5)

Doxylaminesuccinate 1.439 3689369 1.580 2652

SNo

Samplename Change Name RT Area Tailing Plate

count

1 Flow1 0.2 mL/min(1.0-0.2)

Pyridoxinehydrochloride 2.817 7750541 1.321 3725

2 Flow2 0.2 mL/min(1.0+0.2)

Pyridoxinehydrochloride 1.950 5083429 1.484 3663

3 Temp1 5oC(25-5)

Pyridoxinehydrochloride 2.239 6066482 1.434 3426

4 Temp2 5oC(25+5)

Pyridoxinehydrochloride 2.229 6140867 1.462 2879

SNo

Samplename Change Name RT Area Tailing Plate

count

1 Flow1 0.2 mL/min(1.0-0.2) Folic acid 5.577 4537881 1.172 3525

2 Flow2 0.2 mL/min(1.0+0.2) Folic acid 4.205 2799044 1.420 3379

3 Temp1 5oC(25-5) Folic acid 4.443 3590236 1.331 2981

4 Temp2 5oC(25+5) Folic acid 4.300 3617127 1.363 2915

Chapter-III

107

Chromatogram obtain during robustness study were shown in Fig3.3.3.10. The conditions studied were flow rate (altered by ± 0.2 mL /min),column temperature (altered by ± 50C) and use of HPLC columns from differentbatches.

Fig 3.3.3.10: Chromatograms of Robustness study of Doxylamine succinate,Pyridoxine hydrochloride and Folic acid

Chapter-III

108

Estimation of the drug in formulation:

Accurately weighed Quantity of sample powder equivalent to 10 mg of

doxylamine succinate, 10 mg of pyridoxine hydrochloride and 2.5 mg of folic acid

was transferred into 100 mL of volumetric flask added 50 mL of water and

sonicated for 30 mins and make up the volume with mobile phase and filtered

through the 0.45 µm membrane filter paper. From the above solution, take 5mL

into 25 mL volumetric flask make up the volume with mobile phase. An aliquot of

this solution was injected into HPLC system. Peak areas of sample were

measured and compared against the peak areas of the standard solution. The

proposed method was able to estimate Doxylamine succinate, Pyridoxine

hydrochloride and Folic acid in the formulation with an accuracy of 99.9%

(~100%) for Doxylamine succinate, 98.95% (~100%) for Pyridoxine hydrochloride

and 99.8% (~100%) for Folic acid. The results were tabulated and the

percentage assay was reported in Table 3.3.3.19.

Table 3.3.3.19: Estimation of Doxylamine succinate, Pyridoxine hydrochlorideand Folic acid from its formulation

Formulation DosageSampleconc.µg/mL

SampleArea

AmountFoundµg/mL

%assay

Becilan

Doxylaminesuccinate

Pyridoxinehydrochloride

Folic acid

20

20

5

3747649.1

6197298.5

3593276.4

19.98

19.79

4.99

99.9

98.95

99.8

Chapter-III

109

3.4. SUMMARY OF THE RESULTS AND DISCUSSION

The overall results obtained for the proposed method validation were tabulated inTable 3.4.1.

Table 3.4.1: Summary of the proposed method validation

S.No Test parameter Result

1Linearity

(correlation coefficient)D – 0.99 (~1.0)P – 0.99 (~1.0)F - 0.99 (~1.0)

2

Precession(%RSD)

a)Intra-day

b)Inter-day

D – 0.10

P – 0.03

F – 0.09

D- 0.03

P- 0.02

F- 0.06

3Accuracy

(% drug substance)

D – 100.00

P - 100.016

F – 99.86

4 LOD(µg/mL)D – 0.050

P - 0.0370

F - 0.033

5 LOQ(µg/mL)

D – 0.167

P - 0.1233

F - 0.109

D - Doxylamine succinate, P - Pyridoxine hydrochloride, F - Folic acid

Chapter-III

110

To obtain suitable mobile phase combination of methanol and 1% sodium

acetate were tested for the analysis of the selected drug combination. Finally the

0.01 M sodium acetate solution and methanol in the ratio of 600: 400 (v/v) as

mobile phase was give symmetric peak at 247 nm in short runtime (6 min). The

pH was maintained at 5.2 and the chromatogram obtained for the mobile phase

has been showed good affinity towards Doxylamine succinate (Rt = 1.4 min and,

Pyridoxine hydrochloride (Rt = 2.2 min) instead of Folic acid (Rt = 4.4min) which

were similar to the earlier reported methods.

The literature survey on various HPLC methods available suggest that a

low tailing factor of 1.622 for Doxylamine succinate, 1.180 for Pyridoxine

hydrochloride and 1.062 for Folic acid were obtained by using a mixture of

0.01 M sodium acetate solution and methanol in the ratio of 600: 400 (v/v) as

mobile phase.

3.5. CONCLUSION

The statistical evaluation of the proposed method revealed its good

linearity, reproducibility and its validation for different parameters made us to

conclude that the current RP-HPLC method can successfully be used for reliable

determination of Doxylamine succinate, Pyridoxine hydrochloride and Folic acid

in pharmaceutical dosage from and also in bulk drug. The developed method

was found specific to the drug and for dosage from because no interfering

material peaks near the drug peak were observed in the chromatograms

obtained in the study runtime.

Chapter-III

111

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Chapter-III

115

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