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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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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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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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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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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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[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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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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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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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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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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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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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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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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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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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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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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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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(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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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
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