164

7.1 Drug profile

7.1.1 Amitriptyline:

Amitriptyline is a tricyclic antidepressant (TCA). It is the most widely used

TCA and has at least equal efficacy against depression as the newer class of SSRIs

according to a study from early 2001(1). As well as reducing depressive symptoms,

these types of tricyclics also ease migraines, tension headaches, anxiety attacks and

some schizophrenic symptoms. It is also known to reduce aggression and violent

behavior.

Figure 7.a: Structure of Amitriptyline.

IUPAC Name : 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-

5- ylidene)-N,N-dimethylpropan-1-amine

Formula : C20H23N

Molecular Weight : 277.403 g/mol

Drug Bank accession number : DB00321 (APRD00227)

CAS number : 50-48-6

Half life : 10 – 50 hrs

Therapeutic category : Trixyclic anti depressant drug

Solubility : Soluble in Methanol, Slightly in Water

Route : Oral

Excretion : Renal

Solubility : Soluble in Methanol, Water and Acetonitrile

165

Amitriptyline is used for a number of medical conditions including: depressive

disorders, anxiety disorders, attention deficit hyperactivity disorder, migraine

prophylaxis, eating disorders, bipolar disorder, post-herpetic neuralgia, and insomnia (2). Amitriptyline is used in ankylosing spondylitis for pain relief. It is also used as a

preventive for patients with recurring biliary dyskinesia (sphincter of Oddi

dysfunction) (3). Amitriptyline is also used in the treatment of nocturnal enuresis

(bedwetting) in children.

Amitriptyline may be prescribed for other conditions such as cyclic vomiting

syndrome, post-traumatic stress disorder (PTSD)(4), chronic pain, tinnitus, chronic

cough, carpal tunnel syndrome (CTS), fibromyalgia, vulvodynia, interstitial cystitis,

male chronic pelvic pain syndrome, irritable bowel syndrome (IBS), diabetic

peripheral neuropathy, neurological pain, laryngeal sensory neuropathy, chronic

fatigue syndrome and painful paresthesias related to multiple sclerosis. Typically

lower dosages are required for pain modification of 10 to 50 mg daily (5).

Mechanism of action:

Amitriptyline acts primarily as a serotonin-norepinephrine reuptake inhibitor, with

strong actions on the serotonin transporter and moderate effects on the norepinephrine

transporter. It has negligible influence on the dopamine transporter and therefore does

not affect dopamine reuptake, being nearly 1,000 times weaker on it than

on serotonin.

Amitriptyline additionally functions as a 5-HT2A, 5-HT2C, 5-HT3, 5-HT6, 5-HT7, α1-

adrenergic, H1, H2, H4, and mACh receptor antagonist and σ1 receptor agonist. It has

also been shown to be a relatively weak NMDA receptor negative allosteric

modulator at the same binding site as phencyclidine. Amitriptyline inhibits sodium

channels, L-type calcium channels and Kv1.1,Kv7.2, and Kv7.3 voltage-gated

potassium channels, and therefore acts as a sodium, calcium, and potassium channel

blocker as well.

Recently, amitriptyline has been demonstrated to act as an agonist of the TrkA and TrkB

receptors. It promotes the heterodimerization of these proteins in the absence of NGF and

has potent neurotrophic activity both in-vivo and in-vitro in mouse mo dels. These are the

166

same receptors BDNF activate, an endogenous neurotrophin with powerful antidepressant

effects, and as such this property may contribute significantly to its therapeutic efficacy

against depression. Amitriptyline also acts as FIASMA (functional inhibitor of acid

sphingomyelinase).

Adverse effect:

The main two side effects that occur from taking amitriptyline are drowsiness

and a dry mouth. Other common side effects of using amitriptyline are mostly due to

its anti cholinergic activity, including: weight gain, changes in appetite, muscle

stiffness, nausea, constipation, nervousness, dizziness, tremor, blurred vision, urinary

retention, and changes in sexual function. Some rare side effects include seizures,

tinnitus, hypotension, mania, psychosis, sleep paralysis, hypnologic or hypnopompic

hallucinations related to sleep paralysis, heart block, arrhythmias, lip and mouth

ulcers, extra pyramidal symptoms, depression, tingling pain or numbness in the feet or

hands, yellowing of the eyes or skin, pain or difficulty passing urine, confusion,

abnormal production of milk in females, breast enlargement in both males and

females, fever with increased sweating, and suicidal thoughts(6).

7.1.2 Chlordiazepoxide:

Chlordiazepoxide, is a sedative/hypnotic drug and benzodiazepine(7).

Chlordiazepoxide was the first benzodiazepine to be synthesized and the discovery of

chlordiazepoxide was by pure chance (8). Chlordiazepoxide and other benzodiazepines

were initially accepted with widespread public approval but were followed with

widespread public disapproval and recommendations for more restrictive medical

guidelines for its use (9).

Figure 7.b: Structure of Chlordiazepoxide

167

IUPAC Name : 7-chloro-2-methylamino-5-phenyl-3H-1,4-

benzodiazepine-4-oxide

Formula : C16H14ClN3O

Molecular Weight : 299.755 g/mol

Drug Bank accession number : DB00475 (APRD00682) CAS number : 58-25-3

Half life : 5 – 30 hrs

Therapeutic category : Sedative / Hypnotic drug

Routes : Oral

Solubility : Soluble in Methanol, Slightly in Water

Route : Oral

Excretion : Renal

Chlordiazepoxide is indicated for the short term (2–4 weeks) treatment of

anxiety which is severe and disabling or subjecting the person to unacceptable

distress. It is also indicated as a treatment for the management of acute alcohol

withdrawal syndrome (10).

Mechanism of action:

Chlordiazepoxide acts on benzodiazepine subreceptors of the main GABAA receptor

and this results in an increased binding of the inhibitory neurotransmitter GABA to

the GABAA receptor thereby producing inhibitory effects on the central nervous

system and body similar to the effects of other benzodiazepines.Chlordiazepoxide

is anticonvulsant. There is preferential storage of chlordiazepoxide in some organs

including the heart of the neonate. Absorption by any administered route and the risk

of accumulation is significantly increased in the neonate. The withdrawal of

chlordiazepoxide during pregnancy and breast feeding is recommended, as

chlordiazepoxide rapidly crosses the placenta and also is excreted in breast milk.

Chlordiazepoxide also decreases prolactin release in rats. Benzodiazepines act

via micromolar benzodiazepine binding sites as Ca2+ channel blockers and

significantly inhibit depolarization-sensitive Calcium uptake in animal nerve terminal

preparations. Chlordiazepoxide inhibits acetylcholine release in mouse hippocampal

168

synaptosomes in vivo. This has been found by measuring sodium-dependent high

affinity choline uptake in vitro after pretreatment of the mice in vivo with

chlordiazepoxide.

Adverse effects:

Common side effects of chlordiazepoxide include, confusion, constipation,

drowsiness, fainting, altered sex drive, liver problems, lack of muscle coordination,

minor menstrual irregularities, nausea, skin rash or eruptions, swelling due to fluid

retention, yellow eyes and skin(11).

List of brand names of chlordiazepoxide and Amitriptyline:

S.NO Brand Name Molecule Label claim Manufacture

1 Amixide H Chlordiazepoxide 5mg Sun Pharma

Amitriptyline 12.5mg

Table 7.1: List of brand names of Chlordiazepoxide and Amitriptyline

169

7.2 Review of Literature

Very few analytical methods have been reported for the estimation of Chlordiazepoxide and Amitriptyline in different combination forms in pharmaceutical dosage forms. In our regular literature survey no analytical methods have been reported for the estimation of chlordiazepoxide and Amitriptyline in a single combined dosage forms using HPLC.

Kudo K et al (12) developed a selective, sensitive, and reliable method to determine concentrations of amitriptyline and its major metabolite, nortriptyline, in human plasma using high-performance liquid chromatography (HPLC) combined with UV and particle beam mass spectrometry (PBMS). Amitriptyline and nortriptyline were effectively extracted in a three-step solvent extraction procedure. Imipramine was used as the internal standard (IS). Amitriptyline, nortriptyline, and the IS were clearly separated by HPLC on a silica column with the mobile phase of acetonitrile: 0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear in the concentration range of 10-1000 ng/g for both compounds with UV and PBMS detections. The lower limits of detection were 5 ng/g for amitriptyline and 10 ng/g for nortriptyline with UV detection and 2 ng/g for amitriptyline and 5 ng/g for nortriptyline with PBMS detection. The absolute recoveries were 58% for amitriptyline and 47% for nortriptyline at a concentration of 50 ng/g. This method proved most useful in accurately identifying amitriptyline and nortriptyline in tissues from an autopsied individual.

Rafael LindenI et al (13) developed and validated a simple and sensitive HPLC-DAD method for the simultaneous determination of Amitriptyline, nortriptyline, E-10-hydroxyamitriptyline, Z-10-hydroxyamitriptyline, E-10-hydroxynortriptyline, Z-10-hydroxynortriptyline and desmethylnortriptyline in human plasma samples. The method employs a two-step liquid-liquid extraction and a reversed phase separation with isocratic elution. Precision assays showed %R.S.D lower than 2% and accuracy was in the range of 93.1 to 102.5%. Lowest Limit of detection was 5 ng/ml for all analytes. Metabolic ratios of amitriptyline demethylation were evaluated in individuals genotyped for CYP2C19, with clear differences between volunteers with zero or two active alleles. The method was suitable for therapeutic drug monitoring of patients under amitriptyline treatment, also allowing the indication of CYP2C19 activity.

170

Glenda K. Ferguson et al (14) developed a quantitative high-performance liquid chromatography (HPLC) laboratory experiment which entails the isocratic separation and simultaneous determination of the two active components of a commercial antipsychotic tablet has been developed. The prescription formulation used in this experiment contains amitriptyline hydrochloride (a tricyclic antidepressant) and perphenazine (a tranquilizer). The experiment makes use of a straightforward HPLC separation on a cyanopropyl-packed column with an Acetonitrile : methanol: aqueous monopotassium phosphate mobile phase pumped at a flow rate of 2.0 ml/min. Analytes were detected by UV absorbance at 215 nm. These conditions yield highly symmetrical and well-resolved peaks in less than 5 min after the injection of a mixture. In the experiment, students were given amitriptyline hydrochloride-perphenazine tablets without the manufacturer's labeled composition claim and a stock solution mixture with known concentrations of amitriptyline hydrochloride and perphenazine. They prepared four standards and a pharmaceutical sample of unknown concentration, assayed each solution in quadruplicate, and plot average peak areas of the concentrations of the known solutions in the construction of a standard curve. From the mathematical relationships that result, the average masses of amitriptyline hydrochloride and perphenazine in the prescription tablet were determined. Finally, the standard deviations of the mean masses were calculated. The entire laboratory procedure and statistical data analysis can be completed in a single 3-hour period.

GopalGarg et al (15) developed and validated three reliable, rapid and selective methods for the determination of imipramine and chlordiazepoxide in pharmaceutical dosage forms. The first method was spectrophotometric method includes simultaneous estimation method, first order derivative spectrophotometric method, isoabsorptive method and multi wavelength method. All variables affecting the reaction have been investigated and the conditions were optimized. The second method was based on separation of the cited drugs (imipramine Rf =0.45 and chlordiazepoxide Rf = 0.26) followed by densitometric measurement of the intact drug spots at 288 nm by HPTLC. The separation was carried on silica gel plates using toluene: ethyl acetate: ethanol: diethanolamine (70: 15: 4: 1 v/v/v/v) as a mobile phase. The linearity range was 4-10µg for chlordiazepoxide and 5-9µg for imipramine with mean accuracy 99.99+1.02%. The third method was accurate and sensitive stability-indicating HPLC method based on separation of imipramine and chlordiazepoxide on a reversed phase C18 column, using a mobile phase of methanol

171

at temperature 27+2 °C and UV detection at 275nm in an overall analysis time of about 9 min., based on peak area. The results obtained were analyzed by statistically to assess that no significant difference between each of the three methods. The validation was performed according to ICH guidelines.

Sejal K. Patel et al (16) determined a binary mixture of trifluoperazineHCl and chlordiazepoxide was using reversed-phase liquid chromatography method using methanol: water (97:03, v/v) pumped at a flow rate of 1.0 ml/min. Quantification was achieved with ultraviolet detection at 262 nm over concentration ranges of 0.1-1 and 0.5-5 μg/ml; mean accuracies were 101.05±0.47 and 98.97±0.33 %, respectively. The method was successively applied to tablet dosage forms as no chromatographic interferences from the tablet excipients were observed. The method retained its accuracy and precision when the standard addition technique was applied.

Haggag RS et al (17) developed and validated a simple, rapid, and selective RP-HPLC method with UV detection were developed for simultaneous determination of chlordiazepoxide hydrochloride and mebeverine hydrochloride (Mixture I) and carvedilol and hydrochlorothiazide (Mixture II). The chromatographic separation in both mixtures was achieved by using an RP-C8 (octylsilyl) analytical column. For Mixture I, a mobile phase composed of acetonitrile: 0.05 M disodium hydrogen phosphate; triethylamine (50: 50: 0.2, v/v/v), pH 2.5, was used; the detector wavelength was 247 nm. For Mixture II, the mobile phase consisted of acetonitrile: 0.05 M disodium hydrogen phosphate (50:50, v/v), pH 4.0, and the detector was set at 220 nm. Quantification of the analytes was based on measuring their peak areas. Both mixtures were resolved in less than 6 min. The reliability and analytical performance of the proposed HPLC procedures were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. The linear dynamic ranges were 2.5-150 and 2.5-500 µg /mL for chlordiazepoxide HCI and mebeverine HCI, respectively, and 0.25-200 and 0.25-150 µg /mL for carvedilol and hydrochlorothiazide, respectively. The validated HPLC methods were successfully applied to the analysis of their commercial tablet dosage forms, for which no interfering peaks were encountered from common pharmaceutical adjuvant.

172

7.3. Material and Methods

7.3.1 Instrumentation:

Chromatographic separation was performed on a PEAK chromatographic

system equipped with LC-P7000 isocratic pump; Rheodyne injector with 20μl fixed

volume loop, variable wavelength programmable UV detector UV7000 and the output

signal was monitored and integrated by PEAK Chromatographic Software version

1.06. Teccomp UV-2301 double beam UV-Visible spectrophotometer was used to

carry out spectral analysis and the data was recorded by Hitachi software. Sonicator

(1.5L), Ultrasonicator was used for sonicating the mobile phase and samples.

Standard and sample drugs were weighed by using Denver electronic analytical

balance (SI-234) and pH of the mobile phase was adjusted by using Systronics digital

pH meter.

7.3.2 Chemicals and Solvents:

The drug samples, Chlordiazepoxide and Amitriptyline working standard was

obtained as gift sample by Sun Pharma Limited, Sikkim. The pharmaceutical

formulation was procured from local market. Methanol, Acetonitrile and Water used

were HPLC grade and were purchased from Merck Specialties Private Limited,

Mumbai, India.

7.3.3 Preparation of standard stock solution:

Standard stock solution of Chlordiazepoxide and Amitriptyline pure drug

(1mg/ml) was prepared by accurately weighing about 10 mg of each drug in 10 ml

volumetric flask. The drugs were dissolved with few ml of methanol, and sonicated to

dissolve it completely and made up to the mark with the same solvent. The contents

were mixed well and filtered through Ultipor N66 Nylon 6, 6 membrane sample filter

paper. From this stock solution selected concentrations were prepared by selected

dilutions. 1ml from the selected concentrations of both the drugs were mixed and used

as a combined standard solution for the estimation in pharmaceutical formulations.

7.3.4 Preparation of Formulation Solution:

Marketed formulation of Chlordiazepoxide and Amitriptyline were purchased

(Amixide H: Chlordiazepoxide 5mg and Amitriptyline 12.5mg). From this 20 tablets

were powdered and the average weight of the powder was calculated. Form the

powder, an average weight of 10mg of Chlordiazepoxide was weighed and was

173

dissolved in little amount of methanol and mixed well and then make up to 10ml with

methanol. It was filtered through 0.45µm nylon membrane filter paper. This was

further diluted to 10µg/ml of Chlordiazepoxide by selected dilutions. Based on the

label claim of the two drugs, 10 µg/ml of Amitriptyline was obtained. This solution

was used for formulation estimation of Chlordiazepoxide and Amitriptyline in

combined dosage forms.

174

7.4. Method Development

For developing the method, a systematic study of the effect of various factors

was undertaken by varying one parameter at a time and keeping all other conditions

constant. Method development consists of selecting the appropriate wave length and

choice of stationary and mobile phases. The following studies were conducted for this

purpose.

7.4.1 Detection wavelength:

The spectrum of diluted solutions of the Chlordiazepoxide and Amitriptyline

in methanol was recorded. The absorption spectrum of Chlordiazepoxide and

Amitriptyline obtained by scanning the sample separately on UV spectrophotometer

in UV region (200-400nm) in spectrum mode showed that both drugs overlay at

222nm. Hence analysis was carried out by adjusting the UV detector of the HPLC

system at 222nm.

7.4.2 Choice of stationary phase:

Preliminary development trials have performed with octadecyl columns with

different types, configurations and from different manufacturers. Finally the expected

separation and shapes of peak was succeeded Analytical column Zodiac C-18 column

with 250 x 4.6mm internal diameter and 5µm particle size.

7.4.3 Selection of the mobile phase:

Based on the solubility of the two drugs Chlordiazepoxide and Amitriptyline,

different trails have been conducted for separation of the two compounds with high

resolution and obey the system suitability criteria. With a mixture of methanol and

acetonitrile in the ratio of 70:30 and the pH of the mobile phase was adjusted to 4.9

with TEA (Tri Ethyl Amine) was found to be most suitable condition.

7.4.4 Selection of mobile phase flow rate:

Flow rate of the mobile phase was changed at optimized mobile phase and

wavelength condition and the results obtained were compared. At a flow rate of

0.9ml/min was found to be most suitable for the separation of Chlordiazepoxide and

Amitriptyline. In this condition, both the compounds were separated with high

resolution factor and all the parameters under the system suitable criteria. Hence

0.9ml/min was selected as an optimized flow rate.

175

7.4.5 Optimized chromatographic conditions:

After completion of systemic trails, the optimized conditions were validated

for the acceptance of the developed method. The optimized conditions were shown in

the table 7.2. Chromatogram of standards, blank and formulaton were shown in figure

7. c to 7.g.

Parameter Condition

Standard Concentration Chlordiazepoxide 10µg/ml

Amitriptyline 25µg/ml

Pump mode Isocratic

Mobile phase Methanol: Acetonitrile 70:30 (v/v)

Mobile Phase PH 4.9 with TEA

Wavelength 222nm

Column Zodiac C-18 column (250 X 4.6 mm, 5μ)

Column Temp Ambient

Diluent Mobile Phase

Injector Rheodyne

Injection Volume 20μl

Flow rate 0.9ml/min

Retention Time Chlordiazepoxide 4.94min

Amitriptyline 7.53min

Run time 12min

Peak Area Chlordiazepoxide 242395

Amitriptyline 610584

Theoretical plates Chlordiazepoxide 3047

Amitriptyline 4876

Tailing Factor Chlordiazepoxide 1.13

Amitriptyline 1.77

Pump Pressure 11.5±5MPa

Table 7.2: Optimized chromatographic conditions of Chlordiazepoxide and

Amitriptyline

176

Figure 7.c: Chromatogram of Blank

177

Figure7.d: Chromatogram of Chlordiazepoxide (10µg/ml) and Amitriptyline

(25µg/ml)

178

Figure 7.e: Standard Chromatogram of Chlordiazepoxide

179

Figure 7.f: Standard Chromatogram of Amitriptyline

180

Figure 7.g: Chromatogram of Chlordiazepoxide and Amitriptyline Formulation

181

7.5 Method Validation

The proposed method was validated as per ICH guidelines. The parameters studied

for validation were specificity, linearity, precision, accuracy, robustness, and system

suitability, limit of detection and limit of quantification.

7.5.1 System suitability:

The proposed method was tested for System suitabilitycriteria. System

suitability tests were carried out on freshly prepared standard stock solution of

Chlordiazepoxide and Amitriptyline. From the prepared solution 20μl of the sample

was injected into HPLC system and the results obtained were used to express the

system suitability of the developed method. System suitability results were shown in

Table 7.3.

Retention Time

Chlordiazepoxide 4.94min

Amitriptyline 7.53min

Peak Area

Chlordiazepoxide 242395

Amitriptyline 610584

Theoretical plates

Chlordiazepoxide 3047

Amitriptyline 4876

Tailing Factor

Chlordiazepoxide 1.13

Amitriptyline 1.77

Table 7.3: System suitability results of Chlordiazepoxide and Amitriptyline

7.5.2 Specificity:

The selectivity of an analytical method is its ability to measure accurately and

specifically the analyte of interest in the presence of components that may be

expected to be present in the sample matrix. If an analytical procedure is able to

separate and resolve the various components of a mixture and detect the analyte

qualitatively the method is called selective. It has been observed that there were no

peaks of diluents and placebo at main peaks. Hence the chromatographic system used

for the estimation of Chlordiazepoxide and Amitriptyline was very selective and

specific. Specificity studies indicating that the excipients did not interfere with the

analysis. Standard solution show symmetric peak with retention time of 4.94min for

Chlordiazepoxide and 7.53min for Amitriptyline. The chromatogram showed neat

182

base line, without any interference of excipients and high resolution of the separated

compounds was observed. This indicates that the proposed method was specific.

Name of the solution Retention Time in Min

Blank No peaks

Chlordiazepoxide 4.94

Amitriptyline 7.53

Table 7.4: Specificity results of Chlordiazepoxide and Amitriptyline

7.5.3 Linearity:

Calibration curve was stabled by preparing six different concentrations of

Chlordiazepoxide and Amitriptyline based on the label claim of the formulation

dosage form. Chlordiazepoxide of 5-30µg/ml and Amitriptyline of 12.5-75µg/ml

solutions were mixed, from this 20 µl of the sample was injected in to HPLC system.

Peak area responses of the corresponding standards were used in constructing the

calibration curve. It was found that the solutions shown good argument in the linear

graph with regression equation of y = 24447x + 34475 (r²=0.999) for Amitriptyline

and y = 23396x+5711.8(r² = 0.9997) for Chlordiazepoxide. Results were shown in

table 7.5 and Standard graphs were shown in figure 7.h and 7.i for Chlordiazepoxide

and Amitriptyline respectively.

S.No Chlordiazepoxide Amitriptyline

Concentration(µg/ml) Peak Area Concentration(µg/ml) Peak Area

1 5 129365 12.5 365715

2 10 242395 25 628621

3 15 351129 37.5 992583

4 20 479362 50 1263210

5 25 585726 62.5 1572163

6 30 708582 75 1836284

Result:

CC: 0.9997

Slope: 23396

Intercept: 5711.8

CC: 0.9990

Slope: 24447

Intercept: 34475

Table 7.5: Linearity results of Chlordiazepoxide and Amitriptyline

183

Figure 7.h: Linearity graph of Chlordiazepoxide

Figure 7.i: Linearity Graph of Amitriptyline

7.5.4 Accuracy:

Accuracy of the method was determined by standard addition method. A

known amount of standard drug was added to the fixed amount of pre-analyzed tablet

solution. Percent recovery was calculated by comparing the area before and after the

addition of the standard drug. The standard addition method was performed at 50%,

100%and 150% levels of standard concentration for both Chlordiazepoxide and

Amitriptyline. The solutions were analyzed in triplicate at each level as per the

proposed method. The percent recovery and % RSD was calculated and results are

184

presented in Table 7.6 and 7.7 for Chlordiazepoxide and Amitriptyline respectively.

% recovery and %RSD of the recovery were under the acceptance criteria. The values

of recovery justify the accuracy of the method. The % recovery values were obtained

within the standard limit which confirms that the method was accurate and free from

any positive or negative interference of the excipients. This indicates that the

proposed method was accurate.

Sample

Concentration in µg/ml Chlordiazepoxide Target con.,

Spiked conc.,

Final Conc.,

Con, Recovered

% of Recovery

Mean

%RSD

50%

10 5 15 14.86 99.0667 98.82

0.19

10 5 15 14.79 98.6 10 5 15 14.82 98.8

100%

10 10 20 19.86 99.3 98.64

0.31

10 10 20 19.77 98.85 10 10 20 19.71 98.55

150%

10 15 25 24.63 98.52 100.55

1.43

10 15 25 25.42 101.68 10 15 25 25.36 101.44

Table 7.6: Recovery results of Chlordiazepoxide

Sample

Concentration in µg/ml Amitriptyline Target con.,

Spiked conc.,

Final Conc.,

Con, Recovered

% of Recovery

Mean

%RSD

50%

25 12.5 37.5 37.41 99.76 99.57

0.14

25 12.5 37.5 37.32 99.52 25 12.5 37.5 37.29 99.44

100%

25 25 50 50.26 100.52

100.79

0.21

25 25 50 50.52 101.04 25 25 50 50.41 100.82

150%

25 37.5 62.5 62.05 99.28 100.42

0.82

25 37.5 62.5 62.98 100.768 25 37.5 62.5 63.25 101.2

Table 7.7: Recovery results of Amitriptyline

185

7.5.5 Precision:

Precision is the measure of how close the data values to each other for a

number of measurements under the same analytical conditions. Precision of the

method was determined by performing interday variation, intraday variation and

repeatability studies.

Intraday Precision:

Six replicate injections of the specific standard at various time intervals on the

same day were injected into the chromatograph and the value of % RSD calculated. %

RSD was found to be 0.20 for Chlordiazepoxide and 0.15 for Amitriptyline which

was found to be within the limits of acceptance. Results of Intraday precision were

shown in table 7.8 and 7.9 for Chlordiazepoxide and Amitriptyline respectively.

Sample

Conc. (in µg/ml)

Injection No.

Peak Areas

Result

Chlordiazepoxide

10µg/ml

1 242395 SD: 494.718

Mean: 242710

% RSD : 0.20

2 243625 3 242854 4 242162 5 242921 6 242302

Table 7.8: Intraday precision results of Chlordiazepoxide

Sample

Conc. (in µg/ml)

Injection No.

Peak Areas Result

Amitriptyline

25µg/ml

1 628621 SD: 913.304

Mean: 627724

% RSD :0.15

2 627152 3 627092 4 626369 5 628192 6 628915

Table 7.9: Intraday precision results of Amitriptyline

186

Interday precision:

Standard concentrations of Chlordiazepoxide and Amitriptyline were prepared

six times in three different days. From this 20µl of the sample was prepared and

injected into HPLC system. Peak area responses of the prepared concentrations were

taken and %RSD was calculated. % RSD was found to be 1.805 for Chlordiazepoxide

and 0.15 for Amitriptyline which is found to be within the limits of acceptance.

Results of Intraday precision were shown in table 7.10 and 7.11 for Chlordiazepoxide

and Amitriptyline respectively.

Sample

Conc. (in µg/ml)

Injection No.

Peak Areas Result

Chlordiazepoxide

10µg/ml

1 243962 SD: 4385.41

Mean: 242927.5

% RSD :1.805

2 239625 3 237152 4 249936 5 240251 6 246639

Table 7.10: Interday precision results of Chlordiazepoxide

Sample

Conc. (in µg/ml)

Injection No.

Peak Areas Result

Amitriptyline

25µg/ml

1 619632 SD: 6088.48

Mean: 620347

% RSD :0.98

2 612139 3 617745 4 626032 5 616253 6 630281

Table 7.11: Interday precision results of Amitriptyline

187

7.5.6. Robustness:

The evaluation of robustness should be considered during the development

phase and depends on the type of procedure under study. It should show the reliability

of an analysis with respect to deliberate variations in method parameters. Robustness

test was carried out by small variation in the chromatographic conditions and %

change in the results was calculated. Here robustness was performed by change in

mobile phase ratio, mobile phase pH and wavelength of the detector. Standard

solution was analyzed under these changed experimental conditions. % change in the

results was calculated and was found to be within the acceptance criteria of bellow 2.

This indicates that the proposed method is valid. Results were shown in table 7.12.

S.NO

PARAMETER

CONDITION

Chlordiazepoxide Amitriptyline

AREA % OF CHANGE

AREA % OF CHANGE

1 Standard Standard 242395 0 628621 0

2 Mobile phase 75:25 241215 0.49 627153 0.23 60:35 243025 0.26 626321 0.36

3 Mobile phase pH

4.8 246631 1.75 621525 1.13 5.0 242863 0.19 619863 1.39

4 Wavelength 218nm 240253 0.88 617296 1.80 228nm 240193 0.91 636215 1.21

Table 7.12: Robustness results of Amitriptyline and Chlordiazepoxide

7.5.7 Ruggedness:

Inter day variations were performed by using six replicate injections of

standard solution which were prepared and analyzed by different analyst on three

different days over a period of one week. The percent relative standard deviation (%

RSD) was calculated and it was found to be 1.31 for Chlordiazepoxide and 0.43 for

Amitriptyline, which are well within the acceptable criteria of not more than 2.0. It

was concluded that the analytical technique showed good repeatability. Results of

system precision studies were shown in Table 7.13 and 7.14.

188

Sample

Conc. (µg/ml)

Injection No.

Peak Areas Result

Chlordiazepoxide

10µg/ml

1 240963 SD: 3205.91

Mean: 245119

% RSD :1.31

2 241190 3 245930 4 249961 5 247309 6 245362

Table 7.13: Ruggedness results of Chlordiazepoxide

Sample

Conc. (in

µg/ml)

Injection No.

Peak Areas Result

Amitriptyline

25µg/ml

1 632963 SD: 2733.49

Mean: 633177

% RSD :0.43

2 631829 3 633235 4 630921 5 638982 6 631133

Table 7.14: Ruggedness results of Amitriptyline

7.5.8 Limit of Detection:

Determination of the signal-to-noise ratio is performed by comparing

measured signals from samples with known low concentrations of analyte with those

of blank samples and establishing the minimum concentration at which the analyte

can be reliably detected. A signal-to-noise ratio 2:1 is generally considered acceptable

for estimating the detection limit. LOD value was found to be 0.005µg/ml and

0.025µg/ml for Chlordiazepoxide and Amitriptyline respectively.

189

7.5.9 Limit of Quantification:

The limit of quantification is generally determined by the analysis of samples

with known concentrations of analyte and by establishing the minimum level at which

the analyte can be quantified with acceptable accuracy and precision. LOQ value was

found to be 0.016µg/ml and 0.0.08µg/ml for Chlordiazepoxide and Amitriptyline

respectively.

7.5.10. Formulation:

The prepared formulation solution was injected into HPLC system. Peak area

response was compared with the standard values and the % assay was calculated and

it was found that more than 98% assay was obtained for both Chlordiazepoxide and

Amitriptyline.

S.No Drug Brand

Name

Lable

claim

Amount

prepared

Amount

found

%Assay

1 Chlordiazepoxide Amixide-

H

5mg 10µg/ml 9.947µg/ml 99.47

2 Amitriptylin 12.5mg 25µg/ml 24.76µg/ml 99.05

Table 7.15: Formulation results of Chlordiazepoxide and Amitriptyline

190

7.6. Results & Discussion

The development of an analytical method for the determination of drugs by

HPLC has received considerable attention in recent years because of their importance

in quality control of drug products. The objective of this study was to develop a rapid

and sensitive HPLC method for estimation of Chlordiazepoxide and Amitriptyline in

tablet formulations using the most commonly employed RP C-18 column with UV

detection. The mobile phase was optimized with Methanol: Acetonitrile 70:30 (v/v).

From the overlain spectrum of Chlordiazepoxide and Amitriptyline, wavelength was

selected, at 222nm, isoabsorptive point for both the drugs. Good resolution was

carried out at 222nm and both drugs showed good absorbance at this wavelength with

minimum interference of the other drug. Standard and blank chromatograms were

shown in figures 7.c to 7.g. All parameters of these proposed method was validated as

per the ICH guidelines.

No peak was detected at the retention time of Chlordiazepoxide and

Amitriptyline, hence proving the specificity of the method. A linearity experiment

shows correlation coefficient for Chlordiazepoxide and Amitriptyline is 0.9997 and

0.9990 respectively over a range of 5-30μg/ml for Chlordiazepoxide and 12.5-

75μg/ml for Amitriptyline. The regression of Chlordiazepoxide and Amitriptyline in

concentrations over its peak area were found to be y = 24447x + 34475 (r²=0.999) for

Amitriptyline and y = 23396x+5711.8(r² = 0.9997) for Chlordiazepoxide. Results

were shown in table 7.5 and Standard graphs were shown in figure 7.h and 7.i for

Chlordiazepoxide and Amitriptyline respectively. The regression equation was used to

estimate the amount of Chlordiazepoxide and Amitriptyline, either in tablet

formulations or in validation study.

The accuracy of the method was established using recovery technique i.e. by

external addition of standard in to a pre analyzed sample at three different levels. An

accuracy criterion for an assay method is that the mean recovery should desirably be

100±2% at each concentration over the range of 50-150% of target concentration.

Since the mean % recovery varies for both drug from 98-102% and were within the

desirable confidence interval, it can be said that the proposed method was accurate.

Results were shown in table 7.6 and 7.7.

191

In precision experiment, (repeatability study) relative standard deviation for

Chlordiazepoxide and Amitriptyline was found to be 0.2 and 0.15 respectively. It was

concluded that the analytical technique showed good repeatability. Same experiment

was repeated three times in a day at three different concentrations and three different

days at same concentration. These values confirmed the intraday and interday

precision of the method. The intra-day and inter-day % RSD values were calculated

which were found to be in the range of 0.2 and 1.80 for Chlordiazepoxide and 0.15-

0.98 for Amitriptyline. Hence, method at selected wavelength was found to be

precise.

For the robustness testing, the chromatographic conditions were changed and

in all varied chromatographic conditions the resolution between Chlordiazepoxide and

Amitriptyline was greater than 2.0, illustrating the robustness of the method.

Ruggedness was confirmed by precision experiment at three different analysts at

standard concentration. % RSD was calculated and was found to be well within the

acceptance criteria. Hence the proposed method was robust and rugged.

Under the developed conditions, the two drugs obey all the system suitable

criteria. Theoretical plates are above 2500 and tailing factor is less than 2 and

separation resolution is 6.25. Hence in the developed conditions more resolved peaks

were observed. The two drugs Chlordiazepoxide and Amitriptyline obey all the

system suitable criteria in all the validations conditions and all the validation

parameters are under the acceptance criteria. Hence the developed method is valid.

LOD values were found to be near to the Nano-gram levels indicates that the

sensitivity of the method. LOQ values were found to be very less indicating that the

proposed method is sensitive.

The proposed method successfully applied for the estimation of

Chlordiazepoxide and Amitriptyline in the marketed formulations. It was found that

the proposed method is successfully applied for the estimation of Chlordiazepoxide is

99.47% and Amitriptylinein is 99.05%.

192

7.7 References

1. Barbui C, Hotopf M. "Amitriptyline v. the rest: still the leading

antidepressant after 40 years of randomised controlled trials". The British

Journal of Psychiatry: the Journal of Mental Science 178 (2): 129–144,

February 2001.

2. "Amitriptyline Hydrochloride". The American Society of Health-System

Pharmacists.Retrieved 3 April 2011.

3. S. G. Hubscher et al. Functional biliary type pain syndrome. In P. J.

Pasricha, W. D. Willis & G. F. Gebhart (Eds.), ' ' italics' ' Chronic

Abdominal and Visceral Pain' 'italics' '. London: Informa Healthcare, pp.

459-461, 2006.

4. National Institute for Clinical Excellence: The Treatment of PTSD in Adults

and Children

5. British National Formulary 45, March, 2003.

6. Otaka M, Jin M, Odashima M et al. "New strategy of therapy for functional

dyspepsia using famotidine, mosapride and amitriptyline". Aliment.

Pharmacol. Ther. 21 (Suppl 2): 42–46, June 2005.

7. Atsumi; Groshan, K.; Blakely, R. D.; Richelson, E. "Pharmacological

profile of antidepressants and related compounds at human monoamine

transporters". European Journal of Pharmacology 340 (2–3): 249–258,

1997.

8. Rauser; Savage, J. E.; Meltzer, H. Y.; Roth, B. L. "Inverse agonist actions

of typical and atypical antipsychotic drugs at the human 5-

hydroxytryptamine (2C) receptor". The Journal of Pharmacology and

Experimental Therapeutics 299 (1): 83–89. 2001.

9. Barbui C, Hotopf M "Amitriptyline v. the rest: still the leading

antidepressant after 40 years of randomised controlled trials". The British

Journal of Psychiatry: the Journal of Mental Science 178 (2): 129–144.

doi:10.1192/bjp.178.2.129. PMID 11157426, February 2001.

193

10. "Amitriptyline Hydrochloride". The American Society of Health-System

Pharmacists.Retrieved 3 April 2011.

11. S. G. Hubscher et al. Functional biliary type pain syndrome. In P. J.

Pasricha, W. D. Willis & G. F. Gebhart (Eds.), ' ' italics' ' Chronic

Abdominal and Visceral Pain' 'italics' '. London: Informa Healthcare, pp.

459-461, 2006.

12. Kudo K, Jitsufuchi N, Imamura T, Selective determination of amitriptyline

and nortriptyline in human plasma by HPLC with ultraviolet and particle

beam mass spectrometry, J Anal Toxicol. 21(3):185-189, May-Jun, 1997.

13. Rafael LindenI ; Marina VenzonAntunes; Ana LuizaZiulkoski;

MaínaWingert; Paula Tonello; MladenTzvetkov; André ArigonySouto,

Determination of amitriptyline and its main metabolites in human plasma

samples using HPLC-DAD: application to the determination of metabolic

ratios after single oral dose of amitriptyline, J. Braz. Chem. Soc. vol.19

no.1 São Paulo 2008.

14. Glenda K. Ferguson, Quantitative HPLC Analysis of a Psychotherapeutic

Medication: Simultaneous Determination of Amitriptyline Hydrochloride

and Perphenazine, J. Chem. Educ., 75 (12), p 1615, 1998.

15. Surya Prakash Gupta, NeerajUpmanyu and GopalGarg, Development and

validation of spectrophotometric, HPTLC and HPLC methods for the

determination of Imipramine and Chlordiazepoxide in pharmaceutical

dosage forms, Der Pharmacia Sinica, 3 (2):185-192, 2012.

16. Sejal K. Patel and N. J. Patel, Simultaneous RP-HPLC Estimation of

Trifluoperazine Hydrochloride and Chlordiazepoxide in Tablet Dosage

Forms, Indian J Pharm Sci. 71(5): 545–547, Sep-Oct; 2009.

17. Haggag RS, Shaalan RA, Belal TS, Validated HPLC determination of the

two fixed dose combinations (chlordiazepoxide hydrochloride and

mebeverine hydrochloride; carvedilol and hydrochlorothiazide) in their

tablets, J AOAC Int.;93(4):1192-200. Jul-Aug, 2010.