ABSTRACT

This research program was conducted for the determination of packaging efficiency of Norflu (Fluphenazine-Nortriptyline) products. We have done this experiment in various conditions (sunlight, dark, 25 watt & 40 watt bulb) exposure. It was observed that the concentration of Fluphenazine hydrochloride was decreased 65.41%, 8.77%, 9.19% and 20.55% and Nortriptyline hydrochloride was decreased 51.59%, 21.35%, 21.71%, and 3.03% for sunlight, dark, 25 watt and 40 watt light exposure respectively. The results showed degradation of the sample. One reason for this could be for transparent packaging. However, because of contradictory results the reason could be other than packaging. Keywords: Fluphenazine hydrochloride, Nortriptyline hydrochloride, Norflu, Batch, Potency.Chapter OneINTRODUCTION1.1 About antipsychotic medications

Antipsychotics (also known as neuroleptics or major tranquilizers) (Harvey et al., 2009) are a class of psychiatric medication, which are helpful to people (in pediatric and adult populations) with schizophrenia, schizoaffective disorder and some forms of manic depression (bipolar disorder). They are able to reduce, or sometimes eliminate, the distressing and disabling symptoms of psychosis, such as paranoia, confused thinking, delusions and hallucinations, so that the person taking them feels better (Sane Australia, 2014a). They can also be used to help severe anxiety or depression (Timms, 2014a).These medications help with: The experience of hearing voices (hallucinations).

Ideas that distress the patient and don't seem to be based in reality (delusions).

Difficulty in thinking clearly (thought disorder).

The extreme mood swings of manic depression/bipolar disorder.

Some can help with severe depression (Timms, 2014a).People with schizophrenia and other psychotic illnesses often have an imbalance in certain natural chemicals in the brain, especially dopamine. Antipsychotic medications help the brain to restore its usual chemical balance and so reduce symptoms (Sane Australia, 2014a).They dont change the patients personality. While the patient might notice changes in his/her mood and the way he/she cope with stress, antipsychotics will not change the personality. Antipsychotics can relieve the symptoms of psychosis-related disorders; they do not stop people from feeling the normal ups and downs of life. People may find it easier to think more clearly (Sane Australia, 2014).These medications can help to control symptoms, but they do not cure the underlying condition (Centre for Addiction and Mental Health, 2009).Antipsychotics, like many medications, change the way one feels. This means that if a person stops taking the medication he/she may start to feel the way he/she did before the treatment. However, antipsychotic medications are not addictive, and one will not become dependent on them (one does not need to take higher and higher doses to get the same benefits) (Sane Australia, 2014a).While antipsychotic medications can help some people with psychosis and mood disorders, at the same time these drugs can have serious side-effects. The aim of medication treatment is to reduce and control symptoms while keeping side-effects at a minimum. Taking antipsychotic medication is one important step in getting better but is rarely enough on its own. As well as medication, effective treatment for schizophrenia and related disorders usually includes ongoing clinical support in the community, psychological therapies, education about the illness and how to deal with it, psychosocial rehabilitation, and accommodation and employment support (Sane Australia, 2014a).1.1.2 Types of antipsychotics

Antipsychotics are commonly categorized into two drug classes, first-generation antipsychotics (FGAs) and second-generation antipsychotics (SGAs), marking two waves of historical development. FGAs were developed in the 1950s. Although FGAs provide treatment for psychotic symptoms, use of these drugs can result in extrapyramidal symptoms, which are various movement disorders characterized by repetitive, involuntary muscle movements, restlessness, or an inability to initiate movement. Other common side effects are dry mouth and sedation. Neuroleptic malignant syndrome and tardive dyskinesia are rare but serious side effects. SGAs emerged in the 1980s. They generally have lower risk of motor side effects, but are associated with significant weight gain, lipid and prolactin elevation, and the development of type 2 diabetes (Seida et al., 2012).While both are effective, the atypical medications have advantages over the typical ones. These advantages include: fewer side-effects such as trembling or stiffening of muscles

less risk of developing tardive dyskinesia: movement of the mouth, tongue and sometimes other parts of the body over which the person has no control

Some evidence suggests that the newer medications may be effective in improving overall mood, thinking and motivation (Sane Australia, 2014a).

While the atypical antipsychotic medications are used more than the typical, some people find that the typical medications suit them better (Sane Australia, 2014a).Table 1: Food and Drug Administration-approved first-generation antipsychotics (Seida et al., 2012)Generic NameIndicationsAge Group for Which Approved

ChlorpromazineSchizophreniaAdults and children (112 years)

Bipolar disorder (mania)

Hyperactivity

Severe behavioral problems

DroperidolAgitationAdults and children

FluphenazinePsychotic disordersAdults

Haloperidol

SchizophreniaAdults

Tourette syndrome

Hyperactivity

Severe childhood behavioral problems

LoxapineSchizophreniaAdults and children 12 years

PerphenazineSchizophreniaAdults and children 12 years

PimozideTourette syndromeAdults and children 12 years

ProchlorperazineSchizophreniaAdults and children >2 years and >20 pounds

Generalized nonpsychotic anxietyAdults

ThiothixeneSchizophreniaAdults and children 12 years

ThioridazineSchizophreniaAdults and children

TrifluoperazineSchizophreniaAdults and children 6 years

Generalized nonpsychotic anxietyAdults

Table 2: Food and Drug Administration-approved second-generation antipsychotics (Seida et al., 2012)Generic NameIndicationsAge Group for Which Approved

AripiprazoleSchizophreniaAdults and adolescents (1317years)

Bipolar disorder (manic/mixed) monotherapy or adjunctive to lithium or valproateAdults and children (1017 years)

Adjunctive treatment of major depressive disorderAdults

Irritability associated with autistic disorderChildren (617 years)

Acute treatment of agitationAdults

AsenapineAcute schizophreniaAdults

Bipolar disorder type 1 (manic/mixed)

ClozapineTreatment resistant schizophreniaAdults

Reduce risk of suicidal behavior in younger patients with schizophrenia

IIoperidoneAcute schizophreniaAdults

Olanzapine

SchizophreniaAdults and adolescents (1317 years)

Bipolar disorder (manic/mixed)

Bipolar disorder (depressive episode)Adults

Treatment-resistant depression

Agitation associated with schizophrenia and bipolar I mania

PaliperidoneSchizophreniaAdults

Schizoaffective disorder

QuetiapineSchizophreniaAdults and adolescents (1317 years)

Bipolar disorder (acute manic)Adults, children and adolescents (1017 years)

Bipolar disorder (depression)Adults

Bipolar disorder (maintenance)

Major depressive disorder

RisperidoneSchizophreniaAdults and adolescents (1317 years)

Bipolar disorder (manic/mixed)Adults, children and adolescents (1017 years)

Irritability associated with autismChildren (516 years)

ZiprasidoneSchizophreniaAdults

Bipolar disorder (manic/mixed)

Bipolar disorder (maintenance)

Acute agitation in patients with schizophrenia

1.1.3 Mechanism of action of antidepressants

All antipsychotic drugs tend to block D2 receptors in the dopamine pathways of the brain. This means that dopamine released in these pathways has less effect. Excess release of dopamine in the mesolimbic pathway has been linked to psychotic experiences. Various neuroleptics such as haloperidol and chlorpromazine suppress dopamine chemicals throughout its pathways, in order for dopamine receptors to function normally (McDonald and Murphy, 2003).In addition of the antagonistic effects of dopamine, antipsychotics (in particular atypical neuroleptics) also antagonize 5-HT2A receptors. Different alleles of the 5-HT2A receptor have been associated with schizophrenia and other psychoses, including depression. Higher concentrations of 5-HT2A receptors in cortical and subcortical areas, in particular in the right caudate nucleus have been historically recorded. This is the same receptor that psychedelic drugs agonize to various degrees, which explains the correlation between psychedelic drugs and schizophrenia (McDonald and Murphy, 2003; Schmidt et al., 1995).Typical antipsychotics are not particularly selective and also block dopamine receptors in the mesocortical pathway, tuberoinfundibular pathway, and the nigrostriatal pathway. Blocking D2receptors in these other pathways is thought to produce some unwanted side effects that the typical antipsychotics can produce. They were commonly classified on a spectrum of low potency to high potency, where potency referred to the ability of the drug to bind to dopamine receptors, and not to the effectiveness of the drug. High-potency antipsychotics such as haloperidol, in general, have doses of a few milligrams and cause less sleepiness and calming effects than low-potency antipsychotics such as chlorpromazine and thioridazine, which have dosages of several hundred milligrams. The latter have a greater degree of anticholinergic and antihistaminergic activity, which can counteract dopamine-related side-effects (Stahl, 2003).Atypical antipsychotic drugs have a similar blocking effect on D2 receptors, however, most also act on serotonin receptors, especially 5-HT2A and 5-HT2C receptors. Both clozapine and quetiapine appear to bind just long enough to elicit antipsychotic effects but not long enough to induce extrapyramidal side effects and prolactin hypersecretion. 5-HT2A antagonism increases dopaminergic activity in the nigrostriatal pathway, leading to a lowered extrapyramidal side effect liability among the atypical antipsychotics (Stahl, 2003).

1.1.4 Dosage of antipsychotic medicationsSome drugs are very potent and the doctor may prescribe a low dose. Other drugs are not as potent and a higher dose may be prescribed (Mental Health Care, 2014).

Unlike some prescription drugs, which must be taken several times during the day, some antipsychotic medications can be taken just once a day. In order to reduce daytime side effects such as sleepiness, some medications can be taken at bedtime. Some antipsychotic medications are available in "depot" forms that can be injected once or twice a month (Education Medicaid Integrity Contractor, 2013).

1.1.5 Side-effects of antipsychotic medications (Mental Health Care, 2014)The different antipsychotic medicines can have different types of side-effects. Also, sometimes one medicine causes side-effects in some people and not in others. Therefore, it is not unusual to try two or more different medicines before one is found that is best suited to an individual.Common side-effects include: Dry mouth, blurred vision, flushing and constipation. These may ease off when you get used to the medicine.

Drowsiness (sedation), which is also common but may be an indication that the dose is too high. A reduced dose may be an option.

Weight gain which some people develop. Weight gain may increase the risk of developing diabetes and heart problems in the longer term. This appears to be a particular problem with the atypical antipsychotics - notably, clozapine and olanzapine (ok and Gaebel, 2008).

Movement disorders which develop in some cases. These include:

Parkinsonism - this can cause symptoms similar to those that occur in people with Parkinson's disease - for example, tremor and muscle stiffness.

Akathisia - this is like a restlessness of the legs.

Dystonia - this means abnormal movements of the face and body.

Tardive dyskinesia (TD) - this is a movement disorder that can occur if you take antipsychotics for several years. It causes rhythmical, involuntary movements. These are usually lip-smacking and tongue-rotating movements, although it can affect the arms and legs too. About 1 in 5 people treated with typical antipsychotics eventually develops TD.

All antipsychotics can make people put on weight, although not everyone does. The older 'typical' antipsychotics cause a rise in the level of the hormone prolactin, which can lead to a lowered sex drive and breast tissue growth in both men and women, and affect womens periods.Atypical antipsychotic medicines are thought to be less likely to cause movement disorder side-effects than typical antipsychotic medicines. This reduced incidence of movement disorder is the main reason why an atypical antipsychotic is often used first-line. Being on antipsychotic medication increases the risk of developing diabetes, and that risk is greater in younger people. There is also the risk of cardiovascular problems and high blood pressure. People who are on antipsychotics should have regular physical health checks and doctors should monitor for diabetes and cardiovascular disease at least once a year.The higher the dose of antipsychotic medication, the more severe the side effects seem to be.1.2 Antidepressant medications (Timms, 2012b, Sane Australia, 2014b)Antidepressants are drugs used for the treatment of major depressive disorder and other conditions, including dysthymia, anxiety disorders, obsessive compulsive disorder, eating disorders, chronic pain, neuropathic pain and, in some cases, dysmenorrhoea, snoring, migraines, attention-deficit hyperactivity disorder (ADHD), substance abuse and sleep disorders. They can be used alone or in combination with other medications.Antidepressants help relieve the symptoms of depression and associated anxiety. They do not make the patient euphoric, but simply help the patient to react more realistically in his/her emotional responses. Taking antidepressant medication is one important step in getting better but is rarely enough on its own. As well as medication, effective treatment for depression and anxiety-related disorders may include education about the illness and how to deal with it, and psychological therapies such as Cognitive Behavioral Therapy (CBT).1.2.1 Types of antidepressant medications (Mayo Clinic Staff, 2011)There are almost thirty different kinds of antidepressants available today and there are five main types. The different classes of antidepressant medications are:1) Tricyclics: Amitriptyline

Clomipramine (Anafranil)

Doxepin (Silenor, Zonalon)

Imipramine (Tofranil, Tofranil-PM)

Trimipramine (Surmontil)

Desipramine (Norpramin)

Nortriptyline (Pamelor, Aventyl)

Protriptyline (Vivactil)

Amoxapine

Maprotiline2) MAOIs (Monoamine oxidase inhibitors):

Isocarboxazid (Marplan)

Phenelzine (Nardil)

Tranylcypromine (Parnate)

Selegiline (Emsam, Eldepryl, Zelapar)

3) SSRIs (Selective Serotonin Reuptake Inhibitors): Citalopram (Celexa)

Escitalopram (Lexapro)

Fluoxetine (Prozac, Prozac Weekly, Sarafem)

Fluvoxamine (Luvox, Luvox CR)

Paroxetine (Paxil, Paxil CR, Pexeva)

Sertraline (Zoloft)

4) SNRIs (Serotonin and Noradrenaline Reuptake Inhibitors): Venlafaxine (Effexor XR)

Desvenlafaxine (Pristiq)

Duloxetine (Cymbalta)

5) NaSSAs (Noradrenaline and Specific Serotoninergic Antidepressants): Aptazapine (CGS-7525A)

Esmirtazapine (ORG-50,081)

Mianserin (Bolvidon, Norval, Tolvon) Mirtazapine (Remeron, Avanza, Zispin) Setiptiline (Tecipul)1.2.2 Mechanism of action of antidepressants (Yldz et al., 2002, Stimmel et al., 1997)MAOIs were the first class of antidepressants to be developed. They fell out of favor because of concerns about interactions with certain foods and numerous drug interactions. MAOIs elevate the levels of norepinephrine, serotonin, and dopamine by inhibiting an enzyme called monoamine oxidase. Monoamine oxidase breaks down norepinephrine, serotonin, and dopamine. When monoamine oxidase is inhibited, norepinephrine, serotonin, and dopamine are not broken down, increasing the concentration of all three neurotransmitters in the brain.

TCAs have been in use since the 1950s when imipramine (Tofranil) was shown to be effective for treating depression. TCAs primarily work by increasing the level of norepinephrine in the brain and to a lesser extent serotonin levels. Some TCAs also are antihistamines (block the action of histamine) or anticholinergic (block the action of acetylcholine, a neurotransmitter), and these additional actions allow for uses of TCAs other than for treating depression as well as additional side effects.SSRIs were developed after TCAs and are the most widely used class of antidepressants. They work by increasing the level of serotonin in the brain. Unlike MAOIs and TCAs, they do not significantly affect norepinephrine levels in the brain. SSRIs also have fewer and milder side effects, fewer drug interactions, and are much less likely to be effective for committing suicide than TCAs.SNRIs are the newest class of antidepressants. SNRIs work by increasing the levels of serotonin and norepinephrine in the brain. Drug interactions and side effects associated with SNRIs are similar to those seen with SSRIs.NaSSAs act by antagonizing the 2-adrenergic receptor and certain serotonin receptors such as 5-HT2A and 5-HT2C, but also 5-HT3, 5-HT6, and/or 5-HT7 in some cases. By blocking 2-adrenergic autoreceptors and heteroreceptors, NaSSAs enhance adrenergic and serotonergic neurotransmission in the brain involved in mood regulation, notably 5-HT1A-mediated transmission. In addition, due to their blockade of certain serotonin receptors, serotonergic neurotransmission is not facilitated in unwanted areas, which prevents the incidence of many side effects often associated with selective serotonin reuptake inhibitor (SSRI) antidepressants; hence, in part, the "specific serotonergic" label of NaSSAs.1.2.3 Dosage of antidepressant medications (National Health Service, 2013)When prescribing antidepressants the lowest possible dose necessary to improve the symptoms is selected. This approach is intended to reduce the risk of side effects. If this dose proves ineffective, it can be gradually increased. Antidepressants are usually taken in tablet form. Depending on the type of antidepressant prescribed and the severity of the patients depression, he/she will usually have to take one to three tablets a day.1.2.4 Common side effects of antidepressants (Allen, 2011)Antidepressants can sometimes cause a wide range of unpleasant side effects, including:i) nausea increased ii) appetite and weight gain

iii) loss of sexual desire and other sexual problems, such as erectile dysfunction and decreased orgasmiv) fatigue and drowsiness

v) insomnia

vi) dry mouth

vii) blurred vision

viii) constipation

ix) dizziness

x) agitation

xi) irritability

xii) anxiety

Antidepressants may cause withdrawal symptoms if abruptly discontinued. Withdrawal symptoms include nausea, vomiting, dizziness, headache, irritability, sleep disturbance, nightmares, psychosis, and seizures. All antidepressants have a warning about use in children and adolescents. Antidepressants increased the risk of suicidal thinking, and suicidal behavior in short-term studies in children and adolescents with depression and other psychiatric disorders. Anyone considering the use of antidepressant in a child or adolescent must balance this risk of suicide with the clinical need for the drug. Patients who are started on therapy should be closely observed for clinical worsening, suicidal thoughts or unusual changes in behavior.1.3 Fluphenazine (College of Psychiatric and Neurologic Pharmacists, 2013)Fluphenazine is a first generation antipsychotic (FGA) or typical antipsychotic drug. It used to treat psychotic disorders (such as schizophrenia), agitation, and dementia (College of Psychiatric and Neurologic Pharmacists, 2013).It is a phenothiazine with a piperazine side-chain. It works by antagonizing the dopamine D2 receptors in the brain. It is a short-acting piperazine phenothiazine.

Figure: Fluphenazine structurePhenothiazines work by inhibiting the actions of the brain chemicals, dopamine and norepinephrine, which are overproduced in individuals with psychosis.Compared to other phenothiazines, fluphenazine has weak anticholinergic, sedative and hypotensive activity, weak anti-emetic effects and strong extrapyramidal effects. It is the most potent of the phenothiazine antipsychotics.Fluphenazine rebalances dopamine to improve thinking, mood, and behavior. It is available in 1 mg, 2.5 mg, 5 mg, and 10 mg tablets, a liquid concentrate containing 5 mg per 1 mL, a rapid-onset injectable form containing 2.5 mg per 1 mL, and a long-acting injectable form containing 25 mg per 1 mL.1.3.1 Fluphenazine synthesis (Cusic, 1956; Yale and Sowinski, 1960; Merrilland Yale, 1968)Fluphenazine (4-[3-[2-(trifluoromethyl)phenothiazin-10-yl]propyl]-1-piperazineethanol), is synthesized by any of the methods described already for the preparation of trifluoperazine and related antipsychotics. Alkylation of 2-trifluoromethylphenothiazine using 4-formyl-1-piperazineylpropylchloride in the presence of sodamide synthesizes 2-trifluoromethyl-10-[3-(4-formyl-1-piperazinyl)propyl]phenothiazine. Further alkaline hydrolysis removes the N-formyl group, giving 2-trifluoromethyl-10-[3-(1-piperazinyl)propyl]phenothiazine. This is alkylated by 2-bromoethanol-1 acetate, which upon further acidic hydrolysis removes the protecting acetyl group, yielding fluphenazine.

Figure: Synthesis of Fluphenazine1.3.2 Fluphenazine hydrochloride (U.S. National Library of Medicine, 2011)Fluphenazine hydrochloride is a trifluoromethyl phenothiazine derivative intended for the management of schizophrenia. Chemically it is 4-[3-[2-(Trifluoromethyl) phenothiazin-10-yl]propyl]-1-piperazineethanol dihydrochloride which may be represented by the following structural formula:

Figure: Structure of Fluphenazine hydrochlorideMolecular formula of Fluphenazine is C22H26F3N3OS2HCL. Molecular weight of FluphenazineHydrochloride is 510.44. Fluphenazine hydrochloride tablets are indicated in the management of manifestations of psychotic disorders. Fluphenazine hydrochloride has not been shown effective in the management of behavioral complications in patients with mental retardation. Fluphenazine hydrochloride has activity at all levels of the central nervous system as well as on multiple organ systems. The mechanism whereby its therapeutic action is exerted is unknown.1.4 Nortriptyline (U.S. National Library of Medicine, 2014)Nortriptyline is in the class of drugs called tricyclic antidepressants (TCAs) and is used for treating depression. Like all TCAs, nortriptyline increases levels of norepinephrine and serotonin, two neurotransmitters, and blocks the action of acetylcholine, another neurotransmitter. It is believed that by restoring the balance of these different neurotransmitters in the brain depression is alleviated. Nortriptyline was approved by the FDA in November 1964.

Figure: Nortriptyline structure1.4.1 Nortriptyline synthesis (Vardanyan and Hruby, 2006)Nortriptyline is 5-(3-methylaminopropyliden)-10,11-dihydrodibenzcycloheptene. In nortriptyline, the nitrogen atom in the central part of the tricyclic system of desipramine is replaced by a carbon atom, which is bound to a side chain by a double bond. Two suggested methods of nortriptyline synthesis are based on the N-demethylation of amitriptyline. The third way utilizes the reaction of methaylamine with 5-(3-bromopropyliden)-10,11-dihyadro-5H-dibenmz[a,d]cycloheptene.According to the first scheme, demethylation takes places by the reaction of amitriptyline with methyliodide, which leads to the formation of a quartanary ammonium salt, the reaction of which with methylamine at a relatively high temperature gives the desired nortriptyline.

Figure: First method of nortriptyline synthesisIn the second scheme, the specified reaction of amitriptyline with ethtylahloroformate leads to the substitution of a methyl group on the amino group with an ethoxycarbonyl group. Hydrolysis of the product leads to nortiptyline.

Figure: Second method of nortriptyline synthesisAccording to the third scheme, Nortriptyline is synthesized by reacting methylamine with 5-(3-bromoptopyliden)-10,11-dihydro-5H-dibenz[a,d]cycloheptene.

Figure: Third method of nortriptyline synthesisNortriptyline is a drug with a relatively short latent period of action. It is practically devoid of sedative effects. It is used in manic-depressive psychoses, in all forms.

1.4.2 Nortriptyline hydrochloride (U.S. National Library of Medicine, 2014)Nortriptyline hydrochloride, USP is 1-propanamine, 3-(10,11-dihydro-5H dibenzo[a,d]cyclohepten-5-ylidene)-N-methyl-, hydrochloride.

Figure: Structure of Nortriptyline hydrochlorideNortriptyline hydrochloride structural formula C19H21NHCl.Molecular weight is 299.8. It is a white to off-white powder, having a slight, characteristic odor. It is soluble in water and in chloroform; sparingly soluble in methanol; and practically insoluble in most organic solvents.Each capsule, for oral administration, contains nortriptyline hydrochloride, USP equivalent to 10 mg, 25 mg, 50 mg or 75 mg nortriptyline.

1.5 Fluphenazine-Nortriptyline combination

Fluphenazine-Nortriptyline is a combination preparation of two well-known and time tested molecules: Fluphenazine and Nortriptyline. Fluphenazine is a typical antipsychotic drug used for the treatment of psychosis such schizophrenia, manic phases of bipolar disorder, agitation, and dementia. Nortriptyline is a second-generation tricyclic antidepressant (TCA) used in the treatment of major depression. In combination the compound renders a preparation with antidepressant, anxiolytic and activating properties and mutually neutralizes side effects. A chart is given showing the brand names and corresponding price of each combination Fluphenazine-Nortriptyline drug that is sold by the pharmaceutical companies in Bangladesh (Square Pharmaceuticals Ltd., 2014).

Table: Showing different Fluphenazine-Nortriptyline combination drugs sold by various pharmaceuticals in Bangladesh (Health Prior 21, 2014).

Trade NameGeneric NamePharmaceuticalsDosage FormulationQuantityPackagingPrice

AmivalFluphenazine hydrochloride + nortriptyline hydrochlorideAmico Pharmaceuticals Ltd. Tablet0.5mg+10mg/Tab100's pack80 BDT

Amival-fFluphenazine hydrochloride + nortriptyline hydrochlorideAmico Pharmaceuticals Ltd. Tablet0.5mg+10mg/Tab100's pack201 BDT

AnfluFluphenazine hydrochloride + nortriptyline hydrochlorideAlco Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack75 BDT

ApresinFluphenazine hydrochloride + nortriptyline hydrochlorideBeximco Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack105 BDT

AtevalFluphenazine hydrochloride + nortriptyline hydrochlorideZiska Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack100 BDT

EuphorFluphenazine hydrochloride + nortriptyline hydrochlorideBio- Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack80 BDT

FlutripFluphenazine hydrochloride + nortriptylinehydrochlorideGeneral Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack71 BDT

Fresh Fluphenazine hydrochloride + nortriptyline hydrochlorideNipa Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack80 BDT

MoodonFluphenazine hydrochloride + nortriptyline hydrochlorideIbnSina Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab200's pack170 BDT

NorfluFluphenazine hydrochloride + nortriptyline hydrochlorideAcme Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack80 BDT

Norz1n Fluphenazine hydrochloride + nortriptyline hydrochlorideAristo Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab250's pack200 BDT

PermivalFluphenazine hydrochloride + nortriptyline hydrochlorideOpsonin Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab200's pack150 BDT

SanitFluphenazine hydrochloride + nortriptyline hydrochlorideSquare Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack79 BDT

TripnorFluphenazine hydrochloride + nortriptyline hydrochlorideSomatec Pharmaceuticals Ltd.Tablet0.5mg+10mg/Tab100's pack75 BDT

In this research project experiment conducted on sample which was manufactured by ACME Pharmaceuticals (Trade name: Noflu).1.5.1 Pharmacology (Square Pharmaceuticals, 2014)Nortriptyline hydrochloride is a tricyclic antidepressant. Nortriptyline inhibits the uptake of norepinephrine and serotonin at nerve terminals. In contrast to its parent compound amitriptyline which is equally potent in inhibiting the uptake of norepinephrine and serotonin, Nortriptyline has a greater effect on norepinephrine reuptake than on serotonin reuptake. Fluphenazine is a tranquilizer of the phenothiazine type with piperazine side chain. Fluphenazine primarily acts as a neuroleptic drug whose main therapeutic effect is believed to reside in potent dopamine (especially D2) receptor antagonism. Due to the nature of the two active constituents and the larger inter and intra subject variability seen in trials, accurate and consistent pharmacokinetic data are not available. This can be illustrated by the fact that studies of nortriptyline hydrochloride have produced half life values ranging from 16 to 38 hours. In the case of Fluphenazine hydrochloride these values have been 10 to 16 hours.

1.5.2 Indications (Square Pharmaceuticals, 2014)This medicine provides effective therapy in the management of patients exhibiting mild to moderate anxiety; tension and/or agitation with or without co-existing depression.Various forms of neurosis (anxiety, hysteria, depression, neurasthenia), disorder of sleep are amenable to treat with this tablet. In addition, patients exhibiting general neurotic feelings, fear, mild to moderate depression and mild to moderate anxiety have responded well to this tablet.

1.5.3 Dosage and administration (Square Pharmaceuticals, 2014)Adult: One tablet three times daily. The course of the treatment should be limited to three months. If the patient does not respond after 4weeks, an alternative treatment should be given.

Children: Not indicated for the treatment of children.

1.5.4 Contraindication and precaution (Ineson, 1996)Phenothiazines and tricyclic antidepressants have been shown to lower the threshold for electrically induced convulsions in animals; hence, this combination is not recommended for patients with a history of epilepsy or brain damage. This combination is further contraindicated in patients with blood dyscrasias, severe cardiac insufficiency, renal or liver damage. It is not advisable to give monoamine oxidase inhibitors (MAOIs) with this combination, nor should they be given in two weeks after cessation of treatment with MAOIs. This combination should be given with caution to patients with glaucoma and to those who have a propensity for urinary retention. This combination should be used with caution in patients with cardiac failure, especially when there is evidence of rhythm disturbance and in patients with recent myocardial infarction.1.5.5 Side effects (Ineson, 1996)Tardive dyskinesias have been reported in phenothiazine therapy, usually after prolonged courses given at doses adequate to control psychotic illness. Consequently, treatment with this drug should be limited to three months. Dryness of mouth, drowsiness, faintness and constipation. Occasionally tachycardia, nasal congestion, blurred vision and excitement are seen. Extrapyramidal reactions are unlikely to occur with this dose of Fluphenazine alone, and it is probable that the anticholinergic activity of Nortriptyline affords protection against such effects. As with all neuroleptic drugs the presence of unexplained hyperthermia could indicate neuroleptic malignant syndrome. In this event, this combination and associated neuroleptic treatment should be discontinued until the origin of the fever has been determined.

1.5.6 Drug interaction (Square Pharmaceuticals, 2014)Interaction with barbiturates, alcohol and narcotic drugs may occur, so central nervous depressants should be administered with caution. This combination may diminish the anti-hypertensive effect of an adrenergic blocking agent and could potentiate the pressor response to locally injected sympathomimetic agents.

1.5.7 Use in pregnancy and lactation (Ineson, 1996)Do not use during pregnancy, especially in the first and last trimesters unless there are compelling reasons. There is no evidence as to drug safety in human pregnancy, nor are the results of animal studies conclusive. Breast feeding is not recommended for women receiving this combination.

1.5.8 Overdose (Square Pharmaceuticals, 2014)Over dosage should be treated symptomatically and supportively. If the patient is conscious, prompt gastric lavage, dilution of the stomach contents to delay absorption, or stimulation of vomiting should be attempted. An open airway should be maintained. Extrapyramidal symptoms are amenable to anti-parkinsonian drugs. In severe hypotension, all the standard procedures for the management of circulatory shock should be substituted.e.g. vasoconstrictors and/or intravenous fluids. If vasoconstrictors are required, metaraminol, mephentermine or noradrenaline should be administered but not adrenaline, as this will further lower the blood pressure through interaction with the phenothiazine.

Chapter TwoLITERATURE REVIEW2.1 Determination and analysis of nortriptyline and fluphenazine combinations

A rapid, simple, and highly sensitive second-derivative synchronous fluorimetric (SDSF) method has been developed for the simultaneous analysis of binary mixtures of fluphenazine hydrochloride (FLZ) and nortriptyline hydrochloride (NTP) in their co-formulated tablets. The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 120 nm in acetic acid. The different experimental parameters affecting the fluorescence intensity of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.25-3.0 and 1-10 microg/ml for FLZ and NTP respectively, with lower detection limits (LOD) of 0.05 and 0.18 microg/ml and quantitation limits of 0.15 and 0.53 microg/ml for FLZ and NTP respectively. The proposed method was successfully applied for the determination of the studied compounds in their synthetic mixtures and in commercial co-formulated tablets. The results obtained were in good agreement with those obtained by the reference methods (Walash et al., 2009).

The construction and electrochemical response characteristics of polyvinylchloride (PVC), membrane sensors for determination of fluphenazine hydrochloride and nortriptyline hydrochloride are described. The method is based on the formation of the ion-pair complexes between the two drugs cations and sodium tetraphenylborate (NaTPB) or tetrakis (4-chlorophenyl) borate (KtpClPB). Four polyvinylchloride sensors were fabricated. For FluphenazineHCl sensors 1 and 3 were prepared using NaTPB and KtpClPB, respectively. For NortriptylineHCl sensors 2 and 4 were prepared using NaTPB and KtpClPB, respectively. They show linear responses for both drugs over the concentration ranges of 103105, 102105, 103105 and 102105 M with cationic slopes of 58.9, 52.5, 59.3 and 54.3 mV per concentration decade, for sensors 14, respectively. The direct potentiometric determination of fluphenazine and nortriptyline hydrochlorides in their pure forms using the proposed sensors gave recoveries of 98.80.9, 99.00.9, 98.70.8 and 99.40.8% for sensors 14, respectively. This is compared reasonably with the data obtained using the British Pharmacopoeial method (1993). Sensors 14 were also used for determination of both drugs in their pharmaceutical dosage forms and in the presence of their degrades. It is noteworthy to mention that tetrakis (4-chlorophenyl) borate affects significantly the life time of the fabricated sensors of both drugs (El-Ragehya et al., 2000).

A simple, fast, robust, and accurate high-performance liquid chromatography (HPLC) method is described for simultaneous quantification of nortriptyline and fluphenazine in bulk powder and dosage forms. The chromatographic separation was carried out in less than one minute on Chromolith Performance RP-18e column, which consists of monolithic rods of highly porous silica, using isocratic binary mobile phase of MeOH and 25 mM KH2PO4 pH 4.5 in the ratio of 70:30 at 5 mL min1 flow rate and 40C. The high porosity of stationary phase enables it to be used at high flow rates without problems concerning backpressure, these high flow rates in turn lead to strong reduction of analysis time. A diode array detector was used at 254 nm for detection. The method was validated for system suitability, linearity, precision, limits of detection and quantitation, specificity, and robustness. LOD were found to be 0.40 and 0.78 g mL1 for nortriptyline and fluphenazine, respectively. LOQ was 3.125 g mL1 for both analytes. Recovery values of this method were 98.50 and 98.00% for nortriptyline and fluphenazine, respectively, and reproducibility was within 2.36%. Robustness study was done for small changes in KH2PO4 concentration and pH, temperature, flow rate, wavelength of detection, % of MeOH in mobile phase, and injection volume (Hashemab and Jirac, 2013).

Spectrophotometric and high performance liquid chromatographic procedures are described for determination of nortriptyline hydrochloride and fluphenazine hydrochloride. The first procedure is based on application of first derivative of ratio spectra (1DD) for quantitative determination of nortriptyline hydrochloride in presence of fluphenazine hydrochloride. Secondly, an accurate, sensitive and stability indicating method has been introduced for determination of nortriptyline hydrochloride and fluphenazine hydrochloride in both bulk powder and in dosage form. In the derivative ratio method, Beer's law is obeyed in the concentration ranges of 832 g mL1 and 432 g mL1 of nortriptyline hydrochloride at wavelengths 271.4 and 284.2 nm, respectively. In high performance liquid chromatographic method, linear relationship in the range of 0.63.6 g mL1 and 1.24.2 g mL1 for nortriptyline hydrochloride and fluphenazine hydrochloride, respectively, was obtained. The mobile phase used was 0.05 M ammonium acetate : methanol : acetonitrile (4 : 1 : 5 v/v/v), and detection was done spectrophotometrically at 254 nm. Results were statistically analyzed and compared with those obtained by applying the British Pharmacopoeia (2000) method (El-Ragehya et al., 2002).A novel method for the simultaneous high-performance liquid chromatographic determination of nortriptyline hydrochloride and fluphenazine hydrochloride was developed and validated. Fluvastatin sodium was used as internal standard. The determination was performed on a Hypersil Gold C8 column (250 mm 4.6 mm i.d., 5 m particle size) at 25C; the mobile phase, consisting of a mixture of formic acid (0.1 M, pH 2.16)-methanol (33:67, v/v), was delivered at a flow rate of 1.1 mL/min and detector wavelength at 251 nm. The retention time of nortriptyline, fluphenazine and fluvastatin was found to be 5.11, 8.05 and 11.38 min, respectively. Linearity ranges were 5.01350.0 and 10.01350.0 g/mL with limit of detection values of 0.72 and 0.31 g/mL, for nortriptyline and fluphenazine, respectively. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. Correlation coefficients (r2) of the regression equations were greater than 0.999 in all cases. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of nortriptyline and fluphenazine (Ashour and Kattan, 2012).A controlled clinical trial on the efficacy of a nortriptyline-fluphenazine combination was carried out in patients with painful diabetic polyneuropathy. A visual analog scale was used to evaluate the relief of pain or paresthesia. Significant relief of both pain and paresthesia was obtained with this combination. The differences were statistically significant. Side effects were frequent but not usually severe enough to lead to cessation of these medications. (Gomez-Perez et al., 1985) A novel method for the simultaneous high-performance liquid chromatographic determination of nortriptyline hydrochloride and fluphenazine hydrochloride was developed and validated. Fluvastatin sodium was used as internal standard. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of nortriptyline and fluphenazine (Nuha and Safwan, 2012).2.2 Determination and analysis of fluphenazine as a single drug or in combination with other drugs

A simple, stability-indicating liquid chromatographic method has been developed for the assay of flunarizinedihydrochloride in the presence of its acid-induced degradation product. A Bondapak-C18 column was used with a mobile phase consisting of methanol-water (75:25, v/v) containing 0.5% w/v sodium chloride and 0.2% v/v triethanolamine adjusted to pH 6.6 with 30% hydrochloric acid at a flow rate 2 ml min-1. Quantitation was achieved with UV detection at 254 nm based on peak area or peak height ratios. The proposed method was successfully applied to the determination of the drug in laboratory-prepared mixtures in the presence of its degradation product and in capsules. Moreover, the method was utilized to investigate the kinetics of the degradation process at different temperatures and the apparent first-order rate constant, half-life and activation energy calculated (Wahbi et al., 1995).

A simple, stability-indicating, reversed phase liquid chromatographic method has been developed for the determination of fluphenazine hydrochloride in the presence of its oxidative and ultraviolet degradation products. Reversed phase chromatography was conducted using an ODS C18 (150 4.6 mm id) column at ambient temperature with UV-detection at 250 nm. A mobile phase consisting of 0.03 M potassium dihydrogen phosphate bufferacetonitrile (40:60, v/v) adjusted to pH 4 was used for the separation of the studied drug and its degradation products at a flow rate of 1 mL min1. The method showed a good linearity over the concentration range of 2.020.0 g mL1 with a detection limit (LOD) of 0.8 g mL1 and a quantification limit (LOQ) of 1.5 g mL1. The proposed method was successfully validated and applied for the analysis of the drug in its commercial dosage forms; the obtained results were favorably compared with those obtained by the official and comparison methods. Moreover, the method was utilized to investigate the kinetics of the oxidative degradation of the drug. The first-order rate constant, half-life time, and activation energy of the degradation were calculated (Walasha and Wahba, 2014).

A simple, rapid, precise, sensitive, specific and accurate instrumental planar chromatographic method for quantification of fluphenazine hydrochloride in injections has been developed and validated. Chromatographic separation was on precoated silica gel F 254 HPTLC plates. The mobile phase was methanol-purified water 9:1 (v/v). Densitometric analysis was performed at 306 nm. The calibration plots were linear in the range 100 to 500 ng L1 with a correlation coefficient of 0.998. The limits of detection (LOD) and quantification (LOQ) were 1.45 and 4.40 ng, respectively. Intra-assay and inter-assay precision, expressed as relative standard deviation (RSD), were in the range 0.731.77% (n = 3) and 1.181.86% (n = 9), respectively. Recovery of fluphenazine hydrochloride was between 98.29 and 101.53%, with RSD no higher than 1.87%. The method was selective for fluphenazine hydrochloride and the preservatives in the injections. Drug content was within the prescribed limits (95110% of the labeled content of the formulations) when the method was used to quantify fluphenazine hydrochloride in real pharmaceutical samples. Because the method is sensitive, precise, accurate, and selective for the compound tested, it can be used for routine quality control testing of fluphenazine hydrochloride in injections (Mennickent et al., 2010).

The electrochemical behaviour of fluphenazine based on its oxidation at platinum and glassy carbon electrodes was investigated by linear sweep and cyclic voltammetry. The influence of pH, concentration, nature of the buffer and scan rate was carefully examined. At both electrodes, three anodic steps (representing an irreversible oxidation) were obtained. The method was applied to the determination of fluphenazine in sugar-coated tablets (Sentrk et al., 1996).

Amber-coloured syringes designed for the distribution of unit-doses of oral drops were studied for the efficiency of the photoprotectiveness and the possible binding of eleven phenothiazine neuroleptics: alimemazine, chlorpromazine, cyamemazine, fluphenazine, levomepromazine, periciazine, pipotiazine, prochlorperazine, thioproperazine, thioridazine, and trifluoperazine, all very easily oxidized in solution in daylight. Spectrofluorimetry made it possible, in one operation, to determine the remaining concentrations of drugs after storage and to verify the absence of photo-oxidation. The storage was performed up to 13 days at 25 +/- 3 degrees C and without any precaution from daylight. All the drugs studied were stable and none bound on the syringes. However, the stability appeared to be due to the antioxidants in the drug preparations, and not to the coloured material, since oral drops were also stable in uncoloured syringes designed for injection. Nevertheless, the amber-coloured syringes efficiently protect the active principles in pure aqueous solutions, without preservative, and thus this physical protection reinforces the chemical one of the galenical formulation (Airaudo et al., 1995).

The coupling of end-column tris (2,2'-bipyridyl) ruthenium (II) electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the analysis of two antipsychotic drugs, perphenazine (PPH) and fluphenazine (FPH). The parameters related to CE separation and ECL detection, including the detection potential, the buffer pH value and concentration, the separation voltage, and Ru(bpy)(2+) concentration, were investigated in detail. Under optimum conditions, PPH and FPH were well separated and detected within 11 min. The linear ranges were 0.1-5 M for PPH, and 0.1-7.5 M for FPH, respectively. The limits of detection of PPH and FPH were 5 and 10 nM (S/N=3). The relative standard deviations (n=3) of the ECL intensity and the migration time were less than 2.5 and 0.65% in a day, and less than 3.4 and 1.7% in different three days. The proposed method was successfully applied to determine PPH and FPH in spiked urine samples with satisfactory results (Xu et al., 2014).

A thin-layer chromatography (TLC)-densitometry method has been developed to identify and quantify haloperidol, amitriptyline, sulpiride, promazine, fluphenazine, doxepin, diazepam, trifluoperazine, clonazepam, and chlorpromazine in selected psychotropic drugs. Separation was performed on precoated silica gel 60 F254 TLC plates. Chromatograms were developed in various mobile phases, and 8 of 30 tested phases were selected based on spot location and developing time. The identification and quantification were carried out based on ultraviolet densitometric measurements at chosen wavelengths. In addition to retention coefficients, the absorption spectra recorded directly from chromatograms were also used in qualitative analysis. Under established experimental conditions, high sensitivity of the method was achieved. The limit of detection ranged from 0.009 to 0.260 microg, depending on the wavelength selected for measuring. A satisfactory recovery, ranging from 92.99 to 104.70%, was achieved for individual constituents (Malanka and Krzek, 2005).

A new spectrofluorometric method was developed for the determination of a ternary mixture of dexamethasone, dexchlorpheniramine maleate, and fluphenazine hydrochloride in dosage forms where the literature did not reveal any method for analysis of this mixture. The method was based on the use of the first and second derivatives of the ratio of the emission spectra with a zero-crossing technique. The ratio spectra were obtained by dividing the emission spectrum of the mixture by that of one of the components. The concentrations of the other components were then determined from their respective calibration graphs treated similarly. The method can resolve the spectral overlapping of the three components and was applied successfully for the determination of these drugs in synthetic mixtures and in commercial dosage forms (El-Yazbi et al., 2006).

A simple, rapid, and sensitive spectrofluorometric method has been developed for the determination of olanzapine (OLZ) and fluphenazine hydrochloride (FPZ HCl). The proposed method is based on the quantitative quenching effect of the studied drugs on the native fluorescence of eosin at pH 3.4 and 3.2 for OLZ and FPZ HCl, respectively. The fluorescence was measured at 547 nm after excitation at 323 nm. The fluorescence-concentration plots were rectilinear over the range of 0.05-1.0 and 0.10-1.0 g/mL, with lower detection limits of 1.8 10-3 and 1.2 10-3 g/mL, for OLZ and FPZ HCl, respectively. The proposed method was successfully applied to the analysis of commercial tablets and ampules containing the drugs, and the results were in good agreement with those obtained with reference methods. The proposed method was further applied to the determination of OLZ in spiked human plasma. The mean recovery was 98.62 +/- 0.24% (n = 4). The method was also used for stability studies of FPZ HCl upon oxidation with hydrogen peroxide, and the kinetics of the reaction were studied. A proposal for the reaction pathway was postulated (Belal et al., 2008).

The aim of the present study was to develop rapid disintegrating tablets of Flupentixol dihydrochloride, a slightly bitter antipsychatric drug. An attempt has been made to prepare bitterless rapid disintegrating tablet using Eudragit E100 as a taste masking agent. The tablet was prepared with three super disintegrants e.g. sodium starch glycolate, cross carmellose sodium and crospovidone, each one was added in three different concentration 2%, 3% and 4%; mass extrusion was the technique used for the preparation of these tablets. The blend was examined for angle of repose, bulk density, tapped density, compressibility index and Hausners ratio. The compressed tablets were evaluated for hardness, drug content, friability, disintegration time in-vitro and in-vivo, wetting time and dissolution rate. The contents of the prepared tablets were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Different nine formulas showed in-vitro disintegration times ranges from 11.8 sec to 61 sec , but it was 150 sec for F1 ( formula without any super disintegrant) .These results were nearly correlated with in vivo disintegration times for the ten formulas. In vitro dissolution studies showed the release in the following descending order of super disintegrants: Crospovidone>Croscarmellose sodium> Sodium Starch Glycolate. Maximum in vitro dissolution rate was found to be with formulation F10 which contains crospovidone (4%). Thus, F10 was considered the best among the other formulations. The stability study was conducted as the International Conference on Harmonization (ICH) guidelines and the formulations subjected again for changes in hardness, friability, drug content, wetting time and disintegration time. Crospovidone at a concentration of 4% w/w is suitable for preparing rapid disintegrating tablet of Flupentixol dihydrocloride (Elbary et al., 2011).In the year of 1999 a study was performed to check the comparative effectiveness of Fluphenazine decanoate. Dose reduction strategies for the maintenance treatment of schizophrenia are designed to maintain the benefits of antipsychotic drug therapy while reducing risks. Previous strategies with decanoate preparations have been based on the use of lower doses per injection to achieve dose reduction; these strategies have achieved dose reduction but have resulted in some increase in symptoms. The authors tested a new dose reduction approach: increasing the interval between injections during intramuscular decanoate antipsychotic treatment. The two dose regimens did not differ significantly in relapse, symptom, or side effect measures. The every-6-weeks regimen was associated with a significant reduction in total antipsychotic exposure. The use of injections every 6 weeks instead of every 2 weeks may increase compliance and improve patients comfort as well as decrease cumulative antipsychotic exposure, without increasing relapse rates or symptoms. (Carpenter et al., 1999)In another study determination of Fluphenazine Dihydrochloride was done by using simple and cost effective UV-spectroscopy method in pure form and in pharmaceutical formulations. Methanol was selected as the solvent as the drug is highly soluble in it. The result was accurate and precise and there was no interference of the excipients (Yunus et al., 2011).2.3 Determination and analysis of Nortriptylineas a single drug or in combination with other drugs

The objective of this research is to develop a simple, sensitive and validated kinetic spectrophotometric method for the determination of nortriptyline hydrochloride in pure and pharmaceutical formulations. The method is based on derivatization of drug with1-chloro-2,4-dinitrobenzene (CDNB) in dimethyl sulfoxide (DMSO) medium at room temperature (30 10C). The reaction was followed spectrophotometrically by measuring the increase in absorbance at 390 nm as a function of time. Under the optimized experimental conditions, Beers law obeyed in the concentration range of 20-100 g/ml for both initial rate and fixed time methods. The limits of quantitation are 2.975 and 3.680 g/ml for initial rate and fixed time methods, respectively. The method has been successfully applied to the determination of nortriptyline in pharmaceutical formulations. Statistical comparison of the results with the reference method shows excellent agreement and indicates no significant difference in accuracy and precision (Rahman et al., 2014).

A method is described for the determination of nortriptyline in sugar-coated tablets. Nortriptyline is extracted into dilute aqueous hydrochloric acid containing triflupromazine hydrochloride as an internal standard. The acidic extract is then chromatographed by a reversed-phase separation using a basic solvent system in conjunction with a phenyl Corasil column. The main advantages of the method are the simple extraction, the over-all speed of analysis and the degree of precision obtained. The method has been used to monitor the stability of experimental formulations and could readily be modified for the assay of single tablets (Salmon and Wood, 1976).

Amitrityline and its metabolite nortriptiline are tricyclic antidepressant drugs widely used for the treatment of several psychiatric disorders. Several methods have been published for the determination of these two antidepressant drugs in pharmaceuticals, biological materials and environmental samples. In this review some of analytical techniques such as ultraviolet/visible spectrophotometry, fluorimetry, capillary electrophoresis, and chromatographic methods (gaschromatography and high-performance liquid chromatography) were discussed. Although HPLC and capillary electrophoresis methods are extensively employed in spite of that UV/VIS spectrophotometry are still popular because of the inherent simplicity, low cost, and reliability for determination of drugs in pharmaceutical preparations (Khatoon et al., 2013).

A spectrophotometric procedure is described for determination of nortriptyline hydrochloride in pure and dosage form as well as in the presence of its degradate. 3-Methyl-2-benzothiazolinone hydrazone (MBTH) has been used as the chromogenic reagent, where aqueous solutions of the drug and reagent are treated with cerium (IV) ammonium sulphate in an acidic medium. Nortriptyline hydrochloride reacts to give a blue coloured product having two absorption maxima at 619 and 655 nm. Various parameters affecting the reaction have been studied. Beer's law is obeyed in the concentration range of 24-216 mug ml-1 of nortriptyline hydrochloride, with mean percentage recoveries of 100.22 +- 0.870 and 100.66 +- 0.642% for both maxima, 619 and 655 nm, respectively. Results were statistically analyzed and compared with those obtained by applying the British Pharmacopoeia (1993) method (El-Ragehya et al., 2001).

Two simple, fast, and sensitive extractive spectrophotometric methods for determination of nortriptyline hydrochloride (NTPH) in pure and pharmaceutical formulations have been developed. These methods are based on the formation of chloroform-soluble ion-association complexes of NTPH and bromophenol blue (BPB) and bromopyrogallol red (BPR) in NaOAc-HCl buffer of pH 3.29. Absorption maxima of both complexes were recorded at 410 nm and at 425 nm, respectively for BPB and BPR. Reaction conditions were optimized to obtain the maximum color intensity. The systems obeyed Beers law in the analytes concentration ranges of 0.1-7.2 mg mL-1 and 0.5-23.4 mg mL-1 for BPB and BPR, respectively. The proposed methods are simple, accurate, and suitable for analysis of pharmaceutical formulations (Kumar et al., 2007).

Two simple, sensitive and rapid extractive spectrophotometric methods have been developed for the assay of the antidepressant drug nortriptyline (NOR) hydrochloride in pure form and in different dosage forms. The methods involve the formation of colored ion-pairs between the drug and the complex of niobium(V)-thiocyanate (Nb-SCN) or iron(III)-thiocyanate (Fe-SCN) followed by their extraction with butanol or a mixture of butanol and chloroform and quantitative determination at 360 nm and 490 nm, using Nb-SCN and Fe-SCN, respectively. The experimental conditions were optimized to obtain the maximum colour intensity. The methods permit the determination of nortriptyline over a concentration range of 15-100 microg/ml and 5-24 microg/ml with the detection limit of 0.84 microg/ml and 0.32 microg/ml, using Nb-SCN and Fe-SCN, respectively. The proposed methods are applicable for the assay of the investigated drug in different dosage forms and the results are in good agreement with those obtained by the official and HPLC methods. No interference was observed from common excipients present in pharmaceutical formulations. The proposed procedures were applied to determine the amount of nortriptyline hydrochloride as active ingredient in the presence of its degradation product, dibenzosuberone. The extractive spectrophotometric methods can also be used to determine the amount of nortriptyline hydrochloride in tablets after its solid phase extraction (SPE) (Misiuk and Tykocka, 2007).

Two simple, rapid and sensitive extractive spectrophotometric methods have been developed for the assay of nortriptyline hydrochloride (NTPH) in pure and pharmaceutical formulations. These methods are based on the formation of chloroform soluble ion-association complexes of NTPH with bromocresol green (BCG) and with methyl orange (MO) in KCl-HCl buffer of pH 2. The colored species exhibited absorption maxima at 416 and 422 nm for BCG and MO with molar absorptivity values of 2.88 104 and 2.29 104 L/mol cm, respectively. Reaction conditions were optimized to obtain the maximum color intensity. Various analytical parameters have been evaluated and the results have been validated by statistical data. The methods were successfully applied to the analysis of NTPH in pharmaceutical formulations (Manjunatha et al., 2009).

Three simple and selective methods are proposed for the determination of nortriptyline hydrochloride in bulk form and in tablets. The first two methods are based on the formation of charge-transfer complexes between the drug base as a n-donor and quinhydrone or p-chloranil as pi-acceptor. The products exhibit absorption maxima at 497 and 560 nm in acetonitrile for quinhydrone and p-chloranil, respectively. The third method is based on the interaction of N-alkylvinylamine formed from the condensation of the free secondary amine group and acetaldehyde with p-chloranil to give a vinylamino substituted quinone. The coloured product exhibits an absorption maximum at 650 nm in dioxane. All variables were studied to optimize reaction conditions. Beer's law was obeyed and the relative standard deviations were found to be less than 1.5%. The methods have been applied to the analysis of nortriptyline hydrochloride in the bulk drug and in tablets (Attia, 2000).

The effect of adding surface active agents to electrolytes containing nortriptyline hydrochloride on the voltammetric response of a hanging mercury drop electrode (HMDE) was studied. The current signal due to the reduction process was a function of the amount of nortriptyline hydrochloride, pH of the medium, type of the surfactant, and accumulation time at the electrode surface. Addition of Tween-20 to the nortriptyline hydrochloride containing electrolyte enhances the reduction current signal. Voltammograms of the drug with Tween-20 in Britton Robinson buffers of pH 2-11 exhibit a single well-defined reduction peak, which may be due to the reduction of -C horizontal line C group. The reduction process was irreversible over the entire pH range, and the mechanism of reduction was postulated on the basis of controlled potential electrolysis and coulometry. Application of Tween-20 in the electrochemical determination of nortriptyline hydrochloride using square-wave voltammetry at the HMDE enhanced the detection limit of the analyte concentration from 8.24 ng/mL in the absence of surfactant to 0.92 ng/mL when present (Jain et al., 2009).

The release of nortritptyline hydrochloride from oil-in-water (o/w) microemulsions (isopropyl myristate as oil, propylene glycol as cosurfactant, polysorbate 80 as surfactant and phosphate buffer, pH 7.4, as the continuous phase) containing increasing concentrations of polyethylene glycol 400, used to facilitate the diffusion of a drug from the inner oily phase of the microemulsion to the outer aqueous phase of such a dispersion system, was studied by determining the permeability constants of the drug through hydrophilic and lipophilic membranes separating the o/w microemulsions from the receiving aqueous phase (phosphate buffer pH 7.4). The permeability of nortriptyline hydrochloride from microemulsions through the lipophilic membrane increased as the concentration of polyethylene glycol 400 in the disperse system increased. The apparent permeability constant for nortriptyline hydrochloride, from the microemulsion without polyethylene glycol, was 1.36 x 10(-3) cm x h(-1), it increased up to 7.80 x 10(-3) cm x h(-1) in the presence of polyethylene glycol at a concentration of 50% (v/v) of the initial volume of the aqueous phase (Moreno et al., 2000).

In this work a novel method for the determination of nortriptyline in flow-injection systems has been developed. The proposed method was used for the fast determination of nortriptyline in its pharmaceutical formulations. The developed technique is very simple, precise, accurate, time saving, and economical, compared to all of the previously reported methods. The effects of various parameters on the sensitivity of the method were investigated. The best performance obtained at pH value of 2, scan rate value of 30 V/s, accumulation potential of 400 mV, and accumulation time of 0.5 s. The proposed method has some advantages over other reported methods such as, no need for the removal of oxygen from the test solution, a subnanomolar detection limit, and finally the method is sufficiently fast for the determination of any such compound, in a wide variety of chromatographic methods. The potential waveform, consisting of the potential steps for cleaning, accumulation and potential ramp of analyte, was continuously applied on an Au disk microelectrode (12.5 microm in radius). The detection limit of the method was 2.0 x 10(-11) M. The relative standard deviation of the method at 1.2 x 10(-8) M was 2.1% for eight runs (Norouzi et al., 2007).

Chapter ThreeMATERIALS & METHODS3.1 MATERIALS3.1.1 Sample CollectionFor the purpose of experimentation to observe the photolytic degradation of fluphenazine hydrochloride and nortriptyline hydrochloride as well as to assess the packaging efficiency, 300 tablets of Norflu (Fluphenazine 0.5 mg &Nortriptyline 10 mg) were collected from the local drug store in Dhaka as a sample. All the tablets were from the same batch (T1094007). Among them 100 tablets were kept light protected for control tests and the remaining 200 tablets were subjected to various lighting conditions over certain periods of time for conducting experiments to determine their potency.3.1.2 SamplesTable 3.1: Samples used in the experiment

Sample NameSource (Supplier Name)Batch No.

Norflu tabletsAcme pharmaceuticals LTD.T1094007

3.1.3 ReagentsTable 3.2: Reagents used in the experiment including sourceReagents NameSource (Supplier Name)

Concentrated Hydrochloric Acid (35 % )MERK, Germany

Distilled WaterLaboratory (East West University)

3.1.4Equipment & InstrumentsTable 3.3: Lists of equipment used for the experimentSerial No.EquipmentsSource (Supplier Name)Origin

1UV-SpectrophotometerShimadzu UV-1800Japan

2Distill Water PlantSMICChina

3Electronic BalancePrecisa XB120ASwitzerland

3.1.5 Images of Instruments

Some images of important instruments those were used in different tests during research work.

Figure 3.1: [Left to right] Shimadzu UV-1800 Double Beam Spectrophotometer and Electronic Balance

Figure 4.2: Distilled Water Plant3.1.6 ApparatusSome apparatus are listed in the following table those were used throughout the experiments.

Table 3.4: List of Apparatus used throughout this projectSerial No.Apparatus

1Beakers

2Thermometer

3Test tubes

4Volumetric Flasks (25ml,50 ml & 100 ml)

5Electric Bulb (25 Watt&40watt)

6Plastic Containers

7Aluminum foil paper

9Filter Papers

10Mortar & Pestles

11Spatula

13Glass Rod

14Pipette pumper

15Pipette (5 ml & 10 ml)

16Glass and Plastic Funnel

17Lamp

3.2 METHOD3.2.1. Preparation of the solvent (0.1N HCL)

1) Lab solvent (HCL) stock solution was collected and its strength was found to be 35%2) Then the concentration of the lab solvent stock solution was determined in NormalityDetermination of the Concentration of the Lab Solvent (HCL) in Normality (N):100 ml of the lab solvent stock solution contains --- 35 gm of HCL

1 ml of the lab solvent stock solution contains ------ (35 / 100) gm of HCL

1000 ml of the lab solvent stock solution contains -- ((35 x 1000) / 100) gm of HCL

= 350 gm of HCL

So, we know that

36.5 gm of HCL present in 1000 ml = 1 N

1 gm of HCL present in 1000 ml

= (1 / 36.5) N

350 gm of HCL present in 1000 ml= ((1 x 350) / 36.5) N

= 9.589 N (~ 9.6 N)3) After the determination of the concentration of the lab solvent stock solution in Normality (N), the amount of lab solvent (9.7N HCL) stock solution required to make 250 ml of 0.1N HCL solvent was calculated as below.Determination of the amount of 9.7N HCL required to make 250 ml of 0.1N HCL:

Using the V1S1 = V2S2,V1 = (V2S2) / S1=> V1 = (250 ml x 0.1 N) / 9.6 N

=> V1 = 2.604 ml (~ 2.6 ml) of lab solvent (HCL) stock solution

Thus 2.6 ml of 9.6 N HCL is required to be dissolved in 250 ml of water to dilute its concentration to 0.1 N HCL.

4) Then 2.6 ml of 9.6 N HCL was transferred from the lab solvent stock solution to a 250 ml volumetric flask which was then filled with water up to mark to make 250 ml of 0.1 N HCL.

3.2.2 Determination of max& Preparation of the Standard Curve of Fluphenazine hydrochloride and Nortriptyline hydrochloride1) Standards of both Fluphenazine hydrochloride and Nortriptyline hydrochloride was selected, Sanit of Square pharmaceuticals. The potency of both standard compounds were 99.99%

2) The specific max for both Fluphenazine hydrochloride and Nortriptyline hydrochloride, at which the absorbances would be measured, were determined from the UV spectrometer by using the standards that were obtained from.

3) The specific max for Fluphenazine hydrochloride was determined to be 260nm and for Nortriptyline hydrochloride, it was determined to be 239 nm.4) Ten serial concentrations of the Standards of Fluphenazine hyrdrochloride and Noetriptyline hydrochloride were prepared for the purpose of creating a standard curve.

Preparation of the stock solution for both Fluphenazine hydrochloride and Nortriptyline hydrochloride using the standard obtained from:

For Fluphenazine hydrochloride: 50 mg of the standard compound, that is Fluphenazine hydrochloride obtained fromSanitwas weighed and dissolved in 25 ml of 0.1N HCL (which is the solvent) in a 25 ml volumetric flask for the 1st dilution.Thus the concentration was calculated to be:

Concentration of 1st dilution = amount of substance added / volume = (50 / 25) mg/ml

= 2 mg/ml

Then 0.5 ml of that 2 mg/ml Fluphenazine hydrochloride solution was taken and dissolved in 50ml of 0.1N HCLThat 0.5 ml contained 1 mg of Fluphenazine hydrochloride. So the concentration finally turned out to be:Concentration of 2nd dilution = amount of substance added / volume = (1 / 50) mg/ml

= 0.02 mg/ml

Then 5 ml of that 0.02 mg/ml Fluphenazine hydrochloride solution was taken and dissolved in 50ml of 0.1N HCLThat 5 ml contained 0.1 mg of Fluphenazine hydrochloride. So the concentration finally turned out to be:Concentration of the stock solution = amount of substance added / volume = (0.1 / 50) mg/ml

= 0.002 mg/mlFor Nortriptyline hydrochloride: 50 mg of the standard of Nortriptyline hydrochloride was weighed and dissolved in 25 ml of 0.1N HCL in a 25 ml volumetric flask.

Thus the concentration of the 1st dilution was calculated to be:Concentration of 1st dilution = amount of substance added / volume = (50 / 25) mg/ml

= 2 mg/ml

Then 0.5 ml of that 2 mg/mlNortriptyline hydrochloride solution was taken and dissolved in 50 ml of 0.1N HCL.That 0.5 ml contained 1 mg of Melitracen hydrochloride. So the concentration final turned out to be:

Concentration of the stock solution = amount of substance added / volume = 1 / 50 = 0.02 mg/mlPreparation of five serial concentrations of solution for both Fluphenazine hydrochloride and Nortriptyline hydrochloride:

Both Fluphenazine hydrochloride and Nortriptyline hydrochloride each had the concentration of their stock solution as 0.002 mg/ml and 0.02 mg/ml respectively.

Six serial concentrations that were prepared for Fluphenazine hydrochloride were as follows 0.0001 mg/ml, 0.0002 mg/ml, 0.0003 mg/ml, 0.0004 mg/ml 0.0005mg/ml& 0.0006 mg/ml for a final volume of 10 ml. While for Nortriptyline hydrochloride the seven serial concentrations prepared were as follows 0.001 mg/ml, 0.002 mg/ml, 0.003 mg/ml, 0.004 mg/ml 0.005 mg/ml , 0.006 mg/ml & 0.007 mg/ml for a final volume of 10 ml.Table 3.5: List of Concentrations used for preparation of Standard Curve of Fluphenazine hydrochloride &Nortriptyline hydrochloride

Sample NameSample no.Concentration (mg/ml)

Fluphenazine10.0001

20.0002

30.0003

40.0004

50.0005

60.0006

Nortriptyline10.001

20.002

30.003

40.004

50.005

60.006

70.007

The solution that were required from the stock solution to prepare the above concentrations were calculated using S1V1=S2V2 formula, where S1= initial strength or concentration, S2= final strength or concentration, V1= initial volume and V2= final volume. Thus the following concentrations were prepared as such for Fluphenazine hydrochloride and Nortriptyline hydrochloride as per the calculations provided below.For Fluphenazine hydrochlorideV1= S2V2 / S1 = (0.0001 x 10) / 0.002 = 0.5 ml of stock solution required to make 0.001 mg/ml concentration of the final solution of 10 ml (0.5 ml of stock solution + 9.5 ml of 0.1N HCL) of Fluphenazine hydrochloride.V1= S2V2 / S1 = (0.0002 x 10) / 0.002 = 1 ml of stock solution required to make 0.002 mg/ml concentration of the final solution of 10 ml (1 ml of stock solution + 9 ml of 0.1N HCL) of Fluphenazine hydrochloride.V1= S2V2 / S1 = (0.0003 x 10) / 0.002 = 1.5 ml of stock solution required to make 0.003 mg/ml concentration of the final solution of 10 ml (1.5ml of stock solution + 8.5 ml of 0.1N HCL) of Fluphenazine hydrochloride.V1= S2V2 / S1 = (0.0004 x 10) / 0.002 = 2 ml of stock solution required to make 0.004 mg/ml concentration of the final solution of 10 ml (2 ml of stock solution + 8 ml of 0.1N HCL) of Fluphenazine hydrochloride.V1= S2V2 / S1 = (0.0005 x 10) / 0.002 = 2.5 ml of stock solution required to make 0.005 mg/ml concentration of the final solution of 10 ml (2.5 ml of stock solution + 7.5 ml of 0.1N HCL) of Fluphenazine hydrochloride.

V1= S2V2 / S1 = (0.0006x 10) / 0.002 = 3 ml of stock solution required to make 0.006 mg/ml concentration of the final solution of 10 ml (3 ml of stock solution + 7 ml of 0.1N HCL) of Fluphenazine hydrochloride.

For Nortriptyline hydrochlorideV1= S2V2 / S1 = (0.001 x 10) / 0.02 = 0.5 ml of stock solution required to make 0.001 mg/ml concentration of the final solution of 10 ml (0.5 ml of stock solution + 9.5 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.002 x 10) / 0.02 = 1 ml of stock solution required to make 0.002 mg/ml concentration of the final solution of 10 ml (1 ml of stock solution + 9 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.003 x 10) / 0.02 = 1.5 ml of stock solution required to make 0.003 mg/ml concentration of the final solution of 10 ml (1.5 ml of stock solution + 8.5 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.004 x 10) / 0.02 = 2 ml of stock solution required to make 0.004 mg/ml concentration of the final solution of 10 ml (2 ml of stock solution + 8 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.005 x 10) / 0.02 = 2.5 ml of stock solution required to make 0.005 mg/ml concentration of the final solution of 10 ml (2.5 ml of stock solution + 7.5 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.006 x 10) / 0.02 = 3 ml of stock solution required to make 0.006 mg/ml concentration of the final solution of 10 ml (3 ml of stock solution + 7 ml of 0.1N HCL) of Nortriptyline hydrochloride.V1= S2V2 / S1 = (0.007 x 10) / 0.02 = 3.5 ml of stock solution required to make 0.007 mg/ml concentration of the final solution of 10 ml (3.5 ml of stock solution + 6.5 ml of 0.1N HCL) of Nortriptyline hydrochloride.5) Then the Absorbance values were measured using a UV spectrophotometer against those five serial concentrations each for Fluphenazine hydrochloride and Nortriptyline hydrochloride.6) Two standard curves were plotted. One for Fluphenazine hydrochloride and the other for Nortriptyline hydrochloride.7) From those standard curve two straight line equations were obtained which were in the form of y = mx+c, where the components of the equations are described as provided below: m = gradient value, y = absorbance values, x = concentrations and c = y-intercept.3.2.3 Sampling, Analysis by UV-Spectrophotometry & Determination of Potency of the pharmaceutical drugs (fluphenazine + nortriptyline) under various lighting condition

To determine the photo-stability of the drug (Fluphenazine hydrochloride and Nortriptyline hydrochloride) in their packaging, the tablets were subjected to various types of light exposure, which were as follows:

I. Direct Sunlight exposureII. Dark condition

III. Electric Bulb exposure (25 watt& 40 watt)1. Under Sunlight condition

1. 36 tablets were kept exposed to sunlight condition for 7 hours at a stretch.Table 3.6: Direct Sunlight Exposed Sample ListTotal Number of SampleSample collectionInterval (hrs)Temperature (0C)

3691.537

9334

94.532

9630

2. After every 1.5 hours 9 tablets were collected and wrapped up with foil paper to prevent any further exposure to the lighting condition and the temperature noted using a thermometer.

3. The foil papers should be labeled to identify the intervals at which the drugs were collected.

4. The tablets were then used for potency determination to see the effect of the exposure of sunlight to the drug components, fluphenazine and nortriptyline.

5. For potency determination, laboratory analysis was done by using UV spectroscopy technique:a. First, three tablets from those sampled tablets were taken.

b. Then the total weight of those three tablets was noted using an analytical balance and the average weight (which is equal to the weight for one tablet containing both active ingredients and excipients) was calculated using the formula given below:

Average wt. (in grams) =Total wt. of the tablets No. of tablets

c. Then the three tablets were crushed by using mortar and pestle.

d. Next, approximately the weight of 1 tablet of crushed tablet powder was taken and dissolved in 50 ml of the solvent (0.1N HCl) (leading to 50 times dilution).

e. Afterwards that 50 ml solution was filtered and 10 ml of that filtered solution was taken from that 50ml of the solution of tablet powder and solvent and was dissolved in 25ml of the solvent (leading to another 2.5 times dilution; total dilution factor 125 times).

f. Next, 5ml of the solution was taken from that 125 times diluted solution and was dissolved in another 25ml of the solvent (0.1N HCL)(leading to 5 times dilution; total dilution factor = 50 x 2.5 x 5 = 625 times).

g. Then from there the solution was poured into a cuvette/container and was inserted into the UV spectrophotometer to observe the absorbance value for both Fluphenazine hydrochloride and Nortriptyline hydrochloride).

6. Then using the absorbance value obtained from UV spectrophotometer, the value was plotted into the standard curve to obtain the total amount of the drug that is present in one tablet

7. Steps 5 to 6 were repeated again for another sampling hour.

8. Under Dark Condition1. 36 tablets were kept in dark condition for 7 hours at a stretch.Table 3.7: Dark Condition Sample ListTotal Number of SampleSample collectionInterval (hrs)

3691.5

93

94.5

96

2. After every 1.5 hours 9 tablets were collected and wrapped up with foil paper to prevent any further exposure to the lighting condition and the temperature was noted using a thermometer.3. The foil papers should be labeled to identify the intervals at which the drugs were collected.4. The tablets were then used for potency determination to see the effect of the exposure of sunlight to the drug components, fluphenazine and nortriptyline.5. For potency determination, laboratory analysis was done by using UV spectroscopy technique:a. First, three tablets from those sampled tablets were taken.

b. Then the total weight of those three tablets was noted using an analytical balance and the average weight (which is equal to the weight for one tablet containing both active ingredients and excipients) was calculated using the formula given below:

Average wt. (in grams) =Total wt. of the tablets No. of tablets

c. Then the three tablets were crushed by using mortar and pestle.

d. Next, approximately the weight of 1 tablet of crushed tablet powder was taken and dissolved in 50 ml of the solvent (0.1N HCl) (leading to 50 times dilution).

e. Afterwards that 50 ml solution was filtered and 10 ml of that filtered solution was taken from that 50ml of the solution of tablet powder and solvent and was dissolved in 25ml of the solvent (leading to another 2.5 times dilution; total dilution factor 125 times).

f. Next, 5ml of the solution was taken from that 125 times diluted solution and was dissolved in another 25ml of the solvent (0.1N HCL)(leading to 5 times dilution; total dilution factor = 50 x 2.5 x 5 = 625 times).

g. Then from there the solution was poured into a cuvette/container and was inserted into the UV spectrophotometer to observe the absorbance value for both Fluphenazine hydrochloride and Nortriptyline hydrochloride).

6. Then using the absorbance value obtained from UV spectrophotometer, the value was plotted into the standard curve to obtain the total amount of the drug that was present in one tablet.7. Steps 5 to 6 were repeated again for another sampling hour.3. Under 25 W bulb lighting condition1) 36 tablets were exposed to 25 W lighting conditions for 7 hours at a stretch. Table 3.8: Electric (25 W) Bulb Exposed Sample ListTotal Number of SampleSample collectionInterval (hrs)Temperature (0C)

3691.527

9327

94.528

9628

2) After every 1.5 hours 9 tablets were collected and wrapped up with foil paper to prevent any further exposure to the lighting condition and the temperature was noted using a thermometer.

3) The foil papers should be labeled to identify the intervals at which the drugs were collected.

4) The tablets were then used for potency determination to see the effect of the exposure of 25 W bulbs lighting condition to drug ingredients: fluphenazine and Nortriptyline.

5) For potency determination, laboratory analysis was done by using UV spectroscopy technique:

a. First, three tablets from those sampled tablets were taken.

b. Then the total weight of those three tablets was noted using an analytical balance and the average weight (which is equal to the weight for one tablet containing both active ingredients and excipients) was calculated using the formula given below:

Average wt. (in grams) =Total wt. of the tablets No. of tablets

c. Tc. Then the three tablets were crushed by using mortar and pestle.

d. Next, approximately the weight of 1 tablet of crushed tablet powder was taken and dissolved in 50 ml of the solvent (0.1N HCl) (leading to 50 times dilution).

e. Afterwards that 50 ml solution was filtered and 10 ml of that filtered solution was taken from that 50ml of the solution of tablet powder and solvent and was dissolved in 25ml of the solvent (leading to another 2.5 times dilution; total dilution factor 125 times).

f. Next, 5ml of the solution was taken from that 125 times diluted solution and was dissolved in another 25ml of the solvent (0.1N HCl) (leading to 5 times dilution; total dilution factor = 50 x 2.5 x 5 = 625 times).

g. Then from there the solution was poured into a cuvette/container and was inserted into the UV spectrophotometer to observe the absorbance value for both Fluphenazine hydrochloride and Nortriptyline hydrochloride).

6) Then using the absorbance value obtained from UV spectrophotometer, the value was plotted into the standard curve to obtain the total amount of the drug that is present in one tablet

7) Steps 5 to 6 were repeated again for another sampling hour.4. Under 40 W bulb lighting condition1) 36 tablets were exposed to 40 W lighting conditions for 7 hours at a stretch. Table 3.9: Electric (40 W) Bulb Exposed Sample List

Total Number of SampleSample collectionInterval (hrs)Temperature (0C)

3691.532

9334

94.534

9634

2) After every 1.5 hours 9 tablets were collected and wrapped up with foil paper to prevent any further exposure to the lighting condition and the temperature was noted using a thermometer.

3) The foil papers should be labeled to identify the intervals at which the drugs were collected.

4) The tablets were then used for potency determination to see the effect of the exposure of 25 W bulbs lighting condition to drug ingredients: fluphenazine and nortriptyline.

5) For potency determination, laboratory analysis was done by using UV spectroscopy technique:

a. First, three tablets from those sampled tablets were taken.

b. Then the total weight of those three tablets was noted using an analytical balance and the average weight (which is equal to the weight for one tablet containing both active ingredients and excipients) was calculated using the formula given below:

Average wt. (in grams) =Total wt. of the tablets No. of tablets

c. T

c. Then the three tablets were crushed by using mortar and pestle.

d. Next, approximately the weight of 1 tablet of crushed tablet powder was taken and dissolved in 50 ml of the solvent (0.1N HCl) (leading to 50 times dilution).

e. Afterwards that 50 ml solution was filtered and 10 ml of that filtered solution was taken from that 50ml of the solution of tablet powder and solvent and was dissolved in 25ml of the solvent (leading to another 2.5 times dilution; total dilution factor 125 times).

f. Next, 5ml of the solution was taken from that 125 times diluted solution and was dissolved in another 25ml of the solvent (0.1N HCl) (leading to 5 times dilution; total dilution factor = 50 x 2.5 x 5 = 625 times).

g. Then from there the solution was poured into a cuvette/container and was inserted into the UV spectrophotometer to observe the absorbance value for both Fluphenazine hydrochloride and Nortriptyline hydrochloride).

6) Then using the absorbance value obtained from UV spectrophotometer, the value was plotted into the standard curve to obtain the total amount of the drug that is present in one tablet

7) Steps 5 to 6 were repeated again for another sampling hour.Chapter FourRESULT AND ANALYSIS

4.1 Standard curve preparation for NortriptylineThe standard curve was made from Sanit, produced by Square Pharmaceuticals Ltd. For different concentration of Nortriptyline hydrochloride we found different absorption. The results are as follows:Table 4.1: Concentration and Respective Absorbance for Standard Curve of Nortriptyline Hydrochloride

Concentration (mg/ml)Absorbance (at 239 nm)

0.0010.218

0.0020.277

0.0030.332

0.0040.362

0.0050.404

0.0060.466

0.0070.534

By plotting the absorbance against the concentration of Nortriptyline hydrochloride a straight line was found. From this an equation was derived where:

This equation was used to determine the concentration of Nortriptyline hydrochloride from different samples absorbance

Figure 4.1: Plot showing straight line for Absorbance with respect to Concentration for Nortriptyline4.1.1 Result from sample that was exposed to sunlight

For our research purpose we have exposed tablets to the sunlight condition. We found 4 different absorbance of Nortriptyline hydrochloride for four samples exposed to the sunlight and the temperature was recorded respectively 37C, 34C, 32C and 30C each for 1.5 hour time interval and it was observed that the concentration of Fluphenazine hydrochloride was declined in each 1.5 hour time interval.Table 4.1.1: Difference in Concentration and Absorbance in each 1.5 Hour time interval for Nortriptyline hydrochlorideTime IntervalAbsorbance (at 239 nm)Diluted Concentration from Samples in mg/ml (625 times diluted)Original Amount of Drug present

(mg)Potency (%)

0 hour1.0000.01579.8198.13

1.5 hour0.8030.01267.87578.75

3 hour0.7850.01237.68776.88

4.5 hour0.6910.01046.5065.00

6 hour0.4760.00613.81238.12

Figure 4.1.1: Graph showing the difference in Concentration after each 1.5 hours time interval for Nortriptyline hydrochloride4.1.2 Result from sample that was kept in dark

For our research purpose we have kept the tablets in dark condition. We found 4 different absorbance of Nortriptyline hydrochloride for four sample