The International journal of analytical and experimental modal analysis ISSN NO:0886-9367

METHOD DEVELOPMENT AND VALIDATION OF TRAZODONE HYDROCHLORIDE (HCL)

Madhu s. shewale*, Suhas siddheshwar, Mahesh kolhe Department of pharmaceutical quality assurance technique, Pravara rural college of pharmacy , pravaranagar ,Tal-rahata, Dist-ahamadnagar, 413736.

Abstract :

The objective of the present investigation was method development and validation of trazadone hydrochloride using u.v detection was developed and validated using C-18,250 x 4.6mm, 5u column and mobile phase composition was buffer(pot.Dihydrogen ortho phosphate):acetonitrile 65:35 at the flow rate of 1.0 ml/min. monitoring wavelength used was 248 nm, with u.v detection run time was selected to be 7 min because the peaks for trazodone hydrochloride were observed around 3.02 min. the method was validated as per ICH guideline for various parameters like accuracy ,linearity precision , robustness ,ruggedness, LOD, LOQ studies the precision of the system and method were checked and was found to be within the limits these indicates the method is precise correlation coefficient was found to be 0.999. the recovery value of pure drug found between 80.0% to 120.0% which indicates that method is accurate and also reveales that commonly used excipients and additives present in the pharmaceutical formulation were not interfering in the proposed analytical method passed both robustness and ruggedness test .on both cases ,relative standard deviation was well satisfactory . the proposed method can be used for routine analysis of trazodone hydrochloride commercial tablet dosage form in very less time.

KEYWORD : Trazodone Hydrochloride , method development, validation .

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INTRODUCTION :

Method Development And Validation Aspect By Assay Method ​ ​ Accurately reflect the purity characteristic of sample , assay procedure are intended to measure the analyte present . By using Analytical methods development and validation assume imperative parts in the discovery, improvement and preparation of pharmaceuticals. Method development is the way toward demonstrating that a analytical strategy is satisfactory for use to measure the concentration of an active pharmaceutical ingredient (API) in a particular formulated dosage form which enables simplified methods to be utilized to check that an analytical procedure, precisely and reliably will convey a reliable estimation of an active ingredient in an compounded preparation. The analytical strategy validation is important for analytical technique improvement and tested widely for specificity, linearity, exactness, accuracy, precision, range, detection limit, quantization limit, and robustness. In outline, analytical method development and validation permits to affirm that an exact and reliable potency estimation of a pharmaceutical preparation can be performed.

Importance of pharmaceutical analysis This analysis will disclose the categorization, recognition, and purpose of the raw materials, final products, and biological products. The numbers of different types of drugs are increasing rapidly at an alarming rate in the current condition. All these new entrances are either the new entities or the incomplete structural amendments of the exciting ones. To guarantee the safety of new drugs, it is imperative to use an appropriate pharmaceutical analysis.

At the present new analytical methods are approaching up for these new drugs and those amalgamations due to several motives. A few of them are subsequent. High Performance Liquid Chromatography / High Pressure Liquid Chromatography Is a form of liquid chromatography used to separate compound that are dissolved in solution Hplc instruments consist of :

1.Reservoir of mobile phase

2.A pump

3.An injector

4.Separation column

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5.Detector

❖ Reservoir of mobile phase – reservoir can be any clean ,inert container it usually contains

0.5-2L of solvent and it should have a cap that allows for a tubing inlet line which feeds mobile phase to solvent delivery system.

❖ Pumps-pushes mobile phase from reservoir and pulls to detector it helps to maintain

flow rate Columns- are usually long narrow tubes containing stationary phase.

❖ Injection system is way of introducing sample into mobile phase .

Detector in HPLC is used to monitor mobile phase that emerges from column .

MATERIALS AND METHODS:

APPARATUS:

Analysis of the drug was carried out on a 1. HPLC –thermo scientific (all parts) equipped with pump and U.V detector was used and coloumn 250x 4,6mm,C18 , 2. UV Labindia Model no.-T60, 3. IR jasco, Model no. –FTIR 4100, Oven- Oswald, balance make essae ,PH meter –Labindia Balance capacity Min 0.0001g Max220 g.

CHEMICAL AND MATERIAL :

Trazodone hydrochloride sample was obtained from piramal healthcare ltd . solvent used are acetotnitrile HPLC grade which were from qualigen pharma pvt.ltd, buffer (potassium dihydrogen orthophosphate ) AR grade from merck LTD . sonicator for remo val of small suspended gas bubbles ,for purpose of weighing analytical weighing balance (make : ESSAE,VIBRA+,CAPA: 220g to 0.0001 g ) was used

CHROMATOGRAPHIC CONDITION :

Quantification of trazodone hydrochloride was carried out on cromasil C18 column with dimension 250x4.6mm, the mobile phase used was in ratio 65%buffer(potassium dihydrogen ortho phosphate and acetonitrile 35% ,the flow rate of solvent optimised for the analysis was 1.0ml/min and was detected at 248nm wavelength, the injection volume of sample in HPLC system was 10ul .

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PREPARATIONS :

Preparation of Mobile Phase: Take 0.1 M Potassium Dihydrogen orthophosphate and Acetonitrile in a ratio of (65:35%) v/v.

Preparation of buffer : 0.1 pot. Dihydrogen orthophosphate in 1 lit mix and filter through 0.45um filter paper.

Preparation of Stock Solution : Take 5mg of TRAZADON HCL and dilute it upto 50ml of mobile phase which will make a stock solution of 100ppm

Standard solution : Take 10ml from above stock solution and dilute it up to 100ml of mobile phase which will makes a solution of 10ppm.

The primary objective of validation is to form a basis for the written procedure for production and process control which are designed to assure that the drug products have the identity, quality, and purity they purport are represented to possess.

Assurance of Quantity

Government Regulation

1.4.2 Validation parameters

Typical analytical parameters used in validation include:

❖ Specificity

❖ Precision

❖ Accuracy

❖ Linearity

❖ Range

❖ Robustness

❖ Limit of detection

❖ Limit of quantification

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❖ 1.4.2.1 Specificity

❖ It is an ability to assess unequivocally the analyte in the presence of components

which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.

❖ 1.4.2.2 Precision

❖ A Precision may be considered at three levels: repeatability, intermediate precision,

and reproducibility.

❖ Precision should be investigated using homogeneous, authentic samples. However, if

it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution.

❖ The precision of an analytical procedure is usually expressed as the variance, standard

deviation, or coefficient of variation of a series of measurements.

❖ Repeatability

❖ Repeatability expresses the precision under the same operating conditions over a short

interval of time. Repeatability is also termed intra-assay precision.

❖ Intermediate precision

❖ Intermediate precision expresses within-laboratories variations: different days,

different analysts, different equipment, etc.

❖ Reproducibility

❖ Reproducibility expresses the precision between laboratories (collaborative studies,

usually applied to the standardization of methodology).

❖ 1.4.2.3 Accuracy

❖ The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.

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❖ 1.4.2.4 Detection limit

❖ The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an exact value.

❖ Several approaches for determining the detection limit are possible, depending on

whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable.

❖ Based on Visual Evaluation

❖ A visual evaluation may be used for non-instrumental methods but may also be used

with instrumental methods.

❖ The detection limit is determined by the analysis of samples with known

concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.

❖ Based on Signal-to-Noise

❖ This approach can only be applied to analytical procedures that exhibit baseline noise.

❖ 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 between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

❖ Based on the Standard Deviation of the Response and the Slope

❖ The detection limit (DL) may be expressed as:

❖ DL = 3.3 σ / S

❖ where σ = the standard deviation of the response S = the slope of the calibration curve

❖ The slope S may be estimated from the calibration curve of the analyte. The estimate

of σ may be carried out in a variety of ways, for example:

❖ Based on the Standard Deviation of the Blank

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❖ Measurement of the magnitude of analytical background response is performed by

analyzing an appropriate number of blank samples and calculating the standard deviation of these responses.

❖ Based on the Calibration Curve

❖ A specific calibration curve should be studied using samples containing an analyte in

the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation.

❖ 1.4.2.5 Quantitation limit

❖ The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products.

❖ 1.4.2.6 Linearity

❖ The linearity of an analytical procedure is its ability (within a given range) to obtain

test results which are directly proportional to the concentration (amount) of analyte in the sample.

❖ Linearity should be evaluated by visual inspection of a plot of signals as a function of

analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation before the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares should be submitted. A plot of the data should be included.

❖ Also, an analysis of the deviation of the actual data points from the regression line

may help evaluate linearity. Some analytical procedures, such as immunoassays, do not demonstrate linearity after any transformation. In this case, the analytical response

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should be described by an appropriate function of the concentration (amount) of an analyte in a sample.

❖ For the establishment of linearity, a minimum of 5 concentrations is recommended.

Other approaches should be justified.

❖ 1.4.2.7 Range

❖ The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy, and linearity.

❖ 1.4.2.8 Robustness

❖ When a method is deliberately deviated by a small amount the robustness of the

method is a measure of its capacity to remain unaffected and gives the reliability during normal usage.

❖ Method validation is very tedious procedures but it is necessary for the newly

developed method since it shows the quality of data generated and it is directly linked to the quality of the procedure. Time constraints often do not allow for sufficient

21 method validations .​ ​

Introduction To Trazodone Hydrochloride:

Introduction: Trazodone hydrochloride is a hydrochloride salt prepared from equimolar amounts of trazodone and hydrogen chloride. It has a role as a serotonin uptake inhibitor, a ​ ​ ​ ​ H1-receptor antagonist, an adrenergic antagonist, a sedative and an antidepressant. contains a trazodone. Trazodone Hydrochloride is the hydrochloride salt form of trazodone, a ​ ​ ​ ​ synthetic triazolopyridine derivative with antidepressant and sedative properties. Based on studies from animal models, trazodone selectively inhibits the re-uptake of serotonin by ​ ​ synaptosomes in the brain, thereby increasing serotonin levels in the synaptic cleft and ​ potentiating serotonin activity. Trazodone is not a monoamine oxidase inhibitor and, unlike ​ ​ amphetamine-type drugs, does not stimulate the central nervous system. The sedative effect ​ of trazodone is likely via alpha-adrenergic and mild histamine H1 blocking actions. ​ ​

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Molecular formula of trazodone hydrochloride is C19H23Cl2N50 molecular weight : 408.3 g/mol. IUPAC NAME 2-[3-[4-(3-chlorophenyl)piperazin-1-yl]propyl]-[1,2,4]triazolo[4,3-a]pyridin-3-one;hydrochlo ride .

1.1 STRUCTURE OF TRAZODONE HCL .

Trazodone Hydrochloride (TRZ) is a well-known chemical compound that is used as an antidepressant that belongs to selective serotonin reuptake inhibitors (SARI). This ​ medication is used to treat depression. It may help to improve your mood, appetite, and ​ ​ energy level as well as decrease anxiety and insomnia related to depression. ​ ​ Trazodone is available in the form of 25 mg, 50 mg, 100 mg, 150 mg, and 300 mg ​ ​ ​ ​ ​ tablets for oral ingestion.An extended release formulation at 150 mg and 300 mg as tablets is also available. ​ ​

History

Trazodone was developed in Italy, in the 1960s, by Angelini Research Laboratories as a ​ ​ ​ second-generation antidepressant. It was developed according to the mental pain hypothesis, ​ ​ ​ which was postulated from studying patients and which proposes that major depression is ​ associated with a decreased pain threshold. In sharp contrast to most other antidepressants available at the time of its development, trazodone showed minimal effects on muscarinic cholinergic receptors. Trazodone was patented and marketed in many countries all over the

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world. It was approved by the Food and Drug Administration (FDA) in 1981 and was the first ​ non-tricyclic antidepressant approved in the US ​ ​

pharmacodynamics

Trazodone is generally described as acting as a potent serotonin 5-HT2A and α1-adrenergic ​ ​ ​ ​ ​ ​ ​ receptor antagonist, a weak serotonin reuptake inhibitor (SRI), and a weak antihistamine or ​ ​ ​ ​ histamine H receptor inverse agonist. Its 5-HT receptor antagonism and weak serotonin ​ ​1 ​ ​ ​2A reuptake inhibition form the basis of its common label as a serotonin antagonist and reuptake ​

inhibitor (SARI). Trazodone,​ both itself and via its major active metabolite ​ meta-chlorophenylpiperazine (mCPP), also binds to a variety of other receptors.] It is an ​ ​ antagonist at most or all of the receptors it binds to except the 5-HT receptor, where it acts ​ ​1A ​ as a partial agonist similarly to buspirone and tandospirone but with comparatively greater ​ ​ ​ intrinsic activity. Conversely, mCPP is a non-selective agonist of most of the serotonin ​ ​ ​

receptors it binds to. A range of weak affinities (Ki) have been reported for trazodone at the ​ ​ ​

human histamine H1 receptor including 220 nM, 350 nM,500 nM, and 1,100 nM ​ ​ ​ ​ ​ ​

Pharmacokinetics : Absorption: Trazodone is rapidly absorbed in the gastrointestinal tract after oral ​ administration , with a bioavailability ranging from 63-91% and an AUCO-t of 8193.0 ng.h/ML . Food may impact absorption in avariable fashion ,and may sometimes lead to decreases in the Cmax of trazodone.

1.3 Society and culture Generic names

Trazodone is the generic name of the drug and its INN, BAN, and DCF, while trazodone ​ ​ ​ ​ ​ ​ ​ hydrochloride is its USAN, USP, BANM, and JAN. ​ ​ ​ ​ ​ ​ ​ ​

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Brand names

Trazodone has been marketed under a large number of brand names throughout the world. Major brand names include Desyrel (worldwide), Donaren (Brazil), Molipaxin (Ireland, United Kingdom), Oleptro (United States), Trazorel (Canada), and Trittico (worldwide).

In 2019, the global Trazodone Hydrochloride (API) market size is anticipated to rise at a considerable rate during the forecast period, between 2020 and 2026.

❖ Method development :

• Mobile phase preparation with objective • Preparation of Mobile phase : • Take 0.1 M Potassium Dihydrogen orthophosphate and Acetonitrile in a ratio of (65:35%) v/v. ultrasonicated and filtered . • We are here using phosphate buffer because they help to maintain a constant ph level in particular environment , researcher generally try to maintain pH of 7.4 as often as possible because the property closely matches those of the human body . In combination of that we have used acetonitrile because it produce wide range of selectivity option in method development . Secondly because of its low u.v cut off it is used. And therefore by combining it gives accurate peak shape for further procedure

Trial 1. Water : Acetonitrile (60:40) ​

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Remark :

Peak shape is not proper hence method is rejected.

Trial 2. Buffer ( pot. Dihydrogen ortho phosphate: acetonitrile (65:35) ​

Remark : ​

This method is suitable for chromatographic separation, hence it is selected for further method validation.

• ASSAY: ​ • Preparation of sample stock solution: • For preparation of sample stock solution of 100ppm, take 19.81mg (50mg of API) of tablet powder and dilute it up to 50ml of mobile phase solution to gives 100ppm sample stock solution. • Preparation of sample working solution:

• Take 10ml of above sample stock solution and dilute it up to 100ml of mobile phase, It will gives 10ppm solution

• Preparation of standard stock solution: ​ • Take 10 mg of standard drug and dilute it up to 100ml of mobile phase which gives 100ppm stock solution. • Preparation of working standard solution: • Take 1ml of stock solution and dilute it up to 10ml of mobile phase which gives 10 ppm solution.

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ASSAY : Calibration curve of standard and sample ​

1. Calibration Curve For Assay At 10 ppm (Standard) ​

2. Calibration Curve For Assay At 10 Ppm (Sample)

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VALIDATION :

The analytical strategy validation is important for analytical technique improvement and tested widely for specificity, linearity, exactness, accuracy, precision, range, detection limit, quantization limit, and robustness. In outline, analytical method development and validation permits to affirm that an exact and reliable potency estimation of a pharmaceutical preparation can be performed.

Accordingly we have studied the following characteristics in systematic manner

❖ System suitability.

❖ Linearity.

❖ Accuracy.

❖ Precision.

❖ robustness

system suitability: ​ • • System suitability testing is a study planned to discover the performance of the system for analysis of selected analyte. • The study is performed by repeating the experiment in duplicate and assessing the chromatogram observed with respective retention time, peak area, number of theoretical plates, tailing factor, resolution etc • The results of %RSD for retention time and peak area was found to be 0.22 and 0.31 respectively. The outcomes are discovered in accordance with the standards prearranged for system suitability testing as per ICH Q2R1 procedure.

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1. Data observed in system suitability testing .

SR NO. RT AREA T.P. ASYMETRY

1 3.18 176917 5838 1.23

2 3.20 177716 5810 1.23

AVG 3.19 177316.5

SD 0.01 399.5

%RSD 0.31348% 0.225303%

2. Outcomes Recorded In System Suitability Testing

Sr. No. Parameter Mean SD %RSD Acceptance Inference observations criteria

1 Peak Area 177316.5 399.5 0.225303 < 2 Complied

2 Retention 3.19 0.01 0.31348 < 1 Complied time 3 Number of 5824 -- > 2000 Complied Theoretica l plates

4 Tailing 1.23 -- < 2 Complied factor CONCLUSION :

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The results of %RSD for retention time and peak area was found to be 0.22 and 0.31 respectively. The outcomes are discovered in accordance with the standards prearranged for system suitability testing as per ICH Q2R1 procedure.

LINEARITY:

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation before the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line, and residual sum of squares should be submitted. A plot of the data should be included.

Linearity of the method was evaluated by injecting series of standard concentrations of TRAZADON HCL to fitting chromatographic conditions. The study was performed in duplicate for each standard concentration tested by RP-HPLC method. Standard concentrations of TRAZADON HCL were measured in duplicate and mean peak area ​ analogous to each concentration was integrated. Table 1.1 showed the acquired mean peak ​ ​ area equivalent to strength

1. 1 Calibration Curve Of Trazodone HCL

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1. Observation table for linearity

SR.NO. Concentrations

1. 5 PPM 10 PPM 15 PPM 20 PPM 25 PPM 30 PPM

2. 89312 172095 265286 347492 441709 531046

3. 89946 172767 265526 348306 442962 526623

AVG 89629 172431 265406 347899 442335 528836

CONCLUSION :

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The correlation coefficient, y-intercept, slope of the regression line were observed to be 0.999, 606.07, 17598 respectively. Regression coefficient was near to unity, for this reason, it was accomplished that the projected system instituted to be linear.

ACCURACY: The accuracy of an analytical procedure expresses the closeness of ​ ​ agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness.

• Preparation of sample working solution: • Take 10ml of above sample stock solution and dilute it up to 100ml of mobile phase, It will gives 10ppm solution. • Preparation of working solution for 80% Recovery • Take 1 ml of sample stock solution (10ppm) +0.8 ml of standard stock solution (8ppm) and dilute it up to 10 ml, to gives 18 ppm working sample solution. • Preparation of working solution for 100% Recovery • Take 1 ml of sample stock solution (10ppm) +1.0 ml of standard stock solution (10ppm) and dilute it up to 10 ml, to gives 20 ppm working sample solution. • Preparation of working solution for 120% Recovery • Take 1 ml of sample stock solution (10ppm) +1.2 ml of standard stock solution (12ppm) and dilute it up to 10 ml, to gives 22 ppm working sample solution

1.Observation table for accuracy ​

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Sample Amount % SD Amount Sr. % Area of Recovered % Mean NO. Composition Sample in ppm % Recovery Recovery

Sample Amoun Amoun t Added t in in ppm ppm

1 10 8 313057 18.00 100.00 0.149628

10 8 312457 17.96 99.81 99.99 80% Recovery 10 8 313606 18.03 100.18

2 10 10 345613 19.96 99.84 0.163723

10 10 345904 19.98 99.92 99.99 100% Recovery 10 10 346934 20.04 100.22

3 10 12 230675 22.08 100.40 0.684501

10 12 381252 22.12 100.55 99.99 120% Recovery 10 12 375481 21.78 99.03

CONCLUSION : Here we can conclude that the refer value and value found are very close . Therefore accuracy study was performed successfully.

PRECISION :

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The precision of an analytical procedure is usually expressed as the variance, standard deviation, or coefficient of variation of a series of measurements.

• The precision of an analytical procedure expresses the closeness of agreement (degree of scattering) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. • Precision for this method was recognized by setting two quality control standards from corner to corner of the given calibration range of 5 to 30μg/ml.

INTERDAY ( OBSERVATION TABELS) DAY 1.

Sr .No. 7 PPM 17 PPM

RT AREA RT AREA

1 3.16 119858 3.15 294546

2 3.15 120128 3.15 294224

AVG 3.155 119993 3.15 294385

sd 0.005 135 0 161

RSD 0.158 0.112 0 0.05469

DAY 2 ​

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Sr .No. 7 PPM 17 PPM

RT AREA RT AREA

1 3.22 121738 3.19 290523

2 3.21 121261 3.17 295804

AVG 3.215 121499 3.18 293163

sd 0.005 338.5 0.01 2640.5

RSD 0.155 0.196 0.314 0.900629

INTRADAY

1.MORNING : ​

Sr .No. 7 PPM 17 PPM

RT AREA RT AREA

1 3.25 122833 3.22 292900

2 3.25 118567 3.23 287914

AVG 3.24 120618 3.23 292682

sd 0.018 2137.73 0.005774 4662.34

RSD 0.56 1.78 0.18 1.6

EVENING:

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Sr .No. 7 PPM 17 PPM

RT AREA RT AREA

1 3.20 120168 3.21 296546

2 3.21 122297 3.20 298503

AVG 3.21 121232 3.205 297524

sd 0.0 1064.5 0.005 978.5

RSD 0.0 0.878065 0.156006 0.32888

CONCLUSION : system precision was determined on two different days with ​ indistinguishable samples of two quality control standards. From the predetermined calibration range two QC standards were defined viz. 7 and 17ppm correspondingly. The solutions for QC standards were prepared by diluting main stock standard solution of 0.7 and 1.7ml up to 10ml with mobile phase.

ROBUSTNESS : • When a method is deliberately deviated by a small amount the robustness of the method is a measure of its capacity to remain unaffected and gives the reliability

during normal usage.

• The robustness of planned method was obtained by analysis of sample from homogenous sets by differing physical parameters like change in flow rate and wavelength which may differ, but the responses were still within the limits of the assay.

OBSERVATION TABLE FOR ROBUSTNESS:

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Level TRAZODON HCL

Area % Recovery

Change in Flow rate (ml/min)

(0.9ml) 181937 100.00

182319

(1.1ml) 182319 1.17

Change in Wavelength (nm)

(247nm) 173049 1.17

171545

(249nm) 178183 1.17

179075

CONCLUSION :

The robustness of planned method was obtained by analysis of sample from homogenous sets by differing physical parameters like change in flow rate and wavelength which may differ, but the responses were still within the limits of the assay.

Result And Conclusion :

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The presented project work was planned to develop simple, accurate, precise, and cost effective RP-HPLC method for quantification of TRAZODON HCL by ASSAY METHOD ​ ​ in bulk as API and to explore its applicability for marketed formulation. The method as per predefined objectives was successfully developed and validated.

At the beginning, procured reagents and chemicals were characterized for its transparency by determination of melting point and absorption maximum. This was particularly done for API TRAZODON HCL. The RP-HPLC method was executed for setting up chromatographic ​ conditions like organic concentration of the mobile phase, flow rate, aqueous phase and its pH, column oven temperature, injection volume etc. The optimized and final conditions were set as mobile phase composition, flow rate, wavelength, injection volume and temperature as Buffer 65: Acetonitrile 35 at, 1.0ml/min, 248nm and 10μl.

Further system suitability testing was performed in order ascertain the qualifying criterion of the method. The observed results in terms of percent RSD for mean peak area (0.59) and mean retention time (0.24) for this experiment were observed in conformity as per ICH guideline Q2R1.

Moreover, the projected method was validated as per ICH guideline Q2R1 for consequent parameters to confirm the appearance of the process throughout program investigation of TRAZODON HCL in product life cycle. ​ a) Linearity b) Precision c) Accuracy d) Robustness e) Assay The linearity of the method was observed in the standard concentration range of 05 to 30ppm with correlation coefficient of 0.9997. The equation of line was calculated to find out slope and intercept of regression line.

The method was assessed for interchangeable presentation on numerous measurements. The precision of the method was studied by repeatability and intermediate precision experiment. The outcomes attained depicted that the method achieved outstanding results in terms of %RSD. Also correlation coefficients observed on frequent injections of all quality control standards were seen inside acquiescence criterion as per ICH Q2R1 guidelines.

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Robustness of the method was evaluated by examining the outcome of persistent alterations in method conditions.

The applicability of the method was explored in finished product marketed formulation (Tablet dosage form). The %recovery experiment carried out at three levels viz. 80%, 100% and 120% was in agreement with the principles prearranged for TRAZODON HCL. ​ ​ As a result, eventually it was accomplished that, the author attained all predefined objectives by conquering development and validation of ASSAY METHOD by RP HPLC method for ​ quantification of TRAZODON HCL. Besides, the method was successfully tested for its ​ ​ applicability for custom analysis of TRAZODON HCL in tablet dosage form. The method ​ was also proved explicit for estimation of TRAZODON HCL in sample matrix of tablet ​ dosage form (marketed formulation).

8. REFERENCE

1) Harris, Daniel C. (2006), Quantitative Chemical Analysis, 7th Edition, W. H. Freeman 2) Mermet, Jean-Michel, Otto, Matthias and Valcarcel Cases, Miguel (2005), Analytical chemistry : a modern approach to analytical science, 2nd Edition, Wiley-VCH 3) Mikus P, Novotny L, Research & Reviews: Journal of Pharmaceutical Analysis, 2015, pp. 13-14. 4) Townshend A. (1995) Encyclopaedia of Analytical Science, Volumes 1-10, Academic Press, London. 5) Bernard Fried (2003). Handbook of Thin-Layer Chromatography., Marcel Dekker Inc. ISBN 978-0824748661. 6) Schindler H, Journal of Chemical Education, 1957, 34(11), pp. 528. 7) Ninfa Alexander J, (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology. 8) Paul-Dauphin S, Karaca F, Morgan T J, et al., Energy & Fuels., 2007, 21 (6), pp. 3484–3489.

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9) Wilchek M, Chaiken I, 2000, "Chapter 1 An Overview of Affinity Chromatography". In Bailon P, Ehrlich G K, Fung W J, Berthold W, (eds.), Affinity Chromatography. Methods in Molecular Biology, 147, Humana Press. pp. 1–6.

th 10) Skoog D A, Holler F J, Crouch S. R. Instrumental Analysis, 6 ​ ed, Thomson ​ Brook/cole, 2007, pp. 13-16, 378-385, 901-905, 893-900. 11) Gerber, F.; Krummen, M.; Potgeter, H.; Roth, A.; Siffrin, C.; Spoendlin, C, Journal of Chromatography A., 2004, 1036(2), pp. 127–133. 12) https://www.thermofisher.com/in/en/home/industrial/chromatography/liquid- chromatography- lc/hplc-uhplc-components/hplc-uhplc-pumps.html retrieved on 5 ​ June, 2019. 13) Perveen, S, Yasmin A, Khan, K M, The Open Analytical Chemistry Journal, 2009, 3, pp. 1–5. 14) Vander Wal S., Snyder LR. J. Chromatograph. 1983, 225, 463. 15) Kuksis A, Encyclopedia of Separation Science, 2000, pp. 3237-3253. 16) IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2006–) "reversed-phase chromatography 17) Snyder, Lloyd R, Dolan, John W, High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model., 2006, Wiley Interscience. 18) Snyder L R, Dolan J W, (2006). High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model. Wiley Interscience. 19) Scott R P W, Technique and Practice of chromatography, Marcel Dekker, New York, 70, pp. 1-12. 20) United States Pharmacopoeia, First Supplement to USP 40–NF 35 pp. 1-12.

21) ICH, Q2R1, Text on validation of analytical Procedures International Conference on ​ ​ ​ ​ harmonization, Geneva, 1994, October, pp. 1-13. 22) https://www.drugbank.ca/drugs/DB00656

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