Bio-Analytical Method Development and Validation for The Estimation of Tapentadol Hydrochloride in Human Plasma By RP-HPLC Method

Pharmaceutical Analysis is the branch of chemistry involved in separating, identifying and determining the relative amounts of the components making up a sample of matter. It is mainly involved in the qualitative identification or detection of compounds and quantitative measurements of the substances present in bulk and pharmaceutical preparation. Analytical instrumentation plays an important role in the production and evaluation of new products and in the protection of consumers and the environment. This instrumentation provides the lower detection limits required to assure safe foods, drugs, water and air.

Types

There are mainly two types of chemical analysis:

1. Qualitative (Identification)
2. Quantitative (Estimation)

1. Qualitative analysis is performed to establish composition of natural/synthetic substances. These tests are performed to indicate whether the substance or compound is present in the sample or not. Various qualitative tests are detection of evolved gas, formation of precipitates, limit tests, color change reactions, melting point and boiling point test etc.

2. Quantitative analytical techniques are mainly used to quantify any compound or substance in the sample. These techniques are based on (a) the quantitative performance of suitable chemical reaction and either measuring the amount of reagent added to complete the reaction or measuring the amount of reaction product obtained. (b) The characteristic movement of a substance through a defined medium under controlled conditions. (c) Electrical measurement. (d) Measurement of some spectroscopic properties of the compound.

Role Of Bioanalysis in Pharmaceutical Drug Development ⁽⁶⁾

Quantitative determination of drugs and their metabolites in biological fluids is termed as bioanalysis. This technique is used very early in the drug development process to provide support to drug discovery programs on the metabolic fate and pharmacokinetics of chemicals in living cells and in humans. Bioanalytical methods play a major role in estimating the drugs, interferences, metabolites from various matrices such as pure drug, dosage form, intermediates and biological fluids. Drug assay technology is now sufficiently advanced for it to be possible to measure the plasma concentration of majority of drugs used in clinical practices. They are useful to measure plasma concentration of drugs to confirm adequate dosage, to identify signs of possible drug toxicity, the response of patients to drug therapy and drug interactions. When a drug is administered orally it passes through the GIT and enters the systemic circulation undergoes metabolism, finally it is excreted as such or in the form of its metabolites. The studies on biological fluids is very challenging and time consuming , but these studies are necessary and utmost  important because biological fluids like blood, urine, cerebrospinal fluid and milk etc containing a relative quantity of drug and their metabolites can be known. Bio-analytical method which gives accurate and reproducible results has been increased significantly now a day. Therapeutic efficacy of the particular drug can be known by bioanalysis. In Pharma field bioanalysis plays a significant role. Bioanalysis involves the following steps.

5. Selection and collection of biological fluid.
5. Preparation of sample –Analyte extraction from biological matrix.
5. Analyte detection done by various methods.

Chemical profile

Brand name:

Nucynta, Nucynta ER, Niap

Chemical name/IUPAC NAME:

3-[(1R, 2R)-3-(Dimethylamine)-1-ethyl-2-methylpropyl] phenol hydrochloride

Molecular formula:

C₁₄H₂₃NO .HCL

Mol. mass: 221.339 g/mo

Fig 6: Molecular Structure of Tapentadol Hydrochloride

HCL

Pharmacokinetic data:

Bioavailability : 31.9 ± 6.8% (oral)

Half-life: 4 hrs 30 to 50hours.

Metabolism : Hepatic glucuronidation and sulfate conjugation

Half-life: 4 hrs

Excretion: Renal (>95%) and feca

Routes: Oral, Oral, Other ROA Unknown

Description:

White or off-white powder

METHODOLOGY 

1. High Performance Liquid Chromatography

1.1 Materials and Instruments used

1.1.1. Drug sample and Study products-

Tapentadol hydrochloride was obtained from MSN laboratories Ltd, hyderabad, india.

Test product:

Tapentadol hydrochloride (NIAP) (Reddy’s Laboratories Ltd) Tablets were purchased from the local market.

Plasma: Blank plasma was collected from Kovai Medical Center and Hospitals, Coimbatore.

1.1.2. Chemicals and solvents used for estimation-

• HPLC Water -Qualigens, Mumbai, India.
• Acetonitrile- Rankem, Mumbai, India.
• Methanol -Himedia, Mumbai, India.
• Distilled water -Double Distilled water.

• Potassium Di-hydrogen phosphate        Analytical grade, SD fine chemicals, Mumbai.
• Sodium hydroxide -Analytical grade, SD fine chemicals Mumbai.
• Orthophosphoric acid-SD fine chem. Ltd, Mumbai.

Instruments used:

• Elico pH meter LI 127.
• Shimadzu LC-20 AT HPLC.
• SPD-M20A Prominence diode array detector.
• Shimadzu 1700 LC-UV Spectrophotometer.
• Sonica ultrasonic cleaner.
• Solvent filtration unit – Millipore.
• Shimadzu electronic balance AY 220.
  2. Ultra cooling centrifuge – Remi, India

Optimization Of Chromatographic Condition for The Estimation of Tapentadol Hydrochloride ⁽³⁷⁾

Selection of Wavelength-

An uv spectrum of 100 µg/ml tapentadol hydrochloride in methanol was recorded by scanning in the range of 200 nm to 400 nm. A wavelength which gives good response for the drugs to be detected is to be selected. From the uv spectrum a wavelength of 275 nm was selected. Tapentadol hydrochloride showed optimal absorbance at this wavelength.

Selection of chromatographic method

Selection of proper chromatographic method depends on the nature of the sample or its properties like ionic/ionizable/neutral character, its molecular weight and solubility. The drug selected for the present study was polar in nature hence, reverse phase HPLC or ion-pair or ion–exchange chromatography method must be used. Because of its simplicity and suitability for initial separations reverse phase method was selected.

Initial chromatographic conditions for separation of tapentadol hydrochloride

Standard solution: 

100μg/ml of tapentadol hydrochloride was prepared by dissolving in 10 ml of HPLC grade methanol.

Equipment

System: Shimadzu gradient HPLC

Pump: LC-20AT prominence solvent delivery system

Detector: SPD-M20A prominence diode array detector

Injector: Rheodyne 7725i with 20µl loop

Chromatographic conditions – 1

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – water

Solvent B - acetonitrile

Solvent ratio: 50: 50 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

Fig 8 - Chromatographic Conditions – 1

Chromatographic Conditions – 2

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – water 

pH adjusted to 4 (with orthophosphoric acid)

Solvent B - acetonitrile

Solvent ratio: 50: 50 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

Fig 9 - Chromatographic Conditions – 2

Chromatographic Conditions – 3

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – water 

pH adjusted to 4 (with orthophosphoric acid) added triethylamine

Solvent B - Acetonitrile

Solvent ratio: 50: 50 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

Fig 10 - Chromatographic Conditions – 3

Chromatographic Conditions – 4

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – Phosphate buffer 

pH adjusted to 3.8 (with sodium hydroxide)

Solvent B - acetonitrile

Solvent ratio: 60: 40 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

Fig 11 - Chromatographic Conditions – 4

Tapentadol hydrochloride was eluted at retention time of 9.3 minutes with peak tailing, broading.

Chromatographic conditions – 5

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A –     Phosphate buffer 

pH adjusted to 5.8 (with sodium hydroxide)

Solvent B - Acetonitrile

Solvent ratio: 50: 50 (A: B)

Detection wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Tapentadol hydrochloride was eluted at retention time of 3.9 minutes and 6.2 for IS with peak tailing, fronting, and splitting. hence not selected further for method development.

Chromatographic Conditions – 6

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – Phosphate buffer pH adjusted to 6.8 (with sodium hydroxide)

Solvent B – Methanol: Acetonitrile (50: 50)

Solvent ratio: 45: 55 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

Fig 13 - Chromatographic Conditions – 6

Tapentadol hydrochloride was eluted at 6.4mins and internal standard at 8.6mins with perfect peak properties, hence selected for further studies.     

1. 1. 4. Effect of pH

 Using acetonitrile + methanol(50:50) and buffer solution of different pH ranging from 3.5, 5.8, 6.2, 6.8 at 275 nm, the standard solution was runned up to 20 min at a flow rate of 0.8 ml/min, 1.0 ml/min and 1.2 ml/min. The retention time of tapentadol hydrochloride was 11.5, 8.2,5.4, 6.4minutes respectively. For the present study, a pH of 6.8 was selected as the chromatogram obtained with this pH was symmetrical in shape.

1. 1. 5. Effect of ratio of mobile phase

Mixture of phosphate buffer adjusted pH with sodium hydroxide and acetonitrile + methanol(50:50) with 70:30, 60:40, 50:50 and 45:55 ratios were used as the mobile phase. At 45:55 ratios, symmetric peaks were eluted at 6.4 mins and 8.6 mins for tapentadol hydrochloride and internal standard (Ibuprofen) respectively. At 60:40 and 50:50 ratios the peaks were asymmetrical in shape. Thus for the present study 45:55 ratio of phosphate buffer (sodium hydroxide) and acetonitrile+methanol(50:50) was selected as the mobile phase

1. 1. 6. Effect of flow rate

Keeping the mobile phase ratio at (45:55, v/v) phosphate buffer methanol+ acetonitrile, the chromatograms were recorded at a flow rate of 0.8ml/min, 1.0ml/min 1.2ml/min. At flow rate of`1.0ml/min, the peaks were sharp and separated with good resolution. Hence, 1.0ml/min was kept constant for the present analysis.

1. 1. 7. Selection of internal standard

Based upon polarity and solubility, aceclofenac, piroxicam, lamivudine and ibuprofen were selected and chromatographed along with the standard drug. The elution time of ibuprofen was 8.6 min. The peak of ibuprofen was symmetric and well resolved from the peak of the tapentadol hydrochloride. Hence, for the present study ibuprofen was selected as the internal standard.

1. 1. 8. Fixed chromatographic conditions

The following chromatographic conditions were used for the estimation of tapentadol hydrochloride in human plasma.

Stationary phase: Phenomenex C18 column

Mobile phase: Solvent A – phosphate buffer the pH adjusted to 6.8 (with sodium hydroxide)

Solvent B – methanol: acetonitrile (50:50)

Solvent ratio: 45: 55 (A: B)

Detection Wavelength: 275 nm

Flow rate: 1.0 ml/min

Sample size: 20 µl

Needle wash: HPLC grade water

Temperature: Room temperature (25ºC)

1. 3. Pretreatment method for biological fluid:

Method of sample preparation is an important criterion for biological samples. For the present study plasma was obtained from clinical laboratory services of KMCH hospital, coimbatore. solid phase extraction method was selected for the present study.

Extraction by SPE

Into the solid phase extraction cartridge added 1 ml of methanol with vacuum assistance. Added 0.5 ml of plasma that had been in-spike with 0.25ml tapentadol hydrochloride and 0.25ml ibuprofen drop by drop. Added 1 ml tetrahydrofuran. Analyte was eluted with 1ml methanol. Analyte was collected and injected into the HPLC and SPE extraction efficiency was calculated.     The chromatograms of plasma extracted with methanol, acetonitrile and methanol mixture, were recorded using the fixed chromatographic conditions. The chromatogram of blank plasma without any drug was also recorded. Based up on the percentage recovery methanol was selected as eluting solvent for the present study because of its higher percentage recovery.

1. 1. 1. Preparation of standard stock solution:

 Stock solution of tapentadol hydrochloride and internal standard 1mg/ml were prepared separately by dissolving 10mg of each drug in 10ml standard flasks and the volume was made up to 10 ml with the mobile phase.

1. 1. 2. Working standards:

From the stock solution working standard solutions of 100µg/ml was prepared by diluting 1ml to 10ml with mobile phase. Further solutions were made by from the above solution by diluting 0.1ml, 0.2ml, 0.3ml, 0.4ml, and 0.5ml standard solutions to 10ml in a standard flask with mobile phase to get effect concentrations of 1, 2, 3, 4, and 5µg/ml respectively. In the similar way the working standards were prepared for internal standard also.

1. 1. 3. Preparation of standard graph:

Preparation of calibration standards

SPE catridge was conditioned with 1ml of methanol. Later 0.5ml of blank plasma 0.25ml of working standard solution of tapentadol hydrochloride and 0.25ml of internal standard working solutions were added to get concentration of 250, 500, 750, 1000, and 1250 ng/ml respectively. Then passed through SPE catridge. Impurities are removed by passing 1ml of tetra hydrofuran. After washing, analytes are eluted with 1ml of methanol and a quantity of 20µl was injected into the HPLC column and chromatograms were recorded. Standard calibration graph was plotted using ratio of peak area of tapentadol hydrochloride to its concentration.

1. 4. Estimation Of Tapentadol Hydrochloride In Human Plasma:

Recording the chromatogram

The optimized chromatographic conditions were maintained to record the chromatograms of the calibration standards of tapentadol hydrochloride and sample from a clinical study. First, baseline stabilization was done for about 20 minutes. Then standard solutions, calibration standard solutions and sample from clinical study containing tapentadol hydrochloride were injected and chromatograms were recorded.

1. 1. 1. Validation Of The Method

After developing a method its validation is necessary to prove the suitability of the method for the intended purpose. Here the procedure followed for the validation of the developed method is described.

a) Precision:

Intraday and interday precision studies were conducted. In intraday precision plasma sample containing drug at three different concentrations with internal standard were injected and chromatogram was recorded. Similarly interday precision over a two week period time was evaluated.

Acceptance criteria:

RSD of the mean concentration of five readings should be less than 15% for bioanalytical method.

b) Accuracy:

It is the closeness of mean tests results obtained by the method to true concentration of analyte. It is also named as trueness. In this studies the selected concentration of the plasma were injected six times and mean peak area for each concentration was calculated. Concentration of the each injection was calculated and the standard deviation between the readings is calculated. To bioanalytical study the percentage RSD should be less than 15%.

c) Recovery studies:

The relative recovery of drug from plasma was calculated by comparing the readings of concentration obtained from the drug spiked plasma to that of equal concentration from standard sample.  Recovery studies were carried out six times for sample concentration at three levels within the calibration curve.

Acceptance Criteria:

For an assay method, mean recovery should be 85-105% ± 2%.

d) Linearity and Range:      

Linearity and range were estimated by using calibration curve. By using calibration standards prepared by spiking plasma (Tapentadol hydrochloride) and internal standard (Ibuprofen) at different concentrations like 250ng/ml to 1250ng/ml the calibration graph was plotted taking concentration of spiked plasma on x-axis and peak area on y-axis. The linearity is determined from 50% to 150% of the proposed concentration.

Acceptance Criteria:

Coefficient of correlation of the calibration should be not less than 0.99

e) Lower Limit of Quantification (LLOQ):

The LLOQ is determined by using the calibration curve. Limit of quantitation is the concentration of substance in the sample that will give a signal-to-noise ratio of 10:1. Detection limit corresponds to the concentration that will give a signal-to-noise ratio of 3:1. The signal to noise ratio were performed by comparing measured signal of blank plasma sample with those of known low concentration of drug.

f) Specificity:

Specificity of the method was demonstrated by using diode array detector peak purity test. The diode array spectrum of both standard and sample peak were recorded and compared. The other way for doing specificity based in measurement of absorbance ratio of drug peaks at two different levels. The retention time (Rt), resolution factor (Rs) and tailing (T) were noted for the peaks of tapentadol hydrochloride. Peak purity study is done to prove that a developed method is specific for the drug of interest.

Acceptance criteria:

Purity angle should be less than purity threshold i.e.0.99-1.00

 g) Stability:

Stability of the sample, standard and reagent used in HPLC method is required for a reasonable time to generate reproducible and reliable results. Stability of plasma sample spiked with drug were subjected to three freeze-thaw cycles, short term stability at room temperature for 3 hours and long term stability at –20⁰C for four weeks. In addition, stability of standard solution and internal standard were performed at room temperature for 6 hours and under frozen condition for two weeks. The stability of this solution was studied by performing the experiment and looking for changes in separation, retention and asymmetry of the peak which were then compared with the pattern of chromatogram of freshly prepared solutions.

h) Selectivity:

Selectivity is the analytical method ability to differentiate and quantify the analyte in the presence of other components in the sample. The selectivity was established by two different methods.

Method I: Chromatograms of six blank plasma samples were compared with chromatogram obtained from standard solutions. Each chromatogram was tested for interferences due to endogenous plasma component on the retention times of the selected drugs.

Method ??: This method involves the peak purity test method using diode array detector. The PDA spectrum, UV spectrum, absorbance ratio curve and first derivative spectrum of the standard and sample peaks was recorded using PDA detector and compared for the peak purity of drug.

1. High Performance Liquid Chromatographic Method

A bioanalytical RP-HPLC method was developed for the tapentadol hydrochloride. The chromatographic conditions were stabilized in order to provide a good performance of the assay. The standard and internal standard solutions were prepared and chromatograms were recorded. This project proposes a method for the determination of tapentadol hydrochloride by solid phase extraction using RP-HPLC.

Chromatographic separation of standard Tapentadol hydrochloride:

The chromatogram of tapentadol hydrochloride was recorded alone and shown in figure (Fig 14).The standard solution which contains internal standard ibuprofen was injected with the developed chromatographic conditions, and the chromatograms were recorded and shown in figure (Fig 15).

Fig 15 - Chromatogram of IS and standard tapentadol hydrochloride 100µg/ml

The retention time of tapentadol hydrochloride and internal standard (Ibuprofen) was 6.4 and 8.6 min respectively with percent RSD of less than 2%. The results are shown in table (Table no 4). The peak purity study reveals that signal ratios (relative absorbance at different wavelengths) were constant across the peak pro?le of tapentadol hydrochloride. The peaks obtained in the present study were symmetric, good and no interference was observed between the peaks.

Table no 4: Retention time of tapentadol hydrochloride and ibuprofen (IS)

The method developed was advantageous than the reported methods by its lesser precision values and increased accuracy values. The run time of 10 minutes makes the method rapid and economical than the previously reported methods.

Chromatographic separation of tapentadol hydrochloride in biological fluid:

The chromatogram of the blank plasma was recorded at the fixed chromatographic conditions and shown in figure (Fig 16).

Fig 16 - Chromatogram of blank plasma

Various eluting solvents were used for extraction of tapentadol hydrochloride in human plasma. But, out of all eluting solvents acetonitrile-methanol mixture and methanol was proved to be good because of its maximum percentage recovery. The percentage recoveries were calculated and shown in table (Table no 5). The retention time for tapentadol hydrochloride and internal standard (Ibuprofen) were 6.4 and 8.6 minutes respectively as shown in (Fig 15). The peaks were symmetric with straight baseline. The extraction method used for the present study was simple and newer than previous methods. For present study solid phase extraction with methanol as eluting solvent was selected because of its maximum recovery of drug from plasma and it is advantageous.

Table no 5: Recovery study of tapentadol hydrochloride

Method Validation

a) Accuracy and precision:

At two –levels these accuracy and precision studies were conducted i.e. intra-day and inter-day. In this the present developed method, shown the good accuracy and precision. Accuracy ranges from 99.4% to 100.4% with the precision 5.82% to 6.94% in intra-day method. In inter-day method the accuracy ranges from 99.7% to 101.07%   with the precision 6.65% to 7.88%.  Finally the data obtained here, was found to be within limits as per ICH guidelines and method was accurate.

Intra-day studies: In this plasma concentration 250-1250 ng/ml were injected six times and mean peak area was calculated separately for each concentration and from that accuracy and precision percentage RSD values were calculated and shown in table (Table no 6)    

Table no 6: Accuracy and precision studies of tapentadol hydrochloride (Intraday)

              **Average of six determinations.

Inter-day studies: In this the plasma concentrations  of 250-1250 ng/ml were injected into HPLC six times in three different days and mean peak areas were calculated and from that accuracy and precision  percentage RSD were calculated and shown in table (Table no 7). The percentage relative standard deviation of precision for tapentadol hydrochloride was less than 15% for the bioanalytical study. The results obtained were within limits.

Acceptance criteria: The percentage RSD value should be less than 15% for bioanalytical study.

Table no 7:  Accuracy and precision studies of tapentadol hydrochloride (Interday)

**Average of six determinations

b) Linearity and range:

This method proved to be linear between ng/ml of tapentadol hydrochloride  in human plasma, with a typical calibration curve of correlation equation y = 0.005x + 0.085, correlation coefficient > 0.999 shown in table (Table no 8)

Fig 17 - Calibration curve for tapentadol hydrochloride

The chromatograms of the plasma calibration standards with concentrations 250, 500, 750, 1000 and 1250 ng/ml were recorded and shown in figures (Fig 18,19,20,21, and 22) and their peak areas of both drug and internal standard were noted. The calibration curve for tapentadol hydrochloride  was plotted as peak response Vs concentration of the tapentadol hydrochloride  calibration standards in plasma was shown (in Fig :17). As we were using internal standard peak response was calculated for calibration curve. Peak response is the ratio of internal standard peak area to drug peak area. The correlation coefficient of Tapentadol hydrochloride shown was 0.999 which was within limits. This calibration curve plotted was linear and showed that the method had adequate sensitivity to the concentration (250 ng/ml-1250 ng/ml) of the drug. Finally the data obtained, in this was within limits. Coefficient of correlation of tapentadol hydrochloride  was found to be less than 0.99.

 Acceptance criteria: The correlation coefficient should not less than 0.99

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            <img alt="Fig 18.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250607183620-8.png" width="150">
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Fig 18 - Chromatogram of IS and tapentadol hydrochloride in human plasma 250ng/ml

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            <img alt="Fig 21.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250607183620-5.png" width="150">
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Fig 21 - Chromatogram of IS and tapentadol hydrochloride in human plasma 1000ng/ml

c) Lower Limit of Quantification:

The LLOQ is the smallest concentration of the analyte, which shows response that can be accurately quantified and LLOQ = 10 × D/S, where, D is the standard deviation of y – intercepts of regression line and S is the slope of the calibration curve. This signal to noise ratio were performed by comparing measured signal of known low concentration of drug with those of blank plasma sample. The Lower limit of quantification (LLOQ) for tapentadol hydrochloride were separately determined and reported, based on the calibration curve for spiked plasma solutions was found to be 88.5 ng/ml respectively.

d) Recovery from plasma:

A recovery study for tapentadol hydrochloride in plasma using acetonitrile-methanol mixture was shown in table (Table no 9). With concentrations 250 ng/ml, 500 ng/ml, 750 ng/ml of tapentadol hydrochloride recovery was calculated and showed 100.2%, 99.4%, 100.4% relative recoveries and percentage RSD as 8.6%, 7.9% and 7.3% respectively. From the data. Obtained, it was observed that the recovery of drugs in plasma was found to be 9% which is sufficient for bio-analytical study.

Acceptance criteria: For an assay method, mean recovery should be 85-105% ± 2%.

**Average of six determinations.

e) Ruggedness:

It expresses the precision within laboratories variations like different days, different analyst, and different equipments. Ruggedness of the method was assessed by spiking the plasma standard 6 times in two different days with different analyst and the standard solutions were analyzed by a different chemist and same instruments on a different day had been performed the reports were shown in table (Table no 10). The deviation among the results obtained by two chemists on a different day was well within the limits. Hence the method was rugged.

Acceptance criteria: The percentage RSD should be less than 15%.

Table no 10:  Ruggedness studies for tapentadol hydrochloride

**Average of six determinations.

f) Specificity:

For specificity the peak purity studies were done. Here for tapentadol hydrochloride the peak purity index was 000 and the peak properties like peak profile were good for both standard and the sample. The peak purity and peak profiles for Tapentadol Hydrochloride standard and sample were shown in figures (Fig 23, 24, 25 and 26) respectively. By the data obtained in this, the present method developed was specific as values were within limits.

Acceptance criteria: Purity angle should be less than purity threshold i.e.0.99-1.00

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            <img alt="Fig 23.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250607183620-3.png" width="150">
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Fig 23 - Peak profile of standard tapentadol hydrochloride

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            <img alt="Fig 24.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250607183620-2.png" width="150">
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Fig 24 - Peak profile of tapentadol hydrochloride in human plasma

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Fig 25 - Peak purity of standard tapentadol hydrochloride

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            <img alt="Fig 26.png" height="150" src="https://www.ijpsjournal.com/uploads/createUrl/createUrl-20250607183620-0.png" width="150">
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Fig 26 - Peak purity of tapentadol hydrochloride in human plasma

g) System suitability:

These parameters were shown to be within specified limits. Column efficiency (theoretical plates), resolution factor and peak asymmetry factor, HETP, tailing factor, LLOQ are the system suitability parameters. These parameters of the optimized methods were found satisfactory. The results of the system suitability studies in plasma were shown in table (Table no 11). These parameters were shown to be within specified limits