Assay testing of a tramadol 37.5 mg and acetaminophen 325 mg tablet would typically involve the quantitative analysis of both active ingredients to determine their content or potency in the tablet. SOP for Tramadol 37.5 mg and Acetaminophen 325 mg tablet Assay Testing is important for ensuring the quality and consistency of the medication, as well as verifying that the tablet contains the expected amount of each active ingredient.

Assay testing can be performed using a variety of analytical techniques, including high-performance liquid chromatography (HPLC), gas chromatography (GC), and spectrophotometry. The specific method used will depend on factors such as the properties of the active ingredients and the desired level of sensitivity.

During the testing process, a sample of the tablet is usually extracted using a suitable solvent, and the active ingredients are then separated and quantified using the chosen analytical method. The results of the assay test can be compared to the expected values for each active ingredient, as determined by the tablet formulation or regulatory requirements.

Assay testing is an important part of the quality control process for pharmaceuticals and helps to ensure that medications are safe and effective for use by patients.

PURPOSE:

To describes the procedure for analysis at in-process and finished stage of Tramadol 37.5 mg and Acetaminophen 325 mg tablet Assay Testing.

SCOPE:

This SAP gives detailed outline for the finished product analysis of Tramadol 37.5 mg and Acetaminophen 325 mg tablet Assay Testing activities on physical, chemical & instrumental basis.

RESPONSIBILITY:

QC Analyst is responsible for physical / chemical testing and preparing standard analytical procedure.
It is the responsibility of QC Manager to assist and ensure Testing Procedure as per SAP and to make certain that this SAP is followed in its entirety, reviewed regularly and revised as necessary.

REFERENCE:

USP 42, NF 37

MATERIAL AND EQIUPMENT:

• HPLC
• Dissolution apparatus
• Friability apparatus
• Hardness apparatus
• Disintegration Tester
• Vernier Caliper
• Analytical Balance
• pH meter
• Moisture analyzer
• Mortar and pestle
• Spatula
• Filter Paper
• Magnetic Stirrer & Hot Plate
• Sonicator
• Vacuum Pump
• Glassware Ammonium Acetate
• Triethylamine
• Acetonitrile HPLC Grade
• Methanol HPLC Grade
• Distilled water
• Cupric sulfate
• L-isoleucine
• Ammonium Acetate

PROCEDURE:

FINAL MIX

Description:

White to off white color granular powder.

Procedure: Take 2.0 g of the test sample in a watch glass and observe visually with black background. Check the appearance of color, nature and any visible foreign particles.

Identification: (By HPLC)

SOP for Tramadol 37.5 mg and Acetaminophen 325 mg tablet Assay Testing

A- The retention time of the major peak in the chromatogram of the assay sample preparation corresponds to that in the chromatogram of the standard preparation as obtained in the assay.
B- The UV spectrum of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

Loss on Drying: (By Moisture Analyzer)

Procedure:

Turn on instrument 20 minute before the test. Take 2 g powder spread uniformly on Moisture Analyzer plate. Close the lid and press start button.

NMT 5.0% when determined by LOD Apparatus, Use 2.0 g of powder.

Assay:

(Limit: Tramadol Hydrochloride and Acetaminophen Tablets contain NLT 90.0% and NMT 110.0% of the labeled amount of tramadol hydrochloride (C16H2SNO2. HCI) and acetaminophen (C8H9NO2).
Weight of Powder/Tablet: 550 mg /tablet

Procedure:

Mobile phase:

Tetrahydrofuran, triethylamine, water, and trifluoroacetic acid (8: 0.1: 92: 0.1). The apparent pH of the final solvent mixture should be between 2.2 and 2.4.
Diluent: Methanol and water (1:9)

Standard solution:

0.065 mg/mL of USP Acetaminophen RS and 0.075 mg/mL of USP Tramadol Hydrochloride RS in Diluent. Sonication may be used to aid dissolution.

Sample stock solution:

Weigh NLT 20 Tablets, and determine the average Tablet weight. Grind the Tablets in to a fine powder, and transfer an amount equivalent to one Tablet to a 50-mL volumetric flask. Add 30 mL of Diluent with continuous shaking to disperse the powder. Sonicate for 15 min with intermittent shaking, and shake the flask on a mechanical shaker for 30 min. Dilute with Diluent to volume, and mix well. Centrifuge the suspension, and use the supernatant for subsequent dilutions.

Tramadol sample solution:

Nominally 75 ug/mL of tramadol hydrochloride in Diluent from the Sample stock solution

Acetaminophen sample solution:

Nominally 65 ug/mL of acetaminophen in Diluent from the Sample stock solution

Chromatographic system

(See Chromatography (621), System Suitability.)

• Mode: LC
• Detector: UV 216 nm for tramadol hydrochloride and UV 249 nm for acetaminophen
• Column: 4.6-mm x 15-cm; 5-um packing L11
• Column temperature: 50°
• Flow rate: 1 mL/min
• Injection volume: 20 uL
• Run time: 4 times the retention time of acetaminophen

System suitability

• Sample: Standard solution

Suitability requirements

• Resolution: NLT 10.0 between acetaminophen and tramadol
• Tailing factor: NMT 2.0 for each analyte
• Relative standard deviation: NMT 2.0% for each analyte

Analysis

Samples: Standard solution, Tramadol sample solution, and Acetaminophen sample solution Calculate the percentage of the labeled amount of tramadol hydrochloride (C16H25NO2.HCI) in the portion of Tablets taken:
Result= (ru/rs) x (Cs/Cu) x 100
ru= peak response of tramadol from the Tramadol sample solution
rs= peak response of tramadol from the Standard solution
Cs= concentration of USP Tramadol Hydrochloride RS in the Standard solution(mg/mL)
Cu= nominal concentration of tramadol hydrochloride in the Tramadol sample solution (mg/mL)
Calculate the percentage of the labeled amount of acetaminophen (C8H9NO2) in the portion of Tablets taken:
Result= (ru/rs) x (Cs/Cu) x 100
ru=peak response of acetaminophen from the Acetaminophen sample solution
rs=peak response of acetaminophen from the Standard solution
Cs= concentration of USP Acetaminophen RS in the Standard solution(mg/mL)
Cu=nominal concentration of acetaminophen in the Acetaminophen sample solution (mg/mL)

Acceptance criteria:

Tramadol hydrochloride: 90.0%-110.0% of the labeled amount of tramadol hydrochloride (C16H25NO2.HCI)
Acetaminophen: 90.0%-110.0% of the labeled amountof acetaminophen (C8H9NO2)

Calculations:

Average Sample AUC x Standard Concentration x Ave. Weight of Tablet x Potency
Average Standard AUC x Sample Concentration x Label Claim x Factor

Alternate Method By UV

Diluent: Ethanol 10: Water 90

Standard Solution:

Take 21.2 mg of Tramadol HCl and 162.5 mg Paracetamol dissolved in diluent and make up the volume to 100mL with diluent. Shake, and sonicate for 5 minutes to facilitate dissolution
Take 1 mL from standard stock solution and make up the volume to 100mL with diluent.

Sample solution:

Take 275 mg weight of finally grinded granules and dissolved in diluent and make up the volume to 100mL with diluent. Shake by mechanical mean for 10 minutes to disperse and allow any insoluble matter to settle.
Pass a portion of the sample stock solution through a suitable filter of 0.45um pore size.
Transfer the 1 mL of filtrate to a 100mL volumetric flask make up volume with diluent. Take reading at UV spectrophotometer at 271 nm for Tramadol HCl and 249 nm for Paracetamol using diluent as blank.

Calculations:
Sample Absorbance x Standard Concentration x Ave. Weight of Tablet x Potency
Standard Absorbance x Sample Concentration x Label Claim
(Limit: 90%-110%of the labeled amount)

Uniformity of Dosage Units: (By Weight Variation)

Weigh 20 tablets individually and calculate the average weight as:
Average weight = (Weight Of 20 Tablets)/20
Weigh all these tablets individually and mark among these weights maximum (WMax) & minimum (WMin) weight tablets. Calculate the maximum and minimum variation in percent as:
Maximum% age variation = ((WMax) – (WAvg.))/WAvg×100

Minimum % age variation ((WMin) – (WAvg.))/WAvg×100
Standard Limit: ± 5 %

Hardness Test & Dimensions:

Perform the hardness test on 10 tablets and take the average. Power on the instrument and regulate zero adjustment and preset the ‘’HARDNESS’’ and “THICKNESS” mode. Place the tablet on the test plate, begin testing and read the hardness thickness and diameter. Clean the shattered tablet and print out test results, perform the test on 10 tablets and take the average.
Hardness Standard Limit: NMT 10 Kg

Friability Test:

For tablets with a unit mass equal to or less than 650 mg, take a sample of whole tablets corresponding to 6.5 g. For tablets with a unit mass of more than 650 mg, take a sample of 10 whole tablets. The tablets should be carefully dedusted prior to testing. Accurately weigh the tablet sample, and place the tablets in the drum. Rotate the drum 100 times, and remove the tablets. Remove any loose dust from the tablets as before, and accurately weigh. Generally, the test is run once. If obviously cracked, cleaved, or broken tablets are present in the tablet sample after tumbling, the sample fails the test. If the results are doubtful or if the weight loss is greater than the targeted value, the test should be repeated twice and the mean of the three tests determined. A maximum mean weight loss from the three samples of not more than 1.0% is considered acceptable for most products.
% FRIABILITY= W1-W2/W1 x 100

Disintegration Test:

Place the beaker filled with water into the beaker stand inside the bath and. Switch on the instrument, set the temperature at 37ºC± 2º C and wait till the temperature of the beaker reaches to the 37ºC± 2º C. Engage the basket on basket hook put the tablets in 6 tubes individually and place the discs then start the oscillations and timer. Note the disintegration time of all the six tablets.

Note the Disintegration time of 1st tablet to the last tablet.

Disintegration Time: NMT 15 minutes at 370C ± 20C

Acceptance criteria of disintegration for tablets:
If 1 or 2 tablet fail to disintegrate completely repeat the test on 12 additional tablets, not less than 16 of the total of 18 tablets tested disintegrate completely.

Leak Test:

Take 06 blisters of tablet 03 filled and 03 empty. Dip these blisters in the bowl of leak test apparatus containing colored water. Create vacuum 250 mmHg and hold for one minute. Release vacuum and check the penetration of water inside the blisters. All blisters should be free of water.

DISSOLUTION (711)

• Medium: 0.1 N hydrochloric acid; 900 mL
• Apparatus 2: 50 rpm
• Time: 30 min

Buffer solution:

6.8 mg/mL of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 2.50.
Mobile phase: Acetonitrile and Buffer solution (1:4)

Standard solution:

0.36 mg/mL of USP. Acetaminophen RS and 0.04 mg/mL, of USP Tramadol
Hydrochloride RS in Medium

Sample solution:

Pass a portion of the solution under test through a suitable filter of 0.45um pore size.

Chromatographic system
(See Chromatography (621), System Suitability.)

• Mode: LC
• Detector: UV 272 nm
• Column: 4.6-mm x 15-cm; 5um packing L7
• Column temperature: 25°
• Flow rate: 1 mL/min
• Injection volume: 25 uL
• Run time: 2 times the retention time of tramadol

System suitability

Sample: Standard solution
[Note-The relative retention times for acetaminophen and tramadol are about 0.5 and1.0, respectively.]

Suitability requirements
Resolution: NLT 5.0 between the acetaminophen and tramadol peaks
Relative standard deviation: NMT 2.0% for both the acetaminophen and tramadol peaks

Analysis

Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of acetaminophen (C8H9NO2) and tramadol hydrochloride (C16H25NO2.HCI) dissolved:
Result = (ru/rs) x Cs x Vx (1/L) x 100
ru= peak response of acetaminophen or tramadol from the Sample solution
rs= peak response of acetaminophen or tramadol from the Standard solution
Cs= concentration of USP Acetaminophen RS or USP Tramadol Hydrochloride RS in the Standard solution (mg/mL)
V= volume of Medium, 900 mL
L=label claim for acetaminophen or tramadol hydrochloride (mg/Tablet)
Tolerances: NLT 80% (Q) of the labeled amount of acetaminophen (C8H9NO2) and tramadol hydrochloride(C16H25NO2.HCI) is dissolved.

CONTENT UNIFORMITY TEST: (By Weight variation)
Calculation of Acceptance Value (AV):

Limits:
L1= ≤ 15 (For 10 Unit)
L2= ≤ 25 (For 30 Unit)