PHM223S PHARMACEUTICAL ANALYSIS LABORATORY STANDARD OPERATING PROCEDURE (S.O.P.

. PHM001): ASSAY OF ACETAMINOPHEN TABLETS USP 325 mg * Raymond M. Reilly, PhD (* has been modified with permission) Reference Source This laboratory was adapted from the following assays in the United States PharmacopoeiaNational Formulary 2002: Acetaminophen Official Monograph, p. 16. Acetaminophen Tablets Official Monograph, p. 18. Purpose: To determine the amount of acetaminophen in Life Brand and student-manufactured acetaminophen tablets using two different methods: HPLC and UV spectrophotometry. Equipment Required Mortar and pestle Spatula Weighing paper Analytical balance Whatman filter paper 10.0 mL, 25.0 mL, 50.0 mL volumetric pipettes (2 of each) 500 mL beaker 250.0 mL volumetric flask (5) 500.0 mL volumetric flask (3) 1.0 L volumetric flask (2) Liquid scintillation vials (100) Marking pen Magnetic stirring bar and stirrer Plastic transfer pipettes UV-transparent plastic cuvettes (1 box) Kimwipes (box) HPLC Grade Methanol (1 L) HPLC Grade Water (1 L) Sodium Phosphate Monobasic (500 g) Sodium Phosphate Dibasic (500 g) HPLC Solvent Bottles (1 L) (2) Agilent Eclipse XDB-CN 5m, 4.6x150mm (series # USBJ001083; LN# BO4057; PN#: 993967-905) HPLC System with Variable Wavelength UV Detector and Manual Injector Injection Syringe (100 L) The UV/Vis spectrophotometer

Reagents Required Acetaminophen Tablets USP 325 mg, Life Brand and student-manufactured Acetaminophen Powder (>98%) Acetaminophen Reference Standard (RS) Solutions 0.0016-0.032 mg/mL 0.05 M Sodium Phosphate Buffer pH 5.8

Teaching Assistant Preparation 1. Weigh 5 tablets of Acetaminophen USP 325 mg (Life Brand and student-manufactured, respectively), record weight and transfer to a mortar and pestle. Crush the tablets and weigh out accurately an amount of the tablet material equivalent to 325 mg of acetaminophen (i.e. one fifth of the weight of five tablets). Transfer this completely to a 500 mL beaker and add 400 mL of distilled and deionized water (must be Milli-Q). Add a stirring bar and dissolve completely. Transfer the solution completely to a 1.0 L volumetric flask by filtering through a Whatman filter paper. Add water to volume to make Solution A containing ~0.32 mg/mL of acetaminophen. 2. Transfer 25.0 mL of the filtered acetaminophen solution (~0.32 mg/mL) to a 500.0 mL volumetric flask and dilute to volume with distilled and deionized water to make Solution B containing ~0.016 mg/mL. 3. Weigh out 400.0 mg of acetaminophen powder and transfer to a 1.0 L volumetric flask. Dissolve and make to volume with distilled and deionized water to create a standard solution of acetaminophen (0.40 mg/mL). Accurately transfer 50.0 mL of this solution to a 500.0 mL volumetric flask and make to volume with distilled and deionized water to create Acetaminophen Reference Standard (RS) solution containing 0.04 mg/mL. Dilute this solution with sodium phosphate buffer pH 5.8 to prepare 250.0 mL of each of Acetaminophen RS solutions (Table 1).

Table 1. Preparation of Acetaminophen RS Solutions. Acetaminophen RS (0.04 mg/mL) 200.0 mL 100.0 mL 50.0 mL 25.0 mL 10.0 mL 0.05 M Sodium Phosphate Buffer pH 5.8 qs 250.0 mL qs 250.0 mL qs 250.0 mL qs 250.0 mL qs 250.0 mL Final Acetaminophen RS Solution (mg/mL) 0.032 (RS 5) 0.016 (RS 4) 0.008 (RS 3) 0.004 (RS 2) 0.0016 (RS 1)

Student Laboratory Note: The teaching assistant will show you how to operate the spectrophotometer and computer software. Part A Demonstration of HPLC Analysis of Acetaminophen The teaching assistants have analyzed the Acetaminophen RS Solutions and Life Brand Acetaminophen sample prior to the lab, according to the procedure outlined below. This component of the laboratory will be demonstrated by one of the teaching assistants. Please make notes on the demonstration. 1. Set up two HPLC solvent bottles containing 100% HPLC grade methanol or HPLC grade pure water. The HPLC system will mix these in the correct proportions for the analysis (25% methanol/75% water). 2. Standard Preparation: Use Acetaminophen RS Solutions 0.0016 to 0.032 mg/mL (RS 1 RS 5) 3. Sample Preparation: Use Life Brand Acetaminophen Solution B (~0.016 mg/mL) 4. Set up the HPLC system as follows: a. b. c. d. e. Column: C8 reversed-phase Mobile Phase: 25% methanol/75% water Flow Rate: 1.5 mL/min UV Detector: 243 nm Peak Detection: Area

5. Inject 20 L of each Acetaminophen RS solution onto the HPLC system and obtain the chromatogram. (This part was completed by the teaching assistants. The data will be handed out in the laboratory.) 6. Inject 20 L of the Acetaminophen Life Brand solution B sample into the HPLC system and obtain the chromatogram. (This part was completed by the teaching assistants. The data will be handed out in the laboratory.) 7. Label the chromatograms as follows: identify which chromatogram corresponds to which Acetaminophen RS solution (RS 1-5) and state the concentration and; identify which peak on the chromatogram is the Acetaminophen peak. 8. Measure the peak area for each Acetaminophen RS solution and plot the peak area vs. standard concentration to obtain the calibration curve. 9. Calculate the concentration of Acetaminophen in the Life Brand sample by reference to the HPLC calibration curve. 10. Calculate the amount of Acetaminophen in the Life Brand sample using the equations given on Page 5.

Part B Ultraviolet Absorption Spectrum for Acetaminophen 1. Open the Scan application of the CaryWin UV software. 2. Using a plastic transfer pipette, transfer 2-3 mL of distilled and deionized water to a UVtransparent plastic cuvette. With a Kimwipe tissure, gently wipe the outside surface of the cuvette to remove fingerprints and any drops of spilled solution and place the cuvette in the spectrophotometer. Zero the absorbance reading on the instrument. 3. Using a plastic transfer pipette, transfer 2-3 mL of Acetaminophen RS 5 solution to a cuvette and obtain an ultraviolet-visible spectrum in the range of 200 nm to 800 nm. Repeat the same procedure for the Life Brand Acetaminophen Solution B. Print both spectra for inclusion in your lab report. Part C Spectrophotometric Assay 1. Open the Simple Reads application of the CaryWin UV software. 2. Obtain 5 mL of each of the following from one of the teaching assistants: i) distilled and deionized water; ii) Acetaminophen Reference Standard (RS) solutions (0.0016, 0.004, 0.008, 0.016 and 0.032 mg/mL); iii) Life Brand Acetaminophen Solution B and; iv) student-manufactured Acetaminophen Solution B. 3. Using a plastic transfer pipette, transfer 2-3 mL of distilled and deionized water to the UV-transparent plastic cuvette. With a Kimwipe tissue, gently wipe the outside surface of the cuvette to remove fingerprints and any drops of spilled solution and place the cuvette in the spectrophotometer. Zero the absorbance reading on the instrument at a wavelength of 243 nm. 4. Using a plastic transfer pipette, transfer 2-3 mL of the first Acetaminophen RS solution (RS 1, 0.0016 mg/mL) to a cuvette and place in the spectrophotometer. Measure the absorbance three times to obtain and average reading. Repeat to obtain the measurements for Acetaminophen RS solutions 2-5: 0.004, 0.008, 0.016 and 0.032 mg/mL. 5. Using a plastic transfer pipette, transfer 2-3 mL of the acetaminophen assay Solution B (Life Brand and student-manufactured, respectively), to a cuvette. Measure the absorbance three times to obtain an average reading. 6. Identify the samples on your Simple Reads Report for inclusion in your laboratory report. 7. Plot the absorbance vs. standard concentration for the Acetaminophen RS solutions to obtain the calibration curve. 8. Calculate the concentration of Acetaminophen in the Life Brand and student manufactured samples by reference to the UV spectrophotometry calibration curve. 9. Calculate the amount of Acetaminophen in the Life Brand and student manufactured samples using the equations given on Page 5.

Calculation of the amount of acetaminophen in one tablet: (1) Conc. of acetaminophen from UV spec or HPLC: (2) Conc. of acetaminophen in stock solution: ___________mg/mL 20 (dilution factor) __________mg/mL (3) Amount of acetaminophen in one tablet: 1000.0 mL (total volume) __________mg (4) Percent of expected amount (325 mg): __________%

Laboratory Report Prepare a laboratory report (12 point font, normal margins, single-spaced) and hand it in to the receptionist at the front desk at the Faculty of Pharmacy. Reports are due one week after your laboratory before 5 pm on the Wednesday. Please ensure your report is prepared in an independent manner and follow the page guidelines given below. The mark for the laboratory report is 20% of the total marks for the course. Please make sure that you write on the front of the report: Your name Your student number Your group number (i.e. A1 etc.) The date that you performed the laboratory The name of the TA that instructed you The report should contain the following sections: Introduction (1/2 page) What are the objectives of this experiment?

Methods (one sentence) a reference to the method/SOP is sufficient

Materials (3/4 page) manufacturer and lot numbers of all reagents and chemicals used manufacturer, models and types of all equipment used including details of the spectrophotometer (computer software, wavelengths) and the HPLC system (pumps, UV detector, column, mobile phase, flow rate, injection volume, computer software)

Results (1 page) HPLC results should include all HPLC chromatograms labeled with concentration and peak representing acetaminophen, standard curve and

associated calculations, statistical parameters and calculation of the amount of acetaminophen in the Life Brand tablets. Attach the HPLC chromatograms as an Appendix. UV spectrophotometry results should include a printout of the scan from Part B, a labeled Simple Read Reports, standard curve and associated calculations, statistical parameters and calculation of the amount of acetaminophen in the Life Brand and student-manufactured tablets. Attach the UV spectrophotometry printouts as an Appendix. A brief interpretation of the results from HPLC and UV spectrophotometry do the tablets meet USP specifications (potency: 90.0 110.0% of 325 mg). How do the results for the Life Brand tablets compare between the two methods used?

Discussion (0.5 pages per question = 2 pages total) 1. Discuss and comment on whether or not the tablets made by the students (in PHM224) are acceptable for distribution and patient consumption. Explain why they are acceptable, or if not, why they might have deviated from the expected amount of drug in the tablets. If there is a problem with the tablets, what would you suggest to resolve the problem? 2. What would you do, if the tablets had already been distributed and this problem was only brought to your attention by a patient who noticed that the tablets were not helping his/her condition? Please write a brief, simple, understandable opinion and recommendation on this in your own words. 3. Compare the two analytical methods used in the laboratory (HPLC vs. UV spectrophotometry). Discuss both advantages and disadvantages of each technique. 4. Acetanilide is an analgesic that is metabolized into acetaminophen. To determine the extent of acetanilide metabolism into acetaminophen, it is necessary to analyze a sample containing both compounds. You are given a 0.1M HCl solution containing a mixture of acetanilide and acetaminophen. Could UV spectrophotometric analysis be used to determine the concentration of each compound in this solution? Why or why not? How and why would HPLC analysis improve your ability to identify the presence of both compounds in the mixture?

Conclusions (max 3-4 sentences) References (max 1 page) Appendices