Rapid Sample Preparation and Analysis of Acetaminophen by HPLC-UV in Tablets

Rapid Sample Preparation and Analysis of Acetaminophen by HPLC-UV in Tablets

Awhile back, I was involved in a multi car accident, where I was sandwiched in the middle. My car and I both took a beating. This accident left me with major wrist pain to the point that I couldn’t shake a separatory funnel without extreme pain and weakness.

For this pain I was prescribed Acetaminophen, take 2 500 mg tablets every 6 to 8 hours for pain. Well, I was in a lot of pain so I took them around the clock, for about 2 years, before I went to a specialist and had multiple surgeries to fix my wrist.

This excessive dosage of acetaminophen caused me to get an ulcer that was even more painful, so I then had to get a prescription antacid to deal with that, as well as cut out the pain pills. This got me to thinking about acetaminophen and how easy it is to overdose on a common medication.

Acetaminophen, introduced in 1955, has become the most widely used pain reliever in the United States, and is a part of hundreds of over-the-counter and prescription medications used worldwide.
Unfortunately, overdoses occur because people will take more than directed because they either don’t heed the warnings, they don’t understand the dosing directions, or don’t realize that there is acetaminophen in multiple products that they are ingesting.

In the case of cold and flu, many will take acetaminophen in pill form to relieve their headache and fever, and then take more in the cough syrup to ease their cough. Acetaminophen is also found in many nasal decongestants.

With acetaminophen contained in so many over-the-counter medications, accurate analysis of the drug is essential to the safety of consumers. Consumers need to have accurate data to know how much they are taking to ensure that they don’t have an accidental overdose.

I took a look at two different pill forms of acetaminophen, one is the average tablet form consisting of a labeled amount of 500 mg in two pills and a cold medicine with the drug and others to combat the symptoms of the common cold, labeled at 325 mg for the two pills.

All four pills were added to centrifuge tubes and placed into the Lucidity SimplePrep. Flat, Teflon coated magnets were added to the tubes and the run was started.

SimplePrep Method
  Temperature 37 ℃
  Time 60:00 minutes
  Volume 35 mL
  Agitation ON
  Wash Cycles 3
  Pre-Treat Volume 0.0 mL
  Filtering OFF
  Settling 5 minutes

Once the SimplePrep was done running I made up a standard of acetaminophen at 0.2 mg/mL and injected onto the Lucidity HPLC-UV 5 times using the method below. The average area of the standard is 23799.24.


Lucidity LC-UV Method
  Flow Rate 2.0 mL/min
  Column C18 4.6 mm x 100 mm, 3.5 µm
  Oven Temperature Ambient
  Wavelength 207 nm
  Flow Isocratic – 4 minutes
  Mobile Phase Acetonitrile:Water (25:75) pH 2.5 with phosphoric acid

The four samples were diluted 1:10 and run on the HPLC-UV with the same method.  To my surprise the samples were maxing out the detector, and I thought that I had done something wrong on the dilutions.  I looked through my notebook and redid the math.  Everything checked out so I double checked the pills and re-read the labels and found that the tablets were not in fact 500 mg in two tablets, but 500 mg each tablet for the pack being 1000 mg dose!  The same was true for the cold medicine with acetaminophen. 

I re-did the dilutions for the samples as 0.1:10 and ran again on the HPLC-UV and the results averaged together.  The table gave an average dosing of 565 mg and the cold medicine gave a dosing of 407 mg per pill.


  Labeled Amount Reported Amount
  Acetaminophen 500 mg 565 mg
  Cold Medicine 325 mg 407 mg

Even with diligent work and doing the research on overdosing on acetaminophen, I still read the labels wrong and would have easily taken more than I should had I been taking them for pain relief and for a cold at the same time. 

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