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Liquid Chromatography – Mass Spectrometry (LC/MS)

Wednesday 1

Introduction Analgesic medications or commonly known as painkillers are drugs that are used to achieve analgesia. These drugs include paracetamol (known as acetaminophen), the nonsteroidal antiinflammatory drugs (NSAIDs) such as the salicylates, and opioid drugs such as morphine and oxycodone. (1) Acetaminophen is the most commonly used pain reliever and fever reducer in the United States and is approved by FDA to be available over-the-counter.(2) Acetylsalicylic acid (or commonly known as Aspirin) is also in the family of analgesics and is used in treatment of mild to moderate pain.(3) Both these compounds are useful in their own way, aspirin can be more effective when dealing with inflammation however it cannot be given to children because of the risk of Reye’s syndrome.(4) Acetaminophen is a suitable substitute for aspirin for its analgesic or antipyretic uses in patients in whom aspirin is contraindicated.(5) In situation that one can not be used, the other can be substituted. Caffeine is the most widely used psychoactive substance in the world and it has been estimated that 80-95% of Americans use it regularly.(6) Caffeine has been added to analgesic drugs such as the ones containing acetaminophen or aspirin for over 50 years now however, it was not known until the 80s that caffeine actually assists in the pain-relieving process of the main active drug and therefore, analgesics containing caffeine are considered to be more effective.(6) Drug concentrations are usually of very high importance as overdoses can be dangerous. FDA has limited the strength of acetaminophen in prescription combination products to 325 mg/dose.(2) On the package of Tylenol which is one of the most commonly known and used pain-relievers, it is indicated that each tablet includes 500mg of acetaminophen and 65mg of caffeine.(7) The emerging question is then how close these values are to the true amount of the compound actually in the tablet, and the evolving problem would be what methods can be used by drug companies to correctly and rapidly check the concentrations of compounds in their products. LCMS is the combined utilization of two analytical methods, liquid chromatography and mass spectroscopy. Through this method, one is able to separate compounds, quantify their concentration and identify what the compounds exactly are. While liquid chromatography separates the compounds present in a mixture, mass spectrometry identifies the compounds. Therefore this method can be applied to a wide range of fields. One of the important uses of this method is in the studies of pharmaceuticals. To determine how quickly a drug will be cleared from the body organs and the blood flow, certain studies are done in which mass analyzers are of high use due to their short analysis time.(8) In a recent study, a validated LCMS method was used to quantitatively determine the caffeic acid phenethyl amide and caffeic acid phenethyl ester from rat plasma to obtain pharmacokinetic profiles.(9) LCMS is a method that can open doors in many different fields and therefore it is a crucial instrument to an analytical lab. In this experiment, an unknown analgesic medication was analyzed through full MS and MS-MS identification to determine whether it contained acetaminophen or acetylsalicylic acid as its main drug compound. Since some analgesics contain caffeine as a side component in lower concentrations than the main drug, the presence or absence of caffeine was looked out for.

Methods and Materials The binary gradient LC/MS instrument used in this experiment contained a Varian 212-LC subunit (Agilent Technologies, USA) and a Varian 500 MS (Agilent technologies, USA). This instrument was optimized using 40 ug/ml standard solutions that were prepared from 2 mg/mL stock solutions. The standards that the instrument was optimized with included acetaminophen, acetylsalicylic acid, and caffeine. These standards were diluted using a diluting solution containing 10% acetonitrile, 0.1% formic acid. The instrument was optimized on both full MS and MS/MS modes for each standard. To analyze the unknown analgesic medication, HPLC-MS was the method of use. The two solvent pumps of HPLC were as follows: 5% Acetonitrile/Water + 0.1% formic acid for pump A, and 10% Acetonitrile/Water + 0.1% Formic acid for pump B. With 100% solvent A at flow rate of 250 uL/min the column was equilibrated for 15 minutes. To determine the analgesic compound in the unknown, a mixture solution of analgesic compounds (300 ug/mL Acetylsalicylic acid, 50 ug/mL caffeine, and 50 ug/mL acetaminophen) was run through LC-MS full scan along with a solution of unknown. The pump conditions of this analysis were set as follows: 0 min 100% A, 10 min 100% B, 11 min 100% A, 18 min 100% A, with flow rate being 250 ul/min at all times. The retention time of the signals were compared and the main compound in the unknown was determined to be acetaminophen. Using the scan type MS/MS, and the optimized parameters for needle voltage, capillary voltage, precursor ion, CID voltage and Rf for acetaminophen, acetaminophen standards with concentrations 20, 40, 60, and 80 ug/mL and the unknown sample were run and based on the integrated area under each standard curve, a calibration curve was provided through which the concentration of the unknown was determined. Results

Area

The instrument was optimized using the three standards that were the possible compounds in the unknown. The signal obtained from the unknown was compared to the standards and based on the m/z value, it was concluded that the main compound of the unknown solution was acetaminophen. Four different standard concentrations of acetaminophen were prepared and used to obtain the calibration curve of acetaminophen. 1.00E+08 9.00E+07 8.00E+07 7.00E+07 6.00E+07 5.00E+07 4.00E+07 3.00E+07 2.00E+07 1.00E+07 0.00E+00 10

f(x) = 813700 x + 26045000 R² = 0.93

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Standard Concentration (ug/mL)

Figure 1. Calibration curve of Acetaminophen obtained using Acetaminophen standard solution. The

Acetaminophen standard concentrations were prepared using a 2 mg/mL stock solution and a diluent (10% acetonitrile, 0.1% formic acid). Based on the equation obtained from figure 1, and the area of unknown (1.356e8) that was determined using the instrument, the concentration of the unknown is calculated to be 129.78 ug/mL. The retention time of this unknown sample was 2.274 min. Discussion The signal that was received from the unknown was much higher than the highest signal of the standards. The concentration of acetaminophen in the unknown was determined to be 129.78 ug/mL and this value is outside the range of standard concentrations. In order to obtain a more accurate concentration value for the unknown that would be closer to the true value, higher standard concentrations should have been included in the calibration curve. If standards of 100, 120 and 140 ug/ml concentration were included in the calibration curve, the equation obtained from the curve would provide a more accurate unknown concentration. As it is observed in figure 1, the coefficient of determination of the calibration curve of acetaminophen is 0.9275. This shows that the regression line does not perfectly fit the data. For better results, an r2 of closer to 1 is preferred. This can be obtained by adding more concentrations to the curve. The most common acetaminophen containing medication used in Canada is Tylenol.(10) The maximum acetaminophen concentration of Tylenol Extended Relief (ER) has been reported to be 62.3 +/- 16.8 ug/ml in literature.(11) This product contains a very low concentration and is less likely to be the product used as the unknown that was analyzed. The maximum concentration of acetaminophen in Immediate Release products (IR) is measured to be 100.2 +/- 25.3 ug/mL.(11) This product shows a close concentration to our unknown sample and it is possible that the unknown analgesic medication that was analyzed in the lab, was IR Tylenol. Aside from the medicinal ingredients, analgesic medication tablets include other compounds that are considered biologically inactive, but may chemically provide a signal. Examples of such inactive ingredients that are known to be found in IR Tylenol based on literature are colloidal silicon dioxide, crascarmellose sodium, hydroxypropyl cellulose, hypromellose, polyethylene glycol, pocidone, strearic acid and titanium dioxide.(12) Since the unknown sample is a mixture of compounds, it is not possible to simply use mass spectroscopy. LCMS solves this issue as the compounds are initially separated through liquid chromatography. The molecular weight of acetaminophen is reported to be 151.165 g/mol.(14) The literature mass spectrum of this compound is presented in figure 2. Since the signals in mass spectrum represent the m/z ratio, the peak of 152.0707 in figure 2 is representing the pseudomolecular ion. It is expected that the ionized acetaminophen is cationic and protonated since this peak is approximately equal to M+1. The positive ionisation and fragmentation of acetaminophen leads to the elimination of the Nacetyl group of this compound and results in the production of a protonated aminophenol.(15) The peak 110.06 is based on this protonated aminophenol and responds to the m/z ratio of it.

Figure 2. Literature mass spectrum of acetaminophen.(13) During optimization, the Collision induced dissociation (CID) voltage for acetaminophen was determined to be 0.35 volts. When this voltage is applied, the daughter ion peak is observed in the mass spectrum. This can be visualized in the figure 2, in which two peaks are observed and the 110.06 peak is caused by a compound with less molecular weight and therefore it is the peak of the daughter ion. Conclusion Here, an unknown analgesic medication was analyzed and was determined to be an acetaminophen based painkiller without caffeine. The concentration of acetaminophen in the unknown medication was determined to be 129.78 ug/mL. This concentration suggests that the type of the medication analyzed was Immediate-Release Tylenol. Caffeine was not detected to be present in this unknown sample. The determined concentration of the unknown could be improved in accuracy by preparing a better calibration curve with a wider range of standard concentrations.

References (1) Analgesics. Wikipedia. (online) https://en.wikipedia.org/wiki/Analgesic (accessed on november 20th 2017) (2) Acetaminophen Fact Sheet. Consumer Healthcaree Products Association. (online) https://www.chpa.org/AcetFactSheet.aspx (accessed on November 20th,17) (3) Acetylsalicilic acid. DrugBank. (online) https://www.drugbank.ca/drugs/DB00945(accessed on November 20th,17) (4) Acetaminophen vs. Aspirin. Diffen. (online) https://www.diffen.com/difference/Acetaminophen_vs_Aspirin(accessed on November 20th,17) (5) Kacso G., Terezhalmy, G.T. Acetylsalicylic acid and acetaminophen. Dental Clinic North America. 1994. (online) https://www.ncbi.nlm.nih.gov/pubmed/7805939(accessed on November 20th,17) (6) How caffeine intake can affect the treatment of pain. Strulowitz Gargiulo, physical therapy and rehabilitation. (online) http://www.sgptr.com/caffeine-intake/(accessed on November 20th,17) (7) (8) Tylenol Headache Migraine pain products. Tylenol for what matters most. (online)

https://www.tylenol.ca/products/headache-migraine/tylenol-ultra-relief#ingredients (9) LCMS pharmacokinetics. Wikipedia. (online) https://en.wikipedia.org/wiki/Liquid_chromatography %E2%80%93mass_spectrometry#Pharmacokinetics(accessed on November 20th,17) (10)http://onlinelibrary-wiley-com.uml.idm.oclc.org/doi/10.1002/bmc.3011/full (11)Tylenol for infants and children. Tylenol for what matters. (online) https://www.tylenol.ca/products/infants-children/childrens-tylenol-liquid#dosage(accessed on November 20th,17) (12)http://onlinelibrary-wiley-com.uml.idm.oclc.org/doi/10.1111/j.1553-2712.1996.tb03508.x/pdf (13)Acetaminophen Rapid Release Extra Strength. Drugs.com. (online) https://www.drugs.com/otc/101849/acetaminophen-rapid-release-extra-strength.html(accessed on November 20th,17) (14)Acetaminophen mass bank record: UF409101. Massbank. (online) http://www.massbank.jp/jsp/Dispatcher.jsp?type=disp&id=UF409101&site=25(accessed on November 20th,17) (15)https://pubchem.ncbi.nlm.nih.gov/compound/acetaminophen#section=Chemical-and-PhysicalProperties (16)https://watermark.silverchair.com/bks139.pdf? token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAcAwggG8BgkqhkiG9w0 BBwagggGtMIIBqQIBADCCAaIGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMbH8JgrtW3eIlSKFAgEQgIIBcxJTgOD1K1_YpD7x6UdMKPKmgMJSA5_OQQHSjXCW1VZSGeuIjxXgTS1PJjaeaIp9b MtefSAl6OzpaZoHzSCg2foWBIDQg0u3x_c3FDWNrzYKCU_GVejBtaCFNM1fch1My-3SYEBSZ7_7FXEOd8je5H_99Sd6eut4uvQWNcMJPBd8mBQGSpvULv6iYP8sEoZpXJztMVx9SHxtjgKvKDqZ8CQiWUs0tWNp1PlPeiayoQtcypFig8yEQIOyCa3nDQhvOq MEnPXfB4ZdCrkNvc_jn5yVRH4TqXaW5zZt7PrZwvPKVk6t09_FdbhTtlp24hs6220WPnr82vYuAnG8 A_TXh136_YBVstW16Q6bn1hOIbWNeczAGb8FdDiZ-vVMPeQr6shOh9eMI40Sl9m0cfRj14WwzTs1pVyC1pfpIh7UX1g86xfCWKor9QUs_oaxF_uiB4BevEaxQsIdGVYsMvTemObpqCKxS1wpdc88UEkUEnfF

Questions 1. What are the reasons to use LC-MS versus GC-MS for the analysis of pharmaceutical and biological samples? GCMS is applied mainly for the analysis of volatile compounds in complex samples whereas LCMS is used for the analysis of thermally unstable molecules in a complex. Most biological fluids and pharmaceuticals

are thermally unstable and sensitive and are not volatile. They require to be derivatized to be able to be analyzed by GC. But they can directly be analyzed in a solution using LCMS. Therefore using LCMS is less time consuming and more efficient in the analysis of pharmaceuticals.

2. What are some of the problems associated with the use of this instrument for quantitative analysis? The height of the peaks in the mass spectrum are relative to the base peak. The tallest peak is usually given an arbitrary height of 100 and the height of everything else is measured relative to it. And this makes the peak height unreliable for quantitative measurements. Mass is measured indirectly with multiple charging and might not be accurate. Sample must be very pure. 3. Compare advantages and disadvantages of using full scan ions verses product ion from transitions for quantitation, and identification. Full scan product ion are used for qualitative applications to obtain structural information. Selected Reaction Monitoring product ion scans through ms-ms are used for quantitative target analysis. Full scan is not as selective and sensitive, whereas when using ms-ms, selectivity and sensitivity are higher. 4. What parameters would you have to control to produce a library capable of identifying compounds based on their fragmentation pattern? Temperature, gas flow and nebulizer pressure....