Experiment on Paracetamol Analysis in Urine Sample - Assignment Example

Experiment on Paracetamol Analysis in Urine Sample Introduction Commonly, Paracetamol is known by its alternatename acetaminophen. It is a usual drug available over the counter and has brand names such as Tylenol®:.1 It’s mainly used to reduce fever and relieve pain (analgesic) and it has mild side effects. Most people don’t experience any problem when they take paracetamol, though in few cases it may be contraindicated. Adults recommended dose is 4000mg or 1000mg/day6. The drug overdose may result to kidney, brain and liver damage2. Thus, it is critical to measure the drug levels in urine and blood. Several methods including; thin layer titration, fluoremetry, UV-spectrophotometry, high performance liquid chromatography (HPLC) and chromatography have been applied in paracetamol analysis in pharmaceutical preparations4. In this experiment HPLC was used to analyze this drug in urine. Normally, the formulation is in water preparations and simple filtration can be done to eliminate insoluble excipients. Filtrate analysis can be through HPLC with UV detection. However, direct analysis in urine or blood is not practical due to biological fluids have several other molecules that are UV active and do interferes with the analysis3. One way to approach such is to employ methodology based on HPLC and mass spectrometric detection which was used in this experiment. The advantage for this method is that there is identification of the analyte with respect to; mass to charge ratio, retention time and mass spectrum4. Method Preparation of Paracetamol Tablet 500 mg paracetamol tablet was ground to a fine powder with a mortar and pestle. The resulting powder was transferred to a volumetric flask (100 ml).To make sure that all the powder was transferred to the volumetric flask the pestle and mortar was rinsed with deionised water. The volume in the flask was filled up to 100 ml mark with deionised water. The solution was sonicated at ambient temperature for 5 min. The resulting solution was filtered into a 100 ml clean volumetric flask. 10 μl was transferred to a sample vial then diluted with deionised water to 1.0 ml. Diluted solution of 100 μl was transferred to a clean vial and diluted with deionised water to 1.0 ml. An aliquot (5 μl) was later injected onto the HPLC column Preparation of Urine Sample: Urine sample was diluted in deionised water (1:10) HPLC-MS Conditions and Instrumentation: 1liter of formic acid (0.1 %) was prepared in water-mobile phase ‘A’ and 1l formic acid (0.1 %) was as well prepared in acetonitrile-mobile phase ‘B’. The solvents were transferred to corresponding solvent lines on the system of HPLC. The system was purged with the solvents by opening the valve of purge on pump ‘B’ and pump ‘A’.The valves were closed after the purge was complete. The following instrument parameters were set for mass spectrometer and HPLC:- HPLC Conditions: Gradient of the Solvent from 95:5, v/v - 30:70,v/v (A:B),Flow rate 0.21 mlmin-1,Temperature of the Column 35 ⁰C, Volume of injection 5μl,Rinsing solvent (water and acetonitrile) for injection needle(50:50, v/v) Conditions of Mass Spectrometric Nitrogen for nebulising gas at 1.5 L/min flow rate, Temperature of CDL - 200 oC, Temperature of Heat block 200 oC, Detector voltage 1.7 kV. Results The single ion chromatogram was separated from total ion chromatogram by a certain software which has the ability to select specific m/z from total ion chromatogram data (Hence referred to as extracted ion chromatogram). A- Paracetamol Standard Total ion chromatogram B- Single ion chromatogram (m/z of 186.2275) assigned to Paracetamol C- Paracetamol assigned Peak Mass Spectrum (8.3 min retention time). A- Urine sample total ion chromatogram B- Single ion chromatogram (m/z of 186.2241) assigned to paracetamol C- Peak Mass spectrum assigned to paracetamol (8.3 min retention time) Discussion With regards to ion chromatogram profiles of paracetamol standard, the numbers of peaks are less (2) as compared to urine sample which has several. This is due to; on a reversed-phase, Paracetamol and its several metabolites are isocratically separated. The metabolites separated include mercapturic, cysteine, sulfate, glucuronide and paracetamol acid conjugates as well as 3-methylthioacetaminophen, 3-methoxyacetaminophen and 3-hydroxyacetaminophen. There are additional peaks at 188.227 and 187.227 to a peak at 186.227 (Paracetomol).This is because mass spectrometer detector, has the ability to sense eluted compound from the column of HPLC by first ionizing it then later measures it’s mass or it can fragment the compound into smaller unites unique to the compound5. Therefore as seen in the mass spectrum, the pattern of the lines is so unique to paracetamol. In the spectrum, the biggest peak appears at a mass of 186.227, that is a fragment ion generated may be by loss of a certain atom This other peaks in the mass spectrum may have been generated because of the ionization or fragmentation of the paracetamol by the mass spectrometer detector10. In clinical laboratory tests, high performance liquid chromatography (HPLC) has been widely used because of good reproducibility of data and easy automatization6. As an analytical method, HPLC has been employed widely for many years in life science research and pharmaceutical quality control for sample separation7. As a result of its flexibility and power, it has been adopted for several routine applications in clinical laboratory. Some of these applications include: Determination of Catecholamines level, Vitamins level determination, diabetes monitoring among others. The advantage of this method is that it takes short time to produce results unlike in liquid chromatography that uses a high speed pump to force compounds through the column. The results are easy to read as well as are of high resolution. The disadvantage is that it can’t detect two escaping compounds from the column at once; hence this can result to inaccurate compound categorization8. The equipment cost is relatively high. It needs trained technician to operate it because of its complexity. It also has low sensitivity to some compounds. Experiments that can be undertaken to confirm whether the peak 186.227 is really paracetomol includes: use of 2 coupling agents, resorcinol and 1-napthol, employing a sensitive spectrophotometric method. Conclusions This experiment demonstrated the use of HPLC in the analysis of paracetamol and its metabolites in urine. The high resolution enabled sensitive detection of paracetamol and its metabolites regardless of high chromatographic interferences9. The obtained information was from the analysis of a single chromato- graphic, it demonstrated various advantages of high resolution and fast result generation mass spectrometry. Bibliography 1. Ali F.M., Boyer E.W. & Bird S.B. (2008). Estimated risk of hepatotoxicity after an acute acetaminophen 2. Baranowska I., Plonka J. & Baranowski J. (2006). HPLC analysis of methylxanthines and selected drugs in urine samples. Chemia Analityczna 51: 751-760. 3. Gotelli G.R., Kabra P.M. & Marton L.J. (1977). Determination of acetaminophen and phenacetin in plasma by high-pressure liquid chromatography. Clinical Chemistry 23(6): 957-959. Health 43: 601-606. 4. Holland D.T., Godfredsen K.A., Page T. & Conner J.D.(1998). Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 707:105-110. 5. Khatri J., Qassim S., Abed O., Abraham B., Al-Lami A. & Masood S.A. (2001). Novel extractionless hplc fluorescence method for the determination of glyburide in the human plasma: application to a bioequivalence study. Journal of Pharmacy & Pharmaceutical Sciences: 4(2):201-206. 6. Koling S., Hempel G., Lanvers C., Boos J. & Wurthwein G. (2007). Monitoring paracetamol metabolism after single and repeated administration in pediatric patients with neoplastic diseases. International Journal of Clinical Pharmacology and Therapeutics 45: 496-503. 7. Peterson R.G. & Rumack B.H. (1978). Toxicity of acetaminophen overdose. Annals of Emergency Medicine 7(5): 202-205. 8. Quattrone’ A.J. & Putnam R.S. (1981). A single liquidchromatographic procedure for therapeutic monitoring of theophylline, acetaminophen, or ethosuximide. Clinical Chemistry 27(1): 129-132. 9. Walsh A., Edwards H. & Fraser J. (2007). Over-the-counter medication use for childhood fever: a cross-sectional study of Australian parents. Journal of Paediatrics and Child 10. Yin O.Q.P., Lam S.S.L. & Chow M.S.S. (2005). Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies. Rapid Communications in Mass Spectrometry 19: 767-774. Read More