CH314 Experiment 4 PDF

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Experiment 4 – Analytical toxicology: Quantitative Determination of Aspirin and Caffeine in an Analgesic by High Performance Liquid Chromatography (HPLC) Date: 27 Nov 2020 Introduction: The aim of this experiment is to analyse the concentration of aspirin and caffeine in an analgesic drug (Anadin) using HPLC. Under strict pharmaceutical industry guidelines and regulations, the concentration of these compounds in the final drug products must be within a specified range. Under USP procedures, the quantity of the APIs analysed for tablets must be within 85% and 115% of the label claim. HPLC is an analysis method used for separating compounds based on their polarity. HPLC parameters (detector wavelength, column, injection volume, run time etc.) used must be carefully considered so that the most accurate and consistent results can be obtained for a particular experiment. In this experiment, a reverse phase HPLC process is used. This means that the mobile phase is polar and that the stationary phase is non-polar. When the sample in mobile phase passes through the column, the polar compounds will pass through first, hence it will have a shorter retention time. Non-polar compounds will be attracted to the non-polar beads in the column and will take a longer time to pass through the column. As the compounds pass through the column, its concentration is measured using a detector. Thus, by examining the HPLC data and its corresponding chromatogram, the concentration of the polar and non-polar compounds can be measured. The polarity of the compounds tested will depend on the pH of the solution they are dissolved in and its protonation/deprotonation. The salicylic acid has the lowest pka of 2.97, acetylsalicylic acid has a pka of 3.5 and caffeine is the most basic with a pka of 10.4. Procedure: Five labelled flasks were prepared with the following: Flask 1: 10 mg of caffeine and 250 mg aspirin Flask 2: 20 mg of caffeine and 300mg aspirin Flask 3: 130 mg of caffeine and 350 mg aspirin I.S (internal standard) : 3g Salicylic Acid Tablet : 1x finely ground Anadin tablet These compounds were weighed out using a weighing boat and a balance with 4 decimal places. 100ml of methanol was then added to each flask. The contents of each flask were stirred until the solid material dissolved. 10ml of the internal standard solution was added into flasks 1-3 and into the flask with the Anadin tablet. The solutions are mixed again for another few minutes. Some of the solution from flasks 1-3 was poured into HPLC sample flasks. The insoluble excipient material in the tablet flask was filtered using a 10μm filter on a syringe before being placed into a labelled HPLC vial.

The marked vials were run as a sample set using HPLC using the following conditions: Instrument: Waters Alliance HPLC with UV Detector Detector wavelength ( λ): 254nm HPLC software: Empower 3 Column: C18 (non -polar) 100mm 3.5 μm column Mobile phase: H2O : Methanol : Acetic Acid (594:400:6) Injection volume: 10μL Run time: 10min

Results: The chromatogram for the standard solutions in flasks 1-3 and the solution in the tablet flask are shown in figures 1-4 respectively.

Figure 1: HPLC chromatogram for standard solution 1 (10 mg caffeine and 250mg Aspirin) and corresponding peak data.

Figure 2: HPLC chromatogram for standard solution 2 (20 mg caffeine and 300mg Aspirin) and corresponding peak data.

Figure 3: HPLC chromatogram for standard solution 3 (30 mg caffeine and 350mg Aspirin) and corresponding peak data.

Figure 4: HPLC chromatogram for tablet solution (unknown caffeine and aspirin concentration) and corresponding peak data

The peak area readings for caffeine from the chromatograms in figures 1-3 are compiled in table 1. The relative peak is given by peak area ÷ internal standard

Table 1:Peak area for caffeine in standard solutions 1-3, the internal standard and calculated relative peak.

Peak Area (Caffeine)

Concentration (mg)

Internal standard

Relative peak

0

0

0

0

147870

10

474347

0.31173382

348985

20

552807

0.63129628

557018

30

553429

1.00648502

139526

Unknown

631950

0.22078645

A calibration curve was constructed (figure 5) using data from standard solutions in table 1.

Plot of Relative Peak vs. Concentration of caffeine 1.2

Relative Peak

1 y = 0.0334x - 0.0135

0.8 0.6 0.4 0.2 0 -0.2

0

5

10

15

20

25

30

35

Concentration of cafeine (mg)

Figure 5: Calibration curve for the determination of caffeine concentration in tablet.

Using the plot in figure 5 and the data for peak area for the tablet in figure 4, the concentration of caffeine can be deduced. The relative peak of 0.220786 for the unknown sample corresponds to a caffeine concentration of approximately 7.0156 mg. This value was calculated using the equation of the line in figure 5. The peak areas, internal standard (salicylic acid) and relative peak for Aspirin are summarised in table 2. The data in this table was used to construct the calibration curve seen in figure 6. Table 2: Peak area for Aspirin for standard solutions 1-3, the internal standard and calculated relative peak.

Peak Area (Aspirin)

Concentration (mg)

Internal standard

Relative Peak

0

0

0

0

303356

250

474347

0.63952339

447897

300

552807

0.8102231

550519

350

553429

0.99474187

626411

unknown

631950

0.9912350661

Plot of Relatve Peak vs. Concentration of Aspirin 1.2

y = 0.0028x - 0.013

1

Relative Peak

0.8 0.6 0.4 0.2 0 0

50

-0.2

100

150

200

250

300

350

400

Concentration of Aspirin (mg)

Figure 6: Calibration curve for the determination of Aspirin concentration in tablet.

Using the data obtained in the HPLC analysis of the tablet solution, the relative peak for Aspirin was calculated to be 0.9912350661. Following the curve in figure 6, this relative peak value corresponds to an Aspirin concentration of approximately 358.7 mg. Discussion/ Questions: 1. HPLC is an analysis method which separates compounds based on their polarity. A mobile phase is passed through a stationary phase (column) at high pressure. If the stationary phase is polar and the mobile phase is non-polar (normal-phase HPLC), the non-polar compounds will be eluted first. This is because the polar compounds will have an affinity to the beads in the column and bind to it for a longer period of time before leaving the column. Hence in normal phase HPLC the non-polar compounds will have a shorter retention time. It is the opposite in reverse phase HPLC where the mobile phase is polar and the stationary phase is non-polar. As the compounds leave the column during HPLC analysis, a detector is used to measure the intensity of the eluents. The peak shown under the chromatogram for each compound is related to the concentration of that compound. HPLC can be used to analyse samples quantitively and qualitatively. Using standard solutions to create calibration curve of concentration vs. relative peak, the concentration of an unknown sample can be calculated. Combining HPLC retention factors for particular compounds and other methods of analysis, the constituents of a sample can be identified.

2. From data shown in figures 1-4, the average retention time for caffeine was 1.87425 minutes. The average retention time for Aspirin was 8.51425 minutes. 3. An internal standard in HPLC is used as a reference. It is a known concentration of a compound which is present in each sample which is analysed. All the relative peak/ peak ratio values are calculated as a proportion of the internal standard peak. 4. The Anadin tablet label claims to contain 15mg caffeine and 325 mg of aspirin. The caffeine concentration in one Anadin tablet was calculated to be 7.0156 mg. This is 46.77% of the label amount, which is not within USP range. The Aspirin concentration in the tablet was measured to be 358.7 mg, which is 110.3% of the label claim. This value is within USP specifications of 85 – 115%. 5. Aspirin Pharmacology: Prostaglandins recruits immune cells to a site of infection or injury causing inflammation. These prostaglandins also have wide variety of other functions such as blood clotting, mucus production in stomach, sensitising nerves to pain, and stimulating the hypothalamus to increase body temperature. The formation of prostaglandins from arachidonic acid is catalysed by an enzyme called cyclooxygenase (COX). Aspirin inhibits COX which results in temporary reduction of prostaglandin induced pain, fever and inflammation. Aspirin also inhibits thromboxane A2 prostaglandins which is responsible for activating platelets for blood clotting. Aspirin may be used as a medication in low does to prevent disease which can result from blood clots such as strokes or heart attacks. However, aspirin also reduces the layer of mucus which lines the stomach, leading to stomach ulcers and internal bleeding. This dangerous side effect is exacerbated by the reduction of blood clotting platelets. When aspirin is taken at toxic levels (150mg aspirin /kg body weight), it can lead to a range metabolic disorders. It stimulates the cerebral medulla of the brain, leading to hyperventilation and respiratory alkalosis, followed by metabolic acidosis. This results in hemodynamic instability and may be followed by end-organ damage. (Patel, S.R., 2013). Other symptoms may include fatigue, abdominal pain, tinnitus and fever. Caffeine pharmacology: Caffeine molecules act as adenosine antagonists in neurons. High levels of adenosine in the brain result from the breaking down of Adenosine triphosphate (ATP). The adenosine bind to receptors on the neurons in the brain, signalling that the brain needs rest (causing drowsiness). Adenosine and caffeine have very similar molecular structures (Figure 7). This allows caffeine to bind to adenosine receptors on the neurons. This blocks the signal telling the brain that it is tired.

Figure 7: Structure of Caffeine and Adenosine

Caffeine can be absorbed quickly in the stomach, meaning that toxic levels can quickly be reached. When caffeine is present in the body at toxic levels, it can trigger seizures, and cerebral and coronary vasoconstriction. Other symptoms include peripheral vasodilation and hypotension (Emohare et al, 2006). Caffeine also leads to increased urine production, leading to dehydration in the body. 6. Caffeine is included in may analgesic medicines as it is shown to increase its effectiveness. Caffeine ability to inhibit A2A and A2B receptors explains this effect (Sawynok J., 2011). It is also known to have some anti-inflammatory effects from inhibition of NF-κB activation (Chu et al., 2011). NF-κB induces the expressions of many pro-inflammatory genes. 7. The liver metabolites acetaminophen by breaking it down into N-acetyl-pbenzoquinone-imine (NAPBQI). This is toxic to the body. In normal therapeutic doses, NAPBQI reacts with the thiol groups of glutathione to produce non-toxic metabolites. In the cause of an overdose, where the acetaminophen levels are toxic, the high levels of in the liver cause a depletion in glutathione. NAPBQI then begins to react with lever cell proteins, causing necrosis and liver failure (Aldred et al., 2009) 8. In a reverse phase HPLC run, the most polar compounds will be elutes first, and the most non-polar compound will be eluted last. The pH of the mobile phase will have an effect on the retention time. All the compounds used in the experiment are non-neutral, they are ionisable. When dissolved in a low pH solution (pH below pka of compound) , the acetylsalicylic acid and the salicylic acid will be less polar and will have a longer retention time. The caffeine will become protonated and charged. This charge makes it more polar and will hence have a shorter retention time. When the compounds are dissolved in a high pH solution (pH above pka), the reverse effect occurs. The salicylic acid and the acetylsalicylic acid will become deprotonated, making them ionised and more polar, reducing the retention time. Caffeine will have the longest retention time. In the case of this experiment, the mobile phase was composed of water: methanol: acetic acid in a ratio of 594:400:6. The mobile phase was acidic. The order of elution will begin with the compound with the highest pKa (caffeine) and end with the compound with the lowest pka (salicylic acid). This is observed in the experimental data.

Biography: Aldred, EM., Buck, C., Vall, K. Pharmacology A handbook for complementary healthcare professionals. 2009, (7):41-52. Chu, Y.-F. et al.. 2011. Type 2 diabetes-related bioactivities of coffee: Assessment of antioxidant activity, NF-κB inhibition, and stimulation of glucose uptake. Food Chemistry 124(3): 914–920. Emohare O, Ratnam V. Multiple cardiac arrests following an overdose of caffeine complicated by penetrating trauma. Anaesthesia. 2006 Jan;61(1):54-6. Patel SR. Toxicologic emergencies in the intensive care unit: management using reversal agents and antidotes. Crit Care Nurs Q. 2013 Oct-Dec;36(4):335-44 Sawynok J. Methylxanthines and pain. Handb Exp Pharmacol. 2011;(200):311–29. doi: 10.1007/978-3-642-13443-2_11....