ACETYLCYSTEINE BP

CHM 211 REPORT NAMES: Shi Guo WONG LECTURER: Dr Roger Reeve GROUP: B DATE: 01/02/07
INTRODUCTION
Every amino acid exists in an acidic form or basic form. This is depending on the pH of the solution in which the amino acid is dissolved. The carboxyl group of the amino acid has pKa values of approximately 2 and the protonated amino groups have pKa

values of nearly 9. At pH 7, the pH of the solution is greater than the pKa of the COOH group, but less than the pKa of the protonated amino group. The COOH group therefore will be in its basic form and the amino group will be in its acidic form. Therefore, at physiological pH (7.3) an amino acid exists as a dipolar ion, called a zwitterions (Bruice, Paula Yurkanis. Organic Chemistry. Pearson Education International, 2004). Acetylcysteine, which is an N-acetyl derivative of the amino acid L-cysteine, is a precursor in the formation of the antioxidant glutathione in the body. It is also known as N-acetylcysteine (NAC). The thiol (sulfhydryl) group confers antioxidant effects. With this, it is able to reduce free radicals. Acetylcysteine is a pharmacological agent used mainly as a mucolytic and in the management of paracetamol overdose. For these indications, acetylcysteine is available under the trade names Mucomyst (Bristol-Myers Squibb), Acetadote (Cumberland Pharmaceuticals), Fluimucil and Parvolex (GSK). Cysteine in the body change to cystine by two ways, oxidation of two molecule of cysteine by make disulphide bond and via acetylation of acetylcysteine. We performed different assay procedure to find the isoelectric point of acetylcysteine using starch solution, phenol red and phenolphthalein as indicators and potassium iodide solution and sodium hydroxide as titrant. We also performed the solubility test, specific optical rotation test, thin-layer chromatography test and the loss on drying test.

METHOD:
For the solubility test, acetylcysteine BP is dissolved in 8 parts of water and in 2 parts of ethanol (96%). It is practically insoluble in chloroform and ether. In the specific optical rotation, 1.25g of acetylcysteine BP is dissolved in a mixture of 1ml of a 0.1% w/v solution of disodium edetate, 7.5ml of 1M sodium hydroxide and sufficient mixed phosphate buffer pH 7.0 to produce 25ml of +21o to +27o of the solution. For recording optical rotation, the polarimeter was used. Freshly prepared and old solution of acetylcysteine, first blank solution was placed into the polarimeter and record the rotations, then old and fresh preparation of acetylcysteine were placed into the polarimeter and the optical rotation were recorded separately. The thin-layer chromatography is carried out using a cellulose precoated chromatoplate and a mixture of 60 volumes of propan-2-ol, 25 volume of 1M hydrochloric acid and 15 volumes of butanone as the mobile phase but allowing the solvent front to ascend 10cm above the line of application. 2l of each of three solutions containing: 1. 5.0%w/v of the substance being examined in water 2. 0.10%w/v of L-cysteine in water 3. 5mg of L-cystine dissolved in 0.5ml of 2M hydrochloric acid and diluted to 10ml with water. is applied separately to the plate. The plate is dried at 105o for 5 minutes after it has been removed. 0.5% w/v solution of ninhydrin in butan-1-ol is sprayed onto the plate. The plate is then heated at 105o for 10 minutes and it is allowed to dry overnight. For the loss on drying test, the sample is dried at 70oC with a pressure not exceeding 0.7kPa for 4 hours. Loses for the sample should not be more than 1.0% of its weight. 1g of the sample is used.

In the titration assay test, there are three titrations to be carried out. First of all, the burettes, flask and funnel are washed twice with distilled water, then 0.140g acetylcysteine is weighed on the electronic mass balance to two decimal places, and after that it is done again with an analytical balance to four decimal places. It is dissolved with 60 ml of distilled water and 10ml of dilute hydrochloric acid was added to the solution. It was cooled down in iced water, and then 10ml of potassium iodide solution is added. The burette is already filled with 0.05M iodine. Furthermore, 1ml of starch solution was added into the conical flask as indicator. This is the first titration. The other two titration assay tests are done by using 0.1M sodium hydroxide as the titrant and using phenol red and phenolphthalein as indicators. This time, approximately 0.408g of acetylcysteine is weighed accurately with the analytical balance to four decimal places. It is dissolved with 60 ml water and stirred until the sample particles are dissolved. Then six drops of phenolphthalein or phenol red are added into the conical flask and is titrated with 0.1M NaOH. The titration was repeated three times in both cases adding phenol red for the second titration and phenolphthalein for the third titration.

Results
Table 1: The amount of C5H9NO3S added and the amount of titration needed during the first assay. 1st 2nd 3rd Mass of C5H9NO3S (g) 0.1400 0.1400 0.1377 Amount of titration (ml) 8.350 8.330 8.360

The calculation for the amount of C5H9NO3S needed for the assay which using 0.1M sodium hydroxide as the titrant and phenol red and phenolphthalein as indicators. Since, 25ml of standard volume from 25ml pipette and 0.1M NaOH used. Therefore, n = CV = (0.1) (25) = 2.5 mol Change unit of n (mol) into mmol, therefore n = 2.5 x 10-3. Using the equation of n = mass/Mr Therefore, mass = n (Mr) = (2.5 x 10-3) (163.2) = 0.408 g Table 2: The amount of C5H9NO3S added and the amount of titration needed during the assay which using 0.1M sodium hydroxide as the titrant and phenol red as indicator. 1st 2nd 3rd Mass of C5H9NO3S (g) 0.4080 0.4090 0.4090 Amount of titration (ml) 24.00 24.18 24.18

Table 3: The amount of C5H9NO3S added and the amount of titration needed during the assay which using 0.1M sodium hydroxide as the titrant and phenolphthalein as indicator. 1st 2nd 3rd Mass of C5H9NO3S (g) 0.4080 0.4092 0.4034 Amount of titration (ml) 28.75 28.85 27.70 Graph 1: Thin-layer chromatography.

(1) (1) N-Acetyl-L-Cysteine (2) L-Cysteine (3) L-Cystine

(2)

(3)

rt = dspot / dsol =x/y

Table 4: x value, y value and the ratio of the components of N-Acetyl-L-Cysteine, LCysteine, and L-Cystine. Component dspot, x (cm) dsol, y (cm) Ratio, (rt = x/y) N-Acetyl-L-Cysteine 6.000 10.15 0.591 L-Cysteine 3.200 10.15 0.315 L-Cystine 0.200 10.15 0.020 The equation for calculating the specific optical rotation is, [] = 100 / (l) (c) where l is the path length and c is the concentration. Table 5: Numbers of optical rotation for blank, freshly prepared and old solutions of acetylcysteine. Type of preparation Number of optical rotation Blank 0.000 Freshly prepared of acetylcysteine +2.400 Old solution of acetylcysteine -1.340

Discussion
According to Table 1, there is a slight experimental error. Even though the mass of acetylcysteine is consistent 0.1400g for the first two titrates, the titre value is not the same. It varies with 8.35ml and 8.33ml. This might be due to reading error from the burette. There might also be impurities in the conical flask or burette due to incomplete wash on the third titration, the mass is 0.1377g but the titre volume is higher than the first two. The again maybe due to incomplete wash out or the skills of the person carrying out the titration. For the other two titrations, the mass of acetylcysteine to be used is calculated to match the concentration of sodium hydroxide given. The calculations are shown in the results section. According to Table 2, there is an inconsistency in the mass weighed. Although there was inconsistency, the results are still reasonable. For the first titration in Table 2, at 0.4080g, the titre volume is 24.00ml. For the second and third titration in Table 2, at 0.4090g for both titrations, the titre volume is 24.18ml. As observed, when the mass of acetylcysteine increases, the titre volume also increases. According to Table 3, there is almost a same trend as the ones in Table 2. The first titration in Table 3, at 0.4080g, the titre volume is 28.75ml. For the second titration, at 0.4092g, the titre volume is 28.85ml. For the third titration, at 0.4034g, the titre volume is 27.70ml. When the mass of acetylcysteine increases, the titre volume increases and vice versa. As observed in Table 4, N-Acetyl-L-Cysteine travels the furthest at 6.0cm away from the origin compared to the others with a ratio of 0.591. L-Cysteine traveled 3.2cm away from the origin with a ratio of 0.315. L-Cystine traveled the shortest with 0.2cm

away from the origin with a ratio of 0.020. This shows that N-Acetyl-L-Cysteine have the highest solubility in the solvent followed by L-Cysteine and L-Cystine. This also shows the polarity of the compounds in the experiment. The further the compounds travels, the higher the polarity of the compound in the solvent.

Discussion Topics
1. Use and mode of action of acetylcysteine. Acetylcysteine contains a sulfhydryl group which acts to split disulphide linkages in the mucoprotein structures of mucus, thus reducing its viscosity. The mucolytic action of the acetylcysteine is unaffected by the presence of DNA which increases with increasing pH, becoming optimal between pH 7 and 9. Acetylcysteine also reduces the extent of the liver damage following acetaminophen overdosage. Mechanisms of action include restoration or maintenance of glutathione levels and detoxification of serving as an alternative substrate for conjugation of the metabolite (Roger T. Malseed, Hail S. Harrigan. Pharmacology Nursing Care). Acetylcysteine is also used as an adjunctive therapy for the relief of abnormal viscous mucus accumulation associated with a variety of chronic respiratory conditions such as tuberculosis, or amyloidosis of the lung. It is also used post-operatively, as a diagnostic aid, and in tracheotomy care. It is considered ineffective in cystic fibrosis (Rossi, 2006). Oral acetylcysteine may also be used as a mucolytic in less serious cases. It is also used to reduce bronchiolar obstructive complication which is associated with tracheotomy, systic fibrosis, surgery, anesthesia or trauma. Investigational uses of acetylcysteine include ophthalmic administration for treatment of keratoconjunctivitis sicca (dry eye) and as an enema for treating bowel obstruction due to meconium ileums. Intravenous acetylcysteine is indicated for the treatment of paracetamol (acetaminophen) overdose. Oral acetylcysteine for this indication is uncommon as it is poorly tolerated owing to the high doses required which is due to poor oral bioavailability of the drug, unpleasant taste or smell and adverse drug reactions like nausea and vomiting. As an oral drug, acetylcysteine is also used for the prevention of radiocontrastinduced nephropathy. 2. Non-acetylated Amino Acids a. Why is this test required? This test is to see the presence of L-cysteine. It can also show whether L-cysteine is an amino acid or not. It is also to show the differences of cysteine concentration in three different solutions. It also observes to reactivity of cysteine with acid compounds. b. Theory of the test including the role of ninhydrin. The thin layer chromatography is used to separate and differentiate chemical compounds or to identify compounds present in a given substance. Ninhydrin is for the analysis of protein or amino acids involved. It also detects ammonia, primary and secondary amines. Amino acid are hydrolyzed and reacted with ninhydrin. If this reactivity occurs, this shows that there is presence of amino acid in the given substance. This is due to the amines left over after the reaction from peptides and proteins.

c. From the information given in the test, calculate the maximum permitted content (ppm or %w/w) of cysteine and cystine in Acetylcysteine BP. 5.0% w/v of the substance being examined in the water.
5g/100ml = 0.05g/1ml = 0.00005g/1l = 0.00001g/2l 0.10% w/v of L-cysteine in water. 0.1/100ml = 0.001g/1ml = 0.000002g/2l %w/w of cysteine = (0.000002/0.00001) x 100 = 2%w/w 5mg of L-cystine dissolved in 0.5ml of 2M hydrochloric acid and diluted to 10ml with water. [(5mg/10.5ml) = (x/100ml)] x = 47.62mg = 0.04762g 0.04762g/100ml = 0.0004762g/1ml = 0.00000004762/1l = 0.00000009524/2l %w/v for L-cystine = 0.00000009524/2l %w/w of cystine = (0.00000009524/0.00001) x 100 = 0.9524%w/w

3. Stereochemistry a. Give a brief description of the sequence rules and designate acetylcysteine as R or S Each chiral center either R or S is labeled by which its substituents are assigned a priority each, according to the Cahn Ingold Prelog priority rules, based on atomic number. If the center is oriented so that the lowest-priority of the four is pointing away, two possibilities will be observed: if the priority of the other three substitutents decreases in clockwise direction, it is labeled R, if it decreases in counterclockwise direction, it is labeled S. Acetylcysteine is in a R sequence. b. Give a brief description of the D and L convention. An optical isomer can be named by the spatial configuration of its atoms. The D/L labeling is unrelated to (+)/ (); it does not indicate which enantiomer is dextrorotatory and which is levorotatory. The compound's stereochemistry of D/L is related to that of the dextrorotatory or levorotatory enantiomer. The groups which are arranged around the chiral center carbon atom with the hydrogen atom placed away from the viewer, if these groups are arranged clockwise around the carbon atom, then it is the D-form. If these groups are arranged counter-clockwise, it is the L-form. c. Why does the specific optical rotation need to be measured? Compare the values obtained for the fresh and old solutions. Specific optical rotation provides a way to access optical purity of a sample containing a mixture of enantiomers. The specific optical rotation of a pure material is a specific property of that material at a given wavelength and temperature. The formal unit for specific optical rotation values is deg cm g-1 but scientific literature uses just degrees. A negative value means levorotatory rotation and a positive value means dextrorotatory rotation which is also the D and L convention method. The results for the optical

rotations are stated in Table 5 in the results section of this report. The fresh preparation of acetylcysteine is a dextrorotatory (D) rotation because it has a positive value. The old preparation of acetylcysteine is a levorotatory (L) rotation because it has a negative value. 4. Assay results a. Calculate the results for all three assays, remembering that the limits are with respect to the dried substance Assays of C5H9NO3S Mr 163.2 Loss on drying data: Initial weight of acetylcysteine sample = 1.0429g Weight after drying under specified conditions = 1.0358 g Therefore, % of purity for dried substance = (1.0358 / 1.0429) x 100% = 99.32% For assay 1 Samples Mass of C5H9NO3S (g) Amount of titration (ml) 1st sample 1 mol I2 + 2 mol Acetylcysteine [C5H8NO3S]2 500ml 1M I2 163.2 acetylcysteine 1ml 1M I2 0.3264 acetylcysteine 1ml 0.05M I2 0.01632 acetylcysteine 1ml 0.0514M I2 0.01678 acetylcysteine Therefore, 1ml 0.514M I2 0.01678 acetylcysteine And, 8.350ml of 0.514M I2 0.1401 acetylcysteine % of purity = (0.1401 / 0.140) x 100% = 100.1% % of purity with respect to the dried substance = [0.1401 / (0.140 x 99.32)] x 100% = 1.008% w/w 2nd sample 1 mol I2 + 2 mol Acetylcysteine [C5H8NO3S]2 500ml 1M I2 163.2 acetylcysteine 1ml 1M I2 0.3264 acetylcysteine 1ml 0.05M I2 0.01632 acetylcysteine 1ml 0.0514M I2 0.01678 acetylcysteine 1st 0.1400 8.350 2nd 0.1400 8.330 3rd 0.1377 8.360

Therefore, 1ml 0.514M I2 0.01678 acetylcysteine And, 8.330ml of 0.514M I2 0.1398 acetylcysteine % of purity = (0.1398 / 0.140) x 100% = 99.8% % of purity with respect to the dried substance = [0.1398 / (0.140 x 99.32)] x 100% = 1.005% w/w 3rd sample 1 mol I2 + 2 mol Acetylcysteine [C5H8NO3S]2 500ml 1M I2 163.2 acetylcysteine 1ml 1M I2 0.3264 acetylcysteine 1ml 0.05M I2 0.01632 acetylcysteine 1ml 0.0514M I2 0.01678 acetylcysteine Therefore, 1ml 0.514M I2 0.01678 acetylcysteine And, 8.360ml of 0.514M I2 0.1403 acetylcysteine % of purity = (0.1403 / 0.1377) x 100% = 101.89% % of purity with respect to the dried substance = [0.1403 / (0.1377 x 99.32)] x 100% = 1.025% w/w For assay 2 Samples Mass of C5H9NO3S (g) Amount of titration (ml) 1st 0.4080 24.00 2nd 0.4090 24.18 3rd 0.4090 24.18

1st sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+ 1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 24.00ml 0.1030M NaOH 0.4034 acetylcysteine % of purity = (0.4034 / 0.4080) x 100 %

= 98.88 % % of purity with respect to the dried substance = [0.4034 / (0.4080 x 99.32)] x 100% = 0.9955 % w/w 2nd sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+ 1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 24.18ml 0.1030M NaOH 0.4065 acetylcysteine % of purity = (0.4065 / 0.4090) x 100 % = 99.39 % % of purity with respect to the dried substance = [0.4065 / (0.4090 x 99.32)] x 100% = 1.001 % w/w 3rd sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+ 1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 24.18ml 0.1030M NaOH 0.4065 acetylcysteine % of purity = (0.4065 / 0.4090) x 100 % = 99.39 % % of purity with respect to the dried substance = [0.4065 / (0.4090 x 99.32)] x 100% = 1.001 % w/w For assay 3 Mass of C5H9NO3S (g) Amount of titration (ml) 1st 0.4080 28.75 2nd 0.4092 28.85 3rd 0.4034 27.70

1st sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+

1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 28.75ml 0.1030M NaOH 0.4833 acetylcysteine % of purity = (0.4833 / 0.4080) x 100 % = 118.5 % % of purity with respect to the dried substance = [0.4833 / (0.4080 x 99.32)] x 100% = 1.193 % w/w 2nd sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+ 1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 28.85ml 0.1030M NaOH 0.4850 acetylcysteine % of purity = (0.4850 / 0.4092) x 100 % = 118.5 % % of purity with respect to the dried substance = [0.4850 / (0.4092 x 99.32)] x 100% = 1.193 % w/w 3rd sample 1mol of acetylcysteine + 1 mol of NaOH [C4H8NOS]COO-Na+ 1000ml 1M NaOH 163.2g acetylcysteine 1ml 1M NaOH 0.1632g acetylcysteine 1ml 0.1030M NaOH 0.01681 acetylcysteine Therefore, 1ml 0.1030M NaOH 0.01681 acetylcysteine And, 27.75ml 0.1030M NaOH 0.4665 acetylcysteine % of purity = (0.4665 / 0.4034) x 100 % = 115.6 % % of purity with respect to the dried substance = [0.4665 / (0.4034 x 99.32)] x 100% = 1.164 % w/w

b. Compare the results obtained by the three different assay methods. For the 1st assay, the percentages of the purity for three samples are all in the percentage range (98 to 102 percentages) of the C5H9NO3S. Therefore, in acetylcysteine which is N-acetyl-L-cysteine contain the standard of C5H9NO3S. Besides that, the percentages between three samples are roughly 1.013% w/w. For 2nd assay, the results are quite in the standard but for sample 1 is a bit lower but still in the standard range of C5H9NO3S. Same as the percentages between three sample, sample 1 is a bit too low as the loses of mass should not more than 1.0% of its weight but 0.9955% w/w is about there and still consider as slightly standard. But not for assay 3, all of them are much higher than standard (98 to 102 percentages). The average of the three samples is about 117.1% w/w which is higher than 102% w/w even though the percentages of the purity are much higher than 1.0% w/w. c. Explain the reasons for the differences between the values obtained and hence why 0.05M iodine is used in the BP assay. In the 1st assay, the values which with respect to the dried substance is quite equilibrium and this means that both of the assay and the C5H9NO3S, acetylcysteine are very standard and high purity of C5H9NO3S since the average is about 100.6% w/w which is in the range of C5H9NO3S (98 to 102 percentages). Same as 2nd assay, just that average is a bit lower than 1st assay, which is about 99.14% w/w. But the values in 3rd assay for three samples are much higher and provided that there might impurities in the sample. There is a small property that the reading or calculation was carried wrongly during the experiment or experimental errors. And this is why the values in the three samples of 3rd assay were higher which the average is about 117.5% w/w. In the 1st assay, 0.05M iodine is used because of sometimes when following the changes in some inorganic reactions, iodine may be used as an indicator to follow the changes of iodine ion and iodine element when soluble starch solution is added in the assay.