N Acetylcysteine Quality Control

N-acetylcysteine (C5H9NO3S Mr 163.2) is the N-acetyl derivative of the naturally occurring amino acid, l-cysteine. The drug occurs as a white, crystalline powder with a slight acetic odor. N-acetylcysteine is freely soluble in water and in alcohol. N-acetylcysteine is commercially available as aqueous solutions of the sodium salt of the drug. It is used as a mucolytic or as an antidote for paracetamol. The British Pharmacopoeia contains a number of tests for this compound designed to ensure the quality.

N-acetylcysteine acts to reduce mucus viscosity by splitting disulfide bonds linking proteins present in the mucus (mucoproteins). Inhaled N-acetylcysteine is indicated for mucolytic (“mucus-dissolving”) therapy as an adjuvant in respiratory conditions with excessive and/or thick mucus production. Such conditions include emphysema, bronchitis, tuberculosis, bronchiectasis, amyloidosis, pneumonia. It is also used post-operatively, as a diagnostic aid, and in tracheostomy care. It may be considered ineffective in cystic fibrosis (Rossi, 2006). However, a recent paper in the Proceedings of the National Academy of Sciences reports that high-dose oral N-acetylcysteine modulates inflammation in cystic fibrosis and has the potential to counter the intertwined redox and inflammatory imbalances in CF (Tirouvanziam et al., 2006). Oral N-acetylcysteine may also be used as a mucolytic in less serious cases.

N-acetylcysteine also acts to augment glutathione reserves (depleted by toxic paracetamol metabolites) in the body and, together with glutathione to directly bind to toxic metabolites. These actions serve to protect hepatocytes in the liver from toxicity due to paracetamol overdose. Intravenous N-acetylcysteine is indicated for the treatment of paracetamol (acetaminophen) overdose. Oral N-acetylcysteine for this indication is uncommon as it is poorly tolerated owing to the high doses required (due to poor oral bioavailability), unpleasant taste or odour and adverse drug reactions (particularly nausea and vomiting). However, some people have shown an adverse allergy to intravenous N-acetylcysteine which includes extreme breathing difficulty, light-headedness, rashes, severe coughing and sometimes also vomiting. Repeated overdoses will cause the allergic reaction to get worse and worse.

N-acetylcysteine is prone to both hydrolysis and oxidation and some of the impurities from these reactions are shown below.

Scheme 2

2. Experimental:

2.1. Materials:

The materials used in this experiment were N-acetylcysteine powder, disodium edentate solution, 1M sodium hydroxide and mixed phosphate buffer pH 7.0, water, dilute hydrochloric acid, potassium iodine solution, 0.05M iodine, 0.1M sodium hydroxide, starch, phenol red and phenolphthalein as indicators.

The apparatus used were optical rotation analyser, conical flasks, 10mL and 50mL pipettes, burette, electronic weigh balance and beakers.

2.2. Methods:

• a) Specific optical rotation: +21ÌŠ to +27ÌŠ

1.25g N-acetylcysteine powder was weighed and allowed dissolve in a mixture of 1ml of 10g/L solution of disodium edentate, 7.5ml of 1M sodium hydroxide and sufficient amount of mixed phosphate buffer pH 7.0 to 25ml total volume. Optical rotations of the freshly prepared solution and the old solutions of N-acetylcysteine provided were measured and recorded.

• b) ASSAY: 98.0%-101.0% C5H9NO3S (as dried material)

0.14g N-acetylcysteine powder was weighed by difference and poured into a conical flask. 60 ml of water and 10ml dilute hydrochloride acid were measured and added into the conical flask. The conical flask was shaking to ensure the N-acetylcysteine powder was fully dissolved. The solution was left to cool. Another 10ml of potassium iodide solution was added into the cooled solution in the conical flask. The solution was then titrated with 0.05M iodine by using starch as indicator. Second titration was carried out to ensure accurate and precise result.

• c) Assay by titration with 0.1M sodium hydroxide

0.3g N-acetylcysteine powder was weighed by difference and poured into a clean conical flask. Approximately 50 ml of distilled water was measured and added into the conical flask. The conical flask was shaking to ensure the N-acetylcysteine powder was fully dissolved. The solution was titrated with 0.1M sodium hydroxide using phenol red as indicator. Second titration was carried out to ensure accurate and precise result.

0.3g N-acetylcysteine powder was weighed by difference and poured into a clean conical flask. Approximately 50 ml of distilled water was measured and added into the conical flask. The conical flask was shaking to ensure the N-acetylcysteine powder was fully dissolved. The solution was titrated with 0.1M sodium hydroxide using phenolphthalein as indicator. Second titration was carried out to ensure accurate and precise result.

• d) Zinc: Not more than 10ppm Zinc

1.00g of N-acetylcysteine powder was weighed and dissolved in 0.001M hydrochloric acid. The solution was diluted to 50ml with 0.001M hydrochloric acid and solution 1 was obtained.

Three solutions were prepared for analysis. The first solution consists of 10ml solution 1 diluted to 20ml with 0.001M hydrochloric acid, second solution consists of 10ml solution 1 and 1ml of 5ppm zinc standard diluted to 20ml with 0.001M hydrochloric acid and the third solution consists of 10ml solution 1 and 2ml of 5ppm zinc standard diluted to 20ml with 0.001M hydrochloric acid.

The absorbance of each solution was measured at 213.8nm using an atomic absorption spectrophotometer. The absorbance for each solution was tabulated. The zinc content in each sample was calculated using the method of standard addition.

• e) Loss on drying: Not more than 1.0%w/w

A sample of N-acetylcysteine was dried at 70ÌŠ C in vacuo for 3 hours and the data was recorded and the percentage loss on drying of this sample was calculated.

• f) Related substances

The chromatograms obtained from the HPLC analysis of both fresh solution and old solution of N-acetylcysteine was examined.

3. Results:

• a) Specific optical rotation:

Mass of weighing boat(g)

26.6089

Mass of weighing boat + sample (g)

27.8609

Mass of weighing boat + residue (g)

26.6079

Mass of sample transferred (g)

1.253

Table 1: The mass of N-acetylcysteine used to make a solution for measurement of specific optical rotation.

Calculations:

According to British Pharmacopoeia (BP 1999; page 40-41), it states that the specific optical rotation is + 21.0 to + 27.0. To obtain the angle of rotation, the equation below is used,

Where, [α] = specific optical rotation

α = observed angle of rotation

C = concentration of active substance in g/100mL of the solution

l = length of column in 2dcms

For freshly prepared solution:

Angle obtained (α): 2.45⁰

Concentration of N-acetylcysteine (c): 5.012 %w/v

Path length = 2 dm

Specific optical rotation:

= 100 x 2.45⁰

2 x 5.012g/ml

= +24.5⁰

For old solution:

Angle obtained (α): -3.29⁰

Concentration of N-acetylcysteine (c): 5.012 %w/v

Path length = 2 dm

Specific optical rotation:

= 100 x 3.29⁰

2 x 5.012g/ml

= -32.9⁰

• b) ASSAY: 98.0%-101.0% C5H9NO3S (as dried material)

Sample 1

Sample 2

Mass of boat + sample (g)

3.8797

3.8777

Mass of boat + residue (g)

3.7393

3.7398

Mass of Acetylcysteine transferred (g)

0.1404

0.1379

Table 2: The mass of N-acetylcysteine powder in sample 1 and sample 2 for titrations with iodine.

First reading

Second reading

Initial volume (mL)

17.40

26.70

Final volume (mL)

26.40

35.50

Volume of 0.05M iodine used (mL)

9.00

8.80

Table 3: The volume of iodine used for both titration using sample 1 and sample 2 of N-acetylcysteine solution and starch as indicator.

Calculations:

Actual concentration of iodine used: 0.0476M

Molecular weight of N-acetylcysteine (C5H9NO3S): 163.2

The balanced equation for the reaction between N-acetylcysteine and iodine:

2 C5H9NO3S + I2 à C5H8NO3SSC5H8NO3 + 2HI

2KI à I2 + 2K+

According to British Pharmacopoeia, 1mL of 0.05M iodine is equivalent to 16.32mg of C5H9NO3S. This means, 2 mole of C5H9NO3S equal to one mole of iodine.

Therefore when 1mL of 0.05M iodine = 16.32mg of C5H9NO3S,

1mL of 0.0476M iodine = 0.0476M x 16.32mg/ 0.05M

= 15.54mg of C5H9NO3S

First titration:

1mL of 0.0476M iodine = 15.54mg of C5H9NO3S

So, 9.00mL of 0.0476M iodine = 9.00mL x 15.54mg/ 1mL

= 139.86mg

= 0.13986g of C5H9NO3S

Second titration:

1mL of 0.0476M iodine = 15.54mg of C5H9NO3S

So, 8.80mL of 0.0476M iodine = 8.80mL x 15.54mg/ 1mL

= 135.52mg

= 0.13552g of C5H9NO3S

Calculation of Percentage of Purity:

Sample 1 of N-acetylcysteine

Sample 2 of N-acetylcysteine

Mass transferred

Actual mass calculated

Mass transferred

Actual mass calculated

0.1404

0.1399

0.1379

0.1355

According to British Pharmacopoeia (BP), the percentage of purity should be within 98.0 – 101.0% of dried substance.

Equation of the Percentage of Purity:

Sample 1:

Sample 2:

c) Assay by titration with 0.1M of sodium hydroxide

i) Titration by using phenol red indicator

Sample 1

Sample 2

Mass of boat + sample (g)

3.8916

3.9199

Mass of boat + residue (g)

3.5913

3.6198

Mass of N-acetylcysteine transferred (g)

0.3003

0.3001

Table 4: The mass of N-acetylcysteine powder in sample 1 and sample 2 for titrations with 0.1M of sodium hydroxide.

First reading

Second reading

Initial volume (mL)

1.00

1.00

Final volume (mL)

18.15

18.10

Volume of 0.05M iodine used (mL)

17.15

17.10

Table 5: The volume of 0.1M sodium hydroxide used for both titration using sample 1 and sample 2 of N-acetylcysteine solution and phenol red as indicator.

Calculations:

Actual concentration of sodium hydroxide (NaOH) used: 0.1062M

Molecular weight of N-acetylcysteine (C5H9NO3S): 163.2

The balanced equation for the reaction between N-acetylcysteine and sodium hydroxide (NaOH):

C5H9NO3S + NaOH à C5H8NO3SNa + H2O

From the equation, one mole of N-acetylcysteine reacts with one mole of NaOH. So the reaction is a 1:1 ratio. To find out the number of mole of NaOH, the equation below is used:

First titration:

Moles of NaOH = (0.1062M x 17.15mL)/1000

= 1.821 x10-3 moles

As the reaction is 1:1 ratio so the number of moles of N-acetylcysteine is equal to the number of moles of NaOH used which is 1.821 x10-3 mole.

Mass of N-acetylcysteine = 1.821 x10-3 moles x 163.2

= 0.2972g

Second titration:

Moles of NaOH = (0.1062M x 17.10mL)/1000

= 1.816 x10-3 moles

As the reaction is 1:1 ratio so the number of moles of N-acetylcysteine is equal to the number of moles of NaOH used which is 1.821 x10-3 mole.

Mass of Acetylcysteine = 1.816 x10-3 mole x 163.2

= 0.2964g

Calculation of Percentage of Purity:

Sample 1 of N-acetylcysteine

Sample 2 of N-acetylcysteine

Mass transferred

Actual mass calculated

Mass transferred

Actual mass calculated

0.3003

0.2972

0.3001

0.2964

According to British Pharmacopoeia (BP), the percentage of purity should be within 98.0 – 101.0% of dried substance.

Equation of the Percentage of Purity:

Sample 1:

Sample 2:

ii) Titration by using Phenolphthalein as the indicator

Sample 1

Sample 2

Mass of boat + sample (g)

3.8916

3.9195

Mass of boat + residue (g)

3.5915

3.6195

Mass of N-acetylcysteine transferred (g)

0.3001

0.3000

Table 6: The mass of N-acetylcysteine powder in sample 1 and sample 2 for titrations with 0.1M of sodium hydroxide.

First reading

Second reading

Initial volume (mL)

18.20

17.10

Final volume (mL)

36.80

36.95

Volume of 0.05M iodine used (mL)

18.60

19.85

Table 7: The volume of 0.1M sodium hydroxide used for both titration using sample 1 and sample 2 of N-acetylcysteine solution and phenolphthalein as indicator.

Calculations:

Actual concentration of sodium hydroxide (NaOH) used: 0.1062M

Molecular weight of N-acetylcysteine (C5H9NO3S): 163.2

The balanced equation for the reaction between N-acetylcysteine and sodium hydroxide (NaOH):

C5H9NO3S + NaOH à C5H8NO3SNa + H2O

From the equation, one mole of a N-acetylcysteine reacts with one mole of NaOH. So the reaction is a 1:1 ratio. To find out the number of mole of NaOH, the equation below is used:

First titration:

Moles of NaOH = (0.1062M x 18.60mL)/1000

= 1.975 x10-3 mole

As the reaction is 1:1 ratio so the number of moles of N-acetylcysteine is equal to the number of moles of NaOH used which is 1.821 x10-3 mole.

Mass of N-acetylcysteine = 1.975 x10-3 mole x 163.2

= 0.3224g

Second titration:

Moles of NaOH = (0.1062M x 19.85mL)/1000

= 2.108 x10-3 mole

As the reaction is 1:1 ratio so the number of moles of N-acetylcysteine is equal to the number of moles of NaOH used which is 1.821 x10-3 mole.

Mass of N-acetylcysteine = 1.816 x10-3 mole x 163.2

= 0.3440g

Calculation of Percentage of Purity:

Sample 1 of N-acetylcysteine

Sample 2 of N-acetylcysteine

Mass transferred

Actual mass calculated

Mass transferred

Actual mass calculated

0.3001

0.3224

0.3000

0.3440

Calculation of Percentage of Purity:

According to British Pharmacopoeia (BP), the percentage of purity should be within 98.0 – 101.0% of dried substance.

Equation of the Percentage of Purity:

Sample 1:

Sample 2:

d) Zinc: Not more than 10ppm Zinc (Zn):

To determine the concentration of Zinc metal present in a standardised sample, atomic absorption spectrophotometer was applied. This was done so as to comply with the British Pharmacopoeia (BP) standards, where the detected concentration of Zinc should not be more than 10ppm.

Mass of Acetylcysteine sample used: 1.00g

This sample was diluted accordingly and then analysed or measured by an atomic absorption spectrophotometer at a set wavelength of 213.8nm. According to the laboratory transcript, the absorbances were given, so the calculation was carried out to determine the concentrations for each solution.

Solution

Concentration (mg/L)

Absorbance (at 213.8nm)

(a)

0.00

0.056

(b)

0.25

0.115

(c)

0.50

0.173

Table 8: The absorbance of solution a, b and c using atomic absorbance spectrophotometer.

From the table 8 above, a standard additions calibration graph of concentration of zinc in mg/L against absorbance at 213.8nm is plotted. A rather small absorbance indicates that there is a trace or small amount of Zinc (Zn) present in Solution A, which practically contained only the N-acetylcysteine sample. Hence, we can plot a line of best fit and extrapolate to find the concentration of Zn present within our sample. Note that the amount of Zn present is proportional to the absorbance detected at 213.8nm wavelength.

Graph 1: The graph of absorbance against concentration of Zinc.

Extrapolated value= -0.24 Solution A = 0.24ppm Solution 1 = 0.24 Ã- 2 = 0.48 ppm

Solution 1

0.48g in 100 000 mL = 2.4 Ã- 10-4g in 50 mL

If 1g of N-acetylcysteine contains 2.4 Ã- 10-4g of zinc ions, 104g of acetylcysteine will contain 2.4g of zinc ions.

So concentration of zinc ions in N-acetylcysteine = 2.4ppm

Using the calibration graph, we obtained an equation for the line of best fit as shown below:

Using the line of best fit we can calculate the concentration of Zinc (Zn) present within Solution 1. This is determined by the difference between the origin (x = 0) and where the line of best fit intercepts the x-axis. To be more accurate, the equation of the line of best fit can be used by assuming the absorbance of N-acetylcysteine at 213.8nm (y-axis) is 0 (y = 0). We can then calculate and find the exact concentration of Zn added (x-axis in mg/L) which gives an absorbance reading of 0.0562 at the wavelength of 213.8 nm. This calculation is shown below where absorbance y = 0.

Concentration of Zinc in solution (a) where no Zinc is added:-

(Concentration comes in positive value)

Therefore, the diluted Solution 1 contains an exact concentration of 0.2402mgL-1 or 0.2402ppm. We can now use this concentration and work backwards from the dilution to obtain the mass of Zn within the 20mL Solution 1, as shown in the calculation below,

Mass of Zinc in Solution 1:-

From the mass of Zinc present in Solution 1 as calculated, we can say that this equals to the 10mL of N-acetylcysteine sample in Solution (a). This is because Solution 1 was diluted to 20mL using 0.001M hydrochloric acid and contained no other sources of Zinc. Hence, 4.8034μg of Zinc in 20mL of Solution 1 is equal to 4.8034μg of Zinc in 10mL of Solution (a). Now using this mass of 4.8034μg in 10mL of Solution (a) we can find out the total mass of Zinc within 50mL. However, the total mass of Zinc within 50mL of Solution (a) is equivalent to 1.00g of N-acetylcysteine sample which is the original sample mix. Using these data, the mass of Zinc can be calculated as shown in the calculation below,

Mass of Zinc in 1.00g of N-acetylcysteine: –

Hence, 2.4017μgmL-1 of Zinc is present in 1.00g. We can now calculate an exact concentration of Zinc in parts per million (ppm) as shown in the calculation below,

Concentration of Zinc within sample in ppm:-

e) Loss on drying: Not more than 1.0% w/w:-

Initial mass of N-acetylcysteine sample (g)

1.0965

Mass after drying under specified conditions (g)

1.0893

f) Related substances

1) Acetylcysteine: fresh sample 8.57mg/mL

From British Pharmacopoeia, the retention time for the N-acetylcysteine substances as below.

Substance

Retention time (min)

L- cystine

About 2.2

L- cysteine

About 2.4

2-methyl-2 thiazoline-4 carboxylic acid

About 3.3

N,N’-diacetyl-L- cystine

About 12

N,N’-diacetyl-L- cysteine

About 14

acetylcysteine

About 6.4

1) Acetylcysteine: fresh sample 8.57mg/mL

Substance

Retention time (min)

Peak retention time obtained

Concentration

L- cystine

About 2.2

1.93

0.5948

L- cysteine

About 2.4

–

–

2-methyl-2 thiazoline-4 carboxylic acid

About 3.3

3.25

0.0794

N,N’-diacetyl-L- cystine

About 12

–

–

N,N’-diacetyl-L- cysteine

About 14

13.623

0.3944

Acetylcysteine

About 6.4

6.972

94.7507

Calculation of impurities:

Peak area/ Total area x 100

Substance

Area

Concentration

Impurity

L- cystine

238606

0.5948

0.5948

L- cysteine

–

–

–

2-methyl-2 thiazoline-4 carboxylic acid

31861

0.0794

0.0794

N,N’-diacetyl-L- cystine

–

–

–

N,N’-diacetyl-L- cysteine

158211

0.3944

0.3944

Acetylcysteine

38007440

94.7507

94.7507

Total area= 40113072

2) Acetylcysteine: old sample 2.5mg/mL

Substance

Retention time (min)

Peak retention time obtained

Concentration

L- cystine

About 2.2

2.11

0.7214

L- cysteine

About 2.4

–

–

2-methyl-2 thiazoline-4 carboxylic acid

About 3.3

3.256

0.8946

N,N’-diacetyl-L- cystine

About 12

–

–

N,N’-diacetyl-L- cysteine

About 14

13.415

15.3284

Acetylcysteine

About 6.4

6.34

33.7241

Calculation of impurities:

Peak area/ Total area x 100

Substance

Area

Concentration

Impurity

L- cystine

62935

0.7214

0.7214

L- cysteine

–

–

–

2-methyl-2 thiazoline-4 carboxylic acid

78046

0.8946

0.8946

N,N’-diacetyl-L- cystine

–

–

–

N,N’-diacetyl-L- cysteine

1337263

15.3284

15.3284

Acetylcysteine

2942118

33.7241

33.7241

Total area= 8724087

3) Cysteine/ cystine: 0.5mg/mL

Substance

Retention time (min)

Peak retention time obtained

Concentration

L- cystine

About 2.2

2.018

5.2956

L- cysteine

About 2.4

2.323; 2.65

2.3189; 2.384

2-methyl-2 thiazoline-4 carboxylic acid

About 3.3

3.008; 3.207

24.9029; 65.0987

N,N’-diacetyl-L- cystine

About 12

–

–

N,N’-diacetyl-L- cysteine

About 14

–

–

Acetylcysteine

About 6.4

–

–

Calculation of impurities:

Peak area/ Total area x 100

Substance

Area

Concentration

Impurity

L- cystine

87001

5.2956

5.2956

L- cysteine

38097; 39167

2.3189; 2.384

2.3189; 2.384

2-methyl-2 thiazoline-4 carboxylic acid

409128; 1069503

24.9029; 65.0987

24.9029; 65.0987

N,N’-diacetyl-L- cystine

–

–

–

N,N’-diacetyl-L- cysteine

–

–

–

Acetylcysteine

–

–

–

Total area= 1642895

4. Discussion:

a) Specific optical rotation:

The specific rotation of a chemical compound [α] is defined as the observed angle of optical rotation α in stereochemistry, when plane-polarized light is passed through a sample with a path length of 1 decimetre (dm) and a sample concentration of 1 gram (g) per 1 millilitre (mL). The specific rotation of a pure material is an intrinsic property of that material at a given wavelength and temperature. The reading should be accompanied by the temperature at which the measurement was performed and the solvent in which the material was dissolved, and this often assumed to be room temperature. The exact unit for specific rotation values is deg dm−1cm3 g−1 or can use degrees (̊). Levorotatory rotation (l) means a negative reading obtained and the rotation being to be left. While dextrorotatory rotation (d) means a positive reading and the rotation is being to be right. The specific optical rotation for the freshly prepared solution of N-acetylcysteine is +24.5⁰ which it is dextrorotatory rotation and the old solution of N-acetylcysteine is -32.9⁰ which means levorotatory rotation.

Measurement of optical rotation is a way to assess optical purity of a sample containing a mixture of enantiomers. An enantiomer is one of two stereoisomers that are mirror images of each other that are “non-superposable” or not identical much as one’s left and right hands are “the same” but opposite. The specific optical rotation of N-acetylcysteine solution is within the range +21ÌŠ to approximately +27ÌŠ. The freshly prepared of N-acetylcysteine solution is found to be in the range however the old N-acetylcysteine solution is not in the range. This reveals stability alteration occurred in the old N-acetylcysteine solution. The impurities have found in the old N-acetylcysteine solution because the presence of small amount of impurities can affect the rotation of the sample.

The actual optical rotation value for freshly prepared N-acetylcysteine solution is measured by single polarimeter because if the sample is very concentrated or it has very large specific rotation or the sample larger than 180°, single polarimeter cannot be used. The variation of specific rotation with wavelength is the basis of optical rotary dispersion (ORD) which used to elucidate the absolute configuration of certain samples. High performance liquid chromatography (HPLC) is used to determined the enantiomeric ratio with a chiral column because the aggregation in the N-acetylcysteine solution cause optical rotation of a sample maybe not linear dependent due to enantiomeric excess.

b) ASSAY: 98.0%-101.0% C5H9NO3S (as dried material)

From the result obtained above, the mass obtained from the titration of N-acetylcysteine solution with iodine with starch as indicator for first titration is 0.13986g and second titration is 0.13552g. The percentage of purity obtained from the experiment for first sample is 99.64%. The percentage of purity from second sample is 98.26%. According to British Pharmacopoeia (BP), the percentage of should be within 98.0 – 101.0% of dried substance. The percentage of purity for both samples is within the range stated in the BP. BP prefer the iodine titration to a titration using sodium hydroxide because iodine is a very useful oxidising titrant which react with reducing agent ,N-acetylcysteine solution using starch as indicator. Iodine forms an intensely dark blue complex with starch. Starch is an oxidation – reduction indicator that shows a reversible colour change between the oxidised and reduced forms. It is not affected by the presence of iodide (I-). Both starch and iodide must be present for the starch to change colour during the titration. Iodine is consumed by thiosulfate in the titration step. The amount of thiosulfate used is proportional to the amount of iodine liberated from the salt. Sodium hydroxide is a strong base. It is more useful in acid- base titration using weak acid or base indicator.

c) Assay by titration with 0.1M of sodium hydroxide

From the result obtained in this experiment, the mass obtained from the titration of N-acetylcysteine solution with0.1M sodium hydroxide with phenol red as indicator for first titration is 0.2972g and second titration is 0.2964g. The percentage of purity obtained from the experiment for first sample is 98.97%. The percentage of purity from second sample is 98.77%. According to British Pharmacopoeia (BP), the percentage of should be within 98.0 – 101.0% of dried substance. The percentage of purity for both samples is within the range stated in the BP.

The mass obtained from the titration of N-acetylcysteine solution with 0.1M sodium hydroxide with phenolphthalein as indicator for first titration is 0.3224g and second titration is 0.3440g. The percentage of purity obtained from the experiment for first sample is 107.43%. The percentage of purity from second sample is 114.67%. According to British Pharmacopoeia (BP), the percentage of should be within 98.0 – 101.0% of dried substance. The percentage of purity for both samples is out of the range stated in the BP.

Phenol red and phenolphthalein are acid-base indicators. The un-dissociated form of the indicator is a different colour than the iogenic form of the indicator. An Indicator does not change colour from pure acid to pure alkaline at specific hydrogen ion concentration, but rather, colour change occurs over a range of hydrogen ion concentrations. This range is termed the colour change interval. It is expressed as a pH range. The pH range for phenol red is 6.8- 8.4 and phenolphthalein is 8.0- 10.0. The selection of indicator will depend on the actual expected pH at the equivalence point which selects an indicator with a pKa right in the middle of the pH change at the equivalence point. N-acetylcysteine solution has pKa 4.0 and 9.5, and a weak acid indicator has to be used to determine the end point of the titration. Phenol red produce a good result compared to the phenolphthalein as indicator when titrate N-acetylcysteine solution with 0.1M sodium hydroxide.

d) Zinc: Not more than 10ppm Zinc (Zn):

By performing the atomic absorbance technique, we have determined that the N-acetylcysteine sample contained a Zinc concentration of 2.4017ppm. This sample complied with the requirement from the British Pharmacopoeia (BP) monograph standards by not having a Zinc concentration of greater than 10ppm.

Atomic absorbance technique can only detect specifically one heavy metal at a time. So, it is very time consuming to detect a wide spectrum of heavy metal impurities within our sample. Plus, the N-acetylcysteine monograph only indicates the need to monitor the level of Zinc present within the sample by atomic absorbance spectrometry. Therefore, to detect other heavy metals we would prefer to use the more generic “Limit Test C for Heavy Metals” as specified in the British Pharmacopoeia (2008), Volume IV, and Appendix VII.

e) Loss on drying: Not more than 1.0% w/w:-

According to British Pharmacopoeia (BP), it states that there should be no more than 1.0% in mass. This sample is complied with the BP monograph standards with a loss of only 0.66% in mass.

f) Related substances:-

HPLC is used in pharmaceutical analysis to quantitative determinations of drugs in formulations. These analyses do not require long time to optimising mobile phase and selecting columns and detectors. Some formulations contain more than one active ingredient and may present more of an analytical challenge since the different ingredients may have quite different chemical properties and elute at very different times from HPLC column.

5. Conclusions:

Quality control is an essential operation of the pharmaceutical industry. Drugs must be marketed as safe and therapeutically active formulations whose performance is consistent and predictable. A bundle of sophisticated analytical methods are being developed for the drugs evaluation in pharmaceutical industry. Requirements governing the quality control of pharmaceuticals in accordance with the British Pharmacopoeia (BP) or European Pharmacopoeia.

Titration is a procedure used in chemistry in order to determine the molarity of an acid or a base. A chemical reaction is set up between a known volume of