Analytical procedure

5.1

Introduction to the analytical procedure

An analytical procedure can be described as a step-by-step process that is used to qualify (identify), quantify or characterise a certain chemical substance or mixture (American Chemical Society, n.d.). In this case, we are qualifying and quantifying acetylcysteine in the 20 % w/v nebulizing solution.

To qualify and quantify acetylcysteine, High-Performance Liquid Chromatography (HPLC) is used as the primary assay. This is an analytical technique used to separate, identify and therefore quantify specific individual components in a liquid mixture. The process is carried out by passing a mobile phase through a column that is tightly packed under high pressure. The acetylcysteine nebulizing solution is treated as a liquid mixture, and the aim is to separate, identify and quantify the acetylcysteine content present.

This method uses reversed-phase chromatography, meaning the stationary phase is non-polar and the mobile phase is polar. The stationary phase consists of octadecylsilyl silica gel (a C18-bonded silica), a non-polar, chemically modified material tightly packed within the column. The mobile phase is a polar aqueous mixture of methanol and buffered ammonium sulphate solution, flowing consistently through the system.

Separation occurs based on polarity: more polar species interact less strongly with the non-polar stationary phase and are carried through the column more quickly by the polar mobile phase, while less polar species are retained longer. As the sample passes through the column, molecules with greater affinity for the stationary phase move slowly, while those with greater affinity for the mobile phase move more quickly. As a result of these different speeds, the different species separate from one another and reach the detector at different times.

5.2

Materials required

Calibration standards

A bracketing series of calibration standards, prepared from the acetylcysteine CRM stock solution and diluted with the mobile phase, spanning 80–120 % of the target working concentration (0.2 % w/v acetylcysteine, or 2000 μg/ml).

% of target	Concentration (μg/ml)
80 %	1600
90 %	1800
100 %	2000
110 %	2200
120 %	2400

HPLC equipment

Key HPLC terms

5.3

HPLC conditions

Stationary phase	A stainless steel column (25 cm × 5 mm), packed with octadecylsilyl silica gel for chromatography (5 μm particle size)
Elution	Isocratic
Mobile phase	10 volumes of methanol and 90 volumes of 0.5 % w/v ammonium sulphate containing 0.02 M sodium pentanesulphonate; adjusted to pH 2.0 using 2 M hydrochloric acid
Flow rate	1 ml per minute
Detection wavelength	205 nm (UV/Vis)
Injection volume	20 μL per solution
Run time	Three times the retention time of acetylcysteine

5.4

Procedure for the standard solution

It is important to have a standard against which a comparison can be made, to ensure reliability and consistency — it provides a reference point when performing any procedure. Creation of a master stock solution is therefore required.

Procedure

1. 01Accurately weigh 40.0 mg of acetylcysteine CRM.
2. 02Transfer to a 10.0 ml volumetric flask and dissolve in a small amount of mobile phase.
3. 03Dilute to volume with mobile phase to give a 4.0 mg/ml (0.4 % w/v, 4000 μg/ml) stock solution.
4. 04Prepare fresh. Do not store beyond the working session, given the oxidation risk discussed in Section 4.3.

After the creation of this stock solution, the calibration curve is built using the equation C₁V₁ = C₂V₂. For the volume of stock solution required and the working detail for each target concentration, see the table under Section 4.7.

5.5

Procedure for sample preparation

The acetylcysteine nebulizing solution contains 200 mg/ml of acetylcysteine (20 % w/v). According to the BP monograph for Acetylcysteine Injection, the target concentration is 0.2 % w/v (2000 μg/ml). Using C₁V₁ = C₂V₂, a volume of 1.00 ml of the nebulizing solution is required, as shown in the calculations under Section 4.5.

Procedure

1. 01Accurately pipette 1.00 ml of the 20 % w/v nebulizing solution into a 100 ml Class A volumetric flask using a Class A bulb pipette.
2. 02Dilute to volume with mobile phase and invert at least 10 times to ensure homogeneity (do not shake vigorously, to avoid introducing oxygen or air into the solution).
3. 03Resulting in a concentration of 2 mg/ml, which is equal to 2000 μg/ml and 0.2 % w/v.

Because this working concentration matches what is prescribed in the BP, the sample sits at the same concentration as the calibration midpoint. This minimises extrapolation error when reading off the final calibration curve.

5.6

System suitability and limits

System suitability is important in HPLC as it ensures the entire setup of the procedure is working properly and meets the criteria of the BP before testing the samples prepared above. It identifies issues that could exist with the equipment or the process before actual testing occurs, and in the end saves time and resources.

According to the British Pharmacopoeia for Acetylcysteine Injection, when detecting impurities caused by the oxidative degradation products of acetylcysteine, the HPLC test being performed is not valid unless it meets the following criteria:

• —In the chromatogram obtained with the system suitability solution, the height of the trough separating the cysteine and cystine peaks is less than one quarter of the height of the cysteine peak.
• —A peak corresponding to N,N′-diacetylcystine (retention time approximately 13 minutes) appears, with an area greater than the area of any corresponding peak in the chromatogram obtained from the freshly prepared system suitability solution — demonstrating the system's ability to detect this degradation product as it forms.

As with any procedure, limits exist. Limits explain the reliability and accuracy of this particular method; being aware of them ensures there is an understanding of whether the method is actually able to detect any impurities present, qualify and quantify the active pharmaceutical ingredient, and meet the required standards. Limitations related to the detection of impurities, according to the British Pharmacopoeia for Acetylcysteine Injection, are as follows:

5.7

Calibration curve construction

Once the calibration standards have been prepared, the calibration curve is constructed as follows:

1. 01Inject 20 μL of each calibration standard into the HPLC system, using the chromatographic conditions specified in Section 5.3.
2. 02Record the peak area corresponding to each calibration standard.
3. 03Plot the recorded peak areas against their known concentrations to construct the calibration curve, and determine the regression line.

The calibration curve serves as the reference against which the sample's peak area is later compared to determine acetylcysteine content.

5.8

Sample analysis

Once the sample dilution is prepared, it is analysed as follows:

1. 01Inject 20 μL of the prepared sample dilution into the HPLC system, under the same chromatographic conditions used for the calibration standards.
2. 02Record the peak area corresponding to acetylcysteine, together with any other secondary peaks corresponding to cysteine, cystine or N,N′-diacetylcystine.
3. 03Confirm the identity of the acetylcysteine peak by comparing its retention time to that of the calibration standards.

5.9

Data analysis

Quantification of acetylcysteine content is calculated by comparing the sample's peak area against the calibration curve obtained in Section 5.7.

• —Read the sample's acetylcysteine concentration directly from the calibration curve.
• —Calculate the % w/v content, accounting for the dilution factor applied during sample preparation, and compare this to the declared 20 % w/v label claim.
• —Assess related substances by expressing the area of each secondary peak (cysteine, cystine or N,N′-diacetylcystine) as a percentage of the acetylcysteine peak, and compare each against the corresponding limit specified in Section 5.6.
• —Confirm that all system suitability and limit criteria have been satisfied before accepting the result as valid.

5.10

Reporting

Acetylcysteine content is reported as % w/v of the nebulizing solution, alongside the % of label claim. Any related substances detected are reported as a percentage of the acetylcysteine peak area, referenced against the British Pharmacopoeia limits for Acetylcysteine Injection (British Pharmacopoeia Commission, 2022).

5.11

Cross-validation using iodometric titration

Acetylcysteine is a derivative of the amino acid L-cysteine and possesses a sulfhydryl group (-SH). Due to this additional hydrogen atom, acetylcysteine is in a reduced state and has additional electrons that can be donated. Iodine reacts readily with free thiol groups, oxidising them in a redox reaction. However, this reaction is not selective for acetylcysteine specifically: iodine will oxidise any free thiol present in the sample, including thiol-containing degradation products.

This lack of specificity is precisely why iodometric titration is unsuitable as the primary quantification method, and is instead used here as an independent cross-validation check against the HPLC result, following the British Pharmacopoeia's official assay method for Acetylcysteine Injection.

Procedure

1. 01Measure out a volume of the nebulizing solution theoretically containing 0.4 g of acetylcysteine.
2. 02Add 20 mL of glacial acetic acid to create the acidic environment required for the redox reaction.
3. 03Titrate with 0.05 M iodine volumetric solution (VS).
4. 04Iodine is continuously consumed by the free thiol groups present until no further reducible thiol remains. The next drop of iodine added is then in excess, producing a permanent pale yellow colour that marks the endpoint.