PKKK/300/UP/031 Penentuan Lovastatin dalam produk tradisional menggunakan HPLC

Materials and Equipment’s

Instrument :

High Performance Liquid Chromatography with photodiode array (PDA) detector

Analytical & microbalance

Column : Thermo ODS Hypersil 5 µm, 200 x 4.6 mm or equivalent

Reference standard : Lovastatin USP or equivalent

Chemical :

Monobasic Sodium Phosphate

Phosphoric acid

Glacial Acetic Acid (AR Grade)

Sodium hydroxide

Methanol (HPLC Grade)

Acetonitrile (HPLC Grade)

Deionized water

Glassware’s and consumable :

Beakers

Volumetric Flasks (Grade A)

Measuring cylinder

Nylon syringe filter 0.45 µm

Disposable syringe without needle (5 mL)

2 mL glass screw cap vial

Preparation of Solutions

Preparation of diluent

Preparation of 2M sodium hydroxide: Weigh 8 gm sodium hydroxide and dissolve in 100 mL deionized water.

Measure 900 mL of deionized water in a beaker and add 3 mL glacial acetic acid. Adjust pH of the solution to pH4 using 2M sodium hydroxide. Transfer the solution into 1 L volumetric flask and make up volume to 1L with deionized water. Then, prepare a mixture of acetonitrile and the resultant solution with composition 80:20.

Preparation of buffer solution

Preparation of 1M phosphoric acid: Mix 6.8 mL of 85% (w/w) concentrated phosphoric acid solution to 93.2 mL water to prepare 100 mL of 1M phosphoric acid.

Preparation of mobile phase

Preparation of Reference Standard

Standard stock solution

Working standard solution (for calibration curve)

To obtain 0.7 µg/mL, measure 0.175 mL of stock standard solution and make up to 25 ml with diluent.

To obtain 2.0 µg/mL, measure 0.2 mL of stock standard solution and make up to 10 ml with diluent.

To obtain 10.0 µg/mL, measure 1 mL of stock standard solution and make up to 10 ml with diluent.

To obtain 20.0 µg/mL, measure 2 mL of stock standard solution and make up to 10 ml with diluent.

To obtain 38.0 µg/mL, measure 3.8 mL of stock standard solution and make up to 10 ml with diluent.

Working standard solution at LOD level, 0.6 µg/mL (300 mg/kg)

Measure 0.15 mL stock standard solution A and make up to 25 mL with diluent.

Working standard solution 20.0 µg/mL (for calibration check)

Sample handling (sampling)

Sampling processes differ between different types of dosage form. Please refer to the document PKKK/200/018 (Prosedur Persampelan Untuk Analisis Sampel) dan PKKK/300/UAT/001 (Proses Persampelan Ujian Penyaringan).

Average weight of the total tablet content

Calculate the total average weight of the tablets

Sample preparation for analysis

For liquid sample, weigh 200 mg of sample in 100 ml volumetric flask in duplicate.

Leave the solution to cool before making it up to volume of 100 mL with diluent

Previously tested samples (Retained samples with known values)

Samples from Inter-Laboratory Comparison (ILC)

Standard spiking method - Sample spiked with Lovastatin at a final concentration of 20.0 µg/mL (10,000 mg/kg)

For Tablets: Take 20 tablets, crush and mix to homogenize

For Liquids: Shake the container to homogenize and pour a suitable amount into a beaker

Leave the solution to cool before making it up to volume of 100 mL with diluent

Perform spiking by adding 20 mL stock standard solution of Lovastatin, 100 µg/mL

Make up to volume of 100 mL with diluent

IQC sample will be injected twice and percent Recovery, (% Recovery) will be calculated by using the appropriate formula below:

For previously tested sample or sample from ILC:

Assigned Value is obtained from:

For previously tested samples: Refer to the original Certificate of Analysis (COA) for that specific batch.

For ILC samples: Refer to the ILC Lovastatin Current Report. Use the “Assigned Value” stated in the official report.

Sample spiked with Lovastatin at 20.0 µg/mL:

x 100%

IQC check passed if the IQC sample passed with recovery within 80 – 110%.

The HPLC system used for determination of Lovastatin is given in Table 1 below.

Table 1: HPLC system for determination of Lovastatin.

Refer to document PKKK/300/UAT/004 (RRLC 1200 Series), PKKK/300/UAT/012 (HPLC Shimadzu) and PKKK/300/UAT/017 (HPLC Perkin Elmer) on how to operate HPLC system in details.

System suitability

In order to check and ensure the system used is fit for the analysis, carry out a system suitability test using working standard solution 20.0 µg/mL (six injections) before starting any sample injection. The requirements are shown in Table 2 below:

Table 2: Requirement for system suitability test (SST)

System suitability test have to be done using HPLC offline system

At chromatogram, discard unnecessary peak

Next, click

If the system suitability does not meet the specified requirements, repeat the test until it passes. If necessary, prepare a new working standard solution before proceeding with any working standard solution for calibration curve and sample injections.

Standard Injection and Calibration curve

Steps required to generate the calibration curve:

Next click load file icon, select 2.0 µg/mL file

Repeat step 6.5.1. iv to 6.5.1.vi for 10.0 µg/mL, 20.0 µg/mL and 38.0 µg/mL working standard solution

Ensure the correlation coefficient (R2

Type ‘LOVASTATIN’ at calibration table

Then, click calibration Calibration Settings. The calibration setting table will pop up.

Title: CALIBRATION CURVE FOR LOVASTATIN

Default Calibration Curve

Type: Linear

Origin: Ignore

Weight: Equal

Type ‘µg/mL’ at ‘Amount Unit’ column

Sample Injection

Retention time and UV spectrum observation

To observe and check retention time and UV spectrum of the sample and RS, refer to document PKKK/300/UAT/004 (RRLC 1200 Series), PKKK/300/UAT/012 (HPLC Shimadzu) and PKKK/300/UAT/017 (HPLC Perkin Elmer).

Calibration Check After Sample Injections

Perform a calibration check by injecting Working Standard Solution B (20.0 µg/mL) at the following intervals:

After every 5 sample injections

After completing all sample injections (end of analysis)

Calculate the % Recovery and Similarity Factor based on the formula below:

% Recovery for Working Standard Solution B

The calibration check is considered passed if Recovery is within 95 – 105%.

Using Similarity Factor, sf

The similarity factor passes if sf is within 98 – 102%.

The calibration check must fulfil both criteria in order to verify calibration integrity.

If either criterion fails:

Re-inject Working Standard Solution B

If still out-of-tolerance, prepare a fresh Working Standard Solution B and repeat

If failure persists, invalidate the batch analysis and investigate (standard preparation, column / system performance, sample preparation).

Sequence of Injection

Table 3: Sequence injection for determination of Lovastatin

If Lovastatin is detected, the amount of Lovastatin needs to be calculated.

Amount of Lovastatin in %w/w

(NPRA limit: Not more than 1.00 %w/w)

The total amount of Lovastatin in sample calculated by the formula below:

Dose Lovastatin per day (mg/day)

(NPRA limit: Not more than 10.00 mg/day)

Tablet Dosage Form

First, calculate the amount of Lovastatin per tablet (mg/tab) by the formula below:

Then, the dose Lovastatin per day is calculated by the formula below:

ii) Liquid Dosage Form

Given product label: T mg of red yeast rice / Monascus Purpureus per Y mL#

Lovastatin content from 6.8.1: Z % w/w

Formula:

Lovastatin per Y mL dose (mg) = T

Then, the dose Lovastatin per day is calculated by the formula below:

#Number of doses taken per day = Can be obtained from the dosage information at the sample packaging.

Sample consider not detected with Lovastatin if the area of Lovastatin peak in the sample is below the area of the working standard solution at LOD level that is 300 mg/kg (0.6 µg/mL)

Result documented in Certificate of Analysis: