SOP for Operation and Calibration of Jasco HPLC with PDA Detector

PURPOSE

To lay down a Procedure for Operation and Calibration of Jasco High-Performance Liquid Chromatography with a PDA Detector.

SCOPE

This procedure applies to the operation and calibration of Jasco high-performance liquid chromatography with a PDA detector located in the quality control department of Pharmainfo Limited.

RESPONSIBILITY

QC Personnel

• To initiate the Document Change Request and to prepare or revise the SOPs.
• To provide the training as per new or revised SOPs.
• To ensure the calibration, maintenance, and cleaning of the instrument in accordance with the SOP.

QC Head/Designee

• To review the new and revised SOPs. (The person who has prepared shall refrain from reviewing/approving the SOP).
• To ensure that the training is provided to all the required personnel.

QA Manager

• To approve the new and revised SOPs.

QA Personnel

• To prepare the master and control copies of the SOPs.
• To Distribute, Retrieve, Archive and Destruct the controlled copies of SOPs.
• To maintain, control and distribute all the SOPs at site.

PROCEDURE

Operation of High-Performance Liquid Chromatography with PDA Detector:

• Ensure that the instrument and surrounding area are clean and instrument calibrated, if not, calibrate the instrument before use.
• HPLC system comprises of,

1. Interface box (LC NETII/ADC),
2. Quaternary pump,
3. Column oven,
4. Autosampler,
5. Detector,
6. Bottle Stand.

• Switch “ON” the main switch.
• Switch “ON” the interface box first as it connects the system.
• Simultaneously put “ON” the switches that are on the front side of each HPLC module.
• Put “ON” the computer. As the server is connected to the computer all functions of individual modules are controlled on the computer (software-ChromNav).
• Double-click on the “ChromNav” icon on the Desktop.
• Enter the allocated user ID and password and click Log on. The control center window appears.

Preparing HPLC System for Analysis:

In the control center window, click start ChromNav, double click on “Jasco HPLC” or “Start in Analysis mode” The ‘Open Project’ window appears. Click the required project and open it. A system monitor window appears.

Purging of mobile phase:

1. Open the purge valve, and press “PUMP” on the Pump module to start the pump.
2. To enter flow rate, press “FLOW”, on the Pump module and enter the flow. Increase the flow rate gradually for purging to avoid sudden jerk to the pump motor.
3. To stop purging, press “PUMP” on the Pump module to stop the pump, decrease the flow rate gradually to 0.0 and close the purge valve.
4. 3.1.9.3 Flushing of syringe :
5. Press “SHIFT” and then “FLUSH” button twice on the Autosampler module to start flushing.

• Open the column compartment and fix the necessary column required.
• On the `pump’ module, press “PUMP” to start the pump. To enter flow rate, press “FLOW”, on the Pump module and enter the flow. Carry out the activity for column washing and run the mobile phase to steady pump operation.
• On the `Column compartment’ module, enter required temperature value.
• On the `Detector’ module, check the PC link light and D2 light

User Manager:

The HPLC System software has following User Types privileges:

1. Administrator (Standard): This password is controlled by the Manager Quality control/ Asst. Manager Quality Control.
2. User (Analyst): This password is controlled by all the users.

Preparation of Project for Analysis:

Click Management Tools, and click Project. In the Operation icon, click New Project. Enter the project name as Product/Material name-Ingredient-Test (for product) - Method of analysis and click New.

For example:

In Lamivudine Tablets BP 150 mg for Lamivudine Related substances name the Project as ‘LAMIVUDINE TAB- LAMIVUDINE -RS’ and for Lamivudine Assay ‘LAMIVUDINE TAB- LAMIVUDINE -ASSAY’.
In Raw Material for Lamivudine Related substances name the Project as ‘LAMIVUDINE - RS’, for Lamivudine Assay ‘LAMIVUDINE - ASSAY’.

Preparation of Control Method for Analysis:

• In System Monitor, in a file, click method editor and control method. Set the Method time required.
• General: Click Autosampler, Pump, column oven, and PDA detector.

1. Autosampler: Set Injection mode, number of flushes, injection method, loop volume
2. Pump: Set pump mode, flow, Maximum and Minimum pressure.
3. Column Oven: Set the temperature required.
4. PDA detector: Enter wavelength, D2 lamp on.

• Click Save As Enter file name as Product / Material name-Ingredient (for product) - Method of analysis and click save.

For example:

In Lamivudine Tablets BP 150 mg for Lamivudine Related substances name the Control method as ‘LAMIVUDINE TAB- LAMIVUDINE - RS’, and for Lamivudine Assay ‘LAMIVUDINE TAB- LAMIVUDINE - ASSAY’.

In Raw Material for Lamivudine Related substances name the Control method as ‘LAMIVUDINE - RS’, for Lamivudine Assay ‘LAMIVUDINE - ASSAY’.

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Preparation of Sequence for Analysis:

• In the file, click method editor and Acquisition sequence.
• Enter Type, Sample, Volume, Chromatogram name, Acquisition time (acquisition time shall be always less than actual run time) & Control method.
• Click save, Enter the file name as Product/Material name-Ingredient (for a product) - Method of analysis, and save the sequence. Prepare the sequence as per Procedure for Sequence of Injections for HPLC Analysis and Integration Parameters.

For example:

In Lamivudine Tablets BP 150 mg for Lamivudine Related substances name the Sequence as ‘LAMIVUDINE TAB- LAMIVUDINE - RS’ for Lamivudine Assay ‘LAMIVUDINE TAB- LAMIVUDINE - ASSAY’.

In Raw Material for Lamivudine Related substances name the Sequence as ‘LAMIVUDINE - RS’ for Lamivudine Assay ‘LAMIVUDINE - ASSAY’.

• For HPLC Calibration: Type the Sequence name as HPLC Calibration-Parameter and click `SAVE` to save the Sequence.
• For HPLC Column Calibration: Type the Sequence name as Column Calibration and click `SAVE` to save the Sequence.
• For Other analysis e.g. R&D samples, method development, Analytical method Validation, Rinse/Swab samples, Cleaning Validation, Dissolution profile, name the Sequence as per the requirement.

To start Baseline:

• Click Acquisition, and then click Baseline Monitor. Select the control method. Set X- axis as required and click OK.
• To view the baseline in running mode, click Chromatogram monitor. A Chromatogram monitor window appears.

To Start the Analysis:

• In file, click acquisition sequence to open the required sequence to run.
• To start the sequence, click the start icon.
• To view the online chromatogram, click on acquisition, chromatogram monitor. A Chromatogram monitor window appears.

Processing of the Chromatogram:

• On completion of Analysis, Click Analysis. In the file, click open chromatogram. ‘Select chromatogram to be opened’ window appears. Then select the executed sequence and double-click the required chromatogram to be integrated. The ‘Chromatogram view’ window appears.
• Click edit peak method icon, change the parameters, and click the check icon to integrate the chromatogram. Click the Save icon, enter the file name as Product/Material name-Ingredient (for the product)-Method of analysis and click save.

For example:

In Lamivudine Tablets BP 150 mg for Lamivudine Related substances name the peak method as ‘LAMIVUDINE TAB- PARACETAMOL- RS’ for Lamivudine Assay ‘LAMIVUDINE TAB- LAMIVUDINE - ASSAY’.

In Raw Material for Lamivudine Related substances name the peak method as ‘LAMIVUDINE - RS’ for Lamivudine Assay ‘LAMIVUDINE - ASSAY’.

• Click edit peak ID table icon, enter name of the peak, channel, retention time and window.
• Click peak process icon, in channel 1, select peak method, click open. Click execute all.
• To add peak information, In view, click peak information items. ‘Arrange peak info. Table’ window appears. Add or delete the required peak Information items and click OK.
• Right-click on the chromatogram, select view options, click peak name in Annotation 1 and retention time in Annotation 2. Click OK.
• Click Edit report style icon, report style window appears. Select the style and information required in the printer layout and save the report template for the Project.
• Further click on ‘Printer layout’ icon and take print of the chromatograms.
• Chromatogram to be signed by Analyst and checked by Section Incharge / QC Executive.

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Calibration of High-Performance Liquid Chromatography:

Frequency: once in six months.

Check the Test Parameters as mentioned below:

1. Pump Flow rate (Pump Performance)
2. Calibration of Column Oven (Temperature Accuracy)
3. Detector Linearity
4. Precision (System Performance)
5. Wavelength Accuracy
6. Injector Carry Over Test

1. Pump Flow rate (Pump Performance)

• Purge each port (viz. A, B, C, D) with purified water for at least 2 minutes.
• Fix the union instead of the HPLC column. Close the column compartment.
• Use the filtered & degassed purified water as the mobile phase. (With all the four ports (A, B, C, D),
• Set the flow rate of the pump at the rate of 0.5 ml/minute for port A & start the pump. Allow the pump to stabilize for 5 minutes.
• Weigh clean & dry 20 ml glass vials; & note down the weight (W1). Put the outlet tubing into weighed vial & collect the water for 20 minutes.
• Check the time with a calibrated stopwatch.
• Weigh the vial again after collecting purified water (W2) & note down weight in Annexure-1.
• Repeat the steps from 4 to 7 for flow rate 1.0 ml / Minute for 10 minutes & 2.0 ml/minute for 5 minutes.
• Repeat the procedure from 4 to 8 for Port B, C, & D.
• Record the Calibration results in Annexure-1.

2. Calibration of Column Oven (Temperature Accuracy)

• Carry out the calibration of Column Thermostat / Oven at 20°C, 25°C, 30°C, 35°C, 40°C,45°C & 50°C.
• Switch 'ON' the column oven.
• Set a desired temperature & allow it to equilibrate for about 10 minutes.
• Place the calibrated digital multi-thermometer in the column oven. After the desired temperature is attained. Read the temperature displayed on to the screen as well on the thermometer.
• Record the Calibration results in Annexure-1.
• Ensure that the temperature should not deviate with the displayed temperature by more than ± 2°C.

3. Gradient Concentration

• Prepare a 0.001% Caffeine in purified water. Place ports B & D in 0.001% Caffeine & ports A & C into purified water.
• Purge each port for about 2 minutes with a flow rate of 5.0 ml/minute.
• Connect a Union as a column & keep the oven temperature 40°C
• Set the flow rate as 1.0 ml/minute & allow it to stabilize for about 15 minutes.
• Set the program as per the Chromatographic conditions mentioned below.

Chromatographic Conditions:

Column Description	Union
Column Temperature	40°C
Flow Rate	1.0 ml/minute
Detection Wavelength	270 nm
Injection Volume	20 µl

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Set the time program as mentioned below:

Step No.	Time (min)	Function	A %	B %
Initial	-	-	100.0	0.0
1	0.1	1	0.0	100.0
2	12.5	1	0.0	100.0
3	12.6	1	100.0	0.0
4	25.0	1	100.0	0.0
5	25.1	1	90.0	10.0
6	35.0	1	90.0	10.0
7	35.1	1	50.0	50.0
8	45.0	1	50.0	50.0
9	45.1	1	10.0	90.0
10	55.0	1	10.0	90.0
11	55.1	1	0.0	100.0
12	65.0	1	0.0	100.0
13	65.1	1	100.0	0.0

C % and D% = 0

• Sample preparation: Purified water
• Set time program for ports C & D, A & D, as shown in step 6 of gradient concentration.
• Prepare the project, control method & sequence as per procedure & run the sequence.
• Integrate the sequence.
• Calculate % Height concentration.
• Record the results in Annexure-1.

4. Detector Linearity 

• It is the ability to elicit test results directly proportional to the concentration of the analyte in samples within a given range.
• Preparation of Caffeine standard solution (50µg/ml): Weigh accurately 50 mg of Caffeine standard in a 100 ml volumetric flask, add sufficient methanol to dissolve, and dilute to 100 ml with methanol (Solution A: 500µg/ml). Further, dilute 10 ml of the above solution to 100 ml with methanol (50µg/ml)
• Operate the instrument as per below chromatographic condition below,

Flow rate	1.0 ml/min
Detection wavelength	273 nm
Column	250 x 4.6 mm, 5 µ (ODS)
Mobile phase	Methanol
Column temperature	25°C
Run Time	5 mins
Retention time of Caffeine	about 3 mins

• Prepare the project, control method & sequence as per the procedure mentioned in this SOP.
• Check Linearity study for a detector, Inject solutions of Caffeine standard as per given below sequence. Integrate the chromatogram & calculate the mean peak area of Caffeine for each concentration. Plot a graph of the mean peak area of Caffeine Vs injection volume in µl. Calculate the correlation coefficient. Record the Calibration results in the Format No. Annexure-1.
• Sequence of Injection:

Type of solution	No. of Injection	Injection volume	Wavelength
Blank (Mobile phase)	1	20 µl	273 nm
50µg/ml Caffeine standard	2	10 µl	273 nm
50µg/ml Caffeine standard	2	20 µl	273 nm
50µg/ml Caffeine standard	2	30 µl	273 nm
50µg/ml Caffeine standard	2	40 µl	273 nm
50µg/ml Caffeine standard	2	50 µl	273 nm
Blank (Mobile phase)	1	20 µl	273 nm

• Acceptance criteria: The correlation coefficient should not be less than 0.997

5. Precision (System Performance):

• It is the degree of repeatability of the results in a series of experiments run during a single session by a single operator with identical reagents and equipment.
• Operate the instrument as per the chromatographic condition for the Detector linearity test.
• Prepare the project, control method & sequence as per procedure mentioned in this SOP.
• Inject 20 µl of Caffeine standard solution (50µg/ml) in replicate six times
• Sequence of Injection :

Type of solution	No. of Injection	Injection volume	Wavelength
Blank (Mobile phase)	1	20 µl	273 nm
50µg/ml Caffeine standard	6	20 µl	273 nm
Blank (Mobile phase)	1	20 µl	273 nm

• Acceptance criteria: % RSD of an area of Caffeine standard is NMT 2.0 % & % RSD of Retention time of Caffeine is NMT 5.0%.
• Record the Calibration results in Annexure-1.

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6. Wavelength Accuracy: 

• It is the closeness of test results obtained by that method to the true value over the entire HPLC UV working range.
• Operate the instrument as per the chromatographic condition for the Detector linearity test.
• Prepare the project, control method & sequence as per the procedure mentioned in this SOP
• Chromatographic condition as per detector linearity test, but change the program for different wavelengths. Inject 50 µg/ml solution of Caffeine standard in duplicates as per the sequences mentioned below.

Type of solution	No. of Injection	Injection volume	Wavelength
Blank (Mobile phase)	1	20 µl	270 nm
50µg/ml Caffeine standard	2	20 µl	270 nm
Blank (Mobile phase)	1	20 µl	271 nm
50µg/ml Caffeine standard	2	20 µl	271 nm
Blank (Mobile phase)	1	20 µl	272 nm
50µg/ml Caffeine standard	2	20 µl	272 nm
Blank (Mobile phase)	1	20 µl	273 nm
50µg/ml Caffeine standard	2	20 µl	273 nm
Blank (Mobile phase)	1	20 µl	274 nm
50µg/ml Caffeine standard	2	20 µl	274 nm
Blank (Mobile phase)	1	20 µl	275 nm
50µg/ml Caffeine standard	2	20 µl	275 nm
Blank (Mobile phase)	1	20 µl	276 nm
50µg/ml Caffeine standard	2	20 µl	276 nm
Blank (Mobile phase)	1	20 µl	277 nm
50µg/ml Caffeine standard	2	20 µl	277 nm
Blank (Mobile phase)	1	20 µl	278 nm
50µg/ml Caffeine standard	2	20 µl	278 nm
Blank (Mobile phase)	1	20 µl	278 nm
Blank (Mobile phase)	1	20 µl	279 nm
50µg/ml Caffeine standard	2	20 µl	279 nm
Blank (Mobile phase)	1	20 µl	279 nm
Blank (Mobile phase)	1	20 µl	280 nm
50µg/ml Caffeine standard	2	20 µl	280 nm
Blank (Mobile phase)	1	20 µl	280 nm

• Calculate the mean peak area of Caffeine at each wavelength. Record the Calibration results in Annexure-01.
• Acceptance Criteria: The peak obtained in the wavelength range of 274 to 278nm should show maximum.

7. Injector Carry Over Test: 

• To check the Instrument carryover.
• Operate the instrument as per the chromatographic condition for the Detector linearity test.
• Prepare the project, control method & sequence as per procedure mentioned in this SOP.
• Inject 20 µl of Caffeine standard solution (20 µg/ml) as mentioned below.

Type of solution	No. of Injection	Injection volume
Blank (B1)	3	20 µl
20 µg/ml Caffeine standard	6	20 µl
Blank (B2)	1	20 µl

• Integrate the chromatogram blank (B1), (B2) & Caffeine std (20 µg/ml) & Note down the Peak area of Caffeine.
• Acceptance criteria: Last blank should not show any peak response of Caffeine.
• Record the Calibration results in Annexure-01.
• Acceptance criteria: The percentage of carry-over should not be more than 0.1%.
• After Calibration affix the “INSTRUMENT CALIBRATION” status label annexure-2 filled with required details and duly signed on the front side of the Instrument.
• If the Acceptance criteria of Calibration are not met, then do not use the Instrument and affix the “OUT OF CALIBRATION" label annexure-3.
• Inform to QA department and investigate as per “Procedure for Deviation and Failure Investigation”.
• Review the data/results taken from the last date of calibration. Analyze a few of the samples/ batches from the last date of calibration to reassess for the validity of results.
• After rectification recalibrate the instrument and affix the “INSTRUMENT CALIBRATION” status label filled with required details and duly signed.

ANNEXURES

Annexure No.: 1 - Calibration of HPLC

ABBREVIATIONS

HPLC: High Performance Liquid Chromatography
µ: Micron
µl: Microliter
UV: Ultra Violet
ODS: Octadecylsilyl
nm: Nanometer
µg: Microgram
ml: Milliliter
PDA: Photo Diode Array

REVISION HISTORY

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