This document details the procedure for the Operation and Calibration of LC/MS/MS. Calibration of a mass spectrometer. It is the policy of Xyz Limited that a written procedure shall be followed for the Operation and Calibration of LC/MS/MS and its use monitored to ensure smooth operation and its use monitored to obtain consistent and reproducible results, minimize downtime, and meet regulatory requirements.
This procedure is to be applied at the time of Operation and Calibration of LC/MS/MS.
RESPONSIBILITY AND ACCOUNTABILITY
Persons along with their responsibilities and accountability are
given below:
To prepare the SOP. To maintain the records as per SOP - Executive – Corporate Quality Assurance
To follow the SOP accordingly - Trainee Analyst/ Jr. Analyst/ Analyst/ Sr. Analyst
To ensure implementation of SOP - Manager – Quality Assurance
5.0 PROCEDURE
5.1 OPERATION
5.1.1 The 6410 triple quad LC/MS instrument is a Liquid Chromatography (LC) Mass Spectrometer (MS), which incorporates an Atmospheric Pressure Ionization (API) ion source.
5.1.2 Starting up the mass spectrometer. Ensure that the gas supply is flowing to the instrument. The pressure should be regulated to 100 psi in the nitrogen generator and 2 psi for Nitrogen is flowing into the collision cell.
Switch “ON” MS, Pump, Column Oven, Autosampler, UV Detector.
Switch “ON” the Computer System.
Double-click on the Acquisition System Launcher icon.
Click On Run.
Click On Data Acquisition
Create a Method
5.1.9.1 Click
On 1290-ALS.
5.1.9.2 Click
On Set Up.
5.1.9.3 Enter
the optimized value of the Autosampler.
5.1.9.4 Click
On 1290- Pump.
5.1.9.5 Enter
the optimized value of 1290 - Pump.
5.1.9.6 Click
On 1290 - Column.
5.1.9.7 Enter
the optimized value of 1290 - Column.
5.1.9.8 Click
On MSQQQ.
5.1.9.9 Swab
the tune file.
5.1.9.10 Select the Scan type.
5.1.9.11 Click On Acquisition.
5.1.9.12 Enter the optimized value of Acquisition.
5.1.9.13 Click On Source.
5.1.9.14 Enter the optimized value of the Source.
5.1.9.15 Click On Chromatogram.
5.1.9.16 Enter the optimized value of the Chromatogram.
5.1.9.17 Click On File and then Save the ‘Method’.
5.1.10 Create a new Work List
5.1.10.1 Enter the
‘Sample Name’ and ‘Sample Position’.
5.1.10.2 Click On
‘Method’, ‘Data file’, and ‘Sample Type’.
5.1.10.3 Click On the file then Save ‘Word List’.
5.1.11 Start Work List
5.1.11.1 Click On Start Work List.
5.2 THE MAINTENANCE PLAN FOR LC/MS/MS UNIT IS AS BELOW:
5.2.1 Daily Checkpoints (XYZ /CQA/SOP-085/FR-04)
5.2.1.1 Cleaning
of Source.
5.2.1.2 Column
Washing.
5.2.1.3 Vacuum
Pressure (Always maintain 3.0 E-5 torr to 4.0 E-5 torr).
5.2.1.4 Nitrogen
gas (Always maintain 2 kg/cm2)
5.2.1.5 Nitrogen
Generator (80- 100 psi)
5.2.2 Weekly Checkpoints (XYZ /CQA/SOP-085/FR-05).
5.2.2.1 Oil level of Vacuum Pump (the limit of oil should be between the marked minimum and maximum).
5.2.3 Half-yearly Checkpoints (XYZ /CQA/SOP-085/FR-06)
5.2.3.1 Calibration
of Mass Spectrometer.
5.2.3.2 Calibration
of a pump.
5.3.3.3 Calibration
of Oven.
5.3.3.4 Calibration
of UV-VIS Detector.
5.2.3.5 Calibration
of Autosampler.
5.2.4 Maintain the logbook on XYZ /CQA/SOP-085/FR-01 as the Logbook of LC/MS/MS (Appendix I).
6.0 Mass Spectrometry Calibration Standards
6.0.1 Calibration Frequency: Half-yearly
6.1 CALIBRATION OF MASS SPECTROMETER
6.1.1 Tuning
of Mass Spectrometer
6.1.2 Click
On Autotune
6.1.3 Record the Calibration data on XYZ /CQA/SOP-085/FR-02 Calibration Data Sheet (Appendix II)
6.2 CALIBRATION OF HPLC
6.2.1 Calibration of Pump
6.2.1.1 Flow rate accuracy
6.2.1.2 Flow rate
consistency
6.2.1.3 Gradient Composition Accuracy
6.2.2 Calibration of Autosampler
6.2.2.1 Injector Accuracy
6.2.2.2 Injector Precision and Linearity
6.2.2.3 Carryover test
6.2.3 Calibration of Detector (UV-VIS)
6.2.3.1 Linearity
of Detector Response
6.2.3.2 Wavelength
Accuracy
6.2.4 Calibration of Column Oven
6.2.1 CALIBRATION OF PUMP
6.2.1.1 Flow Rate Accuracy
6.2.1.1.1 Use Milli Q water (Degassed & filter) as a mobile phase for
calibration of a pump.
6.2.1.1.2 Before starting the calibration purge the system two or three times to
remove air bubbles.
6.2.1.1.3 Before starting calibration set the pump at constant flow and discard the
volume for five minutes.
6.2.1.1.4 Set the Flow rate to to1.0ml (through channel A) allow the system to
stabilize and measure the flow using a volumetric flask and with the help of a calibrated stopwatch for the delivered
volume10ml at the flow rate of 1.0ml / min and 2.0ml / min. Repeat the same
operation another 4 times for both 1.0ml / min and 2.0ml / min flow rates
respectively and determine % RSD for the observed flow rate (five replicates). Also, determine the % flow accuracy using
the formula.
Volume
collected
Observed Flow rate = --------------------
Observed
time
Observed
flow rate
Flow accuracy =---------- X 100
Set flow rate
6.2.1.1.5 Repeat the steps 6.3.1 to 6.3.1.4 for calibration of pump B.
Acceptance criteria
Flow accuracy: Between
95% and105% of the set flow rate
Flow precision: %RSD of
five measurements at each flow, should not be more than 2.0%
6.2.1.1.6 Record the calibration data on XYZ /CQA/SOP-85/FR-02 Calibration of Pump A & Pump B (Appendix II)
6.2.1.2 Flow Rate
Consistency:
6.2.1.2.1 From the injection under injector precision i.e. (6.3.4) 20µL (5 replicate injections) calculate the %RSD of retention time (RT).
Acceptance criteria
% RSD of RT shall not be
more than 1%.
6.2.1.2.2 Record the Calibration data on XYZ /CQA/SOP-085/FR-02 Flow Rate
Consistency (Appendix II)
6.2.1.3 Gradient Composition Accuracy
6.2.1.3.1Set the
following chromatographic conditions
Column: Restriction
capillary (~0.12mm ID)
Solvent for Pump A: Filtered
and degassed purified water
Solvent for Pump B: 0.3%
Acetone in purified water
Flow rate: 1.0
ml/min
Wavelength: 254
nm
Injection volume: 0.00
µl
Run time: 50
min
Gradient programming:
|
Step |
Time in minute |
% Solvent A |
%Solvent B |
|
1 |
0.01 |
100.0 |
0 |
|
2 |
1.0 |
100.0 |
0.0 |
|
3 |
2.0 |
90.0 |
10.0 |
|
4 |
10.0 |
90.0 |
10.0 |
|
5 |
10.01 |
50.0 |
50.0 |
|
6 |
20.0 |
50.0 |
50.0 |
|
7 |
20.01 |
10.0 |
90.0 |
|
8 |
30.0 |
10 |
90 |
|
9 |
30.01 |
0 |
100 |
|
10 |
40.0 |
10 |
100 |
|
11 |
40.01 |
100 |
0 |
|
12 |
50.0 |
100 |
0 |
6.2.1.3.2 Procedure
Set the flow rate at 1.0ml per minute. Keep the system in this flow rate for some time to equilibrate. Start the gradient programming. After completion of Gradient programming calculate actual concentration by using the formula below:
Actual conc of X % = absorbance
level of set conc(X%) – absorbance level of set conc(0%)
absorbance level of set conc100%- absorbance level of set conc 0%
Where X =10, 50, 90
Acceptance criteria:
At each absorbance level
±1 of the set concentration
6.2.1.3.3 Record the calibration data on XYZ /CQA/SOP-85/FR-02 Gradient Composition Accuracy (Appendix II)
6.2.2 Calibration of Autosampler
6.2.2.1 Injector Accuracy
6.2.2.1.1 Fill a standard
vial with HPLC grade water (MilliQ) and seal with cap.
6.2.2.1.2 Weigh this vial
and record the weight as (W1) at ambient temperature.
6.2.2.1.3 Inject 50µL from the vial and repeat it 5 more times.
6.2.2.1.4 Program HPLC system for a flow rate of 1.0ml/min and run for 1 min using water as per mobile phase.
6.2.2.1.5 After completion of 6 injections, remove the vial and weigh again (W2) at ambient temperature.
6.2.2.1.6 Calculate the average volume injected per injection using the formula:
W3 = [(W1 –W2)/6} *1000 = mg of water withdrawn per injection = µL per injection
6.2.2.1.7 Record the calibration data on XYZ /CQA/SOP-085/FR-02 Autosampler Calibration (Appendix II)
Acceptance criteria
The average volume of injection shall be 50±1.0 µL. (49 µL to 51 µL).
6.2.2.2 Injector Precision and Linearity
6.2.2.2.1Requirements
Acetonitrile (HPLC grade)
Water (HPLC grade) /MilliQ
6.2.2.2.2 Procedure
Set up a chromatographic system using the following parameters:
Flow : 1.0ml/min
Column: C18 250X4.6mm, 5µ (Inertsil ODS
column is suitable)
Wavelength: 273nm
Runtime: 15min
Mobile phase : Water:
Acetonitrile (85:15)
6.2.2.2.3 Prepare a solution of 100 ppm of Caffeine using mobile phase as diluents.
6.2.2.2.4 Inject mobile
phase as blank run.
6.2.2.2.5 Inject Caffeine with injection volume 5 µL (Duplicate), 10 µL (Duplicate), 20 µL (Five replicates), 50 µL. (Duplicate), 100 µL (Duplicate).
6.2.2.2.6 From the data obtained plot a graph of Area vs. injection and calculate the value of the correlation coefficient (r2).
6.2.2.2.7 Record the calibration data on XYZ /CQA/SOP-085/FR-02 as Injection Precision and Linearity (Appendix II)
Acceptance criteria:
Correlation coefficient
(R2).should not be less than 0.999.
%RSD of an area of replicate injection should not be more than 1.
6.2.2.3 Carryover Test:
6.2.2.3.1 Requirements:
Acetonitrile (HPLC grade)
Purified Water (HPLC
grade)
6.2.2.3.2 Procedure:
6.2.2.3.2.1 Set up a chromatographic system using the following parameters.
Flow: 1.0 ml/min
Column: C18 250X4.6mm, 5µ (Inertsil ODS
column is suitable)
Wavelength: 273nm
Run time: 15 min
Mobile phase : Water: Acetonitrile (85:15)
Injection volume: 100 µL
6.2.2.3.2.2 Prepare a
solution of 250ppm of Caffeine using mobile phase as diluent.
6.2.2.3.2.3 Inject 100
µL. of the following solutions:
Pre
blank- mobile phase
Caffeine solution -250ppm
Post blank –mobile phase
6.2.2.3.2.4 The peak area
measured in post blank compared with the peak area of Caffeine solution indicates the amount of carryover.
6.2.2.3.2.5 If there is
any peak area measured in the pre-blank at the same retention time the same shall
be subtracted from the peak area of the post-blank before it is compared with the
Caffeine solution.
6.2.2.3.2.6 Record the calibration data on XYZ /CQA/SOP-085/FR-02 as Carryover Test (Appendix II)
6.2.3 Detector Calibration
6.2.3.1 Linearity of
Detector Response
6.2.3.1.1 Chromatographic conditions:
Column: C18 250 x 4.6mm, 5µ (Inertsil ODS
column is suitable)
Mobile
phase: Acetonitrile: water (15: 85)
Flow rate: 1.0ml/min
Wavelength: 273nm
Run Time: 15min
Column oven temperature: Ambient
6.2.3.1.2 Preparation of standard solution (1000ppm): Weigh accurately about 0.1g of Caffeine in 100ml volumetric flask to dissolve and dilute to volume with the mobile phase.
6.2.3.1.3 Preparation solution 1(100 ppm): Dilute 10 ml of standard solution
to 100 ml with the mobile phase.
6.2.3.1.4 Preparation solution 2(50 ppm): Dilute 25 ml of solution 1 to 50 ml with the mobile phase.
6.2.3.1.5 Preparation solution 3(25 ppm): Dilute 25 ml of solution 2 to 50 ml with the mobile phase.
6.2.3.1.6 Preparation solution 4(10 ppm): Dilute 10 ml of solution 1 to 100 ml with the mobile phase.
6.2.3.1.7 Preparation solution 5(5ppm): Dilute 25ml of solution 4 to 50 ml with the mobile phase.
6.2.3.1.8 Procedure:
After stabilization of the system, Inject the blank (mobile phase) and then inject each of Solutions 5, solution 4 (in duplicate), solution 3 (five times) solution 2, and Solution 1 (in duplicate) injecting the blank at each step and record the chromatograms. Calculate the average peak area of caffeine from the duplicate injections at each level. Plot the linearity graph between the concentration of caffeine (ppm) on the x-axis and the average area of Caffeine at each level on the y-axis.
6.2.3.1.9 Record the Calibration data on XYZ /CQA/SOP-085/FR-02 Linearity of Detector response (Appendix II)
Acceptance
criteria
The linearity coefficient ‘r’ obtained from the linearity graph of caffeine for different levels should not be less than 0.999.
6.2.3.2 Wavelength accuracy
(Caffeine in water)
6.2.3.2.1 Using Caffeine Solution
6.2.3.2.2 Set the
following chromatographic conditions.
Mobile phase : Water: Acetonitrile (85:15)
Flow
rate: 1.0 ml/minute
Column : C18
250 x 4.6mm, 5µ (Inertsil ODS column is suitable)
Run time: 15 min
Injection Volume:
20µl
6.2.3.2.3 Sample preparation: Weigh accurately 25mg of caffeine, transfer into a 100 ml volumetric flask, dissolve, and dilute to volume with purified water. (250 ppm Caffeine in purified water).
6.2.3.2.4 Switch “ON” the
detector UV lamp for at least 1 hour.
6.2.3.2.5 Inject the Caffeine solution in the mobile phase at the wavelength 271 nm, 272 nm, 273 nm, 274 nm & 275 nm and record the results and check the maxima.
Acceptance
Criteria:
|
Standard
wavelength maxima |
Limit |
|
273.0
nm |
± 1.0nm |
6.2.3.2.6 Record the Calibration data on XYZ /CQA/SOP-085/FR-02 as Wavelength Accuracy (Appendix II)
6.2.4 Column
Oven Calibration (Temperature Calibration)
6.2.4.1 Switch “ON” the column oven and set the Temperature
at 40°C.
6.2.4.2 Remove the right side plastic opening center
clip and insert the standard calibrated thermometer up to the middle of the column
oven.
6.2.4.3 Properly pack the side space of the thermometer
with cotton for proper insulation.
6.2.4.4 Now wait up to ready position lamp (up to set
temperature is achieved).
6.2.4.5 Observe the oven temperature displayed reading
(current) and the standard thermometer reading.
6.2.4.6 Record the calibration data on XYZ /CQA/SOP-085/FR-02 Column Oven calibration (Appendix II)
Similarly, repeat the
calibration for 50°C.
Acceptance
Criteria:
The observed reading (current) and standard thermometer reading do not differ by more than ± 2°C from the set temperature.
6.2.4.8 Summarize the calibration data on XYZ /CQA/SOP-085/FR-03 LC/MS/MS Calibration summary sheet (Appendix III).
7.0 GENERATION OF INSTRUMENT CALIBRATION
NUMBER
Generate the instrument calibration number
on the calibration datasheet as INSCALXXYYZZZ where INS denotes instrument, CAL
denotes calibration, XX denotes the year, YY denotes month and ZZZ denotes sequence
number.
8.0 ABBREVIATIONS
XYZ Xyz Limited
SOP Standard Operating Procedure
QC Quality Control
CQA Corporate
Quality Assurance
ppm Parts
per million
AMU Atomic
mass unit
cps Counts
per second
µL Microliter
MCA Multiple
Couple Acquisition
PPG Polypropylene
Glycol
FWHH Full
Width at Half Maximum Height
nm nanometer
RSD Relative Standard Deviation
LC/MS/MS Liquid Chromatography/ Mass Spectrometer/ Mass Spectrometer
HPLC High-Performance Liquid Chromatography
Appendix and Logbook:- Download