Procedure for the Operation and Calibration of LC/MS/MS

This document details the procedure for the Operation and Calibration of LC/MS/MSCalibration of a mass spectrometer. It is the policy of Xyz Limited that a written procedure shall be followed the Operation and Calibration of LC/MS/MS and its use monitored in order to ensure smooth operation and its use monitored to obtain consistent and reproducible results, minimize downtime and to meet regulatory requirements.

This procedure is to be applied at the time of Operation and Calibration of LC/MS/MS.


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



5.1.1 The 6410 triple quad LC/MS instrument is 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 Click On 1290-ALS. Click On Set Up. Enter the optimized value of Autosampler. Click On 1290- Pump. Enter the optimized value of 1290 - Pump. Click On 1290 - Column. Enter the optimized value of 1290 - Column. Click On MSQQQ. Swab the tune file. Select the Scan type. Click On Acquisition. Enter the optimized value of Acquisition. Click On Source. Enter the optimized value of Source. Click On Chromatogram. Enter the optimized value of Chromatogram. Click On File and then Save the ‘Method’.

5.1.10  Create a new Work List Enter the ‘Sample Name’ and ‘Sample Position’. Click On ‘Method’, ‘Data file’, and ‘Sample Type’. Click On file then Save ‘Word List’.

5.1.11  Start Work List Click On Start Work List.


5.2.1    Daily Checkpoints (XYZ /CQA/SOP-085/FR-04) Cleaning of Source. Column Washing. Vacuum Pressure (Always maintain 3.0 E-5 torr to 4.0 E-5 torr). Nitrogen gas (Always maintain 2 kg/cm2) Nitrogen Generator (80- 100 psi)

5.2.2    Weekly Checkpoints (XYZ /CQA/SOP-085/FR-05). 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) Calibration of Mass Spectrometer. Calibration of a pump. Calibration of Oven. Calibration of UV-VIS Detector. Calibration of Autosampler.

5.2.4    Maintain the logbook on XYZ /CQA/SOP-085/FR-01 as Logbook of LC/MS/MS (Appendix I).  

6.0  Mass Spectrometry Calibration Standards

6.0.1    Calibration Frequency: Half-yearly


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) 


6.2.1 Calibration of Pump Flow rate accuracy Flow rate consistency Gradient composition accuracy                                                 

6.2.2  Calibration of Autosampler Injector Accuracy Injector Precision and Linearity Carryover test           

6.2.3  Calibration of Detector (UV-VIS) Linearity of Detector Response Wavelength Accuracy

6.2.4  Calibration of Column Oven

6.2.1 CALIBRATION OF PUMP Flow rate Accuracy Use Milli Q water (Degassed & filter) as a mobile phase for calibration of a pump. Before starting the calibration purge the system two or three times to remove air bubbles. Before starting calibration set the pump at constant flow and discard the volume for five minutes. Set the Flow rate to1.0ml (through channel A) allow the system to stabilize and measure the flow using a volumetric flask and with the help of 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 rate respectively and determine % RSD for 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 Repeat the steps 6.3.1 to 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% Record the calibration data on XYZ /CQA/SOP-85/FR-02 Calibration of Pump A & Pump B (Appendix II) Flow rate Consistency: 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%. Record the Calibration data on XYZ /CQA/SOP-085/FR-02 Flow Rate Consistency  (Appendix II) Gradient Composition Accuracy 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:


Time in minute

% Solvent  A

%Solvent  B
















































0 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 set concentration Record the calibration data on XYZ /CQA/SOP-85/FR-02 Gradient Composition Accuracy (Appendix II)

Calibration of mass spectrometer

6.2.2 Calibration of Autosampler Injector Accuracy Fill a standard vial with HPLC grade water (MilliQ) and seal with cap. Weigh this vial and record the weight as (W1) at ambient temperature. Inject 50µL from the vial and repeat it 5 more times. Program HPLC system for a flow rate of 1.0ml/min and run for 1 min using water as per mobile phase. After completion of 6 injections, remove vial and weigh again (W2) at ambient temperature. Calculate average volume injected per injection using the formula:

W3 = [(W1 –W2)/6} *1000 = mg of water withdrawn per injection = µL per injection 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). Injector Precision and Linearity:          

Acetonitrile (HPLC grade)

Water (HPLC grade) /MilliQ Procedure:

Set up chromatographic system using 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) Prepare a solution of 100 ppm of Caffeine using mobile phase as diluents. Inject mobile phase as blank run. Inject Caffeine with injection volume 5 µL (Duplicate), 10 µL (Duplicate), 20 µL (Five replicates), 50 µL. (Duplicate), 100 µL (Duplicate). From the data obtained plot a graph of Area vs. injection and calculate the value of the correlation coefficient (r2). 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. Carryover Test: Requirements:

Acetonitrile (HPLC grade)

Purified Water (HPLC grade) Procedure: Set up 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 Prepare a solution of 250ppm of Caffeine using mobile phase as diluent. Inject 100 µL. of following solutions:

 Pre blank- mobile phase

Caffeine solution -250ppm

Post blank –mobile phase The peak area measured in post blank compared with the peak area of Caffeine solution indicates the amount of carryover. 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. Record the calibration data on XYZ /CQA/SOP-085/FR-02 as Carryover Test (Appendix II) 

6.2.3 Detector Calibration Linearity of Detector response 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

Wave length                :           273nm

Run Time                    :           15min 

Column oven temperature:     Ambient Preparation of standard solution (1000ppm): Weigh accurately about 0.1g of Caffeine in 100ml   volumetric flask dissolve and dilute to volume with the mobile phase. Preparation solution 1(100 ppm): Dilute 10ml of standard solution to 100 ml with the mobile phase. Preparation solution 2(50 ppm): Dilute 25ml of solution 1 to 50 ml with the mobile phase. Preparation solution 3(25 ppm): Dilute 25ml of solution 2 to 50 ml with the mobile phase. Preparation solution 4(10 ppm): Dilute 10ml of solution 1 to 100 ml with the mobile phase. Preparation solution 5(5ppm): Dilute 25ml of solution 4 to 50 ml with the mobile phase. Procedure:

After stabilization of the system, Inject the blank (mobile phase) and then inject each of solution 5, solution 4 (in duplicate), solution 3 (five times) solution 2, and solution 1 (in duplicate) injecting 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. 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. Wavelength accuracy (Caffeine in water) Using Caffeine Solution 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 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). Switch “ON” the detector UV lamp for at least 1 hour. 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


273.0 nm

± 1.0nm Record the Calibration data on XYZ /CQA/SOP-085/FR-02 as Wavelength Accuracy (Appendix II) 

6.2.4    Column Oven Calibration (Temperature Calibration) Switch “ON” the column oven and set Temperature at 40°C. Remove the right side plastic opening center clip and insert the standard calibrated thermometer up to the middle of the column oven. Properly pack the side space of the thermometer with cotton for proper insulation. Now wait up to ready position lamp (up to set temperature is achieved). Observe the oven temperature displayed reading (current) and the standard thermometer reading. 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. Summarize the calibration data on XYZ /CQA/SOP-085/FR-03 LC/MS/MS Calibration summary sheet (Appendix III).


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.


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

>SOP for calibration of Turbidity meter

>Operation of the Air Handling Unit

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