![Tests for Mass Spectrometer Detectors of
LCMS:
MSD 1. Vacuum Verification
Rationale: A stable, high vacuum is required for high-sensitivity mass
spectrometry.
Procedure: Multiple readings of the vacuum system are taken and an
automated comparison of these values to the known acceptable values is
made. Passing this test is a pre-requisite for the following tests.
MSD 2. Scan Verification
Rationale: Calibration of mass range is critical in qualitative mass
spectrometry.
Procedure: [Agilent LCMS] The built-in Agilent autotune is performed to
determine the proper calibration of the MSD and ensure that masses are
correctly reported across the entire mass range of the instrument. [Non-
Agilent LCMS] A manual tune is made where applicable.](https://image.slidesharecdn.com/lcmsslide-181121091429/85/LCMS-14-320.jpg)
LCMS
- 1. QUALIFICATION OF LC-MS By Ankush Ramrao sule M. Pharmacy 1st year School of Pharmacy, Nanded
- 2. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY •Liquid chromatography-mass spectrometry (LC/MS) is a technique that uses liquid chromatography (or HPLC) with the mass spectrometry. •It is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry.
- 3. PRINCIPLE •The LC-MS technology involves use of an HPLC, wherein individual components in a mixture are first separated followed by ionization and separation of the ions on the basis of their mass/charge ratio. The separated ions are then directed to a photo multiplier tube detector, which identifies and quantifies each ion. The ion source is an important component in any MS analysis, as this basically aids in efficient generation of ions for analysis. To ionize intact molecules, the ion source could be APCI (Atmospheric Pressure Chemical Ionization), ESI (Electronspray Ionization), etc. to name a few popular ones. The choice of ion source also depends on the chemical nature of the analyte of interest i.e. polar or non- polar.
- 5. 1. Ion source, which can convert gas phase sample molecules into ions. Following are the most common ionization methods : i. Electrospray Ionization ii. Atmospheric Pressure Chemical Ionization iii. Atmospheric Pressure Photo-ionisation Components of Mass Spectrometer :
- 6. Components of Mass Spectrometer : 2. Analyzer , where ions are separated according to their mass-to-charge ratio by applying electromagnetic fields. •Its task is to separate ions in terms of their mass-to charge •ratio and to direct the beam of focused ions to the detector. •The key performance parameters of an analyzer include; (a) separation efficiency (b) m/z measurement precision (c) range of the m/z values measured •There are following kinds of mass analyzers that can be used in LC/MS : (a) Quadrupole Analyzer, (b) Time-of-Flight Analyzer, (c) Ion Trap Analyzer
- 7. Components of Mass Spectrometer : 3. Detectors : The detector is used to count the ions emergent from the mass analyzer, and may also amplify the signal generated from each ion. Following are three different kinds of detectors are used in Mass Spectrometry; (a)Electron Multipliers (b)Dynolyte Photomultiplier:
- 8. Tandem Mass Spectrometry • Tandem mass spectrometry (MS/MS) is a system of two combined analyzers of the same type or different types, characterized by high separation efficiency. • The ions produced by the source are separated in the first analyzer (MS1). Ions with the selected m/z value reach the collision cell where, depending on the analysis conditions, they undergo dissociation or remain unchanged. • In comparison with analysis using a single analyzer, tandem analysis shows a considerable improvement in selectivity and considerably increased sensitivity
- 9. OQ—Operational Qualification • Temperature accuracy and stability of column heater/cooler • Holmium oxide wavelength scan (if applicable) • Detector lamp intensity and wavelength accuracy • Detector noise and drift • Pump flow rate accuracy and repeatability • High and low pressure shutdown accuracy • Injector precision • Detector linearity and sampleto-sample carryover • Injection volume linearity • Gradient composition accuracy • Linear gradient tested using IPA and 0.5% Acetone/IPA • Five step gradient tested using IPA and 0.5% Acetone/IPA
- 10. IQ—InstallationQualification • Inventory of instruction manuals, • components and serial numbers • Installation verification
- 11. PQ—Performance Qualification • System performance and robustness • Typically a 5 or 10 μL injection of a low concentration sample, repeated five times • Compare area count and retention time deviations
- 12. Tests for HPLC Systems (Non-MSD) Test Name Setpoints and Parameters Limits Vacuum Verification N/A High vacuum min.: 8E-6 torr (any source) High vacuum max.: 4E-5 torr (any source), Scan Verification All used masses: ± 3.0 ppm (DSES, ES+AJST; N/A for others) Response Linearity Evaluated mass: 156 m/z Injection volume on column: 20 ul* Coefficient of determination (r2): ≥ 0.98000 (DSES, ES+AJST; N/A for others)
- 13. Tests for HPLC Systems (Non-MSD) Test Name Setpoints and Parameters Limits Injection Precision Evaluated mass: 156 m/z Injection volume on column: 20 ul* Area RSD: ≤ 20.00 % (any source) Height RSD: Not applicable (any source) Injection Carry Over Evaluated mass: 156 m/z Injection volume on column: 20 ul* Area & height carry over ≤ 1.00 % (DSES, ES+AJST; N/A for others) Signal to Noise Evaluated mass: 156 m/z Injection volume on column: 20 ul* Signal to noise: ≥ 10 (DSES, ES+AJST; N/A for others)
- 14. Tests for Mass Spectrometer Detectors of LCMS: MSD 1. Vacuum Verification Rationale: A stable, high vacuum is required for high-sensitivity mass spectrometry. Procedure: Multiple readings of the vacuum system are taken and an automated comparison of these values to the known acceptable values is made. Passing this test is a pre-requisite for the following tests. MSD 2. Scan Verification Rationale: Calibration of mass range is critical in qualitative mass spectrometry. Procedure: [Agilent LCMS] The built-in Agilent autotune is performed to determine the proper calibration of the MSD and ensure that masses are correctly reported across the entire mass range of the instrument. [Non- Agilent LCMS] A manual tune is made where applicable.
- 15. Tests for Mass Spectrometer Detectors of LCMS: MSD 3. Response Linearity Rationale: Knowledge of the response curve is critical for quantitative analysis. Procedure: A sulfa drug mix standard of four sulfonamide drugs is injected into the system at five concentrations representing a wide range for LCMS. The ions monitored are appropriate to the system type. The calculated RSQ best-fit regression line and plot of the response curve provides the statistics required to evaluate the instrument’s overall response curve. This allows users to set appropriate calibration ranges and limits in their quantitative application methods. MSD 4. Injection Precision Rationale: System precision is critical for accuracy of quantitation. Autosampler performance and MS ionization contribute to LCMS system precision. Autosampler precision is challenged in the standard LC module tests using a UV detector. A repeat precision test in MS mode further challenges the precision of source ionization and MS detection. Procedure: A blank injection followed by six repeat injections of the sulfa drug mix followed by a final blank injection are made. The %RSD of the six injections is calculated to provide precision statistics.
- 16. Tests for Mass Spectrometer Detectors of LCMS: MSD 5. Carry Over Rationale: Low carry over from a previous injection is critical for accuracy of quantitative and reliability of qualitative analysis. Autosampler performance and MSD condition contribute to LCMS carry over. Autosampler carry over is challenged in the standard LC module tests using a UV detector. A repeat carry over test in MS mode further challenges the full LCMS system carry over performance. Procedure: A blank injection followed by single injection of the highest concentration standard followed by a blank injection. The last blank injection is evaluated for carry over and the result expressed as a percentage of the value for the standard injection. MSD 6. Signal to Noise Rationale: Sensitivity of MS detection is an important performance feature in quantitative and qualitative analysis. A signal-to-noise value of representative compounds and appropriate ions at known concentration provides sensitivity statistics