Basics of LC-MS Shimadzu 8040 and Chromatography Method Development
1. BASICS OF LCMS SHIMADZU MODEL
8040 & CHROMATOGHAPHY METHOD
DEVELOPMENT
BY Prashik Sudhir Shimpi
Mpharm QA
07Nov2020
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2. WHAT IS MASS SPECTROMETER?
“A mass spectrometer is an instrument that
measures the masses of individual
molecules that have been converted to
ions...”
from “What is Mass Spectrometry” by ASMS
“A mass spectrometer is universal ion
detector ... not a universal detector”
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3. LC GIVES ONE DIMENSIONAL INFORMATION
UV - VIS CHROMATOGRAM
INDICATES THE PRESENCE OR ABSENCE OF A COMPOUND AT A
SPECIFIC RETENTION TIME
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4. LC MS
LC-MS ADDS SECOND DIMENSION: MOLECULAR WEIGHT
MS CHROMATOGRAM
INDICATES THE PRESENCE OF A COMPOUND AT A SPECIFIC RT
MASS SPECTRA AFFORDS MOLECULAR WEIGHT
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6. LC – MS WORKING PRINCIPLE
# A HIGH VOLTAGE IS APPLIED TO THE COLUMN ELUATE, AND IT IS ATOMIZED BY NITROGEN GAS.
# THE CHARGED DROPLETS GRADUALLY BECOME SMALLER, AND ION EVAPORATION OCCURS.
# AS THE IONS PASS THROUGH THE MASS SEPARATOR, THEY ARE SEPARATED ACCORDING TO MASS.
# IN THE DETECTOR, THE QUANTITY OF IONS IS DETECTED AS A CURRENT VALUE.
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15. PRECURSOR ION SCAN
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MS1 operates at scan mode while MS2 selects the product ion of a particular m/z formed by CID
in the collision cell. This mode is especially useful for determining the precursor ions that produce
fragment ions of a particular m/z.
16. PRODUCT ION SCAN
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MS1 is fixed at selected m/z while MS2 operates at scan mode. This scan acquires all the product
ions from the fragmentation of a selected precursor ion. The intensity and m/z of all product ions
are captured and displayed in the mass spectrum .
17. MULTIPLE REACTION MONITORING (MRM)
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The transition of a selected precursor ion to a selected product ion is monitored where MS1 and
MS2 are fixed at the specific m/z.
18. LCMS METHOD SET UP
LCMS method set up
Converting LC method to LCMS method:
LC methods are available for many samples
Save time on developing suitable LC conditions
LC methods may need some modifications to be suitable for
LCMS
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19. BASIC CONSIDERATIONS
Convert LC to LCMS method:
Column: semi-micro column (2 mm ID) preferred to analytical
column (4.6 mm ID).
Mobile phase:
Suitable for ionization of target compounds
MS conditions:
Ionization by ESI or APCI
MS and interface operation mode/condition
Develop into quantitative/qualitative method
Method performance evaluation and validation
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20. COLUMN LENGTH AND PEAK SEPARATION
In quantitative analysis, a short column (30-150 mm)
may be used to replace a longer one (150-250 mm),
since the mass-based selective detection does not
need a complete separation of elute peaks.
However, in identification and confirmation analysis,
a complete separation of elute peaks by LC column is
essential and important.
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21. MOBILE PHASE FOR LCMS
ESI: Acetonitrile/ Water and Methanol/Water
APCI: Methanol/Water recommended; Acetonitrile
/Water may cause coking on the corona needle in
negative mode.
The ratio is not so critical, but higher water content
may cause lower ionization efficiency.
Gradient elution with changing aqueous phase
between 0 and 100% can be used.
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23. BUFFER AND PH CONTROL FOR LCMS
Non-volatile buffer like phosphate is not recommended
Using volatile buffer to replace phosphate buffer:
Ammonium acetate/ Acetic acid, Ammonium
format/Formic acid,NH3/TFA, etc…
Use a lower buffer concentration: 10 –50 mM
pH control:
pH 1.8 -2.5 : TFA, conc. < 0.1%
pH 2.5 -4 : Formic Acid, conc. ~ 0.1%
pH 4 -5 : Acetic Acid, conc. 0.1~5%
pH 7 : Ammonium Acetate
pH > 7 : Ammonia aqueous solution
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24. IONIZATION OF TARGET COMPOUND
Compound properties: easy for polar and ionic organic
compounds; pure hydrocarbons (CnHm) cannot be
ionized.
A very high water content may affect ionization efficiency
of some compounds (less polar).
Buffer concentration: Use lower conc. (10-50 mM) to
avoid/reduce ion suppression
pH of mobile phase/solvent:
Acidic :positive ions formation
Basic :negative ions formation
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25. IONIZATION IN POSITIVE OR NEGATIVE MODE
Depend on structures and properties of compounds
High proton affinity: C-O, C-N double and triple bonds,
basic compound tend to form positive ions
Low proton affinity: –COOH, -F, -Cl, -HSO3, phenols and
sugars tend to form negative ions due to stronger tendency
to donate proton
Many compounds form both positive & negative ions due
to multi function groups
Acquire both positive & negative mass spectra in multi
sequence mode and compare sensitivity & spectrum
quality
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