Liquid Chromatography Mass Spectrometry (LC MS): How To Understand

Introduction and outcome

LC-MS (Liquid chromatography mass-spectrometry) or HPLC-MS plays an important role in pharmaceutical development as it is a powerful analytical technique used to separate, identify and quantify known and unknown compounds and elucidate their structure. It is widely used in pharmaceutical industries due to its high sensitivity and selectivity. However, it needs knowledge of analytical chemistry, organic chemistry and expertise to handle this instrument and interpret the LC-MS data. That is why I decided to share my skill-based expertise on this topic. In this article, you will learn about LC-MS in pharmaceutical development, structure elucidation, mobile phase selection, LC-MS ionisation modes, mass analyser, quadrupole, molecular peak, mass resolution, mass accuracy, structure elucidation by LC-MS, nitrogen rule, mass of isotopes, case studies and FAQS

Table of content

Introduction and outcome
LC-MS
Mobile phase selection in LC-MS
Sample Preparation in LC-MS
LC-MS Positive mode
LC-MS Negative mode
Mass spectroscopic terms
Applications of LC-MS in Pharmaceutical Development
Structure Elucidation by LC-MS
Nitrogen Rule
LC-MS of Isotopes
Mass of Isotopes of Chlorine (Cl) and Bromine (Br)
Limitations and disadvantages of LC-MS
Conclusion
FAQs

LC-MS

LC-MS is the integration of the two systems HPLC and MS through LC-MS interface. The function of HPLC is to separate different analytes from a sample mixture and the function of MS is to identify the mass of each analyte.

LC-MS = LC + MS

HPLC MS and UPLC MS

When HPLC is used as liquid chromatography it is called HPLC MS and when UPLC is used as liquid chromatography it is called UPPLC MS.

7 Steps LC-MS Workflow Process

Sample Injection: A sample is injected into the HPLC system.
Separation (HPLC): The sample components are separated in the chromatographic column.
Ionization (MS): As the separated compounds exit the HPLC column, they are ionized (commonly by electrospray ionization, ESI, or atmospheric pressure chemical ionization, APCI) before entering the mass spectrometer.
First MS Analysis: The first mass spectrometer stage (MS1) analyzes the molecular mass of the ions.
Fragmentation (MS/MS): Selected ions are fragmented in a collision cell, generating product ions.
Second MS Analysis: The second mass spectrometer stage (MS2) analyzes the fragment ions, providing detailed structural information about the original compound.
Data Analysis: The data is processed to identify and quantify the compounds based on their retention time in the HPLC and the mass spectrum obtained.

HPLC Function

HPLC involves the use of a mobile phase and a stationary phase to separate the components of a sample. The mobile phase is adjusted to suit the sample, while the stationary phase is adjusted to work well with the mobile phase. The degree of compound separation is based on the compound’s affinity for the mobile phase.

There are two main categories of HPLC methods based on the properties of the stationary and mobile phases. “Normal-phase chromatography” uses a polar stationary phase and a non-polar mobile phase, while “Reverse-phase chromatography” uses a non-polar stationary phase and a polar mobile phase.

In normal-phase HPLC, the column is typically filled with silica particles, which are polar and bind to polar molecules in the mobile phase. This means that the least nonpolar compounds elute first, while the most polar compounds elute last. Normal-phase HPLC is suitable for highly hydrophobic or hydrophilic compounds, as well as compounds that are not soluble in water or may decompose in water. It is particularly useful for the separation of isomers.

On the other hand, in reverse-phase HPLC, the stationary phase usually consists of C8 or C18 silica, which is silica derivatized with alkyl chains. In reverse-phase HPLC, the most polar compounds elute first, while the least polar compounds elute last. The choice of the stationary phase can be tailored to meet specific needs.

Mobile phase selection in LC-MS

In LC-MS analysis a mobile phase without any buffer or a mobile phase with volatile buffer is used.The mobile phase can be a mixture of water and organic solvent or volatile buffers in water such as ammonium acetate, ammonium formate, acetic acid, formic acid and organic solvents such as methanol, ethanol, and acetonitrile.

Sample Preparation in LC-MS

Pharmaceuticals that need to be determined must be dissolved into suitable solvents and filtered before analysis. Sample concentration is decided based on response and type of analysis. LC-MS is a highly sensitive instrument and the compound can be analysed at a very low level.

MS Function

Once the compounds have been separated using HPLC, they are identified by mass spectrometry. Mass spectrometry creates a mass spectrum that is unique for every compound, allowing for precise identification. In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios.

LC-MS Ionisation Methods

The following ionisation methods are used in the pharmaceutical industries:

Electrospray (ESI): Common for polar compounds, including pharmaceuticals and metabolites
Atmospheric Pressure Chemical Ionization (APCI): Suitable for less polar compounds.
Atmospheric Pressure Photo-Ionization (APPI)
New dual sources (ESI/APCI) or (APCI/APPI

ESI is widely used in the industries. More than 90% of drugs are analysed in ESI mode

LC-MS Ionisation Modes

Pharmaceuticals are analysed in positive and negative modes in the Electrospray (ESI) method.

Positive mode

Best suited to basic drugs that can accept the proton
[M+H]⁺ⁿ⁺ is the primary ion formed
[M+nH]⁺ⁿ and [M+Na⁺]⁺ can also be formed
Solvents: Protonated solvents e.g. HCOOH, TFA (Trifluoroacetic acid), CH3COOH are used in the Mobile phase
Na and K adduct formation takes place

Negative mode

Best suited to acidic drugs that can donate the protons and convert them into anions e.g. –COOH containing molecules
[M-H]⁻, [M-nH]−, [M-nH]ⁿ⁻ and [M-I-]⁻, anions are formed
Solvents: Unprotonated solvents e.g. NH₃, CH₃COONH₄ are used in the mobile phase

Mass spectroscopic terms

The following terms are widely used in the LC-MS

Mass analyser
Quadrupole
Triple Quadrupole (TQ)
Ion Trap (IT)
Time of flight (TOF)
Molecular peak
Mass resolution
Mass accuracy
SIM and MRM

Mass analyser

Mass analyzer is the component of mass spectrometer that takes ionized masses and separates them based on charge to mass ratio and sends them to the detector where they are detected and later converted to digital output. Triple Quadrupole (TQ), Ion Trap (IT) and Time of flight (TOF) are commonly used Mass analyser in the KC-MS.

Quadrupole

The quadrupole acts as a mass filter and it separates ions based on the m/z ratio

Triple Quadrupole (TQ)

A triple quadrupole system consists of three quadrupoles (Q1, Q2 and Q3). The first (Q1) and third quadrupole (Q3) are used as analyzers, and can either scan the ion stream or select ions of a certain m/z ratio, while the second quadrupole (Q2) It runs in radio frequency (RF) mode and works as a collision cell.

Ion Trap (IT)

A single ion trap serves as mass analyzer and collision cell

Time of flight (TOF)

In a time of flight (TOF) mass analyzer, a uniform magnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube. Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratio of the ions are determined by their arrival time. TOF mass analyzers have a wide range and can be very accurate in mass measurements.

Molecular peak

The molecular peak is the second-highest peak that corresponds directly to the compound in question. The m/z ratio directly correlates to the molecular weight. The molecular peak of toluene is 92 which is the molecular weight of the same.

Mass resolution

Mass resolution measures how well a mass spectrometer separates ions of different masses. It is calculated by the following formula:

Mass Resolution = Mass/full-width half-height mass

Case study: Let the mass be 500, FWHM be 0.1, then Mass resolution will be 500/0.1 = 5000
Low resolution: Refers to instruments capable of separating only ions that differ in nominal mass; that is ions that differ by at least 1 or more atomic mass units
High resolution: Refers to instruments capable of separating ions that differ in mass by as little as 0.0001 atomic mass unit

The higher the mass resolution better the MS instrument

Mass accuracy

Mass accuracy is calculated by the following formula:

Mass Accuracy (in ppm) = {(True mass – Measured mass) x 1000000}/True mass

Case study: Suppose the true mass of a compound is 400 and the measured mass is 400.002 then
Mass Accuracy will be 0.002 x 1000000 = 5 ppm

SIM

In the SIM (selected ion monitoring) technique, instead of setting the mass spectrometer to scan over a predetermined mass range and record complete mass spectra, it is set to monitor intensities at specific m/z values. It is a widely used technique for trace analysis such as the quantification of nitrosamine impurities in pharmaceuticals

MRM

In MRM or multiple reaction monitoring, ions are first selected to make their way through quadrature and into the collision cell. These ions are known as precursor, or parent, ions. These ion collisions disintegrate in the cell. It is the most common method for quantitating analytes by LC/MS/MS.

Advantages

Applications of LC-MS in Pharmaceutical Development

The following are some main applications of LC-MS in pharmaceutical Development:

Identification of analyte
Mass determination
Structure elucidation
Both quantitative and qualitative analysis
Bioequivalence studies
Identification of degradation impurities during forced degradation impurities
Identification and quantification of drug metabolites
Keto enol tautomerization studie
To check peak homogeneity in specificity test during HPLC method validation

Structure Elucidation by LC-MS

Procedure to Predict Mass Fragments:

Write down the structure
Look for fragments whose breakage is most likely to produce stable cations
Write down all fragments
The most stable cation will be a major fragment

Case study: Mass fragmentation of Benzoic acid

Its molecule weight is 122.21Da
Having seen the structure, the first -OH group may break and then -COOH group will break
{C₆H₅CO}⁺ and {C₆H₅}⁺will be possible fragments.
{C₆H₅CO}⁺ is the most stable fragment and hence it will be a major fragment.

Applications in other industries

Apart from pharmaceutical industries, HPLC-MS is used in several other industries such as basic research, agrochemical studies, forensic laboratories, the food industry, water treatment plant, environmental analysis, biotechnology and petrochemicals etc.

Nitrogen Rule

A molecule with an even number of molecular weights must not contain any nitrogen atom or must contain an even number of nitrogen atom
The molecule with an odd number of molecular weight must not contain an odd number of nitrogen.

LC-MS of Isotopes

The atomic number is the number of protons (+) in the nucleus and determines the element’s identity
Isotopes of an element have a different number of neutrons in the nucleus. Electrons (-) form a cloud and most of the volume of the atom
Electrons weigh very little.
Atomic weight is basically the sum of the number of protons and neutrons
Some of the elements have more than one stable isotope, For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da. The atomic mass of Carbon is 12.000 amu for 12C but 13.3355 for 13C.The atomic mass of Hydrogen is 1.00794 amu for H and 2.0141 for D

Mass of Isotopes of Chlorine (Cl) and Bromine (Br)

The atomic mass of Chlorine is 34.9688 amu for isotope 35 and 36.9659 for isotope 37.
Br of isotope-79 in nature is 50.7% and Br of isotope 81 is 49.3%
A ratio of M to M + 2 of approximately 1:1 indicates the presence of a single Bromine in a compound

Case study: Mas spectra of Chlorobenzene
Its atomic mass is 112.5 and hence it will contain two major fragments of 112.5 and 114.5 with a 3:1 ratio.
Case study: Mas spectra of Bromobenzene:
Its atomic mass is 156 and hence it will contain two major fragments. One will have a mass of 156 and another will have a mass of 158 with a ratio of about 1:1

Limitations and disadvantages of LC-MS

The following are some limitations of the LC-MS:

Some compounds like Phenol does not give fragmentation patterns in mass
Costly instrument and it is impossible to afford by small industries
It needs knowledge of Organic chemistry, Analytical chemistry and high-pressure liquid chromatography
Analysis needs a lot of time

HPLC MS MS

Conclusion

LC-MS is a powerful analytical technique that combines the capabilities of liquid chromatography and mass spectrometry. It offers high sensitivity, specificity, and the ability to identify and quantify components in complex samples. In the pharmaceutical industry, HPLC-MS is extensively used for the analysis of drug substances, intermediates, and related compounds.

I hope this article has helped you understand LC-MS and its pharmaceutical development. Now you can independently perform LC-MS testing and structure characterisation. You may also want to check out other articles on my blog, such as the application of GC-MS and FTIR

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FAQs

What is LC-MS used for?

LC-MS is used for the identification, characterisation and quantification (such as nitroso amine impurity content in API) of pharmaceuticals or their impurities.

What is the difference between HPLC MS and LC-MS?

Both HPLC MS and LC-MS are same.

What is an LC-MS MS blood test for?

It measures drug in the blood

What is the basic principle of LC-MS MS?

LC-MS-MS is a combination of two instruments, HPLC and mass detector. HPLC separates the individual components of the sample solution and the mass detector provides the mass of each peak/component

What is the basic principle of MS?

Once the compounds have been separated using HPLC, they enter into the mass spectrometry detector. Mass spectrometry detector creates a mass spectrum that is unique for every compound.

What is normal phase HPLC-MS?

When LC-MS analysis is performed using the non-polar mobile phase (such as mixture of Hexane and ethanol) and Polar stationary phase or polar column (like Silica column) then it is called normal phase HPLC-MS.

What is the abbreviation for HPLC-MS MS?

High-performance liquid chromatography (HPLC) and mass spectrometry (MS

What are the solvents in LC-MS MS?

Volatile solvents like ethanol, acetonitrile, water or volatile buffers like ammonium acetate, and ammonium formate are used in the LC-MS MS analysis.

What are the advantages of LC-MS over HPLC?

HPLC gives only qualitative or quantitative values of any compound whereas LC-MS provides the mass of the compound in addition to qualitative or quantitative values.

What are the disadvantages for LC-MS/MS?

Some of the compounds like phenol compound can not be analysed by HPLC

Why is LC MS analysis necessary?

LC-MS is necessary to quantify the compound at a very low level such as amino impurities and characterise the compound.

What molecules cannot be identified by LC/MS?

Some of the compounds like phenol compound can not be analysed by HPLC

What is the difference between positive and negative ionization in LC-MS? Which one can we use for fatty acids analysis in LC-MS?

Protonated solvents e.g. HCOOH, TFA (Trifluoroacetic acid), and CH3COOH are used in the Mobile phase) int the positive mode whereas unprotonated solvents e.g. NH3, and CH3COONH4 are used in the mobile phase in the negative mode. Positive mode is suitable for fatty acids.

Who provides training on LC-MS in India?

pharmaknowledgeforum.com provides online training on LC-MS

How is data acquired in LC-MS/MS?

In mass spectrometry, the compounds and their fragments are ionized using either electron or chemical ionization. The sample is then accelerated through a mass analyzer, which includes either a quadrupole or an ion trap, and the ions are identified based on their mass-to-charge (m/z) ratios.

What concentration should a sample for LC-MS be?

There is no single concentration for LC-MS analysis. Sample concentration is decided based on response and type of analysis. LC-MS is a highly sensitive instrument and the compound can be analysed at a very low level.

How can I calculate the concentration of an unknown compound from LC-MS?

Concentration of an unknown compound can be optimised based on the response.

How do I read an HPLC-MS/LC MS result?

HP/LC-MS spectrum, the y-axis (vertical axis) ion intensity and the x-axis (horizontal axis) contains mass-to-charge ratio (m/z)

What is tuning in LC/MS-MS? What it is meant for?

The LC-MS process in which mass spectrometry parameters such as the precursor/product ions, collision energies, and other voltages are optimized for each analyte is called tuning.

What manufacturer should I choose when buying LC/MS/MS equipment?

Sensitivity play an important role in LC/MS/MS equipment. There are several manufacturers of the LC-MS but you should consider your requirement and budget while buying LC/MS/MS equipment

What is the difference between GC MS and HPLC-MS?

GC-MS is used for the separation, identification, mass determination and characterisation of volatile compounds whereas HPLC-MS for the separation, identification, mass determination and characterisation of non-volatile compounds

Is there any online training available for LC-MS for quantitative analysis?

Pharma Knowledge Forum (pharmaknowledgeforum.com) provides online training on LC-MS

What are quantifier and qualifier peaks in Mass Spectrometric data for (GC-MS or LC-MS)?

What is the difference between QTOF LC-MS and Orbitrap LC-HRMS? Which is more sensitive?

QTOF has a range of mass accuracy of about 1-2 ppm and a resolution R = 30000 to 50000. Orbitraps have mass accuracy in the sub-ppm range and are even better in resolution.

Which external/internal standards would you use to aid in the quantification of small molecules using LC-MS?

The main purpose of using internal standards in LC-MS is to improve the accuracy and precision and avoid analytical errors during the quantitation analysis of pharmaceuticals. The internal standards must be separated from the main peak. The external standard of the compound of interest is used. External standard of the compound whose quantity is to be determined is used.

How can we select the mobile phase on the basis of pKa value in an LC-MS/MS method development?

pKa tells about the acidic nature of the molecules and it is very helpful in choosing the mobile phase in LC-MS/MS method development.

Why does liquid chromatography-mass spectrometry have a higher sensitivity compared to HPLC?

HPLC separates compounds based on their physicochemical properties while MS of liquid chromatography-mass spectrometry separates compounds based on mass (specifically their mass-to-charge ratio). It is this dual selectivity that makes LC-MS such a powerful analytical tool and it has higher sensitivity.

What is an adduct in mass spectrometry?

Adduct formation takes place in the positive mode of ionisation. Adduct ions are formed by the interaction of a precursor ion with one or more atoms or molecules, forming an ion that contains all of the component atoms of the precursor ion plus additional atoms from related atoms or molecules.

References

Abbreviations

LC-MS: Liquid chromatography mass-spectroscopy
HPLC: High-performance liquid chromatography
SIM: Selected ion monitoring
MRM: Multiple reaction monitoring