Liquid Chromatography Mass Spectrometry (LCMS) In Drug Development

Liquid Chromatography Mass Spectrometry (LCMS) has become an indispensable tool in pharmaceutical development. This powerful analytical technique allows for the separation, identification, and quantification of both known and unknown compounds, as well as the elucidation of their chemical structures. Its high sensitivity, specificity, and versatility make it a cornerstone in drug discovery, development, and quality control.

However, effective use of LC-MS requires a solid foundation in analytical and organic chemistry, along with practical expertise in instrument handling and data interpretation. Understanding the intricacies of LC-MS can significantly enhance the accuracy and efficiency of pharmaceutical analysis.

In this article, I aim to share my skill-based expertise on LC-MS, providing valuable insights for professionals and students alike. You will learn about its critical role in pharmaceutical development, including key concepts such as structure elucidation, mobile phase selection, ionisation techniques, mass analysers, quadrupole functionality, molecular peaks, mass resolution, mass accuracy, and more. We will also explore important principles like the nitrogen rule and isotope mass patterns, supported by real-world case studies and a dedicated FAQ section.

Whether you’re new to LC-MS or looking to deepen your knowledge, this guide offers a comprehensive overview of its applications and best practices in the pharmaceutical field.

Liquid Chromatography Mass Spectrometry (LCMS)

Liquid Chromatography-Mass Spectrometry (LC-MS) is the integration of the two systems, HPLC and MS, through an 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.

LCMS = 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 UPLC-MS.

Related: How To Control Impurities In Pharmaceuticals: Get Mastery In …

How does Liquid Chromatography Mass Spectrometry (LCMS) work?

The following are the functions of different components in any LCMS:

1. Sample Injection: A sample is injected into the HPLC system.
2. Separation (HPLC): The sample components are separated in the chromatographic column.
3. Ionisation (MS): As the separated compounds exit the HPLC column, they are ionised (commonly by electrospray ionisation, ESI, or atmospheric pressure chemical ionisation, APCI) before entering the mass spectrometer.
4. First MS Analysis: The first mass spectrometer stage (MS1) analyses the molecular mass of the ions.
5. Fragmentation (MS/MS): Selected ions are fragmented in a collision cell, generating product ions.
6. Second MS Analysis: The second mass spectrometer stage (MS2) analyses the fragment ions, providing detailed structural information about the original compound.
7. 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.

How does liquid chromatography (LC) work?

LC/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 derivatised 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 a 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 in suitable solvents and filtered before analysis. Sample concentration is decided based on the response and type of analysis. LC-MS is a highly sensitive instrument, and the compound can be analysed at a very low level.

Why is LC used with mass spectrometry (MS)?

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) ratio.

Liquid Chromatography Mass Spectrometry Ionisation Methods

The following ionisation methods are used in the pharmaceutical industry:

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

ESI is widely used in the industry. 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 the mass spectrometer that takes ionised 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 analysers 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 a mass analyser and collision cell

Time of flight (TOF)

In a time-of-flight (TOF) mass analyser, 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 analysers 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 a quadrupole 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 quantifying analytes by LC/MS/MS.

What are the advantages of Liquid Chromatography Mass Spectrometry?

Liquid Chromatography-Mass Spectrometry (LC-MS) offers an added dimension of selectivity by generating mass spectral data for every point across the chromatogram. Analytes are first separated using High-Performance Liquid Chromatography (HPLC) or Ultra-High-Performance Liquid Chromatography (UHPLC), producing a chromatogram with distinct peaks, each corresponding to a specific retention time.

At each time point, a corresponding mass spectrum is acquired, revealing the mass-to-charge ratio (m/z) of ions eluting at that moment. This enables the identification of analytes based solely on their mass, even in the absence of reference standards. Unlike traditional detectors, LC-MS can resolve co-eluting compounds by their distinct m/z values, offering superior specificity and sensitivity.

Mass detectors outperform many conventional LC detectors, especially for compounds that lack chromophores, which are essential for UV or diode-array detection. Furthermore, LC-MS can extract meaningful information from unresolved chromatographic peaks, reducing dependence on complete chromatographic separation.

When combined with data from other LC detectors, mass spectral information enhances confidence in compound identification, confirmation, and quantification, making LC-MS a vital technique in complex analytical workflows.

Applications of Liquid Chromatography Mass Spectrometry in Pharmaceutical Development

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

• Identification of the unknown analytes/impurities
• Mass determination of analytes
• Structure elucidation of unknown analytes/impurities
• Quantitative and qualitative analysis
• Bioequivalence studies
• Identification of degradation impurities during forced degradation studies
• Identification and quantification of drug metabolites
• Keto enol tautomerization study
• To check peak homogeneity of the peak in the specificity test

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: LC-MS of Benzoic acid

• Its molecular weight is 122.21 Da
• 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 plants, 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.

LCMS 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 amu for 13C. The atomic mass of Hydrogen is 1.00794 amu for H and 2.0141 amu 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 amu 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: Mass 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 of Liquid Chromatography Mass Spectrometry (LC-MS)

The following are some limitations of the LC-MS:

• Some compounds, like Phenol, do 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

HPLC-MS/MS stands for High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry. It’s a powerful analytical technique for impurities identification and quantification. Following is a quick breakdown:

1. HPLC: It separates compounds in a sample based on their interactions with the stationary phase and the mobile phase.
2. MS/MS (Tandem Mass Spectrometry): After separation by HPLC, the individual compounds are ionized and analyzed using mass spectrometry. Tandem MS involves two stages of mass spectrometry, providing more detailed information about the structure of molecules. The first stage (MS1) detects the ions, and the second stage (MS2) fragments these ions to give more specific structural data.

Advantages:

• Sensitivity and Specificity: It is very sensitive and can detect even trace amounts of substances.
• Complex Sample Analysis: It’s great for complex matrices like blood, urine, or environmental samples.
• Quantification and Identification: It can both identify and quantify compounds with high accuracy.

Conclusion

C-MS is an essential analytical tool that combines the separation power of liquid chromatography with the detection capabilities of mass spectrometry. Its high sensitivity, specificity, and versatility make it invaluable for identifying and quantifying compounds in complex pharmaceutical samples. From drug substances and intermediates to impurities and degradation products, LC-MS plays a central role across all stages of pharmaceutical development.

I hope this article has enhanced your understanding of LC-MS and its practical applications. With this foundational knowledge, you are better equipped to perform LC-MS testing and structure elucidation with greater confidence.

If you found this helpful, be sure to explore other articles on my blog covering related techniques, such as the applications of GC-MS and FTIR in pharmaceutical analysis.

Related

• What is the Analytical Method Validation?
• The 7 Key Differences Between Validation and Verification
• How to perform cleaning validation?
• What Is Forced Degradation Studies Of Pharmaceuticals and How To Perform?
• What Is Photostability Testing Of New Drug Substances: Learn in 5 minutes
• What is CSV (Computer System Validation): Learn in 12 Easy Steps

FAQs

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.

How is data acquired in LC-MS/MS?

In mass spectrometry, the compounds and their fragments are ionised using either electron or chemical ionisation. The sample is then accelerated through a mass analyser, 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 the 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?

The 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 is it meant for?

The LC-MS process in which mass spectrometry parameters such as the precursor/product ions, collision energies, and other voltages are optimised 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 is used 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 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. An 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

• <736> Mass spectrophotometry
• Liquid Chromatography Mass Spectrometry

Abbreviations

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