Liquid Chromatography/
Mass Spectrometry Fundamentals

From routine analysis to drug discovery, there’s an LC/MS for the job

Mass spectrometry answers two main questions: “What’s in my sample?” and “How much is in my sample?” Depending on the selectivity and sensitivity required, different types of LC/MS systems may be needed.

Single quadrupole

Single quadrupole LC/MS

Easy-to-use, highly selective single quadrupole LC/MS is ideal for routine quantification and quality control applications. Using mass detection as an alternative, or in addition, to traditional LC detectors has several benefits. A single quadrupole LC/MS can be more sensitive than UV detection. It can discriminate based on mass-to-charge ratio. And it can be connected to other separation techniques like supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and ion chromatography (IC).

Mass spectrometers can operate in either SIM or scan mode. Below are the advantages of each.

Selected ion monitoring mode (SIM)

In SIM mode, the MS parameters are set to monitor a few specific mass-to-charge ratios (m/z). This specificity allows the detector to spend more time sampling each of the target m/z values, dramatically increasing sensitivity. What’s more, the cycle time between data points is often shorter than in scan mode, improving quantitative precision and accuracy through optimal peak-shape profiling.

Since the m/z values to be sampled must be set in advance, SIM is most often used for targeted analysis. For analyses consisting of multiple target compounds, SIM ion sampling choices can be time-programmed to match compound elution time windows.

SIM is rarely used for qualitative analysis, because no data are collected at m/z values other than those sampled.

Scan mode

In scan mode, the instrument detects signals over a mass range (i.e. 50 to 2,000 m/z) during a short period (i.e. two seconds). During this scan period, the MS sequentially reads signals detected within narrower mass intervals until the full mass range is covered. Stored spectra represent the detected signal for the full mass range. Since full mass spectra are recorded, scan mode is typically used for qualitative analysis, or for quantitation when all analyte masses are not known in advance.

Samples may be introduced into an MS by infusion or through HPLC. With HPLC, it’s important to match the peak width and the cycle time, which depends on the scan range. The narrower the peaks, the shorter your total cycle time must be to get proper peak definition. To achieve a short total cycle time, you may need to reduce the scan range.

Triple quadrupole

Triple quadrupole LC/MS

For targeted high-sensitivity analyses, triple quadrupole LC/MS is the technique of choice. It’s also known as tandem quadrupole liquid chromatography/mass spectrometry (or LC/MS/MS, LC/TQ, or LC/QQQ).

The key to its high sensitivity is the ability to isolate an ion with one quadrupole (Q1), fragment it in the collision cell, then isolate one of the fragments in a tandem quadrupole (Q2). This technique is known as MS/MS, and it greatly reduces chemical noise while preserving ions of interest.

Multiple reaction monitoring (MRM) is the primary MS/MS mode used. It provides high selectivity and sensitivity for specific target compounds, making it useful for target quantitation in complex matrices. Here’s how it works:

MRM mode

Precursor ion selection: In MRM, you start by setting the first quadrupole to filter an ion of interest (precursor ion). The precursor ion is transmitted through the first quadrupole, similar to a selected ion monitoring (SIM) experiment.
Collision-induced dissociation (CID): As the precursor ions move into the collision cell, energy is applied and collisions with inert gas molecules occur. This process, known as collision-induced dissociation (CID), reproducibly generates more ion fragments, called product ions.
Product ion isolation: The parameters of the second quadrupole are set to allow only a specific product ion to pass through to the detector. This multistep process is known as an MRM transition, which is highly selective for the target analyte. Typically, the most abundant MRM transition is measured across a chromatogram for quantitation and is referred to as the quantifier or target transition.
Addition of qualifier transitions: To enhance confidence in your method and ensure that the signal is coming from your intended target, you can add more MRM transitions specific to the analyte. These additional transitions are known as qualifier transitions. It’s common to have one to three qualifiers in addition to the quantifier transition.
Automation with software tools: Developing MRM methods can be more complex than SIM or scan methods. However, software tools like MassHunter Optimizer automate this process on Agilent LC/MS/MS instruments.

Time-of-flight

Time-of-flight, high resolution mass spectrometry

Liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF) delivers standout resolving power and mass accuracy, distinguishing it from unit mass spectrometry measurements recorded by LC/MS and LC/MS/MS. LC/Q-TOF instruments provide rich information that can be useful for compound identification.

LC/Q-TOF resolution produces data with accurate mass information containing four or more decimal points. This precision matters, because the mass of an atom⁠—or the molecular mass of a compound⁠—is not a whole number, as sometimes indicated in simplified descriptions. For instance, oxygen has an exact mass of 15.9949 atomic mass units (amu), not 16 amu.

The typical error between measured mass and theoretical mass falls within the range of 1 to 5 parts per million (ppm), allowing species with very similar masses to be distinguished.

Similar to high-resolution video, high-resolution accurate mass spectrometry (HRAM) captures or defines mass spectrum details that might appear blurred or merged with lower-resolution LC/MS and LC/MS/MS.

Time-of-flight (TOF) analyzer

In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied to all the ions at the same time, causing them to accelerate through a flight tube.

Lighter ions travel faster and arrive at the detector first. The mass-to-charge (m/z) ratios of the ions are determined by their arrival times.

Time-of-flight (TOF) mass analyzers can analyze ions over a wide mass range and measure arrival times with extreme precision, resulting in high resolution.

Full scan mode

Full scan mode, also known as total transmission of ions (TTI) or total ion chromatography (TIC), is commonly used in LC/Q-TOF systems. The system is operated without quadrupole isolation, allowing all precursor ions to pass through the flight tube and reach the detector. Since all information is captured, this mode is particularly valuable for retrospective analyses.

LC/Q-TOF systems can also operate in semitargeted or fully targeted modes, enhancing selectivity by using the quadrupole and collision cell for MS/MS experiments. Additionally, they offer reliable measurements for routine quantitative analyses.

LC/MS ionization sources

Almost all LC/MS ion sources work on the principle of adducting a charged species to a neutral molecule. This form of ionization differs from GC/MS in that the molecule itself doesn’t lose an electron. However, it can gain an adduct, which is typically a H+ proton (for a positive ion), or lose a proton (for a negative ion). Therefore, LC/MS can generate ions without degrading the sample analyte, which enables the detection of the intact mass of analytes. This process is also called soft ionization.

Analyte polarity and molecular weight determine the ionization source. Electrospray ionization (ESI) is useful for analyzing samples that become multiply charged (such as proteins, peptides, and oligonucleotides). It can also analyze samples that are singly charged (like small molecule drugs, pesticides, and metabolites). Atmospheric pressure chemical ionization (APCI) is applicable to a wide range of polar and nonpolar analytes that have moderate molecular weights.

Electrospray ionization Jet Stream Atmospheric pressure chemical ionization Multimode source

Gently ionize large and small polar molecules

With electrospray ionization (ESI), a nebulizer is used to create a fine aerosol spray from the solvent stream, and a charge is induced on droplets containing analyte molecules. A heated drying gas sheathes the aerosol spray, helping to evaporate the solvent (desorption). The rapidly evaporating droplet and rising net electrical charge combine to cause a “columbic explosion,” resulting in individual analyte ions paired to an adduct (for example, [M + H]⁺, a protonated molecule). These ions are passed through a capillary sampling orifice into the mass analyzer. ESI has an enhanced ability to generate multiply charged ions, which allows detection of very large molecules (> 150 kDa). It is best suited for polar analytes.

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Maximize sensitivity across many sample types

Agilent Jet Stream high sensitivity ion source (AJS)⁠—an exclusive technology⁠—is based on electrospray ionization. It uses superheated nitrogen to improve droplet desolvation and ion generation for a stronger signal and reduced noise. What’s more, it offers the highest sensitivity for most analytes, and a response that is five-fold higher⁠—or more⁠—relative to standard ESI.

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Confidently analyze compounds not suitable for electrospray

Atmospheric pressure chemical ionization (APCI) works by spraying the LC eluent through a heated vaporizer (typically 250 to 400 °C) at atmospheric pressure. The resulting gas-phase solvent molecules are ionized by electrons discharged from a corona needle. Solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization). These analyte ions pass through a capillary sampling orifice into the mass analyzer. APCI is applicable to a wide range of polar and nonpolar molecules. It is less well-suited than electrospray for analyzing large biomolecules that may be thermally unstable.

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Never compromise between information content and throughput

Agilent multimode source (MMI) is a breakthrough LC/MS technology that combines both ESI and APCI into a single ion source. It essentially acts as an ESI source spraying into a corona discharge needle, which can create chemically ionized species. Performance may be slightly less than either source individually, but it allows both nonpolar and polar analytes to be ionized by a single source without swapping or downtime. MMI operates in ESI-only, APCI-only, or mixed mode for ultimate versatility.

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