Finding the Correct Mass Spectrometry for Small Molecule Analysis: A Helpful Guide for Laboratories

Mass spectrometry (MS) is a valuable technique used by researchers and scientists to advance scientific knowledge and enable groundbreaking discoveries. MS instruments are widely used across diverse scientific fields, from life sciences to environmental studies and from pharmaceutical research to material sciences. They have proven to be indispensable analytical instruments as they can be used for many different applications in biology, chemistry, physics, pharmaceuticals, clinical medicine and even space exploration.

In this helpful guide, intended for small molecule analysis (and for those new to the MS world), we explore three widely used types of MS instruments and some of their applications.

What Is Mass Spectrometry?

Mass spectrometry (MS) is an advanced and powerful qualitative and quantitative analytical technique by which gaseous and charged ions of chemical substances are identified and sorted in electric and magnetic fields according to their mass-to-charge ratios or m/z, where m is the molecular weight of the ion (in Daltons) and z is the number of charges present on the measured ion. When coupled with gas or liquid chromatography, this sensitive and accurate technique provides valuable information about the molecular structures of chemical compounds present in the sample, its confirmation, and its quantification. In addition, all atomic and molecular ions are, in principle, accessible by an MS instrument, making it a universal tool for chemical analysis. 

MS instruments have been marked by an ever-increasing number of applications in science and technology. New applications and technological developments continue to expand to create a more advanced array of modern MS instruments that can provide highly specific chemical information directly related to the chemical structure, such as accurate mass, isotope distribution patterns for elemental formula determination, and characteristic fragment ions for structural elucidation or identification via MS/MS spectral databases. MS instruments are currently routinely used in combination with chromatographic separation, principally in the form of the GC-MS(/MS), LC-MS(/MS), LC-TOF-MS, GC-TOF-MS or LC-IT-MS instruments.

Various industries and scientific fields utilize MS instruments due to their versatility for a wide range of applications, their sensitivity to detect chemical compounds in complex matrices at trace levels and their accuracy in ensuring that only a single elemental composition contributes to the mass spectral peak in question (i.e., the proximity of the experimental measurement to the true value or the exact mass). 

For small molecules analysis, an MS instrument is used in: 

• Scientific research to analyze chemical compounds, study molecular structures, identify unknown substances and investigate some complex biological systems. An MS instrument is also used to study molecular reactions of gas phase ions and determine ion energetics. It can also be used for elemental identification and isotopic abundance measurement (e.g., carbon-14 dating) in geological and archaeological studies.
• The pharmaceutical industry at every juncture of drug development – from the inception of discovery to the quality control of the “end” product. An MS instrument helps identify drug metabolites and differentiate closely related metabolites in a complex matrix, determine drug purity and ensure the effectiveness and safety of pharmaceutical products. An MS instrument also plays a key role in detecting and quantifying impurities that could affect a drug’s efficacy or safety. This is essential because it also enables faster and more accurate screenings in the clinical analysis of patient samples.
• Forensic science to analyze biological samples (such as blood, urine, hair, nails, and tissues: brain, liver, kidney, etc.) to detect and identify illicit drugs, toxins, and other substances, analysis of trace evidence (such as fibers in carpet, polymers in paint), arson investigation (such as fire accelerant), confirmation of drug of abuse, and identification of explosive residues.
• Clinical research in neonatal screening, drug therapy monitoring, diagnosing metabolic disorders and detection of diseases due to biomarkers. Biomarkers are utilized in diagnoses, prognoses, and treatment. Accurate and timely test results can help enable informed treatment decisions. MS instruments can measure biomarkers ranging in size from small molecules to relatively large macromolecules.
• Environmental monitoring to assess trace levels of organic and inorganic pollutants, contaminants in air and drinking water samples, detecting trace elements of heavy metals leaching, soil contamination assessment, carbon dioxide monitoring, screening and quantitation of pesticide residues, PAH and PCB analysis and other harmful contaminants and chemicals. 
• The food and beverage industry to ensure food and beverage safety, quality control, and regulatory compliance. MS instruments play a pivotal role in the identification and quantification of contaminants such as pesticides, mycotoxins, veterinary drugs and antibiotics, hormones, allergens and heavy metals. They are also used to analyze meat speciation and food additives and to scrutinize nutritional content, including vitamins and antioxidants.

Sample Introduction

A sample must be prepared and introduced before using modern MS instruments for routine analysis. Generally, the samples are either in the liquid or gaseous phase. Coupling chromatography to MS instruments offers an excellent solution to complex mixture analyses and has been extensively used in various scientific fields. It offers several advantages, such as reducing matrix effects and ionization suppression, separation of isomers, providing additional and orthogonal data (i.e., retention times) and allowing for more accurate quantification of individual compounds. The widely used types of chromatography techniques that are routinely used to introduce the sample to be ionized are liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE). The two-dimensional LC (LC×LC) and GC (GC×GC) can further separate more complex mixtures, but they are less widely employed.

Liquid Chromatography (LC) Technique

LC technique separates samples based on interactions with the mobile and stationary phases. This can be based on polarities, meaning that if a specimen component has a different polarity than the mobile phase, it will migrate down the analytical column faster. The development of a fast and more efficient Ultra-High-Performance LC (UHPLC) technique, which utilizes higher pressures (15,000–18,000 psi compared with ∼6000 psi for HPLC) and sub-2-μm packing particles, has substantially increased chromatographic resolution and peak capacity. 

Gas Chromatography (GC) Technique

GC technique separates components of a mixture of gases and filters the passage of these molecules based on physical characteristics like shape, size, molecular weight, and boiling point. A sample is diluted and vaporized in the chromatograph, where it is separated. After separation, the gases enter the MS instrument for analysis. The GC technique is ideally suited for the analyses of both volatile and nonvolatile compounds following derivatization, as they need to be thermally stable.

Capillary Electrophoresis (CE) Technique

CE technique separates analytes based on charge and size, which is particularly suitable for analyzing highly polar and ionic metabolites. 

CE-MS instruments have some limitations, such as low sensitivity, poor reproducibility and lack of necessary robustness, and they are the least suitable choice for analyzing complex biological samples.

The Three Core Components of an MS Instrument

After separation by either GC, LC or CE techniques, individual components can be further analyzed in an MS instrument, which includes three essential components that play a crucial role in the MS instrument’s overall operation. Their optimal functioning is crucial for the quality of the data produced and for the reliability of the results.

1. Ion Source

The ion source is responsible for ionizing molecules within a sample. It converts neutral atoms or molecules into charged ions (both positive and negative ions) in the gas phase. This important MS instrument component exists in different types, such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron impact ionization (EI), chemical ionization (CI), matrix-assisted laser desorption ionization (MALDI), etc. Source selection depends on the nature of the sample and the molecules to be analyzed In the LC technique coupled with the MS instrument, the most widely used ionization techniques are ESI and APCI, which generally leave the molecular ion intact in the source (soft ionization). In GC coupled with MS, the most common form of ionization technique used is EI. This technique requires a source of electrons in the form of a filament to which an electric potential is applied, typically at 70 eV. In recent years, several new ionization techniques such as desorption ESI (DESI), direct analysis in real time, and extractive ESI (EESI) have also been used to facilitate the use of direct MS instrument analysis in various fields such as metabolomics.

2. Mass Analyzer

The mass analyzer (MA) separates the produced gaseous ions based on their m/z ratio, making it a critical component of an MS instrument and in the accurate measurement and identification of the compound ions within a sample. These MAs come in various types, and the most widely used ones in routine small molecule analysis are quadrupoles (Q), time-of-flights (TOF) and ion traps (IT). By considering a comprehensive view of the unique advantages and disadvantages of each one of them, it will become possible to navigate complex choices and make decisions that align with the specific goals, needs, and circumstances at hand.

3. Detector

The detector measures the abundance of ions at different m/z ratios. After ions are separated and focused by the MA, the detector detects the ions and converts their signals into measurable electrical signals. These detectors exist in various types: some are more sensitive, and some are faster. Examples include electron multiplier (EM), faraday cup (FC) and multi-channel plate (MCP). The detector type depends on the type of instrument and its intended scientific field and the specific application(s). Detector choice in instrument design will be based on a combination of the desired sensitivity and the speed of analysis. It is generally accepted that the best detectors are those with high amplification, fast time response, low noise, high collection efficiency, low cost, narrow distribution of responses, similar response for all masses, large dynamic range, long-term stability and long life.

It’s clear that the MS is an invaluable analytical instrument that has become essential to modern laboratories in many industries. A good understanding of its function, benefits and drawbacks is very important when selecting the right MS instrument for your laboratory. 

Let’s explore the three MS instrument options for small molecule analysis. 

Types of MS Instruments

When looking for an MS instrument, you will encounter different mass analyzers (MA) with different resolving powers. The MA is the part of the MS instrument that analyzes how a mixture of ionic species is separated according to the m/z ratio. The analysis may be qualitative and/or quantitative. Each MS instrument has its distinct and unique advantages and limitations. Understanding the applications for which you need the MS instrument will help you select the one that meets your current and future laboratory needs. Some MS instruments combine multiple different mass analyzers in one MS instrument to be used for advanced scientific research and complex applications. In this first blog, we will focus on three main types of MS instruments. 

1. Quadrupole MS (Q-MS)

A quadrupole mass spectrometer (Q-MS) uses a set of four parallel rods with applied radio frequency (RF) and direct current (DC) voltages to filter ions based on their m/z ratio. Based on a specific combined DC potential and RF field frequency, only ions of a specific m/z ratio would have stable trajectories between the rods. In contrast, the other ions with unstable trajectories will collide with the rods and will be filtered out. The m/z ratio for each ion is measured with single-digit mass accuracy. Q-MS is most widely used for routine targeted analysis. It allows for the detection of a wide range of compounds in a single analysis. It is an ideal choice for laboratories focusing on food safety testing, environmental monitoring, pharmaceutical, cannabis testing or clinical analyses that need accurate identification of a sample’s specific compounds with rapid scan speeds and high sensitivity. Since Q-MS has fast duty cycles, it can be easily combined with GC and LC techniques. 

Advantages

• Speed of analysis: High-throughput analysis with a straightforward operation that allows for processing more samples efficiently.
• Sensitivity: Crucial for detecting contaminants and other harmful chemicals at trace levels. Better signal-to-noise, which allows for lower limits of quantification (LOQs).
• Selectivity: Enables precise control over ions and can select the analyte out of an intense and complex matrix. It offers the ability to quantify a high number of targeted compounds in a single injection. When multiple quadrupole mass analyzers are used in series, the electric fields and the collision energy can be held constant so only analyte ions with a certain m/z ratio (precursor/ product ion pair) can reach the detector. 
• Affordability: Cost-effective and versatile. Relatively simple, easy to use, highly utilitarian, and offers a variety of interfaces at a relatively low cost.
• Precision: Ability to provide accurate and consistent measurements of ion masses.

Quadrupole mass spectrometry (Q-MS) does not require a very high vacuum condition. It is reasonably stable and thus requires less calibration.

These benefits are particularly useful when aiming for the lowest level of detection and quantification and the fastest time to detect targeted compounds.

Disadvantages

• Mass Range: Limited mass range (~100 to 3000 Daltons) compared to other types of MS instruments. This can be a drawback when analyzing large biomolecules, polymers or complex mixtures with high-mass compounds.
• Mass Resolution: This instrument has a lower mass resolution than other high-resolution MS instruments, such as Fourier transform ion cyclotron resonance MS (FT-ICR-MS), orbitrap MS, and TOF-MS instruments. Its ability to resolve and distinguish ions of closely spaced masses may be limited, which can be a disadvantage when dealing with complex samples or isobaric compounds.
• Interferences and Ion Suppression: Susceptible to interferences from matrix effects, isobaric interferences, and chemical noise. These interferences can impact the accuracy and specificity of the measurements. Despite the selectivity and sensitivity of Q-MS techniques, there are considerable challenges due to matrix effects with regard to reproducibility and accuracy when analyzing complex samples.
• Targeted Analysis: Only provide information about unknown compounds with predefined settings and target identification.

2. Time-of-Flight MS (TOF-MS)

A time-of-flight mass spectrometer (TOF-MS) separates ions based on their differences in flight time through a field-free drift region. Due to their kinetic energies, ions of different mass-to-charge ratios will have different velocities. Lower m/z ions travel faster and have the same kinetic energy as higher m/z ions. As the ions travel at different speeds based on their m/z ratio, they reach the detector at different times. The flight time is measured as the time it takes for each ion to reach the detector at the end of the drift region. The m/z ratio for each ion will be measured in four decimal places.

TOF-MS is known for its high-speed data acquisition capabilities, which allow for rapid analysis and high sensitivity. It is useful for the structural elucidation of novel or unknown compounds in complex samples. TOF-MS is commonly used in various scientific sectors, such as proteomics, metabolomics, lipidomics, pharmaceutical research, environmental and forensic analysis, drug discovery, food and beverage testing, and materials science.

Advantages

• Mass Range: Wide mass range capability, from small ions to relatively large biomolecules and polymers. This makes TOF-MS suitable for analyzing complex samples containing both low and high-molecular-weight compounds, including proteins, peptides, metabolites, and small molecules.
• High-Speed Data Acquisition: Fast data acquisition rates allow for rapid analysis. This high-speed capability makes TOF-MS advantageous for applications requiring high-throughput analysis and screening of large sample sets.
• Interference: Less prone to interferences from isobaric species or matrix effects compared to some other instruments, such as Q-MS. 
• Relatively High-resolving Power: Making TOF-MS suitable for analyses that require precise mass accuracy and molecular rate determination.
• Unknown compound identification: Revealing structure information during general unknown screening experiments.

Disadvantages

• Mass Resolution: TOF-MS traditionally has a lower mass resolution than other high-resolution MS, such as orbitrap MS and FT-ICR-MS instruments. The ability to resolve and distinguish ions of closely spaced masses may be limited, leading to challenges in analyzing complex samples with overlapping peaks or isobaric compounds.
• Complexity and Cost: TOF-MS can be relatively complex and expensive compared to other instruments, such as Q-MS, which may limit their accessibility for some research laboratories or applications with budget constraints.
• Skilled Technical Personnel are required: With increased complexity comes the need for more expertise to operate TOF instruments and to ensure correct operation and accurate results.
• Data Analysis Software: Large amounts of data require sophisticated software analysis tools. 

In addition, TOF-MS requires more maintenance, including regular calibration, to prevent mass accuracy deviation and maintain optimal performance.

3. Ion Trap MS (IT-MS)

An ion trap mass spectrometer (IT-MS) is an analog of the Q-MS that uses electromagnetic fields to trap and manipulate ions, allowing for selective ionization, fragmentation and detection. One of the most significant uses of IT-MS involves sequential trapping and fragmentation of specific ions to produce highly specific multistage MSⁿ spectral data with minimum chemical noise to facilitate the structural identification of unknown chemical compounds in a sample. It is generally used for the discovery and targeted analysis of molecules and molecular reactions in fields such as metabolomics and lipidomics, as well as post-translational modification analysis. This kind of mass analyzer comes in two different types:

• 3D IT-MA: The three-dimensional IT-MA uses three electrodes to trap ions in a small volume. It consists of two hyperbolic electrodes (end caps) facing each other and separated by a hyperbolic ring electrode. A damping gas, such as helium, is introduced to cool energetic ions, and RF and DC voltages are applied to the three electrodes. Mass scanning is performed by increasing the amplitude of the voltages to eject ions of increasing m/z ratio for detection. It provides detailed structural analysis and compound identification in analytical applications.
• Linear 2D IT-MA: The two-dimensional linear IT-MA is composed of two pairs of rods that collect and trap ions using radio frequencies. It has been developed from the conventional Quadrupole mass analyzer and can also be used to collect and inject pulses of ions coming from continuous sources. Simple plate lenses at the ends of the quadrupole provide the DC trapping field to keep the ions confined within the MA. The ion beam is reflected repeatedly between the two electrodes, and a slot is made in one of the rods, allowing ions to be radially ejected.  

Advantages

• Capability for highly detailed and complex structural data analysis: This is useful for challenging matrices that require extensive sample preparation and specifically for large molecules where fragmentation can be used iteratively to elucidate their structures, making IT-MS ideal for compound identification compared to other MS types such as Q-MS. Ions can be stored indefinitely, and multiple fragmentation (MSⁿ) experiments can be performed “in time” to provide structural information not only on the molecular ion but also on resulting fragment ions to construct mass spectral trees. Q-MS and TOF-MS instruments can only perform MS/MS (MS²) experiments.
• Compact and Relatively Inexpensive: Mechanical simplicity in an IT-MS instrument that is capable of high performance.

Disadvantages

• Resolution: It diminishes rapidly as the ion density increases, and there is a capacity for only a limited number of ions at any one time before repulsive charges (known as the space charge effect) cause excess ions to be ejected. 
• Dynamic range, accurate mass measurement, and quantitative precision: Limitations result from space-charge effects when too many ions enter the trapping space. However, the increased volume of a LIT-MA instrument (over a 3D IT-MA) improved the dynamic range.
• Sensitivity: IT-MS instruments do not scan like Q-MS, so using the single ion monitoring (SIM) technique does not improve sensitivity on 3D IT-MS as it does on Q-MS instruments.

Some Considerations for Choosing a MS Instrument

A well-planned investment in the right MS instrument is a necessary requirement for any prosperous research or routine testing laboratory. It can significantly improve productivity and the capability to provide reproducible, reliable and accurate results. When selecting an MS instrument for your laboratory, you should consider the type of analyses you perform, the number of samples to process a day, a month, a year – and the kind of results you expect from them. In addition, consider the software and its user-friendliness, its integration with a laboratory information management system (LIMS) and its compliance needs, the turnkey applications methods package/solution, the right support and service plan that fits your needs, the post-installation service and support and the on-site training. These factors should all be part of the decision-making process. A few essential factors to consider:

1. Applications

Understanding your current and future “planned” applications’ needs plays a major role in selecting an MS instrument. You need to ensure you are acquiring an instrument that best fits the needs of your laboratory. The optimal selection of a particular MS instrument depends on the study’s goals and the main applications. For routine small molecule analysis, it is usually a compromise among the important “3S”: Sensitivity, Selectivity, and Speed. In addition, you need to consider if the MS instrument will be used for direct sample analysis or if it needs to be coupled to chromatography, such as LC, GC or CE techniques.

For small molecule analysis:

• In laboratories with a high workload of samples and focusing on targeted compounds for quantitative analysis such as food and beverages safety testing, environmental monitoring, forensic toxicology or clinical analyses, a quadrupole MS instrument (mostly triple quadrupole MS) will potentially be the instrument of choice based on its robustness, reproducibility, sensitivity and speed of analysis to process as many samples as possible per day. These instruments are generally used in combination with an LC or a GC technique in the form of an LC-MS/MS and/or GC-MS/MS instruments.
• For laboratories focusing on the simultaneous detection and quantification of specific metabolites in targeted metabolomics and the identification of unknown metabolites in biological samples, or the detection and quantification of drug impurities and degradation products, or structural elucidation of drug molecules and their metabolites, or proteins identification and quantification, or the study of post-translational modifications (PTMs), then the TOF-MS instrument is potentially the instrument of choice based on its sensitivity, resolution and precision. It is generally used in combination with an LC, a GC, or MALDI techniques in the form of LC-TOF-MS, GC-TOF-MS, or MALDI-TOF-MS instruments.
• For laboratories focusing on the discovery and targeted analysis of molecules, molecular reactions, screening, confirmation using pesticides, antibiotics, mycotoxins and drugs MS/MS library-matched databases, the combination of Q-MS with LIT-MA technology in the form of an instrument using axial ejection, is particularly interesting because this instrument retains the classical triple quadrupole scan functions such as selected reaction monitoring (SRM), product ion (PI), neutral loss (NL) and precursor ion (PC) while also providing access to sensitive ion trap experiments. Simultaneous quantitative and qualitative analysis can be performed using the same instrument.
• Laboratories focusing on clinical diagnostic testing that develop their own clinical assays might require an MS instrument classified for in vitro diagnostics (IVD) with unique medical device (MD) maintenance and service policies and procedures. 
• Some laboratories use an MS instrument to directly analyze sample crude mixtures without chromatographic separations. This can provide a high throughput screening tool that is often the only practical choice for large sample numbers such as those encountered during clinical trials or screening of large mutant populations or in crude oil samples analysis. FT-ICR-MS, for example, can be a very useful instrument for direct sample analysis due to its ultra-high resolution (>1,000,000) and mass accuracy (<1 ppm). The relatively low resolution and low mass accuracy of Q-MS and IT-MS instruments limit their roles in directly analyzing complex samples.

2. Technical Specifications

Understanding the MS technical specifications needed for your laboratory’s applications, which can be from elemental to large proteins and polymers, is crucial. For an effective and safe usage of the MS instrument, it is also very important to consider the skill level and the knowledge of the potential operators in your team. Some MS instruments may require specialized training as they can be more complex to understand and operate. Some important parameters to consider when specifying an MS instrument include resolution, mass range, mass accuracy, sensitivity, usability and personalized hardware and software training.

Merely relying on vendor specifications or price isn’t enough; a comprehensive evaluation of technical specifications is necessary to ensure that the chosen MS instrument meets the specific analytical requirements and performance criteria essential for accurate and reliable results. This informed assessment helps laboratory directors or managers make the right choice for your laboratory.

3. Support & Service

The critical difference among vendors with similar products is the quality of support and service.  Guaranteed response times and reliable service are essential for maintaining investment functionality and keeping your laboratory running optimally. By minimizing downtime and maximizing efficiency, you can ensure your laboratory operations run smoothly and that your research or analytical tasks are completed without interruptions or delays. Remote troubleshooting can allow the identification of MS instrument issues and potentially resolve any concerns, order parts in advance and decrease downtime.

Service and support extend beyond a single transaction; they form a lasting partnership. Regardless of the instrument type, ongoing commitment and assistance matter most, especially when maintenance is needed later. The right service provider will contribute to the success of your laboratory by helping to ensure consistent and long-term quality and functionality of your MS instruments. Having a reliable partner for MS servicing ensures your instruments’ continuous functionality and gives your end-users training and confidence in troubleshooting the MS instrument.

Conclusion

MS instruments are indispensable tools that provide powerful insights into molecular composition, structure and interactions. Various types of MS instruments exist, each with advantages and disadvantages that must be considered before making a final selection. It’s also imperative to consider your applications, the required specifications, and the right service and support plan that fits your laboratory needs to ensure reliable results.

Professional MS instrument services are indispensable in maintaining and performing your instrument. When you need to service your new MS instruments, consider an experienced multivendor LC-MS service provider. Contact ZefSci today to discuss how we can keep your MS instruments operating optimally for years to come.

ZefSci specializes in servicing multi-vendor LC-MS instruments and offers a wide range of support and services to help your laboratory obtain results faster and maximize instruments’ uptime.