C268 mass spectrometry tosh thematic crop

Mass spectrometry – small samples, high-speed, low cost

Recent years have witnessed the growing use of mass spectrometry (MS) in the clinical laboratory. MS provides massive improvements in the sensitivity and specificity of clinical tests. It does this by using an ionized molecule’s mass/charge (m/z) ratio for identification.
MS has its roots in the screening and fingerprinting of molecules in drugs of abuse. Over the years, the technology has rapidly evolved. Today, it is routinely used to screen for diseases and to precisely identify causes of infections for targeted therapies. The analysis of proteins is also accelerating, with special potential demonstrated by biomarkers such as thyroglobulin.Alongside, limitations to immunoassays have also driven adoption of MS. For example, no immunoassays were approved for the immunosuppressant sirolimus and this compelled laboratories to turn to MS. Another advantage lay in improved assay quality, such as the measurement of testosterone in patients with low endogenous concentrations, such as women and children.

Enticing advantages
One of the most enticing set of advantages of mass spectrometry is that it provides clinically relevant information from relatively small sample volumes, and does this both rapidly and at a reduced cost. Gas chromatography (GC), liquid chromatography (LC) and ion mobility spectrometry (IMS) separation now allow targeting of ever-smaller analyte concentrations. LC-MS/MS (liquid chromatography-tandem mass spectrometry), on its part, offers scope to cut costs further, while continuing to improve accuracy. Other technology trends include integrating MS with low-flow chromatography, ultra-high pressure chromatography and online/multi-dimensional  chromatography.
Nevertheless, MS also adds a new layer of complexity. As a result, close and well-structured communication between laboratories and clinicians is a vital component for the effective use of MS.

A three-step process
Today, there are three principal steps for conducting an analysis by MS: sample preparation, separation by gas-chromatography (GC) or liquid-chromatography (LC), and mass spectrometric analysis.
In MS, a sample ‘matrix’ refers to everything present in a sample, excluding analytes of interest. Differences in behaviour between analytes and matrix components determines the choice of sample preparation. Although sample preparation requires more labour than immunoassays, in-house mass spectrometry-based assays are now considered cost-effective, even for smaller labs.

Sample preparation
The preparation of samples and their subsequent separation by chromatography both use mechanisms which first position molecules (the stationary phase) and then separate analytes from matrix components (the mobile phase).
Preparation firstly depends on the sample type selected for analysis (e.g. blood/serum or urine). Analytes from serum (including blood fractions) require the maximum care in preparation, owing to a relatively low ratio in the concentration of analytes to matrix components. On the other hand, urine analytes are often compatible with simple dilution protocols, due to the concentrating effect of kidneys in the production of urine.

Typical techniques in preparing samples include solid-phase extraction (SPE), immunoextraction and dilution. The choice depends principally on whether the analytes are acidic or alkaline, and if they are heavily protein-bound.

Solid-phase extraction
SPE is based on combining a solid stationary phase with a liquid mobile phase.
Analytes of interest (and matrix components) remain in the liquid phase or associate only temporarily with the solid stationary phase. The amount of time taken up by the latter is based on characteristics such as charge and polarity of the analytes versus matrix components. A binding-and-wash solvent (different from the elution solvent), provides a relatively crude separation of analytes from the (unwanted) components.

Immunoextraction (also known as immunoaffinity purification) is based on the use of antibodies in a solid phase. This separates antibody-bound analytes from free matrix components.

When analytes are present in high concentrations, dilution provides a simple and effective methodology to reduce matrix components. Dilution (often called ‘dilute-and-shoot’) is a common method of sample preparation for comprehensive screening and for confirmatory testing for drugs in urine.

Gas chromatography
GC chromatography uses hydrogen or helium to push molecules into a column (known as the stationary phase). Modifying the column temperature then changes the affinity of molecules in the stationary phase, thereby separating analytes from matrix components (known as the mobile phase). Though largely relevant for volatile, heat-stable compounds, ‘derivatization’ via chemical modification can increase compatibility with GC.
GC mass spectrometry (GC-MS) remains the most common method for comprehensive drug screening in the clinical laboratory.

Liquid chromatography
LC chromatography is largely used for separation of samples before MS analysis. This is largely due to a wide range of LC-compatible analytes and a reduced need for derivatization. The mobile phase in LC uses a combination of organic solvents and water. Adjustments to the ratio between the organics and water redistributes components between the mobile and stationary phases.

Ionization techniques: APCI and electrospray
MS detects charged analytes in the gaseous phase alone. Ionization is required to convert liquid-phase analytes for analysis. The two most common methods in the clinical laboratory consist of atmospheric pressure chemical ionization (APCI) and electrospray ionization.
APCI produces ions by using heat to evaporate the solvent and plasma to ionize the sample. Physical interaction with gaseous analytes leads to formation of negative or positive ions.
Electrospray ionization, on its part, combines electricity, air and heat to produce successively smaller and concentrated droplets from the liquid which elutes off a chromatographic column. This leads to a dramatic increase in charge per unit volume. Ions on the droplet surface desorb from liquid to gas phase, and the latter is introduced into the mass spectrometer.

Sample transfer to mass spectrometer
There are several choices for introducing samples into a mass spectrometer. These range from direct infusion to multidimensional chromatographic separation. The latter enables the staggered delivery of analytes and matrix components. This permits more effective utilization of analyser time by limiting analysis to fractions containing analytes of interest.

Methods of analysis
MS analysis is largely based on quadrupole analysers, time-of-flight (TOF) analysers and tandem mass spectrometers, as well as combinations of the three.

Quadrupole analysers
Linear quadrupole analysers are currently the most common type of mass spectrometer in a clinical laboratory. Called quadrupoles due to the presence of four parallel rods in a square, one pair of (diagonally opposed) rods is positively charged, while the other is negatively charged. The charges are optimized and alternated based on the analyte of specific interest. Via sequential attraction and repulsion, an ion of interest can be programmed to maintain a stable flight path between the rods. The charge and frequency can moreover be rapidly altered to sequentially detect different analytes. Quadrupole analysers have high sensitivity and mass accuracy. On the other hand, they have a limited range in mass/charge (m/z) ratios – which, as noted previously, is a unique identifier for a particular ion.

Time-of-flight analysers
Time-of-flight (TOF) mass spectrometers are based on using an electric field which accelerates gas phase ions to a detector. The time taken for this travel is based on an ion’s m/z ratio, with low m/z ions travelling faster than higher ones.
TOF analysers have an essentially unlimited m/z range and high sensitivity and accuracy, but users face limits in their dynamic range.

Key challenges in clinical MS
Tandem mass spectrometry

Successful identification of an analyte by m/z alone does not always confer specificity. One good example is morphine and hydromorphone. Though the two are distinct, they have identical positive ions, with 286 m/z.
Tandem mass spectrometers (MS/MS) use multiple quadrupoles in series. One typical configuration is to use three quadrupoles. The first and third (denoted Q1 and Q3) use combinations of charge and frequencies as described above (see section on ‘Quadrupole Analysers’). The second quadrupole, denoted q2 (in smaller case), serves as a collision cell with an inert gas (e.g. nitrogen). On entry into q2, ions collide with the inert gas, and fragment into smaller product ions which then pass through Q3 and hit a detector.
In the case of morphine and hydromorphone, q2 entry produces stable product ions (m/z of 153 for the former and 157 for the latter). After this, setting the Q3 charge and frequency to first transmit the product ion for morphine and then change the charge/frequency settings to transmit the  hydromorphone product ion results in a way to measure and differentiate between the two compounds. This approach is also known as multiple reaction monitoring (MRM) and allows a mass spectrometer to scan faster – by targeting specific m/z set points rather than a broad range.

Ion suppression/enhancement and ion ratios
Ion suppression and enhancement are two of the most common problems facing MS. These occur when a substance in a sample interferes with the ionization process of the analytes. These can range from matrix molecules to co-eluting compounds. For example, components in the sample with lower volatility can reduce the efficiency of solvent evaporation, resulting in reduced ion formation.
There are several options to reduce or eliminate such interference, including mobile phase additives to aid ionization.
Another approach is to use ion ratios. When analytes of interest are present alongside structurally similar compounds in complex matrices, interference risks rise due to co-eluting molecules with identical mass. However, ion ratios seek to monitor multiple m/z transitions for each analyte and determine ratios of chromatographic peak area for more abundant fragments to less abundant ones. The use of ion ratios further enhances the specificity of MS.

The future

Although industry has sought to use antibody-mediated detection to overcome the inherent limitations of immunoassays in identifying proteins and small molecules, these have yet to be meaningfully eradicated.
Rising interest in (regular and more-frequent) testing for vitamin D has also driven implementation of LC-MS/MS (liquid chromatography-tandem mass spectrometry), which separates vitamin D2 from vitamin D3 and provide information on its epimeric form. This is not possible with existing immunoassays.
Meanwhile, although steroid hormone assays for diagnostic and forensic testing continue to grow, a lack of specificity and accuracy at low concentrations has hampered the diagnosis of endocrine disorders. This has led several medical groups to recommend mass spectrometry as the preferred method of analysis, in spite of the high degree of technical competence, skill and experience required to achieve meaningful results.

Measurements of the genome and proteome need to be accompanied by quantified data on the metabolome to comprehend differences between disease and healthy status, and provide meaningful diagnosis and monitoring of disease. One of the fastest growing areas for MS in metabolomics is the screening of newborns.

Protein analysis

The success of MS in precise measurement of small molecules has driven interest in using it for peptide and protein analysis for diagnostic testing. In spite of some challenges, quantitative proteomics (covering factors such as isotope dilution and m/z transitions) is an especially exciting application for mass spectrometry.