Laboratory testing and its value has an acknowledged widespread role in clinical decision making, and therefore a role in determining clinical outcome. Value is often expressed in terms of quality, clinical efficacy and effectiveness, timeliness, clinical efficiency, cost effectiveness, productivity affordability and cost.¹

Traditionally, laboratories define quality of service in terms of the precision and accuracy of the data they provide. Accurate, sensitive, and rapid clinical diagnostic tests are greatly desired to foster clinical decision making and improve human healthcare. The continuous development of new analytical methods and techniques over the last few decades has made an important impact on a disease detection and treatments.^2,3

Beside spectrophotometer, the majority of in vitro diagnostics tests involves immunoassays to detect the target analyte. However, certain limitations of those methods (cross-reactivity, requirement of high amount of specimen, low sensitivity, limited dynamic range, and often expensive and time-consuming processing) are forcing us to find a better solution with alternative approaches, which should be more sensitive and specific and offer high throughput.

Gas/Liquid chromatography applications are being used in many fields like food, pharmaceuticals, and environmental testing since decades. Mass spectrometry (MS) has been an evolving technique over the years with performance improvement resulting in lower detection limits and higher sensitivity. And finally, tandem use of chromatography and mass spectrometry has evolved, which has a potential to bring clinical laboratory service to an even higher level of advanced developing technology.

Concepts of MS detection encompass electron impact ionization (EI), chemical ionization (CI), field ionization/desorption, electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI), inductively coupled plasma mass spectrometry (ICP-MS), fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), and atmospheric pressure chemical ionization (APCI).⁴

Electrospray ionization (ESI) is the most popular ionization technique. The electrospray is created by putting a high voltage on a flow of liquid at atmospheric pressure; sometimes this is assisted by a concurrent flow of gas. The created spray is directed to an opening in the vacuum system of the mass spectrometer, where the droplets are de-solvated by a combination of heat, vacuum and acceleration into gas by voltages. Eventually the ions are ejected from the droplets and accelerated into the mass analyzer by voltages. ESI is being used for polar or very polar substances.

Atmospheric pressure chemical ionization (APCI) is a method that is typically done using a similar source as ESI, but instead of putting a voltage on the spray itself, the voltage is placed on a needle that creates a corona discharge at atmospheric pressures. This discharge creates ions, in theory mostly H3O+ or water clusters. The sample is injected into the discharge by a spray created by a flow of liquid combined with a heated gas that volatilizes the sample. The ions are formed by proton transfer from the H3O+ or the water clusters to the sample.  These ions are then extracted into the same opening vacuum that is used for electrospray. APCI is good answer for those who are dealing with slightly polar or non-polar molecules. Recent developments of some equipment manufacturers are allowing both ESI and APCI ionization configurations in the same equipment so they can be used separately or combined, which is a big plus for research and development (R&D) departments of laboratory.

Inductively coupled plasma mass spectrometry (ICP-MS) uses a plasma torch generated by electromagnetic induction to ionize samples. Due to extremely high effective temperature of the plasma, samples are broken down to ions of their constituent elements. This technique is best suited for elemental analysis since all chemical structure is lost. ICP-MS is typically used for analysis of trace elements.

Matrix assisted laser desorption ionization (MALDI). Laser desorption ionization generates ions by ablation from a surface using a pulsed laser. This technique is greatly improved by the addition of a matrix co-crystallized with the sample. As the sample is irradiated, a plume of desorbed molecules is generated.

There is a difference between single quadrupole (MS) and triple quadrupole (MS/MS) detection. In MS detection there is only one ionized component while in MS/MS detection, first ionization product (precursor, Q1) is giving secondary fragment(s) (product, Q3); and determination and quantification are performed from the pair of Q1 and Q3, named multiple reaction monitoring (MRM), therefore gaining a triple quadrupole concept extra specificity over the single quadrupole due to additional analytical dimension. Though the state-of-art MS/MS are capable of simultaneous scanning hundreds of MRM pairs the matrix effect, possible epimers and isobaric interferents should be separated prior to MS/MS detection to avoid misleading measurements. For that, gas/liquid chromatography is where the selection of the chromatography column and mobile phase conditions should be seriously considered. Nowadays there are many different types of liquid chromatography (LC) columns being developed while gas chromatography columns are not due to the nature of technique. This situation is making LC technique preferable over GC in most of cases since the analytes can be separated with the same performance by using several different columns—but sometimes there is the only one due to chemistry of analyte.

Pros of Lc/MSMS

Three-dimensional (3D) chromatography provided by LC/MSMS technique has a number of benefits, like unequalled sensitivity, detection limits, speed, and diversity of its applications. Depending on ionization nature, the limits of the quantification can go down to picomole per liter (pmol/L) levels with a good intra/inter-day repeatability, offering trace-level detection for biomarkers. Single quadrupole (MS) detection, like other detection types (UV, FLD, ELSD) can also provide multianalysis, however, with compromised specificity and selectivity compared to MS/MS detection. On the other hand, MS/MS can run same sets of analytes in a shorter timeframe with better selectivity and shorter run times. For example, a classic amino acid analyzer completes one run in 2 hours while with LC/MSMS it is possible to analyze the same set of amino acids just in 15 minutes in a more economical way without any derivatization step. This also results in shorter turnaround time and lower operational costs at the same time. Sample amount needed for most of treatments prior to injection to LC/MSMS is very low, which results in a much lower amount of specimen—making testing easier for most age groups.

Cons of LC/MSMS

LC/MSMS methodology has certain drawbacks. The major limitation of MS, except for MALDI, is the molecular weight of compounds; 2 kilodaltons (kDa) is the upper limit. However, most clinical diagnostics target analytes are small molecules with molar mass below 2kDa. For larger molecules this issue can be eliminated with analyzing certain fragments (peptides of proteins) and creating their map.

The second biggest issue is a selection and commercial availability of internal standards. The use of an internal standard (IS) not only allows correction for ion suppression/enhancement effects but also facilitates the normalization of results by compensating for matrix effects, sample preparation variability, and instrument fluctuations in ionization efficiencies.⁵ Stable isotopically labeled internal standards are compounds in which several atoms in the analyte are replaced by their stable isotopes, such as ²H (D), ¹³C, ¹⁵N, or ¹⁷O. There are many internal standards available in the market for testing of popular analytes like certain vitamins, amino acids, pollutants, toxins and others. However, scientists may not find it in the market when it comes to development of specialty testing. Then the knowledge of chemistry and potential behavior of the target analyte(s) in MS/MS play the most important role to find a proper “representative” internal standard candidate. Plus, using an internal standard for each analyte is a cost elevating factor. In case of many molecules, they can be grouped accordingly (same stability, similar retention times) versus certain internal standard with no compromise to the quality of the testing.

Traditional liquid chromatography separations are being achieved by interaction of analytes with mobile phase and column active surface and can be improved by using different buffers (phosphates and other salts), ion pair agents (trifluoroacetic acid, heptafluorobutyric acid and others) and acids. In mass spectrometry applications, this number of chemicals is strictly limited to volatile ones. Most ion pair agents are also not desirable factors due to stacking in tubing parts of LC and randomly generating ghost peaks in the system. In accordance with that, the final solution of the extracted sample also should have some volatile agents to avoid issues.

Sensitivity and speed of the state-of-art LC/MSMS instruments have frequently misled scientists in thinking that almost anything is possible with just one injection of the sample into the system. Yes, it is applicable in many cases in pharmaceutical applications; however, clinical diagnostics is a far more complicated area, especially when it comes to vitamin testing. Besides dealing with a complicated composition of matrixes, careful consideration of targeted analytes is the most critical part of R&D in developing a diagnostic test.

For example, thiamine (Vitamin B1) can be measured in any type of biological fluid, and it is very popular to provide its intercellular value which is, in fact, non-informative to either patients or the physicians. However, measurement of vitamin B1’s metabolically active form, thiamine pyrophosphate (TPP), in a whole blood combined with a measurement of thiamine/creatinine ratio in urine provides a good picture of vitamin B1’s metabolic status.

In another instance, correlation studies between traditional measurement of pantothenic acid (vitamin B5) and direct measurement by LC/MSMS may also provide erroneous information. Technically there is no issue in the measurement of pantothenic acid; however, classical methodology involves enzymatic dephosphorylation steps, which release pantothenic acid. Therefore, clinically valuable guidance of vitamin B5 testing from classical approach to LC/MSMS methodology is possible via adapted enzymatic steps only.

There are also certain tests already developed for clinical diagnostics and based on LC/MSMS methodology, which include preparative steps like solid-phase extraction (SPE) or liquid-liquid extraction (LLE) that make the measurement of Vitamin 25-OH D₃, and other fat-soluble vitamins possible. This is essential for elimination of possible interfering substances and matrix effects.

As we tried to explain above, LC/MSMS is becoming a powerful tool in clinical diagnostics field only with the right selection of chromatographic components. Though the development of tests based on LC/MSMS methodology is growing rapidly, there is no recognition from FDA or other official bodies yet—though there are several guidelines related to mass spectrometry already published by Clinical and Laboratory Standards Institute (CLIS). However, when the beneficial sides of mass spectrometry are evaluated further, the future testing would be mainly transitioned to LC/MSMS methodology; and we may expect publications of standardized testing protocols for many tests in clinical diagnostics area in that period, which should be a golden goal for this methodology. At Health Diagnostics and Research Institute (HDRI), we always aim to provide clinically relevant data and have transferred most of our tests to LC/MSMS platform during last few years to be ahead of time.

References

1. Sikaris KA. Enhancing the Clinical Value of Medical Laboratory Testing. Clin Biochem Rev. 2017 Nov; 38(3): 107–114.
2. Swiner et al. Applications of Mass Spectrometry for Clinical Diagnostics: The Influence of Turnaround Time. Anal Chem. 2020 January 07; 92(1): 183–202
3. Banerjee S. Empowering Clinical Diagnostics with Mass Spectrometry. ACS Omega. 2020 Feb 11; 5(5): 2041–2048.
4. Edmond de Hoffmann and Vincent Stroobant, Mass spectormetry  Principles and applications. 3rd ed. 2007 pp 15-79 John Wiley and sons ltd.
5. Tan A, Awaiye K. Use of Internal Standards in LC-MS. Bioanalysis Handbook of LC‐MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations. Chapter 17. August 2013.