Tandem mass spectrometry in the clinical laboratory: A tutorial overview

Tandem mass spectrometry in the clinical laboratory: A tutorial overview

Clinical Mass Spectrometry xxx (xxxx) xxx Contents lists available at ScienceDirect Clinical Mass Spectrometry journal homepage: www.elsevier.com/lo...

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Clinical Mass Spectrometry xxx (xxxx) xxx

Contents lists available at ScienceDirect

Clinical Mass Spectrometry journal homepage: www.elsevier.com/locate/clinms

Tandem mass spectrometry in the clinical laboratory: A tutorial overview J. Grace van der Gugten Provincial Health Services Authority, Vancouver, BC, Canada

a r t i c l e

i n f o

Article history: Received 20 March 2019 Received in revised form 4 September 2019 Accepted 4 September 2019 Available online xxxx Keywords: Tandem mass spectrometry Liquid chromatography Triple quadrupole Clinical mass spectrometry Clinical laboratory Quantitative analysis

a b s t r a c t Tandem mass spectrometry (tandem MS) is a powerful technique that directly measures compounds based on their molecular weight. Liquid chromatography triple quadrupole mass spectrometry (LC-MS/ MS), a type of tandem MS, has been increasingly adopted by clinical laboratories in recent years. While it is complicated and challenging, it has selectivity and accuracy advantages over more traditional laboratory techniques such as immunoassay. Able to measure a wide range of compounds, when correctly utilized and implemented, LC-MS/MS can benefit the clinical laboratory and improve patient care. This mini review is an introduction to clinical LC-MS/MS for the novice user. Crown Copyright Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sample preparation . . . . . . . . . . . . . . . . . . . . . . Liquid chromatography . . . . . . . . . . . . . . . . . . . Triple quadrupole mass spectrometry . . . . . . . Quantitative analysis . . . . . . . . . . . . . . . . . . . . . Challenges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recommended reading and learning resources Declaration of Competing Interest . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The clinical laboratory exists primarily to provide patientspecific relevant results to the physician who uses the data to

Abbreviations: MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid chromatography; LC-MS/MS, liquid chromatography triple quadrupole mass spectrometry; IA, immunoassay; ESI, electrospray ionization; TOF, time of flight; SIL, stable isotope labeled; IS, internal standard; PAR, peak area ratio. E-mail address: [email protected]

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inform diagnosis and treatment [1]. Thus, it is important that the clinical laboratory produce accurate results. The immunoassay (IA) is currently one of the most widely used clinical diagnostic instrument platforms [2], but IA tests can suffer from inaccuracy as a result of poor selectivity [3,4]. Tandem mass spectrometry (tandem MS, MS/MS) has a unique and important role to play in the clinical laboratory for its ability to provide accurate patient results, particularly where it excels over more traditional IAbased methodologies. The focus of this mini-review is to introduce liquid chromatography triple quadrupole mass spectrometry

https://doi.org/10.1016/j.clinms.2019.09.002 2376-9998/Crown Copyright Ó 2019 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). All rights reserved.

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(LC-MS/MS), a type of tandem MS, and how this technology is used in the clinical laboratory. It is non-technical, for those with little or no clinical mass spectrometry experience.

2. Background Mass spectrometry (MS) is an analytical technique that measures charged, gas-phase molecules based on mass-to-charge (m/ z) ratio [5–7]. It has existed for more than a century, but its complexity and hard ionization techniques left it largely relegated to esoteric research projects for most of its existence. These hard ionization techniques caused fragmentation of the compound during ionization, and required the sample to be in gas phase, limiting analysis to volatile and thermally stable compounds [6–9]. Following the perfection of soft ionization techniques in the 1980s, allowing molecules to remain intact during ionization [10–12], mass spectrometry was utilised for analyzing biological compounds, many of which are thermally labile and non-volatile [7,10,11]. These developments also allowed mass spectrometry to be coupled to front-end liquid chromatography – a combination with the power to provide superior analytical accuracy and specificity [4,9,13]. Other developments in the late 20th century resulted in the development of tandem MS, where two or more MS systems are combined [14,15]. Tandem MS offers increased selectivity over other common analytical measurement techniques, such as single stage MS, ultraviolet–visible spectrophotometry (UV–Vis), liquid chromatography (LC) or IA [16–19]. Enhanced selectivity is achieved by selection of a compound-specific precursor ion in the first mass spectrometer, fragmentation of the precursor ion in a collision cell, and subsequent selection of the specific fragment or product ions in a second mass spectrometer [14,20,21]. Liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) is the most prevalent tandem MS construct currently implemented in clinical laboratories [22,23], and has been gradually adopted in concept and practice around the world over the past 20 years [1,5,13,24–29]. LC-MS/MS has a number of advantages over IA including the ability to develop new methods inhouse with relative speed, improvements in accuracy, selectivity and specificity, reduced cost-per-test, and the ability to multiplex (Table 1) [4,5,13,15,30]. Since the first clinical diagnostic tests were run by LC-MS/MS, the number and frequency of tests run on this platform has rapidly expanded [5,13,17,24–28], and it is now considered a ‘gold standard’ technique for certain compounds [22,31], such as low concentration steroid hormones, where IA performs poorly [4,32–36]. Clinical laboratories currently use LC-MS/MS to measure a wide variety of compounds in various disciplines (Table 2) [1,4,5,10,13,17,18,22,26–28,31–46]. Although the terms are often used interchangeably, tandem MS is not limited to triple quadrupole mass spectrometry [6], but also includes ion trap and time of flight (TOF) analyzers [6,47–52]. An ion trap MS holds ions in a defined region by application of magnetic, electrostatic and/or RF electric fields. After a set amount of time, ions are selectively released to the detector. This can provide detailed

Table 2 Examples of LC-MS/MS applications in the Clinical Laboratory. Clinical area

Example target analytes

Newborn Screening/Inborn errors of metabolism

Amino acids Acylcarnitines

Therapeutic Drug Monitoring

Immunosuppressants (i.e. Sirolimus, Tacrolimus, Cyclosporine) Antifungals (i.e. Voriconazole, Posaconazole) Antidepressants Anti-cancer drugs Monoclonal Antibodies (i.e. Infliximab)

Endocrinology

Testosterone Aldosterone Estradiol Cortisol Angiotensin I Metanephrines Catecholamines Thyroid hormones 25-Hydroxyvitamin D 1,25-Dihydroxyvitamin D

Proteomics and Protein Markers

Thyroglobulin Immunoglobulin G subclasses Insulin-like Growth Factor 1 Insulin Apolipoprotein a1

information about the structure of the molecule, and unknown compounds can be identified based on comparison to standard library spectra. TOF analyzers detect ions based on the time it takes for them to travel through a flight tube, with smaller ions traveling faster than larger ions, resulting in high mass accuracy. Both ion trap and TOF MS systems have been primarily used qualitatively for applications that require compound identification, protein sequencing, or broad spectrum drug screening [6,18,19,47–52]. The quantitative tandem MS workhorse in a clinical MS laboratory is LC-MS/MS [22,23], and a detailed description of how it works, including all aspects of sample analysis, follows below. 3. Sample preparation Clinical laboratory testing is performed on biological fluids – matrices that are not amenable to direct analysis by LC-MS/MS and which must first be treated to make them suitable for analysis (Fig. 1). Sample preparation is a critical aspect of the LC-MS/MS method, and has a number of purposes: - isolate the analyte of interest from the biological sample and place it in an LC-MS/MS compatible matrix, - concentrate or dilute the analyte of interest, depending on the detection limit of the LC-MS/MS system being used, and - separate the analyte from matrix components and other compounds that could interfere with the measurement [4,17,53–55]. For each analyte, multiple options must be considered and validated depending on the properties of the analyte itself and the

Table 1 Advantages of LC-MS/MS over immunoassay in the clinical laboratory.

New Assay Development Accuracy, selectivity and specificity Multiplexing Cost-per-test (consumables)

LC-MS/MS

Immunoassay

In house, limited by instrument capability and trained staff Excellent – direct measurement of analytes and wide dynamic range Can analyze many compounds in a single run. Can be very low ($1s/test)

Limited to Manufacturer Test Menu. Can be very poor (depending on the analyte measured) due to interferences and phenomena such as antigen excess. Typically can only analyze one compound at a time. Typically mid to high ($10s+/test)

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interfere with accurate measurement of the analyte (Fig. 2). The possible combinations of mobile phase, stationary phase, and gradient program are extensive, and require optimization to obtain the best possible separation and peak shape for the analyte of interest in order to deliver a reliable and robust analytical test [38,56–59]. 5. Triple quadrupole mass spectrometry

Fig. 1. Flow diagram showing steps required for LC-MS/MS analysis.

matrix within which it resides [4,17,38,54–56]. Sample preparation procedures are varied and available options range from simple (i.e., protein precipitation) to complex (i.e., solid phase extraction). Simpler procedures are appealing from the perspective of technologist time, workload and cost, but assay robustness should be considered when determining the best sample preparation protocol [54]. Selectivity of the LC-MS/MS assay can be enhanced by using sample preparation procedures which are orthogonal to the liquid chromatography portion of the assay. For example, using an ion exchange solid phase extraction for sample preparation with reversed phase LC [54–57].

4. Liquid chromatography Following sample preparation, the sample extract is introduced into the liquid chromatography portion of the LC-MS/MS system (Fig. 1). Liquid chromatography (LC) is comprised of two phases – the moving, or mobile, liquid phase, and the solid stationary phase. The sample extract is injected into the mobile phase flow – a mixture of water and organic solvent – which is being pumped into an analytical column. The column contains the stationary phase – it is a small tube tightly packed with coated silica particles used to separate components of the extract [58,59]. Based on the affinity of each compound for the column’s stationary phase, analytes will ‘stick’ until a change in the mobile phase composition creates conditions that release the analyte into the flow of the mobile phase and, subsequently, into the mass spectrometer. LC allows the analyte to be concentrated on the column, thereby enhancing the signal in the mass spectrometer. Further, LC is used to separate the analyte from co-extracted compounds that could

Mobile phase flowing from the LC enters the mass spectrometer’s ion source through a narrow capillary tube where the components in the LC flow are ionized. Ionization is critical as only charged particles will be detected in the mass spectrometer. There are a number of ionization techniques currently used with LC-MS/ MS, including atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) [51,52,60]. ESI, first used effectively in the 1980’s, is the most common ionization technique in the clinical laboratory as it can be used for a wide variety of both small and large molecules, due to its ability to form multiple charges [11,18,60–62]. ESI occurs in 3 major steps: first, voltage is applied to the capillary as the mobile phase enters the source chamber, and Coulomb forces cause the charged liquid to disperse into a fine spray of charged droplets [11,12,60–64]. Second, application of heat and gas in the source rapidly evaporates the solvent, causing the droplets to shrink and increasing the density of charge on the surface of the droplets. Third, gas phase ions are produced from the highly charged droplets [5,11,12,60–64]. The exact mechanism for ion production in this final phase of ESI is not fully understood, but is thought to happen either by ion evaporation model, or charged residue model (Fig. 3) [11,12,38,60,62,63]. The ions generated in the source are drawn into the mass spectrometer via vacuum pressure, subsequently focused and accelerated through a transfer region and into the first quadrupole, Q1. Intact molecular weight ions, commonly called precursor ions, are selected in Q1 and sent to the collision cell, sometimes referred to as the second quadrupole, Q2, where they are collided with high energy gas and, thereby, fragmented. These fragment, or product ions then pass to the third quadrupole, Q3, where they are selectively sent to the detector [5,26,65]. Ions are drawn through the quadrupoles via a vacuum gradient, and ions oscillate within the quadrupoles by application of RF and DC voltages to opposite pairs of rods (Fig. 4). Molecules that do not have a stable trajectory with these set voltages are expelled from the quadrupole (Fig. 4) [6,65]. The signal obtained at the detector of the product ion selected in Q3, generated from a specific precursor ion selected in Q1, is commonly referred to as a ‘mass transition’, and the experiment is referred to as selected reaction monitoring, ‘SRM’, or multiple reaction monitoring, ‘MRM’. Modern

Fig. 2. Representation of liquid chromatography. Three different compounds (represented by blue, green and red) are loaded onto the column with the mobile phase. The green compound has the least affinity for the stationary phase, and elutes first, followed by the red compound and finally the blue compound, which has the most affinity for the stationary phase.

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6. Quantitative analysis

Fig. 3. Ion formation during electrospray ionization. The ion evaporation model is shown on the left, and the charged residue modeled on the right. Figure has been re-constructed based on Fig. 1 in Adaway et al. [38].

Fig. 4. Representation of a quadrupole. Only ions oscillating between the quadrupole rods that have stable trajectories with optimized RF and DC voltages applied travel through the quadrupole (green path), while ions with unstable trajectories (red path) are expelled from the quadrupole [6,65].

mass spectrometers can scan for, and detect, many mass transitions in a single run, meaning that many analytes can be detected in a single sample extract (Fig. 5) [4,10,22,24,32].

The final output generated by the combined LC-MS/MS experiments is a chromatogram (Figs. 5 and 6) containing the analyte peaks. Multiple analytes detected in the same sample will generate multiple, overlaid chromatographic peaks (Figs. 5 and 6). The area of each peak is the representation of the response, or signal, for that specific mass transition detected by the mass spectrometer. Analyte peak area is proportional to the concentration of analyte in the sample [5]. However, due to the nature of ESI, strict proportionality, which is ideal, is not necessarily achieved. This is because the charge available during the ESI process is finite, and co-extracted molecules in the sample extract can ‘steal’ the available charge, resulting in reduction of ionization of the analyte of interest, a phenomenon referred to as ‘ion suppression’ [17,51,66–68]. One of the approaches employed to compensate for ESI variability and ion suppression is the use of internal standards (IS) [5,24,50,66,67]. Internal standards are compounds that are physically and chemically similar to the analyte of interest. These are preferably stable isotope labeled (SIL) compounds, typically prepared using deuterium (2H), carbon-13 (13C) or Nitrogen-15 (15N) labels. SIL compounds have near-identical chemical and physical properties as the analyte of interest, and thus behave very similarly during sample preparation, liquid chromatography, and in the ion source [5,16,17,24,67]. The SIL IS will co-elute with the un-labeled analyte of interest, but because of its different mass due to isotopic labeling, it is distinguishable from the un-labeled analyte in the mass spectrometer (Fig. 7) [5,16,24,67]. Care should be taken when choosing deuterium (D) labeled ISs. Deuterium-hydrogen exchange is possible [56,69], and when a high number of Ds are present in the IS, the IS may not sufficiently correct for ion suppression if there are resulting differences in LC retention between the IS and the analyte [18,56,57]. 15N and 13C labeled compounds do not suffer from these stability or retention time issues, and are preferable, but are generally more expensive and not as widely available as deuterated compounds [18,69,70]. When a SIL IS is not available, a structural analog of the analyte of interest may be used, although this is a less ideal approach [28,56,57]. Internal standards are added at the same volume and concentration to each standard, quality control, and patient sample at or near the start of the sample preparation process. The IS is used

Fig. 5. Chromatogram of Immunologlobulin G subclasses assay: 5 different peptides corresponding to IgG1, IgG2, IgG3, IgG4 and total IgG are detected with 2–3 MRMs acquired for each peptide, plus MRMs for internal standards for each different peptide. A total of 18 MRMs are acquired by the mass spectrometer for each sample extract in this method.

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Fig. 6. Chromatogram of Testosterone from a patient specimen. Time or chromatographic information on the x-axis: Testosterone retention time is 4.08 min. Mass Spectra or detector information on the y-axis: Signal for the Testosterone MRMs is 2.3e4 counts-per-second. Here, two separate MRMs (one blue, on red) are acquired to confirm detected peaks in the patient samples are testosterone.

Fig. 7. Top pane shows chromatogram of testosterone analyte MRM of 289.3 ? 97.2. Bottom pane shows co-eluting Testosterone-d3 IS MRM of 291.3 ? 97.2. The retention time of the analyte and IS are the same, but they are distinguishable due to their different masses and, therefore, different m/z ratios in the mass spectrometer.

to correct for variances during the entire analytical process, including sample preparation, LC separation and MS analysis. By correcting for variances, the IS enables accurate quantitation [28,56,57,70]. The standard approach for LC-MS/MS quantitation is by using the peak area ratio (PAR), which is the ratio of the analyte peak area to internal standard peak area [57]. The concentration of analyte in any given sample is determined by comparing the PAR of the sample to a calibration curve, which is constructed from the PARs of standard samples and their known concentrations (Fig. 8). Therefore, preparing or obtaining accurate standard samples is critical to the accurate quantitation of analyte(s) in patient specimens [56,57,67,74]. Assessing calibration accuracy can be performed using a number of hierarchical approaches described in various guidelines and publications [24,28,47,48,70–72,74].

7. Challenges LC-MS/MS is a complicated technique, and requires trained and experienced staff to develop, validate, implement and maintain clinical assays on this platform [17,28,30,56–58,73]. Clinical LCMS/MS assays are often lab-developed tests, and method development and validation are extensive undertakings [25,28,48,49,57]. Although capable of being selective and accurate, LC-MS/MS methods must be appropriately developed to take advantage of the benefits that this technology offers [40,56]. For example, ion suppression must be mitigated if demonstrated to be problematic; isobaric compounds must be chromatographically separated in order to be accurately quantified, as they cannot be distinguished in the mass spectrometer [16,24]; and calibration accuracy must be assessed, corrected if necessary, and maintained [70,74].

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Fig. 8. Calibration curve generated using peak area ratios from standard samples. The Analyte concentration in patient samples is determined by input of the sample peak area ratio into the regression equation generate from the calibration curve. X-axis is analyte concentration; y-axis is the analyte peak area ratio (analyte peak area/internal standard peak area).

Care must also be taken when analyzing data, and controls should be in place to minimize human error. LC-MS/MS vendor software typically has a quantitation module whereby peak integration, calibration curve construction and calculation of sample analyte concentrations is performed automatically. However, there exists no perfect software for peak integration, and depending on chromatographic quality, visual inspection of the chromatographic peaks is still required. This can take valuable technologist time, and manual changes to integration parameters are subjective and should be avoided [67,74]. There are ongoing efforts by laboratories and vendors to improve automation of data analysis workflows such as using ‘‘review by exception” based on acceptance criteria rules, or utilizing third party software solutions [73]. Modern IA platforms are random access, highly automated systems requiring minimal user interaction. LC-MS/MS procedures, however, typically require significant hands-on manual intervention, particularly during the sample preparation process where samples are usually prepared in batches. Automated liquid handlers (ALH) are a viable option for improving throughput and increasing capacity, and decreasing the amount of hands-on time required during sample preparation. It should be noted that the required programming can be challenging, and, in the experience of the author, these are not walk-away systems. An alternative automation approach is on-line solid phase extraction (SPE), sometimes referred to as on-line sample preparation. This requires additional pumps and LC equipment, and often an initial sample preparation step offline for sample dilution or protein precipitation is still necessary. Since the LC system is more complex, there are added technical challenges involved with online extraction [54,75]. There are some vendors that provide improved automation by directly connecting an ALH with the LC [54].

8. Future directions Efforts are underway by major instrument manufacturers, to develop and produce mass spectrometry platforms intended to bring the ease of use of mass spectrometry-based systems to the level currently enjoyed by standard auto-analyzers [76–78]. Some fully automated LC-MS/MS platforms will be closed systems, with a limited, set test menu, with new methods developed by the man-

ufacturer [76,77]. Technologists used to the random access plugand-play IA platforms will require little training to operate these LC-MS/MS platforms. Other systems are modular, with the option of user-developed tests [78]. There are other ongoing developments and advancements in clinical mass spectrometry that have been recently summarized by Yang and Herold [47]. 9. Conclusion The combination of sample preparation, liquid chromatography, and tandem mass spectrometry makes LC-MS/MS a highly selective analytical technique for quantitative analysis (Fig. 1). Although it is a complex technique and has its challenges, the ability to generate accurate patient results has made it a valuable addition to the clinical diagnostic laboratory. As this technology continues to advance, it will continue to be a critical tool for providing accurate patient results and improving patient care. 10. Recommended reading and learning resources 1. Nair H, Clarke W (Eds.) Mass Spectrometry for the Clinical Laboratory. 1st ed. Elsevier, London, 2017. 2. Rifai N, Horvath AR and Wittwer CT (Eds.) Principles and Applications of Clinical Mass Spectrometry. Elsevier, London, 2018. 3. Mass Spectrometry Applications for the Clinical Laboratory (MSACL) – online learning centre: https://www.msacl.org/index.php?header=Learning_Center.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] D. Chace, Mass spectrometry in the clinical laboratory, Chem. Rev. 101 (2001) 445–477, https://doi.org/10.1021/cr99007+. [2] A. Wu, A selected history and future of immunoassay development and applications in clinical chemistry, Clin. Chim. Acta 369 (2006) 119–124, https://doi.org/10.1016/j.cca.2006.02.04.

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