Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an impressively adaptable analytical technology that provides significant benefit in clinical analysis [1]. LC-MS/MS has facilitated improvements in quality and throughput for numerous diagnostic assays, including steroid, amino acid, vitamin, peptide, protein, neurotransmitter, cancer biomarker, therapeutic drug monitoring, and toxicological assays [2]. There are numerous publications on novel approaches and assays deployed in the clinical environment, particularly focusing on development and the validation of outcomes. While these articles are of interest to clinicians and laboratory staff, there is a lack of publications on the operational lifespan of LC-MS/MS in the clinic.

Many laboratory processes are applicable to all technologies used in clinical analysis. Activities related to pre-analytical observations, documentation, or electronic medical records apply to the laboratory. Among guidance and best practice documents, resources are similarly broad, with some notable exceptions focused on LC-MS/MS, which are addressed further in this review. I will discuss components of validation and operation specific to LC-MS/MS, with a focus on those aspects of the technology that are true differentiators in clinical analysis.

This review is a follow-up of a previous one [3]. Together, these publications intend to capture the lifecycle of LC-MS/MS from development through validation to the execution and maintenance of a clinical assay. Despite best efforts, no mechanism exists to encapsulate the specifics of individual assays, laboratory setups, or procedures. However, the principles discussed herein are broadly applicable to the processes, experiments, and protocols used to generate high-quality data from clinical LC-MS/MS assays. Readers should note that sections are commonly cross-referenced within this review, reflecting the nature of LC-MS/MS workflows in that practices occur in parallel. For example, data review during calibration verification is a nested process; each subsection represents part of a whole of clinical LC-MS/MS analysis and should be read as such.

Validation has several colloquial meanings, but the International Organization for Standardization (ISO) defines validation as “confirmation, through the provision of objective evidence, that the requirements for a specific intended use or application have been fulfilled” [4]. Regardless of the technology, the validation of quantitative lab tests must demonstrate accuracy, precision, stability, analytical specificity, linearity, and lack of interferences [5]. To support the claims of intended use of LC-MS/MS assay results, the list of validation experiments can be long. Literature reports vary widely in how validation experiments are to be executed. Demonstration of all experimental outcomes of validation may not be necessary to support the hypothesis in a publication. Alternatively, word count limits may constrain exhaustive descriptions of validation assays in reports. Consequently, critical experiments for LC-MS/MS validation may not be fully disclosed in the numerous reports.

Resources for validation guidance are issued by multiple institutions. The US Food and Drug Administration (FDA) Bioanalytical Method Validation (BMV) guidelines prescribe drug development assays focused on MS analysis [6]. In the most recent version of the BMV, specific expectations and guidance on how to address certain endogenous biomarker concerns, such as the use of stripped matrices and commutability, are further clarified [7]. However, the FDA BMV is not designed for diagnostic laboratories and has discrete principles and practices (e.g., incurred sample re-analysis experiments) suited for different purposes. Nevertheless, many experiments utilized for the validation of clinical diagnostic assays are mirrored in these guidelines.

An entire chapter of the Tietz Textbook of Clinical Chemistry is dedicated to a distillation of the FDA BMV, CLSI guidelines, and general best practices for the execution of LC-MS/MS validation [8]. It prescribes batch sizes, replicates, designs, and timeframes for validation experiments. Furthermore, a review of experiments and acceptance criteria has been recently published [9]. As experimental designs, timeframes, and degrees of replication have been addressed elsewhere, this review focuses on the more enigmatic components of LC-MS/MS validation for diagnostic testing.

With a validated method in hand, the laboratory is not yet prepared to test patient samples. Prior to the launch of a new assay, assay translation must be addressed, particularly if the research/development/validation technicians are not actively working in routine patient testing. In a sense, the research/development/validation technicians can be treated as a distinct entity from the operations team, representing two different laboratories. Protocols for inter-laboratory method transfers have been outlined in the context of clinical trials or pharmaceutical analysis and provide important recommendations [42, 43]. These experimental components can also be utilized to satisfy regulatory requirements, such as competency evaluations for staff performing the tests, if the laboratory is subject to such obligations [4, 44].

In method transfer/competency, aspects beyond the extraction/analysis protocol should be assessed. All components of the procedure prepared in the testing facility benefit from this exercise, even if the performing staff are highly skilled and competent. This is because of the concepts of oral transmission of laboratory skills or expectations for integrated knowledge, which may not be translated in a standard operating procedure (SOP). The Aims and Scope of the Current Protocols series from Wiley present this point quite well: “nuances that are critical for an experiment’s success are not captured in the primary literature but exist only as part of a lab’s oral tradition” [45]. This may hold true for SOPs for assays that have not been operationally stressed or attempted by multiple technicians, who each have their own interpretation of written and unwritten laboratory processes.

The SOP must be revised during the transfer process to ensure that sufficient details are included, even if that knowledge is considered extant in the laboratory. Quality system expectations have recently included specific sections addressing knowledge management to ensure that critical information is retained both on paper and within the collective knowledge of the staff [46, 47]. An exercise may be performed wherein staff are asked to perform the assay as described explicitly by the SOP; each question asked by the staff during this process may represent SOP language worth expansion or clarification.

The competency assessment and transfer process should be structured to include critical reagent changes, new calibration material correlation, new quality control (QC) range generation, and patient sample correlation. This effort is similar to lot-to-lot verification, which is discussed in detail below. Broadly, scientists/technicians, calibrators, QCs, and instrumentation involved in validation would be used to compare the laboratory’s normal manufacturing and operational components. Patient samples are to be assayed in parallel with validation materials and samples freshly prepared in the operational environment (within the stability timeframe of the validation materials). Acceptance criteria include standard assay evaluations for batch acceptance, including clean blank samples and transition ratios within tolerance. Comparison of the results for the validation materials and operational samples can be expressed as imprecision/inaccuracy on a per-sample or all-group basis or be based on regression of the dataset (preferably, Deming or Passing–Bablok regression) and review of the slope and correlation [48]. Expectations for these comparisons should be in line with those defined in the assay validation, associated with state-of-the-art criteria or the BV of the measurand.

These data can also be utilized for the validation of laboratory information system (LIS) integration of a new workflow. Analyte identifiers, reference intervals, and reporting units (including decimal points and rounding) are all LIS features to be evaluated for new assay launches. Assay-specific determinations in the LIS, such as pertinent clinical history, scheduling of dilutions, modifications/calculations associated with the test result (e.g., creatinine normalization), and alert values, must be verified during the pre-launch phase [49]. These data should be reviewed and confirmed in a test environment prior to assay deployment.

General laboratory concerns, such as facility maintenance and the inspection of patient samples for pre-analytical deviation, are consistent for all laboratory assays and are therefore not discussed here, apart from mentioning that maintaining a significant quantity of reagents (within the expiry dates) is particularly essential for LC-MS/MS assays. It is not uncommon to have back-orders or material shortages of the many supplies required for testing, such as the global reduction of acetonitrile experienced between 2008 and 2011 [50, 51]. A sufficient supply allows the time required to source, evaluate, and validate necessary modifications to the method when a shortage becomes imminent. Thorough assay validations can indicate the viability of alternative critical reagents where appropriate [52].

Validation should also identify acceptable parameters for the assay to be run at scale within the laboratory. These include the maximum batch size (numbers of samples included in a single run), the frequency of calibration curves, and the number of samples between QCs. In clinical laboratories, it is common to prepare patient samples for LC-MS/MS in parallel, utilizing 96-well plates, multichannel pipettes, and multi-head liquid handlers [53]. However, validation may have been performed using different volumes from those used in the operational assay; in such cases, the laboratory has to determine the standard/QC frequency after the assay has been launched. Often, 96-well plate assays include calibration curve standards at the beginning of each plate and either reanalyze the standards or prepare another set of calibrators at the end of each plate (batch-calibration mode) [54-56]. Less frequent calibration is demonstrated for certain assays [57]. Careful evaluation of the instrument response drift and the implementation of re-calibration/maintenance when QCs fail are essential to realize the gains from non-batch-based calibration. The placement of calibration curve standards at defined intervals (e.g., batches, numbers of samples) allows for the prompt identification and remediation of issues with the assay. Additionally, consistency in calibration placement can reduce the burden on technical staff by providing a reliable expectation of the standard curve frequency. This allows for simple decision-making flow charts when a calibration curve fails to meet certain criteria.

QCs are fundamental to laboratory testing and applied to all types of assays. LC-MS/MS poses specific challenges in terms of QC. Some historical context is appropriate. The widely adopted approach of applying standard deviations to the results to monitor assay performance was originally discussed in 1950 [58]. In 1952, the concept was expanded with the adoption of a patient pool as QC material [59]. The treatment of QC results as used in current practice was published in 1977, 8 years before the first graphing calculator became commercially available [60, 61]. This means that all calculations (means, standard deviations) were likely performed manually using standard statistical tables. The multi-rule approach as described by the CLSI (i.e., 1_3S, 2_2S, R_4S, 4_IS, 10_X determinations) is deployed in numerous clinical tests [62]. These QC treatments are representative of single-measurand assessments, whereas LC-MS/MS can measure multiple compounds in a single sample.

Multiplexed measurement is not uncommon in LC-MS/MS assays for diagnostic purposes. Numerous molecules can be measured in a single sample, regardless of their biological or clinical co-relevance. Amino acids and acyl carnitines by flow injection MS/MS, steroid panels, drug evaluations, neurotransmitter assays, and assays of classes of proteins are all multiplexed LC-MS/MS assays in clinical use [63-67]. If one concedes that statistical rates of randomness used in QC evaluations apply to LC-MS/MS assays, any analysis that measures more than 6–9 molecules presents great difficulties [68]. Multi analyte assays face challenges of compounding randomness (imprecision) that single-analyte assays do not [69, 70]. To address this, there are some practical approaches that can be considered when measuring multiple components from a single QC. These approaches consider the analytical control afforded by isotopically labeled ISs and the richness of LC-MS/MS data applicable to root-cause analysis of errors.

First, we will explore the features of QCs as a means of accepting a batch. A pass/fail status can be applied as a static bias from the expected concentration (measured or theoretical), which is typically 15% for non-LLOQ concentrations and 20% at or near the LLOQ, rather than being assessed from standard deviations from the mean [6, 32]. The establishment of expected concentrations is discussed in “Assay Maintenance” section. Precision is poorly accounted for as each QC analyte is assessed for inaccuracy in an acute manner. Longitudinal evaluations serve to assess drift in concentrations and, if warranted, shift the expected mean. However, consistent drift observed across numerous calibration events for all QCs may indicate a change in calibration or QC concentrations. Such drift must be investigated prior to modifying the expected QC concentration(s). If a QC is determined to fail due to gross error (e.g., undefined concentration because of no IS response), the data should not be included in the longitudinal dataset; all other data should be included. Broader or tighter criteria can be applied based on total allowable error for the molecule(s) of interest, although this would remain an acute determination for each batch [71]. Again, consideration of the number of co-measured molecules is necessary and may result in expansion of the acceptance criteria.

One approach to multiple-analyte QC has been to move state-of-the-art acceptance conditions from a 15% maximum inaccuracy to a 30% maximum inaccuracy [72]. A 30% error in urinary drug levels is typically of little consequence as the intended use is the detection of compounds; however, for assays where quantitation is clinically meaningful, such allowable inaccuracy may be inappropriate. Discussion with appropriate clinical groups responsible for interpreting the results of multiplexed analyses can provide insights into the required degree of reproducibility.

QC results may also be interpreted in the context of the results of other samples in the batch. For example, in a multi-analyte drug assay, a high-concentration QC fails for one drug of interest by measuring more than 115% of the expected value, but just outside of the acceptance range. Two lower-concentration QCs pass for the same analyte. All IS responses for the single analyte are within 20% of the mean of the calibrators for each sample. No sample in the batch produces a signal for the analyte of interest. In this case, there not necessarily is batch failure, and re-analysis is not required given that the low recovery is acceptable and the high-concentration QC failure does not indicate an error that would lead to false-negative results. The context of pass/fail assessments based on QC results in LC-MS/MS should be assay-dependent and leverage the capabilities of the platform for accurate reporting [73]. Given the analytical detail provided in LC-MS/MS, QCs can serve many purposes. The underlying role of QC materials in evaluating drift and error in an assay is key to any approach and must be considered as the minimal framework for QC result interpretation [62].

The frequency of QC analysis for LC-MS/MS analysis depends on numerous factors. Under the Clinical Laboratory Improvement Act (CLIA) issued in the US, a minimum of two liquid levels of QCs must be assayed every 24 hours of analysis for any technology [74]. However, such a QC scheme may underserve LC-MS/MS assays in error detection. Variation in absolute recovery can occur between batches of samples and response function drift is a recognized LC-MS/MS feature. While validation exercises can guide QC limits due to assay calibration drift (distinct from instrument mass calibration drift), other aspects of the laboratory work flow must be considered for risk [75]. For example, solid phase extraction (SPE) can use various versions of manifolds for performing both tube-based and plate-based SPE. The manifolds can hold 8, 12, 16, 24, or even 48 tubes; plate manifolds are often constructed for 96 or 384 wells. As a QC check against variable recovery due to a difference in manifold settings (e.g., excessive vacuum applied at sample loading), at least one QC/manifold/run is recommended in a tube-based SPE protocol. Plate-based SPE (96 sample capacity) would benefit from multiple levels within a single plate [76]. Similarly, batches of manual liquid-liquid extraction analyses would include QCs based on the number of tubes that can be assayed in a contiguous manner as determined by the space available in an evaporation device.

Given the breadth and variety of meanings, a characterization of the term “quality assurance” (QA) is appropriate prior to a more detailed discussion. The International Union of Pure and Applied Chemistry (IUPAC) definition is perhaps most closely tied to our intention, in that QA is designed to protect against the failures of QC [77]. QA should also provide mechanisms to ensure the veracity of not just QCs, but all samples assayed. This encompasses blanks, standards, QCs and, most importantly, patient samples.

QA can be partitioned into evaluations of meaningful pre-analytical, analytical, and post-analytical metrics. These are included in the quality management section of the College of American Pathologist’s all Laboratory Common Checklist [78]. Each evaluation relies on distinct elements to determine whether a system is executing the measurement as intended. All elements should be considered in the context of successful and reliable sample analysis, with identified failures initiating investigation and remediation.

LC-MS/MS assay components are consumables. They include pipette tips, QC solutions, and extraction materials as well as chromatography columns, ionization electrodes, MS source heaters and, to a far lesser extent, the instruments themselves. Each constituent will eventually require replacement. The assay volume may also increase beyond the laboratory’s current capacity, requiring the purchase of new equipment. Patient samples should not be tested before the performance of the new material/equipment has been verified. This is referred to as “lot-to-lot verification” or “between-lot verification.” Guidelines and published papers have reported procedures for performing these evaluations [129, 130]. Guidelines recommend that previously analyzed samples be re-assayed using the new material and the results compared [108]. However, this approach may not be appropriate for LC-MS/MS assays. For example, an analytical column has been degrading over multiple days, leading to failure of separation. In comparison with a new column, a previous batch assayed with the degraded column shows a bias in sample results. It may be difficult to ascertain whether that bias is due to the new column performing poorly or whether the old column is the source of error. Additional variables, such as analyte stability, batch-to-batch calibration variance, and even technical staff differences, can affect comparative outcomes. SST injections may be considered to provide excellent evidence for LC-MS/MS column performance, particularly if known isobaric species are included in the SST.

Lot-to-lot variation studies for each material being replaced should be considered for associated risk, variables, and necessary evidence to provide confidence in the new component. Instrument-based materials, such as new mobile phases, columns, or source electrodes, may be accepted by the use of SST and blank injections. Extraction materials, such SPE stationary media or LLE solvents, may require a more thorough evaluation of a new lot. As a thought experiment, the closer a component is to the actual source of the mass spectrometer, the less likely it is to cause issues with individual samples. Issues proximal to the mass spectrometer may cause errors in all samples, including calibrators and QCs, thus rendering the assay “out of control.” In that sense, simplified assessments via SSTs are used in some instances, whereas sample-based verifications are preferred in others, as shown in Table 7.

Perhaps the most critical materials in LC-MS/MS assays are calibration standards used to generate a calibration curve. On a practical level, the entire LC-MS/MS procedure has no intrinsic accuracy requirement, but rather, within-batch precision is the goal [8]. For example, an imaginary assay SOP states that “50 µL of sample, calibrator, QC, or blank shall be aliquoted to each well of a 96-well plate.” If a technician inadvertently aliquots 55 µL for each of the sample types, this will not result in a 10% bias in the data. The calibrators and samples will have been aliquoted at a precise volume within that batch. Assay accuracy is thus derived from the accuracy of the concentration(s) of the measurand(s) in the calibration materials and the precision (relative to treatment of calibration solutions) of each analytical step in the procedure. Further, when isotopically labeled ISs are used, the only requirements are precision of the sample and IS solution addition—such is the power of LC-MS/MS. However, in instances of poor calibration accuracy, indications of failure are limited. These can include requested follow-up testing to resolve discrepancies between test results and patient presentation, inquiries from clinicians, or a notification of a proficiency test result failure.

New calibration materials in the laboratory must be prepared with exceptional care. Close surveillance of expiry or depletion of a current lot of standards allows for adequate time to prepare, subaliquot, verify and, if necessary, correct a newly prepared lot. At least two weeks prior to the conclusion of the current lot’s utility is the minimum time necessary for new standard preparation, especially for tracing and resolving errors determined in verification. More challenging calibration systems (i.e., those with multiple components) benefit from longer lead times.

The collection and analysis of the blank matrix is a critical first step. The exact matrix type, quality, and manufacturer should be determined in validation and published in the assay SOP [131]. In case of deviations from the validation-established matrix due to availability issues or a change in the preferred vendor, the matrix must be re-validated prior to use [6, 8, 9]. This is particularly important in endogenous assays using a depleted or stripped matrix. Distinct vendors may utilize discrete procedures or sampled populations (e.g., gender, age, geographic location of donors) to manufacture stripped matrices [121]. Pre-screening of the blank matrix should include a comparison of the response function of extracted blanks with the current standard lot LLOQ. Any observed response feature of the analyte(s) at the appropriate retention time represents a contamination. An acceptable level of 20% of the LLOQ signal (integrated peak area, not the calculated concentration) is consistent with guidelines [6, 8]. Unacceptable blank matrix contamination can be managed by three means. Accurate assessment of the concentration via standard addition can be used to adjust stock fortification volumes to achieve accuracy of the expected concentrations [132]. Dilution of the contaminated matrix can be performed until contribution to the analyte(s) is no longer observed, although this should be limited to preserve commutability. Lastly, the contaminated matrix can be returned to the vendor, replaced with a new lot, and retested.

Other essential materials include Class A volumetric flasks and pipettes, a calibrated mass balance, and vessels for sub-aliquoting to assay-specific volumes. The latter component infers that bulk calibration materials are prepared, apportioned, and stored at useful volumes (i.e., one aliquot per batch/shift/day as stability allows). This is distinct from freshly preparing calibration solutions with each batch of standards by spiking at the laboratory bench. If an analyte is stable in matrix, long-term storage of bulk preparations decreases labor and removes the possibility of variation/error from repeat calibration preparation.

Stock solutions for fortification can be prepared in-house from lyophilized materials using gravimetry and dissolution. Alternatively, a solution at a known concentration can be purchased and used for fortification. In either case, review of the certificate of analysis is required to capture the impurities/salts and adjust analyte concentration. If the laboratory has access to appropriate equipment, assessment of the concentration of solutions using alternative methods (e.g., spectrophotometry) prior to fortification can provide confidence in the concentration [133]. Spiking of the fortification solutions must follow some recognized rules. First, Class A glassware is preferred over other means of liquid transfer, especially air-displacement pipettes. Air-displacement volumes are calibrated to water; if organic stock solutions are used in standard preparation, there will be a bias in the final concentration due to density differences [134]. Pipettes must be calibrated to the density of the solution utilized or the delivery volume modified to account for different solutions used in preparation. Efficient preparation and prompt subaliquoting and storage is recommended.

The verification of new calibration solutions has no defined path in regulatory guidelines, but method comparison can be a useful framework [135]. Calibration verification is broadly described as experimentally demonstrating that materials of known concentrations, measured as patient samples, produce the expected concentration [136]. For commercially available test systems, calibration materials are validated by the manufacturer prior to release; only verification of those concentrations is required by the laboratory. In contrast, the laboratory is the manufacturer of most LC-MS/MS tests and abbreviated calibration verification experiments may be insufficient to identify errors in standard curve preparation.

Calibration verification for new in-house prepared materials should include assessment of their inaccuracy and imprecision. When possible, a demonstration of trueness by comparison with a certified reference material (CRM) or a reference method procedure is encouraged [108]. Proficiency test (PT) samples with only peer-group mean values can be used in the certification of new standard concentrations, as long as that material is commutable and within expiry. The analysis sequence should be designed to account for LC-MS/MS-specific variables, such as longitudinal calibration drift. A direct correlation of calibration lots can be performed in a single batch, preferably with at least triplicates of the new lot of calibrators. These must be measured as “unknowns” to not influence the curve regression. The precision of the triplicates should be within the expectation for the assay; deviations require re-preparation of the batch or re-analysis. If the mean of the new calibrators falls within the accepted bias of the assay, additional batches can be prepared using patient samples for result comparison. In batch-calibration mode, a current calibration curve at the beginning of the batch and one for the new lot of standards at the end of the batch is acceptable. Two separate standard curve regressions are used to independently calculate the patient concentrations. Bias plotted by sample index can be used to observe any drift in detection within the batch, leading to instrument maintenance and re-analysis. Multiple batches should be performed (n≥3) with samples containing concentrations across the AMR; Deming or Passing–Bablok regression and a Bland–Altman data plot are useful data reduction techniques. The actual choice of regression technique is up to the laboratory, although as suggested by Westgard, acceptance by both models is a valid criterion [137]. The number of samples and the distribution of their results should be rationalized per assay [138]. Efforts should be made to incorporate samples spanning the AMR as is reasonably possible without unduly influencing the regression [139]. Fortification or dilution of samples may be necessary if a high percentage of samples are in the low or high end, respectively. Acceptance criteria are consistent with method comparisons (e.g., Deming slope between 0.9 and 1.1, correlation coefficient >0.98) and Bland–Altman plots should indicate random bias around the unity line. In cases where a minimum bias differs from the state of the art, criteria are narrowed down to match the expectations for allowable bias. Note that intercept review is generally not indicated as interpretation can be muddled by the dynamic range of the assay; intercept interpretation should be done carefully [140]. Any CRMs or PT samples must back-calculate within the tolerances allowed by the assay or the material provider. Values for QCs measured against the new lot of standards should agree with the previously determined mean within the expected variance for each concentration.

New lots of calibration material must be reviewed longitudinally against previous lots [141]. Comparison of the back-calculated bias across multiple lots can indicate a persistent trend (positive or negative). In the case of a constant negative bias, the stock material should be assessed for stability or, in the case of lyophilized materials used consecutively, hydration of the material over time. Multiple positive biases across lots may indicate instability of the previous lot of calibrators used as standards in the comparison. In this case, the re-assessment of calibrator stability is indicated.

In some unfortunate instances, calibration curve verification fails. Root-cause analysis may indicate the source of the issue, but due to time constraints, a new preparation and verification may not be possible. The transformation of calibration curves (modification of theoretical values) should be performed cautiously. Significant deviations from the AMR, particularly at the LLOQ or ULOQ, are not allowed; such changes must be communicated to assay end-users to determine their effect on clinical decisions. Multiple replicates of the new lot should be assessed against known materials to establish the accurate concentration (n≥10). External reference samples (e.g., CRMs, PTs, external quality assessment [EQAS] materials) should be measured in several batches, using independent calibration with the new lot in each batch, to determine the effect of the changes on reported values. Rapid send-out of those calibration materials to a reference laboratory is recommended after confirmation that the materials are commutable with the external method. If transformed calibrators are implemented, the results from real patient samples must be closely monitored for unexpected shifts in the expected values or reference intervals.

The preparation of new lots of QC is similar to that of new standard curve materials in terms of planning and materials. The manufacturing of QCs should be temporally distinct from that of calibrators [141]. Bridging QCs between calibrator lots prevents the possibility of running out of both standards and QCs at the same time. Separate preparation of new standard and new QC lots also affords uninterrupted within-laboratory traceability to the originally validated method. Separate stock solutions should be utilized in calibrator and QC preparation [142]. Distinct stocks can capture errors in the concentration of a material that may be concealed had the calibrator and QC lots been prepared simultaneously from the same stock solution(s).

For each QC, exogenous analytes should be fortified to known concentrations into authentic matrix [6]. Similar to calibration materials, the blank matrix should be verified prior to fortification. When assayed, concentrations should back-calculate to the expected, allowing for imprecision. Deviations in the accuracy of the QCs may indicate a process error (manufacturing) or a calibration error. For non-commercially available exogenous molecules with specific features, collecting patient samples known to be positive for the analyte(s) and assessing the mean is appropriate. These instances include molecular species that are not easily synthesized, such as rare drug conjugates that hydrolyze prior to LC-MS/MS or biologically modified compounds [143].

Endogenous measurand QCs can be prepared by fortification into stripped matrix or generated as a pool of authentic samples. These two types of QCs can also be combined to provide for checks against accuracy (fortified stripped matrix) and matrix effects (authentic pool) [144]. Authentic sample pools can be over-fortified to generate higher concentrations or mixtures of pools used to generate multiple QC ranges [145-147]. Analogous to calibration preparation, materials should be prepared efficiently, subaliquoted to an appropriate volume for use, and stored under SOP-defined conditions. Assay-specific steps, such as mixing time to ensure equilibrium of the fortified samples, should be followed explicitly to prevent variable QC results, especially for analytes with high-affinity binding partners.

The qualification of new QC lots has been described [62]. Qualification studies can consist of preferably 20 replicates across 20 days (batches) or as few as 10 days (batches). Attempting to capture normal laboratory variation is essential. Importantly to LC-MS/MS, variation in calibration curves is as critical as replication of QCs in determining the mean concentration. If an assay is performed on more than one LC-MS/MS system, QC qualification should be performed for each system, with an expectation of consistent results. When the measurand(s) are fortified to a known concentration, estimated results should be near the expected value. In this instance, the theoretical value can be used as the target, requiring fewer replicates in verification. The verification of commercially available QCs is also recommended, even if the manufacturer provides expected target concentrations, though these can be qualified with fewer replicates if desired. All new QC materials, regardless of their origin, should be evaluated for mean and imprecision. Significant changes in the imprecision (variance) of a QC concentration between lots requires investigation. A common cause of shifts in CVs is attributed to a lack of homogeneity. Post-implementation monitoring of QC results may indicate a required shift in the mean of the QC material if insufficient variation is applied in the qualification phase.

Despite best efforts in method development, no assay is guaranteed to be immune from interferences. The very nature of both MS and biology will not allow an assay to be perfectly selective if used on a sufficient number samples [102]. To address this, alternative procedures may be deployed to manage infrequent interferences by leveraging additional chromatographic fidelity. For example, a method for measuring 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH, the primary metabolite of marijuana) using LC-MS/MS was validated in 2017. The assay was validated against all interferences available at the time. In the beginning of 2019, a number of sample results were rejected due to an unknown interferent closely eluting to THC-COOH. An example of this interferent found in a patient sample is shown in Fig. 1. It was determined to be an isobar of THC-COOH, specifically, the delta-8 isomer, which has seen a recent acceleration in abuse rate in the US [154]. To manage such results, an alternative methodology was developed to increase the chromatographic resolution. The original validated gradient and revised gradient are shown in Table 9. Briefly, the modification was to lower the initial %B and lower the pitch of the gradient to achieve a better resolving power, as visualized in Fig. 2. The modification was validated by re-analysis of 70 THC-COOH-only samples from three different batches using the modified gradient, quantifying the results from the original calibration curve, and comparing the reported concentrations using Deming regression. Transition ratios for all three transitions agreed between the original and modified separations. This method is not applicable to all samples. The prevalence of the interferent is such that additional time spent in analysis by reflex injections is less than the time required to assay each sample with the modified method. Future increases in the prevalence of the interferent would require re-evaluation of the practice to possibly incorporate all samples.

Proficiency tests and EQASs are critical to the harmonization of assays by ensuring the accuracy of test results, regardless of the technology used [155, 156]. Despite its clear advantages over other platforms, LC-MS/MS assays are not assumed to be perfect and proficiency failures do occur. General reasons for PT failures reportedly are calibration errors, reportable range, instability, component failure, method bias, or indeterminate (of unknown origin) [157]. In addition to these root causes, LC-MS/MS presents distinct sources of PT failure, including variations in matrix effects, lot-to-lot variation of components/reagents, poor specificity, and non-commutability of the PT/EQAS materials [158].

PT/EQAS failures require an investigation of the root cause. This often starts with a review of documentation associated with the test sample, followed by sample handling/reconstitution evaluations, sample preparation error investigations, reagent/calibration record examinations, and finally, instrument maintenance records checks [159]. For LC-MS/MS assays, calibration error is often a cause of inaccurate PT/EQAS results, particularly for a measurand for which a reference method procedure or CRM for multiple laboratories to harmonize to is lacking. As most LC-MS/MS assays are developed in-house, sources of calibration material may differ between labs participating in a PT/EQAS scheme. Preparation methods may differ, such as gravimetry from a lyophilized material in one laboratory versus spiking of a liquid stock solution in another. Different sources of standard stock material may have different qualification procedures, variable quality and purity, and distinct assignments of concentrations, resulting in disagreement between assigned concentration(s) [160-162]. Laboratories running the same analyte may benefit from cross-site trades of calibration materials, QCs, and patient samples at intervals to ensure that neither acute nor longitudinal drift has occurred [163].

A lack of commutability of the test sample can result in PT/EQAS failures, particularly in assays with leveraging extraction modes that are more complex than protein precipitation [164]. Additives intended to maintain stability or inhibit bacterial growth may alter the pH, change the solubility of either the measurand(s) or other species, or introduce unusual ionization features [165, 166]. Experimental approaches to determine non-commutable materials in an assay can include multiple steps. Sample dilution (according to the assay SOP) is viable if the concentration is sufficiently high for the AMR [167]. Post-column infusion comparison of a blank injection, authentic matrix, and a PT/EQAS sample can be informative for abnormal ion suppression [168]. This can be performed for endogenous analytes by extracting the samples without IS and using the IS as an infused analyte, thus resolving any signal contribution from the analyte. Admixing of the PT/EQAS material and true human matrix may also identify sample-based biases [17].

Unacceptable PT/EQAS results should be viewed as an impetus for improvement, but this improvement can only occur with thoughtful and honest root-cause determinations. Regardless of the degree of error, PT failures commonly have multiple or non-obvious sources. Compounding errors, e.g., calibration bias providing some portion of the inaccuracy and imprecision providing the remainder, or seemingly simple procedural deviations, such as not adequately mixing reagents, can be difficult to detect without systematic investigations [169].

Of all the resources required to operate an LC-MS/MS laboratory, human capital may be the most essential. The skill sets held by excellent LC-MS/MS technicians are difficult to train and, once trained, become highly valuable to competitive industries (e.g., pharmaceutical bio-analysis) [170]. Laboratory generalists may provide satisfactory technical benchwork, but LC-MS/MS specialists are necessary to perform the complex troubleshooting and maintenance required. Laboratories with sufficient numbers of platforms (i.e., >10) often benefit from internalization of primary service functions [171]. For laboratories with smaller LC-MS/MS footprints, external service contracts are essential to operations, but it should be recognized that facilities historically supported by external services (e.g., research or academia) generally have lower service needs than clinical laboratories [172]. Hospital laboratories and reference facilities may operate continually, even during holidays. Service contracts can be structured to align with the laboratory’s expectations for maximum allowable downtime. Alternatively, redundancy in platforms can be instituted by cross-validation of assays on backup systems, if the volumes of both samples and available instruments allow for extension of the service timelines.

Staff acquisition and retention rely on several factors. Training features highly in both aspects [173]. Extensive training and experience may be required for satisfactory platform performance and should be considered a priority by the responsible management [174]. This can be challenging as general qualifications for clinical laboratory technicians seldom address MS methods; vocational training is the most common in the LC-MS/MS industry. Professional organizations, such as the American Association of Clinical Chemistry, offer online programs for top-level instruction in LC-MS/MS [175]. These lack specific details on instrument repair or maintenance; such particulars can be learned from instrument manufacturers or by spending adequate time with service engineers. Retaining trained staff is a balance of management responsibilities, including providing an appropriate work environment and adequate compensation and growth opportunities [173, 176].

This review intended to capture the lifecycle of LC-MS/MS clinical assays after method development, from validation through to the management of operationalization. Despite sincere efforts to summarize sufficient information and experience of the laboratory processes, this review is not all-encompassing. The diversity of science fields underpinning LC-MS/MS, variation in vendors, and enormous complexity of the target population indicate that a truly comprehensive review is nearly impossible. Each laboratory will experience challenges that are not discussed herein, but hopefully, the content is sufficient to provide experimental paths for solution elucidation.

The potential of LC-MS/MS in clinical testing is enormous. The direct measurement of chemical species with high sensitivity while also providing absolute analytical normalization and adding an integrated specificity assessment are of immense benefit to patients. These advantages overcome many liabilities of other technologies, particularly in terms of selectivity, and offer a means to provide quality data in the age of evidence-based medicine. Hopefully, with ideas derived from this manuscript, continuous growth of the skill set of laboratory staff, and a never-ending aim for better quality, the promising potential of LC-MS/MS can be fulfilled.