What is liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) and when should it be used instead of immunoassays for clinical laboratory testing?

What is LC-MS/MS and When Should It Be Used Instead of Immunoassays?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) should be the preferred method for measuring steroids (especially testosterone and other androgens in women), therapeutic drug monitoring, and toxicology panels because it provides superior specificity and sensitivity compared to immunoassays, which suffer from cross-reactivity and poor accuracy at low concentrations. 1, 2, 3

Technical Overview of LC-MS/MS

LC-MS/MS is an analytical technique that combines three key components to achieve unmatched specificity 1:

• Liquid chromatography separation: Analytes are first separated on a chromatographic column based on their chemical properties 1
• Ionization: Most commonly via electrospray ionization, which works effectively for charged metabolites 1
• Tandem mass spectrometry: Uses triple quadrupole mass spectrometers where the first quadrupole selects the parent ion, the second fragments it, and the third selects characteristic daughter ions (called selected reaction monitoring or SRM) 1

Specificity is achieved through the combination of retention time from the column plus the unique mass-to-charge ratio signature, making it far more specific than antibody-based methods 1.

When LC-MS/MS Should Replace Immunoassays

Steroid Hormone Measurement (Highest Priority)

For measuring testosterone, free testosterone, and other androgens in women, LC-MS/MS is mandatory—not optional—because immunoassays are highly inaccurate at the low concentrations typical of the female range. 2, 4, 3

• Total testosterone measured by LC-MS/MS has 74% sensitivity and 86% specificity for detecting hyperandrogenism 2, 4
• Free testosterone measured by LC-MS/MS has 89% sensitivity and 83% specificity 2, 4
• Direct immunoassay methods for free testosterone should be completely avoided in women due to poor accuracy 2, 4
• The Endocrine Society and International PCOS Guidelines explicitly recommend LC-MS/MS as the preferred method for androgen measurement 2, 4

Therapeutic Drug Monitoring and Adherence Testing

LC-MS/MS is the gold standard for medication adherence testing because it can detect 40-54+ medications simultaneously with lower limits of detection typically <1 ng/mL, far exceeding immunoassay capabilities 1, 5.

• Immunoassays for therapeutic drugs suffer from cross-reactivity with drug classes, requiring MS confirmation of positive results 1
• LC-MS/MS eliminates the need for separate confirmatory testing 1, 6
• The technique is cost-effective at approximately £250,000 ($346,349) for equipment, typically on lease/rental models 1

Toxicology and Drug Screening

LC-MS/MS offers simultaneous detection of large panels of analytes with superior specificity compared to immunoassay screening, which requires confirmatory testing 6.

• Major advantages include improved specificity, flexibility, and sample throughput over gas chromatography-mass spectrometry 6
• The technology has become a de facto gold standard for bioanalytical chemistry due to unmatched sensitivity, selectivity, and specificity 1

Key Advantages Over Immunoassays

Specificity and Cross-Reactivity

Immunoassays measure whatever binds to the antibody, not necessarily the target analyte, leading to false elevations from structurally similar compounds 1, 3.

• Second-generation PTH immunoassays can vary up to 47% between different manufacturers due to cross-reactivity with PTH fragments 1
• LC-MS/MS overcomes these limitations by identifying analytes based on exact molecular mass and fragmentation patterns 1

Sensitivity at Low Concentrations

Immunoassays perform poorly at the low concentrations required for pediatric samples, female hormone measurement, and trace drug detection 2, 3.

• LC-MS/MS achieves detection limits <1 ng/mL routinely 1
• This is critical for measuring androgens in women, where concentrations are 10-20 times lower than in men 3

Multi-Analyte Capability

A single LC-MS/MS run can measure dozens to hundreds of compounds simultaneously, whereas immunoassays require separate tests for each analyte 1, 5.

• High-resolution full-scan MS can detect approximately 10,000 ion peaks per run 1
• This allows comprehensive metabolic profiling or drug panels from a single sample 1

Standardization and Harmonization

LC-MS/MS serves as the reference measurement procedure for standardizing immunoassays, as it provides traceable, accurate measurements 1, 3.

• The IFCC working group uses LC-MS/MS as the candidate reference method for PTH standardization 1
• This frees laboratories from dependence on assay-specific reagent suppliers 3

Important Limitations and Caveats

Technical Complexity

LC-MS/MS requires highly skilled personnel with expertise spanning biology, clinical chemistry, chromatography, and mass spectrometry—there are no formal academic programs specifically for clinical LC-MS/MS 7.

• Most assays are internally developed by laboratories rather than provided as ready-to-use kits 5, 7
• Validation, quality assurance, and troubleshooting require deep technical knowledge 7

Ionization Limitations

LC-MS/MS is blind to metabolites that do not ionize via electrospray, such as cholesterol 1.

• Alternative ionization methods like atmospheric pressure chemical ionization can be used for non-ionizing compounds 1
• This is a fundamental limitation that cannot be overcome without changing ionization technique 1

Cost and Infrastructure

Implementation requires significant capital investment (£250,000-£400,000 for equipment) and ongoing costs for standards and maintenance 1.

• High-resolution mass spectrometry (HR-MS) offers even greater capability but costs approximately £400,000 and requires more experienced personnel 1
• Running costs are largely unaffected by panel size, but each additional analyte requires chemical standards at approximately £150-£209 each 1

Sample Handling Artifacts

Metabolite degradation during sample preparation can lead to false results, particularly for redox-active species like NADPH and glutathione 1.

• Modest oxidation during drying strongly perturbs concentrations of oxidized species 1
• Prequenching handling must be minimized to avoid biological perturbations 1

Practical Implementation Algorithm

When to Choose LC-MS/MS Over Immunoassay

1. Always use LC-MS/MS for:

   • Testosterone and androgen measurement in women 2, 4, 3
   • Therapeutic drug monitoring requiring multi-drug panels 1, 5
   • Toxicology screening where specificity is critical 6
   • Any steroid measurement requiring high accuracy 3

2. Consider LC-MS/MS when:

   • Immunoassay results are discordant with clinical presentation 1, 3
   • Measuring low-abundance analytes 1
   • Standardization or reference measurements are needed 1, 3
   • Multiple related compounds must be measured simultaneously 5, 6

3. Immunoassays remain acceptable for:

   • High-concentration analytes where cross-reactivity is minimal 1
   • Screening applications where positive results will be confirmed by MS 1, 6
   • Point-of-care testing requiring rapid turnaround 5

Quality Assurance Considerations

Laboratories implementing LC-MS/MS must establish rigorous validation protocols, quality control procedures, and troubleshooting frameworks because most assays are laboratory-developed tests 7.

• Validation must address specificity, sensitivity, linearity, precision, and accuracy 7
• Ongoing quality assurance requires participation in proficiency testing programs 7
• Staff training must be comprehensive and continuous given the technical complexity 7