madman
Super Moderator
Clinical LC-MS/MS applications have been in routine service for just over 20 years. The introduction of electrospray ionization first allowed the development of newborn screening methods, but it was not until the start of the new millennium that routine applications for immunosuppressant drugs and then steroids began to emerge. Method development was driven by the increased sensitivity and specificity offered by LC-MS/MS over existing immunoassay (IA) and HPLC methods. Compared to HPLC, the new LC-MS/MS methods were faster, sample preparation was often simpler and drugs such as ciclosporin which have no chromophores could be measured for the first time using chromatographic methods. Immunosuppressant drugs continue to be measured by LC-MS/MS in the larger transplant centers and methods have now evolved to measure finger prick samples collected by the patient at home. This is proving to be a useful strategy in the COVID era with many clinics being performed remotely.1
Vitamin D became an early target for analysis because of the poor performance observed across EQA schemes, mainly due to cross-reactivity with metabolites, but the huge workload has become unmanageable for some departments and many have reverted to IA methods. This has highlighted the main deficiencies of LC-MS/MS compared to established IA methods: lack of automation, expensive equipment, and the requirement for highly trained staff. Other potential analytical targets for LC-MS/MS were the small molecules either difficult to measure or poorly measured by IA. Steroid hormones were obvious candidates and these were initially developed as single test methods, but as LCMS/MS instrumentation has advanced, there is now a trend to move towards multi-steroid panels. Methodological differences including instrumentation, internal standards, sample extraction, and chromatography still mean that interlaboratory comparability is not as good as it should be.2 Typically, it is the steroids that are infrequently measured (such as 17OHP) that suffer the worst comparative performance between laboratories.3
Improvements in interlaboratory performance are ongoing and have been driven by the increasing awareness that robust thoroughly validated methods using high-quality isotopic internal standards are needed.4 The production of commercially available calibration material in recent years has also helped because routine labs find it difficult to make and maintain calibrators, especially for a wide range of analytes. Quality is also being underpinned by initiatives such as the CDC hormone standardization programme5 and vitamin D standardization certification program.6 Having LC-MS/MS target values assigned to EQA material in these schemes allows assessment of bias from the true result and LC-MS/MS assigned target values have since been adopted by other national external quality assessment schemes, for example, UKNEQAS, but as yet only for select steroids. Improvements in formulation and performance of some IAs have been made to address specificity issues, but there remain concerns around specificity in individual methods, for example, many oestradiol IAs perform poorly when measuring in the low concentration range found in children, men, post-menopausal women, and patients taking aromatase inhibitors.7 Importantly, LC-MS/MS methods do not suffer cross-reactivity problems with the estrogen receptor antagonist fulvestrant8 used in breast cancer treatment.
Achieving the necessary sensitivity to measure oestradiol using LC-MS/MS has been difficult without using derivatization, but using the most sensitive instrumentation has enabled the development of assays capable of meeting the required sensitivity of 3.7 pmol/L advocated by the Endocrine Society.9 Derivatisation of analytes to improve detection limits may reduce the specificity of the assay, thus negating the benefits of LC-MS/MS analysis and should be avoided if possible.
The automation of mass spectrometry is still in its early stages and is only available from one or two manufacturers, but whilst the equipment is excellent, the test repertoire is limited, and the systems are expensive. For established LCMS/MS users, financing systems from existing budgets means sacrificing staff or LC-MS/MS instruments, and as a result, uptake for the technology has been slow. This is not surprising because the introduction of automation for IA methods 30 years ago followed a similar course. Other vendors are developing automation and the situation will change, as it did with IA, with increased competition and reduction of costs with volume sales.
*Many would view LC-MS/MS as a powerful analytical tool that has permitted the development of novel methods and provided an interface with clinical researchers to improve diagnostic testing. However, some have had a poor experience with this technology because in reality to make it work effectively requires a lot of support from enthusiastic staff, and this may not always be the case in a routine laboratory struggling for resources. Automation may eventually provide the solution to this problem, but it is difficult to see how it would cope with the more difficult assays requiring some form of enrichment/concentration step to improve sensitivity. Improvements in sensitivity have been impressive over the past 20 years mainly due to improvement in ion transmission and detection, but the main problem with mass spectrometry is in the poor efficiency of electrospray ionization. Consequently, some form of sample clean-up will always be required to improve signal-to-noise in methods requiring high sensitivity.
Improvements in instrument design with improved scan speeds, greater sensitivity, and robustness have improved the scope of laboratories to measure some challenging analytes over the past 20 years. It could be argued that much of the low-hanging fruit has already been picked but technology drives this field and continuing improvements in instrumentation ensure that existing methods can always be improved. LC-MS/MS is also well placed to discover and exploit new diagnostic tests and will have a major part to play in clinical laboratories in the future.
Vitamin D became an early target for analysis because of the poor performance observed across EQA schemes, mainly due to cross-reactivity with metabolites, but the huge workload has become unmanageable for some departments and many have reverted to IA methods. This has highlighted the main deficiencies of LC-MS/MS compared to established IA methods: lack of automation, expensive equipment, and the requirement for highly trained staff. Other potential analytical targets for LC-MS/MS were the small molecules either difficult to measure or poorly measured by IA. Steroid hormones were obvious candidates and these were initially developed as single test methods, but as LCMS/MS instrumentation has advanced, there is now a trend to move towards multi-steroid panels. Methodological differences including instrumentation, internal standards, sample extraction, and chromatography still mean that interlaboratory comparability is not as good as it should be.2 Typically, it is the steroids that are infrequently measured (such as 17OHP) that suffer the worst comparative performance between laboratories.3
Improvements in interlaboratory performance are ongoing and have been driven by the increasing awareness that robust thoroughly validated methods using high-quality isotopic internal standards are needed.4 The production of commercially available calibration material in recent years has also helped because routine labs find it difficult to make and maintain calibrators, especially for a wide range of analytes. Quality is also being underpinned by initiatives such as the CDC hormone standardization programme5 and vitamin D standardization certification program.6 Having LC-MS/MS target values assigned to EQA material in these schemes allows assessment of bias from the true result and LC-MS/MS assigned target values have since been adopted by other national external quality assessment schemes, for example, UKNEQAS, but as yet only for select steroids. Improvements in formulation and performance of some IAs have been made to address specificity issues, but there remain concerns around specificity in individual methods, for example, many oestradiol IAs perform poorly when measuring in the low concentration range found in children, men, post-menopausal women, and patients taking aromatase inhibitors.7 Importantly, LC-MS/MS methods do not suffer cross-reactivity problems with the estrogen receptor antagonist fulvestrant8 used in breast cancer treatment.
Achieving the necessary sensitivity to measure oestradiol using LC-MS/MS has been difficult without using derivatization, but using the most sensitive instrumentation has enabled the development of assays capable of meeting the required sensitivity of 3.7 pmol/L advocated by the Endocrine Society.9 Derivatisation of analytes to improve detection limits may reduce the specificity of the assay, thus negating the benefits of LC-MS/MS analysis and should be avoided if possible.
The automation of mass spectrometry is still in its early stages and is only available from one or two manufacturers, but whilst the equipment is excellent, the test repertoire is limited, and the systems are expensive. For established LCMS/MS users, financing systems from existing budgets means sacrificing staff or LC-MS/MS instruments, and as a result, uptake for the technology has been slow. This is not surprising because the introduction of automation for IA methods 30 years ago followed a similar course. Other vendors are developing automation and the situation will change, as it did with IA, with increased competition and reduction of costs with volume sales.
*Many would view LC-MS/MS as a powerful analytical tool that has permitted the development of novel methods and provided an interface with clinical researchers to improve diagnostic testing. However, some have had a poor experience with this technology because in reality to make it work effectively requires a lot of support from enthusiastic staff, and this may not always be the case in a routine laboratory struggling for resources. Automation may eventually provide the solution to this problem, but it is difficult to see how it would cope with the more difficult assays requiring some form of enrichment/concentration step to improve sensitivity. Improvements in sensitivity have been impressive over the past 20 years mainly due to improvement in ion transmission and detection, but the main problem with mass spectrometry is in the poor efficiency of electrospray ionization. Consequently, some form of sample clean-up will always be required to improve signal-to-noise in methods requiring high sensitivity.
Improvements in instrument design with improved scan speeds, greater sensitivity, and robustness have improved the scope of laboratories to measure some challenging analytes over the past 20 years. It could be argued that much of the low-hanging fruit has already been picked but technology drives this field and continuing improvements in instrumentation ensure that existing methods can always be improved. LC-MS/MS is also well placed to discover and exploit new diagnostic tests and will have a major part to play in clinical laboratories in the future.
