Chromatography
Automation and Liquid Chromatography-Tandem Mass Spectrometry in Therapeutic Drug Monitoring
Author details: Mikaël Levi1, Neil Loftus2
1 MS Business Unit, Life Science Business Department, Analytical & Measuring Instruments Division Shimadzu Corporation, Kyoto, Japan. 2 MS Business Unit Overseas, Life Science Business Department, Analytical & Measuring Instruments Division Shimadzu Corporation, Manchester, UK.
Therapeutic drug monitoring (TDM) has become a key clinical tool to help individualise therapy, check compliance and maximise response while lowering side effects. Liquid chromatography-tandem mass spectrometry has become a major technology in TDM given its inherent specifi city, sensitivity and quantitative capability for small molecule drug analysis. Within the context of a routine clinical pathology environment there are considerable advantages in integrating mass spectrometry into small molecule drug monitoring when compared to immunoassays. This review considers the impact of LC-MS/MS in a routine clinical pathology laboratory compared to conventional immunoassay techniques and highlights mass spectrometry in the analysis of immunosuppressant’s and anticonvulsants.
1. Introduction
Therapeutic drug monitoring (TDM) is a multi-disciplinary science helping to understand the factors that determine the dose-effect relationship and to use this knowledge to optimise drug treatment (maximise effi cacy /minimise side effects). In many routine clinical pathology laboratories, the panels of drugs subject to routine TDM in patients is limited, including several immunosuppressive drugs, antibiotics, antiepileptics, antidepressants, digoxin and methotrexate. This refl ects the need to monitor drug classes that have a narrow therapeutic index, established consequences for under- or over-dosing, a defi ned relationship between blood concentration and clinical/toxic effect, signifi cant variation within and between individuals and for drugs that have a proven knowledge base for clinical management [1-3] (see Table 1).
In most routine clinical pathology laboratories, automated immunoassay platforms dominate bioanalytical drug assays. However, immunoassay techniques may produce results that have a bias due to the cross reactivity of the active metabolites, batch-to-batch heterogeneity in antibodies or reagent quality, high-dose-hook effect and, for some drug immunoassays, a high cost per analysis [4].
As precision medicine emerges as a possible approach to treat a specifi c individual patient with a specifi c disease taking into account individual variability in genes, environment and lifestyle TDM is likely to have a high impact in dose adjustments. However, this is a novel application for TDM and requires extensive assay development and validation together with rapid turnaround times so that assays can be used for drug development and individual patient care. Supporting such analytical and clinical strategies requires methodologies which are versatile and can be easily adapted to a ‘laboratory-developed test’ (LDT) or ‘in-house’ assay.
In this article, we highlight the application of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) widely regarded as the gold standard in TDM for immunosuppressants and anticonvulsants and considers the development of further automation.
2. LC-MS/MS for TDM
From a historical point of view, liquid chromatography techniques have been used for TDM with ultra-violet (UV), photo diode array (PDA) or fl uorescent detection (FLD) systems for several decades. However, despite the high impact of ultra-high- performance liquid chromatography (UHPLC) [5-7] in reducing run times and enhancing separation effi ciency, such detection techniques are limited in terms of specifi city often resulting in extensive sample cycle times and poor sensitivity as many compounds lack a natural chromophore or fl uorophore [1]. More recently mass spectrometry is now regarded as a key technique for routine clinical pathology laboratories delivering robust, rugged platforms with highly selective and sensitive detection [8]. Its capability in the development of assays for individual drugs (‘laboratory-developed tests’ (LDTs) or ‘in- house’ assays) and in multiplexing analyte panels creates new opportunities in expanding the number of drug assays. By increasing the number of drug assays, helping to provide better access to the technology and using novel blood sampling strategies including micro-sampling for pediatric TDM or at home sampling [9-12] also helps position TDM for personalised medicine.
3. Automated Sample Management and Preparation
Biological fl uids are highly complex matrices which present challenges in matrix management as endogenous and exogenous components result in compound and system-specifi c effects in mass spectrometry. Negating the effects of the matrix needs to be carefully considered in all sample preparation and management protocols. Matrix effects can lead to isobaric interferences, particulate clogging, ion suppression or ion enhancement resulting in a difference between the signal intensity detected in a neat standard solution compared to a matrix-matched standard [13-16].
In electrospray ionisation, ion suppression is due to a change in the droplet formation and surface tension which will affect charge transfer effi ciency. Non-volatile compounds such as blood phospholipids, salts, uncharged matrix components, reagent impurities, drugs and metabolites are known to produce ion suppression or enhancement [17]. To help reduce the impact of matrix effects on bioanalytical assays there are several strategies open for the analyst. One of the most important techniques is to use appropriate and validated internal standards, particularly stable-isotope-labelled analogues (SIL-ISTD) to help correct for ion signal changes and handling errors in sampling preparation protocols. However, for many assays the effect of high inter and intra-patient variability in endogenous molecule concentrations also requires sample clean-up using extraction or purifi cation techniques such as liquid-liquid extraction (LLE), solid-phase extraction (SPE) or protein precipitation (PPT). Each technique needs to be considered in the context of assay performance to achieve an acceptable level of accuracy and precision while also taking into account the effi ciency and recovery of the extraction in addition to the ease of use and cost per sample.
Figure 1: General view of the LC-MS/MS system with integrated sample preparation module CLAM-2000.
LAB ASIA - JANUARY/FEBRUARY 2018
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52
