Steroids and Vitamin D Testing by Mass Spectrometry: LC/MS/MS
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1 Steroids and Vitamin D Testing by Mass Spectrometry: LC/MS/MS Dr. Yusra Othman, PhD, DABCC, FACP Senior Clinical Scientist Tawam Hospital ASCP MIDDLEEAST 2014 Abu Dhabi - United Arab Emirates December, 2014
2 Financial Disclosers Nothing to disclose
3 Learning Objectives on LC/MS/MS Simple description of mass spectrometry Why is it challenging to measure steroids including Vitamin D? What to consider when introducing LC/MS/MS? Process for Developing a Clinical Method Issues Unique to LC MS/MS Analyses Common Sample Preparations Things to consider on implementation of LC/MS/MS methodology Update on regulations of LC/MS/MS
4 What is mass spectrometry? A technique that measures molecules in the gas phase charged species are generated and sorted based on the mass to charge ratio
5 What are the components of a mass spectrometry system?
6 Steroid Structure It is steroid structure that makes the technical problem of steroid analysis challenging. All steroids share the same characteristic arrangement of four cycloalkane rings All are derivatives of cholesterol
7 Likewise Structure of Vitamin D
8 The problem of isobars DOC, mw= g/mol 17-OHP, mw= g/mol
9 The problem of isobars Epi-testosterone, mw= g/mol Testosterone, mw= g/mol
10 Testosterone example, Immunoassay Vs MS (worst case) Taieb et al. Clin Chem 2003;49:
11 Testosterone example (best case) Taieb et al. Clin Chem 2003;49:
12 Vitamin D example All evaluated methods except HPLC demonstrated a more or less considerable deviation of individual 25(OH)D3 values compared with LC-MS/MS Roth et al. Annals of Clinical Biochemistry 2008; 45:
13 25-OH-Vit CAP survey (D-Y-A-2012)
14 25-OH-Vit CAP survey (D-Y-B-2014) The measured (analyte) Vitamin D Total Calibrators is traceable to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved isotope dilution mass spectrometry (ID LC MS/MS) reference method procedure (RMP) developed at Ghent University.25,26 This RMP is further traceable to the NIST SRM ,27 Traceability process is based on EN ISO
15 Testosterone-Y-A-2012
16 CAP-Y-A-2014 The measured (analyte) Testosterone Calibrators is traceable to USP reference material. Traceability process is based on EN ISO
17 What to consider when introducing LC/MS/MS
18 LC MS/MS Analyses Make sure to calculate return on investment (ROI) Most methods are home developed Resources: Can be develop from scratch, first time Previously validated method from colleagues Literature Vendors Most variation between labs related to differences in methods & standards CLIA High Complexity Highly specific for parent compounds Capable of being multiplexed
19 Process for Developing a Clinical Method Implementation Plan Method Selection Selection of Key Operator Outline/plan methods Acquisition of materials Set quality goals Validation Monitoring and statistics SOP preparation, staff training Imprecision Recovery Linearity Sensitivity Specificity Interferences Specimen Validate the Method Method Comparison Assay Calibration
20 Issues Unique to LC MS/MS Analyses New Terminology Matrix Effects Differential ionization of Internal Standards or Calibrators In Source Transformation (fragmentation) Isobaric Compounds and Isomers Chromatographic Resolution Carry Over Vogeser & Seger, 2010, Clin Chem
21 Before Introducing LC/MS/MS Needs questions: What steroid do I want to analyze? Is there any other steroid that I can concomitantly measure that will be useful? How often am I going to have to run this? What is my current volume for this test? Has anyone published a method? Collect all the papers, read them, which can be adopted? continue
22 Before Introducing LC/MS/MS Throughput Questions: If this test becomes popular, am I going to be able to handle the throughput? Will the sample prep need automation or can I get away with pipetting by hand? Do I have the onsite expertise to make this work? will I get adequate support from the company?
23 Before Introducing LC/MS/MS Analytical questions tied to technical need: Can I buy standard material? In what form? How will I prepare the standards? Do I have a good IS is it free of analyte? Do I have analyte-free matrix? Method validation questions: Do I have a reliable comparator lab? How am I going to get the comparator specimens? What is a comparison going to cost me?
24 Common Sample Preparations Dilution Protein Crash or Ultrafiltration (PPT) Liquid Liquid extraction (LLE) Solid Phase extraction (SPE) can be offline or online Derivatisations: Certain steroids are often measured with derivatization because they do not ionize well. Examples include 1,25(OH)2 VitD Estradiol
25 Vitamin D, WHY LC MS/MS?: Vitamin D2 and D3 are available as over the counter supplements. The Vitamin D, 25 Hydroxy, by LC/MS/MS is useful for: diagnosing vitamin D deficiency - Can distinguish different form of Vit D, D2, D3 and the epi form monitoring response to vitamin D2 or D3 supplementation, differentiating between vitamin D toxicity and other causes of hypercalcemia.
26 LC/MS/MS advantage is the analysis of multi-analytes and metabolite in one run
27
28
29 Need to justify moving to LC/MS/MS Create a clinical justification Accuracy and precision of patient results, Decrease turn-around time for results, Expand laboratory testing menu, recognize as reference for gold standard methodology in the region, medical necessity of mass spectrometry Create a cost justification Review send out test menu, Identify high cost send out items, Can they be brought in house with MS?, Calculate reveniew on investment (ROI) Create a request for proposal (RFP) Plan for about 6 months to work with vendors and, purchasing, learn about new innovations from various vendors, have questions for needs, Include specific expectations Perform necessary study of requirements, do site visit Describe room modifications required Describe differences between method development and method validation
30 RFP Should address: Space Software Hardware HVAC requirements Interface Training Method development Nitrogen supply, Nitrogen generator (uses room air), Nitrogen filter (requires 110 psi clean air), Liquid nitrogen UPS LC Autosampler Service Contract Mean time between failures Etc
31 Setting up MS lab Begin planning on space early in the process Get site planning guides from several vendors Make sure that your HVAC will handle heat Consult with building engineer Consider ventilations required Close attention required, wiring and electricity connection UPS requirements Plan out sources of gases Instrument placement (workflow, connections, bench?)
32 EP50 Mass Spectrometry in the Clinical Laboratory General overview of mass spectrometry and clinical applications General guidelines on analytical method development and validation Seeks to harmonize some of the international documents A.pdf
33 Two New CLSI Documents in the Works C 57 Mass Spectrometry for Androgen and Estrogen Measurements in Serum C 60 Liquid Chromatography / Mass Spectrometry Methods. Could be ready and available by end of year or early 2015.
34 References Taieb et al. Clin Chem 2003;49: Roth et al. Annals of Clinical Biochemistry 2008; 45: Vogeser & Seger, 2010, Clin Chem, A.pdf Robert L. Fitzgerald, PhD, Professor of Pathology UCSD, Mass Spectrometry in the Clinical Laboratory: Best Practices and Current Applications, October , Justification, Instrument Selection, and Due Diligence for Successful Implementation of MS
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