Successful implementation of online SPE LC-MS/MS in the clinical research laboratory
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1 Successful implementation of online SPE LC-MS/MS in the clinical research laboratory Martijn van Faassen, PhD student Prof. Dr. Ido Kema University Medical Center Groningen Department of Laboratory Medicine, Special Chemistry Groningen, The Netherlands
2 Wonderlab Comic
3 Sample prep LC-MS Dilute-and-shoot Liquid-Liquid Extraction (LLE) Supported Liquid Extraction (SLE) Solid Phase Extraction (SPE) offline/online
4 Department of Laboratory Medicine Subdepartment: Special Chemistry Focus on routine patient care (2 FTE) Low volume, specialized tests Specialized in chromatography, mass spectrometry Several techniques: - HPLC-UV/Fluorometry/ECD - GC-FID, GC-MS and GC-MS/MS - LC-MS/MS - Ion chromatography - Atomic absorption spectroscopy - Infrared spectroscopy Mass spectrometry since 1975, LC-MS since 23
5 Online SPE Pros Decreased total analysis time SPE and LC in parallel No manual sample pretreatment / Reduction human errors Optimal SPE (superior clean-up) Increased accuracy and precision Easy upscaling of production (e.g. research projects) Method development Minimal sample volume needed
6 Online SPE Points of consideration Skilled personnel for development (operation) and trouble shooting Robustness system important Medium sized throughput Carry-over
7 Our toys Systems: 3 x Quattro Premier XE Symbiosis Pharma System (Spark Holland) 1 x Xevo TQ-MS - Symbiosis Pharma System (Spark Holland) 2 x Xevo TQ-s Online SPE Manager (Waters) & ACQUITY UPLC I-Class 1 x 55 Shimadzu HPLC
8 Current LC-MS/MS assays in routine patient care: Catecholamines and metanephrines in plasma Catecholamines, metanephrines, serotonin and 5-hydroxyindoleacetic acid (5HIAA) in urine 25-OH-Vitamin-D3 in plasma Cortisol in urine, saliva and hair Methylmalonic acid in plasma Tryptophan, 5-hydroxytryptophan, serotonin and 5-HIAA in platelet-rich-plasma Methylhistamine and imidazoleacetic acid in urine Vitamin B3 metabolites in urine Testosterone in plasma Androgen profile: androstenedione, 17-OH-progesterone, testosterone, dihydrotestosterone, progesterone in plasma Aldosterone in plasma
9 For research purposes: Testosterone in saliva (male, female and paediatric) Cortisol and melatonin in plasma and saliva 6-Sulfatoxymelatonin in urine Tryptophan, kynurenine, 3-OH-kynurenine in urine/plasma Thyroid hormone profile in plasma (T2, rt2, T3, rt3, T4) Vitamin K in plasma (K1, MK-4) Vitamin D profiling in mother s milk and fat tissue 1,25-diOH Vitamin D3 in plasma (+25-OH Vit D3) IGF-I in plasma Fulvestrant in plasma C4 in plasma (intermediate bile acid) Cortisol and metabolites in urine (THE, THF, allo-thf, cortisone, cortisol)
10 ACQUITY Online SPE Manager Online SPE coupled the UPLC Precipitation is necessary Several SPE chemistries available: - Xbridge C 8 - Xbridge C 18 - Oasis WCX - Oasis HLB FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
11 ACQUITY Online SPE Manager FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
12 High pressure dispenser For extraction and wash solvents Valves for extraction and wash solvents (up to 23) Inline elution to column SPE extraction and wash SPE cartridges 1x1mm 2x96 cartrdiges
13 Online SPE
14 Short movie OSM in action
15 Illustrations Androgen profile in plasma Aldosterone in plasma Overview of applications FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
16 Androgen profile in plasma 21-Hydroxylase Aromatase
17 Androgen profile Instrumentation ACQUITY Online SPE Manager (OSM) Xevo TQ-S Online SPE & LC Xbridge C8, 1µm cartridges (1x1mm) Waters Cortecs UPLC C18, 1.6µm, 2.1xmm -Mobile phase: A).5%FA + 2mM NH4Ac in 1% MeOH B).5%FA + 2mM NH4Ac in MeOH FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
18 Protocol 2µL plasma, 25µL internal standard mix, 25 µl protein disrupting buffer Incubation for 3minutes at room temperature Fill up to µl with water 4µL is injected onto OSM system Xbridge C8 for online SPE extraction: Conditioning: Methanol (.5mL), Magic Mix (1.mL), Water(1.mL ) Extraction: Water (1.mL ) Wash: 2% MeOH +.1% ammonia (.5mL) 3% MeOH +.1% FA (.5mL) FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
19 Androgen profile in plasma Elution time: :9 minute Flow:.4mL/min, temperature column 4 C Total run-time per sample: 6: minutes Time (min) Flow rate (ml/min) % A % B Initial : : : : FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
20 % % % % % Androgen profile in plasma 15513_125 2: MRM of 4 Channels ES > 97.1 (Androstenedione-Quan) 7.7e6 Androstenedione, 5.6 nmol/l _125 1: MRM of 12 Channels ES > 97.5 (Testosterone-Quan) 3.27e7 Testosterone, 18 nmol/l _125 1: MRM of 12 Channels ES > 97.1 (17-OH-Progesterone-Quan) 6.87e6 17α-Hydroxyprogesterone, 4.4 nmol/l _125 3: MRM of 4 Channels ES > (Dihydrotestosterone-Quan) 3.6e5 Dihydrotestosterone, 1.1 nmol/l _125 4: MRM of 4 Channels ES > 97.1 (Progesterone-Quan) 4.1e5 Progesterone,.26nmol/L Time FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
21 % Recovery Chromatography on SPE Androstenedione Testosterone 17-OH-P DHT Progesterone Increase in % organic in SPE wash steps
22 Validation results (Summary) Precision LLOQ Intra-assay n=1 on one day Inter-assay: n=2 replicates on 12 different days (over 5 weeks) On six different days, samples with decreasing concentrations analyzed. LLOQ: CV 2% and S/N >1 Recovery Analyte Androgen Imprecision profile in plasma Intraassay (n = 1) LLOQ (nmol/l) Interassay (n = 12 different days) Mean (nmol/l) CV (%) Mean (nmol/l) CV (%) Progesterone OH-Progesterone Androstenedione Testosterone Progesterone.3 Within % 3.9 for all compounds OH-progesterone Dihydrotestosterone Androstenedione Testosterone Dihydrotestosterone FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
23 Outlook - Reference interval study Samples from population study Lifelines * 12 plasma and urine samples from healthy males, 2-8 yrs, 2 per age decade 12 plasma and urine samples from healthy females, 2-8 yrs, 2 per age decade 4 plasma and urine samples from healthy females using contraception (2-4yrs old) *
24 Aldosterone in plasma Negative ion mode Difficult to measure by LC-MS/MS Low endogenous concentrations (low pmol/l) Ion ratios not stable
25 Aldosterone in plasma Instrumentation Waters Online SPE Manager (OSM) Waters Xevo TQ-S Online SPE & LC XBridge C18, 1µm cartridges (1x1mm) Waters ACQUITY CSH Phenyl-Hexyl, 1.7µm, 2.1xmm -Mobile phase: A) 1% MeOH B) MeOH FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
26 Protocol 3µL plasma + µl internal standard mix for 1min Biotage SLE extraction protocol (96-well 4µL plate) After evaporation dissolve in 2µL 2% MeOH 5µL is injected onto OSM system XBridge C18 for online SPE extraction: Conditioning: Methanol (.5mL), Magic Mix (.5mL), Water(.5mL ) Extraction: Water (1.mL ) Wash: 1% MeOH +.1% ammonia (2.mL) 15% MeOH (.5mL) FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
27 Protocol Elution time: 1: minute Flow:.4mL/min, temperature column 4 C Total run-time per sample: 6:3 minutes Time (min) Flow rate (ml/min) % A % B Initial FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
28 % % N _45 Sm (Mn, 1x1) MRM of 4 Channels ES- S/N:PtP= > (Aldosteron 1) 6.97e4 Aldosterone in plasma: 15 pmol/l 7 fmol/2.5 picogram on column _45 Sm (Mn, 1x1) MRM of 4 Channels ES > (Aldosteron 1-d7) 2.25e FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES Time
29 Validation results (summary) Precision Intra-assay n=2 on one day Inter-assay: n=2 replicates on 2 different days (over 1 weeks) LLOQ aldosterone: 2 pmol/l Recovery Within % Aldosterone in plasma Low (19) Aldosterone (pmol/l) Medium (288) High (1259) Intra-assay % Inter-assay % FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES
30 Method comparison
31 % % % % % % % % % % % Overview current applications/projects OH-vitamin L-DOPA D 3 /D 2 in plasma 33 nmol/l 3.11 Cortisol/cortisone in saliva/urine _Corticosteroids_99 Cortisol =.73 nmol/l Catecholamines & metanephrines in plasma Thyroid hormone profile in plasma (T2, rt2, T3, _Corticosteroids_99 rt3, T4) Dopamine Norepinephrine Epinephrine 14813_Corticosteroids_99 Cortisone 3-Methoxytyramine = 5.6 nmol/l IGF-I (conversion of 2.8offline method to 3.7 online Normetanephrine _Corticosteroids_99 2: MRM of 12 Channels ES nmol/l > (Cortison-d7 Quan.) e7 Area method) nmol/l 2: MRM of 12 Channels ES > (Cortisol Quan.) 3.73e5 Area 2: MRM of 12 Channels ES > (Cortisol-d4 Quan.) 1.33e7 Area nmol/l.13 nmol/l.4 nmol/l 2: MRM of 12 Channels ES > (Cortison Quan.) 3.6e6 Area Time FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES Time
32 Summary Online SPE-LC-MS/MS in our lab Increase in assay portfolio, without extra hands Increase in sample throughput and quality 6 technicians responsible for all routine LC-MS/MS assays Costs are mainly determined by depreciation, maintenance and cartridges Outlook: Consolidation and focus on other multi-analyte assays
33 Acknowledgements UMCG Ido Kema Claude van der Ley Jasper Krijnen Anna van der Veen Hillie Adema Carlijn Boulet Waters Darwin Asa Kendon Graham
34 Thank you for your attention
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