Overcoming the Challenges of Sample Preparation and LC/MS/MS Method Development for Clinical Applications

Transcription

1 Overcoming the Challenges of Sample Preparation and LC/MS/MS Method Development for Clinical Applications Speaker: Sky Countryman, Manager of PhenoLogix and Applied Technologies, Phenomenex Webinar Host: Sonia Nicholas, Clinical Diagnostics Editor of SelectScience

2 Overcoming the Challenges of Sample Preparation and LC/MS/MS Method Development for Clinical Applications Presented by Sky Countryman

3 Learning Objectives How to determine the best sample preparation technique Method development tips for achieving separation of target compounds via LC/MS/MS How to increase throughput without sacrificing results

4 Myths of LC/MS/MS 1. I don t need good resolution 2. I don t need to do any sample clean up

5 Codeine Interferences

6 Endogenous Interferences EtS Intensity 3.2e5 2.4e5 1.6e5 8.0e4 Urine Contaminant EtG Min

7 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 The Matrix Effect 1.1e5 1.0e5 Enhancement In te n s ity, c p s 9.0e4 8.0e4 7.0e4 Suppression Normal 6.0e4 5.0e4 4.0e4 3.0e e4 1.0e Time, min

8 Matrix Effects What contributes to the matrix effect? Disease state Endogenous compounds Exogenous compounds MS source design Sample preparation

9 Poll 1: Which of the following sample preparation techniques do you perform in your lab? Solid Phase Extraction (SPE) Liquid-Liquid Extraction (LLE) Phospholipid Removal Protein Crash/Precipitation Filtration Dilution (Dilute and Shoot)

10 Methods Of Sample Preparation Solid Phase Extraction (SPE) Liquid / Liquid Extraction Phospholipid Removal Protein Crash / Precipitation Filtration Centrifugation Settling and Decanting Dilution Highly Selective Techniques Non-Selective Techniques

11 SPE Clean Up Relatively Clean Urine SPE Extracted Urine

12 What Dies First? $2 $600 $80,000 $350,000

13 Today s Agenda Discuss three case studies where sample prep played an important role in method stability 1. Vitamin D 2. Aldosterone 3. Pain Panel

14 Poll 2: Which of the following applications do you run most often? Vitamin D Analysis Pain Panel (pain medications) Steroids Immunosuppressants Other

15 Case Study 1 25-OH D2 & D3 from Plasma

16 Method Background Matrix: plasma Vitamin D is protein bound High phospholipid content in plasma APCI source Reduces ion suppression (phospholipids) Fast separation Kinetex 2.6µm C18 30 x 3.0 mm Two minute ballistic gradient

17 Sample Prep Strategies Protein Precipitation Crash using organic solution Disrupts protein-analyte binding Limited clean up Phospholipid Removal Crash using organic solution Selectively remove phospholipids using special designed phase Limited interaction with target compounds

18 i t y, I n c I n t p e t s e n n s is t y i t I, y n, I c n t p c e t p s e n s n s is t y i t, y I, c n p c t p s e s n s i t y, c p s XIC of +MRM (5 pairs): / Da ID: D2/1 from Sample 16 (QC2(75ng)-Phree) of P-A batch Phree wiff (Heated Nebu e4 5.0e4 3.0e4 4.0e4 2.0e4 3.0e4 1.0e4 2.0e4 Max cps. 1.0e Time, min 0.0 XIC of +MRM (50.2 pairs): / Da ID: 0.8 D3/2 from 1.0 Sample (QC2(75ng)-PPT) 1.4 of 1.6P-A batch 1.8Phree wiff 2.0 (Heated 2.2 Nebuliz Max. 2.9e4 3.4 cps. Time, min XIC of +MRM (5 pairs): / Da ID: D2/1 from Sample 16 (QC2(75ng)-Phree) of P-A batch Phree wiff (Heated Nebu... Max cps. 6.7e4 6.0e4 1.7e4 1.5e4 4.0e e4 2.0e4 25-OH-Vit D Phospholipid Removal LC/MS/MS Data XIC of +MRM (5 pairs): / Da ID: D2/1 from Sample 21 (QC2(75ng)-PPT) of P-A batch Phree wiff (Heated Nebuliz e5 1.0e5 5.0e4 25-OH-Vit D Max. 1.4e4 cps Time, min XIC of +MRM (5 pairs): / Da ID: D2/1 from Sample 21 (QC2(75ng)-PPT) of P-A batch Phree wiff (Heated Nebuliz... Max. 1.4e4 cps. 4.0e4 Protein Precipitation 25-OH-Vit D2 No major difference slightly higher signal from PPT Choose the cheapest / easiest sample prep method Time, min Time, min

19 What are Phospholipids? Double Chain = Phosphatidyl choline Single Chain = Lysophosphatidyl choline

20 Negative Effects of Phospholipids Signal suppression in positive ESI mode Loss in sensitivity for ESI+ Much less pronounced APCI Dependent on source design (older = more suppression) Retention time shifts as phospholipids build up Tandem Labs reported retention shifts of >1 minute Reduce column life Build up on column reaches critical concentrations Phospholipids crash out of solution and back pressure spikes

21 LLE: Depletion of Phospholipids Errors in Bioanalysis Due to Phospholipids Definitive Measurement, Mechanism and Management ; ASMS 2011 Poster by LabCorp, Russel Grant, Matthew Crawford, Brian Rappold and Stacy Dee. Less solubility in ACN

22 Phosphatidyl Cholines are the major component of lecithin Important Classes of Phospholipids

23 Detection of Phospholipids Look for precursors: Lysos m/z: 496, 522 m/z: 760, 784, 786 Product Ion m/z: 184 Polar head group fragment Mass ~184 The transition provides signal for all phosphatidyl cholines and lysophosphatidyl cholines

24 PPT vs. Phree in ESI+

25 Phospholipid APCI (Extraction from PPT) XIC of +MRM (7 pairs): / Da ID: D2/1 from Sample 11 (Samp1-PPT(ACN-300uL)) of Phree VS PPT(012513)Scan VitD+... Max cps. 8.2e5 8.0e5 7.5e5 7.0e5 Phospholipids? 6.5e5 6.0e5 5.5e5 I n t e n s i t y, c p s 5.0e5 4.5e5 4.0e5 3.5e5 Phospholipids? 3.0e5 2.5e5 25-OH-Vit D2/D3 2.0e5 1.5e5 1.0e5 5.0e Time, min

26 Phospholipid Elution XIC of +MRM (7 pairs): 184.0/184.0 Da ID: PL(Isource) from Sample 11 (30 ug/ml Amoxapine - PPT Infusion) of PostColumn_MeOH_1... Lysos PC-1,4 PC-1 PC-2 Max. 4.9e6 cps. In te n s ity, c p s 4.8e6 4.6e6 4.4e6 4.2e6 4.0e6 3.8e6 3.6e6 3.4e6 3.2e6 3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e Time, min It takes >10 min to elute all Phosphatidyl cholines at 95% MeOH

27 Final Sample Prep Method Dispense: 300 µl of ACN/MeOH (85/15) into each well 100 µl of spiked plasma Aspirate: Manually aspirate or vortex to achieve crash Wait 30 seconds Vacuum: Apply vacuum for 1-2 mins at Hg Collect & inject: Make direct injection on the LC column No need to go through the time consuming dry down step Total time for sample prep is 4 minutes

28 Final Method Table 1. Precision and Accuracy Data for 25 OH Vitamin D2 Expected Conc. (ng/ml) %CV Accuracy Table 2. Precision and Accuracy Data for 25 OH Vitamin D3 Expected Conc. (ng/ml) %CV Accuracy Accuracy and precision based on quantitation against an internal standard

29 Conclusions Removing phospholipids was determined to be critical to long term method stability Methods were adapted to 96-well plate for high throughput analysis Final methods showed high recoveries for Vit D

30 Case Study 2 Aldosterone in Plasma

31 Method Background Required detection limit 10 pg/ml API 5000 Requires sample pre-concentration/clean up Ionizable in both APCI and ESI APCI showed lowest background Negative mode detection

32 Aldosterone Ionization Capable of forming [M+H] +, [M-H] -, [M+Na] +, [M+OCOCH 3 ] - [M-H] gives the most intense signal It exists as three possible tautomers: Source: Yamashita et al, Chem Pharma Bull, 56(6), (2008)

33 Chromatography Initial chemistries used: Kinetex C18, 50x2.1 mm, 2.6 µm Kinetex XB-C18, 50x2.1 mm, 2.6 µm and 30x2.1 mm Kinetex PFP, 50x2.1 mm, 2.6 µm Gemini NX C18, 50x2.0 mm, 3 µm The goal was to separate the Aldosterone from its known isomers: Cortisone & Prednisone XB-C18 provided the best separation between the Aldosterone and its possible interferences

34 Chromatogram XIC of -MRM (3 pairs): / Da ID: Aldo 1 from Sample 4 (5000 pg/ml) of Cal wiff (Heated... Max. 1.3e5 cps. In te n s ity, c p s 1.25e5 1.20e5 1.15e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Notice the presence of impurities or tautomers prior to Aldosterone elution Time, min

35 SPE sorbents tested Strata-X Strata-X-A Strata C18-E SPE Method Strata-X-A provided the cleanest extract Next step: Optimization Strata-X-A

36 Optimization: Acidic Load XIC of -MRM (3 pairs): / Da ID: Aldo 1 from Sample 16 (0.1 ng/ml Plasma Ext-pH 5, #3) of Ext ph-0... Max. 1.5e4 cps. 1.5e e4 1.3e4 1.2e4 1.1e4 High level of matrix background 1.0e4 In te n s ity, c p s Time, min

37 Optimization: Sample Load at ph 7 XIC of -MRM (3 pairs): / Da ID: Aldo 1 from Sample 17 (0.1 ng/ml Plasma Ext-pH 7, #1) of Ext ph-... Max cps In te n s ity, c p s

38 Optimization: Sample Load at ph 9 XIC of -MRM (3 pairs): / Da ID: Aldo 1 from Sample 21 (0.1 ng/ml Plasma Ext-pH 9, #2) of Ext ph-... Max cps In te n s ity, c p s Time, min

39 SPE Optimization Increase in ph during the sample load reduced the amount of the late-eluting matrix components

40 Final SPE Method Strata-X-A 60 mg /3 ml Load: 0.5 ml plasma diluted 1:2 with 25 mm NH 4 HCO 3, ph Wash: 1mL of 25 mm buffer 1mL 25% MeOH in Water Dry the SPE bed Elute: 2 ml of 1.5% NH 4 OH in MeOH (2x 1mL elution) Post SPE : Dry the C Reconstitute with 100 µl of 30:70 MeOH:H 2 O containing ~1 ppm Estriol 100 pg/ml plasma spike is 85% (n=3)

41 Problem: Columns died after as little as <50 injections rapid increase in back pressure / split peaks

42 Pressure (bar) Pressure Trace Injections

43 Investigation SEM images of column frits show build up of proteinaceous material times magnification times magnification Contaminant & particle build up

44 Solution Proteinaceous material was highly methanol / water soluble Very low solubility in organic solvents such as hexane, ethyl acetate, MeCl 2 Changing elution solvent to EA/IPA/NH 4 OH provided elution of Aldosterone but not junk

45 10 pg/ml Aldosterone in Plasma

46 Case Study 3 Pain Panel in Urine

47 Hydrolysis Hydrochloric acid Very efficient & cheap Destroys suboxone and 6-MAM Corrosive to metal system components Beta-glucuronidase / sulfatase Inefficient & costly High specificity Good for all drug classes

48 Sample Prep Strategies Dilute & shoot Cheap & easy Very hard on system components Solid Phase Extraction More costly Requires special equipment Decreases system maintenance Increase column lifetime / method stability Provides sample concentration

49 SPE Extraction Morphine Codeine Diazepam Amphetamine Norbuprenorphine PCP Benzoylecgonine 6-MAM Developing a single SPE method can be a challenge!

50 Optimized Method for 41 Pain Panel Compounds 18 Opiates 12 Benzodiazepines 5 Amphetamines 4 Analgesics 2 Drugs of Abuse For complete method details visit Method readily adaptable to automated formats Good for acid or enzymatic hydrolysis

51 Optimizing Wash Strength

52 Optimized Wash/Elution Wash1: 0.1N HCl Wash2: 100% MeOH Elution: 5% MeOH in NH 4 OH Wash1: 0.1N HCl Wash2: 100% MeOH Elution: NH 4 OH:IPA:Ethyl Acetate Wash1: Buffer Wash2: 100% MeOH Elution: NH 4 OH:IPA:Ethyl Acetate

53 Problems with Enzyme Hydrolyzed samples contain solubilized enzyme that must be removed Centrifugation works well in test tubes Rotor arms that adapt to 96-well plates reduce maximum spin speed Resulting samples can rapidly decrease column lifetime For more details visit

54 Back Pressure Hydrolyzed Samples Processed in 96-Well Plates Number of injection VS. Increase in Back Pressure for Samples Without PPT Back pressure (bar) # Of Injections

55 Beta Glucuronidase Removal Protein Precipitation (PPT) Using Impact Precipitation Plates after centrifugation SPE procedures also remove enzyme

56 Back Pressure PPT Samples: No Increase in Back Pressure # # of of injections vs. VS. increase Increase in back Back pressure Pressure for samples Samples with With PPT (using (Using Impact) Impact-U) Back pressure (bar) # Of Injections

57 Conclusion SPE provides the highest level of clean up Long term = Lowest amount of system maintenance Enzymes must be removed before HPLC analysis Centrifugation in 96-well plates is not effective in removing the solubilized enzyme PPT using impact removes the enzyme and is suitable for high throughput environments Once the enzyme is removed, acceptable column lifetime is observed

58 In Summary LC/MS/MS reduces many of the challenges to chromatographic analysis MS technology continues to improve There is still a need for chromatography to separate isobaric interferences Sample prep can significantly improve method stability

59 Thank You! Questions?

60 Poll Results

61 Which of the following sample preparation techniques do you perform in your lab? Solid Phase Extraction (SPE) 28% Liquid-Liquid Extraction (LLE) 20% Phospholipid Removal 8% Protein Crash/Precipitation 16% Filtration 16% Dilution (Dilute and Shoot) 12%

62 Which of the following applications do you run most often? Vitamin D Analysis 22% Pain Panel (pain medications) 14% Steroids 14% Immunosuppressants 17% Other 33%

63 Poll 2: Which of the following applications do you run most often? Vitamin D Analysis Pain Panel (pain medications) Steroids Immunosuppressants Other

64 Q & A

65 Thank you for attending We hope you found the webinar useful and informative. If you have any further questions please