LCMSMS Solutions For Steroid Analysis

LCMSMS Solutions For Steroid Analysis Dan Blake Manager, Clinical Applications For Research Use Only. Not for use in diagnostic procedures. RUO-MKT-18-1166-A

Agenda Special considerations for steroid analysis Methods of Steroid analysis 1: Simple & Fast Testosterone imethod Online Extraction with 2DLC and simple sample processing Low Level Estrogen imethod Improving Selectivity with Ion Mobility Methods of Steroid Analysis 2: Extending the coverage Steroid Panel imethod Commercial Kits Summary of and conclusions from the different approaches 2 2016 SCIEX RUO-MKT-18-1166 A

Special considerations for steroid analysis

Traditional immunoassays, while convenient, are unreliable for reasons previously discussed Varying sample volumes available Paediatric/neonatal samples Pressures of sample number and turnaround time demand a fast and easy to setup method Again, similar pressures require the use of rapid, automatable extraction procedures Ideally some form of protein precipitation Liquid Liquid extraction is an option but not appropriate for all laboratories SPE is an option but can be costly Derivitisation, for example with dansyl chloride, is an option for challenging steroids but should be considered as a last resort. 4 2016 SCIEX RUO-MKT-18-1166 A

What Steroids to analyse? The LC/MS/MS method should give us as much information as possible However, increasing the number of analytes could potentially increase the complexity of the assay 5 2016 SCIEX RUO-MKT-18-1166 A

The ideal LC/MS/MS Steroid Application The convenience of an immunoassay style kit approach Sensitive enough to assay the lowest level steroids The which instrument part of the question Able to cope with sample volume and throughput demands The highest control Wide dynamic range standard curve Deuterated internal standards, for each analyte if possible Ability to utilise traceable Quality Control material 6 2016 SCIEX RUO-MKT-18-1166 A

Testosterone imethod

Testosterone is one of the most widely measured steroids Measurement of testosterone is used to research a variety of disorders, such as congenital adrenal hyperplasia and hypogonadism. Measurement of the typical levels in men is straightforward; however, measurement of the low levels present in women and children can be especially challenging. SCIEX provides solutions for routine analysis for both circumstances. Simple male testosterone assay on the API3200 platform using protein precipitation Low level (female) assay on the API5000 or Triple Quad 5500 using more thorough sample preparation 8 2016 SCIEX RUO-MKT-18-1166 A

Testosterone imethod, API3200 Data from a 3.4 nmol/l testosterone sample analyzed using an SCIEX API 3200 LC/MS/MS System. 9 2016 SCIEX RUO-MKT-18-1166 A

Testosterone imethod, API5000 Data from a 0.069 nmol/l testosterone sample analyzed using an SCIEX API 5000 LC/MS/MS System. 10 2016 SCIEX RUO-MKT-18-1166 A

Testosterone imethod Online Extraction with 2DLC and simple sample processing

A simple method for analysis of a small number of routinely measured steroids has been proposed The method employs a simple protein precipitation with direct injection onto a 2DLC system (no need for offline sample concentration) The remainder of the sample cleanup process is performed on a trapping column, followed by a reversed flow gradient onto a common C18 HPLC column, with a total runtime of 5 minutes As a consequence, sample preparation time is kept to a minimum and sample throughput is maximised The trap column is reusable, with a lifetime in excess of 1000 injections 12 2016 SCIEX RUO-MKT-18-1166 A

GRADIENT PUMPS A/B Proposed Flow and Plumbing diagram for 2DLC Steroids method TRAPPING COLUMN Column Oven Analytical Column MS System Switching Valve 10 1 2 5 AUTOSAMPLER VALVE 6 ISOCRATIC LOAD PUMP C 9 8 7 5 6 3 4 Valve position A: (Analyte Loading onto Trapping Column) Valve position B: (Analyte Elution from SPE Column onto Analytical Column) Switching Valve: Time(min) 0.0 A 1.0 B 5.5 A Valve Position WASTE WASTE 13 2016 SCIEX RUO-MKT-18-1166 A

Extracted Steroid sample, approx 0.15nmol/L, Analysed by 2DLC approach Testosterone 17-OH-P 11-Deoxycortisol Androstenedione 14 2016 SCIEX RUO-MKT-18-1166 A

Low Level Estrogen imethod

The Importance of Estrogens Estrogen is an entire class of related female sex hormones: estrone (E1) estradiol (E2), and estriol (E3) Estrone (E1) is widespread throughout the body. It is the only one of estrogens that s present in any amount in women after menopause. Estradiol (E2) is the primary sex hormone of childbearing women. It is formed from developing ovarian follicles. Estradiol is responsible for female characteristics and sexual functioning. Also, estradiol is important to women s bone health. Estradiol contributes to most gynecologic problems and even female cancers. Estriol (E3) is made from the placenta. It s important only during pregnancy. 16 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Conditions: Instrumentation API 5000 and SCIEX Triple Quad 5500 Mass Spectrometers Shimadzu Prominence 20A and Agilent 1200 integrated systems Method Information 500 µl sample volume required Liquid-liquid extraction without derivatization Clinically relevant dynamic range of 5-500 pg/ml 6 minute total run time 17 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Lower Limit of Quantitation Estrone 5 pg/ml LLOQ, S/N=22 Estradiol 5 pg/ml LLOQ, S/N=19 18 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Precision and Accuracy Estrone (E1) Sample (pg/ml) %CV % Accuracy Estradiol (E2) Sample (pg/ml) %CV % Accuracy Day 1-3 Day 1-3 LLOQ (5) 9 104 Low QC (15) 3 104 High QC (100) 3 101 ULOQ (500) 3 100 Day 1-3 Day 1-3 LLOQ (5) 10 101 Low QC (15) 4 96 High QC (100) 5 102 ULOQ (500) 4 101 Estrone (inter-day) LLOQ CV s 9%,QC s CV s 5% Accuracy deviation 4% Estradiol (inter-day) LLOQ CV s 10%,QC s CV s 5% Accuracy deviation 4% 19 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Linearity Estrone Estradiol Clinically relevant dynamic range 5-500 pg/ml, R 0.999 20 2016 SCIEX RUO-MKT-18-1166 A

Estrone and Estradiol: Summary The method was verified internally across laboratories for analytical analysis LLOQ with suitable signal to noise (20) Inter-day Precision and Accuracy LLOQ CVs 10%,QCs CV s 5% Accuracy Deviation ±4% Control samples at clinically relevant low and high concentrations (5-500 pg/ml) with linear response r 0.999 Liquid-Liquid extraction without derivatization 6 minutes run time 21 2016 SCIEX RUO-MKT-18-1166 A

Improving Selectivity with Ion Mobility

Ion Mobility to improve selectivity There are many different ways to achieve improved selectivity in applications of LC-MS/MS, including: Improved chromatography Tandem mass spectrometry (MS/MS, or even higher MS 3 ) High-resolution mass spectrometry More up-front sample cleanup In many applications of LC-MS/MS, mass measurements alone (even high resolution) cannot provide sufficient selectivity Isobaric interferences may have the same exact mass as analyte ions Interferences may have common fragment ions Chemical background may pose a challenge The SelexION ion mobility technology provides an orthogonal means of separating / filtering ions prior to, and independent of, MS/MS analysis. 23 2016 SCIEX RUO-MKT-18-1166 A

SelexION Ion Mobility Device Components: Existing ion path New hardware 24 2016 SCIEX RUO-MKT-18-1166 A

SelexION Ion Mobility Device Components: 1. Orifice Plate 2. Ion Mobility Cell 3. Curtain Plate Robust, easy-to-install, hardware components: No tools required No cables No need to break vacuum Installation in about 2 minutes 25 2016 SCIEX RUO-MKT-18-1166 A

Tuning the SelexION Ion Mobility Device ions in transport gas t 2 =0 0 0 to mass spectrometer t 1 Separation Voltage (SV) Compensation Voltage (COV) ( E) K( E) K(0) K(0) Ions migrate towards one of the planar electrodes If an ion s high-field mobility K(E) is larger than its low-field mobility K(0), >0 If an ion s low-field mobility K(0) is larger than it high-field mobility K(E), <0 Any ion can be steered back onto the center-line, by application of a compound-specific DC compensation voltage (CV). 26 2016 SCIEX RUO-MKT-18-1166 A

Tuning the SelexION Ion Mobility Device Rel. Int. (%) 100% 50% The ion residence time, and hence the resolution of the ion mobility device, can be controlled. CV peak widths range from approximately 0.5 2.5 V FWHM. 0% -14-12 -10-8 -6-4 -2 0 2 4 6 8 10 12 14 Compensation Voltage (CV) In infusion mode, the compensation voltage (CV) parameter is ramped in order to determine the optimized value. Note that this is analogous to tuning any other compound-specific parameter, e.g. the collision energy (CE) 27 2016 SCIEX RUO-MKT-18-1166 A

Mixture of 6 androgens in 60:40 Methanol:Water Sample chromatogram from QTRAP 5500 LC/MS/MS system I n t e n s i t y, c p s XIC of +MRM (6 pairs): Exp 1, 287.2/97.0 Da ID: ASD from Sample 15 (Androgens mix 5) of Quant-RestekC18-150msec.wiff (Turbo Spr... 1.29e5 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 1.00e4 5000.00 75% B ASD (~500pg/mL) 0.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min 28 2016 SCIEX RUO-MKT-18-1166 A DHEA (~30ng/mL) T (~400pg/mL) 3.17 5 -ASD (~10ng/mL) DHT (~4ng/mL) AND (~10ng/mL) 80% B 98% B 55% B Max. 8.7e4 cps.

Structures of two androgens, DHEA and T Testosterone, C 19 H 28 O 2 CH 3 H CH 3 O Low ionization efficiencies Common precursor ion masses (m/z 289) Similar fragmentation patterns HO H H Similar chromatographic retention properties Dehydroepiandrosterone (DHEA), C 19 H 28 O 2 CH 3 OH CH 3 H H H O 29 2016 SCIEX RUO-MKT-18-1166 A

MS/MS of DHEA and Testosterone (m/z 289) (A) MS/MS fragmentation pattern (Collision Energy ramp) for DHEA CH 3 O HO CH 3 H H H Used for MRM transition (B) MS/MS fragmentation pattern (Collision Energy ramp) for Testosterone Used for MRM transition CH 3 H CH 3 OH H H O 30 2016 SCIEX RUO-MKT-18-1166 A

Resolving DHEA and Testosterone using the SelexION Ion Mobility Device SelexION device off SelexION device on Interference caused by the presence of Testosterone (a) No interference caused by the presence of Testosterone (b) DHEA (289.0/271.1) DHEA (289.0/253) DHEA (289.0/271.1) DHEA (289.0/253) a) There is a significant interference in the MRM channel for DHEA, caused by the presence of testosterone b) When the SelexION ion mobility device is employed the interference is completely removed. Thus there is no longer a need for chromatographic separation, and run-times can be reduced dramatically. 31 2016 SCIEX RUO-MKT-18-1166 A

Liquid-Liquid Extraction (LLE) LC-MS/MS 289/109 289/97 Interference Testosterone peak in female serum Interference Testosterone peak in female serum Area ratio ~ 23 Expected ratio =1.23 x Liquid-Liquid Extraction (LLE) LC-MS/MS with SelexION Technology 289/109 289/97 Area ratio = 1.28 Expected ratio =1.23 32 2016 SCIEX RUO-MKT-18-1166 A

Protein Precipitation (PPT) LC-MS/MS 289/109 Testosterone 289/97 peak in female serum Testosterone peak in female serum Protein Precipitation (PPT) LC-MS/MS with SelexION Technology 289/109 289/97 33 2016 SCIEX RUO-MKT-18-1166 A

STD Curve in Serum Sample using the SelexION Ion Mobility Device (sample preparation = PPT) Accuracy 96-105% 4 female subjects 4 serum samples from females Sample Neat (N=28) Serum PPT (N=25) Serum LLE (N=25) Sample Neat (n=28) Patient PPT (n=25) Patient LLE (n=25) Average Ion Ratio (97/109) 1.23 1.28 1.24 STDEV 0.04 0.07 0.04 %CV 3.44 5.47 3.12 34 2016 SCIEX RUO-MKT-18-1166 A

SelexIon performance for Estrogen Analysis Serum (40 ul) by PPT and no SelexIon I n t e n s i t y, c p s XIC of -MRM (3 pairs): 275.200/147.100 Da ID: d4-e2 from Sample 1 (PlasmaNew ppt 15) of 111220-NoDms-PlasNew-15.wiff (Turbo Spray) 9000 8000 7000 6000 5000 4000 3000 Max. 6.8e4 cps. 2000 1000 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time, min XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa... Max. 3710.6 cps. XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa... Max. 1.4e4 cps. offset by -2.05 min offset by -2.05 min E2 I n t e n s i t y, c p s 1000 800 600 400 7.827.98 8.72 I n t e n s i t y, c p s 9000 8000 7000 6000 5000 4000 3000 8.68 E1 200 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min 2000 1000 7.76 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min 35 2016 SCIEX RUO-MKT-18-1166 A

SelexIon performance for Estrogen Analysis Serum (40 ul) by PPT with SelexIon I n t e n s i t y, c p s XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sample 6 (plasma 15ppt) of 111222-Plasma15-demifast.wiff (Turbo Spray) 2313 2000 1500 1000 8.66 Max. 2313.0 cps. 500 I n t e n s i t y, c p s 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time, min XIC of -MRM (3 pairs): 271.200/145.100 Da ID: E2 from Sa... Max. 289.3 cps. XIC of -MRM (3 pairs): 269.200/145.100 Da ID: E1 from Sa... Max. 2313.0 cps. 8.64 289 250 200 150 100 E2 I n t e n s i t y, c p s 8.66 2313 2000 1500 1000 E1 50 500 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min 36 2016 SCIEX RUO-MKT-18-1166 A 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time, min

Spiked plasma sample analyzed without SelexION (A) and with SelexION Technology (B). The light blue marked regions defined where signal and noise were collected. E1: S/N (1 sigma): 77 E2: S/N (1 sigma): 27 E1: S/N (1 sigma): 462 E2: S/N (1 sigma): 162 37 2016 SCIEX RUO-MKT-18-1166 A

Steroid Panel imethod

Increasing the number of compounds analysed in an assay is a perfect way of maximising potential return on investment LC-MS/MS is ideally suited to this approach The SCIEX Steroid Panel imethod allows simultaneous analysis of 10 commonly measured steroids and also 25- Hydroxyvitamin D3 Sample preparation is simple protein precipitation with dilution LC separation is required in order to keep the sample preparation to a minimum, run time is 12.5 minutes In order to achieve the required sensitivity for all compounds assayed, this method is designed for use on the API5000 and Triple Quad 5500 instruments 39 2016 SCIEX RUO-MKT-18-1166 A

Steroid Panel imethod, Extracted sample at ULOQ 40 2016 SCIEX RUO-MKT-18-1166 A

Performance Data, Steroid Panel imethod 41 2016 SCIEX RUO-MKT-18-1166 A

SCIEX offers several solutions for steroid analysis Various forces will determine which approach is needed Analysis of less challenging steroids Requirements for analysis of multiple compounds Sensitivity requirements on challenging steroids Desire for the convenience of a kit-based assay Multiplexing with additional applications From this, the appropriate method and instrument can be matched to the analytical requirements 42 2016 SCIEX RUO-MKT-18-1166 A

Trademarks/Licensing SCIEX is doing business as SCIEX. For Research Use Only. Not for use in diagnostic procedures. 2016 SCIEX. The trademarks mentioned herein are the property of SCIEX Pte. Ltd. or their respective owners. SCIEX is being used under license. 43 2016 SCIEX RUO-MKT-18-1166 A