Sensitive steroid analysis on a new High Speed Triple Quadrupole LC/MS/MS. Mikaël LEVI Shimadzu France

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1 Sensitive steroid analysis on a new High Speed Triple Quadrupole LC/MS/MS Mikaël LEVI Shimadzu France Welcome Seminar Barcelona 2013

2 Interest in Steroids assay by LC-MS/MS Steroids measurement is essential for diagnosis of: Congenital adrenal hyperplasia (CAH), Mineralocorticoid excess, Familial hyperaldosteronism type I, Primary aldosteronism, Cushing s disease, Adrenal insufficiency, Sexual differentiation and gonadal function. Precocious puberty, premature thelarche, and polycystic ovary disease, Etc Most methods are based on immunoassays Rapid and easy, Lack of specificity and matrix effects.

3 Interest in Steroids assay by LC-MS/MS Advantages of LC-MS/MS : Specificity (LC separation of steroids and their conjugates), Precision, Sensitivity, low sample volume required for pediatric diagnosis, Automation and high throughput, Possibility to simultaneously assay several steroids for profiling, Speed?

4 Speed and Sensitivity in LC-MS/MS More and more samples are submitted to clinical labs for steroid profiling, Need for fast and reliable assay : Simple sample pre-treatment, Fast analysis to deliver results, Reliable and reproducible results (high precision and accuracy). Fast LC-MS/MS Need for sensitivity : To diagnose some steroid disorders, very low steroid concentrations must be reached. Sensitive LC-MS/MS

5 Sample preatreatment Charcoal stripped plasma was spiked with steroid mix (max organic solvent amount of %), Spiked plasma sample (200 µl) was spiked with ISTD and half diluted with water, Diluted sample was loaded under gravity on ISOLUTE SLE+ 400 cartridges (Biotage), After 5 min contact, steroids were eluted with 2x1 ml of dichloromethane, After evaporation, dry residue was reconstituted in water/methanol 8/2 (50 µl).

6 Analytical conditions UHPLC : System : NEXERA X2 with binary high pressure gradient, Column : YMC Triart C18 1.9µm 50x2mm, Temperature : 45 C, Mobile phases : Water + 5% ammonia / Methanol Flow-rate : 600 µl/min, Gradient : Injected volume : 30µL,

7 Analytical conditions Mass spectrometry : System : LCMS-8050 with Heated ESI, Temperatures: HESI : 400 C, Heater block : 500 C, Desolvatation line : 150 C, Gas flows : Heating Gas : 10 L/min (Air), Nebulizing Gas : 3 L/min (N 2 ), Drying Gas : 10 L/min(N 2 ), Acquisition parameters : Pause time : 1 msec, Dwell time : 4 to 99 msec, Polarity switching : 5 msec,

8 Analytical conditions Mass spectrometry : 2 MRM per target compound / 1 MRM for ISTD, Acquisition window RT± min, Max loop time 0.2 s at least 30 points per peak/mrm

9 Choice of the ionization source Steroids are classically ionized by APCI or more rarely APPI, Shimadzu Heated ESI enables high sensitivity and ruggedness, Softer but efficient conjugated steroid ionized in intact form (see DHEA sulfate).

10 Source parameters optimization Use new Interface Setting Support software, Creates a factorial design of experiments for source parameters : Temperatures and gas flow rates.

11 Source parameters optimization Sample DHEA:Dehydroepiandrosterone m/z 271.2>253.3(Black) AS:Androsterone m/z 273.2>255.3(Pink) Neb L/min DL 150 HB 300 IF 300 Neb 3.0L/min DL 150 HB 300 IF 300 Neb 3.0L/min DL 250 HB 300 IF 300 Analysis Conditions Analytical Column: YMC Triart Mobile Phase A: 25% ammonium hydroxide Mobile Phase B: Methanol /Isopropanol Gradient Program: Time(min) B conc Flow Rate: 0.4 ml/min Injection Volume: 1 L Column Oven Temperature: 45 C Heating Gas Flow: Drying Gas Flow: 10L/min 10L/min Neb L/min DL 150 HB 500 IF 300 Neb L/min DL 150 HB 500 IF 400 Neb 3.0L/min DL 150 HB 500 IF 300 Neb 3.0L/min DL 150 HB 500 IF 400 Neb 3.0L/min DL 250 HB 500 IF 300 Neb 3.0L/min DL 250 HB 500 IF 400 Neb: Neburizer gas flow DL: DL temperature HB: Heat Block temperature IF: Interface temperature Export the method from Data Browser window and apply sample analysis.

12 Results Typical chromatogram DHEA 17a-hydroxyprogestérone Aldostérone Estradiol Cortisol Progestérone DHEA Sulfate 11-désoxycortisol Testostérone Androstènedione Corticostérone min

13 Results High sensitivity and reproducibility : 1 pg/ml standard in plasma (120 fg on column), 3 replicate extracts, Aldosterone Testosterone Androstenedione 17-OHP Progesterone min min min min min

14 Thank s for your attention

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