Integration of steroids analysis in serum using LC-MS/MS with full-automated sample preparation

Transcription

1 PO-CON69E Integration of steroids analysis in serum using LC-MS/MS with full-automated sample preparation MSACL 6 EU Stéphane Moreau, Daisuke Kawakami, Toshikazu Minohata Shimadzu Europe GmbH, Duisburg, Germany, Shimadzu Corporation, Kyoto, Japan

2 Introduction Currently sample preparation for the detection of steroids in serum by liquid chromatography-mass spectrometry (LC-MS/MS) involves complex offline extraction methods such as solid phase extraction or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are time-consuming, often taking one hour or more per sample, and are more vulnerable to variability due to analyst errors during manual preparation. Our approach is offering a high sensitivity steroid detection fully automated for multiple samples. It is using an automated sample preparation coupled to the detection capabilities of a high sensitivity triple stage quadrupole mass spectrometer, that requires no human intervention from loading the samples to obtaining the results. Method steroid hormones (cortisol, aldosterone, -deoxycortisol, corticosterone, 7-alpha-hydroxyl-progesterone (7-OHP), 4-androstene-3,7-dione (androstenedione), dehydroepiandrosterone (DHEA), dehydroepi-androsterone sulfate (DHEAS), progesterone and testosterone) in serum were verified using CHS MSMS Steroids Kit (PerkinElmer, USA). Serum sample was loaded directly into the automated sample preparation system (CLAM- Shimadzu, Japan). The CLAM- was programmed to perform protein precipitation using acetonitrile followed by filtration and sample collection. The sample is then transported using an arm from the CLAM- to the HPLC without human intervention for LC-MS/MS analysis. Fig. CLAM- and LCMS-86 system Dispensing 3 µl of serum Reagent Dispensing 6 µl of ACN with IS Shaking sec Filtration sec To Autor

3 The treated samples were trapped using a MAYI-ODS column and then separated by Core-Shell Biphenyl HPLC column at 4 ºC with a binary gradient system at a flow rate of.3 ml/min in min. Trap Trap column Analysis waste pump C pump A analytical column pump B LCMS Fig. Flow Diagram of Trapping system Table Analytical Condition HPLC Mobile Phase A : mm ammonium fluoride water Mobile Phase B : Methanol Mobile Phase C : mm ammonium formate water Column temperature : 4 ºC Analytical Column : Kinetex Biphenyl (mm L x mm I.D.,.6μm) Guard Column : MAYI-ODS column (mm L x mm I.D.) Injection Volume : 3 µl Gradient Program : Mass (LCMS-86 triple quadrupole mass spectrometry) Ionization : heated ESI Nebulizing Gas Flow : 3 L / min Drying Gas Pressure : 7 L / min Heating gas flow : 3 L/min DL Temperature : ºC BH Temperature : 4 ºC Interface Temperature : 37 ºC MRM parameter : B Conc. (%) FCV(-) FCV(-6) Flow (ml/min) trapping B Conc..6 Pump A/B Flow.4 Pump C Flow

4 Result and discussion We evaluated this system using calibrator and control serum spiked with steroids contained in the kit and carried out concurrent analysis over a range of concentrations for each steroid: cortisol (-3 ng/ml), aldosterone (3-.4 ng/ml), -deoxycortisol (8-8 ng/ml), corticosterone (.9-6 ng/ml), 7-OHP (.-6 ng/ml), androstenedione (8-8 ng/ml), DHEA (.3-6 ng/ml), DHEAS (.9-7 ng/ml), progesterone (.-6. ng/ml) and testosterone (3-7. ng/ml). The calibration curves that were generated had linear regression values of r >.997 for each curve. The reproducibility (N=3) at seven concentrations, including LLOQ of each compounds was excellent (CV<%) r =.997 r =.999 r =.999 r =.998 r = Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Aldosterone Cortisol DHEAS Coricosterone -Deoxycortisol r =.999 r =.999 r =.998 r =.999 r =.999 Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Conc. Ratio Androstenedione Testosterone 7-OHP DHEA Progesterone (x,) 36.>3.(+) >343.(+) CV=6.3% (N=3) (x,) 363.4>.(+) >97.(+). (x,) (x,) (x,) (x,) 7.>3.(+) 347.>.(+) 347.>9.(+) 87.>97.(+) 7.>97.(+) 347.>97.(+) >97.(+).6% 3.9% 4.6%.% 87.>9.(+) 4.6% Aldosterone (3 ng/ml) Cortisol ( ng/ml) DHEAS (.9 ng/ml) Coricosterone (.9 ng/ml) -Deoxycortisol (8 ng/ml) Androstenedione (8 ng/ml) (x,) (x,) (x,) (x,) (x,) (x,,) 89.>97.(+) 33.>97.(+) 7.>3.(+) 3.>97.(+) 39.>89.(-) 367.>97.(-) >9.(+) % 33.>9.(+) 6.% 7.>3.(+) 3.>9.(+) 39.>33.3(-) 6. 6.% 3.9% 6.6%.% Testosterone (3 ng/ml) 7-OHP (. ng/ml) DHEA (.3 ng/ml) Progesterone (. ng/ml) Aldosterone (neg) (3 ng/ml) DHEAS (neg) (.9 ng/ml) Fig. 3 Calibration Curves (L-L7) and MRM Chromatograms (L) of Steroids 4

5 We found that the sample preparation time was reduced from 6 minutes to 6 minutes by the automated system. Thus sample preparation and LC-MS/MS analysis can be performed in parallel to accelerate throughput. Traditional sample preparation (protein precipitation) Add ACN with IS Shake for min Centrifuge for 3 min Transfer the supernatants Dry for >3 min Reconstitute Automated sample preparation process by CLAM- Add ACN with IS Shake for 3 min Filtrate for min Fig. 4 Comparison with a time required for sample preparation injection min injection min injection LC/MS/MS analysis min LC/MS/MS analysis min Preparation Preparation Preparation Fig. Analytical Flow with Parallel Processing

6 Conclusion We completed steroid analysis using the automated sample preparation system coupled to LC-MS/MS. The results shows the capability of the system for large sample set analyses with improved accuracy and precision by eliminating human error associated with manual sample handling. First Edition: October, 6