LCMS-8030 Application Report. Steroids: Testosterone, Progesterone, Estradiol, Ethinylestradiol

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1 LCMS-8030 Application Report Steroids: Testosterone, Progesterone, Estradiol, Ethinylestradiol

2 Summary: Sensitivity was tested for four steroids on the LCMS-8030 triple quadrupole mass spectrometer coupled with a Nexera UPLC. Background: Steroids are powerful hormones that are needed for normal biological activity but may also be present in the environment. Water contaminated with steroids poses a serious environmental and public health threat. Sensitive and rapid methods are needed for accurate quantitative analysis. UPLC-MS-MS methods for analysis of these compounds were developed and the sensitivity for each analyte was tested. Method: Standards for four steroids were obtained at a concentration of 1 mg/ml in methanol. The standards were: Testosterone (T4), Progesterone (P4), Estradiol (E2), and Ethinylestradiol (EE2). Standards were diluted in 50/50 water/acetonitrile for optimization of mass spectrometry parameters. Multiple Reaction Monitoring (MRM) was used for analysis. Collision energy, quadrupole prerod bias voltage, and other MS parameters were optimized by flow injection of the individual standards. T4 and P4 ionized most efficiently using a 0.1% formic acid/acetonitrile mobile phase using the DUIS dual ion spray (ESI with corona discharge) source in positive mode. The MRM transition of m/z was monitored for T4 and the transition of m/z was used for P4. E2 and EE2 ionized most efficiently using a water/acetonitrile mobile phase with atmospheric pressure chemical ionization (APCI) in negative Testosterone Progesterone Level T4 P4 E2 EE2 L L L L L L L L Estradiol Ethinylestradiol Table 1: Dilution levels (ng/ml) Figure 1: Structures of tested steroids Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V) MRM 1 + Testosterone 288.9> MRM 2 + Progesterone 315.4> Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V) MRM 1 - Estradiol 271.4> MRM 2 - Ethinylestradiol 295.4> Measurement Time (min) Measurement Time (min) Table 2: MRM parameters Page 2

3 Area(x1,000,000) P4 r 2 = T4 r 2 = SetEPAx01.lcb Conc SetEPAx01.lcb Conc SetEPAx01_15_EPAMIX2L10_015.lcd 5.0 (x100) 1:315.40>109.10(+) P4 Level SetEPAx01_8_EPAMIX2L12_008.lcd (x100) 2:288.90>97.00(+) T4 65 ng/ml SetEPAx01_29_EPAMIX2L05_029.lcd (x100,000) 1:315.40>109.10(+) P4 Level 2 Figure 2: Calibration curves and selected mass chromatograms for P4 and T SetEPAx01_29_EPAMIX2L05_029.lcd (x100,000) 2:288.90>97.00(+) T4 Level 2 mode. Therefore these compounds were analyzed in a separate method. The MRM transitions for E2 and EE2 were and respectively. A Shimadzu Shimpack-XR DS III column (1.6 µm, 2 50 mm) was used for analysis. A binary gradient of 0.1% formic acid and acetonitrile was used for T4 and P4 while a binary gradient of water and acetonitrile was used for E2 and EE2. The gradient began at 55% acetonitrile and increased to 95% over 1 minute. After a 6 second isocratic hold, the column was re-equilibrated for approximately 1 minute. The column temperature was 40 C and the injection volume was 10 µl. Calibration curves were prepared by serial dilution of a mixed standard as shown in Table 1. Results and Discussion: Representative chromatograms of the T4/P4 and E2/EE2 methods are shown in Figures 2 and 3. The limit of detection (LD, S/N > 3) for P4 was between and 82 ng/ml using a 10 µl injection, and the limit Page 3

4 E2 r 2 > EE2 r 2 > SetEPAx02.lcb Conc SetEPAx02.lcb Conc SetEPAx02_11_E2EE2L09_011.lcd SetEPAx02_30_E2EE2L03_030.lcd (x10) 4.0 1:271.40>183.10(-) (x10,000) 1:271.40>183.10(-) E2 2 ng/ml E2 Level SetEPAx02_10_E2EE2L09_010.lcd SetEPAx02_30_E2EE2L03_030.lcd (x10) 2:295.40>145.00(-) (x10,000) 0 2:295.40>145.00(-) EE2 2 ng/ml EE2 Level 3 Figure 3: Calibration curves and selected mass chromatograms for E2 and EE2 of quantitation (LQ, S/N >10) was approximately ng/ml. For T4, the LD was approximately 65 ng/ml and the LQ was approximately 0.2 ng/ml. For T4 and P4, the calibration curve was constructed between and 370 ng/ml. The curves were linear in the tested range (r 2 > 0.99). For E2, the LD was approximately ng/ml and the LQ was approximately 4.5 ng/ml. The LD and LQ for EE2 was similar. The calibration curves were constructed from 4.57 ng/ml up to 10 µg/ml, and the curves were both linear (r 2 > 0.99) Conclusion: Rapid and sensitive methods for determination of four steroids was developed using UPLC-MS-MS. Page 4

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