Xianrong (Jenny) Wei, Sean Orlowicz, and Kristen Parnell Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501, USA

Increased Capabilities for the Analysis of Hormones in Drinking and Waste Water using SPE and LC/MS/MS

Introduction LC/MS/MS Conditions Table 3. Extracted Matrix Recovery Table 4. Method Limits and Linearity for Each Results and Discussion Analyte Column: Kinetex® 5 µm EVO C18 Sample: The presence of various hormones extraction and separation method, all Dimensions: 100 x 2.1 mm 1. Estriol On the foundation of EPA Method 539, allow an alkaline mobile phase to be Part No.: 00D-4633-AN 2. Equilin 3. Estrone 13C in worldwide drinking water supplies while shortening the run time to less Mobile Phase: A: 0.2% NH OH in Water 3 a fast and reproducible SPE method used without compromising column 4 4. Estrone B: 0.2% NH OH in Methanol 4 for analyzing an expanded hormones lifetime, and increases compound sen- has been of public concern for some than 14 minutes. We expand the sam- 5. 17β – Estradiol D5 Gradient: Time (min) % B 6. β – Estradiol list (Table 1) in drinking and waste wa- sitivity significantly. The lowest detec- time. As the scientific community tries ple preparation technique to employ a 0 30 7. 17α - Ethynylestradiol to identify acceptable exposure limits, more versatile format for ease of use 1 65 8. Norethisterone ter using LC/MS/MS is developed. In tion limits on an AB Sciex 4000 QTRAP 6 65 9. Androstenedione 10. Testosterone D this study, we use Strata C18-E SPE can be reached at 0.05 ng/L for most many new endogenous and synthetic in a variety of water matrices. By uti- 6.5 85 3 11. Testosterone 10.5 85 in tube format to bring benefits such analytes (Table 4). hormones are being discovered. Com- lizing a SPE tube format and exploring 12. Progesterone D9 11 30 13. Progesterone as faster processing, increased sensi- pounds such as ethynylestradiol, the various particle sizes, differing water 13.5 30 To improve sample analysis for high Flow Rate: 200 µL/min tivity, consistency, and extraction effi- active ingredient in a commonly pre- sources can be scaled and processed throughput in the lab, we have opti- Injection Volume: 30 μL ciency (Tables 2 and 3). scribed birth control medication, are more easily. In addition, the final ex- Instrument: Shimadzu® 30-AD and Agilent® 1260 LC mized LC/MS/MS procedures to effec- Detection: AB Sciex API 4000™ QTRAP® known to cause detrimental effects tract concentration levels can be easily AB Sciex API 4500™ The high pH mobile phase greatly im- tively analyze all target compounds in to both aquatic and human life. Many monitored and controlled to be more Samples (n=3) were collected on 26 March 2015 in Torrance, CA 90501, USA. proves the ionization of some of the tar- one injection within 13.5 minutes (Fig- other compounds are currently being appropriate for a variety of different Note: The ionization source was ESI with rapid polarity switching in positive and negative ion mode. Shimadzu 30-AD LC series, inter- Tap water contains all the possible contaminants, so the recoveries of analytes are various, MRL - Method reporting limit get compounds, resulting in improved ures 3, 4 and 5), with mass spectrom- faced with an AB Sciex API 4000 QTRAP, was for lowest detection level test only; all other experiments were done on Agilent 1260 LC but within ±30% of acceptance criteria, respectively. LOD - Lowest of detection level LC/MS/MS sensitivity. Because con- eter polarity switching. The represen- investigated. Due to this public risk, detectors, depending on the required series with an AB Sciex API 4500 mass spectrometer. Linear Regression at 1/X ventional silica-based HPLC media is tatives of linearity of analytes across there is a rapidly growing interest in sensitivity. unstable under alkaline conditions, the all positive and negative analytes are monitoring these compounds. Within choice of analytical columns is very shown in Figures 6 and 7. the United States, EPA Method 539 This study also offers an optimized limited. In this study, we selected a was specifically developed to monitor LC/MS/MS method that explores ion- Figure 2. Structure of the Organo-Silica Figure 3. Representative of TIC Chromatogram Figure 6. Representative of Curve – Testosterone Phenomenex Kinetex 5 µm EVO C18 this growing problem. ization polarities and utilizes a high Kinetex EVO Particle 6.4e6 50 100 x 2.1 mm column, a core-shell or- pH mobile phase in order to maximize gano-silica particle which incorporates 6.0e6 This study builds on the foundation LC/MS/MS response. Because con- 45 Curve range: 0.5 – 50 ng/L uniform stabilizing ethane cross-link- of EPA 539 by providing an expand- ventional silica-based HPLC media is 5.5e6 ing and provides resistance to high pH 40 ed list of target analytes to include not stable under alkaline conditions, Kinetex EVO: a patented 5.0e6 attack, all while maintaining mechani- cal strength (Figure 2). These benefits compounds of current interest (e.g. a core-shell organo-silica column was organo-silica grafting process 35 4.5e6 progesterone) and a more modern used to perform these analyses. which incorporates uniform Analyte Area / IS 4.0e6 30

stabilizing ethane cross-link- Intensity, cps 3.5e6 ing to provide resistance to 25 high pH attack while maintain- 3.0e6 20 Figure 1. Structures of Hormones ing mechanical strength of the 2.5e6 Conclusion With Polarity 15 core-shell particle. 2.0e6 Switching 1.5e6 10 EPA Method 539 is a liquid chromatog- recovery and reproducibility. LC/MS/ 1.0e6 raphy, electrospray ionization, tandem MS analysis time was significantly App ID 22973 5 App ID 22978 17α- Ethynylestradiol -Estradiol Estriol mass spectrometry method for the reduced from 55 minutes to 13.5 min- Stable to pH ~12 5.0e5 0 determination of hormones in finished utes with a Kinetex 5 µ EVO C18 100 x 0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min drinking water. This work presents an 2.1 mm column, which delivers stabil- Analyte Conc. / IS Conc. optimized method with an expanded ity under high pH mobile phase, while

Testosterone Estrone Androstenedione list of hormones that includes com- maintaining excellent linearity and

pounds of current interest (e.g. pro- low detection levels. This method will

gesterone and norethisterone) to suc- significantly increase laboratory pro- Table 1. MRM Transitions Figure 4. Representative of Positive Ion Figure 7. Representative of Curve – β-Estradiol cessfully monitor a growing problem ductivity, efficiency, and throughput. 8.5 in our scientific community. An SPE Chromatogram – 2.0 ng/L 8.0 Equilin Progesterone Norethisterone extraction method using Phenome- 4.7e5 7.5 4.5e5 5 - 6 Curve range: 0.5 – 50 ng/L 7.0 nex Strata C18-E provides excellent

6.5 4.0e5 6.0 Analyte Area / IS 3.5e5 1. Norethisterone 5.5 Experimental Conditions 2. Androstenedione 5.0 3.0e5 4.5 3. Testosterone D3 Intensity, cps Sample Preparation 4. Testosterone 4.0 2.5e5 5. Progesterone D 3.5 Pretreated Samples: 9 3.0 Per the EPA 539 method protocol, 1L water samples are dechlorinated, preserved, collected and stored. 2.0e5 6. Progesterone Acknowledgements All standards and QCs are freshly prepared in 50 % Methanol in water containing 20 ng/mL of working internal standards. 2.5

We evaluated silica-based (Strata® C18-E) and polymer-based (Strata™-X) SPE sorbents. Strata C18-E provides recoveries 1.5e5 2.0 of 91.1 – 103 % across all compounds (Table 3). 1.5 We would like to provide special thanks to Julissa Fernandez for sample preparation 1.0e5 1.0 Optimized Solid Phase Extraction Method and all PhenoLogix team members for their technical assistance. 1 - 2 Cartridge: Strata C18-E, 1 g/20 mL 0.5 5.0e4 3 - 4 App ID 22974 App ID 22979 Part No.: 8B-S001-JEG 0.0 Condition: 10 mL Methanol 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 0.0 Equilibrate: 10 mL Water 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min Analyte Conc. / IS Conc. Load: Pretreated samples Wash: 10 mL 15 % Methanol in water Dry: 5 – 10 minutes under 10” Hg vacuum Elute: 2 x 6 mL Methanol Dry down: Evaporate completely under a stream of nitrogen @ 50 °C Reconstitute: Add 1.0 mL of 50 % Methanol in water containing 20 ng/mL of working internal standards. Table 2. Mini Validation Results Figure 5. Representative of Negative Ion Chromatogram – 2.0 ng/L 9.0e4 3 - 4 8.5e4 8.0e4 1. Estriol SPE Accessories 7.5e4 2. Equilin 7.0e4 13 6.5e4 3. Estrone C3

6.0e4 4. Estrone 5.5e4 5. 17β–Estradiol D Intensity, cps 5 5.0e4 6. β–Estradiol Trademarks STD 2 is Method Reported Limit (MRL) 4.5e4 7. 17α–Ethynylestradiol Strata and Kinetex are registered trademarks, and Strata-X is a trademark of Phenomenex. 4.0e4

3.5e4 Agilent is a registered trademark of Agilent Technologies. Shimadzu is a registered trademark of Shimadzu 3.0e4 Corporation. API 4000 and API 4500 are trademarks, and QTRAP is a registered trademarks of AB SCIEX Pte. 2.5e4 Ltd. AB SCIEX is being used under license. 2.0e4 5 - 6 1.5e4 Kinetex EVO is patented by Phenomenex. U.S. Patent No. 7,563,367 and 8,658,038 and foreign counterparts. 7 1.0e4 1 2 Strata-X is patented by Phenomenex. U.S. Patent No. 7,119,145 5000.0 App ID 22975 0.0 © 2015 Phenomenex, Inc. All rights reserved. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 min PO32020415_W