Determination of ormones in Serum by L/MS/MS Using Agilent Bond Elut Plexa SPE Application Note Bio-analysis, Pharmaceutical, linical Research Authors Megan Juck and William Long Agilent Technologies, Inc. Abstract A method for the determination of 1 hormones and their respective internal standards in serum was developed. ormones were extracted from serum using Agilent Bond Elut Plexa Solid Phase Extraction (SPE) ( mg, 1 ml) cartridges. They were separated on an Agilent InfinityLab Poroshell P- column (.1 mm,. µm), and analyzed by liquid chromatography tandem mass spectrometry (L/MS/MS). ormones were analyzed using both positive and negative electrospray ionization (ESI) using a 1 mm ammonium fluoride mobile phase to improve hormone responses in both modes. verall recoveries ranged from to 1%, with relative standard deviations (RSDs) between. and.%. This application note demonstrates the ease-of-use and effectiveness of Bond Elut Plexa SPE cartridges for hormone determination in serum. Introduction A method using polymeric solid phase extraction (SPE) was developed for the determination of steroid hormones in serum. Table 1 shows the structures and chemical formulas for the 1 analyzed hormones. Agilent Bond Elut Plexa has a unique polymeric architecture with a hydroxylated, nonretentive, amide-free surface. Proteins and lipids bind minimally to the polymeric surface, resulting in cleaner samples and reduced matrix interferences. The nonpolar PS-DVB core is ideal for retaining small molecules such as hormones. This application note demonstrates the effectiveness and ease-of-use of Bond Elut Plexa SPE cartridges.
Table 1. Molecular formulas and structures for 1 analyzed hormones. Isobaric pair information is also included. ormone Molecular formula Structure ormone Molecular formula Structure Aldosterone 1 -Deoxycorticosterone 1 ortisol 1 Androstenedione 1 ortisone 1 Estrone 1 orticosterone 1 1α Progesterone 1 -Deoxycortisol 1 Dihydrotestosterone (DT) 1 β-estradiol 1 Progesterone 1 Testosterone 1 Isobaric pairs: Aldosterone and cortisone -Deoxycorticosterone and 1α progesterone -Deoxycortisol and corticosterone
Experimental Reagents and chemicals All reagents were PL grade or higher. Methanol was bought from oneywell (Muskegon, MI, USA.) Water was purified using an EMD Millipore Milli-Q Integral System (Darmstadt, Germany.) Reagent-grade formic acid (FA, p/n G-) was from Agilent Technologies. Ammonium fluoride was from Sigma-Aldrich (St. Louis, M, USA). -Deoxycorticosterone d was from Toronto Research hemicals (Toronto, ntario, anada). All other hormones and internal standards were from Sigma-Aldrich. Serum (D Mass Spect Gold, MSG) was bought from Golden West Biologicals, Inc. (Temecula, A, USA). Serum was stored at until day of use. Equipment and materials Eppendorf pipettes and repeater oncentration evaporation system Vortexer and multitube vortexers (VWR, Radnor, PA, USA) Agilent Vac Elut SPS manifold with collection rack for 1 1 mm test tubes (p/n 1) Agilent Bond Elut Plexa, mg, 1 ml straight barrel cartridges (p/n 1) Agilent MS analyzed amber screw-top glass vials with write-on spot (p/n 1-1) Agilent bonded screw cap, PTFE/red silicone septa (p/n 1 ) Agilent vial insert, µl, deactivated glass with polymer feet (p/n ) Sample preparation A hormone standard mix and hormone internal standard mix were each prepared in methanol. Table lists the concentrations for each analyte and its respective internal standard. Each internal standard was kept at the same concentration as its respective analyte. For example, aldosterone was at a concentration of ng/ml in the hormone standard mix, and aldosterone-d was ng/ml in the hormone internal standard mix. Table. ormone and internal standard concentrations are provided. Internal standards were spiked at the same concentration as parent analytes. Analyte/internal standard Aldosterone/aldosterone d ortisol/cortisol d ortisone/cortisone d orticosterone/corticosterone d -Deoxycortisol/-deoxycortisol d β-estradiol/β-estradiol d Testosterone/testosterone d -Deoxycorticosterone/-deoxycorticosterone d Androstenedione/androstenedione 1 Estrone/estrone d 1α Progesterone/1α progesterone d DT/DT d Progesterone/progesterone d Standard concentration, each (ng/ml) Instrumentation Analysis was performed on an Agilent 1 Infinity L system consisting of: Agilent 1 Infinity binary pump (GA) Agilent 1 Infinity high performance autosampler (GA) equipped with an Agilent 1 F/ALS thermostat (G1B) Agilent 1 Infinity thermostatted column compartment (G) The L system was coupled to an Agilent A triple quadrupole L/MS/MS system equipped with Agilent Jet Stream Electrospray ionization technology. Agilent Massunter workstation software was used for all data acquisition and analysis.
Sample pretreatment Prior to the SPE protocol, µl of.% formic acid (FA) in water was added to 1 µl of serum. Samples were spiked with µl of hormone standard mix and µl of hormone internal standard mix. The final concentration was 1 ng/ml for β-estradiol, β-estradiol d, estrone, estrone d, DT, and DT d. All other hormones and internal standards were at a final concentration of 1 ng/ml in serum. Samples were vortexed and centrifuged to remove any serum particulates. After SPE, the collected eluent was evaporated to dryness under nitrogen at, then reconstituted with 1 µl of / Me/water. Sample was vortexed, then transferred to a vial with a glass insert for L/MS/MS analysis. SPE Protocol ondition: 1 ml Me +.% FA Equilibrate: 1 ml water +.% FA Load: Pretreated serum sample Wash: 1 ml % Me in water Dry: minutes (minimum 1 inch g) Instrument conditions Parameter PL olumn: Guard: Mobile phase: Flow rate: Value olumn temperature: Autosampler temperature: Injection volume: 1 µl Needle wash: Agilent InfinityLab Poroshell P-,.1 mm mm,. µm (p/n ) Agilent InfinityLab Poroshell P-, UPL guard column,.1 mm mm,. µm (p/n 1 ) A) 1 mm ammonium fluoride in water B) acetonitrile (AN). ml/min Gradient: Time (min) % B Stop time: Post-time: MS Electrospray Ionization (ESI) 1:1:1:1 AN/Me/isopropanol/water with. % FA 1 minutes 1 minute Gas temperature: Gas flow: Nebulizer: L/min psi Sheath gas heater: Sheath gas flow: apillary: Nozzle voltage: Delta EMV (+): Delta EMV ( ): L/min Positive, V; Negative, V Positive V; Negative 1, V V V Elute*:. ml Me *Gravity elution was performed. After the eluent flow stopped, a low vacuum (< inch g, - seconds) was applied to fully collect all eluent. Figure 1. SPE protocol for hormone determination using an Agilent Bond Elut Plexa mg, 1 ml cartridge.
Table. L/MS/MS dmrm parameters and retention times for target analytes. Analyte RT (min) Precursor ion (m/z) Frag (V) Quant ion (m/z) Product ion E (V) Qual ion (m/z) Aldosterone 1. 1. 1. 1 1. 1 Positive ortisol 1.. 1 11.1 1.1 Positive ortisone. 1. 1 1.1 11 Positive orticosterone.1.. 1 11 Positive -Deoxycortisol.. 1 1.1.1 Positive E (V) Ionization mode β-estradiol. 1.1 1 1.1 1 Negative Testosterone.1. 1 1.1.1 Positive -Deoxycorticosterone.1 1. 1 1.1.1 Positive Androstenedione.. 1.1.1 Positive Estrone..1 1 1.1 1 Negative 1α Progesterone.1 1. 1 1.1.1 Positive DT. 1. 1.1.1 Positive Progesterone. 1. 1 1.1.1 Positive Table. L/MS/MS dmrm parameters and retention times for internal standards. Analyte RT (min) Precursor ion (m/z) Frag (V) Product ion Quant ion (m/z) E (V) Aldosterone d 1.. 1. 1 Positive ortisol d 1.. 1 11 Positive ortisone d.. 1 1.1 Positive orticosterone d.1. 1 11 Positive -Deoxycortisol d.. 1 1. Positive Ionization mode β-estradiol d.. 1 1.1 Negative Testosterone d.1. 1.1 Positive -Deoxycorticosterone d.1. 1.1 Positive Androstenedione 1.. 1 1.1 Positive Estrone d..1 1 1.1 Negative 1α Progesterone d.. 1 1.1 Positive DT d.1. 1. 1 Positive Progesterone d.. 1 1.1 Positive
ounts 1 1. 1. 1. 1. 1. 1. 1. 1. 1.1 1.. A Peak ID 1. Aldosterone (1 ng/ml). ortisol (1 ng/ml). ortisone (1 ng/ml). orticosterone (1 ng/ml). -Deoxycortisol (1 ng/ml). β-estradiol (1 ng/ml). Testosterone (1 ng/ml). -Deoxycorticosterone (1 ng/ml). Androstenedione (1 ng/ml) 1. Estrone (1 ng/ml). 1α Progesterone (1 ng/ml) 1. DT (1 ng/ml) 1. Progesterone (1 ng/ml) ounts 1........... 1. 1.. B 1....1..........1.. 1..... 1...1 1. 1. 1. 1............................ Figure. Final dmrm chromatogram of 1 analyzed hormones in serum (A), with a zoomed in chromatogram (B) showing β-estradiol and estrone. oncentrations are provided in the legend. 1 1
ounts 1 1. A 1. 1. 1. 1. 1. 1. 1. 1.1 1... Peak ID 1. Aldosterone d (1 ng/ml). ortisol d (1 ng/ml). ortisone d (1 ng/ml). orticosterone d (1 ng/ml). -Deoxycortisol d (1 ng/ml). β-estradiol d (1 ng/ml). Testosterone d (1 ng/ml). -Deoxycorticosterone d (1 ng/ml). Androstenedione 1 (1 ng/ml) 1. Estrone d (1 ng/ml). 1α Progesterone d (1 ng/ml) 1. DT d (1 ng/ml) 1. Progesterone d (1 ng/ml) ounts 1 B...... 1..... 1. 1.......1..........1.. 1.......1 1 1 1 1. 1. 1. 1............................ Figure. Final dmrm chromatogram of 1 analyzed hormone internal standards in serum (A) with a zoomed in chromatogram (B) showing β-estradiol d and estrone d. oncentrations are provided in the legend.
Results and Discussion SPE ptimization When developing an SPE method, it is important to use a strong enough wash solvent to remove matrix interferences, while ensuring your analytes of interest remain retained on the cartridge. By collecting and analyzing the wash eluent from each of the candidate wash solvents (1% water, 1% Me in water, % Me in water, and so forth), hormone loss can be determined. After evaluation, a % Me in water wash was chosen as the optimal wash for this protocol. Similar to the wash step, the elution solvent can be optimized as well. The goal of the elution step is to choose a solvent that is strong enough to elute all the target analytes, without eluting matrix interferences that may be retained on the sorbent during the SPE procedure. For this application note, % Me in water, % Me in water, 1% Me, and / Me/AN elution solvents were evaluated. The 1% Me elution solvent provided the highest hormone recoveries, and was therefore chosen for this application. L/MS/MS ptimization During initial L/MS/MS method development, hormones were analyzed: aldosterone, cortisol, cortisone, corticosterone, -deoxycortisol, androstenedione, -deoxycorticosterone, testosterone, 1α progesterone, DT (negative mode), and progesterone. Two negative mode analytes (β-estradiol and estrone) were later added to the method, and therefore were not included in method development chromatograms. An Agilent InfinityLab Poroshell 1 E- column (.1 mm,. µm) was compared to an Agilent InfinityLab Poroshell 1 E-1 column (.1 mm,. µm) (Figure ). The shorter alkyl chain of the E- delivered slightly shorter retention times for the hormones, while still separating all isobaric pairs. onditions Mobile phase A: Water +.1% formic acid Mobile phase B: Methanol +.1% formic acid Gradient program: minutes: % mobile phase B to % mobile phase B,.1 minutes: % mobile phase B, hold ounts ounts 1 1 1 1..........1 A Agilent InfinityLab Poroshell 1 E-,.1 mm x mm,. µm 1 B Agilent InfinityLab Poroshell 1 E-1,.1 mm x mm,. µm 1.. 1. 1. 1..................... Figure. omparison of separation and retention for hormones (1 ng/ml) using Agilent InfinityLab Poroshell 1 E and E-1 columns. β-estradiol and estrone were added to the final method after this chromatogram was generated, and are not included in this chromatogram., Total run time: 1 minutes + 1 minute post-time Flow rate:. ml/min Injection volume: 1 µl Peak ID 1. Aldosterone. ortisol. ortisone. orticosterone. -Deoxycortisol. Androstenedione. -Deoxycorticosterone. Testosterone. 1α Progesterone 1. DT 1. Progesterone 1
Formic acid (.1%), ammonium hydroxide (.%, p 1.), and ammonium fluoride (p.) mobile phases were also evaluated (Figures -). igh p mobile phase with ammonium hydroxide (.%) has been shown to improve ionization of hormones analyzed in negative mode [1]. Ammonium fluoride has been shown to improve responses in both positive and negative modes [,]. Since most silica-based L columns are not recommended for mobile phases with a p higher than or, an Agilent InfinityLab Poroshell P- column was used for evaluation. This column is stable up to a p of, and provides very similar selectivity to an InfinityLab Poroshell 1 E- column. Therefore, the InfinityLab Poroshell P- column was used for method optimization with both ammonium hydroxide and ammonium fluoride mobile phases, and chosen for the final analytical method. Ammonium fluoride (1 mm) mobile phase yielded the highest analyte responses, and was used for the final chromatographic method. onditions Mobile phase A: Mobile phase B: ounts 1 Peak ID.% Ammonium hydroxide in water 1. Aldosterone.1% Formic acid in water..... 1... 1. DT............. 1. 1...1% Formic acid in water or.% ammonium hydroxide in water Methanol Gradient program: minutes: % mobile phase B to % mobile phase B minutes: % mobile phase B to % mobile phase B Total run time: 1 minutes + 1 minute post-time Flow rate:. ml/min Injection volume: 1 µl. 1. 1................. 1 ortisol ortisone orticosterone -Deoxycortisol Androstenedione -Deoxycorticosterone Testosterone 1α Progesterone. Progesterone Figure. hromatogram for a 1 ng/ml hormone sample with.1% formic acid in water (green trace) and.% ammonium hydroxide in water (red trace) using an Agilent InfinityLab Poroshell P-,.1 mm mm,. µm column. The L/MS/MS gradient was kept the same for comparison purposes. β-estradiol and estrone were added to the method after these chromatograms were generated, and are not included.
onditions Mobile phase A: Mobile phase B: ounts 1......... 1. 1.. 1 mm ammonium fluoride in water or. % ammonium hydroxide in water Methanol Gradient program: minutes: % mobile phase B to % mobile phase B minutes: % mobile phase B to % mobile phase B Total run time: 1 minutes + 1 minute post-time Flow rate:. ml/min Injection volume: 1 µl 1 mm Ammonium fluoride in water.% Ammonium hydroxide in water 1. 1. 1................ 1 Peak ID 1. Aldosterone. ortisol. ortisone. orticosterone. -Deoxycortisol. Androstenedione. -Deoxycorticosterone. Testosterone. 1α Progesterone 1. DT. Progesterone Figure. Total ion chromatogram for a 1 ng/ml hormone sample with 1 mm ammonium fluoride in water (black trace), and.% ammonium hydroxide in water (red trace). An Agilent InfinityLab Poroshell P-,.1 mm mm,. µm was used. The L/MS/MS gradient was kept the same for comparison purposes. β-estradiol and estrone were added to the method after these chromatograms were generated, and are not included. 1
ounts 1 1. 1. 1. 1. 1.1 1..........1 DT, 1 ng/ml 1 mm Ammonium fluoride in water.% Ammonium hydroxide in water.1% Formic acid in water.........1.........1.........1 Figure. MRM chromatogram overlay for DT (1 ng/ml; negative mode) with three mobile phases: 1 mm ammonium fluoride in water (black),.% ammonium hydroxide in water (red), and.1% formic acid in water (green). An Agilent InfinityLab Poroshell P-,.1 mm mm,. µm was used. Gradient conditions are provided in the descriptions of Figures and. Recovery and reproducibility Recovery and reproducibility were evaluated by extracting prespiked and blank serum samples using the SPE protocol previously described. Serum samples were prespiked with a concentration of 1 ng/ml for β-estradiol, estrone, and DT. The remaining 1 hormones were prespiked at a concentration of 1 ng/ml. Blank and spiked serum samples were extracted using the SPE protocol. Blank samples were postspiked after final dry down at a concentration of 1 and 1 ng/ml, as described earlier. Percent recovery and RSD values were determined by comparing average peak areas for prespiked and postspiked samples (n = ). Table. Percent recovery and RSD values (shown in parentheses) are provided (n = ). Analyte Fortified concentration (ng/ml) % Recovery (RSD) Aldosterone 1 (.) ortisol 1 1 (.) ortisone 1 (.) orticosterone 1 (.1) -Deoxycortisol 1 (.) β-estradiol 1 1 (.) Testosterone 1 (.) -Deoxycorticosterone 1 1 (.) Androstenedione 1 (.) Estrone 1 (.) 1α Progesterone 1 1 (.) DT 1 (.) Progesterone 1 (.)
onclusions A method was developed for the determination of 1 hormones, and their respective internal standards in serum using Agilent Bond Elut Plexa SPE cartridges. Analyte response with an Agilent triple quadrupole L/MS/MS operating in both positive and negative mode was greatly enhanced with the addition of 1 mm ammonium fluoride. An Agilent InfinityLab Poroshell P- (.1 mm mm,. µm) column yielded fast run times and baseline separation of three isobaric pairs, allowing for more reproducible integration and quantification. The wide p range makes it an ideal column choice for method development. The method provided excellent recoveries (-1%) and low RSD values (.-.%). Agilent Bond Elut Plexa SPE products require little method development and are simple to use, making them the ideal choice for biological analysis. References 1. Fu, R.; Zhai, A. Determination of ormones in Drinking Water by L/MS/MS Using an Agilent InfinityLab Poroshell P olumn (EPA ); Application note, Agilent Technologies, Inc. Publication number 1 EN, 1.. indle, R. Improved Analysis of Trace ormones in Drinking Water by L/MS/MS (EPA ) using the Agilent Triple Quadrupole L/MS; Application note, Agilent Technologies, Inc. Publication number 1 EN, 1.. Fiers, T.; asetta, B.; Bernaert, B.; Vandersypt, E.; Debock, M.; Kaufman, J. Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization. Journal of hromatography B. 1,,. For More Information These data represent typical results. For more information on our products and services, visit our Web site at www.agilent.com/chem. www.agilent.com/chem For Research Use nly. Not for use in diagnostic procedures. Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance, or use of this material. Information, descriptions, and specifications in this publication are subject to change without notice. Agilent Technologies, Inc., 1 Printed in the USA May, 1 1-EN