Zicheng Yang (Presenter)
Bruker Daltonics
Bio: Sr. Field application specialist
Authorship: Zicheng Yang, Louis Maljers
Bruker Daltonics Inc., 3500 West Warren Ave, Fremont, CA 94538
| Short Abstract Testosterone concentrations in adult females and children (male and female) are an order of magnitude lower than adult male testosterone concentrations and require a sensitive and specific analytical method to accurately determine the testosterone level. A sensitive method for the quantification of total testosterone in serum was developed using Bruker TQ LC/MS system. Excellent sensitivity, linearity and dynamic range were obtained with a LOD of 1 pg/mL, R2 > 0.99, and greater than four orders of dynamic range. The low pg/mL level detection with wide dynamic detection range will cover the clinical research needs. |
Long Abstract
Introduction
Testosterone concentrations in adult females and children (male and female) are an order of magnitude lower than adult male testosterone concentrations and require a sensitive and specific analytical method to accurately determine the testosterone level. LC-MS/MS is becoming the analytical method of choice over immunoassays due to its sensitivity, specificity and accuracy for analysis of total testosterone in serum. A rapid and sensitive method for the quantification of total testosterone in serum was developed using the Bruker Advance UHPLC coupled to the EVOQ Elite LC-TQ MS system. Excellent sensitivity, linearity and dynamic range were obtained with a limit of detection (LOD) of 1 pg/mL, R2 > 0.99, and greater than four orders of dynamic range (LLOQ: 2.5 pg/mL to ULOQ 50 ng/mL). The total run cycle time was 5 minutes.
Experimental
Following the simple extraction using high purity hexane/ethyl acetate reagent, the Bruker Advance UHPLC coupled to the Bruker EVOQ Elite TQ system equipped with ESI source was used to separate, detect and quantify testosterone in serum. The MRM ion transitions of m/z 289→97 (collision energy (CE) 21v) and m/z 289→109 (CE 21v) were used as quantifier and qualifier ion, respectively for testosterone. The transition of m/z 292.3→112 (CE 23v) was used for the stable labeled internal standard. The scan times for both testosterone and internal standard were 100 ms. MSG3000, testosterone-free Human Serum, was used as matrix for preparing calibration standards.
Calibration: Testosterone was spiked in MSG3000 for calibration solutions at levels of 1 pg/mL, 2.5 pg/mL, 25 pg/mL, 50 pg/mL, 250 pg/mL, 1.0 ng/mL, 5 ng/mL, 25 ng/mL, and 50 ng/mL and triplicate solution for concentration of 5 pg/mL, 500 pg/mL, and 10 ng/mL was prepared as QC standard.
0.20 mL of serum sample or calibration standard in a 2 mL microfuge tube was spiked with 10 µL of 10 ng/mL internal standard and vortex-mixed for 5 s and incubated at room temperature for 5 min. To this 2 mL microfuge tube, 1.0 mL of ethyl acetate:hexane, 10:90, v:v was added and vortex-mixed for about 1 min and left at room temperature for 2 min. then centrifuged at 14,500 rpm for 3 min. 0.85 mL of supernatant was transferred into a new 2 mL microfuge tube and dried by SpeedVac at 45 ̊C for about 20 min. The residue was dissolved in 100 µL of solvent (methanol:water, 50/50, v:v) and transferred into an autosampler vial with a 300 µL fused Insert and analyzed by LC/MS. Sample preparation time was about 30 min and LC/MS run time was 5 min.
LC/MS Conditions
HPLC was performed using YMC-Pack Pro C18 RS, 3 µm, 50mm × 2.0 mm I.D. column. Mobile phase A was 2 mM ammonium formate, 0.1% formic acid (FA) in water) and mobile phase B was 2 mM ammonium formate, 0.1% FA in Methanol. A simple gradient started from 50%b (keep 0.2min) to 95% over 2 min and keep at 95% for 1 min then equilibrate at initial condition for 2 min with flow rate 300 µL/min. Column temperature was at 40 ˚C.
Heated Electron Spray Ionization (HESI) was used. The spray voltage was 4000 V. The cone, probe and nebulizer gases were 15, 40 and 55 units, respectively. The cone and probe temperature were 300 ˚C and 450 ˚C, respectively. The active exhaust was on. The collision gas pressure (Argon) was 1.5 mTorr.
Results and discussion
The LC method used a five-minute run cycle time with a testosterone retention time of 2.63 minutes. The blank shows no interference of the analysis at LLOQ level. The assay was sensitive (LOD 1 pg/mL, S/N 12) and linear (R2 >0.999). The response factor RSD for testosterone concentration range from 2.5 pg/mL to 50 ng/mL was 9.8%. The sensitivity and linear range well covers the testosterone concentrations in children and adults of both genders.
The %RSD of QC standards at levels of 5 pg/mL, 500 pg/mL, and 10 ng/mL were 6.8%, 1.3% and 1.8% (n=3) with accuracy of 119.3%, 101.5%, and 103.8%, respectively. The results for single female donor serum and pooled male serum were 238 pg/mL and 4.17 ng/mL, respectively. The testosterone test results are in the range for adult female and male.
Conclusions
The Bruker EVOQ TQ LC/MS is well suited for the analysis of total testosterone in serum from low to high levels. The system is sensitive enough to detect low pg/mL level of testosterone in serum and provides greater than four orders of dynamic detection range. The research method provides a rapid, simple, high accuracy, high precision, and high selective procedure that can easily be used for the determination of serum testosterone concentrations.
References & Acknowledgements:
Deborah French Development and validation of a serum total testosterone liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay calibrated to NIST SRM 971. Clinica Chinica Acta 415 (2013) p109-117
| Description | Y/N | Source |
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| Salary | yes | Bruker Daltonics |
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IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |