Deborah French1. 1. Department of Laboratory Medicine, University of California-San Francisco, San Francisco, CA 94107, United States. deborah.french@ucsf.edu
Abstract
BACKGROUND: At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house. METHODS: A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed. RESULTS: Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded <15% CV at the limit of quantitation and <7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed. CONCLUSIONS: An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds.
BACKGROUND: At our institution, serum testosterone in adult males is measured by immunoassay while female and pediatric specimens are sent to a reference laboratory for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis due to low concentrations. As this is of significant cost, a testosterone LC-MS/MS assay was developed in-house. METHODS: A 5500 QTRAP® using electrospray ionization and a Shimadzu Prominence with a C18 column were used. Gradient elution with formic acid, water and methanol:acetonitrile at 0.5 ml/min had a 7-min run-time. A liquid-liquid extraction with hexane:ethyl acetate was carried out on 200 μl of serum. Multiple reaction monitoring was employed. RESULTS: Sample preparation took ~80 min for 21 samples. Six calibrators were used (0-1263 ng/dl; concentration assigned by NIST SRM 971) with 3 quality controls (9, 168 and 532 ng/dl). The limits of detection and quantitation were 1 and 2 ng/dl respectively. Extraction recovery was ~90% and ion suppression ~5%. Within-run and total precision studies yielded <15% CV at the limit of quantitation and <7% CV through the rest of the linear range. Isobaric interferences were baseline separated from testosterone. Method comparisons between this assay, an immunoassay, and another LC-MS/MS assay were completed. CONCLUSIONS: An accurate and sensitive LC-MS/MS assay for total testosterone was developed. Bringing this assay in-house reduces turnaround time for clinicians and patients and saves our institution funds.
Authors: Rebecca A Clewell; Jeremy A Leonard; Chantel I Nicolas; Jerry L Campbell; Miyoung Yoon; Alina Y Efremenko; Patrick D McMullen; Melvin E Andersen; Harvey J Clewell; Katherine A Phillips; Yu-Mei Tan Journal: Toxicol In Vitro Date: 2020-04-08 Impact factor: 3.500
Authors: Mohamed S Attia; Safwat A Mahmoud; Amal M Ahmed; Tarek A Amin; Ahmed O Youssef; Mohammed A Amin; Mona N Abou-Omar; Ekram H Mohamed Journal: RSC Adv Date: 2021-10-12 Impact factor: 4.036
Authors: Carolyn J Broccardo; Kevin L Schauer; Wendy M Kohrt; Robert S Schwartz; James P Murphy; Jessica E Prenni Journal: J Chromatogr B Analyt Technol Biomed Life Sci Date: 2013-07-04 Impact factor: 3.205
Authors: Mohamed S Attia; Amal M Ahmed; Tarek A Amin; Ahmed O Youssef; Mohammed A Amin; Ekram H Mohamed; Safwat A Mahmoud; Mona N Abou-Omar Journal: RSC Adv Date: 2021-10-06 Impact factor: 4.036