Abstract:
This dataset contains liquid chromatography tandem mass spectrometry (LC-MS/MS) data from common bottlenose dolphin plasma. These data were collected between January and December 2017 to examine the use of reverse phase solid phase extraction (SPE) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous, precise (< 15 % relative standard deviation), and accurate (between 70 % and 120% recovery of spiked quantities) measurement of steroid hormones in dolphin plasma. This dataset includes data from three individual experiments using three different sample sets. The first is a spike recovery experiment performed with free-ranging bottlenose dolphin plasma collected in September 2017, the second is an endogenous precision experiment using pooled samples collected in 2012 from dolphins maintained by the United States Navy, and the final experiment is a matrix (serum/plasma) comparison utilizing samples collected from free-ranging dolphins between 2014 and 2016. This dataset supports the publication: Galligan, T. M., Schwacke, L. H., Houser, D. S., Wells, R. S., Rowles, T., & Boggs, A. S. P. (2018). Characterization of circulating steroid hormone profiles in the bottlenose dolphin (Tursiops truncatus) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). General and Comparative Endocrinology, 263: 80–91. doi:10.1016/j.ygcen.2018.04.003
Suggested Citation:
Thomas Galligan, Lori Schwacke. 2018. Dataset for: Characterization of Circulating Steroid Hormone Profiles in the Bottlenose Dolphin (Tursiops truncatus) by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7GF0S16
Data Parameters and Units:
Sheet 1 "Spike Recovery": ID, 17-Hydroxyprogesterone (ng/g), Testosterone (ng/g), Androstenedione (ng/g), Progesterone (ng/g), Cortisone (ng/g), Cortisol (ng/g), Corticosterone (ng/g), 11-Deoxycortisol (ng/g), 11-Deoxycorticosterone (ng/g), Estradiol-DC (ng/g), Estrone-DC (ng/g). Sheet 2 Endogenous Precision": D, 17-Hydroxyprogesterone (ng/g), Testosterone (ng/g), Androstenedione (ng/g), Cortisone (ng/g), Cortisol (ng/g), Corticosterone (ng/g). Sheet 3 "Matrix Comparison": ID, Plasma_17-Hydroxyprogesterone (ng/g), Plasma_Testosterone (ng/g), Plasma_Androstenedione (ng/g), Plasma_Progesterone (ng/g), Plasma_Cortisone (ng/g), Plasma_Cortisol (ng/g), Plasma_Corticosterone (ng/g), Serum_17-Hydroxyprogesterone (ng/g), Serum_Testosterone (ng/g), Serum_Androstenedione (ng/g), Serum_Progesterone (ng/g), Serum_Cortisone (ng/g), Serum_Cortisol (ng/g), Serum_Corticosterone (ng/g)
Methods:
Sample IDs labeled as “Spike” in the “Spike Recovery” datasheet were spiked prior to extraction via reverse phase solid phase extraction with progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, estrone, estradiol, cortisol, cortisone, 11-deoxycortisol, corticosterone, 11-deoxycorticosterone. A mixture of these hormones was made for spiking with the following amounnts of each compound: Hormone Concentration (ng/g) Progesterone 29.5644 17-Hydorxyprogesterone 34.354 Estrone 6.6062 Cortisone 12.8888 Estradiol 11.6938 Testosterone 37.4457 Cortiol 54.9831 Corticosterone 28.8083 11-deoxycorticosterone 7.5513 11-deoxycortisol 11.1395 Androstenedione 3.5097 Hormone Mean Spike Mass (ng) Progesterone 9.014 17-Hydroxyprogesterone 10.47 Androstenedione 1.069k Testosterone 11.41 Estrone 2.012 Estradiol 3.562 Cortisol 16.75 Cortisone 3.926 11-Deoxycortisol 3.393 Corticosterone 8.776 11-Deoxycorticosterone 2.300 400 microliters of the spike solution was added gravimetrically to each Spike sample. Approximately 2mL of plasma was used for the solid phase extractions in the spike recovery experiment. Plasma data in the Endogenous Precision data sheet reports values from pooled plasma samples. The number of samples pooled was not recorded. A mixture of isotopically labeled internal standards was added to each sample in the whole study. This mixture included: progesterone-13C3, 17-hydroxyprogesterone-13C3, androstenedione-13C3, testosterone-13C3, estradiol-13C3, cortisol-d4, cortisone-13C3. These standard were used for inter-sample variation in inference and extraction efficiency. 2 mL of plasma was used in solid phase extraction for endogenous precision experiment. Data in the worksheet Matrix Comparison provide comparison of plasma and serum matrices. Approximately 2mL of plasma or serum was used in SPE matrix comparison (quantity added was tracked gravimetrically). A mixture of isotopically labeled internal standards was added to each sample in the whole study. This mixture included: progesterone-13C3, 17-hydroxyprogesterone-13C3, androstenedione-13C3, testosterone-13C3, estradiol-13C3, cortisol-d4, cortisone-13C3. These standard were used for inter-sample variation in inference and extraction efficiency. Solid Phase Extraction information: 100 microliters or 150 microliters of internal standard mixture was added to clean culture tubes and dried under nitrogen gas at 100 – 130 kilopascals in a 40 degree Celsius water bath. 2 mL of serum or plasma or 0.5 mL or 1.0 ml of calibration standard was added. The mass of the internal standard and serum, plasma or calibration standard were measured gravimetrically. Sodium acetate buffer (4 mL, 0.01 mole per Liter, pH f) was added to each tube. Tubes were vortexed and incubated at room temperature for 1 hour. For SPE Supelclean LC-18 6 mL capacity 1 g bed weight cartridges from Sigma-Aldrich were used after being conditioned with 5 mL of methanol, 5 mL of MilliQ water, a 1 mL of sodium acetate buffer (0.01 mole per Liter, pH5). The cartridges were loaded with sample/buffer mixtures and a vacuum was applied. Cartridges were washed with 12 milliliters of MilliQ water followed and 5 milliliters of 80:20 MilliQ water:acetonitrile. Samples were eluted into clean borosilicate culture tubes with 2.5 milliliters of methanol and dried under nitrogen at 100 - 130 kilopascals in a water bath at 40 degrees Celsius. Samples were reconstituted in 200 microliters of methanol, and transferred to amber autosampler vials with 250 microliter glass inserts. Estrogen was measured using Dansyl chloride derivatization. 50 microliters of the final 200 microliter SPE extract was added to 200 microliters of acetone and 500 microliters of sodium bicarbonate buffer (0.1 mole per Liter, pH 10.5). The solution was vortex for 1 minutes. 500 microliters of Dansyl chloride solution (1 mg/mL) in acetone was added and vortexed for 1 minutes. The mixture was incubated a 60 degrees Celsius for 3 minutes and dried under nitrogen at 100-130 kilopascals in a 40 degree Celsius water bath. Dried samples were reconstituted in 2 milliliters of methanol and filter using UniPrep 0.2 micron PTFE syringeless filters. Filtered samples were dried under nitrogen at 100-130 kilopascals in a 40 degree Celsius water bath and reconstituted in 50 microliters of methanol. LC-MS/MS method information: Three different chromatographic separations were performed: 1) biphenyl separation of underivatized steroids, 2) biphenyl separation of derivatized estrogens, and 3) C18 separation to improve detection of corticosteroid. An Agilent 1200 Series HPLC system with binary pump and autosampler linked to an AB Sciex API 400 QTRAP triple quadruple/linear ion trap mass spectrometer. A Restek (Bellefonte, PA) Ultra Biphenyl column (250 mm x 4.6 mm, 5 μm particle size) was used with a gradient of acetonitrile and methanol (both containing 0.1 percent formic acid) beginning at 80 percent methanol which was decreased to 65 percent methanol over 20 min, then decreased to 0 percent methanol over 1 min, held for 5 min, increased to 80 percent methanol over 0.1 min, and held for 9.9 min. For C18 separation, extracts were solvent exchanged into 50:50 methanol:water (volume fraction) by transferring 50 microliter of extract to a clean tube and drying under nitrogen between 100-130 kilopascals in a 40 degree Celsius water bath and constituting with 50:50 methanol: water (volume fraction) and transferring to a new glass insert. Agilent Eclipse Plus C18 column (150 mm x 21 mm, 5.0 micron particle size), and a gradient of methanol and milliQ water (both with 0.1 % acetic acid) was used beginning with 46 percent methanol and held for 10 min, increased to 82.5 percent methanol over 10 min, then increased to 83.3 percent methanol over 5 min. The column was then washed with 100 percent methanol for 5 min, and re-equilibrated to 46:54 methanol:water (volume fraction) for 10 min Injection volume = 10 microliter, Gas = nitrogen, ion source = electrospray ionization, mode = positive ion mode.
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