2017-03-29
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Sigma Aldrich
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Sample Preparation of Serum Prior to Determination of Free Testosterone
The industry’s gold standard for sample preparation of free hormones from serum has been associated with equilibrium dialysis.1 It is the free portion of hormones, including testosterone, that is responsible for the biological activity.2 Free testosterone accounts for approximately 1-2% of total testosterone.3 In males this generally falls in the range of 20 – 230 pg/mL and for females in the range of 0.6 – 10 pg/ mL (0.8-1.4% of total).4 Solid phase microextraction (SPME) is a relatively new method to employ for measurement of free concentration and one that has recently been incorporated into a 96-pin device for use with conventional well plates. Bioanalytical SPME, or BioSPME, has been shown to be a fast sample preparatory technique.5
The BioSPME sample preparation method for the 200 μL samples utilized a Supel™ BioSPME C18 96-pin device with a Hamilton® Starlet system. The method includes multiple steps but was developed to have a total process time of 1 hour (Figure 1). A volume of 500 μL was used for the conditioning, acetonitrile, and wash solution, water. The desorption and derivatization steps were performed off-line. The acetonitrile desorption solution, 50 μL, contained 25 pg/mL D3-testosterone prior to derivatization. Derivatization was performed with an addition of 200 mM hydroxylamine hydrochloride, 100 μL, at 60 °C for 20 min with agitation at 600 rpm.6 The free concentration of testosterone in serum samples was determined using simultaneously extracted calibrators (10 – 200 pg/mL) prepared in phosphate buffered saline and analyzed by an Agilent 1290 LC utilizing an Ascentis® Express C18 (5 cm x 2.1 mm, 2.7 μm) column connected to an AB Sciex 6500+ TripleQuad™. Quantifier and qualifier transitions were utilized for the natural and isotopically labelled testosterone and derivatized testosterone (Table 1). Injection volumes of 60 μL of the final prepared samples of the total 150 μL were used to accommodate reinjections if required. Free testosterone in male serum samples were previously determined by externally validated equilibrium dialysis-based methods at respective laboratories (Lab A, Lab L, and Lab Q). Samples were purchased from Lab A and were previously tested. The serum samples sent to Labs L and Q were collected in collaboration with the Clinical & Translational Science Institute at Penn State University. Aliquots of these samples were submitted for testing with the remainder of samples kept at -80 °C. A second extraction was performed after one month in the -80 oC freezer. A total of 30 unique serums samples were tested using BioSPME in randomized analysis. In general, samples were tested in duplicate or triplicate depending on availability of sample quantity. Images of the actual samples are shown in Figure 3.
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serum-prior-determination-an9202en-mk.pdf
产品名称 订货编号
Supel™ BioSPME C18 96-Pin Devices, 1 pack 59680-U
Supel™ BioSPME C18 96-Pin Devices, 10 pack 59683-U
Positioning Adapter (for Automation of Supel™
BioSPME), 1 pack
59686-U
Phosphate Buffer Solution P5358
Acetonitrile, LC-MS LiChrosolv® 1.1544
Water, LC-MS LiChrosolv® 1.00029
Testosterone solution 1.0 mg/mL in acetonitrile, ampule
of 1 mL, certified reference material, Cerilliant®
T-037
Testosterone-d3 (16, 16, 17-d3) solution 100 μg/
mL in acetonitrile, ampule of 1 mL, certified reference
material, Cerilliant®
T-046
Nunc™ 96 DeepWell™ plate size 96 wells, U-bottom
natural polypropylene wells, maximum volume 1.3 mL,
non-sterile
P8241
Corning® Thermowell PCR 96 well plates 96 well plate,
Thermowell PCR plate, polypropylene, conical bottom,
clear, 25/cs
CLS6551
Plate Glass Coated Microplates 96-Well Microplate, glass
coated, round well; U-Shape, 7.2mm dia
Available
from NS3
MultiTherm™ shaker with heating Z755753
Texan™ reagent reservoir for multichannel pipettes R9259
Zone-Free™ Sealing Films Z721646
SealPlate Film Z369659
Corning® Stripwell™ accessories “egg crate” strip holder CLS2572
Hydroxylamine hydrochloride ReagentPlus®, 99% 159417