Calibrator stock solutions were prepared separately for each steroid by dissolving reference steroids in ethyl acetate. The stock solutions were then pooled to a calibrator standard solution and diluted in 50% methanol/ water. Eight calibrator working solutions were made within the calibration range presented in Supplementary Table 1 (4 (link)). Estradiol, estrone, dihydrotestosterone, progesterone, androstenedione, DHEA, and 17α-hydroxyprogesterone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Testosterone was purchased from Fluka (Buchs, Germany). Calibration was performed by determining the peak area ratio between the target analyte and the isotope-labeled internal standard. Before each run, calibrator samples were freshly prepared by spiking 25 µL calibrator working solution to 225 µL of 0.5% BSA in PBS buffer. Internal standard stock solutions were made separately using 13C3-labeled versions of each steroid, except for DHEA that was labeled with d6. An internal standard working solution with labeled steroid concentrations in the middle of the calibration range was prepared in 50% methanol/water and aliquoted. All calibrator and internal standard stock and working solutions were stored at −80 °C until use.
17α hydroxyprogesterone
Manufactured by Merck Group
Sourced in United States, China
17α-hydroxyprogesterone is a laboratory reagent used in various analytical and research applications. It is a steroidal hormone that plays a role in the biosynthesis of other steroid hormones. The product is used for research purposes only and its core function is to serve as a standard or reference material in analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Progesterone, by Merck Group (7 mentions)
Cortisone, by Merck Group (3 mentions)
Testosterone, by Merck Group (3 mentions)
Corticosterone, by Merck Group (3 mentions)
Dihydrotestosterone, by Merck Group (2 mentions)
Estrone, by Merck Group (2 mentions)
Androstenedione, by Merck Group (2 mentions)
Pregnenolone, by Merck Group (2 mentions)
Cortisol, by Merck Group (2 mentions)
Dehydroepiandrosterone, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Androsterone, by Merck Group (2 mentions)
11 deoxycortisol, by Merck Group (2 mentions)
Estradiol, by Merck Group (1 mentions)
Desmosterol, by Merck Group (1 mentions)
D3 testosterone, by Cerilliant (1 mentions)
D9 progesterone, by Cerilliant (1 mentions)
13c3 estrone, by Cerilliant (1 mentions)
Ammonium iodide nh4i, by Merck Group (1 mentions)
16α hydroxyestrone, by Merck Group (1 mentions)
8 protocols using 17α hydroxyprogesterone
1
Steroid Biomarker Quantification Protocol
2
Comprehensive Steroid Analysis Workflow
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The internal standards (d3-testosterone, d4-cortisol, d9-progesterone, d7-cholesterol, 13c3-androstene-3,17-dione, and 13c3-estrone) were purchased from Cerilliant (Round Rock, TX, USA). L-ascorbic acid, sodium acetate, acetic acid, β-glucosaldosidase/arylsulfatase, ammonium iodide (NH4I), dithioerythritol (DTE), and N-methyl-n-(trimethylsilyl)trifluoroacetamide (MSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Commercial standards of steroids (dehydroepiandrosterone, testosterone, 11-deoxycorticosterone, cortisone, cortisol, 16α-hydroxyestrone, pregnenolone, 17α-hydroxypregnenolone, epipregnanolone, dihydrotestosterone, 21-hydroxyprogesterone, androsterone, epiandrosterone, corticosterone, estrone, 17β-estradiol, estriol, 16-epiestriol, progesterone, 17α-hydroxyprogesterone, desmosterol, cholesterol, etc.) were purchased from Sigma-Aldrich, J&K Chemical Ltd. (Beijing, China), or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chromatographic pure hexane, methanol, and ethyl acetate were purchased from Merck (Fairfield, OH, USA). The Oasis HLB SPE cartridge was obtained from Waters (1.5 ml, 60 mg; Waters, Milford, MA, USA).
3
Quantitative Analysis of Progestins
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Methanol (≥99.9%) and acetonitrile (≥99.9%) were purchased from Sigma-Aldrich (St. Louis, USA). All reagents used were of analytical grade. Anhydrous magnesium sulfate (MgSO4), sodium chloride (NaCl), primary secondary amine (PSA), and alumina-N were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). High-purity (least 98% purity) analytical standards were used. Norethisterone, 17α-hydroxyprogesterone, D-methylnorethindrone, 17α-hydroxyprogesterone acetate, megestrol, medroxyprogesterone, chlorprogestone acetate, medroxyprogesterone acetate, melengestrol acetate, and progesterone were purchased from Sigma-Aldrich (St. Louis, USA).
Individual stock standard solutions (1000 mg/L) of each compound were prepared using Methanol and stored in the refrigerator at −20°C. The mixed standard solution was prepared by mixing the 10 individual stock standard solutions (100 μL for each) and diluting with Methanol to 10 mL in a volumetric flask, and the final concentration of each progestin was 10 mg/L. The mixed standard solutions prepared were stored at 4°C.
Acquity ultra-performance liquid chromatography (Waters USA); Synapt G2-Si quadrupole time-of-flight mass spectrometry (Waters USA); GR22GIII high-speed refrigerated centrifuge (HITACHI Japan); N-EVAPTM 112 nitrogen blowing apparatus (Organomation, USA); and KQ-500DE numerical control ultrasonic cleaner (Kunshan, China).
Individual stock standard solutions (1000 mg/L) of each compound were prepared using Methanol and stored in the refrigerator at −20°C. The mixed standard solution was prepared by mixing the 10 individual stock standard solutions (100 μL for each) and diluting with Methanol to 10 mL in a volumetric flask, and the final concentration of each progestin was 10 mg/L. The mixed standard solutions prepared were stored at 4°C.
Acquity ultra-performance liquid chromatography (Waters USA); Synapt G2-Si quadrupole time-of-flight mass spectrometry (Waters USA); GR22GIII high-speed refrigerated centrifuge (HITACHI Japan); N-EVAPTM 112 nitrogen blowing apparatus (Organomation, USA); and KQ-500DE numerical control ultrasonic cleaner (Kunshan, China).
4
Steroid Hormone Quantification by LC-MS/MS
5
Isolation and Purification of Anti-Cortisol Fab
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Anti-cortisol (17) Fab is a murine antibody
isolated from a VTT steroid-specific antibody library of 1.8 ×
108 unique clones. The discovery, isolation, production,
and purification of the anti-cortisol (17) Fab fragment are described
elsewhere.9 (link) Prior to the native MS measurements,
the protein was desalted by Bio-Rad Micro Bio-Spin 6 columns using
200 mM ammonium acetate as an eluent. For protein concentration determination,
both the Bio-Rad DC (detergent compatible) protein assay (with bovine
serum albumin as the standard) and UV absorbance measurements at 280
nm were utilized. All the studied ligands, cortisol (hydrocortisone;
Sigma–Aldrich, product no. H4001), prednisolone (Sigma–Aldrich,
P6004), cortisone (Sigma–Aldrich, C2755), corticosterone (Sigma–Aldrich,
C2505), 11-deoxycortisol (Sigma–Aldrich, R0500), 17α-hydroxyprogesterone
(Sigma–Aldrich, H5752), 11-deoxycorticosterone (BioNordika,
22916), progesterone (MilliporeSigma, P0130), and testosterone (Sigma–Aldrich,
86500), were carefully weighed and dissolved in MeOH. Additionally,
UV absorbance at 242 nm was used as a control for the concentration
determination of cortisol, using a specific extinction coefficient,
ε1%,1 cm = 445.25
isolated from a VTT steroid-specific antibody library of 1.8 ×
108 unique clones. The discovery, isolation, production,
and purification of the anti-cortisol (17) Fab fragment are described
elsewhere.9 (link) Prior to the native MS measurements,
the protein was desalted by Bio-Rad Micro Bio-Spin 6 columns using
200 mM ammonium acetate as an eluent. For protein concentration determination,
both the Bio-Rad DC (detergent compatible) protein assay (with bovine
serum albumin as the standard) and UV absorbance measurements at 280
nm were utilized. All the studied ligands, cortisol (hydrocortisone;
Sigma–Aldrich, product no. H4001), prednisolone (Sigma–Aldrich,
P6004), cortisone (Sigma–Aldrich, C2755), corticosterone (Sigma–Aldrich,
C2505), 11-deoxycortisol (Sigma–Aldrich, R0500), 17α-hydroxyprogesterone
(Sigma–Aldrich, H5752), 11-deoxycorticosterone (BioNordika,
22916), progesterone (MilliporeSigma, P0130), and testosterone (Sigma–Aldrich,
86500), were carefully weighed and dissolved in MeOH. Additionally,
UV absorbance at 242 nm was used as a control for the concentration
determination of cortisol, using a specific extinction coefficient,
ε1%,1 cm = 445.25
6
Targeted Steroid and Lipid Profiling
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MS-grade water (7732-18-5), methanol (A456-500), acetonitrile (A9554), and acetone (67-64-1) were purchased from Fischer Scientific (Morris Plains, NJ, USA). MS-grade acetic acid (64-19-7) was purchased from Sigma Aldrich (St. Louis, MO, USA). Analytical grade chemical standards were purchased [Progesterone (Sigma-Aldrich, P-069-1ML), THDOC (Sigma-Aldrich, P2016-5MG), Estriol-16-Glucuronide (Sigma-Aldrich, E1877-10MG), DHEA-S (Sigma-Aldrich, Dehydroepiandrosterone-D5-3-sulfate (DHEAS-D5) (2,2,3,4,4,-D5) sodium salt solution, D-066-1ML), PE(P-16:0e/0:0) (Avanti Polar lipids, 852470), Androstane-3,17-diol (Sigma-Aldrich, A7755-100MG), 17α-HydroxyProgesterone (Sigma-Aldrich, H-085-1ML)] and prepared in methanol, except PE(P-16:0e/0:0), which was prepared in chloroform/methanol (8:2).
7
Progesterone Biotransformation by Isaria farinosa
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The substrates, progesterone (1 ), 11α-hydroxyprogesterone (3 ), 17α-hydroxyprogesterone (4 ), 16α,17α-epoxyprogesterone (5 ) and pregnenolone (6 ) were purchased from Sigma-Aldrich.
The biocatalyst was Isaria farinosa KCh KW1.1, a strain obtained from the Department of Chemistry collection, Wrocław University of Environmental and Life Sciences, Poland. Its isolation and genetic determination were described previously [9 (link)].
The biocatalyst was Isaria farinosa KCh KW1.1, a strain obtained from the Department of Chemistry collection, Wrocław University of Environmental and Life Sciences, Poland. Its isolation and genetic determination were described previously [9 (link)].
8
Standardized Herbal Capsule Production
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Glucose powder was purchased from Utopian Co. Ltd., (Samutprakan), Thailand, and 17-α-hydroxyprogesterone was purchased from Sigma, (Shanghai) China. Acetonitrile was analytical grade from RCI Labscan, (Bangkok) Thailand. Methanol was HPLC grade from RCI Labscan, (Bangkok), Thailand.
Dried powder of KP Romkaou strain rhizomes obtained from Phurue, Loei province of Thailand was authenticated and kept as a voucher specimen (No. KP-BS-2010) at the Center for Research and Development of Herbal Health Products, Khon Kaen University, (Khon Kaen), Thailand. KP extract was prepared by maceration in 95% ethanol (following the petty patent of Thailand No. 4048) and the crude extract was obtained at 5.71% yield. The KP extract was analyzed for the content of methoxyflavones using HPLC (Figure 1 ). The tested KP product was prepared in the dosage form of a capsule to contain 90 mg of KP extract and other excipients (Table 1 ). The placebo capsule containing no KP extract was composed of the same excipients and capsule color. The entire study was conducted using a single batch of KP extract to optimize product consistency.
Dried powder of KP Romkaou strain rhizomes obtained from Phurue, Loei province of Thailand was authenticated and kept as a voucher specimen (No. KP-BS-2010) at the Center for Research and Development of Herbal Health Products, Khon Kaen University, (Khon Kaen), Thailand. KP extract was prepared by maceration in 95% ethanol (following the petty patent of Thailand No. 4048) and the crude extract was obtained at 5.71% yield. The KP extract was analyzed for the content of methoxyflavones using HPLC (
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