Mar 09, 2026
Method for profiling twelve steroids in avian samples using LC-MS/MS
- Victoria Betterton1,
- Scott Denham2,
- Joanna Simpson2,
- Ian Dunn3,
- Michael Clinton3,
- Natalie Homer2
- 1University of Edinburgh;
- 2School of Neurological and Cardiovascular Sciences, University of Edinburgh;
- 3Roslin Institute, University of Edinburgh
Protocol Citation: Victoria Betterton, Scott Denham, Joanna Simpson, Ian Dunn, Michael Clinton, Natalie Homer 2026. Method for profiling twelve steroids in avian samples using LC-MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbe8yqgpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 07, 2026
Last Modified: March 09, 2026
Protocol Integer ID: 273157
Keywords: steroids in avian sample, broad steroid profile for avian species, gold standard method for steroid measurement, doors to broad steroid profiling, broad steroid profiling, steroid measurement, estrogens alongside progestogen, major active glucocorticoid in avian species, sensitivity of estrogen, broad steroid profile, estrogen, androgen measurement, extracted steroid, androgens along with glucocorticoid, labelled steroid, recent methods for avian species, androgen, liquid chromatography tandem mass spectrometry, panel of steroid, avian sample, progesterone, steroid, estradiol, avian species, dehydroepiandrosterone, corticosterone, progestogen, biological sample, mineralocorticoid, testosterone, tandem quadrupole mass spectrometer, glucocorticoid, aldosterone, dehydrocorticosterone, major active glucocorticoid
Funders Acknowledgements:
Biotechnology and Biological Sciences Research Council
Grant ID: BB/P013759/1
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Abstract
When seeking methods to measure steroids in avian samples then liquid chromatography tandem mass spectrometry (LC-MS/MS) is an ideal choice. It is already considered a gold standard method for steroid measurement in the clinical setting (1). Recent methods for avian species and rodents focus on one or two (2,3) or a small targeted panel of steroids and do not always cover a broad steroid profile for avian species. Other methods using LC-MS/MS employ derivatisation to boost sensitivity of estrogens (4) but excludes androgen measurement.
Methods that can simultaneously measure estrogens and androgens along with glucocorticoids, progestogens and mineralocorticoids open doors to broad steroid profiling in avian species. This is valuable in a variety of research studies where steroids play an important role such as nesting, reproduction, development and welfare. Here we present a method that measures both androgens and estrogens alongside progestogens, mineralocorticoids - it includes corticosterone, the major active glucocorticoid in avian species.
Biological samples (up to 200 µL) were enriched with isotopically labelled steroids, diluted with water (0.1% formic acid, v/v) and extracted alongside a (0.0025 - 10 ng) calibration curve, by automated 96-well supported liquid extraction (SLE), using dichloromethane and isopropanol as an organic solvent. Extracted steroids were separated on a Shimadzu Nexera uHPLC with gradient elution at a flow rate of 0.5 mL/min on a Kinetex C18 column (150 x 3 mm; 2.6 µm) and a mobile phase of methanol and water (0.05 mM ammonium fluoride in water and methanol). The run time was 16 minutes, followed by mass spectral analysis on an AB Sciex 6500+ tandem quadrupole mass spectrometer operated in multiple reaction mode, positive ionisation. The method measures twelve steroids - corticosterone, 11-dehydrocorticosterone, aldosterone, progesterone, androstenedione, testosterone, 5a-dihydrotestosterone, estrone, 17beta-estradiol, 17α-hydroxyprogesterone, 11-deoxycorticosterone, and dehydroepiandrosterone alongside isotopically labelled internal standards. Validation demonstrated that this method is sensitive, specific, and reliable.
Guidelines
Ensure your training is up-to-date for operating the necessary laboratory instrumentation and equipment.
Materials
Chemicals and reagents
Water (LC-MS grade), acetonitrile (LC-MS grade), methanol (LC-MS grade), dichloromethane (HPLC grade) and propan-2-ol (HPLC and LC-MS grade) and formic acid (LC-MS grade) were from VWR, Lutterworth, UK. Ammonium fluoride was supplied by Sigma-Aldrich, Dorset, UK.
Certified reference materials (1 mg/mL or 100 µg/mL in methanol or acetonitrile) for progesterone, androstenedione, testosterone, dehydroepiandrosterone, 5a-dihydrotestosterone, 17a-hydroxyprogesterone, 11-deoxycorticosterone, 17beta-estradiol, estrone, aldosterone and corticosterone were supplied by Cerilliant/Sigma-Aldrich, Dorset, UK, as were certified reference materials (100 µg/mL in methanol or acetonitrile).
Isotopically labelled internal standards; 2,3,4-[13C3]-testosterone (13C3-T), 2,3,4-[13C3]-androstenedione (13C3-A4), 2,3,4-[13C3]-dihydrotestosterone (13C3-DHT), 2,2,4,4,6,6,21,21,21 -[2H]9-progesterone, 2,3,4-[13C3]-estrone (13C3-E1), 2,3,4-[13C3]-17beta-estradiol (13C3-E2), were from IsoSciences/QMX laboratories, Thaxted, Essex, UK while 2,2,4,6,6,17α,21,21-[2H8]-aldosterone (d8-Aldo) and 2,2,4,6,6,17α,21,21-[2H8]-corticosterone (d8-B) were from CK Isotopes, Unthank, Leicestershire, UK.
11-dehydrocorticosterone (A), was powder from Steraloids Inc, Newport, Rhode Island, USA. Reference standard solutions and powders were stored as directed by the manufacturers.
Chemicals and Analytical Standards Table
| Item | Supplier | Article number | |
| Water (HPLC grade) | Fisher Scientific | C-10449380-X | |
| Acetonitrile (LC-MS grade) | VWR | 83640.320 | |
| Methanol (LC-MS grade) | VWR | 83638.320 | |
| Water (LC-MS grade) | VWR | 83645.320 | |
| Isopropanol (HPLC grade) | VWR | 20880.320 | |
| Dichloromethane (HPLC grade) | Fisher Scientific | C-23373320-X | |
| Progesterone | Sigma-Aldrich/Cerilliant | (P-069) 1 mg/mL in methanol | |
| Corticosterone | Sigma-Aldrich/Cerilliant | (C-107 ) 1 mg/mL in methanol (certified) | |
| Androstenedione | Steraloids | (A-075) 1 mg/mL in acetonitrile (certified) | |
| Testosterone | Sigma-Aldrich/Cerilliant | (T-037) 1 mg/mL in acetonitrile (certified) | |
| 5a-Dihydrotestosterone | Sigma-Aldrich/Cerilliant | (D-073) 1 mg/mL in methanol (certified) | |
| Dehydroepiandrosterone | Sigma-Aldrich/Cerilliant | (D-063) 1 mg/mL in methanol (certified) | |
| Estrone | Sigma-Aldrich/Cerilliant | (E-075) 1 mg/mL in methanol (Certified) | |
| 17beta-Estradiol | Sigma-Aldrich/Cerilliant | (E-060) 1 mg/mL in acetonitrile (certified) | |
| 17a-hydroxyprogesterone | Sigma-Aldrich/Cerilliant | (H-085) 1 mg/mL in acetonitrile (certified) | |
| 11-deoxycorticosterone | "Steraloids, Newport, USA" | Q3460-000 - supplied as powder | |
| Aldosterone | Sigma-Aldrich/Cerilliant | (A-096) 100 ug/mL in acetonitrile | |
| 11-dehydrocorticosterone | Steraloids, Newport, USA | Q3690-000 Supplied as powder | |
| D8-Corticosterone | Cambridge Isotope Laboratories/CK Isotopes | DLM-7347 Supplied as powder | |
| D8-Aldosterone | Cambridge Isotope Laboratories/CK Isotopes | DLM-8438-C Supplied as powder | |
| 13C3-estrone | Sigma-Aldrich/Cerilliant | (E-108) 100 ug/mL in methanol | |
| 13C3-estradiol | Sigma-Aldrich/Cerilliant | (E-073) 100 ug/mL in acetonitrile | |
| 13C3-testosterone | Sigma-Aldrich/Cerilliant | (T-070) 100 ug/mL in acetonitrile (certified) | |
| 13C3-dihydrotestosterone | IsoSciences/QMX labs | 6065 (supplied as powder) | |
| 13C3-androstenedione | Sigma-Aldrich/Cerilliant | (A-084) 100 ug/mL in acetonitrile (certified) | |
| d9-progesterone | Sigma-Aldrich/Cerilliant | (P-070) 100 ug/mL in acetonitrile |
Table M1 - Chemicals and analytical standards for extraction and steroid profiling
Consumables
ISOLUTE‱, SLE+ 400 Supported Liquid Extraction Plates and 2 mL deep well plates were from Biotage (Uppsala, Sweden) and 2 mL collection plates were from Waters (Wilmslow, UK).
The Kinetex C18 150 x 3.0 mm, 2.6 µm liquid chromatography column was supplied by Phenomenex (Macclesfield, UK) and fitted with a KrudKatcher Ultra In-Line Filter (0.5 µm porosity) Phenomenex, UK.
| A | B | C | D | |
| Item | Supplier | Part no. | Quantity | |
| 1.75 mL glass vials with lids | Scientific Laboratory Supplies Ltd | TUB1200 | 10 | |
| 7 mL glass vials with lids | Scientific Laboratory Supplies Ltd | TUB1220 | 5 | |
| Isolute SLE+ 400 96 well plate | Biotage | 820-0400-P01 | 1 | |
| 96-well plate sealing film | VWR | 391-1250 | 1 | |
| Adhesive Plate Seal | Waters | 186006336 | 1 | |
| Kinetex C18 (150 x 3 mm; 2.6 um) | Phenomenex | OOD-4462-YO | 1 | |
| Kinetex KrudKatcher, 0.5 um | Phenomenex | AFO-8497 | 1 | |
| Deep well 96 well collection plate | Biotage | 121-5203 | 1 | |
| Deep well (2 mL) 96 well collection plate | Waters | 186002482 | 1 |
Table M2 - Consumables for steroid extraction by supported liquid extraction (SLE) and steroid analysis by LC-MS/MS
EQUIPMENT
| Item | Model | Supplier | |
| Plate Shaker | 3005 | GFL | |
| Liquid Handling Robot | Extrahera | Biotage | |
| Evaporator | SPE Dry-96 dual | Biotage | |
| Liquid Chromatography Pump | Nexera X2 LC-30AD (x2) | Shimadzu | |
| Column oven | CTO-20AC with column switching | Shimadzu | |
| Autosampler | Nexera X2 SIL-30ACMP | Shimadzu | |
| Mass spectrometer | QTrap 6500+ | Sciex |
Table M3 - Equipment required for extraction and steroid analysis
Solutions Required
- 0.1% formic acid in water. Make up to 200 mL with Water (HPLC grade). Mix thoroughly.
- 98:2 Dichloromethane:Isopropanol (1 L) - Add 20 mL Isopropanol (HPLC grade) to 980 mL Dichloromethane (HPLC grade). Mix thoroughly.
- Methanol (HPLC grade): for preparation of calibration standard/internal standard dilutions.
- Water (HPLC grade): for preparation of calibration standards.
- 70:30 Water:Methanol (100 mL) - Add 30 mL methanol (LC-MS grade) to 70 mL water (LC-MS grade). Mix thoroughly.
Troubleshooting
Safety warnings
Ensure risk assessments are up to date and that all local laboratory guidelines are followed for handling chemicals and biological samples
Ethics statement
Before handling animal samples ensure samples have been collected ethically and that you have detail of the ethical approval
Before start
Ensure that all equipment has been well serviced and calibrated as appropriate, e.g. pipettes, balances and mass spectrometers
Preparation of calibration standard stock solutions
2h
For steroid powders prepare standard solutions at 1 mg/mL by dissolving a suitable amount of steroid powder in methanol. Remove certified reference standards of steroids from the freezer.
1h
Ensure there are 100 µg/mL stock solutions of each of the 12 steroids - corticosterone, 11-dehydrocorticosterone, aldosterone, progesterone, androstenedione, testosterone, 5a-dihydrotestosterone, estrone, 17beta-estradiol, 17α-hydroxyprogesterone, 11-deoxycorticosterone, and dehydroepiandrosterone in methanol.
1h
Prepare a mixed stock of the steroids by using 100 µg/mL stock solutions of each steroid. Do this by adding 50 µL x 100 µg/mL and dilute in methanol to give a 5 µg/mL stock.
Dilute the 5 µg/mL stock Mixed STOCK by 1:10 dilution (100 µL x 5 µg/mL + 900 µL methanol ) to give 500 ng/mL stock
Dilute the 500 ng/mL mixed STOCK by 1:10 dilution (100 µL x 500 ng/mL + 900 µL methanol ) to give 50 ng/mL stock
Dilute the 50 ng/mL mixed STOCK by 1:10 dilution (100 µL x 5 µg/mL + 900 µL methanol ) to give 5 ng/mL stock
Dilute the 5 ng/mL Mixed STOCK by 1:10 dilution (100 µL x 5 µg/mL + 900 µL methanol ) to give 500 pg/mL stock
Dilute the 500 pg/mL Mixed STOCK by 1:10 dilution (100 µL x 5 µg/mL + 900 µL methanol ) to give 50 pg/mL stock
Preparation of working internal standard (WIS) solution
1h
Prepare 100 µg/mL solutions of each isotopically labelled internal standard (d8-corticosterone, 13C3-testosterone, 13C3-androstenedione, 13C3-dihydrotestosterone, 13C3-estrone, 13C3-estradiol, d8-aldosterone and d9-progesterone) in methanol.
1h
Prepare a mixed 5 µg/mL Internal Standard mix stock solution of the isotopically labelled steroids by adding 25 uL x 100 ug/mL of each steroid and diluting up to 500 uL
Prepare a 5 ng/mL Working Internal Standard (WIS) solution by taking 10 µL x 5 µg/mL Working Int Std Mix + 1990 µL methanol.
Preparation of avian samples for extraction in a 96-well plate
2h 30m
Using an electronic sample list pre-select avian samples for extraction (up to 76 per 96-well plate).
30m
Locate and remove avian samples and quality control (QC) samples from the freezer and defrost on ice.
1h
Design and prepare a full batch of standards, quality controls (QCs) and samples using this Editable MS Excel 96-well plate map template. The platemap follows a column-wise plate map design to suit the Biotage Extrahera liquid handling robot (Figure 1).
FIgure 1 - Plate Map - Column-wise plate layout for automated Supported Liquid
Extraction on an Extrahera liquid handling robot (Biotage, Uppsala, Sweden)
1h
Prepare calibration standards directly into the 96-well deep well plate using Table S1 below to aliquot the correct volumes of each stock concentration, into a final volume of 200 uL with water
| Standard name | Amount (ng) | STDs Mix vol (uL) | Vol water (uL) | |
| 0 STD | 0 | 20 uL (x 100 ng/mL) | 0 | |
| 0.0025 STD | 0.0025 | 20 uL (x 100 ng/mL) | 5 uL x 500 pg/mL | |
| 0.0050 STD | 0.0050 | 20 uL (x 100 ng/mL) | 10 uL x 500 pg/mL | |
| 0.010 STD | 0.0100 | 20 uL (x 100 ng/mL) | 20 uL x 500 pg/mL | |
| 0.0250 STD | 0.0250 | 20 uL (x 100 ng/mL) | 5 uL x 5 ng/mL | |
| 0.0500 STD | 0.0500 | 20 uL (x 100 ng/mL) | 10 uL x 5 ng/mL | |
| 0.100 STD | 0.1000 | 20 uL (x 100 ng/mL) | 20 uL x 5 ng/mL | |
| 0.250 STD | 0.2500 | 20 uL (x 100 ng/mL) | 5 uL x 50 ng/mL | |
| 0.500 STD | 0.5000 | 20 uL (x 100 ng/mL) | 10 uL x 50 ng/mL | |
| 1.00 STD | 1.0000 | 20 uL (x 100 ng/mL) | 20 uL x 50 ng/mL | |
| 2.50 STD | 2.5000 | 20 uL (x 100 ng/mL) | 5 uL x 500 ng/mL | |
| 5.00 STD | 5.0000 | 20 uL (x 100 ng/mL) | 10 uL x 500 ng/mL | |
| 10.0 STD | 10.000 | 20 uL (x 100 ng/mL) | 20 uL x 500 ng/mL |
Table S1 - Calibration Standard Preparation Table
Once the calibration standards are in the plate wells then aliquot the now defrosted biological samples and QCs into the correct wells according to the plate map designed for the batch.
Using a multi-step pipette add 20 µL x 5 ng/mL Working Internal Standard into each well of calibration standard, including 0 std and each QC and biological sample. Make sure not to add WIT to the double blanks.
Supported liquid extraction of steroids from calibration standards, QCs and samples
2h 10m
Use the Extrahera liquid handling robot to extract steroids from the samples by supported liquid extraction
2h
Programme the liquid handling robot to aliquot the diluent (200 µL 0.1% formic acid) in water into each well of the 96-well deep well plate containing the samples, QCs and calibration standards.
Programme the Extrahera to transfer 400 µL of liquid from each well (containing sample and the diluent, into a 400 µL volume Supported Liquid Extraction plate (SLE400), pre-placed into the deck on the Extrahera, with a deep well Waters 2 mL deep well collection plate below, pre-labelled with the batch details and date of extraction.
Allow the diluted sample to adsorb onto the SLE extraction bed for 00:05:00 before eluting with eluent (600 µL x 98:2 (v/v) dichloromethane/isopropanol and repeating twice more), each time collecting the eluent into the collection plate
5m
Dry down the eluent collected into the 2 mL collection plate using the SPE Dry down for 96-well plates under nitrogen.
Resuspend in 100 µL x 70:30 water/methanol, seal the plate with a zone-free plate seal and shake on ThermoShaker for 00:05:00 at 300 rpm
5m
Place the plate immediately into the autosampler for LC-MS/MS or store at -20 °C until ready for analysis.
Set up a chromatographic and mass spectrometric method for avian steroid profiling
3h
Set up an acquisition batch in Analyst software using the electronic excel file of the calibration standards and sample list, and either build an LC-MS/MS method as below, or select the Avian Steroid Profiling Method
Set up the Nexera X2 liquid chromatography system (Shimadzu, Japan) and fit with a Krud Katcher and a 150 x 3 mm; 2.6 µm Kinetex C18 liquid chromatography column (Phenomenex, UK), using mobile phase A - water with 0.05 mM ammonium fluoride and mobile phase B - methanol with 0.05 mM ammonium fluoride. Purge the lines and then condition the chromatographic column with mobile phase 55%B, at a flow rate of 0.5 mL/min at temperature of 40 °C.
Set up chromatographic gradient as below (Table S2) with a run time of 16 minutes per sample and ensure the method has divert to the mass spectrometer only at 0.2 mins and return to waste at 12 minutes
| A | B | C | D | |
| Time (min) | Flow (mL/min) | A (%) | B (%) | |
| Initial | 0.5 | 45 | 55 | |
| 4.00 | 0.5 | 45 | 55 | |
| 10.00 | 0.5 | 0 | 100 | |
| 12.00 | 0.5 | 0 | 100 | |
| 12.10 | 0.5 | 45 | 55 | |
| 16.00 | 0.5 | 45 | 55 |
Table S2 - Chromatographic gradient details. A - water w/ 0.05 mM ammonium fluoride; B - methanol w/ 0.05 mM ammonium fluoride. 40oC. Kinetex C18 (150 x 3 mm; 2.6 µm)
1h
Set up the mass spectrometer for a Multiple Reaction Monitoring (MRM) method in polarity switching positive/negative mode, with electrospray ionisation as below (Table S3), with divert of LC flow into the mass spectrometer set at 1 minute and 12 minutes.
Set up the mass spectrometer to monitor for the following multiple reaction monitoring (MRM) transitions for each steroid and each isotopically labelled steroid (Table S5).
| A | B | C | D | E | F | G | H | I | |
| Q1 Mass | Q3 Mass | Retention time (min) | MRM Transition ID | DP (volts) | CE (volts) | CXP (volts) | Polarity | Internal Standard | |
| 347.1 | 121.1 | 6.9 | Corticosterone 1 | 76 | 29 | 8 | + | D8B | |
| 347.1 | 90.9 | 6.9 | Corticosterone 2 | 76 | 75 | 12 | + | D8B | |
| 355.3 | 125.1 | 6.8 | D8-Corticosterone | 56 | 31 | 8 | + | ||
| 345.1 | 121.2 | 5.0 | 11Dehydrocorticosterone 1 | 66 | 31 | 12 | + | D8B | |
| 345.1 | 91.2 | 5.0 | 11Dehydrocorticosterone 2 | 66 | 83 | 40 | + | D8B | |
| 289.1 | 97.0 | 8.2 | Testosterone 1 | 101 | 29 | 12 | + | 13C3T | |
| 289.1 | 109.2 | 8.2 | Testosterone 2 | 101 | 31 | 6 | + | 13C3T | |
| 292.1 | 100.0 | 8.2 | 13C3-Testosterone | 96 | 29 | 12 | + | ||
| 287.0 | 97.0 | 7.7 | Androstenedione 1 | 61 | 27 | 14 | + | 13C3A4 | |
| 287.0 | 78.9 | 7.7 | Androstenedione 2 | 61 | 67 | 10 | + | 13C3A4 | |
| 290.2 | 100.1 | 7.7 | 13C3-Androstenedione | 31 | 27 | 12 | + | ||
| 291.3 | 255.2 | 9.0 | Dihydrotestosterone 1 | 116 | 21 | 30 | + | 13C3DHT | |
| 291.3 | 91.0 | 9.0 | Dihydrotestosterone 2 | 116 | 55 | 10 | + | 13C3DHT | |
| 294.2 | 258.3 | 9.0 | 13C3-Dihydrotestosterone | 61 | 21 | 12 | + | ||
| 289.1 | 253.0 | 8.5 | Dehydroepiandrosterone 1 | 121 | 15 | 46 | + | 13C3DHT | |
| 289.1 | 231.1 | 8.5 | Dehydroepiandrosterone 2 | 121 | 11 | 12 | + | 13C3DHT | |
| 331.2 | 97.0 | 8.0 | 11-deoxycorticosterone 1 | 86 | 29 | 16 | + | D8B | |
| 331.2 | 109.0 | 8.0 | 11-deoxycorticosterone 2 | 86 | 31 | 12 | + | D8B | |
| 331.0 | 109.1 | 8.4 | 17OH-Progesterone 1 | 66 | 31 | 12 | + | D9P4 | |
| 331.0 | 96.9 | 8.4 | 17OH-Progesterone 2 | 66 | 29 | 12 | + | D9P4 | |
| 315.1 | 97.1 | 9.3 | Progesterone 1 | 96 | 25 | 10 | + | D9P4 | |
| 315.1 | 109.1 | 9.3 | Progesterone 2 | 96 | 27 | 10 | + | D9P4 | |
| 324.1 | 100.0 | 9.2 | D9-Progesterone | 151 | 31 | 15 | + | ||
| 359.1 | 188.9 | 3.5 | Aldosterone 1 | -70 | -24 | -21 | - | D8Aldo | |
| 359.1 | 331.0 | 3.5 | Aldosterone 2 | -70 | -22 | -35 | - | D8Aldo | |
| 367.2 | 193.9 | 3.4 | D8-Aldosterone | -75 | -26 | -21 | - | ||
| 269.1 | 144.9 | 7.9 | Estrone 1 | -150 | -48 | -15 | - | 13C3E1 | |
| 269.1 | 142.9 | 7.9 | Estrone 2 | -150 | -70 | -15 | - | 13C3E1 | |
| 272.0 | 147.8 | 7.9 | 13C3-Estrone | -165 | -50 | -17 | - | ||
| 271.1 | 182.9 | 8.0 | 17b-estradiol 1 | -110 | -52 | -19 | - | 13C3E2 | |
| 271.1 | 144.9 | 8.0 | 17b-estradiol 2 | -110 | -52 | -21 | - | 13C3E2 | |
| 274.0 | 185.9 | 8.0 | 13C3-Estradiol | -155 | -54 | -19 | - |
Table S4 - Multiple Reaction Monitoring (MRM) settings and Retention times on a Shimadzu Nexera uHPLC fitted with a Kinetex C18 (150 x 3 mm; 2.6 um) column coupled to a QTrap 6500+ mass spectrometer operated in polarity switching mode
1h
Inject a mid-level standard. Check the chromatography and each steroid retention time is consistent with expected times according to Table S4. For your instrument and multiple batches check that the peak area response is as expected compared to previous batches.
1h
Confirm you are happy with the chromatographic performance before next step
Set batch to analyse steroids
1d 1h
Create batch file name AND data file name to match the batch of samples being analysed and use the maing convention: with todays date, naming the file with yyyymmdd_STUDYCODE_BATCHnumber_initials. Make sure the correct location for the samples has been chosen and make sure the correct LC-MS method has been selected. Check there is sufficient mobile phase A and mobile phase B and the three washes for the LC instrument. Failure to have sufficient solvent will endanger your analysis and can cause damage to the instrumentation, chromatographic column. Begin and end all batches with a blank solvent. Submit the full batch, injecting 20 µL per sample. Note that a full plate of 96-wells will take 25 hours to analyse by LC-MS/MS
1d 1h
Make sure the correct location for the samples in the autosampler has been chosen
Make sure the correct LC-MS method has been selected for avian steroid profiling.
Check there is sufficient mobile phase A and mobile phase B and the three washes for the LC instrument. Failure to have sufficient solvent will endanger your analysis and can cause damage to the instrumentation, chromatographic column.
Submit the full batch, injecting 20 µL per sample. Note that a full plate of 96-wells will take 25 hours to analyse by LC-MS/MS. It will take longer if repeats are required.
Once submitted check the time calculated to run the samples. Check that your booking on PPMS for the mass spectrometer is long enough for the batch submitted
Do not leave the lab until you know your first injection of calibration standard has given appropriate results. Use Analyst Explore view to check the chromatogram.
Method specific data evaluation of avian steroid profiling LC-MS/MS data
2h
Use the data analysis parameters to systematically assess the retention times and peak area integration of the chromatograms for each of the Steroid analytes and their nominated internal standards (Table S5)
| A | B | C | D | |
| Steroid Name | Abbreviation | Retention time (Min) | Internal Standard | |
| Corticosterone | B | 6.9 | D8B | |
| 11-Dehydrocorticosterone 1 | A | 5.0 | D8B | |
| Testosterone | T | 8.2 | 13C3T | |
| Androstenedione | A4 | 7.7 | 13C3A4 | |
| Dihydrotestosterone | DHT | 9.0 | 13C3DHT | |
| Dehydroepiandrosterone | DHEA | 8.5 | 13C3DHT | |
| 11-deoxycorticosterone | 11DOC | 8.0 | D8B | |
| 17OH-Progesterone | 17OHP4 | 8.4 | D9P4 | |
| Progesterone | P4 | 9.3 | D9P4 | |
| Aldosterone | Aldo | 3.5 | D8Aldo | |
| Estrone | E1 | 7.9 | 13C3E1 | |
| 17beta-estradiol | E2 | 8.0 | 13C3E2 | |
| Internal Standards | ||||
| D8-Corticosterone | D8-B | 6.8 | IntStd | |
| 13C3-Testosterone | 13C3-T | 8.2 | IntStd | |
| 13C3-Androstenedione | 13C3-A4 | 7.7 | IntStd | |
| 13C3-Dihydrotestosterone | 13C3DHT | 9.0 | IntStd | |
| D9-Progesterone | D9P4 | 9.2 | IntStd | |
| D8-Aldosterone | D8Aldo | 3.4 | IntStd | |
| 13C3-Estrone | 13C3E1 | 7.9 | IntStd | |
| 13C3-Estradiol | 13C3E2 | 8.0 | IntStd |
Table S5 - Method specific parameters of retention time and specified internal standards for each steroid
2h
Use MultiQuant software (Sciex, UK) or an equivalent to evaluate the LC-MS/MS data.
Use the Table above (Table S5) to define and the retention times and mass transitions of each steroid and Internal standard for the 'processing method' and then process the collected data file.
Define each injection as either a double blank, standard, QC or unknown
Input calibration standard levels according to Table S1 and Plate Map Figure 1
Check that each peak integrated matches to the retention time of the steroid AND the internal standard, as defined by the method (Table S5) and use the metric plot function in MultiQuant to double-check all peaks have been picked.
Ensure accuracy of the calibration standards, (linear regression > 0.99) by checking calculated amount and the 'accuracy' calculated from expected amount (should be 20% for lowest calibration point and 15% for all other calibration points). If not exclude that point.
The concentration of each steroid in the QCs and samples (Unknowns) will be calculated by the software and then extract and sort in Microsoft Excel, as detailed in the protocol below.
Protocol
CREATED BY
Natalie ZM Homer
Protocol references
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Acknowledgements
We acknowledge the support of the Edinburgh CRF, laboratory management of the Centre for Cardiovascular Sciences and the staff in teh Stores of the Queen's Medical Research Institute, University of Edinburgh. We thank the staff of the National Avian Research Facility at the Roslin Institute for animal husbandry services.