Feb 28, 2024
Version 2
LC-MS/MS analysis of 5 steroids in plasma in a clinical study of Congenital Adrenal Hyperplasia V.2
- Catriona Kyle1,2,
- George Just3,
- Scott G Denham3,
- Natalie ZM Homer1,3
- 1University of Edinburgh;
- 2NHS Lothian;
- 3Mass Spectrometry Core, Edinburgh Clinical Research Facility, University of Edinburgh
- Clinical Mass Spectrometry
Protocol Citation: Catriona Kyle, George Just, Scott G Denham, Natalie ZM Homer 2024. LC-MS/MS analysis of 5 steroids in plasma in a clinical study of Congenital Adrenal Hyperplasia. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdrmzlmk/v2Version created by Natalie ZM Homer
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: February 23, 2024
Last Modified: February 28, 2024
Protocol Integer ID: 95685
Keywords: steroid, steroid analysis, quantitative steroid measurement, LC-MS/MS, corticosterone, testosterone, supported liquid extraction, tandem mass spectrometry, steroid mass spectrometry, 17-hydroxyprogesterone, androstenedione, cortisol, congenital adrenal hyperplasia, androstenedione, corticosterone, cortisol, hydrocortisone, 17a-hydroxyprogesterone
Funders Acknowledgements:
NHS Research Scotland
Grant ID: EDCRF
Chief Scientist Office
Grant ID: SCAF/17/02
Abstract
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that affects adrenal steroidogenesis, resulting in deficiency of the glucocorticoid cortisol and in many cases the mineralocorticoid aldosterone1. This resulting lack of glucocorticoid (and mineralocorticoid) activates the hypothalamic-pituitary-adrenal (HPA) axis causing excessive release of adrenocorticotrophic hormone (ACTH) and excess adrenal androgen synthesis.
Diagnosing and treating CAH requires reliable methods for steroid analysis. Tandem mass spectrometry methods coupled with chromatographic separation are considered the gold standard analytical technique for steroid analysis2 with the added benefit of enabling simultaneous analysis of multiple steroids. There are a range of methods that have been developed to measure multiple steroids in CAH3. Here we have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for application to a clinical study that specifically explores the administration of d8-corticosterone as an alternative to hydrocortisone for CAH treatment.
Plasma samples (200 µL) were enriched with isotopically labelled steroids, diluted with water (0.1% formic acid v/v) and extracted alongside a (0.0025 - 400 ng) calibration curve, by automated 96-well supported liquid extraction (SLE), using dichloromethane and isopropanol as an organic solvent, on a Biotage Extrahera automated sample handler. Extracted steroids were separated on a Shimadzu Nexera uHPLC with gradient elution on a Kinetex C18 column (150 x 3 mm; 2.6 µm) and a mobile phase of methanol and water (0.1% formic acid 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 five steroids - hydrocortisone (cortisol) and d8-corticosterone - combined with markers of CAH - androgens and the intermediate 17α-hydroxyprogesterone, alongside the internal standards - in plasma. Validation demonstrated that this method is sensitive, specific, and reliable.
Attachments
Guidelines
Ensure all training is up-to-date for operating the necessary laboratory instrumentation and equipment.
Materials
Consumables Table
| 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 | 00F-4462-Y0 | 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 |
Consumables for homogenisation of bone marrow and steroid extraction by supported liquid extraction (SLE)
Chemicals and Analytical Standards Table
| A | B | C | |
| Item | Supplier | Article no. | |
| 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 | |
| Cortisol | Sigma-Aldrich/Cerilliant | (C-106 ) 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) | |
| 17a-hydroxyprogesterone | Sigma-Aldrich/Cerilliant | (P-069) 1 mg/mL in acetonitrile (certified) | |
| D8-Corticosterone | Cambridge Isotope Laboratories/CK Isotopes | (DLM-7347) Supplied as powder | |
| 13C3-testosterone | Sigma-Aldrich/Cerilliant | (T-070) 100 ug/mL in acetonitrile (certified) | |
| 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 | |
| Formic acid (LC-MS grade) | Fisher Scientific | 10596814 |
Chemicals and analytical standards
Solutions Required
- 0.1% formic acid (aq) (200 mL) 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.
Equipment Table
| A | B | C | |
| Item | Model | Supplier | |
| 2 x Liquid Chromatography Pumps | LC30AD | Shimadzu | |
| Autosampler | SIL-30ACMP | Shimadzu | |
| Column oven , Nexera X2 | CTO-20AC | Shimadzu | |
| QTrap 6500+ mass spectrometer | 5038125-J | AB Sciex | |
| Gilson Repetman | Gilson Repetman | Gilson | |
| Deepwell plate thermoshaker | TS-DW | Grant Scientific | |
| Liquid handling robot | Extrahera | Biotage, Sweden | |
| SPE Dry 96 dual evaporator | SPE Dry | Biotage, Sweden |
Equipment required for homogenisation, extraction and steroid analysis
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
All human studies were approved by the University of Edinburgh NHS Lothian ACCORD Ethical Review Board.
Before start
Ensure all consumables are in stock and all compounds and reagents are freshly prepared
Preparation of human plasma for extraction
Preparation of human plasma for extraction
Remove human plasma samples from the freezer and defrost on ice.
Preparation of calibration standard stock solutions
Preparation of calibration standard stock solutions
Prepare 1 mg/mL stock solution of d8-corticosterone (d8B) from powder then prepare separate 100 µg/mL stock solutions of each steroid - d8B, cortisol (F), 17a-hydroxyprogesterone (17OHP4), testosterone (T) and androstenedione (A4) in methanol.
Prepare a mixed stock of the 5 steroids - D8B, F, 17OHP4, T, A4 - by using 100 µg/mL stock solutions. Do this by adding 50 µL x 100 µg/mL D8B, 50 µL x 100 µg/mL F, 50 µL x 100 µg/mL 17OHP4, 50 µL x 100 µg/mL T and 50 µL x 100 µg/mL A4 + 750 µL 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 go to step #3
Preparation of internal standard solution
Preparation of internal standard solution
Prepare 100 µg/mL solutions of each isotopically labelled internal standard (d4-cortisol, 13C3-testosterone, 13C3-androstenedione and d9-progesterone) in methanol.
Prepare a mixed 5 µg/mL Internal Standard mix stock solution of the three isotopically labelled steroids by adding 25 µL x 100 µg/mL d4-cortisol, 25 µL x 100 µg/mL 13C3-testosterone and25 µL x 100 µg/mL13C3-androstenedione and25 µL x 100 µg/mL d9-progesterone to425 µL methanol.
Prepare a 5 ng/mL Working Internal Standard solution by taking 10 µL x 5 µg/mL Int Std Mix + 1990 µL methanol.
Set up of supported liquid extraction of steroids from calibration standards and samples
Set up of supported liquid extraction of steroids from calibration standards and samples
10m
10m
Label a 2 mL deep well 96-well collection plate (Biotage). Label a Supported Liquid Extraction SLE400 plate with batch details. Label a 2 mL deep well 96-well collection plate (Waters).
Design and prepare batch of standards and samples in Microsoft Excel template (see Files), following a column-wise plate map design as below (Table S1).
Table S1 - Plate Map - Column-wise plate layout for automated Supported Liquid Extraction on an Extrahera liquid handling robot (Biotage, Sweden)
Preparation of calibration standard curve and samples
Preparation of calibration standard curve and samples
Prepare calibration standards directly into the 96-well deep well plate using Table S2 below, for volumes of each stock concentration, into a final volume of 200 µL water.
| A | B | C | D | |
| Standard name | Amount (ng) | STD Mix Vol (uL) | Vol water (uL) | |
| 0 STD | 0 | 0 | 200 | |
| 0.00250 STD | 0.00250 | 5 uL x 500 pg/mL | 195 | |
| 0.00500 STD | 0.00500 | 10 uL x 500 pg/mL | 190 | |
| 0.01000 STD | 0.0100 | 20 uL x 500 pg/mL | 180 | |
| 0.0250 STD | 0.0250 | 5 uL x 5 ng/mL | 195 | |
| 0.0500 STD | 0.0500 | 10 uL x 5 ng/mL | 190 | |
| 0.100 STD | 0.100 | 20 uL x 5 ng/mL | 180 | |
| 0.250 STD | 0.250 | 5 uL x 50 ng/mL | 195 | |
| 0.500 STD | 0.500 | 10 uL x 50 ng/mL | 190 | |
| 1.00 STD | 1.00 | 20 uL x 50 ng/mL | 180 | |
| 2.50 STD | 2.50 | 5 uL x 500 ng/mL | 195 | |
| 5.00 STD | 5.00 | 10 uL x 500 ng/mL | 190 | |
| 10.0 STD | 10.0 | 20 uL x 500 ng/mL | 180 | |
| 25.0 STD | 25.0 | 5 uL x 5 ug/mL | 195 | |
| 50.0 STD | 50.0 | 10 uL x 5 ug/mL | 190 | |
| 100 STD | 100.0 | 20 uL x 5 ug/mL | 180 | |
| 200 STD | 250 | 5 uL x 50 ug/mL | 195 | |
| 400 STD | 400.0 | 8 uL x 50 ug/mL | 192 |
Table S2 - Calibration standard preparation table
Aliquot 200 µL plasma sample into the correct well according to the plate map design.
Supported liquid extraction of steroids from calibration standards and samples
Supported liquid extraction of steroids from calibration standards and samples
10m
10m
Using a multi-step pipette enrich the plate containing calibration standards with WIS by adding 20 µL x 5 ng/mL Working Internal Standard into each calibration standard, including 0 std and each sample (human plasma), except for the double blank and solvent blank.
Using the Extrahera liquid handling robot, set up with the batch labelled SLE400 extraction plate and the deep well extraction plate, containing the calibration standards and samples. Programme Extrahera to aliquot200 µL 0.1% formic acid in water (v/v) into each well of the 96-well deep well plate containing the samples and 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 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 in the autosampler for LC-MS/MS or store at-20 °C until ready for analysis.
Steroid analysis by LC-MS/MS
Steroid analysis by LC-MS/MS
16m
16m
Set up an acquisition batch in Analyst software using the electronic excel file of the calibration standards and sample list. Set to inject 10 µL per sample and use a method of chromatographic separation as described in step 16 and 17 and mass spectrometer settings as outlined in steps 18 and 19.
Set up the Shimadzu Nexera X2 liquid chromatography system and fit with a Phenomenex Krud Katcher and a Phenomenex 150 x 3 mm; 2.6 µm Kinetex C18 liquid chromatography column, using mobile phase A - water with 0.1% formic acid and mobile phase B - methanol with 0.1% formic acid at 0.5 mL/min and 40 °C diverting to the mass spectrometer at 0.2 mins and
Set up chromatographic gradient as below (Table S3) with a run time of00:16:00 per sample
| 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 S3 - Chromatographic gradient details. A - water w/ 0.1% formic acid; B - methanol w/ 0.1% formic acid. 40oC. Kinetex C18 (150 x 3 mm; 2.6 µm)
16m
Set up the mass spectrometer for Multiple Reaction Monitoring (MRM) method in positive mode, with electrospray ionisation as below (Table S4), with divert of LC flow into the mass spectrometer set at 1 minute and 12 minutes.
| A | B | |
| Instrument | Sciex QTrap 6500+ | |
| Source, Ionisation Mode | IonDrive Turbo V Source, ESI | |
| Scan Mode, Polarity | MRM, Positive | |
| Resolution (Q1/Q3) | unit/unit | |
| Mass range | Low mass | |
| Pause Time | 5.007 ms | |
| Acquisition time | 16.0 min | |
| Delay time | 0 sec | |
| Curtain Gas (CUR) (N2) | 30 units | |
| Collision Gas (CAD) (N2) | Medium | |
| IonSpray Voltage (IS) (Positive) | 5500 V | |
| Temperature (TEM) | 600 °C | |
| Ion Source Gas 1 (GS1) (Air) | 40 units | |
| Ion Source Gas 2 (GS2) (Air) | 60 units | |
| Entrance Potential (EP) (Positive) | 10 V | |
| Probe position (x – axis) | 5 | |
| Probe position (y – axis) | 2 |
Table S4 - Mass Spectrometry source settings for positive ion electrospray ionsiation on QTrap 6500+ mass spectrometer
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 | |
| Q1 Mass (Da) | Q3 Mass (Da) | Scan time (msec) | Steroid Name | DP (V) | CE (V) | CXP (V) | |
| 287.1 | 97.0 | 10 | Androstenedione 1 | 61 | 27 | 14 | |
| 287.1 | 78.9 | 10 | Androstenedione 2 | 61 | 67 | 10 | |
| 363.1 | 121.2 | 10 | Cortisol 1 | 66 | 31 | 12 | |
| 363.1 | 91.0 | 10 | Cortisol 2 | 76 | 83 | 10 | |
| 289.1 | 97.0 | 10 | Testosterone 1 | 101 | 29 | 12 | |
| 289.1 | 109.2 | 10 | Testosterone 2 | 101 | 31 | 6 | |
| 333.1 | 109.1 | 10 | 17a-hydroxyprogesterone 1 | 66 | 31 | 12 | |
| 333.1 | 96.9 | 10 | 17a-hydroxyprogesterone 2 | 66 | 29 | 12 | |
| 355.3 | 128.1 | 10 | d8B-Corticosterone 1 | 37 | 45 | 14 | |
| 355.3 | 125.0 | 10 | d8B-Corticosterone 2 | 29 | 56 | 14 | |
| 292.1 | 100.0 | 10 | 13C3-Testosterone | 96 | 29 | 12 | |
| 290.2 | 100.1 | 10 | 13C3-Androstenedione | 31 | 27 | 12 | |
| 324.1 | 100.0 | 10 | d9-progesterone | 151 | 31 | 15 | |
| 367.2 | 121.1 | 10 | d4-cortisol | 80 | 29 | 16 |
Table S5 - Multiple reaction monitoring (MRM) settings for each steroid, including quantitative (1) and qualitative (2) ions for each steroid. DP - declustering potential, CE - collision energy, CXP - collision exit potential
Check the retention times of the steroids are as expected, as shown in the chromatogram in Fig S1:
Expected result
Retention times; cortisol at 4.0 mins, d8-corticosterone at 6.1 mins, androstenedione at 7.3 mins, testosterone at 7.8 mins and 17a-hydroxyprogesterone at 8.1 mins
Figure S1 - Overlaid Ion Chromatogram of MRM transitions for cortisol, D8-corticosterone, androstenedione, testosterone and 17a-hydroxyprogesterone, separated by gradient on a Kinetex C18 (150 x 3 mm; 2.6 um) at 0.5 mL/min with mobile phase 0.1% formic acid in water and methanol
Inject a mid-level standard. Check the chromatography and each steroid retention time is consistent with expected times and peak area response is as expected. Once satisfied then set the batch of samples to analyse, injecting 10 µL per sample.
Method specific data evaluation of LC-MS/MS data
Method specific data evaluation of LC-MS/MS data
Use the data analysis parameters to assess the peak area of the chromatograms for the Steroid analytes and their nominated internal standards (Table S6)
| A | B | C | D | |
| Steroid Name | Abbreviation | Retention Time (min) | Internal Standard | |
| Cortisol | F | 4.0 | d4F | |
| d8-Corticosterone | d8B | 6.1 | d4F | |
| Androstenedione | A4 | 7.3 | 13C3A4 | |
| Testosterone | T | 7.8 | 13C3T | |
| 17a-hydroxyprogesterone | 17OHP | 8.1 | d9P4 | |
| Internal Standards | ||||
| d4-cortisol | d4F | 3.9 | Int Std | |
| 13C3-Androstenedione | 13C3A4 | 7.3 | Int Std | |
| 13C3-Testosterone | 13C3T | 7.8 | Int Std | |
| d9-progesterone | d9P4 | 9.0 | Int Std |
Table S6 - Method specific parameters of retention time and specific internal standard of the steroids
Use MultiQuant software and Microsoft Excel to evaluate the LC-MS/MS data, by defining calibration standard levels, ensuring accuracy of the calibration standards and linear regression > 0.99. Use the Table above, to calculate the concentration of steroids in each sample, as detailed in the protocol below. Remember to account for the volume of sample extracted and express as ng/mL.
Protocol
NAME
Using MultiQuant and Excel software to evaluate and report multi-analyte targeted LC-MS/MS dataCREATED BY
Natalie ZM Homer
Protocol references
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Guasti L, Huebner A, Kortmann BBM, Krone N, Merke DP, Miller WL, Nordenstrom A, Reisch N, Sandberg DE, Stikkelbroeck N, Touraine P, Utari A, Wudy SA & White PC. Congenital Adrenal Hyperplasia-Current Insights in Pathophysiology, Diagnostics, and Management. Endocr Rev 2022 43 91-159.
2 - Handelsman DJ, Wartofsky L. Requirement for mass spectrometry sex steroid assays in the Journal of Clinical Endocrinology and Metabolism. J Clin Endocrinol Metab. 2013 Oct;98(10):3971-3
3 - Rakete S, Schubert T, Vogeser M. Semi-automated serum steroid profiling with tandem mass spectrometry. J Mass Spectrom Adv Clin Lab. 2022 27 40-48.