TITLE: LC-MS/MS analysis of 5 steroids in plasma in a clinical study of Congenital Adrenal Hyperplasia
AUTHORS: Catriona Kyle, George Just, Scott G Denham, Natalie ZM Homer

[DESCRIPTION]
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.

[BEFORE_START]
Ensure all consumables are in stock and all compounds and reagents are freshly prepared

[GUIDELINES]
Ensure all training is up-to-date for operating the necessary laboratory instrumentation and equipment.

[STEPS]
SECTION: Preparation of calibration standard stock solutions
2. 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.
SECTION: Preparation of calibration standard stock solutions
2.1. 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. 
SECTION: Preparation of calibration standard stock solutions
2.2. 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
SECTION: Preparation of calibration standard stock solutions
2.3. 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
SECTION: Preparation of calibration standard stock solutions
2.4. 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
SECTION: Preparation of calibration standard stock solutions
2.5. 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
SECTION: Preparation of calibration standard stock solutions
2.6. Dilute the 500 pg/mL Mixed STOCK by 1:10 dilution (100 µL x 5 µg/mL + 900 µLmethanol ) to give 50 pg/mL stock   

SECTION: Preparation of internal standard solution
3. Prepare 100 µg/mL solutions of each isotopically labelled internal standard (d4-cortisol, 13C3-testosterone, 13C3-androstenedione and d9-progesterone) in methanol.
SECTION: Preparation of internal standard solution
3.1. 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 µLx 100 µg/mL 13C3-testosterone and25 µLx 100 µg/mL13C3-androstenedione and25 µLx 100 µg/mL d9-progesterone to425 µL methanol.

SECTION: Preparation of internal standard solution
3.2. Prepare a 5 ng/mL Working Internal Standard solution by taking 10 µL x 5 µg/mL Int Std Mix + 1990 µL methanol.
SECTION: Preparation of calibration standard curve and samples
5. 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 µLwater.
 
 
 
SECTION: Set up of supported liquid extraction of steroids from calibration standards and samples
4. 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).


 



SECTION: Supported liquid extraction of steroids from calibration standards and samples
6. Using a multi-step pipette enrich the plate containing calibration standards with WIS by adding 20 µLx 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.

SECTION: Supported liquid extraction of steroids from calibration standards and samples
7. 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. 

SECTION: Supported liquid extraction of steroids from calibration standards and samples
8. 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.

SECTION: Supported liquid extraction of steroids from calibration standards and samples
9. Allow the diluted sample to adsorb onto the SLE extraction bed for 5 min 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

SECTION: Supported liquid extraction of steroids from calibration standards and samples
10. Dry down the eluent collected into the 2 mL collection plate using the SPE Dry down for 96-well plates under nitrogen.
SECTION: Supported liquid extraction of steroids from calibration standards and samples
11. Resuspend in 100 µL x 70:30 water/methanol, seal the plate with a zone-free plate seal and shake on ThermoShaker for 5 min at  

SECTION: Supported liquid extraction of steroids from calibration standards and samples
12. Place the plate in the autosampler for LC-MS/MS or store at-20 °Cuntil ready for analysis.

SECTION: Steroid analysis by LC-MS/MS
13. 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.

SECTION: Steroid analysis by LC-MS/MS
14. 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 

SECTION: Steroid analysis by LC-MS/MS
15.  Set up chromatographic gradient as below (Table S3) with a run time of16 min per sample
 

SECTION: Steroid analysis by LC-MS/MS
16. 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.
 
 
 

SECTION: Steroid analysis by LC-MS/MS
17. 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).

 

SECTION: Steroid analysis by LC-MS/MS
18. Check the retention times of the steroids are as expected, as shown in the chromatogram in Fig S1: 
 
 

 


SECTION: Steroid analysis by LC-MS/MS
19. 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.


SECTION: Preparation of human plasma for extraction
1. Remove human plasma samples from the freezer and defrost on ice. 

SECTION: Preparation of calibration standard curve and samples
5.1. Aliquot 200 µLplasma sample into the correct well according to the plate map design.

SECTION: Method specific data evaluation of LC-MS/MS data
20. Use the data analysis parameters to assess the peak area of the chromatograms for the Steroid analytes and their nominated internal standards (Table S6)

 
 
 
SECTION: Method specific data evaluation of LC-MS/MS data
21. 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.