Extraction and LC-MS/MS analysis of four steroids from mouse plasma and bone marrow

Prepare 0.5 mL Eppendorf microtubes for all bone marrow samples by removing the bottom of a 0.5 mL microtube (Eppendorf) with a razor blade

Batch sizes of 20-40 bone marrow samples are most manageable. Isolate bone marrow (BM) from frozen (or fresh) tibia by doing the following:

Remove tibiae from freezer and transfer frozen tibiae from dry ice onto wet ice prior to cutting.

Use a razor blade to cut off the proximal and distal ends of each tibia

Place each cut tibia into a cut-off 0.5 mL microtube, to keep the bone upright, and add this tibia in a 0.5 mL microtube into a 2 mL microtube.

Centrifuge the 2 mL cap-lock microtube and its contents in a microcentrifuge for8000rcf,4°C. The BM pellet will collect at the base of the 2 mL microtube

Record the mass of each BM pellet, using an analytical balance, prior to homogenisation. Multiple BM pellets can be combined at this point. (e.g. 2, 4 , 6 or 8 BM pellets can be homogenised together).

Add 500µL to the bone marrow in the 2 mL microtube

Add a 5 mm stainless steel metal bead into a 2 mL microtube, cap securely and homogenise in the Qiagen bead mill homogeniser at 30 Hz for 0h 0m 30s

Remove and transfer the homogenisation tube containing the homogenate to the freezer for 1h 0m 0s at -20°C

Remove the homogenate tube from the freezer, centrifuge for 0h 5m 0s at 16,100 rcf

Transfer the homogenate to a clean, labelled glass vial using a glass pipette

Add another aliquot of 500µL to the bone marrow pellet, vortex and centrifuge for 0h 5m 0s at 16,100 rcf

Transfer the supernatant from the homogenisation tube and add it to the first supernatant in the glass tube.

Reduced the supernatant to dryness under nitrogen at 40°C

Resuspend the dried down homogenate supernatant ofSample by adding 200µL prior to preparation of calibration standards (4), working internal standard (5) and then supported liquid extraction (See point 7) of steroids

Remove mouse plasma samples from the freezer and defrost on ice. Label up glass culture tubes with mouse sample IDs and aliquot 50µL of each Sample into labelled glass tubes, alongside bone marrow. Record exact volume of plasma aliquoted in plate map.

Prepare 100 µg/mL stock solutions of each steroid - corticosterone (B), 11-dehydrocorticosterone (A), testosterone (T) and progesterone (P4) in 1.75 m

Prepare a mixed stock of the 4 steroids - B, A, T, P4 - by using 100 µg/mL stock solutions. Do this by adding 50 µL x 100 µg/mL B, 50 µL x 100 µg/mL A, 50 µL x 100 µg/mL T and 50 µL x 100 µg/mL P4 + 800 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 (100µL x 5 µg/mL + 900µLmethanol ) to give 50 pg/mL stock

Prepare 100 µg/mL solutions of each isotopically labelled internal standard (d8-corticosterone, ¹³C₃-testosterone and d9-progesterone) in methanol.

Prepare a mixed 5 µg/mL Internal Standard mix stock solution of the three isotopically labelled steroids by adding 25µL x 100 µg/mL d8-corticosterone, 25µL x 100 µg/mL ¹³C₃-testosterone and25µL x 100 µg/mL d9-progesterone to425µL methanol.

Prepare a 5 ng/mL Working Internal Standard solution by taking 10 µL x 5 µg/mL Int Std Mix + 1990 µL methanol.

Prepare calibration standards directly into labelled glass culture tubes using the following table 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

Calibration standard preparation table

Transfer 200 µL of prepared calibration standards, bone marrow homogenate and mouse plasma from glass culture tubes into a 2 mL deep well 96-well collection plate (Biotage), prepared in Microsoft Excel template (see Files) and following a plate map design as below -

Using a multi-step pipette enrich the plate containing calibration standards with IS by adding 20µL x 5 ng/mL Working Internal Standard into each calibration standard, including 0 std and each sample (bone homogenate and mouse plasma), except for the Double blank and solvent blank.

Using the Extrahera liquid handling robot, set up with an SLE400 extraction plate and a deep well extraction plate, aliquot 200 µL 0.5Molarity (M)ammonium hydroxide in water into each well of the 96-well plate.

Transfer 400µL of liquid from each well (containing sample and the diluent, 0.5Molarity (M)ammonium hydroxide) 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 0h 5m 0s before eluting with 600µL x 98:2 (v/v) dichloromethane/isopropanol and repeating twice more, each time collecting the eluent into the collection plate

Dry down the eluent collected into the 2 mL collection plate using the SPE Dry down for 96-well plates under nitrogen.

Resuspend in 100µL x 70:30 water/methanol, seal the plate with a zone-free plate seal and shake on ThermoShaker for 0h 5m 0s at 300rpm

Place the plate in the autosampler for LC-MS/MS or store at-20°Cuntil ready for analysis.

Set up an acquisition batch in Analyst software using the electronic 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.

Set up chromatographic gradient as below with a run time of0h 16m 0s 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

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)

Set up the mass spectrometer for Multiple Reaction Monitoring (MRM) method in positive mode, with electrospray ionisation as below, 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)	4500 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

Mass Spectrometry source settings for positive ion electrospray ionsiation on QTrap 6500+

Set up the mass spectrometer to monitor for the following MRM transitions for each steroid and isotopically labelled steroid.

A	B	C	D	E	F	G
Q1 Mass (Da)	Q3 Mass (Da)	Scan time (msec)	Steroid Name	DP (V)	CE (V)	CXP (V)
347.1	121.1	50	Corticosterone 1	76	29	8
347.1	90.9	50	Corticosterone 2	76	75	12
345.1	121.2	50	11-Dehydrocorticosterone 1	66	31	12
345.1	91.2	50	11-Dehydrocorticosterone 2	66	83	40
289.1	97.0	50	Testosterone 1	101	29	12
289.1	109.2	50	Testosterone 2	101	31	6
315.1	97.1	50	Progesterone 1	96	23	10
315.1	109.1	50	Progesterone 2	96	27	10
355.3	128.1	50	d8B-Corticosterone 1	37	45	14
355.3	125.0	50	d8B-Corticosterone 2	29	56	14
292.1	100.2	50	13C3-Testosterone	101	29	12
324.1	100	50	d9-Progesterone	96	23	10

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 below:

Once the chromatography has been checked and the retention times are consistent, set the batch of samples to analyse. Use MultiQuant software and excel to evaluated the LC-MS/MS data to calculate the concentration of steroids in each sample, as detailed:

Using MultiQuant and Excel software to evaluate and report multi-analyte targeted LC-MS/MS data.