Fig. 1
Typical elution profile for sphingolipids analysis by LC/MS/MS
2 Materials and Equipment
2.1 Samples
Venous blood is drawn into Vacutainer® tubes containing EDTA (Becton, Dickinson and Company, Franklin Lakes, NJ), shipped on cold packs overnight, and held at 4 °C upon arrival. The blood samples are usually spotted within 24 h of collection.
2.2 Solvents and Reagents
1.
Acetonitrile, Chloroform, Methanol, and Water are purchased from Burdick & Jackson (Morristown, NJ).
2.
Trifluoroacetic acid is purchased from EMD Millipore (Billerica, MA).
3.
Dimethyl sulfoxide is purchased from Sigma (St. Louis, MO).
4.
Sample extraction solution (methanol/acetonitrile/water, 80/15/5 %, v/v/v): To make 1 L of solution, combine 800 mL methanol, 150 mL acetonitrile, 50 mL water in a 1 L glass bottle. Mix on a stir plate for 30 min prior to use.
5.
HPLC mobile phase (methanol/acetonitrile/water (80/15/5 %) with 0.5 % trifluoroacetic acid): Combine 800 mL methanol, 150 mL acetonitrile, 50 mL water, and 5 mL trifluoroacetic acid in a 1 L glass bottle. Mix on a stir plate for 30 min prior to use.
6.
2:1 Chloroform/methanol: Combine 600 mL chloroform and 300 mL methanol in a 1 L glass bottle. Mix on a stir plate for 30 min prior to use.
2.3 Standards and Internal Standards
1.
Glucocerebrosides (#1521), N-Octadecanoyl-D35-psychosine (#1914), Ceramide trihexosides (#1067), N-Heptadecanoyl ceramide trihexoside (#1523), and Ceramides (#1056) are purchased from Matreya, LLC (Pleasant Gap, PA).
2.
C17-Ceramide (#860517) is purchased from Avanti Polar Lipids (Alabaster, AL).
2.4 Analytical Equipment and Supplies
1.
Whatman 903 protein saver cards and MiniPax® absorbent packets are purchased from Sigma-Aldrich (St. Louis, MO).
2.
Rocking platform shaker and Multi-Tube Vortexer are purchased from VWR (Radnor, PA).
3.
Hot pocket column heater is purchased from Thermo Scientific (Waltham, MA).
4.
Ultra silica column (#9100552) is purchased from Restek (Bellefonte, PA).
5.
API5000 mass spectrometer is from AB SCIEX (Framingham, MA).
6.
HTC-PAL autosampler is from Leap Technologies (Carrboro, NC).
7.
1200 series binary pump and automated degasser are from Agilent (Santa Clara, CA).
2.5 Standard Solutions Preparation
1.
Individual standard stock solution preparation:
(a)
Standard stock solutions A (1 mg/mL): Weight 5 mg each standard into individual vials and dissolve each in 5 mL 2:1 chloroform/methanol solution. Vortex solutions for 5 min, sonicate for 10 min and then vortex an additional 5 min.
(b)
Standard stock solutions B (10 μg/mL): 100 μL of stock solution A is diluted in 9.9 mL 2:1 chloroform/methanol solution. Vortex solutions for 5 min
2.
Standard mixture solution preparation (2.4 μg/mL Cer, 0.8 μg/mL GL-1, 0.8 μg/mL GL-3): From the Stock B solutions for each standard, 2.4 mL of Cer, 800 μL of GL-3, and 800 μL GL-1 are mixed in a vial and dried under nitrogen. The dried vial is then reconstituted in 10 mL of 2:1 chloroform/methanol. Vortex solution for 5 min, sonicate for 10 min, and then vortex an additional 5 min.
2.6 Internal Standards Solutions Preparation
1.
Individual internal standard stock solution preparation:
(a)
Internal standard stock solutions A (1 mg/mL): Weight 5 mg each internal standard into individual vials and dissolve each in 5 mL 2:1 chloroform/methanol solution. Vortex solutions for 5 min, sonicate for 10 min, and then vortex an additional 5 min.
(b)
Internal standard stock solutions B (10 μg/mL): 100 μL of internal standard stock solution A is diluted in 9.9 mL 2:1 chloroform/methanol solution. Vortex solutions for 5 min.
2.
Internal standard mixture solution preparation (3.75 μg/mL C17-Cer, 1.25 μg/mL N-Octadecanoyl-D35-psychosine, 1.25 μg/mL N-Heptadecanoyl ceramide trihexoside):
From the Stock B solutions for each internal standard, 3.75 mL of C17-Cer, 1.25 mL of N-Octadecanoyl-D35-psychosine, and 1.25 mL of N-Heptadecanoyl ceramide trihexoside are mixed in a vial and dried under nitrogen. The dried vial is then reconstituted in 10 mL of 2:1 chloroform/methanol. Vortex solution for 5 min, sonicate for 10 min, and then vortex an additional 5 min.
2.7 Preparation of Calibrators
Using standard mixture solution and internal standard mixture solution, pipette the volume shown (Table 1) into 1 mL total recovery vials to create calibrators 1–10. Dry calibrators under nitrogen and store at −20 °C. Reconstitute each calibrator in 200 μL pooled DBS extract (preparation shown below). Vortex the calibrator vials for 5 min, sonicate for 10 min, and vortex an additional 5 min.
Table 1
Preparation of calibration standards
Calibration standards | Theoretical Cer/GL-1/GL-3 (ng/mL) | Standard mixture solution (μL) | Internal standard mixture solution (μL) |
|---|---|---|---|
1 | 0 | 0 | 40 |
2 | 150/50/50 | 12.5 | 40 |
3 | 300/100/100 | 25 | 40 |
4 | 600/200/200 | 50 | 40 |
5 | 1200/400/400 | 100 | 40 |
6 | 2400/800/800 | 200 | 40 |
7 | 3600/1200/1200 | 300 | 40 |
8 | 4800/1600/1600 | 400 | 40 |
9 | 6000/2000/2000 | 500 | 40 |
10 | 9000/3000/3000 | 750 | 40 |
2.8 Preparation of Control Samples
1.
Using the standard stock solutions B and internal standard working solution mixture, pipette the volume (Table 2) into 4 mL glass vials to create low, medium, and high controls. Dry controls under nitrogen. Reconstitute each control in 4 mL pooled DBS extract (preparation shown below). Vortex the calibrator vials for 5 min, sonicate for 10 min, and vortex an additional 5 min. It’s important to point out that actual QC level should be the combination of spiked and endogenous lipid.
Table 2
Preparation of controls
Control | Spiked Cer/GL-1/GL-3 (ng/mL) | Cer stock solution B (μL) | GL-1 stock solution B (μL) | GL-3 stock solution B (μL) | Internal standard mixture solution (μL) |
|---|---|---|---|---|---|
Endogenous level | 0/0/0 | 0 | 0 | 0 | 800 |
Low | 240/80/80 | 96 | 32 | 32 | 800 |
Medium | 1200/400/400 | 480 | 160 | 160 | 800 |
High | 4800/1600/1600 | 1920 | 640 | 640 | 800 |
2.
200 μL aliquots of each control are pipetted in total recovery vials, dried under N2, and stored at −20 °C.
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