Keywords

1 Reagents

  • Sphingolipids (Avanti Polar Lipids)

  • C18 sphingosine (Sph)

  • C18 dihydrosphingosine (dhSph)

  • C18 phytosphingosine (pSph)

  • C18 sphingosine-1-phosphate (S1P)

  • C18 dihydrosphingosine-1-phosphate (dhS1P)

  • C18 phytosphingosine-1-phosphate (pS1P)

  • C17 sphingosine (C17-Sph)

  • C17 sphingosine-1-phosphate (C17-S1P)

  • Methanol for LC/MS

  • Chloroform for HPLC

  • Acetonitrile for LC/MS

  • Formic acid for LC/MS

  • Ammonium formate 1 mol/l solution for HPLC

  • Polytyrosine-1,3,6 calibration solution (Thermo Fisher Scientific)

  • Water (ultrapure grade)

2 Equipments

  • High performance liquid chromatography system

  • ESI-triple quadrupole mass spectrometer with analytical software

  • Polyether ether ketone (PEEK) tubing

  • HPLC column (ODS):

  • Example: CAPCELL PAK C18 ACR (3 μm, 1.0 mm i.d. × 150 mm, Shiseido) for analytical column and CAPCELL PAK ACR (3 μm, 1.0 mm i.d. × 35 mm, Shiseido) for trap column

  • Syringe (500 μl, Hamilton Company)

  • Centrifuge

  • Siliconized sample tube (2.0 ml, WATOSON)

  • YMC Duo Filter (0.2 μm, pore size, 4 mm inner diameter, YMC)

  • Micro tube mixer

  • Ultrasonic bath

  • Ultrasonic homogenizer

3 Prepare Stock Solutions and Working Solutions of Sphingolipids

Siliconized sample tubes and pipette tips are used. For quantitative analysis, stable isotope analogue internal standards are added to biological samples. The ratio between the internal standard and the compound of interest can be measured by LC/MS. However, many compounds in which acyl groups differ exist in sphingolipids. The separation and selective detection by MS will be difficult if the stable isotope-labeled compound overlaps the same m/z as the target compounds. Therefore, C17 (C17-Sph and C17-S1P) are used for sphingolipid analysis as the internal standard.

  1. 1.

    Dissolve each powder of S1P, pS1P, C17-S1P, Sph, dhSph, and pSph in an appropriate volume of solution to prepare a stock solution; that is, Sph, C17-Sph, dhSph, and pSph are dissolved in methanol (1 mg/ml); S1P, C17-S1P, and pS1P are dissolved in methanol (0.04 mg/ml); dhS1P is dissolved in chloroform:methanol:H2O = 1.5:1.4:0.2 (0.04 mg/ml). Store the solutions at −80°C.

  2. 2.

    Dilute the stock solutions with methanol to a final concentration of 10.0 μmol/l (solution AS1P, pS1P, dhS1P, Sph, dhSph, pSph), and then use it (them) for the optimization of a mass spectrometer.

  3. 3.

    Prepare seven working solutions (1000, 200, 20.0, 10.0, 2.0, 1.0, and 0.2 nmol/l) using each stock solution (AS1P, pS1P, dhS1P, Sph, dhSph, pSph) as described in Table 26.1. Store at −80 °C.

    Table 26.1 Preparation of seven working solutions
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  4. 4.

    Prepare IS mixture 1 (each 1.0 μmol/l) and IS mixture 2 (each 200 nmol/l) of the internal standard containing C17-S1P and C17-Sph. Store at −80 °C.

  5. 5.

    For each calibration curve, mix equal amounts of working solutions and IS mixture 2 (final concentration of sphingolipids are 500, 100, 10, 5.0, 1.0, 0.5, 0.1 nmol/l, and C17-S1P and C17-Sph are 100 nmol/l) to determine the concentration–response relationship.

  6. 6.

    Prepare quality control (QC) samples by spiking blank plasma or biological samples with stock solution. QC samples should be prepared at four levels: high-level QC (HQC), middle-level QC (MQC), low-level QC (LQC), and lower low-level QC (LLQC).

  7. 7.

    Prepare a deproteinization solution, 0.1 % formic acid in methanol.

4 Protocol for Sample Preparation from Plasma

Before analysis, all frozen samples (−80 °C) should be thawed and allow to equilibrate at 4 °C (or on ice). Do not leave the blood samples on ice before preparing plasma because red blood cells are a source of the plasma S1P. Thus, it is necessary to be prepared for plasma fraction from whole blood as soon as possible. Sample preparation for MS is recommended to utilize deproteinization of an organic solvent such as methanol (Fig. 26.1) as follows:

Fig. 26.1
figure 1

Deproteinization method for sample preparation

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  1. 1.

    Transfer 10 μl plasma into a siliconized sample tube.

  2. 2.

    Add 80 μl deproteinization solution (0.1 % formic acid in methanol).

  3. 3.

    Add 10 μl IS mixture 1 (final concentration of IS is 100 nmol/l).

    Tthe additional volume of IS mixture should be changed to match the standard calibration curve if you need concentration steps for sample preparation.

  4. 4.

    Close the tube and vortex for 30 s.

  5. 5.

    Then, homogenize for 5 min in an ultrasonic bath on a tube floater.

  6. 6.

    Centrifuge at 16,400 g for 10 min at 4 °C and separate supernatant.

  7. 7.

    The supernatant (approximately 100 μl) should be passed through a filter (0.2 μm pore size, 4 mm inner diameter).

  8. 8.

    Inject 10 μl filtered solution into the chromatographic system using an automatic injector. That is, each internal standard is 1 pmol/on column.

5 Protocol for Sample Preparation from Tissue

The sample preparation for tissue is also shown in Fig. 26.1 as follows:

  1. 1.

    Tissue (approximately 50 mg) is placed into a siliconized sample tube (2.0 ml).

  2. 2.

    Add 400 μl deproteinization solution.

  3. 3.

    Add 50 μl IS mixture 1 (final concentration of IS is 100 nmol/l).

    The additional volume of IS mixture should be changed to match the standard calibration curve if you need concentration steps for sample preparation.

  4. 4.

    Close the tube and vortex for 30 s.

  5. 5.

    Then, homogenize for 30 s by an ultrasonic cell disruptor.

  6. 6.

    Centrifuge at 16,400 g for 10 min at 4 °C.

  7. 7.

    Transfer 100 μl supernatant to another siliconized tube (2.0 ml).

  8. 8.

    Pass the supernatant through a filter (0.2 μm pore size, 4 mm inner diameter, YMC).

  9. 9.

    Inject 10 μl filtered solution into the chromatographic system using an automatic injector.

6 Optimization of H-ESI-MS/MS by Infusion of Sphingolipids

A triple quadrupole mass spectrometer equipped with an ESI source is used for sphingolipids analysis such as TSQ Quantum Ultra with heated electrospray ionization (H-ESI) probe (Thermo Fisher Scientific). Because carryover of samples based on interactions with metal components and the phosphate group of lipids is a concern, PEEK tubing and connector are useful and are recommended. To optimize the sensitivity of sphingolipids, several parameters such as spray voltage, sheath gas pressure, auxiliary gas flow rate, capillary temperature, tube lens offset voltage, and vaporizer temperature need to be optimized.

  1. 1.

    Infuse a polytyrosine-1,3,6 calibration solution directly into the ESI source with a syringe pump to tune and calibrate.

  2. 2.

    Save the tune method and calibration files.

  3. 3.

    Fill a clean syringe with 10 μg/ml sphingolipid solution and place in the syringe holder of the syringe pump of MS.

  4. 4.

    Set up the MS detector to tune in H-ESI/MS mode and start infusion. The MS/MS transitions will be detected in the full scan mode (m/z 50–500). Optimal conditions of tube lens offset and collision energy for SRM analysis employ the transition of the [M + H]+ precursor ions to their product ions of S1P, dhS1P, pS1P, Sph, dhSph, and pSph. The parameters of ionization, especially vaporizer temperature, sheath gas pressure, and auxiliary gas pressure, should be retuned after LC condition is fixed because they depend on the flow rate of LC. Example of defined parameters of ionization and MS/MS transitions are described in Table 26.2.

    Table 26.2 Parameters of ionization and MS/MS transition for SRM
    Full size table

7 Separation Condition of Liquid Chromatography (LC)-MS/MS

The basic LC instrumentation for MS consists of degassing equipment, pumps, an automatic injector, columns, and a column oven with PEEK tubing and connector. To check carryover, make a blank injection during the course of multiple runs as needed. For low carryover, washing and cleaning of the needle attached to an automatic injector may be effective with more than one washing/cleaning solvent. An online solid-phase extraction may be valid for ultramicroanalysis. Our LC system for sphingolipids analysis is described in Fig. 26.2. The sample is injected by an autosampler and located to a trap column by eluent C for 7 min at position A (1–6). After changing from position A to B of a six-port valve, trapped compounds are eluted from the trap column and loaded to the analytical column by eluent A and B for 10 min at position B (1–2).

Fig. 26.2
figure 2

(a) The sample is injected by autosampler and located to atrap column by eluent C at position A. (b) The traped compounds to a trap column are eluted and loaded to the analytical column at position B

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Isocratic elution or gradient elution may be chosen. The column selection (phase material, inner diameter, length, etc.) and optimization of the mobile phase are very important for stable analysis. On sphingolipid analysis using reverse-phase chromatography, pH 4.0 of the mobile phase is especially important (Table 26.3).

Table 26.3 HPLC system and run condition
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Generally, an ODS column is selected for sphingolipids analysis. However, a peak-tailing problem may be detected because of an interaction between a phosphate group of sphingolipids and the residual silanol group (imperfect hydration state) or a metal ion inside a ODS column. Therefore, column that has as reduced a residual silanol group as possible to improve the peak-tailing problem should be selected for sphingolipids analysis. To conduct good analysis, the improvement of peak shape should be aimed at evaluating a number of theoretical stages and a symmetry factor carefully, and this leads to the rise of sensitivity.

8 Preparation of the Mobile Phase

  1. 1.

    Mobile phase A [5 mM HCOONH4 in H2O, pH 4.0 (HCOOH)] is prepared by mixing 99.5 ml water and 0.5 ml 1 mol/l ammonium formate solution, and then adjusting to pH 4.0 with formic acid (~9.6 μl).

  2. 2.

    Mobile phase B [5 mM HCOONH4 in H2O/CH3CN = 5/95, pH 4.0 (HCOOH)] is prepared by mixing 95 ml acetonitrile, 4.5 ml water, and 0.5 ml 1 mol/l ammonium formate solution, and then adjusting to apparent pH 4.0 with formic acid (~1160 μl).

9 Condition of Ionization by LC-MS/MS

The parameters of ionization using LC change depending on the flow rate, especially vaporizer temperature, sheath gas pressure, and auxiliary gas pressure. Our optimal parameters for Nanospace SI 2-Quantum Ultra system are described in Table 26.4. SRM chromatograms are shown in Fig. 26.3.

Table 26.4 Parameters of ionization
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Fig. 26.3
figure 3

Selected reaction monitoring (SRM) chromatograms of sphingolipids by LC-MS/MS

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10 Preparation of Calibration Curve and Determination of Sphingolipids by LC-MS/MS

  1. 1.

    All peaks were integrated automatically by Xcalibur software (Thermo Fisher Scientific).

  2. 2.

    The unknown sphingolipid amount is estimated from the calibration curves by the ratios of their peak areas to that of IS, which regression coefficients are given with appropriate software. For example, a calibration curve fitting a straight line with nonlinear regression for S1P and Sph is shown in Fig. 26.4a, b, which was drawn using GraphPad Prism (GraphPad Software, Inc.) from the experimental data with 1/x weighting method, i.e., y = mx + c, where y is area ratio, x is analyte level, m is the gradient of the line, and c is its intercept with the y-axis.

    Fig. 26.4
    figure 4

    (a) Caliburation curve for S1P. (b) Caliburation curve for Sph

    Full size image
  3. 3.

    Estimate the concentration of unknown sample using the line equation.

For the analysis of sphingolipids by MS in various biological samples, ionization of matrix effects are taken into account. A matrix effect is the phenomenon that increases or decreases in the sensitivity of the target compound in a tested sample. To perform an exact determination quantity, a matrix effect has to be reduced as much as possible. Therefore, the validation test needs to be carried out using a practical biological sample, that is, plasma, liver, or brain, for developing the determination method, and evaluated accuracy and precision of reproducibility from intraday and interday assay. The analytical method validation is referenced from generally published guidance, i.e., Bioanalytical Method Validation; Food and Drug Administration.

This section describes a protocol for analysis of sphingolipids by LC-MS/MS in a biological sample. However, the present method was specific for 1-acyl sphingolipids, and there are other sphingo base compounds and many species of diacyl sphingolipids of glycosphingolipids, sphingomyerine, ceramide, and their phosphates. We recommend replacing the analytical column with a C8-based column, or using ultrahigh performance liquid chromatography, because the simultaneous analysis for these compounds takes 1–2 h using an ODS (C18-based) column [1]. The useful methods for sphingolipids and related features are summarized in Table 26.5. Although the performance of sphingolipids profiling depends on the improvement of equipment, this basic protocol is utilized for analysis in future studies.

Table 26.5 Methods of sphingolipids and related features
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