Rapid evaluation of 25 key sphingolipids and phosphosphingolipids in human plasma by LC-MS/MS
- 2.5k Downloads
We report on a new, sensitive, and fast LC-MS/MS method for the simultaneous determination of 25 key sphingolipid components in human plasma, including phosphorylated sphinganine and sphingosine, in a single 9-min run. This method enables an effective and high-throughput coverage of the metabolic changes involving the sphingolipidome during physiological or pathological states. The method is based on liquid–liquid extraction followed by reversed-phase LC-MS/MS. Exogenous odd-chain lipids are used as cost-effective but reliable internal standards. The method was fully validated in surrogate matrix and naive human plasma following FDA guidelines. Sample stability and dilution integrity were also tested and verified.
KeywordsCeramides Sphingomyelins Sphingosine-1-phosphate Sphinganine-1-phosphate LC-MS/MS Human plasma
Materials and methods
Sphingolipid standards were purchased from Avanti Polar Lipids (Alabaster, Alabama USA). Solvents and chemicals were from Sigma-Aldrich (Milan, Italy). UPLC/MS and MS/MS systems and columns were from Waters (Milford, USA).
Human plasma samples
Healthy male and female subjects were enrolled in the MCI/AD Italian prevention project [31, 32] aimed at studying cognitive and neuropsychiatric symptoms and disorders in patients with MCI and AD at the IRCCS Santa Lucia Foundation memory clinic in Rome, Italy. The nature and purpose of the study were presented to patients and caregivers and controls, and written informed consent was obtained. The study was approved by the Ethical Committee of the Santa Lucia Foundation. Human plasma samples were collected from healthy male and female volunteers of all ages. Blood samples were taken by venipuncture in the morning after an overnight fast. Blood was collected into 10 ml tubes containing spray-coated K2EDTA (Vacutainer, Becton Dickinson, Italy). Plasma was then prepared by centrifugation of blood at 400×g for 15 min, then stored at −80 °C before analysis. Blood drawing and sample preparation of human plasma samples were carried out applying the best safety precautions. Plasma samples from all subjects were then pooled together and used as naïve matrix for the present study.
Stock solution preparation
Stock solutions were prepared in methanol and chloroform mixture (1:1). An internal standard (IS) solution (200 nM Cer d18:1/17:0, 200 nM PC 23:0/23:0, 200 nM GlcCer d18:1/12:0, 250 nM SPH d17:1, 250 nM SPH d17:0, 250 nM S1P d17:1, and 250 nM S1P d17:0) was prepared by spiking the corresponding standards in the extraction solvent methanol/chloroform (2:1) added with trifluoroacetic acid (TFA) to a final 0.1 % (v/v) concentration.
Samples were extracted using a Bligh and Dyer method for lipid extraction . Calibration curve (CC), quality control (QC), or test samples (50 μL) were transferred to glass vials. Liquid–liquid extraction (LLE) was carried out using a 1:2 by volume chloroform/methanol mixture (2 ml) added with TFA (final 0.1 % v/v) and spiked with the IS as described above. After mixing for 30 s with a Vortex®, chloroform (0.5 mL) and water (0.5 mL) were sequentially added, thoroughly mixing after each addition. The samples were then centrifuged for 15 min at 3500×g at room temperature. At the end of the process, the aqueous (upper) and organic (lower) phases were separated by a protein precipitate floating at the interface. The organic phase was then transferred to glass vials. To increase the overall recovery, the aqueous fraction was extracted again with chloroform (1 mL). The two resulting organic phases were pooled, dried under a stream of N2, and the residues were redissolved in methanol/chloroform (9:1, by volume; 0.1 mL). After mixing (30 s) and centrifugation (10 min at 5000×g, room temperature), the samples were transferred to glass vials for analyses.
LC-MS/MS analyses of the samples were carried out on an Acquity UPLC system coupled with a Xevo TQ-MS triple-quadrupole mass spectrometer. Chromatographic separation was achieved using a BEH C18 column (2.1 × 50 mm, 1.7 micron particle size) eluted at a flow rate of 0.4 mL/min. Instruments and column were from Waters Inc. Milford, MA, USA. The mobile phase consisted of 0.1 % formic acid in acetonitrile/water (20:80 v/v) as solvent A and 0.1 % formic acid in acetonitrile/2-propanol (20:80 v/v) as solvent B. A step gradient program was developed for the best separation of all metabolites: 0.0–1.0 min 30 % B, 1.0–2.5 min 30 to 70 % B, 2.5–4.0 min 70 to 80 % B, 4.0–5.0 min 80 % B, 5.0–6.5 min 80 to 90 % B, and 6.6–7.5 min 100 % B. The column was then reconditioned to 30 % B for 1.4 min. The total run time for analysis was 9 min, and the injection volume was 3 μL. The mass spectrometer was operated in the positive ESI mode, and analytes were quantified by multiple reaction monitoring (MRM). The capillary voltage was set at 3 kV. The cone voltage was set at 25 V for all transitions, except for some SM. The complete panel of source parameters and MRM transitions are reported in the datasheet in the Electronic Supplementary Material (ESM). The source temperature was set to 120 °C. Desolvation gas and cone gas (N2) flow were set to 800 and 20 l/h, respectively. Desolvation temperature was set to 600 °C. Data were acquired by MassLynx software and quantified by TargetLynx software. Calibration curves were constructed by plotting the analyte to IS peak areas ratio versus the corresponding analyte concentration using weighted (1/x 2) least square regression analysis, as recommended by Gu and colleagues .
As analytes are present endogenously in plasma, 5 % bovine serum albumin (BSA) in saline solution was used as surrogate matrix  for the first part of the validation process (see the “Results and discussion” for additional information). Calibration standards were prepared by spiking the analytes in the surrogate matrix. Eight-point calibration curves (1 to 1000 nM) were prepared by serial dilution into saline solution containing 5 % BSA.
Quality control samples
Quality control samples were prepared at three different levels, as low QC (LQC), medium QC (MQC), and high QC (HQC), using the same procedure described for the standard curve to final concentrations of 19.5, 260, and 650 nM, respectively.
Linearity, precision, and accuracy
Method linearity was tested using the eight-point calibration curve described above. The overall method performance was assessed by evaluating the accuracy and precision of back-calculated concentrations of standards and evaluating the slope, intercept, and coefficient of determination of the 1/(concentration)2 weighted regression line. Following FDA guidelines in this matter, acceptance criteria for the calibration curve were set to ±15 % of the nominal concentration and six out of eight calibration points had to meet the acceptance criteria. Precision and accuracy were determined by assessing the performance of quality control samples (LQC = 19.5 nM, MQC = 260 nM, and HQC = 650 nM). All the QC samples were run in triplicate. Accuracy was evaluated by calculating the percent deviation (% dev.) from nominal concentration. Precision was determined by calculating the coefficient of variation (% CV) of replicates within each batch. Acceptance criteria for precision and accuracy were defined as ≤15 % .
The lower limit of quantification for our method was set to 1 nM for all analytes. At this value, all species showed a S/N value above 10. Six replicate 1-nM calibrators spiked in 5 % BSA were extracted and analyzed. The obtained %CV values for each analyte are reported in the ESM (supplementary datasheet).
Sample recovery and matrix effects
Recovery and matrix effects were evaluated using the method outlined by Matuszewski et al. . Three sets of samples were prepared spiking the standards at three different concentrations. Set 1 consisted of neat samples (standards spiked in reconstitution solution). Set 2 consisted of post-extraction spiked samples (blank matrix was extracted and then spiked with standards). Set 3 consisted of normal extracted samples (standards were spiked in blank matrix and then extracted). Matrix effect (ME) was calculated as (set 2/set 1) × 100. Recovery (RE) was calculated as (set 3/set 2) × 100. Human plasma matrix effect was evaluated using the post-column infusion method: A mixture of analytes diluted in 9:1 MeOH/CHCl3 to a final 10 μM concentration was infused post-column in the LC-MS/MS system using a tee union. Repeated injections of extracted human plasma samples were performed in the system with the aim to investigate significant decreases or increases in the analyte MRM ion currents.
Analyte recovery from plasma was calculated using the same criteria described above: A mixture of standards was added at different concentrations in plasma (from 50 nM to 15 μM) prior or after the extraction (pre- and post-extraction spiking). The recovery was calculated as the ratio between the analyte peak area pre-spiked in plasma and the analyte peak area in post-extraction spiked samples (see “Results and discussion” for detailed information).
Autosampler and plasma stability and dilution integrity
Plasma stability was assessed by spiking odd-chain standards in human plasma and keeping the sample under different temperature and timing conditions: short-term conditions (4 and 25 °C for 6 h) and long-term storage conditions (−20 ° C for 9 days). Sample stability in the instrument autosampler was also evaluated by re-analyzing extracted samples kept under the autosampler condition (18 h at 4 °C). All the stability studies were conducted in triplicate at 500 nM concentration. Method integrity to dilutions was also evaluated by spiking odd-chain standards in human plasma to a final 4000 nM concentration. Samples were then diluted 20-fold in blank human plasma, extracted, and analyzed.
Results and discussion
MS parameter optimization and choice of internal standards
A detailed summary of the MRM transitions and source parameters for all analytes and internal standards is reported in the ESM (supplementary datasheet). Since we are fully aware that the lack of a coeluting internal standard might represent a major problem in LC-MS/MS, we did our best to evaluate possible matrix effects using the post-column infusion method . The results of this experiment are available for download as ESM. Most analytes did not show any relevant matrix effect when extracted human plasma samples were injected. Five analytes showed non-negligible matrix effects with three out of four glucosylceramides among them (16:0, 18:0, and 18:1). It is thus important to point out that, assuming identical matrix effects for galactosylceramides, the evaluation of hexosylceramides content in plasma might be affected, in the absence of commercially available deuterated standards. As expected, injections of extracted plasma caused a positive increase in the post-column infusion trace of those metabolites that are endogenously present in plasma at micromolar concentrations (sphingomyelins mostly). We also investigated the possibility of endogenous plasma phosphatidylcholines coeluting with the four sphingomyelins quantified by our method and having similar m/z values. For the same reasons indicated above (common 184 m/z fragment), this would result in interferences and inaccuracy in sphingomyelins quantification. We addressed this issue by means of high-resolution MS, a brute formula calculation software and LipidMaps database search, and we showed that, at the retention time of the four sphingomyelins targeted by our method, no phosphatidylcholines having a m/z value close to that selected for fragmentation (±0.5 m/z units) is coeluting. The results of this experiment, along with a detailed description of the experiment, are available for download as ESM.
Recovery values by sphingolipid class (mean ± standard deviation) from 5 % BSA and from human plasma samples
BSA 5 %
89.66 ± 2.40
95.74 ± 7.21
91.53 ± 1.70
96.89 ± 5.03
82.97 ± 0.79
97.82 ± 1.36
89.93 ± 2.54
100.93 ± 7.15
116.11 ± 2.94
71.93 ± 4.19
116.86 ± 1.16
93.69 ± 4.90
76.93 ± 2.45
105.01 ± 14.08
75.89 ± 0.71
86.64 ± 4.21
Linearity, precision, accuracy, carryover, stability, and dilution integrity
Our method showed a very good linearity over a 103-fold range (1–1000 nM, eight calibration points) with an r 2 value of 0.9895–0.9973 using 5 % BSA as matrix and 0.9947–1.0000 using human plasma as matrix. A signal-to-noise ratio (S/N) of ≥10 was considered as lowest limit of quantification to determine method sensitivity, although for most analytes the S/N at 1 nM was higher than 10. For this reason, lower limit of quantification (LLOQ) from 5% BSA was set to 1 nM. At this concentration, all analytes showed a %CV below 20 % (see ESM, supplementary datasheet). Precision and accuracy were also determined in 5 % BSA, assessing the performance of three quality control samples (LQC = 19.5 nM, MQC = 260 nM, and HQC = 650 nM). Accuracy was evaluated by calculating the percent deviation from nominal concentrations. Precision was determined by calculating the coefficient of variation of replicates within each batch. All the QC samples were run in triplicate. Acceptance criteria for precision and accuracy were defined as ≤15 % . Intra- and inter-assay precision and accuracy were also tested by analyzing the same QC samples. The full dataset is reported in the ESM (supplementary datasheet). Furthermore, in order to evaluate carryover, a blank sample was injected immediately after the highest standard. Since some carryover was observed with the usual water/methanol/ACN/2-propanol needle washing system, we used acetonitrile/water (1:1) containing 10 % acetone as needle wash solvent. With this expedient, no significant carryover was observed for any of the analytes (less than 0.5 %). Analyte stability evaluation was also included in the experimental layout: Odd-chain standards were spiked in naïve human plasma to a final 500 nM concentration (triplicate samples) and tested for stability in both short-term (4 and 25 °C for 6 h) and long-term conditions (9 days at −20 °C). The stability of extracted samples in the instrument autosampler conditions (4 °C for 6 h) was also evaluated. Analytes are stables in all the abovementioned conditions (our acceptance criteria were set to ±15 % of nominal concentration). Since sphingolipids are present in human plasma at very different concentrations (from low nM to high μM), we also tested the dilution integrity of our method in order to be able to confidently quantify samples above the upper limit of the calibration curve. We then spiked odd-chain standards in naïve human plasma at a final 4 μM concentration, and we diluted the samples 20-fold using naïve human plasma. After sample extraction and analysis, analyte concentrations were calculated using the appropriate multiplication factor. Results demonstrate that our method is tolerant to 20-fold dilution since the calculated concentrations were within the acceptance criteria (precision 0.16–4.82 %, accuracy 83.04–94.25 %). Data on stability and dilution integrity are reported in the ESM (supplementary datasheet). All the sphingolipids that were detected and quantified with our method in naïve human plasma are reported in the supplementary datasheet in the ESM.
In the present report, we describe a new LC-MS/MS method that allows for the rapid identification and quantification of 25 key sphingolipid species in human plasma, including S1P d18:1 and S1P d18:0. We proved that a LogP span of more than 10 units (from 3.43 of S1P d18:1 to 14.42 of Cer d18:1/24:0 can be efficiently explored in a single sample preparation and LC-MS run. While other validated methods for the LC-MS/MS quantification of sphingolipids are available in the literature, none of them offers a comparable level of analyte coverage, rapidity, and validation. For example, Bui et al.  describe a 5-min separation but do not cover sphingomyelins; Kasumov and colleagues  describe a separation of seven species in 15 min. Other methods, though validated, offer an even more limited analyte coverage: Cer d18:1/22:0 and Cer d18:1/24:0  or seven species by nano-LC/MS . The method we propose here is sensitive, linear, robust, and very rapid (9 min per run). Although it suffers from some matrix effect that cannot be fully compensated by the use of odd-chain internal standards, we managed to evaluate this effect and control it, achieving very promising results from human plasma. Future implementations of this method with custom-made deuterated internal standards will hopefully improve the overall method performance, making it suitable for clinical routine. The huge costs for custom synthesis will surely be compensated by the final outcome on health-care systems since sphingolipids and phosphorylated sphingolipids have been proposed as biomarkers for various pathological conditions. The potential of the method presented here is manifested as it permits a complete and reliable analysis of the full sphingolipid core metabolism in less than 10 min. Furthermore, in addition to the whole sphingolipids panel, the possibility to track S1P d18:1 and S1P d18:0 in plasma could be of great importance for clinical research on cancer and immune system disorders.
The authors would like to thank Dr. Gianfranco Spalletta (IRCCS Santa Lucia, Rome, Italy) for kindly providing human plasma samples from healthy donors. The financial support of the National Institutes of Health (grants DK073955 and DA012413 to D.P.) is gratefully acknowledged.
- 2.Teichgräber V, Ulrich M, Endlich N, Riethmüller J, Wilker B, De Oliveira-Munding CC, van Heeckeren AM, Barr ML, von Kürthy G, Schmid KW, Weller M, Tümmler B, Lang F, Grassme H, Döring G, Gulbins E (2008) Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis. Nat Med 14(4):382–391CrossRefGoogle Scholar
- 6.Mielke MM, Haughey NJ, Bandaru VV, Weinberg DD, Darby E, Zaidi N, Pavlik V, Doody RS, Lyketsos CG (2011) Plasma sphingomyelins are associated with cognitive progression in Alzheimer’s disease. J Alzheimers Dis 27(2):259–269Google Scholar
- 7.Mielke MM, Haughey NJ, Ratnam Bandaru VV, Schech S, Carrick R, Carlson MC, Mori S, Miller MI, Ceritoglu C, Brown T, Albert M, Lyketsos CG (2010) Plasma ceramides are altered in mild cognitive impairment and predict cognitive decline and hippocampal volume loss. Alzheimers Dement 6(5):378–385CrossRefGoogle Scholar
- 17.Yano M, Kishida E, Muneyuki Y, Masuzawa Y (1998) Quantitative analysis of ceramide molecular species by high performance liquid chromatography. J Lipid Res 39(10):2091–2098Google Scholar
- 18.Lee S, Lee YS, Choi KM, Yoo KS, Sin DM, Kim W, Lee YM, Hong JT, Yun YP, Yoo HS (2012) Quantitative analysis of sphingomyelin by high-performance liquid chromatography after enzymatic hydrolysis. Evid Based Complement Alternat Med 2012:396218Google Scholar
- 31.Palmer K, Di Iulio F, Varsi AE, Gianni W, Sancesario G, Caltagirone C, Spalletta G (2010) Neuropsychiatric predictors of progression from amnestic-mild cognitive impairment to Alzheimer’s disease: the role of depression and apathy. J Alzheimers Dis 20(1):175–183Google Scholar
- 33.Gu H, Liu G, Wang J, Aubry AF, Arnold ME (2014) Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal Chem 86(18):8959–8966CrossRefGoogle Scholar
- 35.Booth B, Arnold ME, DeSilva B, Amaravadi L, Dudal S, Fluhler E, Gorovits B, Haidar SH, Kadavil J, Lowes S, Nicholson R, Rock M, Skelly M, Stevenson L, Subramaniam S, Weiner R, Woolf E. (2014) Workshop report: crystal city V-Quantitative bioanalytical method validation and implementation: the 2013 revised FDA guidance. AAPS journal. doi: 10.1208/s12248-014-9696-2
- 40.Geis-Asteggiante L, Lehotay SJ, Lightfield AR, Dutko T, Ng C, Bluhm L (2012) Ruggedness testing and validation of a practical analytical method for >100 veterinary drug residues in bovine muscle by ultrahigh performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1258:43–54CrossRefGoogle Scholar
- 42.Chen RF (1967) Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem 242(2):173–181Google Scholar
- 45.Bowen CL, Kehler J, Evans CA (2010) Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 878(30):3125–3133CrossRefGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.