A rapid and quantitative LC-MS/MS method to profile sphingolipids

Abstract

including ceramide, sphingosine (SPH), and SPH-1-phosphate (S1P), have been shown to be involved in different cell functions such as proliferation, differentiation, growth arrest, and apoptosis ( 2 ). Especially the counterregulatory functions of ceramide and S1P, resembling the sphingolipid rheostat, indicate that not only a single metabolite concentration but rather the relative levels of these lipids are important to determine the cell fate ( 2-5 ). Sphingolipids are associated with several diseases such as cancer, obesity, and atherosclerosis ( 1, 2, 6-9 ). Structural diversity and inter-conversion of these sphingolipid metabolites represent technical challenges. Nevertheless, to understand the differential role of sphingolipids in a regulatory network, it is imperative to use specifi c and quantitative methods.During the last decade, LC-MS/MS has become a powerful tool for sphingolipid analysis ( 10-21 ). However, either these methods do not cover a broad spectrum of sphingolipid metabolites or they show disadvantages like laborious sample preparation, time-consuming LC-separation, or separation of analytes and internal standards (ISs).Therefore, we applied, as previously described for lysophosphatidic acid and sphingoid base phosphates, hydrophilic interaction chromatography (HILIC) coupled to MS ( 18 ) to achieve coelution of sphingolipid species and their ISs. We present a fast and simple LC-MS/MS method for the quantifi cation of hexosylceramide (HexCer), lactosylceramide (LacCer), sphingosine (SPH), sphinganine (SPA), phyto-SPH (PhytoSPH), di-and trimethyl-SPH (Di-; TrimetSPH), sphingosylphosphorylcholine (SPC), ceramide-1-phosphate (Cer1P), and dihydroceramide-1-phosphate (dhCer1P). This method was validated and apAbstract Sphingolipids comprise a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signaling molecules. To understand the differential role of sphingolipids in a regulatory network, it is important to use specifi c and quantitative methods. We developed a novel LC-MS/MS method for the rapid, simultaneous quantifi cation of sphingolipid metabolites, including sphingosine, sphinganine, phyto-sphingosine, di-and trimethyl-sphingosine, sphingosylphosphorylcholine, hexosylceramide, lactosylceramide, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Appropriate internal standards (ISs) were added prior to lipid extraction. In contrast to most published methods based on reversed phase chromatography, we used hydrophilic interaction liquid chromatography and achieved good peak shapes, a short analysis time of 4.5 min, and, most importantly , coelution of analytes and their respective ISs. To avoid an overestimation of species concentrations, peak areas were corrected regarding isotopic overlap where necessary. Quantifi cation was achieved by standard addition of naturally occurring sphingolipid species to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness. As an example, sphingol...

Large-scaled human serum sphingolipid profiling by using reversed-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: Method development and application in hepatocellular carcinoma

“…They are SM41:2 (SM (d17:1/24:1) and SM (d18:2/23:0)), SM42:2 (SM (d18:1/24:1) and SM (d18:2/24:0)) as highlighted in Table S1. In contrast to other methods applying NPLC [18,19,37] or HILIC [21,31], it also facilitated quantification by avoiding isotope correction. Moreover, a linear regression model could be constructed by plotting carbon number v.s.…”

“…Efficient separation by ultra high performance RPLC based on chain length and unsaturation, is helpful in a dynamic MRM method embracing up to 90 analytes, compared with by NPLC [18,19,37], or HILIC [21,31], where separation is based on polar group and coelution occurs for each subcategory. Fig.…”

“…Previous sphingolipid extraction procedures employed classic Bligh & Dyer extraction [15,29], isopropanol/ethyl acetate extraction [17,30] and butanolic extraction [21,31]. Besides, efforts have been made, by applying mild alkaline hydrolysis [16,18,19,23,32], or solid phase extraction (SPE) [20,33], aimed at reduced ion suppression, especially in a shotgun pattern.…”

“…In comparison, LC-MS/MS based methods dramatically enhanced sensitivity, structural-specificity and accuracy for detection of intact sphingolipid species. By far, a large number of LC-MS-based methods with various extraction protocols have been established, using normal phase liquid chromatography (NPLC), hydrophilic interaction liquid chromatography (HILIC), or reversed-phase liquid chromatography (RPLC) for sphingolipid separation, coupled with multiple reaction monitoring (MRM) acquisition via electrospray ionization (ESI) or atmospheric-pressure chemical ionization (APCI) [15][16][17][18][19][20][21][22][23]. Nevertheless, no method has been capable of a comprehensive profiling of a large spectrum of sphigolipid species/subcategories in a single method, let along in a single run.…”

Tandem Mass Spectrometry of Sphingolipids: Application in Metabolic Studies and Diagnosis of Inherited Disorders of Sphingolipid Metabolism

“…These product ions have different uses. The 264 m/z fragments are used to analyze sphingolipids, whereas the 282 m/z fragments are used to analyze N-deacylated sphingolipids (lysoderivatives) (Gu, et al, 1997;Lieser, et al, 2003;Olling, et al, 1998;Scherer, et al, 2010). The ceramide fragments mentioned above are commonly used in tandem mass analysis, but other specific sphingolipid structures can also be used for this purpose.…”