Development of an Optimized LC-MS Method for the Detection of Specialized Pro-Resolving Mediators in Biological Samples

Abstract

The cardioprotective and anti-inflammatory effects of long chain omega-3 polyunsaturated fatty acids (n3 PUFA) are believed to be partly mediated by their oxygenated metabolites (oxylipins). In the last two decades interest in a novel group of autacoids termed specialized pro-resolving mediators (SPMs) increased. These are actively involved in the resolution of inflammation. SPMs are multiple hydroxylated fatty acids including resolvins, maresins, and protectins derived from the n3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as lipoxins derived from arachidonic acid (ARA). In the present paper, we developed an LC-MS/MS method for a comprehensive set of 18 SPMs derived from ARA, EPA, and DHA and integrated it into our targeted metabolomics platform. Quantification was based on external calibration utilizing five deuterated internal standards in combination with a second internal standard for quality assessment of sample preparation in each sample. The tandem mass spectrometric parameters were carefully optimized for sensitive and specific detection. The influence of source parameters of the used AB Sciex 6500 QTRAP instrument as well as electronic parameters and the selection of transitions are discussed. The method was validated/characterized based on the criteria listed in the European Medicines Agency (EMA) guideline on bioanalytical method validation and method performance is demonstrated regarding recovery of internal standards (between 78 ± 4% and 87 ± 3% from 500 μL of human serum) as well as extraction efficacy of SPMs in spiked plasma (intra-day accuracy within ±20 and ±15% at 0.1 and 0.3 nM in plasma, respectively). Based on the lower limit of quantification of 0.02–0.2 nM, corresponding to 0.18–2.7 pg on column, SPMs were generally not detectable/quantifiable in plasma and serum supporting that circulating levels of SPMs are very low, i.e., <0.1 nM in healthy subjects. Following septic shock or peritonitis, SPMs could be quantified in the samples of several patients. However, in these studies with a small number of patients no clear correlation with severity of inflammation could be observed.

Methods of the Analysis of Oxylipins in Biological Samples

“…In order to obtain the best results, they tried different compositions and gradients of the mobile phase [102]. Using a Zorbax Eclipse Plus C18 column with a diameter of particles less than 2 microns (150 × 2.1 mm, 1.8 µm; Agilent, Waldbronn, Germany) and an optimized gradient, Kutzner et al separated the maresin (Mar)1 and 7 (S)-Mar1 and protectin (N)PD1 and PDX stereoisomers from human serum and the results were comparable to those achieved by using a chiral column [103]. The high resolution power of approaches using these columns is usually used for separation of known isomers, not for identification of novel compounds.…”

“…Various parameters such as source temperature (ST), collision energy (CE), declustering potential (DP), collision cell exit potential (CEP), collision activated dissociation (CAD), and gas pressure affect the signal intensity in the SRM mode. Simultaneously, caution should be taken with increasing the temperature of nebulizer gas as well as auxiliary gas, because the optimum temperature for some compounds may lead to the thermal degradation of others [103]. To obtain better detection limits and avoid interference from nearby isomers, at the last step of optimization, the most intense and specific fragments and transitions are usually chosen [101,103,163].…”

“…Simultaneously, caution should be taken with increasing the temperature of nebulizer gas as well as auxiliary gas, because the optimum temperature for some compounds may lead to the thermal degradation of others [103]. To obtain better detection limits and avoid interference from nearby isomers, at the last step of optimization, the most intense and specific fragments and transitions are usually chosen [101,103,163]. However, in the case of interference ions, it is better to select more characteristic rather than stronger fragmentation ions.…”

“…For example, when Yang et al developed a method for murine sera and BALF oxylipin profiling, for 8, 9 EET the weaker transition 319.2/123 was chosen, because the stronger transition 319.2/167 overlaps with the transition 319.2/167 for 11, 12 EET [101]. Also, to increase the sensibility of the MS method, the optimization of MS operating conditions should be carried out for each individual compound [15,69,103,131,164]. By the careful selection of transitions, it is possible to optimize sensitivity and selectivity so that the overall sensitivity will improve several times [85,101].…”

Use of DAMPs and SAMPs as Therapeutic Targets or Therapeutics: A Note of Caution

“…Plasma concentrations of SAMPs, here SPM profiles, have also been measured using liquid chromatographytandem mass spectrometry (LC-MS) technology [49,50]. Of note, in a recently published evaluation of SPM profiles using an optimized LC-MS method for the detection of SPMs in biological samples, Kutzner et al [51] concluded, "SPMs were generally not detectable/quantifiable in plasma and serum of healthy individuals, while good recovery rates were found in spiked samples. These results strongly support findings that circulating levels of SPMs are very low, i.e., < 0.1 nM in healthy subjects.…”