The risk of future cardiovascular events in patients already diagnosed with cardiovascular disease (CVD) was assumed to be uniform, but significant variation exists in their 10-year risk of future events, so a means of stratifying secondary risk is highly desirable. Serum and plasma concentrations of ceramides, including Cer (d18:1/16:0), Cer (d18:1/18:0), Cer (d18:1/24:1), and Cer (d18:1/24:0), and phosphatidylcholine (PCs), including PC 14:0/22:6, PC 16:0/22:5, and PC 14:0/22:6 are associated with primary and secondary CVD risk , . A simple, integer-based, scoring system based on their concentration, dubbed CERT2, was developed to predict cardiovascular death in patients with stable CVD and acute coronary syndromes . Here, we report a method for simultaneous measurement of the 4 ceramides and 3 PCs for routine clinical use.
Automated protein precipitation was performed using a VIAFLO96 (Integra) in a 96-well plate format. Calibration standards, quality controls (QCs), and serum specimens were treated with internal standard (IS) (a mixture of the corresponding deuterated ceramides and phosphatidylcholines) in an isopropanol/ethyl acetate mixture, vortexed, centrifuged and supernatants transferred to a new 96-well plate, where they were then diluted with methanol and injected onto the LC-MS/MS system (Agilent StreamSelect LC system coupled to an Agilent 6495C QqQ MS). Analytes were resolved using BEH C18 columns (Waters) and gradients of water to acetonitrile/isopropanol (both mobile phases containing ammonium acetate/formic acid/trifluoroacetic acid). The analytes and IS were monitored using electrospray ionization in positive-ion mode with multiple reaction monitoring (MRM).
Due to the presence of endogenous ceramide and PC species in human serum (including charcoal-stripped serum), calibration standards were prepared in DMF to achieve stable calibration curves. The calibration matrix effect was evaluated by serially diluting the standard mixture (in DMF) with serum, and signal linearity was confirmed over the analytical measurement ranges. Linear calibration curves were achieved over the range of 50–10,000 ng/ml for Cer(d18:1/24:1), Cer(d18:1/24:0), and PC 14:0/22:6; 500–100,000 ng/ml for PC 16:0/22:5, and PC 14:0/22:6; and 5–1,000 ng/ml for Cer(d18:1/18:0). Quadratic fitting was used for Cer(d18:1/16:0) with a calibration range of 5–1,000 ng/ml. All calibrations curves achieved correlation coefficients (R2) of above 0.99.
The imprecision (%CV) was below 10% and the accuracy between 93% and 107% for all seven analytes over 20 separate runs. Spike recovery was determined by spiking analyte standards into serum and calculating the differences between the sample concentrations and spiked sample concentrations. Recoveries ranged from 93% to 108%.
Seven serum samples were used to evaluate various stabilities, including freeze/thaw, room temperature, refrigerator, and frozen long-term storage. Analytes were found to be stable after each of seven freeze/thaw cycles, 24 hours at room temperature, and for at least 3 months at 4 °C, -20 °C, and -70 °C.
Extraction recovery of IS in serum were evaluated by comparison of peak area ratios of pre-spiked samples versus post-spiked samples. Extraction recoveries ranged from 89% to 103%.
Furthermore, concordant CVD risk classification in a previously characterized cohort (N=60) was demonstrated using CERT2 scores independently derived and obtained in the current study, with any discordant scores differing by no more than one risk category – the result of differences between the respective distributions of the reference population (North American vs Northern European).
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