Comitas Institute for Anthropological Study

2.1 Chemicals

Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was from Supelco (Bellefonte, PA), [³H]anandamide (221 Ci/mmol) from New England Nuclear (Wilmington, DE), fatty acyl chlorides (5,8,11,14-eicosatetraenoylchloride, hexadecanoylchloride and 9-octadecenoylchloride) from Nu-Check Prep (Elysian, MN), [²H₄]ethanolamine (isotopic atom enrichment=98%) from Cambridge Isotope Laboratories (Andover, MA). All solvents were from Burdick and Jackson (Muskegon, MI) and all other chemicals from Sigma (St. Louis, MO).

2.2 Synthesis of standard AEs and [²H₄]AEs

Standard AEs were synthesized by the reaction of fatty acyl chlorides with unlabeled or ²H₄-labeled ethanolamine [1]. Fatty acyl chlorides in dichloromethane (10 mg/ml) were mixed with 1 equivalent of ethanolamine, and allowed to react for 15 min at 0–4°C. Reactions were stopped by adding water. After vigorous mixing, the upper aqueous phases were discarded to remove unreacted ethanolamine. The organic phases were washed twice with water, concentrated to dryness under a stream of N₂, and the reaction products were reconstituted in methanol. Identity and chemical purity (>98%) of the synthesized AEs and [²H₄]AEs were determined by GC/MS (see Section 2.4).

2.3 Extraction and fractionation of rat plasma AEs

5 ml of blood was collected from the heart of anesthetized male Wistar rats using a syringe filled with 2 ml Krebs-Tris buffer (NaCl 136 mM, KCl 5 mM, MgCl₂ 1.2 mM, CaCl₂ 2.5 mM, glucose 10 mM, Trizma base 20 mM; pH 7.4) containing EDTA 4.5 mM. Blood samples were centrifuged in Accuspin tubes (Sigma) for 10 min at 18°C (800×g) and the plasma layers were carefully recovered and spiked with [²H₄]AEs (600 pmol each). After acetone precipitation of plasma proteins, the supernatants were collected and subjected to lipid extraction with methanol/chloroform. Enough of each solvent was added to reach a final ratio buffer/methanol/chloroform of 1:1:2 (v/v/v). The chloroform phases were recovered, evaporated to dryness under N₂, reconstituted in chloroform (50 μl) and injected into the HPLC. HPLC fractionations were performed on a Hewlett-Packard 1090 Liquid Chromatograph, equipped with a normal-phase Resolve Silica column (3.9 mm×15 cm, 5 μm, Waters Associates), eluted with a gradient of isopropyl alcohol (B) in n-hexane (A) (100% A initial; 90% A, 10% B for 1 min; 60% A, 40% B for 7 min, 50% A, 50% B for 12 min) at a flow rate of 1.7 ml/min. Under these conditions, all AEs were eluted from the HPLC column between 4.7 and 5.3 min. The AE-containing fractions were collected in glass reaction vessels (Supelco), dried under N₂ and subjected to chemical derivatization (see Section 2.4). To estimate recoveries, in some experiments [³H]anandamide (20 000 dpm) was added to an aqueous solution (1 ml) of non-radioactive anandamide (100 nM), extracted in chloroform/methanol and subjected to HPLC fractionation. The AE-containing fractions were concentrated to a volume of 1 ml under N₂, and radioactivity was determined by liquid scintillation counting.

2.4 GC/MS analysis

The AEs were derivatized by treatment with BSTFA for 15 min at room temperature. The trimethylsilylether (TMS) derivatives produced in this reaction were dried under N₂, reconstituted in n-hexane and injected in the splitless mode into a Hewlett-Packard 5890 GC equipped with an HP-5MS capillary column (30 m; internal diameter, 0.25 mm) and interfaced with a Hewlett-Packard 5972 MS. Starting one minute after the injection, the oven temperature was increased from 150°C to 280°C at a rate of 8°C/min. The injector temperature was kept at 250°C and helium was used as carrier gas. The ion energy was 25 eV and the accelerating voltage was 1.6 V. Under these conditions, the TMS derivatives of [²H₄]AE standards were eluted from the GC at the following retention times: [²H₄]anandamide: 18.6 min; [²H₄]palmitylethanolamide: 15.6 min; [²H₄]oleylethanolamide: 17.3 min. Because of GC isotope discrimination, unlabeled AEs had retention times approximately 0.02 min longer than the corresponding [²H₄]AEs. We monitored, by selected-ion monitoring (SIM), AE fragments produced by the loss of one methyl group ([M-15]⁺).

3.1 Mass spectral properties of AEs and [²H₄]AEs

The electron-impact mass spectra of synthetic unlabeled anandamide (cis20:4), palmitylethanolamide (16:0) and oleylethanolamide (cis18:1) are shown in Fig. 1 . Diagnostic fragments for all compounds were found in the high mass range. They included molecular ions ([M]⁺) as well as ions produced by the loss of one methyl group ([M-15]⁺). Additional informative and prominent fragments were: [M-43]⁺ (loss of one propyl group); [M-90]⁺ (loss of TMSOH group; this fragment was absent from the spectrum of anandamide-TMS which contained instead a fragment at m/z 328, [M-91]⁺); and m/z 175/179 (possibly corresponding to [H₂CCOH–NH–CH₂–CH₂–O–TMS]⁺, which may be produced through McLafferty rearrangement).

3.2 Linearity of the isotope dilution assay

Monitoring the [M-15]⁺ fragments, which are reasonably abundant in the AE-TMS spectra, we observed MS responses that were linear (r ²=0.99) when amounts of unlabeled AEs ranging from 0 to 1000 pmol were injected into the GC/MS together with a fixed amount of ²H₄-labeled standards (600 pmol).

3.3 Precision and accuracy

Precision and accuracy of the method were assessed in five independent determinations by injecting 2.5, 10 or 100 pmol of each AE together with 600 pmol of the corresponding standard. The estimates obtained in these analyses are reported in Table 1 . Percent coefficients of variation (CV) were calculated dividing the standard deviation by the sample mean and multiplying the resulting value by 100.

3.4 Limit of detection (LOD) and limit of quantification (LOQ)

When monitoring the [M-15]⁺ fragments of ²H₄-labeled AE standards by SIM, we obtained the following labeled/unlabeled ratios: 0.012 for anandamide, 0.0009 for palmitylethanolamide and 0.0087 for oleylethanolamide. These blank values were plotted in calibration curves as corresponding to 0 pmol. The LOD, i.e. the injected quantity which produced a signal corresponding to an average blank plus 3 standard deviations, was 0.4 pmol for anandamide, 0.1 pmol for palmitylethanolamide, and 0.1 pmol for oleylethanolamide.

The LOQ, i.e. the lowest quantity which could be measured with acceptable accuracy (arbitrarily set at a CV<20%), was determined by injecting into the GC/MS varying amounts of AEs together with 600 pmol of the corresponding ²H₄-labeled standards. The lowest quantifiable mass was 2 pmol for anandamide (CV=4%; n=5), 1.25 pmol for oleylethanolamide (CV=8.5%; n=3), and 0.6 pmol for palmitylethanolamide (CV=9.6%; n=3).

3.5 Identification and quantification of AEs in rat blood plasma

Synthetic [²H₄]AEs (600 pmol each) were added to plasma samples, proteins were precipitated with acetone, and lipids in the supernatant were extracted with chloroform/methanol. Preliminary tests indicated that the acetone precipitation step was necessary to assure the elimination of contaminating components present in plasma and to obtain a satisfactory chromatographic fractionation. The lipid extracts were subjected to normal-phase HPLC using a method previously published by our laboratory [21], but modified to require a shorter elution time (see Section 2). Under these conditions, all AEs were eluted together from the column at a retention time comprised between 4.7 and 5.3 min. Considering the time necessary to re-equilibrate the column, the fractionation of one sample was completed in approximately 20 min. Although time-consuming, this HPLC step was essential, as it significantly improved GC resolution and prolonged GC column life. The overall recovery of the extraction and fractionation procedures, measured in six independent experiments by using [³H]anandamide as a tracer, was 68.0±2.5%.

In plasma samples from six rats, we observed components which were eluted from the GC at the retention times expected for anandamide, palmitylethanolamide and oleylethanolamide (Fig. 2 and data not shown). These components coeluted with the corresponding ²H₄-labeled AE standards, and displayed the following diagnostic fragments: anandamide, m/z 419, [M]⁺ and m/z 404, [M-15]⁺; palmitylethanolamide, m/z 356, [M-15]⁺; oleylethanolamide, m/z 382, [M-15]⁺ (Fig. 2 and data not shown). These results indicate that deproteinated rat plasma contains detectable levels of the three endogenous AEs. A quantitative estimate of AE plasma concentrations, obtained in 12 independent determinations from six rats, revealed 5.2±1 pmol/ml of anandamide, 16.7±2.7 pmol/ml of palmitylethanolamide and 8.1±1.2 pmol/ml of oleylethanolamide.