Radiotracers
The PET radiotracers used were 11C]PBR28 (radioligand for translocator protein (18 kDa))
[16, 17], 11C]dLop (radiotracer of P-glycoprotein function)
[18, 19], 11C]MePPEP (cannabinoid subtype 1 receptor radioligand)
[20, 21], and 11C](R)-rolipram (radioligand for cAMP phosphodiesterase IV)
[22] (Figure
1A,B,C,D). Each radiotracer was prepared in high radiochemical purity (>99%) and formulated in sterile saline solution, as described previously
[16–22].
HPLC-radiometric measurement of SA
Freshly prepared and formulated radiotracer solution (100 μL), contained in a syringe, was measured for radioactivity in a calibrated ionization chamber (Atomlab 300, Biodex, Shirley, NY, USA) and then injected onto HPLC (Beckman, Fullerton, CA, USA). The radiotracer was eluted isocratically (acetonitrile(aq) 10 to 100 mM HCO2NH4 or 0.1% CF3CO2H) on a reverse-phase column (Onyx, Prodigy, or Luna, Phenomenex, Torrance, CA, USA) equipped with radioactivity and absorbance detectors. The mass of the carrier in the injectate was determined from a linear calibration curve generated from injections of known amounts of authentic non-radioactive standards. The SA was calculated from the decay-corrected activity present in the same volume of injectate, as measured in the calibrated ionization chamber.
LC-MS/MS
Methods for radiotracer analyses were developed on an API 5000 LC-MS/MS system (AB Sciex, Foster City, CA, USA), consisting of Shimadzu LC (Columbia, MD, USA) interfaced via electrospray with triple quadrupole MS/MS operated in positive ionization mode. The technique principally involves quantitative measurement of two ions that differ by two mass-to-charge ratio (m/z) units, one for the 11C-labeled species, denoted [11C]species, and another for the carrier, denoted [M + 1]carrier. [M + 1]carrier ions are generated from isotopologues having predominantly one carbon-13 atom or another heavy stable isotope (2H, 15N, or 17O) of very low abundance. Thus, for example, a solution of PBR28 was infused into the MS/MS, and, upon recording the product ions, compound-dependent and gas parameters were optimized for the m/z 349 [M + H]+ → 121 transition for monitoring [12C]species. An acquisition method for [11C]PBR28 was set up using the same instrument parameters and with the multiple-reaction monitoring table being edited to perform transitions, m/z 348 → 120 and m/z 350 → 122 for measuring [11C]species and [M + 1]carrier, respectively (Figure
1A).
Similarly, MS/MS was tuned with dLop, MePPEP, and (R)-rolipram involving transitions m/z 463 [M + H]+ → 252, m/z 455 [M + H]+ → 351, and m/z 276 [M + H]+ → 191, respectively. Methods were set up to acquire product ions to measure [11C]species and [M + 1]carrier in these radiotracers. The dwell time in these methods ranged between 100 and 175 ms. In each method, the possible cross-talk between transitions was verified by inserting a dummy [M + H]+ → product ion transition between those for [11C]species and [M + 1]carrier. The masses for the dummy transition differed by 5 to 10 amu from those for target ions. Each compound's product ion (Figure
1) was further verified by LC-MSn (n = 2, 3) analysis in an ion-trap mass analyzer (LCQ Deca, Thermo Scientific, San Jose, CA, USA).
Each radiotracer sample was diluted four- to tenfold, and 2 to 5 μL of solution (<500 kBq) was injected with an autosampler (n = 6 or 12) at about 5-min intervals onto LC-MS/MS. Samples of [11C]PBR28, [11C]dLop, and [11C]MePPEP were chromatographed at 40°C on a C18 column (2 × 20 mm, 3 μm; Phenomenex) with a gradient of binary solvents (A/B; 400 μL/min), where A was 10 mM MeCO2NH4 in water/acetonitrile (90:10 v/v) and B was 10 mM MeCO2NH4 in acetonitrile/water (90:10 v/v). In the analysis of [11C]PBR28, for example, the pumps ran 70% A:30% B for 0.1 min and then a gradient reaching 20% A:80% B over 1 min. After 3.5 min, the mobile-phase composition was returned to the initial condition. [11C](R)-Rolipram was chromatographed on the same column eluted with a gradient of water (A) and acetonitrile (B) (A/B; 0.2% acetic acid): 80% A:20% B for 0.1 min, reaching 20% A:80% B over 2 min.
The relative abundance of the M + 1 peak was measured by injecting a solution of PBR28 onto the LC-MS/MS setup to perform m/z 349, 350 [M + H]+ → 121, 122 (12C, M + 1) transitions and thereby to provide m/z 122 to 121 peak area ratio (%). Other compounds were similarly analyzed using respective acquisition methods edited to monitor product ions of 12C and M + 1 species. The measured abundance (%) of M + 1 species relative to 12C was used for converting M + 1 into 12C peak area for radiotracer carrier. The peak area ratio (%) of M + 2 to M + 1 was measured by injecting 40 and 400 pg of PBR28 and acquiring transitions m/z 350, 351 → 122, 123.
Calculations
The SA of the radiotracer (Bq/mol) was calculated as
(1)
where (1) A*
M,M+1 is the sum of the measured 11C, M]species peak area and 11C, M + 1]species area calculated from the relative abundances
[23] of 13C, 2H, 15N, and 17O in the product ion; (2) A
M+1,M is the sum of the measured [M + 1]carrier peak area and 12C]carrier area calculated from the measured M + 1 abundance in the non-radioactive tracer; and (3) SATheoretical is the theoretical SA of carbon-11 (3.413 × 1020 Bq/mol), the product of ln2/t
1/2 and Avogadro's number. The SA of the radiotracer was decay-corrected to give the SA at the end of radiosynthesis (SA0) as SAeλt, where t is the time between the end of synthesis and peak elution in LC-MS/MS and λ is 0.034 min-1 (ln2/20.38). A plot of lnSA (n = 12) versus clock time of peak elution in Prism 5.02 (GraphPad, La Jolla, CA, USA) gave the value for λ and thus t
1/2 (t
1/2 = ln2/λ).