Scientists created a quick test to measure both natural thymosin‑alpha‑1 and its lab‑made version in blood and urine, and found that when the lab version is injected it gains the same N‑terminal acetyl group that the natural peptide has, while the natural peptide doesn’t lose it. This means the two forms end up looking the same in the body.

Thymosin α1 (Thymalfasin, Tα1) is a naturally occurring polypeptide widely used as an immune system enhancer for the treatment of HIV/AIDS, hepatitis B and C, and cancer. Recombinant human Tα1 (rh-Tα1) lacking N-terminal acetylation (NTA) displays similar biological activity to Tα1 and has completed phase III clinical trials in China. To compare the pharmacokinetics of rh-Tα1 and Tα1 and establish whether they undergo mutual transmutation in vivo, we developed a novel bioassay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of the two peptides in human plasma and urine. Sample preparation by protein precipitation using a mixture of methanol and perchloric acid was followed by HPLC on a Zorbax 300SB-C18 column (150 × 4.6 mm, 5 μm) maintained at 40 °C. Detection was by multiple reaction monitoring (MRM) of the precursor-to-product ion transitions at m/z 778.0→316.0 for Tα1, m/z 767.3→955.0 for rh-Tα1 and m/z 832.3→159.2 for the internal standard, eptifibatide. The method was linear in the range 2-100 ng/mL for both analytes with good accuracy and precision. High sample throughput was facilitated by inclusion of a parallel two-column chromatographic system. The method was successfully applied to a comparative pharmacokinetic study involving single subcutaneous injections of either Tα1 or rh-Tα1 to two groups of healthy male volunteers. The results indicate that rh-Tα1 undergoes NTA in vivo to form Tα1 but that Tα1 is not deacetylated to rh-Tα1.