Electron Capture vs Transfer Dissociation for Site Determination of Tryptic Peptide Tyrosine Sulfation: Direct Detection of Fibrinogen Sulfation Sites and Identification of Novel Isobaric Interferences.

Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.

HPLC Method Development for Quantification of Doxorubicin in Cell Culture and Placental Perfusion Media.

Assessment of drug transport across the placenta is important in understanding the effect of drugs on placental and fetal health. These phenomena can be studied in both in vitro cell lines and ex vivo placental perfusions. We have successfully developed a sensitive yet simple high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine the concentration of doxorubicin (DXR) in cell culture media for transport studies in human trophoblast cells (BeWo, b30 clone) and in fetal media for placental perfusion experiments. The method was developed based on a protein precipitation technique and was validated in both media types for linearity, intra-day, and inter-day precision and accuracy. The relationship of peak area to concentration was linear with R2 values of 0.99 or greater obtained over the concentration range of 1.5 to 15,000 ng/mL. Despite the high concentrations of albumin in fetal perfusion media (30 mg/mL), the lower limits of detection and quantification for DXR were found to be 1.5 and 5 ng/mL, respectively. This analytical method may be used to study the transport of DXR across BeWo cells and human placenta during placental perfusion studies.

Investigation of different spectrophotometric and chemometric methods for determination of entacapone, levodopa and carbidopa in ternary mixture.

New, simple, accurate and sensitive UV spectrophotometric and chemometric methods have been developed and validated for determination of Entacapone (ENT), Levodopa (LD) and Carbidopa (CD) in ternary mixture. Method A is a derivative ratio spectra zero-crossing spectrophotometric method which allows the determination of ENT in the presence of both LD and CD by measuring the peak amplitude at 249.9nm in the range of 1-20µgmL-1. Method B is a double divisor-first derivative of ratio spectra method, used for determination of ENT, LD and CD at 245, 239 and 293nm, respectively. Method C is a mean centering of ratio spectra which allows their determination at 241, 241.6 and 257.1nm, respectively. Methods B and C could successfully determine the studied drugs in concentration ranges of 1-20µgmL-1 for ENT and 10-90µgmL-1 for both LD and CD. Methods D and E are principal component regression and partial least-squares, respectively, used for the simultaneous determination of the studied drugs by using seventeen mixtures as calibration set and eight mixtures as validation set. The developed methods have the advantage of simultaneous determination of the cited components without any pre-treatment. All the results were statistically compared with the reported methods, where no significant difference was observed. The developed methods were satisfactorily applied to the analysis of the investigated drugs in their pure form and in pharmaceutical dosage forms.