METHODS: This study examined the composition purity of PCAV through a decomplexation proteomic approach, applying size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tandem mass spectrometry liquid chromatography-tandem mass spectrometry (LC-MS/MS).
RESULTS: SDS-PAGE and SEC showed that the major protein in PCAV (constituting ∼80% of total proteins) is approximately 110 kDa, consistent with the F(ab')2 molecule. This protein is reducible into two subunits suggestive of the light and heavy chains of immunoglobulin G. LC-MS/MS further identified the proteins as equine immunoglobulins, representing the key therapeutic ingredient of this biologic product. However, protein impurities, including fibrinogens, alpha-2-macroglobulins, albumin, transferrin, fibronectin and plasminogen, were detected at ∼20% of the total antivenom proteins, unveiling a concern for hypersensitivity reactions.
CONCLUSIONS: Together, the findings show that PCAV contains a favorable content of F(ab')2 for neutralization, while the antibody purification process awaits improvement to minimize the presence of protein impurities.
METHODS: The mass spectral characterization of the proposed NMs-GSH conjugates was performed with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). The final reaction mixtures were analysed in positive electrospray ionisation (ESI) at different incubation times.
RESULTS: This study identified three types of conjugates in addition to ethanolamines, the hydrolysis products of NMs. Monoglutathionyl, diglutathionyl and phosphorylated conjugates were produced for each of the NMs, bis(2-chloroethyl)ethylamine (HN1), bis(2-chloroethyl)methylamine (HN2) and tris(2-chloroethyl)amine (HN3). The monoglutathionyl conjugates consisted of HN1-GSH, HN2-GSH and HN3-GSH. The spontaneous and primary conjugates of diglutathionyl were HN1-GSH2, HN2-GSH2 and HN3-GSH2. These included phosphorylated conjugates, namely HN1-GSH-PO4 , HN2-GSH-PO4 and HN3-GSH-PO4 , as might have formed due to hydrolysis in phosphate buffer.
CONCLUSIONS: The mass spectral data of all conjugates formed in the presence of all NMs and GSH are reported in this study. These GSH metabolites can be used to confirm NMs toxicity in biological samples such as urine.
RESULTS: A clear separation was only observed between non-organic G and organic Z, which were then selected for further investigation in the fermentation of soybeans (GF and ZF). All four groups (G, Z, GF, ZF) were analyzed using nuclear magnetic resonance (NMR) spectroscopy along with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this way a total of 41 and 47 metabolites were identified respectively, with 12 in common. A clear variation (|log1.5 FC| > 2 and P
METHODS: A total of 401 whole blood samples with a fresh HbA1c measurement were randomly selected from The Malaysian Cohort's (TMC) biobank. The HbA1c measurements of fresh and frozen (stored for 7-8 years) samples were assayed using different high-performance liquid chromatography (HPLC) systems. The HbA1c values of the fresh samples were then calculated and corrected according to the later system. The reproducibility of HbA1c measurements between calculated-fresh and frozen samples was assessed using a Passing-Bablok linear regression model. The Bland-Altman plot was then used to evaluate the concordance of HbA1c values.
RESULTS: The different HPLC systems highly correlated (r = 0.99) and agreed (ICC = 0.96) with each other. Furthermore, the HbA1c measurements for frozen samples strongly correlate with the corrected HbA1c values of the fresh samples (r = 0.875) with a mean difference of -0.02 (SD: -0.38 to 0.38). Although the mean difference is small, discrepancies were observed within the diabetic and non-diabetic samples.
CONCLUSION: These data demonstrate that the HbA1c measurements between fresh and frozen samples are highly correlated and reproducible.
OBJECTIVE: A sensitive method for the estimation of CRM in plasma, as well as fecal matter-based solid self-nano emulsifying drug delivery system (S-SNEDDS), has been reported for the first time.
METHODS: A bioanalytical method was optimized using Box-Behnken Design having 13 runs and 3 responses. The optimized method was developed using methanol and water (70:30 v/v) with a flow rate of 1 mL/min. Quercetin was used as an internal standard. A specificity test was also performed for the developed CRM solid self-nano emulsifying drug delivery system.
RESULTS: The retention time of CRM was found to be 14.18 minutes. The developed method was validated and found to be linear in the range of 50-250 ng/mL with an R2 of 0.999. Accuracy studies indicated that CRM had a percentage recovery of less than 105% and more than 95%, respectively. Precision studies were carried out for inter, intraday, and inter-analyst precision, and the %RSD was found to be less than 2%. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.37 ng/mL and 10.23 ng/mL, respectively. Stability studies for shortterm, long term and freeze-thaw cycles showed a %RSD of less than 2%, indicating the stability of CRM in the plasma matrix. Moreover, the blank fecal microbiota extract slurry did not show any peak at the retention time of CRM in a CRM-loaded solid nanoemulsifying drug delivery system containing fecal microbiota extract indicating its specificity.
CONCLUSION: Hence, the developed method can have clinical implications as it helps estimate CRM in blood samples and also provides a simple and sensitive method for the estimation of plant-based flavonoids along with fecal microbiota extract formulations.