A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Validation of LC-MS/MS Coupled with a Chiral Column for the Determination of 3- or 15-Acetyl Deoxynivalenol Mycotoxins from Fusarium graminearum in Wheat

The major causal agents Fusarium graminearum (F. graminearum) and Fusarium asiaticum could produce multiple mycotoxins in infected wheat, which threatens the health of humans and animals. Specifically, deoxynivalenol (DON) and its derivatives 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) are commonly detected mycotoxins in cereal grains. However, the good chromatographic separation of 3-ADON and 15-ADON remains challenging. Here, an LC-MS/MS method for the chemotype determination of Fusarium strains was developed and validated. 3- and 15-ADON could be separated chromatographically in this study with sufficiently low limits of detection (LODs; 4 μg/kg) and limits of quantification (LOQs; 8 μg/kg). The satisfying intraday and interday reproducibility (both %RSDr and %RSDR were <20%) of this method indicated good stability. The recoveries of all analytes were in the range of 80–120%. In addition, three F. graminearum complex (FGC) strains, i.e., PH-1 (chemotype 15-ADON), F-1 (chemotype 3-ADON) and 5035 (chemotype 15-ADON), were selected to verify the accuracy of the method in differentiating phenotypes. The validation results showed that this LC-MS/MS method based on sample pretreatment is effective and suitable for the chromatographic separation of 3-ADON and 15-ADON in wheat.

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application

A novel, fast and sensitive enantioselective HPLC assay with a new core–shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(−)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1–450 ng/mL (r2 ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(−)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(−)-VER established higher Cmax and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core–shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).

Analysis of α-Tocopherol Stereoisomers in Fortified Infant Formula by Chiral Chromatography

Background Direct measurement of the bioavailable α-tocopherol content presents a significant analytical challenge and requires chiral separation of the α-tocopherol stereoisomers. Objective The objective of the study was to validate an analytical method for the analysis of α-tocopherol stereoisomers in infant formulas and dairy products. Method Samples were saponified at elevated temperature and lipophilic components were extracted into an organic solvent, with subsequent chromatographic separation of the α-tocopherol stereoisomers achieved by HPLC with a chiral column and fluorescence detection. Results The method was shown to be accurate, with spike recoveries of 91.9–108.8% for RRR-α-tocopherol and 90.1–104.7% for α-tocopherol, with no statistical bias against NIST 1849a certified reference material (p-value = 0.54) and an HPLC-UV analytical method (p-value = 0.48). Acceptable precision was confirmed, with repeatabilities estimated at 3.5% RSDr (HorRat = 0.6) for RRR-α-tocopherol and 4.6% RSDr (HorRat = 0.4) for α-tocopherol. Conclusions A straightforward chiral chromatographic method for the analysis of stereoisomeric forms of α-tocopherol is described. In a single analytical run, the method can quantify: (i) the total α-tocopherol content; (ii) the nutritionally important RRR-α-tocopherol and/or 2R,4′-ambo,8′-ambo-α-tocopherol contents; (iii) the amount of all-rac-α-tocopherol, all-rac-α-tocopheryl acetate, or all-rac-α-tocopheryl succinate fortified into the product. Highlights An accurate and precise chiral chromatographic method for the analysis of isomeric forms of α-tocopherol is described. The method is able to distinguish between natural and synthetic tocopherol sources. The method is accurate and precise and is suitable either for routine product compliance testing during product manufacture or as a possible reference method.

Circularly polarized luminescent systems fabricated by Tröger's base derivatives through two different strategies

The Tröger's base derivative rac-TBPP was synthesized and separated into two enantiomers R 2N -TBPP and S 2N -TBPP by chiral column chromatography. These compounds show a strong circularly polarized luminescence with g lum values of +0.0021, and −0.0025, respectively. The second way to fabricate the rac-TBPP-based CPL-active material is to co-gel the fluorescent rac-TBPP with a chiral ᴅ-glutamic acid gelator DGG by co-assembly strategy. At the molar ratio of rac-TBPP/DGG = 1:80, the g lum value of the co-gel was about three times higher than the g lum values of R 2N -TBPP and S 2N -TBPP enantiomers. Interestingly, the CPL handedness of the rac-TBPP/DGG co-gel could be adjusted effectively by changing their stoichiometric ratios.

Circularly Polarized Luminescent Systems Fabricated by Tröger's Base Derivatives through Two Different Strategies

Tröger's base derivative rac-TBPP was synthesized and separated into two enantiomers R2N-TBPP and S2N-TBPP by chiral column, which emit strong circularly polarized luminescence with glum values of +0.0021, and –0.0025, respectively. The different way to fabricate the CPL-active material is to cogel the fluorescent rac-TBPP with a chiral D-glutamic acid gelator DGG by co-assembly strategies. At the molar ratio of rac-TBPP : DGG = 1 : 80, the glum value of the cogel was increased to be about three times higher than that observed from R2N-TBPP and S2N-TBPP enantiomers. Interestingly, the CPL handedness of the rac-TBPP/DGG cogel could be adjusted effectively by changing their stoichiometric ratios.

Separation of RRR-α-Tocopherol by Chiral Chromatography

Background α-Tocopherol can exist as eight possible stereoisomers due to the presence of three chiral carbons. Regulations and industry guidelines necessitate that dietary vitamin E intakes be based on the vitamin E activity of RRR-α-tocopherol. Food products fortified with synthetic all-rac-α-tocopherol or all-rac-α-tocopheryl acetate during manufacturing will require chiral separation of the α-tocopherol stereoisomers for accurate estimation of vitamin E activity. Objective The development of an HPLC method utilizing a chiral column for the chromatographic separation of RRR-α-tocopherol from other α-tocopherol stereoisomers. Method Normal phase liquid chromatographic separation using a polysaccharide-based chiral column with fluorescence detection of α-tocopherol stereoisomers. Results The described chromatographic method achieves baseline resolution of RRR-α-tocopherol from its stereoisomers. Method selectivity, precision, and robustness were evaluated and acceptable performance was achieved. Conclusions The chromatographic method was found to be suitable for application where both RRR-α-tocopherol content and total α-tocopherol content are required for routine compliance testing. Highlights A robust and precise chomatographic method for the baseline resolution of RRR-α-tocopherol from its stereoisomers was acheived.