Development of Certified Reference Material Solutions for Phytochemicals in Ginger and Kava.

BACKGROUND: Dietary supplements derived from botanicals are commonly consumed and investigated in biomedical studies for their potential health benefits. Accurate identification and quantification of key chemical constituents from botanical ingredients is necessary for consistent product preparations and reproducible research results. Manufacturers need quantitative reference materials of the chemical constituents of interest to verify the content of ingredients and products. The rigor and reproducibility of biomedical research is enhanced through thorough characterization of the interventions used in mechanistic, clinical, and safety investigations. Quantitative reference materials enable reliable product quality assessments and reproducible research results. OBJECTIVE: Solution-based certified reference material (CRM) mixes were developed as calibrants for phytochemicals in Ginger and Kava. The Kava CRM contained yangonin, desmethoxyyangonin, dihydrokavain, DL-kavain, methysticin, dihydromethysticin, flavokawain A, flavokawain B, and flavokawain C. The Ginger CRM contained 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, and 10-shogaol. METHODS: Each phytochemical was sourced as an isolated compound and assigned a purity factor by a mass balance approach accounting for residual impurities. The solution standard mixes were formulated by gravimetric addition of each phytochemical incorporating the purity factor and diluting with acetonitrile to the target concentrations of 500 µg/mL for the gingerols and shogaols, 250 µg/mL for the kavalactones and 25 µg/mL for the flavokawains. RESULTS: The concentration accuracy of each component in the solution mixes was analytically verified by HPLC-UV assay comparison to an independently prepared calibration solution. Each component in the Ginger and Kava CRMs were within 5% and 7% of the target concentrations, respectively. CONCLUSIONS: Homogeneous Kava and Ginger phytochemical solution mixes were produced with accurate constituent concentrations and demonstrated good stability over two years. These solution mixes were launched as commercially available CRMs. HIGHLIGHTS: These mixes can be used as accurate concentration stock solutions to prepare calibrators and controls for botanical dietary supplement product testing and standardization.

Determination of psilocybin and psilocin content in multiple Psilocybe cubensis mushroom strains using liquid chromatography - tandem mass spectrometry.

A method for clinical potency determination of psilocybin and psilocin in hallucinogenic mushroom species Psilocybe cubensis was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Five strains of dried, intact mushrooms were obtained and analyzed: Blue Meanie, Creeper, B-Plus, Texas Yellow, and Thai Cubensis. An extraction protocol was developed; this included an evaluation of sample milling technique, extraction solvents, and recovery/stability. Reversed phase chromatography on fused-core particle phases was developed for the determination of the two analytes using internal standard calibration with deuterated isotopologues of each analyte. The separation takes less than 5 min. Matrix effects were investigated by comparing signal response of calibration samples in neat solution and several mushroom matrices; no significant matrix effects were observed. The limit of detection for psilocybin was 1.5 ng/mL (1.5 pg on-column; 300 ng/g mushroom) and for psilocin was 0.15 ng/mL (0.15 pg on-column; 30 ng/g mushroom) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. Assessment of the accuracy and precision of the method indicated percent error and RSD were <6 % at all concentration levels. Three whole, intact mushrooms from each strain were analyzed individually to obtain average content differences both between strains and between mushrooms of the same strain. From most to least potent, the study found that the average total psilocybin and psilocin concentrations for the Creeper, Blue Meanie, B+, Texas Yellow, and Thai Cubensis strains were 1.36, 1.221, 1.134, 1.103, and 0.879 % (w/w), respectively. A subset of these mushrooms was also tested in a separate non-affiliated laboratory, and the results were comparable between the two laboratories. Results from the secondary laboratory showed improved precision when multiple mushrooms were homogenized together, prior to extraction.

Absolute Protein Quantification by Mass Spectrometry: Not as Simple as Advertised.

Stable isotopically labeled (SIL) tryptic peptides, cleavable SIL peptides, and a full-length SIL protein were compared for internal calibration (i.e., as internal calibrators) and external calibration (i.e., as internal standards) when quantifying three forms of unlabeled, human thyroglobulin (Tg) by bottom-up protein analysis. All SIL materials and human proteins were standardized by amino acid analysis to ensure traceability of measurements and allow confident assignment of accuracy. The three forms of human Tg quantified were (1) the primary reference material BCR457-a native protein purified from human thyroids, (2) a commercially available form also purified from human thyroids, and (3) a full-length recombinant form expressed and purified from a human embryonic kidney 293 cell-line. Collectively, the results unequivocally demonstrate the lack of commutability of tryptic and cleavable SIL peptides as internal calibrators across various bottom-up assays (i.e., denaturing/digestion conditions). Further, the results demonstrate the potential during external calibration for surrogate protein calibrators (i.e., recombinant proteins) to produce inaccurate concentration assignments of native protein analytes by bottom-up analysis due to variance in digestion efficiency, which is not alleviated by altering denaturation/digestion stringency and indicates why protein calibrators may not be commutable in bottom-up protein assays. These results have implications regarding the veracity of "absolute" protein concentration assignments by bottom-up assays using peptide calibrators, as well as protein calibrators, given that absolute accuracy was not universally observed. Nevertheless, these results support the use of recombinant SIL proteins as internal standards over SIL peptides due to their ability to better mimic the digestion of human-derived proteins and mitigate bias due to digestion-based matrix effects that were observed during external calibration.

The uncertainty of reference standards--a guide to understanding factors impacting uncertainty, uncertainty calculations, and vendor certifications.

Certified solution standards are widely used in forensic toxicological, clinical/diagnostic, and environmental testing. Typically, these standards are purchased as ampouled solutions with a certified concentration. Vendors present concentration and uncertainty differently on their Certificates of Analysis. Understanding the factors that impact uncertainty and which factors have been considered in the vendor's assignment of uncertainty are critical to understanding the accuracy of the standard and the impact on testing results. Understanding these variables is also important for laboratories seeking to comply with ISO/IEC 17025 requirements and for those preparing reference solutions from neat materials at the bench. The impact of uncertainty associated with the neat material purity (including residual water, residual solvent, and inorganic content), mass measurement (weighing techniques), and solvent addition (solution density) on the overall uncertainty of the certified concentration is described along with uncertainty calculations.