Rapid and Economic Determination of 13 Steviol Glycosides in Market-Available Food, Dietary Supplements, and Ingredients: Single-Laboratory Validation of an HPLC Method.

Steviol glycosides, obtained from leaves of Stevia rebaudiana Bertoni (stevia) or produced via bioconversion and biosynthesis, are diterpenes used by the food/dietary supplement industry as zero-calorie sweeteners derived from natural sources. JECFA 2017 is the most updated international standardized method but it runs for 80 min per sample with suboptimal separations on several critical pairs for its high-performance liquid chromatography-ultraviolet (HPLC-UV) determination. We developed and validated a rapid and economic HPLC-UV method using the superficially porous particle column to determine 13 steviol glycosides (stevioside, dulcoside A, rubusoside, steviobioside, and rebaudioside A-F, I, M, and N). Baseline separation with a minimum resolution of 1.5 for 13 steviol glycosides was achieved within only 14 min of separation time. The hydrocarbon stationary phase with additional steric interactions from the isobutyl side chains on the C18 ligand was shown to be an important contributor to chromatographic selectivity of several critical pairs of steviol glycosides. The method was proven to perform suitably on columns from three different manufacturers and two HPLC instruments. The method was further used to perform a single-lab validation on eight food and supplement products with multiple matrices. The results ranged from 0.05% w/w rebaudioside A for a hard-candy finished product to 100.8% w/w purity for a rebaudioside M raw ingredient. The validation test results showed that the method was linear, suitable, specific, accurate, and precise. The method is therefore suitable to be considered as a new industrial standard for quality control analysis for stevia products.

Determination of Select Nonvolatile Ginger Constituents in Dietary Ingredients and Finished Dosage Forms, First Action 2018.04.

A consensus industrial reference was necessary to be established for meeting the U.S. Food and Drug Administration's current Good Manufacturing Practice Compliance for the manufacture and quality control of dietary ingredients and supplements that contain ginger rhizome, dry extract, and/or purified nonvolatile ginger constituents. An analytical method has been developed, validated, and previously published for identifying and quantifying 6-,8- and 10-gingerols, 6-, 8- and 10-shogaols, 6-paradol, and zingerone. HPLC with UV-Vis detector was used for the determination of nonvolatile ginger constituents by following AOAC Guidelines for Single-Laboratory Validation. Sample was accurately weighed and diluted with acidified water and methanol mixture. The sample solution was then sonicated and filtered through a PTFE filter and analyzed under a linear gradient scheme instrument condition. A reverse-phase superficially porous particle C18 column and an absorption wavelength of 230 nm were used for analyte separation and determination. The method was demonstrated to be selective, linear (R2 > 0.999), specific, accurate (91.1-103.2% spike recovery rate), and precise (RSDr < 5%, RSDR < 8%) and therefore met all AOAC Standard Method Performance Requirements (2017.012) criteria. With a relatively short run time (12 min) and optimized extraction solvent system, the method has been validated to simultaneously determine nonvolatile ginger constituents in a variety of dietary ingredients and dietary supplements matrices including dry extract, powder, tablet, capsule, liquid capsule, softgel capsule, and oleoresin.

Quantitative determination of 58 aromatic amines and positional isomers in textiles by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry.

A straightforward method for simultaneous determination of 58 aromatic amines (AAs) in clothing textiles is described. Of the tested amines, 22 are known carcinogens and regulated under European law. Further unregulated amines, which are suspected to be carcinogenic or mutagenic, are also expected to occur in textiles. The focus of this study was to obtain a multimethod with regulated and unregulated AAs and to separate different positional isomers of specific amines. To achieve this goal, the HPLC parameters such as column phase chemistry and mobile phase pH were optimized. All other amines were separated with the same conditions evaluated for the positional isomers. The sample preparation was reduced to a minimum in accordance to DIN EN 14362-1. After reductive cleavage and dilution of the extracts, the samples were prepared for LC-MS/MS analysis. Further cleanup of the samples with diatomaceous earth was no longer required with this modified sample preparation. The method was validated by spiking the extracts of blank textiles with the chosen amines. Lower Limits of Quantification (LLOQs) ranged from 1 to 10 mg kg-1. Excellent linearity from 10 to 500 µg L-1 was obtained with coefficients (r) higher than 0.99 allowing a one-point calibration (300 µg L-1 corresponding to 60 mg kg-1 in textiles) forced through zero for the samples. Recovery ranged from 88 to 116% with intra- and inter-day precision values of 1.8-12.1%. The accuracy of this method was tested by a certified reference material (iis18A03, Institute for laboratory Studies, the Netherlands) and showed excellent recoveries higher than 95%. Finally, the described method was successfully applied on 150 real samples with different textile fibres and colours. Positive results were confirmed by means of the spectral library and with the ion ratios.

Determination of bioactive nonvolatile ginger constituents in dietary supplements by a rapid and economic HPLC method: Analytical method development and single-laboratory validation.

Most of the validated methods for ginger-containing dietary supplements have long run time and low sensitivity and only analyze gingerols and shogaols. 6-Paradol and zingerone become popular in modern dietary supplement industry as bioactive ginger constituents. Therefore, we developed an efficient HPLC-UV/Vis method to analyze all above major constituents. Compared to 282/280 nm used by the current compendial United States Pharmacopeia (USP) monograph method and International Organization for Standardization (ISO) 13685-1997 method, detection wavelength was optimized to 230 nm which showed a higher sensitivity (signal-to-noise ratio) and better peak resolution. For measuring the ginger constituents in AOAC required matrices, the method was demonstrated to be selective, linear (R2 > 0.999), specific, accurate (91.1-103.2% spike recovery rate) and precise (RSDr < 5%, RSDR < 8%). Among 10 commercial ginger-containing samples that we screened using this method, the results were 80-123% of the products' labeling value. The HPLC running time was successfully shortened from 29 min (USP method) and 40 min (ISO method) to 12 min without the need of using an expensive Mass Spectrometer for analyte separation. The method is the first method that meets all AOAC SMPR 2017.12 requirements and therefore has the potential to be adopted as a consensus industrial reference method for meeting FDA's cGMP Compliance for the manufacture and quality control of dietary supplements and ingredients.

Chromatographic characterisation of 11 phytocannabinoids: Quantitative and fit-to-purpose performance as a function of extra-column variance.

INTRODUCTION: Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids. OBJECTIVE: To develop a rapid, quantitative and broadly applicable liquid chromatography-tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character. METHODOLOGY: Bulk diffusion coefficients were calculated using the Taylor-Aris open tubular method, with four reference compounds used to validate the experimental set-up. Three columns were quantitatively evaluated using van Deemter plots and fit-to-purpose performance metrics. Low (1.2 µL2 ) and standard (3.6 µL2 ) extra-column variance ultra-high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts. RESULTS: Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging Δ8 /Δ9 -tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids. CONCLUSIONS: The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra-column variance UPLC configurations.