Elizabeth Mudge

Analysis of alkylamides in Echinacea plant materials and dietary supplements by ultrafast liquid chromatography with diode array and mass spectrometric detection.

Alkylamides are a class of compounds present in plants of the genus Echinacea (Asteraceae), which have been shown to have high bioavailability and immunomodulatory effects. Fast analysis to identify these components in a variety of products is essential to profile products used in clinical trials and for quality control of these products. A method based on ultrafast liquid chromatography (UFLC) coupled with diode array detection and electrospray ionization mass spectrometry was developed for the analysis of alkylamides from the roots of Echinacea angustifolia (DC.) Hell., Echinacea purpurea (L.) Moench, and commercial dietary supplements. A total of 24 alkylamides were identified by LC-MS. The analysis time for these components is 15 min. Compared to the alkylamide profiles determined in the Echinacea root materials, the commercial products showed a more complex profile due to the blending of root and aerial parts of E. purpurea. This versatile method allows for the identification of alkylamides in a variety of Echinacea products and presents the most extensive characterization of alkylamides in E. angustifolia roots so far.

The Importance of Method Selection in Determining Product Integrity for Nutrition Research

The American Herbal Products Association estimates that there as many as 3000 plant species in commerce. The FDA estimates that there are about 85,000 dietary supplement products in the marketplace. The pace of product innovation far exceeds that of analytical methods development and validation, with new ingredients, matrixes, and combinations resulting in an analytical community that has been unable to keep up. This has led to a lack of validated analytical methods for dietary supplements and to inappropriate method selection where methods do exist. Only after rigorous validation procedures to ensure that methods are fit for purpose should they be used in a routine setting to verify product authenticity and quality. By following systematic procedures and establishing performance requirements for analytical methods before method development and validation, methods can be developed that are both valid and fit for purpose. This review summarizes advances in method selection, development, and validation regarding herbal supplement analysis and provides several documented examples of inappropriate method selection and application.

Quantification of pyrrolizidine alkaloids in North American plants and honey by LC-MS: single laboratory validation.

Pyrrolizidine alkaloids (PAs) are a class of naturally occurring compounds produced by many flowering plants around the World. Their presence as contaminants in food systems has become a significant concern in recent years. For example, PAs are often found as contaminants in honey through pollen transfer. A validated method was developed for the quantification of four pyrrolizidine alkaloids and one pyrrolizidine alkaloid N-oxide in plants and honey grown and produced in British Columbia. The method was optimised for extraction efficiency from the plant materials and then subjected to a single-laboratory validation to assess repeatability, accuracy, selectivity, LOD, LOQ and method linearity. The PA content in plants ranged from1.0 to 307.8 µg/g with repeatability precision between 3.8 and 20.8% RSD. HorRat values were within acceptable limits and ranged from 0.62 to 1.63 for plant material and 0.56–1.82 for honey samples. Method accuracy was determined through spike studies with recoveries ranging from 84.6 to 108.2% from the raw material negative control and from 82.1–106.0 % for the pyrrolizidine alkaloids in corn syrup. Based on the findings in this single-laboratory validation, this method is suitable for the quantitation of lycopsamine, senecionine, senecionine N-oxide, heliosupine and echimidine in common comfrey (Symphytum officinale), tansy ragwort (Senecio jacobaea), blueweed (Echium vulgare) and hound’s tongue (Cynoglossum officinale) and for PA quantitation in honey and found that PA contaminants were present at low levels in BC honey.

Determination of Aloin A and Aloin B in Aloe vera Raw Materials and Finished Products by High-Performance Liquid Chromatography: Single-Laboratory Validation.

A single-laboratory validation (SLV) was conducted on an HPLC method for the detection and quantification of aloin A and aloin B in Aloe vera raw materials and finished products. An extraction procedure using sonication with an acidified solvent was used for solid test materials while liquid test materials only required dilution, if necessary, prior to filtration and analysis. Separation was achieved using a fused core C18 column in 18 min under isocratic elution conditions allowing for a single analyte (aloin A) calibration curve to quantify both aloins. Adequate chromatographic resolution (Rs ≥ 1) was achieved for aloin A and aloin B. The calibration curves for aloin A exhibited coefficients of determination (r(2)) of ≥ 99.9% over the linear range of 0.3-50 μg/mL. The LOD values were 0.092 and 0.087 μg/mL, and LOQ 0.23 and 0.21 μg/mL for aloin A and aloin B, respectively. Repeatability studies were performed on nine test materials on each of 3 separate days, with five of the test materials determined to be above the LOQ having repeatability RSD (RSDr) values ranging from 0.61 to 6.30%. Method accuracy was determined through a spike recovery study on both liquid and solid matrixes at three different levels: low, medium, and high. For both aloins, the recovery in the liquid matrix ranged from 92.7 to 106.3% with an RSDr of 0.15 to 4.30%, while for the solid matrix, the recovery ranged from 84.4 to 108.9% with an RSDr of 0.23 to 3.84%. Based on the results of the SLV study, it is recommended that this method be evaluated for reproducibility through a collaborative study.

Determination of Phenolic Constituents in Echinacea Raw Materials and Dietary Supplements by HPLC-UV: Collaborative Study.

A collaborative study was conducted to evaluate an HPLC method for determining phenolic compounds in Echinacea spp. raw materials, powdered extracts, and tinctures. Eleven collaborating laboratories received three practice samples representing each matrix type, phenolic reference standards, eight test samples as blind duplicates, the validated analytical method, and instructions. Test samples included two raw materials, four extracts (including one in combination with astragalus and reishi), one ethanolic tincture in combination with goldenseal, and one glycerite tincture. Each material was extracted with a 60% methanol aqueous solution, separated on a C18 column, and detected at 330 nm. Results reported by laboratories for total phenolics in Echinacea roots, aerial parts, and extracts ranged from 9.5 to 62.9 mg/g with RSDR ranging from 3.64 and 7.95% and Horwitz ratio (HorRat) values ranging from 1.06 to 2.01. Total phenolics in the ethanolic tincture ranged from 4837 to 5962 μg/mL, with an RSDR of 6.35% and a HorRat value of 1.45. The glycerite tincture showed poor interlaboratory precision with a HorRat value of 3.32, an RSDR of 21.8%, and reported total phenolic values ranging from 257 to 539 μg/mL.

Determination of Mitragynine in Mitragyna speciosa Raw Materials and Finished Products by Liquid Chromatography with UV Detection: Single-Laboratory Validation.

Mitragyna speciosa (kratom) is a tree indigenous to Southeast Asia, and its leaves are used in herbal formulations because they contain indole alkaloids mitragynine and 7-hydroxy (7-OH) mitragynine. An HPLC method was developed, optimized, and validated using single-laboratory validation guidelines to quantify mitragynine in kratom raw materials and finished products. The method optimization evaluated several extraction parameters including solvent type, solvent volume, time, and extraction method. The separation of the mitragynine alkaloids was achieved in 18 min with a fused-core C18 EVO column using gradient separation with ammonium bicarbonate (pH 9.5) and acetonitrile. The calibration range for mitragynine was 1.0-500 μg/mL with correlation coefficients of ≥99.9% throughout method development and validation. The method detection limit and LOQ were 0.2 and 0.6 μg/mL, respectively for mitragynine. Eight test samples were obtained to evaluate method repeatability. RSDr ranged from 0.4 to 1.0%, whereas intermediate precision ranged from 3.7 to 7.3%, with HorRat values from 0.68 to 1.96. 7-OH mitragynine was below the LOQ for all samples, therefore, spikes repeatability sample RSD values were <1%. The validation data presented meet the Standard Method Performance Requirements as specified by the AOAC INTERNATIONAL Kratom Working Group.

Determination of Curcuminoids in Turmeric Raw Materials and Dietary Supplements by HPLC: Single-Laboratory Validation, First Action 2016.16

The AOAC Expert Review Panel (ERP) approved a method for the quantitation of curcuminoids for consideration for First Action Official MethodSM status. The previously published method summarized a single-laboratory validation of three individual curcuminoinds-curcumin, demethoxycurcumin, and bis-demethoxycurcumin-in raw materials and finished products. Method performance was compared with AOAC Standard Method Performance Requirement 2016.003. With repeatability precision ranging from 0.3 to 5.5% and recoveries from 96.6 to 103.3% in the different product matrixes, the ERP adopted the method and provided recommendations for achieving Final Action status.

Determination of Alkaloids in Mitragyna speciosa (Kratom) Raw Materials and Dietary Supplements by HPLC-UV: Single-Laboratory Validation, First Action 2017.14

The AOAC Expert Review Panel (ERP) approved a method for the quantitation of alkaloids in Mitragyna speciosa for consideration as First Action Official MethodSM status. The previously published method summarized a single-laboratory validation of two alkaloids, mitragynine and 7-hydroxymitragynine, in raw materials and finished products. The methods performance was compared with the AOAC Standard Method Performance Requirement 2015.008. With repeatability precision (RSDr) ranging from 0.51 to 0.95% and recoveries from 93.6 to 98.9% in the different product matrices, the ERP adopted the method and provided recommendations for achieving Final Action status.