Optimization and single-laboratory validation of a method for the determination of flavonolignans in milk thistle seeds by high-performance liquid chromatography with ultraviolet detection.

Seeds of milk thistle, Silybum marianum (L.) Gaertn., are used for treatment and prevention of liver disorders and were identified as a high priority ingredient requiring a validated analytical method. An AOAC International expert panel reviewed existing methods and made recommendations concerning method optimization prior to validation. A series of extraction and separation studies were undertaken on the selected method for determining flavonolignans from milk thistle seeds and finished products to address the review panel recommendations. Once optimized, a single-laboratory validation study was conducted. The method was assessed for repeatability, accuracy, selectivity, LOD, LOQ, analyte stability, and linearity. Flavonolignan content ranged from 1.40 to 52.86% in raw materials and dry finished products and ranged from 36.16 to 1570.7 µg/mL in liquid tinctures. Repeatability for the individual flavonolignans in raw materials and finished products ranged from 1.03 to 9.88% RSDr, with HorRat values between 0.21 and 1.55. Calibration curves for all flavonolignan concentrations had correlation coefficients of >99.8%. The LODs for the flavonolignans ranged from 0.20 to 0.48 µg/mL at 288 nm. Based on the results of this single-laboratory validation, this method is suitable for the quantitation of the six major flavonolignans in milk thistle raw materials and finished products, as well as multicomponent products containing dandelion, schizandra berry, and artichoke extracts. It is recommended that this method be adopted as First Action Official Method status by AOAC International.

Determination of major phenolic compounds in Echinacea spp. raw materials and finished products by high-performance liquid chromatography with ultraviolet detection: single-laboratory validation matrix extension.

A method previously validated to determine caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid in echinacea raw materials has been successfully applied to dry extract and liquid tincture products in response to North American consumer needs. Single-laboratory validation was used to assess the repeatability, accuracy, selectivity, LOD, LOQ, analyte stability (ruggedness), and linearity of the method, with emphasis on finished products. Repeatability precision for each phenolic compound was between 1.04 and 5.65% RSD, with HorRat values between 0.30 and 1.39 for raw and dry extract finished products. HorRat values for tinctures were between 0.09 and 1.10. Accuracy of the method was determined through spike recovery studies. Recovery of each compound from raw material negative control (ginseng) was between 90 and 114%, while recovery from the finished product negative control (maltodextrin and magnesium stearate) was between 97 and 103%. A study was conducted to determine if cichoric acid, a major phenolic component of Echinacea purpurea (L.) Moench and E. angustifolia DC, degrades during sample preparation (extraction) and HPLC analysis. No significant degradation was observed over an extended testing period using the validated method.

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.