Determination of Total Phenolic Content Using the Folin-C Assay: Single-Laboratory Validation, First Action 2017.13.

A single-laboratory validation of a method using Folin & Ciocalteu's phenol reagent (Folin-C reagent) for determination of total phenolic content of selected dietary supplement extracts was performed. The method is composed of a water extraction of dried extracts with sonication followed by reaction with the Folin-C reagent. The resulting colorimetric reaction is measured at 765 nm and compared with a standard curve generated with gallic acid standard solutions. The validation results were compared with Standard Method Performance Requirement (SMPR®) 2015.009, developed by the Stakeholder Panel on Dietary Supplements. The method demonstrated acceptable within-day RSDr of 1.96-7.47% for the five matrixes studied (grape seed extract, grape skin extract, black tea extract, green coffee extract, and cocoa extract). When gallic acid was spiked into maltodextrin (a surrogate dietary supplement carrier) at 30 or 70%, the recovery ranged from 91 to 104%, within the acceptable range established by SMPR 2015.009. Selectivity testing with glucose, fructose, and sucrose demonstrated no positive interference by these compounds. Finally, ruggedness studies demonstrated no significant effects due to changes in the heating apparatus, test material weight, read time after reaction, amount of Folin-C reagent, reaction time, reaction temperature, and amount of Na2CO3. The single-laboratory validation results support adoption of the method as First Action Official MethodSM 2017.13 and further evaluation in a collaborative study.

Electrophoretic separation of alginic sodium diester and sodium hexametaphosphate in chondroitin sulfate that interfere with the cetylpyridinium chloride titration assay.

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in cormbination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived fromn CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use methodfor detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CSby CAME and that exhibit CPC titration behavior similar to CS.

Determination of catechins and caffeine in camillia sinensis raw materials, extracts, and dietary supplements by HPLC-uv: single-laboratory validation.

A rapid method has been developed to quantify seven catechins and caffeine in green tea (Camillia sinensis) raw material and powdered extract, and dietary supplements containing green tea extract. The method utilizes RP HPLC with a phenyl-based stationary phase and gradient elution. Detection is by UV absorbance. The total run time, including column re-equilibration, is 13 min. Single-laboratory validation (SLV) has been performed on the method to determine the repeatability, accuracy, selectivity, LOD, LOQ, ruggedness, and linearity for (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-gallocatechin gallate, (-)-epigallocatechin gallate, and (+)-gallocatechin, as well as caffeine. Repeatability precision and recovery results met AOAC guidelines for SLV studies for all catechins and caffeine down to a level of approximately 20 mg/g. Finished products containing high concentrations of minerals require the use of EDTA to prevent decomposition of the catechins.

Enhanced uridine bioavailability following administration of a triacetyluridine-rich nutritional supplement.

BACKGROUND: Uridine is a therapy for hereditary orotic aciduria and is being investigated in other disorders caused by mitochondrial dysfunction, including toxicities resulting from treatment with nucleoside reverse transcriptase inhibitors in HIV. Historically, the use of uridine as a therapeutic agent has been limited by poor bioavailability. A food supplement containing nucleosides, NucleomaxX®, has been reported to raise plasma uridine to supraphysiologic levels. METHODOLOGY/PRINCIPAL FINDINGS: Single- and multi-dose PK studies following NucleomaxX® were compared to single-dose PK studies of equimolar doses of pure uridine in healthy human volunteers. Product analysis documented that more than 90% of the nucleoside component of NucleomaxX® is in the form of triacetyluridine (TAU). Single and repeated dosing with NucleomaxX® resulted in peak plasma uridine concentrations 1-2 hours later of 150.9 ± 39.3 µM and 161.4 ± 31.5 µM, respectively, levels known to ameliorate mitochondrial toxicity in vitro. C(max) and AUC were four-fold higher after a single dose of NucleomaxX® than after uridine. No adverse effects of either treatment were observed. CONCLUSIONS/SIGNIFICANCE: NucleomaxX®, containing predominantly TAU, has significantly greater bioavailability than pure uridine in human subjects and may be useful in the management of mitochondrial toxicity.

Accuracy, precision, and reliability of chemical measurements in natural products research.

Natural products chemistry is the discipline that lies at the heart of modern pharmacognosy. The field encompasses qualitative and quantitative analytical tools that range from spectroscopy and spectrometry to chromatography. Among other things, modern research on crude botanicals is engaged in the discovery of the phytochemical constituents necessary for therapeutic efficacy, including the synergistic effects of components of complex mixtures in the botanical matrix. In the phytomedicine field, these botanicals and their contained mixtures are considered the active pharmaceutical ingredient (API), and pharmacognosists are increasingly called upon to supplement their molecular discovery work by assisting in the development and utilization of analytical tools for assessing the quality and safety of these products. Unlike single-chemical entity APIs, botanical raw materials and their derived products are highly variable because their chemistry and morphology depend on the genotypic and phenotypic variation, geographical origin and weather exposure, harvesting practices, and processing conditions of the source material. Unless controlled, this inherent variability in the raw material stream can result in inconsistent finished products that are under-potent, over-potent, and/or contaminated. Over the decades, natural product chemists have routinely developed quantitative analytical methods for phytochemicals of interest. Quantitative methods for the determination of product quality bear the weight of regulatory scrutiny. These methods must be accurate, precise, and reproducible. Accordingly, this review discusses the principles of accuracy (relationship between experimental and true value), precision (distribution of data values), and reliability in the quantitation of phytochemicals in natural products.

Determination of hydrastine and berberine in goldenseal raw materials, extracts, and dietary supplements by high-performance liquid chromatography with UV: collaborative study.

A multilaboratory collaborative study was conducted on a high-performance liquid chromatographic (HPLC) method utilizing UV detection, previously validated using AOAC single-laboratory validation guidelines for determination of hydrastine and berberine in goldenseal (Hydrastis canadensis L.) raw materials, extracts, and dietary supplements at levels ranging from 0.4 to 6% (w/w). Nine collaborating laboratories determined the hydrastine and berberine content in 8 blind samples. Sample materials included powdered botanical raw materials, whole root material, and 4 finished product dietary supplements containing either goldenseal powdered root material or extract. The materials were extracted with an acidified water and acetonitrile solution. HPLC analyses of the extracts were performed on a C18 column using UV detection at 230 nm. Results for powdered root material and capsule products ranged from about 0.2% (w/w) for each alkaloid to about 4% (w/w) for each alkaloid. Liquid tincture results were approximately 4000-5000 microg/mL for each alkaloid. Reproducibility relative standard deviations (RSDR) for hydrastine ranged from 2.68 to 6.65%, with HorRat values ranging from 0.77 to 1.89. RSDR for berberine ranged from 5.66 to 7.68%, with HorRat values ranging from 1.32 to 2.12. All finished products containing goldenseal extract yielded HorRat values <2.0. Based on these results, the method is recommended for Official First Action for determination of hydrastine and berberine in goldenseal raw materials and dietary supplement finished products containing powdered goldenseal and goldenseal extract.

Determination of synephrine in bitter orange raw materials, extracts, and dietary supplements by liquid chromatography with ultraviolet detection: single-laboratory validation.

A method has been developed to quantify synephrine in bitter orange raw material, extracts, and dietary supplements. Single-laboratory validation has been performed on the method to determine the repeatability, accuracy, selectivity, limit of detection/limit of quantification (LOQ), ruggedness, and linearity for p-synephrine and 5 other biogenic amines: octopamine, phenylephrine (m-synephrine), tyramine, N-methyltyramine, and hordenine, which may be present in bitter orange. p-Synephrine was found to be the primary biogenic amine present in all materials tested, accounting for >80% of the total biogenic amine content in all samples except a finished product. Repeatability precision for synephrine was between 1.48 and 3.55% RSD. Synephrine recovery was between 97.5 and 104%. The minor alkaloids were typically near the LOQ of the method (300-900 microg/g) in the test materials, and between-day precision for the minor compounds was poor because interferences could sometimes be mistakenly identified as one of the minor analytes. Recoveries of the minor components ranged from 99.1 to 103% at approximately 6000 microg/g spike level, to 90.7 to 120% at 300 microg/g spike level.

Determination of ephedra alkaloids in urine and plasma by HPLC-UV: collaborative study.

Ten collaborating laboratories determined the ephedra alkaloid content (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine) in 8 blind duplicates of human plasma and urine using high-performance liquid chromatography (HPLC) with UV detection. In addition to negative urine and plasma controls, urine samples were spiked with individual ephedra alkaloids ranging in concentration from about 1 to 5 microg/mL. Plasma samples were spiked with individual ephedra alkaloids ranging in concentration from about 100 to 400 ng/mL. Sample solutions were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. The ephedra alkaloids were not consistently detected in any of the spiked plasma samples. When ephedra alkaloids were detected in the plasma samples, reproducibility between blind replicate samples was very poor. Repeatability, reproducibility, and accuracy were also very poor for the spiked urine samples. On the basis of these results, the HPLC-UV method for the determination of ephedra alkaloids in human urine and plasma is not recommended for adoption as Official First Action.

Determination of ephedrine alkaloids in botanicals and dietary supplements by HPLC-UV: collaborative study.

An international collaborative study was conducted of a high-performance liquid chromatography (HPLC)-UV method for the determination of the major (ephedrine [EP] and pseudoephedrine [PS]) and minor (norephedrine [NE], norpseudoephedrine [NP], methylephedrine [ME], and methylpseudoephedrine [MP]) alkaloids in selected dietary supplements representative of the commercially available products. Ten collaborating laboratories determined the ephedrine-type alkaloid content in 8 blind replicate samples. Five products contained ephedra ground herb or ephedra extract. These 5 products included ground botanical raw material of Ephedra sinica, a common powdered extract of Ephedra sinica, a finished product containing only Ephedra sinica ground botanical raw material, a complex multicomponent dietary supplement containing Ma Huang, and a high-protein chocolate flavored drink mix containing Ma Huang extract. In addition, collaborating laboratories received a negative control and negative control spiked with ephedrine alkaloids at high and low levels for recovery studies. Test extracts were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. Repeatability relative standard deviations (RSDr) ranged from 0.64-3.0% for EP and 2.0-6.6% for PS, excluding the high protein drink mix. Reproducibility relative standard deviations (RSDR) ranged from 2.1-6.6% for EP and 9.0-11.4% for PS, excluding the high protein drink mix. Recoveries ranged from 84.7-87.2% for EP and 84.6-98.2% for PS. The data developed for the minor alkaloids are more variable with generally unsatisfactory HORRATS (i.e., >2). However, since these alkaloids generally add little to the total alkaloid content of the products, the method gives satisfactory results in measuring total alkaloid content (RSDr 0.85-3.13%; RSDR 2.03-10.97%, HORRAT 0.69-3.23, exclusive of the results from the high protein drink). On the basis of these results, the method is recommended for Official First Action for determination of EP and PS in dietary supplements exclusive of the high protein drinks.