Standard operating procedure for the collection and preparation of voucher plant specimens for use in the nutraceutical industry

A vital part of the development of any standardized protocol for the extraction of plant-derived crude extracts to be used in herbal medicine or nutritional supplementation is proper documentation of the original botanical source of the extract via acquisition of a voucher specimen. The purpose of this document is to serve as an accepted protocol for voucher specimen collection, handling, and storage, with specific guidelines to address commercial and research uses.     

Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

Measuring vitamins and minerals in dietary supplements for nutrition studies in the USA

This article illustrates the importance of having analytical data on the vitamin and mineral contents of dietary supplements in nutrition studies, and describes efforts to develop an analytically validated dietary supplement ingredient database (DSID) by a consortium of federal agencies in the USA. Preliminary studies of multivitamin mineral supplements marketed in the USA that were analyzed as candidates for the DSID are summarized. Challenges are summarized, possible future directions are outlined, and some related programs at the Office of Dietary Supplements, National Institutes of Health are described. The DSID should be helpful to researchers in assessing relationships between intakes of vitamins and minerals and health outcomes.     

Liquid‐phase microextraction combined with liquid chromatography‐mass spectrometry. Extraction from small volumes of biological samples

Liquid‐phase microextraction (LPME) is a sample preparation technique based on disposable polypropylene hollow fibres, which results in efficient sample clean‐up and high preconcentration. The present paper describes the combination of LPME with LC‐MS utilising electrospray ionisation for high sensitivity. Nine antidepressant drugs were extracted from 50 or 500 μL of plasma or whole blood samples, through a thin layer of dodecyl acetate immobilised in the pores of the hollow fibre, and into 15 μL of 200 mM formic acid as acceptor solution inside the hollow fibre. Analyte recoveries in the range 12–68% and 9–52% were obtained from 50 μL of plasma and whole blood respectively. The acceptor solution (15 μL) was diluted with 60 μL of 5 mM ammonium formate pH = 2.7 prior to injection into the LC‐MS system. The system was qualitatively investigated for matrix effects utilising a post‐column infusion system. Whole blood from 5 different persons was cleaned‐up by LPME and injected onto the analytical column while a solution of the 9 model compounds was continuously infused post‐column. No signs of ion suppression were seen for any of the model compounds. Limits of quantification (S/N = 10) were in the low ng/mL range for 6 of the 9 model compounds utilising a whole blood sample volume of only 50 μL. The repeatability of the extractions was investigated utilising paroxetine as internal standard. Acceptable RSDs (%) were obtained (< 20%) for 5 of the antidepressants. By increasing the sample volume from 50 to 500 μL of whole blood RSDs below 20% (3–16%) were observed for all 8 antidepressants.     

An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins

Matrix-assissted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses of complete proteolytic digests are often hampered by contaminations and the complexity of the sample. This results in suppression effects and the formation of adducts which are difficult to assign, thus leading to low scores in database searches. In particular, signals of post-translationally modified peptides such as glycopeptides are often of low intensity or completely suppressed. Online liquid chromatography electrospray ionization mass spectrometry (ESI-MS) can, in part, overcome this problem, but the analytes are completely consumed during the run. Coupling of nano-flow HPLC (nano-LC), microfractionation and MALDI-TOF-MS combines separation and high-sensitivity UV detection with the possibility of collecting fractionated peptides and preserving the sample for detailed mass spectrometric analyses. Here we report on an optimized protocol for nano-LC-MALDI-TOF-MS analyses of glycoproteins. This protocol improves spectral quality, resulting in better protein identification scores in database searches. Furthermore, post-translationally modified peptides could be detected with higher sensitivity by changing the experimental conditions, allowing assignment, localization and characterization of the respective carbohydrate substituents.     

On-line identification of sugarcane (Saccharum officinarum L.) methoxyflavones by liquid chromatography-UV detection using post-column derivatization and liquid chromatography-mass spectrometry

Sugarcane (Saccharum officinarum L., Gramineae) bagasse and leaves were investigated for their flavonoid content and transgenic sugarcane ("Bowman-Birk" and "Kunitz") was compared with non-modified ("control") plants. Analyses were carried out by high-performance liquid chromatography coupled to diode array UV detection (LC/UV), also using post-column addition of shift reagents, and tandem MS (atmospheric pressure chemical ionization-MS/MS and collision-induced dissociation-MS). On-line UV and MS data demonstrated the presence of methoxyflavone glycosides and aglycones in a total of seven compounds. Three naturally occurring flavones glycosides and two unusual erythro- and threo-diastereoisomeric flavolignan 7-O-glucosides were identified together with their aglycones.     

Separation and Determination of Ephedrine Enantiomers and Synephrine by High Performance Capillary Electrophoresis in Dietary Supplements

The chiral separation of ephedrine alkaloids by high performance capillary electrophoresis is of great interest since the enantiomers exhibit quantitative and qualitative differences in pharmacological activity. The isomers of (–)-ephedrine, (+)-pseudoephedrine, (–)-N-methylephedrine, (+)-N-methylpseudoephedrine, (+)-norpseudoephedrine and (–)-norephedrine are the major bioactive components of E. sinica (Ma-Huang) which is a Chinese herb used for weight loss and as an energy booster in the US. However, the compounds stereoisomers are not present in the plant material. The electrophoretic separation was performed using a 110 cm × 50 m I.D. (101.5 cm effective length) fused silica capillary. The samples were injected by pressure for 5 s at 50 mbar and the running voltage was 30 kV at the injector end of the capillary. Within 23 min, nine ephedrine compounds and synephrine were separated at 210 nm. The method was successively applied to the determination of the ephedrine compounds in dietary supplement products. Parameters affecting the resolution between (+) and (–)-enantiomers, such as pH, cyclodextrin concentration, temperature, organic modifier, buffer concentration and capillary dimensions were reported.     

Standard Reference Materials to support US regulations for nutrients and contaminants in food and dietary supplements

To address the measurement and standard needs of the food and nutrition communities, the National Institute of Standards and Technology (NIST) has developed a suite of food-matrix Standard Reference Materials (SRMs) characterized for nutrient concentrations. These food-matrix SRMs include infant formula, baby food, and typical diet composites; meat homogenate, oyster, mussel, and fish tissues; baking chocolate; peanut butter; and spinach. Many of these materials were developed based on recommendations of the food industry to populate a nine-sectored triangle, developed by the Association of Analytical Communities (AOAC) International, in which foods are positioned based on their fat, protein, and carbohydrate contents. Value assignment of proximates, vitamins, and elements of nutritional interest in these food-matrix SRMs has been based primarily on the combination of results from measurements at NIST and from a group of collaborating laboratories involved in food measurements. Food-matrix SRMs are now available that are representative of all nine sectors of the AOAC International food-matrix triangle. Current activities are focused on the development of SRMs for dietary supplements including botanical and multivitamin/multielement materials.Presented at the CCQM Workshop on Comparability and Traceability in Food Analysis, 18–19 November 2003, BIPM, Sèvres, France.     

Rapid surface plasmon resonance immunobiosensor assay for microcystin toxins in blue-green algae food supplements

A surface plasmon resonance (SPR) immunobiosensor assay was developed and validated to detect microcystin toxins in Spirulina and Aphanizomenon flos-aquae blue-green algae (BGA) food supplements. A competitive inhibition SPR-biosensor was developed using a monoclonal antibody to detect microcystin (MC) toxins. Powdered BGA samples were extracted with an aqueous methanolic solution, centrifuged and diluted in HBS-EP buffer prior to analysis. The assay was validated in accordance with the performance criteria outlined in EU legislation 2002/657/EC. The limit of detection (LOD) of the assay was calculated from the analysis of 20 known negative BGA samples to be 0.561 mg kg⁻¹. The detection capability (CCβ) of the assay was determined to be ≤0.85 mg kg⁻¹ for MC-LR. The biosensor assay was successfully applied to detect MC-LR toxins in BGA samples purchased on the Irish retail market. MC-LR was detected in samples at levels ranging from <0.5 to 2.21 mg kg⁻¹. The biosensor results were in good agreement with an established LC-MS/MS assay. The assay is advantageous because it employs a simple clean-up procedure compared to chemical assays and allows automated unattended analysis of samples unlike ELISA.     

Capillary electrophoresis determination of carnitine in food supplements

L-Carnitine is a substance natural for human body which transfers fatty acids to the place of burning-mitochondria and aids the transformation of fats into energy and this way supports overweight reduction and immediate physical performance, increases resistance from physical load and protect heart from overload. In this study are described newly developed electrophoretic methods (ITP, CZE with direct and/or indirect UV detection) for carnitine determination in various samples. The results were compared with results obtained by validated HPLC method. All of these methods gave comparable results. The detection limits of the electrophoretic methods were between 2.4 and 4.7 microg/ml, reproducibility (relative standard deviation, RSD%) was between 1.2 and 4.4% and recoveries were between 91 and 113% in different samples. The shorter analysis and low running cost are the main advantages of CE methods.