Preparation and certification of standard reference material 3278 tocopherols in edible oils.

Standard Reference Material (SRM) 3278 Tocopherols in Edible Oils has been issued for use as a quality assurance tool in the measurement of tocopherols. Like other natural-matrix SRMs, this material can be used in method validation or in assignment of tocopherol values to in-house quality control materials. Because most edible oils contain one predominant tocopherol isoform, the SRM is a blend of sunflower, soy, canola, and safflower oils to provide roughly comparable chromatographic peak heights of the two main tocopherols, γ and α, with smaller amounts of δ and ß. The four tocopherol isoforms were determined by three independent liquid chromatography methods with absorbance and fluorescence detection. Various chromatographic and detection modes are used for assignment of certified values because biases inherent to one method should not be present in the other, and the existence of bias can therefore be identified.

Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials.

As part of a collaboration with the National Institutes of Health's Office of Dietary Supplements and the Food and Drug Administration's Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of beta-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, beta-carotene, and gamma-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, beta-carotene isomers, and delta-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements.

Mass spectrometric determination of the predominant adrenergic protoalkaloids in bitter orange (Citrus aurantium).

The predominant adrenergic protoalkaloid found in the peel and fruit of bitter orange, Citrus aurantium, is synephrine. Synephrine is reputed to have thermogenic properties and is used as a dietary supplement to enhance energy and promote weight loss. However, there exists some concern that the consumption of dietary supplements containing synephrine or similar protoalkaloids may contribute to adverse cardiovascular events. This study developed and validated a positive-ion mode liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of the major (synephrine) and minor (tyramine, N-methyltyramine, octopamine, and hordenine) adrenergic protoalkaloids in a suite of National Institute of Standards and Technology (NIST) bitter orange Standard Reference Materials (SRMs): SRM 3258 Bitter Orange Fruit, SRM 3259 Bitter Orange Extract, and SRM 3260 Bitter Orange Solid Oral Dosage Form. The limit of quantitation (LOQ) for all protoalkaloids is approximately 1 pg on-column, except for octopamine (20 pg on-column). Additionally, the method has a linear dynamic range of > or =3 orders of magnitude for all of the protoalkaloids. Individual, as well as "total", protoalkaloid levels (milligrams per kilogram) in the NIST SRMs were determined and compared to the levels measured by an independent liquid chromatography/fluorescence detection (LC/FD) method. Satisfactory concordance between the LC/MS/MS and LC/FD protoalkaloid measurements was demonstrated. LC/MS/MS analysis of the protoalkaloids in the SRMs resulted in mean measurement imprecision levels of < or =10% coefficient of variation (% CV).

Characterization of a suite of ginkgo-containing standard reference materials.

A suite of three ginkgo-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for flavonoid aglycones, ginkgolides, bilobalide, and selected toxic trace elements. The materials represent a range of matrices (i.e., plant, extract, and finished product) that provide different analytical challenges. The constituents have been determined by at least two independent analytical methods with measurements performed by NIST and at least one collaborating laboratory. The methods utilized different extractions, chromatographic separations, modes of detection, and approaches to quantitation. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

Determination of bitter orange alkaloids in dietary supplement Standard Reference Materials by liquid chromatography with atmospheric-pressure ionization mass spectrometry.

A liquid chromatographic atmospheric-pressure ionization electrospray mass spectrometry (LC-API-ES-MS) method has been developed for the determination of five bitter orange alkaloids (synephrine, octopamine, n-methyltyramine, tyramine, and hordenine) in bitter orange-containing dietary supplement standard reference materials (SRMs). The materials represent a variety of natural, extracted, and processed sample matrices. Two extraction techniques were evaluated: pressurized-fluid extraction (PFE) and sonication extraction. The influence of different solvents, extraction temperatures, and pH were investigated for a plant material and a processed sample. The LC method uses a new approach for the separation of highly polar alkaloids. A fluorinated, silica-based stationary phase separated the five alkaloids and the internal standard terbutaline in less than 20 min. This method enabled the determination of the dominant alkaloid synephrine and other minor alkaloids in a variety of dietary supplement SRMs.

Determination of Bitter Orange alkaloids in dietary supplements standard reference materials by liquid chromatography with ultraviolet absorbance and fluorescence detection.

Four adrenergic amines [synephrine, octopamine, tyramine, and n-methyltyramine] were determined in a variety of Bitter Orange containing dietary supplements. Two extraction techniques were evaluated in detail: Soxhlet extraction and sonication extraction. A liquid chromatographic separation using a reversed-phase C(18) stationary phase and the ion-pairing reagent sodium dodecyl sulfate was developed to separate the Bitter Orange alkaloids. Ultraviolet absorbance detection at 220 nm and fluorescence detection with excitation at 273 nm and emission at 304 nm were used for the alkaloid detection. The method described was used for the assignment of the levels of the predominant alkaloids in three candidate standard reference materials containing Bitter Orange.

Preparation and characterization of standard reference material 3276, carrot extract in oil.

The National Institute of Standards and Technology (NIST), the Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) and Center for Food Safety and Applied Nutrition (CFSAN), and the National Institutes of Health (NIH), Office of Dietary Supplements (ODS) are collaborating to produce a series of standard reference materials (SRMs) for dietary supplements. Standard reference material (SRM) 3276 Carrot Extract in Oil is one in this series, with values assigned for trans-alpha-carotene, trans- and total beta-carotene, delta- and gamma-tocopherol, and twelve fatty acids. Results for carotenoids and tocopherols were obtained by use of combinations of liquid chromatography (LC), on columns of differing selectivity, with absorbance and mass spectrometric (MS) detection. Fluorescence detection also was used for the tocopherols. Results for fatty acids were obtained by use of gas chromatography (GC) with both flame-ionization and mass-spectrometric detection. This material is intended for use as a primary control material when assigning values to in-house (secondary) control materials and for validation of analytical methods for measurement of these analytes in similar matrices.

Application of polymer based stationary phases in high performance liquid chromatography and capillary high performance liquid chromatography hyphenated to microcoil 1H nuclear magnetic resonance spectroscopy.

The increased demand for chromatographic materials that are able to achieve a fast separation of large quantities of structure analogues is a great challenge. It is known that polymer based chromatographic materials have a higher loadability, compared to silica based sorbents. Unfortunately these polymer materials cannot be used under high pressure which is necessary in order to obtain high flow rates, and hence long times are needed to perform a separation. However, by immobilizing a polymer on a mechanically stable porous silica core, this problem can be circumvented and higher flows become feasible on these materials. Especially for capillary liquid chromatography hyphenated with nuclear magnetic resonance a high loadability is of great importance in order to obtain sharp, resolved, and concentrated peaks thus resulting in a good signal to noise ratio in the NMR experiment. Therefore, a highly shape selective chromatographic sorbent was developed by covalently immobilizing a poly(ethylene-co-acrylic) acid copolymer (-CH(2)CH(2)-)(x)[CH(2)CH(CO(2)H)-](y) (x=119, y=2.4) with a mass fraction of acrylic acid of 5% as stationary phase on silica via a spacer molecule (3-glycidoxypropyltrimethoxysilane). First, the loadability of this sorbent compared to C(30) is demonstrated by the HPLC separation of two xanthophyll isomers. Subsequently, it has been successfully employed in the hyphenation of capillary HPLC with microcoil (1)H NMR spectroscopy by separating and identifying a highly concentrated solution of the tocopherol homologues.

Structure elucidation of deoxylutein II isomers by on-line capillary high performance liquid chromatography-(1)H nuclear magnetic resonance spectroscopy.

Thermal and iodine-catalyzed photochemical (Z/E)-isomerization of deoxylutein II [(3R,6'R)-3-hydroxy-3',4'-didehydro-beta,gamma-carotene, anhydrolutein I] (2), the dehydration product of lutein [(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol] (4), yielded multi-component mixtures of (Z)-isomers. By I(2)-catalyzed photoisomerization, (9Z)-2, (9'Z)-2, (13Z)-2, (13'Z)-2 and (15Z)-2 are generated as main products. In addition, this thermodynamic-equilibrium mixture contains traces of (9Z,9'Z)-2 and other (di-Z)-isomers in minor concentrations. Thirteen isomers are chromatographically separated and detected on-line by UV-vis and mass spectrometry. (all-E)-Deoxylutein II (2) and six of its (Z)-configured isomers are separated by capillary HPLC (acetone-d(6)/D(2)O = 85:15) and detected on-line by (1)H NMR spectroscopy in a microprobe. With the microprobe and the active detection volume of 1.5 microl, it is possible to perform structure elucidation with very small amounts available for various (Z)-isomers of deoxylutein II (2) in the isomerization mixture.

Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana).

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.

Hyphenation of capillary high-performance liquid chromatography to microcoil magnetic resonance spectroscopy--determination of various carotenoids in a small-sized spinach sample.

The development of miniaturized hyphenated systems such as capillary high-performance liquid chromatography--and nuclear magnetic resonance spectroscopy (HPLC-NMR) remains challenging in the field of structure elucidation. In combination with a highly specific sample preparation technique, matrix solid-phase dispersion (MSPD), and a highly selective C30 reverse phase HPLC-NMR enables the identification of small amounts of natural compounds. Here, the investigation of five carotenoids in a standard solution and two carotenoids from a spinach sample demonstrate the potential of this new development. The separation of the carotenoids is performed with self-packed fused-silica capillaries with a binary solvent gradient consisting of acetone and water. The miniaturized system allows the use of fully deuterated solvents for on-line HPLC-NMR coupling. The 1H NMR spectra of the various carotenoids obtained in stopped-flow mode gave a high signal-to-noise ratio with a sample amount in the low nanogram range. All necessary parameters for structure elucidation such as multiplet structure, coupling constants and integration values can be detected unambiguously.

Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus.

Most Staphylococcus aureus strains produce the orange carotenoid staphyloxanthin. The staphyloxanthin biosynthesis genes are organized in an operon, crtOPQMN, with a sigma(B)-dependent promoter upstream of crtO and a termination region downstream of crtN. The functions of the five encoded enzymes were predicted on the basis of their sequence similarity to known enzymes and by product analysis of gene deletion mutants. The first step in staphyloxanthin biosynthesis is the head-to-head condensation of two molecules of farnesyl diphosphate to form dehydrosqualene (4,4'-diapophytoene), catalyzed by the dehydrosqualene synthase CrtM. The dehydrosqualene desaturase CrtN dehydrogenates dehydrosqualene to form the yellow, main intermediate 4,4'-diaponeurosporene. CrtP, very likely a mixed function oxidase, oxidizes the terminal methyl group of 4,4'-diaponeurosporene to form 4,4'-diaponeurosporenic acid. CrtQ, a glycosyltransferase, esterifies glucose at the C(1)'' position with the carboxyl group of 4,4'-diaponeurosporenic acid to yield glycosyl 4,4'-diaponeurosporenoate; this compound was the major product in the clone expressing crtPQMN. In the final step, the acyltransferase CrtO esterifies glucose at the C(6)'' position with the carboxyl group of 12-methyltetradecanoic acid to yield staphyloxanthin. Staphyloxanthin overexpressed in Staphylococcus carnosus (pTX-crtOPQMN) and purified was analyzed by high pressure liquid chromatography-mass spectroscopy and NMR spectroscopy. Staphyloxanthin was identified as beta-D-glucopyranosyl 1-O-(4,4'-diaponeurosporen-4-oate)-6-O-(12-methyltetradecanoate).

Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP-70 using microprobe NMR and on-line coupled capillary HPLC-NMR.

The protein kinase ZAP-70 is involved in T-cell activation and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485-496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y-492 and Y-493 tyrosines were specifically synthesized and analyzed by (1)H/(13)C-NMR at 600 MHz using a capillary HPLC-NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 microl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 microg of the analyte were used (corresponding to ca 2 mg in classical 5-mm tubes). Capillary HPLC-NMR spectra were recorded in the stopped-flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ((1)H,(1)H-COSY-90, (1)H/(13)C-HSQC, and (1)H/(13)C-HMBC).

Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by HPLC-MS and 1H NMR.

The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D(2)O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (all-E)-Lutein and (all-E)-beta-carotene in spinach showed partial deuteration from (2)H(1) to (2)H(12), with the abundance maximum at (2)H(5). (all-E)-beta-Carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from (2)H(1) to (2)H(17), with the abundance maximum at (2)H(11). The (1)H NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.

Hyphenation of capillary HPLC to microcoil (1)H NMR spectroscopy for the determination of tocopherol homologues.

Highly selective reversed phases (C(30) phases) are self-packed in 250 microm inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC- NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 microg of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow (1)H NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D (1)H,(1)H COSY NMR spectrum of alpha-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.