Fatty Acids of Ten Commonly Consumed Pulses.

Gas chromatography (GC) with flame ionization detection (FID) and mass spectrometry (MS) detection were used to characterize the fatty acid (FA) compositions of ten commonly consumed (i.e., market class) pulses. Lipids from ground pulses were extracted using a classical chloroform/methanol extraction and quantified by GC-FID with structural confirmation by GC-MS. Principal component analysis (PCA) was applied to FA compositions of the pulse extracts, and the pulses clustered into three distinct groups: one rich in linolenic acid, 18:3 (carbon number:unsaturation, C:U), one rich in 16:0, and one in which 18:1 was highest, along with predominant 18:2. These ten pulses averaged 46.1% linoleic acid (18:2), 22.7% oleic acid (18:1), 18.0% palmitic acid (16:0), and 7.6% linolenic acid (18:3). Individual values ranged widely, with 18:2 ranging from 26.0% in black beans to 48.4% in garbanzo beans. The greatest difference was in 18:3, which ranged from 2.2% in garbanzo beans to 38.8% in pinto beans. Oxo-FA were observed in all ten samples, and the distribution of oxo-FA in the samples also varied. Overall, the very different FA compositions of pulses lead to the possibility of breeding and genetic modification between pulses to produce the most desirable FA composition for nutritional benefit.

The Updated Bottom Up Solution applied to mass spectrometry of soybean oil in a dietary supplement gelcap.

Among the goals of lipidomics applied to triacylglycerols (TAGs) is identification of molecular species, degree and location of unsaturation, and positions of fatty acyl chains (i.e., identification of regioisomers). Toward those ends, we define one, two, and three "Critical Ratios" for Types I, II, and III TAGs that provided different aspects of the desired information. Critical Ratio 1, [MH](+)/Σ[DAG](+), is correlated to the degree of unsaturation ([MH](+) is the protonated molecule and Σ[DAG](+) is the sum of diacylglycerol-like ions, [DAG](+)); Critical Ratio 2, [AA](+)/[AB](+) for Type II TAGs ("ABA/AAB/BAA") and [AC](+)/([AB](+)+[BC](+)) for Type III TAGs ("ABC/CBA/BAC/CAB/ACB/BCA"), is correlated to identification of regioisomers; and Critical Ratio 3, [BC](+)/[AB](+), provides information about those [DAG](+) from Type III TAGs. Furthermore, Critical Ratios are used in the Updated Bottom Up Solution (UBUS) to reproduce the mass spectra of TAGs by atmospheric pressure chemical ionization mass spectrometry applied to analysis of soybean oil in a dietary supplement gelcap. We present a new model for the [MH](+)/Σ[DAG](+) ratio, quantify regioisomers using the [AA](+)/[AB](+) ratio, and describe trends for [BC](+)/[AB](+) that have never been reported before. The UBUS is also applied to other classes of molecules, i.e., vitamin D and DAGs. The amount of vitamin D3 in the gelcap fell from 2011 ± 22 when received to 1689 ± 33 just prior to expiration. The Critical Ratios constitute a compact data set that can provide structural information and also act as a library of mass spectra.

Extract-filter-shoot liquid chromatography with mass spectrometry for the analysis of vitamin D2 in a powdered supplement capsule and standard reference material 3280.

An "extract-filter-shoot" method for the analysis of vitamin D2, ergocalciferol, in a dry powdered dietary supplement capsule containing rice flour excipient and in a National Institute of Standards and Technology standard reference material 3280 is reported. Quantification of vitamin D2 was done by atmospheric pressure chemical ionization mass spectrometry using selected ion monitoring, two transitions of selected reaction monitoring, and extracted ion chromatograms from full scans. UV detection was used for the quantification of Vitamin D2 in the dry powder capsule, whereas interfering species rendered UV detection unreliable for standard reference material 3280. Average values for standard reference material 3280 ranged from 8.27 ± 0.58 to 8.33 ± 0.57 µg/g using internal standard calibration and response factor approaches, compared to the previous National Institute of Standards and Technology internal value for vitamin D2 of 8.78 ± 0.11 µg/g, and the recently updated reference value of 8.6 ± 2.6 µg/g. The powdered supplement capsule was found to contain 28.19 ± 0.35 to 28.67 ± 0.90 µg/capsule for a capsule labeled to contain 25.00 µg. The triacylglycerol composition of the rice flour excipient in the powdered supplement capsule determined by atmospheric pressure chemical ionization mass spectrometry is also reported.

Three-dimensional liquid chromatography with parallel second dimensions and quadruple parallel mass spectrometry for adult/infant formula analysis.

Three dimensions of chromatographic separation, using split-flow two-dimensional liquid chromatography (SF-2D-LC) with two parallel second dimensions, LC × 2LC, combined with quadruple parallel mass spectrometry (LC3MS4) is demonstrated for analysis of NIST SRM 1849a adult/infant formula. The first dimension, 1D, was a conventional non-aqueous reversed-phase (NARP) HPLC separation using two C18 columns in series, followed by detection using an ultraviolet (UV) detector, a fluorescence detector (FLD), with flow then split to a corona charged aerosol detector (CAD), and then dual parallel mass spectrometry (MS), conducted in atmospheric pressure photoionization (APPI) and electrospray ionization (ESI) modes. The first second dimension, 2D(1), UHPLC was conducted on a 50.0 mm C30 column using a NARP-UHPLC parallel gradient for separation of short-chain triacylglycerols (TAGs) from long-chain TAGs, with detection by UV and ESI-MS. The second dimension, 2D(2), UHPLC was conducted using a 100.0 mm C30 column with a NARP-UHPLC parallel gradient for improved separation of TAG isomers, with detection by UV, an evaporative light scattering detector, and high-resolution, accurate-mass (HRAM) ESI-MS. Transferred eluent dilution was used to refocus peaks and keep them sharp during elution in both 2Ds. The separation space in the 2D(2) was optimized using multi-cycle (aka, "constructive wraparound") elution, which employed flow rate programming. In the 1D, calibration lines for quantification of fat-soluble vitamins were constructed. A lipidomics approach to TAG identification and quantification by HRAM-ESI-MS was applied to the 2D(2). These experiments can be represented: LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4, or three-dimensional liquid chromatography with quadruple parallel mass spectrometry.

"Dilute-and-shoot" triple parallel mass spectrometry method for analysis of vitamin D and triacylglycerols in dietary supplements.

A method is demonstrated for analysis of vitamin D fortified dietary supplements that eliminates virtually all chemical pretreatment prior to analysis, which is referred to as a "dilute-and-shoot" method. Three mass spectrometers, in parallel, plus a UV detector, an evaporative light-scattering detector (ELSD), and a corona charged aerosol detector (CAD) were used to allow a comparison of six detectors simultaneously. Ultraviolet data were analyzed using internal standard, external standard, and response factor approaches. The contents of gelcaps that contained 2,000 IU (50 µg) vitamin D(3) in rice bran oil, diluted to 100 mL, were analyzed without the need for lengthy saponification and extraction. Vitamin D(3) was analyzed using UV detection, extracted ion chromatograms, selected ion monitoring (SIM) atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and two transitions of multiple reaction monitoring (MRM) APCI-MS. The internal standard, external standard, and response factor methods gave values of 0.5870 ± 0.0045, 0.5893 ± 0.0041, and 0.5889 ± 0.0045 µg/mL, respectively, by UV detection. The values obtained by MS were 0.6117 ± 0.0140, 0.6018 ± 0.0244, and 0.5848 ± 0.0146 µg/mL by SIM and two transitions of MRM, respectively. The triacylglycerols in the oils were analyzed using full-scan APCI-MS, electrospray ionization (ESI) MS, up to MS(4), an ELSD, and a CAD. The method proved to be very sensitive for vitamin D(3), as well as triacylglycerols (TAGs), allowing identification of intact TAGs containing fatty acids up to 28 carbons in length. LC-ESI-MS of glycerin polymers is also demonstrated.

Timed relay contact closure controlled system for parallel second dimensions in multi-dimensional liquid chromatography.

OBJECTIVE: Short-chain triacylglycerols (TAGs) in lipid extracts of biological samples are not sufficiently resolved using conventional reversed-phase separation on two C18 columns in series, or using a two-dimensional chromatographic separation with a silver ion column as the second dimension (2D). An additional dimension of separation was required. RESULTS: The hardware and software components to allow a second second-dimension (2D) separation and three total separation dimensions were developed. Two contact closure (CC) activated 4-port, 2-position valves (4P2PVs) for ultra-high performance liquid chromatography (UHPLC) were joined together and used for one of two second dimensions in comprehensive two-dimensional liquid chromatography (2D-LC) coupled to four mass spectrometers simultaneously in parallel in an LC1MS2 × (LC1MS1 + LC1MS1) = LC3MS4 configuration. A timed contact closure circuit (TCCC) controlled the two UHPLC valves, operated by repetitive CCs for the 4P2PVs. The TCCC-controlled 4P2PVs were used to direct a portion of the 1D eluent to one of the two 2D's for separation by a quaternary UHPLC system that was not allowed by the commercial 2D-LC system. The 1D separation was a non-aqueous reversed-phase HPLC instrument used for separation of TAGs; the commercial 2D-LC 2D binary UHPLC was used for silver-ion chromatography of unsaturated TAGs; and the CC-controlled second 2D was used for separation of short-chain (SC) saturated TAGs.

Dual parallel liquid chromatography with dual mass spectrometry (LC2/MS2) for a total lipid analysis.

Total lipid extracts containing both polar and non-polar lipids were separated using two liquid chromatographic systems joined together using a column-switching valve. Detection was accomplished using mass spectrometers attached to each of the two liquid chromatographic systems, for a dual liquid chromatography/dual mass spectrometry (LC2/MS2) arrangement. This experimental approach is demonstrated to be useful to identify many classes of polar and non-polar lipids, and numerous individual molecular species within each class, which is a primary goal of lipidomics. Data are presented from a bovine brain total lipid extract and a sand bream fillet extract as examples. The use of both ESI-MS and APCI-MS are demonstrated for analysis of non-polar lipids, and advantages and shortcomings of each technique are discussed. The two parallel chromatographic separations are compared to lipidomics approaches that employ infusion without chromatography. The data show that the LC2/MS2 is one approach to a comprehensive total lipid analysis that can be performed using only commercially available instruments with no fabrication or modification required. The LC2/MS2 approach represents a uniquely valuable tool for monitoring the amounts of precursors and products of molecules involved in cell signaling processes. The dual liquid chromatography/dual mass spectrometry approach represents one of the most powerful tools for lipidomics analysis reported to date.

Liquid chromatography with dual parallel mass spectrometry and (31)P nuclear magnetic resonance spectroscopy for analysis of sphingomyelin and dihydrosphingomyelin. I. Bovine brain and chicken egg yolk.

Liquid chromatography coupled to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) mass spectrometry (MS), in parallel, was used for detection of bovine brain and chicken egg sphingolipids (SLs). APCI-MS mass spectra exhibited mostly ceramide-like fragment ions, [Cer-H(2)O+H](+) and [Cer-2H(2)O+H](+), whereas ESI-MS produced mostly intact protonated molecules, [M+H](+). APCI-MS/MS and MS(3) were used to differentiate between isobaric SLs. APCI-MS/MS mass spectra exhibited long-chain base related fragments, [LCB](+) and [LCB-H(2)O](+), that allowed the sphinganine backbone to be differentiated from the sphingenine backbone. Fragments formed from the fatty amide chain, [FA(long)](+) and [FA(short)](+), allowed an overall fatty acid composition to be determined. The presence of both dihydrosphingomyelin (DSM) and sphingomyelin (SM) sphingolipid classes was confirmed using (31)P NMR spectroscopy.

The Simulacrum System as a Construct for Mass Spectrometry of Triacylglycerols and Others.

A construct called a simulacrum is defined that provides all possible solutions to a sum of two mass spectral abundances, based on values (abundances) or ratios of those values. The defined construct is applied to atmospheric pressure chemical ionization mass spectrometry (MS) of triacylglycerols (TAG). A simulacrum has precisely defined components, specifically a simulacrum sum, four Possibilities to Observe, two Cases, and eight solutions. A simulacrum with no restrictions is the First General Form of a Simulacrum. When one value is specified to be 1 (as in MS), the construct is called a Unit Simulacrum, also called the First Specified Form of a Simulacrum. When one value is 1 and no value can be greater than 1 (the two specifications dictated by mass spectrometry), the construct is called the Second Specified Form of a Simulacrum, or the Mass Spectrometry Simulacrum. Simulacra are used with three Critical Ratios calculated from raw abundances in mass spectra of TAG to provide structural information about the degree of unsaturation in TAG, the identity and quantity of regioisomers, and other structural characteristics. Three-level-deep nested simulacrum solutions yield the recently reported Updated Bottom Up Solution, from which the protonated molecule, [MH](+), and all diacylglycerol-like fragments, [DAG](+), of TAG can be reproduced from the Critical Ratios. Thus, the simulacrum solutions constitute a reduced data set in which more information is provided in fewer values than raw abundances, such that the Critical Ratios constitute a compact library of mass spectra.

The bottom-up solution to the triacylglycerol lipidome using atmospheric pressure chemical ionization mass spectrometry.

Presented here is an approach to representing the data from atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG) using a set of one, two, or three Critical Ratios. These Critical Ratios may be used directly to provide structural information concerning the regioisomeric composition of the triacylglycerols (TAG), and about the degree of unsaturation in the TAG. An AAA-type, or Type I, TAG has only one Critical Ratio, the ratio of the protonated molecule, [M + H]+, to the DAG fragment ion, [AA]+. The Critical Ratio for a Type I TAG is [MH]+/Z[DAG]+, and the mass spectrum of a Type I TAG can be reproduced from only this one ratio. An ABA/AAB/BAA, or Type II, TAG has two Critical Ratios, the [MH]+/sigma[DAG]+ ratio and the [AA]+/[AB]+ ratio. The [AA]+/[AB]+ ratio for a single TAG or TAG mixture can be compared with the [AA]+/[AB]+ ratios of pure regioisomeric standards, and the percentage of each regioisomer can be estimated. The abundance of the protonated molecule and the abundances of the two [DAG]+ fragment ions can be calculated from the two Critical Ratios for a Type II TAG. To calculate the abundances, the Critical Ratios are processed through the Bottom-Up Solution to the TAG lipidome. First, Critical Limits are calculated from the Critical Ratios, and then the Critical Ratios are classified into Cases by comparison with the Critical Limits. Once the Case classification is known, the equation for the abundance of each ion in the mass spectrum is given by the Bottom-Up Solution. A Type III TAG has three different FA and three Critical Ratios. The [MH]+/Z[DAG]+ ratio is the first Critical Ratio, the [AC]+/([AB]+ + [BC]+) ratio is the second Critical Ratio, and the [BC]+/[AB]+ ratio is the third Critical Ratio. The second critical ratio for a Type III TAG can be compared with regioisomeric standards to provide an estimate of the percentage composition of the regioisomers. The three Critical Ratios for a Type III TAG can be processed through the Bottom-Up Solution to calculate the four ion abundances that make up the APCI-MS mass spectrum. The Critical Ratios constitute a reduced data set that provides more information in fewer values than the raw abundances.

Construction of a wireless communication contact closure system for liquid chromatography with multiple parallel mass spectrometers and other detectors.

A contact closure system was constructed that uses two contact closure sender boards that communicate wirelessly to four contact closure receiver boards to distribute start signals from two or three liquid chromatographs to 14 instruments, pumps, detectors, or other components. Default high, closed low, TTL logic (5-volt) start signals from two autosamplers are converted to simple contacts by powered relay boards that are then connected to two 16-channel wireless contact closure sender boards. The contact closure signals from the two sender boards are transmitted wirelessly to two pairs of eight-channel receiver boards (total of 32 contact signals) that distribute the start signal to 14 switches that allow selection of which start signal is sent to which instrument, pump, or detector. The contact closure system is used for quadruple parallel mass spectrometry experiments in which four mass spectrometers, using three different atmospheric pressure ionization modes, plus a UV detector, an evaporative light-scattering detector, a corona charged aerosol detector, and two syringe pumps supplying electrolyte, are all synchronized to start simultaneously. A wide variety of liquid chromatography-mass spectrometry experiments using multiple liquid chromatographs and mass spectrometers simultaneously, LCx/MSy, including column-switching experiments, can be reconfigured simply by toggling switches.

Effects of UV-B Radiation Levels on Concentrations of Phytosterols, Ergothioneine, and Polyphenolic Compounds in Mushroom Powders Used As Dietary Supplements.

Compositional changes of powder dietary supplements made from mushrooms exposed to different levels of UV-B irradiation were evaluated for the bioactive naturally occurring mushroom antioxidant, ergothioneine; other natural polyphenolic compounds, e.g., flavonoids, lignans, etc.; and selected phytosterols. Four types of mushroom powder consisting of white, brown (Agaricus bisporus), oyster (Pleurotus ostreatus), and shiitake (Lentinula edodes) mushrooms from three different treatment groups (control, low and high UV-B exposures) were evaluated. Ergothioneine concentrations found in mushroom powders were 0.4-10.4 mg/g dry weight (dw) and were not appreciably affected by UV-B radiation. No individual polyphenols were detected above 0.1 µg/g. Phytosterols ergosterol (2.4-6.2 mg/g dw) and campesterol (14-43 µg/g dw) were measured in mushroom powder samples. Ergosterol concentrations decreased significantly with the increased level of UV-B treatment for all mushroom powder types, except for white. These results provide some new information on effects of UV-B radiation on these important natural bioactive compounds in mushrooms.

Quadruple parallel mass spectrometry for analysis of vitamin D and triacylglycerols in a dietary supplement.

A "dilute-and-shoot" method for vitamin D and triacylglycerols is demonstrated that employed four mass spectrometers, operating in different ionization modes, for a "quadruple parallel mass spectrometry" analysis, plus three other detectors, for seven detectors overall. Sets of five samples of dietary supplement gelcaps labeled to contain 25.0µg (1000 International Units, IU) vitamin D3 in olive oil were diluted to 100mL and analyzed in triplicate by atmospheric pressure chemical ionization (APCI) mass spectrometry (MS), atmospheric pressure photoionization (APPI) MS and electrospray ionization (ESI) MS, along with an ultraviolet (UV) detector, corona charged aerosol detector (CAD), and an evaporative light scattering detector (ELSD), simultaneously in parallel. UV detection allowed calculation by internal standard (IS), external standard (ES), and response factor (RF) approaches, which gave values of 0.2861±0.0044, 0.2870±0.0059, and 0.2857±0.0042µg/mL, respectively, which were not statistically significantly different. This indicated an average amount of vitamin D3 of 14.5% over the label amount. APCI-MS analysis by selected ion monitoring (SIM) and two transitions of selected reaction monitoring (SRM) provided values of 0.2849±0.0055, 0.2885±0.0090, and 0.2939±0.0097µg/mL, respectively, relative to vitamin D2 as the IS. The triacylglycerol (TAG) composition was determined by APCI-MS, APPI-MS and ESI-MS, and the fatty acid (FA) compositions calculated from the TAG compositions were compared to the FA composition determined by gas chromatography (GC) with flame ionization detection (FID) of the FA methyl esters (FAME). APCI-MS provided the FA composition closest to that determined by GC-FID of the FAME. A previously reported approach to TAG response factor calculation was employed, which brought all TAG compositions into good agreement with each other, and the calculated FA compositions into excellent agreement with the FA composition determined from GC-FID of the FAME.

Dual parallel mass spectrometry for lipid and vitamin D analysis.

There are numerous options for mass spectrometric analysis of lipids, including different types of ionization, and a wide variety of experiments using different scan modes that can be conducted. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) provide complementary types of information that are both desirable. However, the duty cycle of the mass spectrometer places limits on the number of experiments that can be performed, and instruments usually employ only one type of ionization at a time. This work describes the approaches we have used that employ two mass spectrometers in parallel or in a column-switching configuration that allows multiple ionization modes and types of experiments to be conducted simultaneously during a single chromatographic run. These data demonstrate how use of two systems can reduce or eliminate the need for repeat injections and repetitive experiments. Approaches are described that employ two mass spectrometers connected in parallel as detectors for a single chromatographic system (LC1/MS2) or that employ two liquid chromatographs and two mass spectrometers in a column-switching arrangement (LC2/MS2). Examples of LC1/MS2 analyses of triacylglycerols (TAGs), sphingolipids, and vitamin D are given, as well as an example of an LC2/MS2 experiment that is used to perform analysis of both polar and non-polar lipids in a total lipid extract.