High-performance Liquid Chromatography Nuclear Magnetic Resonance Research Articles

Quantification of Mixtures of C2 and C3 Hydroxy Acids and Products of Glycerol Oxidation by High‐Performance Liquid Chromatography and Quantitative 13C NMR

AbstractThe aim of this study was to develop an improved general method for detecting and quantifying mixtures of hydroxy acids and other products of glycerol oxidation in aqueous media, to prevent the confusions that can occur due to similarities and interactions between these compounds depending on media conditions. Standard potential products of glycerol oxidation—glycerol, glyceraldehyde, dihydroxyacetone, glyceric acid, lactic acid, glycolic acid, glyoxylic acid, oxalic acid, tartronic acid, and mesoxalic acid—were analyzed by high‐performance liquid chromatography (HPLC) and quantitative 13C nuclear magnetic resonance (NMR), in mixtures of known composition. The results obtained were concordant with the known compositions tested. HPLC was more accurate than quantitative 13C NMR for simple mixtures, but 13C NMR was required for complex mixtures containing dihydroxyacetone and glycerol, oxalic acid and mesoxalic acid, or glyoxylic acid and tartronic acid, pairs of compounds not well separated or detected by HPLC. As proof‐of‐concept, an unknown mixture generated by glycerol oxidation was analyzed by HPLC and quantitative 13C NMR. The results obtained were concordant and allowed accurate determination of the composition of the sample, which contained mesoxalic acid as the major product, with oxalic acid, tartronic acid, and glyceric acid as by‐products.

Aggregation Behavior and Thiol-Thioester Exchange for Cationic Surfactants with Propylthioacetate Side Chain.

A series of cationic surfactants containing the thioacetate group, [CnH2n+1N(CH3)2(CH2)3SCOCH3] Cl (Cn3SAc, n = 12, 14, 16), were prepared and their properties in aqueous solution were investigated by conductivity, fluorescence, and dynamic light scattering measurements. The critical micelle concentrations (CMCs) of Cn3SAc decreased to about half the value of the corresponding alkyltrimethylammonium chloride. Thioacetate was eliminated with the addition of dithiothreitol (DTT) as well as NaOH. HPLC (high performance liquid chromatography) analysis and NMR (nuclear magnetic resonance) spectroscopy showed that thiol surfactants, [CnH2n+1N(CH3)2(CH2)3SH]Cl (Cn3SH), were generated upon the addition of DTT in aqueous solution via thiol-thioester exchange, whereas gemini surfactants, [CnH2n+1N(CH3)2(CH2)3SS(CH2)3N(CH3)2CnH2n+1]2Cl (2Cn3SS), were generated upon incubation in alkaline solution via hydrolysis and air oxidation.

Ardipusilloside-I Metabolites from Human Intestinal Bacteria and Their Antitumor Activity.

Ardipusilloside-I (ADS-I) is a triterpenoid saponin extracted from Ardisia pusilla DC, and has been demonstrated to have potent antitumor activity. However, ADS-I metabolism in humans has not been investigated. In this study, we studied the biotransformation of ADS-I in human intestinal bacteria, and examined the in vitro antitumor activity of the major metabolites. Ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) was used to detect ADS-I biotransformation products, and their chemical structures were identified by high performance liquid chromatography–nuclear magnetic resonance (HPLC–NMR). The antitumor activity of the major metabolites was determined by the MTT assay. Here, we show that main reaction seen in the metabolism of ADS-I in human intestinal bacteria was deglycosylation, which produced a total of four metabolites. The structures of the two major metabolites M1 and M2 were confirmed by using NMR. MTT assay showed that ADS-I metabolites M1 and M2 have the same levels of inhibitory activities as ADS-I in cultured SMMC-7721 cells and MCF-7 cells. In conclusion, this study demonstrates deglycosylation as a primary pathway of ADS-I metabolism in human intestinal bacteria, and suggests that the pharmacological activity of ADS-I may be mediated, at least in part, by its metabolites.

The application of HPLC and microprobe NMR spectroscopy in the identification of metabolites in complex biological matrices.

Nuclear magnetic resonance (NMR)-based metabolomics can be used directly to identify a variety of metabolites in biological fluids and tissues. Metabolite analysis is an important part of life science and metabolomics research. However, the identification of some metabolites using NMR spectroscopy remains a big challenge owing to low abundance or signal overlap. It is important to develop a method to measure these compounds accurately. Two-dimensional NMR spectroscopy, metabolite prediction software packages, and spike-in experiments with authentic standards are often used to solve these problems, but they are costly and time-consuming. In this study, methods were developed to identify metabolites in complex biological mixtures using both high-performance liquid chromatography (HPLC) and off-line microprobe NMR spectroscopy. With use of these methods, 83 and 73 metabolites were identified in Sprague Dawley rat urine and feces, respectively. Among them, 40 and 45 metabolites, respectively, could not be identified with traditional NMR methods. Our research revealed that the combination of HPLC and NMR techniques could significantly improve the accuracy of trace and overlapped metabolite identification, while offering an effective and convenient approach to identify potential biomarkers in complex biological systems.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-8556-y) contains supplementary material, which is available to authorized users.

Structure elucidation of metabolites of gambogic acid in vivo in rat bile by high-performance liquid chromatography–mass spectrometry and high-performance liquid chromatography–nuclear magnetic resonance

Gambogic acid (GBA), the main component of Gamboge, possesses significant anti-tumour activity. Due to its structural complexity, little is known about GBA metabolism. Here, we investigate the metabolism of GBA in vivo in rat bile. Identification of the metabolites formed was elucidated using high-performance liquid chromatography (HPLC) with UV–vis detection, HPLC/ion trap electrospray ionization-mass spectrometry, as well as HPLC/nuclear magnetic resonance. Four main metabolites were determined. Two phase I metabolites, 10-hydroxygambogic acid and 9,10-epoxygambogic acid, were oxides on the 9,10-olefinic bond of GBA. The others phase II metabolites, were 9,10-epoxygambogic acid-30- O-glucuronide and 10-hydroxylgambogic acid-30- O-glucuronide.

A Modified Colorimetric Method for Phytic Acid Analysis in Soybean

A quantitative, reproducible, and efficient phytic acid assay procedure is needed to screen breeding populations and support genetic studies in soybeans [Glycine max (L.) Merr.]. The objective of this study was to modify the colorimetric Wade reagent method and compare the accuracy and applicability of this new method in determining seed phytic acid content in soybean with three well‐established phytic acid assay methods: anion exchange column (AEC), high‐performance liquid chromatography (HPLC), and 31P nuclear magnetic resonance (NMR). The CV for repeated measurements of a low phytic acid soybean mutant, CX1834‐1‐6, ranged from 1.8 to 4.2% (n = 5), indicating the results were precise and reproducible. Phytic acid content of 42 soybean genotypes as determined by this method showed a correlation of 93.7 to 96.6% with the measurements by AEC, HPLC, and NMR. According to analysis of covariance, using inorganic P content as a predictor, phytic acid P content in a given sample analyzed by the four assay methods can be estimated with four linear regression models at the α = 0.01 level. Compared with HPLC, AEC, and 31P NMR, this modified colorimetric method is simpler and less expensive for assaying a large number of samples, allowing its effective application in breeding and genetic studies of low phytic acid soybean.

Application of high-performance liquid chromatography–nuclear magnetic resonance coupling to the identification of limonoids from mahogany tree ( Switenia macrophylla, Meliaceae) by stopped-flow 1D and 2D NMR spectroscopy

Separation and characterization of limonoids from Switenia macrophylla (Meliaceae) by HPLC–NMR technique has been described. Analyses were carried out using reversed-phase gradient HPLC elution coupled to NMR (600 MHz) spectrometer in stopped-flow mode. Separated peaks were collected into an interface unit prior to NMR measurements, which were performed with suppression of solvent signals by shaped pulses sequences. Structure elucidation of the limonoids was attained by data obtained from 1H NMR, TOCSY, gHSQC and gHMBC spectra without conventional isolation that is usually applied in natural products studies.

Characterization of the metabolites of alosetron in experimental animals and human

The metabolism of radiolabelled alosetron was studied in rat, dog, rabbit, mouse and human. The metabolism in rat and dog was studied at a low and an elevated dose designed to generate sufficient quantities of metabolite for definitive identification. A strategy for the characterization of metabolites in cases of extensive metabolism was developed and demonstrated for alosetron. Semi–preparative high–performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), nuclear magnetic resonance (NMR) and liquid chromatography–nuclear magnetic resonance (HPLC–NMR) enabled the isolation and characterization of 28 metabolites of alosetron. The characterization of the metabolites in animal excreta facilitated the identification of human systemic metabolites.

Metabolome analysis: the potential of in vivo labeling with stable isotopes for metabolite profiling

Metabolome analysis technologies are still in early development because, unlike genome, transcriptome and proteome analyses, metabolome analysis has to deal with a highly diverse range of biomolecules. Combinations of different analytical platforms are therefore required for comprehensive metabolomic studies. Each of these platforms covers only part of the metabolome. To establish multiparallel technologies, thorough standardization of each measured metabolite is required. Standardization is best achieved by addition of a specific stable isotope-labeled compound, a mass isotopomer, for each metabolite. This suggestion, at first glance, seems unrealistic because of cost and time constraints. A possible solution to this problem is discussed in this article. Saturation in vivo labeling with stable isotopes enables the biosynthesis of differentially mass-labeled metabolite mixtures, which can be exploited for highly standardized metabolite profiling by mass isotopomer ratios.

High-performance liquid chromatographic–nuclear magnetic resonance investigation of the isomerization of alachlor–ethanesulfonic acid

The metabolism of the acetanilide herbicide alachlor in soils leads to the formation of alachlor–ethanesulfonic acid (alachlor–ESA) as one of the major transformation products of this compound. The unique structure of alachlor and its metabolites allows the formation of two diastereomers ( s- trans and s- cis) due to the hindered rotation of the amide bond connected to a rigid aromatic ring. Although these stereoisomers do interconvert by rotation about the amide bond, the rate of interconversion is slow allowing separation of the isomers on the chromatographic time scale. Once separated, the unique nuclear magnetic resonance signals of each isomer can be used to monitor the rate of isomerization. This paper reports the on-line separation and detection of the rotational diastereomers using high-performance liquid chromatography–nuclear magnetic resonance (HPLC–NMR) to efficiently measure the isomerization rate of alachlor–ESA.

Identification of major metabolites in Aloe littoralis by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy.

Examination of the leaf exudate of the South African species Aloe littoralis by reversed-phase HPLC revealed the presence of two major metabolites. The identification of the two compounds without isolation was attempted by HPLC-NMR based on separation using a C18 column eluting with a deuterium oxide:acetonitrile solvent gradient and an inverse HPLC-NMR probe. For each compound, one-dimensional proton spectra, and two-dimensional homonuclear COSY and TOCSY, and heteronuclear HSQC and HMBC, spectra were collected. On the basis of the data obtained, the metabolites were characterised as 10-hydroxyaloin B and deacetyllittoraloin.

Synthesis, high-performance liquid chromatography–nuclear magnetic resonance characterization and pharmacokinetics in mice of CD271 glucuronide

Retinoic acid-glucuronides are known as retinoids with activity in acne therapy, limited placental transfer and reduced retinoid adverse effects. We synthesized the glucuronide of a novel retinoid, CD271 (adapalene), used for the treatment of moderate acne. The synthesis product (“CD271 glucuronide”, CD271G) was purified by preparative HPLC. It undergoes in aqueous solution, like other glucuronides, rapid acyl-migration of the bound aglycone leading to position isomers. Thus characterization of purified CD271G could be only achieved by HPLC–NMR coupling. A subfraction (“CD271GB”) consisting essentially of 2′- and 3′-CD271G was used for pharmacokinetic studies. After a single subcutaneous injection at a dosage of 30 mg/kg the substance showed considerable uptake and metabolism to CD271 indicating that CD271GB could serve as a prodrug for CD271.

Phenylpropanoid interconversion in Anigozanthos preissii observed by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy.

HPLC-NMR spectroscopy was employed to observe isotopically labelled intermediates of the phenylpropanoid metabolism in Anigozanthos preissii. Hydroxylation of dihydrocinnamic acid to dihydro-p-coumaric acid and the reversible interconversion between phenylpropanoids and phenylpropenoids were detected, suggesting the operation of a metabolic network in early phenylpropanoid biosynthesis in this plant.

Observation of exchangeable protons by high-performance liquid chromatography–nuclear magnetic resonance spectroscopy and high-performance liquid chromatography–electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natural products

The first high-performance liquid chromatography–nuclear magnetic resonance (HPLC–NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H 2O– 1H 2O–MeCN (deuterium oxide–water–acetonitrile) or 2H 2O–MeCN and, after direct injection or chromatographic separation, examined in a 60-μl NMR flow probe. Exchangeable amino protons initially detected by HPLC–electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC–NMR for the investigation and structure elucidation of natural products.

Identification and quantification of metabolites of 2,3,5,6-tetrafluoro-4-trifluoromethylaniline in rat urine using 19F nuclear magnetic resonance spectroscopy, high-performance liquid chromatography–nuclear magnetic resonance spectroscopy and high-performance liquid chromatography–mass spectrometry

The urinary excretion profile and identity of the metabolites of 2,3,5,6-tetrafluoro-4-triflouromethylaniline, following i.p. administration to the rat at 50 mg kg −1, were determined using a combination of 19F-NMR, HPLC–NMR and HPLC–MS. A total of 38% of the dose was eliminated in the urine as five metabolites. The major routes of metabolism were N-glucuronidation, sulfation and oxidation with a significant amount of metabolic defluorination to give a number of ortho-ring hydroxylated metabolites. The oxidised metabolites were excreted as glucuronide and/or sulfate conjugates.

Lignans from Torreya jackii identified by stopped-flow high-performance liquid chromatography–nuclear magnetic resonance spectroscopy

Coupled reversed-phase HPLC–NMR spectroscopy has been applied to the rapid detection and identification of plant metabolites of Torreya jackii, a species of Taxaceae. Analysis consisted of gradient HPLC elution and directly coupled 1H NMR (500 MHz) spectroscopic detection in a stopped-flow mode. Seven lignans were detected and their structures were elucidated, based on their HPLC– 1H NMR spectra and MS data. The structures were confirmed by isolation of the single components followed by conventional NMR measurements.

High-performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy: Application to the ecdysteroids of Silene otites

High-performance liquid chromatography (HPLC) coupled to nuclear magnetic resonance (NMR) spectroscopy has been used to obtain 1H NMR spectra of ecdysteroids present in an extract of the plant Silene otites. Reversed-phase gradient chromatography was performed using a 2H 2O–acetonitrile–trifluoroacetic acid-based solvent system. Diagnostic NMR spectra were obtained for 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone using the stopped-flow HPLC–NMR technique. The compounds were present in the extracts in amounts between ca. 12 and 135 μg on-column (as determined using HPLC–UV).

Application of Directly Coupled High-performance Liquid Chromatography–Nuclear Magnetic Resonance–Mass Spectrometry to the Detection and Characterisation of the Metabolites of 2-Bromo-4-trifluoromethylaniline in Rat Urine

Directly coupled HPLC–NMR–MS was used to identify the major urinary metabolite of 2-bromo-4-trifluoromethylaniline in the rat as the sulfate conjugate of 2-bromo-4-trifluoromethyl-6-hydroxyaniline. This metabolite could not have been fully characterised using HPLC–NMR or HPLC–MS alone as the sulfate group is ‘NMR silent’ and MS would not have enabled the position of substitution on the aromatic ring to have been assigned. The need to carefully select HPLC solvents for directly coupled HPLC–NMR–MS so that NMR and MS data can both be obtained is also shown.

Application of on-line capillary high-performance liquid chromatography-nuclear magnetic resonance spectrometry coupling for the analysis of vitamin A derivatives

The direct on-line coupling between capillary high-performance liquid chromatography (capillary HPLC) and proton high-field nuclear magnetic resonance (NMR) spectrometry has been used to derive structural information about constituents of a mixture of vitamin A derivatives. 1H NMR spectra were recorded in the stopped-flow and continuous-flow mode within a 180 μm I.D. capillary column mounted in a micro probe on a 600 MHz NMR spectrometer. The resolution of the 1H NMR spectra obtained in capillary HPLC-NMR coupling experiments is sufficient to determine coupling constants in the order of 1.5 Hz. The detection limit is in the lower nanogram range. A stopped-flow 2D-TOCSY experiment of a 1% solution of vitamin A acetate acquired within 4 h reveals that the acquisition of 2D NMR spectra is possible in the nanoliter detection scale without any loss of structural information.

Nuclear magnetic resonance and high-performance liquid chromatography-nuclear magnetic resonance studies on the toxicity and metabolism of ifosfamide.

A combination of high-resolution nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC)-NMR spectroscopic methods has been used to analyse urine from humans and rats treated with the anticancer drug ifosfamide. It was possible to detect a range of abnormal endogenous metabolites in urine after ifosfamide administration to human subjects undergoing cancer therapy and to relate the metabolic perturbations to the nephrotoxic effects of the drug. Changes observed by 1H NMR included increases in levels of urinary glucose, glycine, alanine, histidine, lactate, acetate, succinate, and trimethylamine-N-oxide and decreases in the levels of hippurate and citrate. Additional evidence was gained that ifosfamide-induced nephrotoxicity might be related to the level of oxidation of the coadministered drug mesna. By using both directly coupled continuous-flow 31P HPLC-NMR spectroscopy to determine the retention times of the phosphorus-containing metabolites and, subsequently, stop-flow 1H HPLC-NMR of the urine, it was possible to isolate and identify on-line the metabolites ifosfamide mustard, 4-hydroxy-ifosfamide, 2-dechloroethylifosfamide, and the parent compound itself. These studies illustrate the potential of combining 1H NMR spectroscopy of biofluids and HPLC-NMR spectroscopy for the investigation of drug metabolism and toxicity in humans and animals.