Reversed-phase Liquid Chromatography Mass Spectrometry Research Articles

Determination of Vitamin B 5 in a range of fortified food products by reversed-phase liquid chromatography–mass spectrometry with electrospray ionisation

Methods for Vitamin B 5 determination in food products remain limited by their low sensitivity and poor selectivity. Here, we have developed a liquid chromatography–mass spectrometry (LC–MS) method for Vitamin B 5 determination in wide range of fortified food products. Vitamin B 5 was extracted from food samples by heat treatment and analysed by LC–MS in the positive mode using electrospray ionisation (ESI). Vitamin B 5 was quantified using hopantenic acid (HOPA) as internal standard after their separation on a C 18 narrow-bore column with a gradient of mobile phase made of water/acetonitrile and trifluoroacetic acid (TFA) 0.025%. MS with single ion monitoring mode at mass m/z 220 was used for Vitamin B 5 quantification. Calibration curve between 0.5 and 10 μg/ml of Vitamin B 5 was linear ( r 2=0.9993) and the detection limit was determined to be 800 pg. The overall quantitative efficiency of the method was evaluated using Nestlé reference sample (infant formula). The intra-assay RSD was 4.8% ( n=8), the inter-assay RSD 6.4% ( n=4) and the recoveries of the spiked samples were above 95%. Application of the LC–MS method to Vitamin B 5 determination in wide range of fortified food products including three US National Institute of Standards and Technology (NIST) reference samples (RM 8435, RM 8415 and SRM 1546) shows consistent results with those obtained by microbiology and recoveries of Vitamin B 5 between 93 and 104% for the spiked samples.

Determination of hyperforin in mouse brain by high-performance liquid chromatography/tandem mass spectrometry.

Hyperforin is one of the essential active ingredients of St. John's wort extract, which is used as an antidepressant for mild to moderately severe depressions. In vitro and in vivo data as well as several clinical studies and meta analyses have confirmed the pharmacological effect of treatment with hyperforin-containing preparations. However, little is known about the brain availability of hyperforin until now. Accordingly, a highly sensitive and selective LC/MS method for this purpose was developed and validated. This method proved suitable for the determination of hyperforin in mouse brain, after oral administration of hyperforin sodium salt and St. John's wort extract. This method involves liquid-liquid extraction of hyperforin with ethyl acetate followed by separation with rapid reversed-phase high-performance liquid chromatography and tandem mass spectrometry detection using electrospray ionization. Excellent linearity was obtained for the entire calibration range from 0.25 to 10 ng/mL (corresponding to 2.5-100 ng/g brain tissue concentration, calculated with the factor derived from sample processing) with an average coefficient of correlation of 0.9992. The recovery of hyperforin from mouse brain homogenates was between 71.4 and 75.3% with a relative standard deviation of less than 3%. Validation assays for the lower limit of quantitation yielded an accuracy of 5.8%. Intraday accuracy and precision for the developed method were between 4.6 and 10.6% and 4.3-8.4%, respectively, while the interday parameters varied between 6.7 and 12.2% for accuracy and 2.0-5.0% for precision. After the method validation, hyperforin brain levels in mice, treated with 15 mg/kg hyperforin (either as the sodium salt or as 5% St. John's wort extract), were investigated. The average concentration of hyperforin found for the sodium salt group was 28.8+/-10.1 ng/g of brain (n = 8), which was somewhat higher than the hyperforin concentration of 15.8+/-10.9 ng/g of brain (n = 8), determined in the extract-treated group. This method is robust, selective, and highly sensitive and represents an appropriate tool to further prove the occurrence and distribution of hyperforin in mouse brain.

Determination of antibiotics from soil by pressurized liquid extraction and liquid chromatography–tandem mass spectrometry

A method for the analysis of several macrolide and ionophore antibiotics, as well as tiamulin, from soil was developed using pressurized liquid extraction (PLE), reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (LC–APCI +-MS–MS). The analytes were extracted from soil by PLE in 30 min and the extracts were cleaned up by solid-phase extraction (SPE) on a diol SPE cartridge. Liquid chromatographic (LC) separation of the antibiotics was achieved in 35 min. Recovery experiments were performed using spiked soil and concentrations varying from 1 to 2000 μg/kg. By using a macrolide internal standard the recovery rates for the macrolides erythromycin and roxithromycin ranged from 43 to 94% (RSD 20–23%), for the ionophore salinomycin the recovery rate was 76% (RSD 29%), while the pleuromutilin tiamulin was completely recovered. The limits of detection ranged from 0.2 to 1.6 μg/kg. In soil samples a maximum concentration of 0.7 μg/kg tiamulin was found.

Selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of alfuzosin in human plasma

A selective, sensitive and rapid liquid chromatography–tandem mass spectrometry method for the determination of alfuzosin in plasma was developed. A PE Sciex API 2000 triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, using TurboIonSpray with positive ionisation was used. Using prazosin as an internal standard, liquid–liquid extraction was followed by C 18 reversed-phase liquid chromatography and tandem mass spectrometry. The mean recovery for alfuzosin was 82.9% with a lower limit of quantification set at 0.298 ng/ml, the calibration range being between 0.298 and 38.1 ng/ml. This assay method makes use of the increased sensitivity and selectivity of tandem mass spectrometric (MS–MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of alfuzosin in human plasma than has previously been described. The assay method was used to quantify alfuzosin in human plasma samples generated in a multiple-dose (5 mg bd.) study at steady state.

A versatile approach towards regioselective platinated DNA sequences.

Undesired N(7) platination of 2'-deoxyguanosine residues at predetermined sites in an oligodeoxynucleotide (ODN) sequence is prevented by applying the sterically demanding diphenylcarbamoyl (DPC) as an O(6)-protecting group. The presence of a base-labile oxalyl linker between the immobilized 3'-nucleotide and controlled pore glass (CPG) allows cleavage of the protected ODN from the support and leaves DPC protection unaffected. This method provides an ODN with specifically blocked guanine-N(7) sites for platination. In the hexanucleotides prepared in this study, 5'-GGBGGT-3'(for B=T, C and A), a platinum GG adduct is introduced at G4,G5. These site-specific platinated hexamers were isolated in a yield of 65 %, and were fully characterized by using reversed-phase HPLC (high performance liquid chromotography), LCMS (liquid chromatography-mass spectrometry), MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), PAGE (polyacrylamide gel electrophoresis) and Maxam-Gilbert sequencing analysis.

Determination of antibiotics such as macrolides, ionophores and tiamulin in liquid manure by HPLC-MS/MS.

A method for the analysis of several macrolide and ionophore antibiotics as well as tiamulin in liquid manure was developed. Reversed-phase liquid chromatography and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry was used for detection.High-performance liquid chromatographic (HPLC) separation of the antibiotics was achieved in 35 min. The analytes were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on a diol SPE cartridge. Recovery experiments with spiked liquid manure concentrations varying from 6 to 2,000 microg kg(-1) gave constant recovery rates. The recovery rates for the macrolides erythromycin, roxithromycin and oleandomycin were 75-94%, that for the ionophore salinomycin was 119%, while that for the pleuromutilin tiamulin was 123%, when using a macrolide internal standard. The relative standard deviation was found to be 15-36% and the limits of detection were 0.4-11.0 micro g kg(-1). The maximum concentrations found in manure samples were 43 micro g kg(-1) for tiamulin and 11 micro g kg(-1) for salinomycin.

LC/MS DETERMINATION OF A SULFOLANE METABOLITE IN WETLAND VEGETATION EXPOSED TO GAS-CONDENSATE CONTAMINATED GROUND WATER

Groundwater containing the process chemicals sulfolane (tetrahydrothiophene 1,1-dioxide) and diisopropanolamine (DIPA) has contaminated a wetland in the vicinity of a sour-gas natural gas processing facility. Residues of the chemicals are found in wetland soil, water and vegetation. While it is known that DIPA undergoes transformation in wetlands plants, little is known regarding the degree of transformation of sulfolane. This is due in part to the lack of analytical methods suitable for the determination of transformation products of sulfolane in vegetation. Described is a new procedure, employing electrospray ionization with reverse-phase liquid chromatography mass spectrometry for the determination of the 3-hydroxysulfolane metabolite in plant tissue. The metabolite was determined using filtered water extracts of the vegetation, preconcentrated using solid-phase extraction with no further clean up or derivatization steps. Under positive ion electrospray conditions, there was preferential formation of a series of adducts with minimal (M+H)+ formation. The same was true for negative-ions, for which the molecular ion was not observed. However, the latter was more selective for crude extracts of vegetation. Quantification was performed using selected ion monitoring of the m/z 197.03 (M+61)− adduct, elucidated as the (M−1)(CN·(H2O)2)− negative-ion. Confirmations were based on comparison of the retention time and product ion scan of an authentic standard. Instrumental detection limits were attainable in the picogram range (50–100 pg injected). Successful application of the procedure is demonstrated for the determination of the metabolite in 22 samples, collected from a sour-gas contaminated wetland and a control site. For roots, shoots, berries, seeds, grasses, and leaves, the recovery was 117±5.6% r.s.d with a detection limit of 50 ng/g. In general, the technique was rugged (based on a study period of 18 months), and relatively fast (∼25 min per sample for a batch of 10 samples). For the study period, it appears that sulfolane is also transformed in the plants in a manner similar to DIPA, suggesting that degradation of both process chemicals occurs in wetland plants.

Enhanced mass detection of oligonucleotides using reverse-phase high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry.

A new method providing enhanced sensitivity for the analysis of oligonucleotides using an on-line coupled system of reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization ion-trap mass spectrometry (ESI-MS) has been developed. The presented method allows the use of the standard gradient elution of 0.1 M triethylammonium acetate (TEAA) buffer (adjusted to pH 7.0 with acetic acid) and acetonitrile that is typically used for the separation of oligonucleotides in RP-HPLC. An added feature of this method is the ability to combine and mix additional 0.1 M imidazole in acetonitrile after the separation column for improved ESI-MS performance. This is similar to the post-column reaction method in liquid chromatography (LC) and the liquid sheath flow method in LC/ESI-MS, both of which offer the advantage of not compromising the chromatographic separation conditions. The application of this new method is demonstrated to afford improved sensitivity for the analysis of oligonucleotides (20-50 mer) via on-line coupled HPLC/ESI-MS analysis and purification systems.

Extractionless and sensitive method for high-throughput quantitation of cetirizine in human plasma samples by liquid chromatography–tandem mass spectrometry

Following a single 10-mg oral dose of cetirizine dihydrochloride to 24 healthy volunteers, the analyte was quantified in human plasma. Protein precipitation using acetonitrile (ACN) was followed by reversed-phase liquid chromatography and tandem mass spectrometry. The MS/MS method was optimised using a PE Sciex API 2000 triple quadrupole mass spectrometer in selected reaction monitoring (SRM) mode, using electrospray with positive ionisation. Oxybutynin was used as the internal standard. The assay method represents a robust, high-throughput, highly specific and sensitive quantitative assay procedure, with 0.5 ng/ml being the lowest plasma concentration that could be reliably quantified. The procedure involves minimal sample preparation, and is well suited to clinical studies of the drug involving large numbers of generated samples. Pre-dose as well as post-dose samples up to and including 48 h were quantified, and the data generated were used to determine the pharmacokinetic profile of the drug.

Determination of selected sulfonamide antibiotics and trimethoprim in manure by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry

A method for the determination of sulfonamides and trimethoprim in the complex matrix liquid manure has been developed using reversed-phase liquid chromatography and atmospheric pressure chemical ionization (APCI) tandem mass spectrometry. A comparison was made between electrospray and atmospheric pressure chemical ionization. APCI proved to be more robust and less sensitive to matrix effects. High-performance liquid chromatographic (HPLC) separation of the analytes was achieved in less than 7 min. The compounds were extracted with ethyl acetate and the extracts were cleaned up by solid-phase extraction on an aminopropyl column. Recoveries were not dependent on the concentration level. The mean recoveries were as follows: trimethoprim 79.0%, sulfadiazine 80.5%, N(4)-acetylsulfadiazine 91.0%, sulfamerazine 78.6%, sulfadimidine 77.2% and sulfamethoxazole 82.8%. Linearity was established over a concentration range of 5 to 5000 microg/kg with correlation coefficients greater than 0.99. The method had a limit of quantitation (LOQ) of 5 microg/kg manure.

Liquid chromatography–mass spectrometry and capillary electrophoresis combined approach for separation and characterization of multicomponent peptide mixtures: Application to crude products of leuprolide synthesis

A sequential combination of reversed-phase liquid chromatography–mass spectrometry (LC–MS) and capillary electrophoresis (CE) has been explored in order to perform separation and characterization of a multicomponent peptide mixture from the synthesis of leuprolide. The mixture was first analyzed and fractionated by LC–MS, and the collected fractions were subsequently separated by CE. Unambiguous identification of the electrophoretic peaks was achieved by injecting the collected fractions separately and spiking the leuprolide crude mixture. Furthermore, structural information about the components of the mixture provided by several semi-empirical migration models has been used to check the accuracy of the structures previously proposed by LC–MS. Combination of the two orthogonal techniques results in an enhancement of their individual selectivity characteristics.

Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure.

The effects of the mutation beta9(A6)Ser --> Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the beta9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher-molecular-weight oligomeric fraction (30%). Reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ESI-MS) under denaturing conditions showed that the tetrameric fraction contained only normal alpha- and beta-chains, whereas the oligomeric fraction contained only normal alpha-chain and disulfide-linked beta(PA) dimer. Under native conditions, ESI-MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 A diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA-CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer-dimer allosteric interface. None of the other variants investigated formed such a large oligomer.

Preliminary application of liquid chromatography–electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C 18-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC–MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/ml.

Detection and localisation of disulphide bonds in a synthetic peptide reproducing the sequence 1-30 of Par j 1.0101 by electrospray ionisation mass spectrometry.

The structural characterisation of a synthetic peptide reproducing the sequence 1-30 of Par j 1.0101, a major allergenic protein present in the pollen of Parietaria judaica, by combined use of chemical and enzymatic cleavage, reversed-phase high-performance liquid chromatography and electrospray ionisation mass spectrometry (ESI-MS), is described. Direct ESI-MS of the synthetic peptide after reaction with methyl iodide showed that the product is a mixture of two peptides: one form in which two out of the four cysteine residues present in the sequence are oxidised and a minor amount of another form in which all the cysteines are fully reduced. It was ascertained, using the combined procedure indicated above and without prior separation of the two species, that the disulphide bond in the partially oxidised form is located between cysteines 29 and 30. These results show the usefulness of this approach for characterising synthetic peptides containing multiple cysteine residues in the sequence.

Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.

A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.

Detection and Localisation of Disulphide Bonds in a Synthetic Peptide Reproducing the Sequence 1–30 of Par j 1.0101 by Electrospray Ionisation Mass Spectrometry

The structural characterisation of a synthetic peptide reproducing the sequence 1–30 of Par j 1.0101, a major allergenic protein present in the pollen of Parietaria judaica, by combined use of chemical and enzymatic cleavage, reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation mass spectrometry (ESI-MS), is described. Direct ESI-MS of the synthetic peptide after reaction with methyl iodide showed that the product is a mixture of two peptides: one form in which two out of the four cysteine residues present in the sequence are oxidised and a minor amount of another form in which all the cysteines are fully reduced. It was ascertained, using the combined procedure described above and without prior separation of the two species, that the disulphide bond in the partially oxidised form is located between cysteines 29 and 30. These results show the usefulness of this approach for characterising synthetic peptides containing multiple cysteine residues in the sequence.

Analysis of triacylglycerols with non-aqueous reversed-phase liquid chromatography and positive ion electrospray tandem mass spectrometry.

A non-aqueous reversed-phase liquid chromatographic method coupled to electrospray ionisation (ESI) tandem mass spectrometry was developed for the analysis of triacylglycerols (TGs). The synthetic TGs studied were separated according to their equivalent carbon number with a gradient of methanol (containing 0.01% (v/v) formate adjusted to pH 5.3 with ammonia) and chloroform. ESI mass spectra of TGs yielded positive ion current signals for [M + NH(4)](+) and [M + NH(4)-RCOONH(4)](+). The mass spectra also showed signals believed to arise from [M + K](+). Collision-induced dissociation (CID) of the [M + NH(4)](+) precursor ion yielded [M + NH(4) - RCOONH(4)](+), [RCO + 74](+) and [RCO](+) product ions as aids for the structural elucidation of the TGs. In addition, [RCO - 18](+) and small amounts of [RCO - 2](+) product ions were also found. The latter ions were observed only for TGs containing unsaturated fatty acids. CID of ammoniated 1-stearoyl-2-oleoyl-3-linoleoyl-glycerol (18:0/18:1/18:2) indicated that neutral loss of the sn-2 fatty acid was energetically less favourable than loss of the fatty acid from the sn-1 or sn-3 position.

Rapid and sensitive analysis of azadirachtin and related triterpenoids from Neem ( Azadirachta indica) by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

Based on reversed-phase high-performance liquid chromatography (RP-HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry, a HPLC–MS method was developed to permit the rapid qualitative and quantitative analysis of azadirachtin and related tetranortriterpenoids from seeds and tissue cultures of Neem ( Azadirachta indica). APCI+ standard scanning mass spectra of the major Neem triterpenoids were recorded and utilized to select suitable ions for selected ion monitoring (SIM). Transitions for selective reaction monitoring (SRM) were based on MS–MS experiments. Using SIM, major Neem triterpenoids were detected in callus culture material and seed kernels of A. indica. The limit of detection for azadirachtin in extract samples (∼1 ng ml −1 or 10 pg in SIM mode) was determined to be (with respect to injected absolute amounts) approximately 1000-times lower than values quoted in the literature for existing HPLC methods (∼200 ng ml −1 or 10 ng). In addition to high sensitivity, the HPLC–MS method is able to tolerate minimal sample preparation and purification, dramatically reducing total analysis time.

Determination of Residues of Pirimicarb and Its Desmethyl and Desmethylformamido Metabolites in Fruits and Vegetables by Liquid Chromatography–Electrospray/Mass Spectrometry

A method was developed for the simultaneous determination of residues of pirimicarb (I) and its desmethylformamido (II) and desmethyl (III) metabolites in plums, peas, green beans, broad beans, carrots, and swedes. The compounds were extracted with ethyl acetate and determined, without cleanup, by reversed-phase liquid chromatography and electrospray mass spectrometry (MS). MS and MS/MS were used concurrently to monitor the protonated molecules and their common collision-induced dissociation product. The limit of detection (signal-to-noise ratio of >3) was 1 ng/mL, corresponding to crop concentrations of <0.0015 mg/kg. All 3 compounds were determined in plums, broad beans, and green beans by MS without interference. Interferences which affected the determination of desmethylformamido-pirimicarb in peas, and to a lesser extent in carrots and swedes, were eliminated by MS/ MS. Recoveries for all 3 compounds, at 0.05 mg/kg for plums and 0.005 mg/kg for other commodities, were in the range 83-124%. No interconversion of I, II and III, occurred during extraction, and the compounds were stable in extracts for > or = 7 days under appropriate conditions.

Solid-phase extraction followed by high-performance liquid chromatography–ionspray interface–mass spectrometry for monitoring of herbicides in environmental water

In this work we developed a sensitive and specific multiresidue method, based on reversed-phase liquid chromatography–mass spectrometry, with an ionspray interface (LC–ISI-MS), for determining 52 of most representative compounds of herbicides in water samples. The procedure used involved passing 0.5 l of surface water, 2 l of ground water and 4 l of drinking water samples, respectively, through a 0.5 g graphitized carbon black (GCB) extraction cartridge. Base–neutral and acid herbicides were differential eluted from GCB cartridge and follow analyzed by HPLC–ISI-MS apparatus. A conventional 4.6-mm-ID reversed-phase LC C 18 column, operating with a mobile phase flow-rate of 1 ml/min, was used to chromatograph the analytes. A flow of 100 μl/min of the column effluent was diverted to the ISI source. The study demonstrates the sensitivity of the technique, with detection limit under 10 ng/l in drinking water samples. Performance data for the method such as recovery and precision are also reported.