Calcinogenic factor in Solanum malacoxylon: evidence that it is 1,25-dihydroxyvitamin D3-glycoside.

After glycosidic cleavage of the water-soluble vitamin D-like principle of the calcinogenic plant Solanum malacoxylon, the active lipophilic portion was purified by column chromatography and analyzed by combined gas chromatography and mass spectrometry. It was identified as 1,25-dihydroxyvitamin D3, the active form of vitamin D. Thus this active metabolite of vitamin D exists in the plant world, and its presence probably accounts for pathologic calcification in grazing animals ingesting Solanum malacoxylon.

LC/MS and LC/MS/MS determination of protein tryptic digests.

Tryptic digests were analyzed by means of online microbore liquid chromatography combined with mass spectrometry (LC/MS) for some common proteins. Following conventional enzymatic digestion with trypsin, the freeze-dried residues were dissolved in high-performance liquid chromatography (HPLC) eluent and subjected to gradient reversed-phase microbore HPLC separation with mass spectrometric detection. The latter was done in the full-scan single or tandem (MS/MS) mass spectrometry mode. The formation of gas-phase ions from dissolved analytes was accomplished at atmospheric pressure by pneumatically assisted electrospray (ion spray) ionization. This produced field-assisted ion evaporation of dissolved ions, which could then be mass-analyzed for molecular mass or structure. In the full-scan LC/MS mode, the masses for the peptide fragments in the tryptic digests can be determined as either their singly or multiply charged ions. When the molecular weights of the peptides lie outside the mass range of the mass spectrometer, the multiply charged feature of these experimental conditions still provides reliable molecular weight determinations. In addition, collision-activated dissociation (CAD) on selected peptide precursor ions provides online LC/MS/MS sequence information for the tryptic fragments. Results are shown for the tryptic digests of horse heart cytochrome c, bovine ß-lactoglobulin A, and bovine ß-lactoglobulin B.

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation.

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collisioninduced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc(+) (m/z 204) and HexHexNAc(+) (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.

Ion spray-tandem mass spectrometry of supramolecular coordination complexes.

Double-helical [M2L2] (n+), triple-helical [M2L3] (n+), and toroidal [M3L3] (n+) (M = Cu, Co, Fe, Ni, La, Eu, Gd, Tb, or Lu) supramolecular complexes have been fully characterized by ion spray mass spectrometry (IS-MS). The IS-MS spectra from pure acetonitrile solutions reflect the nature of the cations present in solution with conservation of the charge state and allow an efficient qualitative speciation of the compounds. The mass spectrometry results can be correlated with other powerful techniques (nuclear magnetic resonance and electronic spectroscopy) for the characterization of supramolecular complexes in solution, Structural information is obtained by collision-induced dissociation, which strongly depends on the metal ions used in the supramolecular complexes and on the various connectivities and topologies of the ligands. When the ligand contains 3,5dimethoxybenzyl groups bound to the benzimidazole rings, the partial fragmentation of the complexes is associated with a decrease of the total charge of the complexes and the appearance of the characteristic fragment at m/z 151 that corresponds to the 3,5-dimethoxybenzyl cation. A detailed analysis of the fragmentation pathways of these supramolecular complexes suggests that the metal-nitrogen coordination bonds are very strong in the gas phase.

Studies on heme binding in myoglobin, hemoglobin, and cytochrome c by ion spray mass spectrometry.

The ion spray mass spectra of three representative heme-containing proteins were studied, with an emphasis on results obtained under neutral (pH 7) aqueous conditions. The noncovalently bound heme in myoglobin and hemoglobin may be readily distinguished from the covalently bound heme prosthetic group attached to cytochrome c by using collisioninduced dissociation in the free-jet expansion region of the mass spectrometer as well as in the collision quadrupole with premass selection. The charge state of iron in the expelled heme from myoglobin and hemoglobin appears to be 3+ but 2f for heme expelled from cytochrome c.

Detection of noncovalent FKBP-FK506 and FKBP-Rapamycin complexes by capillary electrophoresis-mass spectrometry and capillary electrophoresis-tandem mass spectrometry.

The well known biospecific noncovalent receptor-ligand association complexes between the immunophilin FKBP and the immunosuppressive drugs FK506 and Rapamycin (RM) were investigated by on-line capillary electrophoresis-mass spectrometry (CE-MS) under selected ion monitoring (SIM) conditions and by CE-MS with tandem mass spectrometry (CE-MS/MS) under selected reaction monitoring (SRM) conditions. Solutions of hFKBP (33.3 µM) were dissolved in 50 mM ammonium acetate at pH 7.5. Samples that contained 100 µM of FK506 or RM also were prepared under the same solution conditions. By using these aqueous pH neutral conditions, samples were analyzed by SIM CE-MS and SRM CE-MS and the target complexes were separated by CE with mass spectrometer detection of the individual complexes between FKBP and FK506 [hFKBP + FK506 + 7HJ(7+) as well as FKBP and RM [hFKBP + RM + 7HJ(7+). In an experiment where a mixture of FK506 and RM was analyzed in the presence of FKBP, a nine-to-one ratio of ion current abundances between the RM and FK506 complexes was observed as reported in the literature from other studies. These results suggest that CE-MS and CE-MS/MS may be yet another analytical method for studying noncovalent interactions of biologically important macromolecules under physiological conditions.

Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and that using solid-phase extraction from 100 to 5000 pg ml-1. The lower level of quantitation (LLQ) using liquid-liquid and solid-phase extraction was 50 and 200 pg ml-1, respectively. The lower level of detection for reserpine by LC/MS/MS was 10 pg ml-1. The intra-assay accuracy did not exceed 13% for liquid-liquid and 12% for solid-phase extraction. The recoveries for the LLQ were 68% for liquid-liquid and 58% for solid-phase extraction.

Determination of LTE4 in human urine by liquid chromatography coupled with ionspray tandem mass spectrometry.

A sensitive and specific high-performance liquid chromatographic/ionspray tandem mass spectrometric (LC/ionspray MS/MS) method was developed and validated to quantitate leukotriene E4 (LTE4) in human urine. This method involves solid-phase extraction with Empore membrane disks to isolate LTE4 and its internal standard, LTE4-d3, from the urine stabilized with antioxidant and metal ion chelating agent. The reconstituted extracts were analyzed by LC/ionspray MS/MS in the selected reaction monitoring (SRM) mode. The assay has a lower level of quantitation (LOQ) of 50 pg ml-1 for LTE4 based on 5 ml aliquots of urine. The calibration graphs were linear from 50 pg ml-1 to 10 ng ml-1 for LTE4 extracted from urine. The inter- and intra-assay precision (RSD) and accuracy (DEV) did not exceed 11% and 8% at any level of the calibration standards and quality control (QC) samples, respectively. The recovery of LTE4 using the Empore disk solid-phase extraction technology was independent of LTE4 concentration in human urine. The overall extraction recovery for LTE4 was 72% (RSD = 2.14%).

Identification of betamethasone and a major metabolite in equine urine.

Betamethasone and its major unconjugated metabolite, 6-beta-hydroxybetamethasone, were detected in equine urine by thin-layer chromatography and characterized by micro-liquid chromatography/mass spectrometry (micro-LC/MS). Their structures were confirmed by a combination of infrared spectroscopy and nuclear magnetic resonance spectroscopy.

Direct analysis of microcystins by microbore liquid chromatography electrospray ionization ion-trap tandem mass spectrometry.

Microcystins are a group of structurally similar cyclic heptapeptide hepatotoxins and tumor promoters, produced by cyanobacteria. A microbore liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-ITMS) method has been developed which is capable of separating and detecting trace amounts of microcystin variants in environmental samples. Extracted water sample was loaded onto a LC trapping column and, using a column switching technique, the compounds of interest were back-flushed onto a 1-mm LC column. Structural elucidation was achieved using ion-trap with tandem mass spectrometry in the data dependent scan mode. Collision-induced dissociation to MS3 allowed tentative identification of these cyclic peptides. Full-scan LC-ESI-MS mass spectrum was obtained when 250 pg of the authentic compound was injected onto the HPLC column, which represents the detection limit for microcystin-LR. This study demonstrated that LC-ESI-ITMS is a reliable and sensitive technique for analysing trace levels of microcystins.

Qualitative and quantitative analysis of hydrochlorothiazide in equine plasma and urine by high-performance liquid chromatography.

A sensitive, quantitative method has been developed for the determination of hydrochlorothiazide in equine plasma and urine. Thin-layer chromatography is used to screen for the presence of the drug in unknown samples. The TLC screening methods described provide minimum detection limits of 50 ng/mL in plasma and 25 ng/mL in urine. A silica micro chromatography column is used to clean up ethyl acetate extracts for HPLC analysis and mass spectral confirmation. An internal standard, trichloromethiazide, is used to derive quantitative data at concentrations as low as 25 ng/mL for plasma disappearance curves and urinary excretion rates.

Distribution of zeranol in bovine tissues determined by selected ion monitoring capillary gas chromatography/mass spectrometry.

Bovine tissues, including liver, muscle, kidney, bile, serum, and urine, have been quantified by selected ion monitoring capillary gas chromatography/mass spectrometry to establish the distribution of the anabolic drug, zeranol, and its metabolites, taleranol and zearalanone, after administration of zeranol to 9 bovine animals. The method used to isolate, confirm, and quantify zeranol is undergoing validation by the United States Department of Agriculture, Food Safety Inspection Service (FSIS). Application of this method demonstrates utility in determining residue levels of zeranol in a variety of tissues with levels ranging over 4 orders of magnitude (i.e., 100 parts per trillion (ppt) to 1 part per million (ppm]. The analyte levels determined in this study complement previously reported pharmacokinetic data on the distribution of zeranol in addition to providing more specific information for taleranol and zearalanone. In this quantitative study it is shown that the liver is the main organ of deposition for zeranol, taleranol, and zearalanone, that taleranol is the main metabolite in the bovine, and that zeranol is efficiently eliminated.

Packed-column supercritical fluid chromatography/mass spectrometry via a two-stage momentum separator.

The practical utility of a two-stage momentum separator for combining packed-column supercritical fluid chromatography (SFC) with mass spectrometry (MS) is described. A Hewlett-Packard model 1084B liquid chromatograph modified for packed-column SFC is connected to a linear fused-silica capillary restrictor housed in a heated probe held at 60 degrees at the terminus. A makeup of coaxial helium gas (1.5 L/min) or dissolved solvent (0.2-0.4 mL/min) can be introduced at the point of supercritical fluid expansion. The latter SFC effluent (0.3-2.0 mL/min) is expanded into a heated (44 degrees) desolvation chamber and directed through a nozzle positioned at the entrance of a two-stage momentum separator. Enrichment of the analyte relative to the volatile gases allows the transfer of sample particles to the MS ion source to produce electron ionization of flash-volatilized eluates. On-line SFC/MS separation and detection of low microgram levels of involatile, thermally labile analytes in synthetic mixtures is accomplished. Identification of an unknown compound in a drug tampering incident and the identification of an unknown metabolite isolated from horse urine is also accomplished.

Capillary electrophoresis/mass spectrometry determination of inorganic ions using an ion spray-sheath flow interface.

The determination of inorganic cations and anions by capillary electrophoresis/mass spectrometry (CE/MS) is reported using an ion spray-sheath flow interface coupling. A twelve-component synthetic mixture of cations which included the positive ions of K, Ba, Ca, Mn, Cd, Co, Pb, Cr, Ni, Zn, Ag, and Cu was loaded into the capillary column at levels ranging from 30 to 300 pg, separated by CE, and detected by indirect UV and in the full-scan (m/z 35-450) positive ion CE/MS mode using an aqueous buffer containing 30 mM creatinine and 8 mM alpha-hydroxyisobutyric acid, pH 4.8. Creatinine forms adducts with the cations which are observed in the gas phase and requires rather high (120 electron volts) declustering energy to dissociate. This produces a reduction in charge state to form the free, singly charged, inorganic cations which are observed in the mass spectra. CE/MS analysis of an aqueous acidic extract of used aircraft engine oil revealed high levels of lead as well as lower levels of chromium and nickel. CE-indirect UV analysis of a synthetic mixture containing 300 pg each of 11 inorganic ions, which included the anions of Br, Cl, NO2, NO3, S2O3, N3, SCN, SO4, SeO4, oxalate, and MoO4, is shown. The running buffer which affected this separation contained 5 mM ammonium dichromate, 10 mM ammonium acetate, and 20 mM diethylenetriamine at pH 9.3. Although indirect UV detection revealed good separation of these anions, CE/MS analysis of this mixture was complicated by interfering ion current signals from the cluster ions formed by the interaction between the additives and the analytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of drugs from urine by on-line immunoaffinity chromatography-high-performance liquid chromatography-mass spectrometry.

A method for rapid extraction and identification of drugs in urine is described. The system utilizes a high-performance protein G immunoaffinity column coupled to a reversed-phase analytical column by use of a trapping column and switching valve. A small amount of antibody (5 micrograms drug-specific) is used for each analysis to extract either propranolol or lysergic acid diethylamide (LSD) from human urine. Urine diluted with phosphate-buffered saline is pumped directly through the protein G column thus eliminating time- and solvent-consuming sample preparation procedures. On-line ultraviolet or mass spectral analysis provides the means of drug detection and identification. With ultraviolet detection propranolol may be detected in spiked urine at the 250 pg/ml level. A Hewlett-Packard mass spectrometer modified for atmospheric pressure ionization and equipped with an ion spray source allows detection of propranolol in urine at 2.5 ng/ml and LSD at 500 pg/ml using single ion monitoring. The potential applicability of the technique for drug confirmations is discussed.

Determination of aminoglycoside antibiotics by reversed-phase ion-pair high-performance liquid chromatography coupled with pulsed amperometry and ion spray mass spectrometry.

This work constitutes a preliminary investigation of a high-performance liquid chromatographic (HPLC)-mass spectrometric (MS) method for confirming aminoglycoside residues in bovine tissues. A reversed-phase ion-pair HPLC method for the separation of four aminoglycosides was developed using volatile ion-pairing agents and optimized for detection with an ion spray HPLC-MS interface. The method is also compatible with a commercial pulsed amperometric detector that was used for HPLC method development and that may be useful for the screening and quantification phases of a regulatory method. Several column phases, eluent compositions, and pairing ions were evaluated for optimum HPLC-MS sensitivity. Detection limits are in the low nanogram range with the pulsed amperometric detector and with HPLC-MS in the selected ion monitoring mode. Results with bovine kidney, fortified to 20 ppm and extracted by matrix solid-phase dispersion, obtained using both detectors are presented.

Determination of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid by microbore high-performance liquid chromatography and capillary zone electrophoresis coupled to tandem mass spectrometry.

The performance of microbore high-performance liquid chromatography and capillary zone electrophoresis, both equipped with on-line tandem mass spectrometric detection capability, was evaluated critically for the determination of endogenous amounts of leucine enkephalin and methionine enkephalin in equine cerebrospinal fluid. Using an identical sample clean-up and enrichment procedure, capillary zone electrophoresis-mass spectrometry is limited in its concentration detection capacity owing to its much smaller injection volume. Leucine enkephalin was identified in post-mortem equine cerebrospinal fluid at the 1-5 ng/ml level by liquid chromatography-mass spectrometry.

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry.

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated from normal human urine was achieved by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urine.