Analysis of natural corticosteroids in adrenal extracts and in biological fluids by high-performance liquid chromatography

A liquid chromatographic procedure is described for the analysis of the principal natural corticosteroids in extracts of adrenal glands. Microparticulate silicic acid columns and gradients of methanol in chloroform are used: conditions are described for the quantitative analysis of the single principal steroidal components of adrenal extracts for pharmaceutical use and of adrenal extracts of rats. In the last case, the use of a 5-micron silica column with the appropriate gradient allows the determination of corticosterone and of 18-hydroxydeoxycorticosterone, which were identified by means of mass spectrometry on their eluates. A single analysis can be performed on the extract of 15 mg of rat adrenal tissue. For the last type of analysis, isocratic conditions on a 10-micron LiChrosorb Diol column are also described. The application of the gradient elution procedure to the analysis of steroidal compounds in human plasma is also described.     

Determination of thiodyglycol in groundwater using solid-phase extraction followed by gas chromatography with mass spectrometric detection in the selected-ion mode

A highly sensitive analytical procedure is described for determining thiodiglycol in groundwater. Samples are initially fortified with 3,3'-thiodipropanol (surrogate), then both species are extracted using sequential solid-phase extraction with both C18 and Ambersorb 572 columns. The C18 column, which removes extraneous groundwater components, is discarded; the Ambersorb 572 column is dried thoroughly before eluting polar components with a small volume of dichloromethane. The extract is taken to dryness using dry flowing nitrogen, and the resulting residue is derivatized using N-(tert.-butyldimethylsilyl)-N-methyltrifluoroacetamide and pyridine. The derivatized products are diluted to a final volume with toluene, chromatographed using a fused-silica capillary column, and detected with a quadrupole mass spectrometric detector in its selected-ion mode. Two independent, statistically unbiased, procedures were used to evaluate the detection limits for thiodiglycol; the values ranged between 4 and 16 microg(-1) groundwater.     

A simplified device for protein identification by microcapillary gradient liquid chromatography-tandem mass spectrometry

A simplified device and procedure have been developed for microcapillary gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS). This procedure has proved useful in identifying low level quantities of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel bands. Microelectrospray needles are packed with reversed-phase resin and function both as a high performance liquid chromatography (HPLC) column and a nanospray mass spectrometer tip when interfaced between an HPLC and ion trap mass spectrometer. Variable submicroliter flow rates are generated by flow splitting between the microelectrospray capillary and an HPLC system. A manual injector is used to inject a protein digest mixture that binds to the column and is then washed at a high flow rate (2 microL/min post split). Gradient elution of bound peptides was initiated by the injection of a filled loop of 70% v/v methanol (5 microL) concomitant with a reduction of flow rate (0.1 microL/min post split). This forms a diffusion-dependent gradient of variable length (typically 15-30 min in length) depending upon the final flow rate. Chromatographic separations of a standard solution digest demonstrate that this diffusion-dependent gradient provides reasonable separations such that multiple peptide identifications by MS/MS can be obtained. Application of this methodology to the analysis of several in-gel-digested gel-separated proteins is presented to demonstrate its utility.     

On-line desalting-mass spectrometry system for the structural determination of hydrophilic metabolites, using a column switching technique and a volatile ion-pairing reagent

A novel desalting method, using a column switching technique and a volatile ion-pairing reagent, pentadecafluorooctanoic acid, was developed. This system allows hydrophilic and cationic compounds in a nonvolatile buffer to be directly introduced into a mass spectrometer for structural elucidation. The desalting procedure consists of four steps: (1) the fractionation of a target compound from a separation column, (2) the removal of salts with pentadecafluorooctanoic acid on the trap column, (3) the desorption of the compound from the trap column, and (4) the re-equilibration of the trap column with a pentadecafluorooctanoic acid solution. In this procedure, we investigated the methods for optimizing the desalting and re-equilibration steps. Various amino acids, including branched chain amino acids, aromatic amino acids, basic amino acids and methionine, after separation with phosphate buffer on a cation-exchange column, were successively desalted by this method, and were observed as protonated ions by mass spectrometry. This desalting system could be useful for the structural elucidation of unknown hydrophilic compounds eluted by conventional high-performance liquid chromatography methods, such as ion-exchange chromatography, with mobile phases containing nonvolatile salts. As an example, we present the structural elucidation of unknown metabolites in bovine serum.     

Evaluation of high-performance liquid chromatography column retentivity using macromolecular probes. II. Silanophilic interactivity traced by highly polar polymers

A new method of HPLC column retentivity testing utilizes polymeric probes instead of conventional sets of low molar mass substances. The procedure allows at least semiquantitative, separate and independent evaluation of adsorption and partition properties of column packings. In this present work, the method is applied for comparison of the polar interactivities of selected silica gel C18 HPLC columns. It is shown that free silanols which remained on the surface of the end-capped silica C18 column packings are accessible for interaction with highly polar macromolecules. High molar mass polymeric test probes are adsorbed on the surface silanols and their retention volumes increase. As result, deviations from regular size-exclusion chromatographic (SEC) behavior are observed. The extent of retention volume changes depends on both the nature of polymer probes and on column packing type. Adsorption of macromolecules can be suppressed by addition of a highly polar substance to the mobile phase. The amount of polar additive which is needed to attain regular SEC elution of the polymer probe depends on the column packing type and can be used as a characteristic of silanophilic column interactivity. Courses of dependences of retention volumes on sizes of macromolecules indicate the presence of "U-turn" adsorption which allows two and more silanols situated among C18 groups to be occupied simultaneously with the same macromolecule.     

Optimization of capillary parameters for gas chromatography

An HETP equation for the capillary column is developed that takes into account the dependence of gaseous diffusion on pressure, the compressibility of the mobile phase, together with the unique relationship between mobile phase velocity, and the resistance to mass transfer in the stationary phase. The equation is used to develop a procedure for column optimization and expressions are derived that allow the optimum column radius and optimum column length to be calculated for a given fixed inlet pressure. It is shown that fast, simple separations are optimally achieved using relatively short small diameter columns. Conversely, optimum performance for the separation of complex mixtures requiring higher efficiencies requires the use of long columns with relatively large diameters.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

The Application of Perdeuterated Polycyclic Aromatic Hydrocarbons (PAH) as Internal Standards for the Liquid Chromatographic Determination of PAH in a Petroleum Crude Oil and Other Complex Mixtures

Abstract

A sequential liquid chromatographic (LC) procedure for the determination of polycyclic aromatic hydrocarbons (PAH) in a petroleum crude oil and other complex mixtures is described. The procedure includes normal-phase LC on an aminosilane column to isolate fractions containing isomeric PAH and reversed-phase LC on a polymeric C₁₈ column to separate the individual PAH isomers. Appropriate perdeuterated PAH are added to the sample so that each isomeric fraction will contain one internal standard. The perdeuterated PAH are excellent internal standards for this sequential LC procedure. Perdeuterated PAH have normal-phase and reversed-phase LC retention characteristics similar to those of the parent PAH. In the normal-phase LC separation, the perdeuterated PAH elute in the same fraction as the parent PAH. In the reversed-phase LC separation, the perdeuterated PAH elute first and are generally resolved from the parent PAH. The optimized spectrofluorometric detection of each PAH analyte is accomplished by programming appropriate sets of excitation and emission wavelengths to correspond with the elation time of each analyte on the polymeric C₁₈ column. The analytical results obtained from this procedure for the analysis of a shale oil sample [Standard Reference Material (SRM) 1580] and a petroleum crude oil (SRM 1582) are compared to values obtained by gas chromatography - mass spectrometry.     

Appropriate column configurations for the rapid analysis and semipreparative purification of the radiolabeled drug flutamide by high-performance liquid chromatography

Flutamide, marketed as Eulexin, is used for treatment of metastic prostatic carcinoma. Purity of a radiolabeled batch for metabolism studies was first determined by reversed-phase HPLC on a 5 microm, 150x4.6 mm analytical column. The separation was then scaled up to give a semipreparative column (5 microm, 250x10 mm) purification procedure. Fraction analysis was done on a short rapid analysis (5 microm, 50x3.0 mm) column. Analysis of the final product was performed on the analytical column. All columns were YMC-Pack ODS-AQ. The analytical work involved large mass injections in order to have the required amounts of radioactivity needed for accurate impurity profile determinations, and the preparative work involved masses much larger than the calculated scale-up values. Ultraviolet and radiochromatograms of the drug on the various column configurations are compared. A 95.7% recovery of product was obtained, with radiochemical purity increased from 95.0 to 99.8%.     

Trace-level determination of pesticides in food using difficult matrix introduction-gas chromatography-time-of-flight mass spectrometry

A procedure for the fully automated analysis of food samples by means of difficult matrix introduction-gas chromatography-time-of-flight mass spectrometry (DMI-GC-ToF MS) is discussed. After extraction, samples require very little clean-up and are injected in a micro- or micro-vial which is held in a liner. Next, the liner is placed in the injector and the contents of the vial are thermally desorbed and led directly to the capillary GC column. After GC-ToF MS analysis, the data are processed automatically using a peak deconvolution algorithm. The practicability of the procedure was demonstrated by analysing spiked grape and pineapple samples down to the 1-10 ng/g concentration level.     

Liquid chromatography of polymers under limiting conditions of desorption II. Tandem injection and quantitative molar mass determination

Liquid chromatography under limiting conditions of desorption (LC LCD) is a method which allows molar mass independent elution of various synthetic polymers. A narrow, slowly moving zone of small molecules, which promotes full adsorption of one kind of polymer species within column (an adsorli) acts as an impermeable barrier for the fast moving macromolecules. The latter accumulate on the barrier edge and elute nearly in total volume of liquid within column. At the same time, transport of less adsorptive macromolecules is not hampered so that these are eluted in the size exclusion (SEC) mode. As result, polymers differing in their polarity and adsorptivity can be easily separated without molar mass interference. Three methods of barrier creation are discussed and compared. It is shown that a fraction of sample may elute unretained if the adsorli sample solvent is used as a barrier in connection with a narrow-pore column packing. One part of excluded macromolecules likely breaks-out from the adsorli zone and this results in partial loss of sample and distortion of the LC LCD peaks. This problem can be avoided if the adsorli zone is injected immediately before sample solution. Applicability of the LC LCD method for polymer separation has been demonstrated with a model mixture of poly(methyl methacrylate) (adsorbing polymer) and polystyrene (non adsorbing polymer) using bare silica gel as a column packing with a combination of tetrahydrofuran (a desorption promoting liquid -a desorli) and toluene (adsorli). It has been shown that the LC LCD procedure with tandem injection allows simple and fast discrimination of polymer blend components with good repeatability and high sample recovery. For quantitative determination of molar masses of both LC LCD and SEC eluted polymers, an additional size exclusion chromatographic column can be applied either in a conventional way or in combination with a multi-angle light scattering detector. A single eluent is used in the latter column, which separates the mixed mobile phase, system peaks and the desorli zone from the polymer peaks so that measurements are free from disturbances caused by the changing eluent composition. The resulting LC LCD x SEC procedure has been successfully applied to poly(methyl methacrylate) samples.     

Confirmation of gentamicin and neomycin in milk by weak cation-exchange extraction and electrospray ionization/ion trap tandem mass spectrometry

A new procedure for the confirmation of two aminoglycoside antibiotics in milk was developed and validated. This work is among the early applications of ion trap mass spectrometry for regulatory methodology, and it incorporates a novel weak cation-exchange extraction. The procedure was validated for the confirmation of both gentamicin and neomycin at 30 ng ml(-1) and above. Milk is first treated with acid and centrifuged. The supernate, excluding the fat layer, is buffered with sodium citrate to neutral pH. The extract is applied to a weak cation-exchange solid-phase extraction column. Aminoglycosides are eluted with acidified methanol. Following separation by ion-pair liquid chromatography, analytes are ionized with an electrospray interface. Protonated molecular ions are selectively stored in an ion trap mass spectrometer, then collisionally dissociated to yield unique product ion spectra. Confirmation is based on matching spectral responses between samples and comparison standards consisting of a bona fide standard spiked into control extracts. Method performance was demonstrated with replicate samples of control milk, fortified milk, and milk containing incurred residues of each compound.     

Liquid chromatography of synthetic polymers under critical conditions of enthalpic interactions 4: Sample recovery

Reduced sample recovery is a frequent feature of LC of macromolecules under critical conditions of enthalpic interactions (LC CC). Several methods of assessment of LC CC sample recovery are compared. A novel approach is based on an online combination of the. The LC CC column with a noninteractive SEC column. It provides not only the amount but also the molar mass of the eluted/withheld polymer. The procedure was tested with poly(methyl methacrylate), bare silica gel column packings, and “critical eluent” tetrahydrofuran/toluene. It was shown that macromolecules with higher molar masses were preferentially trapped within the LC CC column packing so that the eluted part of the sample was no longer representative. The incomplete polymer elution can make the LC CC polymer analyses susceptible to significant experimental errors.     

Multi-stage chromatographic procedure for the isolation of a single oligomeric species (vinyl chloride tetramer) from poly(vinyl chloride) resin used for food packaging applications

A multi-stage chromatographic procedure is described whereby low-molecular-weight material initially obtained by soxhlet diethyl ether extraction from food grade poly(vinyl chloride) (PVC) resin, is fractionated to yield firstly a mixture of vinyl chloride oligomers and then a single species free from other impurities. A number of column chromatographic procedures were evaluated, and a combination of high-performance size exclusion columns using initially tetrahydrofuran solvent followed by toluene solvent were shown to produce the most effective separation. The isolation and purification of a vinyl chloride tetramer was monitored by capillary column gas chromatography with specific chlorine detection (Hall electrolytic conductivity detector) and the identification confirmed by mass spectrometry. Using this newly developed procedure 0.5 mg of vinyl chloride tetramer was isolated from a PVC base resin.     

Reversed phase high performance liquid chromatography of glycopeptides

A practical procedure for isolating and purifying glycopeptides is described, viz. enzymatic hydrolysis-gel permeation chromatography-ion exchange chromatography-reversed phase HPLC. Using this procedure 28 glycopeptides from hen ovalbumin have been isolated some of which hitherto have not been identified. Water was a suitable mobile phase for preparing pure glycopeptides, and control of column temperature was important for good separations and reproducible retention times. Structural confirmation was by fast atom bombardment mass spectrometry.     

Isolation and identification of the glucuronide conjugate of 2-hydroxydesipramine by preparative liquid chromatography

A semi-preparative column liquid chromatographic procedure for the isolation and purification of milligram quantities of the glucuronide conjugate of 2-hydroxydesipramine, a major metabolite of desipramine, is presented. Urine from patients receiving desipramine was collected and passed through a column of XAD-2 resin. The methanolic extract was chromatographed on a reversed-phase octadecyl semi-preparative column followed by further purification on a silica gel column of the same dimension, yielding a product 95% pure. Fast atom bombardment and thermospray mass spectroscopy, as well as ultraviolet photodiode-array spectroscopy and hydrolysis with beta-glucuronidase confirmed the identification and purity of 2-hydroxydesipramine glucuronide. This important glucuronide metabolite will be a useful tool as an authentic standard for pharmacokinetic and metabolism studies and for determining its pharmacological characteristics in laboratory animals.     

HPLC Determination of Glyphosate,Aminomethylphosphonic Acid,and Glufosinate Using a Hypercarb Porous Graphite Adsorbent

A method for the HPLC determination of glyphosate, aminomethylphosphonic acid, and glufosinate using the gradient separation of analytes on a Hypercarb porous graphitized carbon adsorbent and an aqueous solution of ammonium formate and ammonia as a mobile phase is proposed. Analytes are detected using quadrupole and three-quadrupole mass spectrometers. In order to increase the retention of the analytes, the chromatographic column is washed with water before the injection of a sample solution. This procedure results in a three- to fourfold increase in the retention factors of the analytes in comparison with the analogues described in the publications.     

Determination of amino acids by gas chromatography using a wide bore capillary column

A procedure is described in which a wide bore capillary column is used as an alternative to the more traditional packed column for the quantitative analysis of amino acids as their N-heptafluorobutyryl isobutyl ester (HBB) derivatives. The column, installed in a gas chromatograph previously configured for use with a packed column, is shown to give good reproducibility by repeated determination of amino acid response factors (RSD values for all amino acids are below 3%). A number of problems, encountered during the use of this column, are discussed and suitable techniques to overcome them are reported.     

Application of high-performance liquid chromatography to the purification, disintegration and molecular mass determination of pyruvate dehydrogenase multi-enzyme complexes from different sources

The pyruvate dehydrogenase complex is associated with the inner mitochondrial membrane. A gentle and rapid purification procedure, especially for the very unstable pyruvate dehydrogenase complex from the extremely thermophilic organism Thermus aquaticus, is described. This procedure is based essentially on a combination of hydrophobic interaction and of adsorption chromatography by the rapid fast protein liquid chromatographic technique. Applying the same method, a relative molecular mass of 9.1 . 10(6) daltons was obtained by gel filtration on Superose 6 HR 10/30 for the pyruvate dehydrogenase complex from T. aquaticus. The same column served to resolve the pyruvate dehydrogenase complex into its enzyme components.     

High-performance liquid chromatography of proteins on short capillary columns

A microchromatographic procedure for highly sensitive analysis of proteins has been developed by using short capillary columns with reverse-phase, ion-exchange, and hydrophobic interaction sorbents. The high analytical mass sensitivity on the level of several nanograms was attained by the miniaturization of the column and the optimization of gradient steepness.