John F. Holland

Rapid computerized identification of compounds in complex biological mixtures by gas chromatography—mass spectrometry

Abstract A new method has been developed for rapid identification of compounds in complex mixtures by gas chromatograph—mass spectrometer—computer system. The method uses a gas chromatographic retention index in conjunction with a small set of discriminating ions to identify each compound. The retention index predicts to within 1% the location of a time-dependent “window” within which a specific compound may appear, while the accuracy of the peak identification is dependent primarily upon the specificity of the ion set chosen. Optimum specificity is obtained by selecting ions which not only are chracteristic of the compound to be identified, but which also differentiate it from other compounds likely to elute at similar retention times. Utilizing the data files created by repetitive scanning during a single elution by gas-liquid chromatography, the present algorithm permits the automated identification of approximately eight compounds per minute. Examples of the identification of acidic urinary metabolites are given; however, the method is sufficiently general to be applied to any group of volatile compounds.

Models for mass-independent space and energy focusing in time-of-flight mass spectrometry

Abstract Various models for improved resolving power in time-of-flight mass spectrometry have been considered. A model for dynamic post-source acceleration has been developed in an attempt to achieve mass-independent space and energy focusing. The model incorporates all pertinent parameters including the extraction voltage, the accelerating and field-free region lengths, and the dynamic acceleration function. Mathematical analysis and computer simulations based on equations from this model have been used to develop a new post-source acceleration technique. Simulations indicate that concurrent space and energy focusing is possible, and that unit resolving power can be achieved up to at least 2000 u using typical gaseous source conditions and readily attainable instrumental parameters.

Design, construction, and evaluation of an integrating transient recorder for data acquisition in capillary gas chromatography/time‐of‐flight mass spectrometry

An integrating transient recorder (ITR) has been designed, constructed, and evaluated to accomplish time‐array detection in gas chromatography time‐of‐flight mass spectrometry (GC‐TOFMS) applications. The ITR consists of a 200‐MHz flash analog‐to‐digital converter, 16 high‐speed 100K emitter‐couple logic (ECL) summing boards, three parallel processors for real‐time data reduction, instrument control and routing functions, and a 300‐Mbyte mass storage device. The ITR is capable of recording 80 μs bursts of transient information with a time resolution of 5 ns. For each transient, up to 16 384 sequential time‐resolved channels may be recorded. An operator‐selectable number of sequential transients may be summed in a locked time registry creating a summed scan file while maintaining the integrity of the transient time resolution. The information from each transient is read, summed, and stored in one of two summing registers (16×1024×24 bits). While incoming information is being stored in one summing register,...

Beam deflection for temporal encoding in time-of-flight mass spectrometry.

The pulsed ion sources used in conventional time-of-flight mass spectrometry (TOFMS) generally do not provide adequate resolving power across the mass range required for applications such as gas chromatography combined with mass spectrometry (GC/MS). Theoretical and experimental aspects of beam deflection techniques, which provide time encoding for TOFJMS with continuous ions sources, are explored here. In this approach, ion source conditions do not affect resolving power, allowing for a greater variety of ionization modes to be used. Theoretical predictions for the resolving power attainable with beam deflection, which are satisfactory for GC/MS applications, agree well with experimentally determined values. The combination of GC-beam deflection-TOFMS with time-array detection is evaluated, and the capabilities of this system are compared to those of scanning mass spectrometers.

Automated qualitative and quantitative metabolic profiling analysis of urinary steroids by a gas chromatography-mass spectrometry-data system.

A computer system (MSSMET), using methylene unit retention indices for an off-line reverse library search analysis of selected ion chromatograms from gas chromatography-mass spectrometry data, has been applied to the qualitative and quantitative determination of urinary steroids. Several published methods for the isolation and derivatization of urinary steroids were evaluated for reproducibility using fused silica capillary column gas chromatography. Using a procedure that gave the greatest reproducibility, MSSMET analyses of urinary steroids were evaluated with packed (3-m 3% OV-101) and capillary (50-m OV-101 WCOT fused silica) columns. Most urinary steroids could be accurately quantitated using the packed column. However, urinary steroids with similar mass spectra and retention behavior on a packed column (i.e., androsterone and etiocholanolone, or 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 beta-pregnane-20-one and 3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 alpha-pregnane-20-one) were completely separated using the capillary column and could be reproducibly quantitated with a 2-sec scan cycle time (10-15 data points across a peak) but not with a longer scan cycle time. Overloading was the major problem encountered with the fused silica capillary column.

A segmented ring, cylindrical ion trap source for time-of-flight mass spectrometry.

An ion trap source has been designed for use with time-of-flight (TOF) mass analysis. Two thin diaphragms make up a segmented ring electrode; the end cap electrodes are planar wire mesh. The potential field produced by the rf voltage applied between the ring and end cap electrodes resembles that of the cylindrical ion trap. The trapped ion population for ions created by electron impact exhibits linear growth against a first-order loss that has a time constant of about 50 µs; no ion loss occurs when the electron beam is off. The observed value of qz at low-mass cutoff for rf ion storage is −0.84. Pulsed extraction of all ions is accomplished by switching the trap electrodes from rf to voltages required to provide a linear dc extraction field. The TOF flight path includes a wide energy range reflectron. Better than unit mass resolution is achieved through m/z 500 without collisional ion cooling. With an extraction rate of 1 kHz and a recording rate of 4 spectra per second, a linear working curve is obtained between 36 pg and 18 ng of chlorobenzene delivered chromatographically. The system has demonstrated the potential to achieve a very high sample utilization efficiency at high spectral generation rates.

Advanced instrumentation and strategies for metabolic profiling

This review summarizes the instrumental techniques applicable for metabolic profiling analysis. Since its origin with the work of Roger Williams, use has been made of planar chromatography, column liquid chromatography, electrophoresis, gas-liquid chromatography, and mass spectrometry to obtain analyte patterns. Strategies for the analysis of various metabolites including organic acids, steroids, drugs, bile acids, carbohydrates, and proteins are discussed.

Mass dependence of time-lag focusing in time-of-flight mass spectrometry—an analysis

Abstract When using mass spectral detection strategies in which wide ranges of m/z values are monitored, it is important for ions of all masses of interest to be in focus during the data collection interval. In conventional time-of-flight mass spectrometry of gaseous samples using dual field source extraction for spatial compensation, ion focus is a function of the time lag and the mass-to-charge ratio of the ion. The mass dependence of ion focus in a CVC 2000 time-of-flight mass spectrometer has been studied using a computer simulation in order to determine the mass ranges over which the losses in mass resolution and peak intensity are acceptable. As few as three different values of time lag are required to cover the m/z range from 50 to 700 u. Selection of optimum extraction voltages involves a trade-off between the width of the mass windows for any given time lag setting and the total mass range available with unit-mass-resolution.

MS/MS by time-resolved magnetic dispersion mass spectrometry

Abstract The modification of a magnetic sector mass spectrometer to provide measurement of Ion flight time results in a novel instrument which can provide all the data presently obtained from tandem MS/MS instrumentation. The combination of ion momentum and velocity analysis provides unambiguous mass assignments for parent and daughter ions while flight time comparison establishes parent-daughter relationships. Data has been obtained from a modified LKB-9000 which demonstrates the feasibility of this technique. Suggestions for an upgraded instrument wlth automated scanning are made and the possibility of achieving full MS/MS data collection on the chromatographic time scale through the use of an integrating transient recorder is presented.

Physical properties of murine fibroblasts with altered acyl chain unsaturation.

Abstract Murine fibroblasts, LM cells, were cultured in suspension with laurate (12:0), myristate (14:0), palmitate (16:0), palmitoleate (16:1), or palmitate + palmitoleate (16:0 + 16:1) bound to fatty acid-free bovine serum albumin. Supplementation with saturated fatty acids decreased the ratio of unsaturated/saturated fatty acids in membrane phospholipids as much as 3.4-fold (palmitate-enriched cells). Concomitantly fluorescence polarization, absorption-corrected fluorescence, and relative fluorescence efficiency of the fluorescence probe molecule, β-parinaric acid, increased 1.5-, 2.9-, and 1.8-fold, respectively, in the membrane phospholipids. Unsaturated fatty acid (palmitoleate) increased the unsaturated/saturated fatty acid ratio by 20% but did not significantly alter the fluorescence parameters. When the cells were fed mixtures of palmitate and palmitoleate, the unsaturated/saturated fatty acid ratio of the membrane phospholipids and the above fluorescence parameters had values intermediate between those if each fatty acid had been fed separately. All fatty acid supplements caused a loss of two characteristic temperatures in Arrhenius plots of relative fluorescence efficiency. However, no shifts or appearance of new characteristic temperatures occurred. The break points at approximately 42, 37, and 22 °C were essentially un-altered. The data were consistent with the possibility that LM cells were unable to maintain constant fluidity, as indicated by fluorescence polarization, when supplemented with different fatty acids. A good correlation could be made between the phospholipid unsaturated/ saturated fatty ratio, the fluorescence polarization, and the toxicity elicited by different fatty acid supplements.