CA1082311A - Method to generate correlative data from various products of thermal degradation of biological specimens - Google Patents
Method to generate correlative data from various products of thermal degradation of biological specimensInfo
- Publication number
- CA1082311A CA1082311A CA277,624A CA277624A CA1082311A CA 1082311 A CA1082311 A CA 1082311A CA 277624 A CA277624 A CA 277624A CA 1082311 A CA1082311 A CA 1082311A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/12—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using combustion
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0468—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components with means for heating or cooling the sample
- H01J49/0472—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components with means for heating or cooling the sample with means for pyrolysis
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Combustion & Propulsion (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Electron Tubes For Measurement (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analyzing Materials Using Thermal Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A B S T R A C T
In a method generating correlative data from various products of thermal degradation of biological specimens and comprising sequential steps for each specimen of degrading such specimen by heating such specimen so as to cause various products of thermal degradation of such specimen to be evolved, ionizing the products of thermal degradation of such specimen by a technique causing negligible fragmentation, detecting ion currents for such specimen, and record-ing such detected ion currents, an improvement is attained wherein each specimen is heated in an identical nonisothermal time-dependent healing sequence, wherein a three-dimensional array comprising a large and suffi-cient number of ion currents, which correspond to substantially all detectible ratios of mass-to-charge within a range at a large and sufficient number of successive instants during the respective healing sequences, are detected and recorded for each specimen, and representative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of the specimens.
Therein, one dimension of each array represents ion currents, another dimension of such array represents mass-to-charge ratios, and another dimension of such array represents specimen temperatures.
In a method generating correlative data from various products of thermal degradation of biological specimens and comprising sequential steps for each specimen of degrading such specimen by heating such specimen so as to cause various products of thermal degradation of such specimen to be evolved, ionizing the products of thermal degradation of such specimen by a technique causing negligible fragmentation, detecting ion currents for such specimen, and record-ing such detected ion currents, an improvement is attained wherein each specimen is heated in an identical nonisothermal time-dependent healing sequence, wherein a three-dimensional array comprising a large and suffi-cient number of ion currents, which correspond to substantially all detectible ratios of mass-to-charge within a range at a large and sufficient number of successive instants during the respective healing sequences, are detected and recorded for each specimen, and representative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of the specimens.
Therein, one dimension of each array represents ion currents, another dimension of such array represents mass-to-charge ratios, and another dimension of such array represents specimen temperatures.
Description
--`` 108Z39 ~L -This application represents a restatement of substantial aspects of the invention clisclose~ ln United States Patent No. 4,075,475 which issued on Eebruary 21, 1978.
.~
BACKGROUND 0~ THE INVENTION
This invention pertains generally to mass spectrometTy. This in~
vention pertains particularly to a method employing mass spectrometry to generate correlative data from various products of thermal degradation of biological specimens so as to facilitate their classification and their iden-tification.
a Prior methods employing mass spectrometry to generate correlative data concerning biological specimens are discussed in two significant pub-lished references:
, .
~1) H.L.C. Meuzelaar et al., "A Technique for Past and Reproducible ~ingerprinting of Bacteria by Pyrolysis Mass Spectrometry,"
_alytical ChemistryJ Vol 45, No. 3, March 1973, pages 587 ., .
et ~2) John P. Anhalt et al., "Identification of Bacteria Using Mass Spectrometry," Analytical Chemistry, Vol. 47, No. 2, February 1975, pages 219 et seq., 2Q also in:
3) Henry L. Friedman et al.; a paper presented to the American Chemical Society, Second Western Regional Mettings, October 16 - 19, 1966, as summarized in Chemical ~ En~ineering News, September 5, 1966, Thermochimica Acta 1 (1970), pages 199 l et seq. at 223 - 224; and Thermal ~ , 1 (1969~, pages ;i 405 et seq. at 408 - 409, .,j _ ;I Prior applications o~ mass spectrometr~ to other studies of re-~ latsd interest are discussed in three additional published references:
... , .
4) ~verett K. Gibson, Jr., et al., "Thermogravimetric-Quadrupole 3Q Mass-Spectrometric Analysis of Geochemical Samples,"
~i T ermochimica Acta, 4 (1972) pages 49 et seq.;
~5~ Everett K. Gibson, Jr., "Thermal Analysis-Mass Spectrometer ", -- 1 --.- ~
- ~)8231~
Comput~r System and its Application to the Evolved Gas Analysis o~ Grcen River Shale and Lunar Soil Samples,"
Thermochimica Acta, 5 ~1973), pages 243 et seq.; and (6) Alfred L. Yergey et al., "Nonisothermal Kinetics Studies of the Hydrodesulfurization of Coal," Industrial ~
En~ineering Chemistry, Process Design ~ Development, ~` Vol. 13, July 1974, pages 233 et seq.
,; As described generally in references (1) and ~2), prior methods to generate correlative data concerning bacterial specimens reflect such s~eps employing mass spectrometry for each specimen as degrading such speci-men by heating such specimen so as to cause various products of thermal degra-dation of such specimen to be evolved, ionizing such products, detecting ion.
~` currents corresponding to different ratios of mass-to-charge among such pro-. ,~. .
~ ducts, and recording such detected ion currents. Reference t2) describes a I methodology wherein each specimen contained in a melting point capillary tube was introduced into a heated ~on source operated at 300 - 350C. Reerence ~ ,, (1) describes a methodology wherein ferromagnetic wires of Curie points of 510C were used to heat a specimen. Reference ~3) discloses unsatisfactory ~`, results, which are evident from the summary in Thermal Analysis.
Reference ~1) suggests, at page 590, that ionization techniques causing negligible fragmentation such as field ionization, chemical ioniza-tion, or low-voltage electron-impact ionization may be useful. Further reference may be made to United States Patent No. 3,555,272, which issued January 12, 1971 to Munson et al, and which describes chemical ionization in substantial detail.
~` Re~erence ~4) and reference ~5) commonly disclose mass spectrometry ~' .
as applied to monltor and ldentify released gases, whose spectra are known, from individual samples of geochemical substances. Reference ~6) discloses -~ kenetic studies of hydrogen sulfide, whose spectra are known, as evolved from non-isothermally heated coal.
Herein, the term "correlative" means susceptible to cross-correla-tion by known manual and automated techniques.
~` 2 -5~
:, :
~ol3231~
~ hen bacterlal specimens and other complex biological specimers are heated gradually, such specimens have been .found to decompose in an orderly and reproducible manner, which has led to a substantially lmproved method for rapid identification Or such specimens. The method also has been found to be useful to study both normal and leukemic lymphocytes and other white blood cells.
The present invention provides in a method generating correlative data from various prod~cts, either known or unknown products, of thermal degradation of biological specimens and compromising sequential steps for each specimen of:
.; (a) degrading such specimen by heating such specimen so as to cause such products of thermal degradation of such specimen to be evolved;
(b) ionizing such products of thermal degradation of such speci-. _..... men by a technique causing negligible fragmentation;
(c) detecting ion currents corresponding to different mass-to-~; charge ratios among the products of thermal degradation of such specimen, and - ..
(d) recording such detected ion currents for such specimen;
. an improvement wherein:
~ 20 (e) each specimen is heated in an identical non-isothermal time-.. - dependent heating sequence;
; (f) a three-dimensional array comprising a large and suf~lclent number of ion currents, which correspond to substantlally all detectible ~..
ratlos of mass-to-charge wlthin a range at a large and su~ficlent number . .
of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, wherein one dimension ~ o~ each three-dimenslonc~l array represents ion~currents, another dimension `: of such three-dimensional array represents mass-to-charge ratios, and another dimension of such three-dimensional array represents specimen -::
:. 30 temperatures as a ~unction of process time; and tg) represen~ative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative _ ~_ ,: :
,:`
.: ~ , . . . . .
.: - :
Z3~
data from the three-dimensional array thus recorded for each other of such specimens.
The method follo~s prior methods insofar as sequential steps for each specimen of degrading such specimen by heating such specimen so as to cause various products of thermal degradation o such specimen to be evolved, ioni~ing such products by a technique causing negligible fragmentation, de-tecting ion currents corresponding to different ratios of mass-to-charge among such products, and recording such detected ion currents for such speci-men.
However, the method is improved wherein each specimen is heated in an identical non-isothermal time-dependent heating sequence, wherein a three-dimensional array comprising a large and sufficient number of ion currents, ` whlch correspond to substantially all detectible ratios of mass-to-charge ~; within a-range a~ alarge and suficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, and wherein representative data rom the three-dimensional array thus recorded for each one of such specimens are correlated to represen-tative data from the three-dimensional array thus recorded or each other of , 1 such specimens.
One dimension of each three-dimensional array represents ion cur-:
rents. Another dimension represents mass-to-charge ratios. Another dimension represents the specimen temperaturesias a unction o process time. Beore any correlations are made, the data rom each specimen precrably are re-constructed as representative two-dimensional arrays, wherein one dimension represents ion currents and another dimension represents specimen temperatures, for representative mass-to-charge ratio .
The method as thus improved has been successully used to discri-- minate certain bacteria and certain lymphocytes, as explained below, and is -,~,'"t expected to be generally useful to discriminate many types of biological organisms including bacteria, yeasts, molds, ungi, viruses, and unicellulars, , as well as biological tissues including lymphocytes, leukocytes, phagocytes, erythrocytes, and platelets.
~ .
~. ~
:' ~ 23~ ~
As an example of its utility, correlative data of ion current versus time-temperature have been successfully used to differentiate indivi-dual species (Arthrobacter oxydans and Arthrobacter ~lobiformis as actual examples) of a common bac~erial genus at one mass-to-charge ratio (m/e 549 in such examples), which appears to represent characteristic differences for such species, although plural ratios may have to be studied for successful differentiation of other specimens.
The foregoing and other objects, features, and advantages of this invention are made evident in the descriptions below of exemplary modes to carry out this invention, with the aid of the several accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic-block diagram of a typical arrangement of conventional apparatus which may be used in practicing this invention. Fig-ure 2 is a perspective view of a special heated solids probe tip which may be used with this invention in the apparatus of Figure 1.
Figures 3, 4, 5, and 6 are graphical depictions of typical data which may result from this invention and of possible utilization of such data to ~acilitate classification and identification of biological specimens.
Figures 7 through 10 depict the total ion current versus time or ,~.,~, temperature and one exemplary ion mass spectrum taken at a time corresponding to the leading edge portion of the total ion plot Eor two particular types oE
' ! I
bacteria in a process according to this invention. Figures 11 and 12 are plots of relatlve lon intensity versus time or temperature or various speci-Eic lon masses for each of the ~wo types of bacteria utilized and referenced in Figures 7 through 10.
~ igure 13 is a schematic-block diagram of an exemplary sequence that may be utilized or processing the recorded data resulting from this invention and classifying and distinguishing an unknown bacteria. Figure 14 is a schematic depiction of an exemplary sequence for grouping according to genus such types of bacteria giving rise to the data set forth in Figures 7 through 10.
.
.
~108Z3~L~
DETAIL~D DESCRIPTION OF ~XEMPLARY MODES
Figure 1 shows, in block diagram, one arrangement o~ conventional apparatus that may be used to practice the substantially improved method of this invention. The apparatus includes a quadrupole mass spectrometer 100, preferably a BIOSPECT chemical ionization mass spectrometer commercially avail-able formerly from Scientific Research Instruments Corporation of Baltimore, Maryland, and presently from its successor, Chemetron Corporation, Medical Products Division, which has a chemical ionization source 102, an ion analyzer-detector 104, and a heated solids probe 106 adapted to heat a solid-phase sample. An elec~rical heater ~not shown) is incorporated with the probe106. The spectrometer 100 is associated with a conventional control console 108.
As required by any such chemical ionization mass spectrometers, ~hich are characterized by negligible fragmentation of parent ions being ionized, a reagent gas input 110 is provided for a suitable reagent gas, which may be methane vapor, water vapor, or other reagents. In the preferred BIOSPECT spectrometer, such gas is directed internally of the source 102 to . ~ .
pass directly over the tip of the probe 106, so as to sweep molecular fragments being produced directly from the tip of the probe 106 into a zone wherein such ~ragments are ionized within the source 102.
As shown in Figure 1, a thermocouple output line 112 from the probe 106 is presented as one input to a comparator 114, and another input to the ~ comparator 114 is provided by a function signal generator 116. Thus, the tem-; perature of the probe 106 is compared with a time-dependent ~unction, which is predetermined and non-isothermal and preferably is a linear ramp function, and an~ deviation causes an output from the comparator 114 to a supply 118 of elec-trical energy to the heater ~not shown) so as to cause an increased or de-creased supply of electrical energy to the heater as necessary to cause the ~` temperature of the probe 106 and thus the temperature of a sample carried by the probe 106 to track the function provided by the generator 116.
The electrical output signals from the spectrometer are presented to a conventional recording system 122, which is controlled by a conventional ~.;
~, . . .
: . .... . : . . ;
```-` lV8Z311 keyboard unit 124, so as to record selected data corresponding to the analyzed and det~cted ions. The unit 124 may be associated with a cathode-ray-tube display uni~ and a hard-copy unit 126. Suitable equipment is com-mercially available from Systems Industries of Sunnyvale, California, and also from Hewlett-Packard and from the Kerns Group.
An alternative to the aforesaid probe is shown in Figure 2 to com-prise two half-cylinders 200 and 202 ~preferably formed of stainless steel) having mating semi-circular recesses 204 and 206 forming a well to receive a small glass cylinder 203 containing a solid-phase sample. A disc 210, which preferably is made of kovar , is provided as a suitable heating element at a lower end portion of the cylinder 208. A thermocouple 216, which has elec-trical leads 218 communicating through suitable passages 220 in the probe, is spot welded to a central bottom portion of the disc 210. The leads 212 and 214, ~hlch also serve physically to position the tip of the probeJ preferably are formed of approximately 20 gauge stainless steel. In the preferred ex-emplary emobidment, the tip has a diameter of approximately 0.156 inch, the slot has a height o approximately 0.175 inch and a width of approximately 0.132 inch and the disc or heating element has a thickness of approximately 0.0015 inch.
~ 20 When either the arrangment of conventional apparatus of Figure 1 or any other suitable apparatus are used to generate correlative data from various products~of thermal degradation of biological specimens, known methodology is employed lnsofar as each specimen is heated and thus degraded so as to cause varlous products oE thermal ~egradation of such specimen to be ,: l '~l evolved, such products are ionized by a technique causing negligible fragmen-~, ., ,j ~rl tatlon~ lon currents corresponding to different mass-to-charge ratios among ,`~ such products are detected, and such detected ion currents are recorded.
, `~ l ~ o~ever, these steps are substantially improved, as discussed below, by this ,;~ invention wherein it does not matter whether such products are known.
Each specimen is heated in an identical non-isothermal time-dependent sequence. A three-dimensional array comprising a large and suffi-.;,, *
Trademark ~: _ 7 _ .
~ 1(J823~L1 cient number o~ lon curr~nts, ~hlch correspond to substantially all detcct-ible ratios of mass-to-charge within a range at a large and suEficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen. For each three-dimensional array, one dimension represents ion currents, another dimension represents mass-to-charge ratios, and another dimension represents specimen temperatures as a function of process time.
Herein, various references to a large and sufficient number of ion currents at a large and sufficient number of successive instants means that a sufficiently large number of discrete data are detected and recorded that a surface may be visualized when the data are plotted in convenient coordinates.
-Actually, such a visualized surface is illusory, for no reason to allow the data to be interpolated is seen.
Representative data from the three-dimensional array thus recorded Por each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of such specimens. Be-fore any correlations are made, the data from each specimen may conveniently be reconstructed as representative two-dimensional arrays, wherein one dimen-sion represents ion currents and another dimension represents specimen tem-peratures for representative mass-to-charge ratios.
When the specimens are bacterial specimens, it has been found ad-vantageous for specimen temperature to be varled from a~bient temperature to approximately 400C at a gradient af 20C per minute, and for 8.3 second scans to be made within a range of detected mass-to-charge ratios from ap-proximately 250 atomic mass units to approximately 750 atomic mass units.
Approximately 75,000 data may thus be collected and displayed in various , :, ` 1arrays that may be analyzed to find characteristic plots for particular bac-terla.
- .:
'Por simplified initial approximation, it has been found advantage-1 ,, ous to display such two-dimensional arrays for each specimen, at diferent mass-to-charge ratios, whereby those arrays that may be posited as character-, ; .
istic may be visually isolated, and correlating representative data from such 10823~
arrays for respective specimens by known techniques (linear correlation as an example) at confidence levels appropriate for the volume of collected data for each specimen. ~or full implementation, computerized pattern-recognition techniques and other highly sophisticated techniques may be ~arranted.
It has been demonstrated, by actual experimentation with the sub-stantially improved method of this invention, that individual species of a common bacterial genus exhibit both common and dissimilar spectra in different ranges, that the genus may thus be fingerprinted for replicate identifications, and that individual species may thus be differentiated from one another with-in the genus. Comparable results were obtained for both normal and leukemic lymphocytes.
Example 1 ~ ive genera of bacteria, representing a diverse group of organisms, ~.
were studied. There were also related types, as the group also included two strains each of two species.
Organism ATCC no. Sequence Pseudomonas putida 12633 A
Pseudomonas putida 15073 B
Pseudomonas fluorenscens 13525 C
Escherichia coli 4147 D
Escherichia coli 11775 E
:! Baclllus megaterium 14581 ; Baclllus subtilis 6051 G
Arthrobacter oxydans14358 H
- Arthrobacter globiformis 8010 Erwinia amylovora 15580 J
Table 1 lists ten organisms obtained from American Type Culture Collection (ATCC~, Rockville, Maryland, along with the accession number of each organism. These ten organisms were prepared at the ATCC by using a 10%
innoculation of 250-ml nutrient broth, culturing for 24 h, harvesting the organisms, spinning down the cells, washing in 0.1 M Phosphate buffer, and :, _ g _ .
,: - . ~ , .
~)IS23~
lyopholyzing for 18 h in two vials. Upon receipt, the vials were s~ored at 4C. Samples for analysis were prepared by opening a sample vial, extract-ing a por~:ion of dried cells with a flamed sp~tula, placing th~ cells onto a flamed watch glass, replugging the sample vial, placing the vial into a flamed test tube, and sealing the tube with a sterile gauze swatch. Only one sample vial was open in the room at a time, and all items in contact with the cells were flamed between openings.
The results showed not only that similar bacteria can be grouped together in terms of their overall decomposition patterns, but that they could be distinquished ~rom each other. For example, Arthrobacter oxydans and Arthrobacter globiformis has similar plots at m/e 261, 2g9, 311, 314, and 523, but could clearly be distinguished by their differing plots at m/e 549.
- The method was even sensitive to differences between strains of the same species; one strain o Escherichia coli could be identified from its plot at . .
m~e 314, another Erom its plot at m/e 523. Other studies indicated that such results were highly reproducible.
Bxample II
In lymphocyte studies, white cells from leukemic patients and also ~rom normal donors, when analyzed as described above, showed significant dif-
.~
BACKGROUND 0~ THE INVENTION
This invention pertains generally to mass spectrometTy. This in~
vention pertains particularly to a method employing mass spectrometry to generate correlative data from various products of thermal degradation of biological specimens so as to facilitate their classification and their iden-tification.
a Prior methods employing mass spectrometry to generate correlative data concerning biological specimens are discussed in two significant pub-lished references:
, .
~1) H.L.C. Meuzelaar et al., "A Technique for Past and Reproducible ~ingerprinting of Bacteria by Pyrolysis Mass Spectrometry,"
_alytical ChemistryJ Vol 45, No. 3, March 1973, pages 587 ., .
et ~2) John P. Anhalt et al., "Identification of Bacteria Using Mass Spectrometry," Analytical Chemistry, Vol. 47, No. 2, February 1975, pages 219 et seq., 2Q also in:
3) Henry L. Friedman et al.; a paper presented to the American Chemical Society, Second Western Regional Mettings, October 16 - 19, 1966, as summarized in Chemical ~ En~ineering News, September 5, 1966, Thermochimica Acta 1 (1970), pages 199 l et seq. at 223 - 224; and Thermal ~ , 1 (1969~, pages ;i 405 et seq. at 408 - 409, .,j _ ;I Prior applications o~ mass spectrometr~ to other studies of re-~ latsd interest are discussed in three additional published references:
... , .
4) ~verett K. Gibson, Jr., et al., "Thermogravimetric-Quadrupole 3Q Mass-Spectrometric Analysis of Geochemical Samples,"
~i T ermochimica Acta, 4 (1972) pages 49 et seq.;
~5~ Everett K. Gibson, Jr., "Thermal Analysis-Mass Spectrometer ", -- 1 --.- ~
- ~)8231~
Comput~r System and its Application to the Evolved Gas Analysis o~ Grcen River Shale and Lunar Soil Samples,"
Thermochimica Acta, 5 ~1973), pages 243 et seq.; and (6) Alfred L. Yergey et al., "Nonisothermal Kinetics Studies of the Hydrodesulfurization of Coal," Industrial ~
En~ineering Chemistry, Process Design ~ Development, ~` Vol. 13, July 1974, pages 233 et seq.
,; As described generally in references (1) and ~2), prior methods to generate correlative data concerning bacterial specimens reflect such s~eps employing mass spectrometry for each specimen as degrading such speci-men by heating such specimen so as to cause various products of thermal degra-dation of such specimen to be evolved, ionizing such products, detecting ion.
~` currents corresponding to different ratios of mass-to-charge among such pro-. ,~. .
~ ducts, and recording such detected ion currents. Reference t2) describes a I methodology wherein each specimen contained in a melting point capillary tube was introduced into a heated ~on source operated at 300 - 350C. Reerence ~ ,, (1) describes a methodology wherein ferromagnetic wires of Curie points of 510C were used to heat a specimen. Reference ~3) discloses unsatisfactory ~`, results, which are evident from the summary in Thermal Analysis.
Reference ~1) suggests, at page 590, that ionization techniques causing negligible fragmentation such as field ionization, chemical ioniza-tion, or low-voltage electron-impact ionization may be useful. Further reference may be made to United States Patent No. 3,555,272, which issued January 12, 1971 to Munson et al, and which describes chemical ionization in substantial detail.
~` Re~erence ~4) and reference ~5) commonly disclose mass spectrometry ~' .
as applied to monltor and ldentify released gases, whose spectra are known, from individual samples of geochemical substances. Reference ~6) discloses -~ kenetic studies of hydrogen sulfide, whose spectra are known, as evolved from non-isothermally heated coal.
Herein, the term "correlative" means susceptible to cross-correla-tion by known manual and automated techniques.
~` 2 -5~
:, :
~ol3231~
~ hen bacterlal specimens and other complex biological specimers are heated gradually, such specimens have been .found to decompose in an orderly and reproducible manner, which has led to a substantially lmproved method for rapid identification Or such specimens. The method also has been found to be useful to study both normal and leukemic lymphocytes and other white blood cells.
The present invention provides in a method generating correlative data from various prod~cts, either known or unknown products, of thermal degradation of biological specimens and compromising sequential steps for each specimen of:
.; (a) degrading such specimen by heating such specimen so as to cause such products of thermal degradation of such specimen to be evolved;
(b) ionizing such products of thermal degradation of such speci-. _..... men by a technique causing negligible fragmentation;
(c) detecting ion currents corresponding to different mass-to-~; charge ratios among the products of thermal degradation of such specimen, and - ..
(d) recording such detected ion currents for such specimen;
. an improvement wherein:
~ 20 (e) each specimen is heated in an identical non-isothermal time-.. - dependent heating sequence;
; (f) a three-dimensional array comprising a large and suf~lclent number of ion currents, which correspond to substantlally all detectible ~..
ratlos of mass-to-charge wlthin a range at a large and su~ficlent number . .
of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, wherein one dimension ~ o~ each three-dimenslonc~l array represents ion~currents, another dimension `: of such three-dimensional array represents mass-to-charge ratios, and another dimension of such three-dimensional array represents specimen -::
:. 30 temperatures as a ~unction of process time; and tg) represen~ative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative _ ~_ ,: :
,:`
.: ~ , . . . . .
.: - :
Z3~
data from the three-dimensional array thus recorded for each other of such specimens.
The method follo~s prior methods insofar as sequential steps for each specimen of degrading such specimen by heating such specimen so as to cause various products of thermal degradation o such specimen to be evolved, ioni~ing such products by a technique causing negligible fragmentation, de-tecting ion currents corresponding to different ratios of mass-to-charge among such products, and recording such detected ion currents for such speci-men.
However, the method is improved wherein each specimen is heated in an identical non-isothermal time-dependent heating sequence, wherein a three-dimensional array comprising a large and sufficient number of ion currents, ` whlch correspond to substantially all detectible ratios of mass-to-charge ~; within a-range a~ alarge and suficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, and wherein representative data rom the three-dimensional array thus recorded for each one of such specimens are correlated to represen-tative data from the three-dimensional array thus recorded or each other of , 1 such specimens.
One dimension of each three-dimensional array represents ion cur-:
rents. Another dimension represents mass-to-charge ratios. Another dimension represents the specimen temperaturesias a unction o process time. Beore any correlations are made, the data rom each specimen precrably are re-constructed as representative two-dimensional arrays, wherein one dimension represents ion currents and another dimension represents specimen temperatures, for representative mass-to-charge ratio .
The method as thus improved has been successully used to discri-- minate certain bacteria and certain lymphocytes, as explained below, and is -,~,'"t expected to be generally useful to discriminate many types of biological organisms including bacteria, yeasts, molds, ungi, viruses, and unicellulars, , as well as biological tissues including lymphocytes, leukocytes, phagocytes, erythrocytes, and platelets.
~ .
~. ~
:' ~ 23~ ~
As an example of its utility, correlative data of ion current versus time-temperature have been successfully used to differentiate indivi-dual species (Arthrobacter oxydans and Arthrobacter ~lobiformis as actual examples) of a common bac~erial genus at one mass-to-charge ratio (m/e 549 in such examples), which appears to represent characteristic differences for such species, although plural ratios may have to be studied for successful differentiation of other specimens.
The foregoing and other objects, features, and advantages of this invention are made evident in the descriptions below of exemplary modes to carry out this invention, with the aid of the several accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic-block diagram of a typical arrangement of conventional apparatus which may be used in practicing this invention. Fig-ure 2 is a perspective view of a special heated solids probe tip which may be used with this invention in the apparatus of Figure 1.
Figures 3, 4, 5, and 6 are graphical depictions of typical data which may result from this invention and of possible utilization of such data to ~acilitate classification and identification of biological specimens.
Figures 7 through 10 depict the total ion current versus time or ,~.,~, temperature and one exemplary ion mass spectrum taken at a time corresponding to the leading edge portion of the total ion plot Eor two particular types oE
' ! I
bacteria in a process according to this invention. Figures 11 and 12 are plots of relatlve lon intensity versus time or temperature or various speci-Eic lon masses for each of the ~wo types of bacteria utilized and referenced in Figures 7 through 10.
~ igure 13 is a schematic-block diagram of an exemplary sequence that may be utilized or processing the recorded data resulting from this invention and classifying and distinguishing an unknown bacteria. Figure 14 is a schematic depiction of an exemplary sequence for grouping according to genus such types of bacteria giving rise to the data set forth in Figures 7 through 10.
.
.
~108Z3~L~
DETAIL~D DESCRIPTION OF ~XEMPLARY MODES
Figure 1 shows, in block diagram, one arrangement o~ conventional apparatus that may be used to practice the substantially improved method of this invention. The apparatus includes a quadrupole mass spectrometer 100, preferably a BIOSPECT chemical ionization mass spectrometer commercially avail-able formerly from Scientific Research Instruments Corporation of Baltimore, Maryland, and presently from its successor, Chemetron Corporation, Medical Products Division, which has a chemical ionization source 102, an ion analyzer-detector 104, and a heated solids probe 106 adapted to heat a solid-phase sample. An elec~rical heater ~not shown) is incorporated with the probe106. The spectrometer 100 is associated with a conventional control console 108.
As required by any such chemical ionization mass spectrometers, ~hich are characterized by negligible fragmentation of parent ions being ionized, a reagent gas input 110 is provided for a suitable reagent gas, which may be methane vapor, water vapor, or other reagents. In the preferred BIOSPECT spectrometer, such gas is directed internally of the source 102 to . ~ .
pass directly over the tip of the probe 106, so as to sweep molecular fragments being produced directly from the tip of the probe 106 into a zone wherein such ~ragments are ionized within the source 102.
As shown in Figure 1, a thermocouple output line 112 from the probe 106 is presented as one input to a comparator 114, and another input to the ~ comparator 114 is provided by a function signal generator 116. Thus, the tem-; perature of the probe 106 is compared with a time-dependent ~unction, which is predetermined and non-isothermal and preferably is a linear ramp function, and an~ deviation causes an output from the comparator 114 to a supply 118 of elec-trical energy to the heater ~not shown) so as to cause an increased or de-creased supply of electrical energy to the heater as necessary to cause the ~` temperature of the probe 106 and thus the temperature of a sample carried by the probe 106 to track the function provided by the generator 116.
The electrical output signals from the spectrometer are presented to a conventional recording system 122, which is controlled by a conventional ~.;
~, . . .
: . .... . : . . ;
```-` lV8Z311 keyboard unit 124, so as to record selected data corresponding to the analyzed and det~cted ions. The unit 124 may be associated with a cathode-ray-tube display uni~ and a hard-copy unit 126. Suitable equipment is com-mercially available from Systems Industries of Sunnyvale, California, and also from Hewlett-Packard and from the Kerns Group.
An alternative to the aforesaid probe is shown in Figure 2 to com-prise two half-cylinders 200 and 202 ~preferably formed of stainless steel) having mating semi-circular recesses 204 and 206 forming a well to receive a small glass cylinder 203 containing a solid-phase sample. A disc 210, which preferably is made of kovar , is provided as a suitable heating element at a lower end portion of the cylinder 208. A thermocouple 216, which has elec-trical leads 218 communicating through suitable passages 220 in the probe, is spot welded to a central bottom portion of the disc 210. The leads 212 and 214, ~hlch also serve physically to position the tip of the probeJ preferably are formed of approximately 20 gauge stainless steel. In the preferred ex-emplary emobidment, the tip has a diameter of approximately 0.156 inch, the slot has a height o approximately 0.175 inch and a width of approximately 0.132 inch and the disc or heating element has a thickness of approximately 0.0015 inch.
~ 20 When either the arrangment of conventional apparatus of Figure 1 or any other suitable apparatus are used to generate correlative data from various products~of thermal degradation of biological specimens, known methodology is employed lnsofar as each specimen is heated and thus degraded so as to cause varlous products oE thermal ~egradation of such specimen to be ,: l '~l evolved, such products are ionized by a technique causing negligible fragmen-~, ., ,j ~rl tatlon~ lon currents corresponding to different mass-to-charge ratios among ,`~ such products are detected, and such detected ion currents are recorded.
, `~ l ~ o~ever, these steps are substantially improved, as discussed below, by this ,;~ invention wherein it does not matter whether such products are known.
Each specimen is heated in an identical non-isothermal time-dependent sequence. A three-dimensional array comprising a large and suffi-.;,, *
Trademark ~: _ 7 _ .
~ 1(J823~L1 cient number o~ lon curr~nts, ~hlch correspond to substantially all detcct-ible ratios of mass-to-charge within a range at a large and suEficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen. For each three-dimensional array, one dimension represents ion currents, another dimension represents mass-to-charge ratios, and another dimension represents specimen temperatures as a function of process time.
Herein, various references to a large and sufficient number of ion currents at a large and sufficient number of successive instants means that a sufficiently large number of discrete data are detected and recorded that a surface may be visualized when the data are plotted in convenient coordinates.
-Actually, such a visualized surface is illusory, for no reason to allow the data to be interpolated is seen.
Representative data from the three-dimensional array thus recorded Por each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of such specimens. Be-fore any correlations are made, the data from each specimen may conveniently be reconstructed as representative two-dimensional arrays, wherein one dimen-sion represents ion currents and another dimension represents specimen tem-peratures for representative mass-to-charge ratios.
When the specimens are bacterial specimens, it has been found ad-vantageous for specimen temperature to be varled from a~bient temperature to approximately 400C at a gradient af 20C per minute, and for 8.3 second scans to be made within a range of detected mass-to-charge ratios from ap-proximately 250 atomic mass units to approximately 750 atomic mass units.
Approximately 75,000 data may thus be collected and displayed in various , :, ` 1arrays that may be analyzed to find characteristic plots for particular bac-terla.
- .:
'Por simplified initial approximation, it has been found advantage-1 ,, ous to display such two-dimensional arrays for each specimen, at diferent mass-to-charge ratios, whereby those arrays that may be posited as character-, ; .
istic may be visually isolated, and correlating representative data from such 10823~
arrays for respective specimens by known techniques (linear correlation as an example) at confidence levels appropriate for the volume of collected data for each specimen. ~or full implementation, computerized pattern-recognition techniques and other highly sophisticated techniques may be ~arranted.
It has been demonstrated, by actual experimentation with the sub-stantially improved method of this invention, that individual species of a common bacterial genus exhibit both common and dissimilar spectra in different ranges, that the genus may thus be fingerprinted for replicate identifications, and that individual species may thus be differentiated from one another with-in the genus. Comparable results were obtained for both normal and leukemic lymphocytes.
Example 1 ~ ive genera of bacteria, representing a diverse group of organisms, ~.
were studied. There were also related types, as the group also included two strains each of two species.
Organism ATCC no. Sequence Pseudomonas putida 12633 A
Pseudomonas putida 15073 B
Pseudomonas fluorenscens 13525 C
Escherichia coli 4147 D
Escherichia coli 11775 E
:! Baclllus megaterium 14581 ; Baclllus subtilis 6051 G
Arthrobacter oxydans14358 H
- Arthrobacter globiformis 8010 Erwinia amylovora 15580 J
Table 1 lists ten organisms obtained from American Type Culture Collection (ATCC~, Rockville, Maryland, along with the accession number of each organism. These ten organisms were prepared at the ATCC by using a 10%
innoculation of 250-ml nutrient broth, culturing for 24 h, harvesting the organisms, spinning down the cells, washing in 0.1 M Phosphate buffer, and :, _ g _ .
,: - . ~ , .
~)IS23~
lyopholyzing for 18 h in two vials. Upon receipt, the vials were s~ored at 4C. Samples for analysis were prepared by opening a sample vial, extract-ing a por~:ion of dried cells with a flamed sp~tula, placing th~ cells onto a flamed watch glass, replugging the sample vial, placing the vial into a flamed test tube, and sealing the tube with a sterile gauze swatch. Only one sample vial was open in the room at a time, and all items in contact with the cells were flamed between openings.
The results showed not only that similar bacteria can be grouped together in terms of their overall decomposition patterns, but that they could be distinquished ~rom each other. For example, Arthrobacter oxydans and Arthrobacter globiformis has similar plots at m/e 261, 2g9, 311, 314, and 523, but could clearly be distinguished by their differing plots at m/e 549.
- The method was even sensitive to differences between strains of the same species; one strain o Escherichia coli could be identified from its plot at . .
m~e 314, another Erom its plot at m/e 523. Other studies indicated that such results were highly reproducible.
Bxample II
In lymphocyte studies, white cells from leukemic patients and also ~rom normal donors, when analyzed as described above, showed significant dif-
2~ ferences in their respective decomposition patterns. The findings suggest ;~ that the genetlc and morphological differences seen in lymphocyte~ examined by other methods reflect the differences in molecular composition observed by the substantially improved method of this invention.
In ~igures 13 and 14, the dlamonds represent points where separa-tions occur, the organisms separated out appear in circles, and the organisms appearing in ~oxes are organisms unseparated.
~ gure 13 is an exemplary flow char~ for differentiationsO For the .. . .
single ion plots at m/e 259, it is clear that organisms E and F are different ~rom all the others present and from each other, organisms H and I are dif-3a ferent from the remainlng 6, but similar to each other, and that organisms A, B, C, D, E, and J resemble each other. For the single ion plot of m/e 261, it is clear that organism A is differentiated from B, C, D, E, and J, . :
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1al~23~
but no separation of ~l and I occurs. Those separated previously are no longerconsidered, but are carried on the chart for uniformity. For the single ion plot of m~e 299, organism C is separat0d, and so forth for the other plots.
It is clear from the diagram that the organisms listed in Table 1 can be dis-tinguished from each other.
It is also possible to classify or group the data into genera. Fig-ure 14 shows the flow chart for this grouping. The speci~ic ion time or tem-perature profiles can be used to obtain a posteriori, the normal taxonomic re-lationships between the organisms. For the single ion plot of m/e 259, it is clear that organisms ~ and I are similar to each other and different from all others as found for the two representatives of genus Arthrobacter (this pair-; ing is seen in most of the other single ion plots), organisms F and G are dif-ferent from each other, but different in a rather specific way from the other six as well. Finally, organisms A, B, C, D, E, and J are all fairly similar.
The plot for m/e 276 separates organism J from organisms A, B, C, D, and E
.,..~, - ; (looking especially at the higher scan numbers) and is the representative of genus Erwinia. As found, m/e 308 shows the grouping for organisms F and G, the genus Bacillus organisms, m/e 313 shows that organisms D and E, genus .
Escherichia, are grouped thereby leaving the three organisms in genus Pseudomonas grouped by default. To summarize, Pseudomonas has a particular shape at m/e 257 that is shared by Escherichia and Erwinia, but is difEerent at m/e 276, which groups Erwinia, and at m/e 313, which groups Escherichia.
Both the differentiation and grouping of ~igures 13 and 1~ are ln-tended to show the operational prlnclples of the pattern recognition tech-nlques and are not meant to be limitive in the use of any particular mass number.
The substantlally improved method of this invention is expected to be significant to the practice of clinical bacteriology. By classical methods, it can take from two days to three months to identify an organism.
,, , ~ 30 The substantially improved method of this invention may allow bacterial iden-;
` tification in less than twenty minutes after a colony of cells have been ' taken from a suitable nutrient medium. It also may be easily adaptable to ` ' , .
:;
" - ~ -31~
rapid identi~ication of other organisms s-uch as yeasts, mold, fungi, and viruses.
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In ~igures 13 and 14, the dlamonds represent points where separa-tions occur, the organisms separated out appear in circles, and the organisms appearing in ~oxes are organisms unseparated.
~ gure 13 is an exemplary flow char~ for differentiationsO For the .. . .
single ion plots at m/e 259, it is clear that organisms E and F are different ~rom all the others present and from each other, organisms H and I are dif-3a ferent from the remainlng 6, but similar to each other, and that organisms A, B, C, D, E, and J resemble each other. For the single ion plot of m/e 261, it is clear that organism A is differentiated from B, C, D, E, and J, . :
-r' ~ ' ! ' . ;
1al~23~
but no separation of ~l and I occurs. Those separated previously are no longerconsidered, but are carried on the chart for uniformity. For the single ion plot of m~e 299, organism C is separat0d, and so forth for the other plots.
It is clear from the diagram that the organisms listed in Table 1 can be dis-tinguished from each other.
It is also possible to classify or group the data into genera. Fig-ure 14 shows the flow chart for this grouping. The speci~ic ion time or tem-perature profiles can be used to obtain a posteriori, the normal taxonomic re-lationships between the organisms. For the single ion plot of m/e 259, it is clear that organisms ~ and I are similar to each other and different from all others as found for the two representatives of genus Arthrobacter (this pair-; ing is seen in most of the other single ion plots), organisms F and G are dif-ferent from each other, but different in a rather specific way from the other six as well. Finally, organisms A, B, C, D, E, and J are all fairly similar.
The plot for m/e 276 separates organism J from organisms A, B, C, D, and E
.,..~, - ; (looking especially at the higher scan numbers) and is the representative of genus Erwinia. As found, m/e 308 shows the grouping for organisms F and G, the genus Bacillus organisms, m/e 313 shows that organisms D and E, genus .
Escherichia, are grouped thereby leaving the three organisms in genus Pseudomonas grouped by default. To summarize, Pseudomonas has a particular shape at m/e 257 that is shared by Escherichia and Erwinia, but is difEerent at m/e 276, which groups Erwinia, and at m/e 313, which groups Escherichia.
Both the differentiation and grouping of ~igures 13 and 1~ are ln-tended to show the operational prlnclples of the pattern recognition tech-nlques and are not meant to be limitive in the use of any particular mass number.
The substantlally improved method of this invention is expected to be significant to the practice of clinical bacteriology. By classical methods, it can take from two days to three months to identify an organism.
,, , ~ 30 The substantially improved method of this invention may allow bacterial iden-;
` tification in less than twenty minutes after a colony of cells have been ' taken from a suitable nutrient medium. It also may be easily adaptable to ` ' , .
:;
" - ~ -31~
rapid identi~ication of other organisms s-uch as yeasts, mold, fungi, and viruses.
. ,` ' ~, ;'. ~:
' . .:
; ~ :
, ~ ~
: ~ :
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Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. In a method generating correlative data from various products, either known or unknown products, of thermal degradation of biological specimens and compromising sequential steps for each specimen of:
(a) degrading such specimen by heating such specimen so as to cause such products of thermal degradation of such specimen to be evolved;
(b) ionizing such products of thermal degradation of such specimen by a technique causing negligible fragmentation;
(c) detecting ion currents corresponding to different mass-to-charge ratios among the products of thermal degradation of such specimen; and (d) recording such detected ion currents for such specimen;
an improvement wherein:
(e) each specimen is heated in an identical non-isothermal time-dependent heating sequence;
(f) a three-dimensional array comprising a large and sufficient number of ion currents, which correspond to substantially all detectible ratios of mass-to-charge within a range at a large and sufficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, wherein one dimension of each three-dimensional array represents ion currents, another dimension of such three-dimensional array represents mass-to-charge ratios, and another dimension of such three-dimensional array represents specimen tem-peratures as a function of process time; and (g) representative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of such specimens.
(a) degrading such specimen by heating such specimen so as to cause such products of thermal degradation of such specimen to be evolved;
(b) ionizing such products of thermal degradation of such specimen by a technique causing negligible fragmentation;
(c) detecting ion currents corresponding to different mass-to-charge ratios among the products of thermal degradation of such specimen; and (d) recording such detected ion currents for such specimen;
an improvement wherein:
(e) each specimen is heated in an identical non-isothermal time-dependent heating sequence;
(f) a three-dimensional array comprising a large and sufficient number of ion currents, which correspond to substantially all detectible ratios of mass-to-charge within a range at a large and sufficient number of successive instants during the respective time-dependent heating sequences, are detected and recorded for each specimen, wherein one dimension of each three-dimensional array represents ion currents, another dimension of such three-dimensional array represents mass-to-charge ratios, and another dimension of such three-dimensional array represents specimen tem-peratures as a function of process time; and (g) representative data from the three-dimensional array thus recorded for each one of such specimens are correlated to representative data from the three-dimensional array thus recorded for each other of such specimens.
2. The improvement of claim 1 wherein, before any correlations are made, the data from each specimen are made, the data from each specimen are reconstructed as representative two-dimensional arrays, wherein one dimension represents ion currents and another dimension represents specimen tempera-tures, for representative mass-to-charge ratios.
3. The improvement of claim 2 wherein the specimens are bacteria.
4. The improvement of claim 3 wherein the range of detected mass-to-charge ratios extends from approximately 250 atomic mass units to approximate-ly 750 atomic mass units.
5. The improvement of claim 2 wherein the specimens are lymphocytes.
6. The improvement of claim 1 wherein the specimens are from a sub-group of a group of biological organisms consisting essentially of these subgroups:
(i) bacteria; (ii) yeasts; (iii) molds; (iv) fungi; (v) viruses; and (vi) unicellulars.
(i) bacteria; (ii) yeasts; (iii) molds; (iv) fungi; (v) viruses; and (vi) unicellulars.
7. The improvement of claim 1 wherein the specimens are from a sub-group of a group of biological tissues consisting essentially of the sub-groups: (i) lymphocytes; (ii) leukocytes; (iii) phagocytes; (iv) erythrocytes; and (v) platelets.
8. The improvement of claim 1 wherein the range of detected mass-to-charge ratios extends from approximately 250 atomic mass units to 750 atomic mass units.
9. The improvement of claim 1 wherein the data from some specimens are reference data.
10. The improvement of claim 1 wherein the total ion current as a function of the temperature of the specimen also is detected and recorded for each one of the specimens and also is correlated to the total ion current detected and recorded in like manner for each other of the specimens.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/682,781 US4075475A (en) | 1976-05-03 | 1976-05-03 | Programmed thermal degradation-mass spectrometry analysis method facilitating identification of a biological specimen |
| US682,781 | 1976-05-03 | ||
| US05/777,366 US4122343A (en) | 1976-05-03 | 1977-03-14 | Method to generate correlative data from various products of thermal degradation of biological specimens |
| US777,366 | 1996-12-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1082311A true CA1082311A (en) | 1980-07-22 |
Family
ID=27102962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA277,624A Expired CA1082311A (en) | 1976-05-03 | 1977-05-02 | Method to generate correlative data from various products of thermal degradation of biological specimens |
Country Status (3)
| Country | Link |
|---|---|
| JP (2) | JPS531081A (en) |
| CA (1) | CA1082311A (en) |
| DE (1) | DE2718880A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3907120B2 (en) * | 2004-02-05 | 2007-04-18 | 株式会社リガク | Sample support device for X-ray analysis and X-ray analysis device |
| JP5170650B2 (en) * | 2008-02-29 | 2013-03-27 | 株式会社リガク | Gas quantitative analysis method and gas quantitative analysis apparatus |
-
1977
- 1977-04-28 DE DE19772718880 patent/DE2718880A1/en not_active Ceased
- 1977-05-02 CA CA277,624A patent/CA1082311A/en not_active Expired
- 1977-05-04 JP JP5172477A patent/JPS531081A/en active Pending
- 1977-05-04 JP JP5172577A patent/JPS52138992A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS52138992A (en) | 1977-11-19 |
| DE2718880A1 (en) | 1977-11-24 |
| JPS531081A (en) | 1978-01-07 |
| JPS611708B2 (en) | 1986-01-20 |
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