Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND APPARATUS FOR DETERMINING
ANALYTES IN VARIOUS MATRICES
1. FIELD OF THE INVENTION
The present invention relates generally to methods and apparatus (e.g.,
devices, systems, test kits) for conducting chemical analyses, and more
particularly to:
a) analytical systems (e.g., test kits which include
apparatus, membranes} and reagent(s)) whereby membranes}
are utilized to separate selected analyte(s) from other matter
present in a complex matrix (e.g., a foodstuff, oil,
pharmaceutical/cosmetic preparation, biological fluid, etc.);
b} analytical apparatus (e.g., sample processing apparatus
and other hardware components in combination with
membrane(s)) useable to qualitatively or quantitatively determine
one or more analytes in a complex matrix; and,
c} analytical methods for qualitatively or quantitatively
determine one or more analytes in a complex matrix;
d) novel chemical tests for qualitative and/or quantitative
determination of certain analytes.
11. RELATED APPLICATIONS
This patent application is a continuation-in-part of United States Patent
Applications Serial No. 08/723,636 filed on October 2, 1996, and claims
priority
to United States Provisional Patent Application Serial 60/063,038 filed on
October 22, 1997, the entire disclosures of which are expressly incorporated
herein by reference.
III. BACKGROUND OF THE INVENTION
Applicant's earlier-filed United States Patent Application Serial No. -
08/723,636, (sometimes referred to herebelow as the "parent application")
describes certain methods and apparatus for determining the presence of one
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or more analytes in a complex matrix (i.e., a matrix which includes many
diverse
physical and/or chemical species, some or all of which may interfere with the
intended analysis). The types of complex matrices in which applicant's
analytical
methods and apparatus may be used include foods, biological fluids (e.g. blood
cerebrospinal fluid), cosmetic preparations, pharmaceutical preparations, etc.
The methods and apparatus described in parent application Serial No.
08/723,636 include a test apparatus which generally comprise a) a first sample-
receiving chamber, b) a second filtrate-receiving chamber fluidly connected to
the first sample receiving chamber, and c) one or more membranes positioned
l0 between the first and second chambers. Initially, a quantity of the
flowable,
analyte-containing matrix is dispensed into the first chamber. The sample is
then caused to flow through the membranes) which remove selected matter
(particles, large molecules, secondary analytes, etc.) from the matrix, and
the
resultant filtrate is allowed to pass into the second chamber. After the
filtrate has
entered the second chamber, reagents) is/are added to such filtrate to
facilitate
qualitative or quantitative determination (spectrophotometric, visual, etc.)
of
primary analyte contained within the filtrate. In instances where multiple
membranes have been employed, one or more of those membranes may have
been used for the purpose of capturing one or more secondary analyte(s) which
were present within the matrix along with the primary analyte. In those
instances, the analyte-capturing membranes may subsequently be removed, and
the secondary analyte(s) may then be eluted (e.g, released, washed} from those
capture membranes and into secondary receiving chamber(s). Appropriate
reagents are then added to the eluant(s) contained within the secondary
receiving chambers) to facilitate qualitative or quantitative determination
(e.g.,
spectrophotometric, visual) of the secondary analyte(s).
Applicant has now devised a number of improvements, additions and
modifications to the test methods and apparatus described in parent
application
Serial No. 08/723,636, and such improvements, additions and modifications are
described and claimed in this continuation-in-part application.
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IV. SUMMARY Of= THE INVENTION
The present invention provides apparatus, systems and methods for
determining analytes in various types of samples (i.e., matrices).
In accordance with the invention, there are provided certain apparatus for
non-electrophoretic testing of samples, such apparatus generally comprising a)
one or more vessels) for receiving samples}, b) one or more membrane
modules which are positioned in alignment with the sample vessels) such that
sample will pass through the membrane(s), and c) one or more filtrate
receiving
vessels positioned in alignment with the membrane modules, to receive filtrate
which has passed through the membranes. Various numbers of membrane
modules may be used, stacked one upon another, to remove particles,
interferants or other unwanted matter from the sample and/or to capture
certain
analyte(s) for subsequent elution from the capture membrane and determination
by suitable visual or analytical means. These test apparatus may include
positive or negative pressure apparatus to crate differential pressure within
the
apparatus for driving the sample(s0 through the membranes. Also, these
apparatus may have a) specialized pressure equalization ports to ensure
efficient and complete processing of all samples, b) selective engagement
apparatus for engaging and disengaging the membrane modules and other
components to/from one another and to form substantially air tight seals
therebetween when assembled, c) specific configurations to allow the membrane
modules and other components to nest or register with one another in a manner
which facilitates proper orientation and functional positioning of all
components,
d) specific construction and mounting of membranes to deter tearing or rupture
of the membranes during operation, and to maximize the functional surtace area
of the membrane(s), and e) structural attributes which hold multiple membranes
in close-spaced, stacked relation to each other during operation.
Further in accordance with the invention, there are provided systems and
test kits as listed in Appendix I. The systems and test kits comprise specific
membranes}, preparation reagent(s), eluant(s)(if necessary} and analytical
reagent(s) for use in connection with the above-sumarized apparatus, in
determining specific analyte(s) in specific types of matrices.
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Still further in accordance with the invention, there are provided certain
novel chemical tests for histamine, sulfite and/or bisulfite, free fatty
acids, and
lipid peroxides, as detailed herein and shown in Appendix I.
Further aspects and particulars of the present invention will become
apparent to those of skill in the art upon reading and understanding the
following
detailed description of the preferred embodiments and examples and
consideration of the accompanying drawings.
V. BRIEF DESCRIPTION OF THE DRAWING S AND APPENDICES
A. Ft ores
Figure 1 is a flow diagram of a general method of the present invention,
for detecting a single analyte.
Figure 2 is a flow diagram of a general method of the present invention,
for detecting multiple analytes.
Figure 3 is a flow diagram of a general method of the present invention,
for detecting an anafyte which is present at low (e.g., sub-detectable)
concentration in a complex matrix.
Figure 4 is a flow diagram of a general method for utilizing one or more
of the analytical methods of Figures 1, 2 and/or 3 to obtain a prediction as
to the
shelf life or other parameter of the sample matrix.
Figure 5 is a perspective view of a first embodiment of a test apparatus
of the present invention.
Figure 5a is an exploded perspective view of the apparatus of Figure 5.
Figure 6 is a cut-away, side elevational view of the apparatus of Figure
5, showing the manner in which varying numbers of membranes may be
employed in order to determine varying numbers of analytes.
Figure 7 is top plan view of a secondary membrane module useable in the
apparatus of Figure 5.
Figure 8 is a top plan view of a primary membrane module useable in the
apparatus of Figure 5.
Figure 9 is a transverse sectional view of the secondary membrane
module of Figure 7.
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Figure 10 is an exploded perspective view of a second embodiment of a
test apparatus of the present invention.
Figure 10a is a showing of the test apparatus of Figure 10 from an angle
which allows one to visualize the undersides of the component parts of the
apparatus, and wherein modified plate-type membrane modules have been
incorporated.
Figure 11 is a schematic, sectional view of the a third embodiment of a
test apparatus of the present invention.
Figure 12 is an exploded, side elevational view of a fourth embodiment
of a test apparatus of the present invention.
Figure 12a is a bottom plan view of one of the membrane modules of the
apparatus of Figure 12.
Figure 13 a is an exploded view of an alternative membrane module
useable in the apparatus of Figure 12.
Figure 13a is an enlarged, cut-away, perspective view of a single
membrane cell of the alternative membrane module of Figure 13.
Figure 14a is a perspective view of a vacuum base apparatus useable
with some of the test apparatus of the present invention, wherein the top
cover
of the vacuum base apparatus is in an open position.
Figure 14b is a perspective view of a vacuum base apparatus of Figure
14a, with its top cover in a closed position.
Figure 15a is a perspective view of one component of a fifth embodiment
of a test apparatus of the present invention.
Figure 15b shows the component of Figure 15a from an angle which
allows one to see the test tube-receiving cavities formed within that
component.
Figure 15c is a perspective view of another component of the fifth
embodiment of the test apparatus shown in Figures 15a-15b.
Figure 15d showes the component of figure 15c from an angle which
allows one to see the underside of that component.
Figure 15e shows yet another component of the fifth embodiment of the
test apparatus shown in Figures 15a-15d.
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Figure 16 is a perspective view of a multi-use vacuum base apparatus
which is useable in conjunction with various ones of the test apparatus of the
present invention.
Figure 17 is a schematic showing of a dipstick testing apparatus of the
present invention.
B. Appendices
In addition to Figures 1-17, the following appendices are also included
within this patent application:
Appendix I is a table listing a number of preferred test methods/kits of the
present invention.
Appendix II is a key to the acronyms used to designate specific
membranes, reagents, and substances in the table of Appendix I.
Appendix III is a table listing commercially available membranes useable
in the test methods/kits of Appendix I.
Appendix IV is a table listing algor'tthms which are useable in conjunction
with certain test kit & methods of the present invention to predict or discern
certain parameters, such as shelf life, presence of contaminants, potential
for
oxidative degradation, etc, in accordance with the general method diagram of
Figure 4.
VI. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Throughout the following detailed description, the preferred embodiments
and examples referred to should be considered as exemplars, rather than
limitations on the apparatus and methods of the present invention. Although
applicant has described certain exemplary embodiments herebelow, it will be
apparent to those having ordinary skill in the art that a number of changes,
modifications, or alterations to the invention as described herein may be
made,
none of which depart from the spirit of the present invention. All such
changes,
modifications and alterations should therefore be seen as within the scope of
the
present invention.
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A, General Methodoloov
The methods of the present invention range in complexity from a basic
method whereby the presence of a single analyte may be qualitatively
determined to a complex method whereby a plurality of different analytes may
be quantitatively determined from a single analytical sample.
i, General Method for DetermJning a Single Analyte
Figure 1 shows a flow diagram of a basic method of the present invention
wherein a single analyte may be qualitatively and/or quantitatively determined
within a complex matrix (i.e., a matrix which contains one or more materials
other than the analyte).
fnitialiy, the complex matrix is prepared and, if necessary, is combined
with added solvent or liquid to form a prepared matrix for subsequent
processing. In instances where the complex matrix is a solid material (e.g.,
food)
it will typically be necessary to grind or chop the complex matrix and to add
a
solvent, digestant, or other carrier liquid such that the "prepared matrix"
will be
in the form of a slurry or suspension.
For many applications of the invention, and in particular those wherein it
is desired to detect specific analytes present in solid matrices such as
foods, a
digester/stabilizer solution including enzymes) and/or stabilizers) and/or
chelator(s) may be added to the matrix during the preparation step to extract
or
dissolve the desired analyte(s). Examples of digesters which may be included
in such solution include lipase enzymes and protease enzymes, and certain
proprietary digester/stabilizer formulations as described in parent
application
Serial No. 08/723,636. Examples of chelators which may be included in such
solution include EDTA. One particular digester/stabilizer solution which may
be
utilized has the following formulation:
Isopropanol........70% by weight
Tween 20...........2.0% by weight
EDTA................. 0.1% by weight
Mannitol...............10 mM
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After the matrix sample has been prepared to a ffowable state, it is
passed through a membrane which removes or retains extraneous matter (e.g.,
solid particles or interfering substances such as proteins) while allowing a
filtrate,
which contains the analyte, to pass therethrough. In many instances, the
membrane will be in the form of a micro-porous cellulose or polymer film
having
a desired pore size (e.g., 0.2-0.6 microns, and typically about .45 microns)
which
will filter out large proteins and relatively large solid particles while
allowing
relatively small solid particles and the accompanying liquid containing the
analyte to pass therethrough. One example of a membrane which may be used
for this purpose is a membrane formed of mixed cellulose ester film having
0.45
micron pores formed therein (e.g., ME-25 Membrane, Schleicher & Schuell
GmbH, P.O. Box 4, D37582, Dassel, Germany).
The analyte-containing filtrate which passes through the membrane is
subsequently mixed with one or more reagents to provide a filtrate/reagent
admixture from which the desired qualitative and/or quantitative determination
of the analyte may be performed.
Thereafter, the filtrate/reagent admixture is subjected to the desired
analytical or measurement techniques to provide the intended qualitative
and/or
quantitative determination of the analyte. In some instances, this
determination
of the analyte may be made by a simple chemical test whereby a visual
indicator
{e.g., a color change) will indicate the presence and/or concentration of the
analyte. In other instances, the determination of the analyte will be carried
out
by one or more analytical instruments, such as a colorimeter,
spectrophotometer, optical densitometer, fluorometer, etc.
Thus, the general method illustrated in the flow diagram of Figure 1
provides a means for qualitatively and/or quantitatively measuring an analyte
which is present within a complex matrix.
ii. General Method For Detecting Multiple Analytes
Figure 2 shows a more elaborate genera! method of the present invention
wherein it is desired to analyze two (2) separate analytes present within a
complex matrix. The complex matrix in this example may be the same as that
described hereabove with respect to Figure 1 (e.g., food), and the method of
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preparing the complex matrix and the optional addition of solvent or liquid
may
be carried out in the same manner.
Thereafter, the prepared matrix is passed through a first membrane which
retains or removes extraneous matter while allowing a filtrate, which contains
both analytes a and b, to pass therethrough. As described hereabove, the first
membrane may comprise a microporous membrane having known pore size so
as to remove particles of solid matter which are larger than the membrane pore
size, while allowing smaller particles of solid matter and the accompanying
liquid
containing Analytes A and B, to pass therethrough. As in the example of Figure
1, one such membrane may be formed of mixed cellulose ester film (e.g., ME-25
Membrane, Schleicher & Schuell GmbH, P.O. Box 4, D37582, Dassel,
Germany).
Thereafter, the filtrate which has passed through the first membrane will
be subsequently passed through a second membrane. This second membrane
is adapted to capture and hold Analyte B, while allowing a sub-filtrate
containing
Analyte A to pass therethrough. in this manner, the second membrane serves
to separate and remove Analyte B from Analyte A.
The Analyte A-containing sub-filtrate which has passed through the
second membrane will be thereafter combined with a reagent to provide a sub-
filtrate/reagent admixture from which qualitative and/or quantitative
determination
of Analyte A may be performed.
Thereafter, the desired qualitative and/or quantitative determination of
Analyte A is performed on the sub-filtrate/reagent admixture in the same
manner
as described hereabove with respect to Figure 1.
The second membrane, which contains Analyte B, may be removed or
relocated and a flush solution, capable of releasing and carrying Analyte B
from
the second membrane, will be passed therethrough. Such passage of the flush
solution through the second membrane will provide an eluant of known volume,
which contains Analyte B.
Thereafter, the eluant containing Analyte B is combined with a reagent to
provide an eluant/reagent admixture from which Analyte B may be qualitatively
and/or quantitatively determined.
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Thereafter, the qualitative and/or quantitative determination of Analyte B
is pertormed on the eluant/reagent admixture in the manner described
hereabove with respect to Figure 1. Thus, the example shown in Figure 2
provides a method whereby two separate analytes may be qualitatively and/or
quantitatively determined in a complex matrix.
It will be appreciated that, although Figure 2 provides an example wherein
only two analytes (e:g., Analyte A and Analyte B) are determined, it will be
possible to determine any desired number of analytes in accordance with the
present invention by providing additional secondary membranes in series with
the "second membrane" shown in Figure 2, so as to capture and collect each of
the desired analytes. Thereafter, flush solutions may be passed through each
of these secondary membranes to provide eluants containing each of the
individual analytes. Those eluants may then be combined with reagents and
subjected to the desired qualitative and/or quantitative determinations for
the
desired analytes.
iii. General Method For Detecting Analyte(s)
Present At Low Concentrations
Figure 3 shows another example of a method of the present invention
wherein it is desired to qualitatively or quantitatively determine the
presence of
a single analyte, which is present in a complex matrix at a concentration
below
the detection limits for the analytical procedure to be used.
In the example shown in Figure 3, the complex matrix is prepared and
optionally combined with solvent or liquid in the same manner as described
hereabove with respect to Figures 1 and 2.
Thereafter, the prepared matrix is passed through a first membrane which
will retain extraneous matter, while allowing a filtrate containing the
Analyte A to
pass therethrough. This first membrane may be the same type of first
membrane described hereabove with respect to Figures 1 and 2.
Thereafter, the filtrate, which contains Analyte A, is passed through a
second membrane. The second membrane is operative to capture and hold
Analyte A, while allowing the remaining fractions) of the filtrate to pass
therethrough as a sub-filtrate, which is subsequently discarded.
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The second membrane, which contains Analyte A, is then relocated and
positioned over a well or containment vessel, and a known volume of flush
solution is passed therethrough. The volume of flush solution which is passed
through the second membrane will be less than the volume of filtrate which had
previously been passed through the first membrane. Passage of this flush
solution through the second membrane will release and carry Analyte A from the
second membrane. In this manner, there is provided an eluant/reagent
admixture wherein Analyte A is contained at a concentration which is higher
that
the original concentration of the Analyte A in the filtrate which passed
through
l0 the first membrane. Thus, Analyte A is now present in the eluant at a
concentration which is high enough to be detected or measured by the desired
analytical procedure or method.
Accordingly, the desired qualitative and/or quantitative determination of
Analyte A is performed on the eluant/reagent admixture, in the manner
described hereabove with respect to Figures 1 and 2.
Thereafter, well known mathematical principles may be utilized to
calculate the concentration at which Analyte A was present in the original
complex matrix, although Analyte A was subsequently concentrated into the
eluant/reagent admixture at higher concentrations capable of being detected or
determined by the desired analytical procedure.
lv. General Methodology for Predlctlng
Changes In a Sample
Figure 4 shows a bock diagram of a general method whereby the test
methods and apparatus of the present invention may be used to predict the
occurrence of certain changes (e.g., oxidation, other degradation, spoilage)
which a sample is likely to undergo within a given time period. These
techniques
may be used as predictors of . shelf life, propensity for oxidative
degradation,
presence of contaminants, etc. Specific examples of this general method are
set forth in detail herebelow.
In these predictive procedures, the sample is initially prepared (e.g,
ground, chopped, macerated, digested, dissolved, etc) as necessary and is
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optionally combined with a solvent or liquid in the same manner as described
hereabove with respect to Figures 1, 2 and 3.
Thereafter, aliquots of the prepared sample are placed in separate
vessels. One sample is subjected to a stress (e.g., heat, light, air, etc.)
Which
is known to promote the particular change which is sought to be predicted.
(e.g.,
oxidation, degradation, etc.)
Thereafter, one or more analytes indicative of the change sought to be
predicted, are determined in the stressed and un-stressed aliquots, using one
or more of the general methods shown in Figures 1, 2 and 3 and generally
described hereabove.
The results of the analyte determinations are then processed by way of
an algorithm or formula, to arrive at the desired prediction as to whether the
sample will undergo the particular change ((e.g., oxidation, degradation,
etc.)
Within a particular time period. Examples of specific algorithms which are
useable in this regard are shown in the table of Appendix IV.
In this manner, the test kits/methods of the present invention may be
adapted and used to provide predictions of shelf fife, stability, color
longevity,
etc.
B. Preferred Ap ap ratus
Figures 4-16 show various embodiments of apparatus which are useable
to perform the analytical methods of applicant's invention. Set forth
herebelow
are detailed descriptions of each of the exemplary embodiments shown in the
drawings.
i. First Embodiment of test Apparatus
Referring to Figures 4-9, the first embodiment of the test apparatus 10
generally comprises the following components: a) a vacuum base 16, b) a test
tube rack 14, c) a cover 12, d) membrane modules) 18, 20, and e) lids 24. As
described in the following paragraphs, these components of the apparatus 10
are configured and constructed to be assembled and disassembled in a -
particular manner to facilitate the pertormance of analytical tests in
accordance
with applicant's above-described methodologies.
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The vacuum base 16 comprises a housing having a cavity 17 formed
therein and opening though the top of the base 16. A vacuum port 32 is formed
in the base 16 to permit a vacuum line to be attached to the base for the
purpose of drawing a partial vacuum within the cavity 17. A seal 30, such as
an
oval-shaped O-ring, is mounted about the upper opening of the cavity 17, as
shown.
The test tube rack 14 has a plurality of test-tube receiving slots into which
test tubes 15 are inserted. The test tube rack 14 with the test tubes 15
inserted
therein is then inserted downwardly into the cavity 17 of the base, as can be
appreciated from the exploded view of Figure 5. Finger passage notches 34 are
. formed on either side of the cavity 17 to permit the users fingers to pass
freely
into the cavity 17 on either side of the test tube rack 14 when inserting or
removing the test tube rack 14.
The cover 12 comprises a generally flat member having a series of
sample ports 13 formed therein. The sample ports are located and configured
such that they will be in direct alignment with the mouths of the test tubes
15,
when the cover 12 and test tube rack 14 are properly mounted within the
apparatus 10. Also, the sample ports 13 have rims 28 which are configured to
receive and hold one or more membrane modules 18, 10 thereon.
The membrane modules 18, 20 are of two (2) basic types--primary
membrane modules 20 and secondary membrane modules 18. The primary
membrane module 20 has a sample-receiving well 21 formed therein and
incorporates a membrane 52 a which typically serves to remove particles, large
molecules or other unwanted matter from the matrix as the sample passes
therethrough. The secondary membrane modules) 18 incorporate
membranes) 52b which typically serve either to a) capture secondary analyte(s)
for subsequent analysis, b) capture a primary analyte which is present in the
matrix at low (e.g., sub-detectible) concentrations to permit such analyte to
be
subsequently concentrated and determined (i.e., qualitatively detected or
quantitatively analyzed), or c) remove specific contaminants (e.g., metals)
which
were not removed by the first membrane and which require a different type of
membrane to be captured and removed. Thus, the primary membrane module
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20 is used in most if not all applications of the apparatus 10, while the
secondary
membrane modules) 18 are used only when a) two or more analytes are to be
determined or b) the primary analyte is present in the matrix in low
concentrations and must be subsequently concentrated to permit its
determination.
As shown specifically in Figures 5, 6, 7 and 8, the primary and secondary
membrane modules 20, 18 are formed partially of a hard polymer HP such as
polypropylene, polystyrene or polyethylene and partially of an eiastomer EM
such as a natural or synthetic rubber or similar material. This dual resin
construction may be accomplished by co-molding techniques whereby the first
(i.e., hard) resin is shot into the mold and, thereafter, the second (i.e.,
elastomeric) material is shot into the same mode so as to become adherent
upon or fused with the first (i.e., hard) resin. In this manner the preferred
two-
material construction described above, can be accomplished in a single mold
with minimal manual operation and handling. Aiternativelt, this dual resin
construction may be accomplished by a two (2) step "over molding" process
which is known in the art of injection molding.
The elastomeric EM portions of the membrane modules 20, 18 are
configured and located to abut against the adjacent membrane modules) 20,
18 and/or against the adjacent sample port rim 28, to effect a substantially
air-
tight seal therebetween. The sealing contact between the membrane modules
20, 18 and the sample port rims 28 may be facilitated by the interaction of
connector members 40, 42 formed thereon. In this regard, the rim 28 of each
sample port 13, and of each secondary membrane module 18, are provided with
first connector members such as projections 40. Each primary and secondary
membrane module 20,18 is also provided with corresponding second connector
members such as slots 42, into which the first connector members 40 will
insert
and engage to thereby hold the primary and secondary membrane modules 20,
18 in stacked, sealing contact upon each sample port 13, as shown.
The number of secondary membrane modules 18 mounted on each -
sample port 13 may vary (i.e. from zero upward) depending on the number of
analytes to be determined. In this regard, the primary membrane module 20 is
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typically located on the top of the stack such that the flowing matrix will
pass
through the membrane 52a of the primary membrane module before passing
through the membranes 50b of the secondary membrane modules) 18.
Because different types of membranes 52a, 52b are used to perform different
tests, the primary and secondary membrane modules 20,18 may be color coded
or otherwise marked for easy identification of the type of membrane 52a, 52b
present hereon. The membrane 52a, 52b or each membrane module 20, 18 is
attached (e.g., by heat fusion, adhesive or other acceptable means) to
membrane support structure such as a ring, flange or cross-members 50a, 50b
formed within each membrane module 20, 18. A central attachment projection
41 extends downwardly from support corss-members 50a, 50b, and such
projection 41 is fused or affixed to the membrane 52a, 52b of that membrane
module 18, 20. In this manner, as shown in Figure 9, the center of each
membrane 52a, 52b is suspended from the attachment projection 41 and the
membrane 52a, 52b is thereby deterred from rupturing or blowing out as the
flowable sample is being drawn downwardly through the membrane 52a, 52b.
At the same time, however, the membrane will remain substantially unattached
to the undersides of the cross-members 50a, 50b and flowable sample is
permitted to flow into and occupy a gap 43 which exists between the membrane
52a, 52b and the adjacent cross-members 50a, 50b. This serves to avoid the
diminution in effective surface area of the membrane 50a, 50b as would occur
if the membranes 52a, 52b were fused or affixed directly to the cross-members
50a, 50b. Such maximization of the effective area of the membrane 52a, 52b
will serve to promote rapid flow of filtrate (or sub-filtrate) through each
membrane 52a, 52b..
The lids 22 are mountable in sealing contact on the rim 20 or each
primary membrane module 20. A limited air inflow port 24 is formed in each lid
22 to permit a controlled amount of make-up air to pass into each sample-
receiving well. These contrplled flow ports 24 may comprise holes with
segments of tubing inserted therewithin. The size of the lumen of each such
segment of tubing may be selected to provide the desired limitation or
constriction on the flow of air which enters each sample-receiving well 21. In
the
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particular.embodiment shown, which is designed for simultaneous processing
of six (6) samples, the inflow rate through each flow port 24 is preferably no
greater than 5/6 the capacity of the vacuum pump used to pull negative
pressure
within the apparatus 10, as described more fully below. In this manner, the
provision of these controlled flow ports 24 will ensure that, even when the
liquid
within five (5) of the six (6) sample-receiving wells 21 has been fully drawn
through the membranes 52a, 52b and into the test tubes 15, the amount of
make-up air received through those five (5) depleted sample-receiving wells 21
will not be so large as to completely nullify the capability of the vacuum
pump to
pull adequate negative pressure to draw the remaining liquid through the
filter
and/or membranes of the remaining sixth sample-receiving well 21.
It will be appreciated that although the apparatus 10 shown in the
attached drawing is designed for simultaneous processing of six (6) samples,
the
apparatus 10 may alternatively be designed to process any desired number of
samples. However, since this particular embodiment of the apparatus requires
handling and mounting of the individual membrane modules 20, 18 and lids 22,
it will typically be used for relatively small numbers of samples (e.g., less
than
24). Another embodiment 1 Oa (described herebelow and shown in Figure 7) is
more sued for simultaneous processing of large numbers (e.g., more than 24)
samples.
In operation of the first embodiment of the apparatus 10 shown in Figures
4-9, a suction or vacuum tube is connected to the vacuum port 32 of the base
16, an d a test tube rack 14 containing clean test tubes 15 is inserted into
the
cavity 17 of the base 16. Thereafter, the desired primary and secondary
membrane modules 20, 18 are mounted in firm sealing engagement on the
sample ports 13, and the cover 12 is mounted in firm sealing contact on the
base
16. fn some applications clamps, rubber bands, screws, or other connector
apparatus (not shown) may be applied to hold the cover 12 in firm sealing
contact with the seal member 30 of the base 16. In other applications, the
cover
12 may be constructed to snap fit or othervvise mount in sealing contact with
the
seal member 30 without the use of such connector apparatus.
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After the cover 12 has been mounted on the base 16, quantities of the
flowable samples) are dispensed into the sample-receiving cavity 21 of each
primary membrane module 20, and the lids 22 are applied. Thereafter, the
vacuum source is actuated and negative pressure is formed within the cavity 17
of the base 16. This negative pressure within the apparatus 10 causes the
quantities flowable samples) dispensed into the sample-receiving cavities 21
to
flow downwardly through the first membrane 52a, through and secondary
membranes) 52b, and the resultant filtrate then collects within the test tubes
15.
Thereafter, the cover 12 is removed, and the test tube rack 14 (with the
filtrate-containing test tubes 15) is removed. The desired reagent (s) is/are
then
mixed with the filtrate contained in the test tubes 15, and the reagent-
filtrate
admixture is then subjected to the appropriate analytical technique (e.g.,
spectrophotometry, visual comparison to color chart or color wheel, etc.} to
qualitatively or quantitatively determine the first analyte in the filtrate.
Thereafter, clean test tubes 15 may be inserted into the rack 14 and the
rack 14 replaced in the cavity 17 of the base 16. The first membrane modules
are removed and discarded. The cover 12, having the second membrane
modules 18 mounted on its sample ports 13 is then once again mounted in
sealing contact upon the base 16. A quantity of an agent or eluant capable of
20 releasing or eluting the second analyte from the second membrane 52b, is
then
dispensed into the release agent receiving cavities 19 of the secondary
membrane modules 18, and the lids 22 are placed in sealing contact upon the
second membrane modules 18. The vacuum pump is then used to once again
draw negative pressure within the apparatus 10, thereby causing the eluant to
flow downwardly through the second membranes 52b and thereby eluting the
second anaiyte from the second membranes 52b. The eluant/second analyte
mixture is then received within the clean test tubes 15. The vacuum pump is
turned off, the test tube rack 14 is removed, and appropriate reagents) are
then
mixed with the eluant/second analyte contained within the test tubes 15. The
desired reagent (s) is/are then mixed with the eluant/second analyte contained
in the test tubes 15, and the eluant/second anaiyte/reagent admixture is then
subjected to the appropriate analytical technique (e.g., spectrophotometry,
visual
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-1 &
comparison to color chart or color wheel, etc.) to qualitatively or
quantitatively
determine the second analyte in the eluant.
It will be appreciated that this process may then be repeated for each
additional secondary membrane module 20 used, to determine N additional
analytes within the samples.
if. Second Embodiment of Test Apparatus
Referring to Figure 10 a second embodiment of the test apparatus 10a
generally comprises a) a vacuum base 100, b) a receiving unit 102 having 24
filtrate-receiving wells 109 formed therein, c} plate-type membrane modules
l0 104a, 104b, 104c, each having multiple (e.g. twenty-four(24)) cavities with
bottom openings and membranes 108a, 108b, or 108c mounted transversely
within such botom openings, and d} a cover 106 having 24 individual air inlet
ports 115 formed therein.
The receiving unit 102 is inserted into the base 100, and the membrane
modules 104a, 104b, 104c are stacked upon the receiving unit such that the
individual cavities and membranes of each membrane module 104 are in direct
alignment with each other and with the filtrate-receiving wells 109 of the
receiving unit. Quantities of sample are initially deposited in sample-
receiving
wells 107 formed on the upper surface of the first membrane module 104a and
the lid 106 is placed in sealing contact with the rim 111 of the base 100, and
each individual air inlet port 115 formed in the lid 106 is positioned to
provide an
air inlet into one of the sample-receiving wells 107 of the first (upper)
membrane
module 104a. . Thereafter, a source of negative pressure is connected to a
port
(not shown} formed in the base so as to create negative pressure within the
cavity 113 of the base 100. This negative pressure causes each sample to be
drawn downwardly through the membranes 108a, 108b and 108c positioned
under that receiving well 107, and the resultant filtrate to be received in
the
filtrate-receiving well 109 positioned under those membranes. In this manner,
this second embodiment of the test apparatus 10a may be used to
simultaneously process up to 24 separate samples.
Typically, the membranes 108a of the first membrane module 104a are
for the purpose of filtering out or removing interferants, particles or other
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.19- . _
unwanted matter while the membranes of any secondary membrane modules
104b, 104c are for capturing analytes for subsequent concentration and/or
analysis. Accordingly, after the initial filtrate has been received in the
filtrate
receiving wells 109, the vacuum source is terminated or disconnected,
differential pressure within the apparatus 10a is allowed to equalize to a
point
where removal of the lid 115 will not cause substantial upward buldging or
rupture of the membranes 108a,108b,108c, and the lid 115 is removed. All of
the membrane modules 104 are then removed and the first membrane module
104a with the captured particles, interferants and/or other unwanted matter is
discarded.
Thereafter, the receiving unit 102 is removed and appropriate reagents)
are added to the filtrate contained within the filtrate receiving wells 109 to
provide
a filtrate-reagent admixture from which a desired first analyte (Analyte A)
may
be qualitatively or quantitatively determined.
In applications where secondary plate-type membrane modules 104b
and/or 104c are used, such secondary membrane modules 104b, 104c will
typically have captured secondary analyte(s) (Analytes B, C, etc...) which are
to
be subsequently released from the membranes 108b, 108c and thereafter
concentrated and/or determined. In furtherance of this, a clean receiving unit
102 may be inserted into the cavity 113 of the base 100, and one of the
secondary membrane modules 104b or 104c is then positioned on top of the
new receiving unit 102 such that each membrane 108b or 108c is positioned
over a receiving well 109. A known volume of flush solution or eluant os then
placed in the cavity above each membrane 108b or 108c, and the lid 115 is
replaced such that it is in sealing contact with the base 100 and the air
inlet
openings 115 are in alignment with each cavity on the membrane module 104b
or 104c. The vacuum source is then reenergized or reconnected to the base to
cause a differential pressure to be once again established within the
apparatus
1 Oa. In this manner the flush solution or eluant is drawn downwardly through
the
membranes 108b or 108c so as to extract or release the captured analyte(s)
from the membranes 108b or 108c. An eluant/analyte mixture is thus received
within each receiving well, and the above described procedure is repeated to
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qualitatively or quantitatively determine that analyte in the elunt/analyte
mixture
within each receiving well.
The same procedure is then repeated for each secondary membrane
module 104b, 104c until all analytes have been determined.
Modified Plate-type Membrane Modules
Figure 10a shows another view of the above-described second
embodiment of the test apparatus 10a(mod) wherein modified plate-type
membrane modules 104a',104b', 104c' have been incorporated. Each of these
modified plate-type membrane modules 104a', 104b', 104c' are formed of two
(2) materials--a hard polymer HP and an elastomer EM. Specific examples of
the preferred hard polymer HP and elastomer EM are referred to above in
relation to the first embodiment (Figures 8-9). As shopwn, an annulus or ring
of
elastomer EM is formed about the underside of each membrane cavity, so as to
abut with the wall of the membrane cavitiies of the module 104b', 104c'
positioned therbelow. In this manner, the elastomer EM serves to form a
substantially air tight seal between adjacent membrane modules 104a', 104b',
104c'. Also, elastomer EM pads 119 are formed on the underside of the lid 106,
around each air inlet port 115, and such pads 119 abut against the upper
surface of the membrane module 104a', 104b', 104c' positioned therebelow to
form a discreet, substantially air tight seal therebetween. This effectively
isolates
each sample flowpath, and prevents escape or leakage of air pressure which
could interrupt the desired pressure diferential used to propel the sample
through the membranes 108a', 108b', 108c'.
Also, optional handles 120a,120b are formed on the membrane modules
104a', 104b', 104c' to facilitate separation of the modules 104a', 104b',
144c'
after the initial filtration has been completed.
Additionally, orientation registry members, such as a post 122 and
apertures 124a, 124b, 124c may be formed as shown to prevent the membrane
modules 104a', 104b', 104c' from being installed in the incorrect rotational
orientation.
iii, Third Embodiment of Test Apparatus
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Figure 11 shows a third embodiment of a test apparatus 10c which
comprises a) a vacuum base 150 having a cavity 176 formed therein, b) a
receiving unit 152 having a plurality of receiving wells 174 formed therein,
c) a
support member 154 having a plurality of apertures 172 formed therein, d)
plate-
type membrane modules 156a, 156b and 166c, each having a plurality of
cavities 171 a, 171 b, 171 c with open bottoms and membranes 170a, 170b, 17Qc
disposed transversely over the open bottom of each cavity 171 a, 171 b, 171 c,
e)
a sample receiving unit 158 having a plurality of sample receiving wells 178
formed therein, and f) a lid 160 which may be placed in sealing contact on top
of the sample receiving unit and which may have a plurality of limited air
inlet
openings (not shown) of the type described above with respect to the first and
second embodiments (see item nos. 24 on Fig. 5a and 115 on Fig. 10). These
components may be assembled in a stacked array, as shown. Each component
is provided with a spring loaded, pivoting, latch member 162 which is
configured
to engage and latch with a notch 164 in the component positioned immediately
therebelow.
In routine operation, the receiving unit 152 is inserted into the cavity 176
of the base 150, and the su~ort member 154 is mounted in the base such that
it is in sealing engagement with the o-ring 153 which surrounds the top
opening
of the base cavity 176 and each aperture 172 is positioned over a receiving
well
174. The membrane modules 156a, 156b, 156c are stacked upon the support
unit 152 such that each cavity 171 a, 171 b, 171, c and its membrane 170a,
170b,
170c are in alignment over an aperture 172 of the support member 154. The
latches 162 of the bottom membrane module 156c are engaged with the notches
164 formed in the support the support member 152, and the latches 162 of the
upper membrane modules 156a,156b are engaged with the notches 164 of the
neighboring membrane modules 156b, 156c positioned therebeneath. The
sample receiving unit 158 is mounted on the upper-most membrane module
156a such that each sample reservoir 178 is positioned over top of a cavity
.171 a, and the latches 164 of the sample receiving unit are engaged with the
notches 164 on the upper-most membrane module 156a.
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Quantities of sample are initially deposited in sample-receiving reservoirs
178 and the lid 160 is mounted in seating contact on top of the sample
receiving
unit 158 with the latches of the lid 160 in engagement with with the notches
164
of the sample receiving unit 158. Thereafter, a source of negative pressure is
connected to a port (not shown} formed in the base 150 so as to create
negative
pressure within the cavity 113 of the base 100. This negative pressure causes
each sample to be drawn downwardly through the rnernbranes 170a, 170b and
170c positioned under that sample reservoir 178, and the resultant filtrate to
be
received in the particular receiving well 174 positioned under those
particular
membranes. In this manner, this third embodiment of the test apparatus 10b
may be used to simultaneously process a plurality (e.g., 24 or 48 separate
samples).
Typically, the membranes 171 a of the first membrane module 156a are
for the purpose of filtering out or removing interferants, particles or other
unwanted matter while the membranes of any secondary membrane modules
170b, 170c are for capturing analytes for subsequent concentration and/or
analysis. Accordingly, after the initial filtrate has been received in the
filtrate
receiving wells 174, the vacuum source is terminated or disconnected,
differential pressure within the apparatus 10a is allowed to equalize to a
point
where removal of the lid 115 will not cause substantial upward bulging or
rupture
of the membranes 170a, 170b, 170c, and the lid 160 is unlatched and removed.
Ali of the membrane modules 156a, 156b, 156c are then removed and the first
membrane module 156a (along with the particles, interferants and/or other
unwanted matter removed by its membranes 170a) is discarded.
Thereafter, the receiving unit 152 is removed and appropriate reagents)
are added to the filtrate contained within the receiving wells 174 to provide
a
filtrate-reagent admixture from which a desired first analyte (Analyte A) may
be
qualitatively or quantitatively determined.
In applications such as that shown in Figure 11, where secondary plate-
type membrane modules 156b and/or 156c are used, such secondary
membrane modules 156b,156c will typically have captured secondary analyte(s)
- (Analytes B, C, etc...) which are to be subsequently released from the
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membranes 170b, 170c and thereafter concentrated and/or determined. In
furtherance of this, a clean receiving unit 152 may be inserted into the
cavity 176
of the base 150, and one of the secondary membrane modules 156b or 156c is
then positioned on top of the new receiving unit 1152 such that each membrane
170b or 170c is positioned over a receiving well 174. A known volume of flush
solution or eluant is then placed in the cavity 171 b or 171 c above each
membrane 170b or 170c, and the lid 160 is replaced such that it is latched to
the
notches in the membrane module in use 156b or 156c and in sealing contact
with the support member 154. The vacuum source is then re-energized or
reconnected to the base 150 to cause a differential pressure to be once again
established within the apparatus 1 Ob. In this manner the flush solution or
eluant
is drawn downwardly through the membranes 170b or 170c so as to extract or
release the captured analyte(s) from the membranes 170b or 170c. An
eluant/analyte mixture is thus received within each receiving well 174, and
the
above described procedure is repeated to qualitatively or quantitatively
determine that analyte in the eluant/analyte mixture within each receiving
well
174.
The same procedure is then repeated for each additional secondary
membrane module 156b, 156c, until ail analytes have been determined.
iv. Fourth Embodiment of Test Apparatus
Figures 12 and 12 a show a top-pressurized fourth embodiment of a test
apparatus 10c of the present invention. This fourth embodiment utilizes
positive
pressure applied to the top of the apparatus 10c rathen than negative pressure
applied to the bottom of the apparatus as in the above-set-forth first, second
and
third embodiments.
This apparatus 1 Oc generally comprises a) a base 190, b) a receiving unit
192 having a plurality of receiving wells (not shown) formed therein, c) a
support
hood 194 having a plurality of apertures 196 formed therein, d) first and
second
membrane modules 198a, 198b, and e) a positive pressure lid 200.
Each membrane module 198a, 198b has a plurality of individual sample
passage channels 210 formed therein. A membrane 216 is disposed
transversely within each sample passage channel 210. Membrane support
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cross-members 214, such as those described hereabove with respect to the first
embodiment (see item nos. 50a, 50b and 41 of Figures 7-9) may optionally be
formed within the sample passage channels 210 to support ant deter tearing or
rupture of the membranes 216.
The operation of this test apparatus 10c is generally consistent with that
described hereabove in reference to the first, second and third embodiments
10,
10a, 10b, except that rather than drawing the sample through the membranes
210 by way of negative pressure applied beneath the membranes, this
apparatus 10c pushes the sample through the membranes 210 by way of
positive pressure applied to the positive pressure lid 200.
Modified Membrane Module for Fourth Embodiment
Figures 13 and 13a shows a modified "top loaded" membrane module
198a' which comprises a housing 220 having a plurality of cylindrical bosses
formed downwardly therein such that the wall 221 of each cylindrical boss
defines a sample passage channel 224. Each channel 224 has a membrane
support floor 240 formed transversely therein. A filtrate-flow opening 242 is
formed through each membrane support floor 240, and a plurality of raised
membrane mounting surfaces 244 are formed on the upper surface of each
membrane support flor 240. Disc shaped membranes 228 are placed flat upon
the membrane mounting surfaces 224, and o-rings or seals 230 are then passed
downwardly into each channel 224 and are disposed in contact with the wall of
the channel 224, on top of and in contact with the periphery of each membrane
228. Sealing ring members 232 are then inserted downwardly into each channel
224 and are affixed to the wall of the channel 224 to compress the o-rings or
seals 230 and to thereby hold the membranes 228 in captured, fixed position
between the o-rings or seals 230 and the underlying membrane support floor
240. The areas between the raised membrane mounting surfaces 244 provide
spaces through which filtrate which passes downwardly through each membrane
228 may drain through filtrate flow openings 242.
Elastomeric sealing rings 226 (e.g., o-rings) are then passed around the -
outer surface of the wall 221 of each cylindrical boss to form a seal between
that
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membrane module 198a and the neighboring membrane module or support unit
154 positioned therebelow.
v. A Negative Pressure Base Unit Adaptable
for Use With Various Embodiments of Test Apparatus
Figures 14a and 14 b show a self contained negative pressure base unit
300 which is adaptable to replace the negative pressure base units of certain
embodiments of the test apparatus, such as base units 16 (Figure 5) and 100
(Fig. 10). This self-contained negative pressure base unit 300 incorporates an
internal vacuum pump (not shown) so as to eliminate the need for use of a
i0 separate vacuum source.
This self-contained negative pressure base unit 300 comprises a housing
302 having a cavity 304 formed therein and a lid 312 which, when closed, forms
a substantially air tight seal of the cavity 304. An elastomeric pad 308 is
formed
on the underside of the lid 312. Such elastomeric pad 308 abuts and seals
against the
component of the test apparatus (e.g., the upper membrane module 104a,
104a'or 156a). A plurality of limited air inlet openings 310 are formed at
locations in the lid 312 to operate in the same manner and perform the same
function as the air inlet openings 24, 115 of the first and second embodiments
described above. A make up air manifold (not shown) connects each air inlet
opening 310 to a single make-up air port 311 formed in the side of the lid
312.
In operation, the filtrate receiving and membrane module components of
the test apparatus are inserted into the cavity 304, the lid 312 is closed,
and the
internal vacuum pump (not shown) of the base apparatus 300, is used to draw
the sample through the membranes) as described repeatedly hereabove.
When all samples have been drawn through the respective membranes, the
vacuum pump (not shown) is de-energized, the pressure differential within the
apparatus is allowed to equalize, and the lid 312 is opened to allow the
operator
to remove the test apparatus and proceed with determination of the analyte{s)
in accordance with the invention.
vi. A Fifth Embodiment of Test Apparatus
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Figures 15a-15e show yet another (i.e., fifth) embodiment of the test
apparatus of the present invention, which is useable in conjunction with the
membrane modules 18, 20 and lids 22 of the above-described first embodiment
10. (see Figures 5-9). This test apparatus 1 Od is constructed for
simultaneous
analysis of multiple (e.g., six (6) ) samples, and comprises a base unit 500
having a plurality of test tube receiving cavities 502 formed therein. A lid
504 is
mountable in sealing contact on the base 500, and such lid 504 incorporates a
plurality of sample ports 506 having sample passage channels 508 extending
downwardly therethrough. As shown, the primary and secondary membrane
modules 18, 20a, 20b (Figures 7-9) are engageable with the sample ports 506
of this apparatus, in the same manner and to perform the same function as
described above with reference to the first embodiment of the test apparatus
10.
A vacuum source is connectable to the base 500 to draw the desired vacuum
within the cavity
Alternative Self Contained Vacuum Base Unit for Fifth Embodiment
The base 500 of the fifth embodiment 10d, may be replaced by a self-
contained base unit 510 of the type shown in Figure 15e. This self-contained
vacuum base unit 510 has a plurality of test tube receiving cavities 502'
formed
therein, as shown. After clean test tubes have been inserted into the cavities
502', the above-described lid 504, membrane modules i 8, 20a, 20b and Lids 22
are applied and utilized in the manner fully described elsewhere in this
application.
vll. A Self Contained Combinatlon Base Unit
Figure 16 shows a self-contained combination base unit 510 a which is
useable with several different embodiments of the test apparatus, such as the
second 10a and fifth 10d embodiments described above. This combination
base unit 51 Oa comprises a housing 511 having a cavity 304' and all of the
same
elements as the self contained negative pressure base unit 300 shown in
Figures 14a and 14b, but additionally including a vacuum station 512 which is
designed to provide negative pressure to the teast apparatus 500 shown in
Figures 15a-15e. In this manner, a vacuum connection nipple 514 is formed in
the vacuum station, and is insertable into a corresponding vacuum connection
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fitting (not shown) on the base 500 of the test apparatus 10d. Shoulders 516
are configured to hold the test apparatus 10d on the vacuum station 516, when
in use. An internal check valve or cap is used to close off the vacuum
connection nipple 514 when the test apparatus 10d is not mounted theron.
vfii. A Dip Stick Test Apparatus
. Figure 17 shows a sixth embodiment of the test apparatus of the present
invention. This sixth embodiment comprises a dipstick 700 having a handle 702,
a first (i.e., outer) membrane 704 and a second (i.e., inner) membrane 706.
The
second membrane 706 is substantially surrounded and enclosed by the first
membrane 704 such that only filtrate which has passed through the first
membrane 704 will come into contact with the second membrane 706. The first
(i.e., outer) membrane is typically a micro-porous membrane which serves to
prevent particles or large molecules which exceed a certain molecular weight
from passing therethrough. Examples of molecular weight cut-off membranes
which may be useable as the first membrane 704 include the SartoriousT""
1000MW cut off, 3000MW cut off, or 5000MW cut-off, as specified in the table
of Appendix III. The second (Le., inner) membrane is typically an indicator
membrane which is impregnated with or which bears an indicator substance,
such as a dye, which will undergo some perceptible change (e.g., a color
change) when contacted by a certain analyte or a predetermined concentration
of a certain analyte. The second membrane 706 may be adapted for a)
qualitative determination of a particular analyte (e.g., the second membrane
56
undergoes a single color change occurs in the presence of a certain analyte
irrespective of the concentration in which that analyte is present; b) semi-
qualitative determination of a certain analyte (e.g.,the second membrane
undergoes a single color change only if contacted by a certain analyte which
is
present at or above a predetermined threshold concentration, or c)
quantitative
determination of the concentration of a particular analyte (e.g., the second
membrane 56 undergoes a scaled color change such that the shade or color of
the second membrane is indicative of the concentration at which the analyte is
present.
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In operation, the user grasps the handle 702 of the dipstick apparatus 700
and dips the end of the dipstick apparatus 700 opposite its handle 702, into a
liquid or gaseous matrix (e.g., a solubilized food product, an oil, a
biological fluid,
etc.) Such that the first (i.e., outer) membrane 704 is fully or partially
immersed
in the matrix. A filtrate of the matrix then permeates the first (e.g., outer)
membrane 704 and comes into contact with the second (i.e., inner) membrane
706. The second (i.e., inner) membrane then undergoes an indicative change
(e.g., a color change) which correlates to the presence of the target analyte
(or
the predetermined concentration of the analyte.
C. Saeclffc Test Kfts & Methods
The table of Appendix I sets forth a number of test kits/assay methods of
the present invention, and provides specific information as to the analyte(s),
membrane(s), reagents) and detection methods) used in each such test
kit/assay method. In the table of Appendix I, each horizontal row sets forth a
particular test kit/method of the present invention. The columns of each
horizontal row are, from left to right, as follows:
Fie~st Column: the first column indicates the anaiyte(s)
which are determined;
Second Column: the second column indicates the typical
matrices in which the analyte(s) are contained;
Third Column: the third major column labeled
"membranes" indicates the type of (i) first membrane (M,), (ii)
second membrane (M2), (iii) third membrane (M3), and (iv) fourth
membrane (M4);
Fourth Column: the fourth major column labeled
"reagents" indicates the (i) first reagent (R,) to be combined with
the first filtrate in the first vessel for detection of the first analyte,
(ii) second reagent (R2) to be combined with eluant from the
second membrane {if any) in a second vessel for detection of the
second analyte (if any), (iii) third reagent (R3) to be combined with
eluant from the third membrane (if any) in a third vessel for
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detection of the third analyte (if any), and (iv) forth reagent (R4) to
be combined with eluant from the fourth membrane (if any) in a
fourth vessel for detection of the fourth analyte (if any);
Fifth Column: the fifth column indicates the preferred
analytical method or instrument used to determine each analyte;
and
Sixth Column: the sixth column sets forth other information
which is particular to that test kit/method.
The table of Appendix II is a key to the acronyms used to designate the
various analytes, membranes, reagents and detection methods in the table of
Appendix I.
Appendix III provides a list of commercially available membranes which
correspond to the acronyms used to refer to the membranes in Appendix I.
Appendix IV is a table listing algorithms which are useable in conjunction
with
certain test kit & methods of the present invention to predict or discern
certain
factors such as shelf life, presence of contaminants, potential for oxidative
degradation, etc., as described more particularly herebelow with respect to
certain assays which are of predictive value.
I. Examples of Test KltslMethods for Qualltatfve and/or
Quantitative Determination of Selected Analytes:
The following are detailed examples of the use of specific test
kits/methods of the present invention, which may be performed using the test
apparatus of the present invention. The term "protectants" as used in the
following examples means compounds) capable of inhibiting or preventing the
occurrence of certain changes in the analyte(s), such as one or more
antioxidants (e.g., ascorbic acid 1 %, BHT 0.1 %, or tocopherols 0.01-1.0%)
capable of deterring oxidation and/or compounds capable of chelating or
binding
metals (e.g., EDTA <0.1 %). The term "stabilizers" as used in the following
examples means one or more substances capable of preventing denaturation -
of a proteinaceous analyte (e.g. albumen 0.1-10.0%) or
conformational/structural
changes of any anaiyte. The term "solubilizers" as used herein means one or
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more surfactants or other substances capable of promoting dissolution of an
analyte (e.g., Tween 80, Tween 20, sodium dodecyl sulfate (SDS), benzyl
(BAC), etc.)
a. EXAMPLE 1: Free Fatty Acids in Oils or Oil Components:
A test kit/method for determining the amount of free fatty acids in oils and
oil components either qualitatively or quantitatively. The oils or oil
components
may be present in a matrix such as a food, personal care product, cosmetic or
other complex matrix. This example is performed in accordance with row 1 of
the table of Appendix I.
A. A sample of the matrix is initially diluted with a diluent such as
isopropanol with or without protectants. The sample may, or may not be,
processed through a membrane to remove particles, proteins or other
interferants, depending on whether such matter is present in the matrix. For
clean oils, such membrane processing may be unnecessary.
B. A dye which is sensitive to concentration of free fatty acid for its
spectral properties (e.g., Xylenol Orange) is solubilized in a diluent such as
isopropanol with or without protectants.
C. A control or standard is prepared by dissolving known concentrations
(e.g., 0.00% to 5.00 %) of the analyte (e.g.free fatty acids) in a diluent
such as
isopropanol.
D. The solutions prepared in steps A and B or C and B above, are
combined and read spectrophotometrically at the peak most sensitive to acidity
of the dye and results of samples are compared to results obtained from the
standards.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this peak
is between 540 and 600 nM with the optimal choice at 570nm. A decrease in the
absorption at this peak increases with acidity on a logarithmic basis and this
is
used to determine the free fatty acid for the oil ( i.e. a log-logit curve
plot).
F. This can be done utilizing any spectral device measuring absorption at
the wavelength for that dye.
G. Sample blanks can be run if necessary for very colored substances,
as can blanks for standards.
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b. EXAMPLE 2: Free Fatty Acids in Oils and Other Matrices.
A test kit/method for determining the amount of free fatty acids in oils and
oil components in food, personal care, cosmetics and other matrices which
contains the following reagents for analyzing liquids undiluted or diluted in
reagents based in solvents, solvent mixtures, or water or water/solvent
mixtures.
This example is performed in accordance with Row 1 of the table of Appendix
A. The oil or oil containing extract is dissolved or disbursed in a diluent
(e.g., methanol, isopropanol, hexane or combinations thereof) with or without
protectants, and may be processed through a membrane if needed, in
accordance with row 1 of the taboe of Appendix I..
B. A dye sensitive to concentration of acid for its spectral properties (e.g.
Xylenol Orange) is solubilized in a diluent e.g., methanol, isopropanol,
hexane
or combinations thereof) with protectants as necessary.
C. A control or standard prepared from free fatty acids or prepared oil and
standard compounds in isopropanol or any solvents listed above at specified
level of free fatty acids of 0.00% to 5.00 % free fatty acids.
D. Where A and B or C and B are combined and read at the peak most
sensitive to acidity of the dye and results of samples are compared to results
obtained from the standards.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol or any of
the solvents fisted above including water or water/isoporpanol mixtures or
water/solvent mixtures this peak is between 540 and 600 nm with the optimal
choice at 570nm. A decrease in the absorption at this peak increases with
acidity
on a logarithmic basis and this is used to determine the free fatty acid for
the oil
( i.e. a log-logit curve plot). This can be done utilizing any spectral device
measuring absorption at the particular wavelength.
F. Sample blanks can be run if necessary for very colored substances as
can blanks for standards.
c. EXAMPLE 3: Free Fatty Acids in Oils and Oil Components in the
Presence of particles, proteins, andlor other interferants:
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A test k'rtlmethod for determining the amount of free fatty acids in oils and
oil components in food, personal care, cosmetics and other matrices. The test
kit contains the following reagents for analyzing liquids undiluted or
diluted, and
utilizes a single or stacked membrane preparation of the matrix to remove
particles, protein, or other interferants (e.g., metals). This example is
performed
in accordance with row 1 of the table of Appendix I.
A. A sample of the matrix is dissolved or mixed (e.g., by vortexing) in a
diluent (e.g., methanol, isopropanol, hexane or combinations thereof) with or
without protectants.
l0 B. The diluted sample is passed through a first membrane such as an
MCE membrane to remove particulate matter.
C. The filtrate which passes through the first membrane is then passed
through a second membrane such as a metal capturing membrane (e.g., an
imino-diacetic acid membrane (IDA) as referred to in Appendix IV), if
necessary,
to remove additional compounds which would bind with the substrate sensitive
to acidity or to bind inorganic acids as to contribute background acidity
levels.
D. A dye sensitive to concentration of acid for its spectral properties (e.g.,
Xylenol Orange) is solubilized in isopropanol with protectants, as necessary.
E. A control and/or standards containing known concentrations of free
fatty acids (e.g., 0.00% to 5.00 %) may be prepared from free fatty acids or
prepared oil and standard compounds in isopropanol.
F. The solutions obtained in steps (C and D) and ( E and D) are
combined, and are read spectrophotometrically at the peak most sensitive to
acidity of the dye, and results of samples are compared to results obtained
from
the standards.
G. For Xylenol Orange between .001 % to 10.0% in isopropanol this peak
is between 540 and 600 nm with the optimal choice at 570nm. A decrease in the
absorption at this peak increases with acidity on a logarithmic basis and this
is
used to determine the free fatty acid for the oil. (e.g., a log-logit curve
plot). This
can be done utilizing any spectral device measuring absorption at the
particular -
wavelength
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H. Sample blanks can be run if necessary for very colored substances as
can blanks for standards.
d. EXAMPLE 4: Dip Stick Test for Free Fatty Acids
in Oils and Other Matrices
A dip stick test kit/method for determining the amount of free fatty acids
in oils and oil components in food, personal care, cosmetics and other
matrices.
The test kit contains the following reagents for analyzing liquids, undiluted
or
diluted.
A. A sample of the matrix is dissolved or mixed in a preparation reagent
such as isopropanol with or without protectants.
B. A dye sensitive to concentration of acid for its spectral properties (e.g.,
Xyienol Orange or Thymol Blue) or other dyes) which undergo color changes
in the range of pH 6 to pH 8 is/are attached to a membrane of a dip stick
(e.g.,
the inner membrane if an outer filtering membrane is present on the dip stick)
of
the type shown in Figure i 7.
C. A control and/or standards containing known concentrations of free
fatty acids (e.g., 0.00% to 5.00 %) may be prepared from free fatty acids or
prepared oil and standard compounds, in isopropanol.
D. The dip sticks are dipped in the solutions obtained in steps A and C,
and the color of the dye in the dip stick dipped into each sample is compared
to
the color of the dye of the dipsticks dipped into the standard solutions, to
obtain
a semi-qualitative determination of the concentration of free fatty acids in
the
samples.
e. EXAMPLE 5: A One Vial Test for Free Fatty Acid
in Oils and Oil Components:
A semi-quantitative, one-vial test kit/method for determining the amount
of free fatty acids in oils and oil components in food, personal care,
cosmetics
and other matrices. The test kit contains the following reagents for analyzing
liquids, undiluted or diluted. This example is carried out in accordance with
row
1 of the table of Appendix I.
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A. A sample of the matrix (e.g., a sample from a bottle of salad oil at
home or in a restaurant, a sample of oil obtained during manufacture/bottling}
is dissolved or mixed in isopropanol with or without protectants, and may or
may
not be processed through a filtering membrane depending on whether particles
or other interferants are believed to be present..
B. A dye sensitive to concentration of acid for its spectral properties, such
as Xylenol Orange, solubilized in isopropanol with protectants, as necessary,
is
provided in pre-filled vials.
C. A control and/or standards containing known concentrations of free
fatty acids (e.g., 0.00% to 5.00 %) may be prepared from free fatty acids or
prepared oil and standard compounds in isopropanol.
D. The solutions obtained from steps (A and B) and (C and B) are
combined, and equal amounts of each such mixture are dispensed into the dye-
containing vials. The resultant color change in each vial is read visually and
results of samples are compared to results obtained from the standards, if
necessary, or to a visual chart or color wheel.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this color
is first blue but changes to yellow in the presence of at least a
predetermined
concentration (e.g. 3.0%) of free fatty acid. Thus, this test kit with such
concentrations of Xylenol Orange can be used to determine whether a certain
sample of olive oil may be labeled as "extra virgin" (i.e., contains less than
3.0%
free fatty acids) or whether a sample of used cooking oil should be deemed no
longer usable (i.e., contains more than 3.0 % free fatty acids).
F. An adjustment in the Xylenol Orange concentration can be made to
allow the test kit to be used to determine any free fatty acid concentration
between 1.0% and 3.~0%.
f. EXAMPLE 6: Free Patty Acid in Olives or Olive Oils:
A test kit for determining whether a sample of olive oil qualifies as "extra
virgin", "virgin" or "virgin corrente" based on the concentration of free
fatty acids
present therein, or for determining whether aged oils are acceptable for human
-
consumption, or for pre-testing of olives to select those olives which will
provide
the highest quality oil. The test kit contains the reagents and membranes (if
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membranes are needed) as specified herebelow. This example is in accordance
with row 1 of the table of Appendix 1.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants, and
may
be processed through a filtering membrane if so required. For clean oils, such
membrane may be unnecessary..
B. A dye sensitive to concentration of acid for its spectral properties, such
as Xylenol Orange, solubilized in isopropanol with protectants, as necessary.
C. A control and/or standards containing known concentrations of free
fatty acids (e.g., 0.00% to 5.00 %) may be prepared from free fatty acids or
prepared oil and standard compounds in isopropanol.
D. The solutions obtained from steps (A and B) and (C and B) are
combined, and equal amounts of each such mixture are dispensed into the dye-
containing vials. The resultant color change in each vial is read visually and
results of samples are compared to results obtained from the standards, to
determine free fatty acid concentration.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this peak
is between 540 and 600 nm with the optimal choice at 570nm. A decrease in the
absorption at this peak increases with acidity on a logarithmic basis and this
is
used to determine the free fatty acid for the oil (i.e. a log-logit curve
plot). This
can be done utilizing any spectral device measuring absorption at the
particular
wavelength
F. Sample blanks can be run if necessary for very colored substances as
can blanks for standards.
G. The free fatty acid concentrations determined by this test are then
used to catagorize the olive oil or oil containing olive extract, in one of
the
following categorys:
~ O to 1°~ FPA.......... extra virgin
~ 1 to 2 %...................virgin
~ 2 to 3 °~ ..................virgin corriente
(syn. "virgin common")
~ more than 3%.........not for human
consumption
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g. EXAMPLE 7: Free Fatty Acid and Potyphenols in Olive Oiis or
Olives to Determine Oil Quality and Long Tem Stability:
A test kit for qualitatively determining the amount of free fatty acids in
oils and oil components in foods in combination with a polyphenol test which
together determines a) oil quality (e.g., extra virgin, virgin, virgin
corriente as
described in Example #6 above and b) long term stability based on
polyphenol content (the higher the polyphenol concentration the longer the
stability). This example is in accordance with row 11 on the table of Appendix
I.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. and
processed through the membranes shown on row 11 of the table of Appendix
B. A dye sensitive to concentration of acid for its spectral properties,
such as Xylenol Orange, solubilized in isopropanol, with protectants as
necessary, is provided for free fatty acid determination.
C. A dye sensitive to phenol such as folin ciocalteau reagent in
water/isopropanol with sodium carbonate, is provided to determine the
polyphenol concentration.
D. A control and/or standards containing known concentrations of
free fatty acids (e.g., 0.00% to 5.00 %) and polyphenols (e.g., 2 to 200
micrograms/gram) may be prepared from free fatty acids or prepared oil and
standard compounds in isopropanol.
E. The solutions obtained from steps A and D above are dispensed
into vials .containing the Xylenol Orange and folin ciocalteau reagents. The
resultant colored sample solutions are read visually or spectrally, and the
samples are compared to the standard solutions to determine free fatty acid
and polyphenol concentrations.
F. A Xylenol Orange/isopropanoi solution having a dye concentration
between .001 % and 10.0% will initially be of a blue color, but will change to
yellow in the presence of more than about 1 % free fatty acid. Such
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discernment of free fatty acid concentrations in excess of 1 % allows the
operator to immediately determine whether an olive oil should be labeled as
"not extra virgin" or a cooking oil should be labeled as "no longer usable".
If it
is desired to diferentiate between higher concentrations of free fatty acids
(e.g., 2% or 3%) the Xylenol Orange concentration may be increased so that
the solution will change to a yellow color at the higher concentration (e.g.,
2%
or 3%) of free fatty acids.
G. Generally, in this example, a deep blue color of the sample solution
indicates good stability with substantial amounts of polyphenol and
antioxidant present, whereas a clear solution is very unstable.
H. Polyphenols from 2 to 200 micrograms per gram are determined
I. The free fatty acid values which can be obtained for olive oil either
extra virgin, virgin, or virgin common are shown in #6. Polyphenol
concentrations in excess of 100 micrograms/gram indicate excellent shelf life,
50 to 100 micrograms/gram indicates very good shelf life, 20 to 50
micrograms/gram indicates good shelf life, and less then 20 micrograms/gram
indicates poor shelf life.
h. EXAMPLE 8: Lipid Peroxides and Free Fatty Acids
in Oils and Oil Components:
A test kit for determining the amount of lipid peroxides and free fatty
acids in oils and oil components either qualitatively or quantitatively in
food,
personal care, cosmetics and other matrices which contains the following
reagents for analyzing liquids undiluted or diluted. This example may be
performed in accordance with either row 2 or row 3 of the table of Appendix I.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. This
sample may be processed through membranes in accordance with rows 2 or
3 of the table of Appendix I.
B. A first dye sensitive to concentration of acid for its spectral
properties, such as Xylenol Orange, solubilized in isopropanol, with
protectants as necessary, is provided for determination of free fatty acids. A
second dye, such as Xylenol Orange or non-oxidized hemoglobin in the
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presence of certain prooxidants such as acidified iron, is provided for
determination of lipid peroxides. The preferred embodiment utilizes 0.1
Xylenol Orange and ferrous sulfate (5-200 mM and preferably about 25 mM)
in combination with sulfuric acid at 50 to 500 mM (optimum at 140m1) for
determination of lipid peroxides. For determination of free fatty acids, 2.25
ml
of the 0.1 %Xylenol Orange solution from step B is added to 42.5 ml of
isopropanoi with 0.1 % BHT, to form the fatty acid reagent.
C. A control or standard prepared from free fatty acids or prepared oil
and standard compounds in isopropanol at specified concentrations of free
i0 fatty acids from 0.00% to 5.00 % and controls or standards or controls with
lipid peroxides prepared from hydrogen peroxide or cumeneperoxide or other
stable or relatively stable peroxides at concentrations of 1 nmol/ml to 1000
nmol/ml.
D. The solutions from steps ( A and B) and (C and B) are combined
and read spectrally at the peak most sensitive to acidity of the dye, and the
results of such readins are compared to results obtained from the standards
and the peroxide reaction is read at that peak for the electron recipient at
570nm.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 nm with the optimal choice at 570nm. A
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acid (e.g., by a log-logit
curve plot). For lipid peroxides the absorption of Xylenol Orange-Fe Complex
increases at 570 nrn as it receives electrons. This absorption can be read
utilizing any spectral device measuring absorption at the particular
wavelength
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
i. EXAMPLE 9: Lipid Peroxides and Free Fatty Acids
in Oils and Oil Gomponents:
A test kit for determining the amount of lipid peroxides and free fatty
acids in oils and oil components either qualitatively or quantitatively in
food,
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personal care, cosmetics and other matrices which contains the following
reagents for analyzing liquids, undiluted or diluted. This example is carried
out in accordance with rows 2 and 3 on the table of Appendix I.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. This
sample may be prcessed through membranes in accordance with rows 2 or 3
of the table of Appendix I.
B. A first dye sensitive to concentration of acid for its spectral
properties such as Xylenol Orange soluablized in isopropanol with
i0 protectants, as necessary, or in other solvents such as isopropanol/water
mixtures, hexane, methanoll isopropanol mixtures.
C A second dye such as Xylenol Orange or non-oxidized hemoglobin
combined with certain pro-oxidants (e.g., acidified iron) such that it will
react
with lipid peroxides. is solubilized in the same solvent system as was used
for
the and the same solvent system used for the first dye in paragraph B
(above) of this example. a second reagent.
D. A control or standard prepared from free fatty acids or prepared oil
and standard compounds in isopropanol at specified concentrations of free
fatty acids from 0.00% to 5.00 % and controls or standards or controls with
lipid peroxides prepared from hydrogen peroxide or cumeneperoxide or other
stable or relatively stable peroxides at concentrations of 1 nmollml to 1000
nmol/ml.
E. The solutions from steps ( A and B), (A and C), (D and B) and (D
and C) are combined and read spectrally at the peak most sensitive to acidity
of the dye, and the results of such readings are compared to results obtained
from the standards and the peroxide reaction is read at that peak for the
electron recipient at 570nm.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 nm with the optimal choice at 570nm. A
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acid (e.g., by a log-logit
curve plot). For lipid peroxides the absorption of Xylenol Orange-Fe Complex
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increases at 570 nm as it receives electrons. This absorption can be read
utilizing any spectral device measuring absorption at the particular
wavelength
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
j. EXAMPLE 10: Lipid Peroxides and Free Patty Acids
in Oils and Oil Components
A test kit for qualitative or semi-quantitative determination of lipid
peroxides and free fatty acids in oils and/or oil components of food, personal
care, cosmetics and other matrices. The test kit contains the reagents and
membranes set forth herebelow and in rows 2 or 3 of the table of Appendix I.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. This
sample is then processed through membranes in accordance with rows 2 or 3
of the table of Appendix I. Such membrane processing may be performed
using a test apparatus of the present invention, as described above.
B. A first dye sensitive to concentration of acid for its spectral
properties such as Xylenol Orange soluablized in isopropanol with
protectants, as necessary, or in other solvents such as isopropanol/water
mixtures, hexane, methanol/ isopropanol mixtures.
C A second dye such as Xylenol Orange or non-oxidized hemoglobin
combined with certain prooxidants (e.g., acidified iron) such that it will
react
with lipid peroxides. is solubilized in the same solvent system as was used
for
the and the same solvent system used for the first dye in paragraph B
(above) of this example. a second reagent.
D. A control or standard prepared from free fatty acids or prepared oil
and standard compounds in isopropanol at specified concentrations of free
fatty acids from 0.00% to 5.00 % and controls or standards or controls with
lipid peroxides prepared from hydrogen peroxide or cumeneperoxide or other
stable or relatively stable peroxides at concentrations of 1 nmol/ml to
1 OOOnmol/ml.
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E. The solutions from steps ( A and B), (A and C), (D and B) and (D
and C) are combined and read spectrally at the peak most sensitive to acidity
of the dye, and the results of such readings are compared to results obtained
from the standards and the peroxide reaction is read at that peak for the
electron recipient at 570nm.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 nm with the optimal choice at 570 nm. A
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acid (e.g., by a log-logit
curve plot). Far lipid peroxides the absorption of Xylenol Orange-Fe Complex
increases at 570 nm as it receives electrons. This absorption can be read
utilizing any spectral device measuring absorption at the particular
wavelength
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
k. EXAMPLE 11: Lipid Peroxides and Free Fatty Acids
in Oils and Oil Components:
A test kit for utilizing a novel chemical test to qualitatively or
quantitatively determine lipid peroxides and free fatty acids in oils or oil
components of foods, personal care products, cosmetics and other matrices.
The test kit includes the reagents and membranes specified below and in row
3 of the table of Appendix I. A. A sample of the oil or oil containing
extracts is dissolved or mixed in a preparation reagent such as isopropanol,
with or without protectants. This sample may or may not be processed
through a membrane, in accordance with row 3 of the table of Appendix I. If
performed, such membrane processing may be carried out using a test
apparatus of the present invention, as described above.
B. A first dye sensitive to concentration of acid for its spectral
properties such as Xylenol Orange or Thyrnol blue (or another dye with
sensitivity to small pH changes in the pH 6 to pH 8 range) is solubilized in a
solvent such as isopropanol, with protectants as necessary.
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C. A second dye such as Xylenol Orange or non oxidized hemoglobin
in the presence of a prooxidant such as acidified iron, is solubilized in the
same solvent system as the first dye of paragraph B of this example. This
second dye will react with lipid peroxides or can be altered by lipid
peroxides
and then interact with XO, Hemoglobin or other sensitive reagents.
D. A control or standard prepared from free fatty acids or prepared oil
and standard compounds in isopropanol at specified concentrations of free
fatty acids from 0.00% to 5.00 % and controls or standards or controls with
lipid peroxides prepared from hydrogen peroxide or cumeneperoxide or other
stable or relatively stable peroxides at concentrations of 1 nmollml to 1000
nmol/ml.
E. Concentration is determined by visual comparison of the color of
the samples to standard solutions or a color wheel or chart. For free fatty
acids the reagent is initially blue but turns yellow as the acidity increases.
For
i5 lipid peroxides, the dye is initially yellow but turns blue as lipid
peroxide
concentration ioncreases--reaching a deep blue at 20 Meq/kg.
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
I. EXAMPLE 12: Semi-Quantitative Test for Lipid Peroxides
and Free Fatty Acids in Oils or Oii Components:
A test kit for semi-quantitative determination of lipid peroxides and
free fatty acids in oils or oil components of a food, personal care product,
cosmetic or other matrix, using a color wheel. The test kit includes the
reagents and membranes (if necessary) described herebelow and in rows 2
or 3 of the table of Appendix 1. This test is particularly useful for
analyzing
liquids, undiluted or diluted, and may be used to classify samples of olive
oil
(i.e., extra virgin, virgin, virgin corriente) or to sub-categorize samples of
olive
oil within a particular class based on expected shelf life.
A. A sample of the oil or oif containing extracts is dissolved or mixed in
3o a preparation reagent such as isopropanol, with or without protectants.
This
sample may or may not be processed through a membrane, in accordance
with row 3 of the table of Appendix 1. If performed, such membrane
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processing may be carried out using a test apparatus of the present
invention, as described above.
B. A first dye sensitive to concentration of acid for its spectral
properties such as Xylenol Orange or Thyrnol blue (or another dye with
sensitivity to small pH changes in the pH 6 to pH 8 range) is solubilized in a
solvent such as isopropanol, with protectants as necessary.
C. A second dye such as Xylenol Orange or non oxidized hemoglobin
in the presence of a prooxidant such as acidified iron, is solubilized in the
same solvent system as the first dye of paragraph B of this example. This
second dye will react with lipid peroxides or can be altered by lipid
peroxides
and then interact with XO, Hemoglobin or other Sensitive reagents.
D. A control or standard prepared may from free fatty acids or
prepared oil and standard compounds in isopropanol at specified
concentrations of free fatty acids from 0.00% to 5.00 % and controls or
standards or controls with lipid peroxides prepared from hydrogen peroxide or
cumeneperoxide or other stable or relatively stable peroxides at
concentrations of 1 nmol/ml to 1 OOOnmol/ml.
E. The solutions from steps ( A and B) and (A and C) are combined
and the colors which develop in those admixtures are visually compared to
those of a color wheel or color chart. Alternatively, a spectral determination
could be used, in which case the solutions from steps (D and B) and (D and
C) will also be combined and mixed with the reagents, and the absorption of
the sample solutions will be compared to the absorptions of the standard
solutions to arrive at determinations of lipid peroxides and free fatty acids
in
the samples.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between blue and when acidified is yellow.
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
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m. EXAMPLE 13: A Test for Lipid Peroxides and Free f=atty Acids in
Olive Oils to Predict Sheif Life and Quality;
A test kit for qualitative or quantitative determination of lipid peroxides
and free fatty acids oils or oil components of a food, personal care product,
cosmetic or other matrix, using a spectrophotometer. The test kit includes
the reagents and membranes (if necessary) described herebelow and in
rows 2 or 3 of the table of Appendix I. This test is particularly useful for
analyzing liquids, undiluted or diluted, and may be used to classify samples
of olive oi! (i.e., extra virgin, virgin, virgin corriente) or to sub-
categorize
samples of olive oil within a particular class based on expected shelf life.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. This
sample may or may not be processed through a membrane, in accordance
with row 3 of the table of Appendix I. If performed, such membrane
processing may be carried out using a test apparatus of the present
invention, as described above.
B. A first dye sensitive to concentration of acid for its spectral
properties such as Xylenol Orange or Thyrnol blue (or another dye with
sensitivity to small pH changes in the pH 6 to pH 8 range) is soiubilized in a
solvent such as isopropanol, with protectants as necessary.
C. A second dye such as Xylenol Orange or non oxidized hemoglobin
in the presence of a pro-oxidant such as acidified iron, is solubilized in the
same solvent system as the first dye of paragraph B of this example. This
second dye will react with lipid peroxides or can be altered by lipid
peroxides
and then interact with XO, Hemoglobin or other sensitive reagents.
D. A control or standard prepared may from free fatty acids or
prepared oil and standard compounds in isopropanol at specified
concentrations of free fatty acids from 0.00% to 5.00 % and controls or
standards or controls with lipid peroxides prepared from hydrogen peroxide or
cumeneperoxide or other stable or relatively stable peroxides at
concentrations of 1 nmol/ml to 1000 nmol/ml.
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WO 99/20396 PGT/US98/22186
-45-
E. The solutions from steps ( A and B) and (A and C} are combined
and those admixtures are read spectrophotometrically at 570nm or at the
wavelength for hemoglobin. The absorption of the test samples is compared
to the absorption of the standards to determine the concentration of free
fatty
acids and lipid peroxides.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 urn with the optimal choice at 570nm. A
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acid for the oil (i.e. a
log-logit
curve plot}. This can be done utilizing any spectral device measuring
absorption at the particular wavelength
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
H. The quality and shelf life of each sample is then classified as
follows:
Quality Classification Shelf Life Prediction
FFA= O-1 %... extra virgin LPO= 0- 6 Meq/Kg...l8 months
FFA= 1-2 %...virgin LPO= 6-12 Meq/Kg...l2 months
FFA= 2-3 %...virgin common LPO=12-20 Meq/Kg... 6 months
FFA= >3%.....not consumable LPO= >20 Meq/Kg....not
consumable
I. It will be appreciated that, as an alternative to spectral
determinations, semi-quantitative determinations of FFA and LPO may be
made using colored standards, color charts or a color wheel, and the quality
classification and shelf life prediction can be arrived at based on a sheme of
visual color combinations or shades.
n. EXAMPLE 14: A Test for Free Fatty Acids, Lipid Peroxides,
and Poiyphenols in Oil and Oil Components to Determine if
the Oil is Adulterated or Aged:
A test kit for qualitatively determining the amount of free fatty acids and
LPO in oils and oil components in foods, in combination with a potrphenol test
which together determines if the olive oil has been adulterated and is aged.
CA 02305613 2000-04-04
WO 99/20396 PCT/US98/22186
-46-
This test is performed in accordance with row 30 of the table of Appendix I
and the test kit includes the reagents and membranes described below and in
row 30 of Appendx I.
A. A sample of the oil or oil containing extracts is dissolved or mixed in
a preparation reagent such as isopropanol, with or without protectants. and
processed through the membranes shown on row 30 of the table of Appendix
B. A first dye sensitive to concentration of acid for its spectral
properties, such as Xylenol Orange, solubilized in a solvent such as
i0 isopropanol, with protectants as necessary, is provided for determination
of
free fatty acids.
C. A second dye sensitive to polyphenol, such as folin ciocalteau
reagent in water/isopropanol with sodium carbonate, is provided to determine
the polyphenol concentration.
D. A third dye or indicator, such as Xylenol Orange combined with
acidified iron, which is sensitive to free electron transfer from lipid
peroxides
is provided to determine lipid peroxides.
E. A control or standard prepared may from free fatty acids or
prepared oil and standard compounds in isopropanol at specified
concentrations of free fatty acids from 0.00% to 5.00 % and controls or
standards or controls with lipid peroxides prepared from hydrogen peroxide or
cumeneperoxide or other stable or relatively stable peroxides at
concentrations of 1 nmol/ml to 1000 nmol/ml.
F. The solutions of (A and B) and ( A and C) and (A and D) are
combined, and the color of each of the resulting admixtures is determined
spectrally, or by visual comparison to known standards, color chart or color
wheel.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol this
color is first blue and then at 1.0% free fatty acid yellow so that an olive
oil
can be immediately labeled as not extra virgin or a cooking oil can be labeled
as no longer usable.
CA 02305613 2000-04-04
WO ~~~~ PCT/US98122186
-47-
G. An adjustment in the Xylenol Orange concentration and a change
for blue to yellow can be seen for 1.0 to 3.0 free fatty acid. Polyphenois
from
2 to 200 micrograms per gram are determined
H. The results of this test allow the oil to be categorized as follows:
1. Quality Based on FFA Concentration:
FFA= O-1 %... extra virgin
FFA= 1-2 %...virgin
FFA= 2-3 %...virgln common
FFA= >3°~.....not consumable
2. Astina Based on LPO Concentration:
LPO= 0- 6 MeqIKg...Minimal Aging--18 months left
LPO= 6-12 Meq/Kg..Some Aging--12 months left
LPO=12-20 Meq/Kg..Maximum Acceptable Aging-- 6 months left
LPO= >20 Meq/Kg....Aged--not consumable
3. Adulteration Based on Polvphenol X FFA:
PPxFFA=75...................... Unadulterated Extra Virgin
PPxFFA=125.................... Unadulterated Virgin
PPxFFA=150.................... Unadulterated Virgin Common
PPxFFA=37.......................50% adulterated Extra Virgin
PPxFFA=35...................... 50% adulterated Virgin
PPxFFA=75...................... 50% adulterated Virgin Common
PPxFFA=1.5.................... 90% adulterated Extra Virgin
PPxFFA=3Ø.................... 90% adulterated Virgin
PPxFFA=9Ø.................... 90% adulterated Virgin Common
0. EXAMPLE 15: Ltpid Peroxides and Free Fatty Acids
_ in Oils and Oil Components:
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WO 99/20396 PCT/US98122186
-48- , _ _
A test kit for determining the amount of lipid peroxides and free fatty
acids in oils and oil components either qualitatively or quantitatively in
food,
personal care, cosmetics and other matrices which contains the following
reagents for analyzing liquids undiluted or diluted and which allow assignment
to categories for olive oil as well as levels within extra virgin which have
longer expected shelf life or within virgin or within virgin common using
colorwheels
A. The oil or oil containing extracts in isopropanol with or without
protestants
B. A dye sensitive to concentration of acid for its spectral properties
such as Xylenol Orange is soubilized in isopropanol, with protectants as
necessary. A second indicator reagent, such as Xylenol Orange or non
oxidized hemoglobin in the presence of a pro-oxidant, is provided to
determine lipid peroxides. This lipid peroxide reagent typically requires a
pro-oxidant such as acidified iron or iron complex to initiate the transfer of
electrons from the lipid peroxides to the final substrate.
C. A control or standard prepared may from free fatty acids or
prepared oil and standard compounds in isopropanol at specified
concentrations of free fatty acids from 0.00% to 5.00 %. Controls or
standards for lipid peroxides are prepared from hydrogen peroxide or
cumeneperoxide or other stable or relatively stable peroxides at
concentrations of 1 nmol/ml to 1000 nmol/ml.
D. The solutions from steps ( A and B) and ( C and B) are combined
and the color developed in those solutions are compared to the standards or
to a color wheel to determine free fatty acids and lipid peroxides.
E. For Xylenol Orange at concentrations between .001 % to 10.0% in
isopropanol, the indicator solution is initially blue and changes to yellow in
the
presence of a predetermined concentration of free fatty acids. When Xylenol
Orange is also used (w/ acidified iron) to indicate polyphenols, the solution
is
initially yellow but changes to deep blue in the presence of polyphenols at 20
Meq/Kg or more.
CA 02305613 2000-04-04
WO 99/20396 PCT/US98~2186
9 ._
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards
p. EXAMPLE 16: Lipid Peroxides and Free Fatty Acids
in Oils and Oil Components:
A test kit for determining the amount of lipid peroxides and free fatty
acids in oils and oil components either qualitatively or quantitatively in
food,
personal care, cosmetics and other matrices which contains the following
reagents for analyzing liquids undiluted or diluted and which allow assignment
to categories for olive oil as well as levels within extra virgin which have
longer expected shelf life or within virgin or within virgin common based on
LPO and FFA.
A. The oil or oil containing extracts in isopropanol with or without
Protectants.
B. A dye sensitive to concentration of acid for its spectral properties
such as Xylenol Orange solubilized in isopropanol with protectants as
necessary and a second reagent such as Xylenol Orange or non-oxidized
hemoglobin which in the presence of certain prooxidants can react with lipid
peroxides. The lipid peroxide reagent requiring acidified iron or iron complex
to initiate the transfer of electrons from the lipid peroxides to the final
substrate.
C. Controls or standards may be prepared for from free fatty acids or
prepared oil and standard compounds, in isopropanol at specified
concentrations of free fatty acids from 0.00% to 5.00 %. Controls or
standards for lipid peroxides are prepared from hydrogen peroxide or
cumeneperoxide or other stable or relatively stable peroxides at
concentrations of 1 nmol/ml to 10.00 nmol/ml.
D. The solutions from steps ( A and B) and ( C and B) are combined
and the color developed in those solutions and in the standards are read at
that peak for the electron recipient either 570nm or the wavelength for
hemoglobin.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 nm with the optimal chaice at 570nm. A
CA 02305613 2000-04-04
w0 99/20396 PCT/US98/2Z186
-50-
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acrd for the oil (i.e. a
log-logit
curve plot). This can be done utilizing any spectral device measuring
absorption at the particular wavelength
F. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
H. Based on the results obtained, the oil samples may be classified as
follows:
Free Fatty Acid Livid Peroxide
O to 1% = extra virgin 0 to 6 MeqIKg = long shelf life
1 to 2 % = virgin 6 to 12 MeqIKg = med. shelf life
2 to 3 % = virgin common 12 to 20 MegIKg = short shelf life
> 3% = not consumable > 20 MeqIKg = not consumable
q. EXAMPLE 17: Free Fatty Aclds in Oils and Oil Components:
A test kit for determining the amount of free fatty acids in oils and oil
components in food, personal care, cosmetics and other matrices which
contains the following reagents for analyzing liquids undiluted or diluted.
Utilizing a single or stacked membrane preparation of the matrix to remove
particufates, protein, or other interferents.
A. The oil or oil containing extracts are solubilized in isopropanol, with
or without protectants, and passed through a first membrane (e.g., MCE 0.45
micron or Durapore 0.45 micron) with or without a second membrane. The
test apparatus of the present invention may be used for this membrane
processing.
B. A second membrane being used if necessary to remove additional
compounds which would bind with the substrate sensitive to acidity or to bind
in organic acids which could contribute background acidity levels.
C. A dye sensitive to concentration of and for its spectral properties
such as Xylenol Orange solubilized in isopropanol with protectants as
necessary.
CA 02305613 2000-04-04
WO 99/20396 PCT/US98/22186
.5 ~ . .
D.. A control or standard prepared from free fatty adds or prepared oil
and standard compounds in isopropanol at specified level of free fatty acids
of 0.00% to 5.00% free fatty adds.
E. Where A and B or C and B are combined and read at the peak most
sensitive to acidity of the dye and results of samples are compared to results
obtained from the standards.
F. For Xylenol Orange between .001 % to 10.0% in isopropanol, this
peak is between 540 and 600 nm with the optimal choice at 57 nm. A
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis, and this is used to determine the free fatty acid for the oil (i.e. a
log-logit curve plot.) This can be done utilizing any spectral device
measuring
absorption at the particular wavelength.
G. Sample blanks can be run if necessary for very colored substances
as can blanks for standards.
r. EXAMPLE 18: Free Fatty Acids in Oils and Oil Components:
A test kit for determining the amount of free fatty acids in oils and oil
components in food, personal care, cosmetics and other matrices which
contains the following reagents for analyzing liquids undiluted or diluted.
A. The oil or oil containing extracts are soluabilized in isopropanol,
with or without proteceants.
B. A dye sensitive to concentration of acid for it's spectral properties,
such as Xylenol Orange or Thymol Blue (or other dyes which undergo color
changes in the pH 6 to 8 range) is solubilized in isopropanol, with
Protestants
as necessary, and with buffering to increase sensitivity.
C. A control or standard prepared from free fatty acids or prepared oil
and standard compounds in isopropanol at specified level of free fatty acids
of 0.00% to 5.00% free fatty ends.
D. Where A and B or C and B are combined and read at the peak most
sensitive to acidity of the dye and results of samples are compared to results
obtained from the standards.
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this
peak is between 540 and 600 nm with the optimal choice at 570nm. A
CA 02305613 2000-04-04
WO 99/20396 PCTNS98/22186
-52-
decrease in the absorption at this peak increases with acidity on a
logarithmic
basis and this is used to determine the free fatty acid for the oh (i.e. a log-
logit
curve plot.) This can be done utilizing a spectral device measuring absorption
at the particular wavelength.
F. Sample blanks can be non if necessary for very colored substances
as can blanks for standards.
s. EXAMPLE 19: Qualitative Determination of Free Patty Acids
in Oils and Oil Components.
A test kit for qualitatively determining the amount of free fatty acids in
oils and oil components in food, personal care, cosmetics and other matrices
which contains the following reagents for analyzing liquids undiluted or
diluted.
A. The oil or oil containing extracts is solubilized in isopropanol, with or
without Protestants. The oil sample may be obtained from a bottle of oil at
restaurant, at home, during preparation etc.
B. A dye sensitive to concentration of acid for its spectral properties
such as Xylenol Orange is solubilized In isopropanol, with protectants as
necessary.
C. A control or standard, if necessary, is prepared from free fatty acids
or prepared oil and standard compounds in isopropanol, at specified free fatty
acid concentrations (e.g., 0.00% to 5.00 %).
D. The solutions obtained in steps (A and B) and ( C and B} are
combined and the color shift in each such solution is read visually. The
results of samples are compared to results obtained from the standards, if
necessary, or to a visual chart or color wheel..
E. For Xylenol Orange between .001 % to 10.0% in isopropanol this
color is first blue and then at 1.0% free fatty acid yellow so that an olive
oil
can be immediately labeled as not extra virgin or a cooking oil can be labeled
as no longer usable
F. An adjustment in the Xylenol Orange concentration and a change
for blue to yellow can be seen for 1.0 to 3.0 free fatty acid.
CA 02305613 2000-04-04
WO 99/20396 PCT/US98I22186
-53-
It will be appreciated that the invention has been described hereabove
with reference to certain preferred embodiments and examples. It is to be
appreciated however, that these preferred embodiments and examples are
not exhaustive, and no effort has been made to specifically describe each
and every embodiment or example of the invention. It is, however, intended
that all embodiments and examples which are within the spirit and scope of
the invention, be included within the scope of the following claims.
15
25
_
CA 02305613 2000-04-04
WO 99/20396 PCT/US98/22186
_ 54 _ . -
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CA 02305613 2000-04-04
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CA 02305613 2000-04-04
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CA 02305613 2000-04-04
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CA 02305613 2000-04-04
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CA 02305613 2000-04-04
WO 99IZ0396 PCT/US98f12186
63 _ ,_
Appendix II
I~ey to Acror~ns
AOS................................................... .. Antioxidant Status
ADP .............................................. Adenosine Triphosphate
AMP ..........................................Adenosine Monophosphate
ATP ............................................... Adenosine Triphosphate
DAO......................................................... Diamine Oxidase
FFA ........................................................... Free Fatty
Acids
HA ................................................... . ........ ..
...Hcstatninc
FBI'............................................Horseradish Perpoxidase
I,.....................................................................Iodine
Vapor
I, ......................................................................
Triodide Ion
IDA ...................................Iminodi Aoedtic Add Membrane
LDL.............................................Low Density Lipoproteins
LDL-.............................Oxidized Low Density Lipoproteins
LPO.............................................................Lipid
Peroxides
Mab.................................................... Monoclonal Antibody
MCE .................................................. Mixed Cellulose Ester
MDA .......................................................... Mzlonaldehydes
ML..................................................................
Methylindole
SP ....................................................... Sun Protector
Factor
TBPB ........................:................... Tetra Bromophenol Blue
TG ....................................................................
Triglyceride
TL...................:................................................. Total
Lipids
SF ............................................................... ..........
Sulfite
VLDL .................................. Very Low Density Lipoproteins
XO ............................................................. Xylenol
Orange
CA 02305613 2000-04-04
WO 99/20396 PCTNS98/2~186
_ 64 ._
Appendix III
sI~.EIa-~Ft 8c SQ-ICIEU,. ~pJPIICAT1UN
Gt;tbH
I
r.o. sx 4. D37se2. Dattel, Rdtt~>I f aol~~~anu, wtint
Gert~Y ~ >.4stt~t
a
:. C~I~se Acaue. 0.45 ttm Removal of solid mono.
i 25 mm discs 23710 rotems
2. rolvvumrfident Fluoride Antibody cwtin
0.2 tun i. is mm disks -
4 t)OOS
3. NA45 DEAF Cdhtlose Manbt>,te.
0.45 um i,
25 mm discs - 23310
v
4. NA45 D1:AG Cellulose Membrute,~ of malondddtvde
0.45 um i, 4x51/4 utcltes sutines
- sulfito-bound aldeh
de~
21410 ,
,
y
~ 5. Nvkm. 0.45 em's. 25tam kanoval of solid motto,
aiscs - 00130 roans > .45 mm
i 6. Nylon. 0.2 tun i. 25 Reeaowi of solid motto
term dsxs - 0003C proteins > .2 mm
i 7. NL rdYamide Capture Ore ~ s - j
I 8. rC Polvarbonaee true aldeh es
~'p~etia C.ooorauon - - AI~G'fION
'
I 111 A
Ave., Livermore. G 94550
1. Miuorrep. rTFL, rr.. NS, Ca tore comootutds havinx
0.2 uta r. 13 mat - 97844 imr acd domes ' ' erwtides
~
' 2. MieroS -
' Nylon, 0.45 um a. Micro-Cmt.Fteatoval of solid snmu
tubes - 97795 raeint
1. L)bra-S ' , Cl' rr s lOk Ranoval o soft mono. tuoceint
MWOD Miero t Tubes - 97771
4. Silva Meatbrsmes 0.4 tun oI vo niks '--
i. 25mm - 51133
5. rolyarboaue Alembaancs,
0.4 em's, 25 mm, rVr
Fm - 11030
6. r ate Membanes. 0.4 um dtloria~d moleatlts
i. 25 mm AOX- 11027
~ 7. rolvtarbottuc Manbaaness
0.4s um i 47 mm, Loa av.
. 13035
I 8. rolrnrbwtue Membtartes,~ Capntre s
0.2 ttm i, 8" x 10", PVl'
Free - 19116
OD'~'~
~~ APPI1GTION
TI~I
BO A Rd, Bedford Ma 01730-2271
1. lso 0 0.t em's, 25 mm Ranoval of solid matte
discs - VCIP 025 00 eins
2. lmmobiloa-CD, 0.45 em's. g~"~ of solid matter
25mrrt disc, roteins
Cationinllv cLarsal (hydropht7icp
PVDF7 - I~M 025 00
~3. Low warn Extratxx6le gf solid snucu withotsr
(I'~ filters. 0.45 tmi s, o
25 i
bmd>n
l
d
I mm disc - HATE 025 00 8
rEao
e mo
ea
es
4. H ~ ~c 0.45 em's. 25 mm ~Rae>ov>I o so ~ muter
diz<s - IiV1.025 OC cotcns
s. It~ttobr~ (ltyaropbbie
rvDr7 >u~ pan
bindin . 0.45 tun i. 25 mm
disc - ISE 025 00
6. Isoporc, HTTP (poly~eonue),
0.4 tun's, 25 mm
discs - HTTP 025 00
7. Lnntobibn.r Translu Membrs>naCoatias with aeaibodies
(PVDF). 0.45 to capnue m r~ove specific
-
ttm i 15 an x 15 em - IPVIi tom s
151 50
8. Immobibn Transfer MembnnaCoatint wd6 atnibodies
(t'VDF). 0.4s to apace or remove ami
specific
'
tun's. 1 S mt x 15 an - ICDMs
15 t 50
9. Immobilon NC tore. 0.22 C~g ~ t or remove antibody
uni s, IS cm x 15 an - INCr specific
151 SO
tom s
10. Lttmobilon.NC (SurfactantC.ouitts welt antibodies
m). 0.45 um i,15 to capture or n'move asui6ody
specific
an x 1 S cm HK1T 151 50 tom s
t t. MultiSamt - DEAE. Anion
Exchut8e rapcr
a a 96 well lutes - MADE
NOB t0
12. MultiSueen -1'hospho
Cdhtlose Catiott
B;t,d ~d for nptsve
Exdun a ra er a 96 well tes
MATH NO8 I0
13. SC X -- MW Goof s Lima
1
14. r tone _ _- _.
t5. IGN-6 Mioobes
16. ICE 450 Bird ntstfeotides DNA
s
~
131IiearlandBlvd
.,o NYtt717
_
1. $an0b111d S BUId mOnO ' OIIabC atltlbOdltt.
"--'-'-- aG.
2. Sanoband C Fatdmam remove!
3. Sanoband
i
Se crate roteins anines
4. Sutoband D DN~'f>T' AMP
5. SanobandIDA _ ~ eations -"-'-.
Gebnam/raL '
IGTIONS
600 South ~%a ter Road. Ann ~
Arbor, MI 48103-9019 -)t
t. Versa r rre(iltacosttamiuants
2. Ubrxbind Os450 Bind mon amibodies ac.
I 3. B a C ins
4. Bi a B Endotoxins nucleotide s
CA 02305613 2000-04-04
WO 99/20396 PCT/US98i''12186
- 65 .-
Appendbc N
Predictive Algorithms
FFA X Polyphenol
i - Numerical i
Prediction
of
Olive
Oil
Adulteration
using
product
She i
1. > 50
~ d
FFA l
X d i
Polyphenol
i
l not a
Please u
refer terate
to
row
29
of
Appendix
I.
MDA/LPO is a scale
0 to 5
0-0.5 696 shelf life
remains
2. Shelf Life Prediction based 0.5-I 3396 shelf
on MDA/LPO ruio life ranains
I-2 1596 shelf life
remains
> 2 5% shelf life
remains
96 change related
to shelf life
Shelf Life Prediaron based 0-1096 > 18 months
stress with petnxyl
3' generator IO-3096 12-18 months
j
30-506 6-12 months
>506 < 6 months
' r Ratio Freeze aw i
0-0.2 one
4. ~ Freeze/Thaw Prediction using0.2-0.4 two
ratio Aadity/LPO I
0.4-0.6 three
( 0.6-0.8 four
Prediction of time to Myeotoxin
contamination using
5 LI'O value LPO
. Please refer to row 33 of i
Aupendix I.
Time to Contamination
Food non-irradiated
has expected
FFA/LPO of < 1
6. Prediction if food is Irradiatal
using FFA/IT'O ratio
Food Irradiated increases
FFA/LPO
>1
