U.S. patent application number 14/776702 was filed with the patent office on 2016-02-11 for detection and quantification of acetylamantadine in urine samples.
This patent application is currently assigned to BIOMARCK TECHNOLOGIES INC.. The applicant listed for this patent is BIOMARCK TECHNOLOGIES INC.. Invention is credited to Aftab Ahmed, Rashid Bux, Brian Cheng, Reuven Gordon, Fraser Alan Hof, Bram Ramjiawan.
Application Number | 20160041145 14/776702 |
Document ID | / |
Family ID | 51535742 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160041145 |
Kind Code |
A1 |
Gordon; Reuven ; et
al. |
February 11, 2016 |
Detection And Quantification Of Acetylamantadine In Urine
Samples
Abstract
A method for quantifying acetylamantadine in a urine sample
comprises eluting acetylamantadine from the urine sample using
solid phase extraction and quantifying the acetylamantadine eluted
from the urine sample using Raman spectroscopy.
Inventors: |
Gordon; Reuven; (Victoria,
CA) ; Cheng; Brian; (Chesterfield, MO) ; Bux;
Rashid; (Vancouver, CA) ; Ramjiawan; Bram;
(Winnipeg, CA) ; Ahmed; Aftab; (Waterloo, CA)
; Hof; Fraser Alan; (Victoria, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOMARCK TECHNOLOGIES INC. |
Richmond |
|
CA |
|
|
Assignee: |
BIOMARCK TECHNOLOGIES INC.
Richmond
BC
|
Family ID: |
51535742 |
Appl. No.: |
14/776702 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/CA2014/050273 |
371 Date: |
September 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61785159 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
436/64 ;
436/111 |
Current CPC
Class: |
G01N 21/65 20130101;
G01N 33/493 20130101; A61B 5/0075 20130101; G01N 27/74 20130101;
G01N 33/57484 20130101; G01N 21/658 20130101; G01N 15/1434
20130101; G01N 33/574 20130101; G01N 1/4055 20130101; C12Q 1/48
20130101; G01N 15/0205 20130101; G01N 27/44721 20130101 |
International
Class: |
G01N 33/493 20060101
G01N033/493; G01N 21/65 20060101 G01N021/65 |
Claims
1. A method for quantifying acetylamantadine in a urine sample, the
method comprising: eluting acetylamantadine from the urine sample
using solid phase extraction; and quantifying the acetylamantadine
eluted from the urine sample using substrate based, surface
enhanced Raman spectroscopy.
2. The method as claimed in claim 1 wherein the solid phase
extraction includes eluting acetylamantadine with methanol.
3. The method as claimed in claim 1 wherein quantifying the
acetylamantadine eluted from the urine sample using Raman
spectroscopy includes quantifying the acetylamantadine based on a
1600 cm-1 band.
4. The method as claimed in claim 1 wherein quantifying the
acetylamantadine eluted from the urine sample using Raman
spectroscopy includes quantifying the acetylamantadine with a
sensitivity of 1 mg/mL.
5. Use of the method as claimed in claim 1 to screen a patient for
a pathological condition based on the quantification of
acetylamantadine in the urine sample.
6. Use of the method as claimed in claim 1 to screen a patient for
cancer based on the quantification of acetylamantadine in the urine
sample.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the detection and
quantification of biomarkers and, in particular, to the detection
and quantification of acetylamantadine in urine samples.
[0003] 2. Description of the Related Art
[0004] Liquid chromatography mass spectrometry has been
successfully employed to detect and quantify extremely low
concentrations of acetylamantadine in biological samples such as
urine. This may facilitate the diagnosis of cancer at an early
stage as the quantification of acetylated forms of
spermidine/spermine N.sup.1-acetyltransferase (SSAT) including
amantadine may be used to detect various pathological conditions
including cancer as disclosed in U.S. Pat. No. 6,811,967 which
issued to Sitar et al. on Nov. 4, 2004, and the full disclosure of
which is incorporated herein by reference. However, the detection
and quantification of acetylamantadine using liquid chromatography
mass spectrometry is relatively time consuming and costly. There is
accordingly a need for an efficient and cost effective method for
detecting and quantifying acetylamantadine to allow for rapid
economical testing.
SUMMARY OF THE INVENTION
[0005] It is an object of the present invention to provide an
improved method for detecting and quantifying acetylamantadine in
urine samples.
[0006] There is accordingly provided a method for quantifying
acetylamantadine in a urine sample. The method comprises eluting
acetylamantadine from the urine sample using solid phase extraction
and quantifying the acetylamantadine eluted from the urine sample
using Raman spectroscopy.
[0007] The solid phase extraction may include eluting
acetylamantadine with methanol. The quantification of the
acetylamantadine eluted from the urine sample using Raman
spectroscopy may include the use of substrate based,
surface-enhanced Raman spectroscopy.
[0008] The method disclosed herein may be used to screen a patient
for a pathological condition based on the quantification of
acetylamantadine in the urine sample. The method disclosed herein
may also be used to screen a patient for cancer based on the
quantification of acetylamantadine in the urine sample.
[0009] The method disclosed herein may be used to detect and
quantify acetylamantadine at a low cost.
BRIEF DESCRIPTIONS OF DRAWINGS
[0010] The invention will be more readily understood from the
following description of the embodiments thereof given, by way of
example only, with reference to the accompanying drawings, in
which:
[0011] FIG. 1 shows the results of open air evaporation and slow
evaporation of acetylamantadine in a methanol drop coated on a
Surface Enhanced Raman Scattering (SERS) substrate;
[0012] FIG. 2 shows quantification of acetylamantadine using a SERS
substrate;
[0013] FIG. 3 shows Raman spectra for different concentrations of
acetylamantadine in a methanol; and
[0014] FIG. 4 shows a calibration curve based on the Raman spectra
of FIG. 3.
DESCRIPTIONS OF THE PREFERRED EMBODIMENTS
[0015] Disclosed herein is the use of Raman spectroscopy to
identify and quantify acetylamantadine, a product of
spermidine/spermine N.sup.1-acetyltransferase (SSAT) metabolism, in
urine samples. Urine is a concentrated solution of many salts,
polar metabolites and multiple non-polar steroids. Expected
concentration of acetylamantadine is about 1000 times smaller than
that of amantadine in urine samples. The distinction between
amantadine and acetylamantadine can be based on the vibrational
band of a carbonyl group at an approximately 1600 cm.sup.-1
wavenumber. There are a few other differences between the spectra
of amantadine and acetylamantadine, but this Raman band may be of
particular interest as it is present only in the spectrum of
acetylamantadine.
[0016] A urine sample was prepared and different constituents of
the urine sample were separated using solid phase extraction (SPE).
The urine sample is accordingly pre-treated using solid phase
extraction to remove impurities prior to using Raman spectroscopy
to identify and quantify acetylamantadine present in the urine
sample.
Urine Sample
[0017] Artificial urine comprising the following components of
urine NaCl 8.00 g/L, KCl 1.64 g/L, K.sub.2SO.sub.4 2.63 g/L, urea
13.40 g/L, and creatinine 1.50 g/L was used to prepare a urine
sample having corticosterone 16.7 mM, amantadine 3.3 mM, and
acetylamantadine 3.3 uM.
Solid Phase Extraction (SPE)
[0018] The urine sample was treated using solid phase extraction to
remove salts and polar impurities, increase the acetylamantadine to
amantadine ratio, and minimize contamination from non-polar
steroids. The following protocol achieved all three aims using
Strata X, Polymeric Reversed Phase from Phenomenex Inc of 411
Madrid Avenue, Torrance, Calif., 90501-1430.
[0019] (1) Prime: 2 mL MeOH, 2 mL deionized H.sub.2O, 2 mL 50 mM pH
7.0 phosphate buffer.
[0020] (2) Load: Combine 2 mL of urine sample with 2 mL of 50 mM pH
7.0 phosphate buffer and load onto SPE cartridge.
[0021] (3) Wash 1: 2 mL deionized H.sub.2O, 2.times.1.5 mL 50 mM pH
7.0 phosphate buffer (salts and polar metabolites elute with this
fraction).
[0022] (4) Wash 2: 2.times.2 mL 40% methanol in H.sub.2O.sub.2O
(amantadine elutes with this fraction while acetylamantadine and
the less polar steroid corticosterone is retained).
[0023] (5) Wash 3: 2 mL 100% methanol (acetylamantadine elutes with
this fraction while corticosterone is retained).
[0024] (6) Dry column by flushing air through it for a few
minutes.
[0025] (7) Eluent: 2 mL ethyl acetate (corticosterone elutes).
Coating of acetylamantadine over Surface Enhanced Raman Scattering
(SERS) Substrate
[0026] Acetylamantadine in methanol, obtained from Wash 3 of SPE
protocol above, is drop coated on the SERS substrate for Raman
measurements. In this example, the SERS substrate was a
Klarite.RTM. SERS substrate from Renishaw Inc. of 5277 Trillium
Boulevard, Hoffman Estates, Ill., 60192. Uniform coating of
acetylamantadine over the SERS substrate assists in reliable
quantification. It was observed that slow evaporation of methanol
results in improved coating of acetylamantadine over the substrate.
FIG. 1 shows the results of open air evaporation and slow
evaporation where the air flow is restricted. It can be seen that
slow evaporation results in uniform coating.
Raman Measurements
[0027] In this example, 30 uL of acetylamantadine in methanol was
drop coated on the SERS substrate and allowed to dry slowly. A
Raman map of 170 mesh points was collected with 1 second of
integration at each mesh point. Out of the 170 spectra, only those
were retained which showed Raman peaks, the rest were neglected.
FIG. 2 shows the quantification based on the 1600 cm.sup.-1 band.
The required resolution and limit of detection of 1 ng/mL is
achieved with adequate signal to noise ratio. It will however be
understood by a person skilled in the art that it is desirable to
use a number of different peaks to create a calibration curve
because different peaks will result in result in calibration curves
having slightly different slopes.
Raman Data and Analysis Used to Create a Calibration Curve
[0028] FIG. 3 shows Raman measurements for acetylamantadine in
methanol in the following concentrations 1 ng/mL, 5 g/mL, 10 ng/mL,
25 ng/mL and 50 ng/mL which were prepared using standard chemistry
techniques to dissolve acetylamantadine in methanol. Five peaks in
the Raman spectra were chosen for each concentration, namely, 738
cm.sup.-1, 776.8 cm.sup.-1, 1198 cm.sup.-1, 1210 cm.sup.-1 and 1436
cm.sup.-1.
[0029] Each peak was separated into a peak area and an adjacent
area. Ten points were chosen in each peak area and adjacent area.
The points were integrated and the number sum of peak area minus
number sum of its adjacent area was used to get the intensity for
each peak. It was then possible to get the Raman intensity for each
concentration by integrating the five peaks as shown below.
n = 5 ( n_peak = 10 I peak - n_adjacent = 10 I adjacent )
##EQU00001##
[0030] Where I.sub.peak is the intensities in peak area and
I.sub.adjacent is the intensities in adjacent area. The sum of
intensities for each concentration were then plotted to create the
calibration curves shown in FIG. 4 which also shows the
revalidation of the analysis. The calibration curve may be used to
detect and quantify the acetylamantadine in a urine sample.
Conclusion
[0031] Results demonstrate that acetylamantadine can be extracted
from urine samples using solid phase extraction. Raman spectroscopy
can then be used to simultaneously detect and quantify the
acetylamantadine with a sensitivity of 1 ng/mL in the urine sample
to screen a patient for a pathological condition such as
cancer.
[0032] It will be understood by a person skilled in the art that
many of the details provided above are by way of example only, and
are not intended to limit the scope of the invention which is to be
determined with reference to the following claims.
* * * * *