# US 11,674,965 B2 · Method for preparing and analyzing fluorescent compounds in plasma

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- Number: US 11,674,965 B2
- Document ID: US11674965B2
- Status: Granted
- Kind: B2
- Application number: 16/192,113
- Filed: 2018-11-15
- Published: 2019-05-23
- Granted: 2023-06-13
- Earliest claimed: 2017-11-20
- Assignees: MediBeacon Inc.
- Applicants: MediBeacon, Inc.
- Inventors: Jeng J. Shieh
- Web page: https://patentgrep.com/patent/US11674965B2

## CPC

- G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00})
- G01N 33/582: {with fluorescent label}
- G01N 30/14: by elimination of some components
- C07D 229/00: Heterocyclic compounds containing rings of less than five members having two nitrogen atoms as the only ring hetero atoms
- G01N 1/38: Diluting, dispersing or mixing samples
- G01N 30/06: Preparation
- G01N 30/22: in high pressure liquid systems
- G01N 30/74: Optical detectors {(measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J)}
- G01N 2800/347: Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
- Y10T 436/147777: Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Disclosed herein is a method for analyzing the concentration of a fluorescent compound in the plasma of a patient. The method includes collecting a sample of plasma from a patient, diluting the sample with a solvent and analyzing the diluted sample by HPLC. The sample does not need to be dried down during sample preparation nor is an internal standard required.

## Claims

### Claim 1

A method for measuring the amount of a fluorescent compound in plasma, the method comprising:
diluting a sample of plasma comprising the fluorescent compound with an aqueous buffer; and
analyzing the diluted sample by high performance liquid chromatography (HPLC) thereby measuring the amount of the fluorescent compound in the sample of plasma;
wherein the sample of plasma is not dried before HPLC analysis, and no internal standard is added to the sample,
wherein the method does not include a protein precipitation step that comprises causing proteins to precipitate from the sample of plasma and removing the precipitated proteins from the sample prior to HPLC analysis,
wherein the fluorescent compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein
each of X1 and X2 is independently —CO2R1, —CONR1R2, —CO(AA) or —CONH(PS);
each of Y1 and Y2 is independently selected from the group consisting of —NR1R2; and
Z1 is a single bond, —CR1R2—, —O—, —NR1—, —NCOR1—, —S—, —SO—, or —SO2—;
each of R1 to R2 are independently selected from the group consisting of H, —CH2(CHOH)aH, —CH2(CHOH)aCH3, —CH2(CHOH)aCO2H, —(CHCO2H)aCO2H, —(CH2CH2O)cH, —(CH2CH2O)cCH3, —(CH2)aSO3H, —(CH2)aSO3−, —(CH2)aSO2H, —(CH2)aSO2−, —(CH2)aNHSO3H, —(CH2)aNHSO3−, —(CH2)aNHSO2H, —(CH2)aNHSO2−, —(CH2)aPO3H2, —(CH2)aPO3H−, and —(CH2)aPO32−;
AA is a peptide chain comprising one or more amino acids selected from the group consisting of natural and unnatural amino acids, linked together by peptide or amide bonds and each instance of AA may be the same or different than each other instance;
PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units connected by glycosidic linkages; and
‘a’ is a number from 1 to 10, ‘c’ is a number from 1 to 100, and each of ‘m’ and ‘n’ are independently a number from 1 to 3.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the aqueous buffer comprises a phosphate buffering agent.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the aqueous buffer is phosphate buffered saline (PBS).

### Claim 4 (depends on 1)

The method according to claim 1, wherein the sample of plasma is diluted with the aqueous buffer in a dilution ratio (v/v) of from 1:5 to 1:1000 (sample:buffer).

### Claim 5 (depends on 1)

The method according to claim 1, wherein the fluorescent compound is
or a pharmaceutically acceptable salt thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the sample of plasma exhibits signs of blood hemolysis.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the sample of plasma comes from a human, an animal or an in vitro study.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the method further comprises preparing a linear calibration curve for HPLC analysis of the sample of plasma, the linear calibration curve prepared using calibration standards containing the fluorescent compound in a concentration of from 0.4 ng/mL to 400 ng/mL, and wherein the diluted sample analyzed by HPLC comprises the fluorescent compound in a concentration of from 0.4 ng/mL to 400 ng/mL.

### Claim 9 (depends on 8)

The method according to claim 8, wherein the diluted sample analyzed by HPLC comprises the fluorescent compound in a concentration of 0.4 ng/mL, and wherein the fluorescent compound is detected with a signal to noise ratio greater than 15:1.

### Claim 10

A method for measuring the amount of a fluorescent compound in plasma, the method comprising:
diluting a sample of plasma comprising the fluorescent compound with an aqueous buffer comprising a phosphate buffering agent, wherein the sample of plasma is diluted with the aqueous buffer in a dilution ratio (v/v) of from 1:5 to 1:1000 (sample:buffer); and
analyzing the diluted sample by high performance liquid chromatography (HPLC) thereby measuring the amount of the fluorescent compound in the sample of plasma;
wherein the sample of plasma is not dried before HPLC analysis, and no internal standard is added to the sample,
wherein the method does not include a protein precipitation step that comprises causing proteins to precipitate from the sample of plasma and removing the precipitated proteins from the sample prior to HPLC analysis,
wherein the fluorescent compound is a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein
each of X1 and X2 is independently —CO2R1, —CONR1R2, —CO(AA) or —CONH(PS);
each of Y1 and Y2 is independently selected from the group consisting of —NR1R2; and
Z1 is a single bond, —CR1R2—, —O—, —NR1—, —NCOR1—, —S—, —SO—, or —SO2—;
each of R1 to R2 are independently selected from the group consisting of H, —CH2(CHOH)aH, —CH2(CHOH)aCH3, —CH2(CHOH)aCO2H, —(CHCO2H)aCO2H, —(CH2CH2O)cH, —(CH2CH2O)cCH3, —(CH2)aSO3H, —(CH2)aSO3−, —(CH2)aSO2H, —(CH2)aSO2−, —(CH2)aNHSO3H, —(CH2)aNHSO3−, —(CH2)aNHSO2H, —(CH2)aNHSO2−, —(CH2)aPO3H2, —(CH2)aPO3H−, and —(CH2)aPO32−;
AA is a peptide chain comprising one or more amino acids selected from the group consisting of natural and unnatural amino acids, linked together by peptide or amide bonds and each instance of AA may be the same or different than each other instance;
PS is a sulfated or non-sulfated polysaccharide chain comprising one or more monosaccharide units connected by glycosidic linkages; and
‘a’ is a number from 1 to 10, ‘c’ is a number from 1 to 100, and each of ‘m’ and ‘n’ are independently a number from 1 to 3.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the fluorescent compound is
or a pharmaceutically acceptable salt thereof.

### Claim 12 (depends on 10)

The method according to claim 10, wherein the aqueous buffer is phosphate buffered saline (PBS).

### Claim 13 (depends on 10)

The method according to claim 10, wherein the sample of plasma exhibits signs of blood hemolysis.

### Claim 14 (depends on 10)

The method according to claim 10, wherein the sample of plasma comes from a human, an animal or an in vitro study.

### Claim 15 (depends on 10)

The method according to claim 10, wherein the sample of plasma comes from a human.

### Claim 16 (depends on 10)

The method according to claim 10, wherein the sample of plasma is diluted with the aqueous buffer in a dilution ratio (v/v) of 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, or 1:1000 (sample:buffer).

### Claim 17 (depends on 10)

The method according to claim 10, wherein the sample of plasma is diluted with the aqueous buffer in a dilution ratio (v/v) of 1:100 (sample:buffer).

### Claim 18 (depends on 10)

The method according to claim 10, wherein the aqueous buffer does not cause decomposition of the diluted sample during HPLC analysis.

### Claim 19 (depends on 10)

The method according to claim 10, wherein the method further comprises preparing a linear calibration curve for HPLC analysis of the sample of plasma, the linear calibration curve prepared using calibration standards containing the fluorescent compound in a concentration of from 0.4 ng/mL to 400 ng/mL, and wherein the diluted sample analyzed by HPLC comprises the fluorescent compound in a concentration of from 0.4 ng/mL to 400 ng/mL.

### Claim 20 (depends on 19)

The method according to claim 19, wherein the diluted sample analyzed by HPLC comprises the fluorescent compound in a concentration of 0.4 ng/mL, and wherein the fluorescent compound is detected with a signal to noise ratio greater than 15:1.

### Claim 21 (depends on 10)

The method according to claim 10, wherein the pH of the diluted sample is between about 7.0 and about 7.4.

### Claim 22 (depends on 10)

The method according to claim 10, wherein plasma proteins in the diluted sample do not have a peak eluted during HPLC analysis that interferes with a peak of the fluorescent compound eluted during the HPLC analysis.

### Claim 23 (depends on 10)

The method according to claim 10, wherein diluting the sample of plasma includes a single diluting step.