Quantitative acetaminophen analytics

Abstract

A method for quantitatively determining the amount of acetaminophen in a sample with greater precision, greater sensitivity and fewer interactions and fewer spectral and chemical interferences with other compounds contained in the sample. The method includes acetaminophen being hydrolyzed and the resulting p-aminophenol being reacted with a compound of general formula (III): ##STR00001##
in the presence of an oxidant to form a compound of general formula (IV): ##STR00002##
wherein R1 and R2, independently of one another, are selected from H, CH.sub.3, and OCH.sub.3, R3 is C.sub.2H.sub.5 and R4 is a C.sub.1-4 alkyl moiety with a terminal sulfonate group, with the proviso that at least one of R1 and R2 is OCH.sub.3 and/or R4 additionally has at least one OH substituent, and then the amount of the compound of general formula (IV) in the reaction mixture being photometrically determined.

Claims

1. A method for quantitatively determining an amount of acetaminophen in a sample, comprising: producing a reaction mixture by hydrolyzing acetaminophen having formula (I) ##STR00008## resulting in p-aminophenol having formula (II) ##STR00009## and reacting the p-aminophenol having formula (II) with a compound of general formula (III) ##STR00010## in the presence of an oxidant to form a compound of general formula (IV) ##STR00011## wherein, in formulae (III) and (IV), R1 and R2, independently of one another, are selected from H, CH.sub.3, and OCH.sub.3, R3 is C.sub.2H.sub.5 and R4 is a substituted C.sub.1-4 alkyl moiety with a terminal sulfonate group, with the proviso that at least one of R1 and R2 is OCH.sub.3 and/or R4 additionally comprises at least one OH substituent, and then an amount of the compound of general formula (IV) in the reaction mixture is photometrically determined.

2. The method according to claim 1, wherein the oxidant is selected from the group consisting of hydrogen peroxide, an organic peroxide, an iron-cyanide complex, a periodate salt, and a periodate complex.

3. The method according to claim 1, wherein an absorption maximum of the compound of general formula (IV) lies in a wavelength range of from 650 nm to 800 nm.

4. The method according to claim 1, wherein the method is carried out in a presence of N-ethylmaleimide and/or maleimide.

5. The method according to claim 1, wherein the method is carried out in a presence of EDTA.

6. The method according to claim 1, wherein the sample is a liquid sample and has a volume of 5 ?L or less.

7. The method according to claim 1, wherein the method is carried out in a presence of a phosphate buffer.

8. The method according to claim 1, wherein the method is carried out in a presence of boric acid.

9. The method according to claim 1, wherein the method is carried out at a pH of from 8 to 10.5.

10. The method according to claim 1, wherein the sample is a liquid sample and is selected from blood, blood serum or blood plasma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph showing curve progressions of the photometric evaluation of different coupling agents over the wavelength of from 500 nm to 800 nm; and

(2) FIG. 2 is a wavelength scan representing results of a photometric determination.

EXAMPLES

1. Preliminary Tests

(3) In order to be able to achieve a still greater precision, a still greater sensitivity and still fewer interactions and fewer spectral and chemical interferences with other compounds contained in the sample for the method according to the invention for quantitatively determining the amount of acetaminophen, different compounds of general formula (III) were tested as coupling agent for the reaction with the p-aminophenol formed from the hydrolysis of acetaminophen.

(4) For this purpose, a sample was used the concentration of which was 100 mg/L acetaminophen, and, after the hydrolytic cleavage by aryl acylamidase, the reaction with compounds Nos. 1 to 7 specified in the following table was effected.

(5) TABLE-US-00001 Abs. No. Name Type R1 R2 R3 R4 (? = 700 nm) 1 ALPS quinoline H H C.sub.2H.sub.5 C.sub.3H.sub.5SO.sub.3Na 0.08 2 ADPS quinoline OCH.sub.3 H C.sub.2H.sub.5 C.sub.3H.sub.6SO.sub.3Na 0.59 3 TOOS quinoline CH.sub.3 H C.sub.2H.sub.5 C.sub.3H.sub.5(OH)SO.sub.3Na 0.13 4 DAOS quinoline OCH.sub.3 OCH.sub.3 C.sub.2H.sub.5 C.sub.3H.sub.5(OH)SO.sub.3Na 0.27 5 reference o-cresol 0.01

(6) Compound No. 1 does not have the constitution required according to the invention, as neither R1 nor R2 is OCH.sub.3 and R4 does not have an OH substituent either. Compounds Nos. 2 to 4 are to be described as according to the invention in comparison herewith.

(7) Compound No. 2 is characterized in that R1 is OCH3. Compound No. 2 thus has the constitution required according to the invention. Compounds Nos. 3 and 4 also have a constitution according to the invention as R4 has an OH substituent in both compounds. Compound No. 4 is in particular also characterized in that both R1 and R2 are OCH3.

(8) Compound No. 5 represents the state of the art in which o-cresol (=ortho-cresol or 2-methylphenol) is used as coupling agent.

(9) The photometric evaluation of the coupling agents tested here revealed that, at the wavelength of ?=700 nm that is of particular interest for the purposes of the invention, compounds No. 2 to No. 4 according to the invention achieved the highest absorbance values. Admittedly, coupling agent No. 5 (o-cresol) also lies in a good range. In particular, the very good absorbance values of the coupling agents No. 2 and No. 4 according to the invention are not achieved, however. In addition, o-cresol has the disadvantage that it is toxic and of limited stability, which can lead to the fact that the measurement reliability in some cases cannot be reproduced over the entire diagnostically relevant range with sufficient reliability.

(10) The complete curve progressions of the photometric evaluation of the different coupling agents over the wavelength range of from 500 nm to 800 nm are represented in the attached FIG. 1.

2. Embodiment Example

(11) In the embodiment described here, the different reagents used for the reaction are provided in the form of a reagent kit with the following solutions, which are stored in separate containers. Here R1 denotes a first reagent solution and R2 denotes a second reagent solution.

(12) R1 contains, in aqueous solution, periodate (1.88 mmol/L) N-ethylmaleimide (10 mM)

(13) and has a phosphate-buffered pH of 10.65.

(14) R2 contains, in aqueous solution, aryl acylamidase (7 u/ml) ADPS (10 mmol/L)

(15) and has a phosphate-buffered pH of 7.00.

3. Test Example

(16) The reagent kit in accordance with the reagent kit described under paragraph 2. is used in a method for determining acetaminophen in the following way.

(17) Whole blood samples with different acetaminophen concentrations are haemolyzed and the reagent solutions according to embodiment example 2 are then added.

(18) Then, a photometric determination of the p-aminophenol quantitatively reacted with the coupling agent is effected.

(19) In FIG. 2 the results of the photometric determination are represented in the form of a wavelength scan. Here, it is apparent that at a wavelength of 700 nm the inherent absorption of the whole blood cannot exert an interfering influence on the acetaminophen determination.