Note: Descriptions are shown in the official language in which they were submitted.
1293676
RAN 4091/15
The present invention is concerned with a method for
the determination of total bilirubin in a biological
sample according to the diazo method as well as with
reagents which are suitable therefor.
The determination of total bilirubin according to the
diazo method i8 a technique which is very well known to
the person skilled in the art. This method as well as the
10 reagents which are used therefor do, however, have certain
disadvantages, in particular it has not been possible to
date to carry out this method in automatic analyses
according to the so-called autoblanking erocedure.
The method in accordance with the present invention
and, re~pectively, the reagents provided therefor result
in a test which is di~tinguished by the following
surprising advantages:
- high linearity
- favourable reaction kinectics which enable the
autoblanking system to be used
- simple reagent preparation
- - long stability of the solution which i8 used and
- good precision and good reproducibility.
As iB known, the diazo method for the determination of
bilirubin comprises reacting the bilirubin which is
present in the biological sample with a diazonium compound
30 to give a colouring substance, the intensity of which
corresponds to the amount of bilirubin in the sample.
In the scope of the pre6ent invention it has now
surprisingly been found that the above-mentioned advant-
Klt/24.7.86
~k
12~3676
ages can be achieved when one or more detergents of thegeneral formula
(R2)
¦~R3
R1 - N\
R4
wherein Rl signifies straight or branched alkyl with
10-16 C-atoms, R2 signifies oxygen or straight or
branched alkyl with 1-4 C-atoms; R3, insofar as n =
0, signifies hydrogen or straight or branched alkyl
with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is
a whole number of 1-5, R3, insofar as n = 1 and R2
signifies oxygen, signifies straight or branched alkyl
with 1-4 C-atoms or -(CH2-CH2-0)xH in which x is
a whole number of 1-5, R3, insofar as n = 1 and R2
signifies straight or branched alkyl with 1-4 C-atoms,
signifies straight or branched alkyl with 1-4 C-atoms;
R4, insofar as n = 0, signifies hydrogen or straight
or branched alkyl with 1-4 C-atoms or
-(CH2-CH2-0)xH in which x is a whole number of
1-5, R4 insofar as n = 1 and R2 signifies oxygen,
signifies straight or branched alkyl with 1-4 C-atoms
or -(CH2-CH2-0)xH in which x is a whole number
of 1-5, R4, insofar as n = 1 and R2 signifies
straight or branched alkyl with 1-4 C-atoms, signifies
straight or branched alkyl with 1-10 C-atoms; or R2,
R3 and R4 together with the nitrogen atom form a
pyridinium group, whereby in this case as well as in
the case where n = 1 and R2 signifies straight or
branched alkyl with 1-4 C-atoms a corresponding anion
is present,
is/are used in the bilirubin determination according to
35 the diazo method.
1~93~i76
-- 3
~ Preferred deteegents talso referred to hereinafter as
accelerator reagents~ of formula I are N,N-dimethyl-decyl-
amine, N,N-dimethyl-dodecylamine, N,N-dimethyl-tetradecyl-
amine, N,N-dimethyl-hexadecylamine, didecyldimethyl-
ammonium chloride, dodecyltrimethylammonium chloride,
dodecyltrimethylammonium bromide, dodecylethyldimethyl-
ammonium bromide, dodecylpyridinium chloride, tetradecyl-
trimethylammonium bromide as well as N,N-dimethyldodecyl-
amine oxide. Dodecylethyldimethylammonium bromide is an
10 especially preferred detergent of general formula I.
According to a first aspect, the present invention is
concerned with an aqueous reagent for the determination of
total bilirubin in a biological sample, said reagent
16 containing on the one hand
a) one or more detergents of the general formula
20 (R2)n
Rl I ~ 3
~ R4
wherein Rl signifies straight or branched alkyl
with 10-16 C-atoms, R2 signifies oxygen or
straight or branched alkyl with 1-4 C-atoms;
R3, insofar as n = 0, signifies hydrogen or
straight or branched alkyl with 1-4 C-atoms or
-~CH2-CH2-0)xH in which x is a whole number
of 1-5, R3, insofar as n = 1 and R2 signifies
oxygen, signifies straight or branched alkyl with
1-4 C-atoms or -(CH2-CH2-0)xH in which x is
a whole number of 1-5, R3, insofar as n = 1 and
R2 signifies straight or branched alkyl with
1-4 C-atoms, signifies straight or branched alkyl
with 1-4 C-atoms: R4, insofar as
1~3676
-- 4
n =0, signifies hydrogen or straight or branched
alkyl with 1-4 C-atoms or -(CH2-CH2-0)xH in
which x is a whole number of 1-5, R4 insofar as
n = 1 and R2 signifies oxygen, signifies
straight or branched alkyl with 1-4 C-atoms or
-(CH2-CH2-0)xH in which x i8 a whole number
of 1-5, R4, insofar as n = 1 and R2 signifies
straight or branched alkyl with 1-4 C-atoms,
signifies straight or branched alkyl with 1-10
C-atoms; or R2, R3 and R4 together with the
nitrogen atom form a pyridinium group, whereby in
this case as well as in the case where n = 1 and
R2 signifies straight or branched alkyl with
1-4 C-atoms a corresponding anion is present,
b) an aromatic amino compound which can be converted
with nitrite into a diazonium compound which is
customary in the determination of bilirubin,
c) a strong acid, and on the other hand
d) an aqueous nitrite solution.
According to a second aspect, the present invention is
25 concerned with an aqueous reagent for the determination of
total bilirubin in a biological sample, said reagent
containing
a) one or more detergent~ of general formula I,
b) a diazonium compound which is customary in the
determination of bilirubin and
c) a strong acid.
In the aforementioned aqueous reagent~ the detergent
35 of general formula I is preferably present in a concentra-
1~93676
tion of 0.25-lOS, especially in a concentration of 1-3%.
The aromatic amino compound is preferably present in a
concentration of 0.5-40 mmol, especially preferably in a
concentration of 1-30 mmol.
Amidosulphuric acid, hydrochloric acid and sulphuric
acid are the preferred strong acids.
The concentration of amidosulphuric acid preferably
lies in the range of 0.025-1 mol/l, especially in a range
of 0.10-0.25 mol/l, and the pH of the reagent i8
preferably between 1 and 3.
When hydrochloric acid is u~ed, the concentration i5
preferably in the range of 0.01-0.5 mol/l, especially in
the range of 0.05-0.2 mol/1, and the pH of the reagent is
preferably 1-3.
When sulphuric acid is used, the concentration prefer-
ably lies in the range of 0.01-0.5 mol/l, especially in
the range of 0.05-0.25 mol/l, and the pH of the reagent is
preferably between i and 3.
The aromatic amino compound, which can react with the
nitrite solution to give the corre~ponding diazonium
compound, is preferably 4-aminobenzenesulphonic acid,
- 2,4- or 2,5-dichloroaniline, 4-nitroaniline, 2-methoxy-4-
-nitroaniline or 2-methoxy-5-nitroaniline.
The previou61y mentioned nitrite solution i~ prefer-
ably pre6ent in a concentration of 4-100 mmol/l.
Insofar as the reagent already contain~ the formed
diazonium compound, this is preferably diazotized 4-amino-
benzenesulphonic acid or diazotized 2,4- or 2,5-dichloro-
lZ93~76
aniline, 4-nitroaniline, 2-methoxy-4-nitroaniline or
2-methoxy-5-nitroaniline.
The concentration of diazonium compound in the aqueous
solution preferably lies in the range of 0.10-5.0 g/l,
especially ih a range of 0.15-3.0 g/l.
The preferred detergents of general formula I have
already been mentioned earlier, but in addition to this it
can also be remarked that the substituents R3 and R4
in general formula I preferably have the same significance.
~ ccording to a third aspect, the pLesent invention is
concerned with a stabilized and lyophilized reagent for
the determination of total bilirubin in a biological
sample, said reagent containing diazotized 4-aminobenzene-
sulphonic acid or diazotized 4-nitroaniline, 2-methoxy-4-
-nitroaniline, 2-methoxy-5-nitroaniline, 2,4-dichloro-
aniline or 2,5-dichloroaniline, as well as one or more
detergents of general formula I.
According to a fourth aspect, the present invention is
concerned with a method for the determination of total
bilirubin in a biological sample according to the diazo
method at a strongly acidic pH, which method comprises
using one or more detergents of general formula I.
According to a fifth aspect, the present invention is
3 concerned with the use of a detergent of general formula 1
in a method for the determination of total bilirubin in a
biological sample according to the diazo method at a
strongly acidic pH.
According to a sixth aspect, the present invention is
concerned with a method for the determination of total
bilirubin in a biological sample according to the diazo
~2936 ~
method at a strongly acidic pH according to the auto-
blanking procedure, which method comprises using one or
more detergents of general formula 1.
The fo}lowing Examples illustrate the invention:
ExamDle
- Reaaent solutions:
The reagent solution 1 contains 20 g of dodecylethyl-
dimethylammonium bromide and 5.195 g of sulehanilic acid
dissolved in one litre of O.O5N hydrochloric acid. The
reagent 2 i~ a 6.25 mmol aqueous sodium nitrite solution.
The reagent 1 is mixed with reagent 2 in the ratio 5 to 1.
The reagent mixture is ready-for-use after 1 minute at RT.
- Test Derformance on Cobas-Bio0
The reagent used is mixed with serum or bilirubin
standard in the ritio 17.5:1 at a determined temperature
(25C, 30C or 37C). The extinction is measured at 550 nm
exactly after 4.5 seconds and again after 30g.5 seconds.
- Evaluation:
- by com~arsion of the extinction diffe~ence with a
bilirubin standard or a control serum:
~E2P-ElP) -RB
- x bilirubin concentration (S)
(E2S-ElS)-RB
P = sample
S = standard or control serum.
~ 93~76
Reagent blank value tRB) is measured and deducted
systematically by the apparatus.
by factor: mg/dl = 18.09 x ~E
~mol/l , 309 x ~E
Figure 1 6hows the verification of linearity with a highly
concentrated bilirubin control serum t42.8 mg/dl).
ExamDle 2
- Reaaent solutions
see Example 1
- Test Performance: manual Drocedure
The reagent solutlons are pipetted into cuvettes
tlayer thickness 1 cm) at room temperature in the
following anner:
~nalvsis (A) Analvsis blank
value tAB)
- Reagent solution 1 - 1.0 ml
- Reagent mixture 1.0 ml
- Sample 0.10 ml 0.10 ml
The mixture is mixed well and then left to stand at RT
for 10 minutes. In the following 60 minutes the extinc-
tions of the sample against analysis blank value are read
off at 546 nm or 550 nm.
The reagent blank value can be disregarded with fresh
reagent mixture tl day at RT or 5 days at 4C).
i;~93~ ~6
- Evaluation
by comparison o~ the extinction difference with
bilirubin standard or control serum:
E(A~-E(AB) x bilirubin concentration (S)
E(S)-E(SB)
by factor 12.4 x ~E (mg/dl)
212 x ~E (~mol/l).
Exam~le 3
- Rea~ents
- LvoDhilized dia20 reaqent
In a 25 mmol aqueous amidosulphuric acid solution
there are di6601ved in 6equence:
4 g of D-mannitol
1 g of Dextran 32 and
2.4 g of diazotized sulphanilic acid tetrafluoro-
borate salt.
The solution is made up tD 1 litre and divided into 3 ml
portions for the lyophilization. When the temperature in
the lyophilizator rearheç -40C, the vacuum i6 adju6ted
(0.1 mm Hg). The plate6 are then heated to 10C under a
- vacuum for about 16 hour~. Finally, the lyophilizate i6
dried at 20C for 2 hours under a vacuum.
- Accelecator rea~ent
To a 0.25M aqueous amidosulphuric acid 601ution there
are added in 6equence:
* trade mark
~'
. ..~.
1293676
-- 10 --
75 ml of dodecyldimethylamine oxide (30% aqueous
solution)
1 g of methylparaben and
25 g of D-mannitol.
The solution is made up to 1 litre.
This solution can be used as the liquid reagent or, a~
above, can be lyophilized after division into 6 ml
portions.
- Rea~ents used
The lyophilized diazo reagent i8 dissolved in 3 ml of
deionized water. Insofar as the accelerator reagent is
lyophilized, it i6 dissolved in 6 ml of deionized water.
- Test Performance on Cobas BioO
The accelerator reagent solution is mixed with a 6erum
sample (P) or a bilirubin standard (S) in the ratio 12.5:1
at a determined temperature (25C, 30C or 37C). ~fter an
incubation period of 10 seconds the extinction is measured
at 550 nm (. analysis blank value). 40 microlitres of
diazo reagent solution are then added to the cuvette and
the extinction is read off after 1 second and again after
301 seconds.
- Evaluation:
By comparison of the extinction difference with a
- bilirubin standard or with a control serum:
(E2P-ElP)-~B-RB
_ __ x bilirubin concentration (S)
(E2S-ElS)-SB-RB
36~6
-- 11
P = sample
S = standard
AB - analysis blank value, SB = standard blank value
RB - reagent blank value measured and deducted system-
atically by the apparatu6
by factor: 16.10 x ~E for mgJdl
275 x ~E for ~mol/l
- Fiaure 2: Kinetics of the reaction with a dilution
series of control serum mixture (40.4 mg bilirubin/dl).
ExamDle 4
- Reaaents
- lyophilized diazo reagent
see Example 3
- accelerator reagent
- Reaaents used: see Example 3
- te t Derformance: manual procedure
25. The reagent solutions are pipetted into cuvettes
(layer thickness 1 cm) at room temperature in the
following manner:
- sample or standard: 0.10 ml
-.accelerator reagent solution 1.00 ml
mixed well and the extinction (El) is read off at
546 nm or 550 nm against water.
- Diazo reagent solution 0.20 ml
..
3676
- 12 -
is added, mixed well and after 6 minutes the
extinction (E2) i8 read off against water.
- Evaluation
- by comparison of the extinction difference with a
bilirubin standard or with a control serum:
E2P-ElP
x bilirubin concentration (S)
E2 S-El S
- by factor: 16.3 x ~E (mg/dl)
279 x ~E (~mol/l)
- Fiaure 3 Accuracy with control sera (concentration in
mg/dl).
ExamDle 5
- Reaaents
- lvo~hilized diazo reaaent see Example 3
- accelerator reaaent
- Reaaents used
- Insofar as the accelerator reagent is lyophilized,
it is dissolved in 6 ml of deionized water.
-
- The diazo reagent is dissolved in 18 ml of acceler-
ator reagent solution.
- Test Performance on Cobas BioO
The reagent used is mixed with sérum or bilirubin
-
3t~7f~
- 13 -
standard in the ratio 17.5:1 at a determined temperature
(25C, 30C or 37C). The extinction i8 measured exactly
after 4.5 seconds at 550 nm and aqain after 304.5 6econds.
- Evaluation:
- comparison of the extinction difference as in
Example 3
- with factor , 16.80 x ~E (mg/dl)
, 287 x AE (~mol/l)
- Fiaure 4:
Xinetics of the reaction with the dilution series of a
control serum mixture (40.4 mg/dl).
Exam~le 6
- Reaa~ents see Example 5
- Test ~erformance: (manual procedure)
The solutions given below are pipetted into cuvettes
(layer thickness 1 cm) at room temperature as follows:
AnalYsis (A) Analvsi6 blank
value (AB)
- Accelerator reagent golution - 1.0 ml
- Reagent solution used 1.0 ml
- Serum or standard 0.10 ml 0.10 ml
The mixture is mixed well and after 6 minutes the
extinction is read off against the extinction (AB) at
546 nm or 500 nm.
36'76
- 14 -
.
A reagent blank value is measured for each series
(reagent solution used).
- Evaluation
- by extinction difference with a standard
EtA~ E~(AB R x bilirubin concentration (S)
E(S)-E(SB)-RB
with factor: 13.20 x ~E (mg/dl)
226 x ~E (~mol/l)
ExamDle 7
- LYoDhilized monoreaaent
To a 0.333M aqueous amidosulphuric acid solution there
are added and admixed in sequence
60 g of dodecylethy}dimethylammonium bromide
3 g of Dextran 32 and
1 g of diazotized sulphanilic acid tetrafluoroborate
salt.
The solution is made up to 1 litre and divided into 2 ml
portions for the lyophilization. The lyophilization i8
carried out as described in Example 3.
- Reaaent used:
The lyophilizate is disgolved iA 6 ml of deionized
water. This reagent is especially favourable for automatic
analyses.
. "
`-' lZ93676
- 15 -
- Test Performance on Cobas Bio
as described in Example 1.
- Evaluation:
- by comparison with standard or control serum; see
Example 1
- by factor 16.95 x aE (mg/dl)
290 x ~E (~mol/l).
- Fiaure 5 Recovery study with control serum mixtures
(23-366 ~mol/l).
Example 8
Lyophilized monoreagent with diazotized
2,4-dichloroaniline
20 - LYohilized monoreaaent
To a 0.33M aqueous amidosulphuric acid solution there
are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide
6 g of Dextran 32 and
1.4 g of diazotized 2,4-dichloroaniline stabilized
with naphthalenedisulphonic acid sodium ~alt.
The solution is made up to 1 litre and divided into 2 ml
30 portions for the lyophilization (see Example 3).
- Reaaent used
The lyophilizate i8 di6601ved in 6 ml of deionized
3~5 water-
3t~76
- 16 -
- Test Procedure on Cobas-Bio
as described in Example 1
- Evaluation-
- by comparison with standard or control serum as
described in Example 1
- with factor: 19.80 x ~E (mg/dl)
- Fiaure 6:
Linearity verification with a highly concentrated
bilirubin control serum (37.7 mg/dl).
15
ExamPle 9
Lyophilized monoreagent with diazotized
2,5-dichloroaniline
- LvoPhilized monoreaaent
To a 0.333M aqueous amidosulphuric acid solution there
are added and admixed in sequence:
30 g of dodecylethyldimethylammonium bromide
6 g of Dextran 32 and
1.4 g of diazotized 2,5-dichloroaniline stabilized with
naphthalenedisulphonic acid sodium salt.
The solution is made up to 1 litre and divided into 2 ml
portions for the lyophilization (see Example 3).
- Reaaent used
The lyophilizate i8 dissolved in 6 ml of deionized
water.
, .
1293676
- 17 -
- Test Procedure on Cobas-Bio~
.
The reagent used is mixed with serum or bilirubin
standard in the ratio 17.5:1 at 25C. The extinction is
measured at 550 nm exactly after 4.5 seconds and again
after 304.5 seconds.
- Evaluation
- by comparison of the extinction difference with a
bilirubin standard or a control serum
- by factor: mg/dl = 21.11 x aE
~mol/l = 361 x aE.
ExamPle 10
Lyophilized monoreagent with 4-nitrobenzenediazonium
tetrafluoroborate salt
- LYoPhilized monoreaaent
In a 0.333M aqueous amidosulphuric acid solution there
are added and admixed in sequence:
54 ml of dodecyldimethylamine
6 g of Dextran 32 and
0.6 g of 4-nitrobenzenediazonium tetrafluoroborate
salt.
The solution is made up to 1 litre and divided into 2 ml
portions for the lyophilization (see Example 3).
- Reaaent uged
The lyophilizate is dissolved in 6 ml of deionized
water.
1~93676
- 18 -
- Test ~rocedure on Cobas-Bio
as described in Example 9.
- Evaluation
- by comparison of the extinction difference with a
bilirubin standard or a control serum
- by factor: mg/dl = 18.44 x ~E
~mol/l = 315.1 x ~E.
