Chapter- 3
MBTH-KIO4-Bilirubin system
Simple and sensitive method for the quantification of total bilirubin
in human serum using 3-methyl-2-benzothiazolinone hydrazone
hydrochloride as a chromogenic probe
Experimental
3.1 Reagents
3.1.1 3-Methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH)
MBTH (Molecular formula=C8H9N3S. HCl, Molecular mass=215.7) was first
synthesized by Bestom. MBTH is an electrophillic coupling reagent employed earlier
in the quantification of aromatic amines and hetero aromatic compounds [1]. Later,
this was extended for the determination of a large number of organic compounds
including those containing methylene groups, as also compounds containing carbonyl
groups such as formaldehyde [2], Schiff’s base, aromatic hydrocarbons, sacharrides,
steroids, olefins, phenols, furfural and heterocyclic bases. MBTH is also used in the
analysis of several compounds of clinical [3], biochemical [4], pharmaceutical [5],
and insecticidal importance [6]. MBTH responds to the enzymatic activity of some of
the enzymes like peroxidase [7], lactase [8], alcohol oxidase [9], and toluene-4monooxygenase [10] in the presence of corresponding substrates. MBTH is readily
soluble in water. The following structure has been assigned to MBTH.
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Chapter- 3
MBTH-KIO4-Bilirubin system
In this study, the author has made an attempt for the first time to develop a
chromogenic probe for the quantification of total bilirubin based on the cleavage of
bilirubin liberating formaldehyde which reacts with diazotized 3-methyl-2benzothiazolinone hydrazone hydrochloride (MBTH) to yield an intense blue
coloured chromogenic product. The optimization of reaction conditions have been
carried out. In the evaluation of this method the author performed linearity, precision,
accuracy, standard deviation and interference studies. Application of the proposed
method for the determination of total bilirubin in human serum sample has been
carried out and compared with the reported Jendrassik-Grof method. The final product
isolated was subjected to IR and 1H NMR spectral analysis for the confirmation of the
structure.
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3. 2 Instrumentation
A JASCO model UVIDEC-610 UV-Vis spectrophotometer with 1.0 cm
matched cells was used for all the absorbance measurements. A water bath shaker
(NSW 133, New Delhi, India) was used to maintain constant temperature for color
development. The FT-IR spectra were recorded using KBr discs on FT-IR Jasco 4100
infrared spectrophotometer. 1H NMR data were obtained using Bruker 400 MHz
instrument.
3.3 Reagents and solutions
All chemicals used in the assay were of analytical grade and double distilled
water was used throughout the assay.
3.3.1 3-Methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH)
MBTH was purchased from Sigma Aldrich. Solution of 9.27 mM was
prepared by dissolving 200 mg of MBTH in 100 ml of double distilled water. The
prepared solution was protected from sunlight by wrapping in a carbon paper.
3.3.2 Potassium periodate
Potassium periodate was purchased from S.D. Fine Chem. Ltd, India. Solution
of 3.369 mM was prepared by dissolving 75 mg of potassium periodate in 100 ml of
distilled water.
3.3.3 Bilirubin
Bilirubin was purchased from Sisco Research Laboratory Mumbai, India.
Solution of 171 µM was prepared by dispersing 1mg bilirubin in 0.1 ml
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dimethylsulfoxide, followed by 0.1 ml of 0.01 M NaOH to obtain clear orange red
solution. The solution was made up to 10 mL using distilled water to get yellow
colored solution. The solution was stable for 2 days at -4 oC in amber colored standard
flasks. This solution was further diluted as per the requirement.
3.3.4 Quality control material
Quality control material used as calibrator in Roche/Hitachi Systems used for
Jendrassik-Grof method, Lot No. 179800, was purchased from Cobas, UK, and
diluted to required concentration using distilled water during experimental study.
3.3.5 Blood samples
Blood samples were collected both from hospitals and clinical laboratories.
Fifty samples were used from discarded samples after routine diagnostic purpose;
other samples were taken from patients with suspected hyperbilirubinemia, as per the
suggestion of attending physician.
3.4 Results and Discussion
3.4.1 Quantification of bilirubin
To a final 10 ml of the reaction mixture containing 0.927 mM MBTH, 0.337
mM of potassium periodate and 5.235 M of glacial acetic acid, various concentrations
of bilirubin were added. The change in absorbance of the coloured solutions with
reference to control containing all the reagents except bilirubin was recorded at 630
nm. The product was highly stable at room temperature. Beer’s law obeyed in the
range of 0.068 μM to 17.2 μM of bilirubin with good linearity.
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MBTH-KIO4-Bilirubin system
The calibration graph for bilirubin by the proposed method was constructed
under the optimal conditions, duplicate measurements were made and the mean values
were plotted on the graph shown in Figure.3.1. There was a good linearity between
increase in absorbance with increase in concentration of bilirubin. The calibration
graph of bilirubin was linear over the range of 0.068 μM to 17.2 μM. The linearity
graph yielded y = 0.044 Cbil + 0.003. The micro molar absorption coefficient was
0.0445 L mol-1 cm-1, Sandell sensitivity was 0.006872 g/cm2, and the RSD was
0.3882% (n = 10). Limit of detection was 0.0161 μM and limit of quantification was
0.0484 μM. Precision and accuracy were studied by analyzing solution containing
known amounts of bilirubin within the Beer’s law range. Within day precision range
was 0.3% – 1.2% (n = 11, 12) and day-to-day precision of the method ranged from
1% - 6% (n = 12).
The calibration graph of bilirubin by JG method was linear over the range of
3.4 μM to 34 μM. Resultant data are plotted on the graph Figure 3.1 which shows y =
0.018 Cbil + 0.025. The micro molar absorption coefficient was 0.0178 L mol-1 cm-1.
Figure.3.1. Calibration graph of bilirubin obtained by -▲- Jendrassik-Grof method
(Y= 0.018 - 0.025 R2= 0.996), -●- Proposed method (Y= 0.044x + 0.003: R2= 0.999)
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3.4.2 Extraction of product
Reaction mixtures contained 10 ml of 92.7 mM MBTH, 10 ml of 33.7 mM
potassium periodate and 20 ml of glacial acetic acid, and 10 ml of 1.72 mM of
bilirubin. The obtained product was isolated using acetone-chloroform (1:1) [11]
mixed solvent in a separatory funnel. The extract was quickly dehydrated by adding 2
g of anhydrous sodium sulfate. The suspended particles in the extract were filtered off
through dry filter paper. The extract was concentrated by evaporating on water bath,
and allowed to dry at room temperature. The obtained dry product was spectrally
analyzed.
3.5 Optimization of reaction conditions
3.5.1 Effect of temperature
The temperature effect on reaction product was studied by preincubating 0.927
mM of MBTH, 0.337 mM of potassium periodate and 5.235 M of glacial acetic acid
and 5.16 μM of bilirubin in the temperature range of 10 – 80 oC. The results indicated
that the coloured product formed was stable in the temperature range of 20- 40 oC and
the results were reproducible. Any further increase in the temperature initiated the
decomposition process with the corresponding decrease in the absorbance values. The
decrease in temperature decreased the time for completion of reaction. Hence all
analyse were carried out at room temperature which was found optimum. Effect of
temperature on reaction product is shown in Figure.3.2.
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Chapter- 3
MBTH-KIO4-Bilirubin system
Figure.3.2. Effect of temperature on reaction
3.5.2 Effect of acetic acid
Since the reaction did not show any colour above pH 6 the reaction was
studied in various acidic buffers and with acids. The buffers and acids used for
optimisation were: dipotassium hydrogen phosphate/ potassium dihydrogen
phosphate, acetic acid/ sodium acetate, and hydrochloric acid. None of the buffers
showed maximum absorbance as that of glacial acetic acid whereas other acids did
not give any colour. Thus the reaction was optimised for glacial acetic acid. The
reaction showed increase in absorbance up to 5.235 M of glacial acetic acid and
remained constant above this concentration. Thus 5.235 M of glacial acetic acid (17.5
N) was used.
3.5.3 Effect of MBTH
The effect of various concentrations of MBTH was studied under optimized
concentration of other reagents, the intensity of the colored product increased on
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Chapter- 3
MBTH-KIO4-Bilirubin system
increasing the concentration of MBTH and decreased beyond 0.927 mM. Hence the
final concentration was set at same level for further assay procedure.
3.5.4 Effect of potassium periodate
The effect of different concentrations of potassium periodate was studied
under optimized concentration of other reagents, the intensity of the colored product
increased on increasing the concentration of potassium periodate and decreased
beyond 0.337mM. Hence the final concentration was set at same level for further
assay procedure.
3.5.5 Spectral characteristics of the colored product
Spectral characteristics of the intense blue colored product were ascertained by
scanning in the visible region between 400 and 700 nm against the control without
either bilirubin. The absorption spectra of the colored species showed an absorption
maximum at the wavelength of 620 nm. Figure.3.3 shows absorption spectra of the
colored product.
Figure.3.3. Absorption spectra of the colored product and the blank containing all the
reagents except bilirubin
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Chapter- 3
MBTH-KIO4-Bilirubin system
3.6 Proposed reaction mechanism
A similar reaction for the cleavage of bilirubin in Vanden Berg Snapper
reagent has been reported by D. W. Hutchinson and B. Johnson. Bilirubin under
strong acetic acid medium and MBTH gets cleaved at the central methylene bridge to
form two isomeric azopigments (II and III). The central methylene bridge carbon
atom of bilirubin (I) is released as formaldehyde during the second step of the reaction
[12]. The formaldehyde formed reacts with diazotized MBTH cation forming highly
resonance stabilized cationic dye [13]. The proposed reaction scheme is presented in
Figure.3.4. The final product is blue cationic dye with maximum absorption at 630
nm.
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Chapter- 3
MBTH-KIO4-Bilirubin system
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Chapter- 3
MBTH-KIO4-Bilirubin system
3.7 Precision
Precision study was carried out for both within day and day-to-day studies.
Absorbance readings were recorded with the optimized concentration of the reagents
along with four different concentrations of standard bilirubin within Beer’s law range.
Reagents were freshly prepared for day to day precision. The result obtained is
presented in Table.3.1.
Table.3.1. Precision of serum bilirubin
Within day precision
X μM
SD
1.36
Day to day precision
n
X μM
SD
CV
n
0.000711 1.1330
11
1.36
0.00415
5.8066
12
3.42
0.000710 0.4459
11
3.42
0.003548 2.2244
12
6.5
0.001203 0.3882
12
6.5
0.00376
1.202
12
10.26
0.001076 0.2336
12
10.26
0.005848 1.269
12
CV
3.8 Characterization of the product
The extracted compound was spotted on TLC and eluted with ethyl acetate acetone mixture in the ratio of 1:1. A single spot was observed through the UV
chamber confirming the extracted product to be almost pure.
The FT-IR spectra obtained for the extracted compound was compared with
formaldehyde - MBTH product. The spectra of both the products merely showed
same IR bands which supports that the product formed is similar. The absence of NH2
bands in the spectra of the resulted products strongly supports the formation of
MBTH cationic dye. The absence of CO band at υmax 1754 cm-1 clearly indicates that
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MBTH-KIO4-Bilirubin system
bilirubin is not directly coupled with diazotized MBTH cation. υmax 2923 (CH
stretching), υmax 2357 (N=C-N conjugated), υmax 1057(C-S bending) are some
important peaks representing the product formed.
1
H NMR (400 mHz) was obtained by using DMSO as solvent. Two singlet
peaks corresponding to N- CH3 group was observed at δ 3.59 and δ 3.98, all
respective aromatic protons at δ 7.32 – δ 7.44 as multiplet was observed. A proton
having singlet at δ 7.59 correspond to the CH group showing the coupling at N-CH-N.
3.9 Interference studies
The extent of interference from foreign substances was studied by taking 5.16
μM fixed concentration of bilirubin. A deviation of  3% from the original value in
the absorbance reading was considered tolerable. The resultant tolerance ratio are
summarised in Table.3.2. It can be clearly observed that some compounds like
creatinine, glucose, ammonia, nitrate, urea and common inorganic ions showed a
reliable tolerance under the given conditions, whereas hemoglobin, ascorbic acid, Fe
(II), (III), uric acid, nitrite and some drugs showed a low tolerance. Sulphamic acid
was helpful to mask nitrite and increase its tolerance ratio to a good extent.
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Table.3.2. Interference Study
Tolerance ratioa
Interferants
Gentamycin, Diazepam, Amoxycillin
3
Aminophylline
9
Nickel, Calcium, Iron (II), (III), Nitrite, Phosphate
15
Uric acid
17
Theophylline, Phenobarbital
20
Magnesium, Aluminum, Sodium, Potassium
30
Hemoglobin, Ascorbic acid
35
Ammonia
70
Bicarbonate
190
EDTA
200
Creatinine
260
Glucose
310
Nitrate
380
Nitrite*
510
Sulphamic acid
**
950
Carbonate
980
Chloride
1650
Urea
a
39,176
Tolerance ratio corresponds to the ratio of limit of inhibiting species concentration to
that of 5.16 μM bilirubin used.
*
After masking, **Masking agent
3.10 Applications with serum sample and recovery
Serum samples collected from hospitals and clinical laboratories were
previously analyzed and the data were collected and cross checked with the
Jendrassik-Grof method performed manually and was found to be accurate. The
samples were measured in duplicate by the proposed method and mean results are
presented in Figure.3.5. From the results it is clear that there is a close linear
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MBTH-KIO4-Bilirubin system
correlation between both methods. The plot showed the slope = 0.994, intercept =
0.015 and correlation coefficient = 0.997.
Figure.3.5. Comparison of bilirubin content of blood as obtained with JendrassikGrof (Jendrassik-Grof) and the proposed method (n = 2).
The results obtained by these two methods are summarized in Table.3.3.
Serum samples used for recovery studies were previously analysed in clinical
laboratories by their method and then by the proposed method. Recovery studies were
carried out by spiking serum samples with known quantity of standard bilirubin
solution. Recovery % was calculated using the equation, [(final concentration – initial
concentration)/added concentration]. The recovery study exhibited minimal
interference and good reproducibility of the assay procedure.
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Chapter- 3
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Chapter- 3
MBTH-KIO4-Bilirubin system
3.11 Conclusion
This novel method on formation of formaldehyde from the cleavage of
bilirubin in strong acetic acid medium and coupling with MBTH has not been
reported so far. The reagents used in this assay are not costly and they are relatively
stable, making the assay efficient and affordable. The intensely blue colored
chromogenic product obtained by the coupling of MBTH with bilirubin is stable and
having high molar extinction co-efficient. The procedure requires small quantity of
serum sample, colorimetric reagents, and utilizes less time to give accurate and
reproducible results. Moreover, in order to demonstrate the usefulness and feasibility
of this assay we quantified bilirubin in human serum samples.
The lower limit of detection and quantification clearly indicates high
sensitivity of the method. Good precision and accuracy indicate that the method is
reproducible with very negligible error. Neither MBTH nor bilirubin shows
absorption in the range of 450 – 750 nm. The analysis could be completed in 2 min,
unlike Jendrassik-Grof method, which needs about 30 min for each analysis.
The proposed method measures the bilirubin even at a low concentration of
0.068 μM which is not quantified by Jendrassik-Grof method. Interference by foreign
substances is comparatively very low. The method requires only a small amount of
blood sample, can be easily performed manually and with some modifications the
method can be easily modified for adoption using automated instruments.
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