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For analysis by MS, please see the following:

Occult blood detection in biological samples by laser desorption and matrix-assisted laser desorption/ionization mass spectrometry for biomedical applications

US 7109038 B2

ABSTRACT

Methods are described for detecting and quantifying occult blood in a biological sample using laser desorption mass spectrometry (LD MS). Biological samples that can be analyzed using various embodiments of the present invention include stool (fecal occult blood, FOB), and any bodily fluid including urine, cerebrospinal fluid and other bodily fluids. If the heme or heme metabolite is bound to protein, the sample is treated with acid before analysis to release the porphyrin. Some of the methods use LD MS with a time of flight analyzer (TOF) to detect and measure unbound heme, other hemoglobin metabolites and other molecules that have a porphyrin-based structure, e.g., bilirubin, biliverdin, protoporphyrin IX, and Zinc protoporphyrin in the biological sample. In other methods, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is used to detect and quantify the individual α- and β-polypeptide chains of hemoglobin.

For the full publication, please use the following link:

https://www.google.com/patents/US7109038

2-Analysis by HPLC and MS:

cta Neurochir Suppl. Author manuscript; available in PMC 2009 Jul 15.

Published in final edited form as:

Acta Neurochir Suppl. 2008; 104: 43–50.

PMCID: PMC2710979

NIHMSID: NIHMS115187

Bilirubin oxidation products (BOXes): synthesis, stability and chemical characteristics

W. L. Wurster,1 G. J. Pyne-Geithman,1 I. R. Peat,2 and J. F. Clark1

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Summary

Bilirubin oxidation products (BOXes) have been a subject of interest in neurosurgery because they are purported to be involved in subarachnoid hemorrhage induced cerebral vasospasm. There is a growing body of information concerning their putative role in vasospasm; however, there is a dearth of information concerning the chemical and biochemical characteristics of BOXes. A clearer understanding of the synthesis, stability and characteristics of BOXes will be important for a better understanding of the role of BOXes post subarachnoid hemorrhage.

We used hydrogen peroxide to oxidize bilirubin and produce BOXes. BOXes were extracted and analyzed using conventional methods such as HPLC and mass spectrometry. Characterization of the stability BOXes demonstrates that light can photodegrade BOXes with a t1/2 of up to 10 h depending upon conditions. Mixed isomers of BOXes have an apparent extinction coefficient of ε = 6985, and a λmax of 310 nm.

BOXes are produced by the oxidation of bilirubin, yielding a mixture of isomers: 4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX A) and 3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide (BOX B). The BOXes are photodegraded by ambient light and can be analyzed spectrophotometrically with their extinction coefficient as well as with HPLC or mass spectrometry. Their small molecular weight and photodegradation may have made them difficult to characterize in previous studies.

Keywords: Bilirubin, reactive oxygen species, HPLC, mass spectrometry, molecular weight, photo-degradation, blood product, photolysis

There is extensive study of the oxidations (including peroxidations) of unsaturated lipids and proteins in biological systems, including the resultant compounds produced [1, 26, 39, 41]. There is also evidence that bilirubin is a biologic antioxidant [34, 43−46, 51]. The oxidation of (unconjugated) bilirubin has largely focused on the degradation of bilirubin between the pyrroles [5], with little discussion concerning the putative biological activity of the products of bilirubin oxidation [49]. We recently reported on a new family of bilirubin oxidation products found following subarachnoid hemorrhage [3, 22, 25, 38]. It appears that bilirubin oxidation products (BOXes) can be formed by the direct, nonenzymatic oxidation of bilirubin with hydrogen peroxide [22]. In Fig. 1a we see the relationship between the EE form of bilirubin and the resultant BOXes produced by oxidation (adapted from Kranc et al. [22]. Cleavage of bilirubin at the pyrrole, rather than between the pyrrole rings produces an apparently reactive monopyrrole amide [22]. However, to date there has been relatively little information concerning the stability or characteristics of BOXes, which is important in understanding the role BOXes may play in subarachnoid haemorrhage induced vasospasm. Here we present our latest findings concerning the chemical characteristics of BOXes produced by the oxidation of bilirubin. The relatively unique characteristics of BOXes and their nonspecific, nonenzymatic production provides important insight into the putative role of unconjugated bilirubin and its oxidation products in human pathology.

Fig. 1a

In this figure the EE form of bilirubin is represented along with the structures of BOXes that are likely to be produced. BOX A and BOX B can be produced by the oxidation of bilirubin. The resultant BOXes also have the E stereoisomer connecting the pyrrole ...

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Methods and materials

All materials are available from common commercial sources and unless noted were ACS Grade or better. Bilirubin (mixed isomers) was purchased from Sigma/Aldrich Chemical Company. Sodium hydroxide, hydrochloric acid, acetonitrile (HPLC grade), H2O2 30% and Whatman I filter paper were obtained from Fisher Scientific. Water (18 mΩ) was generated using a Biocel Millipore system.

Synthesis method for BOXes

In order to prepare BOXes by non-enzymatic oxidation, 100 mg of mixed isomers of bilirubin in is dissolved in 50 ml of water containing 10 g of sodium hydroxide (in 50 ml cell culture bottle with lid, taped or covered with foil). The solubilization is performed protected from light at room temperature and is completed in 24 h. The subsequent steps are carried out in the dark or protected from light. After 24 h, 23 ml of concentrated hydrochloric acid is added to the solubilized solution. The resultant solution is stirred and allowed to cool. Upon cooling to room temperature, the pH of the solution is taken. The solution is then brought to pH 7.5 by the careful addition of dilute hydrochloric acid.

After adding the hydrochloric acid to neutralize the solution, 1 PBS tablet (capable of making 200 ml of PBS aqueous) is added to the bilirubin solution. The resultant solution, once neutralized to pH 7.5, has sufficient buffering capacity to maintain the pH at 7.5 during the oxidation. If the solution is unbuffered, due to the Fenton-like nature of the reaction, the pH will drift up from 7.5 to 8.2 during the course of the reaction.

Next, the volume of the solution is measured in a graduated cylinder and hydrogen peroxide added to a final concentration of ∼10% hydrogen peroxide. After 24 h of stirring at room temperature, the resultant yellowish solution is frozen at −80°C. The frozen solution is then lypholyzed overnight, producing a yellow solid. The yellowish solid is then thoroughly triturated with 100 ml of chloroform. The chloroform solution is then filtered using Whatman 1 and a Hirsch Funnel. The filtrate is evaporated under nitrogen gas. This produces about 4% by weight of BOXes.

HPLC analysis

HPLC analysis was performed using a Waters Chromatography System consisting of a 2790 Separations Module and 2487 Variable Wavelength Detector running Millennium 32 Data Analysis Software. Separation was achieved on a Symmetry C18, 5 μm 4.6 × 150 mm, column. The elutant was monitored at 310nm. Column temperature was held at 35 degrees centigrade with a flow rate of 1.0 ml/min. The mobile phase consisted of acetonitrile/water with a gradient of 5% to 50% acetonitrile being used.

Mass spectrmetry

HPLC-MS analysis was performed on a Bruker Daltronics system using conditions identical to the above, with the exception of the gradient being 20% to 63% acetonitrile over 15 min with the acetonitrile being held constant until the end of the run. The MS was performed using air pressure chemical ionization in the negative ion mode with simultaneous monitoring at 254 nm.

Photostability study

Approximately 0.1 mg of BOXes was dissolved in 1 ml of phosphate buffered saline (PBS) at pH 7.5. The solution was exposed to various experimental conditions. Samples were taken at 30min intervals for 120min and the resultant samples were diluted 50:50 with PBS and the spectra read from 250 to 550 nm. For spectrometry, we used a μ-Quant plate reader and BD Falcon 96 well microplate. The data reported used the OD at 310 nm.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2710979/

3-The following is the procedure for HPLC analysis which should be similar to that of LC-MS:

II. Preparation of Standard Solutions

The stock standard solution of bilirubin with a concentration

of 1,000 μg/mL was prepared by dissolving 10 ±

0.5 mg bilirubin in 10 mL of DMSO and this was stored at

4°C. The working standard solutions with bilirubin concentrations

of 10, 5, 2.5, 1.25 and 0.625 μg/mL were prepared

by diluting the stock standard solution to the corresponding

concentrations with mobile phase. These working standards

were used to prepare the standard calibration curve.

III. Sample Preparation

The pig liver samples were processed according to the

following procedure under reduced light intensity to reduce

photodegradation. The liver samples were intermittently

blended at low speed in a blender with stainless steel blades

for 1 min to obtain a homogeneous paste. Aliquots of 2 ±

0.01 g of blended liver sample were accurately weighed into

a 15-mL polypropylene centrifuge tube. A volume of 2 mL

of DMSO was added to the sample which was then homogenized

by vortex for 2 min. Subsequently, 2 mL of acetonitrile

was added into the centrifuge tube and the mixture was

homogenized for 1 min. The sample was then centrifuged

at a speed of 6,000 rpm for 5 min at 5°C. Two milliliters of

the supernatant was collected in a 2-mL micro centrifuge

tube and centrifuged at 15,000 rpm for 10 min at 5°C. The

extracted supernatant was collected for further analysis,

but if still cloudy, it was filtered through a polyvinylidene

fluoride (PVDF) 0.22-μm pore size syringe membrane filter

before analysis.

IV. HPLC System

HPLC separations were performed using a Hitachi

7000 series LC system (Tokyo, Japan) consisting of a binary

pump (L-7100), an autosampler (L-7200) and a diode array

detector (L-7455). A reversed-phase Nova-Pak C18 column

(150 × 3.9 mm, 4 µm) coupled to a C18 guard column (20 ×

3.9 mm), both purchased from Waters (Milford, MA, USA),

were used for the analysis. Signals and data were processed

by Hitachi Model D7000 Chromatography Data Station

Software (version 3.1). The mobile phase was a mixture of

DMSO : acetic buffer (pH = 5.0) : acetonitrile (6 : 6 : 6.5,

v/v/v). Separation was achieved by isocratic solvent elution

at a flow rate of 1.0 mL/min. An injection volume of 20 μL

was used. The detection wavelength for bilirubin was set at

496 nm.

To view the full paper, please see attached file.

Hoping this will be helpful,

Rafik

Dear Rafik,

Thank you for this information, it was helpful.

Sumeia

Dear Sumeia,

If what I provided was helpful, why you downvote my answer.

Rafik