AU2017100545A4 - Method of determining histamine in a sample and kit for doing the same - Google Patents

Abstract

A method of determining histamine in a sample comprising steps of: a) providing a sample; b) preparing a mixture by adding a derivatizing agent to the sample, wherein 5 the derivatizing agent is an alkyl phosphite, and subjecting the mixture to conditions where the derivatizing agent reacts with the histamine present in the sample to form a histamine derivative; c) separating the histamine derivative from the mixture so as to determine the presence and/or amount of the histamine derivative, wherein the amount of the histamine derivative corresponds to the amount of the histamine in the 10 sample. Also disclosed herein is a kit for determining histamine in a sample comprising: a first mixture comprising a derivatizing agent which is capable of reacting with histamine present in the sample to form a histamine derivative; and a second mixture comprising a deprotonating agent. eN) co (0 co 0 r L 961 -=ma c4 co o No (0 U 6 " n 0 n ,2U' V Ln MU U- u O 0i co r~m w LO Ir'N C x4

Description

METHOD OF DETERMINING HISTAMINE IN A SAMPLE AND KIT FOR DOING

THE SAME

TECHNICAL FIELD

The present application relates to a method of determining histamine in a sample, in particular but not exclusively a method of determining the presence and/or amount of histamine in a sample. The present invention also relates to a kit for determining histamine in a sample.

BACKGROUND OF THE INVENTION

Histamine is a vital biogenic amine which exerts various effects in humans. Histamine is involved in the tasks of the immune and neuroendocrine systems, neurotransmission, gastric secretion, cell life and death, and development. It is therefore a potent mediator of numerous biological reactions. The alterations in histamine-related factors may lead to various conditions and diseases including allergy, asthma, anaphylactic shock, cerebral ischemia, urticarias, cancers and so on. Therefore, the determination and quantification of histamine in a biological sample is useful for the diagnosis of diseases and determination of the pathological conditions of a subject.

Various approaches had been developed to determine histamine in different kinds of matrix samples, e.g. enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and so on. Generally, the amount of histamine in the sample is low (e.g. 25 - 65 ng/mL in blood) and therefore mass spectrometric approaches (MS) are widely applied for the quantification of histamine owing to its high sensitivity, specificity, and accuracy. For instance, the LC-MS/MS approaches were developed to quantify histamine in the cell line and plasma samples, and the lowest limits of quantitation (LLOQ) were 0.3 and 0.2 ng/mL, respectively. However, histamine is unstable in the biological samples, and could be rapidly inactivated by histamine-/V-methyltransferase or diamine oxidase. The half-life of histamine is only about 1 min in the extracellular fluid. Besides, histamine has high polarity and therefore can hardly be separated completely from other endogenous components.

Some existing methods include derivatization, for example by using methyl chloroformate, propionic anhydride, and benzoyl chloride as a derivatization reagent. However, the sensitivity of the current methods is still not satisfactory, and the microdialysate must be collected for at least 5 min in order to determine histamine in the brain. As the clinical samples are usually in a small volume, it is necessary to develop a more sensitive method to determine the trace amount of histamine in a small volume of sample.

Accordingly, there remains a need for an improved method for determining histamine in a sample which has a relatively high sensitivity and specificity to histamine and allows a determination of trace amount of histamine in a small volume of sample.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of determining histamine in a sample comprising steps of: a) providing a sample; b) preparing a mixture by adding a derivatizing agent to the sample, wherein the derivatizing agent is an alkyl phosphite; and subjecting the mixture to conditions where the derivatizing agent reacts with the histamine present in the sample to form a histamine derivative; c) separating the histamine derivative from the mixture so as to determine the presence and/or amount of the histamine derivative, wherein the amount of the histamine derivative corresponds to the amount of the histamine in the sample.

The sample is preferably a biological fluid or obtained from cells or a cell population.

The derivatizing agent is preferably an alkyl phosphite having the Formula (II),

Formula (II) wherein the R and X are independently selected from an alky group or a hydrogen atom. In particular, the derivatizing agent is diisopropyl phosphite of Formula (III).

Formula (III)

In a preferred embodiment, the histamine derivative preferably has a structure of Formula (IV)

Formula (IV).

Step c) of the method preferably comprises an ion exchange, liquid chromatography and/or mass spectrometry.

In a further embodiment, an analog of histamine, preferably deuterated histamine, is added to the sample before step b) and it acts as an internal reference for the quantification of histamine.

In a second aspect, the present invention pertains to a kit for determining histamine in a sample comprising: - a first mixture comprising a derivatizing agent which is capable of reacting with histamine present in the sample to form a histamine derivative, wherein the derivatizing agent is an alkyl phosphite; and - a second mixture comprising a deprotonating agent.

The method and the kit of the present invention allows for a highly advantageous approach for determining the presence and/or amount of histamine in a sample. The inventors surprisingly found that the method of the present invention can be applied to a sample containing relative low amount of histamine, for example as low as in pico- scale, with an exceptional promising results compared with the existing approaches (refer to Table 1 below). The LLOQ reached 0.1 pg/mL, which was low enough to detect histamine in one single cell, and low nano-liter of serum and CSF in theory.

The combined use of derivatization of histamine and analog of histamine -deuterated histamine increases the sensitivity and specificity of the quantity and quality analysis of histamine. Accordingly, it provides an improved approach for the diagnosis of diseases, and for determination of health conditions or pathological conditions of a subject. It is exceptionally useful for the determination of histamine level in a baby or child as it can detect histamine in a pico-scale which means that only a small amount of biological sample is required from the subject. It would be also appreciated that the method and kit as disclosed herein are also useful in laboratory research and other clinical studies.

Table 1. Comparison of the results obtained by the existing approaches and the method of the present invention in the determination of histamine in a biological sample.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications. The invention also includes all steps and features referred to or indicated in the specification, individually or collectively, and any and all combinations of the steps or features.

Other features and aspects of the invention will become apparent by consideration of the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A is a base peak chromatogram (BPC) of histamine (4.2ng/mL) in DMEM without derivatization. Fig. 1B is a base peak chromatogram (BPC) of histamine (4.2ng/mL) in DMEM with derivatization. Fig. 1C is a base peak chromatogram (BPC) of histamine (4.2ng/mL) in DMEM with derivatization and followed by a solid-phase extraction (SPE).

Fig. 2 shows the amounts of a histamine derivative, histamine-DIPP, produced under different reaction conditions. “T” refers to the test carried out under customary reaction conditions.

Fig. 3 shows the retention times of histamine and amino acids in DMEM before and after derivatization.

Fig. 4 is a plot showing the responses of histamine and amino acids in a mass spectrometry in DMEM without derivatization, with derivatization and in the absence of SPE, and with derivatization and SPE.

Fig. 5A shows the histamine release in C48/80 and triton X-100 treated cell line and control. Fig. 5B shows the histamine release in sinomenine treated cell line and control.

Fig. 6 shows the histamine contents in children suffering from allergy and healthy children (n = 15).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one skilled in the art to which the invention belongs.

As used herein, “comprising” means including the following elements but not excluding others. “Essentially consisting of” means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components or solvents. “Consisting of means that the material solely consists of, i.e. is formed by the respective element. As used herein, the forms “a,” “an,” and “the,” are intended to include the singular and plural forms unless the context clearly indicates otherwise.

The present invention in the first aspect provides a method of determining histamine in a sample, in particular a method of determining the presence and/or amount of histamine in a sample. The method comprises steps of: a) providing a sample; b) preparing a mixture by adding a derivatizing agent to the sample, wherein the derivatizing agent is an alkyl phosphite; and subjecting the mixture to conditions where the derivatizing agent reacts with the histamine present in the sample to form a histamine derivative; c) separating the histamine derivative from the mixture so as to determine the presence and/or amount of the histamine derivative, wherein the amount of the histamine derivative corresponds to the amount of the histamine in the sample.

Histamine, having a chemical name of 2-(1F/-imidazol-4-yl) ethan-1-amine, is an organic compound represented by the following Formula (I).

Formula (I)

In general, histamine is involved in various immune responses and functions as a neurotransmitter in neuroendocrine systems. Histamine is also an analyte of interest of diseases diagnosis. With regard to the structure, histamine possesses an aliphatic amino group and an alky amino group. In embodiments of the present invention, the alkyl amino group is susceptible to modification or a substitution by a derivatizing agent so as to form a derivative of histamine for subsequent determination and measurement.

The term “derivative” used herein refers to a compound produced from an original compound directly, by modification or by partial substitution of the core structure of the original compound. The derivative may have a similar core structure with the original compound with a side chain modified or substituted.

In the present invention, a derivatizing agent is applied to convert histamine to form a histamine derivative. Preferably, the derivatizing agent is an alkyl phosphite with the following Formula (II)

Formula (II) wherein the R and X are independently selected from an alky group or a hydrogen atom.

The term “alkyl group” as used in the present invention refers to a straight chain or branched hydrocarbyl radical comprising carbon and hydrogen atoms. Accordingly, "C1-C5 alkyl group" refers to a hydrocarbyl radical comprising from 1 to 5 carbon atoms and "C2-C5 alkyl group" refers to a hydrocarbyl radical comprising from 2 to 5 carbon atoms. "Straight chain or branched alkyl" includes all linear or branched alkyl groups. For example, C1-C5 alkyl group includes methyl, ethyl, n-propyl, isopropyl, butyl and its isomers (e.g. n-butyl, /'sobutyl, sec-butyl and ferf-butyl), pentyl and its isomers (n-pentyl, tert-pentyl neopentyl, /'sopentyl, sec-pentyl, 3-pentyl). The alkyl group is preferably a C1-C5 alkyl group.

In embodiments, the alkyl phosphite is mono-alkyl phosphite or di-alkyl phosphite. “Mono-alkyl phosphite” refers to an alkyl phosphite having one of R or X being an alkyl group, and “di-alkyl phosphite” refers to an alkyl phosphite with R and X being an alkyl group. The R and X in a di-alkyl phosphite may be the same or different alkyl group. The term “alkyl group” is as defined above. Preferably, the alkyl phosphite is a di-alkyl phosphite.

In an embodiment of the present invention, diisopropyl phosphite of Formula (III)

Formula (III) is applied as a derivatizing agent to react with histamine by introducing an alkyl phosphite group to histamine. A histamine derivative having the following Formula (IV) is formed thereafter.

Formula (IV)

Without intending to be limited by theory, it is believed that the histamine derivative of Formula (IV) is more stable than histamine in sample preparations and may provide a better response in mass spectrometry compared with histamine. It is believed that the introduction of the neutral phosphoryl group leads to an increase in gas-phase proton affinity. The increase in gas-phase proton affinity improves the ionization efficiency of the histamine derivative and thus most of or at least a substantial amount of histamine derivative can be ionized efficiently for detection in a mass spectrometer. Moreover, the histamine derivative is more hydrophobic than histamine and therefore the retention in the liquid chromatography is improved.

The inventors also found out that histamine derivative provides an enhanced response in mass spectrometry. It is believed that such an enhancement is due to a decrease in ion suppression effect.

The sample as used in the present invention may be a biological fluid obtained from a subject, preferably a mammal such as a human, or may be obtained from cells or a cell population. In an embodiment, the sample may be a biological fluid selected from the group consisting of serum, blood, urine, transcellular fluid, interstitial fluid, saliva, exudates, fluid collected through microdialysis and other fluids from mammal susceptible of containing histamine. Preferably, the sample is serum, urine, transcellular fluid or fluid collected through microdialysis. In a particular embodiment, the sample is serum, or fluid collected through microdialysis.

In an embodiment of the present invention, step b) comprises: preparing a mixture by adding a derivatizing agent as defined above to the sample, and - subjecting the mixture to conditions where the derivatizing agent reacts with the histamine present in the sample to form a histamine derivative.

Preferably, a deprotonating agent is also added to the mixture in step b) for deprotonating histamine and facilitating the reaction between the derivatizing agent and histamine. In an embodiment of the present invention, the deprotonating agent is a tertiary amine or a trialkyamine, preferably a tertiary amine and in particular triethylamine (herein denoted as TEA). The person skilled in the art would appreciate that other amines which are suitable as a deprotonating agent may also be applied in the present invention.

After the addition of the derivatizing agent and optionally the deprotonating agent as described above to the sample, the mixture is incubated at about 20°C to about 25°C for about 0.5 min to about 1 h, about 0.5 min to about 30 min, about 0.5 min to about 20 min, about 0.5 min to about 10 min, or about 1 min, for completing the reaction between the derivatizing agent and histamine. In a particular embodiment, after the addition of the derivatizing agent and the deprotonating agent, the mixture is optionally mixed and incubated at about 20°C for 1 min. In another embodiment, after the addition of the derivatizing agent and the deprotonating agent, the mixture is immediately subject to incubation at 20°C for 1 min, without being subject to a preincubation at 4°C or below 4°C between the two steps.

After the reaction, the mixture may then be subject to a solvent removing step. Preferably, the one or more solvents contained in the mixture, for example but not limiting to the solvent for dissolving the derivatizing agent and the solvent for dissolving the deprotonating agent, are removed by solvent evaporation under reduced pressure. The residue obtained may be re-dissolved with a solution having a pH value less than 7. In an embodiment, said solution is a formic acid with a low concentration.

Step c) preferably comprises steps of: (i) introducing the mixture obtained in step b) to an ion exchange column to obtain a resultant mixture comprising the histamine derivative; and (ii) performing a liquid chromatography to separate the histamine derivative form the resultant mixture for detection and quantification.

Preferably, the ion exchange column is a cation exchange column which is capable of binding the histamine derivative and other alkaline components present in the mixture obtained in step b) under acidic condition. In an embodiment, the ion exchange column is an ion exchange resin cartridge. After introducing the mixture to the ion exchange column, a washing solution such as an alcohol may be added to the column to wash out most of or at least some of the alkaline components bound to the column. Preferably, the washing solution is a C1-C4 alcohol selected from methanol, ethanol, n-propanol, /'so-propanol, n-butanol, /'so-butanol or tertiary butanol. Subsequently, the bound histamine derivative may be collected by adding an eluting solution to the column. Preferably, the eluting solution is a base. In an embodiment, the eluting solution is ammonia solution in methanol, preferably the percentage of ammonia in the solution is about 2% to about 6% or about 3% to about 5% or about 5% by volume. The person skilled in the art is aware of other alkaline eluting solutions which are suitable for extracting components from a cation exchange column. Accordingly, the mixture in step b) is at least partially purified to remove a substantial amount of unwanted components present in the mixture, and thus a resultant mixture comprising the histamine derivative is obtained.

In step (ii), a liquid chromatography is performed to further separate the histamine derivative form the resultant mixture for subsequent detection and quantification. Preferably, step (ii) further comprises performing multiple-reaction monitoring mass spectrometry for quantifying the amount of the histamine derivative after the liquid chromatography. The liquid chromatography may be high performance liquid chromatography (HPLC) or ultra-high performance chromatography (UHPLC) which may or may not be coupled with a mass spectrometer. The mass spectrometer may be a triple quadrupole mass spectrometer (QQQ or TQ MS) which is able to run multiple-reaction monitoring.

The results obtained from the liquid chromatography and the mass spectrometry are then analyzed, preferably with reference to the standard solutions of histamine at different concentrations, to determine the presence and/or amount of the histamine derivative in the resultant mixture thereby determining the same of histamine original present in the sample.

In an advanced embodiment, the method further comprises a step of adding an analog of histamine to the sample before step b). The analog of histamine may be deuterated histamine. The deuterated histamine refers to a compound having a structure of histamine but with one or more hydrogen atoms being replaced by deuterium which is an isotope of hydrogen. Preferably, the deuterated histamine is of the following Formula (V)

The addition of the analog of histamine is advantageous in the quantification of histamine, in particular the analog of histamine acts as an internal standard for the quantification of histamine in the mass spectrometry.

In the second aspect, the present invention provides a kit for determining histamine in a sample, in particular for determining the presence and/or the amount of histamine in a sample. As described above, the sample may be a biological sample or obtained from cells or a cell population.

The kit comprises: - a first mixture comprising a derivatizing agent which is capable of reacting with histamine present in the sample to form a histamine derivative, wherein the derivatizing agent is an alkyl phosphite; and - a second mixture comprising a deprotonating agent preferably a tertiary amine or a trialkylamine .

Preferably, the derivatizing agent and the deprotonating agent in the kit are as those described above. In an embodiment, the derivatizing agent is an alkyl phosphite selected from the group consisting of mono-alkyl phosphite and di-alkyl phosphite, preferably is di-alkyl phosphite of Formula (II)

Formula (II) wherein the R and X are independently selected from an C1-C5 alkyl group, or a hydrogen atom.

In a particular embodiment, the first mixture comprises diisopropyl phosphite and an organic solvent for example but not limiting to carbon tetrachloride (CCU), and the second mixture comprises triethylamine and a C1-C4 alcohol selected from methanol, ethanol, n-propanol, iso-propanol, n-butanol, /'so-butanol or tertiary butanol. Preferably, the volume ratio of diisopropyl phosphite to the organic solvent is about 1:10, about 1:8, about 1:5, about 1:3, or about 1:1, preferably about 1:5. The volume ratio of triethylamine to C1-C4 alcohol is from about 1:3 to about 3:1, or about 1:2 to about 2:1, preferably about 1:1.

In an advanced embodiment, the kit further comprises a third mixture comprising an analog of histamine as described above, in particular deuterated histamine. Furthermore, the kit may also comprise a standard solution of histamine with a known concentration. This standard solution may be applied to prepare a series of standards of histamine with different concentrations so as to construct a calibration curve for use in quantification.

Still further, the kit may comprise an ion exchange column for preliminary removing most of or at least some of the unwanted components from the sample. Preferably, the ion exchange column is an ion exchange resin cartridge, more preferably is a cation exchange resin cartridge.

The method and the kit of the present invention allows for a highly advantageous approach for determining the presence and/or amount of histamine in a sample, in particular for a sample containing trace amount of histamine. The method of the present invention is capable for determining histamine present in pico- scale. The method is also easy to operate.

Accordingly, the present invention provides an improved approach for the diagnosis of diseases, and for determination of health conditions or pathological conditions of a subject. It is exceptionally useful for the determination of histamine level in a baby or a child. It would be also appreciated that the method and kit as disclosed herein are also useful in laboratory research and other clinical studies.

EXAMPLES EXAMPLE 1

Preparation of standard references and internal reference

Histamine, histamine-a,a,p,p-d4 dihydrochloride (herein denoted as histamine-d4), and carbon tetrachloride (CCU) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Diisopropyl phosphite (DIPP), and absolute ethanol were obtained from Alfa Aesar Chemical Ltd (Lancashire, United Kingdom). Triethylamine (TEA), LC-MS grade acetonitrile, methanol and water were provided by Tedia (Fairfield, OH). Oasis MCX 1cc (30 mg) cartridge was produced by Waters Corporation (Milford, MA).

The stock solutions of histamine and histamine-d4 were prepared by dissolving the corresponding standards in water (LC-MS grade) at a concentration of 10 pg/mL. The working solutions of histamine were prepared by serially diluting with water at the concentrations of 0.02, 0.1, 0.2, 0.5, 2, 8, 16, and 32 pg/mL, and the concentration of histamine-d4 was 25 pg/mL. Two reaction solutions A and B for derivatization, or called A/-phosphorylation labeling, were prepared by mixing ethanol, TEA, DIPP and CCI4 according to the following volume ratio, respectively: reaction solution (A): ethanol:TEA=1:1; and reaction solution (B): DIPP:CCl4=1:5. All solutions were kept at -20 °C for further use.

EXAMPLE 2A

Preparation of a sample from a primary mast cell culture

Peritoneal fluid from anesthetized male SD rat (300 g ± 20) was collected by washing the peritoneal cavity with D-Tyrode’s salt solution (D-TSS, 137 mM NaCI, 2.7 mM KCI, 0.36mM NaH2P04*H20, 6.1 mM D-Glucose; pH 7.4) as follows. D-TSS (15 mL) was injected into the peritoneal cavity, and the abdomen was gently massaged for approximately 5 min. The peritoneal cavity was carefully opened, and the fluid containing peritoneal cells was aspirated by Pasteur pipettes. The peritoneal cells were sedimented by centrifugation at 4 °C, 300 g for 10 min and re-suspended in 0.75 mL TSS (137 mM NaCI, 2.7 mM KCI, 1.8 mM CaCI2, 0.49 mM MgCI2-6H20, 0.36 mM NaH2P04-H20, 12 mM NaHCOs, 5.5 mM D-Glucose; pH 7.4). Then 3.5 mL of 1.110 g/mL Percoll was added and gently mixed, and 0.5 mL TSS was added flat on the surface, centrifuged at 4 °C, 130 g for 10 min, the cells remained at the bottom of the Percoll gradient were collected. The collected cells were washed for 3 times with 3 mL of TSS by centrifugation at 4 °C, 200 g for 10 min.

The content of mast cells was determined by Toluidine Blue staining, and the mast cells with the purities of over 90% were re-suspended in DMEM and seeded in streaming tube. Different concentrations of sinomenine, C48/80 or Triton X-100 were added in the drug-treated groups, while TSS was added in the control group. All cells were incubated at 37 °C for 30 min. After centrifugation, the supernatants were collected for further use.

EXAMPLE 2B

Preparation of human serum samples

Venous blood samples were collected from 30 1- to 13-year-old children, including 15 healthy children and 15 children suffering allergy. Sera were obtained by centrifuging the collected blood samples at 3000 g for 10 min at 4 °C and were kept at -80 °C until use.

EXAMPLE 2C

Preparation of a sample of brain microdialysis

Sprague-Dawley rats (300-350 g) were anesthetized and then a microdialysis guide cannula was implanted into the prefrontal cortex with the aid of stereotaxic apparatus (CMA, Stockholm, Sweden). After recovery for 3-4 days, a microdialysis probe (4 mm) was put in and perfused with artificial cerebrospinal fluid (CSF, 140 mM NaCI, 3.0 mM KCI, 1.2-3.4 mM CaCI2, 1.0 mM MgCI2, 1.2 mM Na2HP04, 0.27 mM NaH2P04, 7.2 mM glucose, PH = 7.4) at 2pL/min. Each microdialysate was collected for 1 min and continued for 30 min. In the collecting tube, 48 pL distilled water, 2 pL histamine-d4, 100 pL solution A, and 60 pL solution B were added in advance to derivatize histamine in microdialysate. EXAMPLE 3 Processing the samples A modified Atherton-Todd reaction was used for /V-phosphorylation derivatization. Briefly, the individual working solution of histamine, diluted serum or DMEM (50 pL) was mixed with histamine-d4 (2 pL), solution A (100 pL), and solution B (60 pL) and reacted for 1 min at room temperature before evaporation to dryness under reduced pressure. The residues were dissolved in 250 μΙ_ of 0.1% formic acid-containing H20 and extracted by Waters Oasis MCX cartridge as follows.

The cartridges were conditioned with methanol (1 mL) and equilibrated by 0.1 N HCI (1 mL). Then above reaction products were loaded onto the cartridges and washed with 1 mL of 0.1 N HCI and 1 mL of methanol successively followed by eluting with 1 mL of 5% ammonia-containing methanol to provide the analytes. Finally, the trap solution was removed using nitrogen and the products were dissolved in 50 pL of methanol prior to LC-MS analysis.

EXAMPLE 4 Performing LC-MS A quantification method was developed using UHPLC-QQQ-MS/MS. An Agilent 1290 Ultra-high Performance Liquid Chromatography (UHPLC) consisting of an autosampler, thermostatted column compartment and binary pump and equipped with a Thermo Syncronis aQ column (2.1 * 100 mm; 1.7 μίτι) was employed for the separation of components. The column temperature was maintained at 40 °C. The injection volume was 1 μί. The mobile phase comprised of 0.1% acetic acid and 20mM ammonium acetate (A) and 0.1% acetic acid-containing acetonitrile (B) with the following gradient, 0-0.5 min: 5% B, 0.5-4.5 min: from 5% to 25% B, 4.5-6.5 min: from 25% to 95% B, 6.5-6.99 min: 95% B; 7 min: 5% B. The optimization of separation and derivatization was conducted on an Agilent 6550 Quadrupole-Time of Flight mass spectrometry, while the quantification was conducted on an Agilent 6490 iFunnel Triple Quadruple mass system with a dual Jet Stream electrospray ion source (dual AJS ESI) in positive mode. The quantification was carried out by multiple reaction monitoring (MRM) mode with two transitions for each compound, i.e. m/z 276.1-»111.9 and 276.1-»94.9 for histamine-DIPP and m/z 280.1->115.9 and 280.1-»98.9 for histamine-d4-DIPP at the collision cell energy of 30 eV. Other MS parameters were optimized by using the labeled histamine and set as follows: gas temperature at 225 °C, gas flow at 13 L/min, nebulizer pressure at 25 psig, sheath gas temperature at 275 °C, sheath gas flow at 12 L/min, capillary at 4000 V, nozzle at 350 V.

The data were acquired and processed with MassHunter Workstation B.06.00 (Agilent). The calibration curves were constructed by plotting the peak area ratios of histamine-DIPP to the internal standard of histamine-d4-DIPP against the concentrations. The ratio of the quantifier and qualifier ions was applied for the confirmation of histamine-DIPP and histamine-d4-DIPP.

The ratios of histamine at m/z 276.1->111.9 to histamine-d4 at m/z 280.1 ->115.9 were set as Y-variations for the construction of calibration curve. A good linearity (r2 = 0.9979) was obtained by weighted least-squares regression (weighting 1/x) in the range from 0.1 to 32 pg/mL, and the equation was y = 1.015444x - 0.152330. The lowest limit of quantification (LLOQ) was considered as the lowest concentration at the calibration curve with the precision and accuracy of 80-120%, i.e. 0.1 pg/mL. It was around 800 times lower than that reported before. EXAMPLE 5

Optimization of the method

The reaction time was optimized by collecting the reaction solution at different times (0, 0.5, 1, 2, 5, 10, 20, and 30) after reacting at room temperature followed by LC-MS analysis. Ethyl acetate and methanol were tried to precipitate the proteins in the biological samples and the efficiency was compared based on the MS responses. The effect of solid phase extraction (SPE) using ion exchange column was evaluated by three groups of experiments, i.e. no SPE purification, MCX cartridge purification, and HLB cartridge purification. Four kinds of ultra-performance liquid chromatography columns, Waters Acquity BEH C18 (2.1 χ 100 mm; 1.7 pm), Agilent Eclipse C18 (2.1 x 100 mm; 1.8 pm), Thermo Syncronis aQ (2.1 χ 100 mm; 1.7 pm), and Phenomenex Luna Omega (2.1 χ 100 mm; 1.6 pm), were tested to obtain better separation.

Precision was determined by the intraday and inter-day variations from replicate analyses of samples at three concentrations of 0.1, 1 and 20 pg/mL on three consecutive days. Accuracy was evaluated by the percentage ratios of measured concentrations to the real concentrations of quality control samples at 0.1, 1 and 20 pg/mL. Recovery was performed by spiking the working solutions of histamine and histamine-d4 into DMEM, blank serum, and CSF at three concentrations, 0.1, 1 and 20 pg/mL, respectively, and calculated by the equation of (concentration of spiked sample - non-spiked concentration)/ spiked concentration χ 100.

The stability of histamine-DIPP in DMEM, serum, and CSF was determined at 4 °C within 72 hours. The measured concentration was compared with that of the freshly prepared sample. The results show that the samples were stable at 4 within 72 hours. The measured concentrations were between 90% and 107% of the freshly prepared sample. EXAMPLE 6 Results analysis

Histamine has high polarity and poorly retains on HPLC column even hydrophilic interaction liquid chromatography (HILIC) and amide column. With reference to Fig. 1 A, the structure of histamine and its retention time on column are very similar to that of amino acids. Therefore, a large amount of amino acids in cell culture medium and biological samples significantly interfere the detection of histamine, e.g. histamine at 4.2 ng/mL was buried in the peaks of amino acids in DMEM.

The Atherton-Todd reaction is applied to improve the sensitivity of histamine by the formation of dialkylphosphite. That is, an alkyl phosphite group is incorporated to histamine in this invention.

The reaction condition was first optimized. Although the reaction was usually conducted at 4 °C for 15 min followed by 30 min at room temperature, the results as shown in Fig. 2 indicate that there is no significant difference in the yield when reacting for different period of times from 0.5 to 30 min at room temperature or using the traditional method. Accordingly, it is believed that an appropriate reaction time is from about 0.5 min to 30 min, preferably from about 0.5 to about 20 min, from about 0.5 to 10 min or from 1 min to 5 min. More preferably, the reaction time is about 1 min.

With reference to Fig. 1B, after derivatization, the retention time of histamine-DIPP is extended to 5.6 min, while histamine was flushed out at about 1.5 min. Although the amino acids in the DMEM also form derivatives, their retention times were different to that of histamine-DIPP as shown in Fig. 3. Therefore, as shown in Fig. 1B, histamine stands out from the matrix. At the same time, the MS response also significantly increased after derivatization, referring to Fig. 1A, 1B and 1C. It is because the incoprated neutral phosphoryl group had high gas-phase affinity.

It appears that the impurities in the reaction system interfered the determination of histamine. Therefore, four different kinds of columns were tested to remove the impurities. Although a Thermo Syncronis aQ column gave a better result, some impurities still remained. Therefore, a clean-up procedure was conducted to remove the side products and/or endogenous components. Two kinds of SPE columns, Oasis HLB and MCX, were tested. HLB is a polymeric SPE sorbent and widely used to purify the compounds with acidic, basic, or neutral properties from a wide spectrum of matrices. However, the result for histamine was not satisfied (data not shown). The cation-exchange column is a better choice as it can absorb histamine and alkaline components in acidic condition to remove the side products or other components in biological samples. Next, the weak alkaline components, e.g. amino acids, can be eluted out with methanol, while histamine will stay in the column until a stronger base solution is applied. For example, 5% ammonia-containing methanol may be applied. As expected, with reference to Fig. 4, most of amino acids and impurities were removed or dramatically decreased except arginine and histidine by Oasis MCX, a mixed-mode cation-exchange SPE column, and the peak area of histamine had no significantly difference. The strong retention of arginine and histidine may be because of the existence of alkaline group, guanidine and imidazole, respectively. The remaining amino acids appeared at different retention times, and had no interference to the determination of histamine.

Inter-day and intra-day variations at three concentrations were determined to assess the precision, and the variations were less than 15%. The accuracies at three concentrations were between 88 and 106%. High recoveries (around 90%) were obtained at high, medium, or low concentration spiked in serum, microdialysate, and DMEM, respectively, which implied that other components in the real samples had no evident influence on the whole procedure, including the reaction, SPE clean-up and LC-MS analysis.

Table 2. Precision, accuracy, and recovery of histamine in DMEM and human serum samples.

EXAMPLE 7 Applications

The developed method was applied to determine histamine in the cell line, human sera, and rat CSF samples. A detection of histamine in samples obtained from rat primary mast cell culture was performed. Mast cell is a kind of granulocyte, and contains many granules. After being stimulated by pathogens or other allergens, mast cell releases the mediators, e.g. histamine, to defense by degranulation. In the present invention, two reagents (C48/80 and triton X-100) promoting the release of histamine were first used to assess the feasibility of this method. With reference to Fig. 5A, C48/80 and triton X-100 evidently increased the release of histamine for about 2 and 4 times, respectively. The inventors further employed this method to determine the effect of sinomenine on the histamine release. Sinomenine is derived from a traditional Chinese medicine Sinomenium acutum and used to treat rheumatoid arthritis in clinic. However, occasional allergy cases were reported. It was found that sinomenine could induce RBL-2H3 cell degranulation by the particle analysis assay. Using the method of the present invention, histamine released into the medium was determined.

It was found that the release of histamine increased along with the concentrations of sinomenine from 1 to 400 μΜ. The density of cell was 40,000/well, accordingly the average release amount of each cell was calculated to be about 0.12 pg/mL, which is higher than the LLOQ. It means that the sensitivity is enough to determine the histamine released by one single cell. A detection of histamine in human serum samples was performed. The method of the present invention was applied to determine histamine in the healthy children and children suffering from allergy. With reference to Fig. 6, the concentration of histamine in children suffering from allergy was significantly higher than that in healthy children. More importantly, in this test, the serum samples were diluted 5000 times for the measurement of histamine, which shows that the present method only requires about 0.01 μΙ_ of serum to determine the presence and/or amount of histamine. A detection of histamine in brain microdialysate samples was performed. Histamine is also a key neurotransmitter in central nervous system, and the microdialysis approach is usually utilized to monitor the change of histamine. The method of the present invention was used to determine histamine in the rat CSF. The brain microdialysate was collected for 1 min at 2 pL/min in each tube, in which the reaction reagents were added. After clean-up by SPE cartridge, histamine in the microdialysate was found to be about 120 pg/mL. No significant difference of histamine was observed within 30 min. The results indicated that the present invention can be applied for the determination of histamine in 50 nano-liter of microdialysate collected in about 2 s, i.e. this method has the potential to be used in a real time determination of histamine in CSF.

Claims (19)

1. A method of determining histamine in a sample comprising steps of: a) providing a sample; b) preparing a mixture by adding a derivatizing agent to the sample, wherein the derivatizing agent is an alkyl phosphite; and subjecting the mixture to conditions where the derivatizing agent reacts with the histamine present in the sample to form a histamine derivative; c) separating the histamine derivative from the mixture so as to determine the presence and/or amount of the histamine derivative, wherein the amount of the histamine derivative corresponds to the amount of the histamine in the sample.

2. The method according to claim 1, wherein the alkyl phosphite is monoalkyl phosphite or di-alkyl phosphite.

3. The method according to claim 1, wherein the derivatizing agent is diisopropyl phosphite.

4. The method according to claim 1, wherein step c) comprises steps of: (i) introducing the mixture obtained in step b) to an ion exchange column to obtain a resultant mixture comprising the histamine derivative; and (ii) performing a liquid chromatography to separate the histamine derivative form the resultant mixture for detection and quantification.

5. The method according to claim 4, wherein a cation exchange column is used in step (i).

6. The method according to claim 4, wherein step (ii) further comprises performing multiple-reaction monitoring mass spectrometry for quantifying the amount of the histamine derivative after the liquid chromatography.

7. The method according to claim 1, wherein the histamine derivative is of Formula (IV):

Formula (IV).

8. The method according to claim 1, wherein the sample is a biological fluid selected from the group consisting of serum, blood, urine, transcellular fluid, interstitial fluid, saliva, exudates, fluid collected through microdialysis and other fluids from mammal susceptible of containing histamine.

9. The method according to claim 1, wherein the sample is obtained from cells or a cell population.

10. The method according to any one of claims 1 to 9, further comprises a step of adding an analog of histamine to the sample before step b).

11. The method according to claim 10, wherein the analog of histamine is deuterated histamine.

12. A kit for determining histamine in a sample comprising: - a first mixture comprising a derivatizing agent which is capable of reacting with histamine present in the sample to form a histamine derivative, wherein the derivatizing agent is an alkyl phosphite; and - a second mixture comprising a deprotonating agent.

13. The kit according to claim 12, wherein the alkyl phosphite is mono-alkyl phosphite or di-alkyl phosphite.

14. The kit according to claim 12, wherein the deprotonating agent is a tertiary amine or a trialkylamine.

15. The kit according to claim 12, wherein the derivatizing agent is diisopropyl phosphite and the deprotonating agent is triethylamine.

16. The kit according to claim 12, further comprises a third mixture comprising an analog of histamine.

17. The kit according to claim 16, wherein the analog of histamine is deuterated histamine.

18. The kit according to claim 12, further comprises a standard solution of histamine with a known concentration.

19. The kit according to any one of claims 12 to 18, further comprises an ion exchange resin cartridge.