AU2018100592A4 - Method and Kit for Detecting Carboxyl-Containing Compound - Google Patents
Method and Kit for Detecting Carboxyl-Containing Compound Download PDFInfo
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- AU2018100592A4 AU2018100592A4 AU2018100592A AU2018100592A AU2018100592A4 AU 2018100592 A4 AU2018100592 A4 AU 2018100592A4 AU 2018100592 A AU2018100592 A AU 2018100592A AU 2018100592 A AU2018100592 A AU 2018100592A AU 2018100592 A4 AU2018100592 A4 AU 2018100592A4
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Abstract
A method for detecting a carboxyl-containing compound in a first sample. The method comprises a step of adding a first derivatizing agent to the first sample to form a first mixture, wherein the first derivatizing agent comprises a compound of Formula (1). A kit 5 for determining the amount of a carboxyl-containing compound in a sample. The kit comprises the first derivatizing agent. -- hr Phe 6000000 - His 3000000 - - - 1 5 10 30 60 90 120 240 600 Reaction Tmie (min) -- Sue 20000000 - Sue - --- -a-- ---- Lac 1 5 10 30 60 90 120 240 600 Reaction Time (min) ---- BA 150000 - P S300000 -- VA 1 5 10 30 60 90 120 240 600 Reaction Time (min) Fig. 1a
Description
TECHNICAL FIELD
The present invention relates to a method for detecting a carboxyl-containing compound in a sample in particular a biological sample. The present invention also pertains to a kit for carrying out the method.
BACKGROUND OF THE INVENTION
Various carboxyl-containing compounds are present in a living organism as essential components involved in growth, development and reproduction of the living organism, whereas some carboxyl-containing compounds may not be involved in normal growth process, but for survival under specific environment. These compounds may be called carboxyl-containing metabolites (CCMs). Generally, CCMs widely exist in an organism. CCMs contain, but not limiting to, amino acids (AAs), tricarboxylic acid cycle intermediates (TCAs), short-chain fatty acids (SCFAs), long-chain fatty acids (LCFAs), bile acids and so on. These CCMs can be biomarkers for disease diagnosis, study of a pathogenic process, metabolism investigation, synthesis of protein, cell growth, or the like. For instance, AAs and TCAs are used in cancer studies. Shortchain fatty acids such as butyric acid (BA) and propionic acid (PA) can be used to evaluate the effects of a drug or potential compound on anti-inflammation. Long-chain fatty acids such as hydroxyeicosatetraenoic acids (HETEs, ω-6), prostaglandins (PGs, ω-6), thromboxanes (TXs, ω-6), and hydroperoxyeicosapentaenoic acids (HEPEs, ω3), play vital roles in disorders such as cancer, inflammation, diabetes, immune cell behavior and other biological activities and therefore can act as a biomarker.
Therefore, the detection of CCMs is important in the development of methods for diagnosis of diseases and clinical studies.
Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) have been used for CCMs analysis. However, the thermal instability of some CCMs hinders the use of GC-MS. Also, the significant polarity differences between hydrophilic CCMs such as AAs, TCAs and SCFAs and hydrophobic CCMs such as LCFAs and bile acids affect the separation of these two groups of CCMs on LC column. Recently, a reversed-phase liquid chromatography (RPLC) column combined with hydrophilic interaction liquid chromatography (HILIC) column or the use of ion pairing agents with reverse-phase column was developed to overcome the significantly polarity difference between hydrophilic and hydrophobic
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CCMs. However, each of these approaches has drawbacks for processing global metabolites. The combination of RPLC and HILIC column are generally associated with broader peak shapes, which will result in the decrease of separation efficiency.
The use of ion pairing agents is general incompatibility with mass spectrometry and causes contamination affecting MS analysis.
Accordingly, there remains a strong need for developing improved method to detect carboxyl-containing compounds for simultaneous analysis and identification of CCMs, and for differentiating hydrophilic and hydrophobic CCMs.
SUMMARY OF THE INVENTION
The first aspect of the present invention relates to a method for detecting a carboxylcontaining compound in a first sample comprising a step of adding a first derivatizing agent to the first sample to form a first mixture, wherein the first derivatizing agent comprises a compound of Formula (I):
/R2
H2N R1 — N
I
R3
Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
Preferably, the first derivatizing agent comprises a compound of Formula (II):
CX3
XsC^ CX3 Formula (II) wherein X is hydrogen or deuterium.
In an embodiment, the method comprises a step of preparing a second mixture comprising a second derivatizing agent and a second sample, wherein the second derivatizing agent is different from the first derivatizing agent and comprises a compound of Formula (I) as described.
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In the second aspect, there is provided a kit for detecting a carboxyl-containing compound, i.e. determining the amount of a carboxyl-containing compound in a sample, comprising a first derivatizing agent comprising a compound of Formula (I):
Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
Preferably, the kit further comprises a second derivatizing agent. In an embodiment, the first derivatizing agent comprises a compound of Formula (Ila) and the second derivatizing agent comprises a compound of Formula (lib):
CH3
.CD
D3C ^cd3
Formula (lib).
Those skilled in the art will appreciate that the invention described herein is 20 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.
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BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 refers to plots of peak area against reaction time or reaction temperature of Compound 1a-derivatized carboxyl-containing standards. Fig. 1a shows the influence of reaction time on Compound 1a-derivatized carboxyl-containing standards of phenylalanine (Phe), threonine (Thr), histidine (His), propionic acid (PA), butyric acid (BA), valeric acid (VA), fumaric acid (Fum), succinic acid (Sue) and lactic acid (Lac). Fig. 1b shows the influence of reaction time on Compound 1 a-derivatized carboxylcontaining standards of 11-dehydro TXB2, 15(S)-HETE, PGE2, 13(S)-HOTrE, 15(S)10 HEPE, 5(S)-HEPE, cholic acid (CA), chenodeoxycholic acid (CDCA) and glycocholic acid (GCA). Fig. 1c shows the influence of reaction temperature on Compound 1aderivatized carboxyl-containing standards of Phe, Thr, His, PA, BA, VA, Fum, Sue and Lac. Fig. 1d shows the influence of reaction temperature on Compound 1aderivatized carboxyl-containing standards of 11-dehydro TXB2, 15(S)-HETE, PGE2,
13(S)-HOTrE, 15(S)-HEPE, 5(S)-HEPE, CA, CDCA and GCA.
Fig. 2 refers to LC-MS chromatograms of carboxyl-containing standards and their Compound 1a-derivatives. Fig. 2a shows the LC-MS chromatograms of carboxylcontaining standards prior to Compound 1a-derivatization. Fig. 2b shows the LC-MS chromatograms of carboxyl-containing standards after Compound 1a-derivatization. After derivatization, the carboxyl-containing standards in region A1 and A2 appeared in region B1 and B2, respectively.
Fig. 3 refers to extracted ion chromatograms (EIC) obtained after derivatization of ten carboxyl-containing standards or carboxyl-containing metabolites in a serum sample obtained from healthy or CRC patient. Fig. 3a shows an EIC of Compound 1aderivatized standards. Fig. 3b shows an EIC of Compound 1 b-derivatized standards. The CCM standards include Trp, Fum, 15(S)-HETE (1), 9(S)-HPODE (2), 11(S)HETE (3), 12(S)-HETE (4), 8(S)-HETE (5), 5(S)-HETE and 14(15)-EET (6), and
11(12)-EET (7). Fig. 3c shows an EIC of Compound 1 a-derivatized healthy human serum. Fig. 3d shows an EIC of Compound 1 b-derivatized CRC patient serum.
Fig. 4 refers to LC-MS chromatograms of CCMs in different samples determined with non-derivatization and Compound 1a-derivatization approaches. Fig. 4a shows the
LC-MS chromatograms of CCMs of cell line. Fig. 4b shows the LC-MS chromatograms of CCMs of human serum. Fig. 4c shows the LC-MS chromatograms
2018100592 09 May 2018 of CCMs of rat serum. Fig. 4d shows the LC-MS chromatograms of CCMs of rat feces Fig. 4e shows the LC-MS chromatograms of CCMs of cerebrospinal fluid (CSF). Fig. 4f shows the LC-MS chromatograms of CCMs of tea.
DETAILED DESCRIPTION OF THE INVENTION 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. The expression that a material is certain element is to be understood for meaning “essentially consists of’ said 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.
In the first aspect of the present invention, there is provided a method for detecting a carboxyl-containing compound in a sample in particular a biological sample. The method can be applied to detect the presence or absence and/or the amount of one or more target carboxyl-containing compounds in a sample.
The term “carboxyl-containing compound” refers to any compounds having one or more carboxyl groups. In one embodiment, the carboxyl-containing compound is selected from the group consisting of an amino acid, a saturated or unsaturated fatty acid, a dicarboxylic acid, a tricarboxylic acid, and a steroid acid such as a bile acid.
The phrase “target carboxyl-containing compound” refers to a particular carboxylcontaining compound of interest. The carboxyl-containing compound of interest may be a metabolite produced by a human, an animal, an alga, a fungus, a yeast, a bacterium or a plant, and the metabolite may be primary or secondary metabolite.
The sample is preferably a biological sample obtained or derived from an animal, a plant, an alga, a fungus, an insect, a yeast, a bacterium or the like. Preferably, the
2018100592 09 May 2018 sample is obtained or derived from a mammal such as a human, or a plant. In an embodiment where the sample is obtained from a human, the sample may comprise blood, a portion of a tissue, an extract, cells, serum, body fluid such as saliva, urea and cerebrospinal fluid, feces or the like. In an embodiment where the sample is obtained or derived from a plant, the sample may comprise a portion of a processed or non-processed tissue, an extract, cells, a secretion or the like. The sample may be in solid form such as dried or lyophilized form before applying the method of the invention.
Turning to the method, the method comprises a step of adding one or more derivatizing agent to one or more samples to form one or more mixtures for detection of a carboxyl-containing compound in the one or more samples. The term “derivatizing agent” used herein refers to an agent that can alter a functional group of a compound to form a corresponding derivative. In particular, the derivatizing agent may alter the carboxyl group of the carboxyl-containing compound to form a corresponding derivative for detection. Preferably, the derivatizing agent comprises a compound of Formula (I):
/r2
H2N-R1—N
I
R3
Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
In an embodiment, R1 may be unsubstituted straight C1-C6 alkyl chain such as methyl, ethyl, propyl, butyl, pentyl or hexyl, or branched C1-C6 alkyl chain, or C1-C6 alkyl chain substituted by a substituent selected from a halo, an alkoxyl or a phenyl group. Preferably, R1 is an unsubstituted straight alkyl chain having 1 to 6 carbon atoms, in particular R1 is a pentyl group.
R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group. R2 and R3 may be each independently methyl, ethyl, propyl or isopropyl group with one or more hydrogen atoms substituted by deuterium. In one embodiment, R2 and R3 are each independently unsubstituted or substituted isopropyl
2018100592 09 May 2018 group, where one or more hydrogen atoms of the isopropyl group may be substituted by deuterium.
In a particular embodiment, the derivatizing agent comprises a compound of Formula 5 (H):
cx3
X3C ^cx3 Formula (II) wherein X is hydrogen or deuterium, i.e. the derivatizing agent may comprise a compound of Formula (Ila) or Formula (lib):
CH3
Formula (Ila)
CD3
d3c^ ^cd3
Formula (lib).
Each of the compounds of Formula (Ila) and Formula (lib) can alter the functional 15 group of a carboxyl-containing compound to form the corresponding derivative. Due to the difference between hydrogen and deuterium, the derivatives produced from the two compounds can be separately identified for example by a mass spectrometer.
After adding the derivatizing agent to the sample to form a mixture, the mixture is 20 subject to conditions where a reaction in particular condensation occurs between the derivatizing agent and the carboxyl-containing compound in the sample. In an embodiment where condensation is required to convert the carboxyl-containing compound, a condensating agent is added into the sample.
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The term “condensating agent” refers to any agents that can facilitate or involve in a condensation reaction between two molecules. Preferably, the condensating agent as used herein comprises 1-hydroxybenzotriazole (abbreviated as HOBt), 1[bis(dimethylamino)methylene]-1 /7-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (abbreviated as HATU), or a combination thereof. In an embodiment, both HOBt and HATU are added to the sample, preferably HOBt and HATU are separately provided in a solvent such as dimethyl sulfoxide (DMSO) before addition to the sample. A person having the ordinary skills in the art would appreciate that other suitable condensating agents may also be applied in accordance with the present disclosure.
In an embodiment, the mixture is prepared by mixing the sample, a derivatizing agent as described above and a condensating agent as described above before subjecting the mixture to conditions where condensation occurs. The derivatizing agent may be provided with a base such as triethylamine (TEA) for addition to the sample. Preferably, the sample is first mixed with HOBt, then the derivatizing agent, followed by HATU at room temperature. The mixture is then incubated at room temperature for about 30 seconds, 1 minute, 2 minutes or more before conducting mass spectrum analysis to detect the presence, absence and/or the amount of the derivative of the carboxyl-containing compound in the sample, i.e. corresponding to the presence, absence and/or the amount of the carboxyl-containing compound in the sample.
The method comprises a step of conducting mass spectrum analysis preferably liquid chromatography-mass spectrum (LC-MS) analysis of the mixture. The LC-MS analysis includes, but not limiting to, high-performance liquid chromatography-mass spectrum (HPLC) analysis, ultra-high performance liquid chromatography-mass spectrum (UHPLC-MS) analysis, and ultra-high performance liquid chromatographyquadrupole rod time-of-flight mass spectrum (UHPLC-Q-TOF/MS) analysis. In a preferred embodiment, a UHPLC-Q-TOF/MS is conducted to obtain a mass spectrum of the mixture. It would be appreciated that an internal standard may be added to the mixture before derivatization and conducting the MS analysis.
The inventors unexpectedly found that the derivatizing agent of the present invention can successfully convert one or more carboxyl-containing compounds in the sample,
i.e. forming derivatives to be detected by the mass spectrometer. In particular, the derivatization enhances the separation efficiency and sensitivity for the detection of
2018100592 09 May 2018 the carboxyl-containing compounds. It also allows effective identification and differentiation between hydrophilic carboxyl-containing compounds and hydrophobic carboxyl-containing compounds. The detection can be completed within 1 hour in particular within 44 min with improved performance compared to the existing direct detection of carboxyl-containing compounds.
In an embodiment where more than one derivatizing agent in particular two derivatizing agents are applied, there are provided a first derivatizing agent comprising a compound of Formula (I) as described above, and a second derivatizing agent comprising a compound of Formula (I) which is different from that in the first derivatizing agent. Preferably, the first derivatizing agent comprises a compound of Formula (Ila) and the second derivatizing agent comprises a compound of Formula (Mb):
ch3
d3c^ ^cd3
Formula (lib).
The first sample and the second sample are different from each other. In one embodiment, the first sample may be derived from an individual suffering from a disorder and the second sample may be derived from a healthy individual, or vice versa. The individual is preferably a mammal such as a human. The disorder includes, but not limiting to, an inflammatory disease, an autoimmune disease, a cardiovascular disease, a gastrointestinal disease, and a cancer. Preferably, the individual may be a human suffering from cancer such as colon cancer, lung cancer, liver cancer, breast cancer, prostate cancer, skin cancer or the like. In an embodiment, the first sample may be obtained from a human suffering from colon cancer, whereas the second sample may be obtained from a healthy human. Alternatively, the first sample may be
2018100592 09 May 2018 obtained from a human suffering from a disorder, and the second sample may comprise a pre-determined amount of one or more target carboxyl-containing compounds.
Preferably, the method comprises steps of:
- adding the first derivatizing agent as described above to the first sample to form a first mixture, and
- preparing a second mixture comprising the second derivatizing agent as described above and the second sample, in particular the second derivatizing agent is different from the first derivatizing agent;
wherein each of the first and second mixture comprises a condensating agent as described above to facilitate the reaction between the derivatizing agent and the carboxyl-containing compound. In an embodiment, the first and second samples are obtained from humans.
Next, after subjecting the first and second mixtures to conditions where condensation occurs, the first mixture and the second mixture are combined to form a third mixture, preferably the first mixture and the second mixture are combined at a volume ratio of about 1:1. Subsequently, the third mixture is subject to mass spectrum analysis as described above, i.e. the method further comprises a step of conducting liquidchromatography-mass spectrum analysis of the third mixture.
The application of a second derivatizing agent allows simultaneous identification and comparison of the level of one or more carboxyl-containing compounds between two samples. This is particularly useful in investigation of the health condition of an individual, diagnosis of a disease in an individual, quality or quantitative assessment of a product, and the like. In other words, the method of the present invention is suitable for various medical applications, and quality control/assurance of a product.
In the second aspect of the invention, there is provided a kit for determining the amount of a carboxyl-containing compound in a sample. The kit comprises one or more derivatizing agents as described above, in particular the kit comprises a first derivatizing agent. The first derivatizing agent comprises a compound of Formula (I) /r2
H2N-R1—N
I
R3
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Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group. In an embodiment, R1 may be unsubstituted straight C1-C6 alkyl chain, or C1-C6 alkyl chain substituted by a substituent selected from a halo, an alkoxyl or a phenyl group. Preferably, R1 is an unsubstituted straight alkyl chain having 1 to 6 carbon atoms, in particular R1 is a pentyl group. R2 and R3 may be each independently methyl, ethyl, propyl or isopropyl group with one or more hydrogen atoms substituted by deuterium. In one embodiment,
R2 and R3 are each independently unsubstituted or substituted isopropyl group, where one or more hydrogen atoms of the isopropyl group may be substituted by deuterium.
Preferably, the first derivatizing agent comprises a compound of Formula (II):
cx3
X3C ^cx3
Formula (II) wherein X is hydrogen or deuterium.
More preferably, the kit further comprises a second derivatizing agent comprising a compound of Formula (I) which is different from that of the first derivatizing agent. In an embodiment, the first derivatizing agent comprises a compound of Formula (Ila) and the second derivatizing agent comprises a compound of Formula (lib):
H3C^ 'ch3 Formula (Ila)
CD3
,CD
D3C ^cd3
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Formula (lib).
The two derivatizing agents can be applied separately to react with one or more carboxyl-containing compounds in different samples.
In an advanced embodiment, the derivatizing agent is provided in a base solution.
In addition, the kit further comprises a condensating agent as described above. In particular, the condensating agent comprises HOBt, HATU, or a combination thereof.
Preferably, the kit comprises HOBt and HATU provided in a solvent such as DMSO.
It would be appreciated that suitable solvents for the derivatizing agent, and the condensating agent may also be included in the kit.
EXAMPLES
Chemicals and reagents
All long-chain carboxylic metabolites standards mixtures were purchased from Cayman Chemical (Ann Arbor, Ml). They are (S)-hydroxyeicosatetraenoic acid (HETE) HPLC mixture containing 5(S)-, 8(S)-, 11(S)-, 12(S)-, and 15(S)-HETE; hydroperoxy
HPLC mixture containing 5(S)-, 12(S)-, and 15(S)-hydroperoxyeicosatetraenoic acid (HPETE), 9(S)- and 13(S)-hydroperoxyoctadecadienoic acid (HPODE); ω-3 hydroxy acid HPLC mixture containing 5(S)-, 12(S)-, and 15(S)-hydroxyeicosapentaenoic acid (HEPE), 13(S)-hydroxyoctadecatrienoic acid (HOTrE), and 15(S)Hydroxyeicosatrienoic acid (HETrE); cyclopentenone prostaglandin (PG) HPLC mixture containing PGA2, PGB2, PGD2, PGE2 and PGJ2; prostaglandin HPLC mixture containing PGE1, PGE2, PGF1a, 6-keto PGF1a and PGF2a; vasoactive eicosanoid HPLC mixture containing thromboxane B2 (TXB2), 11-dehydro TXB2, 6keto PGF1a, 2,3-dinor-6-keto PGF1a and 12(S)-hydroxyheptadecatrienoic acid (HHTrE); prostaglandin metabolite HPLC mixture containing 13,14-dihydro-15-keto
PGD2 (DK-PGD2), 13,14-dihydro-15-keto PGE2 (DK-PGE2), 1ip-PGF2a, 13,14dihydro-15-keto PGF2a (DK-PGF2a) and PGF2a; Leukotriene B4 (LTB4); 11(12)epoxyeicosatrienoic acid (11(12)-EET). Bile acids containing cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycocholic acid (GCA), ursodeoxycholic acid (UDCA), glycochenodeoxycholic acid (GCDCA); amino acids mixtures containing L-alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine
2018100592 09 May 2018 (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr), tryptophan (Trp), glutamine (Gin) and valine (Val); TCA cycle intermediates containing aconitic acid, malic acid (Mai), fumaric acid (Fum), aketoglutaratic acid (α-KG), lactic acid (Lac) and succinic acid (Sue); short-chain fatty acids containing butyric acid (BA), isobutyric acid (IBA), propionic acid (PA), valeric acid (VA) and isovaleric acid (IVA) were purchased from Sigma-Aldrich Laboratories, Inc. (St. Louis, MO). The internal standard LTB4-d4 were obtained from Cayman Chemical (Ann Arbor, Ml) and 4-chloro-DL-phenylalanine were obtained from SigmaAldrich (St. Louis, MO).
5-(Diisopropylamino)amylamine (abbreviated as DIAAA, denoted as Compound 1a) was synthesized and purified by prepared HPLC before derivatization. 1Hydroxybenzotriazole hydrate (abbreviated as HOBt), 1[bis(dimethylamino)methylene]-1 /7-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (abbreviated as HATU), 2-iodopropane, 2-iodopropane-d7, triethylamine (abbreviated as TEA) and DMSO were also purchased from SigmaAldrich Laboratories, Inc. (St. Louis, MO). /V-Z-1,5-pentanediamine hydrochloride was purchased from Technology Consulting, Inc. Acetonitrile of LC/MS grade were purchased from Anaqua Chemicals Supply Inc., Ltd. (Houston, TA, USA). Deionized water was prepared using a Millipore water purification system (Millipore Corp, USA). MS grade formic acid and other chemical reagents were purchased from SigmaAldrich Laboratories, Inc. (St. Louis, MO).
EXAMPLE 1
Synthesis and characterization of Compound 1a and 1b
Compound 1a and 1b of the present invention were prepared according to Reaction Scheme 1:
TFA, ch2ci2 Room Temp.
NH2 + Y _XX DMF' Κ2θθ3 X3C SCX3
H2N °C
N ι
XX cx3
XX ex.
X3C
CX3 .,,ΧΧ i1 CX3 XX Xcx3 cx3
Reaction Scheme 1 wherein X is hydrogen or deuterium.
2018100592 09 May 2018 /V-Z-1,5-pentanediamine hydrochloride (170 mg) was added to a stirring solution of DMF (1.5 mL) containing 1 g of K2CO3 at 50°C, followed by the addition of 1 mL of 2iodopropane or 2-iodopropane-d7. The mixture was allowed to react for 46 h and then concentrated. The carboxybenzyl (Cbz) protected Compound 1a or 1b was purified by HPLC using ACN:H2O (20:60) as eluent. TFA:CH2Cl2 (in a volume ratio of 9:1) was added dropwise to a CH2CI2 solution of the Cbz protected compound 1a or 1b. The mixture was stirred for 1 h at room temperature, and then concentrated in vacuo, the residue was washed with 5% Na2CO3 until the pH of the residue turns to pH 8-9. The supernatant was dried under rotary evaporation to afford Compound 1a or 1b.
EXAMPLE 2 Sample Preparation
Cold methanol was added to each serum sample at a volume ratio of 4:1 (methanol:
serum sample) for protein precipitation. The mixtures were subject to centrifugation at 13000 rpm for 5 min at 4°C prior to derivatization. These steps were repeated for 3 times and the supernatants were dried under a nitrogen stream. The residue was stored at -20 °C before derivatization.
Derivatization
Derivatization of a carboxyl-containing compound is carried out according to Reaction Scheme 2:
I
R OH h2n' cx3 ,c
HOBt, HATU x3c
N X .,cx cx3
CX3 min, Room Temp.
wherein X = H or D.
In the process, HATU and HOBt are used as the condensating reagent due to their high condensation efficiency and quick coupling times. Stock solutions of HOBt (20 mM) and HATU (20 mM) were prepared by dissolving the same in DMSO respectively.
A stock solution of Compound 1a was prepared by dissolving 100 pmol of Compound 1a and 200 pmol of TEA in 1 mL of DMSO. Similarly, a stock solution of Compound 1b was prepared by dissolving 100 pmol of Compound 1b and 200 pmol of TEA in 1 mL of DMSO.
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Representative carboxyl-containing compounds such as phenylalanine (Phe), threonine (Thr), histidine (His), propionic acid (PA), butyric acid (BA), valeric acid (VA), fumaric aicd (Fum), succinic acid (Sue), lactic acid (Lac), 11-dehydro TXB2, 15(S)5 HETE, PGE2, 13(S)-HOTrE, 15(S)-HEPE, 5(S)-HEPE, cholic acid (CA), chenodeoxycholic acid (CDCA), and glycocholic acid (GCA) were used as standard compounds.
pL of HOBt, 5 pL of Compound 1a solution or Compound 1b solution, and 5 pL of
HATU were sequentially added to the dried residue of standards or real samples having carboxyl-containing metabolites, and mixed. The mixture was incubated at room temperature for 1 min for derivatization. Subsequently, 35 pL of acetonitrile was added to make up to the mark. Aliquot (1 pL) of the resultant solution was directly injected into UHPLC-Q-TOF/MS for analysis.
UHPLC-Q-TOF/MS Analysis
An Agilent 1290 Infinity LC system (UHPLC, Santa Clara, CA) and binary pump with Waters ACQUITY UPLC HSS T3 column (2.1 x 100 mm, 1.8 pm, Waters Corp., MA, USA) was employed for the separation of components. The column temperature was maintained at 40°C and the autosampler was set at 4°C. The injection volume was 1 pL and the flow rate was 0.3 mL min'1. Mobile phase A and B were 0.1% formic acidcontaining water and 0.1% formic acid-containing acetonitrile, respectively, and the gradient was set as follows: 0-0.5 min, 2% to 5% B; 0.5-2.5 min, 5%-6% B; 2.5-4.5 min, 6%-7% B; 4.5-5.5 min, 7%-7.3% B; 5.5-7.5 min, 7.3%-7.8% B; 7.5-11 min, 7.8%25 9% B; 11-13 min, 9%-14% B; 13-18 min, 14%-23% B; 18-19 min,; 23%-25% B; 19-26 min, 25%-33% B; 26-26.5 min, 33%-35%; 26.5-34.5 min, 35%-47% B; 34.5-38 min, 47%-60% B; 38-40 min, 60%-95% B; 39-43.9 min, 95%; 44 min, 2% B.
The mass spectrometry was conducted on an Agilent 6550 UHD accurate-mass Ci30 TOF/MS system with a dual Jet stream electrospray ion source (dual AJS ESI). The instrument was operated in positive and negative mode. The MS parameters were set as: dry gas temperature at 250°C, dry gas flow at 15 L min'1, sheath gas temperature at 300°C, sheath gas flow at 11 L min'1, nebulizer pressure at 20 psig, capillary voltage at 5000 V, and nozzle voltage at 500 V for positive ion mode and 1500 V for negative ion mode. The mass spectra were recorded across the range of 100-1000 m/z for derivatized samples and 50-1000 m/z for non-derivatized samples. Accurate
2018100592 09 May 2018 mass measurements were obtained by using a low flow of TOF reference mixture, containing the internal reference masses at m/z 922.0098 (C18H18F24N3O6P3) for positive ion mode and 966.0007 (HP-921) for negative ion mode. For MS/MS acquisition, automated and target MS/MS were applied and the collision cell energy was set at 30 eV.
With reference to Fig. 1a to 1 d, the results obtained from the standard compounds show that the derivatization almost finished within 1 min at room temperature and the derivatization did not exhibit obvious time-dependent or temperature-dependent changes along with the reaction times (1, 5, 10, 30, 60, 90, 120, 240 and 600 min) and temperatures (20, 30, 40, 50 and 60 °C). Accordingly, the derivatization is preferably conducted for at least 1 min and may be conducted at room temperature.
Referring to Figs. 2a and 2b, after derivatization, the sensitivity for standard hydrophilic and hydrophobic carboxyl-containing compounds enhanced using LC-MS approach. More importantly, the inventors unexpectedly found that the retention times of hydrophilic carboxyl-containing compounds prolonged from 0.5-3 min to 0.5-16.5 min (region A1 to B1) after derivatization, while conversely the retention times of hydrophobic carboxyl-containing compounds shortened from 27-41 min to 21.5-39 min (region A2 to B2). Particularly, the isomers of HETEs and epoxyeicosatrienoic acids (EETs) could achieve baseline separation, which is essential for the identification and quantification. That is to say the derivatization of the present invention does not only achieve the simultaneous determination of hydrophilic and hydrophobic carboxyl-containing compounds but also improved the separation efficiency and sensitivity of different hydrophilic or hydrophobic carboxyl-containing compounds. As a result, 68 fatty acid standards could be well separated and determined within 40 min, while only 45 could be detected with very weak MS signals and heavy peak overlap using non-derivatization method even in higher concentrations.
EXAMPLE 3
Derivatization of carboxyl-containing metabolites in biological samples The derivatization method was applied to determine the change of carboxylcontaining metabolites (CCMs) in human colorectal cancer (CRC) patients.
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First, a derivatizing agent - Compound 1b was prepared and added to the biological sample obtained from the patients. A comparison sample was set by using compound 1a with tryptophan (Trp), Fum, 5(S)-, 8(S)-, 11 (S)-, 12(S)-, and 15(S)-HETE, 11(12)EET, and 9(S)-HPODE as standards. Second, a mixture containing the standards was equally divided into two vials and dried. One residue was reacted with Compound 1a, while another was incubated with Compound 1b under the same condition.
With reference to Figs. 3a and 3b, UHPLC-O-TOF/MS analysis indicated that Compound 1 a-derivatives (derivatives formed by reacting with Compound 1a) and
Compound 1 b-derivatives (derivatives formed by reacting with Compound 1b) had the same retention times. Moreover, the MS responses of these derivatives had no significant difference. That is, the reaction efficacy of Compound 1b with CCMs had no significant difference with that of Compound 1a. Accordingly both Compound 1a and 1b are suitable for labeling derivatization method and can be constructed as an isotope labeling pair.
Compound 1a and Compound 1b were separately reacted with healthy and CRC human serum samples. Then, the two resultant mixtures were mixed in equal volume followed by LC-MS analysis in a single run. Referring to the results in Figs. 3c and 3d, the relative abundance of almost all CCMs in CRC patient serum decreased compared to that of healthy human. Using this method, the analysis time was reduced to half, which can increase the throughput. In addition, since the two samples were simultaneously determined in the same run, the error was decreased to a large extent,
i.e. the accuracy and reliability of this endogenous CCMs analysis can be improved.
Still further, the present method can be applied to analyze the metabolites in other biological samples such as in vitro cells, serum, feces, cerebrospinal fluid and plant tissues or product such as tea. Figs. 4a to 4f are LC-MS chromatograms of CCMs in different samples determined with or without derivatization of the present invention.
All results show that much more metabolites can be sensitively detected after derivatization.
2018100592 09 May 2018
Claims (4)
1. A method for detecting a carboxyl-containing compound in a first sample comprising a step of adding a first derivatizing agent to the first sample to form a first mixture, wherein the first derivatizing agent comprises a compound of Formula (I):
/R2
H2N-R1—N
I
R3
Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
2. The method of claim 1, wherein the first derivatizing agent comprises the compound of Formula (I) with R1 being an unsubstituted straight alkyl chain having 1 to 6 carbon atoms, and wherein R2 and R3 are each independently unsubstituted or substituted isopropyl group.
3. The method of claim 1, wherein the first derivatizing agent comprises the compound of Formula (I) with R1 being a pentyl group, and wherein R2 and R3 are each independently unsubstituted or substituted isopropyl groups.
20 4. The method of claim 1, wherein the first derivatizing agent comprises a compound of Formula (II):
cx3
X3C^ ^cx3 Formula (II) wherein X is hydrogen or deuterium.
5. The method of claim 1, further comprising a step of subjecting the first mixture to conditions where condensation occurs between the first derivatizing agent and the carboxyl-containing compound in the first sample, wherein the first mixture further comprises a condensating agent.
2018100592 09 May 2018
6. The method of claim 5, further comprising a step of conducting liquidchromatography-mass spectrum analysis of the first mixture.
7. The method of claim 5, wherein the condensating agent comprises 15 hydroxybenzotriazole, 1-[bis(dimethylamino)methylene]-1/7-1,2,3-triazolo[4,5bjpyridinium 3-oxide hexafluorophosphate, or a combination thereof.
8. The method of claim 1, wherein the carboxyl-containing compound is selected from the group consisting of an amino acid, a saturated or unsaturated fatty acid, a
10 dicarboxylic acid, a tricarboxylic acid, and a steroid acid.
9. The method of claim 1, wherein the first sample is a biological sample.
10. The method of claim 1 further comprises a step of preparing a second mixture
15 comprising a second derivatizing agent and a second sample, wherein the second derivatizing agent is different from the first derivatizing agent and comprises a compound of Formula (I):
/r2
H2N-R1—N
I
R3
Formula (I),
20 wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
11. The method of claim 10, further comprises a step of combining the first
25 mixture and the second mixture to form a third mixture.
12. The method of claim 11, wherein the first mixture and the second mixture are combined at a volume ratio of 1:1.
30 13. The method of claim 10, wherein the first derivatizing agent comprises a compound of Formula (Ila) and the second derivatizing agent comprises a compound of Formula (lib):
2018100592 09 May 2018 ch3 h2n h2n h3c
Formula (Ila)
CD3
N
CD3
D3C DD3 Formula (lib).
14. The method of claim 10, wherein the second sample comprises a carboxylcontaining compound.
15. The method of claim 10, wherein the first sample is derived from an individual suffering from a disorder and the second sample is derived from a healthy individual.
16. The method of claim 14, wherein the second sample comprises a predetermined amount of carboxyl-containing compound.
17. The method of claim 15, wherein the disorder is cancer or inflammatory disease.
18. The method of claim 15, wherein the first and second samples are obtained from humans.
19. The method of claim 11, further comprises a step of conducting liquidchromatography-mass spectrum analysis of the third mixture.
20. A kit for determining the amount of a carboxyl-containing compound in a sample, comprising a first derivatizing agent comprising a compound of Formula (I):
H2N-R1—
I
R3
2018100592 09 May 2018
Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
21. The kit of claim 20, wherein the first derivatizing agent comprises the compound of Formula (I) with R1 being an unsubstituted straight alkyl chain having 1 to 6 carbon atoms, and wherein R2 and R3 are each independently unsubstituted or substituted isopropyl group.
22. The kit of claim 20, wherein the first derivatizing agent comprises the compound of Formula (I) with R1 being a pentyl group, and wherein R2 and R3 are each independently unsubstituted or substituted isopropyl group.
15 23. The kit of claim 20, wherein the first derivatizing agent comprises a compound of Formula (II):
cx3
X3C ^cx3 Formula (II) wherein X is hydrogen or deuterium.
24. The kit of claim 20, further comprises a second derivatizing agent being different from the first derivatizing agent, wherein the second derivatizing agent comprises a compound of Formula (I):
/R2
H2N-R1—N
I
R3
25 Formula (I), wherein R1 is an unsubstituted or substituted straight or branched alkyl chain having 1 to 6 carbon atoms, and R2 and R3 are each independently unsubstituted or substituted methyl, ethyl, propyl or isopropyl group.
2018100592 09 May 2018
25. The kit of claim 24, wherein the first derivatizing agent comprises a compound of Formula (Ila) and the second derivatizing agent comprises a compound of Formula (lib):
ch3 .CD
D3C ^cd3
Formula (lib).
26. The kit of claim 20, further comprises a condensating agent.
27. The kit of claim 26, wherein the condensating agent comprises 1hydroxybenzotriazole, 1 -[bis(dimethylamino)methylene]-1 /7-1,2,3-triazolo[4,5b]pyridinium 3-oxide hexafluorophosphate, or a combination thereof.
2018100592 09 May 2018
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Fig. 1b
2018100592 09 May 2018
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2018100592 09 May 2018
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2018100592 09 May 2018
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2018100592 09 May 2018
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2018100592 09 May 2018
Fig. 4a i J ) 4 ί » ' t f ii ii i; u h ι; ii r is i> » ’i ΐ a y Λ r ;i » » π « » h jj « J! m 40 4i Retention Time (min,
Fig. 4b
2018100592 09 May 2018
Fig. 4c
Retention Time (min,
Fig. 4d
2018100592 09 May 2018
Retention Time (min)
Fig. 4e
Fig. 4f
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115420825A (en) * | 2022-08-31 | 2022-12-02 | 北京大学第三医院(北京大学第三临床医学院) | Bile acid detection method and bile acid derivative |
CN115812912A (en) * | 2022-09-29 | 2023-03-21 | 中国人民解放军海军军医大学 | Preparation method of fermented soybean with controlled content of isoflavone and carboxyl-containing compound |
-
2018
- 2018-05-09 AU AU2018100592A patent/AU2018100592A4/en not_active Ceased
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115420825A (en) * | 2022-08-31 | 2022-12-02 | 北京大学第三医院(北京大学第三临床医学院) | Bile acid detection method and bile acid derivative |
CN115420825B (en) * | 2022-08-31 | 2023-12-22 | 北京大学第三医院(北京大学第三临床医学院) | Method for detecting bile acid and bile acid derivative |
CN115812912A (en) * | 2022-09-29 | 2023-03-21 | 中国人民解放军海军军医大学 | Preparation method of fermented soybean with controlled content of isoflavone and carboxyl-containing compound |
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