CN113376264A

CN113376264A - Method for detecting monosaccharides in sample

Info

Publication number: CN113376264A
Application number: CN202010790935.XA
Authority: CN
Inventors: 张曦; 郭玮; 温冬; 陈智; 杨文静
Original assignee: Shanghai Puen Haihui Medical Laboratory Co ltd; Zhongshan Hospital Fudan University
Current assignee: Shanghai Dunhui Medical Technology Development Co ltd
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2021-09-10

Abstract

The invention discloses a method for detecting monosaccharide composition in human saliva or plasma by pre-column derivatization combined with ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS), which comprises the step of derivatizing monosaccharide in a sample by using 1-phenyl-3-methyl-5-pyrazolone (PMP); and a step of analyzing the treated sample for one or more monosaccharides. The detection method has the advantages of high detection sensitivity, strong specificity, rapidness and the like, and can be used for accurately detecting various monosaccharides in food samples, plant samples and body fluid samples, particularly human saliva and plasma.

Description

Method for detecting monosaccharides in sample

Technical Field

The present invention relates to the field of analytical testing; in particular, the invention relates to methods for detecting one or more monosaccharides in a sample, such as a bodily fluid.

Background

Saliva detection is an emerging medical inspection mode in recent years, and is a medical inspection research hotspot with the most potential and application prospect in the field of noninvasive inspection. Compared with urine samples, saliva has the advantage of being sampled in real time; compared with blood samples, saliva has the advantages of no wound, no risk of spreading of blood-borne diseases, no pain of patients and the like. For the population such as infants, the elderly, hemophiliacs and the like, the noninvasive acquisition is more easily accepted. In addition, the saliva detection also improves the compliance of patients on the monitoring of clinical treatment effect and disease progress which need frequent sampling, does not need special instruments and specially trained blood sampling personnel, reduces the risk of cross infection of medical care personnel and patients, and particularly reduces the infection rate of hepatitis virus and Human Immunodeficiency Virus (HIV). The vast amount of biological information contained in saliva can be applied: early diagnosis of diseases, monitoring of disease progression, formulation of treatment regimens, and determination of treatment effects.

Currently, monosaccharides used for clinical diagnosis are mainly glucose, 1, 5-anhydroglucitol (1,5-AG) and galactose. Plasma glucose detection is one of the main indicators for diabetes diagnosis, and the detection methods include an o-toluidine method, a glucose oxidase method, a hexokinase method, a glucose dehydrogenase method and the like, wherein the hexokinase method is an internationally recommended reference method. Blood plasma or blood serum 1,5-AG is also one of the detection indexes of diabetes, and the detection method is mainly an enzyme method. There are reports in the literature on the detection of glucose or 1,5-AG alone by the LC-MS method. The congenital lactose intolerance is clinically identified by detecting urine galactose, and the detection method mainly comprises qualitative determination of a galactose oxidase method and the like. The levels of fucose in serum or urine have been reported in the literature to be associated with tumors.

Besides the three types, monosaccharides in a human body also comprise glucose isomer mannose, structural analogues and the like, and the traditional detection technology is difficult to separate the compounds with similar structures, so that the interference is large, the specificity is poor, and the detection result is unreliable. At present, the development of sugar derivative reagents, high performance liquid chromatography separation technology and liquid chromatography-mass spectrometry technology make it possible to accurately detect the substance with strong polarity and no luminous group in a complex system.

Therefore, there is a need in the art for suitable derivatizing reagents for derivatizing a monosaccharide of interest in a bodily fluid sample, thereby establishing a robust and reliable method for detecting one or more monosaccharides in a bodily fluid sample.

Disclosure of Invention

The object of the present invention is to provide a method for the detection of monosaccharides in a sample, such as a sample of body fluid, which method is stable and reliable and which is capable of detecting one or more monosaccharides in a sample.

In a first aspect, the present invention provides a method of detecting one or more monosaccharides in a sample, comprising the steps of:

1) sample pretreatment: derivatizing monosaccharide in a sample by using 1-phenyl-3-methyl-5-pyrazolone (PMP) as a monosaccharide derivatization reagent in the presence of Lewis base;

2) optionally neutralizing with a lewis acid after derivatizing the monosaccharide in the sample; and

3) analyzing the treated sample for one or more monosaccharides.

In particular embodiments, the lewis base comprises: triethylamine, diethylamine, ethylamine, ammonia water, ammonium bicarbonate, etc.; the Lewis acid includes: formic acid, acetic acid, oxalic acid, trifluoroacetic acid, heptafluorobutyric acid, and the like.

In specific embodiments, the lewis base is ammonium bicarbonate or triethylamine; the Lewis acid is formic acid.

In particular embodiments, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol, xylose, rhamnose, arabinose, N-glucose, N-galactose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, iduronic acid, ribose, etc.

In particular embodiments, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol.

In a preferred embodiment, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose.

In a preferred embodiment, the sample treated in step 1) is analyzed in step 3) for one or more monosaccharides using a combination of liquid chromatography-fluorescence detection, liquid chromatography-ultraviolet-visible light detection, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry, preferably liquid chromatography-mass spectrometry.

In preferred embodiments, the samples include, but are not limited to: food samples, plant samples, body fluid samples.

In preferred embodiments, the bodily fluid sample is a human bodily fluid sample; including but not limited to saliva, blood, urine, cerebrospinal fluid, interstitial fluid.

In a preferred embodiment, the body fluid sample is a human saliva sample or a human blood sample.

In a preferred embodiment, the monosaccharide is mannose or glucose, the lewis base is ammonium bicarbonate or ammonia, the lewis acid is formic acid or acetic acid; preferably ammonium bicarbonate/acetic acid or ammonia/acetic acid;

the monosaccharide is galactose, the Lewis base is ammonium bicarbonate, triethylamine or ammonia water, and the Lewis acid is formic acid or acetic acid; preferably ammonium bicarbonate/formic acid or triethylamine/formic acid;

the monosaccharide is fucose or 1, 5-anhydroglucitol, the Lewis base is ammonium bicarbonate or triethylamine, and the Lewis acid is formic acid; preferably ammonium bicarbonate/formic acid or triethylamine;

the monosaccharide is a combination of mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol; the Lewis base is ammonium bicarbonate or triethylamine; the lewis acid is formic acid.

In particular embodiments, step 1) is optionally preceded by a step of treating the sample to dissociate the bound sugars, and/or a step of treating the sample by protein precipitation and liquid-liquid extraction.

In a preferred embodiment, perchloric acid, concentrated sulfuric acid, trifluoroacetic acid, hydrochloric acid; the sample is preferably treated with perchloric acid to dissociate the bound sugars.

In a preferred embodiment, the conditions for centrifugation in the protein precipitation and liquid-liquid extraction processes are: centrifuging at 10000-12700 rpm for 3-5 minutes.

In a preferred embodiment, the step of dissociating the bound sugar is performed, followed by the steps of treating the sample with protein precipitation and liquid-liquid extraction.

In a preferred embodiment, PMP as the monosaccharide-derivatizing reagent is a 0.2-0.5% solution of PMP in methanol.

In a preferred embodiment, the volume ratio of the methanol solution of PMP to the sample is 1:1 to 2: 1.

In a preferred embodiment, the ratio of the Lewis acid and Lewis base to the sample is 1:1 to 1:3 by volume.

In a preferred embodiment, the method of the present invention further comprises making a qualitative judgment and a quantitative analysis based on the detection result of step 2).

In a preferred embodiment, the qualitative judgment and quantitative analysis is to judge the existence of the target monosaccharide according to the retention time of the target monosaccharide and the corresponding isotope internal standard substance and the abundance ratio of the qualitative and quantitative ion pair; and (3) quantifying by using an isotope internal standard method, establishing a standard curve by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of corresponding monosaccharide in human saliva or plasma according to the peak area ratio of the target monosaccharide to the corresponding internal standard substance on the internal standard curve.

In a specific embodiment, the monosaccharide detection sensitivity of the method is: 0.1 pg/ml-10 ng/ml; preferably 0.1pg/ml to 1 ng/ml; more preferably 0.1pg/ml to 100 pg/ml; more preferably 0.1pg/ml to 10 pg/ml; most preferably 0.1pg/ml to 1 pg/ml.

In a preferred embodiment, the method has the sensitivity of detecting mannose at 5-40 pg/mL, the sensitivity of detecting glucose at 5-30 pg/mL, the sensitivity of detecting galactose at 5-20 pg/mL, the sensitivity of detecting fucose at 0.5-5 pg/mL, and the sensitivity of detecting 1, 5-anhydroglucitol at 0.5-5 ng/mL; more preferably, the sensitivity for detecting mannose is 10-20 pg/mL, the sensitivity for detecting glucose is 5-15 pg/mL, the sensitivity for detecting galactose is 5-10 pg/mL, the sensitivity for detecting fucose is 0.5-1 pg/mL, and the sensitivity for detecting 1, 5-anhydroglucitol is 0.5-1 ng/mL.

In a preferred embodiment, the method of the invention has an in-batch precision of < 8%, preferably < 5%; the batch-to-batch precision is less than 8%; the monosaccharide recovery rate of the method is 85-110%; the CV of the method is 1-9%.

In a second aspect, the present invention provides a kit for detecting one or more monosaccharides in a sample, said kit comprising:

1) 1-phenyl-3-methyl-5-pyrazolone (PMP);

2) a lewis base comprising: triethylamine, diethylamine, ethylamine, ammonia water, ammonium bicarbonate, etc.;

3) optionally a Lewis acid, said Lewis acid comprising: formic acid, acetic acid, oxalic acid, trifluoroacetic acid, heptafluorobutyric acid, and the like; and

4) instructions for using the 1-phenyl-3-methyl-5-pyrazolone (PMP), the Lewis base, and the Lewis acid to derivatize one or more monosaccharides in the sample and to detect the derivatized one or more monosaccharides.

In a specific embodiment, the lewis base is ammonium bicarbonate or triethylamine and the lewis acid is formic acid.

In a preferred embodiment, the kit further comprises other necessary reagents for derivatizing one or more monosaccharides in the sample.

In a preferred embodiment, the kit further comprises a standard for the one or more monosaccharides.

In a preferred embodiment, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol, xylose, rhamnose, arabinose, N-glucose, N-galactose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, iduronic acid, ribose, etc.; more preferably, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol; more preferably, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose; most preferably, the monosaccharide is a combination of mannose, glucose, galactose, fucose and 1, 5-anhydroglucitol.

In a third aspect, the present invention provides an apparatus for detecting one or more monosaccharides in a sample, said apparatus comprising the detection kit of the second aspect.

In a preferred embodiment, the apparatus further comprises means for performing liquid chromatography-fluorescence detection, liquid chromatography-ultraviolet-visible light detection, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a total ion flux chromatogram of a target monosaccharide and an internal standard in a blank saliva matrix;

FIG. 2 shows a total ion flux chromatogram of a target monosaccharide and an internal standard in a saliva sample;

FIG. 3 shows a total ion flux chromatogram of a target monosaccharide and an internal standard in a blank plasma matrix;

FIG. 4 shows a total ion flux chromatogram for a monosaccharide of interest and an internal standard in a plasma sample;

FIGS. 5A-5E show the effect of different Lewis bases, Lewis acids, on the extraction efficiency of low and high concentrations of monosaccharides; wherein NH₄HCO₃Denotes ammonium hydrogen carbonate, NH₄OH represents ammonia, TEA represents triethylamine, FA represents formic acid, and HAc represents acetic acid; man watchD-mannose, Glc D-glucose, Gal D-galactose, Fuc L-fucose, 1,5-AG 1, 5-anhydroglucitol; the blue or left band in each pair of bands is low in concentration.

Detailed Description

The inventors have conducted extensive and intensive studies, and unexpectedly found that, after a monosaccharide in a body fluid sample is derivatized with a specific derivatization reagent in combination with a specific lewis base and/or lewis acid, separation and detection results of the monosaccharide can be significantly improved by combining with a detection analysis means applied to saccharide substances, thereby successfully applying to analysis of the monosaccharide and oligosaccharide. The present invention has been completed based on this finding.

The monosaccharide detection method of the invention

In clinical diagnosis, monosaccharides in body fluids are often associated with specific diseases, and screening or diagnosis of the associated diseases can be achieved by detecting various monosaccharides in body fluids, such as human saliva or plasma. However, monosaccharides have strong polarity and similar structure, do not have ultraviolet or fluorescent groups, lack optical activity, and cannot be directly detected by an ultraviolet detector and a fluorescent detector, so the detection mode is limited by the structure of monosaccharides. The chemical derivatization technology is a tool which is important for avoiding the problem, and meanwhile, the mass spectrum ionization efficiency of the monosaccharide can be enhanced and the matrix interference can be reduced through derivatization, so that the monosaccharide can be better detected.

However, there are many derivatization reagents suitable for high performance liquid pre-column derivatization of sugar substances, and an ideal derivatization reagent can improve detection sensitivity and does not pollute a subsequent mass spectrometry system. The inventor of the invention has conducted extensive and intensive research to determine that a specific derivatization reagent, namely 1-phenyl-3-methyl-5-pyrazolone (PMP), has a mild derivatization process, does not need catalysis, does not have stereoisomers in products, and after sugar is derivatized by PMP, the hydrophobicity of the sugar is improved, so that the sugar is applied to HPLC analysis of saccharides, can obtain better separation and detection results, and is successfully applied to monosaccharide analysis and oligosaccharide analysis.

Further, the present inventors have determined process conditions for derivatizing monosaccharides with PMP. According to the process conditions, the Lewis acid and the Lewis base are used, so that the separation degree and the detection sensitivity of the target substance are improved, the mass spectrum pollution is avoided, and the working efficiency is obviously improved.

Based on the teachings of the present invention, one skilled in the art will appreciate that the methods of the present invention are applicable not only to bodily fluid samples, but also to the detection of monosaccharides in food samples or plant samples.

The method realizes the simultaneous detection of various monosaccharides in human saliva or blood plasma, thereby being capable of promoting the screening or diagnosis and treatment of related diseases. The methods of the invention are equally applicable to other bodily fluid samples including, but not limited to, saliva, blood, urine, cerebrospinal fluid, interstitial fluid samples.

Based on the teachings of the present invention, one skilled in the art can determine monosaccharides suitable for processing and detection by the methods of the present invention. In particular embodiments, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol, xylose, rhamnose, arabinose, N-glucose, N-galactose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, iduronic acid, ribose. In a preferred embodiment, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose and 1, 5-anhydroglucitol. In a further preferred embodiment, the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose.

While PMP is used for derivatizing monosaccharide, the invention also combines ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to detect various monosaccharides in human saliva or plasma, thereby overcoming various defects in the prior art. Other specific monosaccharide detection methods may also be employed by those skilled in the art based on the teachings of the present invention, including but not limited to high performance liquid chromatography-fluorescence detection techniques, high performance liquid chromatography-ultraviolet-visible light detection techniques, gas chromatography-tandem mass spectrometry.

In a specific embodiment, the invention provides a method for detecting various monosaccharides in human saliva or plasma by pre-column derivatization and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), which comprises the following steps:

1) preparing a mixed monosaccharide standard product: taking a proper amount of D-mannose (D-mannose, Man), D-glucose (D-glucose, Glc), D-galactose (D-galactose, Gal), L-fucose (Fuc) and 1, 5-anhydroglucitol (1,5-Anhydro-D-glucitol,1,5-AG), co-dissolving in deionized water to obtain a mixed monosaccharide standard stock solution, and storing at 4 ℃ for later use;

2) preparation of stable isotope internal standard: taking a proper amount of L- [ UL-13C6] fuse (13C6-fuc), D-glucose-13C6(13C6-glc) and 1,5-anhydro-D- [ UL-13C6] glucitol (13C6-1,5-AG), dissolving in deionized water to obtain a mixed isotope internal standard stock solution, and storing at 4 ℃ for later use;

3) sample pretreatment:

respectively adding an internal standard into a sample to be detected and a mixed monosaccharide standard substance, adding methanol or acetonitrile after mixing, oscillating, mixing uniformly, centrifuging, taking supernatant, drying by using nitrogen (35-45 ℃), adding a certain volume of deionized water for redissolving, adding 0.2-0.5% PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution with the volume of 1-2 times, adding Lewis base with the volume ratio of 1: 1-1: 3, fully shaking, carrying out water bath derivatization at 60-100 ℃, taking out derivatives after 0.5-3 hours, cooling to room temperature, adding 1: 1-1: 3 Lewis acid with the volume ratio of 1: 1-1: 3, mixing uniformly, adding 1-5 times volume of chloroform for extraction, centrifuging, removing an organic layer, repeatedly extracting for 1-3 times, centrifuging to obtain an upper layer aqueous solution, taking supernatant, diluting 10-20 times by using methanol/water or acetonitrile/water, to be tested;

wherein the volume ratio of the internal standard solution to the human saliva or plasma sample is 1: 1-1: 4; the volume ratio of the human saliva or blood plasma, the methanol or acetonitrile and the double solvent is 1:3: 1-1: 5: 3; the volume ratio (V: V) of the diluent methanol/water or acetonitrile/water is 1: 9-3: 7.

The step is to pretreat a saliva or plasma sample, and to aim at the characteristic of strong polarity of monosaccharide compounds, the monosaccharide compounds are derived by adopting a PMP derivatization technology, so that the monosaccharide compounds have better separation selectivity. PMP can be subjected to condensation dehydration reaction with monosaccharide under mild alkaline conditions to quantitatively synthesize PMP-monosaccharide derivatives, and the product is stable and has no stereoisomerism.

While the monosaccharide in the sample is derivatized by PMP, a liquid-liquid extraction technology is adopted to enrich the target monosaccharide compound in the sample in the pretreatment process of the sample, and redundant PMP and other interfering substances are removed.

Since monosaccharides exist in body fluids in free form, they exist in a form that combines with proteins or lipids to form bound sugars. In the pretreatment process of the sample, the sample can be subjected to protein precipitation and liquid-liquid extraction so as to further remove some small molecular compounds and low molecular proteins or polypeptides in the body fluid sample, thereby achieving the purpose of enriching free monosaccharides and removing a large number of matrix interferents. The centrifugation condition in the pretreatment step is generally 10000-12700 rpm for 3-5 minutes.

In the sample treatment of the bound sugar, a reagent such as perchloric acid is added to the sample to dissociate the bound sugar, and then the other steps of the present invention are carried out to perform the analysis of the bound sugar.

4) UPLC-MS/MS quantitative analysis:

according to the chemical and physical characteristics of the PMP-monosaccharide derivative, performing separation analysis on the saliva or plasma monosaccharide derivatization treatment solution obtained in the step 3) and the mixed monosaccharide standard product derivatization solution by adopting an ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry to determine the composition and content of monosaccharide in saliva or plasma.

In a specific embodiment, the liquid chromatography conditions in the methods of the invention comprise:

c18 chromatographic column, 100mm × 2.1mm,1.7 μm; column temperature of the chromatographic column: 25-40 ℃; mobile phase composition: a, 2-10 mM ammonium formate or ammonium acetate aqueous solution containing 0.1-0.5% ammonia water; b, acetonitrile; flow rate: 0.4 mL/min; sample introduction volume: 5 mu L of the solution; gradient elution was used: the acetonitrile proportion is 15% in 0-1.5 min, the acetonitrile proportion rises to 25% after 0.1min, the acetonitrile proportion rises to 100% from 25% in 3.2-4.5 min, the initial mobile phase proportion returns after 1.0min, the initial mobile phase proportion is maintained for 1-2 min, and the time of the whole gradient is 5-8 min;

the mass spectrum detection conditions are as follows:

ion source and collision cell parameters: in electrospray ionization (ESI) positive and negative ion mode, a Multiple Reaction Monitoring (MRM) ion scanning mode is employed. The voltage of the positive ion mode capillary tube is 2.5-3.5 kV, and the voltage of the negative ion mode capillary tube is 1.0-2.5 kV; sampling taper hole voltage is 10-40V; the desolventizing gas is nitrogen, the flow rate of the desolventizing gas is 800-1000L/h, and the temperature of the desolventizing gas is 500-550 ℃; the collision gas is argon, and the collision energy is 10-30V.

The quantitative parent-child ion pair for simultaneously monitoring the PMP derivative of the target monosaccharide comprises: the mass-to-charge ratio of parent ions of the derivatives of mannose, glucose and galactose is 509.1-509.5, and the mass-to-charge ratios of corresponding daughter ions are 335.1-335.5 and 215.1-215.5; the mass-to-charge ratio of parent ions of the fucose and the 1, 5-anhydroglucitol derivative is 495.1-495.5, and the mass-to-charge ratios of corresponding daughter ions are 175.0-175.5 and 201.1-201.5; the mass-to-charge ratio of parent ions of 13C 6-glucose is 515.1-515.5, and the mass-to-charge ratios of corresponding daughter ions are 217.0-217.5 and 341.1-341.5; the mass-to-charge ratio of parent ions of the 13C 6-fucose is 501.1-501.5, and the mass-to-charge ratios of corresponding daughter ions are 219.0-219.5 and 175.1-175.5; the mass-to-charge ratio of parent ions of the 13C6-1, 5-anhydroglucitol is 501.1-501.5, and the mass-to-charge ratios of corresponding daughter ions are 203.0-203.5 and 175.1-175.5; wherein the internal standards of mannose, glucose and galactose are all 13C 6-glucose. Conditions such as sampling Cone Voltage (CV), Collision Energy (CE) and the like of each target compound are systematically optimized so as to achieve higher detection sensitivity and stability.

To remove the influence of matrix effects in the liquid chromatography, it is most effective to select an appropriate sample pretreatment method. According to the invention, the combination of protein precipitation, derivatization and liquid-liquid extraction is selected, 5 monosaccharides are simply and rapidly enriched, and the interference of proteins, polypeptides, fats and some small molecular substances is eliminated to a great extent; lewis base and acid are respectively adopted in a derivatization experiment, so that the response of a target compound is obviously improved, the matrix interference is reduced, and the pollution of a mass spectrometer is avoided. In addition, the method for improving the chromatographic analysis conditions is also the most important method for eliminating the matrix effect, and the invention selects a proper chromatographic column, a mobile phase composition and gradient elution conditions to effectively perform chromatographic separation on the target PMP-monosaccharide derivative and other substances in saliva or plasma.

5) Qualitative judgment and quantitative analysis

And (3) judging whether the monosaccharide exists in the saliva and plasma samples according to the retention time of the target monosaccharide and the PMP derivative of the stable isotope internal standard substance and the abundance ratio of the quantitative ion pair.

Performing qualitative analysis on the sample by using ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry: under the same experiment condition, the chromatographic retention time of the target substance to be detected in the sample is consistent with that of the corresponding substance in the standard solution; and (3) judging that the corresponding target compound exists in the sample if the deviation of the relative abundance of the selected detection ion pair in the total ion current chromatogram of the sample and the relative abundance ratio of the detection ion of the standard solution with the corresponding concentration does not exceed a specified range. And (3) calculating the monosaccharide content in the sample by using the peak area ratio of the PMP-monosaccharide derivative and the PMP-internal standard substance derivative thereof by using a stable isotope internal standard quantitative method.

The invention has the advantages that:

compared with the prior art, the invention has the following advantages:

1. the present invention can be applied to the detection of various samples, including saliva and plasma;

2. the invention can simultaneously detect a plurality of target objects in one analysis;

3. the method has high separation degree, and the ultra-high performance liquid chromatography instrument system is combined with the high resolution separation performance of the chromatographic column filled with the submicron particle packing, so that the structure and similar isomers or structural analogues thereof can be separated, and the detection accuracy is ensured;

4. the method has high flux, and can simultaneously and rapidly detect a plurality of monosaccharides in saliva or plasma within 5-8 min by high resolution and rapid separation of ultra-high performance liquid chromatography and combination of a serial four-stage rod MRM scanning technology;

5. the method has high sensitivity, eliminates a large amount of matrix interference by derivatization treatment, utilizes the high resolution separation capability of ultra-high performance liquid chromatography and mass spectrum MRM technology through two-stage ion selection, greatly reduces the chemical background of a total ion current chromatogram, obviously improves the signal-to-noise ratio of a target detection object, and even can detect several pg/mL trace monosaccharides in saliva or plasma;

6. the method has high accuracy and good reproducibility, and can detect monosaccharide substances meeting the set requirements by using the characteristic of high specificity of the MRM technology, so that the false positive rate in the analysis process is greatly reduced, and the data reproducibility is greatly improved by combining the UPLC high-resolution separation technology and the stable sample pretreatment technology. The method can further eliminate errors brought by the pretreatment and detection processes of the sample by adopting isotope internal standard quantification, thereby achieving accurate quantification;

7. the method of the invention not only improves the separation degree and the detection sensitivity of the target substance, but also avoids mass spectrum pollution and improves the working efficiency.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989) or in the general reference books or literature in the field of organic synthesis or according to the conditions recommended by the manufacturer.

Examples

Example 1.

1. Sample processing

Adding monosaccharide standard solution with appropriate concentration into blank saliva and blank saliva, respectively taking 50 mu L of saliva sample, respectively placing into 1.5mL centrifuge tubes, sequentially adding 50 mu L of internal standard mixed solution with appropriate concentration and 150 mu L of acetonitrile, mixing uniformly, centrifuging at 12700rpm for 5min, taking 50 mu L of supernatant, drying at 40 ℃ with nitrogen, adding 50 mu L of deionized water for redissolving, sequentially adding 0.3% PMP methanol solution and 100 mu L of triethylamine, mixing uniformly, placing in 70 ℃ water bath for derivatization for 2h, taking out, cooling to room temperature, adding 100 mu L of formic acid, mixing uniformly, extracting twice with 500 mu L of chloroform, respectively, discarding the organic layer, taking 50 mu L of supernatant, diluting with 950 mu L of 25% methanol solution, and storing at 4 ℃ for testing.

UPLC-MS/MS analysis

1) Chromatographic conditions are as follows:

chromatograph is Waters ACQUITY UPLC I-Class, and chromatographic column is C18 reversed phase chromatographic column ACQUITY UPLC BEH C18100 mm × 2.1mm,1.7 μm; the column temperature was 40 ℃; the mobile phase A is 5mM ammonium acetate aqueous solution containing 0.3% ammonia water; the mobile phase B is acetonitrile; the flow rate is 0.4 mL/min; the injection volume was 5. mu.L. The mobile phase gradient elution conditions are shown in table 1.

TABLE 1 gradient elution conditions

2) Mass spectrum conditions: the mass spectrometer is Waters Xevo TQ-S, ionization source: electrospray Ionization (ESI); an ionization mode: positive and negative ion modes; the scanning mode is as follows: multiple Reaction Monitoring (MRM) was used. Gas: the desolventizing gas is nitrogen, and the collision gas is argon. Capillary voltage: 3.0kV (+), 2.5kV (-); taper hole voltage: 10-40V; desolventizing air flow rate: 1000L/h; back blowing of the taper hole: 100L/h; desolventizing gas temperature: 500 ℃; the MRM ion pairs, collision energies and retention times of the 5 target monosaccharides and their isotopic internal standard PMP derivatives were monitored simultaneously and are detailed in table 2.

TABLE 2 LC-MS/MS parameters of PMP derivatives of five monosaccharides and isotopes

3. Method verification result

1) The specificity of the method: the total ion flow chromatogram of the target and internal standards in the blank saliva matrix spiked sample is shown in FIG. 1. As can be seen from the figure, the target substances to be detected in the saliva matrix sample are separated and have symmetrical peak shapes, and basically no interference peak exists, which indicates that the method has specificity and can be used for accurate quantitative analysis of monosaccharide in saliva.

2) Calibration curve: and (3) establishing a standard curve by adopting a stable isotope internal standard quantitative method and taking the concentration ratio of the monosaccharide standard substance to the internal standard substance as an X axis and the peak area ratio of the monosaccharide standard substance to the internal standard substance as a Y axis, and calculating the concentration of the target monosaccharide in the quality control substance or the saliva sample. Each curve was examined 3 times separately and the average, recovery and coefficient of variation for each curve point for 5 monosaccharides were calculated. The linear correlation coefficient of the linear fitting equation of the 5 monosaccharides in the respective concentration range is above 0.995, and the quantitative requirement is met. As can be seen from the results of the measurements in Table 3, the LOQ values of the 5 monosaccharides Man, Glc, Gal, Fuc and 1,5-AG obtained according to the protocol of the example were 0.43, 0.41, 0.02 and 3.6ng/mL, respectively, and the recovery rates were all within 85-110%.

TABLE 3.5 Linear regression equation, correlation coefficient, quantitative limit detection table of monosaccharides in saliva matrix

3) Accuracy and precision experiments:

blank saliva is taken, low, medium and high concentration monosaccharide mixed samples are respectively added, each concentration monosaccharide mixed sample is 3 parts, each concentration monosaccharide sample is subjected to single batch parallel determination for 15 times, and after the detection is carried out according to the method of the embodiment, the batch accuracy and precision of the detection result are calculated. The results are shown in Table 4.

Blank saliva is taken, low, medium and high concentration monosaccharide mixed samples are respectively added, each concentration monosaccharide mixed sample is 3 parts, each concentration monosaccharide sample is subjected to single batch parallel determination for 3 times, and after the detection is carried out according to the method of the embodiment, the batch accuracy and precision of the detection result are calculated.

The results are shown in Table 4, and it can be seen that the precision in each batch is less than 5% and the precision between batches is less than 8%.

TABLE 4 accuracy and precision test results in saliva base batch to batch

4) Recovery and matrix Effect test

Blank saliva is taken, low-concentration, medium-concentration and high-concentration monosaccharide mixed liquor is respectively added, each sample with each concentration level is 3 parts, each sample with each concentration level is subjected to single batch parallel measurement for 5 times, after the detection is carried out according to the method of the embodiment, the detection result is compared with the detection result of the standard solution with the same concentration, and the recovery rate and the matrix effect of the detection result are respectively calculated.

The results are shown in Table 5, the recovery rate of 5 monosaccharides is 92-105%, and CV of 5 repeated experiments is in the range of 2% -9%; the recovery rate of matrix effect is 94-110%, and CV of 5 times of repeated experiments is 1% -9%. The method of the example is shown to have better recovery and reproducibility.

TABLE 5 sample recovery of 5 monosaccharides in saliva matrix and matrix Effect

4. Saliva sample testing

Taking a saliva sample, obtaining a sample to be detected according to the sample pretreatment steps in the embodiment, and carrying out data acquisition on the processed saliva sample according to the UPLC-MS/MS analysis conditions in the embodiment. The detection results are shown in FIG. 2.

Example 2.

1. Sample pretreatment:

adding 50 mu L of a monosaccharide standard solution with a proper concentration into blank plasma, respectively taking a plasma sample, respectively placing the blank plasma and the plasma sample into 1.5mL centrifuge tubes, sequentially adding 50 mu L of an internal standard mixed solution with a proper concentration and 150 mu L of acetonitrile, uniformly mixing, centrifuging at 12700rpm for 5min, taking 50 mu L of supernatant, drying the supernatant in nitrogen at 40 ℃, adding 50 mu L of deionized water for redissolving, sequentially adding 100 mu L of a methanol solution of 0.3% PMP and triethylamine, uniformly mixing, placing the mixture in a water bath at 70 ℃ for derivatization for 2h, taking out the mixture, cooling the mixture to room temperature, then adding 100 mu L of formic acid, uniformly mixing, respectively extracting the mixture twice with 500 mu L of chloroform, discarding an organic layer, taking 50 mu L of supernatant, diluting with 950 mu L of a 25% methanol solution, and storing the supernatant at 4 ℃ for testing.

The detection method of UPLC-MS/MS is the same as that of example 1.

3. Method verification result

1) The specificity of the method: the total ion flow chromatogram of the target monosaccharide and the internal standard in the blank plasma matrix labeled sample is shown in FIG. 3. As can be seen from the figure, the separation resolution ratio of the target object to be detected in the plasma sample is high, the peak shape is symmetrical, and basically no interference peak exists, which indicates that the method has specificity and can be used for accurate quantitative analysis of monosaccharide in plasma.

2) Calibration curve: and (3) establishing a standard curve by adopting a stable isotope internal standard quantitative method and taking the concentration ratio of the monosaccharide standard substance to the internal standard substance as an X axis and the peak area ratio of the monosaccharide standard substance to the internal standard substance as a Y axis, and calculating the concentration of the target monosaccharide in the quality control substance or the saliva sample. Each curve was examined 3 times separately and the average, recovery and coefficient of variation for each curve point for 5 monosaccharides were calculated. The linear fitting equation of the 5 monosaccharides in the respective concentration range has a linear correlation coefficient of more than 0.995, and meets the quantitative requirement. As can be seen from the results of the measurements in Table 5, LOQ of the 5 monosaccharides Man, Glc, Gal, Fuc and 1,5-AG obtained according to the protocol of the example were 0.44, 0.38, 0.40, 0.02 and 3.24ng/mL, respectively, and the recovery rates were all within 85-110%.

TABLE 5.5 Linear regression equation, correlation coefficient, quantitation limit of monosaccharides in plasma matrix

3) Accuracy and precision experiments:

taking blank plasma, respectively adding 3 parts of monosaccharide mixed samples with low, medium and high concentration levels, carrying out single-batch parallel determination 15 times on each sample with the concentration level, and calculating the batch accuracy and precision of the detection result after detection according to the method of the embodiment.

Taking blank plasma, respectively adding low, medium and high concentration level monosaccharide mixed samples, wherein each concentration level sample is 3 parts, each concentration level sample is subjected to single batch parallel determination for 3 times, and after the detection is carried out according to the method of the embodiment, the batch accuracy and precision of the detection result are calculated.

The results are shown in Table 6, and it can be seen from the results that the precision in and between batches was < 8%.

TABLE 6 accuracy and precision test in plasma matrix batch to batch

4) Recovery and matrix Effect test

Taking blank plasma, adding 3 parts of monosaccharide derivative mixed solution with low, medium and high concentrations respectively, measuring each sample with 3 concentration levels in a single batch for 5 times in parallel, detecting according to the method of the embodiment, comparing with the measuring result of the standard solution with the same concentration, and respectively calculating the recovery rate and the matrix effect of the detection result.

The results are shown in Table 7, the recovery rate of 5 monosaccharides is 92-105%, and CV of 5 repeated experiments is in the range of 2% -9%; the recovery rate of matrix effect is 94-110%, and CV of 5 times of repeated experiments is 1% -9%. The method of the example is also shown to have better recovery rate and reproducibility when applied to plasma samples.

TABLE 7 sample recovery of five monosaccharides in plasma matrix and results of matrix effects

4. Plasma sample testing

Plasma samples were taken. The treatment was performed according to the method of the examples, and the treated plasma samples were subjected to data collection. The detection results are shown in FIG. 4.

Discussion of the related Art

The above examples 1-2 simultaneously detected 5 monosaccharides in human saliva or plasma by UPLC-MS/MS. The LC-MS/MS can simultaneously detect the peak-out time and ion pairs of various target objects, has high sensitivity, high separation degree and strong specificity, and can greatly avoid the interference of cross reaction. Meanwhile, the isotope internal standard method is adopted for quantification, so that the interference of the matrix can be further eliminated, the influence of condition changes such as a pretreatment process, a chromaticness instrument analysis process and the like can be counteracted, and the requirement of accurate quantification can be met.

In addition, in order to improve the performances of retention, separation, ionization efficiency and the like of the analyte, PMP and monosaccharide are subjected to condensation reaction to generate monosaccharide derivatives by utilizing a pre-column derivatization method, so that the separation performance and sensitivity of the target object are greatly improved, and the detection requirement of some trace monosaccharides in saliva or plasma can be met by combining with the UPLC-MS/MS method.

Comparative example 1.

In the study, the present inventors also derivatized monosaccharides in the sample with conventional derivatization reagents 2-AB, ABEE. 2-AB and ABEE reduces oligosaccharides or monosaccharides by a reductive amination process, and labeling occurs by the expected mechanism of open-chain carbohydrate production by protected and unprotected glucose derivatives, rather than by the formation of any heterocyclic materials.

However, comparative analysis shows that the 2-AB or ABEE derivatives of mannose, glucose and galactose have poor separation performance on a reversed phase chromatographic column or a HILIC chromatographic column, and the mannose, the glucose and the galactose are difficult to separate. In contrast, as shown in the above examples, PMP derivatives of these three monosaccharides showed enhanced retention and good separation resolution on the reverse phase chromatography column.

In addition, the 1,5-AG derivatives of 2-AB or ABEE are inferior in detection sensitivity to PMP derivatives and cannot satisfy the detection of 1,5-AG in a trace amount in plasma.

Therefore, even with the most suitable process conditions, the use of 2-AB or ABEE as derivatizing reagents still does not allow the detection of monosaccharides in body fluids.

Comparative example 2.

In the study, the present inventors also used the traditional PMP derivatization method to derivatize monosaccharides in a sample. However, the conventional PMP derivatization method generates a large amount of salt, thereby contaminating the mass spectrometry system and affecting the sensitivity of mass spectrometry detection. Therefore, an additional desalting process is required using the conventional PMP derivatization method.

In contrast, as shown in the above embodiments, the sample pretreatment method of the present invention can directly perform mass spectrometry without desalting treatment, which not only avoids contamination to the mass spectrometer, but also improves the detection sensitivity.

EXAMPLE 3 examination of Lewis acid base

According to the method described in example 1 or 2, the present inventors examined the effect of different lewis bases, lewis acids, on the extraction efficiency of monosaccharides using the low concentration and high concentration samples of example 1.

The results are shown in FIGS. 5A-5E, where it can be seen that:

for D-mannose, the extraction rate of ammonium bicarbonate/formic acid is reduced by about 10 percent compared with ammonia water/acetic acid;

for D-glucose, the extraction rates of ammonia water/formic acid, ammonium bicarbonate/acetic acid and ammonia water/acetic acid are not different greatly; the extraction rate of ammonium bicarbonate/formic acid is reduced by about 10 percent compared with that of ammonia water/acetic acid;

for D-galactose, the extraction rate of ammonium bicarbonate/formic acid or triethylamine/formic acid is improved by about 10 percent compared with that of ammonia water/acetic acid, and the extraction effect of only triethylamine is not much different from that of ammonia water/acetic acid;

for L-fucose, the extraction rate of ammonia water/formic acid is improved by 3 times compared with that of ammonia water/acetic acid, which shows that the effect of formic acid is better than that of acetic acid; the extraction rate of ammonium bicarbonate is improved by about 5 times compared with that of ammonia water/acetic acid; the extraction rate of ammonium bicarbonate/formic acid or triethylamine/formic acid is improved by about 6 times compared with that of ammonia water/acetic acid; the extraction rate of triethylamine is improved by about 8 times compared with that of ammonia water/acetic acid;

for 1, 5-anhydroglucitol, the extraction rate of ammonium bicarbonate/formic acid is improved by about 50 percent compared with that of ammonia water/acetic acid; in the extraction of the target substance of the low concentration sample, only triethylamine or ammonium bicarbonate is used, but ammonium bicarbonate/formic acid is preferred in consideration of the extraction effects of the low concentration sample and the medium concentration sample.

Summary and discussion of data:

aiming at the extraction of Man and Glc, comprehensively, Lewis base added in the derivatization reaction is ammonium bicarbonate or ammonia water, and Lewis acid is preferably formic acid or acetic acid; ammonium bicarbonate/acetic acid or ammonia/acetic acid are preferred.

Aiming at the extraction of Gal, in the derivatization reaction, compared with ammonia water/acetic acid, ammonium bicarbonate/formic acid or triethylamine/formic acid is added, and the extraction rate is improved by about 10 percent; therefore, the Lewis base is preferably ammonium bicarbonate, triethylamine or ammonia water, and the Lewis acid is preferably formic acid or acetic acid; ammonium bicarbonate/formic acid or triethylamine/formic acid are preferred.

Extraction for Fuc and 1, 5-AG: in the Fuc extraction process, the extraction efficiency of ammonia water/formic acid is improved by 3 times compared with that of ammonia water/acetic acid, which shows that the effect of formic acid is better than that of acetic acid; the extraction efficiency of ammonium bicarbonate is improved by about 5 times compared with that of ammonia water/acetic acid; the extraction efficiency of ammonium bicarbonate/formic acid or triethylamine/formic acid is improved by about 6 times compared with that of ammonia water/acetic acid, and the extraction efficiency of triethylamine is improved by about 8 times compared with that of ammonia water/acetic acid. Therefore, in the derivatization reaction aiming at the two compounds, the added Lewis base is ammonium bicarbonate or triethylamine, the added Lewis acid is formic acid, or only the Lewis base ammonium bicarbonate or triethylamine is selected; ammonium bicarbonate/formic acid or triethylamine is preferred. Furthermore, it can be seen from the figure that the extraction effect of the two target compounds during derivatization of low and high concentration samples using triethylamine is different.

Considering 5 monosaccharides in combination, the lewis base is preferably ammonium bicarbonate or triethylamine, and the lewis acid is preferably formic acid.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method of detecting one or more monosaccharides in a sample, comprising the steps of:

3) analyzing the treated sample for one or more monosaccharides.

2. The process of claim 1, wherein the lewis base comprises: triethylamine, diethylamine, ethylamine, ammonia water, ammonium bicarbonate, etc.; the Lewis acid includes: formic acid, acetic acid, oxalic acid, trifluoroacetic acid, heptafluorobutyric acid, and the like.

3. The process according to claim 2, characterized in that the lewis base is ammonium bicarbonate or triethylamine; the Lewis acid is formic acid.

4. The method of claim 1, wherein the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol, xylose, rhamnose, arabinose, N-glucose, N-galactose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, iduronic acid, ribose, etc.

5. The method of claim 4, wherein the monosaccharide is one or more selected from the group consisting of: mannose, glucose, galactose, fucose, 1, 5-anhydroglucitol.

6. The method of claim 1, further comprising, prior to step 1), optionally a step of treating the sample to dissociate bound sugars, and/or a step of treating the sample by protein precipitation and liquid-liquid extraction.

7. The method of any one of claims 1 to 6, wherein the monosaccharide detection sensitivity of the method is: 0.1 pg/ml-10 ng/ml; preferably 0.1pg/ml to 1 ng/ml; more preferably 0.1pg/ml to 100 pg/ml; more preferably 0.1pg/ml to 10 pg/ml; most preferably 0.1pg/ml to 1 pg/ml.

8. A kit for detecting one or more monosaccharides in a sample, comprising:

1) 1-phenyl-3-methyl-5-pyrazolone (PMP);

9. The kit of claim 8, wherein the lewis base is ammonium bicarbonate or triethylamine and the lewis acid is formic acid.

10. A device for detecting one or more monosaccharides in a sample, said device comprising a detection kit according to claim 8 or 9.