CN113203805A - Liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood - Google Patents
Liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood Download PDFInfo
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Abstract
The invention discloses a liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood, which comprises the following steps: preparing a standard substance mixed solution; preparing an internal standard substance extracting solution; adding the internal standard substance extracting solution into the standard substance mixed solution, and performing liquid chromatography tandem mass spectrometry detection; respectively establishing a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve according to the detection result of the liquid chromatography-tandem mass spectrometry; adding the internal standard substance extracting solution into a blood sample to be detected, and performing liquid chromatography tandem mass spectrometry detection; and respectively substituting the detection results of the blood sample to be detected into a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve to obtain the glucose concentration, the fructose concentration and the 1, 5-deoxyglucitol concentration in the blood sample to be detected. The invention can accurately realize the simultaneous quantitative detection of glucose, fructose and 1, 5-deoxyglucitol in blood with high flux and high sensitivity.
Description
Technical Field
The present invention relates to the field of analytical testing. More particularly, the invention relates to a liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood.
Background
Diabetes Mellitus (DM) has become the third killer of human after cardiovascular diseases and cancers, and is a global serious public health problem, blood sugar detection is one of important monitoring indexes of Diabetes mellitus, 1, 5-deoxyglucitol (1, 5-AG) in serum can reflect the blood sugar state for 2 days to 2 weeks, and serum 1, 5-AG is listed as an index for assisting blood sugar monitoring in 2015 Chinese blood sugar monitoring clinical application guide. In addition, more and more researches show that the monitoring of the fructose level is also extremely important, because the fat synthesis capability of human livers is obviously improved due to a large amount of fructose, so that obesity is caused, insulin resistance, non-alcoholic fatty liver disease and the like are easily caused by obesity, and the adverse effect on the whole body metabolism is obvious. However, only mass spectrometry for detecting 1, 5-AG and glucose in blood is available, but mass spectrometry for simultaneously monitoring three indexes of glucose, fructose and 1, 5-AG levels is not reported. Therefore, it is necessary to establish an accurate detection method which can simultaneously carry out glucose, fructose and 1, 5-deoxyglucitol.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to provide a liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood, which can accurately, highly flux and highly sensitively realize simultaneous quantitative detection of glucose, fructose and 1, 5-deoxyglucitol in blood.
To achieve these objects and other advantages in accordance with the present invention, there is provided a liquid chromatography tandem mass spectrometry method for simultaneously determining glucose, fructose, and 1, 5-deoxyglucitol in blood, comprising:
preparing a plurality of standard substance mixed liquids containing glucose, fructose and 1, 5-deoxyglucitol at the same time, wherein the glucose concentration in the plurality of standard substance mixed liquids is different, the fructose concentration in the plurality of standard substance mixed liquids is different, and the 1, 5-deoxyglucitol concentration in the plurality of standard substance mixed liquids is different;
is prepared by13C6Glucose, glucose,13C3Fructose and13C6extracting solution of an internal standard substance of 1, 5-deoxyglucitol;
respectively adding the internal standard substance extracting solution into the plurality of standard substance mixed solutions, and respectively carrying out liquid chromatography tandem mass spectrometry on the plurality of standard substance mixed solutions mixed with the internal standard substance extracting solution;
respectively establishing a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve according to the detection result of liquid chromatography tandem mass spectrometry of a plurality of standard substance mixed solutions mixed with an internal standard substance extracting solution;
adding the internal standard substance extracting solution into the blood sample to be detected, and performing liquid chromatography tandem mass spectrometry on the blood sample to be detected mixed with the internal standard substance extracting solution;
and respectively substituting the detection results of the liquid chromatography tandem mass spectrometry of the blood sample to be detected mixed with the internal standard substance extracting solution into a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve to obtain the glucose concentration, the fructose concentration and the 1, 5-deoxyglucitol concentration in the blood sample to be detected.
Preferably, the standard substance mixed solution mixed with the internal standard substance extracting solution is subjected to vortex and centrifugation, then the supernatant is taken for liquid chromatography tandem mass spectrometry, the blood sample to be detected mixed with the internal standard substance extracting solution is subjected to vortex and centrifugation, and then the supernatant is taken for liquid chromatography tandem mass spectrometry.
Preferably, the vortex time is 1-5 min, the centrifugal rotating speed is 14000-16000 rpm, the centrifugal temperature is 1-5 ℃, and the centrifugal time is 8-12 min.
Preferably, the equipment used for the detection of the liquid chromatography tandem mass spectrometry is an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer.
Preferably, the chromatographic separation conditions adopted in the detection process of the liquid chromatography-tandem mass spectrometry are as follows: the chromatographic column is ACQUITY
BEH Amide 1.7 μm, 3.0 × 150mm, flow rate of 0.3mL/min, column temperature of 35 deg.C; isocratic elution, wherein the mobile phase is 74.95-84.85% acetonitrile water solution containing 0.05-0.15% ammonia water.
Preferably, the mass spectrum conditions adopted in the detection process of the liquid chromatography tandem mass spectrum are as follows: ESI negative ion mode, ion source temperature 350 ℃, atomizer pressure 45psi, auxiliary pressure 50psi, air curtain pressure 40psi, spray voltage-4500V, multiple reaction monitoring mode.
Preferably, the parent ion-daughter ion pairs for mass spectrometry in the detection process of liquid chromatography tandem mass spectrometry are: glucose, m/z 178.9-m/z 59.01; fructose, m/z 178.9-m/z 59.01; 1, 5-deoxyglucitol, m/z 162.9-m/z 101;13C6glucose, m/z 184.9-m/z 92;13C3fructose, m/z 181.9-m/z 92;13 C 61, 5-deoxyglucitol, m/z 168.9-m/z 105.
Preferably, the concentration range of glucose in the standard substance mixed solution is 5-500000 ng/mL, the concentration range of fructose in the standard substance mixed solution is 5-500000 ng/mL, and the concentration range of 1, 5-deoxyglucitol in the standard substance mixed solution is 5-500000 ng/mL.
Preferably, the volume ratio of the internal standard substance extracting solution to the blood sample to be detected is (1-4): 1.
preferably, the preparation method of the standard substance mixture comprises:
respectively preparing glucose standard product mother liquor, fructose standard product mother liquor and 1, 5-deoxyglucitol standard product mother liquor with specific concentrations, mixing the glucose standard product mother liquor, the fructose standard product mother liquor and the 1, 5-deoxyglucitol standard product mother liquor, and diluting step by step to prepare a plurality of standard product mixed liquor, wherein the diluent adopts a phosphate buffer solution of bovine serum albumin with the mass volume fraction of 1%;
the preparation method of the internal standard substance extracting solution comprises the following steps: respectively prepared at specific concentrations13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6taking mother liquor of 1, 5-deoxyglucitol internal standard substance13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6mixing the 1, 5-deoxyglucitol internal standard substance mother liquor, diluting the mixed liquor step by step to a preset concentration to prepare an internal standard substance mixed liquor, wherein the diluent adopts 74.95-84.85% acetonitrile aqueous solution containing 0.05-0.15% ammonia water, and then mixing the internal standard substance mixed liquor with the same diluent according to a preset volume ratio to prepare an internal standard substance extracting solution.
The invention at least comprises the following beneficial effects: the invention successfully establishes the mass spectrum method for simultaneously detecting the glucose, the fructose and the 1, 5-AG in the blood by the liquid chromatography-tandem mass spectrum for the first time, the serum can be analyzed on a computer by simple extraction, the detection process is simple, convenient and quick, and the pretreatment of the sample to be detected in the method is simple, the derivation is not needed, and the serum dosage is less.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a chromatogram of a mixed solution 4 of a standard substance according to the present invention;
FIG. 2 is a standard curve of glucose, fructose and 1, 5-deoxyglucitol according to the present invention, FIG. 2a is a standard curve of 1, 5-deoxyglucitol, FIG. 2b is a standard curve of glucose, and FIG. 2c is a standard curve of fructose.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example >
The invention provides a liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood, which comprises the following steps:
step A, preparing a plurality of standard substance mixed liquids simultaneously containing glucose, fructose and 1, 5-deoxyglucitol, wherein the concentrations of the glucose in the plurality of standard substance mixed liquids are different, the concentrations of the fructose in the plurality of standard substance mixed liquids are different, and the concentrations of the 1, 5-deoxyglucitol in the plurality of standard substance mixed liquids are different;
specifically, glucose standard product mother liquor, fructose standard product mother liquor and 1, 5-deoxyglucitol standard product mother liquor with specific concentrations are respectively prepared, the glucose standard product mother liquor, the fructose standard product mother liquor and the 1, 5-deoxyglucitol standard product mother liquor are mixed and then diluted step by step to prepare a plurality of standard product mixed liquor, and the diluent adopts phosphate buffer solution of bovine serum albumin with the mass volume fraction of 1%;
more specifically, the concentration of glucose in the mother liquor of the glucose standard product was 1mg/mL, the concentration of fructose in the mother liquor of the fructose standard product was 1mg/mL, the concentration of 1, 5-deoxyglucitol in the mother liquor of the 1, 5-deoxyglucitol standard product was 100. mu.g/mL, the ratio of 1: 1: after 1 volume ratio gets glucose standard substance mother liquor, fructose standard substance mother liquor and 1, 5-deoxyglucose alcohol standard substance mother liquor and mixes (here get standard substance mother liquor according to volume ratio and do not restrict the standard substance mother liquor quantity, but in order to make the proportion that three kinds of ingredient concentration ratio in the standard substance mixed liquor are close to in the human blood, can adjust according to actual demand), use the phosphate buffer solution of bovine serum albumin that the quality volume fraction is 1% to dilute step by step, prepare 8 standard substance mixed liquor 1 ~ 8, as shown in table 1:
TABLE 1
Step B, preparing the mixture simultaneously13C6Glucose, glucose,13C3Fructose and13C6extracting solution of an internal standard substance of 1, 5-deoxyglucitol;
specifically, the specific concentrations are respectively prepared13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6taking mother liquor of 1, 5-deoxyglucitol internal standard substance13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6mixing the 1, 5-deoxyglucitol internal standard substance mother liquor, diluting the mixed liquor to a preset concentration step by step (the preset concentration can be adjusted according to actual requirements), preparing an internal standard substance mixed liquor, mixing the internal standard substance mixed liquor and the same diluent according to a preset volume ratio, wherein the diluent adopts 74.95-84.85% acetonitrile aqueous solution containing 0.05-0.15% ammonia water;
more specifically, the present invention is to provide a novel,13C6in mother liquor of glucose internal standard substance13C6The concentration of glucose is 1mg/mL,13C3in the internal standard substance mother liquor of fructose13C3The concentration of the fructose is 1mg/mL,13 C 61, 5-deoxyglucitol internal standard substance mother liquor13C6The concentration of 1, 5-deoxyglucitol was 100. mu.g/mL, as 1: 1: 1 volume ratio of13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6after the 1, 5-deoxyglucitol internal standard substance mother liquor is mixed (the internal standard substance mother liquor is taken according to the volume ratio, the use amount of the internal standard substance mother liquor is not limited, but can be adjusted according to actual requirements in order to enable the concentration ratio of three components in the internal standard substance mixed liquor to be close to the proportion in human blood), 0.1 percent ammonia water is usedDiluting the 75 percent acetonitrile water solution step by step to13C6The concentration of glucose is 10000ng/mL,13C3The concentration of the fructose is 10000ng/mL,13C6The concentration of 1, 5-deoxyglucitol is 1000ng/mL, and the internal standard substance mixed solution and 75% acetonitrile aqueous solution containing 0.1% ammonia water are mixed according to the proportion of 2: 13 (the volume ratio can be adjusted according to actual requirements) to prepare the internal standard substance extracting solution.
Step C, respectively adding the internal standard substance extracting solution into the plurality of standard substance mixed solutions, and respectively carrying out liquid chromatography tandem mass spectrometry on the plurality of standard substance mixed solutions mixed with the internal standard substance extracting solution;
specifically, the standard substance mixed solution mixed with the internal standard substance extracting solution is firstly subjected to vortex and centrifugation, and then the supernatant is taken to be subjected to liquid chromatography tandem mass spectrometry detection;
specifically, the vortex time is 1-5 min, the centrifugal rotation speed is 14000-16000 rpm, the centrifugal temperature is 1-5 ℃, and the centrifugal time is 8-12 min;
specifically, the equipment used for the detection of the liquid chromatography tandem mass spectrometry is an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer;
more specifically, the method comprises the following steps:
(1) transferring 100 mu L of standard substance mixed solution 1 into a 1.5mL centrifuge tube, adding 300 mu L of internal standard substance extracting solution, carrying out vortex mixing for 5min, centrifuging for 10min at the temperature of 4 ℃ and the speed of 15000rpm by using a centrifuge, transferring supernatant to a 96-hole microporous plate, putting the 96-hole microporous plate into an automatic sample injector of a liquid chromatography tandem mass spectrometry system, opening application software, calling a designed acquisition file, establishing a sample list, and carrying out on-machine detection;
(2) respectively carrying out on-machine detection on the standard substance mixed liquid 2-8 according to the step (1);
more specifically, the chromatographic separation conditions adopted in the detection process of the liquid chromatography-tandem mass spectrometry are as follows: the chromatographic column is ACQUITY
BEH Amide 1.7 μm, 3.0 × 150mm, flow rate of 0.3mL/min, column temperature of 35 deg.C; isocratic elution with a mobile phase of75% acetonitrile in water containing 0.1% ammonia; in the elution process, the retention time of 1, 5-deoxyglucitol is 4.87min, the retention time of fructose is 5.85min, the retention time of glucose is 6.56min, the total chromatographic time is 8min, and the typical spectrogram obtained by detection is shown in figure 1.
More specifically, the mass spectrum conditions adopted in the detection process of the liquid chromatography tandem mass spectrum are as follows: ESI negative ion mode, ion source temperature 350 ℃, atomizer air pressure 45psi, auxiliary air pressure 50psi, air curtain air pressure 40psi, spray voltage-4500V, using multi-reaction monitoring mode, specific quantitative parent ion-daughter ion pairs and corresponding collision energy as shown in Table 2:
TABLE 2
Step D, respectively establishing a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve according to the detection result of the liquid chromatography tandem mass spectrometry of the mixed solution of the plurality of standard products mixed with the internal standard substance extracting solution;
specifically, a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve are respectively obtained by using data processing software of a liquid chromatography-tandem mass spectrometry system, as shown in fig. 2:
FIG. 2a is a graph showing a 1, 5-deoxyglucitol standard curve in which the abscissa represents the concentration of 1, 5-deoxyglucitol in the mixed solution of the standard substance and the ordinate represents the signal intensity and signal intensity of the 1, 5-deoxyglucitol standard substance13C6The ratio of signal intensity of 1, 5-deoxyglucitol internal standard substance, 1, 5-deoxyglucitol standard curve equation is y-0.01438 x +0.01158 (r-0.99994, r2＝0.99989)；
FIG. 2b is a glucose standard curve, in which the abscissa represents the glucose concentration in the mixed solution of the standard substance and the ordinate represents the signal intensity of the glucose standard substance and13C6the ratio of the signal intensity of the glucose internal standard substance and the standard curve equation of glucose are that y is 4.86952 multiplied by 10-4x-0.00642(r＝0.99969，r2＝0.99938)；
FIG. 2c is a standard fructose curve, in which the abscissa is the fructose concentration in the mixed solution of the standard substance and the ordinate is the signal intensity of the standard fructose and13C3the ratio of the signal intensity of fructose internal standard substance and the standard curve equation of fructose are that y is 0.00127x +0.01118(r is 0.99991, r2＝0.99982)。
Step E, adding the internal standard substance extracting solution into the blood sample to be detected, and then carrying out liquid chromatography tandem mass spectrometry on the blood sample to be detected mixed with the internal standard substance extracting solution;
specifically, a blood sample to be detected mixed with an internal standard substance extracting solution is firstly subjected to vortex and centrifugation, and then supernatant is taken to be subjected to liquid chromatography tandem mass spectrometry detection;
specifically, the vortex time is 1-5 min, the centrifugal rotation speed is 14000-16000 rpm, the centrifugal temperature is 1-5 ℃, and the centrifugal time is 8-12 min;
specifically, the equipment used for the detection of the liquid chromatography tandem mass spectrometry is an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer;
more specifically, the method comprises the following steps:
transferring 100 mu L of a blood sample to be detected into a 1.5mL centrifuge tube, adding 300 mu L of an internal standard substance extracting solution, carrying out vortex mixing for 5min, centrifuging for 10min at the temperature of 4 ℃ and the speed of 15000rpm by using a centrifuge, transferring supernatant to a 96-hole microporous plate, putting the 96-hole microporous plate into an automatic sample injector of a liquid chromatography tandem mass spectrometry system, opening application software, calling a designed acquisition file, establishing a sample list, and carrying out on-machine detection;
more specifically, the chromatographic separation conditions adopted in the detection process of the liquid chromatography-tandem mass spectrometry are as follows: the chromatographic column is ACQUITY
BEH Amide 1.7 μm, 3.0 × 150mm, flow rate of 0.3mL/min, column temperature of 35 deg.C; isocratic elution, the mobile phase is 75% acetonitrile water solution containing 0.1% ammonia water.
More specifically, the mass spectrum conditions adopted in the detection process of the liquid chromatography tandem mass spectrum are as follows: ESI negative ion mode, ion source temperature 350 ℃, atomizer pressure 45psi, auxiliary pressure 50psi, gas curtain pressure 40psi, spray voltage-4500V, using multi-reaction monitoring mode, specific quantitative parent ion-daughter ion pairs and corresponding collision energy are shown in Table 2.
And F, respectively substituting the liquid chromatogram tandem mass spectrum detection result of the blood sample to be detected mixed with the internal standard substance extracting solution into the glucose standard curve, the fructose standard curve and the 1, 5-deoxyglucitol standard curve to obtain the glucose concentration, the fructose concentration and the 1, 5-deoxyglucitol concentration in the blood sample to be detected.
Specifically, the intensity of a glucose signal obtained by detecting a blood sample to be detected mixed with an internal standard substance extracting solution by liquid chromatography-tandem mass spectrometry is compared with the intensity of the glucose signal obtained by detecting the internal standard substance extracting solution by liquid chromatography-tandem mass spectrometry13C6Substituting the ratio of the signal intensity of the glucose internal standard substance into the standard curve in the graph 2b, and calculating the glucose concentration in the blood sample to be detected;
detecting fructose signal intensity and fructose signal intensity obtained by liquid chromatography tandem mass spectrometry of blood sample to be detected mixed with internal standard substance extracting solution13C3Substituting the ratio of the fructose internal standard substance signal intensity into the standard curve in fig. 2c, and calculating the fructose concentration in the blood sample to be detected;
detecting the signal intensity and the signal intensity of the 1, 5-deoxyglucitol obtained by the liquid chromatography tandem mass spectrometry of the blood sample to be detected mixed with the internal standard substance extracting solution13C6The ratio of the signal intensity of the 1, 5-deoxyglucitol internal standard substance is substituted into the standard curve in fig. 2a, and the concentration of 1, 5-deoxyglucitol in the blood sample to be tested can be calculated.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. The liquid chromatography tandem mass spectrometry method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol in blood is characterized by comprising the following steps:
preparing a plurality of standard substance mixed liquids containing glucose, fructose and 1, 5-deoxyglucitol at the same time, wherein the glucose concentration in the plurality of standard substance mixed liquids is different, the fructose concentration in the plurality of standard substance mixed liquids is different, and the 1, 5-deoxyglucitol concentration in the plurality of standard substance mixed liquids is different;
is prepared by13C6Glucose, glucose,13C3Fructose and13C6extracting solution of an internal standard substance of 1, 5-deoxyglucitol;
respectively adding the internal standard substance extracting solution into the plurality of standard substance mixed solutions, and respectively carrying out liquid chromatography tandem mass spectrometry on the plurality of standard substance mixed solutions mixed with the internal standard substance extracting solution;
respectively establishing a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve according to the detection result of liquid chromatography tandem mass spectrometry of a plurality of standard substance mixed solutions mixed with an internal standard substance extracting solution;
adding the internal standard substance extracting solution into the blood sample to be detected, and performing liquid chromatography tandem mass spectrometry on the blood sample to be detected mixed with the internal standard substance extracting solution;
and respectively substituting the detection results of the liquid chromatography tandem mass spectrometry of the blood sample to be detected mixed with the internal standard substance extracting solution into a glucose standard curve, a fructose standard curve and a 1, 5-deoxyglucitol standard curve to obtain the glucose concentration, the fructose concentration and the 1, 5-deoxyglucitol concentration in the blood sample to be detected.
2. The method according to claim 1, wherein the standard mixture mixed with the internal standard extract is subjected to vortexing and centrifugation, and then the supernatant is taken for the LC-MS/MS detection, and the blood sample to be tested mixed with the internal standard extract is subjected to vortexing and centrifugation, and then the supernatant is taken for the LC-MS/MS detection.
3. The method according to claim 2, wherein the vortexing time is 1-5 min, the centrifugation speed is 14000-16000 rpm, the centrifugation temperature is 1-5 ℃, and the centrifugation time is 8-12 min.
4. The method of claim 1, wherein the apparatus for simultaneous determination of glucose, fructose and 1, 5-deoxyglucitol in blood is an ultra high performance liquid chromatography triple quadrupole tandem mass spectrometer.
5. The method of claim 1, wherein the chromatographic separation conditions used in the tandem mass spectrometry detection are: the chromatographic column is ACQUITY
BEH Amide 1.7 μm, 3.0 × 150mm, flow rate of 0.3mL/min, column temperature of 35 deg.C; isocratic elution, wherein the mobile phase is 74.95-84.85% acetonitrile water solution containing 0.05-0.15% ammonia water.
6. The method of claim 1, wherein the mass spectrometric conditions used during the simultaneous determination of glucose, fructose and 1, 5-deoxyglucitol in blood are: ESI negative ion mode, ion source temperature 350 ℃, atomizer pressure 45psi, auxiliary pressure 50psi, air curtain pressure 40psi, spray voltage-4500V, multiple reaction monitoring mode.
7. The method of claim 1, wherein the parent ion-daughter ion pair of mass spectrometry used in the detection of the liquid chromatography tandem mass spectrometry is: glucose, m/z 178.9-m/z 59.01; the fructose is used as the raw material of the fruit sugar,m/z 178.9-m/z 59.01; 1, 5-deoxyglucitol, m/z 162.9-m/z 101;13C6glucose, m/z 184.9-m/z 92;13C3fructose, m/z 181.9-m/z 92;13C61, 5-deoxyglucitol, m/z 168.9-m/z 105.
8. The method according to claim 1, wherein the concentration of glucose in the mixture of standard substance is 5-500000 ng/mL, the concentration of fructose in the mixture of standard substance is 5-500000 ng/mL, and the concentration of 1, 5-deoxyglucitol in the mixture of standard substance is 5-500000 ng/mL.
9. The method for simultaneously measuring glucose, fructose and 1, 5-deoxyglucitol according to claim 1, wherein the volume ratio of the internal standard substance extract to the blood sample to be measured is (1-4): 1.
10. the method of claim 1, wherein the method comprises the steps of:
respectively preparing glucose standard product mother liquor, fructose standard product mother liquor and 1, 5-deoxyglucitol standard product mother liquor with specific concentrations, mixing the glucose standard product mother liquor, the fructose standard product mother liquor and the 1, 5-deoxyglucitol standard product mother liquor, and diluting step by step to prepare a plurality of standard product mixed liquor, wherein the diluent adopts a phosphate buffer solution of bovine serum albumin with the mass volume fraction of 1%;
the preparation method of the internal standard substance extracting solution comprises the following steps: respectively prepared at specific concentrations13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6taking mother liquor of 1, 5-deoxyglucitol internal standard substance13C6Mother liquor of glucose internal standard substance,13C3Internal standard substance mother liquor of fructose and13C6mixing the 1, 5-deoxyglucitol internal standard substance mother liquor, diluting the mixed liquor step by step to a preset concentration to prepare an internal standard substance mixed liquor, wherein the diluent adopts 74.95-84.85% acetonitrile aqueous solution containing 0.05-0.15% ammonia water, and then mixing the internal standard substance mixed liquor with the same diluent according to a preset volume ratio to prepare an internal standard substance extracting solution.
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| CA2291912A1 (en) * | 1998-12-11 | 2000-06-11 | Kyowa Medex Co., Ltd. | Method and reagent for quantitative determination of 1,5-anhydroglucitol |
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