CN115901909A - Method for quantitatively detecting myoinositol in blood - Google Patents
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Abstract
The invention discloses a method for quantitatively detecting myoinositol in blood, which adopts an electrochemical method for detection and specifically comprises the following steps: a. preparing a series of working solutions with standard concentrations; b. preparing a blank sample solution; c. respectively sucking blank sample solution and series concentration standard working solutions, and drawing a standard curve by adopting a Differential Pulse Voltammetry (DPV) method; d. preparing a sample solution to be detected; e. and (3) measuring the content of myoinositol in the sample to be measured. The experiment proves that the method has accurate and reliable result and can be used for clinical blood detection.
Description
Technical Field
The invention relates to a method for quantitatively detecting myoinositol in blood.
Background
Myoinositol (Myo-inositol), also known as hexahydroxycyclohexane, is a glucose-derived polyol. Myoinositol plays a crucial role in regulating a variety of cellular functions, and is the structural basis of secondary messengers in eukaryotic cells, particularly as precursors to inositol phosphates, phosphatidylinositols, and phosphatidylinositol phosphate lipids. It therefore contributes to important functional and structural roles including cell growth and survival, osteogenesis, reproduction, development of the nervous system. Dysmyoinositol metabolism has been implicated in the development of several disease states, such as neural tube defects, down's syndrome, alzheimer's disease and diabetic glomerular disease. Previous studies have shown that myoinositol has chemopreventive and chemotherapeutic properties in human cancer cells and animal cancer models, including prostate, breast, colon, pancreatic, liver and lung cancer. Meanwhile, myoinositol has been tested as a pharmacological intervention for a variety of conditions, including depression, panic disorder, obsessive compulsive disorder, and the like. Therefore, the potential application of myoinositol as a pharmacological agent and a metabolic marker requires the establishment of a method for measuring myoinositol in serum.
Myoinositol in blood is maintained at a concentration by incorporation into cells or excretion, resorption and renal oxidation, with normal serum myoinositol concentrations at 18-41umol/L. Currently, various analytical methods have been developed to detect myoinositol, including gas chromatography-mass spectrometry (GC/MS), high Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (HPLC/MS), liquid chromatography pulsed amperometric detection, and enzyme cycling detection. Most of these detection techniques are generally complex and time consuming, expensive instruments, bulky, and technically demanding for the operator. Furthermore they require cumbersome sample pre-treatment and are therefore limited in application. Compared with the instruments and methods, the electrochemical method has the characteristics of simple and convenient operation, small sample consumption, high sensitivity, easy miniaturization and the like, shows wide application prospect in the aspect of ultrasensitive detection of small molecular compounds, becomes a new hotspot of methodology research, but no report of applying electrochemistry to the content detection of myoinositol in blood exists at present.
Disclosure of Invention
In order to solve the problems, the invention provides a method for quantitatively detecting myoinositol in blood, which adopts an electrochemical method to detect myoinositol in blood.
Further, it includes measuring the response current value of myoinositol solution, blank serum solution and blood sample solution to be tested by differential pulse voltammetry (DPV method), and calculating the myoinositol content in the blood sample according to the myoinositol standard curve established by the concentration of myoinositol solution and blank serum solution and the corresponding current value.
Furthermore, the method specifically comprises the following steps:
(1) Establishment of standard curve of myoinositol
a. Preparation of standard working solutions of a series of concentrations: dissolving myoinositol in water, diluting to obtain series concentration standard solutions, respectively collecting series concentration standard solutions, adding blank serum, mixing, adding acetonitrile, centrifuging, collecting supernatant, drying, and dissolving residue in glycine-NaOH buffer solution to obtain series concentration standard working solutions;
b. preparation of blank sample solution: taking blank serum, adding acetonitrile, mixing uniformly, centrifuging, taking supernatant, drying, and dissolving residues in glycine-NaOH buffer solution to obtain blank sample solution;
c. respectively sucking a blank sample solution and a series of concentration standard working solutions, adding an oxidized nicotinamide adenine dinucleotide solution (NAD +) and myoinositol, uniformly mixing the oxidized nicotinamide adenine dinucleotide solution (NAD +) and the myoinositol through an inositol dehydrogenase solution (IDH), reacting for 10-30 min, taking a reaction mixed solution, attaching the reaction mixed solution to a screen printing carbon electrode, accessing an electrochemical workstation, measuring a response current value by adopting a Differential Pulse Voltammetry (DPV) method, and drawing a myoinositol standard curve by taking the concentration of the myoinositol as an X axis and the difference value of the response current values of the series of concentration standard working solutions and the blank sample solution as a Y axis;
(2) Measuring the content of myoinositol in the sample to be tested:
d. preparation of sample solution to be tested
B, taking a serum sample, and preparing by the same method in the step b to obtain a sample solution to be detected;
e. determination of myoinositol content in sample to be tested
And (c) taking a sample solution to be detected, measuring the response current value by the same method of the step c, and calculating the myoinositol content in the sample to be detected according to the standard curve of the step (1).
Further, the volume ratio of the series of concentration standard solutions, the blank serum and the acetonitrile in the step a is 1:9:40.
further, the concentration of the series of concentration standard solutions is 50-5000.0 μmmol/L, preferably 50.0 μmmol/L,100.0 μmmol/L,200.0 μmmol/L,500.0 μmmol/L,1000.0 μmmol/L,2000.0 μmmol/L,4000.0 μmmol/L and/or 5000.0 μmmol/L.
Further, the volume ratio of the blank serum and the acetonitrile in the step b is 9-13: 30 to 50, preferably 10:40.
further, the blending in the step a and the step b is vortex blending; the centrifugation temperature is 2-8 ℃, the rotation speed is 10000-20000 rpm/min, the time is 8-15 min, the preferred centrifugation temperature is 4 ℃, the rotation speed is 13300rpm/min, and the time is 12min.
Further, the drying in the step a and the step b is nitrogen flow drying; the temperature of the nitrogen stream drying is 60-100 ℃, preferably 80 ℃.
Further, the glycine-NaOH buffer solution added in the step a and the step b is 1/10 to 1/15, preferably 1/12 of the volume of the acetonitrile.
Further, the pH value of the glycine-NaOH buffer solution is 9-11, wherein the glycine content is 500-600 mmol/L, preferably 10.3, and the glycine content is 550mmol/L.
Further, the blank sample solution or the working solution of the series of concentrations of the standard in step c is mixed with NAD + The volume ratio of the solution to the IDH solution is 25-45: 3 to 7:3 to 7, preferably 40:5:5.
further, the NAD + The concentration of the solution is 100-200 mmol/L, the concentration of the IDH solution is 4000-5000U/L, and NAD is preferred + The concentration of the solution is 160mmol/L, and the concentration of the IDH solution is 4500U/L.
Further, the reaction temperature in the step c is 20-30 ℃ and the reaction time is 20min.
Furthermore, the volume of the reaction mixture attached to the screen-printed carbon electrode in step c is 20 to 60. Mu.L, preferably 40. Mu.L.
Furthermore, the parameters of the DPV method in the step c are voltage 0.1-0.73V, increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s.
Compared with the existing myoinositol analysis and detection method, the method for quantitatively detecting myoinositol in blood has the advantages of simple and quick preparation method, good stability, low detection cost, suitability for field detection and the like, and can be used for detecting myoinositol in blood serum.
The method has the advantages of high sensitivity, high analysis speed, wide linear range, good reproducibility and strong anti-interference capability, the detection limit is as low as 1.0 mu M (S/N = 3), the quantification limit is 2.5 mu M (S/N = 10), and the analysis and detection range is 5.0-500.0 mu mol/L. Three working solutions, namely low (10.0 mu mol/L), medium (100.0 mu mol/L) and high (200.0 mu mol/L), are prepared for the standard myoinositol solutions with different concentrations, five times of measurement are continuously carried out on the same day, five days of measurement are continuously carried out simultaneously, the intra-day precision is 3.2-6.2%, and the inter-day precision is 7.1-9.0%. The recovery rate in the day is 96.6-106%, and the recovery rate in the daytime is 90.3-103%.
The method of the invention determines the myoinositol concentrations of the serum sample without adding bilirubin and hemoglobin and the serum sample with different concentrations of bilirubin (0.0-200.0) mu mol/L and hemoglobin (0.0-16.0) mg/mL, and the result shows that the hemoglobin and bilirubin in the serum have no obvious interference to the determination of the serum myoinositol when respectively (0.0-8.0) mg/mL and (0.0-160.0) mu mol/L, and the result is accurate and reliable and can be used for clinical blood detection.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 response to changes in current △ Working curve of I and serum myoinositol concentration
Detailed Description
Example 1 measurement of the content of myoinositol in blood
(1) Establishment of myoinositol Standard Curve
a. Preparation of a series of concentration standard working solutions: dissolving myoinositol solid powder with ultrapure water to prepare a myoinositol stock solution of 200mmol/L, diluting with ultrapure water to prepare a series of myoinositol standard solutions (50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, 4000.0, 5000.0) mu mmol/L, taking 100 mu L of myoinositol standard solutions with different concentrations, mixing with 900 mu L of blank serum by vortex to prepare a standard-added mixed serum solution with a myoinositol concentration of (5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 400.0, 500.0) mu mol/L, taking 120 mu L of standard-added mixed serum solution, slowly adding 133mu L of acetonitrile, mixing, vortex for 30s, centrifuging at 4 ℃, 00rpm/min, centrifuging for 12min, taking supernatant, drying at 80 ℃ under nitrogen flow, and dissolving residues in 40 mu L of glycine-buffer solution (NaOH/L, pH = 10.3) to obtain a series of standard working solution with concentration;
b. preparation of blank sample solution: taking 120 mu L of blank serum, slowly adding 480 mu L of acetonitrile, mixing, vortexing for 30s, centrifuging the mixture at 4 ℃ and 13300rpm/min for 12min, then taking supernatant, drying the supernatant under 80 ℃ nitrogen flow, and dissolving residues in 40 mu L of glycine-NaOH buffer solution (550 mmol/L, pH = 10.3) to obtain blank sample solution;
c. respectively sucking 40 mu L of blank sample solution and series concentration standard working solution, adding 5 mu L of oxidized nicotinamide adenine dinucleotide solution (NAD +,160 mmol/L) and 5 mu L of myoinositol, uniformly mixing the oxidized nicotinamide adenine dinucleotide solution and myoinositol through inositol dehydrogenase solution (IDH, 4500U/L), carrying out enzyme reaction for 20min at room temperature, taking 40 mu L of reaction mixed solution, attaching the reaction mixed solution to a screen-printed carbon electrode, accessing an electrochemical workstation (a reference electrode AgCl ink of the electrochemical workstation, a working electrode and a counter electrode: carbon ink), measuring a response current value by adopting a differential pulse voltammetry (DPV method), wherein parameters are voltage (0.1V to 0.73V), increment of 0.005V, amplitude of 0.10V, pulse width of 0.05s and pulse period of 0.2s, taking the concentration of the myoinositol as an X axis, and taking the difference value of the response current value of the series concentration standard working solution and the blank sample solution as a Y axis, and drawing a myoinositol standard curve;
(2) Measuring the content of myoinositol in the sample to be tested:
d. preparation of sample solution to be tested
B, preparing a serum sample by the same method in the step b to obtain a sample solution to be detected;
e. determination of myoinositol content in sample to be tested
And (d) taking a sample solution to be detected, measuring the response current value by the same method of the step c, and calculating the myoinositol content in the sample to be detected according to the standard curve of the step (1).
The advantageous effects of the present invention are described below by way of experimental examples.
EXAMPLE 1 detection of tagged myoinositol in serum samples
Taking myoinositol stock solution (200 mmol/L), and diluting to prepare serial myoinositol standard solutions (50.0, 100.0, 200.0, 500.0, 1000.0, 2000.0, 4000.0 and 5000.0) mu mmol/L. 100 mu L of standard myoinositol solutions with different concentrations are taken to be mixed with 900 mu L of mixed blank serum, and after vortex mixing, standard mixed serum samples with myoinositol concentration of (5.0, 10.0, 20.0, 50.0, 100.0, 200.0, 400.0, 500.0) mu mol/L are prepared. Add 120. Mu.L of spiked mixed serum sample to the centrifuge tube, then add 480. Mu.L of acetonitrile slowly and mix vortexed for 30s. The mixture was centrifuged at 13300rpm/min for 12min at 4 ℃. Subsequently, the supernatant was dried under a nitrogen stream at 80 ℃; the residue was dissolved in 40. Mu.L of glycine-NaOH buffer (550 mmol/L, pH = 10.3). Respectively taking 40 mu L of the double solution and 5 mu L of NAD + (160 mmol/L), 5. Mu.L of IDH solution (4500U/L) was vortexed and enzymatically reacted at room temperature for 20min. 40 μ L of the reaction mixture solution was dropped onto the SPCE working electrode and connected to an electrochemical workstation (reference electrode AgCl ink, working electrode and counter electrode of the electrochemical workstation: carbon ink). The electrochemical workstation parameters were voltage (0.1V to 0.73V), increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s, DPV scans were performed, each experiment was repeated 5 times, and the response current was recorded. Blank serum response current I at myoinositol concentration 0 0 The response current of the standard sample containing myoinositol to be measured is I X Drawing a delta I-C working curve by the increase value delta I of the response current and the myoinositol concentration of the spiked mixed serum sample, and collectingAnd obtaining a delta I-C linear regression equation by using a linear regression method. Within the range of 5.0-500.0 mu mol/L, the DPV peak current and the serum myoinositol concentration have good linear relation, and a regression equation is obtained: y = 0.007225X +0.1893 (Y is the oxidation peak current μ A of NADH, X is the myoinositol concentration μmol/L), and the linear correlation coefficient is 0.9981. Repeating the experiment for more than 5 times by taking the concentration corresponding to the current signal which is 3 times larger than the noise signal as the lowest detection limit, wherein the lowest detection limit of the method is 1.0 mu M (S/N = 3); the concentration corresponding to the current signal 10 times higher than the noise signal is used as the lowest detection limit, and the experiment is repeated for more than 5 times to obtain that the lowest quantification limit is 2.5 μ M (S/N = 10), which is shown in fig. 1 in detail.
Experimental example 2 determination of precision and recovery of serum myoinositol by electrochemical method
The low (10.0 mu mol/L), medium (100.0 mu mol/L) and high (200.0 mu mol/L) working solutions are prepared by adding myoinositol standard solutions with different concentrations into blank serum respectively, 120 mu L of mixed serum samples are added into a centrifuge tube, and then 480 mu L of acetonitrile is slowly added for mixing and vortex for 30s. The mixture was centrifuged at 13300rpm/min for 12min at 4 ℃. Subsequently, the supernatant was dried under nitrogen flow at 80 ℃; the residue was dissolved in 40. Mu.L of glycine-NaOH buffer (550 mmol/L, pH = 10.3). Respectively taking 40 mu L of the compound solution and 5 mu L of the compound solution + (160 mmol/L), 5. Mu.L of IDH solution (4500U/L) were vortexed and mixed, and enzymatic reaction was performed at room temperature for 20min. 40. Mu.L of the reaction mixture solution was dropped onto the SPCE working electrode and connected to an electrochemical workstation. The electrochemical workstation parameters were voltage (0.1V to 0.73V), increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s, DPV scans were performed, each experiment was repeated 5 times, and the response current was recorded. Regression equation obtained by experimental example 1: y = 0.007225X +0.1893 (Y is oxidation peak current μ A of NADH, and X is myoinositol concentration μmol/L), and calculating the concentration of serum myoinositol. The myo-inositol in the same serum sample was tested five times in succession on the same day and the relative recovery = (measured concentration of myo-inositol after spiking-initial concentration of myo-inositol before spiking) ÷ myo-inositol addition concentration x 100% for five consecutive days. The result shows that the precision in the day is 3.2-6.2%, and the precision in the day is 7.1-9.0%; daily recoveryThe rate is 96.6% -106%, and the daytime recovery rate is 90.3% -103% (see table 1).
TABLE 1 electrochemical determination of serum myoinositol precision and recovery
Experimental example 3 Effect of hemoglobin and bilirubin on electrochemical detection of serum myoinositol
Hemoglobin standard and bilirubin standard were added to the same serum concentration to prepare mixed serum samples containing 0.0, 2.0, 4.0, 8.0, 12.0, 16.0mg/ml hemoglobin and 0.0, 40.0, 80.0, 160.0, 180.0, 200.0. Mu. Mol/L bilirubin, respectively. Add 120. Mu.L of the pooled serum sample to the centrifuge tube, then slowly add 480. Mu.L of acetonitrile mix vortex for 30s. The mixture was centrifuged at 13300rpm/min for 12min at 4 ℃. Subsequently, the supernatant was dried under a nitrogen stream at 80 ℃; the residue was dissolved in 40. Mu.L of glycine-NaOH buffer (550 mmol/L, pH = 10.3). Respectively taking 40 mu L of the compound solution and 5 mu L of the compound solution + (160 mmol/L), 5. Mu.L of IDH solution (4500U/L) were vortexed and mixed, and enzymatic reaction was performed at room temperature for 20min. 40. Mu.L of the reaction mixture solution was dropped onto the SPCE working electrode and connected to an electrochemical workstation. The electrochemical workstation parameters were voltage (0.1V to 0.73V), increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s, DPV scans were performed, each experiment was repeated 5 times, and the response current was recorded. Regression equation obtained by experimental example 1: y = 0.007225X +0.1893 (Y is oxidation peak current μ A of NADH, X is myoinositol concentration μmol/L), and the concentration of myoinositol in serum before and after hemoglobin or bilirubin is calculated and defined as X C And X T . Calculating an interference value (X) T -X C ) Below 1.96s, no significant interference is indicated, denoted as N; when the calculated interference value is higher than 1.96s, obvious interference exists and is marked as I. The results showed that serum hemoglobin and bilirubin at (0.0-4.0) mg/mL and (0.0-160.0) μmol/L, respectively, did not significantly interfere with the measurement of serum myoinositol (Table 2).
TABLE 2 influence of hemoglobin and bilirubin on the electrochemical detection of serum myoinositol
Note: the concentration of myo-inositol in serum before and after the addition of hemoglobin or bilirubin was defined as XC and XT, respectively. When the calculated interference value XT-XC is lower than 1.96s, the interference is expressed as no obvious interference and is marked as N; when the calculated interference value is higher than 1.96s, obvious interference exists and is marked as I. )
In conclusion, the method has the advantages of high sensitivity, high analysis speed, wide linear range, good reproducibility and strong anti-interference capability, the detection limit is as low as 1.0 mu M (S/N = 3), the quantification limit is 2.5 mu M (S/N = 10), and the analysis and detection range is 5.0-500.0 mu mol/L. Three working solutions, namely low (10.0 mu mol/L), medium (100.0 mu mol/L) and high (200.0 mu mol/L), are prepared for the standard myoinositol solution with different concentrations, five times of measurement are continuously carried out on the same day, five days of measurement are continuously carried out simultaneously, the precision in the day is 3.2-6.2%, and the precision in the day is 7.1-9.0%. The daily recovery rate is 96.6-106 percent, and the daytime recovery rate is 90.3-103 percent.
The results of the measurement of the myoinositol concentrations of the serum samples without adding bilirubin and hemoglobin and the serum samples with different concentrations of bilirubin (0.0-200.0) mu mol/L and hemoglobin (0.0-16.0) mg/mL show that the hemoglobin and bilirubin in the serum have no obvious interference on the measurement of the serum myoinositol when respectively being (0.0-4.0) mg/mL and (0.0-160.0) mu mol/L, the results are accurate and reliable, and the method can be used for clinical blood detection.
Claims (10)
1. A method for quantitatively detecting myoinositol in blood, which is characterized by comprising the following steps: it is used to detect myoinositol in blood by electrochemical method.
2. The method of claim 1, wherein: the method comprises the steps of measuring response current values of a myoinositol solution, a blank serum solution and a blood sample solution to be measured by a Differential Pulse Voltammetry (DPV) method, and calculating the content of myoinositol in the blood sample according to a myoinositol standard curve established by the concentrations of the myoinositol solution and the blank serum solution and corresponding current values.
3. The method of claim 2, wherein: the method specifically comprises the following steps:
(1) Establishment of myoinositol Standard Curve
a. Preparation of standard working solutions of a series of concentrations: dissolving myoinositol in water, diluting to obtain series concentration standard solutions, respectively collecting series concentration standard solutions, adding blank serum, mixing, adding acetonitrile, centrifuging, collecting supernatant, drying, and dissolving residue in glycine-NaOH buffer solution to obtain series concentration standard working solutions;
b. preparation of blank sample solution: taking blank serum, adding acetonitrile, mixing, centrifuging, taking supernatant, drying, and dissolving residues in glycine-NaOH buffer solution to obtain blank sample solution;
c. respectively sucking a blank sample solution and a series of concentration standard working solutions, adding an oxidized nicotinamide adenine dinucleotide solution (NAD +) and myoinositol, uniformly mixing the oxidized nicotinamide adenine dinucleotide solution (NAD +) and the myoinositol through an inositol dehydrogenase solution (IDH), reacting for 10-30 min, taking a reaction mixed solution, attaching the reaction mixed solution to a screen printing carbon electrode, accessing an electrochemical workstation, measuring a response current value by adopting a Differential Pulse Voltammetry (DPV) method, and drawing a myoinositol standard curve by taking the concentration of the myoinositol as an X axis and the difference value of the response current values of the series of concentration standard working solutions and the blank sample solution as a Y axis;
(2) Measuring the content of myoinositol in the sample to be measured:
d. preparation of sample solution to be tested
B, taking a serum sample, and preparing by the same method in the step b to obtain a sample solution to be detected;
e. determination of myoinositol content in test sample
And (c) taking a sample solution to be detected, measuring the response current value by the same method of the step c, and calculating the myoinositol content in the sample to be detected according to the standard curve of the step (1).
4. The method of claim 3, wherein: the volume ratio of the series of concentration standard solutions, the blank serum and the acetonitrile in the step a is 1-3: 8-10: 30 to 50, preferably 1:9:40; the concentration of the series of concentration standard solutions is 50-5000.0 mu mmol/L, preferably 50.0 mu mmol/L,100.0 mu mmol/L,200.0 mu mmol/L,500.0 mu mmol/L,1000.0 mu mmol/L,2000.0 mu mmol/L,4000.0 mu mmol/L and/or 5000.0 mu mmol/L.
5. The method of claim 3, wherein: the volume ratio of the blank serum to the acetonitrile in the step b is 9-13: 30 to 50, preferably 10:40.
6. the method of claim 3, wherein: the step a and the step b are evenly mixed in a vortex mode; the centrifugation temperature is 2-8 ℃, the rotation speed is 10000-20000 rpm/min, the time is 8-15 min, the preferred centrifugation temperature is 4 ℃, the rotation speed is 13300rpm/min, and the time is 12min; and/or, the drying in step a and step b is nitrogen flow drying; the temperature for drying the nitrogen flow is 60-100 ℃, and the preferential temperature is 80 ℃.
7. The method of claim 3, wherein: the addition amount of the glycine-NaOH buffer solution in the step a and the step b is 1/10 to 1/15, preferably 1/12 of the volume of the acetonitrile; the pH value of the glycine-NaOH buffer solution is 9-11, wherein the glycine content is 500-600 mmol/L, preferably 10.3, and 550mmol/L.
8. The method of claim 3, wherein: step c, the blank sample solution or the series of concentration standard working solutions and NAD + The volume ratio of the solution to the IDH solution is 25-45: 3 to 7:3 to 7, preferably 40:5:5; and/or, the NAD + The concentration of the solution is 100-200 mmol/L, the concentration of the IDH solution is 4000-5000U/L, and NAD is preferred + The concentration of the solution is 160mmol/L, and the concentration of the IDH solution is 4500U/L.
9. The method of claim 3, wherein: the reaction temperature in the step c is 20-30 ℃, and the reaction time is 20min; and/or the volume of the reaction mixture attached to the screen-printed carbon electrode in the step c is 20 to 60. Mu.L, preferably 40. Mu.L.
10. The method of claim 3, wherein: the parameters of the DPV method in the step c are voltage 0.1-0.73V, increment 0.005V, amplitude 0.10V, pulse width 0.05s and pulse period 0.2s.
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| EP1491636B1 (en) * | 2002-03-29 | 2008-05-21 | Asahi Kasei Pharma Corporation | Method of detecting mild impaired glucose tolerance or insulin hyposecretion |
| CN102645414B (en) * | 2012-04-06 | 2014-04-02 | 上海蓝怡科技有限公司 | Reagent kit capable of determining urine inositol |
| JP2014153243A (en) * | 2013-02-12 | 2014-08-25 | Tanita Corp | Biosensor and measurement method employing the same |
| JP2017104049A (en) * | 2015-12-09 | 2017-06-15 | 日本化薬株式会社 | Measurement method for component in biological sample |
| CN105842356A (en) * | 2016-03-23 | 2016-08-10 | 张霆 | Inositol detection method |
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