CN111189933A - 1 α, 25-dihydroxy vitamin D high performance liquid chromatography-mass spectrometry combined detection kit - Google Patents

Abstract

The invention provides a 1 α, 25-dihydroxy vitamin D2 and 1 α, 25-dihydroxy vitamin D3 high performance liquid chromatography mass spectrometry detection kit, which consists of a reference substance, a quality control product, an isotope internal standard extracting solution, a diluent, a conversion solution, a complex solution, a mobile phase additive A, a mobile phase additive B, a 96-well reaction plate, a 96-well filter plate and an instruction.

Description

1 α, 25-dihydroxy vitamin D high performance liquid chromatography-mass spectrometry combined detection kit

Technical Field

The invention belongs to the field of detection, and particularly relates to a detection kit and a detection method for high performance liquid chromatography-mass spectrometry of 1 α, 25-dihydroxy vitamin D.

Background

Vitamin D is sterol derivative, ergosterol, and ergocalciferol, and has effects of resisting rickets, also known as vitamin for resisting rickets. Members of the vitamin D family include vitamins D1, D2, D3, D4, and D5, with D2 (ergocalciferol) and D3 (cholecalciferol) being more closely related to health. It is not only a fat-soluble vitamin, but also a hormone with multiple physiological activities, and has wide physiological metabolic activity. Vitamin D deficiency can cause rickets, tetany, osteomalacia and other diseases, but excessive vitamin D intake for a long time can cause calcification of blood vessels and organs. Therefore, the regular detection of the vitamin D level in the human body has important significance for measuring the vitamin D supplement effect of the human body and maintaining the health of the human body.

Vitamin D can be produced by skin synthesis or food intake, has a half-life of about 3 hours and is associated with vitamin D binding proteins, 1 α,25- (OH)₂The half-life of the vitamin D2/D3 is about 24 hours, and the content of the vitamin D in blood is extremely low; 25-hydroxyvitamin D is a blood-detectable marker because it has a half-life of about 3 weeks, is relatively stable, and is the main storage form of vitamin D in humans, and it can be detected to determine the overall vitamin D status. Vitamin D is a major element in maintaining bone health. A severe deficiency of vitamin D in childhood will lead to skeletal deformity, i.e. rickets. Vitamin D deficiency is a common cause of secondary hyperparathyroidism. In recent years, vitamin D has been found to be relevant to cancer, cardiovascular diseases, diabetes and the like, and a sufficient level of vitamin D in vivo has a certain preventive effect on cancer, multiple sclerosis, diabetes and the like.

The vitamin D content of the people with vitamin D deficiency can be increased by food, sunlight and vitamin supplements, and the food sources comprise egg yolk, salmon, tuna, fish liver oil, beef liver, margarine, yoghourt, cheese and the like; the supplement provides vitamin D in two forms: d2 and D3. Both forms are effective, each of which can ensure adequate vitamin D levels. But 2 and 3 are not equivalent. D3 is a human-produced form, and recent studies have shown that D3 rapidly increases vitamin D levels by up to 3-fold and persists longer. The supplementary dose depends on age and risk factors. The recommended dietary standard is 600 IU/day for 70 years old and 800 IU/day for 71 and above. Some researchers have suggested that high vitamin D levels may bring a variety of health benefits, but too much may cause harm. According to the american medical institute, the risk of harm increases beyond 4000 IU/day.

lα，25(OH)₂D is often used for treating chronic renal function injury, hypoparathyroidism and hypercalcemia, etc., and can accurately quantify serum l α,25 (OH)₂D level has important effect on disease diagnosis and prognosis monitoring, however, the traditional immunological detection method can not detect l α,25 (OH) distinctively₂D2 and l α,25 (OH)₂D3 is affected by both isomer and vitamin D binding protein, and lacks sufficient detection accuracy and specificity.

In view of the above, there is an urgent need in the art to develop a detection kit and a detection method that have short analysis time, sensitivity, accuracy and capability of simultaneously quantitatively detecting 1 α, 25-dihydroxyvitamin D2 and 1 α, 25-dihydroxyvitamin D3.

Disclosure of Invention

The invention aims to provide a detection kit and a detection method which have short analysis time, are sensitive and accurate and can simultaneously and quantitatively detect 1 α, 25-dihydroxyvitamin D2 and 1 α, 25-dihydroxyvitamin D3.

In a first aspect of the present invention, there is provided a high performance liquid chromatography-mass spectrometry detection kit, comprising:

(1) a quality control material comprising 1 α, 25-dihydroxyvitamin D2 and 1 α, 25-dihydroxyvitamin D3;

(2) an isotope internal standard extracting solution, which contains 1 α, 25-dihydroxy vitamin D2-D6 and 1 α, 25-dihydroxy vitamin D3-D6;

(3) a diluent which is saturated monohydric alcohol;

(4) a conversion solution comprising 4-phenyl-1, 2, 4-triazoline-3, 5-dione (PTAD);

(5) a compound solution, wherein the compound solution is water;

(6) a mobile phase additive A which is a saturated monocarboxylic acid; and

(7) a mobile phase additive B, wherein the mobile phase additive B is a C1-C3 amine compound.

In another preferred example, the concentration of 1 α, 25-dihydroxy vitamin D2-D6 and the concentration of 1 α, 25-dihydroxy vitamin D3-D6 in the isotope internal standard extracting solution are 0.5-2 ng/mL and 0.5-2 ng/mL respectively.

In another preferred example, the concentration of 1 α, 25-dihydroxy vitamin D2-D6 in the isotope internal standard extract is the same as the concentration of 1 α, 25-dihydroxy vitamin D3-D6.

In another preferred example, the isotope internal standard extracting solution contains 1ng/mL of 1 α, 25-dihydroxy vitamin D2-D6 and 1ng/mL of 1 α, 25-dihydroxy vitamin D3-D6.

In another preferred example, the concentration of 4-phenyl-1, 2, 4-triazoline-3, 5-dione in the conversion solution is 0.01mg/g to 10 mg/g.

In another preferred embodiment, the test kit further comprises (8) a control, wherein the control comprises 1 α, 25-dihydroxy vitamin D2 and 1 α, 25-dihydroxy vitamin D3.

In another preferred embodiment, the mobile phase additive B is selected from the group consisting of: methylamine, ethylamine, propylamine, or combinations thereof.

In another preferred embodiment, the mobile phase additive B is methylamine.

In another preferred embodiment, the saturated monocarboxylic acid is selected from the group consisting of: formic acid, acetic acid, propionic acid, butyric acid, or a combination thereof.

In another preferred embodiment, the saturated monocarboxylic acid is formic acid.

In another preferred embodiment, the saturated monoalcohol is selected from the group consisting of: methanol, ethanol, or a combination thereof.

In another preferred embodiment, the saturated monohydric alcohol is methanol.

In a second aspect of the invention, there is provided a method for determining the amount of one or more dihydroxyvitamin D metabolites in a taken biological sample by high performance liquid chromatography-mass spectrometry comprising:

(i) ionizing the one or more dihydroxyvitamin D metabolites and an internal standard by an electrospray ion source (ESI) to produce at least one precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively;

(ii) generating one or more fragment ions of the precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively; and

(iii) (iii) comparing the amount of the one or more dihydroxyvitamin D metabolites and the one or more ions of the internal standard produced in step (i) or (ii) or both to determine the amount of the one or more dihydroxyvitamin D metabolites in the biological sample;

wherein, C1-C3 amine compound is added into the mobile phase during the chromatographic detection.

In another preferred embodiment, methylamine is added to the mobile phase during chromatographic detection.

In another preferred example, the mass spectrum is a tandem mass spectrum.

In another preferred embodiment, the one or more dihydroxyvitamin D metabolites include 1 α, 25-dihydroxyvitamin D2, and wherein the parent ion of the 1 α, 25-dihydroxyvitamin D2 has a mass/charge ratio of 635.4 + -0.5, and/or

The one or more dihydroxyvitamin D metabolites include 1 α, 25-dihydroxyvitamin D3, and wherein the mass/charge ratio of the parent ion of the 1 α, 25-dihydroxyvitamin D3 is 623.4 ± 0.5.

In another preferred embodiment, wherein said one or more fragment ions comprise one or more ions selected from the group consisting of ions having a mass/charge ratio of 314.4 ± 0.5.

In another preferred example, the method further comprises: derivatizing the one or more dihydroxyvitamin D metabolites and the internal standard with a conversion solution prior to ionizing the one or more dihydroxyvitamin D metabolites and the internal standard by an electrospray ion source (ESI).

In another preferred example, in the chromatographic detection process, the liquid chromatogram has one or more of the following chromatographic conditions:

(i) the mobile phase comprises a mobile phase A and a mobile phase B;

wherein the mobile phase A is water containing a mobile phase additive A and a mobile phase additive B; the mobile phase B is methanol containing the mobile phase additive A;

(ii) the temperature of the chromatographic column is 30-50 ℃;

(iii) the flow rate is 0.2-1.0 ml/min.

In another preferred example, during the chromatographic detection, the gradient is:

0-0.5 min: the mobile phase B accounts for 10-50 percent, and the balance is the mobile phase A;

0.5-3 min: the mobile phase B accounts for 50% -95%, and the balance is the mobile phase A;

3.0-4.5 min: the mobile phase B accounts for 95 percent, and the balance is the mobile phase A;

4.5-5.0 min: 95-10% of mobile phase B and the balance of mobile phase A;

5.0-6.0 min: mobile phase B was 10% and the remainder mobile phase a.

In another preferred embodiment, the mobile phase additive a is a saturated monocarboxylic acid; and the mobile phase additive B is a C1-C3 amine compound.

In another preferred example, in the mobile phase a, the ratio of mobile phase additive a: mobile phase additive B: the volume ratio of water is (0.5-2): (0.5-2): 1000, parts by weight; preferably, 1: 1: 1000.

in another preferred example, in the mobile phase a, the ratio of mobile phase additive a: the volume ratio of methanol is (0.5-2): 1000, parts by weight; preferably, 1: 1000.

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 standard chromatograms of 1 α, 25-dihydroxyvitamin D2 (top panel) and internal isotope chromatograms of 1 α, 25-dihydroxyvitamin D2-D6 (bottom panel).

FIG. 2 shows standard chromatograms of 1 α, 25-dihydroxyvitamin D3 (top panel) and internal isotope chromatograms of 1 α, 25-dihydroxyvitamin D3-D6 (top panel).

FIG. 3 is a graph of the standard curve for 1 α, 25-dihydroxy vitamin D2.

FIG. 4 is a graph of the standard curve for 1 α, 25-dihydroxy vitamin D3.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have developed for the first time a high performance liquid chromatography-mass spectrometry detection method capable of simultaneously quantitatively detecting 1a, 25-dihydroxyvitamin D2 and 1a, 25-dihydroxyvitamin D3 in a single assay through a large number of screenings, for example, screening of diluents, complex solutions, etc., and the detection method has a short assay time, high sensitivity and accuracy of detection results, and high reproducibility of results.

Term(s) for

As used herein, the terms "1 α, 25-dihydroxy vitamin D2-D6" and "1 α, 25-dihydroxy vitamin D2-²H' is used interchangeably to refer to a deuteron formed by substituting H in 1 α, 25-dihydroxy vitamin D2 with deuterium isotope, and in the present invention, it can be used as an internal standard for mass spectrometric detection of 1 α, 25-dihydroxy vitamin D2.

As used herein, the terms "1 α, 25-dihydroxy vitamin D3-D6" and "1 α, 25-dihydroxy vitamin D3-²H' is used interchangeably to mean the deuteron formed after H in 1 α, 25-dihydroxy vitamin D3 is replaced by deuterium isotope, which can be used as an internal standard for mass spectrometric detection of 1 α, 25-dihydroxy vitamin D3 in the present invention.

As used herein, the terms "1 α, 25-dihydroxy vitamin D2", "1 α,25- (OH)₂VD2 "and" 1 α,25- (OH)₂Vitamin D2 "is used interchangeably.

As used herein, the terms "1 α, 25-dihydroxy vitamin D3", "1 α,25- (OH)₂VD3 "and" 1 α,25- (OH)₂Vitamin D3 "is used interchangeably.

As used herein, the terms "precursor ion" and "parent ion" are used interchangeably to refer to an ion that can undergo further decomposition reactions to produce fragment ions.

As used herein, the terms "fragment ion" and "daughter ion" are used interchangeably and refer to those resulting from a single molecular fragmentation reaction of a molecular ion or larger fragment ion.

As used herein, the terms "mobile phase a", "phase a", or "aqueous mobile phase" are used interchangeably to refer to the mobile phase obtained after adding mobile phase additive a, mobile phase additive B, respectively, to ultrapure water in a ratio of 1:1000 in liquid phase conditions.

As used herein, the terms "mobile phase B", "B phase" or "organic mobile phase" are used interchangeably to refer to a mobile phase resulting from the addition of mobile phase additive a to methanol in a ratio of 1:1000 under liquid phase conditions.

As used herein, "liquid chromatography" (LC) refers to process retardation in which one or more components of a fluid solution are selectively delayed as the fluid is uniformly filtered through a column of finely divided materials or through capillary channels due to the distribution of the components of the mixture between the one or more stationary phases and the bulk fluid (bulk fluid) (i.e., mobile phase) as the fluid moves relative to the stationary phase(s) "liquid chromatography" includes Reverse Phase Liquid Chromatography (RPLC), High Performance Liquid Chromatography (HPLC), and High Turbulence Liquid Chromatography (HTLC).

As used herein, the term "HPLC" or "high performance liquid chromatography" refers to liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a tightly packed column.

As used herein, "mass spectrometry" (MS) refers to analytical techniques for identifying compounds by their mass MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass to charge ratio (m/z) the compound can be ionized and detected by any suitable means "mass spectrometers" generally include ionizers and ion detectors.

The term "electron ionization" as used herein refers to a method in which the interaction of an analyte of interest in a gaseous or vapor phase with a stream of electrons and the analyte produces analyte ions, which can then be used in mass spectrometry techniques.

The term "chemical ionization" as used herein refers to a method in which a reagent gas (e.g., ammonia) is used for electron collisions and analyte ions are formed by the interaction of the reagent gas ions with analyte molecules.

The term "ionizing" as used herein refers to a process in which negative ions that produce analyte ions having a net charge equal to one or more electron units are ions having a net negative charge of one or more electron units, while positive ions are ions having a net positive charge of one or more electron units.

The term "about" as used herein in reference to a quantitative measurement means that the indicated value is plus or minus 10%.

Reagent kit

The invention provides a 1 α, 25-dihydroxyvitamin D liquid chromatography-mass spectrometry combined detection kit (namely a liquid-mass spectrometry combined method), which comprises a quality control product, an isotope internal standard extracting solution, a diluent, a conversion solution, a complex solution, a mobile phase additive A and a mobile phase additive B, preferably, the detection kit also comprises a reference substance, a 96-well reaction plate, a 96-well filter plate, an instruction and the like.

Specifically, in the kit of the present invention, the reference substance and/or the quality control substance comprises 1 α, 25-dihydroxy vitamin D2 and 1 α, 25-dihydroxy vitamin D3, the isotope internal standard extract comprises 1 α, 25-dihydroxy vitamin D2-D6 and 1 α, 25-dihydroxy vitamin D3-D6, the diluent is saturated monohydric alcohol (such as methanol), the conversion solution is 4-phenyl-1, 2, 4-triazoline-3, 5-dione (PTAD), the complex solution is ultrapure water, the mobile phase additive a is saturated monocarboxylic acid (such as formic acid, acetic acid, propionic acid, butyric acid or a combination thereof), the mobile phase additive B is a C1-C3 amine compound (such as methylamine), and the kit is stored at 2 to 8 ℃.

Detection method

The invention also provides a detection method for detecting dihydroxyvitamin D metabolites represented by 1 α, 25-dihydroxyvitamin D.

In general, the method of the invention comprises the steps of:

(i) ionizing the one or more dihydroxyvitamin D metabolites and an internal standard by an electrospray ion source (ESI) to produce at least one precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively;

(ii) generating one or more fragment ions of the precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively; and

(iii) (iii) comparing the amount of the one or more dihydroxyvitamin D metabolites and the one or more ions of the internal standard produced in step (i) or (ii) or both to determine the amount of the one or more dihydroxyvitamin D metabolites in the biological sample;

wherein, C1-C3 amine compound is added into the mobile phase during the chromatographic detection.

The method of the invention is particularly suitable for detecting 1 α, 25-dihydroxy vitamin D2, especially for detecting 1 α, 25-dihydroxy vitamin D2. with high sensitivity and is also suitable for detecting 1 α, 25-dihydroxy vitamin D3.

In one embodiment, a more specific detection method comprises the steps of:

(1) preparation of samples to be tested

(1.1) preparation of isotope internal standard extraction solution:

and (3) calculating the amount of the required isotope internal standard extraction working solution according to the amount of the test sample, diluting the concentrated isotope internal standard extraction solution by using a diluent according to the ratio of 1:100, and sufficiently and uniformly mixing.

(1.2) extraction

Adding 400 μ L of sample (such as human serum sample) into 2ml of EP tube, adding 50 μ L of diluted internal standard extract, mixing for 20 s, adding 1ml of diluent, mixing thoroughly, centrifuging, adding 900 μ L of supernatant into 2ml of 96-well deep-well plate, adding 600 μ L of diluent into the rest sample, mixing thoroughly, centrifuging, adding 500 μ L of supernatant into 2ml of 96-well deep-well plate, mixing with the first supernatant, and blowing with nitrogen.

(1.3) derivatization

To a blow-dried 96-well plate, 100. mu.L of the transformation solution was added, and after thorough mixing, derivatization was carried out at room temperature for 0.5 hour.

(1.4) redissolution

Adding 50 μ L of the redissolution, mixing, transferring the serum sample to a 96-well protein filter plate (a 1ml 96-well deep-well plate is placed under the protein filter plate), centrifuging in a centrifuge, placing the 1ml 96-well deep-well plate with the sample, and covering with a cover pad for standby.

(2) Liquid phase conditions

Mobile phase preparation

Phase A: adding a mobile phase additive A and a mobile phase additive B into ultrapure water according to the ratio of 1:1000 respectively.

Phase B: mobile phase additive a was added to methanol in a ratio of 1: 1000.

Wherein the mobile phase additive A is a saturated monocarboxylic acid, preferably formic acid, acetic acid, propionic acid, butyric acid or a combination thereof, such as formic acid; the mobile phase additive B is C1-C3 amine compound, such as methylamine.

A chromatographic column: the octaalkylsilane bonded silica is a filler or equivalent.

Column temperature of the chromatographic column: at 40 ℃.

Sample introduction amount: 5 μ L.

Flow rate: 0.4 mL/min.

Gradient: 0-0.5 min: the mobile phase B accounts for 10 to 50 percent; 0.5-3 min: the mobile phase B is 50-95%; 3.0-4.5 min: the mobile phase B is 95%; 4.5-5.0 min: 95% -10% of mobile phase B; 5.0-6.0 min: the mobile phase B was 10%.

(3) Conditions of Mass Spectrometry

Multi-reaction monitoring ion pairs (MRMs) and corresponding voltage parameters

Other Mass Spectrometry parameters

An ion source: electrospray (ESI) ion source.

Capillary voltage: 3.0 kV.

Taper hole voltage: 30V.

Ion source temperature: at 150 ℃.

Desolventizing gas temperature: at 500 ℃.

Flow rate of desolventizing gas: 1000L/Hr.

(4) Detection on machine

And injecting the processed reference substance, quality control substance and detection sample solution into a high performance liquid chromatography-tandem mass spectrometer for detection, and recording peak areas of a chromatogram and detection samples 1 α, 25-dihydroxyvitamin D2, 1 α and 25-hydroxyvitamin D3 and peak areas of isotope internal standards 1 α, 25-dihydroxyvitamin D2-D6 and 1a, 25-dihydroxyvitamin D3-D6.

(5) Quantitative analysis

The standard curve drawing method comprises the following steps: and (3) to 10 (preferably 4 to 7) reference substance labeled concentrations are used as abscissa (x), and the ratio of the actual detection peak area of the 7 reference substances to the respective internal standard peak area is used as ordinate (y), so as to draw a standard curve.

Fitting of standard curve equation: the peak area ratio (y) of 3-10 (preferably 4-7) reference samples is used to perform linear regression on the labeled concentration (x). A regression equation can be obtained: y is a + bx, where y is the ordinate, x is the abscissa, a is the intercept, and b is the slope.

And (3) calculating a detection sample result: and substituting the ratio of the actual peak area of the detection sample to the peak area of the internal standard into the standard curve equation, and calculating the concentration of the compound to be detected in the detection sample.

The main advantages of the invention include:

(a) the test kit can detect 1 α, 25-dihydroxy vitamin D2 and 1 α, 25-dihydroxy vitamin D3 respectively, does not need extraction in operation, so the process is simple and convenient, the time consumption is short, the mass/charge ratio of parent ions of 1 α, 25-dihydroxy vitamin D2 is 635.4 +/-0.5, the mass/charge ratio of parent ions of 1 α, 25-dihydroxy vitamin D3 is 623.4 +/-0.5, and the detection sensitivity is high.

(b) The kit has high linear correlation of standard curves for detecting 1 α, 25-dihydroxy vitamin D2 and 1 α, 25-dihydroxy vitamin D3, so that the detection result has high accuracy

(c) When the kit is used for detection, water is used as a complex solution, and the derivatization reaction can be completely terminated, so that the measurement repeatability is good and the precision is high.

(d) The kit of the invention has excellent detection sensitivity of 1 α, 25-dihydroxy vitamin D2.

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. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1

The assay for 1 α, 25-dihydroxyvitamin 1 α, 25-dihydroxyvitamin D3 was carried out by standard curve.

Preparation of samples to be tested

Preparation of internal standard extract

Diluting 10 μ L of isotope internal standard extract with 990 μ L mL of diluent, and mixing well.

Extraction of

Adding 400 mu L of human serum sample into a 2ml EP tube, adding 50 mu L of diluted internal standard extracting solution, mixing for 20 seconds, adding 1ml of diluent, mixing uniformly, centrifuging, adding 900 mu L of supernatant into a 2ml 96-hole deep-hole plate, adding 600 mu L of diluent into the rest sample, mixing uniformly, centrifuging, adding 500 mu L of supernatant into a 2ml 96-hole deep-hole plate, mixing with the first supernatant, and then blowing with nitrogen.

Derivatisation

To a blow-dried 96-well plate, 100. mu.L of the transformation solution was added, and after thorough mixing, derivatization was carried out at room temperature for 0.5 hour.

Redissolving

Adding 50 μ L of the redissolution, mixing, transferring the serum sample to a 96-well protein filter plate (a 1ml 96-well deep-well plate is placed under the protein filter plate), centrifuging in a centrifuge, placing the 1ml 96-well deep-well plate with the sample, and covering with a cover pad for standby.

Liquid phase conditions

Mobile phase preparation

Phase A: adding a mobile phase additive A and a mobile phase additive B into ultrapure water according to the ratio of 1:1000 respectively.

Phase B: mobile phase additive a was added to methanol in a ratio of 1: 1000.

Wherein the mobile phase additive A is formic acid; the mobile phase additive B is methylamine.

A chromatographic column: the octaalkylsilane bonded silica is a filler or equivalent.

Column temperature of the chromatographic column: at 40 ℃.

Sample introduction amount: 5 μ L.

Flow rate: 0.4 mL/min.

Gradient: 0-0.5 min: the mobile phase B accounts for 10 to 50 percent; 0.5-3 min: the mobile phase B is 50-95%; 3.0-4.5 min: the mobile phase B is 95%; 4.5-5.0 min: 95% -10% of mobile phase B; 5.0-6.0 min: the mobile phase B was 10%.

Conditions of Mass Spectrometry

Multi-reaction monitoring ion pairs (MRMs) and corresponding voltage parameters

Other Mass Spectrometry parameters

An ion source: electrospray (ESI) ion source.

Capillary voltage: 3.0 kV.

Taper hole voltage: 30V.

Ion source temperature: at 150 ℃.

Desolventizing gas temperature: at 500 ℃.

Flow rate of desolventizing gas: 1000L/Hr.

Detection on machine

And (3) setting parameters of a liquid chromatograph-mass spectrometer according to chromatographic conditions and mass spectrum conditions, injecting the processed reference substance, quality control substance and sample solution to be detected into an instrument for detection, and recording peak areas of chromatograms and detection samples 1 α, 25-dihydroxy vitamin D2, 1 α and 25-dihydroxy vitamin D3 and peak areas of isotope internal standards 1 α, 25-dihydroxy vitamin D2-D6, 1 α and 25-dihydroxy vitamin D3-D6 (as shown in figures 1 and 2).

Quantitative analysis

The standard curve drawing method comprises the following steps: and (3) drawing a standard curve by taking the labeled concentration of 4-7 reference substances as an abscissa (x) and taking the ratio of the actual detection peak area of the 4-7 reference substances to the peak area of each internal standard as an ordinate (y), as shown in fig. 3 and 4.

And fitting a standard curve equation, namely performing linear regression on the labeled concentration (x) by using the peak area ratio (y) of 4-7 control substances, wherein y is a + bx, y is an ordinate, x is an abscissa, a is an intercept, and b is a slope, and the regression equation of 1 α and 25-dihydroxyvitamin D2 in the example is y is 1.522x + -0.00364, as shown in figure 3, and the regression equation of 1 α and 25-dihydroxyvitamin D3 is y is 0.223x + -0.0152, as shown in figure 4.

And (3) calculating a detection sample result: substituting the ratio of the actual peak area of the detection sample to the internal standard peak area into the standard curve equation, and calculating the concentration of the compound to be detected in the detection sample, which is shown in tables 1-1 and 1-2.

TABLE 1-11 α, 25-dihydroxy vitamin D2

TABLE 1-21 α, 25-dihydroxy vitamin D3

Name of Compound	Sensitivity of detection	Time of detection
			1 α, 25-dihydroxy vitamin D3	0.1ng/ml	3.48min
1 α, 25-dihydroxy vitamin D2	0.01ng/ml	3.53min

Experimental results show that under the condition, the linear relation of 1 α,25- (OH)2-VD2 is excellent, the lower limit of quantification reaches 0.01ng/mL, and 1 α,25- (OH) are difficult to detect in other detection methods known in the prior art₂VD2, with a typical detection sensitivity of about 1ng/mL, whereas the method of the invention unexpectedly detects 1 α, 25-dihydroxyvitamin D2 with a very high detection sensitivity with a lower detection sensitivity limit of up to 0.01 ng/mL.

In addition, the method is also suitable for simultaneously detecting 1 α,25- (OH)₂And VD3, the detection sensitivity is very high and reaches 0.1 ng/ml.

In contrast to the prior art methods, the lower limit of detection sensitivity of 1 α, 25-dihydroxyvitamin D3 is generally lower than the lower limit of detection sensitivity of 1 α, 25-dihydroxyvitamin D2. in the present method, the lower limit of detection sensitivity of 1 α, 25-dihydroxyvitamin D2 is much lower than the lower limit of detection sensitivity of 1 α, 25-dihydroxyvitamin D3. this indicates that the present invention is well suited for detecting very low levels of 1 α, 25-dihydroxyvitamin D2.

Example 2

The test conditions were substantially the same as in example 1 except that the flow rate of the mobile phase was 0.5 ml/min.

From the experimental results, it was found that the flow rate of the mobile phase had substantially no effect on the detection, as in the case of the detection in example 1.

Comparative example 1

Methanol was used as a reconstituted solution, and other detection conditions were substantially the same as in example 1.

According to experimental results, when the complex solution is methanol, the repeatability of the detection result is poor, the signal-to-noise ratio is low, the base line of a chromatographic pattern is high, and the methanol cannot completely stop the derivatization reaction.

Comparative example 2

The effect of the use of MeOH (methanol) and ACN (acetonitrile) in the organic mobile phase (i.e., mobile phase B) on the experimental results was examined and the peak shape was found to be better when MeOH was used in the organic phase.

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 (10)

1. The high performance liquid chromatography-mass spectrometry combined detection kit is characterized by comprising:

(1) a quality control material comprising 1 α, 25-dihydroxyvitamin D2 and 1 α, 25-dihydroxyvitamin D3;

(2) an isotope internal standard extracting solution, which contains 1 α, 25-dihydroxy vitamin D2-D6 and 1 α, 25-dihydroxy vitamin D3-D6;

(3) a diluent which is saturated monohydric alcohol;

(4) a conversion solution comprising 4-phenyl-1, 2, 4-triazoline-3, 5-dione (PTAD);

(5) a compound solution, wherein the compound solution is water;

(6) a mobile phase additive A which is a saturated monocarboxylic acid; and

(7) a mobile phase additive B, wherein the mobile phase additive B is a C1-C3 amine compound.

2. The test kit of claim 1, further comprising (8) a control comprising 1 α, 25-dihydroxyvitamin D2 and 1 α, 25-dihydroxyvitamin D3.

3. The test kit of claim 1, wherein the mobile phase additive B is methylamine.

4. The test kit of claim 1, wherein the saturated monocarboxylic acid is selected from the group consisting of: formic acid, acetic acid, propionic acid, butyric acid, or a combination thereof.

5. The test kit of claim 1, wherein the saturated monocarboxylic acid is formic acid.

6. The test kit of claim 1, wherein the saturated monohydric alcohol is selected from the group consisting of: methanol, ethanol, or a combination thereof.

7. The test kit of claim 1, wherein the saturated monohydric alcohol is methanol.

8. A method for determining the amount of one or more dihydroxyvitamin D metabolites in a taken biological sample by high performance liquid chromatography-mass spectrometry, comprising:

(i) ionizing the one or more dihydroxyvitamin D metabolites and an internal standard by an electrospray ion source (ESI) to produce at least one precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively;

(ii) generating one or more fragment ions of the precursor ion of the one or more dihydroxyvitamin D metabolites and the internal standard, respectively; and

(iii) (iii) comparing the amount of the one or more dihydroxyvitamin D metabolites and the one or more ions of the internal standard produced in step (i) or (ii) or both to determine the amount of the one or more dihydroxyvitamin D metabolites in the biological sample;

wherein, C1-C3 amine compound is added into the mobile phase during the chromatographic detection.

9. The method of claim 8, wherein the one or more dihydroxyvitamin D metabolites comprises 1 α, 25-dihydroxyvitamin D2, and wherein the parent ion of the 1 α, 25-dihydroxyvitamin D2 has a mass/charge ratio of 635.4 ± 0.5, and/or

The one or more dihydroxyvitamin D metabolites include 1 α, 25-dihydroxyvitamin D3, and wherein the mass/charge ratio of the parent ion of the 1 α, 25-dihydroxyvitamin D3 is 623.4 ± 0.5.

10. The method of claim 8, wherein the one or more fragment ions comprise one or more ions selected from the group consisting of ions having a mass/charge ratio of 314.4 ± 0.5.

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