CN101446582A - 1, 5-anhydroglucitol assay kit and assay method thereof - Google Patents
1, 5-anhydroglucitol assay kit and assay method thereof Download PDFInfo
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- CN101446582A CN101446582A CNA2008102020095A CN200810202009A CN101446582A CN 101446582 A CN101446582 A CN 101446582A CN A2008102020095 A CNA2008102020095 A CN A2008102020095A CN 200810202009 A CN200810202009 A CN 200810202009A CN 101446582 A CN101446582 A CN 101446582A
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Abstract
A 1, 5-anhydroglucitol assay kit comprises a reference substance (1) containing 0.02-100mug/ml of 1,5-AG and 0.4-40mug/ml of 80 percent methanol solution of internal standard ((UL-<13>C6)1,5-AG), a quality control substance (2) containing 0.06-80mug/ml of 1,5-AG and 0.4-40mug/ml of 80 percent methanol solution of internal standard ((UL-<13>C6)1,5-AG) and a protein precipitator (3) containing 0.5-50mug/ml of methanol or acetonitrile of internal standard ((UL-<13>C6)1,5-AG). The liquid chromatography tandem mass spectrometry determination method is used for assay. The invention has the advantages that the prepared reference substance and the quality control substance solvent can be directly used for sample injection without treatment, and the proposal of adopting the protein precipitator to combine the internal standard not only simplifies the operation steps, but also ensures the accuracy of the determination result. The invention also optimizes the liquid chromatography conditions and the mass spectrometry conditions, thereby being capable of obtaining better peak shape, sensitivity and specificity. The method is simple, accurate, fast and stable and has no need of derivatization, enzymatic conversion and other complicated steps, the assay result is not interfered by endogenous substances, and the solvent has low cost, thereby being applicable to the assay of a large number of clinical samples.
Description
Technical field
The invention belongs to the clinical biochemical detection range, be specifically related to a kind of with liquid chromatography tandem mass spectrum technical measurement 1, the detection kit of 5-dewatered grape sugar alcohol.
Background technology
U.S.'s diabetes control points out to prevent the importance of diabetic complication to be strict glycemic control with complication research group (DCCT).The efficiency index of current glycemic control mainly comprises glycosylated hemoglobin, saccharification haemocyanin, fasting blood-glucose and postprandial blood sugar, and these indexs respectively have characteristics on the reflection blood sugar level.Glycosylated hemoglobin is a goldstandard of measuring long-term blood sugar level (2～March), as the diabetic complication risk profile factor, widespread use clinically.But this index is influenced by red blood cell life span, can not reflect recent glycemic control situation.The average blood sugar level of saccharification haemocyanin reflection 1-2 before week, it is subjected to the influence of albumin amount, and the fluctuation of every day is bigger.With the moment blood sugar level of 2h blood sugar reflection after the meal, be the common counter of diabetes diagnosis screening, but then can't provide information on an empty stomach the glycemic control situation of a period of time.
Clinical research confirmation, 1, (1,5-anhydroglucitol is called for short 1 to 5-dewatered grape sugar alcohol, 5-AG) can reflect the situation of change of blood glucose fluctuation situation and postprandial hyperglycemia in a couple of days fast, delicately, can be used as the index of parameters of short term glycemic control.
1,5-AG is the Cl-deoxidation form of glucopyranose, claim 1 again, the 5-anhydro sorbitol, be be present in polyvalent alcohol glucide in human cerebrospinal fluid, serum and the blood plasma a kind of, content is only second to glucose, is suppressed by glucose is competitive when renal tubule heavily absorbs, so in the diabetes human blood 1,5-AG content obviously descends.There are some researches show, when diabetes in the blood 1, the 5-AG density loss, be a kind of reliably, not examined condition effect, individual event result can be used as the efficiency index of diagnosing diabetes glycemic control.Japan is with 1, and 5-AG detects the conventional index of classifying diabetes management as, and the clinical testing of USA in recent years has also confirmed 1, the feasibility of 5-AG aspect parameters of short term glycemic control.
Originally studies show that 1,5-AG and existing blood glucose target all present remarkable negative correlation, blood plasma 1, the minimizing degree of 5-AG and diabetes order of severity significant correlation.1,5-AG and the most significant difference of other indexs are that the cycle of reaction change of blood sugar is different, and lack the blood sugar monitoring index of short-term at present in diabetes blood sugar monitoring index, 1,5-AG has then remedied the deficiency of blood sugar monitoring index, and it can reflect within the next few days the blood sugar level to 1 week.Bibliographical information is arranged in short-term 1, the susceptibility of 5-AG is apparently higher than HbA1C.1, the 5-AG horizontal span is bigger, especially at blood sugar during near the euglycemia level.When HbA1C controls when good, and 1,5-AG not necessarily controls well.If 1, hurried deterioration appears in the independent explanation diabetes control degree on the low side of 5-AG, and perhaps the fluctuation of plasma glucose levels strengthens, and this phenomenon sees the type i diabetes people especially.1,5-AG is as the New Set of diabetes glycemic control, can reflect the parameters of short term glycemic control level sensitive, exactly, it is measured and not to be subjected to sampling condition to influence that (whole blood, serum or plasma specimen all can, it is significantly relevant that this three is, correlation coefficient r〉0.98), but the sample long preservation is concentrated mensuration, and be not subjected to age, diet, obesity, motion, stress and take various medicines etc. and influence, have good clinical value.There are some researches show 1,5-AG can effectively monitor postprandial hyperglycemia, can be used as the sharp index of diagnosing diabetes close friend commentaries on classics and degradating trend, is effectively replenishing the diabetes comprehensive monitoring.Therefore select 1,5-AG is as the blood sugar monitoring index, and in conjunction with other monitoring indexes such as HbA1C, PPG, assessment glycemic control situation will help the blood sugar monitoring more fully to the diabetic, make glycemic control in desirable scope.
At present both at home and abroad mainly adopt the holoenzyme method to measure 1,5-AG,, the approval that obtained U.S. FDA in 2003 based on the GlycoMark detection kit of this principle.In the holoenzyme method testing process, need a series of enzyme to participate in detecting to eliminate the glucose interference and to generate coloring matter, main pyranose oxidase (PROD) source difficulty, and reagent costs an arm and a leg, detect interference such as being subjected to endogenous material such as glucose, mating type cholerythrin, haemoglobin easily, its specificity remains further to be improved.In the uremia patient, the concentration of serum creatinine obviously raises, and utilizes the holoenzyme method to measure, and causes too high evaluation serum 1, the concentration of 5-AG.
Summary of the invention
The purpose of this invention is to provide a kind of situation of change index 1 that can reflect blood glucose fluctuation situation and postprandial hyperglycemia in a couple of days fast, delicately, the detection kit of 5-AG and detection method.
Disclosed by the invention 1, the 5-AG detection kit is formed and is comprised:
1. reference substance: contain 0.02～100 μ g/ml 1, mark ([UL-in 5-AG and 0.4～40 μ g/ml
13C
6] 1,80% methanol solution 5-AG).(be equivalent to blood sample concentration 0.1～500 μ g/ml 1,5-AG).
2. quality-control product: contain 0.06～80 μ g/ml 1, mark ([UL-in 5-AG and 0.4～40 μ g/ml
13C
6] 1,80% methanol solution 5-AG).(be equivalent to blood sample concentration 0.3～400 μ g/ml 1,5-AG).
3. protein precipitant: mark ([UL-in the 0.5-50 μ g/ml
13C
6] 1, methyl alcohol 5-AG) or acetonitrile.
Adopt kit of the present invention to carry out 1, the step that 5-AG detects comprises:
1. blood specimen collection: gather venous blood to sample hose, room temperature is placed, centrifugal separation plasma in 8 hours.
2. blood sample pre-service: get blood plasma to be measured in the 1.5ml centrifuge tube, add 2～10 volume protein precipitant vortexs vibrations 10sec, centrifugal (protein precipitation of 15000g * 3min) is got the supernatant sample introduction and is measured.
3. the preparation of typical curve: get reference substance and quality-control product direct injected.
4. liquid chromatography tandem mass spectrum (LC-MS/MS) condition determination
4.1 chromatographic condition
High performance liquid chromatograph (HPLC) comprises modules such as pump, injector, column oven and online degassing instrument.Analytical column adopts silica gel bonded phase filling post, comprises type analysis posts such as nh 2 column, silicagel column, sugared post, cyano group post, and column internal diameter is 1.0～5.0mm, and column length is 30～250mm.Moving phase is acetonitrile or methyl alcohol by A phase+B phase composition: A mutually, and B is water mutually, A: the B volume ratio is (20-90%): (10-80%), adopt isocratic elution, flow velocity 0.3mL/min adopts isocratic elution; Perhaps gradient elution (, design and adjust) according to dissimilar analytical columns with the elution time graded, for nh 2 column, when A is an acetonitrile, when B water is, but reference table 1 proportioning is carried out:
Moving phase A and B volume ratio during table 1 gradient elution
4.2 mass spectrum condition
Electron spray ionisation source (ESI), negative ion multiple-reaction monitoring scanning (MRM).Electron spray voltage-4500V, 450 ℃ of ion source temperatures.Ion selector channel (Q1/Q3): 1,5-AG m/z 163〉101, IS m/z 169〉105.The ion channel scope can be ± 1.0amu.
5. density calculating method and detectability
With peak area or peak height method, calculate 1, the content of 5-AG in blood by internal standard method.
Compared with prior art, the present invention has following useful result:
The present invention 1, the 5-AG detection kit, be equipped with and be used to measure the reference substance of content and the quality-control product of method of inspection stability, the reference substance and the quality-control product solvent of preparation, but need not to handle direct injected, adopt protein precipitant to merge interior target scheme, both simplified operation steps, guaranteed the correctness of measurement result again.The present invention also optimizes liquid-phase condition and mass spectrum condition, is analytical column with the nh 2 column, and acetonitrile-water is a moving phase, and isocratic elution under negative ion mode, carries out quantitatively can obtaining peak shape, sensitivity and specificity preferably with multiple-reaction monitoring (MRM).In 1～50 μ g/ml scope 1,5-AG linear relationship good (r〉0.999), in batch, betweenrun precision (RSD%) respectively≤6.37% and≤8.75%, accuracy is 96.2～103.1%.
This method is easy, accurate, quick, stable, high specificity, highly sensitive, determinand is directly detected, need not complex steps such as derivatization, enzymatic conversion, testing result is not subjected to the interference of endogenous material, and reagent cost is cheap, is applicable to the detection requirement of clinical great amount of samples.
Embodiment
The present invention is further elaborated below in conjunction with embodiment, but do not limit the present invention.
Embodiment 1
The application of kit on clinical sample is measured
With this kit measurement 159 routine diabetes B patients and 290 routine contrast crowd blood plasma 1,5-AG content, control group comprise 267 routine health check-up crowds and 23 routine old NDs, its age, sex and 1, the 5-AG measurement result sees Table 2.Diabetic's blood plasma 1,5-AG significantly is lower than young and aged control (P＜0.001) by sex and aggregate level.Because diabetes B is the common disease of elderly population, see in that young crowd is fewer.In young control and aged control, 1, there is significant sex difference in 5-AG content, and the male sex is significantly higher than women (P＜0.05), does not then show statistical discrepancy on the sex in the diabetes group.Prompting 1,5-AG content and diabetes, sex are relevant, have nothing to do with the age.Merging young control and aged control is the normal control group, between normal control group gender 1, the measurement result of 5-AG all is normal distribution, be remarkable statistical discrepancy between the gender, be respectively 28.80 ± 7.91 μ g/ml and 19.03 ± 5.77 μ g/ml (P＜0.001), so the male sex's normal reference value is 13.30～44.30 μ g/ml, the women be 7.72～30.34 μ g/ml (normal reference value be calculated as mean value ± 1.96SD).
Embodiment 2
Testing conditions and method example
1. the preparation of kit:
(1) reference substance: concentration is 0.2,0.5,1,5,10 μ g/ml 1,5-AG and add mark ([UL-in the 4 μ g/ml respectively
13C
6] 1,80% methanol solution 5-AG), 5 concentration point, each 200 μ l.
(2) quality-control product: concentration is 0.6,3,8 μ g/ml 1,5-AG and add mark ([UL-in the 4 μ g/ml respectively
13C
6] 1,80% methanol solution 5-AG), 3 concentration point, each 1000 μ l.
(3) protein precipitant: mark ([UL in the 5 μ g/ml
-13C
6] 1, methyl alcohol 5-AG), cumulative volume 20ml.
Table 2 control group and diabetes group blood plasma 1,5-AG content
Annotate:
##Compare with aged control and diabetes group P＜0.001;
*Compare with young control and aged control P＜0.001.
2. blood specimen collection:
Gather venous blood to sample hose, room temperature is placed, centrifugal separation plasma in 8 hours.
3. sample preparation:
Get blood plasma 50 μ l to be measured in the 1.5ml centrifuge tube, add 200 μ l protein precipitant vortexs vibrations 10sec, centrifugal (protein precipitation of 15000g * 3min) is got 20 μ l supernatant sample introductions and is measured.
4. the preparation of typical curve:
Get reference substance and quality-control product direct injected, be equivalent to blood sample 1,5-anhydroglucose determining alcohol 1,2.5,5,25,50 and 3,15,40 μ g/ml.
Liquid chromatography tandem mass spectrum (LC-MS/MS) condition determination
Shimadzu series liquid chromatograph instrument (day island proper Tianjin company) comprises LC20ADvp pump, SIL-HTc automatic sampler and the online degassing instrument of DGU-20A3.Analytical column: CAPCELL PAK NH2 UG80 S5 column (150mm * 2.0mm i.d., 5 μ m, Shiseido).Guard column: Phenomenex NH2 (4.0mm * 3.0mm i.d.5 μ m).Moving phase: acetonitrile: water (80: 20, v/v).Flow velocity: 0.3mL/min.The bulk analysis time: 5min.
The electron spray ionisation source, the scanning of negative ion multiple-reaction monitoring.Electron spray voltage-4500V, 450 ℃ of ion source temperatures.Ion selector channel (Q1/Q3): 1,5-AG 163〉101 m/z, and IS 169〉105 m/z.The ion channel scope can be ± 1.0amu.
5. density calculating method
With peak area or peak height method, calculate 1, the content of 5-AG in blood by internal standard method.
Embodiment 3
Concentration is selected
Present embodiment has been selected different reagent concentrations, presses testing conditions and the method for embodiment 2, has measured 1, the linear relationship of 5-AG detection kit, accuracy and precision.
The concentration of reference substance is 0.05,0.5,5,50 and 100 μ g/ml 1,5-AG and add mark ([UL-in the 30 μ g/ml respectively
13C
6] 1,80% methanol solution 5-AG).5 concentration point, each 200 μ l.The concentration of quality-control product is 0.1,5,80 μ g/ml 1,5-AG and add mark ([UL-in the 30 μ g/ml respectively
13C
6] 1,80% methanol solution 5-AG).3 concentration point, each 1000 μ l.Protein precipitant is mark ([UL-in the 37.5 μ g/ml
13C
6] 1, methyl alcohol 5-AG).Cumulative volume 20ml.
Measurement result shows 1, and the linear relationship of 5-AG is Y=0.0662X+1.216, and the correlation coefficient r value is 0.9993, and accuracy 96.3-102.5%, precision %CV are 1.35-6.43%.Obtain accurate, reliable measurement result.
Embodiment 4
Chromatographic column is selected
The collection of kit, blood sample and the method for processing, and mass spectrum condition and embodiment 2 with, chromatographic condition center pillar adopt nh 2 column CAPCELL PAK NH2 UG80 S5 column (150mm * 2.0mm i.d., 5 μ m, Shiseido).With this understanding, 1,5-AG and interior target retention time are 3.6min, as seen chromatogram has the identical crest of a figure at this retention time place under the representational isocratic elution condition, does not have tangible endogenous material and disturbs.
Embodiment 5
The methodology checking
Adopt embodiment 2 listed testing conditions and method, assay method has been carried out the methodology checking with kit.In blood plasma adds 1～50 μ g/ml concentration range, 1,5-AG presents good linear relationship (r 〉=0.9990), and lowest detectable limit can reach 0.1 μ g/ml.In batch, betweenrun precision RSD respectively≤6.37% and≤8.75%, accuracy is 96.2～103.1%.The recovery is 98.0～103.8%, RSD≤3.3%, medium effect are 94.5～100.9%, 1, the 5-AG stock solution was placed 2 months down at 4 ℃, at room temperature place 24h after the sample preparation, plasma sample is at room temperature placed 6h through 3 multigelations, with preservation under-20 ℃ 3 months, study on the stability is the result show, 1, and 5-AG is stable in above-mentioned condition.The presentation of results LC-MS/MS of methodology checking can be sensitive, accurately and rapidly clinical sample in enormous quantities is detected.
1. typical curve and quantitatively scope
Be mixed with concentration by embodiment 2 methods and be respectively 1,2.5,1 of 5,10,25,50 μ g/ml, the 5-AG typical curve repeats sample introduction 3 times, altogether 3 cover typical curves.With 1,5-AG concentration is the x axle, 1, and 5-AG is the y axle with the ratio of interior mark peak area, carries out linear regression, sets up regression equation.The result shows: the typical curve equation is Y=0.0553X+0.197, and the r value of 3 batches of bioassay standard curves is respectively 0.9998,0.9997,0.9991.See table 3 for details.The lowest detection of this law is limited to 0.1 μ g/ml.
Table 31,5-AG standard curve determination result (3 batches, every crowd of n=2)
Annotate: CV: the coefficient of variation; Bias: bias.
2. accuracy and precision
Be mixed with concentration by embodiment 2 methods and be respectively 1,3,1 of 15,40 μ g/ml, 5-AG quality-control product.Every kind of concentration is prepared 6 parts, handles and analyzes by above-mentioned sample treatment and liquid chromatography tandem mass spectrum condition on the same day, calculates withinrun precision and accuracy.As stated above, divide 3 days preparation 3 batch samples to handle and analyze, calculate betweenrun precision and accuracy.The results are shown in Table 4, batch interior, betweenrun precision CV%≤10% are between the accuracy 90～110%.
Table 4 accuracy and precision result (3 batches, every crowd of n=6)
3. the recovery and matrix effect
Sample concentration, disposal route and sampling condition all with under precision and the correctness item, record the recovery and the results are shown in Table 5, and the recovery is all between 90～110%.The matrix effect influence is very little.
Table 5 recovery and matrix effect result (n=6)
Table 6 stability result
(table 6 is continuous)
4. stable
Sample concentration, disposal route and sampling condition are all with under precision and the accuracy item, investigate in the automatic sampler 24 hours, freeze thawing 3 times, room temperature place 6 hours ,-20 ℃ and placed 3 months, record precision and accuracy the results are shown in Table 6, precision CV%≤10% is between the accuracy 90～110%.
Embodiment 6
1,5-AG and other blood glucose target correlation analysiss
This research has been measured fasting blood-glucose (FBG), postprandial blood sugar (PPG), glycosylated hemoglobin (HbA1c), saccharification haemocyanin (FA) simultaneously and has been waited other blood sugar test indexs, analyzes 1, the correlativity of 5-AG and these indexs.As a new blood sugar test index, 1,5-AG and above-mentioned traditional index all demonstrate tangible correlativity.1, the correlation coefficient r of 5-AG and FBG, PPG, HbA1c and FA is respectively-0.351 (n=77, P＜0.005) ,-0.408 (n=81, P＜0.001) ,-0.635 (n=166, P＜0.001) ,-0.539 (n=63, P＜0.001).
Because clinical blood sugar monitoring in most cases, adopts easier finger to get blood and carries out blood sugar detection, be used for diabetes patient's diagnosis and treatment effectiveness evaluation.Clinical sample is common serum and plasma sample, and 44 routine diabetics' serum, blood plasma and finger blood gathered in this research simultaneously, 1 in the working sample, and 5-AG content is analyzed the correlativity between the three.The result shows three kinds of blood samples 1, and 5-AG does not have statistical discrepancy (P＜0.05), but is significantly relevant (P＜0.001), and correlation coefficient r is respectively 0.994 (serum and blood plasma), 0.984 (serum and finger blood), 0.980 (blood plasma and finger blood).
Claims (2)
1,1,5-dewatered grape sugar alcohol detection kit is characterized in that composition comprises:
(1) reference substance: contain 0.02-100 μ g/ml1, mark ([UL-in pure and mild 0.4-40 μ g/ml of 5 anhydroglucose
13C
6] 1,5 dewatered grape sugar alcohol) and 80% methanol solution;
(2) quality-control product: contain 0.06-80 μ g/ml1, mark ([UL-in 5-AG and 0.4-40 μ g/ml
13C
6] 1,5 dewatered grape sugar alcohol) and 80% methanol solution;
(3) protein precipitant: mark ([UL-in the 0.5-50 μ g/ml
13C
6] 1,5 dewatered grape sugar alcohol) and methyl alcohol or acetonitrile.
2,1, the detection method of 5-dewatered grape sugar alcohol detection kit may further comprise the steps:
(1) blood specimen collection: gather venous blood to sample hose, room temperature is placed, centrifugal separation plasma in 8 hours;
(2) blood sample pre-service: get blood plasma to be measured in the 1.5ml centrifuge tube, add 2-10 volume protein precipitant vortex vibration 10sec, centrifugal 15000g * 3min protein precipitation gets the supernatant sample introduction and measures;
(3) preparation of typical curve: get reference substance and quality-control product direct injected;
(4) the liquid chromatography tandem mass spectrum is measured;
(4.1) chromatographic condition adopts silica gel bonded phase filling post, and moving phase is acetonitrile or methyl alcohol by A phase+B phase composition: A mutually, and B is water mutually, A: the B volume ratio is (20-90%): (10-80%), adopt isocratic elution; Perhaps gradient elution, moving phase are formed proportioning with the elution time graded;
(4.2) mass spectrum condition electron spray ionisation source ESI, negative ion multiple-reaction monitoring scanning MRM, ion selector channel Q1/Q3:1,5 dewatered grape sugar alcohol m/z163〉101, ISm/z169〉105.The ion channel scope can be ± 1.0amu;
(5) density calculating method and detectability with peak area or peak height method, calculate the content of 1,5 dewatered grape sugar alcohol in blood by internal standard method.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110988165A (en) * | 2019-11-29 | 2020-04-10 | 上海市第六人民医院 | Saliva noninvasive detection method of 1,5-anhydroglucitol and application thereof |
| CN112485340A (en) * | 2019-11-27 | 2021-03-12 | 南京品生医学检验实验室有限公司 | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN112611814A (en) * | 2020-11-27 | 2021-04-06 | 大连润生康泰医学检验实验室有限公司 | Method for determining 1, 5-anhydroglucitol in dried blood slices |
| CN113748341A (en) * | 2017-12-20 | 2021-12-03 | 安德烈斯·黑兰-维韦斯 | Method for determining mean concentration of cortisol and glucose using cerumen |
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2008
- 2008-10-30 CN CNA2008102020095A patent/CN101446582A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113748341A (en) * | 2017-12-20 | 2021-12-03 | 安德烈斯·黑兰-维韦斯 | Method for determining mean concentration of cortisol and glucose using cerumen |
| CN112485340A (en) * | 2019-11-27 | 2021-03-12 | 南京品生医学检验实验室有限公司 | Method for detecting 1, 5-sorbitan in plasma by ultra-high performance liquid chromatography tandem mass spectrometry |
| CN110988165A (en) * | 2019-11-29 | 2020-04-10 | 上海市第六人民医院 | Saliva noninvasive detection method of 1,5-anhydroglucitol and application thereof |
| CN112611814A (en) * | 2020-11-27 | 2021-04-06 | 大连润生康泰医学检验实验室有限公司 | Method for determining 1, 5-anhydroglucitol in dried blood slices |
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Application publication date: 20090603 |