CN115902039A - Method for detecting concentration of melatonin in blood plasma - Google Patents
Method for detecting concentration of melatonin in blood plasma Download PDFInfo
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- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical compound COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 title claims abstract description 92
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Abstract
The invention provides a method for detecting concentration of melatonin in blood plasma. The method comprises the steps of carrying out protein precipitation pretreatment, enriching, concentrating and extracting by using a C18-E solid phase extraction column to obtain a sample supernatant, analyzing a target object by using a liquid chromatography-tandem mass spectrum (LC-MS/MS), and calculating the concentration of melatonin in blood plasma by establishing a standard curve through an internal standard method, thereby realizing a more comprehensive method for detecting the concentration of melatonin in blood plasma. The method has the advantages of high sensitivity, high flux, simplicity, convenience and rapidness and high extraction recovery rate, meets the quantitative requirements of various mass spectrum devices, and can be applied to the detection of the concentration of the melatonin clinically.
Description
Technical Field
The invention belongs to the technical field of test analysis, and particularly relates to a method for detecting concentration of melatonin in blood plasma.
Background
Melatonin (MEL) is a potent neurotransmitter and hormone secreted by the pineal gland, primarily to help regulate circadian rhythm and sleep. Its role in sleep regulation is particularly important for Autism Spectrum Disorder (ASD) patients. The endogenous concentration of melatonin was determined by LC-MS/MS to be below 0.02-0.2ng/mL and varied with time and age. After oral administration of 6mg dose to 60 female subjects, the mean peak concentration was 12ng/mL and the peak time was approximately 0.75 hour. The male subjects took a 10mg dose of melatonin, with an average concentration of 9.8ng/mL. Major side effects of melatonin include drowsiness and lethargy (from MayoC lnic). While the reference range given by the Quest diagnosis is 2.0-80pg/mL in the daytime; the concentration of the solution at night is 18.5-180.0pg/mL. Therefore, it is necessary to develop a method for detecting the concentration of melatonin with high sensitivity and a wide quantitative range.
A Chinese patent (application number: 201610220614. X) applied in 2016 discloses a method and a kit for detecting melatonin in saliva by a high performance liquid chromatography tandem mass spectrometry technology, and a Chinese patent (application number: 202110651856.5) applied in 2021 Nanjing Protehealth medical science and technology Limited company discloses a method for detecting concentrations of 5-hydroxytryptamine and melatonin in serum, wherein both the two methods adopt a liquid-liquid extraction pretreatment mode, and extract liquid has high toxicity and is not suitable for large-flux application and scientific research.
Disclosure of Invention
The invention aims to provide a method for detecting the concentration of melatonin in blood plasma. According to the method, protein precipitation pretreatment is carried out, a C18-E solid phase extraction column is used for enrichment and concentration extraction to obtain sample supernatant, and a liquid chromatography tandem mass spectrum (LC-MS/MS) is used for analyzing a target object, so that the liquid mass analysis time required by a single sample is only 2 min, and the detection flux is greatly increased; the C18-E solid phase extraction column has low cost, high extraction recovery rate, high sensitivity and no obvious matrix effect, meets the quantitative requirements of mass spectrum detection equipment of multiple models, and can be applied to clinical detection work.
The method for detecting the concentration of the melatonin in the blood plasma comprises the following steps:
preprocessing a plasma sample containing melatonin to be detected to obtain a target sample solution;
and testing the melatonin of the target sample solution, and calculating the concentration of the melatonin in the plasma sample.
The operation of preprocessing the plasma sample containing melatonin to obtain a target sample solution comprises the following steps:
collecting a plasma sample containing melatonin to be detected, adding an internal standard solution, carrying out vortex oscillation, and adding ZnSO 4 And taking the solution and acetonitrile or methanol or a mixed solution of methanol and acetonitrile as a precipitant, uniformly mixing by vortex, centrifuging, taking supernatant, adding diluent, uniformly mixing, enriching by adopting a solid phase extraction column, collecting eluent, drying by nitrogen, redissolving, and taking supernatant to obtain the target sample solution.
The step of testing the melatonin of the target sample solution and calculating the concentration of the melatonin in the plasma sample comprises the following steps:
preparing standard curve sample solutions containing different melatonin with known concentration, and drawing a standard curve of the standard curve sample solutions;
testing the target sample solution by using a high performance liquid chromatography tandem mass spectrum, and obtaining the content of the melatonin in the target sample solution according to the standard curve so as to obtain the content of the melatonin in the plasma sample containing the melatonin to be tested;
the step of preparing standard curve sample solutions containing different melatonin with known concentrations and drawing a standard curve of the standard curve sample solutions comprises the following steps of:
first, a standard containing different known melatonin concentrations was preparedAdding the internal standard solution into the curve sample, performing vortex oscillation, and adding ZnSO 4 Taking the solution and acetonitrile or methanol or a mixed solution of methanol and acetonitrile as a precipitant, carrying out vortex mixing, centrifuging, taking supernatant, adding diluent, mixing uniformly, enriching by adopting a solid phase extraction column, eluting, collecting eluent, drying the eluent, redissolving, carrying out vortex oscillation, centrifuging, and taking supernatant, thereby obtaining a series of standard curve sample solutions containing different melatonin with known concentration;
secondly, testing the standard curve sample solution by using high performance liquid chromatography tandem mass spectrometry, calculating the peak area ratio of melatonin to an internal standard, and establishing a standard curve by using an internal standard method.
In the method, the internal standard solution is MEL-d4 solution, and the concentration of the internal standard solution can be 0.3-0.5ng/mL;
the volume ratio of the plasma sample or standard curve sample containing melatonin to be detected to the internal standard solution can be 10:1;
ZnSO 4 the concentration of the solution is 0.2-3.3mol/L;
plasma sample or standard curve sample containing melatonin to be detected and ZnSO 4 The volume ratio of the solution can be 1:1-1:2;
the volume ratio of the plasma sample or the standard curve sample containing the melatonin to be detected to the methanol or the acetonitrile or the mixed solution of the methanol or the methanol and the acetonitrile can be 1:2-1:4;
the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile can be 1-2:8-9, specifically 2:8 or 1:9.
Adding ZnSO 4 The solution and methanol or acetonitrile or the mixed solution of methanol or methanol and acetonitrile are mixed evenly by vortex under the condition of 2000rpm for 5min;
the centrifugation condition can be 15000g,4 ℃, 5 minutes;
the diluent can be water;
the solid phase extraction column is a C18-E solid phase extraction column, and can be specifically an unblocked C18-E solid phase extraction column;
the steps of adopting a solid phase extraction column for enrichment, eluting and collecting eluent are as follows: pretreating the solid phase extraction column, loading, eluting with eluent 1 and eluent 2, eluting with eluent, collecting eluent,
the pretreatment comprises the steps of firstly adopting an activating solution to activate a solid-phase extraction column, and then balancing the activated solid-phase extraction column by using a balancing solution;
the activating solution is methanol; the equilibrium solution is 0.01 to 1 percent ammonia water, the leacheate 1 is water, the leacheate 2 is acetonitrile,
the eluent is acetonitrile;
the solvent for redissolution is a mixed solution of methanol and water with the volume ratio of 1:1;
after redissolution, vortex shaking was carried out at 2000rpm 1min, centrifugation was carried out at 15000g,4 ℃ for 5 minutes.
The liquid chromatography conditions were: and (3) chromatographic column: waters, ACQU I TY UPLC BEH C18.7 μm I VD 2.1X 50mm; the column temperature is 40 ℃; the injector temperature was 4 ℃; mobile phase a was water (containing 0.05mmo L/L ammonium fluoride), B was methanol, and gradient elution (0-0.5 min, mobile phase B increased from 30% to 35%;0.5-0.8min, mobile phase B increased from 35% to 75%;0.8-1.49min, mobile phase B was 75%;1.49-1.50min, mobile phase B decreased from 75% to 30%;1.51-2.00min, mobile phase B was 30%); the flow rate is 0.3mL/min; the amount of the sample was 10. Mu.L.
The mass spectrum parameters are as follows: ionization mode: ES I +; the flow rate of the atomizing gas (N) is 3L/min; heater flow rate (F): 12L/mi n; interface temperature (I) is 400 ℃; DL temperature (D) is 150 ℃; the temperature (H) of the heating block is 150 ℃; the flow rate of the drying gas (G) is 8L/min; multiple reaction detection (MRM);
the quantitative range of melatonin of the standard curve is as follows: 0.8pg/mL-400pg/mL; the reported range is 0.0008ng/mL to 40ng/mL.
The invention has the beneficial effects that:
(1) The invention sets up a detection method with wider reporting range from clinical actual requirements, can simultaneously consider the requirements of different concentration ranges of normal people and people needing to take melatonin, is qualitative and quantitative, is beneficial to unifying detection standards and saves experimental resources;
(2) The invention adopts an uncapped C18 solid-phase extraction column-C18-E, has better sensitivity, the lower limit of quantification is 0.8pg/mL, and the method can be suitable for the detection requirements of various mass spectrum devices; the price of C18-E is far lower than that of waters HLB, so that the cost of clinical application and scientific research is greatly reduced;
(3) In the invention, the liquid mass analysis time is only 2 min, so that baseline separation can be achieved, no interference of miscellaneous peaks is caused, and the detection flux of a clinical laboratory is greatly increased;
the detection method provided by the invention meets the verification requirements, verifies the interference acceptance standards of hemolysis, lipemia and jaundice, and better meets the clinical requirements.
According to the method, from the clinical practical requirements, the solid-phase extraction column is adopted to enrich the melatonin, and the LC-MS/MS analysis time is 2.0 min, so that the detection flux is greatly increased; meanwhile, the invention meets the methodological requirements of '2020 version Chinese pharmacopoeia-9012 biological sample quantitative analysis method verification guide principles' and 'liquid chromatography-mass spectrometry clinical application suggestions (guidelines and consensus)' (J. China inspection medicine, 2017,40 (10): 770-779), has high extraction recovery rate and high sensitivity, meets the quantitative requirements of mass spectrometry detection equipment of multiple models, and can be applied to clinical actual detection work.
Drawings
Fig. 1 is a chemical structural formula of melatonin;
FIG. 2 is a chromatogram of melatonin and its internal standard in plasma;
fig. 3 is a chromatogram of gradient 1 melatonin;
fig. 4 is a chromatogram of gradient 2 melatonin.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 establishment and validation of a method for detecting melatonin in plasma
1. Materials and reagents
Melatonin (MEL) (TCI, the purity is more than or equal to 98 percent) and melatonin-d 4 (MEL-d 4) (TRC, the purity is more than or equal to 98 percent); 10 × 0.01m × pbs (So l arbi o), ammonium fluoride (HPLC grade, si gma), acetonitrile (chromatographically pure, 99.9% purity, FI SHER), methanol (chromatographically pure, 99.9% purity, merck), water (homemade ultrapure water), zinc sulfate heptahydrate (HPLC grade, mirelin), ammonia (HPLC grade, sigma).
2. Instrument and apparatus
LCMS-8060NX CE high performance liquid chromatography-tandem mass spectrometer, wherein the detector is triple quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; a Mi l iQ water purifier; a vortex mixer; a nitrogen blowing instrument; a solid phase extractor; the filler used for the solid phase extraction column: C18-E (50 mg); and (3) chromatographic column: waters, ACQU I TY UPLC BEH C181.7 μm IVD 2.1X 50mm
3. Preparing a standard curve and a quality control base working solution:
preparing various series of standard curves and quality control working solutions according to requirements, and performing vortex mixing to obtain standard curves and quality control samples with various concentrations. Finally, the standard curve concentrations and quality control concentrations are shown in table 1 below:
TABLE 1 Final concentrations and formulations of standard curves and quality control plasma samples
| ng/mL | MEL | Source of formulation | Stock solution (mu L) | 50% methanol-water (μ L) |
| WS A | 0.008 | WS E | 5 | 95 |
| WS B | 0.016 | WS E | 10 | 90 |
| WS C | 0.04 | WS E | 20 | 60 |
| WS D | 0.08 | WS E | 50 | 50 |
| WS E | 0.16 | WS J | 10 | 240 |
| WS F | 0.4 | WS J | 10 | 90 |
| WS G | 0.8 | WS J | 20 | 80 |
| WS H | 1.6 | WS J | 32 | 48 |
| WS I | 2 | WS J | 50 | 50 |
| WS J | 4 | MEL:100ng/ |
12 | 288 |
| WS QC LLOQ | 0.008 | WS QC M | 16 | 464 |
| WS QC L | 0.02 | WS QC M | 37.5 | 412.5 |
| WS QC M | 0.24 | WS QC H | 37.5 | 462.5 |
| WS QC H | 3.2 | MEL:100ng/mL | 19.2 | 580.8 |
| WS QC UL | 4 | WS QC UH | 4 | 356 |
| WS QC UH | 360 | MEL:10μg/mL | 18 | 482 |
4. Standard curve, quality control plasma sample pretreatment process:
standard curve, quality control plasma sample preparation by taking 180. Mu.L of blank matrix (0.01M PBS), adding 20. Mu.L of base working solution, adding 20. Mu.L of internal standard base working solution (MEL-d 4 is 0.5 ng/mL), and vortexing for standby.
Plasma sample preparation, 200. Mu.L of sample was taken, 20. Mu.L of internal standard base working solution (MEL-d 4 0.5 ng/mL) was added, and vortexed until ready for use.
Precipitation process, taking standard curve or plasma sample, adding 200 μ LZnSO 4 The solution (concentration 3.3 mo/L) was added with 400. Mu.L of methanol, centrifuged at 2000rpm 5min, 15000g,4 ℃ for 5 minutes, and 400. Mu.L of the supernatant was taken, added with 800. Mu.L of water, and vortexed at 2000rpm 30s to give SPE samples.
And (3) SPE (solid phase extraction) process: activating a C18-E solid phase extraction column by using 300 mu L of methanol (activation solution), balancing by using 300 mu L of 0.01% ammonia water (balance solution), loading 1200 mu L of precipitated sample, leaching by using 500 mu L of water (eluent 1), leaching by using 50 mu L of acetonitrile (eluent 2), finally eluting by using 400 mu L of acetonitrile (eluent), receiving the eluent, blowing dry by using nitrogen at 40 ℃, adding 80 mu L of methanol-water (1, v/v), carrying out vortex oscillation at 200rpm 1mi n,15000g,4 ℃, centrifuging for 5 minutes, and sampling 50 mu L of supernatant for analysis.
5. Detection of melatonin: the following chromatographic and mass spectrometric conditions were used for this experiment:
liquid chromatography conditions: a chromatographic column: waters, ACQU I TY UPLC BEH C18.7 μm IVD 2.1X 50mm; the column temperature is 40 ℃; the sample injector temperature is 4 ℃; mobile phase a was water (containing 0.05mmol/L ammonium fluoride), B was methanol, and gradient eluted (0-0.5 min, mobile phase B increased from 30% to 35%;0.5-0.8min, mobile phase B increased from 35% to 75%;0.8-1.49min, mobile phase B75%; 1.49-1.50min, mobile phase B decreased from 75% to 30%;1.51-2.00min, mobile phase B30%); the flow rate is 0.3mL/min; the amount of the sample was 10. Mu.L.
The mass spectrum parameters are as follows: ionization mode: ES I +; the flow rate of the atomizing gas (N) is 3L/min; heater flow rate (F): 12L/min; interface temperature (I) is 400 ℃; DL temperature (D) is 150 ℃; the temperature (H) of the heating block is 150 ℃; the flow rate of the drying gas (G) is 8L/min; multiple reaction detection (MRM);
6. evaluation of methodology
6.1 the invention is verified according to the verification guide principle of the quantitative analysis method of the biological samples in '2020 edition' Chinese pharmacopoeia-9012 'and' clinical application suggestion (guide and consensus) of liquid chromatography-mass spectrometry (J.Zhonghua inspection medicine, 2017,40 (10): 770-779) and the verification guide of the performance of the CNAS-GL037 clinical chemistry quantitative inspection program: including specificity and selection, interference, linear range, sensitivity, precision, accuracy (spiking recovery), matrix effects, stability, and dilution reliability.
(1) Specificity and selectivity Studies
After the double blank, single blank and LLOQ samples are processed according to the items of '4, standard curve and quality control plasma sample pretreatment process', LC-MS/MS analysis is carried out, and the result shows that under the experimental condition, each object to be detected and the internal standard peak have good shapes and higher specificity. And (3) no interference of an impurity peak exists on two sides of a chromatographic peak, and the requirements that no background peak exists or the interference peak area is less than 20% of the quantitative lower limit peak area of an analyte and less than 5% of the internal standard peak area are met.
TABLE 2 melatonin Selectivity and specificity
(2) Interference
After the treatment is carried out according to the items of '4, standard curve and quality control plasma sample pretreatment process', the interference result is shown in a table 3; after the plasma sample is treated, the relative deviation of the measurement result after the plasma sample is treated by adding the interfering substances is less than 15%, and the result shows that the severe hemolysis, the severe lipemia and the moderate jaundice (combination and non-combination) have no influence on the detection result.
TABLE 3 interference measurement results
(3) Linear range
Processing a standard curve plasma sample according to the item of '4, standard curve and quality control plasma sample pretreatment process', performing LC-MS/MS analysis, performing weight linear regression on the drug concentration X according to the ratio Y (Y = As/Ai) of each drug peak area As and internal standard peak area Ai, fitting the standard curve, wherein the weight is 1/X 2 (X is concentration value), the standard curve expressed as Y = slope X + intercept is shown in table 4.
TABLE 4 Standard Curve and Linear Range
(4) Precision and accuracy
And (3) processing the quality control sample according to the item of '4, standard curve and quality control plasma sample pretreatment process', performing LC-MS/MS analysis, parallelly preparing 6 parts of each concentration in each batch, continuously preparing for three days, and preparing one batch every day. The ratio Y of the drug peak area As to the internal standard peak area Ai (Y = As/Ai) was substituted into the daily follow-up standard to calculate the sample concentration, thereby calculating the intra/inter-batch precision (CV <15% is qualified) and accuracy (CV <15% is qualified) and the precision and accuracy data of each drug are shown in table 5.
TABLE 5 precision and accuracy
(5) Matrix effect
Preparing a pure solution sample: a1.5 mL EP tube was taken and the solution WS QCM 40. Mu.L was taken, and 360. Mu.L of 50% aqueous methanol was added and vortexed for 30 seconds. And (4) performing liquid chromatography-tandem mass spectrometry analysis on the supernate according to the process operation of '4, standard curve and quality control plasma sample pretreatment process'. In parallel 6 portions are made. Calculating the mean value y of the ratio of the response values of the substance to be detected and the internal standard in the pure solution sample s 。
Preparing a plasma sample: a1.5 mL EP tube was used to collect 200. Mu.L of 6 different human plasma samples (including severe hemolysis, severe lipemia, and moderate jaundice samples). And (3) performing liquid chromatography-tandem mass spectrometry on the supernatant according to the process operation of '4, standard curve and quality control plasma sample pretreatment process'. Calculating the mean value y of the ratio of the response values of the to-be-detected object and the internal standard in the plasma sample x 。
Plasma: preparation of a pure solution mixture (3: a1.5 mL EP tube was used, and 60. Mu.L and 140. Mu.L of the above two samples were taken, respectively, and vortexed for 30 seconds. According to the operation of the process of sample pretreatment, taking the supernatant to carry out liquid chromatography-tandem mass spectrometry. Calculating the mean value of the ratio of the response values of the to-be-detected object and the internal standard in the mixture sample to be 7 s+x 。
Plasma: preparation of a pure solution mixture (7: a1.5 mL EP tube was used, and 140. Mu.L and 60. Mu.L of each of the above two samples were taken, and vortexed for 30 seconds. According to the operation of the process of sample pretreatment, taking the supernatant to carry out liquid chromatography-tandem mass spectrometry. Calculating the analyte and internal standard in the mixture sampleMean value of ratio of response values 3 s+x 。
Each group is corresponded to y x ，y s And y s+x Substituting into a formula: deviation (%) = |1- (y) s+x )/(y x *0.3+y s *0.7 100%, deviation (%) = |1- (y) s+x )/(y x *0.7+y s *0.3 And) 100% to obtain the relative matrix effect deviation of the object to be detected. The results are shown in Table 6:
TABLE 6 matrix Effect
(6) Stability survey
Adopting low-concentration and high-concentration QC plasma samples, calculating the concentration of each drug by substituting the samples into the following standard curve on the same day after analyzing the samples, and respectively investigating the stability of the samples after being placed for 3 days at room temperature, the stability of the samples after being placed for 7 days at 2-8 ℃, the stability of freeze-thaw triple circulation, the stability of the samples after being placed for 30 days at-20 ℃ and the stability of the samples after being placed for 48 hours at 4 ℃ of an automatic sample injector after being processed, wherein the results are shown in Table 7;
TABLE 7 stability
(7) Dilution reliability
Blank control plasma samples: 200 μ L of pooled human plasma was used as a blank plasma sample. And performing liquid chromatography-tandem mass spectrometry according to the operation of the sample pretreatment process. And (5) calculating the ratio y (y = As/Ai) of each peak area As of the object to be measured in the plasma sample to the peak area Ai of the internal standard, and substituting the ratio y into the daily follow working curve to calculate the sample concentration blank.
Adding a standard plasma sample: taking 200 mu L of standard quality control basic working solution (WS QCUL, WS QCUH), adding 1.80mL of mixed human plasma, and performing vortex oscillation to prepare plasma with the Melatonin (MEL) addition standard concentration of 0.4 and 36ng/mL in the background.
Dilution of the spiked plasma samples: when the dilution factor is 50, 40 μ L of the 0.4ng/mL plasma is taken, and 1.96mL blank matrix is added to prepare the plasma with the Melatonin (MEL) concentration of 8pg/mL in the background. When the dilution factor is 100, 20 mu L of the 36ng/mL plasma is taken, 1.98mL blank matrix is added, and plasma with the Melatonin (MEL) concentration of 360pg/mL in the background is prepared. 6 parts of the total amount of each concentration are parallelly prepared, and liquid chromatography-tandem mass spectrometry is carried out according to the operation of a sample pretreatment process. And (5) calculating the ratio y (y = As/Ai) of each peak area As of the object to be measured in the plasma sample to the peak area Ai of the internal standard, and substituting the ratio y into the daily follow-up working curve to calculate the concentration C of the sample for adding the standard.
Diluting the sample extracting solution: in the background, 6 parts of Melatonin (MEL) is added into plasma with standard concentrations of 0.4ng/mL and 36ng/mL in parallel, and after the operation according to the process of 'sample pretreatment' and when the dilution factor is 50, 5 mu L of supernatant of the plasma with the standard concentration of 0.4ng/mL is taken, and 245 mu L of IS dilution liquid is added; when the dilution factor is 100, 5. Mu.L of the supernatant of the 36ng/mL plasma was taken and 495. Mu.L of IS dilution was added.
And substituting the C adding mark, the C blank and the C theory corresponding to each group into a formula: the normalized recovery rate (%) = C normalized/((C blank + C theory)/50) × 100% and the normalized recovery rate (%) = C normalized/((C blank + C theory)/100) × 100%, and the normalized recovery rate was calculated. The results are shown in Table 8;
TABLE 8 dilution reliability
The above items meet the validation guidelines of the quantitative analysis method of biological samples in ' 2020 edition ' Chinese pharmacopoeia-9012 ' and the clinical application suggestions (guidelines and consensus) of liquid chromatography-mass spectrometry (Chinese journal of medical inspection, 2017,40 (10): 770-779) and CNAS-GL037, and can be applied to the detection work of melatonin.
Example 2 screening of extraction conditions for pretreatment of melatonin
1. Materials and reagents
Melatonin (MEL) (TCI, purity is more than or equal to 98 percent) and melatonin-d 4 (MEL-d 4) (TRC, purity is more than or equal to 98 percent); 10 × 0.01M × PBS (So l arbio), ammonium fluoride (HPLC grade, sigma), acetonitrile (chromatographically pure, 99.9% purity, FI SHER), methanol (chromatographically pure, 99.9% purity, merck), water (homemade ultrapure water), zinc sulfate heptahydrate (HPLC grade, michelin), ammonia (HPLC grade, sigma)
2. Apparatus and device
LCMS-8060NX CE high performance liquid chromatography-tandem mass spectrometer, wherein the detector is triple quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; a Mi l iQ water purifier; a vortex mixer; a nitrogen blowing instrument; a solid phase extractor.
3. Detection of melatonin: the following chromatographic and mass spectrometric conditions were used for this experiment:
liquid chromatography conditions: a chromatographic column: waters, ACQUI TY UPLC BEH C18.7 μm IVD 2.1X 50mm; the column temperature is 40 ℃; the injector temperature was 4 ℃; mobile phase a was water (containing 0.05mmo L/L ammonium fluoride), B was methanol, and gradient elution (0-0.5 min, mobile phase B increased from 30% to 35%, 0.5-0.8min, mobile phase B increased from 35% to 75%, 0.8-1.49min, mobile phase B decreased from 75%, 1.49-1.50min, mobile phase B decreased from 75% to 30%, 1.51-2.00min, mobile phase B30%); the flow rate is 0.3mL/min; the sample size was 10. Mu.L.
The mass spectrum parameters are as follows: ionization mode: ESI +; the flow rate of the atomizing gas (N) is 3L/min; heater flow rate (F): 12L/mi n; interface temperature (I) is 400 ℃; DL temperature (D) is 150 ℃; the temperature (H) of the heating block is 150 ℃; the flow rate (G) of the drying gas is 8L/min; multiple reaction detection (MRM);
4. comparison of extraction conditions before melatonin
(1) Condition 1: a sample of 200. Mu.L of plasma (standard curve or quality control plasma) was taken in a 1.5mL tube, 20. Mu.L of internal standard solution (MEL-d 4 at 0.3 ng/mL) was added, and vortexed. Taking a standard curve or a plasma sample, and adding 200 mu LZnSO 4 The solution (3.3 mol/L) was added with 400. Mu.L of methanol, vortexed at 2000rpm5mi n,15000g,4 ℃ and centrifuged for 5 minutes, and 400. Mu.L of the supernatant was taken with 800. Mu.L of water, vortexed at 2000rpm 30s.
C18-E (50 mg) SPE procedure: activating a C18-E solid phase extraction column by using 300 mu L of methanol (activation solution), balancing by using 300 mu L of 0.01% ammonia water (balance solution), loading 1200 mu L of precipitated sample, leaching by using 500 mu L of water (eluent 1), leaching by using 50 mu L of acetonitrile (eluent 2), finally adding 400 mu L of acetonitrile eluent, receiving the eluent, drying by using nitrogen at 40 ℃, adding 80 mu L of methanol-water (1, v/v), carrying out vortex oscillation at 2000rpm1mi n,15000g,4 ℃, centrifuging for 5 minutes, and sampling and analyzing 50 mu L of supernatant.
(2) Condition 2: a200. Mu.L sample of plasma (standard curve or quality control plasma) was placed in a 1.5mL tube, 20. Mu.L of internal standard solution (MEL-d 4 at 0.3 ng/mL) was added, and vortexed. A standard curve or a plasma sample is taken, 400 mu L of methanol is added, vortex oscillation is carried out for 2000rpm for 5min, 15000g is carried out at 4 ℃, centrifugation is carried out for 5min, and 400 mu L of supernatant and 800 mu L of water are taken, vortex oscillation is carried out for 2000rpm for 30s. SPE process: see condition 1. (results are shown in Table 9)
TABLE 9 comparison of Condition 1 and Condition 2 results
(3) Condition 3: a sample of 200. Mu.L of plasma (standard curve or quality control plasma) was taken in a 1.5mL tube, 20. Mu.L of internal standard solution (MEL-d 4 at 0.3 ng/mL) was added, and vortexed. Taking a standard curve or a plasma sample, and adding 200 mu LZnSO 4 The solution (3.3 mol/L) was added with 400. Mu.L of methanol, and vortexed at 2000rpm1mi n. The rest processes are as follows: see condition 1. (results are shown in Table 10)
TABLE 10 comparison of Condition 1 and Condition 3 results
(4) Condition 4: C18-E (50 mg) SPE procedure: activating solution: 300 μ L of methanol, equilibrium: 300 μ L of water, rest procedure: see condition 1. (results are shown in Table 11)
TABLE 11 comparison of Condition 1 and Condition 4 results
(5) Condition 5: the SPE procedure was performed on C8 (50 mg) and the rest was seen in Condition 1. (results are shown in Table 12)
TABLE 12 comparison of Condition 1 and Condition 5 results
Analysis of results
Comparing 5 different extraction conditions, the result shows that the ZnSO is not added in the condition 2 4 The solution response will decrease by about 2-fold; the condition 3 is that the vortex mixing time is short, which directly causes the lower recovery rate of the added standard; in the condition 4, no ammonia water is added into the equilibrium liquid, so that the response is reduced by nearly one time; the C8 solid phase extraction column is adopted under the condition 5, and the response is reduced by about 2 times; the condition 1 adopts an unblocked C18-E solid phase extraction column, has strong nonpolar interaction force and polar interaction, and has better retention for a medium-polarity compound such as melatonin, so that the response and the extraction recovery rate of the condition 1 are both in accordance with requirements.
Example 3 screening of melatonin chromatographic gradient conditions
1. Materials and reagents
Melatonin (MEL) (TCI, the purity is more than or equal to 98 percent) and melatonin-d 4 (MEL-d 4) (TRC, the purity is more than or equal to 98 percent); 10 × 0.01M × PBS (So l arbio), ammonium fluoride (HPLC grade, sigma), acetonitrile (chromatographically pure, 99.9% purity, FI SHER), methanol (chromatographically pure, 99.9% purity, merck), water (homemade ultrapure water), zinc sulfate heptahydrate (HPLC grade, michelin), ammonia (HPLC grade, sigma)
2. Instrument and apparatus
LCMS-8060NX CE high performance liquid chromatography-tandem mass spectrometer, wherein the detector is triple quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; a Mi l iQ water purifier; a vortex mixer; a nitrogen blowing instrument; a solid phase extractor.
3. Melatonin pretreatment:
a200. Mu.L sample of plasma (standard curve or quality control plasma) was placed in a 1.5mL tube, 20. Mu.L of internal standard solution (MEL-d 4 at 0.3 ng/mL) was added, and vortexed. Taking a standard curve or a plasma sample, and adding a precipitant 1 (ZnSO) 4 Solution 3.3 mol/L), adding precipitator 2 (methanol), whirling and vibrating at 2000rpm5mi n,15000g,4 ℃, centrifuging for 5 minutes, taking supernate, adding diluent, whirling and vibrating at 2000rpm 30s. C18-E (50 mg) SPE procedure: activating solution: 300 μ L, equilibration fluid: 300 μ L, sample loading, adding eluent 1 (500 μ L water), adding eluent 2 (50 μ L acetonitrile), adding eluent (400 μ L acetonitrile), receiving eluent, drying with nitrogen at 40 ℃, adding complex solution (80 μ L methanol-water (1, v/v)), vortex shaking 2000rpm1mi n,15000g,4 ℃, centrifuging for 5 minutes, and taking supernatant for sample injection analysis.
4. Detection of melatonin: the following chromatographic and mass spectrometric conditions were used for this experiment:
liquid chromatography conditions: a chromatographic column: waters, ACQU I TY UPLC BEH C18.7 μm IVD 2.1X 50mm; the column temperature is 40 ℃; the injector temperature was 4 ℃; the mobile phase A is water (containing 0.05mmol/L of ammonium fluoride) and the mobile phase B is methanol
The mass spectrum parameters are as follows: ionization mode: ES I +; the flow rate of the atomizing gas (N) is 3L/min; heater flow rate (F): 12L/min; interface temperature (I) is 400 ℃; DL temperature (D) is 150 ℃; the temperature (H) of the heating block is 150 ℃; the flow rate of the drying gas (G) is 8L/min; multiple reaction detection (MRM);
comparison of chromatographic gradient conditions
(1) Gradient 1:0-0.5 min, and the content of the mobile phase B is increased from 30% to 35%;0.5-0.8min, and the mobile phase B is increased from 35% to 75%;0.8-1.49min, and 75% of mobile phase B; 1.49-1.50min, and the content of the mobile phase B is reduced from 75% to 30%;1.51-2.00min, 30% mobile phase B (see figure 3 for separation results);
(2) Gradient 2:0-0.5 min, the mobile phase B is increased from 25% to 60%;0.5-0.8 min, the mobile phase B is increased from 60% to 95%;0.8-1.49 min, and 95% of mobile phase B; 1.49-1.50min, the content of mobile phase B is reduced from 95% to 25%;1.51-2.00 min, 25% mobile phase B (see figure 4 for separation results);
analysis of results
Comparing two different gradient conditions, selecting a single human plasma sample as the sample, and displaying the result by a gradient 1, wherein when the mobile phase B is increased from 35% to 75%, the melatonin and an impurity peak reach a baseline separation; gradient 2 shows that melatonin cannot be baseline separated from the impurity peaks. Thus, gradient 1 is selected.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A method for detecting melatonin concentration in plasma, said method comprising the steps of:
preprocessing a plasma sample containing melatonin to be detected to obtain a target sample solution;
testing the melatonin of the target sample solution, and calculating the concentration of the melatonin in the plasma sample;
the operation of preprocessing the plasma sample containing melatonin to obtain a target sample solution comprises the following steps: collecting a plasma sample containing melatonin to be detected, adding an internal standard solution, performing vortex oscillation, and adding ZnSO 4 Mixing the solution and methanol or acetonitrile or the mixture of methanol and acetonitrile as precipitant, vortex mixing, and separatingAnd (4) taking the supernatant, adding the diluent, mixing uniformly, enriching by using a solid phase extraction column, collecting the eluent, drying by using nitrogen, redissolving, and taking the supernatant to obtain the target sample solution.
2. The method of claim 1, wherein: the step of testing the melatonin of the target sample solution and calculating the concentration of the melatonin in the plasma sample comprises the following steps:
preparing standard curve sample solutions containing different melatonin with known concentrations, and drawing a standard curve of the standard curve sample solutions;
and testing the target sample solution by using a high performance liquid chromatography tandem mass spectrum, and obtaining the content of the melatonin in the target sample solution according to the standard curve so as to obtain the content of the melatonin in the plasma sample containing the melatonin to be tested.
3. The method according to claim 1 or 2, characterized in that: the step of preparing standard curve sample solutions containing different melatonin with known concentrations and drawing a standard curve of the standard curve sample solutions comprises the following steps of:
firstly, respectively preparing standard curve samples containing different melatonin with known concentration, adding internal standard solution, carrying out vortex oscillation, and adding ZnSO 4 Taking the solution and methanol or acetonitrile or a mixed solution of the methanol and the acetonitrile as a precipitator, carrying out vortex mixing, centrifuging, taking supernatant, adding diluent, mixing uniformly, adopting a solid phase extraction column for enrichment, eluting, collecting eluent, drying the eluent, redissolving, carrying out vortex oscillation, centrifuging, and taking supernatant, thus obtaining a series of standard curve sample solutions containing different melatonin with known concentration;
secondly, testing the standard curve sample solution by using a high performance liquid chromatography tandem mass spectrum, calculating the peak area ratio of the melatonin to an internal standard, and establishing a standard curve by using an internal standard method.
4. The method according to any one of claims 1-3, wherein: the internal standard solution is MEL-d4 solution, and the concentration of the internal standard solution is 0.3-0.5ng/mL;
the volume ratio of the plasma sample or the standard curve sample containing the melatonin to be detected to the internal standard solution is 10:1;
ZnSO 4 the concentration of the solution is 0.2-3.3mol/L;
plasma sample or standard curve sample containing melatonin to be detected and ZnSO 4 The volume ratio of the solution is 1:1-1:2.
5. The method according to any one of claims 1-4, wherein: the volume ratio of the plasma sample or the standard curve sample containing the melatonin to be detected to the methanol or the acetonitrile or the mixed solution of the methanol and the acetonitrile is 1:2-1:4;
the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 1-2:8-9;
adding ZnSO 4 The solution and methanol or acetonitrile or the mixture of methanol and acetonitrile are mixed by vortex for 5min under the condition of 2000 rpm.
6. The method according to any one of claims 1-5, wherein: the solid phase extraction column is a C18-E solid phase extraction column,
the steps of adopting a solid phase extraction column for enrichment, eluting and collecting eluent are as follows: pretreating the solid phase extraction column, loading, eluting with eluent 1 and eluent 2, eluting with eluent, collecting eluent,
the pretreatment comprises the steps of firstly adopting an activating solution to activate the solid-phase extraction column, and then balancing the activated solid-phase extraction column by using a balancing solution.
7. The method of claim 6, wherein: the activating solution is methanol; the equilibrium solution is 0.01 to 1 percent ammonia water, the leacheate 1 is water, the leacheate 2 is acetonitrile,
the eluent is acetonitrile;
the solvent for redissolution is a mixed solution of methanol and water in a volume ratio of 1:1.
8. The method according to any one of claims 1-7, wherein: the liquid chromatography conditions were: and (3) chromatographic column: waters, ACQUITY UPLC BEH C18.7 μm IVD 2.1X 50mm; the column temperature is 40 ℃; the injector temperature was 4 ℃; mobile phase a was water (containing 0.05mmol/L ammonium fluoride), B was methanol, gradient elution: the time is 0-0.5min, and the mobile phase B is increased from 30% to 35%; the time is 0.5-0.8min, and the mobile phase B is increased from 35% to 75%;0.8-1.49min, and 75% of mobile phase B; 1.49-1.50min, reducing the mobile phase B from 75% to 30%;1.51-2.00min, and 30% of mobile phase B; the flow rate is 0.3mL/min; the amount of the sample was 10. Mu.L.
9. The method according to any one of claims 1-8, wherein: the mass spectrum parameters are as follows: ionization mode: ESI +; the flow rate of atomizing gas (N) is 3L/min; heater flow rate (F): 12L/min; interface temperature (I) is 400 ℃; DL temperature (D) is 150 ℃; the temperature (H) of the heating block is 150 ℃; the flow rate of the drying gas (G) is 8L/min; multiple reaction detection (MRM).
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