阅读说明：本技术 一种检测人血浆左乙拉西坦浓度的hplc-uv方法 (HPLC-UV method for detecting human plasma levetiracetam concentration ) 是由 桑奕雯 陈萌 陶志华 王旭楚 高嵩 刘伟伟 于 2021-01-04 设计创作，主要内容包括：本发明公开了一种检测人血浆左乙拉西坦浓度的HPLC-UV方法,本发明通过采用甲醇直接沉淀蛋白法结合HPLC-UV检测人血浆左乙拉西坦浓度,只需要极少量血浆即可检测左乙拉西坦浓度,线性范围2-100μg/mL(R～2>0.99),批内与批间精密度均≤3.58％,准确度范围是96.09％-97.86％,本发明检测限低、选择性佳且具有极好的特异性和灵敏度,整个检测时间短,残留、干扰及提取回收率都满足要求,同时具有检测结果稳定的优点。(The invention discloses an HPLC-UV method for detecting human plasma levetiracetam concentration, which detects the human plasma levetiracetam concentration by combining a methanol direct precipitation protein method with HPLC-UV, and can detect the levetiracetam concentration only by a very small amount of plasma, wherein the linear range is 2-100 mu g/mL (R is 2-100 mu g/mL) 2 >0.99), the precision between batches is less than or equal to 3.58 percent, the accuracy range is 96.09 percent to 97.86 percent, the invention has low detection limit, good selectivity and excellent specificityAnd the sensitivity is high, the whole detection time is short, the residue, interference and extraction recovery rate meet the requirements, and the method has the advantage of stable detection result.)

1. An HPLC-UV method for detecting the concentration of human plasma levetiracetam is characterized in that: the method comprises the following steps:

s1 chromatographic conditions: a chromatographic column: diamonsil C18 column, 150mm × 4.6mm ID, 5 μm; mobile phase: the ratio of acetonitrile to 10mM potassium dihydrogen phosphate is 10: 90; flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction volume: 15 mu L of the solution; detection wavelength: 210 nm;

s2, solution preparation: weighing levetiracetam standard substance, dissolving with methanol to obtain levetiracetam stock solution with concentration of 1.0mg/mL, and refrigerating at 4 ℃ for later use;

s3, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions by using methanol according to a proportion, and adding blank plasma to prepare a levetiracetam standard curve plasma sample; the plasma sample concentrations of the levetiracetam standard curve are respectively as follows: 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 70. mu.g/mL, and 100. mu.g/mL; then respectively taking 50 mu L of plasma sample, adding 150 mu L of methanol, uniformly mixing for 1min by vortex, centrifuging at 15000r for 10min at high speed, and taking 15 mu L of supernatant for chromatographic sample injection;

s4, performing linear regression on the 7 concentration points subjected to chromatographic sampling in the step S3 by adopting 1/x weighting, namely performing a least square method on the sample concentration by adopting the peak area of levetiracetam to obtain a levetiracetam standard curve regression equation:

y＝2.16×10^-4x+0.816，(r²＝0.998)；

in the formula: y is the concentration of levetiracetam in plasma, and x is the area of the peak of levetiracetam;

s5, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions according to a proportion by using methanol, and adding blank plasma to prepare high, medium and low plasma quality control samples; the concentrations of the plasma quality control samples are respectively 4 mug/mL, 35 mug/mL and 80 mug/mL, 50 mug L of the plasma quality control samples are respectively taken, 150 mug L of methanol is added, vortex mixing is carried out for 1min, high-speed centrifugation is carried out for 10min at 15000r, and 15 mug L of supernatant is taken for chromatographic sample injection; continuously detecting for 3 days, and calculating the precision within and during the day;

s6, diluting levetiracetam stock solution with methanol in proportion into 5 parts of standard solutions with the concentrations of 4 mu g/mL, 35 mu g/mL and 80 mu g/mL respectively, then taking 50 mu L of standard solutions respectively, adding 150 mu L of methanol, uniformly mixing in a vortex manner for 1min, centrifuging at a high speed of 15000r for 10min, taking 15 mu L of supernatant, and carrying out chromatography sample injection; after the detection, the ratio of the peak area in the step S4 to the peak area in the present step was compared to determine the extraction recovery rate.

2. The HPLC-UV method for detecting the concentration of human plasma levetiracetam according to claim 1, characterized in that: in step S4, the minimum quantification limit of the plasma levetiracetam concentration is determined to be 2 μ g/mL according to RSN 10 and clinical diagnostic requirements.

Technical Field

The invention relates to the technical field of medical treatment, in particular to an HPLC-UV method for detecting the concentration of human plasma levetiracetam.

Background

Levetiracetam is a novel antiepileptic drug, can be used for single treatment of partial epileptic seizures, can also be used as adjuvant treatment of partial myoclonus and primary generalized tonic clonus seizures, and is introduced into China in 2007. The structure and the antiepileptic mechanism of the compound are different from other medicines, and mainly play a role in selectively inhibiting the supersynchrony of epileptiform burst discharge and the spread of epileptic seizures. The pharmacokinetics of levetiracetam vary between children and adults, with the pharmacokinetic processes of absorption, distribution, metabolism, and excretion changing with growth. Although levetiracetam is easy to take and tolerated, drug monitoring is required in many clinical treatments (suspected noncompliance, pregnancy, childhood dose, renal failure, and high dose ineffectiveness). At present, an HPLC method or an LC-MS/MS method is recommended to be adopted in the levetiracetam determination method, and the LC-MS/MS method is complex and time-consuming in sample pretreatment and expensive in instrument, and is difficult to popularize and apply to daily clinic. Therefore, it is necessary to develop a simple and convenient detection method using HPLC.

Disclosure of Invention

The invention aims to provide an HPLC-UV method for detecting the concentration of human plasma levetiracetam. The invention has the advantages of rapid detection, high sensitivity and high specificity.

The technical scheme of the invention is as follows: an HPLC-UV method for detecting the concentration of human plasma levetiracetam comprises the following steps:

s1 chromatographic conditions: a chromatographic column: diamonsil C18 column, 150mm × 4.6mm ID, 5 μm; mobile phase: the ratio of acetonitrile to 10mM potassium dihydrogen phosphate is 10: 90; flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction volume: 15 mu L of the solution; detection wavelength: 210 nm;

s2, solution preparation: weighing levetiracetam standard substance, dissolving with methanol to obtain levetiracetam stock solution with concentration of 1.0mg/mL, and refrigerating at 4 ℃ for later use;

s3, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions by using methanol according to a proportion, and adding blank plasma to prepare a levetiracetam standard curve plasma sample; the plasma sample concentrations of the levetiracetam standard curve are respectively as follows: 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 70. mu.g/mL, and 100. mu.g/mL; then respectively taking 50 mu L of plasma sample, adding 150 mu L of methanol, uniformly mixing for 1min by vortex, centrifuging at 15000r for 10min at high speed, and taking 15 mu L of supernatant for chromatographic sample injection;

s4, performing linear regression on the 7 concentration points subjected to chromatographic sampling in the step S3 by adopting 1/x weighting, namely performing a least square method on the sample concentration by adopting the peak area of levetiracetam to obtain a levetiracetam standard curve regression equation:

y＝2.16×10^-4x+0.816，(r²＝0.998)；

in the formula: y is the concentration of levetiracetam in plasma, and x is the area of the peak of levetiracetam;

s5, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions according to a proportion by using methanol, and adding blank plasma to prepare high, medium and low plasma quality control samples; the concentrations of the plasma quality control samples are respectively 4 mug/mL, 35 mug/mL and 80 mug/mL, 50 mug L of the plasma quality control samples are respectively taken, 150 mug L of methanol is added, vortex mixing is carried out for 1min, high-speed centrifugation is carried out for 10min at 15000r, and 15 mug L of supernatant is taken for chromatographic sample injection; continuously detecting for 3 days, and calculating the precision within and during the day;

s6, diluting levetiracetam stock solution with methanol in proportion into 5 parts of standard solutions with the concentrations of 4 mu g/mL, 35 mu g/mL and 80 mu g/mL respectively, then taking 50 mu L of standard solutions respectively, adding 150 mu L of methanol, uniformly mixing in a vortex manner for 1min, centrifuging at a high speed of 15000r for 10min, taking 15 mu L of supernatant, and carrying out chromatography sample injection; after the detection, the ratio of the peak area in the step S4 to the peak area in the present step was compared to determine the extraction recovery rate.

In step S4, the HPLC-UV method for detecting human plasma levetiracetam concentration determines the minimum limit of quantitation of plasma levetiracetam concentration to be 2 μ g/mL according to RSN 10 and clinical diagnosis requirements.

Compared with the prior art, the method provided by the invention has the advantages of rapidness in detection, high sensitivity and high specificity by adopting a methanol direct precipitation protein method and combining with HPLC-UV to detect the concentration of human plasma levetiracetam. The invention can detect the levetiracetam blood concentration of a patient, avoid poor curative effect or adverse reaction of clinical medication, provide an optimized administration scheme for clinic and provide reliable experimental basis for individualized medication of the patient. Experimental results show that the levetiracetam concentration can be detected only by a very small amount of blood plasma, and the linear range is 2-100 mu g/mL (R)²>0.99), the precision between batches is less than or equal to 3.58 percent, the accuracy range is 96.09-97.86 percent, the invention has the advantages of low detection limit, good selectivity, excellent specificity and sensitivity, short whole detection time, satisfying requirements of residue, interference and extraction recovery rate, and stable detection result.

Drawings

FIG. 1 is a blank plasma chromatogram;

FIG. 2 is a chromatogram of blank plasma plus levetiracetam standard;

FIG. 3 is a patient plasma chromatogram;

FIG. 4 is a schematic representation of the chromatograms of FIGS. 1, 2 and 3 superimposed.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.

Example (b): an HPLC-UV method for detecting the concentration of human plasma levetiracetam, the instrument adopted by the implementation comprises:

HPLC-UV system: e2695 high performance liquid chromatography (Waters, usa) and 2489 ultraviolet detector (Waters, usa);

EmpowerTM (Waters, USA);

a Forma-86C ultra-low temperature refrigerator (Thermo, usa);

SL102N electronic balance (MINQIAO, china);

XP26 microanalysis balance (mettler-toledo, switzerland);

allegra 6R, 64R ultra high speed cryogenic centrifuge (Beckman, USA);

MS3 basic vortexer (IKA, germany);

solvent filters (jin teng, china).

The standard substance and the reagent for detecting the levetiracetam concentration by the technology comprise:

levetiracetam control (Toronto Research Chemicals Inc, batch number: 3-JSH-3-1, CAS: 102767-28-2, content 98%);

acetonitrile (HPLC grade, Merck);

methanol (HPLC grade, Merck corporation);

potassium dihydrogen phosphate (HPLC grade, research and development center for chemical reagent engineering technology in guangdong province);

Milli-Q ultrapure water (made by house).

The method comprises the following steps:

s1 chromatographic conditions: a chromatographic column: diamonsil C18 column, 150mm × 4.6mm ID, 5 μm; mobile phase: the ratio of acetonitrile to 10mM potassium dihydrogen phosphate is 10:90 (mass ratio); flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction volume: 15 mu L of the solution; detection wavelength: 210 nm;

s2, solution preparation: precisely weighing 2.35mg of levetiracetam standard, dissolving with methanol to obtain levetiracetam stock solution with the concentration of 1.0mg/mL, and storing in a refrigerator at 4 ℃ for later use;

s3, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions in proportion by using methanol, and adding blank plasma to prepare a levetiracetam standard curve plasma sample, wherein the plasma sample concentrations of the levetiracetam standard curve are respectively as follows: 2. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL, 70. mu.g/mL, and 100. mu.g/mL; then respectively taking 50 mu L of plasma sample, adding 150 mu L of methanol, uniformly mixing for 1min by vortex, centrifuging at 15000r for 10min at high speed, and taking 15 mu L of supernatant for chromatographic sample injection; as shown in fig. 1 and fig. 2, the retention time of levetiracetam is 4.7min, 6 different blank plasmas and plasmas with levetiracetam standard are randomly examined, and the results are respectively shown in fig. 1 and fig. 2, it can be seen from fig. 1 and fig. 2 that blank plasmas are well chromatographically separated, and endogenous substances do not interfere with the peak emergence of levetiracetam;

s4, performing linear regression on the 7 concentration points subjected to chromatographic sampling in the step S3 by adopting 1/x weighting, namely performing a least square method on the sample concentration by adopting the peak area of levetiracetam to obtain a levetiracetam standard curve regression equation:

y＝2.16×10^-4x+0.816，(r²＝0.998)；

in the formula: y is the concentration of levetiracetam in plasma, and x is the area of the peak of levetiracetam;

determining the lowest limit of quantitation of the plasma levetiracetam concentration to be 2 mug/mL according to the RSN of 10 and the requirements of clinical diagnosis;

s5, taking levetiracetam stock solution, diluting the levetiracetam stock solution into a series of standard solutions according to a proportion by using methanol, adding blank plasma to prepare high, medium and low plasma quality control samples, wherein the plasma quality control sample concentrations are 4 mu g/mL, 35 mu g/mL and 80 mu g/mL respectively, then taking 50 mu L of plasma quality control samples respectively, adding 150 mu L of methanol, uniformly mixing by vortex for 1min, centrifuging at a high speed of 15000r for 10min, taking 15 mu L of supernatant fluid to perform chromatography sample injection; continuously detecting for 3 days, and calculating the precision within and during day (obtaining concentration by substituting peak area according to the above formula, and obtaining precision by analyzing concentration result);

s6, diluting levetiracetam stock solution with methanol in proportion into 5 parts of standard solutions with the concentrations of 4 mu g/mL, 35 mu g/mL and 80 mu g/mL respectively, then taking 50 mu L of standard solutions respectively, adding 150 mu L of methanol, uniformly mixing in a vortex manner for 1min, centrifuging at a high speed of 15000r for 10min, taking 15 mu L of supernatant, and carrying out chromatography sample injection; after the detection, the ratio of the peak area in the step S4 to the peak area in the present step was compared to determine the extraction recovery rate.

Specifically, the precision and recovery of the measurement of the present invention are shown in Table 1:

TABLE 1

In the table: conc. mu.g/mL: concentration (. mu.g/mL); inter-day precision: the precision in the day; intra-day precision: the daytime precision; extraction recovery: recovery rate; mean ± SD: mean ± standard deviation; RSD: a mutation system.

The applicant also places the plasma sample for 6 hours at room temperature, places the supernatant sample for 24 hours in an automatic sampler, performs three times of freeze-thaw cycle, stores the plasma sample for 30 days at-20 ℃ and 95 days at-80 ℃, then respectively takes 50 mu L, adds 150 mu L of methanol, vortexes and mixes for 1min, centrifuges at high speed of 15000r for 10min, takes 15 mu L of supernatant for chromatographic sample injection; a standard curve is freshly prepared to evaluate the accuracy, and the concentration deviation between the mean value and the theoretical value of the same concentration detection is less than or equal to 15 percent.

Further, the applicant utilizes the present invention to actually detect the levetiracetam concentration in the plasma of the patient, the result is shown in fig. 3, and the superimposed effect graph shown in fig. 4 is obtained by superimposing fig. 1 to fig. 3, as can be seen from fig. 3 and fig. 4, the present invention can accurately detect the levetiracetam concentration in the plasma of the patient, and has the advantages of rapid detection, high sensitivity and high specificity.

In conclusion, the levetiracetam concentration can be detected only by a very small amount of blood plasma, and experiments prove that the linear range of the levetiracetam concentration detection kit is 2-100 mu g/mL (R)²>0.99), the precision between batches is less than or equal to 3.58 percent, the accuracy range is 96.09-97.86 percent, the invention has the advantages of low detection limit, good selectivity, excellent specificity and sensitivity, short whole detection time, satisfying requirements of residue, interference and extraction recovery rate, and stable detection result.