CN108169370B - Method for determining ethyl pyruvate and metabolite pyruvate in biological sample - Google Patents
Method for determining ethyl pyruvate and metabolite pyruvate in biological sample Download PDFInfo
- Publication number
- CN108169370B CN108169370B CN201711378269.3A CN201711378269A CN108169370B CN 108169370 B CN108169370 B CN 108169370B CN 201711378269 A CN201711378269 A CN 201711378269A CN 108169370 B CN108169370 B CN 108169370B
- Authority
- CN
- China
- Prior art keywords
- pyruvic acid
- biological sample
- plasma
- content
- organism
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a method for measuring pyruvic acid ethyl ester and metabolite pyruvic acid in a biological sample, which uses high performance liquid chromatography of ultraviolet detection, which can be abbreviated as LC-UV method or HPLC-UV method, and uses the pyruvic acid content in the biological sample as a detection index, uses the endogenous pyruvic acid content in the biological sample obtained from an organism not administered with pyruvic acid ethyl ester as a background, measures the metabolic pyruvic acid content in the biological sample obtained from the organism administered with pyruvic acid ethyl ester, and further measures the dynamics of pyruvic acid ethyl ester in the organism. The HPLC-UV method established by the invention has strong selectivity, simple operation and quick analysis time, and is particularly suitable for analyzing a large batch of plasma samples in the pharmacokinetics or toxicological kinetics research of ethyl pyruvate.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a reliable method for determining the content of ethylpyruvate and the metabolite pyruvic acid thereof in a biological sample such as blood after an organism such as a rat is administered with the ethylpyruvate so as to evaluate the behavior of the ethylpyruvate in vivo. In particular, the invention relates to a method for determining pyruvate and its metabolite pyruvate in a biological sample, such as rat plasma. The HPLC-UV method established by the invention has strong selectivity, simple operation and quick analysis time, and is particularly suitable for analyzing a large batch of plasma samples in the pharmacokinetics or toxicological kinetics research of ethyl pyruvate.
Background
Ethyl Pyruvate (EP) is an esterified product of pyruvic acid, and is an oily compound having an aromatic odor. It was initially recognized that the pharmacological effects of ethylpyruvate stem from the study of pyruvic acid. As early as 1904, it was discovered that pyruvate can eliminate endogenous H2O2 and has antioxidant effect in Holleman [ Brand K.Aerobic catalysis by stimulating cells: protective organic oxidative stress at the depend of energy yield [ J ]. J Bioenerg biomembr, 1997, 29(4): 355-. The pharmacological actions of ethylpyruvate are also known from its antioxidant action [ shihong, bangwei, antiinflammatory action and mechanism of ethylpyruvate [ J ]. repanda (medical edition), 2012, 39 (3): 309-312]. With the progress of research, people successively discover other pharmacological actions such as anti-inflammation, anti-oxidative stress, anti-tumor and anti-sepsis of ethyl pyruvate [ Qiaojingui, Zhaofu, research progress of anti-inflammation action of ethyl pyruvate [ J ], college of Long-term therapeutics, 2005, 19(4): 318-; protection effect of Wanghao, He Yi boat, Luo Zheng pyruvic acid ethyl ester on oxidative stress injury of liver cells and mechanism research [ J ] Chinese clinical medicine 2015, 22(2): 195) 198; inhibition of ethyl pyruvate on human pancreatic cancer cells [ J ] proceedings of Shandong university (medical edition), 2011, 49(5): 48-53; the study of the effect and effect of ethyl pyruvate on the expression of serum HMGB1 in septic rats [ J ]. chinese emergency medicine, 2009, 29 (5): 442-.
At present, there are reports on the HPLC-UV method for determining the content of ethyl pyruvate [ Liyanzhi, Sunyao, Zhang Chun Branch ] in microcapsule preparation and the content detection [ J ] in microcapsule, Anhui agricultural science, 2010, 38(36):20518 20520] and the gas chromatography method for determining the content of ethyl pyruvate [ Wangxiang, Buckoo, Wangbaig ] in ethyl pyruvate injection [ J ] by GC method, Chinese pharmacist, 2016, 19 (5): 1021-; GC method to determine EP content [ J ] in pyruvic acid ethyl ester injection, modern biomedical progress, 2012, 12 (15): 2822-2825]. In addition, the literature reports that the HPLC-UV method is used for measuring the pyruvic acid [ J ] in the fermentation liquor of pyruvic acid [ Kyowa, Wudi, Zhangjian, reversed phase high performance liquid chromatography (RP-HPLC) in the fermentation liquor, the university of Tianjin science and technology, 2006, 21 (l): 21-24%, and [ Chenzhi, Wang, Anjiayan-Yan, HPLC and spectrophotometry ] determination of pyruvic acid [ J ] in wine, food industry, 2015, 36 (8): 290-292) and chitosan nanoparticles [ Xufeng, Zhaofengxin, Sun Zhao-mao ] reversed phase high performance liquid chromatography method for determining content of ethyl pyruvate in chitosan nanoparticles [ J ]. Chinese tissue engineering research, 2014, 18 (8): 1205-1210] pyruvic acid content.
However, no method for measuring the content of ethyl pyruvate or the metabolite thereof, i.e., pyruvate, in a biological sample has been reported.
During the development of the preparation of the ethyl pyruvate, the in vivo behavior of the ethyl pyruvate needs to be evaluated, and typical preclinical work includes pharmacokinetic and toxokinetic evaluation and the evaluation of the in vivo behavior of the drug in the subsequent clinical stage. These in vivo behavioral assessments will all involve measuring the amount of ethylpyruvate or its metabolite pyruvate in a biological sample, such as a plasma sample.
Therefore, there is still a need in the art for an objective and accurate method for measuring pyruvate or its metabolite pyruvate in a biological sample, such as a plasma sample, which can help the development of drugs related to pyruvate ethyl.
Disclosure of Invention
The invention aims to provide a method for objectively and accurately measuring ethyl pyruvate or a metabolite pyruvate thereof in a biological sample such as a plasma sample, thereby providing help for the development work of medicines related to ethyl pyruvate. It has been surprisingly found that the method of the invention is fully capable of satisfying the determination of ethylpyruvate or its metabolite pyruvate in a biological sample, such as a plasma sample, and the invention has been completed based on this finding.
To this end, the present invention provides a method for measuring the pyruvic acid content of ethylpyruvate or a metabolite thereof in a biological sample, which uses high performance liquid chromatography for ultraviolet detection (which may be abbreviated as LC-UV method or HPLC-UV method in the present invention) to measure the metabolic pyruvic acid content in the biological sample obtained from an organism to which ethylpyruvate is administered, using the pyruvic acid content in the biological sample as a detection index, using the endogenous pyruvic acid content in the biological sample obtained from the organism to which ethylpyruvate is not administered as a background, and further measuring the kinetics of ethylpyruvate in the organism; the high performance liquid chromatography comprises the following items or steps:
(1) chromatographic analysis conditions
The chromatographic column is a C18 column and is matched with a C18 protective column for use;
KH2PO4 (containing 0.6-0.8% H3PO4) buffer solution with a mobile phase of 0.025-0.075 mol/L;
the flow rate is 0.8-1.2 ml/min, and isocratic elution is carried out; the sampling amount is 10-50 mu L; the ultraviolet detection wavelength is 205-220 nm;
(2) preparation and assay of plasma substitutes
Accurately weighing 300mg of bovine serum albumin, dissolving the bovine serum albumin in 10mL of physiological saline, and uniformly mixing the bovine serum albumin with the physiological saline in a vortex manner to obtain blank plasma substitute;
taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of precipitant acetonitrile, vortexing for 3min, centrifuging (for example, 12000rpm, 10min), taking 10 mu L of supernatant, and performing HPLC-UV analysis and determination to determine that the blank plasma substitute has no pyruvic acid interference;
(3) preparation and determination of pyruvic acid standard solution
Accurately weighing 50.0mg of pyruvic acid reference substance in a 10mL volumetric flask, dissolving with ultrapure water, fixing volume, shaking up to prepare stock solution with the concentration of 5.0mg/mL, and refrigerating and storing in a refrigerator at 4 ℃; precisely measuring a certain volume of stock solution, and diluting the stock solution into a series of standard solutions with the concentrations of 25, 50, 100, 250, 500, 1000 and 2500 mu g/mL by using water;
taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, adding 10 mu L of pyruvic acid series standard solution to prepare a plasma substitute sample with the concentration of 5, 10, 20, 50, 100, 200 and 500 mu g/mL, adding 150 mu L of precipitant acetonitrile, vortexing for 3min, centrifuging (for example 12000rpm and 10min), taking 10 mu L of supernatant to perform HPLC-UV analysis and determination, and determining a standard curve according to a chromatographic response value and pyruvic acid sample addition amount;
(4) determination of pyruvate content in biological samples of organisms not dosed with ethylpyruvate
Taking 50 mu L of plasma of a biological sample of an organism (such as a rat) which is not administered with ethyl pyruvate, placing the biological sample into a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, vortexing for 3min, centrifuging (such as 12000rpm for 10min), taking 10 mu L of supernatant, carrying out HPLC-UV analysis and determination, and determining the content of endogenous pyruvic acid in the biological sample as the background content of pyruvic acid according to a chromatographic response value and a standard curve;
(5) determination of pyruvate content in a biological sample of an organism administered ethylpyruvate
Taking 50 mu L of plasma of a biological sample of an organism (such as a rat) given ethyl pyruvate, putting the biological sample into a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, vortexing for 3min, centrifuging (such as 12000rpm for 10min), taking 10 mu L of supernatant, performing HPLC-UV analysis measurement, and determining the metabolic pyruvic acid content in the biological sample according to the chromatographic response value and a standard curve as the pyruvic acid content in the biological sample of the organism given ethyl pyruvate;
(6) and (5) determining the content of the pyruvic acid in the biological sample, which is contributed by the organism to give the ethyl pyruvate according to the results of the step (4) and the step (5), and optionally determining the kinetics of the ethyl pyruvate in the organism according to the sampling time of the biological sample.
The method for determining the content of the ethyl pyruvate or the metabolite pyruvic acid in the biological sample comprises the following steps of determining the content of the pyruvic acid in the biological sample, wherein the chromatographic column is a chromatographic column with the column length of 20-30 mm, the inner diameter of 4.6mm and the filler granularity of 5 mu m.
The method for determining the content of the pyruvic acid ethyl ester or the metabolite pyruvic acid of the biological sample is characterized in that the chromatographic column is a Discovery brand C18 column of American chromatography.
The method for determining the content of the ethyl pyruvate or the metabolite pyruvate of the ethyl pyruvate in the biological sample comprises the step of measuring the content of the pyruvate in the biological sample, wherein the protection column is a 4X 3.0mm I.D.C. 18 protection column with the specification of Phenomenex company in the United states.
The method for determining the content of the ethyl pyruvate or the metabolite pyruvate thereof in the biological sample comprises the following steps of determining the content of the ethyl pyruvate or the metabolite pyruvate thereof in the biological sample, wherein the mobile phase KH2PO4 buffer solution contains 0.6-0.8% of H3PO 4.
The method for determining the content of ethylpyruvate or the metabolite pyruvate thereof in a biological sample according to the invention, wherein the organism is selected from the group consisting of: human, rat, mouse, dog, monkey, etc.
The method for determining the content of ethyl pyruvate or the metabolite pyruvate thereof in the biological sample according to the invention is characterized in that the biological sample is selected from the group consisting of: whole blood, plasma, urine, etc.
The method for determining the content of ethyl pyruvate or the metabolite pyruvate thereof in a biological sample according to the invention, wherein the centrifugation is performed at 12000rpm for 10 min.
The method for determining the content of ethyl pyruvate or the metabolite thereof, namely pyruvic acid in a biological sample is disclosed, wherein the flow rate is 1 ml/min.
According to the method for determining the content of the ethyl pyruvate or the metabolite pyruvate of the ethyl pyruvate in the biological sample, the sample injection amount is 10-20 mu L.
The method for determining the content of the ethyl pyruvate or the metabolite pyruvate thereof in the biological sample comprises the step of sampling 10 mu L.
The method for determining the content of the ethyl pyruvate or the metabolite pyruvate of the ethyl pyruvate in the biological sample has the ultraviolet detection wavelength of 210 nm.
In the above-described method steps of the present invention, although the specific steps described therein may be distinguished in some detail or in language specific to the steps described in the examples of the detailed description section below, those skilled in the art can, nevertheless, readily generalize the above-described method steps in light of the detailed disclosure of the present invention as a whole.
Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
The inventor has found that the pyruvic acid ethyl ester is extremely unstable in the plasma of rats, and a curve of the plasma concentration of the pyruvic acid ethyl ester changing with time after the pyruvic acid ethyl ester is added into the plasma of the rats is monitored by an HPLC-UV method, so that the pyruvic acid ethyl ester is metabolized by 50% in the plasma of the rats after 2min, and can not be detected after 10 min.
The inventor has found that endogenous pyruvic acid contained in rat blank plasma interferes with the determination of pyruvic acid content, while artificial plasma substitute does not interfere with the determination of pyruvic acid, and the methodology verification can be carried out by adopting the artificial plasma substitute to replace the rat blank plasma.
The present inventors have found that in the measurement of the linear range and the lowest quantitative limit, a standard curve, which is a linear regression equation obtained by performing regression operation using a weighted least square method with the concentration of a specimen in plasma as an abscissa and the peak height of pyruvic acid of the specimen as an ordinate, is treated with a pyruvic acid standard series solution and measured. The measurement result according to the standard curve shows that the linear range of the method for measuring the concentration of the pyruvic acid plasma substitute is 5-500 mug/mL, the lowest limit of quantitation is 5 mug/mL, and the quantitative detection of the pyruvic acid in a biological sample of an organism such as rat plasma can be completely met.
The inventor has found that in the precision and accuracy measurement, the pyruvic acid quality control solution is used for measurement, and the result shows that the LC-UV method has excellent accuracy and precision when measuring the pyruvic acid in the rat plasma.
The inventor has found that in stability examination, solutions such as stock solutions and quality control working solutions have good stability and can meet the general measurement requirements. In addition, the pyruvic acid in various plasmas (including plasma substitutes and plasma of biological samples) also has excellent stability, for example, the pyruvic acid in the samples has excellent stability after being treated in the modes of 4-24 h, room temperature-6 h, repeated freeze thawing for 3 times, long-term storage at-20 ℃ and the like.
It has been found that ethylpyruvate is very unstable in rat and dog plasma and that the crude drug ethylpyruvate is not detectable in plasma 10 minutes after administration. The present invention therefore attempts to detect its active metabolite pyruvate in several assays. The inventor tries to establish an LC-MS/MS method to determine the concentration of pyruvic acid, tries a pretreatment method of precipitated protein and liquid-liquid extraction, and a sample prepared by the precipitated protein method has extremely strong matrix inhibition effect when being detected by the LC-MS/MS method, so that the content of pyruvic acid in plasma can not be accurately quantified by the LC-MS/MS technology. While pyruvic acid can be detected by adopting a liquid-liquid extraction method (ethyl acetate: isopropanol-9: 1), the method has poor linear relation, high quantitative limit and extraction recovery rate of less than 10 percent, and is not suitable for analysis of biological samples. Furthermore, the inventor uses the HPLC-UV method to monitor the concentration of the metabolite pyruvic acid of the ethyl pyruvate in the plasma, and the HPLC-UV method for measuring the concentration of the pyruvic acid in the plasma is established and verified through continuous exploration. According to the method for determining the content of ethyl pyruvate or the metabolite pyruvate thereof in a biological sample, calcium chloride and propylene glycol, which are respectively called salt and alcohol in the invention, are also added along with the amount of the precipitant acetonitrile; in one embodiment, the amounts of the above two substances which are also added with the amount of the precipitant acetonitrile are 0.2mg and 10 μ l, respectively; in one embodiment, both are added with the acetonitrile after dissolving together to form a hydroalcoholic solution. It has been surprisingly found that the recovery of pyruvic acid can be significantly improved when the two additional additive salts and alcohol are used in combination, but not when either or both of them are not used. The salts and alcohols have no ultraviolet absorption and do not affect the measurement. In the first supplementary experiment, recovery test was performed, as described in section "1.5.2. pyruvic acid assay rat plasma sample pretreatment" of example 1 of the present invention, 2. mu.g/mL, 20. mu.g/mL, and 200. mu.g/mL pyruvic acid was additionally added to a plasma sample containing a drug collected from a rat to which ethylpyruvate was administered, then 10. mu.L of ultrapure water and 150. mu.L of acetonitrile precipitant were sequentially added, vortexed for 3min, centrifuged (12000rpm) for 10min, and 10. mu.L of the supernatant was taken for HPLC-UV analysis, and the sample recovery was measured from the chromatographic response value in accordance with the other procedure of example 1; the results showed that the sample recovery rates of the three sample concentrations were 57.2. + -. 4.8%, 47.7. + -. 6.4%, and 53.1. + -. 5.3%, respectively, and that the RSD values of the three recovery rates were all within the ideal range of 6.6-8.2% which is less than 10%, as compared with the method of example 1. The recovery test was conducted in a second supplementary test, and referring to the first supplementary test, except that the recovery of the three sample concentrations was 92.5. + -. 8.1%, 89.3. + -. 9.2% and 91.5. + -. 7.3%, respectively, with addition of 150. mu.L of acetonitrile precipitant and 10. mu.L of a saline solution (prepared from 0.2mg of salt and 10. mu.L of alcohol), indicating that the recovery was significantly improved when the saline solution was supplemented during sample treatment, as shown by the results of the test. The recovery test was conducted in a third supplemental run, referred to above as the second supplemental run, except that no salt was added; the recovery test was conducted in a fourth supplemental run, referred to above as the second supplemental run, except that no alcohol was added (water was used instead of dissolved salts); the recovery test was conducted in a fifth supplemental run, referred to above as the second supplemental run, except that the alcohol was changed to an equivalent amount of glycerol; in the third to fifth supplementary tests, the recovery rates of the three sample-adding concentrations are all in the range of 44-61%, the RSD is less than 10%, and the recovery rates are obviously low. In addition, in the second to fifth supplementary tests described above, it has been found that the specificity, linear range and minimum quantitation limit, precision and accuracy, solution stability, stability of plasma surrogate sample and stability of plasma sample of these supplementary tests are substantially the same as those of example 1. In addition, through the examination of the blank rat plasma, the inventor finds that pyruvic acid is an endogenous substance in the rat plasma, a certain baseline value exists in a blank matrix, and in order to deduct the influence of the endogenous pyruvic acid in the matrix on the measurement, the inventor uses artificial blank plasma (bovine serum albumin with proper concentration [ Sun Lily, Meng Huan, Yu Lin, Guo Tao Shen. liquid chromatography-tandem mass spectrometry for measuring the concentration of progesterone in the rat plasma [ J ] International journal of pharmaceutical research, 2015,42 (1): 107-111]) to replace the rat blank plasma to establish a standard curve, and verifies the methodology.
In order to separate pyruvic acid with larger polarity, the inventor selects a C18 chromatographic column with the length of 25cm, and when a mobile phase is optimized, the inventor finds that a better peak shape can be obtained by adding a proper amount of KH2PO4, the peak position is shifted backwards when phosphoric acid is more, and conversely, the peak position is shifted forwards when phosphoric acid is less, so that the pyruvic acid cannot be completely separated from impurities and solvent peaks. Finally, the inventor selects 0.05mol/L KH2PO4 (containing 0.7% H3PO4) buffer water solution as mobile phase for isocratic elution, and obtains good chromatographic peak pattern while obtaining better separation degree, and the analysis time of each sample is 4.5 min.
The pretreatment of a biological sample is very important for the determination of an object to be detected, the commonly used pretreatment method comprises protein precipitation and liquid-liquid extraction, and methanol and acetonitrile are firstly tried as precipitating agents, so that the precipitation effect of the acetonitrile is found to be better. Due to the high polarity of pyruvic acid, liquid-liquid extraction with organic solvents is not suitable. In addition, the protein precipitation method is simple to operate and high in extraction recovery rate. The inventor considers the dosage of the precipitating agent, and finally adopts acetonitrile with 3 times of plasma amount as the precipitating agent to treat the plasma sample.
The HPLC-UV method established in the research has strong selectivity, simple operation and quick analysis time, and is suitable for analyzing a large batch of plasma samples in the pharmacokinetics and/or toxokinetics research of the ethyl pyruvate.
Drawings
FIG. 1: the plasma concentration of ethylpyruvate was monitored by HPLC-UV as a function of time after addition of ethylpyruvate to rat plasma.
FIG. 2: chromatogram of pyruvic acid in plasma substitute and rat blank plasma (A. blank plasma chromatogram; B. blank plasma with pyruvic acid reference chromatogram; C. blank rat plasma chromatogram; D. plasma sample chromatogram after intramuscular injection of pyruvic acid ethyl ester injection to rat).
Detailed Description
The invention will be further described by the following examples, which are, however, provided for the purpose of illustration only and are not intended to, nor should they be construed as limiting the invention in any way. Those skilled in the art will recognize that conventional variations and modifications can be made to the following embodiments without departing from the spirit or scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.
Example 1: method for determining pyruvic acid content of ethyl pyruvate or metabolite thereof in biological sample
1. Assay methods and procedures
1.1. Provision of test articles
Providing rat plasma without administration of ethylpyruvate, providing rat plasma taken at different times after administration of ethylpyruvate; other substances and test materials are provided according to conventional requirements.
1.2. Chromatographic analysis conditions
The chromatographic column for the assay of the ethyl pyruvate plasma samples was Eclipse XDB-C8 (4.6X 150mm, 5 μm, Agilent, USA), C18 guard column (4X 3.0mm I.D., Phenomenex, USA); the mobile phase was water-acetonitrile (60:40, v/v).
The chromatographic column for measuring the pyruvic acid plasma sample is a Discovery C18 column (4.6X 250mm, 5 μm, America chromatography company), a C18 protective column (4X 3.0mm I.D., America Phenomenex company); the mobile phase was 0.05mol/L KH2PO4 (containing 0.7% H3PO4) buffer.
The flow rate is 1ml/min, and isocratic elution is carried out; the sampling amount is 10 mu L; the ultraviolet detection wavelength is 210 nm.
1.3. Preparation of stock solution, preparation and measurement of standard solution
Preparing a pyruvic acid ethyl ester solution: accurately weighing 20.0mg of ethyl pyruvate reference substance, placing the ethyl pyruvate reference substance in a 10mL volumetric flask, dissolving the ethyl pyruvate reference substance with acetonitrile, fixing the volume, shaking up the ethyl pyruvate reference substance to prepare a stock solution of 2.0mg/mL, and freezing and storing the stock solution at the temperature of minus 20 ℃. Precisely measuring a certain volume of stock solution, and diluting the stock solution into a series of standard solutions with the concentrations of 50, 100, 250, 500, 1000 and 2000 mu g/mL by using acetonitrile. And weighing ethyl pyruvate reference substances, preparing quality control working solutions with the concentrations of 80, 400 and 1600 mu g/mL according to the same method, and storing the solutions in a refrigerator at 4 ℃ for later use. Taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, adding 10 mu L of ethyl pyruvate series standard solution or quality control working solution, adding 150 mu L of precipitating agent acetonitrile, whirling for 3min, centrifuging (12000rpm, 10min), taking 10 mu L of supernatant, performing HPLC-UV analysis and determination, and calculating related parameters according to a chromatographic response value.
Preparing a pyruvic acid control strain solution: accurately weighing 50.0mg of pyruvic acid reference substance in a 10mL volumetric flask, performing constant volume with ultrapure water, shaking up to prepare stock solution with the concentration of 5.0mg/mL, and refrigerating and storing in a refrigerator at 4 ℃. A certain volume of stock solution is precisely measured and diluted with water into a series of standard solutions with the concentrations of 25, 50, 100, 250, 500, 1000 and 2500 mug/mL. Quality control working solutions with concentrations of 40, 400 and 2000 mug/mL were prepared according to the same method and stored in a refrigerator at 4 ℃ for further use. Taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, adding 10 mu L of pyruvic acid series standard solution or quality control working solution, adding 150 mu L of precipitating agent acetonitrile, whirling for 3min, centrifuging (12000rpm, 10min), taking 10 mu L of supernatant, performing HPLC-UV analysis and determination, and calculating related parameters according to a chromatographic response value.
1.4. Preparation and assay of plasma substitutes
And precisely weighing 300mg of bovine serum albumin, dissolving the bovine serum albumin in 10mL of physiological saline, and uniformly mixing by vortex to obtain blank plasma substitute.
Blank plasma substitute 50. mu.L is taken and placed in a 1.5mL centrifuge tube, 10. mu.L of ultrapure water and 150. mu.L of precipitant acetonitrile are sequentially added, vortexed for 3min, centrifuged (e.g., 12000rpm, 10min), and 10. mu.L of supernatant is taken for HPLC-UV analysis to determine that there is no pyruvic acid interference in the blank plasma substitute.
1.5. Pretreatment of plasma samples
1.5.1. Pretreatment of ethyl pyruvate determination rat plasma sample
Taking 50 mu L of rat plasma, placing the rat plasma in a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, vortexing for 3min, centrifuging (12000rpm) for 10min, taking 10 mu L of supernatant, carrying out HPLC-UV analysis, and calculating related parameters according to a chromatographic response value.
1.5.2. Pretreatment of pyruvic acid determination rat plasma sample
Taking 50 mu L of rat plasma, placing the rat plasma in a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, vortexing for 3min, centrifuging (12000rpm) for 10min, taking 10 mu L of supernatant, carrying out HPLC-UV analysis, and calculating related parameters according to a chromatographic response value.
2. Results of the measurement
2.1. Ethyl pyruvate plasma sample measurements
The experiment shows that the ethyl pyruvate is extremely unstable in the plasma of rats, and the curve of the plasma concentration of the ethyl pyruvate changing with time after the ethyl pyruvate is added into the plasma of the rats by the HPLC-UV method is shown in figure 1. As can be seen from FIG. 1, ethylpyruvate was metabolized to 50% in rat plasma for 2min, and was not detected at 10 min.
2.2 methodological validation of the measurement of the pyruvate concentration in rat plasma by HPLC-UV method
2.2.1. Specificity
Taking 50 μ L of blank plasma substitute, operating according to 1.5.1, injecting 10 μ L of sample, and recording chromatogram (FIG. 2A); adding 10 μ L of pyruvic acid standard solution (equivalent to 500 μ g/ml plasma concentration) into blank plasma substitute, operating according to the same method, and recording chromatogram (FIG. 2B);
sampling blank rat plasma according to 1.5.2 method, introducing sample of 10 μ l, and recording chromatogram (FIG. 2C); 10. mu.L of pyruvic acid quality control solution (equivalent to 400. mu.g/ml) was added to rat blank plasma, and the chromatogram was recorded by the same method (FIG. 2D). The result shows that the rat blank plasma contains endogenous pyruvic acid which can interfere the content determination of the pyruvic acid, while the artificial plasma substitute has no interference to the determination of the pyruvic acid, and the artificial plasma substitute is required to replace the rat blank plasma for carrying out the methodological verification.
2.2.2. Linear range and lowest quantitative limit
Substituting 50 μ L of plasma, adding 10 μ L of pyruvic acid standard series solution, preparing plasma substitute samples with concentration of 5, 10, 20, 50, 100, 200, 500 μ g/mL, operating the rest according to 1.5.1, injecting 10 μ L, and recording chromatogram. Taking the concentration of the substance to be detected in the blood plasma as an abscissa and the peak height of pyruvic acid of the substance to be detected as an ordinate, performing regression operation by using a weighted least square method, and obtaining a linear regression equation which is a standard curve. According to the standard curve, the linear range of the pyruvic acid plasma substitute concentration determination method is 5-500 mug/mL, the lowest quantitative limit is 5 mug/mL, and the quantitative determination of pyruvic acid in rat plasma can be met.
2.2.3. Precision and accuracy
50 mu L of substituted plasma is added with 10 mu L of pyruvic acid quality control solution to prepare a sample (QC) of the minimum quantitative limit, low, medium and high quality control plasma substitute with the concentration of 5, 8, 80 and 400 mu g/mL, the operation is carried out according to the method of 2.2.1, the sample and the standard curve are simultaneously measured, each concentration is analyzed by 6 samples, the continuous measurement is carried out for 3d, and the concentration of each quality control point is calculated by standard curve. The concentrations of the respective quality control points were subjected to variance analysis to determine the precision and accuracy of the method, and the results are shown in Table 1.
Table 1: accuracy and precision of determination of pyruvic acid in rat plasma by LC-UV method (n ═ 18)
The results show that the precision in the day is less than or equal to +/-5.46%, the precision in the day is less than or equal to +/-11.91%, and the RE is less than or equal to +/-10.30%, which meet the requirements of relevant regulations [ FDA. Bioanalytical method evaluation (draft) [ EB/OL ] (2012-01-30) [2016-12-13]. http: // www.fda.gov/downloads/Drugs/guide ].
2.2.4. Stability survey
2.2.4.1. Stability of solution
The long-term stability of the stock solution when the stock solution is placed at 4 ℃ is investigated in the experiment, and the result shows that the sample is stable (RE is less than or equal to +/-0.35%) when the stock solution is placed at 4 ℃ for a long time. The stability of the quality control working solution with low concentration and high concentration (40 mu g/mL and 2000 mu g/mL) for 4 hours at room temperature and the long-term stability of the working solution at 4 ℃ are considered, and the results show that the sample is stable (RE is less than or equal to +/-0.07%) when the working solution is placed at room temperature for 4 hours, and the sample is stable (RE is less than or equal to +/-0.03%) when the working solution is placed at 4 ℃ for a long time.
2.2.4.2. Plasma surrogate sample stability
Low and high (8 and 400 mu g/mL) plasma substitute samples are considered, and the plasma substitute samples are placed at room temperature for 6 hours, placed at room temperature for 24 hours after treatment and frozen and thawed for 3 cycles of stability.
Table 2: stability study of pyruvic acid in plasma substitute (n ═ 6)
| Marker concentration (μ g/mL) | The samples were treated at 4 ℃ for 24h | Standing the plasma at room temperature for 6h | Repeatedly freezing and thawing blood plasma for 3 times |
| 8.0 | 8.51±0.44 | 8.76±0.196 | 8.72±0.127 |
| 400 | 408.4±34.3 | 379.1±10.78 | 404.6±3.226 |
The results show that the plasma substitute sample is stable after being placed at room temperature for 6h (RE is less than or equal to +/-9.49 percent), the sample is stable after being placed at room temperature for 24h after treatment (RE is less than or equal to +/-6.37 percent), and the plasma substitute sample is stable after being frozen and thawed for 3 cycles (RE is less than or equal to +/-10.17 percent).
2.2.4.3. Plasma sample stability
Two low and high (8, 400 mug/mL) concentration rat plasma samples are considered, and the rat plasma samples are placed for 6 hours at room temperature, placed for 24 hours at room temperature after treatment, frozen and thawed for 3 cycles and placed for a long time at-20 ℃.
Table 3: stability of pyruvic acid in plasma (n ═ 6)
| Marker concentration (μ g/mL) | The samples were treated at 4 ℃ for 24h | Standing the plasma at room temperature for 6h | Repeatedly freezing and thawing blood plasma for 3 times | Standing at-20 deg.C for a long time |
| 8.0 | 9.70±0.315 | 8.54±0.53 | 8.34±1.04 | 8.25±0.84 |
| 400 | 470.6±42 | 396.1±32.0 | 360.4±3.45 | 401.5±15.9 |
The results show that the rat plasma sample is stable after being placed at room temperature for 6h (RE is less than or equal to +/-6.75), is unstable after being placed at room temperature for 24h after treatment (RE is more than or equal to +/-21.25), is stable after freeze-thaw for 3 cycles (RE is less than or equal to +/-9.91) and is stable after being placed at 20 ℃ for a long time (RE is less than or equal to 3.10).
In the initial stage of an experiment, the inventor firstly uses an LC-MS/MS technology to determine the contents of ethyl pyruvate and pyruvic acid in rat plasma, a sample matrix treated by a protein precipitation method has strong inhibition, and the concentration of an object to be detected cannot be detected by mass spectrometry; the linear relation of samples treated by the liquid-liquid extraction method is poor, and the extraction recovery rate is less than 10%. While the LC-MS/MS technique is a common method for measuring drug concentration in biological samples, mass spectrometry detectors are not suitable for accurate quantification of either ethyl pyruvate or pyruvic acid when determined.
Considering that pyruvate is an endogenous substance in rats and a blank plasma sample of rats contains a certain amount of pyruvate, the inventors deduct endogenous pyruvate in a plasma sample of rats when measuring the actual plasma sample of rats. In carrying out the methodological validation of HPLC-UV measurement of the concentration of ethyl pyruvate and its metabolites in biological samples, the present inventors used artificial plasma substitutes in place of rat blank plasma.
The invention aims to establish an HPLC-UV method for measuring the concentration of pyruvic acid and ethyl pyruvate in rat plasma, and is beneficial to new drug development and clinical research work of ethyl pyruvate in the future.
The spirit of the present invention is described in detail by the preferred embodiments of the present invention. It will be understood by those skilled in the art that any modification, equivalent change and modification made to the above embodiments in accordance with the technical spirit of the present invention fall within the scope of the present invention.
Reference material: the national food and drug administration: the guidelines for non-clinical pharmacokinetic studies of chemical drugs, 2005.3.
Claims (7)
1. A method for measuring the content of pyruvic acid ethyl ester or a metabolite thereof in a biological sample, which uses a high performance liquid chromatography of ultraviolet detection, which is abbreviated as HPLC-UV method, and measures the metabolic pyruvic acid content in the biological sample obtained from an organism to which pyruvic acid ethyl ester is administered by taking the pyruvic acid content in the biological sample as a detection index and the endogenous pyruvic acid content in the biological sample obtained from the organism to which pyruvic acid ethyl ester is not administered as a background, thereby measuring the kinetics of pyruvic acid ethyl ester in the organism; the high performance liquid chromatography comprises the following items or steps:
(1) chromatographic analysis conditions
The chromatographic column is C18Column, and co-ordination C18The protective column is used;
KH with the mobile phase of 0.025-0.075 mol/L2PO4A buffer solution;
the flow rate is 0.8-1.2 ml/min, and isocratic elution is carried out; the ultraviolet detection wavelength is 205-220 nm;
(2) preparation and assay of plasma substitutes
Accurately weighing 300mg of bovine serum albumin, dissolving the bovine serum albumin in 10mL of physiological saline, and uniformly mixing the bovine serum albumin with the physiological saline in a vortex manner to obtain blank plasma substitute;
taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of precipitant acetonitrile, vortexing for 3min, centrifuging, taking 10 mu L of supernatant, and performing HPLC-UV analysis and determination to determine that the blank plasma substitute is free from pyruvic acid interference;
(3) preparation and determination of pyruvic acid standard solution
Accurately weighing 50.0mg of pyruvic acid reference substance in a 10mL volumetric flask, dissolving with ultrapure water, fixing volume, shaking up to prepare stock solution with the concentration of 5.0mg/mL, and refrigerating and storing in a refrigerator at 4 ℃; precisely measuring a certain volume of stock solution, and diluting the stock solution into a series of standard solutions with the concentrations of 25, 50, 100, 250, 500, 1000 and 2500 mu g/mL by using water;
taking 50 mu L of blank plasma substitute, placing the blank plasma substitute in a 1.5mL centrifuge tube, adding 10 mu L of pyruvic acid series standard solution to prepare a plasma substitute sample with the concentration of 5, 10, 20, 50, 100, 200 and 500 mu g/mL, adding 150 mu L of precipitant acetonitrile, vortexing for 3min, centrifuging, taking 10 mu L of supernatant to perform HPLC-UV analysis and determination, and determining a standard curve according to a chromatographic response value and the pyruvic acid sample addition amount;
(4) determination of pyruvate content in biological samples of organisms not dosed with ethylpyruvate
Taking 50 mu L of biological sample plasma of an organism without ethyl pyruvate, placing the biological sample plasma in a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, carrying out vortex for 3min, centrifuging, taking 10 mu L of supernatant, carrying out HPLC-UV analysis and determination, and determining endogenous pyruvic acid content in the biological sample as the background content of pyruvic acid according to a chromatographic response value and a standard curve;
(5) determination of pyruvate content in a biological sample of an organism administered ethylpyruvate
Taking 50 mu L of biological sample plasma of an organism given with ethyl pyruvate, placing the biological sample plasma into a 1.5mL centrifuge tube, sequentially adding 10 mu L of ultrapure water and 150 mu L of acetonitrile precipitant, vortexing for 3min, centrifuging, taking 10 mu L of supernatant to perform HPLC-UV analysis and determination, and determining the content of metabolic pyruvic acid in the biological sample according to a chromatographic response value and a standard curve to serve as the content of pyruvic acid in the biological sample after the ethyl pyruvate is given;
(6) determining the content of pyruvic acid contributed by the organism to give the ethyl pyruvate according to the results of the step (4) and the step (5), and determining the dynamics of the ethyl pyruvate in the organism according to the sampling time of the organism sample,
the method is characterized in that 0.2mg of calcium chloride and 10 mu l of propylene glycol are also added together with the precipitant acetonitrile, and the calcium chloride and the propylene glycol are dissolved together to form a salt alcohol solution and then are added together with the acetonitrile.
2. The method according to claim 1, wherein the column is a column having a length of 20 to 30mm, an inner diameter of 4.6mm, and a packing particle size of 5 μm.
3. The process according to claim 1, wherein said mobile phase is KH2PO4The buffer solution contains 0.6-0.8% of H3PO4。
4. The method of claim 1, wherein the organism is selected from the group consisting of: human, rat, mouse, dog, monkey.
5. The method according to claim 1, wherein the centrifugation is at 12000rpm for 10 min.
6. The method of claim 1, wherein the flow rate is 1 ml/min.
7. The method of claim 1, wherein the ultraviolet detection wavelength is 210 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711378269.3A CN108169370B (en) | 2017-12-19 | 2017-12-19 | Method for determining ethyl pyruvate and metabolite pyruvate in biological sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711378269.3A CN108169370B (en) | 2017-12-19 | 2017-12-19 | Method for determining ethyl pyruvate and metabolite pyruvate in biological sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108169370A CN108169370A (en) | 2018-06-15 |
| CN108169370B true CN108169370B (en) | 2020-09-04 |
Family
ID=62522609
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711378269.3A Active CN108169370B (en) | 2017-12-19 | 2017-12-19 | Method for determining ethyl pyruvate and metabolite pyruvate in biological sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108169370B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157630B (en) * | 2018-11-07 | 2022-12-06 | 天津药业研究院股份有限公司 | Method for detecting benzyl oleate and benzyl oleate in progesterone injection |
| CN117030918B (en) * | 2023-10-08 | 2023-12-08 | 万舒（北京）医药科技有限公司 | Method for determining concentration of progesterone in human plasma by using liquid chromatography-tandem mass spectrometry |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107247095A (en) * | 2017-05-25 | 2017-10-13 | 武汉大学 | A kind of construction method of the rat model for oxidation-resisting and caducity drug screening based on metabonomic analysis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1837657A1 (en) * | 2006-03-24 | 2007-09-26 | Metanomics GmbH | Means and method for predicting or diagnosing diabetes |
| CN102466663A (en) * | 2010-11-17 | 2012-05-23 | 中国石油化工股份有限公司 | Liquid chromatography method for determining carbonyl compound content in methylrhenium trioxide (MTO) aqueous product |
| CN103675129A (en) * | 2013-12-05 | 2014-03-26 | 柳州联海科技有限公司 | Gas chromatography-mass spectrometry detection method for intracellular carbohydrates and organic acids |
| MX2017016041A (en) * | 2015-06-11 | 2018-04-24 | Agios Pharmaceuticals Inc | Methods of using pyruvate kinase activators. |
-
2017
- 2017-12-19 CN CN201711378269.3A patent/CN108169370B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107247095A (en) * | 2017-05-25 | 2017-10-13 | 武汉大学 | A kind of construction method of the rat model for oxidation-resisting and caducity drug screening based on metabonomic analysis |
Non-Patent Citations (7)
Also Published As
| Publication number | Publication date |
|---|---|
| CN108169370A (en) | 2018-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Muzzio et al. | Determination of resveratrol and its sulfate and glucuronide metabolites in plasma by LC–MS/MS and their pharmacokinetics in dogs | |
| Gottås et al. | Determination of dopamine concentrations in brain extracellular fluid using microdialysis with short sampling intervals, analyzed by ultra high performance liquid chromatography tandem mass spectrometry | |
| CN108169370B (en) | Method for determining ethyl pyruvate and metabolite pyruvate in biological sample | |
| Trexel et al. | Macrophage-mediated clofazimine sequestration is accompanied by a shift in host energy metabolism | |
| Citti et al. | Untargeted rat brain metabolomics after oral administration of a single high dose of cannabidiol | |
| CN108362807B (en) | Hemodialysis liquid detection method and application thereof in pharmacokinetics-pharmacodynamics of pulse-activating injection | |
| Zhuang et al. | Simultaneous determination of triptolide and its prodrug MC002 in dog blood by LC–MS/MS and its application in pharmacokinetic studies | |
| Liu et al. | A HPLC-UV method for the determination of puerarin in rat plasma after intravenous administration of PEGylated puerarin conjugate | |
| Nayebare et al. | Analysis of terephthalate metabolites in human urine by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) | |
| Zhang et al. | Liquid chromatography–tandem mass spectrometry for the determination of paclitaxel in rat plasma after intravenous administration of poly (l-glutamic acid)-alanine-paclitaxel conjugate | |
| de LT Vieira et al. | Simultaneous HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: Application to a pharmacokinetic study | |
| Jia et al. | Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion studies of brazilin in rats | |
| Li et al. | Analysis of CoQ10 in rat serum by ultra-performance liquid chromatography mass spectrometry after oral administration | |
| Yang et al. | Gender-related pharmacokinetics and absolute bioavailability of diosbulbin B in rats determined by ultra-performance liquid chromatography-tandem mass spectrometry | |
| Wang et al. | Preclinical pharmacokinetics, tissue distribution and excretion studies of a potential analgesics–corydaline using an ultra performance liquid chromatography–tandem mass spectrometry | |
| Putnam et al. | Bioanalytical method development and validation of a liquid chromatography-tandem mass spectrometry method for determination of β-lapachone in human plasma | |
| Qiao et al. | Microdialysis combined with liquid chromatography–tandem mass spectrometry for the determination of 6-aminobutylphthalide and its main metabolite in the brains of awake freely-moving rats | |
| JP2019530883A (en) | Identification and use of very long chain dicarboxylic acids for disease diagnosis, chemoprevention, and treatment | |
| He et al. | LC–MS/MS method for the simultaneous quantitation of three active components derived from a novel prodrug against schistosome infection | |
| Zheng et al. | Pharmacokinetics and tissue distribution study of camellianin A and its major metabolite in rats by liquid chromatography with tandem mass spectrometry | |
| Momper et al. | Determination of cidofovir in human plasma after low dose drug administration using high-performance liquid chromatography–tandem mass spectrometry | |
| Li et al. | Determination of antazoline hydrochloride in Beagle dog plasma by HPLC–UV and its application to pharmacokinetics | |
| Wang et al. | Identification and assay of 3′-O-methyltaxifolin by UPLC–MS in rat plasma | |
| Clavijo et al. | Development and validation of a semi-automated assay for the highly sensitive quantification of Biolimus A9 in human whole blood using high-performance liquid chromatography–tandem mass spectrometry | |
| Zhao et al. | A validated UHPLC-MS/MS method for measurement of pharmacokinetics and tissue distribution of trolline in rat |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |