| Gas chromatography is a chromatographic method using gas as mobile phase.It has the advantages of high separation efficiency,minimal sample consumption,rapid turnaround times,and simple operation.With the emergence of high performance chromatographic columns and high sensitive detectors,gas chromatography has been widely used to detect most organic compounds in agriculture,industry,food,health,medicine,environment and other fields.When gas chromatography is used to detect substances with high boiling point,high polarity or poor thermal stability,derivatization methods are often needed to convert them into derivatives suitable for analysis.The commonly used derivatization reagents including silylation reagents,alkylation reagents,and acylation reagents.Lactic acid(LA)and pyruvic acid(PY)are important metabolic products of glucose.They can be reversibly transformed under the catalysis of lactate dehydrogenase to maintain the body’s stability.At the cellular level,as the final metabolite of anaerobic respiration of glucose,lactic acid can be used to reflect the function of mitochondria and the state of glycolysis of cells.It can also be the energy source of cancer cells.In clinic,lactic acid and pyruvic acid can be used as the most effective screening indicators for various diseases.The detection of lactic acid and pyruvic acid has important physiological and pathological significance.In this paper,a derivatization gas chromatography method was developed for the determination of lactic acid and pyruvic acid in biological samples.The main contents are as follows:PART 1 Detection of lactic acid in cell culture media by derivatization gas chromatographyObjective:A new method for the determination of lactic acid in cell culture media by derivatization gas chromatography was established.It was used to quantify the lactic acid secreted by cardiac myocytes exposed to different doses of mono-2-ethylhexyl phthalate(MEHP)and cancer cells cultured at different times.Method:After the establishment of two cell models,the cell culture media was collected and diluted with deionized water.Lactic acid was reacted with ethyl chloroformate,and the derivative was detected by gas chromatography-flame ionization detector(GC-FID)under the optimized chromatography and derivatization conditions.Results:Under the optimum conditions,a good linear relationship was obtained from 0.100 to 5.00 mmol/L.The linear regression equation is Y=93.191+850.95X,and the linear correlation coefficient(r)is 0.9997.The limit of detection was 0.67μmol/L(S/N=3)and the limit of quantitation was2.20μmol/L(S/N=10).The average recoveries of the derivative in cell culture media were 99.60%~100.76%at all three concentrations and RSD was less than 5.49%.Conclusion:A derivatization gas chromatography method was established to detect lactic acid in cell culture media.This method is simple,rapid and sensitive,and it can be applied for studies on mitochondrial function and cell glycolysis conditions.PART 2 Simultaneous determination of lactic acid and pyruvic acid in tissue by gas chromatography after in situ derivatization-ultrasound-assisted emulsification microextractionObjective:Combining liquid phase microextraction with derivatization,in situ derivatization-ultrasound-assisted emulsification microextraction was established to detect lactic acid and pyruvic acid in tissue.It also successfully applied to quantify pyruvic acid and lactic acid in the animal hypoxia models.Method:After the establishment of animal hypoxic model,myocardial tissue was homogenated with 1.2%perchloric acid.Using methoxyamine and isobutyl chloroformate as derivatization reagents,Lactic acid and pyruvic acid were detected by gas chromatography-flame ionization detector(GC-FID)under the optimized chromatography and derivatization conditions.Results:Under the optimum conditions,good linear relationships were obtained from 50.0~1.00×10~3μmol/L for lactic acid and 1.00~100.0μmol/L for pyruvic acid.The linear regression equations are Y=199.04+3.7173X and Y=2.3100X-1.1838,respectively.The limit of detection(LOD)was 0.12μmol/L,the limit of quantitation(LOQ)was 0.41μmol/L for lactic acid.The limit of detection(LOD)was 0.29μmol/L,the limit of quantitation(LOQ)was 0.97μmol/L for pyruvic acid.The recoveries of the two analytes were between 93.60%and 102.80%,and RSD was less than 6.20%.Conclusion:A derivatization gas chromatography method was established to detect lactic acid and pyruvic acid in cardiac myocytes.This method is simple,rapid and sensitive,and it was successfully applied to quantify pyruvic acid and lactic acid in the animal hypoxia models which provided an auxiliary means for the diagnosis of mitochondrial diseases. |
