malondialdehyde ( mda ) is an end - product of the radical - initiated oxidative decomposition of poly - unsaturated fatty acids and , therefore , it is a frequently measured biomarker of oxidative stress . , mda is generated as a side product of thromboxane a2 synthesis   and by gamma irradiation of dna . in case of thromboxane a2 synthesis , mda is formed during the conversion of the endoperoxide prostaglandin h2 ( pgh2 ) by thromboxane synthase . particularly , mda s high reactivity and capability of forming adducts with multiple biological molecules such as proteins or dna has attracted major attention over the last decades [ 7 , 8 ] . mda s high reactivity is mainly based on its electrophilicity making it strongly reactive toward nucleophiles , such as basic amino acid residues ( i.e. , lysine and histidine ) . this reactivity is not only based on mda s aldehydic nature but is also influenced by its 1,3-dialdehydic structure making it possible to form mesomerically stabilized schiff bases . as will be discussed below , especially this high reactivity towards nucleophilic molecules has been extensively exploited during the last years to develop several derivatization strategies for mda . furthermore , mda s high reactivity has for example also been used for the controlled modification of basic amino acids in proteomics studies . as examples the structure of a meta - stable lysine and a stable arginine adduct with mda 1mda adducts of a lysine  n--lysyl - aminoacrolein and b arginine  n-(2-pyrimidyl)-l - ornithine [ 10 , 11 ] as mda adduct formation mainly occurs on the protein level as shown in the -amino carboxylic acid group would be incorporated into a peptide bond mda adducts of a lysine  n--lysyl - aminoacrolein and b arginine  n-(2-pyrimidyl)-l - ornithine [ 10 , 11 ] as mda adduct formation mainly occurs on the protein level as shown in the -amino carboxylic acid group would be incorporated into a peptide bond however , not only in the life sciences field but also in food analysis , mda serves as a biomarker of lipid oxidation ( oxidative decomposition ) [ 12 , 13 ] . mda s potential mutagenicity , atherogenicity and cancerogenicity   additionally underline the importance of monitoring this substance . taken together , these facts point out that mda is not simply a biomarker for the oxidative decomposition of poly - unsaturated fatty acids but instead might also be derived from other sources and more importantly shows physiological relevance as it is capable of forming dna and protein adducts [ 8 , 15 ] . several different strategies have been pursued for measuring mda in a variety of different matrices ( i.e. , plasma , urine , and saliva ) . the employed analytical strategies can roughly be sub - divided into derivatization - based and label - free methodologies . particularly for the analysis of biological matrices , these strategies have been coupled to separation techniques such as liquid chromatography ( lc ) or gas chromatography ( gc ) . due to its high reactivity , the majority of present mda might be bound to plasma proteins and other species . hence , it is important to emphasize whether the free or total mda level is determined . referring to this fact , it is also essential to choose suitable sample preparation conditions particularly hydrolysis conditions when total mda is to be analyzed and to consider the release and/or formation of mda when harsh derivatization conditions are to be applied . an overview about selected analysis techniques for mda is given in table  1.table  1overview of lc- and gc - based methods for the measurement of mdadetection techniqueseparation technologyderivatization reagent ( derivatization time)matrixsample preparationlodcommentreferencesuvrp lc ( c18)serum ( s)protein precipitation with hclo412 nmtotal mda levels ( bound and unbound mda)uvrp  lc ( c18)diaminonaphthalene ( 180  min)plasma ( p)sprotein precipitation ( metaphosphoric acid)0.05 nmfree and total mda levelsuvrp  lc ( c18)dnph ( 60  min)urine ( u)liquid liquid extraction ( lle ) after derivatization56 nmfree mda levels , standard addition quantitationuvrp lc ( c18)dnph ( 10 min)pprotein precipitation with hclo4 , lle after derivatization2.1  mtotal mda levels ( bound and unbound mda)ms(ms)rp  lc ( porous graphite  hypercarb)exhaled breath condensate ( ebc)upebc and u  spe , p protein precipitation0.4 nm ( ebc)1.3 nm ( u)0.4 nm ( p)free mda levelsmsrp  lc ( c18)4-apc ( 240  min)pup  protein precipitation ( acn)on - line spe ( wcx)0.5 nm ( standard solution)free mda levelsms(ms)rp  lc ( c18)dnph - d3-dnph ( 120  min)ebc n.d.aida quantificationfdrp  lc ( cn)2-aminoacridone ( 90  min)u1.8 nmfree mda levels , standard addition quantificationfdrp  lc ( c18)tba ( 60  min)uplle after derivatization128 nmtotal mda levels ( free and bound mda)fdrp  lc ( c18)dansylhydrazinetissue homogenate ( mouse)automated spad0.02 g / mlautomated spad system ( standard addition quantification)fdrp  lc ( c18)fmoc - hydrazine ( 240  min)pprotein precipitation ( acn)4.0 nmfree and total mda levelsmsgcpentafluorophenylhydrazine ( 10  min)uhead space injection0.56 nm  ms ( sim)gcphenylhydrazine ( 30  min)pmicrosomeslle with or without previous hydrolysis / protein precipitation5  pmol injectedfree and total mda levelsmsgc2,2,2-trifluoroethylhydrazine ( 40  min)bloodhead space  spme5.6 nm spme0.01  m  ecdgctcph ( 60  min)p ( defatted)lle after derivatization  +  drying step over sodium sulfate0.05  m  ecdgco-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine ( 60  min)u ( rat)lle after derivatization  +  drying step over sodium sulfate50 fmoltotal mdan.d . not determined , sim selected ion monitoring , rp reversed phase overview of lc- and gc - based methods for the measurement of mda n.d . several possibilities for the label - free analysis of mda exist , ranging from simple uv - based methodologies to lc malondialdehyde has a pka value of approximately 4.5 and a melting point of around 72  c . the rather low pka value of mda can be explained from its mesomeric structure ( fig .  2 ) . basically , mda can be drawn either as a di - aldehyde or as a vinylogenic carboxylic acid to explain the low pka value observed for this substance . based on its mesomeric structure , which possesses a ,-unsaturated carbonyl function , mda can spectrophotometrically be determined under acidic conditions at 245  nm or under alkaline conditions at 267  nm [ 18 , 19].fig . 2mesomeric structures of mda mesomeric structures of mda it is evident that such an ultraviolet ( uv ) absorbance - based methodology carried out at low wavelengths suffers from rather poor sensitivity and selectivity , especially when biological matrices are involved . hence , separation techniques such as reversed - phase liquid chromatography ( lc )   or capillary electrophoresis ( ce )   have been used to separate mda from matrix constituents and to facilitate its detection by uv - absorbance . while karatas et al .   used perchloric acid ( hclo4 ) for protein precipitation and the release of bound mda in human serum samples , wilson et al .   applied a combined centrifugation , filtration approach to prepare plasma samples for analysis . an lc  ms(ms)-based methodology employing a triple - quadrupole mass spectrometer ( ms ) and a porous graphite column ( hypercarb ) for the measurement of mda and other biomarkers of oxidative stress in exhaled breath condensate , urine and plasma the method showed very low limits of detection ( lod ) for all mentioned matrices and can be applied without the necessity of labeling mda . although the method of syslov et al . did not demand any derivatization allowing the detection of mda , solid - phase extraction ( spe ) , incorporating methylmalondialdehyde as internal standard , was applied to exhaled breath condensate ( ehb ) and urine samples , while plasma samples underwent protein precipitation as sample pretreatment . limits of detection ( lod ) as low as 32  pg / ml ( 0.4 nm ) in exhaled breath condensate could be reached . 3.fig .  3chromatogram of a urine sample obtained with the label - free lc mda malondialdehyde , memda methylmalondialdehyde , hne hydroxynonenal , 8-iso pgf2 8-iso prostaglandin f2 chromatogram of a urine sample obtained with the label - free lc  ms / ms method , described by syslov et al . mda malondialdehyde , memda methylmalondialdehyde , hne hydroxynonenal , 8-iso pgf2 8-iso prostaglandin f2 the vast majority of analytical approaches for the determination of mda make use of some kind of derivatization . most strategies are based on the  aldehydic - reactivity  of mda , hence employing hydrazine - based derivatization reagents . classic  2-thiobarbituric acid ( tba ) assay   in which two molecules of tba condense with one molecule of mda to give a colored reaction product ( cf fig .  4 ) , which can be measured spectrophotometrically at 535  nm , or by fluorescence detection with excitation at 530  nm and emission at 550  nm . the tba assay intrinsically is not specific for mda , and therefore , it is frequently combined with lc separation and fluorescence detection of the formed products . still , the tba assay including the lc step was reported to overestimate urinary mda concentrations by almost tenfold . this might be related to the harsh conditions ( 100  c , acidic conditions ) needed to yield the colored reaction product . in a recent study , examining the inter - laboratory variation in the measurement of oxidative stress biomarkers , which was mainly accomplished by the combined lc - tba assay , breusing et al . could show huge variations between the actual measured mda levels in irradiated human plasma samples . surprisingly , in this study the lc - tba assay proved to give the lowest mda levels when compared to other assay formats.fig . 4reaction of mda with different derivatization reagents , a tba assay , b labeling with 2,2,2-trifluoroethylhydrazine , c labeling with 2-aa ( reprinted with permission from [ 33 , 45 ] reaction of mda with different derivatization reagents , a tba assay , b labeling with 2,2,2-trifluoroethylhydrazine , c labeling with 2-aa ( reprinted with permission from [ 33 , 45 ] due to the drawbacks of the classic tba assay being non - specific for mda and not allowing a distinction between free and bound mda , several novel approaches for the specific determination of mda have been developed . especially hydrazine - based derivatization reagents , such as dinitrophenylhydrazine ( dnph ) , fmoc - hydrazine , or dansylhydrazine , have been employed in the lc analysis of mda . while dnph has been used in combination with uv detection , the later reagents were used for the fluorescence detection of the resulting derivatives . all of these reagents have been used to determine mda in human plasma , dnph has additionally been applied to determine mda in human urine . dansylhydrazine in combination with a so - called solid - phase analytical derivatization ( spad ) where the derivatization is carried out on a solid support has also been applied for the analysis of mda in mouse tissue homogenates . sample preparation in the cited dnph - based method , included alkaline hydrolysis for mda release , followed by a hclo4-based protein precipitation and derivatization . finally , the derivatives had to be extracted with hexane , dried and reconstituted before analysis . another study involving the dnph reagent to investigate urinary mda levels , described a combined derivatization extraction step , involving pentane as the extraction solvent . especially for the preparation of the derivatization reagent solution , the authors describe a rather elaborate protocol , including chloroform extractions subsequent to preparing the reagent solution in 4  n hcl . in case of the fmoc reagent , free mda could be analyzed by protein precipitation with subsequent derivatization prior to analysis . a more automated extraction and derivatization protocol is presented in the spad - based analysis platforms [ 29 , 30 ] , which have been used to determine mda in tissue homogenates as well as plasma samples . although the spad analysis is a fully automated process , its complexity might be a complication for its applicability to large sample cohorts . three non - hydrazine - based reagents which were used for the determination of mda are : 1-methyl-2-phenylindole , which is a colorimetric reagent , forming a chromophore with a uv absorbance maximum at 586  nm . diaminonaphthalene , which was used for the lc - uv based determination of free and bound mda in human plasma samples , and 2-aminoacridone ( 2-aa ) , which was used for the lc  fluorescence detection ( fd ) of free mda in human urine . in the case of methyl-2-phenylindole , the reagent is directly added to the sample of interest ( i.e. , plasma and tissues homogenate ) , the mixture is incubated and centrifuged before the absorbance is read . in the case of diaminonaphthalene , protein precipitation with or without release of mda from proteins was applied . the authors described the limit of detection for their method to be better than 50  pm being very low for a uv - based methodology . urine samples derivatized with 2-aa did not undergo any sample pretreatment . in case of the 2-aa reaction , the formed derivative showed distinct changes in the conjugated -system , and , respectively , in its uv - absorbance as well as fluorescence properties , thereby possibly allowing the development of a specific plate reader - based assay ( see fig . other advantages of this methodology are : ( a ) the simple derivatization protocol , which basically consists of mixing the urine sample with citrate buffer and reagent solution followed by an incubation step ( 90  min ) and ( b ) the possibility of isocratic analysis , thereby allowing a rather high throughput as sample analysis times in this case were described to be less than 4  min . besides the fact that most mda derivatives are analyzed by lc one such approach uses dnph and deuterium - labeled d3-dnph to apply a novel isotope dilution technique called alternate isotope - coded derivatization assay ( aida ) for the determination of mda and other biomarkers of oxidative stress in exhaled breath condensate . another reagent used for the lc  ms detection of mda and other aldehydes is 4-(2-(trimethylammonio)ethoxy)benzenaminium halide ( 4-apc ) , which is coupled via reductive amination to the aldehyde group . this reagent was used in combination with an on - line weak cation exchange ( wcx ) solid - phase extraction lc ms system , for the detection of mda and other aldehydes in urine and plasma . in case of urine samples , a simple centrifugation step was carried out as sample pretreatment , while plasma samples underwent protein precipitation with acetonitrile before being subjected to derivatization and subsequent analysis by on - line spe lc  ms . as for the lc - based determinations , hydrazine labels dominate the employed strategies for the gc - based methodologies as well . phenylhydrazine has been used to analyze mda in plasma and microsomal samples , as due to very mild reaction conditions free as well as total mda levels can be determined . pentafluorophenylhydrazine has been used for the head space - based analysis of mda in urine . a combined head space  solid phase micro extraction ( spme ) approach for the determination of mda in human blood incorporated trifluoroethylhydrazine as derivatization reagent   ( see fig . along these lines also methylhydrazine has to be named , which was used for the gc nitrogen phosphorus detector ( npd)-based detection of mda in various matrices [ 2 , 39 ] . another hydrazine - based label which was used in combination with an electron capture detector ( ecd ) is 2,4,6-trichlorophenylhydrazine ( tcph ) . two non - hydrazine - based reagents which have been used for the analysis of mda by gc ms(ms ) analysis in the selected reaction monitoring mode ( m / z 253    177 and m / z 251    175 ) . the second reagent , o-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine was used to determine mda in rat urine by a gc  ecd based method . in the case of plasma samples , liquid extraction ( lle ) employing hexane after derivatization , with or without previous protein precipitation using acetonitrile ( acn ) . in case of microsomal incubations , shin et al . as well as fujioka and shibamoto could successfully circumvent the need of protein precipitation or lle in urine , blood and cod liver oil samples by employing head space or head space spme analysis after mda derivatization [ 3739 ] . when the tcph reagent was applied to defatted plasma samples , the sample preparation involved lle and a drying step over anhydrous sodium sulfate before subsequent drying under nitrogen was carried out . employed a toluene extraction of the dried residue after derivatizing whole plasma samples . de zwart et al .   analyzed total mda after acid hydrolysis in rat urine samples . after derivatization , lle employing heptane and a drying step over sodium sulfate were applied . several possibilities for the label - free analysis of mda exist , ranging from simple uv - based methodologies to lc malondialdehyde has a pka value of approximately 4.5 and a melting point of around 72  c . the rather low pka value of mda can be explained from its mesomeric structure ( fig .  2 ) . basically , mda can be drawn either as a di - aldehyde or as a vinylogenic carboxylic acid to explain the low pka value observed for this substance . based on its mesomeric structure , which possesses a ,-unsaturated carbonyl function , mda can spectrophotometrically be determined under acidic conditions at 245  nm or under alkaline conditions at 267  nm [ 18 , 19].fig . 2mesomeric structures of mda mesomeric structures of mda it is evident that such an ultraviolet ( uv ) absorbance - based methodology carried out at low wavelengths suffers from rather poor sensitivity and selectivity , especially when biological matrices are involved . hence , separation techniques such as reversed - phase liquid chromatography ( lc )   or capillary electrophoresis ( ce )   have been used to separate mda from matrix constituents and to facilitate its detection by uv - absorbance . while karatas et al .   used perchloric acid ( hclo4 ) for protein precipitation and the release of bound mda in human serum samples , wilson et al .   applied a combined centrifugation , filtration approach to prepare plasma samples for analysis . an lc  ms(ms)-based methodology employing a triple - quadrupole mass spectrometer ( ms ) and a porous graphite column ( hypercarb ) for the measurement of mda and other biomarkers of oxidative stress in exhaled breath condensate , urine and plasma the method showed very low limits of detection ( lod ) for all mentioned matrices and can be applied without the necessity of labeling mda . although the method of syslov et al . did not demand any derivatization allowing the detection of mda , solid - phase extraction ( spe ) , incorporating methylmalondialdehyde as internal standard , was applied to exhaled breath condensate ( ehb ) and urine samples , while plasma samples underwent protein precipitation as sample pretreatment . limits of detection ( lod ) as low as 32  pg / ml ( 0.4 nm ) in exhaled breath condensate could be reached . 3.fig .  3chromatogram of a urine sample obtained with the label - free lc mda malondialdehyde , memda methylmalondialdehyde , hne hydroxynonenal , 8-iso pgf2 8-iso prostaglandin f2 chromatogram of a urine sample obtained with the label - free lc  ms / ms method , described by syslov et al . the vast majority of analytical approaches for the determination of mda make use of some kind of derivatization . most strategies are based on the  aldehydic - reactivity  of mda , hence employing hydrazine - based derivatization reagents . classic  2-thiobarbituric acid ( tba ) assay   in which two molecules of tba condense with one molecule of mda to give a colored reaction product ( cf fig .  4 ) , which can be measured spectrophotometrically at 535  nm , or by fluorescence detection with excitation at 530  nm and emission at 550  nm . the tba assay intrinsically is not specific for mda , and therefore , it is frequently combined with lc separation and fluorescence detection of the formed products . still , the tba assay including the lc step was reported to overestimate urinary mda concentrations by almost tenfold . this might be related to the harsh conditions ( 100  c , acidic conditions ) needed to yield the colored reaction product . in a recent study , examining the inter - laboratory variation in the measurement of oxidative stress biomarkers , which was mainly accomplished by the combined lc - tba assay , breusing et al . could show huge variations between the actual measured mda levels in irradiated human plasma samples . surprisingly , in this study the lc - tba assay proved to give the lowest mda levels when compared to other assay formats.fig . 4reaction of mda with different derivatization reagents , a tba assay , b labeling with 2,2,2-trifluoroethylhydrazine , c labeling with 2-aa ( reprinted with permission from [ 33 , 45 ] reaction of mda with different derivatization reagents , a tba assay , b labeling with 2,2,2-trifluoroethylhydrazine , c labeling with 2-aa ( reprinted with permission from [ 33 , 45 ] due to the drawbacks of the classic tba assay being non - specific for mda and not allowing a distinction between free and bound mda , several novel approaches for the specific determination of mda have been developed . especially hydrazine - based derivatization reagents , such as dinitrophenylhydrazine ( dnph ) , fmoc - hydrazine , or dansylhydrazine , have been employed in the lc analysis of mda . while dnph has been used in combination with uv detection , the later reagents were used for the fluorescence detection of the resulting derivatives . all of these reagents have been used to determine mda in human plasma , dnph has additionally been applied to determine mda in human urine . dansylhydrazine in combination with a so - called solid - phase analytical derivatization ( spad ) where the derivatization is carried out on a solid support has also been applied for the analysis of mda in mouse tissue homogenates . sample preparation in the cited dnph - based method , included alkaline hydrolysis for mda release , followed by a hclo4-based protein precipitation and derivatization . finally , the derivatives had to be extracted with hexane , dried and reconstituted before analysis . another study involving the dnph reagent to investigate urinary mda levels , described a combined derivatization extraction step , involving pentane as the extraction solvent . especially for the preparation of the derivatization reagent solution , the authors describe a rather elaborate protocol , including chloroform extractions subsequent to preparing the reagent solution in 4  n hcl . in case of the fmoc reagent , free mda could be analyzed by protein precipitation with subsequent derivatization prior to analysis . a more automated extraction and derivatization protocol is presented in the spad - based analysis platforms [ 29 , 30 ] , which have been used to determine mda in tissue homogenates as well as plasma samples . although the spad analysis is a fully automated process , its complexity might be a complication for its applicability to large sample cohorts . three non - hydrazine - based reagents which were used for the determination of mda are : 1-methyl-2-phenylindole , which is a colorimetric reagent , forming a chromophore with a uv absorbance maximum at 586  nm . diaminonaphthalene , which was used for the lc - uv based determination of free and bound mda in human plasma samples , and 2-aminoacridone ( 2-aa ) , which was used for the lc  fluorescence detection ( fd ) of free mda in human urine . in the case of methyl-2-phenylindole , the reagent is directly added to the sample of interest ( i.e. , plasma and tissues homogenate ) , the mixture is incubated and centrifuged before the absorbance is read . in the case of diaminonaphthalene , the authors described the limit of detection for their method to be better than 50  pm being very low for a uv - based methodology . urine samples derivatized with 2-aa did not undergo any sample pretreatment . in case of the 2-aa reaction , the formed derivative showed distinct changes in the conjugated -system , and , respectively , in its uv - absorbance as well as fluorescence properties , thereby possibly allowing the development of a specific plate reader - based assay ( see fig . other advantages of this methodology are : ( a ) the simple derivatization protocol , which basically consists of mixing the urine sample with citrate buffer and reagent solution followed by an incubation step ( 90  min ) and ( b ) the possibility of isocratic analysis , thereby allowing a rather high throughput as sample analysis times in this case were described to be less than 4  min . besides the fact that most mda derivatives are analyzed by lc one such approach uses dnph and deuterium - labeled d3-dnph to apply a novel isotope dilution technique called alternate isotope - coded derivatization assay ( aida ) for the determination of mda and other biomarkers of oxidative stress in exhaled breath condensate . ms detection of mda and other aldehydes is 4-(2-(trimethylammonio)ethoxy)benzenaminium halide ( 4-apc ) , which is coupled via reductive amination to the aldehyde group . this reagent was used in combination with an on - line weak cation exchange ( wcx ) solid - phase extraction lc ms system , for the detection of mda and other aldehydes in urine and plasma . in case of urine samples , a simple centrifugation step was carried out as sample pretreatment , while plasma samples underwent protein precipitation with acetonitrile before being subjected to derivatization and subsequent analysis by on - line spe lc  ms . as for the lc - based determinations , hydrazine labels dominate the employed strategies for the gc - based methodologies as well . phenylhydrazine has been used to analyze mda in plasma and microsomal samples , as due to very mild reaction conditions free as well as total mda levels can be determined . pentafluorophenylhydrazine has been used for the head space - based analysis of mda in urine . a combined head space  solid phase micro extraction ( spme ) approach for the determination of mda in human blood incorporated trifluoroethylhydrazine as derivatization reagent   ( see fig . also methylhydrazine has to be named , which was used for the gc  nitrogen phosphorus detector ( npd)-based detection of mda in various matrices [ 2 , 39 ] . another hydrazine - based label which was used in combination with an electron capture detector ( ecd ) is 2,4,6-trichlorophenylhydrazine ( tcph ) . two non - hydrazine - based reagents which have been used for the analysis of mda by gc ms(ms ) analysis in the selected reaction monitoring mode ( m / z 253    177 and m / z 251    175 ) . the second reagent , o-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine was used to determine mda in rat urine by a gc ecd based method . in the case of plasma samples , cighetti et al .   applied a liquid liquid extraction ( lle ) employing hexane after derivatization , with or without previous protein precipitation using acetonitrile ( acn ) . in case of microsomal incubations , shin et al . as well as fujioka and shibamoto could successfully circumvent the need of protein precipitation or lle in urine , blood and cod liver oil samples by employing head space or head space spme analysis after mda derivatization [ 3739 ] . when the tcph reagent was applied to defatted plasma samples , the sample preparation involved lle and a drying step over anhydrous sodium sulfate before subsequent drying under nitrogen was carried out . employed a toluene extraction of the dried residue after derivatizing whole plasma samples . de zwart et al .   analyzed total mda after acid hydrolysis in rat urine samples . after derivatization , lle employing heptane and a drying step over sodium sulfate were applied . due to the drawbacks of the classic tba assay being non - specific for mda and not allowing a distinction between free and bound mda , several novel approaches for the specific determination of mda have been developed . especially hydrazine - based derivatization reagents , such as dinitrophenylhydrazine ( dnph ) , fmoc - hydrazine , or dansylhydrazine , have been employed in the lc analysis of mda . while dnph has been used in combination with uv detection , the later reagents were used for the fluorescence detection of the resulting derivatives . all of these reagents have been used to determine mda in human plasma , dnph has additionally been applied to determine mda in human urine . dansylhydrazine in combination with a so - called solid - phase analytical derivatization ( spad ) where the derivatization is carried out on a solid support has also been applied for the analysis of mda in mouse tissue homogenates . sample preparation in the cited dnph - based method , included alkaline hydrolysis for mda release , followed by a hclo4-based protein precipitation and derivatization . finally , the derivatives had to be extracted with hexane , dried and reconstituted before analysis . another study involving the dnph reagent to investigate urinary mda levels , described a combined derivatization extraction step , involving pentane as the extraction solvent . especially for the preparation of the derivatization reagent solution , the authors describe a rather elaborate protocol , including chloroform extractions subsequent to preparing the reagent solution in 4  n hcl . in case of the fmoc reagent , free mda could be analyzed by protein precipitation with subsequent derivatization prior to analysis . a more automated extraction and derivatization protocol is presented in the spad - based analysis platforms [ 29 , 30 ] , which have been used to determine mda in tissue homogenates as well as plasma samples . although the spad analysis is a fully automated process , its complexity might be a complication for its applicability to large sample cohorts . three non - hydrazine - based reagents which were used for the determination of mda are : 1-methyl-2-phenylindole , which is a colorimetric reagent , forming a chromophore with a uv absorbance maximum at 586  nm . diaminonaphthalene , which was used for the lc - uv based determination of free and bound mda in human plasma samples , and 2-aminoacridone ( 2-aa ) , which was used for the lc  fluorescence detection ( fd ) of free mda in human urine . in the case of methyl-2-phenylindole , the reagent is directly added to the sample of interest ( i.e. , plasma and tissues homogenate ) , the mixture is incubated and centrifuged before the absorbance is read . in the case of diaminonaphthalene , the authors described the limit of detection for their method to be better than 50  pm being very low for a uv - based methodology . urine samples derivatized with 2-aa did not undergo any sample pretreatment . in case of the 2-aa reaction , the formed derivative showed distinct changes in the conjugated -system , and , respectively , in its uv - absorbance as well as fluorescence properties , thereby possibly allowing the development of a specific plate reader - based assay ( see fig . other advantages of this methodology are : ( a ) the simple derivatization protocol , which basically consists of mixing the urine sample with citrate buffer and reagent solution followed by an incubation step ( 90  min ) and ( b ) the possibility of isocratic analysis , thereby allowing a rather high throughput as sample analysis times in this case were described to be less than 4  min . one such approach uses dnph and deuterium - labeled d3-dnph to apply a novel isotope dilution technique called alternate isotope - coded derivatization assay ( aida ) for the determination of mda and other biomarkers of oxidative stress in exhaled breath condensate . another reagent used for the lc  ms detection of mda and other aldehydes is 4-(2-(trimethylammonio)ethoxy)benzenaminium halide ( 4-apc ) , which is coupled via reductive amination to the aldehyde group . this reagent was used in combination with an on - line weak cation exchange ( wcx ) solid - phase extraction lc ms system , for the detection of mda and other aldehydes in urine and plasma . in case of urine samples , a simple centrifugation step was carried out as sample pretreatment , while plasma samples underwent protein precipitation with acetonitrile before being subjected to derivatization and subsequent analysis by on - line spe lc  ms . as for the lc - based determinations , hydrazine labels dominate the employed strategies for the gc - based methodologies as well . phenylhydrazine has been used to analyze mda in plasma and microsomal samples , as due to very mild reaction conditions free as well as total mda levels can be determined . pentafluorophenylhydrazine has been used for the head space - based analysis of mda in urine . a combined head space solid phase micro extraction ( spme ) approach for the determination of mda in human blood incorporated trifluoroethylhydrazine as derivatization reagent   ( see fig .  4 ) . along these lines also methylhydrazine has to be named , which was used for the gc nitrogen phosphorus detector ( npd)-based detection of mda in various matrices [ 2 , 39 ] . another hydrazine - based label which was used in combination with an electron capture detector ( ecd ) is 2,4,6-trichlorophenylhydrazine ( tcph ) . two non - hydrazine - based reagents which have been used for the analysis of mda by gc ms(ms ) analysis in the selected reaction monitoring mode ( m / z 253    177 and m / z 251    175 ) . the second reagent , o-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine was used to determine mda in rat urine by a gc  ecd based method . in the case of plasma samples , cighetti et al .   applied a liquid liquid extraction ( lle ) employing hexane after derivatization , with or without previous protein precipitation using acetonitrile ( acn ) . in case of microsomal incubations , shin et al . as well as fujioka and shibamoto could successfully circumvent the need of protein precipitation or lle in urine , blood and cod liver oil samples by employing head space or head space spme analysis after mda derivatization [ 3739 ] . when the tcph reagent was applied to defatted plasma samples , the sample preparation involved lle and a drying step over anhydrous sodium sulfate before subsequent drying under nitrogen was carried out . employed a toluene extraction of the dried residue after derivatizing whole plasma samples . de zwart et al .   analyzed total mda after acid hydrolysis in rat urine samples . after derivatization , lle employing heptane and a drying step over sodium sulfate were applied . malondialdehyde has been analyzed in a wide variety of different matrices in numerous studies dealing with oxidative stress parameters or the modification of endogenous substances such as proteins , or dna . several technologies for the determination of free and total mda which are more sensitive and selective compared to the classic tba assay have been developed during the last decade . although the superior performance of all these methods has been proven , the tba assay is still widely applied , even though its unspecific nature is known today . the most likely reason for this might be its convenient utilization in particular to large sample numbers , needing only a plate reader instrument . hence , it seems to be crucial to develop a selective and sensitive derivatization strategy for mda , which can easily be applied to large sample cohorts . such a reagent could possibly evolve from a non - hydrazine - based reagent , such as 2-aminoacridone , where the coupling of mda causes distinct changes in the spectroscopic properties and thereby might lead to a selective plate reader - based detection of this important substance . another strategy to develop such a plate reader - based analysis platform might be the development of frster resonance energy transfer ( fret)-based derivatization strategies   for mda . such reagents could possibly be developed on the basis of the aromatic amine binding properties of mda ( cf 2-aa ) , changing the spectroscopic properties of a primary fluorescent molecule , now allowing fret , involving a secondary fluorescent part of the reagent . additional possibilities for the development of advanced detection technologies for mda might also arise from so - called f - trap reagents , which due to their fluorine containing nature allow the selective removal of excess reagent via fluorous solid phases . the method of syslov et al .   offers a highly sensitive analysis method for mda , which does not require a derivatization step . the main drawback of the method is the need for highly expensive equipment , such as the employed triple - quadrupole ms . within the derivatization - based methods , the gc-(head space ) spme methodologies and the 2-aa based method for urine samples seem to be superior as they demand minimal sample pretreatment and show low limits of detection .
malondialdehyde ( mda ) is an end - product of lipid peroxidation and a side product of thromboxane a2 synthesis . 
 moreover , it is not only a frequently measured biomarker of oxidative stress , but its high reactivity and toxicity underline the fact that this molecule is more than  just  a biomarker . 
 additionally , mda was proven to be a mutagenic substance . 
 having said this , it is evident that there is a major interest in the highly selective and sensitive analysis of this molecule in various matrices . in this review 
 , we will provide a brief overview of the most recent developments and techniques for the liquid chromatography ( lc ) and gas chromatography ( gc)-based analysis of mda in different matrices . while the 2-thiobarbituric acid assay still is the most prominent methodology for determining mda , several advanced techniques have evolved , including gc 
 ms(ms ) , lc 
 ms(ms ) as well as several derivatization - based strategies .