Simultaneous determination of DL-lactic acid and DL-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC-ESI-MS/MS.
A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12 fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.
Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
Haruhito
H
Tsutsui
Toshiki
T
Mochizuki
Toshio
T
Maeda
Ichiro
I
Noge
Yutaka
Y
Kitagawa
Jun Zhe
JZ
Min
Kenichiro
K
Todoroki
Koichi
K
Inoue
Toshimasa
T
Toyo'oka
D020155:3-Hydroxybutyric Acid / Q000737:chemistry* / Q000378:metabolism; D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003920:Diabetes Mellitus / Q000378:metabolism*; D005260:Female; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012463:Saliva / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D019344:Lactic Acid; D020155:3-Hydroxybutyric Acid