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Development of a New LC-MS/MS Method for the Quantification of Keto Acids

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Development of a New LC-MS/MS Method for the Quantification of Keto Acids Chromatography Original Paper Development of a New LC-MS/MS Method for the Quantification of Keto Acids Kazuyoshi NOGUCHI, Toshimi MIZUKOSHI*, Hiroshi MIYANO, Naoyuki YAMADA Fundamental Technology Labs. Institute For Innovation, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan Abstract Keto acids are known to be key intermediates in various metabolic pathways. The development of an accurate method for the determination of keto acids is therefore important for diagnosing metabolic disorders as well as elucidating cellular metabolic processes in the TCA cycle, glycolysis and amino acid biosynthesis. In this study, we have developed a comprehensive and reliable LC-MS/MS method for the analysis of biological samples using a pre-column derivatization process. Ten keto acids, including - !-keto acids, were converted to the corresponding O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives and analyzed by LC-MS/MS. Oxaloacetic acid, which is generally considered to be unstable, was also successfully derivatized under mild reaction conditions. The pretreatment procedure used in this study was simple and did not require any difficult extraction or evaporation processes. The separation and detection of the derivatized keto acids was achieved using an LC-MS/MS system in multiple reaction monitoring mode. This newly developed method was applied to the analysis of keto acids in rat plasma, and showed good reproducibility (1.1–4.7% as CV) and recovery (96–109%) rates. This method also exhibited a low limit of detection in the range of 0.01–)r2 > 0.997) over a wide concentration range ) Based on these performance characteristics, this method could be readily applied to the comprehensive analysis of keto acids in biological samples. Keywords: Keto acid; Quantification method; O-(2,3,4,5,6-Pentafluorobenzyl)oxime; LC-MS/MS 1. Introduction [4], where it is accumulated during fatty acid metabolism Keto acids contain both carboxylic acid and ketone when glucose is not available. Oxaloacetic acid and moieties within their structure, and are formed as -ketoglutaric acid are known to play key roles in amino intermediates during the metabolism of amino acids, sugars acid metabolism and the TCA cycle [5]. and carboxylates. The development of accurate methods for However, no reliable methods have been reported in the the measurement of keto acids has attracted considerable literature for the simultaneous analysis of multiple keto acids attention because the quantification of these compounds with high sensitivity. The main reason for the lack of could be used to provide an in-depth understanding sensitive analytical methods for keto acids have been pathophysiological disorders and metabolic processes. For attributed to their non-specific UV absorption properties and instance, pyruvic acid is a marker of pyruvic acidemia [1], hydrophilic nature. For this reason, significant research whereas -keto-isovaleric acid, -keto-isocaproic acid and efforts have been directed towards the development of -keto-!-methylvaleric acid (branched chain keto acid) are pre-column derivatization methods, which can be used to markers for maple syrup urine disease [2]. Furthermore, provide high level of sensitivity with significant UV phenylpyruvic acid is a marker for phenylketonuria [3], and absorption or fluorescent, as well as making the compounds acetoacetic acid plays an important role in diabetes mellitus more hydrophobic. Several methods have been developed *Corresponding author: Toshimi MIZUKOSHI Received: 4 August 2014 Tel: +81-44-210-5832; Fax: +81-44-210-5872 Accepted: 11 October 2014 E-mail: [email protected] J-STAGE Advance Published: 24 November 2014 DOI: 10.15583/jpchrom.2014.017 Chromatography for the analysis of keto acids using phenylenediamine type O-(2,3,4,5,6-Pentafluorobenzyl)hydroxylamine (PFBHA) reagents in conjunction with HPLC-fluorescence analysis &"&! $X -keto-glutarate, sodium [6-8], HPLC-UV analysis [9] and LC-MS/MS analysis [10]. -keto-$<! $X -keto-isocaproate, sodium Lower limits of quantificatio)!) -keto-!-X&<! $X -keto-butyrate, lithium 15 nM have been achieved with these methods. Although acetoacetate, and phenylpyruvic acid were purchased from these methods showed high levels of sensitivity for the Sigma–Aldrich (St. Louis, MO, USA). Oxaloacetic acid, "#"$!&"'-keto sodium pyruvate, 4-hydroxyphenylpyruvic acid, and HPLC acids, with unstable keto acids such as oxaloacetic acid grade distilled water were purchased from Wako Pure decomposing under the severe reaction conditions. Chemical Industries, Ltd. (Osaka, Japan). Acetone and With regards to the simultaneous detection of different HPLC grade acetonitrile were purchased from Junsei keto acids, Sternowsky et al. [11], Lee et al. [12], Fu et al. Chemical Co., Ltd. (Tokyo, Japan). [13], Cry et al. [14], and Nguyen et al. [15] have all reported the development of GC-MS methods involving the use of an 2.2. Preparation of standard keto acid solutions and PFBHA oxime-silyl dual derivatization technique. Although this solution <= "$$ ?$ - !-keto acids to be Individual 10 mM stock solutions of keto acid A (i.e., derivatized using the same procedure, it requires multiple <" "! ^"" "! -keto-glutaric acid, steps. The first of these steps involves the reaction of the -keto-'""!-keto-$<""!-keto-isocaproic ketone group with hydroxylamine to give the corresponding "!-keto--methylvaleric acid and acetoacetic acid) were oxime derivative, which can be purified by solvent prepared by dissolving the materials in distilled water. extraction or cation-exchange column chromatography. The Individual 1 mM stock solutions of keto acid B (i.e., carboxylic acid group can then be silylated under standard phenylpyruvic acid and 4-hydroxyphenylpyruvic acid) were conditions, and the resulting product can be subjected to prepared by dissolving these materials in 0.1% (w/w) NaOH. GC-MS analysis. However, the requirement for complicated `)X^&#"$ was prepared by mixing pretreatment processes and the decomposition of the {"&Xstock solution of keto acid A, 4.5 mL resulting derivatives during GC analysis has limited the of each stock solution of keto acid B and 2.4 mL of distilled application of this technique and led to low rates of recovery water to give a standard stock solution. Standard solutions of with some substrates. Furthermore, this derivatization 0.01– ) ? ' & $'$| technique affords two stereoisomers (i.e., the syn and anti & ) stock solution of keto acids with a 1:1 (v/v) forms) of the derivatives, which make the chromatograms mixture of acetonitrile and 0.1% (wt/wt) NaOH solution. All more complicated. In order to improve the stereoselectivity, &#"$$$?$%~ Lee et al. have reported oxime derivatization with being used. A PFBHA solution was prepared at a O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) concentration of 10 mg/mL by dissolving the material in a [12]. 1:1 (v/v) solution of acetonitrile and 0.1% (wt/wt) NaOH In this study, we have developed a reliable method for the solution. analysis of keto acids in biological samples by combining O-(2,3,4,5,6-pentafluorobenzyl)oxime (O-PFBO) 2.3. LC-MS/MS instrumentation and conditions derivatization with highly selective and sensitive LC-MS/MS analysis was performed on a Shimadzu LC-MS/MS detection. This method was developed using ten HPLC Prominence System (Shimadzu, Kyoto, Japan) different keto acids and exhibited several distinct advantages, equipped with an AB Sciex API 4000TM System (Sciex, including: (1) no complicated purification stages were Framingham, USA). The auto-sampler temperature and required following the derivatization process; (2) unstable " <X ? $ { )! $"< keto acids such as oxaloacetic acid were stably derivatized Liquid chromatography was performed at 40 °C using a &X"\'&- -keto acids can be Unison UK-&"X&XX!)X\X# effectively determined using same procedure. The method Corporation, Kyoto, Japan) and a gradient elution system has been successfully applied to the analysis of rat plasma, with the mobile phase consisting of solvent A (50 mM where it allowed for the simultaneous detection of 10 keto formic acid in water) and solvent B (acetonitrile). The flow acids. Based on these results, we believe that this method ?$X{)X&&&$$ could be readily applied to the analysis of keto acids in a The gradient elution conditions were 0–2 min (10% B), 2.5 variety of different areas of pathophysiological and min (35% B), 13.5 min (40% B), and 13.6–17.5 min (95% biological research. B). The system was then returned to the initial conditions (10% B) for 4 min to allow for a period of equilibration prior 2. Experimental to the next sample injection. The MS was operated in a 2.1. Materials multiple reaction monitoring (MRM) mode with negative Chromatography detection. Ionization was carried out by electrospray process was optimized by performing the derivatization ionization (ESI). The ion source temperature was set to experiment at 0 °C (ice water) and room temperature for 30 300 °C. The curtain gas, ion source gas1, ion source gas2 min. The reaction time was optimized by incubating the and collision gas were set at 40, 50, 80 and 5 psi, reaction mixture at 0 °C for 5, 15, 30, 60 and 120 min. respectively. The ion source voltage and entrance potential ?$%{%!$"<&Xass values 2.6. Derivatization of rat plasma and transition processes that were monitored using this Rat plasma samples (female Sprague-Dawley, 20 weeks analytical technique are described in the results and old) were stored at -80 °C, and dissolved on ice water before discussion section. X$X $X ) ?$ = ?& 1:1 (v/v) mixture of acetonitrile and 0.1% (wt/wt) NaOH 2.4. Derivatization procedure of standard samples ${)!&$X^?$" ` ) & $ $X $ at 15,000×g for 5 mi&$)?$ containing keto acids (i.e., pyruvic acid, oxaloacetic acid, collected and transferred to a new test tube, where it was -keto-" "! -keto-'" "! -keto-isovaleric subjected to the standard derivatization procedure described "! -keto-$"" "! -keto--methylvaleric acid, above. acetoacetic acid, phenylpyruvic acid and 4-hydroxyphenylpyruvic acid) was diluted with a 1:1 (v/v) 2.7.
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