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 ?$%{%‡!$"<ˆ&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 €ˆ&$?$ 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. Method validation for the analysis of the keto acids mixture of acetonitrile and 0.1% (wt/wt) NaOH solution (45 ` $ $ ?&"h was O-PFBO ! & $ X^ ?$ X^ -mL test derivatized was stored at 4 °C in an auto-sampler and tube. Twenty microliters of the mixture was then transferred repeatedly analyzed at 6 h intervals. The peak areas of the to a new test tube, followed by a 10 mg/mL PFBHA solution O-PFBO derivatives were then measured to provide an !?&ich allowed for the conversion of the keto acids evaluation of their stability over time. The repeatability of to the corresponding O-PFBO derivatives. The reaction the analytical process was verified by analyzing a standard mechanism of O-PFBO derivatization is shown in Fig. 1. $ ?&"& ?$ O-PFBO derivatized six times The reaction for the formation of the O-PFBO derivatives in continuous manner. was conducted at 0 °C (ice water) or room temperature for 5, The linearity of the analytical method was confirmed by 15, 30, 60 or 120 min. The derivatization reaction was analyzing standard solutions which was O-PFBO derivatized quenched by the addition of a 1:1 (v/v) mixture of at concentrations in the range of 0.01– ˆ& ? " " ! & $ X^ limit of quantification (LLOQ) was defined as the was diluted with a 1:1 (v/v) mixture of acetonitrile and 0.1% concentration at which the SN ratio became greater than 10. ?†? ‰Š $ ˆ& X^ ?$ The intra- and inter-day reproducibility characteristics of then stored at 4 °C in the auto-sampler of the LC system for the analytical method were verified by performing six the LC-MS/MS measurements. sequential O-PFBO derivatization procedures and LC-Œ†Œ X$X$ << $ solutions and rat plasma samples under the same conditions within the same day and over three different days. The recovery rates were verified by performing five sequential LC-MS/MS measurements using rat plasma $X$$#?&$$?&"&?$ O-PFBO derivatized.

3. Results and discussion 3.1. Strategy of comprehensive analysis of various keto acids We have successfully developed a new method for the simultaneous analysis of various keto acids using an O-PFBO derivatization strategy, with the resulting derivatives being detected by a triple quadrupole tandem Fig. 1. Reaction mechanism of O-PFBO derivatization and mass spectrometer. This new method allowed for the example of specific cleavage for MRM detection. $X$ " $< - -keto acids, including the unstable oxaloacetic acid, with high levels of sensitively and selectively. The mild conditions required for 2.5. Optimization of reaction temperature and reaction time the effective derivatization of the keto acids have been The reaction temperature for the O-PFBO derivatization described below, together with the analytical conditions, Chromatography

including the HPLC separation and MS/MS detection derivatization process was then investigated, with the conditions. This newly developed method has been validated reaction being conducted at 0 °C for 5, 15, 30, 60 and 120 in terms of its linearity, lower limit of quantification, min. The results of these experiments are shown in Table 2 reproducibility and rate of recovery, and successfully applied as the normalized peak areas of the 10 different keto acid to the analysis of keto acids in rat plasma. derivatives relative to those from a reaction time of 30 min. These results show that the peak areas of the O-PFBO 3.2. Optimization of O-PFBO derivatization reaction derivatives reached to maximum in 15 min, and tended to conditions decrease after 30 min. Based on this result, a reaction time Experiments for the optimization of the reaction of 30 min appeared to give the best results for the O-PFBO temperature for the O-PFBO derivatization process were derivatization reaction of all of the keto acids tested. Taken carried at 0 °C (ice water) and room temperature. The together, these results indicated that the optimum conditions reaction time was fixed at 30 min, with the reaction being for the O-PFBO derivatization reaction required a reaction quenched by the addition of acetone. The reaction mixture temperature of 0 °C and a reaction time of 30 min. did not need to be concentrated prior to analysis, and was diluted before being injected into the LC-MS for analysis. 3.3. Stability of O-PFBO derivatives Table 1 shows the normalized peak areas for the different The keto acids were reacted with PFBHA to give the keto acids at the different two temperatures. The normalized corresponding O-PFBO derivatives, and the reaction mixture peak areas were smaller in all cases when the reaction was was quenched by the addition of acetone. The resulting "" X X! ^" -ketoglutaric mixture was then diluted with a 1:1 (v/v) mixture of acid, where the peak area was the same at both temperatures. acetonitrile and a 0.1% (wt/wt) NaOH solution before being These results suggested that the decomposition of keto acids placed in the auto-sampler of the LC system (4 °C, was reduced or suppressed at the lower temperature. SIL-20AC). The mixture in the auto-sampler was then analyzed every 6 h to evaluate the stability of the O-PFBO Table 1. Effect of the reaction temperature on the derivatives. Table 3 shows the normalized peak areas of the derivatization of the keto acids. 10 different keto acid derivatives at time points of 0, 6, 12, Normalized valuesa 18 and 24 h. These data clearly show that all of the keto 0 °C Room temp. acids tested in the current study were stable for at least 24 h. oxaloacetic acid 1.00 0.98 pyruvic acid 1.00 0.85 #"" 1.00 1.00 Table 3. Stability of O-PFBO derivatives.

#'"" 1.00 0.90 a 4-hydroxyphenylpyruvic acid 1.00 0.94 Normalized values acetoacetic acid 1.00 0.97 0 h 6 h 12 h 18 h 24 h #$<"" 1.00 0.96 oxaloacetic acid 1.00 0.98 0.99 1.00 0.98 #$""" 1.00 0.99 pyruvic acid 1.00 1.00 1.01 1.01 0.99 #X&<"" 1.00 0.97 #"" 1.00 1.02 1.01 1.02 1.01 #'"" 1.00 1.01 1.04 0.99 1.00 phenylpyruvic acid 1.00 0.99 4-hydroxyphenylpyruvic acid 1.00 1.01 0.99 1.01 0.99 a All of the measurements have been expressed as the normalized acetoacetic acid 1.00 1.02 1.03 1.02 1.02 values for the ratio of the peak area to corresponding value at #$<"" 1.00 1.00 1.01 1.02 1.00 0 °C. #$""" 1.00 1.03 1.02 1.01 1.02 #X&<"" 1.00 1.02 1.01 1.00 1.00 Table 2. Effect of the reaction time on the derivatization of the phenylpyruvic acid 1.00 1.01 1.03 1.02 1.03 keto acids. a a All measurements have been expressed as the ratio of the Normalized values normalized peak area values to the corresponding values at 0 h. 5 min 15 min 30 min 60 min 120 min oxaloacetic acid 0.99 0.98 1.00 0.98 0.97 pyruvic acid 1.02 1.03 1.00 0.98 0.90 3.4. Optimization of MS/MS detection condition #"" 1.02 1.02 1.00 1.00 1.00 #'"" 1.00 0.98 1.00 0.98 0.92 The tandem mass spectrometry experiments were 4-hydroxyphenylpyruvic acid 0.92 0.96 1.00 1.01 0.92 conducted with electrospray ionization (ESI) in the negative acetoacetic acid 0.97 0.99 1.00 0.98 0.96 ion mode. The MRM mode was used for the detection of the #$<"" 0.97 0.97 1.00 1.01 0.94 O-PFBO derivatives because this particular mode provided #$""" 0.98 1.00 1.00 1.01 0.96 #X&<"" 0.97 1.00 1.00 1.01 0.98 the necessary high levels of specificity and sensitivity. Table phenylpyruvic acid 0.98 1.02 1.00 1.03 0.97 4 shows the parameters for each derivative, which were aAll measurements have been expressed as the normalized values optimized using the direct infusion method. An m/z value of of the peak area ratios to the corresponding values at 30 min. 167 was selected as the product ion for all of the keto acid <<$!^"^"""!-keto-glutaric acid The effect of the reaction time on the outcome of the (di-"'^"#"""""-keto acid). Chromatography

– The ion was estimated to be [C6F5] , and was characteristic of the derivatives of the mono "'^" -keto acid. Distinct ions were observed for oxaloacetic acid, -keto-glutaric acid and acetoacetic acid with m/z values 84, – 98 and 212, which were attributed to [CN(CH2)COO] , – – [CN(CH2CH2)COO] and [NHOCH2C6F5] , respectively.

Table 4. Selected ions and transitions for the 10 keto acids in Fig. 3. SIM (Q1 326) mass chromatogram of the O-PFBO the MRM mode by LC-MS/MS. derivative of oxaloacetic acid (a), and the MRM (Q1/Q3 326/84) mass chromatogram of the O-PFBO derivative of oxaloacetic Q1 (Da) Q3 (Da) DPa (V) CEb (V) CXPc (V) acid (b). oxaloacetic acid 326 84 -30 -14 -7 pyruvic acid 282 167 -55 -12 -9 The O-‚Ž<<$ -keto butyric acid (peak 4) #"" 340 98 -30 -18 -5 and acetoacetic " #$  a < & $X #'"" 296 167 -55 -14 -7 4-hydroxyphenylpyruvic acid 374 167 -60 -14 -7 precursor ion (Q1), but were successfully separated using acetoacetic acid 296 212 -55 -12 -13 different MRM transition channels by selecting distinct #$<"" 310 167 -45 -12 -13 product ions (Table 4). The O-‚Ž <<$ -keto #$""" 324 167 -30 -18 -5 #X&<"" $"" " # ~ -keto--methylvaleric acid phenylpyruvic acid 358 167 -60 -12 -9 (peak 9) had the same Q1 and Q3 precursor ions, but were separated completely on the column. It is noteworthy that aDeclustering potential. bCollision energy. cCollision cell exit potential. this type of derivatization can potentially yield syn and anti stereoisomers during the formation of the C=N oxime bond. However, the O-PFBO derivative of acetoacetic acid was the 3.5. Optimization of the separation conditions only O-PFBO derivative in the current study to show a The conditions used for the separation of the 10 O-PFBO mixture of syn/anti oxime peaks. One possible reason why derivatives were optimized using a reversed phase column the other derivatives existed as a single peak is that the (Phenyl column). The hydrophobic properties of PFBHA peaks for the individual stereoisomers could not be separated meant that the O-PFBO derivatives of the keto acids were under the current separation conditions. Several other retained for longer periods of time on the column. Together separation conditions were also investigated using a variety with the highly selective and sensitive MRM method, all 10 of different mobile phases, gradient programs and columns derivatives could be effectively separated within a 14 min (e.g., C8, ODS and other phenyl columns). However, all of elution period (Fig. 2). these conditions resulted in the elution of these nine O-PFBO derivatives as single peaks (data not shown), which indicated that no stereoisomer peaks existed in this method. Another possible reason for seeing only single peaks is that this reaction favors the formation of only one stereoisomer. A higher level of steric hindrance would be observed between the O-(2,3,4,5,6-pentafluorobenzyl) group and the carboxylic acid group of the keto acid in the syn stereoisomer [12] (Fig. 2). The insertion of an extra carbon unit between the carboxylic acid group and the oxime moiety would reduce the amount of steric hindrance in the acetoacetic acid derivative, which could explain why this derivative shows a pair of peaks for the syn/anti oxime stereoisomers. The use of selected ion monitoring (SIM) detection was also investigated to monitor the formation of precursor ions (Q1). In this case, two peaks were eluted for oxaloacetic acid (Fig. 3) and acetoacetic acid. This result which suggested that oxaloacetic acid generated syn/anti Fig. 2. MRM mass chromatograms of the O-PFBO derivatives stereoisomers, but that the use of the MRM mode effectively of the 10 keto "$&$X^”^"" "! ” <" "! ” -#" "! {” -keto-butyric simplified peak detection. acid, 5: 4-&^&<""!a”""""! •” -keto-$<" "! ~” -keto-isocaproic acid, 9: 3.6. Linearity and lower limits of quantification -keto--methylvaleric acid, and 10: phenylpyruvic acid. The linearity and quantification range of this LC-MS/MS Chromatography

method were verified by analyzing standard solutions which which provided a lower level of baseline noise and allowed were O-PFBO derivatized at concentrations in the range of the dynamic range of the linearity to be extended by four 0.01–ˆ'ˆ&<$?"" digits. as the concentrations where the signal to noise ratio (S/N) was 10. 3.7. Reproducibility of the LC-MS/MS method for the analysis of the O-PFBO derivatives of 10 keto acids Table 5. Linearity and quantification range of the LC-MS/MS method for the analysis of O-PFBO derivatives. The intra- and inter-day reproducibility characteristics of the current method were verified by performing six LLOQa HLOQb r2 value sequential analyses of the O-‚Ž<<$ oxaloacetic acid 0.01 300 0.997 standard solution (Table 6) and rat plasma sample (Fig. 4, pyruvic acid 0.25 300 1.000 Table 7) under the same conditions. The inter-day #"" 0.01 300 0.999 reproducibility experiments gave a variation coefficient of #'"" 0.05 300 1.000 less than 3.7% for the standard sample and less than 5.9% 4-hydroxyphenylpyruvic acid 0.10 100 0.999 for the rat plasma sample. The intra-day reproducibility was acetoacetic acid 0.10 300 0.999 #$<"" 0.05 300 0.999 measured over days and expressed as CVs under 4.7% for #$""" 0.03 300 1.000 the standard sample and CVs under 9.9% for the rat plasma. #X&<"" 0.03 300 1.000 Good intra- and inter-day reproducibility characteristics phenylpyruvic acid 0.05 300 1.000 were therefore obtained for all of the keto acids tested in the current study. aLower limit of quantification. bHigher limit of quantification. Table 6. Reproducibility of the LC-MS/MS method for the With the exception of pyruvic acid, the LLOQ values analysis of the O-‚Ž<<$X^$ solution. were determined to be in the range of 0.01– ! 0.67–6.7 fmol per injection). The LLOQ for pyruvic acid Inter-day Intra-day, CV (%) was determined to be much higher because of contamination CV (%) day1 day2 day3 resulting from the water used in the experiment. Even oxaloacetic acid 3.5 2.9 3.3 2.5 though several different commercial water products were pyruvic acid 1.8 3.6 1.4 2.1 #"" 2.7 2.5 1.6 2.1 tested, including distilled water and ion exchanged water, it #'"" 1.6 3.6 3.2 3.5 was not possible to remove this influence completely. The 4-hydroxyphenylpyruvic acid 1.3 2.6 2.2 3.4 standard curves were linear with r2 values of greater than acetoacetic acid 3.7 4.7 3.3 2.7 0.995 for all of the keto acids tested using our newly #$<"" 1.9 1.5 2.4 2.5 developed analytical method. The ionization experiments in #$""" 1.9 1.1 1.5 3.5 #X&<"" 2.3 4.1 2.9 1.5 the current method were performed in the negative mode, phenylpyruvic acid 0.1 3.1 3.9 4.4

Fig. 4. MRM mass chromatograms of rat plasma which was O-PFBO derivatized. 1: oxaloacetic acid, 2: pyruvic acid, 3: -#""!{”-keto-butyric acid, 5: 4-&^&<""!a”""""!•”-keto-isovaleric acid, ~”-keto-$"""!–”-keto--methylvaleric acid, and 10: phenylpyruvic acid. Chromatography

Table 7. Reproducibility of the LC-MS/MS method for the derivatization process; (2) unstable keto acids such as analysis of the O-PFBO derivatives in rat plasma. oxaloacetic acid could be readily analyzed under the mild derivatization "$\'&- -keto acids can be Conc. Inter-day Intra-day, CV (%) effectively evaluated using this method; (4) good levels of CV (%) day1 day2 day3 oxaloacetic acid 0.6 1.9 8.5 4.9 4.7 reproducibility (1.1–4.7% as CV); (5) good recovery rate pyruvic acid 233.6 3.4 2.9 2.8 1.4 (95.8–108.4%); and (6) good sensitivity (LLOQ 0.01–0.25 #"" 38.3 4.2 1.8 4.0 3.5 2 > 0.997$ #'"" 1.2 5.9 5.0 3.8 2.7 these findings, we believe that this method could be widely 4-hydroxyphenylpyruvic acid 0.4 3.1 9.9 6.9 4.3 applied to the profiling of targeted keto acids in biological acetoacetic acid 46.7 5.3 4.2 5.0 3.3 #$<"" 20.5 4.0 3.0 3.1 2.8 samples. #$""" 37.8 4.7 2.9 3.0 0.7 #X&<"" 35.3 4.4 3.7 2.4 2.4 References phenylpyruvic acid - a - a - a - a - a [1] Toshima, K.; Kuroda, Y.; Hashimoto, T.; Ito, M.; aDetected as trace. Watanabe, T.; Miyao, M.; Ii, K. Pediatr. Res. 1982, 16, 430-435. [2] Burlina, A. B.; Bonafé, L.; Zacchello, F. Semin. 3.8. Recovery rates of keto acids from rat plasma Perinat. 1999, 23, 162-173. The recovery rates of the 10 keto acids from rat plasma [3] Chalmers, R. A.; Watts, R. W. E. Clin. Chim. Acta 1974, were evaluated using plasma samples that had been spiked 55, 281-294. ?& mixed standard solution (Table 8). The [4] Laffel, L. Diabetes Metab. Res. Rev. 1999, 15, results of this analysis revealed the recovery rates to be in 412-426. the range of 95–109%. These results therefore represent the [5] Brière, J. J.; Favier, J.; Gimenez-Roqueplo, A. P.; first reported account of a method capable of the Rustin, P. Am. J. Physiol. Cell. Physiol. 2006, 291, comprehensive and precise analysis of various types of keto C1114-C1120. acids, including the highly unstable oxaloacetic acid. One of [6] Koike, K.; Koike, M. Anal. Biochem. 1984, 141, the key advantages of our newly developed method is the 481-487. mild derivatization conditions. This method also avoids the [7] Pailla, K.; Blonde-Cynober, F.; Aussel, C.; Bandt, J. P. requirement for further silylation and isolation processes D.; Cynober, L. Clin. Chem. 2000, 46, 848-853. during the preparation of the keto acid derivatives [12]. [8] Fuches, M.; Engel, J.; Campos, M.; Matejec, R.; Henrich, M.; Harbach, H.; Wolff, M.; Weismüller, K.; Table 8. Recovery of keto acids spiked to rat plasma. Meges, T.; Heidt, M. C.; Walters, I. D.; Krull, M.; Recovery (mean ± SD), % Hempelmann, G.; Mühling, J. Amino Acids 2009, 36, n=5 1-11. oxaloacetic acid 108.4 ± 1.1 [9] Mahar,K.P.; Abbasi,K.U.; Khuhawar,M.Y.; Mastoi, pyruvic acid 98.8 ± 3.6 G. M.; Channer, A. H.; Sahito, S. B.; Kandhro, A. J. #"" 99.9 ± 5.4 #'"" 98.5 ± 2.3 Pak. J. Anal. Environ. Chem. 2013, 14, 16-25. 4-hydroxyphenylpyruvic acid 98.6 ± 3.5 [10] Olson, K. C.; Chen, G.; Lynch, C. J. Anal. Biochem. acetoacetic acid 101.1 ± 4.0 2013, 439, 116-122. #$<"" 99.4 ± 1.7 [11] Sternowsky, H. J.; Roboz, J.; Hutterer, F.; Gaull, G. #$""" 99.4 ± 3.8 Clin. Chim. Acta 1973, 47, 371-379. #X&<"" 100.6 ± 3.6 [12] Lee, S. H.; Kim, S. O.; Chung, B. C. J. Chromatogr. B phenylpyruvic acid 95.8 ± 1.9 1998, 719, 1-7. $$?$$#&$X<X [13] Fu, X.; Kimura, M.; Iga, M.; Yamaguchi, S. J. plasma. Chromatogr. B 2001, 758, 87-94. [14] Cry, D.; Giguère, R.; Villain, G.; Lemieux, B.; Drouin, 4. Conclusion R. J. Chromatogr. B 2006, 832, 24-29. We have successfully developed a new method for [15] Nguyen, D. C.; Lee, G.; Paik, M. J. J. Chromatogr. B determining the concentration of keto acids using 2013, 913-914, 48-54. pre-column O-PFBO derivatization and LC-MS/MS detection. This method was developed using ten different keto acids and exhibited several distinct advantages, including: (1) no complicated purification stages were required following the