Separation and determination of the enantiomers of lactic acid and 2-hydroxyglutaric acid by chiral derivatization combined with GC–MS

Xuemei Ding1, Shuhai Lin2, Hongbo Weng3※,Jianying Liang1※

Abstract

Lactic acid and 2-hydroxyglutaric acid are chiral metabolites that have two distinct D and L enantiomers with distinct biochemical properties. Perturbations of single enantiomeric form have been found to be closely related to certain diseases. The ability to differentiate the D and L enantiomers is therefore important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2-hydroxyglutaric acid enantiomers by chiral derivatization (with L-menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of enantiomers mentioned above exhibited linear calibration curves with correlation coefficient (R2) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17–102.30% with inter-day and intra-day precisions (relative standard deviations) in the 4.23–17.26% range. The limits of detection for D- lactic acid, L-lactic acid, D-2-hydroxyglutaric acid and L-2-hydroxyglutaric acid were 0.13, 0.11, 1.12 and 1.16 μM, respectively. This method was successfully applied to analyze mouse plasma. The D-lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t-test) than those of normal mice, suggesting that D-lactic acid may serve as an indicator for type 2 diabetes mellitus.

Key words
Chiral derivatization; diabetes; Gas chromatography; Lactic acid; mass spectrometry

1. Introduction

Abnormal metabolic states are involved in many human diseases, perturbing the normal physiology and leading to severe dysfunction. Understanding these metabolic outliers is a primary focus for disease-oriented research [1]. Interestingly, chiral metabolites, such as lactic acid (LA) and 2-hydroxyglutaric acid (2HG), have two distinct mirror-image forms. Although these pairs of enantiomers have identical physical and chemical properties, they have different biochemical properties and activate different metabolic pathways.
In humans, lactic acid (LA) exists predominantly in the levorotatory isoform (L-LA), and is derived from pyruvic acid during anaerobic glycolysis. Its dextrorotatory isoform (D-LA) exists at lower levels (1–5%), compared to those of L-LA, and arises through methylglyoxal metabolism or bacterial fermentation in the gastrointestinal tract or diet [2, 3]. Studies have suggested that elevated levels of D-LA in the blood and urine are associated with type 2 diabetes mellitus (T2DM) [4–6] and its complications: diabetic ketoacidosis and diabetic nephropathy [2, 7]. Patients suffering from infection [8], ischemia [9], traumatic shock [10], short bowel syndrome [3], and schizophrenia [11] were also found to have increased levels of D-LA. 2-Hydroxyglutaric acid (2HG) is another chiral metabolite. Its D isoform has been identified as an onco-metabolite with oncogenic properties [12]. Studies have shown that cancer-associated cytosolic isocitrate dehydrogenase 1 and 2 mutations result in the production of D-2HG, but not L-2HG [13]. In addition, the excess D-2HG that accumulates in vivo contributes to cancer initiation and maintenance [14].
Therefore, the metabolic perturbations of single enantiomers of LA and 2HG play important roles in the development of certain diseases. Methods that can separate and determine the absolute configurations of LA and 2HG are important for studies of these diseases.
Both LA and 2HG are alpha hydroxy acids (AHAs). A variety of analytical methods for the separation and determination of the enantiomers of LA, 2HG, and other AHAs have been reported, including HPLC [7, 15, 16], HPLC–MS [17–19], CE [20, 21], GC [22, 23] and GC–MS [24], with a chiral or achiral stationary phase. GC–MS is considered to be superior to other techniques due to its high sensitivity and robustness. Since chiral columns are expensive, an achiral column with pre-column chiral derivatization is much more widely used.
The most widely utilized method for the chiral derivatization of AHAs involves the esterification of carboxylic acid moieties with chiral alcohols such as L-menthol, (S)-3-methyl-2-butanol, or (R)-2-butanol, followed by acetylation of any remaining hydroxyl groups. Kyoung-Rae et al. used (S)-3-methyl-2-butanol and trifluoroacetic anhydride (TFAA) for the chiral derivatization of AHAs. However, LA and other short-chain hydroxy fatty acids were lost during work-up due to the volatilities of the O-TFA (S)-3-methyl-2-butyl esters [22]. L-Menthol and TFAA were subsequently used for derivatization [23]. It is notable that the derivatives of 2HG enantiomers were not resolved on a DB-5 column which is widely used in metabolomics research, using either method. Chalmers. et al. used (R)-2-butanol and acetic anhydride to derivatize the 2HG enantiomers, but the D- and L-2HG derivatives were still poorly resolved in the subsequent GC–MS analysis [25], as has been reported by others using this method [26, 27]. Moreover, (R)-2-butanol is expensive and the preparation of (R)-2-butanolic hydrochloride (1 M HCl) reagent is tedious.
The present study aims to establish a method for the separation and quantification of LA and 2HG enantiomers based on chiral derivatization combined with GC–MS, using L-menthol and acetyl chloride as the derivatizing reagents. The applicability of this method in biological systems was assessed by using normal and T2DM mouse plasma. It is intended that this method overcomes the shortcomings of the previous methods and is fully validated as a confirmation tool for qualitative and quantitative research related to LA and 2HG enantiomers.

2. Materials and Methods

2.1 Chemicals and reagents

L-Menthol, acetyl chloride, ethyl acetate, trifluoroacetic anhydride, pyridine, acetic anhydride, (R)-(–)-2-butanol, tridecanoic acid, D-LA, L-LA, DL-LA, L-2-HG disodium salt, D-2HG disodium salt and DL-2-HG disodium salt were obtained from Sigma–Aldrich (St. Louis, MO, USA). Toluene and chloroform (GC grade) were obtained from Sinopharm (Beijing, China). Methanol and acetonitrile were obtained from Merck (Darmstadt, Germany), and ultrapure water was prepared using a Milli-Q system (Millipore, Billerica, MA). Stock solutions of D-LA, L-LA, DL-LA, D-2HG disodium salt, L-2HG disodium salt and DL-2HG disodium salt were prepared in ultrapure water at concentrations of 10 mM. A stock solution of tridecanoic acid, the internal standard (IS), was prepared in methanol at a concentration of 1 mM. Working standard solutions were prepared weekly by diluting the stock solutions. All stock solutions were stored at -20°C, while all the working solutions were stored at 4°C.

2.2 Animal study

2.2.1 Animals and ethics statements

Eight-week-old male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal (SPF II Certificate; No. SCXK2012-0002), and kept under specific pathogen-free normal housing conditions in a 12 h light and dark cycle. The human care of the mice complied with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All the protocols in this experiment were approved by the Animal Ethical Committee of School of Pharmacy, Fudan University (Permit Number: 2013-1).

2.2.2 Induction of type 2 diabetes mellitus in mice

The T2DM mouse models were induced according to a previous report [28]. Briefly, after fasting for 16 h, the C57BL/6 mice were given intraperitoneal injections with streptozotocin (100 mg/kg body weight in a 0.1 M citrate buffer) for two successive days. The control mice were injected with the buffer alone. A week after the final streptozotocin injection, the blood glucose levels were assayed using blood glucose test strips (Abbott). Mice with blood glucose levels higher than 16.8 mmol/L were chosen for experiments.

2.3 Sample collection and preparation

After T2DM mice had been successfully induced, their blood was collected in sodium heparin tubes and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant plasma was collected and stored at −80°C. Before analysis, the samples were thawed on ice for 15 min, and 100 μL of methanol (including 30μM IS) was added to 30 μL of plasma and vortex-mixed for 5 min to precipitate proteins, followed by centrifugation (12000 rpm, 10 min, 4°C). A 100 μL sample of the supernatant was transferred to a GC vial and stored for derivatization.

2.4 Chiral derivatization

L-Menthol and acetyl chloride were used to derivatize the LA and 2-HG enantiomers. In general, 100 μL of a 200 μg/ μL solution of L-menthol in ethyl acetate was added to a GC vial containing the analyte. Ethyl acetate and methanol were evaporated to dryness under a gentle flow of nitrogen gas at room temperature (25°C). Then, 60 μL of toluene and 5 μL of acetyl chloride were added and the mixture was incubated at 80°C for 2 h followed by the removal of excess reagent under a gentle flow of nitrogen gas. Subsequently, 50 μL of acetyl chloride was added and the mixture was incubated at room temperature for 1 h. The excess reagent was removed under a gentle flow of nitrogen gas. The residue was dissolved in 30 μL of chloroform and 1 μL of this sample was injected into the GC–MS instrument for analysis.

2.5 GC–MS analysis

The derivatized samples were analyzed by GC–MS (Agilent 7890B gas chromatograph coupled with a 5977A quadrupole mass spectrometer). A 1 μL aliquot of each derivatized sample was injected onto a DB-5 MS capillary column (30 m × 0.25 mm × 0.25 μm), with helium as the carrier gas at a flow rate of 1.2 mL min-1 and a spilt ratio of 20:1. The temperatures of the inlet, interface and ion source were set to 300, 280 and 230°C, respectively. The following temperature gradient was used: 60°C for 2 min, followed by heating to 300°C at 17°C min-1, and this temperature was held there for 2 min. A single run took 18.12 min to complete. The solvent delay time was set to 9 min. The gain factor was set to 2.0, and electron impact (70 eV) was used to ionize the samples. The 33−650 m/z range was examined. During qualitative analyses, the mass spectrometer was operated in full-scan mode, while selected-ion monitoring (SIM) mode was used during quantitative analyses. MassHunter Qualitative Analysis (version B.06.00) and Agilent MassHunter Quantitative Analysis software (version B.06.00) were used for data analysis.

2.6 Method validation

This method was validated in terms of linearity, sensitivity, accuracy, precision, and stability for D-LA, L-LA, D-2HG and L-2HG analysis.

2.6.1 Linearity

To construct calibration curves, mixed working standard solutions were prepared at six concentrations to cover the 3–200, 300–10000, 3–100, and 3–100 μM ranges for D-LA, L-LA, D-2HG, and L-2HG, respectively. Measurements were carried out in triplicate for each concentration level. Calibration curves for all the four compounds were obtained by linear regression of the As/Ai (As: peak area of derivatized product, Ai: peak area of derivatized internal standard) as functions of the nominal concentration.

2.6.2 Precision and accuracy

Accuracy and precision was determined using normal mouse plasma samples spiked with the standards at four concentrations. The accuracy was evaluated in terms of the calculated recoveries; the intra-day and inter-day precisions were tested over two separate days using three batches and were reported in terms of RSDs.

2.6.3 Stability

The stabilities of freeze/thaw cycles, storage at 4°C for 24 h, and storage at –80°C for a week were evaluated. Low-concentration (8.13, 312.50, 8.00 and 8.00 μM for D-LA, L-LA, D-2HG and L-2HG, respectively) and high-concentration (162.67, 6982.68, 80.00 and 80.00 μM for D-LA, L-LA, D-2HG and L-2HG, respectively) mixed-standard samples and spiked samples were used for assessment.

3. Result and Discussion

3.1 Chiral derivatization

Both LA and 2HG contain easily-derivatized alcohol and carboxylic acid functional groups. Upon esterification of the carboxylic acids with L-menthol and subsequent acetylation of the hydroxyl groups with acetyl chloride, the LA and 2HG enantiomers were converted into the corresponding O-acetylated L-menthyl esters. Tridecanoic acid (IS) was converted into the corresponding L-menthyl ester, as shown in Figure 1A.
Derivatized standard samples were first analyzed by GC–MS in the full-scan mode and the raw data was imported to the MassHunter Qualitative Analysis software (version B.06.00) for the mass spectra deconvolution and the expected product identification. Figure 1B displays the mass spectra of the derivatized LA, 2HG, and tridecanoic acid. The fragmentation pattern of each compound is consistent with that expected for the product. Figure 2A displays a typical selected-ion monitoring (SIM) chromatogram of the derivatized standard mixture, and partial enlargements of the peaks of the LA, 2HG, and tridecanoic acid derivatives, which reveal that both the derivatized LA and 2HG enantiomers are separable.
The monitored selected ions are listed in Table 1. In this study, L-menthol and TFAA [23] were also used to label the LA enantiomers. We found that in addition to the O-trifluoroacetylated L-menthyl ester products, this method produced O-acetylated L-menthyl ester byproducts. This is because acetyl chloride, which is used as a catalyst for esterification, could also react with alcohol functional groups of LA enantiomers. This resulted in a reduction in sensitivity and difficulties during quantification analysis. (R)-2-Butanol and acetic anhydride [25] were also used to label the 2HG enantiomers. However, even after the temperature gradient was optimized, poor separation was still observed as previously reported [25–27], with a resolution of 0.6. The method proposed here provided a superior resolution of 1.3 without the need for (R)-2-butanolic hydrochloride (1 M HCl), which is difficult to prepare.

3.2 Optimization of the derivatization conditions

The reaction time and temperature for both the esterification of the acid and acetylation of the alcohol, were optimized using L-LA as the representative compound, to obtain good derivatization efficiencies. Optimization experiments were carried out at different temperatures and times for esterification (80, 100, and 120°C; 1, 2, and 3 h) and acetylation (25, 40, and 60°C; 20, 40, 60, and 80 min). These results indicate that incubation at 80°C for 2 h provides the best esterification efficiency (Figure 3A), while the peak area of the acetylated product plateaued when incubated at 60°C for 20min, 40°C for 60 min, or 25°C for 60 min (Figure 3B). For operational convenience, the incubation conditions of 25°C (room temperature) for 60 min were used for acetylation.
The reaction yield under the optimized conditions was evaluated as 67.9% by GC–MS using synthesized DL-LA O-acetylated L-menthyl ester （Figure S1） as standard control. No racemization was observed after derivatization of the high-concentration standard samples of L-LA, D-LA, L-2HG and D-2HG as shown in Figure S2. Although the equivalent of L-menthol is far more than the analytes, there is still possibility of ester formation between analytes and themselves. Fortunately, following method validation shows satisfied linearity, accuracy and precision which reveals that the As/Ai could accurately reflect the analyte concentrations.

3.3 Method validation

3.3.1 Linearity, limit of detection and lower limit of quantification

Calibration curves for the four compounds (D-LA, L-LA, D-2HG and L-2HG) were constructed by linear regression, and exhibited coefficients (R2) in excess of 0.99. (Table 2). The LOD and LLOQ of each compound was determined in water according to the International Union of Pure and Applied Chemistry recommendations. The LLOQs of D-LA, L-LA, D-2HG and L-2HG were 0.43, 0.35, 3.72 and 3.89 μM, respectively. And the LODs of D-LA, L-LA, D-2HG and L-2HG were 0.13, 0.11, 1.12 and 1.16 μM, respectively. In comparison with other methods proposed for the determination of LA and 2HG enantiomers，the method here reported is more sensitive than that based on CE [20], HPLC with achiral column [15] and HPLC with chiral columns [29] where LOD values for D-lactic acid were reported as 80, 5.24 and 1 μM, respectively.

3.3.2 Precision and accuracy

The precision and accuracy were determined from spiked mouse plasma and the corresponding values are listed in Table 3. The measured values were accurate and precise, with an acceptable recovery range of 88.17–102.30% and inter-day and intra-day precisions (RSDs) in the 4.23–17.26% range.

3.3.3 Stability

The stability results are displayed in Table 4. The samples in either water or mouse plasma, were stable over four freeze/thaw cycles, with recoveries in the 88.36–103.24% range. These samples were also stable for 24h at -4℃ and a week at −80℃, with acceptable recoveries of 87.81–102.04 and 88.78–103.17%, respectively.

3.4 Determination of D- and L-lactic acid, and D- and L-2-hydroxyglutaric acid in type 2 diabetes mellitus mouse plasma samples

To assess the applicability of the proposed method, the concentrations of D-LA, L-LA, D-2HG and L-2HG were determined in the plasma of normal mice (n = 9) and T2DM mice (n = 9). A typical SIM chromatogram of derivatized mouse plasma along with the partial enlargements of peaks of the LA, 2HG, and tridecanoic acid derivatives, is shown in Figure 2B. Differences between the groups were evaluated using the unpaired two-tailed t-test; P values of less than 0.05 were considered to be statistically significant. The data were statistically processed with SPSS 19.0 software (SPSS).
The results reveal that both the D- and L-LA levels in the plasma of T2DM mice were significantly (P < 0.05) higher than those of normal mice (Figure 4A, B), which is consistent with the literature [30–31]. There are other studies have reported that the L-LA levels increased slightly without significance [32–33]. While significant increases of D-LA were reported in all studies involving the LA enantiomers and T2DM [4–6, 29–32], suggesting that D-LA may serve as an indicator for T2DM.
The concentration results of D-2HG reveal no significant difference between the groups (Figure 4C). And the L-2HG concentrations were close to the LLOQ or under it that could not be quantified. There are a few reports suggest a relationship between 2HG and T2DM [34–36]. However, since no accurate conclusions or general consensuses have been achieved by far, whether 2HG or its enantiomers play important roles in the development of T2DM still needs to be further explored.

4. Conclusions

A method for the separation and determination of the LA and 2HG enantiomers was developed, which used chiral derivatization combined with GC–MS. The proposed method overcame the shortcomings of previous methods, and was validated through satisfactory linearity, accuracy and precision. It was also successfully applied to the analysis of mouse plasma samples. The established method is an efficient assay and will benefit the disease research involving LA and 2HG. In addition, we propose that this method can also be applied to the analysis of other AHA enantiomers.

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