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Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry
Journal of Chromatography B, 962 (2014) 9–13
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry Xiuwei Wang a , Ting Zhang a , Xin Zhao b , Zhen Guan a , Zhen Wang a , Zhiqiang Zhu a , Qiu Xie a , Jianhua Wang a,∗ , Bo Niu a,c,∗ a
Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing 100020, China Chines Academy of Inspection & Quarantine, Beijing 100023, China c Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China b
a r t i c l e
i n f o
Article history: Received 13 January 2014 Accepted 8 May 2014 Available online 20 May 2014 Keywords: UPLC/MS/MS Neural tube defects Folic acid One-carbon metabolism
a b s t r a c t Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r2 > 0.998), sensitivity (limit of detection ranged from 0.05 to 0.200 ng/mL), intra- and inter-day precision (both CV < 6%) and recovery (each analyte was >90%). The total analysis time was 7 min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P < 0.05) and 5-FoTHF (P < 0.05), and higher concentrations of Hcy (P < 0.05) and SAH (P < 0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Folate is converted to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR) once transported into the cell and functions as the carrier for one carbon unit involved in various biological processes particularly nucleotide metabolism, DNA damage repair and methylation action [1,2]. Reports have shown that folate deficiency is a risk factor for a variety of diseases such as megaloblastic anemia [3], cancer [4–6], congenital heart disease [7,8], and neural tube defects (NTDs) [9]. Therefore, it is of vital significance to develop a sensitive and fast method for detecting folate levels in the human body. The analysis of folate provides a great challenge owing to its poor stability and extremely small amounts in biological matrix samples, and its compounds are complicated. There have been many
∗ Corresponding authors at: Capital Institute of Pediatrics, Beijing 100020, China. Tel.: +86 10 85695545; fax: +86 10 85631504. E-mail addresses: [email protected] (J. Wang), [email protected] (B. Niu). http://dx.doi.org/10.1016/j.jchromb.2014.05.023 1570-0232/© 2014 Elsevier B.V. All rights reserved.
methods developed for the detection of folate such as microbiological assay [10], radioimmunoassay [11,12], capillary electrophoresis [13] and chromatography [14,15]. The most useful methods are microbiological assay and radioimmunoassay in clinical practice. However, microbiological assays are time costly and lack repetition. Radioimmunoassay has the advantage of fast and simple for sample detection, but its results vary wildly with different kits. These methods detect the total folate, that is folate plus its derivatives, with low selectivity, and different results are found by different methods used for the same sample. When high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) was used for folate analysis, it not only rapidly and effectively separated the folate compounds, but could meet the requirements of a low detection limit owing to the high sensitivity of mass spectrometer, and show great advantages in qualitative and quantitative analysis [16,17]. Therefore, HPLC–MS has great application potential for the folate compounds in serum. Liquid chromatography tandem mass spectrometry (LC/MS/MS) has been shown to provide a more efficient assessment of abnormal one-carbon metabolism [18,19]. In 2008, Zhang et al. established the LC/MS/MS
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method to detect the 10 compounds in folate- and homocysteinemediated one-carbon metabolism [20]. Separating and detecting specific folate vitamers may be one of the greatest advantages of this work. With the development of high performance liquid chromatography (HPLC), the emergence of ultraperformance liquid chromatography (UPLC) prompted the development of chromatography. Some reports have shown that the ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) has the advantage of being faster, more accurate, with higher sensitivity and specificity, compared with LC/MS/MS [21,22]. The use of UPLC is the emerging methodology of choice for folate determination in human health [23], the key folate forms including folic acid, tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-MeTHF), 5formyltetrahydrofolate (5-FoTHF), 5,10-methenylTHF [24,25] and S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) [26] were detected by use of the isotope dilution UPLC–MS/MS, which is demonstrated to have better accuracy (recovery) and precision. However, it has not been reported that UPLC–MS/MS were used to simultaneously quantify folic acid, 5-MeTHF, 5-FoTHF, homocysteine (Hcy), SAM and SAH in the same system. Here, we established simple, fast and sensitive UPLC/MS/MS method to simultaneously examine and compare the major components involved in the maternal folate–homocysteine (Hcy) metabolism. The potential of this method is to help investigate the underlying mechanism of diseases such as NTDs.
2. Materials and methods 2.1. Equipment UPLC-quadrupole tandem mass spectrometer XEVOTM TQ MS (Waters, Milford, MA, USA) was used for UPLC–MS/MS analysis. Separation was performed on a BEH C18 analytical column (2.1 mm × 50 mm I.D., 1.7 m, Waters, USA) at 40 ◦ C. Ultrapure water was prepared with a Milli-Q water purification system (Millipore, France).
2.2. Chemicals and reagents The following compounds were obtained from Sigma–Aldrich (St. Louis, MO, USA): folic acid, 5-methyltetrahydrofolate (5MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), and dithiothreitol (DTT). HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Waltham, MA, USA). Ascorbic acid and citric acid monohydrate were purchased from Beijing Chemical Company (Beijing, China).
2.3. UPLC–MS/MS The mobile phases were set as follows: 0.1% (V/V) formic acid in water (eluent A), and acetonitrile (eluent B). The following linear elution gradient was used (flow rate, 400 L/min): 0–1 min, 99.9%–99.9% A; 1–3 min, 99.9% A–82% A; 3–4 min, 82% A–10% A, 4–5 min, 10% A–10% A; 5–6 min, 10%–99.9%. The equilibration time was 1 min. The total analysis time was 7 min and the injection volume was 5 L in each run. The MS/MS operating parameters were obtained and optimized under positive-ion (ESI + ) with multiple reactions monitoring (MRM). The capillary voltages of 3000 V and source temperature of 150 ◦ C were adopted. The desolvation temperature was 400 ◦ C. The collision gas flow was set at 0.13 mL/min. The cone gas and desolvation gas flow were 30 L/h and 900 L/h, respectively.
2.4. Validation of the method 2.4.1. Preparation of calibration standards and quality control sample Stocked solutions for each standard were prepared at a concentration of 100 g/mL in 50:50 (V/V) methanol/water (containing 100 g/mL each of ascorbic acid, citric acid and DTT to inhibit oxidation) and stored at −80 ◦ C. Calibrants were prepared by diluting the stocked solution with acetonitrile/water (1/9) (containing ascorbic acid, citric acid and DTT as above), resulting in concentrations of 0.5, 1, 2, 5, 10, 50 ng/mL for folic acid, 5-MeTHF, 5-FoTHF, and SAH; 2, 5, 10, 50, 100, 200 ng/mL for SAM; and 0.05, 0.1, 0.25, 0.5, 1, 2 g/mL for Hcy. Quality control (QC) samples were spiked by adding low, medium, and high concentrations of standards into blank serum to obtain serum spiking solutions. All stocked solutions, working solutions and QC samples were stored at −80 ◦ C and brought to room temperature before use. 2.4.2. Linearity The calibration curves were obtained from plots of the peak-area versus the concentration of the standards. The concentrations of the metabolites in serum samples were determined by the equations of linear regression obtained from the calibration. 2.4.3. Intra-day and inter-day precision and recovery Intra-day precision was evaluated by analysis of QC samples at different times on the same day. Inter-day precision were determined by repeated analysis of QC samples over five consecutive days. The calibration curves were calibrated every day to ensure the precision of the results. The extraction recoveries were determined by analysis of the blank serum spiked with standards. Three concentrations were studied: the center was the endogenous level, the low and high were 50% and 200% of the center, respectively. 2.4.4. Sensitivity and stability The sensitivity of the assay was estimated using the limit of detection (LOD) and the limit of quantitation (LOQ) of the six compounds. The LOD and LOQ were defined as the minimum detectable values with signal-to-noise levels of 3 for LOD and 10 for LOQ. The compound stability for 0, 2, 4, 8, 16 and 24 h at −20 ◦ C in serum was evaluated by repeated analysis at the medium concentration of QC samples. The precision and accuracy were determined by calculating the CVs (coefficient of variations). 2.5. Sample selection and pretreatment The serum samples were collected from the NTD-affected pregnant women, and the details have been described previously [20]. 50 L dithiothreitol (DTT) (10 mg/mL) was added to 100 L serum, vortexed for 1 min, and then treated with 500 mL of methanol containing 100 g/mL each of ascorbic acid and citric acid. The mixture was vortexed for 2 min and then centrifuged at 12,000 rpm for 20 min at 4 ◦ C. The supernatant was transferred to a 1.5 mL Eppendorf tube and dried under nitrogen at room temperature. The residue was dissolved in 60 L aliquots of acetonitrile:water (1:9) (containing 100 g/mL of ascorbic acid, citric acid, and 10 mg/mL DTT), and stored at −80 ◦ C before analysis. 2.6. Statistical analysis Linear regression analysis was used to verify the linearity of the calibration curves. Folic acid, 5-MeTHF, 5-FoTHF, Hcy, SAM, and SAH concentrations were expressed as mean ± standard deviation (s.d.) and examined by Student’s t-test. All statistical analyses were performed with SPSS16.0 (SPSS Inc., Chicago, IL, USA).
X. Wang et al. / J. Chromatogr. B 962 (2014) 9–13
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Table 1 Multiple reaction monitoring in ESI+ of the compounds. Compound
Precursor ion
Production
Cone voltage (V)
Collision energy (eV)
Dwell time (s)
Folic acid 5-MeTHF 5-FoTHF Hcy SAM SAH
442.24 460.30 474.28 136.072 399.24 385.24
295.05 180.12 166.10 90.16 250.09 136.20
30.0 30.0 30.0 16.0 30.0 30.0
14.0 22.0 18.0 12.0 16.0 20.0
0.060 0.077 0.060 0.044 0.024 0.077
3. Results 3.1. Optimization of sample preparation To eliminate interference from the serum matrix that suppresses ionization or interferes with analyte detection, sample extraction was necessary for UPLC/MS/MS analysis. Based on previous studies [20,27], methanol was chosen as the protein precipitation agent for its intermiscibility with analytes and protein precipitation. Because of the instability of folates and homocysteine, it was necessary to add antioxidants to the system to prevent degradation. Based on literature review [20,27], the optimal combinations and concentrations of antioxidants were the combination of ascorbic acid (100 g/mL), citric acid (100 g/mL) and DTT (15 mg/mL). The protective effect of the optimal antioxidant system for folates in 24 h was evaluated. The results show that stabilities of folates obviously increased with the presence of the antioxidants (data not shown). 3.2. Optimization of chromatography and mass spectrometry conditions To minimize ion suppression from both matrix effects and interference from co-eluted compounds in the system, it was essential to increase chromatographic separation. Therefore, the hydrophilic column was chosen over standard reverse-phase columns because of its better adsorption capacity of polarity compounds. Based upon hydrophilic interaction liquid chromatography, we evaluated five different hydrophilic chromatography columns including Ultimate AQ-C18 (250 mm × 4.6 mm I.D., 5 m particles, Welch Materials, MD, USA), Acquity UPLC HSS T3 column (2.1 mm × 50 mm I.D., 1.8 m, Waters, USA), Acquity UPLC HSS T3 column (2.1 mm × 100 mm I.D., 1.8 m, Waters, USA), Acquity UPLC BEH C18 column (2.1 mm × 100 mm I.D., 1.7 m, Waters, USA), and Acquity UPLC BEH C18 column (2.1 mm × 50 mm I.D., 1.7 m, Waters, USA). Finally, the A Acquity UPLC BEH C18 column (2.1 mm × 50 mm I.D., 1.7 m waters, USA) was chosen for its better retention and separation of the analytes than the other four columns. The mobile phases included organic solvents and volatile aqueous buffers at various pH values and ionic strengths. Considering the volatile buffers (ammonium formate, formic acid and acetic acid) was investigated. The influences of temperature, pH, and mobile phase flow rate on retention were also studied. The final chromatography conditions were determined as described in Section 2.3. Under the optimal UPLC conditions, folic acid, 5-MeTHF, 5FoTHF, Hcy, SAM, and SAH eluted at 2.83, 2.48, 2.78, 0.66, 0.36, 0.47 min, respectively (Fig. 1). Carryover was not obvious in blank matrices (less 10% of LOQ). Blank plasma samples from six different lots of plasma showed no interference for the compound analytes. To obtain the highest selectivity and lowest limit of quantification, the ion source temperature, ionspray voltage, collision gas, and nebulizer gas were optimized in turn by the manual sample injector. The flow rate and injection volume were the same as sample
Fig. 1. UPLC/MS/MS analysis of six compounds from serum in a single run with MRM mode. FA, folic acid, 5-MeTHF, 5-methyltetrahydrofolate; 5-FoTHF, 5-formyltetrahydrofolate; Hcy, homocysteine; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; UPLC/MS/MS, ultraperformance liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring.
analysis for each analyte. In addition, many ion source parameters interact with each other, which require repeated modulation. Optimal MRM conditions were obtained in the positive electrospray ionization mode, and typical m/z transitions of the compounds are shown in Table 1.
3.3. Validation of the UPLC–MS/MS method 3.3.1. Calibration curve, linearity, and sensitivity Under the optimized conditions, six compounds (folic acid, 5MeTHF, 5-FoTHF, Hcy, SAM, and SAH) could be separated and detected using the UPLC/MS/MS method, and calibration curves were obtained. The correlation between analyte concentration and peak area was linear. The regression equation of calibration curves and their correlation coefficients (r) are shown in Table 2. All the calibration curves were suitable for the analysis of samples. The limit of detection (LOD) and the limit of quantification (LOQ) are displayed for all the metabolites in Table 2.
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Table 2 Regression equations and limits of detection and quantitation of six compounds. Measured compound
Regression equation
Folic acid 5-MeTHF 5-FoTHF Hcy SAM SAH
y = 2.684x − 12.71 y = 51.74x − 83.27 y = 15.99x + 30.84 y = 142.3x + 180.8 y = 3.586x − 0.399 y = 18.89x − 30.53
Linear range (ng/mL) 10–500 2–100 0.5–50 50–2000 4–200 4–200
r2
LOD (ng/mL)
LOQ (ng/mL)
0.999 0.999 0.998 0.999 0.998 0.999
0.05 0.05 0.05 0.05 0.20 0.10
0.20 0.15 0.20 0.50 0.50 0.20
3.3.2. Inter-day, Intra-day reproducibility and stability The data from QC samples were calculated to estimate the intraday precision, inter-day precision, and extraction recoveries. The results are displayed in Table 3. The QC samples showed no significant degradation observed in 24 h at room temperature (CV < 8.5%) (data not shown).
concentrations of 5-MeTHF (P < 0.05), 5-FoTHF (P < 0.05) and higher concentrations of Hcy (P < 0.05), and SAH (P < 0.05) compared with the serum samples from controls.
3.4. Application of the method
Folate deficiency is considered a risk factor for many diseases including NTDs [9,28]. In the folate metabolic process, unmetabolized dietary folic acid, THF, 5-MeTHF, and 5-FoTHF are the main folate metabolic intermediate products. Furthermore, folate is involved in homocysteine-mediated one-carbon metabolism, and a low-folate status can result in the accumulation of Hcy [29]. Additionally, high Hcy levels result in the accumulation of Sadenosyl-homocysteine (SAH). Elevated SAH concentrations will reduce the ratio of S-adenosyl-methionine (SAM):SAH, also called “methylation index” [30,31]. THF could not be detected because of its extremely low concentrations in human serum or plasma [32]. Therefore, we chose folate and its main metabolites (5-MeTHF, 5-FoTHF, Hcy, SAM and SAH) as the objects of study. Isotope dilution UPLC–MS/MS has been regarded as the ‘gold standard’ and a small number of isotope dilution UPLC–MS/MS methods have been reported for the analysis of folates in serum [24–26], which will result in better accuracy (recovery) and precision. However, some general shortcomings have to be mentioned. Firstly, it is expensive to use stable isotope labeled standards; secondly, isotope exchange between the standard and sample is possible, and interferences in biological systems can occur; thirdly, one important disadvantage is that most of the labeled standards were not commercially available [23,26]. According to the NIST SRM 1955, assigned values for 5-FoTHF, SAM, SAH are lacking, nevertheless, values for folic acid, 5MeTHF, and Hcy (https://www-s.nist.gov/srmors/view detail.cfm?srm=1955) are possessed and measuring these folate levels in such a material would support accuracy claims based on recovery of the assigned value. Furthermore, the detection of the six compounds levels in human serum can help us to monitor the folate metabolic pathways more comprehensively, and provide us a chance to find abnormalities in these pathways. Therefore, we developed a UPLC–MS/MS method for the simultaneous quantification of folate metabolites in human serum. The new method is low cost, simple to operate, fast to detect the six compounds. Besides, it reduced sample injections for folate metabolites detection. The total analysis time was only 7 min and the injection volume was 5 L. Additionally, the sensitivity (detection limit ranged from 0.05 to 0.200 ng/mL) of our method is less than the above-mentioned method, but the linearity (r2 > 0.998), precision (CV < 6%) and accuracy (each analyte was >90%) in our study are consistent with the previous study [24–26]. Therefore, the linearity, sensitivity, accuracy and precision of our method can meet the sample testing requirementWe used our method to analyze the NTD samples, and found that serum samples from NTD cases had lower concentrations of 5-MeTHF (P < 0.05) and 5-FoTHF (P < 0.05), and higher concentrations of Hcy (P < 0.05) and SAH (P < 0.05) compared with the serum samples from controls. The results are consistent with the previous LC/MS/MS method reported by Zhang et al. [20].
Folate deficiency is closely related to developmental abnormalities, especially NTDs. The method was used to investigate the relationship between disturbed maternal folate and homocysteine metabolism and the risk of having pregnancies affected by NTDs. Six compounds, including folic acid, 5-MeTHF, 5-FoTHF, Hcy, SAM and SAH could be separated and detected simultaneously using the established UPLC/MS/MS method. Serum concentrations for each component and the ratio of SAM:SAH are summarized in Table 4. The serum samples from these NTD cases showed lower Table 3 Precisions and recoveries of spiked QC serum samples. Compound
Intra-day (n = 6)
Inter-day (n = 9)
Spiked concentration (ng/mL)
Recovery (%)
CV (%)
CV (%)
Folic acid
20 40 160
100.3 103.8 105.0
5.21 5.08 4.69
4.11 4.00 3.46
5-MeTHF
10 20 40
96.4 99.3 100.9
4.89 3.61 1.83
5.64 3.43 2.44
5-FoTHF
5 10 20
90.1 93.2 98.2
5.59 3.17 2.89
5.43 3.60 2.72
400 800 1600
95.7 96.4 99.6
5.32 4.55 4.01
4.78 3.95 2.45
SAM
10 20 40
90.3 93.4 94.2
5.82 5.58 5.84
3.90 3.66 2.21
SAH
8 16 32
92.0 99.5 105.3
4.03 3.79 2.97
5.60 3.62 3.50
Hcy
Table 4 Serum component concentrations between case and controls for pregnant women. Control (n = 50) Folic acid(ng/mL) 5-MeTHF (ng/mL) 5-FoTHF (ng/mL) Hcy (ng/mL) SAM (ng/mL) SAH (ng/mL) SAM:SAH (ng/mL)
37.62 77.51 3.69 798.95 49.41 12.36 4.83
± ± ± ± ± ± ±
15.76. 24.93 1.51 229.62 19.13 5.49 3.17
NTD Case (n = 50) 39.52 38.55 1.69 1255.67 43.37 18.23 2.86
± ± ± ± ± ± ±
14.65 15.44* 0.93* 477.16* 16.92 7.25* 1.77
* P < 0.05 NTDs versus control, adjusted for age, gestational week, folate supplementation, gravidity, education level and household income.
4. Discussion
X. Wang et al. / J. Chromatogr. B 962 (2014) 9–13
A simple, sensitive, specific, accurate and repeatable method for determination of six compounds of folate–Hcy metabolism in serum was optimized, validated and applied to the analysis of serum from NTD-affected pregnancies, and six metabolites were quantified. The high analysis speed, short analysis time, low sample cost and simple pretreatment procedure permit the use of this method for large-scale population-based applications. In conclusion, our presented method will provide a solid foundation for prenatal diagnosis and prevention of NTDs, as well as some other diseases related to disturbed folate–Hcy metabolism such as cancer or congenital heart defects. Acknowledgements This study was supported by The Ministry of Science and Technology of China, National “973” Project on Population and Health (Project 2013CB945404) and the National Natural Science Foundation of China (Project 81070491). We thank the staff and all participants who joined in and contributed to the laborious field work and also appreciate the participation of the female volunteers in this study. References [1] A.E. Beaudin, P.J. Stover, Birth Defects Res. C: Embryo Today 81 (3) (2007) 183–203. [2] S.J. James, B.J. Miller, D.R. Cross, L.J. McGarrity, S.M. Morris, Environ. Health Perspect. 101 (Suppl 5) (1993) 173–178. [3] B.N. Ames, Ann. N. Y. Acad. Sci. 889 (1999) 87–106. [4] S.J. Duthie, S. Narayanan, G.M. Brand, L. Pirie, G. Grant, J. Nutr. 132 (8 Suppl.) (2002) 2444S–2449S. [5] E. Knock, L. Deng, N. Krupenko, R.D. Mohan, Q. Wu, D. Leclerc, S. Gupta, C.L. Elmore, W. Kruger, M. Tini, R. Rozen, J. Nutr. Biochem. 22 (11) (2011) 1022–1029. [6] Y.I. Kim, K. Fawaz, T. Knox, Y.M. Lee, R. Norton, S. Arora, L. Paiva, J.B. Mason, Am. J. Clin. Nutr. 68 (4) (1998) 866–872. [7] D.G. Weir, J.M. Scott, Nutr. Res. Rev. 11 (2) (1998) 311–338. [8] J.C. Huhta, J.A. Hernandez-Robles, Fetal Pediatr. Pathol. 24 (2) (2005) 71–79.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Quantification of folate metabolites in serum using ultraperformance liquid chromatography tandem mass spectrometry Xiuwei Wang a , Ting Zhang a , Xin Zhao b , Zhen Guan a , Zhen Wang a , Zhiqiang Zhu a , Qiu Xie a , Jianhua Wang a,∗ , Bo Niu a,c,∗ a
Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing 100020, China Chines Academy of Inspection & Quarantine, Beijing 100023, China c Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China b
a r t i c l e
i n f o
Article history: Received 13 January 2014 Accepted 8 May 2014 Available online 20 May 2014 Keywords: UPLC/MS/MS Neural tube defects Folic acid One-carbon metabolism
a b s t r a c t Folate deficiency is considered a risk factor for many diseases such as cancer, congenital heart disease and neural tube defects (NTDs). There is a pressing need for more methods of detecting folate and its main metabolites in the human body. Here, we developed a simple, fast and sensitive ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method for the simultaneous quantifications of folate metabolites including folic acid, 5-methyltetrahydrofolate (5-MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The method was validated by determining the linearity (r2 > 0.998), sensitivity (limit of detection ranged from 0.05 to 0.200 ng/mL), intra- and inter-day precision (both CV < 6%) and recovery (each analyte was >90%). The total analysis time was 7 min. Serum samples of NTD-affected pregnancies and controls from a NTD high-risk area in China were analyzed by this method, the NTD serum samples showed lower concentrations of 5-MeTHF (P < 0.05) and 5-FoTHF (P < 0.05), and higher concentrations of Hcy (P < 0.05) and SAH (P < 0.05) compared with serum samples from controls, consistent with a previous study. These results showed that the method is sensitive and reliable for simultaneous determination of six metabolites, which might indicate potential risk factors for NTDs, aid early diagnosis and provide more insights into the pathogenesis of NTDs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Folate is converted to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR) once transported into the cell and functions as the carrier for one carbon unit involved in various biological processes particularly nucleotide metabolism, DNA damage repair and methylation action [1,2]. Reports have shown that folate deficiency is a risk factor for a variety of diseases such as megaloblastic anemia [3], cancer [4–6], congenital heart disease [7,8], and neural tube defects (NTDs) [9]. Therefore, it is of vital significance to develop a sensitive and fast method for detecting folate levels in the human body. The analysis of folate provides a great challenge owing to its poor stability and extremely small amounts in biological matrix samples, and its compounds are complicated. There have been many
∗ Corresponding authors at: Capital Institute of Pediatrics, Beijing 100020, China. Tel.: +86 10 85695545; fax: +86 10 85631504. E-mail addresses: [email protected] (J. Wang), [email protected] (B. Niu). http://dx.doi.org/10.1016/j.jchromb.2014.05.023 1570-0232/© 2014 Elsevier B.V. All rights reserved.
methods developed for the detection of folate such as microbiological assay [10], radioimmunoassay [11,12], capillary electrophoresis [13] and chromatography [14,15]. The most useful methods are microbiological assay and radioimmunoassay in clinical practice. However, microbiological assays are time costly and lack repetition. Radioimmunoassay has the advantage of fast and simple for sample detection, but its results vary wildly with different kits. These methods detect the total folate, that is folate plus its derivatives, with low selectivity, and different results are found by different methods used for the same sample. When high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) was used for folate analysis, it not only rapidly and effectively separated the folate compounds, but could meet the requirements of a low detection limit owing to the high sensitivity of mass spectrometer, and show great advantages in qualitative and quantitative analysis [16,17]. Therefore, HPLC–MS has great application potential for the folate compounds in serum. Liquid chromatography tandem mass spectrometry (LC/MS/MS) has been shown to provide a more efficient assessment of abnormal one-carbon metabolism [18,19]. In 2008, Zhang et al. established the LC/MS/MS
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method to detect the 10 compounds in folate- and homocysteinemediated one-carbon metabolism [20]. Separating and detecting specific folate vitamers may be one of the greatest advantages of this work. With the development of high performance liquid chromatography (HPLC), the emergence of ultraperformance liquid chromatography (UPLC) prompted the development of chromatography. Some reports have shown that the ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) has the advantage of being faster, more accurate, with higher sensitivity and specificity, compared with LC/MS/MS [21,22]. The use of UPLC is the emerging methodology of choice for folate determination in human health [23], the key folate forms including folic acid, tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-MeTHF), 5formyltetrahydrofolate (5-FoTHF), 5,10-methenylTHF [24,25] and S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) [26] were detected by use of the isotope dilution UPLC–MS/MS, which is demonstrated to have better accuracy (recovery) and precision. However, it has not been reported that UPLC–MS/MS were used to simultaneously quantify folic acid, 5-MeTHF, 5-FoTHF, homocysteine (Hcy), SAM and SAH in the same system. Here, we established simple, fast and sensitive UPLC/MS/MS method to simultaneously examine and compare the major components involved in the maternal folate–homocysteine (Hcy) metabolism. The potential of this method is to help investigate the underlying mechanism of diseases such as NTDs.
2. Materials and methods 2.1. Equipment UPLC-quadrupole tandem mass spectrometer XEVOTM TQ MS (Waters, Milford, MA, USA) was used for UPLC–MS/MS analysis. Separation was performed on a BEH C18 analytical column (2.1 mm × 50 mm I.D., 1.7 m, Waters, USA) at 40 ◦ C. Ultrapure water was prepared with a Milli-Q water purification system (Millipore, France).
2.2. Chemicals and reagents The following compounds were obtained from Sigma–Aldrich (St. Louis, MO, USA): folic acid, 5-methyltetrahydrofolate (5MeTHF), 5-formyltetrahydrofolate (5-FoTHF), homocysteine (Hcy), S-adenosyl-methionine (SAM), S-adenosyl-homocysteine (SAH), and dithiothreitol (DTT). HPLC-grade methanol and acetonitrile were purchased from Fisher Scientific (Waltham, MA, USA). Ascorbic acid and citric acid monohydrate were purchased from Beijing Chemical Company (Beijing, China).
2.3. UPLC–MS/MS The mobile phases were set as follows: 0.1% (V/V) formic acid in water (eluent A), and acetonitrile (eluent B). The following linear elution gradient was used (flow rate, 400 L/min): 0–1 min, 99.9%–99.9% A; 1–3 min, 99.9% A–82% A; 3–4 min, 82% A–10% A, 4–5 min, 10% A–10% A; 5–6 min, 10%–99.9%. The equilibration time was 1 min. The total analysis time was 7 min and the injection volume was 5 L in each run. The MS/MS operating parameters were obtained and optimized under positive-ion (ESI + ) with multiple reactions monitoring (MRM). The capillary voltages of 3000 V and source temperature of 150 ◦ C were adopted. The desolvation temperature was 400 ◦ C. The collision gas flow was set at 0.13 mL/min. The cone gas and desolvation gas flow were 30 L/h and 900 L/h, respectively.
2.4. Validation of the method 2.4.1. Preparation of calibration standards and quality control sample Stocked solutions for each standard were prepared at a concentration of 100 g/mL in 50:50 (V/V) methanol/water (containing 100 g/mL each of ascorbic acid, citric acid and DTT to inhibit oxidation) and stored at −80 ◦ C. Calibrants were prepared by diluting the stocked solution with acetonitrile/water (1/9) (containing ascorbic acid, citric acid and DTT as above), resulting in concentrations of 0.5, 1, 2, 5, 10, 50 ng/mL for folic acid, 5-MeTHF, 5-FoTHF, and SAH; 2, 5, 10, 50, 100, 200 ng/mL for SAM; and 0.05, 0.1, 0.25, 0.5, 1, 2 g/mL for Hcy. Quality control (QC) samples were spiked by adding low, medium, and high concentrations of standards into blank serum to obtain serum spiking solutions. All stocked solutions, working solutions and QC samples were stored at −80 ◦ C and brought to room temperature before use. 2.4.2. Linearity The calibration curves were obtained from plots of the peak-area versus the concentration of the standards. The concentrations of the metabolites in serum samples were determined by the equations of linear regression obtained from the calibration. 2.4.3. Intra-day and inter-day precision and recovery Intra-day precision was evaluated by analysis of QC samples at different times on the same day. Inter-day precision were determined by repeated analysis of QC samples over five consecutive days. The calibration curves were calibrated every day to ensure the precision of the results. The extraction recoveries were determined by analysis of the blank serum spiked with standards. Three concentrations were studied: the center was the endogenous level, the low and high were 50% and 200% of the center, respectively. 2.4.4. Sensitivity and stability The sensitivity of the assay was estimated using the limit of detection (LOD) and the limit of quantitation (LOQ) of the six compounds. The LOD and LOQ were defined as the minimum detectable values with signal-to-noise levels of 3 for LOD and 10 for LOQ. The compound stability for 0, 2, 4, 8, 16 and 24 h at −20 ◦ C in serum was evaluated by repeated analysis at the medium concentration of QC samples. The precision and accuracy were determined by calculating the CVs (coefficient of variations). 2.5. Sample selection and pretreatment The serum samples were collected from the NTD-affected pregnant women, and the details have been described previously [20]. 50 L dithiothreitol (DTT) (10 mg/mL) was added to 100 L serum, vortexed for 1 min, and then treated with 500 mL of methanol containing 100 g/mL each of ascorbic acid and citric acid. The mixture was vortexed for 2 min and then centrifuged at 12,000 rpm for 20 min at 4 ◦ C. The supernatant was transferred to a 1.5 mL Eppendorf tube and dried under nitrogen at room temperature. The residue was dissolved in 60 L aliquots of acetonitrile:water (1:9) (containing 100 g/mL of ascorbic acid, citric acid, and 10 mg/mL DTT), and stored at −80 ◦ C before analysis. 2.6. Statistical analysis Linear regression analysis was used to verify the linearity of the calibration curves. Folic acid, 5-MeTHF, 5-FoTHF, Hcy, SAM, and SAH concentrations were expressed as mean ± standard deviation (s.d.) and examined by Student’s t-test. All statistical analyses were performed with SPSS16.0 (SPSS Inc., Chicago, IL, USA).
X. Wang et al. / J. Chromatogr. B 962 (2014) 9–13
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Table 1 Multiple reaction monitoring in ESI+ of the compounds. Compound
Precursor ion
Production
Cone voltage (V)
Collision energy (eV)
Dwell time (s)
Folic acid 5-MeTHF 5-FoTHF Hcy SAM SAH
442.24 460.30 474.28 136.072 399.24 385.24
295.05 180.12 166.10 90.16 250.09 136.20
30.0 30.0 30.0 16.0 30.0 30.0
14.0 22.0 18.0 12.0 16.0 20.0
0.060 0.077 0.060 0.044 0.024 0.077
3. Results 3.1. Optimization of sample preparation To eliminate interference from the serum matrix that suppresses ionization or interferes with analyte detection, sample extraction was necessary for UPLC/MS/MS analysis. Based on previous studies [20,27], methanol was chosen as the protein precipitation agent for its intermiscibility with analytes and protein precipitation. Because of the instability of folates and homocysteine, it was necessary to add antioxidants to the system to prevent degradation. Based on literature review [20,27], the optimal combinations and concentrations of antioxidants were the combination of ascorbic acid (100 g/mL), citric acid (100 g/mL) and DTT (15 mg/mL). The protective effect of the optimal antioxidant system for folates in 24 h was evaluated. The results show that stabilities of folates obviously increased with the presence of the antioxidants (data not shown). 3.2. Optimization of chromatography and mass spectrometry conditions To minimize ion suppression from both matrix effects and interference from co-eluted compounds in the system, it was essential to increase chromatographic separation. Therefore, the hydrophilic column was chosen over standard reverse-phase columns because of its better adsorption capacity of polarity compounds. Based upon hydrophilic interaction liquid chromatography, we evaluated five different hydrophilic chromatography columns including Ultimate AQ-C18 (250 mm × 4.6 mm I.D., 5 m particles, Welch Materials, MD, USA), Acquity UPLC HSS T3 column (2.1 mm × 50 mm I.D., 1.8 m, Waters, USA), Acquity UPLC HSS T3 column (2.1 mm × 100 mm I.D., 1.8 m, Waters, USA), Acquity UPLC BEH C18 column (2.1 mm × 100 mm I.D., 1.7 m, Waters, USA), and Acquity UPLC BEH C18 column (2.1 mm × 50 mm I.D., 1.7 m, Waters, USA). Finally, the A Acquity UPLC BEH C18 column (2.1 mm × 50 mm I.D., 1.7 m waters, USA) was chosen for its better retention and separation of the analytes than the other four columns. The mobile phases included organic solvents and volatile aqueous buffers at various pH values and ionic strengths. Considering the volatile buffers (ammonium formate, formic acid and acetic acid) was investigated. The influences of temperature, pH, and mobile phase flow rate on retention were also studied. The final chromatography conditions were determined as described in Section 2.3. Under the optimal UPLC conditions, folic acid, 5-MeTHF, 5FoTHF, Hcy, SAM, and SAH eluted at 2.83, 2.48, 2.78, 0.66, 0.36, 0.47 min, respectively (Fig. 1). Carryover was not obvious in blank matrices (less 10% of LOQ). Blank plasma samples from six different lots of plasma showed no interference for the compound analytes. To obtain the highest selectivity and lowest limit of quantification, the ion source temperature, ionspray voltage, collision gas, and nebulizer gas were optimized in turn by the manual sample injector. The flow rate and injection volume were the same as sample
Fig. 1. UPLC/MS/MS analysis of six compounds from serum in a single run with MRM mode. FA, folic acid, 5-MeTHF, 5-methyltetrahydrofolate; 5-FoTHF, 5-formyltetrahydrofolate; Hcy, homocysteine; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; UPLC/MS/MS, ultraperformance liquid chromatography tandem mass spectrometry; MRM, multiple reaction monitoring.
analysis for each analyte. In addition, many ion source parameters interact with each other, which require repeated modulation. Optimal MRM conditions were obtained in the positive electrospray ionization mode, and typical m/z transitions of the compounds are shown in Table 1.
3.3. Validation of the UPLC–MS/MS method 3.3.1. Calibration curve, linearity, and sensitivity Under the optimized conditions, six compounds (folic acid, 5MeTHF, 5-FoTHF, Hcy, SAM, and SAH) could be separated and detected using the UPLC/MS/MS method, and calibration curves were obtained. The correlation between analyte concentration and peak area was linear. The regression equation of calibration curves and their correlation coefficients (r) are shown in Table 2. All the calibration curves were suitable for the analysis of samples. The limit of detection (LOD) and the limit of quantification (LOQ) are displayed for all the metabolites in Table 2.
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Table 2 Regression equations and limits of detection and quantitation of six compounds. Measured compound
Regression equation
Folic acid 5-MeTHF 5-FoTHF Hcy SAM SAH
y = 2.684x − 12.71 y = 51.74x − 83.27 y = 15.99x + 30.84 y = 142.3x + 180.8 y = 3.586x − 0.399 y = 18.89x − 30.53
Linear range (ng/mL) 10–500 2–100 0.5–50 50–2000 4–200 4–200
r2
LOD (ng/mL)
LOQ (ng/mL)
0.999 0.999 0.998 0.999 0.998 0.999
0.05 0.05 0.05 0.05 0.20 0.10
0.20 0.15 0.20 0.50 0.50 0.20
3.3.2. Inter-day, Intra-day reproducibility and stability The data from QC samples were calculated to estimate the intraday precision, inter-day precision, and extraction recoveries. The results are displayed in Table 3. The QC samples showed no significant degradation observed in 24 h at room temperature (CV < 8.5%) (data not shown).
concentrations of 5-MeTHF (P < 0.05), 5-FoTHF (P < 0.05) and higher concentrations of Hcy (P < 0.05), and SAH (P < 0.05) compared with the serum samples from controls.
3.4. Application of the method
Folate deficiency is considered a risk factor for many diseases including NTDs [9,28]. In the folate metabolic process, unmetabolized dietary folic acid, THF, 5-MeTHF, and 5-FoTHF are the main folate metabolic intermediate products. Furthermore, folate is involved in homocysteine-mediated one-carbon metabolism, and a low-folate status can result in the accumulation of Hcy [29]. Additionally, high Hcy levels result in the accumulation of Sadenosyl-homocysteine (SAH). Elevated SAH concentrations will reduce the ratio of S-adenosyl-methionine (SAM):SAH, also called “methylation index” [30,31]. THF could not be detected because of its extremely low concentrations in human serum or plasma [32]. Therefore, we chose folate and its main metabolites (5-MeTHF, 5-FoTHF, Hcy, SAM and SAH) as the objects of study. Isotope dilution UPLC–MS/MS has been regarded as the ‘gold standard’ and a small number of isotope dilution UPLC–MS/MS methods have been reported for the analysis of folates in serum [24–26], which will result in better accuracy (recovery) and precision. However, some general shortcomings have to be mentioned. Firstly, it is expensive to use stable isotope labeled standards; secondly, isotope exchange between the standard and sample is possible, and interferences in biological systems can occur; thirdly, one important disadvantage is that most of the labeled standards were not commercially available [23,26]. According to the NIST SRM 1955, assigned values for 5-FoTHF, SAM, SAH are lacking, nevertheless, values for folic acid, 5MeTHF, and Hcy (https://www-s.nist.gov/srmors/view detail.cfm?srm=1955) are possessed and measuring these folate levels in such a material would support accuracy claims based on recovery of the assigned value. Furthermore, the detection of the six compounds levels in human serum can help us to monitor the folate metabolic pathways more comprehensively, and provide us a chance to find abnormalities in these pathways. Therefore, we developed a UPLC–MS/MS method for the simultaneous quantification of folate metabolites in human serum. The new method is low cost, simple to operate, fast to detect the six compounds. Besides, it reduced sample injections for folate metabolites detection. The total analysis time was only 7 min and the injection volume was 5 L. Additionally, the sensitivity (detection limit ranged from 0.05 to 0.200 ng/mL) of our method is less than the above-mentioned method, but the linearity (r2 > 0.998), precision (CV < 6%) and accuracy (each analyte was >90%) in our study are consistent with the previous study [24–26]. Therefore, the linearity, sensitivity, accuracy and precision of our method can meet the sample testing requirementWe used our method to analyze the NTD samples, and found that serum samples from NTD cases had lower concentrations of 5-MeTHF (P < 0.05) and 5-FoTHF (P < 0.05), and higher concentrations of Hcy (P < 0.05) and SAH (P < 0.05) compared with the serum samples from controls. The results are consistent with the previous LC/MS/MS method reported by Zhang et al. [20].
Folate deficiency is closely related to developmental abnormalities, especially NTDs. The method was used to investigate the relationship between disturbed maternal folate and homocysteine metabolism and the risk of having pregnancies affected by NTDs. Six compounds, including folic acid, 5-MeTHF, 5-FoTHF, Hcy, SAM and SAH could be separated and detected simultaneously using the established UPLC/MS/MS method. Serum concentrations for each component and the ratio of SAM:SAH are summarized in Table 4. The serum samples from these NTD cases showed lower Table 3 Precisions and recoveries of spiked QC serum samples. Compound
Intra-day (n = 6)
Inter-day (n = 9)
Spiked concentration (ng/mL)
Recovery (%)
CV (%)
CV (%)
Folic acid
20 40 160
100.3 103.8 105.0
5.21 5.08 4.69
4.11 4.00 3.46
5-MeTHF
10 20 40
96.4 99.3 100.9
4.89 3.61 1.83
5.64 3.43 2.44
5-FoTHF
5 10 20
90.1 93.2 98.2
5.59 3.17 2.89
5.43 3.60 2.72
400 800 1600
95.7 96.4 99.6
5.32 4.55 4.01
4.78 3.95 2.45
SAM
10 20 40
90.3 93.4 94.2
5.82 5.58 5.84
3.90 3.66 2.21
SAH
8 16 32
92.0 99.5 105.3
4.03 3.79 2.97
5.60 3.62 3.50
Hcy
Table 4 Serum component concentrations between case and controls for pregnant women. Control (n = 50) Folic acid(ng/mL) 5-MeTHF (ng/mL) 5-FoTHF (ng/mL) Hcy (ng/mL) SAM (ng/mL) SAH (ng/mL) SAM:SAH (ng/mL)
37.62 77.51 3.69 798.95 49.41 12.36 4.83
± ± ± ± ± ± ±
15.76. 24.93 1.51 229.62 19.13 5.49 3.17
NTD Case (n = 50) 39.52 38.55 1.69 1255.67 43.37 18.23 2.86
± ± ± ± ± ± ±
14.65 15.44* 0.93* 477.16* 16.92 7.25* 1.77
* P < 0.05 NTDs versus control, adjusted for age, gestational week, folate supplementation, gravidity, education level and household income.
4. Discussion
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A simple, sensitive, specific, accurate and repeatable method for determination of six compounds of folate–Hcy metabolism in serum was optimized, validated and applied to the analysis of serum from NTD-affected pregnancies, and six metabolites were quantified. The high analysis speed, short analysis time, low sample cost and simple pretreatment procedure permit the use of this method for large-scale population-based applications. In conclusion, our presented method will provide a solid foundation for prenatal diagnosis and prevention of NTDs, as well as some other diseases related to disturbed folate–Hcy metabolism such as cancer or congenital heart defects. Acknowledgements This study was supported by The Ministry of Science and Technology of China, National “973” Project on Population and Health (Project 2013CB945404) and the National Natural Science Foundation of China (Project 81070491). We thank the staff and all participants who joined in and contributed to the laborious field work and also appreciate the participation of the female volunteers in this study. References [1] A.E. Beaudin, P.J. Stover, Birth Defects Res. C: Embryo Today 81 (3) (2007) 183–203. [2] S.J. James, B.J. Miller, D.R. Cross, L.J. McGarrity, S.M. Morris, Environ. Health Perspect. 101 (Suppl 5) (1993) 173–178. [3] B.N. Ames, Ann. N. Y. Acad. Sci. 889 (1999) 87–106. [4] S.J. Duthie, S. Narayanan, G.M. Brand, L. Pirie, G. Grant, J. Nutr. 132 (8 Suppl.) (2002) 2444S–2449S. [5] E. Knock, L. Deng, N. Krupenko, R.D. Mohan, Q. Wu, D. Leclerc, S. Gupta, C.L. Elmore, W. Kruger, M. Tini, R. Rozen, J. Nutr. Biochem. 22 (11) (2011) 1022–1029. [6] Y.I. Kim, K. Fawaz, T. Knox, Y.M. Lee, R. Norton, S. Arora, L. Paiva, J.B. Mason, Am. J. Clin. Nutr. 68 (4) (1998) 866–872. [7] D.G. Weir, J.M. Scott, Nutr. Res. Rev. 11 (2) (1998) 311–338. [8] J.C. Huhta, J.A. Hernandez-Robles, Fetal Pediatr. Pathol. 24 (2) (2005) 71–79.
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