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Simultaneous determination of seven endogenous aldehydes in human blood by headspace gas chromatography–mass spectrometry
Journal of Chromatography B 1118–1119 (2019) 85–92
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Simultaneous determination of seven endogenous aldehydes in human blood by headspace gas chromatography–mass spectrometry
T
Yi Weia, Menghan Wanga, Huixia Liua, Ying Niua, Shaomin Wanga, , Fuyi Zhanga, Hongmin Liub ⁎
a b
College of Chemistry and Molecular Engineering, Zhengzhou University, PR China Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, PR China
ARTICLE INFO
ABSTRACT
Keywords: Endogenous aldehydes Gas chromatography-mass spectrometry Static headspace technique Blood Bladder cancer
Endogenous aldehydes (EAs) formed by the free-radical-mediated reaction are regarded as potential biological markers of several diseases. In this work, an automated and solvent-free analytical method was developed for quantitative analysis of seven EAs (C1–C7) in human blood by using gas chromatography–mass spectrometry coupled with a headspace generator sampler (HS-GC–MS). A 1:4 (blood:water) dilution and the 1,2‑dibromopropane internal standard were introduced to reduce the influence of the matrix effect from complex biological fluids. The sample preparation steps were simplified. Various experimental parameters for derivatization and extraction conditions were studied such as HS extraction temperature and time, the amount of derivatization reagent, pH and the salt effect. Under these optimum conditions, seven low-mass aldehydes were separated and analyzed within 10 min. Additionally, this method achieved limits of detection in the range of 0.0692–0.864 μg/L, an excellent linearity with correlation coefficients higher than 0.9996 and appropriate repeatability and reproducibility values (RSD < 12% at low, middle and high levels). The HS-GC–MS method was applied to measure the concentrations of the seven aldehydes in blood from bladder cancer patients (n = 15) and control subjects (n = 15). Compared with the control subjects, the levels of butanal (p < 0.01), formaldehyde, acetaldehyde, propanal, pentanal, hexanal and heptanal (all p < 0.001) were increased significantly, indicating that EAs may be useful as biomarkers in the early diagnosis of bladder cancer.
1. Introduction Endogenous aldehydes (EAs) are formed by the metabolism of amino acids, carbohydrates, lipids, biogenic amines, vitamins and steroids [1]. The lipid peroxidation process in which a variety of reactive oxygen/nitrogen species (ROS/RNS) attack polyunsaturated fatty acid residues of phospholipids is a major source of endogenous aldehydes, proving that the free-radical-mediated reaction occurs during the formation of EAs [1,2]. Human tumor cells produce large amounts of radicals that lead to an increase in the aldehyde concentration [3,4], and remarkable aldehyde levels were found in the blood and urine of cancer patients [5,6]. Therefore, various aldehydes can be regarded as potential biomarkers of cancer, and accurate quantification of EAs in biological fluids is expected to be an important part of clinical early diagnosis of diseases in the future. Chromatographic separation techniques have been commonly used for the determination of aldehydes. A derivatization step prior to the extraction and chromatographic analysis is necessary because the aldehydes are highly polar, chemically unstable, volatile and do not
⁎
contain chromophores and fluorophores [7]. 2,4‑Dinitrophenylhydrazine (DNPH) is the most frequently used derivatization reagent for liquid chromatography (LC) separation followed by diode array or mass spectrometric (MS) detection [8–12]. However, several drawbacks preclude the use of DNPH in the determination of aldehyde compounds; for example, formaldehyde cannot be accurately quantified due to the high uncertainty of the MS signals of its DNPH derivative [11], and methylglyoxal required > 12 h for reacting with DNPH [13]. Gas chromatography/mass spectrometry (GC–MS) has been widely applied to the analysis of low-molecular-mass aldehydes [14–17] because of its rapid analysis and high sensitivity. Several derivatization reagents used in GC have been reported such as 2,3‑diamino‑2,3‑dimethylbutane (DDB) [18], 2,4,6‑trichlorophenylhydrazine (TCPH) [19], pentafluorophenylhydrazine (PFPH) [20] and O‑2,3,4,5,6‑(pentafluorobenzyl) hydroxylamine (PFBHA) [15,16,21–24]. Because aldehydes yield stable and volatile oximes at room temperature by reaction with PFBHA, GC-PFBHA analysis methods are regarded as a useful alternative to LC-DNPH analysis. Due to the presence of various interferences and trace amounts of
Corresponding author at: College of Chemistry and Molecular Engineering, Zhengzhou University, 75 University Road, Zhengzhou 450052, PR China. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.jchromb.2019.04.027 Received 29 January 2019; Received in revised form 9 April 2019; Accepted 11 April 2019 Available online 12 April 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
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aldehydes in biological samples, cleanup and enrichment procedures are adopted to reduce the matrix effect and improve the method sensitivity. Due to the well-known disadvantages of conventional sampling techniques for the determination of aldehydes, microextraction techniques such as solid phase microextraction (SPME) [22,25] and single drop microextraction (SDME) [19,23] have attracted increasing interest. However, SPME is prone to sample carryover and is relatively expensive due to limited lifetime and fragility of the fiber [26], while SDME requires more elaborate manual operations. The headspace sampling (HS) technique is a fast, automated and solvent-free sample preparation method. The use of HS with GC can minimize the sample treatment and avoid the interferences of non-volatile components in the matrices [27]. Recently, a sensitive HS-GC–MS analytical method using diluted blank urine for the detection of twelve aldehydes in urine has been introduced by Serrano et al. [24]. Based on this method, an automatic, low-cost, solvent-free analytical method for EAs has been realized and the strong effect of the matrix protein was minimized. Some reports have focused on the presence of aldehydes in urine [11,24,27,28] for the detection of several diseases because of the noninvasive nature of the detection method. Only a few have been reported in the blood, which can directly relate to the internal activities of the human body. In these studies, hexanal and heptanal have been commonly detected [7,12,22,23]. Based on the considerations mentioned above, this research focused on the following aspects: (i) developing a solvent-free and automated HS-GC–MS method to quantify seven aldehydes including hexanal and heptanal in the blood of bladder cancer patients; (ii) reducing the matrix effect of blood samples with an appropriate dilution ratio and internal standard (IS); and (iii) simplifying the sample preparation step with a derivatization step and HS extraction only. The obtained results showed that the objectives of this work have been realized for the seven EAs in human blood. 2. Material and methods 2.1. Material Fig. 1. Chromatogram of the blank blood (A) produced by heating for 4 h and (B) produced under freeze-drying conditions. Peak identification: 1, formaldehyde; 2, PFBHA.
Formaldehyde (C1, 36%–38%, w/v solution in water), propanal (C3, purity > 98%), and butanal (C4, > 98.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetaldehyde (C2, 40%, w/v solution in water) was purchased from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Hexanal (C6, > 95%) and heptanal (C7, > 95%) were purchased from Tokyo chemical industry (Tokyo, Japan). Methanol (99.9%), pentanal (C5, 97%), o‑2,3,4,5,6‑pentafluorobenzylhydroxylamine hydrochloride (PFBHA, 98%), and the internal standard 1,2‑dibromopropane (5.0 mg/ mL, solution in methanol) were supplied from J&K chemical (Beijing, China). Sodium bicarbonate (> 99.5%) was purchased from Sitong Chemical Factory (Tianjin, China). Sodium carbonate (> 99.8%) was purchased from Hengxing Chemical Reagent (Tianjin, China). FD-1C-50 freeze dryer was purchased from Bilon (Shanghai, China). EDTAK anticoagulant tubes were purchased from Hepeng Biological (Shanghai, China). 2.2. Standards and blood samples Individual stock solutions were prepared with purified water using a Milli-Q system (Millipore, France) containing the compounds at concentrations of 3.75–5.42 mg/mL. The internal standard was diluted with methanol to the intermediate stock solution concentration, and the obtained solutions were stored in amber glass vials at −30 °C. A PFBHA solution was obtained by the dilution of the neat chemical with water to 50 mg/mL. Working solutions were prepared daily by the appropriate dilution of each stock solution in water with addition of 50 μL of IS. The blood samples of 15 bladder cancer patients and 15 healthy subjects were collected by venipuncture into blood collection vials
Fig. 2. Optimization of the dilution factor of the blood. Line identification: C1, formaldehyde; C2, acetaldehyde; C3, propanal; C4, butanal; C5, pentanal; C6, hexanal; C7, heptanal. 86
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Fig. 4. Influence of the salting-out effect on the aldehyde peak areas.
Fig. 3. Influence of (A) pH values and (B) the amount of derivatization reagent on the derivatization efficiency of seven aldehydes, with the other conditions kept the same.
containing EDTAK as the anticoagulant from the First Affiliated Hospital of Zhengzhou University. Subsequently, the blood samples were stored at −80 °C in the dark and were measured within 10 days after collection. 2.3. Sample preparation 1,2‑Dibromoprane (50 μL, 0.5 μg/L) and mixed standard solution were added to blood (1 mL) in a headspace vial (20 mL), and then the solution was diluted with water to 5 mL. Next, buffer salts (0.5 g) and PFBHA (2 mg, 50 mg/mL) were added to the solution. Then, the vial was immediately sealed and vortexed for 10 s and derived for one hour at 50 °C. After cooling down to room temperature, the vial was placed into an autosampler, was introduced into an HS oven for incubating for 30 min at 80 °C, and then was subjected to GC analysis.
Fig. 5. Effect of the equilibrium (A) temperature and (B) time on HS extraction.
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Table 1 Characteristic ions, retention time (tR), determination coefficients (R2), limits of detection (LOD) and limits of quantitation (LOQ) for the target compounds with the HS-GC–MS method. Compound
m/za
tR (min)
R2
Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal IS PFBHA
181, 195, 117 181, 209, 239 181, 236, 223 181, 239, 195 181, 239, 207 181, 239, 114 181, 239, 128 121, 123 181, 161, 195
5.57 6.43, 6.50 7.14, 7.19 7.84, 7.89 8.54, 8.57 9.20, 9.23 9.83, 9.84 4.36 6.30
0.9998 0.9996 0.9998 0.9998 0.9998 0.9997 0.9997 – –
a
Linear range (μg/mL)
LOD (μg/L)
LOQ (μg/L)
0.1–4 0.01–0.4 0.01–0.4 0.01–0.4 0.01–0.4 0.02–0.8 0.02–0.8 – –
0.0692 0.0867 0.161 0.198 0.401 0.392 0.864 – –
0.231 0.289 0.538 0.662 1.34 1.31 2.88 – –
Target ions depicted in bold are used for quantification.
3. Results and discussion
Table 2 Recoveries and repeatability at three different levels obtained with the HSGC–MS method. Compound
Concentration (μg/ mL)a
Recovery (%, n = 3)
Intra-day Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal
0.1 0.5 2 0.02 0.1 0.4 0.02 0.1 0.4 0.02 0.1 0.4 0.02 0.1 0.4 0.04 0.2 0.8 0.04 0.2 0.8
99.3 96.2 92.1 104.8 105.7 94.5 96.8 102.3 103.4 96.6 106 94.4 102.3 92.0 96.2 103.4 97.3 99.2 95.9 93.8 96.5
3.1. Stripping matrix preparation
Repeatability (RSD%, n = 3)
1.9 2.9 6.7 7.9 7.1 3.8 7.1 6.7 8.9 4.4 6.1 8.5 9.7 4.7 9.2 3.1 6.0 9.7 2.1 6.7 8.2
b
An experiment was designed to compare two methods of producing stripping matrix “blank blood”. In the first method, after the frozen blood sample was completely thawed, healthy human blood was placed into a water bath at 60 °C under continuous stirring for 2 h. Small amounts of formaldehyde-PFBHA derivatives were observed, proving that formaldehyde was not removed completely. We continued the stirring for an additional 2 h, obtaining the result displayed in Fig. 1A. Formaldoxime was still present, and the color of the blood was deeper than that of fresh blood. Therefore, this method was abandoned due to the change in the matrix composition and contamination from laboratory air. In the other method, the blood sample was placed into a vacuum freeze-drying oven for 24 h after freezing in an ultra-low temperature freezer at −80 °C for 24 h. A satisfactory result was obtained. No peaks other than that of PFBHA appeared in the chromatogram (Fig. 1B). Therefore, in subsequent experiments, the stripping matrix “blank blood” was prepared using the second method.
c
Inter-day 4.7 6.7 9.5 8.8 10.6 5.6 8.6 8.3 9.4 3.4 7.1 10.5 9.4 6.4 11.9 3.9 6.0 10.2 4.4 7.7 11.9
3.2. Matrix effect The determination of EAs was severely affected by the strong interactions between the target aldehydes and matrix components such as protein. Dilution of the matrix can be used to overcome this problem [29]. A lower viscosity and a higher diffusion coefficient can be realized with increased dilution to improve the HS extraction efficiency. However, dilution of the samples will lead to an increase in the detection limits. Therefore, it is necessary to find a suitable level of dilution to reduce the matrix effect while ensuring analytical sensitivity. The mixed standard solution including seven aldehydes was added to blank blood and then was diluted 2, 4, 5, 6 and 10 times with ultrapure water, respectively. Fig. 2 shows the analytical responses measured by HS-GC–MS for the samples with different dilution ratios. The peak areas of seven aldehydes continue to increase and come to the top when the ratio reached 1:4 (blood:water). Therefore, the 1:4 dilution ratio was selected for the analysis of EAs. With the exception of formaldehyde and acetaldehyde, similar peak areas were observed for the aldehydes with 1:1 and 1:9 dilutions, suggesting that aldehydes suffer a strong matrix effect that cannot be eliminated by simply diluting the sample. Hence, internal calibration in diluted blank blood was used for the quantification of EAs. There was no significant difference observed among the recoveries (between 92% and 106%) obtained from human blood and blank blood with the 1:4 dilution.
a Low spiked concentration, intermediate spiked concentration and high spiked concentration of each aldehyde. b Measured in triplicate in one day. c Measured in three consecutive day.
2.4. HS-GC–MS analysis Sample analysis was performed using a TRACE ISQ™ gas chromatograph-mass spectrometry instrument coupled with a Triplus HS autosampler and an Xcalibur data processing system. The compounds were separated using an HP-5MS capillary column (5% diphenyl‑95%‑dimethylpolysiloxane, 25 m × 0.20 mm i.d., 0.33 μm, Agilent Technologies). The operating conditions were as follows: the vial equilibration time was programmed to be 30 min, and the vial temperature was kept at 80 °C; electron impact ionization was applied at 70 eV; the mass spectrometer was operated in the selected ion monitoring (SIM) mode; and the transfer line temperature and ion source temperature were kept at 250 °C. Injection was carried out in the split mode (split ratio 50:1) with an inlet temperature of 200 °C; helium carrier gas was passed at a rate of 1 mL/min, and a solvent delay of 4 min was used. The column temperature was programmed to change from 40 °C (held for 2 min) to 110 °C at a rate of 20 °C/min, then ramped up at a rate of 10 °C/min to 125 °C, followed by increasing at a rate of 20 °C/ min to 250 °C and holding for 2 min.
3.3. Optimization of the derivative reaction Aldehydes are transformed into the corresponding oximes by reversible nucleophilic addition. The sample pH plays a key role in oxime 88
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Fig. 6. Extracted ion current chromatograms at m/z 181 obtained in the analysis of (A) blood from a healthy subject and (B) blood from a bladder cancer patient by GC–MS (SIM mode). Peak identification: 1, formaldehyde; 2, acetaldehyde; 3, propanal; 4, butanal; 5, pentanal; 6, hexanal; 7, heptanal; R, PFBHA.
formation. The influence of pH on this reaction was studied in the pH range from 8.5 to 10.7 because oximes can be prepared in basic media as described in the literature [17,26]. Different proportions of sodium bicarbonate and sodium carbonate salts were used to adjust the pH. The salts were added to the stripping matrix “blank blood” spiked with the mix standard solution before the target aldehydes were derived. Fig. 3A shows the peak areas obtained at different pH levels. An appreciable increase in the analytical signal was observed at pH 10.34, and then the
response signal decreased due to the possible decomposition of oximes to the corresponding alcohols and nitriles [30]. The effect of the amount of PFBHA on derivatization efficiency was studied in 1.5, 2, 2.5, and 3 mg. For all of the oxime compounds except for butyraldehyde oxime, the highest peak areas were obtained with 2 mg of PFBHA. Considering the content of the excess derivatization reagent, 2 mg of PFBHA was chosen for this derivatization reaction (Fig. 3B). 89
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Fig. 7. The box-plot of the concentration (μg/mL) of seven EAs in the blood of the control group and cancer patients.
pentanal and hexanal, the peak areas were improved by a factor of 2–3. Therefore, the salting-out effect was required to improve the sensitivity of this method, and a salt content of 0.1 g/mL was an appropriate choice for further experiments.
Table 3 Comparison of seven EAs in the blood of the bladder cancer patients and the control group. Compound
Normal people (n = 15) Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal
P-valuea
Concentration (Mean ± SD) (μg/mL)
1.52 0.083 0.023 0.014 0.028 0.049 0.036
± ± ± ± ± ± ±
0.49 0.043 0.010 0.010 0.010 0.008 0.023
Bladder cancer patients (n = 15) 3.44 0.421 0.195 0.071 0.206 1.74 0.219
± ± ± ± ± ± ±
0.43 0.221 0.111 0.050 0.117 1.45 0.098
3.5. Optimization of the HS extraction The vapor pressure and partition coefficient between the liquid and gas phases are affected by the equilibrium temperature and equilibrium time. These parameters were selected for HS extraction optimization. Fig. 5A shows the effect of equilibrium temperature on the peak areas. The signal increased slowly before 70 °C. After that, a rapid growth was observed at 80 °C, but temperatures higher than 80 °C were not used in order to avoid the possible denaturalization of the protein and decomposition of the target aldehydes. The response signals of EAs at different equilibrium times with the same temperature of 80 °C are displayed in Fig. 5B. It is observed that the amount of aldehydes increased with time until 30 min. Based on these results, 80 °C and 30 min were chosen as the optimal extraction conditions.
< 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001
a T-test of two independent samples was used to calculate the P values of each group; the differences are extremely significant when P < 0.001, and significant differences are obtained when P < 0.01.
3.4. Salting-out effect In HS analysis, ionic strength has a great impact on the volatility of the target components. In this case, the added salts can compensate for the ionic strengths, which may vary considerably between the different samples in the blood [29]. Sodium bicarbonate and sodium carbonate were added to the solution in order to evaluate the effect of ionic strength on the blood samples. Compared to the extraction with a salt content of 0.02 g/mL, the extraction efficiency of all aldehydes improved with the addition of 0.1 g/mL salts (Fig. 4). For butanal,
3.6. Validation of the method The target aldehydes in the blood were detected by measurement of the corresponding oximes. The efficient separation of oxime compounds under optimal conditions was realized within 10 min. Specific ions were used to identify each aldehyde as shown in Table 1. In addition to formaldehyde, two products, namely, Z-oxime and E-oxime, were 90
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obtained after the aldehydes were derivatized with PFBHA. Therefore, the sum of the Z and E isomer peak areas was used for quantification. The ratio of the peak areas of target aldehydes to that of the IS was used for calibration. Method properties such as linearity, detection limits, precision and recovery were evaluated at the optimal chemical and instrumental conditions (Tables 1 and 2). The calibration curves show excellent linearity in the range of 0.01–4 μg/mL with correlation coefficients higher than 0.9996. LOD and LOQ were calculated using the Xcalibur software on the basis of the signal-to-noise ratios of three and ten, respectively. The LOD values ranged from 0.0692 to 0.864 μg/L. Precision expressed by the relative standard deviation (RSD) was studied at low, medium and high concentration levels on the same day and three different days. An examination of the data shows that good repeatability (intra-day) and reproducibility (inter-day) were obtained, with RSD average values of 1.9–9.7% and 3.4–11.9%, respectively. A recovery test was used to evaluate the accuracy of this method by adding known amounts of each aldehyde at three different concentration levels in triplicate, and the result (recovery ranging from 92 to 106%) shows that no matrix effect was observed in the determination of EAs in human blood for this method.
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3.7. Analysis of endogenous aldehydes in human blood The proposed HS-GC–MS method was applied to determine seven EAs in 30 samples from different individuals (15 bladder cancer patients and 15 healthy people). Figs. 6 and 7 show the chromatograms and box-plots obtained from the blood of cancer patients and healthy people, respectively. C1–C7 aldehydes were detected in the patients' blood at higher concentration levels compared to the control group, particularly for propanal and hexanal. A statistical analysis of the data was performed to evaluate the possible differences between bladder cancer patients and normal people even though the number of the samples was limited (Table 3). Some comments can be made from the data: with the exception of formaldehyde, the average concentrations of the EAs in patient blood were several times higher than the normal concentrations; formaldehyde, acetaldehyde, propanal, pentanal, hexanal and heptanal showed extremely significant differences (p < 0.001) and butanal showed a significant difference (p < 0.01). 4. Conclusions In this work, a simple, automated and environmentally friendly method based on HS with GC–MS was developed for the determination of seven EAs in bladder cancer patient blood. In the proposed method, the aldehydes were detected with good accuracy and precision by using diluted human blood and an internal standard to avoid the influence of the matrix effect, and buffer salts were applied to improve the sensitivity of this method. To the best of our knowledge, this is the first report on the use of HS-GC–MS for simultaneous determination of these EAs in bladder cancer patient blood. The results show significant differences in the concentrations of these aldehydes compared to the blood of the healthy subjects, and these aldehydes can be regarded as the biomarkers of bladder cancer. Acknowledgements The authors thank the volunteers who participated in this experiment. The work was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consent of each subject was obtained. We thank Dongkui Song and Lei Shi of the Department of Urology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, for the assistance of blood sample collection. The project was supported by the National Science Foundation of China (No. 21772180), and all authors 91
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Y. Wei, et al. [23] N. Li, C. Deng, X. Yin, N. Yao, X. Shen, X. Zhang, Gas chromatography-mass spectrometric analysis of hexanal and heptanal in human blood by headspace single-drop microextraction with droplet derivatization, Anal. Biochem. 342 (2005) 318–326, https://doi.org/10.1016/j.ab.2005.04.024. [24] M. Serrano, M. Gallego, M. Silva, Analysis of endogenous aldehydes in human urine by static headspace gas chromatography-mass spectrometry, J. Chromatogr. A 1437 (2016) 241–246, https://doi.org/10.1016/j.chroma.2016.01.056. [25] L.K. Silva, G.A. Hile, K.M. Capella, M.F. Espenship, M.M. Smith, V.R.D. Jesús, B.C. Blount, Quantification of 19 aldehydes in human serum by headspace SPME/ GC/high-resolution mass spectrometry, Environ. Sci. Technol. 52 (2018) 10571–10579, https://doi.org/10.1021/acs.est.8b02745. [26] M. Serrano, M. Gallego, M. Silva, Static headspace gas chromatography-mass spectrometry for the one-step derivatisation and extraction of eleven aldehydes in drinking water, J. Chromatogr. A 1307 (2013) 158–165, https://doi.org/10.1016/j. chroma.2013.07.065. [27] A.P. Antón, A.M. Ferreira, C.G. Pinto, B.M. Cordero, J.L.P. Pavón, Headspace
generation coupled to gas chromatography-mass spectrometry for the automated determination and quantification of endogenous compounds in urine. Aldehydes as possible markers of oxidative stress, J. Chromatogr. A 1367 (2014) 9–15, https:// doi.org/10.1016/j.chroma.2014.09.038. [28] J.M. Fernández-Molina, M. Silva, LC–MS analytical method for biomonitoring of aliphatic and aromatic low-molecular-mass aldehydes in human urine, Chromatographia 78 (2015) 203–209, https://doi.org/10.1007/s10337-0142824-4. [29] M. Alonso, M. Castellanos, E. Besalú, J.M. Sanchez, A headspace needle-trap method for the analysis of volatile organic compounds in whole blood, J. Chromatogr. A 1252 (2012) 23–30, https://doi.org/10.1016/j.chroma.2012.06. 083. [30] S. Salahuddin, O. Renaudet, J.L. Reymond, Aldehyde detection by chromogenic/ fluorogenic oxime bond fragmentation, Org. Biomol. Chem. 2 (2004) 1471–1475, https://doi.org/10.1039/b400314d.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Simultaneous determination of seven endogenous aldehydes in human blood by headspace gas chromatography–mass spectrometry
T
Yi Weia, Menghan Wanga, Huixia Liua, Ying Niua, Shaomin Wanga, , Fuyi Zhanga, Hongmin Liub ⁎
a b
College of Chemistry and Molecular Engineering, Zhengzhou University, PR China Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, PR China
ARTICLE INFO
ABSTRACT
Keywords: Endogenous aldehydes Gas chromatography-mass spectrometry Static headspace technique Blood Bladder cancer
Endogenous aldehydes (EAs) formed by the free-radical-mediated reaction are regarded as potential biological markers of several diseases. In this work, an automated and solvent-free analytical method was developed for quantitative analysis of seven EAs (C1–C7) in human blood by using gas chromatography–mass spectrometry coupled with a headspace generator sampler (HS-GC–MS). A 1:4 (blood:water) dilution and the 1,2‑dibromopropane internal standard were introduced to reduce the influence of the matrix effect from complex biological fluids. The sample preparation steps were simplified. Various experimental parameters for derivatization and extraction conditions were studied such as HS extraction temperature and time, the amount of derivatization reagent, pH and the salt effect. Under these optimum conditions, seven low-mass aldehydes were separated and analyzed within 10 min. Additionally, this method achieved limits of detection in the range of 0.0692–0.864 μg/L, an excellent linearity with correlation coefficients higher than 0.9996 and appropriate repeatability and reproducibility values (RSD < 12% at low, middle and high levels). The HS-GC–MS method was applied to measure the concentrations of the seven aldehydes in blood from bladder cancer patients (n = 15) and control subjects (n = 15). Compared with the control subjects, the levels of butanal (p < 0.01), formaldehyde, acetaldehyde, propanal, pentanal, hexanal and heptanal (all p < 0.001) were increased significantly, indicating that EAs may be useful as biomarkers in the early diagnosis of bladder cancer.
1. Introduction Endogenous aldehydes (EAs) are formed by the metabolism of amino acids, carbohydrates, lipids, biogenic amines, vitamins and steroids [1]. The lipid peroxidation process in which a variety of reactive oxygen/nitrogen species (ROS/RNS) attack polyunsaturated fatty acid residues of phospholipids is a major source of endogenous aldehydes, proving that the free-radical-mediated reaction occurs during the formation of EAs [1,2]. Human tumor cells produce large amounts of radicals that lead to an increase in the aldehyde concentration [3,4], and remarkable aldehyde levels were found in the blood and urine of cancer patients [5,6]. Therefore, various aldehydes can be regarded as potential biomarkers of cancer, and accurate quantification of EAs in biological fluids is expected to be an important part of clinical early diagnosis of diseases in the future. Chromatographic separation techniques have been commonly used for the determination of aldehydes. A derivatization step prior to the extraction and chromatographic analysis is necessary because the aldehydes are highly polar, chemically unstable, volatile and do not
⁎
contain chromophores and fluorophores [7]. 2,4‑Dinitrophenylhydrazine (DNPH) is the most frequently used derivatization reagent for liquid chromatography (LC) separation followed by diode array or mass spectrometric (MS) detection [8–12]. However, several drawbacks preclude the use of DNPH in the determination of aldehyde compounds; for example, formaldehyde cannot be accurately quantified due to the high uncertainty of the MS signals of its DNPH derivative [11], and methylglyoxal required > 12 h for reacting with DNPH [13]. Gas chromatography/mass spectrometry (GC–MS) has been widely applied to the analysis of low-molecular-mass aldehydes [14–17] because of its rapid analysis and high sensitivity. Several derivatization reagents used in GC have been reported such as 2,3‑diamino‑2,3‑dimethylbutane (DDB) [18], 2,4,6‑trichlorophenylhydrazine (TCPH) [19], pentafluorophenylhydrazine (PFPH) [20] and O‑2,3,4,5,6‑(pentafluorobenzyl) hydroxylamine (PFBHA) [15,16,21–24]. Because aldehydes yield stable and volatile oximes at room temperature by reaction with PFBHA, GC-PFBHA analysis methods are regarded as a useful alternative to LC-DNPH analysis. Due to the presence of various interferences and trace amounts of
Corresponding author at: College of Chemistry and Molecular Engineering, Zhengzhou University, 75 University Road, Zhengzhou 450052, PR China. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.jchromb.2019.04.027 Received 29 January 2019; Received in revised form 9 April 2019; Accepted 11 April 2019 Available online 12 April 2019 1570-0232/ © 2019 Elsevier B.V. All rights reserved.
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aldehydes in biological samples, cleanup and enrichment procedures are adopted to reduce the matrix effect and improve the method sensitivity. Due to the well-known disadvantages of conventional sampling techniques for the determination of aldehydes, microextraction techniques such as solid phase microextraction (SPME) [22,25] and single drop microextraction (SDME) [19,23] have attracted increasing interest. However, SPME is prone to sample carryover and is relatively expensive due to limited lifetime and fragility of the fiber [26], while SDME requires more elaborate manual operations. The headspace sampling (HS) technique is a fast, automated and solvent-free sample preparation method. The use of HS with GC can minimize the sample treatment and avoid the interferences of non-volatile components in the matrices [27]. Recently, a sensitive HS-GC–MS analytical method using diluted blank urine for the detection of twelve aldehydes in urine has been introduced by Serrano et al. [24]. Based on this method, an automatic, low-cost, solvent-free analytical method for EAs has been realized and the strong effect of the matrix protein was minimized. Some reports have focused on the presence of aldehydes in urine [11,24,27,28] for the detection of several diseases because of the noninvasive nature of the detection method. Only a few have been reported in the blood, which can directly relate to the internal activities of the human body. In these studies, hexanal and heptanal have been commonly detected [7,12,22,23]. Based on the considerations mentioned above, this research focused on the following aspects: (i) developing a solvent-free and automated HS-GC–MS method to quantify seven aldehydes including hexanal and heptanal in the blood of bladder cancer patients; (ii) reducing the matrix effect of blood samples with an appropriate dilution ratio and internal standard (IS); and (iii) simplifying the sample preparation step with a derivatization step and HS extraction only. The obtained results showed that the objectives of this work have been realized for the seven EAs in human blood. 2. Material and methods 2.1. Material Fig. 1. Chromatogram of the blank blood (A) produced by heating for 4 h and (B) produced under freeze-drying conditions. Peak identification: 1, formaldehyde; 2, PFBHA.
Formaldehyde (C1, 36%–38%, w/v solution in water), propanal (C3, purity > 98%), and butanal (C4, > 98.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Acetaldehyde (C2, 40%, w/v solution in water) was purchased from Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Hexanal (C6, > 95%) and heptanal (C7, > 95%) were purchased from Tokyo chemical industry (Tokyo, Japan). Methanol (99.9%), pentanal (C5, 97%), o‑2,3,4,5,6‑pentafluorobenzylhydroxylamine hydrochloride (PFBHA, 98%), and the internal standard 1,2‑dibromopropane (5.0 mg/ mL, solution in methanol) were supplied from J&K chemical (Beijing, China). Sodium bicarbonate (> 99.5%) was purchased from Sitong Chemical Factory (Tianjin, China). Sodium carbonate (> 99.8%) was purchased from Hengxing Chemical Reagent (Tianjin, China). FD-1C-50 freeze dryer was purchased from Bilon (Shanghai, China). EDTAK anticoagulant tubes were purchased from Hepeng Biological (Shanghai, China). 2.2. Standards and blood samples Individual stock solutions were prepared with purified water using a Milli-Q system (Millipore, France) containing the compounds at concentrations of 3.75–5.42 mg/mL. The internal standard was diluted with methanol to the intermediate stock solution concentration, and the obtained solutions were stored in amber glass vials at −30 °C. A PFBHA solution was obtained by the dilution of the neat chemical with water to 50 mg/mL. Working solutions were prepared daily by the appropriate dilution of each stock solution in water with addition of 50 μL of IS. The blood samples of 15 bladder cancer patients and 15 healthy subjects were collected by venipuncture into blood collection vials
Fig. 2. Optimization of the dilution factor of the blood. Line identification: C1, formaldehyde; C2, acetaldehyde; C3, propanal; C4, butanal; C5, pentanal; C6, hexanal; C7, heptanal. 86
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Fig. 4. Influence of the salting-out effect on the aldehyde peak areas.
Fig. 3. Influence of (A) pH values and (B) the amount of derivatization reagent on the derivatization efficiency of seven aldehydes, with the other conditions kept the same.
containing EDTAK as the anticoagulant from the First Affiliated Hospital of Zhengzhou University. Subsequently, the blood samples were stored at −80 °C in the dark and were measured within 10 days after collection. 2.3. Sample preparation 1,2‑Dibromoprane (50 μL, 0.5 μg/L) and mixed standard solution were added to blood (1 mL) in a headspace vial (20 mL), and then the solution was diluted with water to 5 mL. Next, buffer salts (0.5 g) and PFBHA (2 mg, 50 mg/mL) were added to the solution. Then, the vial was immediately sealed and vortexed for 10 s and derived for one hour at 50 °C. After cooling down to room temperature, the vial was placed into an autosampler, was introduced into an HS oven for incubating for 30 min at 80 °C, and then was subjected to GC analysis.
Fig. 5. Effect of the equilibrium (A) temperature and (B) time on HS extraction.
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Table 1 Characteristic ions, retention time (tR), determination coefficients (R2), limits of detection (LOD) and limits of quantitation (LOQ) for the target compounds with the HS-GC–MS method. Compound
m/za
tR (min)
R2
Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal IS PFBHA
181, 195, 117 181, 209, 239 181, 236, 223 181, 239, 195 181, 239, 207 181, 239, 114 181, 239, 128 121, 123 181, 161, 195
5.57 6.43, 6.50 7.14, 7.19 7.84, 7.89 8.54, 8.57 9.20, 9.23 9.83, 9.84 4.36 6.30
0.9998 0.9996 0.9998 0.9998 0.9998 0.9997 0.9997 – –
a
Linear range (μg/mL)
LOD (μg/L)
LOQ (μg/L)
0.1–4 0.01–0.4 0.01–0.4 0.01–0.4 0.01–0.4 0.02–0.8 0.02–0.8 – –
0.0692 0.0867 0.161 0.198 0.401 0.392 0.864 – –
0.231 0.289 0.538 0.662 1.34 1.31 2.88 – –
Target ions depicted in bold are used for quantification.
3. Results and discussion
Table 2 Recoveries and repeatability at three different levels obtained with the HSGC–MS method. Compound
Concentration (μg/ mL)a
Recovery (%, n = 3)
Intra-day Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal
0.1 0.5 2 0.02 0.1 0.4 0.02 0.1 0.4 0.02 0.1 0.4 0.02 0.1 0.4 0.04 0.2 0.8 0.04 0.2 0.8
99.3 96.2 92.1 104.8 105.7 94.5 96.8 102.3 103.4 96.6 106 94.4 102.3 92.0 96.2 103.4 97.3 99.2 95.9 93.8 96.5
3.1. Stripping matrix preparation
Repeatability (RSD%, n = 3)
1.9 2.9 6.7 7.9 7.1 3.8 7.1 6.7 8.9 4.4 6.1 8.5 9.7 4.7 9.2 3.1 6.0 9.7 2.1 6.7 8.2
b
An experiment was designed to compare two methods of producing stripping matrix “blank blood”. In the first method, after the frozen blood sample was completely thawed, healthy human blood was placed into a water bath at 60 °C under continuous stirring for 2 h. Small amounts of formaldehyde-PFBHA derivatives were observed, proving that formaldehyde was not removed completely. We continued the stirring for an additional 2 h, obtaining the result displayed in Fig. 1A. Formaldoxime was still present, and the color of the blood was deeper than that of fresh blood. Therefore, this method was abandoned due to the change in the matrix composition and contamination from laboratory air. In the other method, the blood sample was placed into a vacuum freeze-drying oven for 24 h after freezing in an ultra-low temperature freezer at −80 °C for 24 h. A satisfactory result was obtained. No peaks other than that of PFBHA appeared in the chromatogram (Fig. 1B). Therefore, in subsequent experiments, the stripping matrix “blank blood” was prepared using the second method.
c
Inter-day 4.7 6.7 9.5 8.8 10.6 5.6 8.6 8.3 9.4 3.4 7.1 10.5 9.4 6.4 11.9 3.9 6.0 10.2 4.4 7.7 11.9
3.2. Matrix effect The determination of EAs was severely affected by the strong interactions between the target aldehydes and matrix components such as protein. Dilution of the matrix can be used to overcome this problem [29]. A lower viscosity and a higher diffusion coefficient can be realized with increased dilution to improve the HS extraction efficiency. However, dilution of the samples will lead to an increase in the detection limits. Therefore, it is necessary to find a suitable level of dilution to reduce the matrix effect while ensuring analytical sensitivity. The mixed standard solution including seven aldehydes was added to blank blood and then was diluted 2, 4, 5, 6 and 10 times with ultrapure water, respectively. Fig. 2 shows the analytical responses measured by HS-GC–MS for the samples with different dilution ratios. The peak areas of seven aldehydes continue to increase and come to the top when the ratio reached 1:4 (blood:water). Therefore, the 1:4 dilution ratio was selected for the analysis of EAs. With the exception of formaldehyde and acetaldehyde, similar peak areas were observed for the aldehydes with 1:1 and 1:9 dilutions, suggesting that aldehydes suffer a strong matrix effect that cannot be eliminated by simply diluting the sample. Hence, internal calibration in diluted blank blood was used for the quantification of EAs. There was no significant difference observed among the recoveries (between 92% and 106%) obtained from human blood and blank blood with the 1:4 dilution.
a Low spiked concentration, intermediate spiked concentration and high spiked concentration of each aldehyde. b Measured in triplicate in one day. c Measured in three consecutive day.
2.4. HS-GC–MS analysis Sample analysis was performed using a TRACE ISQ™ gas chromatograph-mass spectrometry instrument coupled with a Triplus HS autosampler and an Xcalibur data processing system. The compounds were separated using an HP-5MS capillary column (5% diphenyl‑95%‑dimethylpolysiloxane, 25 m × 0.20 mm i.d., 0.33 μm, Agilent Technologies). The operating conditions were as follows: the vial equilibration time was programmed to be 30 min, and the vial temperature was kept at 80 °C; electron impact ionization was applied at 70 eV; the mass spectrometer was operated in the selected ion monitoring (SIM) mode; and the transfer line temperature and ion source temperature were kept at 250 °C. Injection was carried out in the split mode (split ratio 50:1) with an inlet temperature of 200 °C; helium carrier gas was passed at a rate of 1 mL/min, and a solvent delay of 4 min was used. The column temperature was programmed to change from 40 °C (held for 2 min) to 110 °C at a rate of 20 °C/min, then ramped up at a rate of 10 °C/min to 125 °C, followed by increasing at a rate of 20 °C/ min to 250 °C and holding for 2 min.
3.3. Optimization of the derivative reaction Aldehydes are transformed into the corresponding oximes by reversible nucleophilic addition. The sample pH plays a key role in oxime 88
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Fig. 6. Extracted ion current chromatograms at m/z 181 obtained in the analysis of (A) blood from a healthy subject and (B) blood from a bladder cancer patient by GC–MS (SIM mode). Peak identification: 1, formaldehyde; 2, acetaldehyde; 3, propanal; 4, butanal; 5, pentanal; 6, hexanal; 7, heptanal; R, PFBHA.
formation. The influence of pH on this reaction was studied in the pH range from 8.5 to 10.7 because oximes can be prepared in basic media as described in the literature [17,26]. Different proportions of sodium bicarbonate and sodium carbonate salts were used to adjust the pH. The salts were added to the stripping matrix “blank blood” spiked with the mix standard solution before the target aldehydes were derived. Fig. 3A shows the peak areas obtained at different pH levels. An appreciable increase in the analytical signal was observed at pH 10.34, and then the
response signal decreased due to the possible decomposition of oximes to the corresponding alcohols and nitriles [30]. The effect of the amount of PFBHA on derivatization efficiency was studied in 1.5, 2, 2.5, and 3 mg. For all of the oxime compounds except for butyraldehyde oxime, the highest peak areas were obtained with 2 mg of PFBHA. Considering the content of the excess derivatization reagent, 2 mg of PFBHA was chosen for this derivatization reaction (Fig. 3B). 89
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Fig. 7. The box-plot of the concentration (μg/mL) of seven EAs in the blood of the control group and cancer patients.
pentanal and hexanal, the peak areas were improved by a factor of 2–3. Therefore, the salting-out effect was required to improve the sensitivity of this method, and a salt content of 0.1 g/mL was an appropriate choice for further experiments.
Table 3 Comparison of seven EAs in the blood of the bladder cancer patients and the control group. Compound
Normal people (n = 15) Formaldehyde Acetaldehyde Propanal Butanal Pentanal Hexanal Heptanal
P-valuea
Concentration (Mean ± SD) (μg/mL)
1.52 0.083 0.023 0.014 0.028 0.049 0.036
± ± ± ± ± ± ±
0.49 0.043 0.010 0.010 0.010 0.008 0.023
Bladder cancer patients (n = 15) 3.44 0.421 0.195 0.071 0.206 1.74 0.219
± ± ± ± ± ± ±
0.43 0.221 0.111 0.050 0.117 1.45 0.098
3.5. Optimization of the HS extraction The vapor pressure and partition coefficient between the liquid and gas phases are affected by the equilibrium temperature and equilibrium time. These parameters were selected for HS extraction optimization. Fig. 5A shows the effect of equilibrium temperature on the peak areas. The signal increased slowly before 70 °C. After that, a rapid growth was observed at 80 °C, but temperatures higher than 80 °C were not used in order to avoid the possible denaturalization of the protein and decomposition of the target aldehydes. The response signals of EAs at different equilibrium times with the same temperature of 80 °C are displayed in Fig. 5B. It is observed that the amount of aldehydes increased with time until 30 min. Based on these results, 80 °C and 30 min were chosen as the optimal extraction conditions.
< 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001 < 0.001
a T-test of two independent samples was used to calculate the P values of each group; the differences are extremely significant when P < 0.001, and significant differences are obtained when P < 0.01.
3.4. Salting-out effect In HS analysis, ionic strength has a great impact on the volatility of the target components. In this case, the added salts can compensate for the ionic strengths, which may vary considerably between the different samples in the blood [29]. Sodium bicarbonate and sodium carbonate were added to the solution in order to evaluate the effect of ionic strength on the blood samples. Compared to the extraction with a salt content of 0.02 g/mL, the extraction efficiency of all aldehydes improved with the addition of 0.1 g/mL salts (Fig. 4). For butanal,
3.6. Validation of the method The target aldehydes in the blood were detected by measurement of the corresponding oximes. The efficient separation of oxime compounds under optimal conditions was realized within 10 min. Specific ions were used to identify each aldehyde as shown in Table 1. In addition to formaldehyde, two products, namely, Z-oxime and E-oxime, were 90
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obtained after the aldehydes were derivatized with PFBHA. Therefore, the sum of the Z and E isomer peak areas was used for quantification. The ratio of the peak areas of target aldehydes to that of the IS was used for calibration. Method properties such as linearity, detection limits, precision and recovery were evaluated at the optimal chemical and instrumental conditions (Tables 1 and 2). The calibration curves show excellent linearity in the range of 0.01–4 μg/mL with correlation coefficients higher than 0.9996. LOD and LOQ were calculated using the Xcalibur software on the basis of the signal-to-noise ratios of three and ten, respectively. The LOD values ranged from 0.0692 to 0.864 μg/L. Precision expressed by the relative standard deviation (RSD) was studied at low, medium and high concentration levels on the same day and three different days. An examination of the data shows that good repeatability (intra-day) and reproducibility (inter-day) were obtained, with RSD average values of 1.9–9.7% and 3.4–11.9%, respectively. A recovery test was used to evaluate the accuracy of this method by adding known amounts of each aldehyde at three different concentration levels in triplicate, and the result (recovery ranging from 92 to 106%) shows that no matrix effect was observed in the determination of EAs in human blood for this method.
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3.7. Analysis of endogenous aldehydes in human blood The proposed HS-GC–MS method was applied to determine seven EAs in 30 samples from different individuals (15 bladder cancer patients and 15 healthy people). Figs. 6 and 7 show the chromatograms and box-plots obtained from the blood of cancer patients and healthy people, respectively. C1–C7 aldehydes were detected in the patients' blood at higher concentration levels compared to the control group, particularly for propanal and hexanal. A statistical analysis of the data was performed to evaluate the possible differences between bladder cancer patients and normal people even though the number of the samples was limited (Table 3). Some comments can be made from the data: with the exception of formaldehyde, the average concentrations of the EAs in patient blood were several times higher than the normal concentrations; formaldehyde, acetaldehyde, propanal, pentanal, hexanal and heptanal showed extremely significant differences (p < 0.001) and butanal showed a significant difference (p < 0.01). 4. Conclusions In this work, a simple, automated and environmentally friendly method based on HS with GC–MS was developed for the determination of seven EAs in bladder cancer patient blood. In the proposed method, the aldehydes were detected with good accuracy and precision by using diluted human blood and an internal standard to avoid the influence of the matrix effect, and buffer salts were applied to improve the sensitivity of this method. To the best of our knowledge, this is the first report on the use of HS-GC–MS for simultaneous determination of these EAs in bladder cancer patient blood. The results show significant differences in the concentrations of these aldehydes compared to the blood of the healthy subjects, and these aldehydes can be regarded as the biomarkers of bladder cancer. Acknowledgements The authors thank the volunteers who participated in this experiment. The work was approved by the Medical Research Ethics Committee of the First Affiliated Hospital of Zhengzhou University and written informed consent of each subject was obtained. We thank Dongkui Song and Lei Shi of the Department of Urology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, for the assistance of blood sample collection. The project was supported by the National Science Foundation of China (No. 21772180), and all authors 91
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