Tandem derivatization combined with salting-out assisted liquid–liquid microextraction for determination of biothiols in urine by gas chromatography–mass spectrometry

Accepted Manuscript Title: Tandem derivatization combined with salting-out assisted liquid-liquid microextraction for determination of biothiols in urine by gas chromatography-mass spectrometry Authors: Chia-Ju Tsai, Fang-Yi Liao, Jing-Ru Weng, Chia-Hsien Feng PII: DOI: Reference:

S0021-9673(17)31450-4 https://doi.org/10.1016/j.chroma.2017.09.069 CHROMA 358898

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

4-5-2017 28-9-2017 28-9-2017

Please cite this article as: Chia-Ju Tsai, Fang-Yi Liao, Jing-Ru Weng, Chia-Hsien Feng, Tandem derivatization combined with salting-out assisted liquid-liquid microextraction for determination of biothiols in urine by gas chromatography-mass spectrometry, Journal of Chromatography A https://doi.org/10.1016/j.chroma.2017.09.069 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tandem derivatization combined with salting-out assisted liquidliquid microextraction for determination of biothiols in urine by gas chromatography-mass spectrometry Chia-Ju Tsaia, Fang-Yi Liaoa, Jing-Ru Wengb, Chia-Hsien Fenga, c, d, e* a

Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan b Department of Marine Biotechnology and Resources, National Sun Yatsen University, Kaohsiung, 80424, Taiwan c Ph.D. Program in Toxicology, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan d Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung, 80424, Taiwan e Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan *Corresponding author E-mail address: [email protected] Full postal address: 100, Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan Tel.: 886-7-3121101-2805 Fax: 886-7-321-0683

Highlights  Tandem derivatization of biothiols with multi-polar groups using BrTFB was achieved.  Excess derivatizing reagent can be recovered and used again by SALLME.  This analytical method enhanced the detectable signal for biothiol analysis by GC-MS in urine.  A fast and environment-friendly derivatization and microextraction technique was developed. Abstract Detection of polar organic compounds (POCs) using gas chromatography (GC) is not straightforward due to high polarity, 1

hydrophilicity, and low volatility of POCs. In this study, we report a tandem microwave-assisted derivatization method combined with salting-out assisted liquid-liquid microextraction (SALLME) to modify successively the polar groups of POCs in protic and aprotic solvents. Biothiols (cysteine and homocysteine) served as a proof of concept for this method because they possess three polar groups (thiol, amine, and carboxyl); the derivatizing reagent was 3,4,5-trifluorobenzyl bromide (Br-TFB) for alkylation. The solubility of the POCs in the protic or aprotic reaction medium affected the number of TFB molecules attached. Using the tandem derivatization with Br-TFB, the thiol and amine groups of biothiols were alkylated in the protic system, and the carboxylic groups of biothiols were alkylated in the aprotic system. The developed method was then successfully applied to measure biothiols in human urine. Because of the complex urine matrix and the lack of urine samples without endogenous biothiols, the standard addition method was utilized to avoid the matrix effect, check the recovery, and calculate the initial biothiol content in the urine. Regarding the linearity of the standard addition curves, the coefficient of determination was ˃0.996, and the linear regression showed satisfactory reproducibility with a relative standard deviation ˂3.9% for the slope and ˂8.8% for the intercept. The levels of cysteine and homocysteine in healthy human urine ranged from 28.8 to 111 μmol L-1 and from 1.28 to 3.73 μmol L-1, respectively. The proposed method effectively increased the sensitivity of GC-MS assays of water-soluble compounds in human urine.

Keywords: Tandem microwave-assisted derivatization, salting-out assisted liquid-liquid

microextraction,

polar organic

compound,

homocysteine, gas chromatography-mass spectrometry.

2

cysteine,

1. Introduction Determination of polar organic compounds (POCs) is a problem area in analytical chemistry. Issues such as bad peak shape, poor retention, adsorption on the column, or poor separation are usually encountered in the common chromatographic techniques [1]. Therefore, chemical derivatization is performed because it can modify the structure of the compound, and thus change its chromatographic retention; moreover, the chemical and physical properties of the compound are altered [2, 3]. This results in the delivery of large amounts of the analytes to the detector in a narrow peak shape, thus increasing the sensitivity, specificity, and selectivity. POCs often have more than one polar group, including hydroxyl, thiol, amine, and carboxyl groups. However, chemical reactions in most of the previous derivatization methods usually involve a single group; thus, other polar groups can affect the gas chromatography (GC) analysis. Therefore, liquid chromatography (LC) has become the favorable chromatographic technique for POC determination [4-6]. There has been a great concern to develop “green” analytical chemical methods in recent years. GC is much more environment-friendly than LC because it does not involve the use of an organic solvent, does not produce waste, and ensures better separation efficiency [7]. Thus, there is a need to develop a method for detection of POCs using GC. The polar nature, hydrophilicity, low volatility, and low thermal stability of POCs require derivatization before GC analysis. There are many GC derivatization methods, which can produce suitable volatile and chemically and thermally stable derivatives [8-11]. One of the most used derivatization methods is silylation [12, 13] because it can simultaneously react with various groups. However, silylation has two drawbacks [11, 14, 15]. First, silylating reagents and silyl derivatives are susceptible to 3

hydrolysis; thus, in situ aqueous derivatization is unsuitable. Therefore, aqueous samples require a prior extraction or drying step, which complicates the experimental procedure. Secondly, they can generate byproducts, which interfere with the chromatographic analysis. A few studies reported that silylation of POCs in aqueous matrices using a large excess of silylating reagents could handle these weaknesses [16-20]. However, these methods were inapplicable for trace analysis. The first attempt [21] for direct gradual silylation using hexamethyldisilazane (HMDS) and bis(trimethylsilyl)trifluoroacetamide (BSTFA) in aqueous matrices was developed to improve the detection limits of compounds. In addition, to improve the limits of detection, the majority of the approaches [22-27] used for determination of POCs usually combine multistep derivatization with extraction methods, such as liquid-liquid extraction (LLE), solid-phase extraction (SPE), and stir bar sorptive extraction (SBSE). However, the reaction time in the previous methods was too long. To overcome these problems, including the high consumption of reagents and long derivatization time, establishing a green, simple, and rapid in situ aqueous derivatization method that involves multiple groups is crucial. Recently, environment-friendly sample pretreatment methods, which use small amounts of low-toxicity reagents, produce less waste, and consume less time, have been developed, such as liquid-phase microextraction (LPME) and solid-phase microextraction (SPME) [28-31]. LPME is a straightforward technique that lowers the expenses of the analysis by using smaller amounts of the sample and organic solvent and reducing the waste generated. Compared with LPME, SPME is a solventless process that integrates several operations, including extraction, isolation, cleanup, and enrichment of analytes from sample matrices; however, this technique might be costly with poorly specific absorption 4

areas, lower absorption rate, and a carry-over effect. Hence, LPME is more preferred for sample preparation compared to SPME. Generally, it is challenging to extract POCs from aqueous matrices into water-immiscible solvent using conventional LPME. Nevertheless, salting-out assisted liquid-liquid extraction (SALLE) was developed by Matkovich and Christian in 1973 [32]. This method couples sample cleanup with preconcentration; in addition, the extract produced is compatible with many analytical techniques; thus, evaporation and residue reconstitution are no longer required. Alternatively, the extract can be directly injected into analytical instruments after extraction. SALLE is carried out based on the salting-out effect to separate the partially water-miscible organic solvent from the aqueous matrix. Salting out is a procedure that involves addition of an inorganic or organic salt into an aqueous solution, which allows the disruption or weakening of the solvation forces, thus, enhances the distribution ratio of specific solutes, particularly hydrophilic compounds, in the organic phase [33, 34]. In this study, a new mode of multistep derivatization, called “tandem microwave-assisted derivatization (tMAD)” was designed to ensure that all the polar groups of compounds were alkylated with alkyl halides. For derivatization, first, the strong nucleophilic groups of the analytes were derivatized in an aqueous solution, which resulted in a decrease in hydrophilicity to increase the partition coefficient of the analytes in organic solvents. Then, the intermediate derivatives were extracted and the derivatizing reagent was resumed into the non-aqueous medium using SALLME. The weak nucleophilic groups of the analytes were derivatized in an aprotic reaction system that reduced the polarity and improved the volatility, thermal stability, separation ability, and sensitivity of the analytes prior to GC-MS analysis. This study aimed to develop a green, rapid, and 5

simple method for the determination of POCs in complex matrix samples. To verify this concept, two biothiols were selected as model analytes, cysteine and homocysteine, because they contain thiol, amine, and carboxyl groups. Cysteine and homocysteine play vital roles in a variety of physiological processes, especially in maintaining redox homeostasis in biological systems [35]. The thiol group of cysteine and homocysteine, possessing high nucleophilicity, can be easily oxidized to form disulfides. The thiol group often serves as a nucleophile and participates in enzymatic reactions. The formation of disulfides from protein cysteines (to form cystine) is involved in maintaining the protein structure and function. Cystinosis [36] and cystinuria [37] are inherited diseases which are caused by disorders of cysteine metabolism and the cystine that accumulates in the body, resulting in crystal formation and the disease symptoms. There are four types of homocysteine in the human body: the free form, homocystine, homocysteine-cysteine disulfide, and the protein-binding form [38]. Clinical monitoring of homocysteine concentration uses the sum of the four types. Hyperhomocysteinemia leads to endothelial cell damage and reduced flexibility of vessels, resulting in cardiovascular disease and its complications [39]. In addition, they act as biomarkers in human physiology, particularly, the inadequacy of cysteine is involved in liver damage, leucocyte loss, and psoriasis, whereas excess homocysteine is involved in Alzheimer’s and folate and cobalamin (vitamin B12) deficiencies [40]. Thus, estimation of their levels may help in early diagnoses. In this study, we determined the concentrations of these biothiols in human urine.

2. Materials and methods

6

2.1. Reagents and Chemicals Cysteine (Cys, pKa = 1.71 (COOH), pKa = 8.33 (SH), pKa = 10.8 (NH2), logKow = -3.05), homocysteine (Hcy, pKa = 2.22 (COOH), pKa = 8.87 (SH), pKa = 10.9 (NH2), logKow = -2.56), 4-amino-3-methylbenzoic acid (AMBA) as an internal standard (IS), sodium borohydride (NaBH4), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), heptafluorobutyric acid (HFBA), and potassium hydroxide (KOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN), acetone (ACT), tetrahydrofuran (THF), potassium hydrogen carbonate (KHCO3), potassium carbonate (K2CO3), sodium chloride (NaCl), and trifluoroacetic acid (TFA) were purchased from Merck (Darmstadt, Germany). Br-TFB, used as a derivatizing reagent, was obtained from TCI (Tokyo, Japan). Pentafluoropropionic acid (PFPA), nonafluoropentanoic acid (NFPA), and undecafluorohexanoic acid (UFHA) were purchased from Alfa Aesar (Lancashire, UK). Deionized water was obtained using a Millipore Milli−Q Lab system (Bedford, MA, USA). For standard analysis, the stock solutions of Cys (2 mmol L-1), Hcy (2 mmol L-1), and AMBA (250 μmol L-1) were individually prepared in deionized water. A standard mixture of both analytes was prepared by mixing the stock solutions at a final concentration of 1 mM, and all the working solutions with desired concentrations were prepared by appropriately diluting the standard mixture solution with deionized water (concentration range from 1 to 40 μmol L-1) before addition of human urine. Br-TFB (900 mmol L-1) solution was prepared in ACN. When not in use, all solutions were stored at 4℃ in a refrigerator. The respective reductant solutions were freshly prepared by adding appropriate amounts of these compounds to an alkaline aqueous solution before use. The individual base catalyst and ion-pairing agent solutions were prepared by dissolving each 7

reagent in deionized water, and all of them were placed at room temperature (25℃).

2.2. Sample Preparation Urine samples from five healthy female subjects, aged 21-30 years, were collected into 15-mL plastic containers without addition of any preservative. The samples were stored at -20℃ until analysis. Before the experiments, the samples were thawed at room temperature. The urine samples were analyzed according to developed procedure after fivefold dilution via addition of 0.1 mL of 250 μmol L-1 AMBA and 0.7 mL of the standard mixture aqueous solution in various amounts (0, 70, 700, 1400, and 2800 pmol).

2.3. Microwave-assisted derivatization (MAD) of biothiols and their extraction Tandem derivatization was applied in protic and aprotic reaction systems using Br-TFB to alkylate the polar groups (thiol, amine, and carboxyl groups). The first step involved derivatization in protic system (1) as follows: 100 μL of diluted urine sample solution was drawn into a 1.5mL eppendorf tube and then, 5 μL of 10 mmol L-1 TCEP in 50 mmol L-1 K2CO3 aqueous solution and 100 μL of 600 mmol L-1 Br-TFB in ACN were successively added. After mixing, MDA was performed in a microwave oven (Panasonic NN-ST677) at 1000 W for 5 min. Subsequently, the second step involved derivatization in aprotic system (2) as follows: 5 μL of 1 mol L-1 NFPA aqueous solution and 30 mg NaCl were added. The tube was vortexed until a little amount of salt remained insoluble, for approximately 2 min. The aqueous phase and organic phase were separated by centrifugation for 1 min at 14,800 rpm. The upper phase, constituting 8

approximately 50 μL was transferred to another 1.5-mL eppendorf tube containing 5 mg of solid KHCO3, and the mixture was heated in a microwave oven at 750 W for 7 min. After cooling, 1 μL of the resulting solution was injected into the GC-MS apparatus. For comparison, a single derivatization in either protic or aprotic reaction system was also performed using Br-TFB. Single derivatization in protic system was carried out as described for step (1) in the previous paragraph. Later, the mixture was continuously heated in the microwave oven at 750 W for 7 min. After reaction, 5 μL of 1 mol L-1 NFPA aqueous solution and 30 mg NaCl were added, and the mixture was vortexed for 2 min. Thereafter, the aqueous phase and organic phase were separated by centrifugation for 1 min at 14,800 rpm. Finally, 1 μL of the supernatant was injected into the GC-MS apparatus. Single derivatization in aprotic system involved drying of 100 μL of the diluted urine sample solutions in a 1.5-mL eppendorf tube using a centrifugal evaporator at 35℃ (for approximately 5 h). Then, 5 mg of KHCO3 crystals, 5 μL of 10 mmol L-1 DTT, and 100 μL of 600 mmol L-1 Br-TFB in ACN were successively added to the residue. The mixture was vortexed for 2 min and was then heated at 1000 W for 5 min in the microwave oven. Later, the mixture was continuously heated at 750 W for 7 min. After reaction, 100 μL of deionized water, 5 μL of 1 mol L-1 NFPA aqueous solution, and 30 mg NaCl were added, and the mixture was vortexed for 2 min. Thereafter, the aqueous phase and organic phase were separated by centrifugation for 1 min at 14,800 rpm. Finally, 1 μL of the supernatant was injected into the GC-MS apparatus.

2.4. Instrumentation and Conditions Biothiols were analyzed by GC-MS using BRUKER 450−GC coupled 9

with a SCION mass spectrometer detector. Separation was accomplished using a VF−5MS fused-silica capillary column (30 m × 0.25 mm i.d.; 0.25μm film thickness, Agilent Technologies) at a constant helium flow rate of 1 mL min-1. A sample volume of 1 µL was injected with split mode at a split ratio of 10:1. The injector temperature was maintained at 290℃. The temperatures of the transfer line and ion source were set at 290 and 270℃, respectively. The oven temperature was initially set at 50℃ for 0.5 min, and then rapidly heated to 290 ℃ at a rate of 120 ℃ /min where the temperature was fixed for 7.5 min (total elution time, 10 min). For GC-MS detection, an electron ionization system with ionization energy of 70 eV was used. The mass spectrometer was operated in selective ion monitoring mode (SIM). The retention time, qualifier and quantitation ions (m/z) obtained are listed in Table 1. In addition, Figure S1 shows the mass spectra of the target TFB derivatives.

3. Results and discussion

3.1. Derivatization The

difference

between

tandem

derivatization

and

single

derivatization in protic and aprotic solvents, which can be depicted by comparing the alkylation of biothiols with Br-TFB, and the procedures of these methods are described in Figure S2. Derivatization was required to reduce the polarity and to enhance the volatility and detection sensitivity of biothiols. Bromomethylation reagents are less hydrolyzed and have better stability than silylation reagents. For this reason, the commercial derivatizing reagent, Br-TFB was selected in this study. Besides, this study used microwave energy to directly heat the 10

target analytes to accelerate the production of derivatives. Alkylation with Br-TFB through MAD in a single reaction is still problematic because of the different ability of different groups to form TFB derivatives. The alkylation reaction rate is affected by solvation of the reactant nucleophiles, particularly weak nucleophiles (e.g. OH-, -CN, and RCO2-). As shown in Figure S2 and Table 2, derivatization of biothiols in protic solvents is convenient because it allowed direct alkylation in the sample matrix without any pretreatment; however, only few triTFB derivatives were formed. On the contrary, SN2 reactions performed much well in aprotic solvents because no hydrogen bonds were formed with the anionic nucleophiles, which could improve the alkylation reaction. Nevertheless, biothiols have low solubility in organic solvent because of their high hydrophilicity. Thus, it is difficult to use a polar aprotic solvent to efficiently extract the compounds from the biological sample or to redissolve the residues after drying processes. For this reason, the triTFB derivatives generated were still few. By using a tandem reaction with BrTFB in protic solvent for alkylation of strong nucleophiles, thiols and amines, and in aprotic solvent for alkylation of weak nucleophiles, carboxyl groups, we could achieve the highest analytical responses during measurements. Table 2 compares the efficiency of the individual alkylation methods, where a relative response of 100 % indicates the largest peak area of the derivatized compounds. The efficiency of single derivatization in protic solvent was the least because the solvent formed a barrier around the nucleophiles that hindered the ability of nucleophiles to attack the electrophilic carbon. Besides, the single reaction in aprotic system exhibited the worst repeatability because the biothiols were difficult to redissolve expeditiously in the polar aprotic solvent. The tandem 11

derivatization was better than the single-reaction procedures; therefore, it was used in the further experiments. To obtain a perfect derivatization efficiency and reproducibility in the tandem reaction, various factors were optimized. These factors are listed, and the optimum results are shown in Table 3. After optimizing the tandem derivatization conditions, the derivatives of Cys and Hcy were quantified by nano LC-MS/MS (the detailed account is provided in the Supplemental Data). When the peak areas for cysteine triTFB and homocysteine triTFB were 100%, the cysteine monoTFB and homocysteine monoTFB were all 0%, and the cysteine diTFB and homocysteine diTFB were 1.22% and 0.19%, respectively. Cysteine triTFB, homocysteine triTFB, and AMBA diTFB were also identified by high-resolution nano LC-MS/MS (Figure S3).

3.1.1. Optimization of tandem derivatization—reaction in a protic system The first step of tandem derivatization was carried out in a protic system. A reductant was used because the majority of biothiols are present in the body linked to proteins or to other biothiols by disulfide bond [41]. The abilities of three reductants (10 mmol L-1 DTT, TCEP, and NaBH4) to break the disulfide bonds were compared. As shown in Figure S4A (Supplemental Data), TCEP exhibited the highest reducing efficiency. Different TCEP concentrations (0.5-40 mmol L-1) were tested. Results showed that the reducing ability increased with increasing the concentration of TCEP (Figure S4B in Supplemental Data). However, the relative response decreased when TCEP concentration exceeded 10 mmol L-1, which was possibly owing to Wittig reaction [42]. Thus, 10 mmol L-1 TCEP was considered optimal. An alkaline medium was used to aid the reaction of the haloalkane with the nucleophiles. Diverse base catalysts (50 mmol L-1), including 12

KHCO3, K2CO3, and KOH, were assessed. As shown in Figure S5A (Supplemental Data), the highest derivatization yield was obtained when K2CO3 was used. This indicated that the suitable basicity of K2CO3 enhanced the nucleophilic attack with Br-TFB. In addition, as shown in Figure S5B (Supplemental Data), the yield was proportional to K2CO3 concentration up to 50 mmol L-1; however, when the concentration of K2CO3 exceeded 50 mmol L-1, derivative production was abated. This reduced yield might be attributed to the hydrolysis of the derivatizing reagent because of the excessively high pH [43]. Accordingly, 50 mmol L1

K2CO3 was selected as the optimal concentration. To ensure that the amount of the derivatizing reagent was sufficient

for the formation of biothiol derivatives, solutions containing various BrTFB concentrations (ranging from 5 to 900 mmol L-1) were examined. Figure S6 (Supplemental Data) shows that the derivatization yield incrementally increased as Br-TFB concentration increased from 5 to 600 mmol L-1. However, when Br-TFB concentration exceeded 600 mmol L-1, the yield of the derivatives slightly diminished because a two-phase supersaturated Br-TFB solution was formed, which reduced the collision frequency between the analyte and reagent molecules in the medium. To accelerate the derivatization reaction, the effect of microwave energy of different powers on the reaction time was evaluated (from 1 to 9 min, at 400, 700, and 1000 W, respectively). The maximum yield was accomplished at 1000 W for 5 min (Figure 1A). Nevertheless, a higher irradiation power resulted in reaction times ˃ 5 min; however, a lower response was achieved because of vaporization of the organic compounds. Thus, a reaction time of 5 min and an irradiation power of 1000 W were selected.

13

3.1.2. Optimization of tandem derivatization—reaction in an aprotic system Aprotic solvents are preferred for use in SN2 reactions because they can reduce the free energy by decreasing the solvation of the reactant nucleophiles. Three aprotic solvents, ACN, ACT, and THF, were evaluated. As shown in Figure S7 (Supplemental Data), ACT and THF exhibited less reaction yield compared to that of ACN. Therefore, ACN was used for further application. In addition, alkaline conditions can accelerate alkylation reactions in aprotic systems; thus, different base catalysts (5 mg of KHCO3, CH3COOK, and K2CO3) were evaluated. Figure S8A shows that KHCO3 resulted in the best response (Supplemental Data). KHCO3 provided a proper basic environment, which favored the ionization of the carboxyl groups that, in turn, facilitated the derivatization reaction. Moreover, as shown in Figure S8B (Supplemental Data), the derivatization yield was proportional to the amount of KHCO3; however, a plateau occurred when the base amount exceeded 5 mg. Different microwave energies (300, 550, and 750 W) and reaction times (ranging from 1 to 9 min) were also compared. As shown in Figure 1B, regardless of the irradiation energy, the derivatization yield increased proportionately with the reaction time. The maximum derivatization yield was obtained within 7 min using an irradiation power of 750 W. Consequently, derivatization of all samples was implemented at 750 W for 7 min in aprotic systems.

3.2. Optimization of extraction conditions—salting-out assisted liquidliquid microextraction (SALLME) A base catalyst is required in derivatization reactions. After the first step, the biothiol derivatives were present in alkaline solution. These derivatives were most likely ionized because biothiols exhibit amphoteric 14

properties and their carboxyl groups have not been alkylated yet. If this aqueous solution was injected into a non-polar GC column directly, the signal intensity of the biothiol derivatives would not be detectable because there were many adsorption-active intermediate derivatives or interfering species in the sample matrix. Separation of the active intermediate derivatives and recovery of the surplus derivatizing reagent into an organic phase were needed for GC-MS; in addition, a second step was carried out to produce the less adsorption-active derivatives. After the first step, the biothiol intermediate derivatives were still dissolved in the aqueous solution even when the sample solution was acidified. To improve the extraction efficiency of the intermediate derivatives from the aqueous layer to the organic layer, an ion-pairing reagent (IP) was used to block the basic groups via formation of ion-pair complexes of higher hydrophobicity than that of the original biothiol intermediate derivatives [44]. In this study, IP was employed for two purposes; to acidify the protic reaction solution, and to increase of “hydrophobic interaction” between the extractant and solute ion-pair complexes. The effects of various IPs, including TFA, PFPA, HFBA, NFPA, and UFHA, at 1000 mmol L-1 were compared. This showed that there was a bad response in the absence of IP; however, all the examined IPs improved the partition coefficients of the derivatives into the organic layer, of which NFPA resulted in the highest extraction efficiency (Figure 2A). In addition, the concentration of IP affected the extraction ability owing to its influence on the distribution of counter ions. Thus, the effect of different concentrations of NFPA (ranging from 50 to 2000 mmol L-1) on the extraction efficiency was evaluated. This showed that the maximum response was reached at NFPA concentration of 1000 mmol L-1 (Figure 2B). In this derivatization procedure, the derivatizing reagent was 15

dissolved in ACN. It was challenging to isolate the biothiol derivatives from the aqueous system. Nevertheless, salting out was helpful to separate the water-miscible organic solvent into two phases via addition of a sufficient amount of salt [33]. Consequently, the effect of the amount of NaCl added (ranging from 10 to 50 mg) on the extraction efficiency was studied. As shown in Figure 2C, the responses were enhanced when the amount of NaCl was increased from 10 to 30 mg and remained practically unchanged with higher amounts of NaCl. These results showed that salt addition decreased the water solubility of the derivatives and changed the physical properties of the Nernst diffusion layer; thus, it increased the mass transfer to the organic layer. Evaluating the recovery of SALLME was difficult by GC-MS, because the signals of the intermediate derivatives of cysteine and homocysteine (still contain polar groups) were hard to detect. Therefore, this study used nano LC-MS/MS to compare the amounts of the derivatives of cysteine and homocysteine in the urine sample before and after SALLME. Since the experiment had not proceeded to the aprotic derivatization step, the mono and double TFB derivatives were dominant. However, substantial suppression of the ESI ionization from complex sample matrices was observed, and the correct recovery of SALLME was unable to be obtained.

3.3. Method validation Standard addition method was applied to quantify the biothiols in human urine because the target analytes are endogenous. Urine samples were spiked with biothiols at a concentration range from 0 to 40 μmol L-1. Derivatization and extraction using tD-SALLME coupled with GC-MS method was performed. The calibration curves were established by plotting 16

biothiol peak area ratio versus the added concentration of the biothiols. Good linearity was achieved with a coefficient of determination (r2) > 0.996 for both Cys and Hcy in human urine. The precision of the slope and intercept was estimated by calculating the relative standard deviation (RSD) of five replicates. The RSD of the slope and intercept were lower than 3.9 % and 8.8 %, respectively. All results are summarized in Table 4.

3.4. Comparison of the present method with other reported methods The developed method was compared with other methods used for multistep derivatization by GC-MS (Table 5). The most commonly used derivatizing reagents are silylation reagents [21, 23-27], whereas the most common extraction methods for compounds containing multifunctional groups are LLE [22, 24, 25] and SPE [24, 26, 27]. Besides, there are two derivatizing reagents being used in each previous method. The consumption of the sample, derivatizing reagent, and solvent, as well as the reaction and extraction times of the previously developed methods were similar to or even higher than this method. In addition, some of the old methods require additional drying procedure [23, 24-27] for the following reasons: (1) to enrich the desired compounds; (2) to remove the excess derivatizing reagent; and (3) to prevent the hydrolysis of the derivatizing reagent or derivatives. This drying step consumes considerable time, results in a longer total analysis time. In contrast, tD-SALLME, used in this study, is a relatively novel method with shorter reaction time (12 min) and extraction time (2 min), which does not involve a drying step. Moreover, this method is a green one because it not only utilizes one derivatizing reagent but also consumes small volumes of the derivatizing reagent (8 μL) and organic solvent (100 μL). Based on these results, this method is a rapid, easy, and reliable technique for multistep derivatization. 17

3.5. Application of the analytical method for human urine samples The applicability of this method was evaluated via assay of biothiols in urine samples. The nucleophilic compounds in urine samples will react with the Br-TFB, including all endogenous amino acids present in the urine. However, the derivatives of other amino acids would not cause interference in SIM mode analysis, because the molecular masses of these amino acids are different from that of Cys and Hcy; the masses of their derivatives are also not the same as that of cysteine triTFB and homocysteine triTFB. Moreover,

we

utilized

the

Human

Metabolome

Database

(http://www.hmdb.ca/) to search for compounds present in humans that have the same molecular weight as Cys (m/z 121), Hcy (m/z 135), cysteine triTFB (m/z 553), and homocysteine triTFB (m/z 567). The search found benzamide (m/z 121), 1-phenylethylamine (m/z 121, CAS number 98-840), phenylethylamine (m/z 121, CAS number 64-04-0), methylcysteine, (m/z 135), adenine (m/z 135), amphetamine (m/z 135), and 1docosahexaenoyl-glycero-3-phosphocholine (m/z 567). However, these compounds would not cause interference in the analysis because their qualifier ions are different from those of cysteine triTFB and homocysteine triTFB listed in Table 1. In this study, urine samples were obtained from five healthy adult volunteers. The initial content of the sample was calculated from the intercept divided by the slope of the regression equation. All urine samples were analyzed. The total levels of Cys and Hcy were detectable, ranging from 28.8 to 111 μmol L-1 and from 1.28 to 3.73 μmol L-1, respectively (Table 6). These results were comparable to those reported in other studies, which showed that the urinary concentrations of total Cys and total Hcy in healthy adults were 27.5–117 μmol L-1 [45] and 1.1–4.0 μmol L-1 [45-47], respectively. Figure 3 shows the chromatograms of urine samples with or without spiked biothiols. In this study, a method was developed to evaluate the total levels (reduced and oxidized forms) of Cys and Hcy, by adding TCEP as a reducing agent. Without TCEP, only the levels of the free, 18

reduced forms of Cys and Hcy would be assayed. 4. Conclusions A method for determination of biothiols in urine samples using SALLME combined with tMAD, which integrated in situ aqueous alkylation and non-aqueous alkylation followed by GC-MS, was developed. This method exhibits many attractive advantages, such as easy operation; low sample, organic solvent, and derivatizing reagent consumption; short reaction and extraction times; and good precision. Introduction of derivatization using Br-TFB improved the sensitivity and detection limit of GC-MS. Moreover, no reagent addition was required in the second step. In comparison with the previous methods, tandem derivatization used in this study was considered a proper alternative derivatization method for determination of compounds containing multifunctional groups because of being rapid, efficient, environmentfriendly, and economic. This method was favorably applied for analysis of biothiols in human urine samples. Acknowledgments This project was supported by a grant from the Ministry of Science and Technology (grant no. MOST 105-2113-M-037-015 and 106-2113-M037-006), by Aim for the Top Universities from Kaohsiung Medical University (grant no. KMU-TP105PR05) and by NSYSU-KMU Joint Research Project (grant no. NSYSUKMU 106-P005). The Research Center for Environmental Medicine, Kaohsiung Medical University, is greatly appreciated for instrumental support.

19

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23

Figure captions Figure 1. Influence of the microwave energy and reaction time on the efficiency of the alkylation reaction in the protic system (A) and aprotic system (B) for formation of the biothiol derivatives. Figure 2. Influence of the type of ion-pairing reagent (A), the concentration of NFPA (B), and the amount of NaCl (C) on the extraction of alkylated derivatives of biothiols in SALLME. Figure 3. The chromatograms of biothiol triTFB derivative under optimized conditions, non-spiked (blue) and 10 μmol L-1 spiked (red) urine. The derivative peak of 1, Cys; 2, Hcy, and IS, AMBA.

24

25

26

27

Table legends Table 1.

The retention times (RT), molecular weights (MW), quantitation, and qualifier ion values are related to the alkylated derivatives of analytes.

Table 2.

Comparison of the efficiency of alkylation methods in different reaction systems with respect to the relative response of the desired derivatives in urine spiked with 40 μmol L-1 biothiols.

Table 3.

Optimal tandem derivatization conditions for GC-MS analysis of biothiols in urine.

Table 4.

Analytical parameters for within-run and between-run analysis of biothiols in urine by GC-MS.

Table 5.

Comparison between different multistep derivatization methods for determination of compounds with multifunctional groups by GC-MS analysis.

Table 6.

Determination of biothiol concentration in urine by GC-MS.

Table 1. The retention times (RT), molecular weights (MW), quantitation, and qualifier ion values are related to the alkylated derivatives of analytes. Analytes Cysteine triTFB Homocysteine triTFB AMBA diTFB

RT (min) MW (g mol-1) 8.46 553.44 9.45 567.47 8.62 439.39

Quantitation (qualifier) ions, m/z values 145 (362, 125, 95) 145 (125, 200, 422) 145 (278, 173, 439)

Table 2. Comparison of the efficiency of alkylation methods in different reaction systems with respect to the relative response of the desired derivatives in urine spiked with 40 μmol L-1 biothiols. Biothiols

Single derivatization Protic RSDa Cysteine triTFB 9.08 2.32 Homocysteine triTFB 8.08 1.65 a Relative standard deviation, %

Aprotic 46.2 23.7

a

RSD 2.77 7.52

Tandem derivatization Protic+Aprotic RSDa 100 0.54 100 1.07

Table 3. Optimal tandem derivatization conditions for GC-MS analysis of biothiols in urine. Factor

Studied types/ ranges

Optimal condition

Protic system Reductant

DTT, TCEP, NaBH4

TCEP

Concentration of TCEP (mmol L-1)

0.5-40

10

Base catalyst

KHCO3, K2CO3, KOH

K2CO3

Concentration of K2CO3 (mmol L-1)

5-100

50

Concentration of Br-TFB (mmol L-1)

5-900

600

Reaction energy (Watt)

400-1000

Reaction time (min)

1-9

1000 5

Aprotic system Medium

ACN, ACT, THF

ACN

Base catalyst

KHCO3, CH3COOK, K2CO3

KHCO3

Amount of KHCO3 (mg)

1-9

Reaction energy (Watt)

300-750

Reaction time (min)

1-9

5 750 7

Table 4. Analytical parameters for within-run and between-run analysis of biothiols in urine by GC-MS. Slope (RSDa) Within-run Between-run Cysteine 0.00038 (0.77) 0.00039 (2.70) Homocysteine 0.00027 (1.03) 0.00028 (3.91) a Relative standard deviation, % ; n=5 Biothiol

Intercept (RSDa) Within-run Between-run 0.80746 (5.17) 0.81816 (1.78) 0.01347 (8.83) 0.00964 (2.16)

Determination coefficient (r2) Within-run Between-run 0.997 0.997 0.998 0.996

Table 5. Comparison between different multistep derivatization methods for determination of compounds with multifunctional groups by GC-MS analysis. Compound (functional group) Alanine (amine, carboxyl) PUT, SPD, SPM (amine) Eβ2 (hydroxyl)

Sample matrix (amount) Cell (0.02 mL) Postmortem brain (0.1 g) River water (10 mL) River water (200 mL) Plasma, AF (0.5, l mL) Urine (2 mL)

Extraction Solventa Ref. method/time (volume, mL) Reacted at 50℃ for 30 min+ Unused ACN (0.3) [21] 80℃ for 30 min Reacted at RT for 30 min in LLE/ Et2O (7)+ EA (0.3) [22] pH 10+ 75℃ for 60 min 10 min AAA (100)+ BSTFA (1) Reacted at RT for 120 min in SBSE/ Unused [23] pH 11+ 280℃ for 5 min 120 min Br-PFB (4)+ TMSI (50) Reacted at 60℃ for 60 min SPE/ - + ACT (13.1) + [24] in pH 11+ RT for 30 min LLE/ Hexane (1.95) PFBHA (100)+ Reacted at RT for 60 min in LLE/ Et2O (4)+ EA (4) [25] BSTFA (100) pH 1+ 70℃ for 60 min HMDS (50)+ Reacted at 40℃ for 10 min+ SPE/ MeOH (2)+ EA (0.02) [26] MBHFBA (20) 80℃ for 5 min Derivatizing reagent (volume, μL) HMDS (300)-TFA (2)+ BSTFA (400) ECF (50)+ TFAA (200)

Derivatization conditions

E1, Eβ2, Eα2, EE2, E3 (hydroxyl) SA (carbonyl, carboxyl ) DA, HVA, Dopa, NE, VMA (amine, hydroxyl, carboxyl) MA, PPA, CC, BZE, Skin MTBSTFA-1% TBDMCS Reacted at 80℃ for 20 min+ SPE/ MeOH (7)+ DMC [27] EME, CD, MP (amine, (0.05 g) (20)+ BSTFA-1% TMCS 80℃ for 45 min (3.2)+ IPA (0.8)+ hydroxyl, carboxyl) (20) ACN (0.52) Cys, Hcy (amine, thiol, Urine Br-TFB (8) Reacted at 1000W for 5 min SALLME/ ACN (0.1) This carboxyl) (0.02 mL) in pH 10+ 750W for 7 min 2 min work PUT, putrescine; SPD, spermidine; SPM, spermine; Eβ2, 17β-estradiol; E1, estrone; Eα2, 17α-estradiol; EE2, 17α-ethynyl estradiol; E3, estriol; SA, succinylacetone; DA, dopamine; HVA, homovanillic acid; Dopa, L-3,4-dihydroxy-l-phenylalanine; NE, D,L-noradrenaline; VMA, 4hydroxy-3-methoxymandelic acid; MA, Methamphetamine; PPA, amphetamine; CC, cocaine; BZE, benzoylecgonine; EME, ecgonine methyl ester; CD, codeine; MP, morphine; AF, amniotic fluid; TFA, trifluoroacetic acid; ECF, ethylchloroformate; TFAA, trifluoroacetic acid anhydride; AAA, acetic acid anhydride; Br-PFB, pentafluorobenzyl bromide; TMSI, N-trimethylsilylimidazole; PFBHA, O-(2,3,4,5,6pentafluorobenzyl)hydroxylamine; MBHFBA, N-Methyl-bis(heptafluorobutyramide); MTBSTFA, N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide; TBDMCS, tert-butyldimethylchlorosilane; TMCS, trimethyl- chlorosilane; Et2O, diethyl ether; EA, ethyl acetate; MeOH, methanol; DCM, methylene chloride; IPA, 2-propanol; RT, room temperature; -, not reported in the method. a Used in the process of derivatization and extraction.

Table 6. Determination of biothiol concentration in urine by GC-MS. Concentration (μmol L-1) Urine Samples 1

a

Cysteine (RSDa) 59.2 (0.92)

Homocysteine (RSDa) 1.94 (3.7)

2

28.8 (2.0)

1.28 (2.5)

3

102 (3.3)

3.73 (5.2)

4

92.5 (1.3)

3.61 (3.9)

5

111 (0.41)

3.17 (3.2)

Relative standard deviation, % ; n= 3