Investigation of changes in endocannabinoids and N-acylethanolamides in biofluids, and their correlations with female infertility

Accepted Manuscript Title: Investigation of Changes in Endocannabinoids and N-acylethanolamides in Biofluids, and their Correlations with Female Infertility Authors: Jun Ding, Xiao-Tong Luo, Yan-Ru Yao, Hua-Ming Xiao, Ming-Quan Guo PII: DOI: Reference:

S0021-9673(17)30879-8 http://dx.doi.org/doi:10.1016/j.chroma.2017.06.029 CHROMA 358599

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

19-1-2017 18-4-2017 11-6-2017

Please cite this article as: Jun Ding, Xiao-Tong Luo, Yan-Ru Yao, Hua-Ming Xiao, Ming-Quan Guo, Investigation of Changes in Endocannabinoids and Nacylethanolamides in Biofluids, and their Correlations with Female Infertility, Journal of Chromatography Ahttp://dx.doi.org/10.1016/j.chroma.2017.06.029 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.

Investigation of Changes in Endocannabinoids and Nacylethanolamides in Biofluids, and their Correlations with Female Infertility Jun Ding1,2†, Xiao-Tong Luo2†, Yan-Ru Yao3†, Hua-Ming Xiao2, Ming-Quan Guo1* †

Equal contributors

1., Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences; Sino-Africa Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China. 2. Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China 3. Department of Obstetrics and Gynecology, Medicine Center for Human Reproduction, Zhongnan Hospital of Wuhan University, Wuhan, Hubei Province, 430071, People's Republic of China *To whom correspondence should be addressed. Tel: +86-27-87518018; fax: +86-2787518018. E-mail address: [email protected] Highlights 

Magnetic liquid microextraction using toluene as extractant was employed for eCBs and NAEs enrichment.

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MS sensitivities of eCBs and NAEs were improved by 4-(N,N-dimethyamino)benzoyl chloride labeling.

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Isomerization of 2-AG was prevented by the method.

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ECBs and NAEs contents in the biofluids from 143 patients and healthy controls were investigated.

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The correlation of eCBs and NAEs with female infertility was discussed.

Abstract Female infertility is a worldwide medical problem, and the scarcity of infertility biomarkers has hindered the ability to launch preventive and therapeutic measures in a timely manner. Intriguingly, alterations in endocannabinoids (eCBs) and Nacylethanolamides (NAEs) have been observed in the biofluids of infertile females. Therefore, a hypothesis of using eCB and NAEs in biofluids as infertility biomarkers was proposed by several researchers; however, little evidence exists to verify the hypothesis. To investigate their correlations with female infertility, we developed a magnetic liquid microextraction-chemical derivatization (MLME-CD) method coupled with liquid chromatography-tandem mass spectrometry for the quantification of eCBs and NAEs in biofluids. The target compounds were first purified with magnetic toluene as sorbents, and then labeled with 4-(N,N-dimethyamino)benzoyl chloride (4-DMABC). The MLME-CD method offered several advantages, including reliable quantification results by preventing the isomerization of eCB, high throughput by requiring 20 min for sample preparation, and good sensitivity with limits of detection at 3.0-54.3 fmol. The intra-day and inter-day relative standard deviations were below 14.5 %, and the recoveries were 87.4%–117.9%. Concentrations of eCB and in the serum of 49 infertile women and 53 fertile women (controls), and in the ovarian follicular fluid of 21 infertile women and 20 controls were then quantified. Using unpaired t test analysis indicated significant differences in AEA and PEA in serum, and OEA in follicular fluid between infertile women and healthy controls, and the areas under the curve were in the range of 0.605-0.707.

Keywords: Endocannabinoids; Female infertility; Magnetic liquid microextraction; Chemical derivatization; Liquid chromatography-mass spectrometry.

1. Introduction Infertility is a condition of the reproduction system that has been recognized as a public health issue worldwide by the World Health Organization [1], and affects onesixth of the couples attempting to conceive, with more than 50% of cases due to female factors [2]. Early diagnosis of infertility, however, may provide an opportunity for timely and appropriate intervention [3-5]. Therefore, it would be of great significance to discover infertility biomarkers that could be used to diagnose reproductive defects, or that reveal any possible pathologic changes in women at an early stage. Endocannabinoids (eCBs) constitute a group of bioactive lipid mediators released from membrane phospholipids and produced on demand by cells [6]. The best-characterized eCBs are N-arachidonoyl ethanolamine, known as anandamide (AEA), and 2-arachidonoyl glycerol (2-AG). Their biosynthesis is catalyzed by the Narachidonoylphosphatidyl-ethanolamine-specific phospholipase D (NAPE-PLD) and diacylglycerol lipase [7, 8], and terminated by hydrolysis with the serine hydrolases fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase [9, 10]. As neurotransmitters, the eCBs occur in an exquisitely regulated balance between synthesis and metabolism, and bind to type-1 and type-2 cannabinoid receptors (CB1 and CB2) and GPR55 (a recently discovered putative “CB3”) to perform a series of physiologic functions[11, 12], both in the central and peripheral nervous systems and in peripheral organs[13, 14]. Over the past few decades, comprehensive studies indicated that all female reproductive events, from oocyte development to parturition,

are regulated by the eCB system, and alterations in FAAH activity or eCB concentrations were observed in reproductively related diseases [15-18]. The enhancement of FAAH, inhibition of NAPE-PLD, or agonism of the eCB receptor is also known to be beneficial in promoting successful human pregnancies. In addition, the N-acylethanolamides (NAEs) (including N-palmitoylethanolamine [PEA] and Noleoylethanolamine [OEA]), have also been shown to be important in reproductively related diseases, as they act as entourage compounds by enhancing the activity of AEA and/or 2-AG[19]. Based upon this evidence, serum and tissue eCBs and NAEs were suggested by several researchers to be diagnostic biomarkers for predicting the reproductive potential of women [20-23]; however,, there is no statistical evidence (based on serum or tissue eCB and NAE concentrations) to confirm their feasibility as biomarkers. It is clear that the development of a reliable analytical method for the analysis of eCBs and NAEs would contribute to the quantification of these substances in patients and healthy subjects, thus providing a tool for studying the correlation between eCB and NAE concentrations and female infertility. High performance liquid chromatography-mass spectrometry (HPLC-MS) has been considered a robust detection platform for the analysis of eCBs and NAEs due to this method’s high sensitivity and selectivity[24]. However, even with the most sophisticated LC-MS technology, there are still some questions regarding the determination of eCBs and NAEs in biologic samples, including, but not limited to: 1) eCBs and NAEs occurring at a low endogenous concentration in serum or tissue, at the threshold of the pM/nM range; 2) the complicated matrix of serum or tissue

diminishing MS detection sensitivity; 3) eCBs and NAEs lacking ionizable groups in their molecular structures, thus making their MS responses unsatisfactory; 4) 2-AG isomerizing spontaneously to 1-AG[25], which is an inactive form (this may affect the accuracy of quantification results); and 5) the fact that a large number of samples are involved when performing a statistical analysis, thus requiring a high-throughput assay of eCBs and NAE. Many previous LC-MS methods of detecting eCBs and NAEs were developed by combining with various sample pretreatment strategies, such as solvent extraction[26, 27], solid phase extraction (SPE)[28-30], microSPE[31], microdialysis[32, 33], or on-line sample trapping[34], etc [24, 35]. Although enrichment and purification of eCBs and NAEs from biologic samples have been achieved using these methods, the acyl transmigration from 2-AG to 1-AG during the sample preparation procedure may lead to inaccurate results[36]. However, a LLE strategy utilizing toluene as the extractant has succeeded in purifying eCBs and NAEs, while preventing 2-AG isomerization[37]; but the requirement for improving MS sensitivity and throughput is still unfulfilled. Magnetic liquid microextraction (MLME) is a newly developed dispersive microextraction procedure that combines the superiority of magnetic solid phase extraction and dispersive liquid-liquid microextraction[38]. In this technique, the extraction medium (“magnetic extractant”) is easily formed by coating a thin layer of extraction solvent on magnetic nanoparticles through the physical adsorption that results from the high surface energy of magnetic nanoparticles. The “magnetic extractant” is then dispersed in the immiscible sampling solution to perform

extraction with the assistance of vortexing. After extraction, the “magnetic extractant” is readily collected by applying an external magnet, and analytes can be easily desorbed by adding a small amount of desorption solution. The MLME strategy is simple and high throughput, because the thin solvent layer on the magnetic core guarantees a fast mass transfer between sample solution and sorbents, and the magnetic retrieval property simplifies the operation. In addition, the selection of extractant is flexible, as long as sample solution is immiscible with the extractant. By selecting an appropriate extractant, the isomerization of 2-AG to 1-AG might possibly be prevented, thus assuring the accuracy of the quantification results. Moreover, several pioneers in this area have revealed the versatility and performance of MLME by successfully analyzing pesticides and 3-monochloropropane-1, 2-diol in edible oils, and polyaromatic hydrocarbons and triazine herbicides in water [39-42]. Nevertheless, its application in biologic samples has yet to be explored. To overcome the inherently poor MS response of eCBs and NAEs, chemical derivatization (CD) has become an effective approach. This technique improves the ionization efficiency of analytes by incorporating a MS-sensitive group into the structure through a chemical reaction; as ECBs and NAEs contain a hydroxyl group, thus providing a reaction moiety for further derivatization. In the present study, a method for the determination of eCBs and NAEs in serum and tissues was established based upon MLME-CD coupled with ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The MLME that involves toluene as an extractant assured high throughput, satisfactory purification

efficiency, and reliability of quantification results; while CD using 4-(N,Ndimethyamino)benzoyl chloride (4-DMABC) as labeling reagent improved the detection sensitivity more than 10-fold. We further assessed the eCB and NAE content in the serum of 49 infertile women and 53 fertile women (controls), and the follicular fluid from 21 infertile women and 20 healthy controls. Based upon the eCB and NAE levels obtained, we investigated the relationship between female infertility and endogenous eCBs and NAEs. 2. Experimental methods 2.1 Chemicals and reagents ECB and NAE standards, including anandamide (AEA, purity ≥98%), Npalmitoylethanolamine (PEA, purity ≥98%), N-oleoylethanolamine (OEA, purity ≥98%), 2-arachidonoylglycerol (2-AG, purity ≥95%, as a 9:1 mixture of the 2-AG and 1-AG); and stable isotope-labeled standards, including d4-AEA (purity ≥98%) and d5-2-AG (purity ≥95%, as a 9:1 mixture of the d5-2-AG and d5-1-AG), were purchased from Cayman Chemical Co. (Michigan, USA). Formic acid (FA, 88%), trichloromethane, ethyl acetate, ferric chloride hexahydrate, sodium acetate, ethylene glycol, ethylenediamine, and ethyl alcohol were all AR grade and purchased from Sinopharm Chemical Reagents (Shanghai, China). 4-DMABC (97 %) was purchased from J&K Scientific (Beijing, China), and acetonitrile (ACN, HPLC grade) and toluene (HPLC grade) were obtained from Merck KGaA (Darmstadt, Germany). Ultra-pure water used throughout the study was purified with a Milli-Q system (Milford, MA, USA).

2.2 Collection of serum and follicular fluid The serum from infertile women (n=49, mean [SD]; 8.9 [4.9] years of age, range 25–47 years) and controls (n=53; 32.2 [5.2] years of age; range, 22–45 years); and follicular fluid from infertile women (n=21; 40.3 [4.1] years of age; range, 29–47 years) and controls (n=20; 32.4 [4.5] years of age; range, 28–45 years) were collected from October 2015 to August 2016 at Zhongnan Hospital of Wuhan University. The study was performed in accordance with the approved guidelines of the ethics committee of Zhongnan Hospital of Wuhan University, and informed consent was obtained from each participant at the time of biofluid collection. 2.3 Synthesis of Fe3O4 magnetic nanoparticles The magnetic core—Fe3O4 magnetic nanoparticles were synthesized according to a previously reported method. Briefly, 5.0 g of FeCl3•6H2O was dissolved in 100 mL of EG. Then, 15.0 g NaAC and 50.0 mL ethylene glycol (ETH) were added to the solution. After stirring for 30 min, a homogeneous mixture was obtained and transferred to a 200 mL teflon-lined stainless-steel reaction kettle, which was heated to 200°C for 8 h. When the mixture was cooled down to room temperature, we separated the product from the solution with a magnet. Finally, the product underwent alternating washes with water and ethanol several times and vacuum-dried at 60°C for 6 h. 2.4 Magnetic liquid microextraction-chemical derivatization procedure

The MLME procedure and the chemical derivatization reaction are depicted in Fig. 1. Briefly, 20 mg of Fe3O4 nanoparticles were placed in a 15-mL vial, and 200 μL of toluene was added. After vortexing for 30 s, Fe3O4 nanoparticles were uniformly dispersed in toluene to form the magnetic toluene through physical adsorption. Then, 50.0 μL of serum or follicular fluid was mixed with internal standards (IS) d4-AEA (0.4 ng) and d5-2-AG (0.4 ng) for quantification, and diluted with 10.0 mL of water. The diluted sample was added to the 15-mL vial that contained magnetic toluene, and vortexed for 5 min to achieve efficient extraction. The supernatant was discarded by placing a Nd-Fe-B rare-earth permanent magnet on the bottom of the vial for several seconds to isolate the magnetic toluene. Subsequently, the remaining sample matrix that was adsorbed on magnetic toluene was washed with 2 mL of water and vortexed for 2 min. The eCBs and NAEs in the magnetic toluene were desorbed with 1 mL of acetone by vortexing for 3 min, and collected by placing a magnet to the bottom of the vial for several seconds. The desorption solution was evaporated to dryness under a mild stream of nitrogen gas, and the CD process was performed. The residue from the desorption solution was re-dissolved with 250 μL toluene that contained 4DMABC at 1 mg/mL. The reaction then took place under the catalysis of 3 mg of anhydrous K2CO3 for 2 h at 30°C. Afterwards, the supernatant was collected and evaporated to dryness by a mild stream of nitrogen gas. Finally, the sample was redissolved in ACN/water (7/3, v/v, 100μL), and 5 μL was injected for UPLC-MS/MS analysis. Importantly, the used magnetic nanoparticles could be reused after

alternating washes with water and ethanol several times to remove sample matrix, and the solution was vacuum-dried at 60°C for 6 h. 2.5 UPLC-MS/MS analysis of eCBs and NAEs Analyses of the eCBs and NAEs were performed on a UPLC-ESI (+)-MS/MS system consisting of a Shimadzu 8040 triple-quadrupole mass spectrometer with an electrospray ionization source, a Shimadzu LC-30AD UPLC system with 2 30AD pumps, a SIL-30AC auto-sampler, a CTO-30A thermostat column compartment, and a DGU-20A5R degasser. Data acquisition and processing were performed using Lab Solutions 5.53 SP2 software. The UPLC separation was performed on a Waters Acquity UPLC BEH C18 column (100.0 mm × 2.1 mm, i.d.; 1.7 μm) at 40°C. Mobile phases A and B were 0.1% formic acid in water and acetonitrile, respectively, and the flow rate was set at 0.3 mL/min. The gradient program of 0-5 min at 30% for B, 5-20 min from 30% B to 70% B, 20-25 min at 5% B, and 25-30 min at 30% B was used to separate the eCBs from NAEs. All eCB and NAE derivatives and internal standard derivatives were analyzed by multiple reaction monitoring (MRM) in the positive mode. A mixture containing eCB and NAE derivatives at 1.0 μg/mL was employed to optimize the MRM and ionization source parameters. The optimal conditions for the ionization source were as follows: DL temperature, 250°C; heat block temperature, 400°C; nebulizing gas,

3L/min; and drying gas, 15L/min. MRM mass spectrometric parameters are summarized in Table S1. 2.6 Statistical analysis We performed all statistical analyses using SPSS 19.0 software (SPSS Inc.). The paired t test was performed to evaluate the differences in eCB and NAE concentrations in serum or follicular fluid between infertile women and healthy controls. All P values were 2-sided, and, generally, P values <0.05 were considered to have statistical significance. A receiver-operating characteristic (ROC) analysis was performed to evaluate the ability of eCBs and NAEs in discriminating infertile women from healthy controls. 3. Results and discussion 3.1 Optimization of MLME conditions ECBs and NAEs exist in serum and follicular fluid at very low concentrations. Therefore, a pre-purification and enrichment process is the prerequisite to assuring the detection of these endogenous molecules. However, 2-AG, an important eCB, may spontaneously turn into 1-AG during conventional sample preparation processes, which can engender inaccuracies in detection results. The majority of the reported methods are presently time-consuming, not suitable for large-sample screening, and, yet, the introduction of an appropriate sample preparation system is essential. MLME has already been proven to be high throughput, and the flexibility of the technique in selecting extractants is beneficial for maintaining the stability of 2-AG. We therefore

determined the feasibility of MLME in the current assay. To maximize the performance of MLME, several parameters affecting extraction efficiencies, throughput, and stability of 2-AG were investigated. The type of extractant is the core element of MLME, as it exerts crucial influences on both the extraction efficiency and the accuracy. Therefore, a careful consideration of the type of extractant should be made before optimizing the MLME conditions. The eCBs and NAEs are hydrophobic compounds, and can be effectively extracted from aqueous serum or follicular fluid samples into organic phases. From this point of view, any water-immiscible solvent, such as n-hexane, chloroform, ethyl acetate, toluene, etc., is a workable solvent. We compared the extraction efficiency of these solvents as magnetic extractants and the stability of 2-AG upon using these solvents as extractants. As shown in Fig. 2A, magnetic toluene exhibited the highest extraction efficiencies toward eCBs and NAEs among all the sorbents investigated because toluene is the most hydrophobic, with a log P at 2.69; and is also polar due to its asymmetrical structure. According to the theory of “like dissolves like,” eCBs and NAEs (hydrophobic compounds with polar moieties), should have a greater solubility in toluene than in other solvents. Moreover, a mixture containing 2-AG and 1-AG at a molar ratio of 4/1 was extracted with magnetic toluene, ethyl acetate, n-hexane, and chloroform, respectively; and the peak area ratios of 2-AG/1-AG in the desorption solutions were compared with a standard solution of a 2-AG/1-AG mixture to investigate the stability of 2-AG in these extraction systems. As shown in Fig. 2B, the peak area of 2-AG/1-AG obtained from magnetic toluene was identical to that of the

standard solution, while it was decreased in the magnetic n-hexane and increased in both the magnetic chloroform and ethyl acetate. The results indicated that 2-AG may isomerize to 1-AG with magnetic n-hexane as sorbent, and 1-AG be converted to 2AG in chloroform and ethyl acetate. This phenomenon was similar to that in a previously reported study [37], but the underlying mechanism(s) is yet to be elucidated. The extraction using magnetic toluene as sorbent was then able to sustain the stability of 2-AG. Based on both the extraction efficiency and accuracy, magnetic toluene was selected as the extraction medium for the subsequent experiments. The MLME method follows the principle of microextraction, as a small volume of extractant is used. In the microextraction mode, the extraction efficiencies depend upon the volume of the extractant; the larger the extractant volume, the better the extraction efficiencies that can be achieved. In this case, toluene in the range of 0300.0 μL was coated on the magnetic core to extract the eCBs and NAEs in water, and the extraction performances were investigated. As shown in Fig. S1A, the extraction efficiencies of eCBs and NAEs increased when the extractant volume increased from 0 to 200.0 μL, and then efficiencies remained constant. We therefore chose 200.0 μL of toluene for the subsequent analysis. Magnetic core Fe3O4 was then responsible for retrieving toluene from the sampling solution. When the volume of extractant is determined, there should be an appropriate amount of Fe3O4 so as to realize the efficient retrieval of the extractant. To this end, the amount of Fe3O4 was investigated in the range of 10.0-100.0 mg. As shown in Fig. S1B, the recoveries of eCBs and NAEs increased with increasing Fe3O4

from 10.0 mg to 20.0 mg, and then values reached a plateau. It was expected that when the amount of Fe3O4 was less than 20.0 mg, a large proportion of toluene would be unable to be retrieved by Fe3O4. In our case, the extracted eCBs and NAEs in the toluene were lost along with unrecoverable toluene, resulting in decreased recoveries. Thus, 20.0 mg of Fe3O4 was selected for subsequent experiments. Under these conditions, 105 μL of toluene could be retrieved by 20 mg Fe3O4 after MLME, which was calculated to be 5.3 μL toluene per milligram Fe3O4 (experimental details are detailed in Supporting Information). NaCl was added to the sampling solution in the range of 0-150.0 mM to investigate the effect of ionic strength on extraction efficiencies. As shown in Fig. S1C, different tendencies were observed for different analytes. For 2-AG, the extraction efficiency increased concomitant with increasing NaCl concentrations. The reason for this might be ascribed to 2-AG being a neutral compound, and according to a salting-out effect on its solubility, it would positively correlate with ionic strength. For AEA, PEA and OEA, we observed an initial decrease followed by an increase in extraction efficiency. Due to the partial ionic properties of AEA, PEA and OEA, the salting-in effect was dominated at relatively lower salt concentrations (below 100.0 mM); and thus the extraction efficiency was suppressed by enhancing the solubility of analytes. When salt concentrations increased above 150.0 mM, the increase in extraction efficiency was attributed to a salting-out effect [43-45]. For the sake of simplicity, no salt was added to the sampling solution.

The sampling process was assisted by vortexing, and the extraction time profile in the range of 0.5-8.0 min was investigated. As shown in Fig. S1D, a slight increase in the recoveries of eCBs and NAEs was observed in the range of 0.5-5.0 min, and then values reached a plateau. Therefore, 5.0 min was chosen as the sampling time. Similarly, the desorption time was investigated, and 3.0 min was chosen as the desorption time based on the results shown in Fig. S1F. The optimal conditions for the MLME process were as follows: 200.0 μL of toluene was coated on 20 mg of magnetic core, and no salt was added to the sampling solution. The extraction and desorption times were 5.0 and 3.0 min, respectively. Under these conditions, the isomerization of 1-AG to 2-AG was prevented, and thus the reliability of the quantification results could be ensured. The entire sample preparation time was below 20 min, which was considered to be high throughput. 3.2 Optimization of the derivatization reaction The eCBs and NAEs showed poor detection sensitivity using MS for 2 reasons: 1) they lacked ionization groups, and thus their ionization efficiencies were relatively low; and 2) because their structures are rigid, they achieved a poor fragmentation pattern in the collision-induced dissociation (CID) cell. For example, 2-AG was broken into small fragments with similar intensity, and no main fragment could be observed (Fig. 3A). Through chemical derivatization (CD), an ionization group could then be incorporated into the analytes, and the fragmentation pattern could potentially be improved, which might eventually improve the MS sensitivity. Fortunately, eCBs and NAEs all possess a hydroxyl group in their structures, which provides a suitable

reaction moiety for chemical derivatization. Considering that acyl chloride can efficiently react with a hydroxyl group, and the MS detection sensitivity for eCBs and NAEs could be improved after labeling an ionizable group tertiary amine into the structure, 4-DMABC (which structurally contains an acyl chloride group and a tertiary amine) was selected as the derivatization reagent. The derivatization conditions, including the reaction solvent, amount of 4-DMABC used, reaction time, and reaction temperature, were all optimized. The solvent environment may influence both the derivatization efficiency and the stability of 2-AG. Thus, the derivatization reaction was performed in acetonitrile, toluene, acetone, and n-hexane, respectively; and the derivatization efficiencies were compared. As shown in Fig. S2A, the highest peak area ratios of analytes/IS were obtained using toluene as the reaction solvent, indicating the highest reaction efficiency in toluene. Moreover, toluene carries advantages in preventing 2-AG from isomerizing into 1-AG. Therefore, toluene was selected as the reaction solvent in the subsequent experiment. An excess of derivatization reagents is commonly required to ensure high yields of the derivatization reaction. In addition, serum or follicular fluid might contain other hydroxyl compounds that may consume extra derivatization reagent as well. Therefore, to obtain the highest sensitivity possible, eCBs that were spiked in serum at 10.0 ng/mL were purified with MLME, and then reacted with 4-DMABC in the range of 12.5-300.0 μg. The effect of the amount of 4-DMABC on reaction efficiencies was then investigated. As shown in Fig.S2B, the peak areas ratios for

eCB/IS increased with 4-DMABC in the range of 12.5-200.0 μg, and then reached a plateau. This indicated that 200.0 μg 4-DMABC would provide the highest derivatization efficiencies in the sample matrix, and we therefore used 200.0 μg of 4DMABC for subsequent experiments. We subsequently investigated the effect of temperature on the derivatization efficiencies by performing reactions at 4°C, 30°C, 40°C, 50°C, and 60°C, respectively. As shown in Fig. S3C, the highest peak area ratio of eCBs and NAEs was obtained at 30°C. We concluded that the esterification reaction between analytes and 4-DMABC was exothermic, and thus could be promoted under a relatively low temperature; whereas a lower temperature (4°C) might slow the reaction. Therefore, the derivatization reaction was performed at 30°C. Moreover, the investigation of the reaction time profile was conducted in the range of 10.0 min-3.0 h. As shown in Figure S4D, the peak area ratios of eCB/IS increased until 2 h, and then remained constant. Therefore, 2 h was chosen as the reaction time. Through the derivatization using 4-DMABC, eCBs and NAEs were labeled with a tertiary amine, which was beneficial for improving the ionization efficiencies; and the fragment pattern of eCB derivatives was also investigated. Fig. 3B shows the product ion mass spectrum for 4-DMABC-labeled 2-AG, and 2 dominant fragment ions at m/z of 148.1 and 166.1 can be easily discerned. These results implied that the fragmentation mainly occurred between the ester bond of 2-AG and 4-DMABC. Since

the fine fragmentation pattern of eCBs and NAEs may further promote the improvement of MS detection sensitivity, in order to demonstrate the superiority of CD, we compared the detection sensitivity of eCBs and NAEs before and after derivatization, both in the standard solution and sample matrix under the optimal conditions. As shown in Table 1, the limits of detection of 4 analytes were improved by 5.0-209-fold in standard solution and by 6.4-307.0-fold in serum. This indicated that 4-DMABC labeling can greatly improve the detection sensitivity of eCBs and NAEs. 3.3 Evaluation of matrix effect Apart from the inherent properties of the target analyte, the sample matrix also has a negative influence on MS detection sensitivity. ECBs and NAEs co-exist with various matrix interferents in serum and follicular fluid, and a suppression of ionization efficiency might occur if they co-eluted with eCBs and NAEs. The purpose of the MLME-CD process was to elimilate or reduce the effect of these matrices to subsequent detection, and thus their performance in urine, serum, and follicular fluid were evaluated by matrix effect. To this end, 2 biologic samples were treated with the MLME-CD method, and one of the samples was spiked with eCB and NAE derivatives at 10 ng/mL after sample pretreatment. Both of the real samples and the eCB and NAE standard derivatives (10 ng/mL) were analyzed by UPLC-MS/MS. The matrix effect was calculated by substracting the peak areas of endogenous eCBs or NAEs in blank biologic samples from the spiked real sample, and then compared to those in the standard sample. As shown in Table 2, the matrix effects in the 2 biologic

samples (serum and follicular fluid) were in the range of 84.2-129.8%, which indicated that after sample preparation, the signals for eCB and NAE derivatives in biologic samples were similar to those in standard solution, and the matrix interferents had been mostly removed. 3.4 Method validation We evaluated the linearity of the method by analyzing solutions containing the eCB standard in the range of 0.17-615.0 nM, with d4-AEA at a fixed concentration of 0.4 ng and d5-2-AG at 4 ng. As there were only 2 available IS, 4 analytes shared the 2. d4-AEA was employed to quantify AEA, PEA, and OEA, while d5-2-AG was used to quanfiy 2-AG. The calibration curves were constructed by plotting the peak area ratios of analyte/IS against eCB concentrations. As listed in Table 3, good linear correlations were obtained for 4 analytes with correlation coefficients (R) larger than 0.9965. The limits of detection (LODs) and limits of quantification (LOQs), calculated as the amounts of the analytes at a signal-to-noise ratio (S/N) of 3 and 10, respectively, ranged from 3.0-54.3 fmol and 10.0–181.1 fmol, respectively. The reproducbility of the method was evaluated by calculating inter- and intraday precision. Both inter- and intra-day relative standard deviations (RSDs) were calculated from serum samples (50 μL) spiked at 3 different concentrations. Five samples treated with the proposed method over 1 day provided the intra-day RSDs, and samples that were independently prepared for a continuous 3 days provided interday precision. As shown in Table 4, acceptable precision was obtained with RSD values below 14.5%, indicating good reproducibility of the proposed method.

The reliability of the method was assessed by recoveries, which were obtained by analyzing serum samples spiked at concentrations of 5 ng/mL, 20 ng/mL, or 50 ng/mL. First, the endogenous eCBs and NAEs in blank serum and the total eCBs and NAEs in spiked-serum samples were calculated from the calibration curves; and the spiked amounts of analytes in serum were then calculated by substracting the endogenous amounts from the total amounts. Finally, the recovery was obtained by comparing the calculated spiked amounts of eCB or NAE to the actual spiked amounts. As shown in Table 4, the relative recoveries were in the range of 87.4%– 117.9%, demonstrating that the accuracy of the proposed method was acceptable. In addition, we evaluated the feasibility of the method in terms of the quantification of endogenous eCBs and NAEs in serum and follicular fluid samples, and found that all target analytes could be successfully detected with good MS signals in 50 μL of serum or follicular fluid. Fig. S3 displays the MRM chromatograms of the detected endogenous eCBs and NAEs in the 2 samples, and their contents are summarized in Table S2. The results demonstrated the utility of the method in analyzing endogenous eCBs and NAEs in different sample matrices, and that it could be employed as a reliable tool for assessing the levels of eCBs and NAEs in biofluids from both reproductively fertile and infertile women. 3.5 ECB contents in biofluids correlate with female infertility Female infertility has affected many couples attempting to concieve. Early diagnosis of the condition might provide a time window for earlier implementation of treatment strategies to manage fertility problems. In the present case, the exploration

of potential biomarkers of female infertility is of paramount importance. There is evidence that alterations in the eCB system are associated with infertility, and that any enhancements to the eCB system are benefical in promoting pregnancy[18]. Thus, eCBs and NAEs have been proposed by several investigators as potential biomarkers of female infertility [20-23]. However, a role for eCBs or NAEs as biomarkers has not been fully confirmed for several reasons: 1) the low detection sensitivity and the instability of eCBs and NAEs have resulted in difficulties in quantifying these molecules; and 2) we have read no report in the literature of a large statistical survey of eCB and NAE contents in the biofluids of infertile women and controls to verify such a hypothesis. We have developed a MLME-CD-UPLC-MS/MS method for the determination of eCBs and NAEs in peripheral serum and ovarian follicular fluids. The MLME process accomplishes the extraction and purification of eCBs and NAEs from complicated biologic sample matrices within 20 min, which constitutes a highthroughput methodology. More importantly, it could efficiently prevent 2-AG from isomerizing to 1-AG, ensuring the accuracy of the method. Subsequent CD has been shown to improve MS detection sensitivity by improving both the ionization efficiencies and the fragmentation partterns of eCBs and NAEs, and this method could allow for the detection of endogenous eCBs and NAEs in as little as 50 μL. With a reliable, sensitive, and high-throughput method as the analytical tool, quantification reliability can be realized, and a statistical survey using a large number of samples can be completed within a short time. In our study, we employed this

method to quantify eCBs and NAEs in the serum of 49 infertile women and 53 healthy controls, as well as in the follicular fluid of 21 infertile women and 20 healthy controls; and analyzed the data obtained by statistically appropriate methods. The eCB and NAE contents in the serum of 49 infertile women and 53 healthy controls are summarized in Table S3. The means (standard deviation [SD]) for 2-AG in the serum of all infertile women and healthy controls were 22.13 nM (13.11 nM) and 18.35 nM (8.76 nM), respectively (Fig. 5B); and the means (SD) for OEA in the serum of all infertile women and healthy controls were 10.39 nM (5.25 nM) and 9.37 nM (5.22 nM), respectively (Fig. 5D). The results in Fig. 7B and 7D showed that there was no significant difference in 2-AG or OEA in serum between infertile women and healthy controls, with P values at 0.087 and 0.330, respectively. Conversely, the means (SD) for AEA in the serum of all infertile women and healthy controls were 2.68 nM (1.34 nM) and 2.09 nM (0.72 nM), respectively (Fig. 5A), which was significantly different based upon an unpaired t test analysis (P=0.005). Mean (SD) for PEA in the serum of infertile women was also significanlty higher than controls (22.17 nM [8.19 nM] vs. 19.25 nM [5.53 nM], respectively; P=0.036, unpaired t test) (Fig. 5C). It is well known that high BMI and age are also risk factors for causing female infertility. Therefore, we performed a statistical analysis to evaluate the correlation of 4 eCBs in the serum of infertile women and healthy controls with respect to BMI and age. According to the Pearson correlation coefficient, there was no correlation between any of the the 4 eCB contents in serum and BMI or age. The detailed data are

summarized in Table S4. The results indicated that factors such as BMI and age exerted only minor influences on eCB and NAE content in serum. Based on the differences in AEA and PEA contents in infertile women and healthy controls, we further evaluated the possibility of using AEA and PEA in serum as biomarkers for the early detection and prognosis of female infertility by performing receiver operating characteristic (ROC) analysis. As shown in Fig. 5E-F, AEA and PEA contents in serum were of only limited value in the detection of female infertility, with the areas under the curve (AUC) being 0.637 and 0.605, respectively. We also analyzed the eCB and NAE contents in ovarian follicular fluid of 21 infertile women and 20 healthy controls, and the quantification results are summarized in Table S5. The mean concentrations of AEA in the follicular fluid of the infertile women and healthy controls were 1.50 nM (0.47 nM) and 1.77 nM (0.38 nM), respectively (Fig. 6A); and the means for 2-AG in infertile women and healthy controls were 17.96 nM (8.32 nM) and 24.13 nM (11.66 nM), respectively (Fig. 6B). The mean contents of PEA in the follicular fluid from infertile women and healthy controls were 6.37 nM (2.64 nM) and 7.51 nM (2.36 nM), respectively (Fig. 6C). The results in Fig. 6A-C suggested that there were no significant differences in AEA, 2AG, or PEA in follicular fluid between infertile women and fertile controls, with P values of 0.055, 0.057 and 0.153, respectively; whereas the OEA content in follicular fluid was significantly lower in infertile women than in healthy controls (5.16 nM [1.13 nM] vs. 6.11 nM [1.43 nM], respectively; P=0.023, unpaired t test) (Fig. 6D).

Similarly, eCBs in follicular fluid from infertile women and healthy controls-with respect to BMI and age--were also investigated using the Pearson correlation coefficient. The result in Table 5 indicated that there was no correlation between the eCB and NAE contents in follicular fluid and BMI or age. The possibility of using OEA in follicular fluid as a biomarker for the early detection and prognosis of female infertility was further evaluated by performing ROC analysis. As shown in Fig. 6E, OEA content in serum showed a modest value for the detection of female infertility, with the AUC being 0.707. To summarize, the statistical results indicated that there was a correlation between eCB and NAE levels and female infertility, which was consistent with previously published reports. However, ROC analysis revealed that AEA and PEA in serum and OEA in follicular fluid were of only weak or modest value in the diagnosis of female infertility. Therefore, eCBs and NAEs in serum or follicular fluids cannot serve as suitable biomarkers of female infertility. However, their concentrations might be considered indicators of altered female fertility.

4. Conclusions In the present study, we developed a MLME-CD method for the determination of eCBs and NAEs in biofluids using UPLC-MS/MS. MLME employing magnetic toluene as sorbents ensured high throughput and stability of eCBs and NAEs, and CD using 4-DMABC as labeling reagent improved MS sensitivity. This method was further employed as an analytical tool to investigate the relationship between eCBs

and female infertility. Statistical analysis of eCB and NAE concentrations in 102 serum samples and 41 follicular fluid samples confirmed an increase in AEA and PEA content in serum, and we observed a reduction in OEA content in follicular fluid from infertile women compared to healthy controls. Our evidence based on ROC analysis suggests that eCBs and NAEs should not be considered as biomarkers of female infertility due to inadequate AUC values. It is therefore necessary to uncover other potential biomarkers for the prediction of reproductive potential in women. Acknowledgments The authors wish to acknowledge financial support from “the Hundred Talents Project” awarded by the Chinese Academy of Sciences (29Y429291a0129), the SinoAfrica joint research project (SAJC20160233), the National Natural Science Foundation of China (21605117), and the Fundamental Research Funds for the Central Universities (2042016kf0035).

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Figure Captions Fig. 1. Schematic diagram of MLME (A), and the derivatization reaction between eCB/NAE and 4-DMABC (B). Fig. 2. Effect of the type of magnetic extractants on the recoveries of eCBs and NAEs (A), and the stability of 2-AG (B). AEA, PEA and OEA were spiked in sampling solutions at 0.1 ng/mL, and 2-AG was spiked at 20 ng/mL. Fig. 3. Product ion scan spectrograms and the fragmentation ions of 2-AG (A) and the 2-AG-4-DMABC derivative (B). Fig. 4. MRM chromatogram of 4 eCB and NAE derivatives. Peaks: 1, AEA; 2, PEA; 3, 2-AG; 4, OEA. Fig. 5. Quantification and statistical analysis of eCB and NAE concentrations in the serum of infertile women and healthy controls. AEA content in the serum of infertile women and healthy controls, (A); 2-AG content in the serum of infertile women and healthy controls, (B); PEA content in the serum of infertile women and healthy controls, (C); OEA content in the serum of infertile women and healthy controls, (D); ROC curve for serum AEA scores of female infertility, (E); and ROC curve for serum PEA scores for female infertility, (F). Fig. 6. Quantification and statistical analysis of eCB and NAE concentrations in ovarian follicular fluid from infertile women and healthy controls. AEA content in follicular fluid from infertile women and healthy controls, (A); 2-AG content in follicular fluid from infertile women and healthy controls, (B); PEA content in

follicular fluid from infertile women and healthy controls, (C); OEA content in follicular fluid from infertile women and healthy controls, (D); and ROC curve for OEA scores of female infertility (E). Table 1. Improvement in sensitivity of eCB and NAE assay after derivatization with 4-DMABC in both standard solution and serum. Analytes AEA PEA OEA 2-AG

Sensitivity improvements In standard solution In serum 9.9 12.9 5.0 6.4 5.5 6.8 209.0 307.0

Table 2. Matrix effect of eCBs and NAEs in serum and ovarian follicular biofluid. Matrix effect Analyte In serum (%)

In follicular fluid (%)

AEA

94.8

110.6

PEA

129.6

124.0

OEA

114.3

126.1

2-AG

115.2

129.8

1

Table 3. Calibration curves, LODs, and LOQ data of 4 eCBs and NAEs. Linear range

Regression data

LOD

LOQ

Analytes (nM)

2 3

Slope (error)

Intercept (error)

R-value

(fmol)

(fmol)

AEA

0.3-288.0

0.0646 (0.0005) -0.0595 (0.0591)

0.9996

8.4

27.9

PEA

0.2-234.0

0.0814 (0.0004)

0.0892 (0.0290)

0.9999

3.0

10.0

OEA

0.3-615.0

0.0785 (0.0020) -0.3856 (0.4064)

0.9965

7.6

25.2

2-AG

1.8-185.0

0.0575 (0.0012) -0.1206 (0.0972)

0.9984

54.3

181.1

Table 4. Precision (intra- and inter-day) and recoveries of 4 eCBs and NAEs in serum at 3 different concentrations. Intra-day precision

Inter-day precision

(RSD, %, n= 5)

(RSD, %, n=3)

Recovery (%, n=5) Analyte

Lowa Mediumb Highc

a

Medium

High

Low

Medium

High

AEA

5.5

7.1

4.9

6.9

4.5

3.5

87.4

96.9

111.0

PEA

14.5

8.6

10.3

12.8

5.3

1.1

98.9

103.6

116.2

OEA

7.5

8.8

3.0

7.6

6.3

2.7

117.9

100.5

105.9

2-AG

10.6

14.3

12.1

6.9

12.3

10.3

103.9

92.3

104.6

For the low concentrations, AEA, PEA, OEA, and 2-AG were spiked with 14.4, 16.7, 15.4, and 13.2 nM in 50 μL serum, respectively.

b

c

Low

For the medium concentrations, AEA, PEA, OEA, and 2-AG were spiked with 57.6, 66.8, 61.5 and 52.9 nM in 50 μL serum, respectively.

For the high concentrations, AEA, PEA, OEA, and 2-AG were spiked with 144.0, 167.1, 153.7 and 132.2 nM in 50 μL serum, respectively.

Figr-1

Figr-2

Figr-3

Figr-4

Figr-5

Figr-6