A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry

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A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry Zhen-peng Kai a , Yue Yin a,b , Zhi-ruo Zhang c , Juan Huang d,e , Stephen S. Tobe d , Shan-shan Chen b,∗ a

School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai, 201418, PR China Institute of Agro-Food Standards and Testing Technologies, Shanghai Academy of Agricultural Science, Shanghai, 201403, PR China c School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, 201418, PR China d Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, ON, M5S 3G5, Canada e Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Beijing, 100101, PR China b

a r t i c l e

i n f o

Article history: Received 30 July 2017 Received in revised form 12 January 2018 Accepted 15 January 2018 Available online xxx Keywords: Juvenile hormone GC–MS/MS JH biosynthesis Inhibitory mechanisms Diploptera punctata

a b s t r a c t A method for rapid quantitation of insect juvenile hormones (JH) and intermediates in the biosynthetic pathway, both in vitro and in vivo (hemolymph and whole body), has been developed using GC–MS/MS. This method is as simple as the radiochemical assay (RCA), the most commonly used method for measurement of JH biosynthesis in vitro, without need for further purification and derivatization, or radioactive precursors or ligands. It shows high sensitivity, accuracy and reproducibility. Linear responses were obtained the range of 1–800 ng/mL (approximately 4–3000 nM). Recovery efficiencies for farnesol, farnesal, methyl farnesoate and JH III were approximately 100% in vitro and over 90% in vivo, with excellent reproducibility at three different spike levels. Titer of JH III in the hemolymph was relatively low at day 0 (adult female emergence) (79.68 ± 5.03 ng/mL) but increased to a maximum of 1717 ng/mL five days later. In whole body, JH III quantity reached a maximum on day 4 (845.5 ± 87.9 ng/g) and day 5 (679.7 ± 164.6 ng/g) and declined rapidly thereafter. It is in agreement with the hemolymph titer changes and biosynthetic rate of JH in vitro. Comparison with the results of inhibition of JH biosynthesis by two known inhibitors (allatostatin (AST) mimic H17 and pitavastatin) using RCA and GC–MS/MS, showed that there was little difference between the two methods In contrast to other methods, the present method with GC–MS/MS can be used to elucidate the mechanism of inhibition by inhibitors of JH biosynthesis without any derivatization and purification. This method is applicable to screening of JH inhibitors and the study of inhibitory mechanisms with high sensitivity and accurate quantification. It may also be useful for the determination of JH titer in other Arthropods. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Juvenile hormones (JHs) are acyclic sesquiterpenoids that play a central role in insect life. They are synthesized and released by the paired corpora allata (CA). [1]. There are 11 JH homologues known in insects and the structures have been elucidated and confirmed [2]. These JH homologues were identified in different insect species and show different actions and activity. The most common

∗ Corresponding author. E-mail address: [email protected] (S.-s. Chen).

JH in insects is JH III [3]. The biosynthesis of JH III is divided into two biosynthetic pathways: the mevalonate (MVA) pathway and the JH specific pathway. The MVA pathway is responsible for the conversion of acetyl-CoA to farnesyl pyrophosphate (FPP) [4], and the JH specific pathway (Fig. 1) involves the farnesyl diphosphate hydrolysis to farnesol, oxidation to farnesal and then farnesoic acid, followed by a methyl transfer and epoxidation [5]. In some species, particularly the Lepidoptera, epoxidation often precedes methylation [6]. Because of its instability, physicochemical characteristics and low concentration in insects, the quantification of JHs has proven difficult. Four methods have been utilized to quantify JH: 1) Radio-

https://doi.org/10.1016/j.chroma.2018.01.030 0021-9673/© 2018 Elsevier B.V. All rights reserved.

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the presence of a high sample matrix background. GC–MS/MS has quickly become established as the technology of choice for bioanalytical applications. At present, it is widely used in environmental analysis, and in the determination of pesticide residues. Quantitative assays for JHs, JH degradation products and farnesoic acid have been reported [18]. In this paper, we report the development of a quantitative assay for JHs and their biosynthetic pathway, using GC–MS/MS. Using both GC–MS/MS and RCA, we have compared the results of JH biosynthesis in vitro and in vivo. 2. Materials and methods 2.1. Animals Diploptera punctata, kept at 27 ± 0.5 ◦ C and relative humidity 50 ± 2% were fed with standard lab cockroach food and water. Day 0 (newly emerged) mated female D. punctata were isolated, placed in containers with food and water.

Fig. 1. Scheme of the JH specific biosynthetic pathway. Adapted from Belles et al. [4].

chemical Assay (RCA), 2) Chromatographic methods, including gas chromatography coupled with mass spectrometry (GC–MS), high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC–MS/MS) and HPLC coupled to fluorescence detection (HPLC-FD), 3) Radioimmunoassay (RIA), 4) JH Binding Protein Assay (JHBP). Radiochemical Assay (RCA) is the most commonly used method for determination of JH biosynthesis in vitro. It does not measure JH concentration, but rather measures the rate of incorporation of the methyl moiety from [3 H]- or [14 C]-methyl methionine into JH by freshly dissected CA in vitro [7]. The use of RCA is confined to assays in vitro and must be accompanied with a blank control. Radioimmunoassays (RIA) have high accuracy, but the crossreactivity of antibodies against other JHs or precursors makes it inappropriate in the presence of JH homologues [8]. JH Binding Protein Assay (JHBP), using binding protein to replace antiserum, has similar precision to the RIA [9]. Moreover, the procedure for this assay can be more straight-forward than RIA, but both RIA and the JHBP assay lack universal applicability [10]. With the development of physicochemical methods, more modern analytical techniques have been used to quantify JH [11–13]. Each method has advantages and disadvantages. In addition to the above methods, HPLC-FD is a sensitive method for the detection of picomolar or femtomolar concentrations of JH [14]. However, it does not directly measure the JH concentration, because JH lacks natural fluorescence. Therefore, derivatization employing fluorescent tags is essential using this approach. However, timeconsuming derivatization and the expense of fluorescent labeling reagents limit the applicability of this method [15]. To date, chromatography coupled to MS is increasingly used for of JH analysis because it provides unequivocal identification and quantification [16]. Until 1955, mass spectrometry (MS) was most commonly used for the direct analysis of volatiles. Gas chromatography (GC) was coupled to MS for the first time with the aim of expanding the analytical capabilities of MS to cover complex mixtures of unknowns [16]. Subsequent technological developments involved the introduction of hybrid mass analyzers such as the triple stage quadrupole (TSQ) mass spectrometers and the use of tandem MS (MS/MS) as a high specificity technique for routine quantitative analysis of complex mixtures [17]. The TSQ mass spectrometers can sometimes be used to quantify low levels of target compounds in

2.2. Reagents and chemicals JH III was purchased from Toronto Research Chemicals (Toronto, ON, Canada). (E, E) Farnesoic acid and methyl farnesoate were obtained from Echelon Biosciences (Salt Lake City, UT). Farnesol, farnesal, citronellol, geranylgeraniol, clotrimazole, pitavastatin, HPLC-grade n-hexane, isooctane and acetonitrile were from SigmaAldrich (St. Louis, MO). Allatostatin mimic H17 was a gift from Dr. Xin-ling Yang, (China Agricultural University, Beijing, China) and was synthesized from Rink Amide-AM resin using the standard Fmoc/tBu chemistry and HBTU/HOBt protocol [19]. Stock standard solutions of citronellol, JH III and its biosynthetic precursors were prepared in hexane and stored at −20 ◦ C (1000 mg/L). The working multi-standard solutions and internal standard solution at the appropriate concentrations were prepared daily by dilution with hexane. 2.3. GC–MS/MS analysis The GC–MS/MS system consisted of a Thermo Trace 1300 series GC coupled with an AI/AS 1310 autosampler and a TSQ 8000 triple-quadrupole mass spectrometer. The system was controlled by TraceFinder software, version 3.1 for data acquisition and processing. (a) GC Parameters. Citronellol, JH III and its biosynthetic precursors were separated with Agilent HP-5 MS UI capillary columns (0.25 mm i.d. × 30 m, 0.25 ␮m film thickness). Helium (purity = 99.999%) was used as the carrier gas with a constant flow rate 1.2 mL/min. Inlet temperature was 230 ◦ C, 2.0 ␮L pulsed splitless injection volume with the purge flow rate at 50 mL/min for 1 min. The column oven was initially held at 60 ◦ C for 1 min, then increased to 160 ◦ C at a rate of 25 ◦ C/min, finally by a 12 ◦ C/min ramp to 280 ◦ C, the total analysis time was 15 min. (b) Triple-quadrupole MS Parameters: The temperature of transfer line and ion source were held at 280 ◦ C. The mass spectrometer was working in EI mode (70 eV), and the filament current was 50 ␮A. Electron multiplier voltage (EMV) was gained by automatic MS/MS tuning, and argon gas were used as the collision gas for default instrument settings in the collision cell. The optimal quantitation and confirmation transitions from parent ions to daughter ions and collision energy for SRM (selectedreaction monitoring) of each compound were achieved with Auto-SRM study tests furnished by the software.

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Table 1 Selected-reaction Monitoring (SRM) Program used for the Analysis of JH III, Intermediates, Internal Standard and Biosynthetic Inhibitors. Analyte

Citronellol Farnesol Farnesal Methyl Farnesoate JH III Farnesoic acid Geranylgeraniol a b

Rta (min)

5.38 8.99 9.18 9.48 10.36 9.75 12.64

Confirmation 1

Quantification

Confirmation 2

Confirmation 3

Confirmation 4

Transition

CEb (eV)

Transition

CEb (eV)

Transition

CEb (eV)

Transition

CEb (eV)

81 → 79.1 93 → 77.1 84.1 → 83.1 114.1 → 83.1 85.1 → 59.1 100.1 → 99.1 68.9 → 41

10 15 10 10 10 40 10

95.1 → 67.1 69 → 41.1 69.1 → 41.1 69.1 → 41.1 81 → 79.1 69.1 → 67.4 81 → 79.1

10 10 10 10 5 35 10

123.1 → 81.1 81 → 79.1 92.9 → 77.1 81 → 79.1 94.9 → 67.1 81 → 79.2 107 → 91.1

10 10 10 10 10 40 10

121 → 93.1 94.9 → 67.1 121.1 → 77.1 120.9 → 93 122.9 → 120

10 10 20 10 45

Transition

CEb (eV)

120.9 → 105.1

15

Retention time. Collision energy.

Quantitative analysis by GC–MS/MS was carried by TraceFinder software using an internal standard method. Identification of JH III and its biosynthetic precursors in samples was confirmed by comparing the consistency of expected retention time and the quantitation and confirmation transitions with standards. The specific SRM transitions and other parameters for all the test JH compounds are given in Table 1. 2.4. Method validation Recovery and reproducibility studies were carried out to validate the accuracy and precision of the methodology with biological samples produced both in vitro and in vivo (from hemolymph). CA from day 7 mated females were incubated at 30 ◦ C in 100 ␮L of M199 for three hours. Following incubation, this biological sample (CA in medium) was spiked before extraction with known amounts of farnesol, farnesal, methyl farnesoate and JH III (0, 2, 10, 80 ng of farnesal, methyl farnesoate or JH III; 0, 4, 20, 160 ng of farnesol) as well as 20 ng of citronellol as an internal standard. This medium was then extracted with 200 ␮L n-hexane. A volume of 50 ␮L hemolymph was collected in a glass centrifuge tube containing 50 ␮L acetonitrile and 50 ␮L 0.9% sodium chloride solution. The tube was agitated on a Vortex mixer for 5 min, and then known amounts of farnesol, farnesal, methyl farnesoate, JH III and 20 ng of citronellol as an internal standard were added as above. The mixture was mixed vigorously before extraction with 200 ␮L n-hexane. The quantitative assay for JH III and intermediates was determined by GC–MS/MS. We calculated per cent recovery by subtracting the endogenous amount of analyte (only JH III was found in CA incubation medium and hemolymph), divided by the spiked amount and multiplied by 100. The linearity of JH III and precursors was verified in the range of 1–800 ng/mL with nine calibration points (1, 5, 10, 20, 50, 100, 200, 400, 800 ng/mL) in triplicate. Intraday analysis assesses the reproducibility in one day using eight separately extracted samples at three spike amounts. Interday analysis was estimated by separately extracting and analyzing twenty four different samples on three different days at three spike amounts (0, 2, 10, or 80 ng of farnesal or methyl farnesoate or JH III; 0, 4, 20, or 160 ng of farnesol). The reproducibility was measured by means of relative standard deviation (RSD, %). 2.5. Bioassays for JH III and intermediates in vitro using GC–MS/MS For the JH biosynthetic assay in vitro, a pair of CA were incubated at 30 ◦ C for 3 h in 100 ␮L of M199 (GIBCO) with Hanks’ salts, 25 mM HEPES buffer (pH 7.2), l-glutamine, 2% (w/v) Ficoll and 1.3 mM CaCl2 in dark with shaking [20,21]. The internal standard citronellol (20 ng) was added to the medium. 200 ␮L n-hexane was mixed with the medium and centrifuged for 5 min. The upper layer organic

phase was transferred to new vials with microvolume inserts for subsequent analysis. The quantitative assays for JH III and intermediates were determined by GC–MS/MS as above. Allatostatin mimic H17, a farnesol dehydrogenase inhibitor, geranylgeraniol [22] and the general cytochrome P450 inhibitor, clotrimazole (inhibits the epoxidation of methyl farnesoate in JH III biosynthesis) [23], were dissolved in M199 for the detemination of JH biosynthesis, respectively. Relative JH biosynthesis inhibition was determined as the percentage activity compared to the control. 2.6. JH biosynthesis assays in vitro using RCA The radiochemical assay in vitro for JH biosynthesis was used to determine rates of biosynthesis [20,21]. CA were incubated in M199 (1.3 mM CaCl2 , plus Ficoll (20 g/L), methionine-free; 100 ␮L) containing l [14 C-S-methyl] methionine (Amersham) for 3 h. Samples were extracted with isooctane and measured using liquid scintillation spectrometry. 2.7. Quantitative assays of JH III and intermediates in hemolymph and whole body using GC–MS/MS D. punctata were anaesthetized by chilling for 15 min on crushed ice. The hemolymph was collected in a microsyringe after clipping the femur. A volume of 50 ␮L hemolymph from 3 to 4 animals was immediately transferred to a 500 ␮L glass centrifuge tube containing 50 ␮L acetonitrile, 50 ␮L 0.9% (w/v) sodium chloride solution and 20 ng of citronellol as an internal standard [24]. The sample was extracted twice with 100 ␮L hexane following vigorous vortexing and centrifuged at 2500 × g for 5 min. The hexane phase (upper layer) was removed and transferred to a new glass vial. This protocol is described in Supplemental Fig. S1. The content of JH III and intermediates was determined by GC–MS/MS. Five insects, washed with ddH2 O, were placed in stainless steel grinding jars with balls of Restch MM400. The grinding jars were chilled in liquid nitrogen for 30 min, and then homogenized with a frequency of 20 Hz for 0.5 min. The homogenate was immediately transferred to a 10 mL glass centrifuge tube containing 1 mL acetonitrile, 1 mL 0.9% (w/v) sodium chloride solution and 20 ng of citronellol as an internal standard, ultrasonicated for 1 min, and then vortexed and extracted twice with 2 mL hexane (Fig. S2). Further processing was as described above. 2.8. Bioassays for JH III and intermediates in vivo using GC–MS/MS For the JH biosynthetic assay in vivo, injections of H17, pitavastatin, geranylgeraniol and clotrimazole were carried out using a microsyringe. The needle was inserted into the membrane between the coxa and femur of the metathoracic leg. Control groups were similarly injected with water. The hemolymph was collected and

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Table 2 Recovery, Intraday and Interday Precision of Quantity of JH III and Intermediates in CA. Analyte

Spike amount (ng)

RSDa (%)

Average Recovery (%)

b

Farnesol

Farnesal

Methyl Farnesoate

JH III

4 20 160 2 10 80 2 10 80 2 10 80

101.28 99.71 99.95 99.44 100.45 103.37 106.62 102.31 100.65 91.70 99.43 97.50

c

Intraday (n = 8)

Interday (n = 24)

5.20 2.72 2.79 3.99 2.40 2.42 4.89 4.39 2.93 4.03 4.82 4.36

7.92 5.87 3.67 10.08 5.88 3.01 9.51 6.27 5.67 9.98 7.81 8.56

LODd (ng/mL)

LOQe (ng/mL)

10

20

1

10

1

10

1

10

A biological sample comprising CA was spiked before extraction with known quantities of farnesol, farnesal, methyl farnesoate and JH III. Recovery was calculated by subtracting the endogenous quantity of analyte (only JH III was found in CA incubation medium), divided by the amount spiked and multiplying by 100. a Relative standard deviation. b Eight separately extracted samples in one day. c Twenty-four separately extracted samples in three days. d Limit of detection. e Limit of quantification.

Table 3 Recovery, Intraday and Interday Precision of Quantity of JH III and Intermediates in hemolymph. Analyte

Farnesol

Farnesal

Methyl Farnesoate

JH III

Spike amount (ng)

4 16 160 1 4 40 1 4 40 2 8 80

Average Recovery (%)

90.07 91.61 89.97 88.77 92.49 96.61 89.21 93.90 94.74 92.08 95.22 97.34

RSDa (%) Intradayb (n = 8)

Interdayc (n = 24)

4.54 5.16 2.26 6.55 5.46 3.75 4.52 6.86 5.17 4.35 5.84 4.89

6.60 5.31 3.77 7.51 6.09 4.45 6.65 7.94 5.75 5.51 7.49 5.46

LODd (ng/mL)

LOQe (ng/mL)

10

20

1

5

1

5

1

10

Hemolymph samples were spiked before extraction with known amounts of farnesol, farnesal, methyl farnesoate or JH III. The recovery was calculated by subtracting the endogenous amount of analyte (only JH III was found in hemolymph), divided by the amount spiked and multiplying by 100. a Relative standard deviation. b Eight separately extracted samples in one day. c Twenty four separately extracted samples in three days. d Limit of detection. e Limit of quantification.

measured as described using the quantitative assay of JH and intermediates in hemolymph. 3. Results and discussion 3.1. Recovery and reproducibility efficiency, limit of detection and limit of quantification for JH III and intermediates by the GC–MS/MS method The efficiency of JH III recovery was over 90% following incubation of CA in medium for 3 h and then spiked with JH III (0, 2, 10 and 80 ng). The recovery of farnesol, farnesal and methyl farnesoate was approximately 100% at three different spike levels in the assay in vitro (Table 2). For the assay of JH III and intermediates in hemolymph, the recovery of JH III, farnesol, farnesal and methyl farnesoate was approximately 90% at three different spike levels (Table 3). Linear curves were fitted by least-squares regression of concentration versus peak area ratio. Linearities for farnesol, from 10 to 800 ng/mL, or farnesal, methyl farnesoate and JH III, from 1 to 800 ng/mL, with R2 ≥ 0.99 are shown in Fig. 2. The limits of detec-

tion (LOD) for the assay in vitro were 1 ng/mL for farnesal, methyl farnesoate and JH III, 10 ng/mL for farnesol, and 10 ␮g/mL for farnesoic acid, respectively. The LOD for the assay using hemolymph were the same as the LOD for the assay in vitro (Table 3). The limits of quantification (LOQ) in vitro were 10 ng/mL for farnesal, methyl farnesoate and JH III, and 20 ng/mL for farnesol, respectively (Table 2). For hemolymph, the LOQ were 5 ng/mL for farnesal and methyl farnesoate, 10 ng/mL for JH III, and 20 ng/mL for farnesol, respectively (Table 3). The reproducibility of the method was evaluated for intraday and interday precision. The former reflected the repeatability of eight separately extracted samples in one day, and the latter established the replicability by processing twenty four independently extracted biological samples on three different days. Table 2 shows the intraday and interday precision of the assay in vitro. Intraday precision (RSD, %) was between 2.40% and 5.20% for JH III and precursors at three spike amounts, respectively. Interday precision (RSD, %) was less than 7.92%, 10.08%, 9.51% and 9.98% for farnesol, farnesal, methyl farnesoate and JH III, respectively (Table 2). For the assay in hemolymph, intraday precision (RSD, %) was less than 5.16%, 6.55%, 6.86% and 5.84% for farnesol, farnesal, methyl farne-

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Fig. 2. Relationship between concentrations of farnesol (A), farnesal (B), methyl farnesoate (C) and JH III (D) and area ratios. Data show the means ± SD of three independent experiments.

soate and JH III, respectively; and interday precision (RSD, %) was less than 6.60%, 7.51%, 7.94% and 7.49% for farnesol, farnesal, methyl farnesoate and JH III, respectively (Table 3). 3.2. Comparison of RCA and GC–MS/MS for determination of JH biosynthesis in vitro JH III biosynthesis rates in vitro on different days (days 0–7) after adult emergence were analyzed using both RCA and GC–MS/MS methods. Fig. 3 shows that the values for GC–MS/MS were similar to those determined with the RCA, although HPLC-FD showed consistently higher values than RCA. Dilution of the radiolabeled methionine by endogenous unlabeled methionine within the CA, losses during extractions and liquid scintillation quantification are likely responsible for the lower values using RCA. JH biosynthesis shortly after adult emergence was low, rose thereafter, reaching a peak on day 3–4 and then declined on day 5, returning to a low level on day 7 (Fig. 3).

3.3. Effect of H17 and pitavastatin on JH biosynthesis in vitro To validate the ability of the GC–MS/MS method to determine the effect of biosynthetic inhibitors of JH, we assayed two inhibitors using both RCA and GC–MS/MS methods. FGLamide allatostatins (ASTs) are a family of insect neuropeptides first isolated from brains of D. punctata that inhibit JH production [25]. We have previously demonstrated that an AST mimic, H17, significantly reduces JH biosynthesis by cockroach CA [19]. The dose response curves in vitro for H17 treatment, determined using RCA and GC–MS/MS (Fig. S3A). The IC50 value for H17 treatment was 12.2 nM for RCA and 15.6 nM for GC–MS/MS, respectively. HMG-CoA reductase (E.C. 1.1.1.34), a key regulatory enzyme in the MVA pathway, can be regarded as an eco-friendly insect growth regulator target candidate. Li et al. determined the IC50 values of three statins in reducing JH biosynthesis in D. punctata [26]. We compared the IC50 values of pitavastatin using RCA and GC–MS/MS. The dose response curves are shown in Fig. S3B; the IC50 value was 397.4 nM by RCA and 347.2 nM by GC–MS/MS, respectively. There was no difference in inhibition of JH biosynthesis by H17 or pitavastatin at all concentrations employed, using either RCA or GC–MS/MS (P values were more than 0.05; Student’s t-test). Both IC50 values and dose response curves for the JH inhibitors were similar whether they were determined using RCA or GC–MS/MS. This suggested that the GC–MS/MS method can be used for the determination of JH biosynthesis and for screening of potential inhibitors. 3.4. Inhibition of JH biosynthesis by geranylgeraniol and clotrimazole and mode of action

Fig. 3. JH biosynthesis in vitro during the first gonadotrophic cycle of D. punctata determined with GC–MS/MS and RCA. Measurements of rates of JH biosynthesis per pair CA were taken every day after the final molt (day 0–day 7). Each point shows mean ± SEM.

The biosynthesis of JHIII can be divided into the MVA pathway (resulting in the production of farnesyl diphosphate) and the JH-specific pathway. The JH-specific pathway involves four intermediates (farnesol, farnesal, farnesoic acid and methyl farnesoate) and five enzymes (farnesyl diphosphate pyrophosphatase, farnesol dehydrogenase, farnesal dehydrogenase, juvenile hormone acid methyltransferase, and methyl farnesoate epoxidase CYP15A1)

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Fig. 4. Effects of NADP+ -dependent farnesol dehydrogenase inhibitor geranylgeraniol and the general cytochrome P450 inhibitor clotrimazole on JH biosynthesis from day 7 mated female D. punctata in vitro determined by GC–MS/MS. (A) The inhibitory effects in vitro of geranylgeraniol and clotrimazole on JH biosynthesis by a pair of CA. (B) Quantitative assay of farnesol in control and geranylgeraniol-treated samples. (C) Quantitative assay of methyl farnesoate in control and clotrimazole-treated samples. Each bar represents the mean ± SEM (N = 8). Asterisks indicate significant differences between inhibitor- and control groups of animals as determined by Dunnett’s multiple comparison test following one-way ANOVA: **, 0.001 < P < 0.01; ***, p < 0.001.

[23]. Previous quantitative assay methods for JH could not show the changes in quantity of JH and intermediates at the same time in one sample. Thus, it was impossible to determine the mechanism of inhibition by inhibitors of JH biosynthesis (i.e. which enzymes are affected by the inhibitor) using previous quantitative assay methods. A farnesol dehydrogenase inhibitor, geranylgeraniol [22], and the general cytochrome P450 inhibitor, clotrimazole [23], were used to asses their action on JH biosynthesis from day 7 mated D. punctata in vitro by GC–MS/MS to reveal at which step in biosynthesis the inhibitors exerted their effect. Identification and quantitation of JH III and its precursors farnesol and methyl farnesoate was determined by comparing the consistency of the retention time and the specific SRM transitions in Table 1 (Fig. S4). For the control, only JH III was found in the medium (Figs. S4A and S4B). No JH III was detected in the geranylgeraniol-treated sample (concentration 1 mM), but in the same sample, farnesol was detected (Figs. S4C and S4D). For the clotrimazole-treated sample (concentration 0.5 mM), both JH III and methyl farnesoate were found in the sample (Figs. S4E and S4F). However, the JH III peak for the clotrimazole-treated sample was significantly lower than that of the control. Fig. 4A–C show the quantitative changes in JH III, farnesol and methyl farnesoate of control, the geranylgeraniol-treated sample and the clotrimazole-treated sample, respectively. In Fig. 4A, geranylgeraniol at 1 mM almost completely inhibited JH III biosynthesis. In the presence of geranylgeraniol at 0.1 mM, the rate of JH biosynthesis was 14 pmol per hour per pair CA, which was 40% of the control (JH biosynthesis rate of the control was 36 pmol per hour per pair CA). The amount of farnesol in the geranylgeraniol-treated sample significantly increased relative to the control (the amount of farnesol in the control was undetectable. Thus, high concentrations of geranylgeraniol almost completely inhibited JH III biosynthesis and resulted in the accumulation of the precursor farnesol simultaneously. In the clotrimazole-treated sample, the rate of JH biosynthesis at 0.5 and 0.1 mM was 5 and 12 pmol per hour per pair CA, respectively. This result demonstrated that clotrimazole has a significant effect on JH biosynthesis, relative to the control. The accumulation rate of methyl farnesoate at 0.5 and 0.1 mM was 16 and 5 pmol per hour of per pair CA, respectively. JHs and their intermediates in JH specific biosynthetic pathway are acyclic sesquiterpenoids that are very similar in their structures. The structural similarity, susceptibility to degradation and low concentration in insects highlights the difficulty in simultaneously determining the quantity of JHs and their sesquiterpene precursors in a sample. JHs regulate multiple physiological processes including embryogenesis, growth, development, metamorphosis, reproduction, migration, diapause and polymorphism. The discrete enzymes of JH biosynthesis can therefore be regarded as potential insecticide

Fig. 5. Hemolymph JH III titer in adult female D. punctata during the first gonadotrophic cycle. Each point shows mean ± SEM, N = 5.

targets. Developing a fast quantitative assay method for JH-related sesquiterpenoids with high sensitivity, precision and accuracy is essential for the screening of new biosynthetic inhibitors of JH. The quantitative changes in JH III and its intermediates determined with this GC–MS/MS method clearly demonstrate that it is possible to measure the quantities of intermediates and JH in the presence of inhibitors. For example, the targets of geranylgeraniol and clotrimazole are farnesol dehydrogenase and methyl farnesoate epoxidase, respectively. Our method also reveals there are dose-dependent effects of these inhibitors, both in terms of inhibition of JH biosynthesis and accumulation of intermediates. Thus, this GC–MS/MS method can be used to dissect the mechanisms of action of these biosynthetic inhibitors.

3.5. JH III titer in hemolymph and whole body JH III was quantified from day 0 to day 7 after adult emergence in female hemolymph. Only JH III was found in D. punctata hemolymph. Titer was relatively low at day 0 (79.68 ± 5.03 ng/mL) but began to increase and reached a maximum of 1717 ng/mL five days later. Titer then declined dramatically in the following 24 h period to approximately 320 ng/mL at day 6 and day 7 (Fig. 5). These values are in close agreement with those in a previously reported study [28]. The quantity of JH III in whole body was also measured from day 0 to day 7 during the first gonadotrophic cycle of adult female D. punctata. JH III quantity reached a maximum on days 4 (845.5 ± 87.9 ng/g) and 5 (679.7 ± 164.6 ng/g), and declined rapidly thereafter (Fig. 6A). We then calculated JH III quantity in whole body extracts on a per animal basis (Fig. 6B).

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Fig. 6. JH III levels in adult female D. punctata whole body extracts in ng/g (A) and on a per animal basis (B). Each point shows mean ± SEM, N = 5.

Fig. 7. Effect of inhibitors on hemolymph JH III titer of female D. punctata. Animals were injected with 5 ␮L of inhibitor on day 1, and hemolymph JH III titer determined on day 3. The concentrations of H17, pitavastatin, geranylgeraniol and clotrimazole were 10 ␮M, 1000 ␮M, 1000 ␮M and 10 ␮M, respectively. Each bar represents the mean ± SEM (N = 5). Asterisks indicate significant differences between inhibitor- and control groups of animals as determined by Dunnett’s multiple comparison test following one-way ANOVA: ****, p < 0.0001.

3.6. Reduction in JH titer in hemolymph by treatment with H17, pitavastatin, geranylgeraniol and clotrimazole H17, pitavastatin, geranylgeraniol or clotrimazole (10 ␮M, 1000 ␮M, 1000 ␮M and 10 ␮M, respectively in 5 ␮L.) was injected into D. punctata females at day 1, and animals were assayed for JH biosynthesis at day 3. Fig. 7 shows that the inhibitors had significant effects on JH titer following treatment in vivo. It suggested that the GC–MS/MS method can be used for assay of JH release into the hemolymph. Application of the GC–MS/MS technique to analyze extracts from in vitro incubations of CA demonstrated that it is applicable to the analysis of natural JH and its intermediates. However, we were also interested in determining if it could be applied to analysis of hemolymph titer and whole-body JH content. Hexane extracts of hemolymph and whole bodies from females were analyzed directly, without further purification, to determine whether we could identify JH in the presence of large amounts of other lipids in the hemolymph and whole bodies. In the measurement of the whole-body JH content of D. punctata, homogenization is difficult because of the cuticle. Our experimentation demonstrated that ball milling with liquid nitrogen can provide a homogeneous whole body sample. As there is high endogenous JH esterase activity in the hemolymph of many insect species, addition of the watersoluble acetonitrile to the hemolymph results in the inactivation of the JH esterase. In addition, the JH III is very well separated from

proteins, including JH binding proteins, using this solvent system [10]. This method has been used for the measurement of JH titer in hemolymph or JH content in whole body of Manduca sexta [27], Drosophila melanogaster and MF in Procambarus clarkii (data not shown). To date, determinations of titers of JH and JH precursors have been performed using several different methods. Each method has advantages and disadvantages. Chromatography tandem mass spectrometries are important techniques in modern analytical methods. Since JHs and their precursors in the JH-specific pathway are lipid-soluble, non-polar organic solvents (for instance, hexane and isooctane) are preferred for extraction. The hexane or isooctane phase can be directly used in the GC, GC–MS and GC–MS/MS analysis, whereas it cannot be used in LC and LC hyphenated protocols (LC–MS or HPLC-FD) immediately following extraction. For LC assays, the hexane or isooctane phase must be dried completely, and then reconstituted with either acetonitrile or methanol. Thus, we used GC–MS/MS in our present work to avoid the cumbersome process of drying and resolubilization. Compared with other methods, our method using GC–MS/MS requires no further purification or derivatization after extraction with hexane. In addition, analysis of each sample with GC–MS/MS takes only fifteen minutes. This technique can also reveal the mechanisms of action for biosynthetic inhibitors in the same GC–MS/MS analysis with the same sample. It can measure JHs and their precursors both in vitro and in hemolymph. However, because only the intermediates farnesol, farnesal and methyl farnesoate can be quantitatively assayed at very low concentrations in GC–MS/MS, our method only can evaluate whether the JH biosynthetic inhibitors affect the farnesol dehydrogenase or the farnesal dehydrogenase or the methyl farnesoate epoxidase. If other mechanisms of inhibition require identification, the method using HPLC-FD labeled with different fluorescent tags should be used [15]. 4. Conclusions GC–MS/MS offers several important advantages for the measurement of titers of JH and its intermediate both in vitro and in hemolymph or whole body. It is easy, quick, sensitive, specific and reproducible. The assay procedure is as simple as RCA without further purification and derivatization, and does not require radioactive materials. This method measures 4 samples in 1 h. Compared with LC–MS, LC–MS/MS and HPLC-FD, it also has the advantages of low operating and maintenance costs. The technique reported here should prove to be a valuable tool for the determination of JH and precursor content across the Arthropods as well as for the screening of JH inhibitors as well as for studies on the mode of action of inhibitors.

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Please cite this article in press as: Z.-p. Kai, et al., A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.01.030