Common structural features facilitate the simultaneous identification and quantification of the five most common juvenile hormones by liquid chromatography-tandem mass spectrometry

Abstract

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB₃) and JH III skipped bisepoxide (JHSB₃). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

Keywords: Homologs; Juvenile hormone; Liquid chromatography; MRM; Quantification.

Fig. 1:. Chemical structures of JH homologs:

JHB₃: juvenile hormone III bisepoxide. JHSB₃: juvenile hormone III skipped bisepoxide. JH III: juvenile hormone III. JH II: juvenile hormone II. JH I: juvenile hormone I. MF: methyl farnesoate. Epoxide groups are in red. Methyl esters groups are in blue. Ethyl groups are in magenta.

Fig. 2:. LC separation of JH homologues:

Typical LC-MS/MS peaks of JH homologues and MF. It shows the relationships between the retention times in minutes (X axis) and the signal intensity (cps; counts per second) (Y axis). Injected mass was 125 pg (325 pg for MF and 32 pg for the internal standard –ISTD-). Retention times are in minutes. Black lines represent the signal intensity of the primary transition, and red lines represent the intensity of the secondary transition. The inset shows the corresponding chromatograms for JH III-D3 (blue is the primary and green is the secondary transitions).

Fig. 3:. STD curves:

relationships between the concentration of each of the five JH standards (C), in parts per billions (ppb) (X-axis), and the signal intensities expressed as the ratio between the JH standard and the internal standard (IS, JH III-D3) (Y-axis). JH III (black circle). JH II (red inverted triangle). JH I (green square), JHSB3 (yellow diamond) and JHB3 (green triangle). Calibration curves for each of the JH homologs were constructed using between 21 and 54 different standard curves replicates. Linearity were observed over a wide concentration range. The blue lines represent the 95% confidence bands, depicting the upper and lower confidence bounds for all points on a fitted line within the range of data.

Fig. 4:. Analysis of JH homologs from biological samples:

Typical ion extracted chromatograms comparing the relationships between retention times in minutes (X-axis) and signal intensities (cps; counts per second) (Y-axis) for JHs from biological samples (solid line) and from standard solutions (dashed lines). MRM transitions: primary (black) and secondary (red). JH I and II are from B. mori larval hemolymph. JH III is from Ae. aegypti adult female hemolymph. JHB3 is from D. melanogaster larval hemolymph. JHSB3 is from Dipetalogaster maxima adult female hemolymph. MF was synthesized in vitro by the CA of Ae. aegypti 4th instar larvae. Primary transitions masses evaluated are in black and secondary transitions are in red.