Simultaneous Determination of Hormone Residues in Aquatic Products by UPLC-ESI-MS-MS

Hormones, such as androgens (AS), estrogens (ES), and progestogens (PS), play an important role in organisms. AS, the main male sex steroids, which are the critical factors responsible for the development of the male phenotype during embryogenesis and for the achievement of sexual maturation at puberty, are generally used as therapeutic agents for restoration of the size and strength of muscle.1 It is known that estrogens have direct effects on the areas of the brain that control mood and cognition.2 Today, most formulations of contraceptive pills contain ethinyl estradiol, often in combination with a PS as the active ingredient. Estrogens combined with progestogens are also used to treat climacteric syndrome.3

Drug abuse has an adverse impact on aquatic products. Hormones can apply to animal sex differentiation, shortening the animal's growth cycle.4-6 Bisphenol A caused apoptotic cell death in swordtail testes and increased the mortality of medaka eggs when males were exposed to waterborne bisphenol A before spawning.7 Diethylstilbestrol has a range of actions on disruption of the thyroid system in the sea bream, such as encompassing the pituitary, thyroid tissue, and deiodinases.8 Certain kinds of hormones are often added to feed to enhance the efficiency of aquatic product breeding. Hormones are stable and less susceptible to degradation, yet still have strong biological activity and potential carcinogenicity when introduced into body tissues through the food chain, inducing neutral fat, immunodeficiency, and osteoporosis.9,10

Many methods have been used to effectively monitor and detect hormones in animal tissues. These can generally be divided into two major groups: 1) immunological methods , which are highly selective due to the antibody–antigen specificity interaction, although their applications in the samples are limited to some extent due to the instability of natural antibodies, and can easily result in false positive results,11,12 and 2) chromatographic methods, such as HPLC,13,14LC-MS,15,16 and GC-MS,17,18 which are the techniques most commonly used for detecting hormones. They, too, have some shortcomings despite their commonality. HPLC can only be used as a conventional method, but not for characterization. GC-MS often requires a derivatization step prior to chromatographic separation, which is mandatory for most of these compounds because they are thermally unstable, nonvolatile, or polar. However, derivatization makes sample preparation laborious and time-consuming, and may lead to losses or degradation of the target steroids.

UltraPerformance LC® (UPLC®) (Waters Corp., Milford, MA) with electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS-MS) has the virtue of efficient separation and accurate characterization, and has become the primary method of determining hormones.19-24 The authors established a method to simultaneously determine 24 hormones in aquatic products. The result was satisfactory after systematized optimization of sample pretreatment methods and instrumental analysis conditions.

Experimental

Materials

Cyproterone acetate and levonorgestrel were purchased from Sigma Chemical Co. (St. Louis, MO). 4-n-Octylphenol was obtained from Supelco (Bellefonte, PA). Bisphenol A, 4–nonylphenol, diethylstilbestrol, estrone, 17α-ethinyl estradiol, 17β-estradiol, estriol, megestrol acetate, trenbolone, norethisterone acetate, ethisterone, progesterone, zeranol, dihydrodiethylstilbestrol, chlormadinone acetate, testosterone, boldenone, 19-nortestosterone, methyltestosterone, dydrogesterone, and dienestrol were all obtained from Dr. Ehrenstorfer (Augsburg, Germany). Methanol, methyl tertiary butyl ether, and acetonitrile were all of HPLC grade and from Tedia (Fairfield, OH). Water was deionized ultrapure water. All other reagents used in the experiment were of analytical grade.

Preparation of stock and standard solutions

Figure 1 - Representative total ion chromatograms of 24 hormones. Names and retention times are as follows: estriol (2.63 min), trenbolone (3.98 min), boldenone (4.14 min), nandrolone (4.29 min), bisphenol A (4.15 min), zeranol (4.36 min), 17β-estradiol (4.38 min), testosterone (4.58 min),17α-Ethinyl estradiol (4.74 min), dihydrodiethylstilbestrol (4.88 min), methyltestosterone (4.91 min),ethisterone (4.98 min), diethylstilbestrol (5.13 min), dienestrol (5.24 min), estrone (5.25 min), levonorgestrel (5.36 min), dydrogesterone (5.73 min), cyproterone acetate (6.09 min), megestrol acetate (6.15 min), progesterone (6.22 min), norethisterone acetate (6.23 min), chlormadinone acetate (6.28 min), 4–nonylphenol (7.93 min), and 4-n-octylphenol (8.08 min).

Individual stock solutions of 100 µg/mL for all compounds were prepared in HPLC-grade methanol and kept at –20 °C. Standard solutions containing all compounds were mixed and diluted with methanol, and working solutions of all compounds and calibration concentrations were prepared by appropriate dilution of the stock solutions on the day of analysis. The total ion chromatograms of 24 hormones (100 µg/L) are shown in Figure 1.

Preparation and purification of samples

Samples (5 g) were homogenized for 30 sec in 20 mL of ethyl acetate, to which 3 mL 10% sodium carbonate was added, and this was ultrasonicated for 10 min at room temperature. The homogenates were then centrifuged at 7000 rpm for 10 min. The supernatant was evaporated at 40 °C and the residue was dissolved in 10 mL methanol/water (1:9, v/v), ready for loading.

The solid-phase extraction (SPE) process with a Waters HLB column can be summarized as follows: 1) Activation with 5 mL ethyl acetate, 5 mL methanol (both steps at 3 mL·min-1); 2) loading 10-mL sample at 1.2 mL·min-1; 3) rinsing with 5 mL methanol/water (2:8, v/v) and drying under vacuum for 5 min; and 4) elution with 10 mL methanol. The eluent was then evaporated to dryness under a gentle stream of nitrogen at 40 °C and the residue was redissolved with a volume of 1 mL of acetonitrile/ultrapure water (1:1, v/v) and syringe filtered using a 0.22-µm filter into an autosampler vial.

UPLCMS-MS conditions

The UPLC-MS-MS system comprises a Waters ACQUITY® UPLC system connected in-line to a Quattro Premier tandem mass spectrometer (Waters).The column used in the experiment was an ACQUITY UPLC BEH C18 (2.1—100 mm, 1.7 µm particle size) maintained at 40 °C. The ACQUITY phase was A (acetonitrile) and B (water). After sample injection (10 µL), a linear gradient was programmed for 8 min from 20:80 A–B to obtain 95:5 A–B composition; then the composition was held for 2 min. Finally, the concentration of A was decreased to 20% and held for 2 min. The total analysis time was 12 min, with 2 min required to reestablish and equilibrate the initial conditions. The flow rate was set at 0.25 mL·min-1 during the chromatographic process, and the temperature of the analytical column was 40 °C. The entire eluent was electrosprayed, ionized, and monitored by MS-MS detection in multiple reaction monitoring (MRM) mode. Ionization was positive for AS and PS and negative for ES. For this purpose, the MS polarity was switched by time segments according to the retention of the target analytes. The flow rate and temperature of the drying gas (N2) were L·hr-1 and 350 °C, respectively. The collision gas (Ar) flow was 0.12 mL/min, and the capillary voltage was 2850 V. The dwell time was set at 100 msec.

Validation procedures

Standard calibration curves and QC samples were analyzed over a period of three consecutive days. Linearity of calibration curves based on the analyte area as a function of the nominal concentration was assessed by weighted (1/C2) least-squares regression. Linearity correlation coefficients were calculated in the experiment.

Results and discussion

Optimization of LC–MS-MS

Table 1 - UPLC-ESI-MS-MS parameters for 13 hormones in ESI+ mode

Each tuning solution was introduced into the electrospray source by direct infusion (10 µL·min-1). The main ions produced in MS and MS-MS were identified in positive and negative ionization modes. The diagnostic fragment ions were selected and all mass spectrometer parameters were optimized to increase sensitivity. Tables 1 and 2 show the precursor and daughter ions for each steroid as well as the optimum values of MS-MS parameters: voltage of the first quadrupole for isolation of the precursor ion and collision energy for efficient fragmentation. In the authors' study, two daughter ions were routinely monitored. This fulfills the recommendations of the European Union (EU) concerning identification, since two MRM transitions from the ionized molecule of the target compound give four points in the scale—a value regarded as sufficient for unequivocal identification. The commonly used mobile-phase compositions such as acetonitrile/water and methanol/water were optimized in the experiment. In terms of consumption and ion response, methanol is a better choice.