| As growth promoters, steroid hormones are usually used to improve the economicbenefits in livestock for weight gain, more lean meat, synchronized estrus and improvementof feed conversion efficiency. The residues of these hormones in food products could result inmetabolic disorder, abnormal growth, and potential risk of carcinogenesis and teratogenesis.At present, the only way to evaluate steroid hormone abuse is based on their physiologicalconcentrations for classical approach based on GC/MS or LC-MS/MS. When the syntheticsteroid hormones are identical to naturally occurring steroid hormones, the decision onwhether there is misuse is much more difficult because they are also naturally produced byfood producing animals. At present, gas chromatography/combustion/isotope ratio massspectrometry (GC/C/IRMS) is the effective method to elucidate the origin of steroidhormones. However, there is yet no research on the origin of residual hormones inanimal-derived foods at home and abroad. Therefore, it is necessary to establish theidentification technique. Simultaneously, it will provide a technical support to China forsafety monitoring of animal-derived foods.In this paper, a quantitative detection method for determination of14steroid hormoneresidues (testosterone (T), epitestosterone (EpiT), androsterone (A), etiocholanolone (E),dehydroepiandrosterone (DHEA), progesterone (P), pregnanediol (PD), pregnanetriol (PT),estrone (E1),/β-estradiol (/β-E), estriol (E3), hydrocortisone(H) and testosteronepropionate (TP)) in animal-derived foods and an identification technique base on GC/C/IRMSfor the origin of12residual hormones (T, EpiT, A, E, DHEA, P, PD, PT, E1,-E, β-E, E3) inanimal-derived foods were developed. The quantitative detection method is a prerequisite andbasis for the identification technique. The identification test was unnecessary performed byGC/C/IRMS when real samples with no target analytes or the very low levels of targetanalytes detected.A HPLC-MS/MS quantitative method was developed for the simultaneous determinationof14steroid hormone residues in animal-derived foods. The mobile phase consisted of water(A) and acetonitrile (B). Glucocorticoid, androgen and progestin were separated on an AgilentPoroshell120EC-C18(150×2.1mm,2.7μm). The system was run in a linear gradient:0.0min (35%B)12.0min (70%B)21.0min (100%B)21.1min (35%B)26.0min (35%B).The flow rate was200μL/min. Estrogen separations were achieved on an YMC HydrosphereC18column (100×2.0mm,3μm). Linear gradient was show as:0.0min (35%B)12.0min(70%B)13.0min (70%B)13.1min (35%B)18.0min (35%B). The flow rate was250 μL/min. The LODs was0.004–1.60μg/kg, indicating that the developed method exhibitedhigh sensitivity.A sample preparation method was developed for the simultaneous extraction of14steroid hormone residues in animal-derived foods. The method was applicable to animalmuscle tissue (raw meat), milk and egg for sample preparation. Sample preparation:sample enzymatically digest extraction with acetonitrile lipid removal with ZnCl2purification with C18-Si-NH2-SPE cartridges detection with HPLC-MS/MS. Goodaverage recoveries (77.7-119.0%), purification effect and minimize matrix effect (beefsample, ME=-1.1%~-22.4%) were obtained.A method was developed for the determination of residual steroid hormones in buttersamples by gel permeation chromatography (GPC). The mobile phase was ethylacetate/cyclohexane (1/1, v/v). The flow rate was4.7mL/min. The column pre-clean, GPC-forerun,GPC-main fraction and GPC-tailing times were0.17min,10.7min,33.3min and4min,respectively. Sample preparation: butter extraction with ethylacetate/cyclohexane oscillation centrifugation supernatant purification with GPC collection fraction dryness and dissolving detection with HPLC-MS/MS. The operation of the method issimple. Good average recoveries (64.1-110.0%) and lipid removal effect were obtained.A concentration and purification technology was developed for the determination ofsteroid hormones by Pre-HPLC. A reversed phase C18column was used for Pre-HPLC. Themobile phase was acetonitrile (B) and water (A). Linear gradient was show as:0.0min(40%B)20.0min (80%B)25.0min (100%B)30.0min (100%B). Good separation wereobtained for steroid hormones. The purified sample met the purity need for GC/C/IRMSmeasurement.Qualitative and quantitative analyses were performed by GC/MS. The samples wereintroduced in the splitless mode. Both the transfer line and the injector temperature weremaintained at280℃. The source and quadrupole temperatures were230℃and150℃,respectively. The flow rate was1mL/min (constant flow). The initial oven temperature wasset at80℃(1min), increased to250℃(2min) at15℃/min, then to272℃at2℃/minand held for5min. Good separation and no chromatographic peak cross interference ofsteroid hormones met the specificity and precision need for GC/C/IRMS measurement.The origin of residual steroid hormones in animal-derived foods was investigated usingGC/C/IRMS. The GC column and the temperature program were identical to that used forGC/MS. The injector temperature and flame ionization detector (FID) temperature were set at 250℃and200℃, respectively. The temperatures of the combustion furnace and interfacewere set at850℃and350℃, respectively. Good separation and no chromatographic peakcross interference provide a good guarantee for the13C values measurement of steroidhormones.The13C values of exogenous steroid hormones in animal-derived foods were found inthe range from-28‰to-38‰and the13C value of endogenous steroid hormones wereusually higher than-27‰. For example, The13C values of endogenous progesterone inbutters were found in the range from-26.770.26‰to-23.720.28‰and the13C value ofexogenous progesterone spiked in the sample was about-30.570.12‰. One-way Analysis ofVariance (ANOVA, P-value=0.003<0.05) indicated that there was a highly significantdifference between the endogenous and exogenous13C values of progesterone. The13Cvalues of endogenous progesterone in porcine ovarian were found in the range from-22.32±0.13‰to-20.44±0.16‰and the13C value of exogenous progesterone spiked in thesample was about-30.66±0.15‰. ANOVA (P-value=0.001) indicated that there was also ahighly significant difference. The13C values of endogenous progesterone in chicken eggswere found in the range from-17.09±0.45‰to-25.84±0.38‰and the13C value of pureexogenous progesterone spiked in standard solution was about-30.72±0.03‰. ANOVA alsoindicated that there was a highly significant difference. The sensitivity of the GC/C/IRMSmethod for all steroid hormones was determined to be from10to100ng. The within-batchprecisions of the method ranged from0.09to0.15‰and the between-batch precision was0.11‰. The precisions were below the acceptable value of0.5‰, indicating that no isotopicdiscrimination occurred in one year of the steroid hormone standards spiked in methanol.However, the GC/C/IRMS working solution dissolved by cyclohexane was unstable afterstoring at room temperature or at4℃for three days and indicating the working solutionshould be analysis immediately. We found the Pre-HPLC fraction collection step was a criticalfactor affecting the isotopic fractionation and measurement accuracy. Therefore, GC/C/IRMScan be a powerful tool to investigate the origin of residual steroid hormones in animal-derivedfoods. |
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