Rapid Analysis of Steroid Hormones by GC/MS
Using the New Rxi-1ms Column
- Resolve 6 common steroid hormones in less than 25 minutes.
- Ultra-low bleed column greatly reduces background interferences.
- Stable performance at 300°C or above.
Determinations of urinary steroid hormones are widely used for diagnosing and monitoring many health conditions, including bio-identical hormone replacement, menopause, Cushing’s syndrome, Addison's disease, adrenal fatigue, and others [1]. Many clinical laboratories use gas chromatography and mass spectrometry (GC/MS) as the primary analytical method for identification and quantification. A capillary GC column with a thin film (0.25µm or less) of 100% dimethylpolysiloxane is the column of choice for many analysts, because this stationary phase has the highest operating temperature available. Temperatures exceeding 300°C are required to elute the high molecular weight (250-400 Dalton) hormones in a reasonable analysis time while maintaining Gaussian peak shape and resolution. A phase film thickness of 0.25µm or less minimizes column bleed at these high temperatures. Also, in order to provide reliable quantification, the column must exhibit the inertness necessary to produce symmetric peaks and reproducible results.
Our new Rxi-1ms column, designed for GC-MS applications, provides the ultra-low bleed and exceptional inertness needed for analyzing urinary steroid hormones. For this application we derivatized six sex hormones, using methoxylamine HCl and trimethylsilyl imidazole to improve chromatography. Figure 1 shows this variety of derivatized steroid sex hormones, analyzed in less than 25 minutes by using an Rxi-1ms column. Note that these compounds elute at temperatures near or above 300°C and that bleed from the Rxi-1ms column is negligible at these temperatures. The Rxi-1ms column exhibits the inertness needed to produce Gaussian peaks and excellent resolution.
Because GC/MS analysis of urinary steroid hormones is a demanding application, it is important to use the lowest bleed, most inert column available. The new Rxi-1ms column meets these requirements better than any column we have tested, and we recommend it as the column of choice for this application.
Figure 1: Negligible bleed, Gaussian peaks, and fast results characterize analyses of derivatized steroids on an Rxi-1ms column.
| Peaks | |
|---|---|
| 1. | Androsterone |
| 2. | Dehydroepiandrosterone (DHEA) |
| 3. | 17α-Estradiol |
| 4. | Estrone |
| 5. | 17β-Estradiol |
| 6. | Testosterone |
| 7. | derivatization by-product |
| Column | Rxi-1ms, 30 m, 0.25 mm ID, 0.25 µm (cat.# 13323) |
|---|---|
| Standard/Sample | Each hormone in methanol or ethanol; compounds derivatized using 2% methoxylamine HCl (CH3ONH2) in pyridine, then N-trimethylsilylimidazole (TMSI), then analyzed |
| Conc.: | 100 µg/mL |
| Injection | |
| Inj. Vol.: | 1.0 µL splitless (hold 0.5 min) |
| Liner: | 3.5mm single gooseneck inlet liner |
| Inj. Temp.: | 250 °C |
| Oven | |
| Oven Temp.: | 100 °C to 320 °C at 10 °C/min (hold 10 min) |
| Carrier Gas | He, constant flow |
| Flow Rate: | 1 mL/min |
| Detector | MS-Scan: Shimadzu 17A with QP5000 |
|---|---|
| Transfer Line Temp.: | 280 °C |
| Ionization Mode: | EI |
| Scan Range: | 40-700 amu |