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Clinical/Forensics
Rapid Analysis of Steroid Hormones by GC/MS
Using the New Rxi™-l ms Column
By Kristi Sellers, Clinical/Forensic I nnovations Chemist
• Resolve 6 common steroid hormones in less than 25 minutes.
Ultra-low bleed column greatly reduces background int erferences.
• Stable performance at 300°C or above .
Determi nations of urinary steroid hormones are
widely used for diagnosing and monit oring many
health conditions, including bio-identical hormone
replacement , menop ause, Cushing's syndrome,
Addison's disease, adrenal fatigue, and others'. Many
clinical laboratories use gas chromatography and
mass spectrometry (GCl MS) as the primary analyti­
cal method for identification and quantification. A
capillary GC column with a thin film (0.25flm or
less) of 100% dimethylpolysiloxane is the column of
choice for many analysts, because this stationary
phase has the highest operating temperatur e avail­
able. Temp eratures exceeding 300°C are required to
elute the high molecular weight (250-400 Dalton)
hormones in a reasonable analysis time while main­
taining and Gaussian peak shape resolution. A phase
film thickness of O.25flm or less minimizes column
bleed at these high temperatures. Also, in order to
provide reliable quantification, the column must
exhibit the inertness necessary to produ ce symme tric
peaks and reprod ucible results.
Our new RxiTM- lms column, designed for GC-MS
applications, provides the ultra-low bleed and excep­
tional inertness needed for analyzing urin ary steroid
horm ones. For this app lication we derivatized six sex
ho rmone s, using meth oxylam ine HCl and
trimethylsilyl imid azole (FigureI) to improve chro ­
matography. Figure I shows this variety of deriva­
tized steroid sex hormones, analyzed in less than 25
minutes by using an Rxi™- lms column. Note that
these compounds elute at tempe ratures near or
above 300°C and that bleed from the RxiTM-l ms col­
umn is negligible at these temperatures. The RxiTM_
Ims column exhibits the inertness needed to pro­
duce Gaussian peaks and excellent resolution.
Because GC/MS analysis of urin ary steroid hormones
is a demand ing application, it is impor tant to use the
lowest bleed, most inert colum n available. The new
RxiTM-l ms column meets these requirements better
than any column we have tested, and we recommend
it as the column of choice for this application.
Reference
1. steroid_dept.html
Rxi™-1 msColumn (fusedsilica)
(Crossbond" 100% dimethyl polysiloxane)
ID df (um) temp. limits length cat.# price
-60 to 3)0/350°C 30-Me",te 13323 $435 0.2Smm 0.25 ",-r---",~",--",-,,,,,,-_
For other dimensions, please vist ourwebsite.
2006.03
Figure 1 Negligible bleed, Gaussian peaks, and fast results
characterize analyses of derivatized steroids on an
Rxi™ -l ms column.
1. androsterone
2. dehydroepiandrosterone (DHEA)
3. 17-u-estradiol
4. estrone
5. 17-~ -estradiol
6. testosterone
7. derivatizationby-product
300°C
j "I
20.0
56
3
1 2 Column:
Sample:
Inj.:
Inj. temp.:
Carrier gas:
Flowrate:
Oven temp.:
Del:
Transfer line
temp.:
Scan range:
Ionization:
Mode:
: bl...... 'OO+.C~~ir l,
10.0 I 20.0
GCPH00372
Rxi--lms 30m, 0.2SmmID,
0.2Sllm (cat.# 13323)
I OOllg/ mLeachhormone inmethanol
or ethanol; compoundsderivatized
using 2%rnethoxylarnineHCI (CH30NH,)
in pyridine,thenN-trimethylsilylimidazole
(TMSI), thenanalyzed
l.0Il Lsplitless (hold 0.5 min.),
3.Smmsingle gooseneck
inlet liner (cat.# 20961)
250°C
helium, constant flow
I mL/min.
100°Cto 320°C@ lOoC/ min.
(hold10 min.)
MS: Shimadzu17AwithQPSOOO
280°C
40-700amu
EI
scan
320°C
/
mm.
_ / CH,QTMS
CH,ON~
~ TMS: !P>OTMS
2.TMSI ~
CH,ON #
Derivati zation Reacti on
Our Allure~ Biphenyl column uses n-rt interactions to provide superior resolution of steroids,
or other unsaturatedmolecules, compared to C18, cyano, or phenyl phases. Tosee compar­
isons, request Allure~ Biphenyl HPLC Columns (lit. cat.# 580015) andImproved HPLC
Analysis of Steroids (lit. cat.# 580020)- or review downloadable pdf files from our website.
Allure"Biphenyl HPLC Columns
EnhancedSelectivity for unsaturated compounds
Corticosteroids, contraceptive steroids,and endogenous steroid hormones illustrate the
uniqueseparation mechanismof the Allure~ Biphenyl phase for molecules that differ in the
numberor positionsof multiplebonds. lit. cat.# 580015
Improved HPLCAnalysis ofSteroids
Using Restek's Unique Allure" Biphenyl Column
Steroidsanalysesshow the Allure" Biphenyl phaseis more selective than a C18, cvano, or
phenyl phase for differencesin the numberor positions of multiple bonds. lit. cat.# 580020
Website : E-mail : TelNo : 03 9762 2034 . . . in AUSTRALIA