Jennifer Simeone and Paul Rainville

Figure 1. UPC2/MS/MS analysis of 20 steroids.

GOAL

To rapidly separate 20 steroid compounds,

including three sets of isobaric species,

by using UltraPerformance Convergence

Chromatography™ with mass spectrometry

(UPC2®/MS/MS).

BAC KG ROU N D

The ability to separate complex mixtures

of structurally similar compounds, such

as steroids, within one method is highly

desirable. Additionally, compounds of related

structures may also be isobaric with similar

fragmentation, rendering them unidentifiable

by MS/MS. In these cases, it is essential that

these compounds be baseline separated

chromatographically.

T H E SO LU T IO N

Convergence chromatography is known to be a

good method for separating structurally similar

compounds more efficiently than other methods,

such as reversed-phase LC. In this application,

20 steroids (Figure 1) were separated on an

ACQUITY UPC2™ BEH 2-EP Column in under

7 minutes, using a co-solvent of IPA/MeOH

modified with formic acid. This steroid mix

includes a number of isobaric species that

have the same precursor to product transition;

because of this, the compounds must be

completely separated chromatographically.

Convergence Chromatography enables the separation

of complex mixtures, such as steroids, without

copious amounts of time spent in developing a

suitable method.

Fast Separation of 20 Steroids Including Isobaric Species using UPC2/MS/MS

Time2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

0

Progesterone

Androstenedione

Estradiol

DHT

Deoxycorticosterone

11KA4

Testosterone

17-OH-Prog

16-OH-Prog

11-OH-Test

11-DHC

11-OH-Androstenedione

18-OH-Corticosterone

11KT

Estrone

Deoxycortisol

Corticosterone

Aldosterone

Cortisone

Cortisol

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, The Science of What’s Possible, and UPC2 are registered trademarks of Waters Corporation. ACQUITY UPC2 and UltraPerformance Convergence Chromatography are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2014 Waters Corporation. Produced in the U.S.A. April 2014 720005021EN TC- PDF

Figure 2. Example separation of three compounds that differ only in the location of the hydroxyl group: either on C16, C17, or C19 (16-OH-progesterone, 17-OH-progesterone, and deoxycorticosterone).

Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

%

0

4.35

3.69

3.153.1

Deoxycorticosterone

17-hydroxyprogesterone

16-hydroxyprogesteroneFigure 2 shows the separation of three isobaric

species that differ only in the location of the

hydroxyl group, either on C16, C17, or C19.

By traditional reversed-phase LC, complete

separation of these three compounds is quite

difficult; however, using UPC,2 each species

is resolved by more than 0.5 min.

SUMMA RY

Convergence chromatography is a great technique for

separating compounds of structural similarity, such

as a complex steroid mix, without copious amounts of

time developing a suitable LC separation. UPC2 also

provides complete separation of isobaric species that

cannot be uniquely identified using MS/MS.

Waters would like to thank Dr. Marietjie Stander from Stellenbosch University for the donation of the steroid samples.