7/28/2019 SSI-008

1/4

Summary

Sensitivity was tested for four steroids on the LCMS-

8030 triple quadrupole mass spectrometer coupled

with a Nexera UHPLC.

Background

Steroids are powerful hormones that are needed fornormal biological activity but may also be present in

the environment. Water contaminated with steroids

poses a serious environmental and public health

threat. Sensitive and rapid methods are needed for

accurate quantitative analysis. UHPLC-MS-MS meth-

ods for analysis of these compounds were developed

and the sensitivity for each analyte was tested.

Method

Standards for four steroids were obtained at a concen

tration of 1 mg/mL in methanol. The

standards were: Testosterone (T4), Progesterone (P4

Estradiol (E2), and Ethinylestradiol (EE2). Standards

were diluted in 50/50 water/acetonitrile for optimizatio

of mass spectrometry parameters.

Multiple Reaction Monitoring (MRM) was used for

analysis. Collision energy, quadrupole prerod bias

voltage, and other MS parameters were optimized by

flow injection of the individual standards. T4 and P4

ionized most efficiently using a 0.1% formic

acid/acetonitrile mobile phase using the DUIS dual io

Figure 1: Structures of tested steroidsTable 1: Dilution levels (ng/mL)

Table 2: MRM parameters

Testosterone Progesterone

Estradiol Ethinylestradiol

O

OH

H

H

H

O

O

H

H

H

HO

OH

H

H

H

HO

OH

H

H

H

Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V)

MRM 1 + Testosterone 288.9>97 80 -14 -25 -10

MRM 2 + Progesterone 315.4>109.1 80 -12 -30 -20

Type Event# +/- Compound Name (m/z) Dwell Time (msec) Q1 Pre Bias(V) CE Q3 Pre Bias(V)

MRM 1 - Estradiol 271.4>183.1 50 13 41 30

MRM 2 - Ethinylestradiol 295.4>145 50 14 47 28

Measurement Time (min)

0-2

0-2

Measurement Time (min)

0-2

0-2

Level T4 P4 E2 EE2

L1 370.3 370.3 10000 10000

L2 123.5 123.5 3333 3333

L3 41.17 41.17 1111 1111

L4 13.72 13.72 370.3 370.3

L5 4.57 4.57 123.5 123.5

L6 1.52 1.52 41.17 41.17

L7 0.582 0.582 13.72 13.72

L8 4.57 4.57

Steroids: Testosterone, Progesterone, Estradiol,

Ethinylestradiol

ApplicationNews

No. SSI-008

Liquid Chromatography Mass Spectrometry

7/28/2019 SSI-008

2/4

spray (ESI with corona discharge) source in positive

mode. The MRM transition ofm/z288.997 was

monitored for T4 and the transition ofm/z315.4109.1 was used for P4.

E2 and EE2 ionized most efficiently using a

water/acetonitrile mobile phase with atmospheric

pressure chemical ionization (APCI) in negative mode.

Therefore these compounds were analyzed in a

separate method. The MRM transitions for E2 and EE2

were 271.4183.1 and 295.4145.0 respectively.

A Shimadzu Shimpack-XR ODS III column (1.6 m, 2

50 mm) was used for analysis. A binary gradient of

0.1% formic acid and acetonitrile was used for T4 an

P4 while a binary gradient of water and acetonitrilewas used for E2 and EE2. The gradient began at 55

acetonitrile and increased to 95% over 1 minute. Aft

a 6 second isocratic hold, the column was

re-equilibrated for approximately 1 minute. The colu

temperature was 40 C and the injection volume wa

10 L.

Calibration curves were prepared by serial dilution o

mixed standard as shown in Table 1.

110504.SetEPAx01_

15_

EPAMIX2L10_

015.lcd

P4 r2 = 0.999 T4 r 2 = 0.991

Figure 2: Calibration curves and selected mass chromatograms for P4 and T4

P4 Level 7

P4 Level 2

T4 0.065 ng/mL

T4 Level 2

0 100 200 300 Conc.0.0

1.0

2.0

3.0

Area(x100,000)

0 100 200 300 Conc0.00

0.25

0.50

0.75

1.00

Area(x1,000,000)

0.0 0.5 1.0 1.5

0.0

1.0

2.0

3.0

4.0

5.0(x100)

1:315.40>109.10(+)

0.

992

0.5 1.0 1.5

0.0

0.5

1.0

1.5

(x100)2:288.90>97.00(+)

0.

616

110504.SetEPA

x01_

8_

EPAMIX2L12_

008.lcd

0.0 0.5 1.0 1.5

0.00

0.25

0.50

0.75

1.00

(x100,000)1:315.40>109.10(+)

0.

998

110504.SetEPAx01_

29_

EPAMIX2L05_

029.lcd

110504.SetE

PAx01_

29_

EPAMIX2L05_

029.lcd

0.5 1.0 1.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0(x100,000)

2:288.90>97.00(+)

0.

620

110504.SetEPAx01.

lcb

110504.SetEPAx0

1.lcb

pplicationews

No.SSI-008

7/28/2019 SSI-008

3/4

Results and Discussion

Representative chromatograms of the T4/P4 andE2/EE2 methods are shown in Figures 2 and 3. The

limit of detection (LOD, S/N > 3) for P4 was between

0.194 and 0.582 ng/mL using a 10 L injection, and the

limit of quantitation (LOQ, S/N >10) was approximately

0.5 ng/mL. For T4, the LOD was approximately

0.065 ng/mL and the LOQ was approximately

0.2 ng/mL. For T4 and P4, the calibration curve was

constructed between 0.194 and 370 ng/mL. The curves

were linear in the tested range (r2 > 0.99).

For E2, the LOD was approximately 1.5 ng/mL and th

LOQ was approximately 4.5 ng/mL. The LOD and LOfor EE2 was similar. The calibration curves were

constructed from 4.57 ng/mL up to 10 g/mL, and the

curves were both linear (r2 > 0.99)

Conclusion

Rapid and sensitive methods for

determination of four steroids was developed using

UHPLC-MS-MS.

E2 r2 > 0.999 EE2 r2 > 0.999

Figure 3: Calibration curves and selected mass chromatograms for E2 and EE2

E2 1.52 ng/mL

E2 Level 3

EE2 1.52 ng/mL

EE2 Level 3

0 2500 5000 7500 Conc.0.0

1.0

2.0

Area(x100,000)

0 2500 5000 7500 Conc.0.0

0.5

1.0

1.5

Area(x100,000)

110505.SetEP

Ax02.lcb

110505.SetEPA

x02.lcb

0.0 0.5 1.0 1.5

0.0

1.0

2.0

3.0

4.0

(x10)1:271.40>183.10(-)

0.

209

0.0 0.5 1.0 1.5

0.0

0.5

1.0

1.5

2.0

2.5

(x10,000)1:271.40>183.10(-)

0.

214

110

505.SetEPAx02_

30_

E2EE2L03_

030.lcd

110505.SetEPAx02_

30_

E2EE2L03_

030.lcd

110505.S

etEPAx02_

11_

E2EE2L09_

011.lcd

0.5 1.0 1.5

0.00

0.25

0.50

0.75

1.00

1.25

1.50(x10,000)

2:295.40>145.00(-)

0.

234

0.5 1.0 1.5

0.0

0.5

1.0

1.5

2.0

2.5

(x10)2:295.40>145.00(-)

0.

234

110505.SetE

PAx02_

10_

E2EE2L09_

010.lcd

pplicationews

No.SSI-008

7/28/2019 SSI-008

4/4

SHIMADZU SCIENTIFIC INSTRUMENTS

7102 Riverwood Drive, Columbia, MD 21046, USA

Phone: 800-477-1227/410-381-1227, Fax: 410-381-1222

URL: www.ssi.shimadzu.com

For Research Use Only. Not for use in diagnostic procedures.

The contents of this publication are provided to you as is without warranty of any kind, and are subject to change without notice. Shimadzu does

not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication.

Shimadzu Corporation, 201

First Edition: November 20

www.shimadzu.com/an/

pplicationews

No.SSI-008