SteroIDQ-Kit: High-Throughput and Standardized LC-MS/MS Steroid-Quantification for Clinical Metabolomics

Therese Koal1, Diane Schmiederer1, Hai Pham-Tuan1, Cornelia Rhring1, Alexandra Gruber1, and Manfred Rauh2
2Kinder1BIOCRATES Life Sciences AG, Innrain 66, 6020 Innsbruck, Austria und Jugendklinik, Universittsklinikum Erlangen, Loschgestr. 15, 91054 Erlangen, Germany

Introduction
Steroid hormones are derived from cholesterol. Due to their fat-soluble characteristics these molecules can pass easily through the cell membrane and cause changes within the cell. Many endocrine diseases are caused by disorders in steroid metabolism. For diagnostic purposes, therefore, an accurate and high-throughput quantification of steroid hormones is of utmost importance. Up till now immunoassays are lab standards but they often deliver unsatisfactory results due to inadequate antibody specificity. Moreover, a single parameter for each measurement leads to a low cost-efficiency when a wider panel of steroids are requested. These drawbacks can be successfully overcome with the application of SteroIDQ Kit on a LCMSMS platform. The 16 steroids included in this Kit are given below:
steroid hormone biosynthesis
(with compounds incl. in SteroIDQ Kit)
Pregnanediol Allopregnanolone Pregnanetriol

Results
Method development: Solid Phase Extraction vs. Protein Precipitation
XIC of +MRM (35 pairs ): 255.1 /159 .3 Da ID : E2 from ... 5200 4000 2000 0 9.10 9.25 9.30 9.2 9.4 9.6 9.73 9.8 9.54

XIC of +MRM (45 pairs ): 255.1/159.3 Da ID: 17B Es tra... 9.49 Intensity, cps 6000 4000 2000 0

Max. 6666.7 cps .

E2 with SPE
10.16 10.45 9.91 10 10.

In te n sity, cp s

Calibrator Cal4 CSS

Calibrator Cal1
9.20

E2 with PP
10.25 9.94 10.2 10.39 10.4 Max. 4.4e4 cps .

10.0 10.2 10.4 Time, min XIC of +MRM (35 pairs ): 255.1/159.3 Da ID: E2 from S...
1.5e4 1.0e4 5000.0 0.0 9.2 9.4 9.6 9.8 10.0 10.2 Time min 10 .4

9.0 9.2 9.4 9.6 9.8 10.0 Time, min XIC of +MRM (45 pairs ): 255.1/159.3 Da ID: 17B Es trad...
4000 3000 2000 1000 9.0 9.2 9.4 9.6 9.8 Time, min 10.0

In te n sity, cp s

9.55

Intensity, cps

Serum re

Serum rep2

10.26

10.

10.2

10.4

SPE 500 L: - Detectable: E2, Aldosterone - Clean extract: long column life

PP 100 L: - Not detectable: E2, Aldosterone - Dirty extract: short column life

Internal Validation:
1. 2. 3. 4. 5. 6. LOD, LLOQ, ULOQ Linear range, R2>0.99 Selectivity Precision (Intra-, Interday) Accuracy (Intra-, Interday) Recovery (70-100% for all compounds) 7. Matrix effects: less than 50% due to clean extract from SPE, fully compensated by isotop labeled IS. 8. Stability: stock solution, sample freeze-thaw, sample and extract storage at 4C, -20C, open SPE plate. 9. Kit stability (shelf life, transport)

Cholesterol

Pregnenolone

Progesterone

11-Deoxycorticosterone

Corticosterone

Aldosterone

17-OH Pregnenolone

17-OH Progesterone 21-Deoxycortisol

11-Deoxycortisol

DHEA-S

DHEA

Androstenedione

Cortisol

Cortisone

Estrone (E1) E1-S Etiocholanolone Androsterone Testosterone 2-OH Estrone

Estradiol (E2) Dihydroandrosterone DHT Estriol (E3)

7-Dehydroestrone

External Validation: Proficiency test (Ringversuch DGKL)

Traditional Immunoassay Cobas e411 delivers too high values

Assay Workflow
The SteroIDQ Kit has been developed and validated for a LC-MS system consisting of a CTC PAL autosampler, an Agilent HPLC 1100/1200 binary pump, an Agilent column oven and an ABSciex 4000 / 4000QTRAP mass spectrometer with an ESI source. Other HPLC systems (for example from Shimadzu) have been also tested. Optionally the ABSciex 5500QTRAP mass spectrometer can be used to improved the sensitivity. The following workflow and estimated time scale is applied for a full 96-well plate configuration (80 wells for samples, the rest for calibrators, blank and QCs). With smaller numbers of samples(1/2 plate for 36 samples or 1/3 plate for 20 samples) the measurement time (step 3) will be reduced accordingly. 1. Preparation of the HPLC-MS/MS System
Prepare: mobile phases, wash solution, blank and Test mix, Purge HPLC-MS/MS instrument, Condition HPLC column, Perform System Suitability Test.

2 hours
(Only when changing from other assay)

SteroIDQ is the only LC-MSMS based method with Estradiol (E2) in its pannel and delivers perfectly accurate results

External Validation: beta testing in external laboratories

4 hours

2. Sample Preparation and Solid Phase Extraction

Prepare samples, IS, Calibrators and QCs, Condition SPE plate, Transfer samples, Calibrators and QCs onto SPE plate, Dry SPE plate under vaccum and N2 flow, Elute SPE plate with DCM (1st extract) and ACN (2nd extract).

3. Performing the HPLC-MS/MS Assay

Generate Analyst batch files, Perform HPLC-MS/MS runs with alternating injections, Total runtime: 20 min / sample

30 hours
(Depending on number of samples)

4 different laboratories, Different HPLC systems: Shimadzu and Agilent, Samples measured in 5 replicates, 3 levels of concentrations: low, medium, high. All operators deliver perfectly similar results

150%

Accuracy QC1
Operator1 Operator2

100%

50%

Operator3 Operator4

0% 150%

Accuracy QC2
Operator1 Operator2

100%

50%

Operator3 Operator4

0% 150%

Accuracy QC3
Operator1 Operator2

100%

50%

Operator3 Operator4

0%

12 2,3 15

7 6

8 10 9

4 5 1

14 11 13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Aldosterone Cortisol Cortisone Corticosterone 11-DOCSt E2 Testosterone 11-DOCSt E1 DHEA Androstenedione 17a-OHP Etiocholanolone Androsterone Progesterone

16

16 DHEAS

Conclusion
With 16 steroid hormones in ist pannel the SteroIDQ Kit is the most versatile LC-MSMS based platform to deliver high-throughput and accurate quantitative steroid measurement. Extensive internal and external validation have shown that the Kit is very reliable and robust.
BIOCRATES Life Sciences AG Innrain 66/2 6020 Innsbruck, Austria Tel: +43-512-57 98 23 Email:therese.koal@biocrates.com Website: www.biocrates.com

4. Data processing up to 4 hours

Perform data quantification with Analyst software, optionally with MultiQuant, Generate report of analysis. (Depending on number of samples)