CAH-Steroid profiling by LC-MS/MS

Kit for Quantitative Determination of 17-hydroxyprogesterone, Androstenedione, Cortisol,

11-deoxycortisol and 21-deoxycortisol in DRIED BLOOD SPOT Samples

Second-tier test
Congenital Adrenal Hyperplasia in newborns

KAT.003.1 / 25.10.2019 / Rev.0

Introduction
Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders that impair steroid
biosynthesis. CAH represents a continuous phenotypic spectrum with over 90% of all cases caused by
21-hydroxylase deficiency. CAH is the most common cause of genital ambiguity in the newborn and is present in
about 1 in 15,000 live births worldwide.

CAH kit allows for the simultaneous specific determination of 17-OHP and other steroids related to CAH such as
androstenedione, cortisol, 11-deoxycortisol, and 21-deoxycortisol in dried blood samples using LC-MS/MS.
Application of this technology to the determination of the above mentioned specific steroids in newborn blood spots
significantly enhances the correct identification of patients with CAH and reduces the number of false-positive
screening results when implemented as a second-tier analysis performed prior to reporting of initial newborn screen
results.

Sample Preparation

2 x 3.2 mm punch from DBS sample Add 200 μL Reagent E.

(calibrator, control, patient) Mix for 30 minutes at 250 rpm

Add 100 μL of Reagent D. Take 150 μL of extract.

Vortex for 30 seconds Evaporate until dryness at 40 °C

Centrifuge at 12.000 rpm for 5 minutes.

Inject 20 μL to LC-MS/MS system
Transfer 80 μL of clean supernatant to vial

Performance Data
Linearity Recovery
Between LLOQ and highest value measured Calculated by using certified reference materials

Analyte Linearity Range Analyte Recovery Rate

17-OHP 0.4-120 ng/mL 17-OHP %105.11

Cortisol 1.3-500 ng/mL Cortisol %93.76

Androstenedione 0.4-120 ng/mL Androstenedione %95.05

11-deoxycortisol 0.5-100 ng/mL 11-deoxycortisol %101.18

21-deoxycortisol 0.7-150 ng/mL 21-deoxycortisol %101.45

Reproducibility LC-MS/MS Parameters

*Intra-assay **Inter-assay System Requires LC-MS/MS

Analyte with sufficient sensitivity
precision (%CV) precision (%CV)
17-OHP % 5.6 % 8.7 Ionization ESI positive

Cortisol % 3.2 % 7.1 Flow 0.42 mL/min, gradient

Androstenedione % 6.2 % 7.8 Column Oven 40 °C
11-deoxycortisol % 4.8 % 8.1 Injection Volume 20 μL
21-deoxycortisol % 6.5 % 9.7
Run Time 9 minutes

Sample Chromatogram
(x100,00)

4,00 Cortisol

3,50

Androstenedione
3,00 17-OHP

2,50

2,00

11-DOC
1,50

1,00

21-DOC
0,50

0,00
5,5 5,7 5,9 6,1 6,3 6,5 6,7 6,9 7,1 7,3 min

Ordering Information

BR130022 CAH Kit 100 Tests

BR130023 CAH Kit 500 Tests
BR130023C1 CAH Kit Analytical Column
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