Quantitation of Steroids in Serum by UHPLC-Triple Quadrupole Mass Spectrometry

This study demonstrates a simple, rapid, and reliable method for the simultaneous quantitation of 8 steroid hormones and their metabolites in serum using the Bruker Elute™ UHPLC coupled to the EVOQ™ LC-TQ Elite MS/MS system. Sample preparation was performed via protein precipitation using a ClinMass® Complete Kit for Steroids in Serum/Plasma.

Introduction

Steroids are biologically active compounds found in animals, plants, and fungi. They can act as hormones, serving as neuro- endocrine regulators of both

physiological development and critical bodily functions, including metabolic control, stress and immune response, and main-tenance of water and mineral balance. All steroids are fat-soluble lipids which pass through cell

membranes to be distributed through the blood stream, and they share a core structure of three cyclohexane rings and a cyclopentane ring.

Keywords: Steroids, serum, quantitation

Authors: Rafaela Martin, Zoltan Czentnar; Bruker Daltonik GmbH, Bremen, Germany

Steroid hormones are divided into two broad classes, corticosteroids and sex steroids, with sub-classifications based on the receptors to which they bind. Corticosteroids include glucocorticoids (e.g., cortisol) and mineralocorticoids (e.g., aldosterone). Sex steroids include androgens (e.g., testosterone), estrogens (e.g., estradiol), and progestogens (e.g., progesterone). These classi-fications can refer to endogenous steroid hormones naturally produced from cholesterol or synthetic hormones produced as pharma- ceutical medications.

Many genetic disorders are related with abnormalities in steroid hormone production, and other disease states (or treatments for the disease) often have indirect effects on the endocrine system. Dramatic shifts in steroid hormone levels can also be observed during growth, maturation, and aging processes, or from environmental exposure to endocrine disrupting chemicals (EDCs) or steroid abuse. The accurate quantitation of steroid hormone levels, particularly as affected by a disease state or the administration of therapeutics, is a key element of modern clinical research.

The method described in this manu-script focuses on the rapid and reliable quantitation of steroid hormones in human serum by UHPLC-triple quadrupole mass spectrometry.

ExperimentalThe analysis was performed on an Bruker EVOQ™ LC-TQ Elite system coupled to an Elute UHPLC using the ClinMass® Complete Kit for Steroids in Serum/Plasma (RECIPE Chemicals + Instruments GmbH, Munich, Germany, MS12000) which included the mobile phase, autosampler washing solution, precipitation reagent, and the HPLC column.

Table 1: Mass Spectrometry Method Conditions

Liquid Chromatography

Instrument Bruker Elute UHPLC

Column ClinMass MS12030 (RECIPE)

Mobile Phase A Included within the ClinMass MS12000 Kit (RECIPE)

Mobile Phase B Included within the ClinMass MS12000 Kit (RECIPE)

Gradient Time [min] Mobile Phase B [%] Flow rate [ml/min]

0.00 27 0.5

4.50 57 0.5

4.51 100 0.5

4.64 100 0.5

4.75 100 0.8

5.35 100 0.8

5.36 27 0.8

5.65 27 0.8

5.66 27 0.5

6.00 27 0.5

Injection Volume 20 µL

Column Oven 40°C

Mass Spectrometry

Instrument Bruker EVOQ™ LC-TQ Elite

Ion Source VIP H-ESI positive and negative, 4500 V

Probe Gas 50 units at 450°C

Cone Gas 20 units at 350°C

Nebulizing Gas 50 units

Active Exhaust on

Collision Gas Argon, 1.5 mTorr

MRM Transitions see Table 2

Table 2: Retention times, MRM transitions, calibration ranges, and RSD RF of calibration curves

Analyte Retention time (min)

Precursor Ion

Product Ion 1

CE 1 (V) Product Ion 2

CE 2 (V) Calibration Range [µg/L]

RSD RF [%]

11-Deoxycortisol 3.18 347.1 109.2 25 97.2 23 0.100 – 13.4 6.8

21-Deoxycortisol 2.84 347.2 311.2 14 121.1 24 0.117 – 14.7 11.7

17-Hydroxyprogesterone 4.56 331.2 97.2 21 109.1 24 0.109 – 12.5 4.2

21-Hydroxyprogesterone 4.38 331.2 97.2 20 109.2 25 0.196 – 24.4 9.0

Androstendione 4.37 287.1 97.2 20 109.2 22 0.117 – 12.7 3.8

Cortisol 2.08 363.2 121.1 30 97.1 33 2.75 – 325 4.4

DHEAS* 2.64 367.1 97.1 27 80.2 73 43.9 – 4851 2.2

Testosterone 4.19 289.2 97.2 22 109.2 24 0.099 – 11.0 4.9

Samples and Sample Preparation

Serum calibrators (MS12013) and three ClinChek® quality con-trol samples (low, medium, high concentration; MS12083) were provided by RECIPE. In addition, the serum sample of a test individual (healthy female, 33 years old)

was analyzed. The quality control samples and the serum test sample were measured in quadruplicate as technical replicates.

Following the protocol of the ClinMass® kit, 50 µL serum samples were added to 75 µL precipitation reagent containing the isotopically

labelled internal standards and vortexed for 1 minute. After precipi- tation for 10 minutes and a 10 second vortex, the samples were centri-fuged for 10 minutes at 14,000 x g. The supernatants then were transferred to HPLC vials for analysis by UHPLC-MS/MS.

Results and Discussion

Following fast and simple sample preparation requiring only 50 µL of serum, the chromatographic separation of the eight analytes was performed within 6.0 minutes using the Elute UHPLC system. Figure 1 illustrates an overlay of the MRM traces for all measured analytes.

The quantitation of the analytes was performed using six isotopically labelled internal standards. Calibra-tion curves included six calibrator levels and provided excellent linearity with RSD RF values from 2.2 – 11.7 % (Table 2). In a perfect calibration curve, the RF (response factor = relative area/amount) is the same for each level. The RSD [%] of the RF values of all calibrators is useful to

*DHEAS measured in ESI negative mode, all other analytes in ESI positive mode

Figure 1: Overlaid MRM traces of all analytes (lowest calibrator level)

kCps

7.5

5.0

2.5

0

1.5 2.0 2.5 4.03.0 3.5 4.5Minutes

Table 3: Quantitative results, bias and relative standard deviation of fourfold measurement of Quality Controls I – III and test sample

Sample QC I QC II QC III Test Sample

AnalyteActual Value [µg/L]

Bias [%] RSD [%]Actual Value [µg/L]

Bias [%] RSD [%]Actual Value [µg/L]

Bias [%] RSD [%]Actual Value [µg/L]

RSD [%]

11-Deoxycortisol 0.204 -2.3 5.9 0.644 -0.4 4.8 3.58 0.1 2.9 0.304 2.1

21-Deoxycortisol 0.237 3.2 5.7 0.752 9.0 5.8 4.18 13.2 3.6 < LOQ -

17-Hydroxyprogesterone 0.197 -6.2 5.0 0.571 -6.4 3.5 3.46 2.3 3.8 1.57 2.6

21-Hydroxyprogesterone 0.323 -11.3 2.5 1.13 7.2 5.1 5.98 -0.5 9.0 < LOQ -

Androstendione 0.223 -4.3 8.1 0.613 -0.2 2.3 3.19 -1.3 5.4 1.27 8.8

Cortisol 5.18 6.2 2.2 14.5 -0.1 1.1 79.9 -0.2 2.4 124 1.9

DHEAS 79.0 -4.4 2.4 224 -0.9 2.4 1240 1.1 0.5 1853 2

Testosterone 0.193 -0.1 2.6 0.534 -1.6 3.5 3.03 4.4 2.1 0.395 2.2

evaluate the quality of a calibration curve. Acceptable values for RSD RF are < 15 – 20%. The calibration curve of 11-Deoxycortisol is shown as an example in Figure 2.

Quality controls (QC) in serum with low (QC I), middle (QC II) and high (QC III) concentrations, as well as

a serum sample of a test individual were measured four times. The RSD of ≤ 9% for all analytes underlines the very good precision of the instrumen-tation used. The experiments also showed a high accuracy with a bias within ±9% for 91% of all determi-nations (max. value 13.2%). Detailed results are presented in Table 3. The

values for precision and accuracy are well within the range required by common guidelines for quantitative results. In the sample of the test individual, all targeted analytes could be detected, and all quantitative results were within reference ranges reported in the literature.

H H

H

O

O

OH

OH

Sig

nal (

rela

tive

peak

are

a)

9.0

4.0

5.0

6.0

7.0

8.0

3.0

2.0

1.0

0

Concentration (µg/L)14.013.00 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

Figure 2: Calibration curve of 11-Deoxycortisol. Calibration range 0.1 – 13.4 µg/L

11-Deoxycortisol, linear (R2: 0.99868) (σRF 6.79)CalibrantQC

Conclusions

• The Bruker Elute UHPLC coupled to the Bruker EVOQ™ LC-TQ Elite system and the ClinMass® Complete Kit for Steroids in Serum/Plasma provide a quick and reliable method to easily detect and quantitate eight steroids in serum.

• Low sample requirements (50 µL serum), easy preparation, and short run time (6.0 minutes) support high sample throughput. Linearity of calibration, precision, and accuracy were outstanding, supporting the use of this combined system in clinical research workflows.

Acknowledgement

The authors acknowledge RECIPE Chemicals + Instruments GmbH (Munich, Germany) for providing the ClinMass kit.

Further Reading

To learn more about the Bruker EVOQ™ LC-TQ Elite and Elute LC systems, please see: https://www.bruker.com/products/mass-spectrometry-and-separations/lc-ms/evoq/overview.html https://www.bruker.com/products/mass-spectrometry-and-separations/lc-ms/liquid-chromatography/elute-lc-series/overview.html

Please see Application Notes LCMS-138, LCMS-139, LCMS-145, LCMS-146, and LCMS-147 for examples of quantitation of clinically relevant drug panels using the ClinMass TDM Platform.

Quantitation of Neuroleptics in Serum by UHPLC-Triple Quadrupole Mass Spectrometry

This study demonstrates a simple, rapid, and reliable method for the simultaneous detection and quantitation of 28 neuroleptics in serum using the Bruker Elute™ UHPLC coupled to the EVOQ LC-TQ Elite triple quadrupole MS/MS system. Sample preparation was performed with protein precipitation using the ClinMass® TDM kit system.

Introduction

Neuroleptics are psychoactive drugs used for the treatment of psychoses such as hallucinations or paranoia, as well as schizophre-

nia and bipolar disorder. They are also applied as sedatives against restlessness or anxiety, especially for elderly people in continuous care facilities. They are the second most prescribed class of

psychotropic drugs following anti-depressants. Several substance groups, including phenothia- zines, butyrophenones and dibenzazepines, are classified as neuroleptics.

Keywords: Neuroleptics, serum, quantitation, therapeutic drug monitoringClinical Research, Forensic Toxicology, EVOQ LC-TQ MS/MS, Elute UHPLC

Author: Rafaela Martin, Bruker Daltonik Applications Quadrupole MS, Bremen, Germany

Quantitation of Antidepressants in Serum by UHPLC-Triple Quadrupole Mass Spectrometry

This study demonstrates a simple, rapid, and reliable method for the simultaneous quantitation of 26 antidepressants in serum using the Bruker Elute™ UHPLC coupled to the EVOQ™ LC-TQ Elite triple quadrupole mass spectrometry system. Sample preparation was performed via protein precipitation using the ClinMass® TDM kit system.

Introduction

Antidepressants are psycho- active drugs mainly used for the

treatment of major depressive disorders, but also for mild depression, anxiety, eating dis-orders, and pain relief. They are

the most prescribed psychotropic drugs, with a growing number of prescriptions throughout the world.

Keywords: Antidepressants, serum, quantitation, therapeutic drug monitoring, clinical research, forensic toxicology, EVOQ LC-TQ Elite, Elute UHPLC

Author: Rafaela Martin, Bruker Daltonik Applications Quadrupole MS, Bremen, Germany

Quantitation of Antiepileptics in Serum by UHPLC-Triple Quadrupole Mass Spectrometry

This study demonstrates a simple, rapid, and reliable method for the simultaneous quantitation of 26 antiepileptics in serum using the Bruker Elute UHPLC coupled to the EVOQ LC-TQ Elite MS/MS system. Sample preparation was performed via protein precipitation using the ClinMass TDM kit system.

Introduction

Antiepileptics, also known as anticonvulsants, are used for the treatment of epileptic seizures which are characterized by convulsions and reduced con-sciousness. A person undergoing a seizure is at risk of severe injury

due to falling or striking other objects. As seizures can happen at any time and often result in significant muscle fatigue, epilepsy causes both psychologi-cal and physical strain. Due to the repeating rhythm of the seizures, antiepileptics must be taken permanently, and the best effects

are observed at a stable blood concentration. As each individual metabolizes drugs at a different rate, it is important to regularly monitor drug (and often metabolite) concentrations in blood to ensure the correct dosage.

Keywords: Antiepileptics, serum, quantitation, therapeutic drug monitoring

Authors: Rafaela Martin, Zoltan Czentnar; Bruker Daltonik GmbH, Bremen, Germany

Quantitation of Benzodiazepines in Serum by UHPLC-Triple Quadrupole Mass Spectrometry

This study demonstrates a simple, rapid, and reliable method for the simultaneous detection and quantitation of 35 benzodiazepines in serum using the Bruker Elute UHPLC coupled to the EVOQ LC-TQ Elite MS/MS system. Sample preparation was performed via protein precipitation using the ClinMass TDM kit system.

Introduction

Benzodiazepines and the so-called z-drugs are among the most prescribed drugs worldwide as they have sedative, hypnotic, anxiolytic, anticonvulsant and muscle relaxant properties; however, they also bear a risk

of dependence and have a very high rate of abuse. Therefore, they are not suitable for per-manent treatment except as an antiepileptic agent. During benzodiazepine therapy, the ability of patients to drive is strongly impaired as reaction times are negatively affected, making this

group of analytes of interest for forensic toxicology as well.

The half-life of benzodiazepines varies from a few hours to some days. Often the metabolites are also pharmacologically effective, and those with a long half-life can accumulate in the body.

Keywords: Benzodiazepines, serum, quantitation, therapeutic drug monitoring

Authors: Rafaela Martin, Zoltan Czentnar; Bruker Daltonik GmbH, Bremen, Germany

Quantitation of Tricyclic Antidepressants in Serum by UHPLC-Triple Quadrupole Mass SpectrometryThis study demonstrates a simple, rapid, and reliable method for the simultaneous quantitation of 13 tricyclic antidepressants and their metabolites, as well as two atypical neuroleptics, in serum using the Bruker Elute UHPLC coupled to the EVOQ LC-TQ Elite MS/MS system. Sample preparation was performed via protein precipitation using the ClinMass TDM kit system.

Introduction

Antidepressants are psycho- active drugs mainly used for the treatment of major depressive disorders, but also for anxiety or pain relief. Various classes of antidepressant drugs are prescribed today, including

selective serotonin and serotonin- noradrenalin-reuptake inhibitors, monoamine oxidase inhibitors, and tricyclic compounds. Tricyclics were the first group of antidepressants, developed in the 1950s, and have a common tricyclic ring structure with different substituents.

Antidepressants are usually taken for an extended period of time, with the best effects observed at a stable blood concentration. Due to the narrow therapeutic range of tricyclic antidepressants, it is important to monitor their concentration in blood. Overdosing can lead

Keywords: Tricyclic antidepressants, serum, quantitation, therapeutic drug monitoring

Authors: Rafaela Martin, Zoltan Czentnar; Bruker Daltonik GmbH, Bremen, Germany

Bru

ker

Dal

toni

cs is

con

tinua

lly im

prov

ing

its p

rodu

cts

and

rese

rves

the

rig

ht

to c

hang

e sp

ecifi

catio

ns w

ithou

t no

tice.

© B

ruke

r D

alto

nics

09

-201

9, L

CM

S-1

50, 1

86

624

5

Bruker Daltonik GmbH

Bremen · GermanyPhone +49 (0)421-2205-0

Bruker Scientific LLC

Billerica, MA · USA Phone +1 (978) 663-3660

For Research Use Only. Not for Use in Clinical Diagnostic Procedures.

ms.sales.bdal@bruker.com – www.bruker.com

Learn MoreYou are looking for further Information? Check out the link or scan the QR code to learn more about Bruker’s complete line of scientific products and instrumentation.

www.bruker.com