All stocks and calibration levels were prepared in water: methanol (50:50) v/v to cover range of all steroid concentrations (refer Table 1).

Transcription

1 Application LCMS-8040 Simultaneous determination of 11 steroids and Vitamin D2/D3 in human serum using LC/MS/MS - Introduction Quantification of endogenous hormonal steroids and their precursors is essential for diagnosing a wide range of endocrine disorders. Historically, these analyses have been carried out using immunoassay, but such methods are problematic, especially for low-concentration analytes, due to assay interference by other endogenous steroids. MS offers improved specificity over immunoassay and can be highly sensitive. GC-MS, with use of stable isotopically labeled internal standards, is considered the Gold standard' method for serum steroid analysis. GC-MS is the method of choice for profiling steroid metabolites in urine, but these techniques are not appropriate for routine use in clinical laboratories owing to a need for extensive sample preparation, as well as analytical expertise. LC/MS/MS compares well to GC-MS in terms of accuracy, precision and sensitivity, but allows simplified sample preparation. While most publications have featured only one or a limited number of steroids, steroid paneling (which is proposed as the preferred term for multitargeted steroid analysis) has great potential to enable clinicians to make a definitive diagnosis. It is adaptable for use in a number of matrices, including serum, saliva and dried blood spots. However, LC/MS/MS-based steroid analysis is not straightforward, and understanding the chemical and analytical processes involved is essential for implementation of a robust clinical service. This application news discusses specific challenges in the measurement of endogenous steroids using LC/MS/MS, and provides examples of the benefits it offers. Experimental Preparation of standards All stocks and calibration levels were prepared in water: methanol (50:50) v/v to cover range of all steroid concentrations (refer Table 1). Analytical Conditions Nexera Parameters Column Column oven temp. Injection volume Mobile phase Flow rate Run time : Phenomenex C8 (30 mm L x 3 mm I.D. ; µ) : 40 ⁰C : 50 ul : A - 0.1% formic acid in water B - 0.1% formic acid in methanol : 0.4 ml/min : 6.0 min LCMS-8040 parameters MS interface : Atmospheric Pressure Chemical Ionisation (APCI) Polarity : Positive Nebulizing gas flow : L/min; Drying gas flow : L/min Interface temp. : 450 ºC Desolvation line temp. : 250 ºC Heat block temp. : 300 ºC Gradient Program : Time A Conc % B Conc % Stop

2 Application Sr. Protocol for sample preparation 250 µl of human serum samples were taken in 2 ml microcentrifuge tubes. 500 µl of acetonitrile was added and vortexed for 30 seconds. The microcentrifuge tubes were centrifuged for 10 mins at rpm. 500 µl of this supernatant with 500 µl of distilled water was taken in autosampler vials and mix thoroughly. These were then injected into the LC/MS/MS system. Table 1: Linearity levels of 11 steroids and vitamin D2/D3 Compounds STD1 STD2 STD3 STD4 STD5 STD6 STD7 STD8 ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 1 Androstenedione Cortisol Corticosterone Deoxycortisol DHEA DHEAS Estradiol Hydroxyvitamin D Hydroxyvitamin D Progesterone Hydroxyprogesterone Testosterone Aldosterone Table 2: Optimized MRM events of 11 steroids and vitamin D2/D3 Sr. Compounds MRM Transitions Q1 PreBias CE Q3 PreBias 1 Androstenedione > Cortisol 360> Corticosterone > Deoxycortisol > DHEA 270> DHEAS 270> Estradiol 250> Hydroxy vitamin D > Hydroxy vitamin D > Progesterone > Hydroxy progesterone > Testosterone > Aldosterone > Results and discussion LC/MS/MS results LCMS-8040 has a feature of Optimisation of method in which the mass spectrometer automatically selects the best product ion(s) and optimises voltages and collision energies for the precursor to product transition. Utilizing this feature, the MRM transitions were optimised (shown in Table 2) and used to determine quantitative sensitivity of all steroids and vit D2/D3. All 13 analytes were separated by UHPLC within 6 min. The feature of staggered MRM was used for data acquisition whereby the total run time was divided into different time segments for MRM acquisition based on retention times of the analytes.

3 Application Limit of Detection (LOD) / Limit of Quantitation (LOQ) The LOD and LOQ values as calculated through software from calibration curves for all analytes are tabulated in Table 3. LOD and LOQ were found well below lower levels of therapeutic range. Accuracy of linearity was also found in expected limits. Table 3: LOD, LOQ, therapeutic range and accuracy percentages of all 13 analytes Steroid Accuracy (%) Therapeutic Range (ng/ml) LOD (ng/ml) LOQ(ng/mL) Cortisol Corticosterone Deoxycortisol Androsteronedione Testosterone Progesterone Hydroxyprogesterone DHEA DHEAS HydroxyVit D HydroxyVit D Aldosterone Estradiol QC Sample Analysis The lyophilized BioRad QC samples made for immunoassay were reconstituted with distilled water and used for this analysis [1, 2, 3, 4 and 5]. The results of the QC samples as shown in Table 4 were well within the mentioned ranges. Table 4: Results of BioRad QC samples as analysed by LC/MS/MS Sr.no. Analyte Level 1* Level 2* Level 3* Result Range Result Range Result Range 1 Cortisol Androstenedione Testosterone Hyroxyvitamin D Progesterone DHEAS hydroxy progesterone Estradiol deoxycortisol < DHEA Aldosterone * All concentrations are in ng/ml.

4 Application 7.5 Corticosterone R 2 = Deoxycortisol (R 2 = 0.995) Conc. Progesterone (r 2 = 0.999) 4.0 Conc. Vit D3 (r 2 = 0.999) Conc. 4.0 Hydroxyprogesterone (r 2 =0.996) Conc. Vit D2 (r 2 = 0.999) 7.5 Conc. DHEA (r 2 =0.995) Conc. Aldosterone (r 2 = 0.998) 1 1 Conc. Area(x10,000,000) DHEAS (r 2 = 0.995) Conc. 1 1 Conc. 3.5 Estradiol (r 2 = 0.996) 1 1 Conc. The Linearity curves for all 13 analytes have been shown in Figure 1.

5 Application 7.5 Cortisol ( r 2 = 0.998) Androsteronedione (r 2 =0.999) Conc Conc. Testosterone (r 2 = 0.998) Conc. Conclusion Figure 1: Calibration curves of all 13 analytes An application was successfully developed on LC/MS/MS system for simultaneous analysis of 11 steroids along with vit D2/D3 from human serum with the ultra high sensitivity and accuracy. The outstanding features of LCMS-8040 system like UF-Scanning (15,000 u/sec) and Ultrafast MRM transition capability (555 MRM transitions/sec) along with lowest dwell time (0.8msec) and pause time (1 msec) ensured the acquisition of good quality data and reliable results even with the use of UHPLC analysis with short runtimes. The high specificity and ease of maintenance of LCMS-8040 system, enables less time for serum sample preparation. References 1. Koal T, Schmiederer D, Pham-Tuan H, Röhring C., Rauh M. J Steroid Biochem Molecul Biolog 129 (2012) Van den Ouweland JM, Beijers AM., J Chromatogr B Analyt Technol Biomed Life Sci May 1;878(15-16): Susan S.C. Tai and Michael J. Welch Development and evaluation of a Candidate Reference material for the Determination of Total Cortisol in Human Serum Anal. Chem.2004,76, Robert L. Taylor, Dwaine Machacek, et. Al. Validation of a High-Throughput Liquid Chromatography- Tandem Mass Spectrometry Method for Urinary Cortisol and Cortisone Clin.Chem : Song-Ja Park, Yun-Je Kim et. Al. Analysis of Corticosteroids in Urine by HPLC and Thermospray LC/MS Journal of Anal. Toxicology Vol.14 March/April 1990 Shimadzu Analytical (India) Pvt.Ltd. 1 A/B, Rushabh Chambers, Makwana Road, Marol, Andheri (East), Mumbai , India. Tel: Fax: Copyright 2013 SHIMADZU ANALYTICAL (INDIA) PVT. LTD. All rights reserved. No part of this document may be reproduced in any form or by any means without permission in writing from SHIMADZU ANALYTICAL (INDIA) PVT. LTD.