MSACL 2016 EU Abstract

Quantification of Serum Androstanediol Glucuronide by LC-MS/MS

Caroline Le Goff (Presenter)
University Hospital of Liege

Bio: I studied pharmacology at the University of Liège (Belgium). After my graduation in 2003, I followed a specialization in Clinical Laboratory Medicine. During this period, I obtained my certification in clinical pharmacy, in RIA and in Blood Draw. Starting on October 1st 2008, at 28 years old, I became a clinical Biologist/Pharmacist at the CHU of Liège in the clinical chemistry department. My main interests in clinical chemistry are, in particular endocrinology, cardiac, inflammatory and oxidative stress biomarkers and their application in mass spectrometry. In 2009, I began a PhD on the impact of intensive strenuous exercise on the liberation of specific cardiac biomarkers. My daily job is measuring different hormones and steroids such as cortisol, aldosterone, renin vitamin D and metabolites, catecholamines, metanephrines and their metabolites. I’m truly enthusiastic about the mass spectrometry development in clinical chemistry laboratory in Belgium. I am member of various scientific societies such as the Belgian Society of Clinical Biology, the Belgian Society of Clinical Chemistry and the European Society of Cardiology. Her activities have led to about 30 scientific papers and contributions in books as author (10) or co-author (22). I have presented 42 scientific communications at national and international congresses as author and 40 as co-author.

Authorship: C.Le Goff (1), N.Fabregat-Cabello (1), V.Stjokoski (1), T.Yilmaz (1), L. Vroonen(2),E.Cavalier(1)
(1) Department of Clinical Chemistry, University of Liège, CHU Sart-Tilman, Liège, Belgium (2) Department of Endocrinology, University of Liège, CHU Sart-Tilman, Liège, Belgium

Short Abstract

Plasma androstanediol-glucuronide (ADG) is considered by many authors to be a highly reliable parameter of peripheral androgenicity. Until recently, immunoassays were the only methods used for ADG determination. We validated a quantification method of the androsterone glucuronide to switch from the immunoassays to Liquid Chromatography triple quadrupole mass spectrometry. The optimization of the different steps (transitions, source, mobile phase injection volume and solide phase extraction) was performed. After, a validation of the method and a comparison between serum and plasma was carried out. Quantification was achieved by standard addition of natural ADG to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness.

Long Abstract

Introduction: Plasma androstanediol-glucuronide(ADG) is considered to be a highly marker of peripheral androgenicity(1).The quantification of steroidal glucuronide conjugates by indirect methods of immunoassays may underestimate some conjugates since hydrolysis is needed in sample processing(2). Since the early 1970s steroids have been typically analysed by immunoassays. Antibody–based methods can have good sensitivity but lack specificity, particularly in the competitive formats required for small molecule analysis (3–6). The major challenge relates to the antibody’s inability to recognize a single molecular structure to the exclusion of the metabolites of the target analyte and other related molecules present in the matrix (7–9). To overcome these limitations, which plague the analysis of many small molecules, clinical laboratories have increasingly turned to Liquid Chromatography (LC) coupled to Tandem Mass Spectrometry (MS/MS) (10–12) because of its high sensitivity, specificity, and an excellent reproducibility. LC-MS/MS has the additional advantage of simultaneous measurement of multiple analytes, high-throughput, and potential for very significant cost savings. These features explain the increasing popularity of the approach. LC-MS/MS instruments however require very specialized and specifically-trained technicians and are not impervious to analytical challenges, namely ion suppression (in electrospray mode) and coeluting isobaric compounds(10).

Aim of the Study: The aim of this work was to thoroughly validate a LC-MS/MS method for ADG determination in plasma and serum and to compare it with our previously employed ELISA.

Material and Methods:

Reagents and Materials.

ADG and d3-ADG were purchased from National Measurement Institute (Australia). Natural and labeled stock solutions were prepared by dissolving the corresponding standards in methanol LC-MS (1mg/L). All stock solutions were stored at -20 °C and employed to prepare daily diluted working standard solutions (50 and 5 ng/mL) in mobile phase (methanol: water (30:70)). From these stock solutions, plasma calibrators at concentrations of 0, 0.1, 0.5, 1.5 and 10 ng/mL were prepared in a blank plasma matrix (Golden West Biologicals Double–Stripped Plasma). The labeled internal standard was added to calibrators and samples at 20 ng/mL. Ammonium formate, formic acid were purchased from Fischer Scientific.

Sample preparation

A volume of 200 µL of calibrators and serum sample were placed into an eppendorf tube and mixed for 10 seconds with 20 µL of labeled internal standard at 20 ng/mL. After, samples were then allowed to rest 30 min at RT. Next, 200 µl of 2%formic acid solution was added and vortexed. A 96-well microSPE (2mg/well-Phenomenex, Torrance, USA) plate was conditioned with 200µL of 100% methanol followed by 200µL of 100nM ammonium formate buffer at a gravity flow. Samples were then loaded and some pressure applied. After washing with 200µL of 100mM ammonium formate followed by 200µL of 25% methanol, samples were eluted with 2X30µL of a 90/10methanol/water solution. Finally, 140µL of LCMS water was added before injection in the LC system.

Liquid chromatography and electrospray tandem mass spectrometry

We used a HPLC system AD20XR Shimazu consisting of vacuum degasser, auto sampler and a tertiary pump, equipped with a C18 Acquity HPLC BEH (1.7µm particle size, 2.1x3.00 mm) (Waters, USA). Injection volume was 30µL for prepared samples. The mobile phase A was composed of ammoniac, 1mM ammonium formate and LC-MS water. The mobile phase B was composed of 0.01% formic acid solution and 1mM ammonium formate in methanol.

The flow rate was 0.3ml/min. The mobile phase composition (30% B) was initially held constant for 2 min. Then the gradient was linearly ramped to 65% B to the 13.5 min mark. Mobile phase B was then increased to 95% for 1.5 min. Finally the column was re-equilibrated for 2 minutes at the initial conditions (30% B). The retention and cycle time were 10.93 and 18 min respectively.

The HPLC system was connected to triple quadrupole mass spectrometer TQ5500 (ABSCIEX, Framingham, Massachusetts, USA). The detection mode was MRM in negative mode. For ADG, the MRM transitions were: 486.35→257.2 (quantifier) and 486.35→275(qualifier). For the d3-ADG, the MRM transitions were: 489.4→278.2(quantifier) and 489.4→260.2(qualifier).Quadrupoles Q1 and Q3 were tuned to unit resolution and the MS parameters optimized for maximum signal intensity for each mass transition. Data acquisition and processing was carried out using Analyst 1.6.2 and calibration curves were prepared using 1/x.

LC–MS/MS validation and comparison with DiaSource ELISA(3-alpha-Diol)

The e-noval software (Arlenda, Belgium) was used to perform the more complex statistical calculations described below.

A calibration curve was prepared using 5 points (0.1, 0.5, 1, 5, 10 ng/mL) and responses were determined by calculating the integrated peak area ratio between endogenous ADG to d3- ADG. Three water samples and three serum samples depleted in steroids were spiked with a known concentration (0.2 ng/mL, 1ng/mL and 5 ng/mL) of ADG; these samples were run in triplicate on 3 different days to evaluate within and between-run CV. According to these results the precision(intra and inter-run) and an accuracy profile were established. In this profile, we set the acceptance limits at ±15% for serum for each level tested. We settled at each level, β-expectations with a probability of 95%. These β-expectations allow calculation of, with a probability of 95%, the variation at each tested point. To be considered as valid, the β-expectations of the method should be within the pre-defined tolerance limits.

Linearity of the calibration curves for serum was assessed by performing linear regression.

We evaluated recovery by spiking ADG from standard solutions into serum free steroids to achieve three final concentrations: low: 0.2 ng/mL, 1ng/mL and 5 ng/mL. Recoveries were calculated as the percentage difference between the quantity of ADG recovered from the spiked and unspiked sample divided by the quantity of the ADG added. By preparing three identical solutions in water (0.2 ng/mL, 1 ng/mL and 5 ng/mL), an experiment to assess matrix effects could also be performed.

The limit of detection (LOD) and limit of quantification (LOQ) were calculated with the lowest concentration that we tested (approximately 0.1 ng/mL for serum). LOD and LOQ were respectively defined as 3:1 and 10:1 signal/noise ratio respectively.

Finally, for method comparison, results of ADG obtained by LC-MS/MS in 10 serum and plasma from routine were compared together and with the DiaSource ELISA.


Validation of the LC-MS/MS.

To avoid coelution and so erroneous quantification, we tested the androsterone glucuronide and etiocholanolone glucuronide , indeed, they are alfa and beta isomers at carbon5. With our column and mobile phase, we obtained a good separation with different retention times for these different compounds. For ADG, the intra-run precision (CV) was 2.5-6.3% and between-run precision (CV) was 4.7-7.4%. Recoveries based on spiking experiments into natural matrix (95%CI: 94.3-107.5) and water (95%CI:101.2-111) did not differ significantly from 100%.

Within the calibration ranges, the linear regression model is fitted as a function of the introduced concentrations n order to obtain the following equation: Y=0.03078+0.9867X.

The LOD was determined using the lowest calibration point at a median concentration of 0.018 (+/-0.002) µg/L (n = 5) and the LOQ at 0.059(+/-0.006) µg/L (n = 5).

Ion suppression was calculated by comparing analyte peak areas obtained from spiked water samples solutions with those obtained from spiked serum samples, in both cases added before extraction. We observed 64±11% of ion suppression occurred for ADG.

For the comparison between LC-MS/MS and RIA, the Passing-Bablok test gave the following regression equation: Y=1.14+1.31X.The average median was 2.57 µg/L (95%CI: 1.18-6.3) for LC-MS/MS and 4.33 µg/L (95%CI: 2.53-10.3) for RIA. The difference between both was 4.6%. Between the serum and plasma in LC-MS/MS, the regression equation was: Y=0.09+0.92X , the difference was only 4.2% in average ( median average serum 2.57 µg/L (95%CI: 1.18-6.3) compared with average median in plasma 2.46 µg/L (95%CI:1.21-6.3)).

Discussion: The within run and total CVs calculated across the clinically important ranges were always lower than 7.4%. With respect to sensitivity, the LOQ obtained with LC-MS/MS (0.018µg/L) was much lower than these reported with ELISA methods (0.1 µg/L).

Recovery by spiking experiments results, either in water and matrix did not, on average, differ significantly from 100% and there was no evidence of endogenous interference observed with the ADG peak at 10.38 min retention.

Compared with LC-MS/MS, ELISA showed a marked positive bias in serum. Antibodies by their nature detect a family of structurally similar molecules, meaning that spurious elevations due to cross-reacting compounds become more of a problem in modern homogenous automated and manual immunoassays as compared to older methods, which required an initial extraction step.

Conclusions: We have validated the method by LC-MS/MS. We noted a significant bias between ELISA and LC-MS/MS. The limitations of our work were that we have recruited a limited number of samples and we have not determined the reference values yet, so more investigations are required. Finally, we urge the Clinical Chemistry community to develop an international standard reference material for steroids and a candidate reference method for LC-MS/MS.

References & Acknowledgements:


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2. Ke Y, Gonthier R, Isabelle M, Bertin J, Simard J-N, Dury AY, et al. A rapid and sensitive UPLC–MS/MS method for the simultaneous quantification of serum androsterone glucuronide, etiocholanolone glucuronide, and androstan-3α, 17β diol 17-glucuronide in postmenopausal women. J Steroid Biochem Mol Biol. Elsevier Ltd; 2015;149:146–52.

3. Dorrian CA, Toole BJ, Alvarez-Madrazo S, Kelly A, Connell JM WA. A screening procedure for primary aldosteronism based on the Diasorin Laision automated chemilumuniscent immunoassay for direct renin. Ann Clin Biochem. 2010;47:195–200.

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7. Fredline VF, Taylor PJ, Dodds HM, Johnson a G. A reference method for the analysis of aldosterone in blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. Anal Biochem. 1997;252(252):308–13.

8. Becker S, Kortz L, Helmschrodt C, Thiery J, Ceglarek U, Büttler RM, et al. Clinica Chimica Acta Measurement of dehydroepiandrosterone sulphate ( DHEAS ): A comparison of Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry ( ID-LC-MS / MS ) and seven currently available immunoassays. Clin Chim Acta. Elsevier B.V.; 2013;424(May 2012):68–75.

9. Allen KR, Azad R, Field HP BD. Replacement of immunoassay by LC tandem mass spectrometry for the routine measurement of drugs of abuse in oral fluid. Ann Clin Biochem. 2005;42:277–84.

10. Vogeser M, Kirchhoff F. Progress in automation of LC-MS in laboratory medicine. Clin Biochem. The Canadian Society of Clinical Chemists; 2011;44(1):4–13.

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12. Becker S, Kortz L, Helmschrodt C, Thiery J, Ceglarek U. LC–MS-based metabolomics in the clinical laboratory. J Chromatogr B. Elsevier B.V.; 2012;883-884:68–75.

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