Michael J Wright (1), Renee C Sahertian (1), Chris P White (1) (2)
1) Department of Clinical Chemistry and Endocrinology, SEALS, Prince of Wales Hospital, Sydney, Australia, 2) The Royal Hospital for Women, Sydney, Australia
Estradiol is arguably the most potent sex steroid in human biology requiring relatively small concentrations to exert large physiological effects in comparison to its precursor, testosterone. However, the delivery of a clinical diagnostic estradiol service is a significant challenge facing many laboratories. In this study we look at a number of sample extraction options, chromatographic techniques and multistage fragmentation (MRM3) using quadrupole linear ion trap instruments to create robust LC-MS methods that meet the requirements of low level measurement. In this presentation we propose three different methods to meet the needs of our diverse patient population.
Estradiol is arguably the most potent sex steroid in human biology requiring relatively small concentrations to exert large physiological effects in comparison to its precursor, testosterone. It is crucial for the development of female secondary sexual characteristics, growth and skeletal maturation, maintaining cyclic menses, libido and pregnancy.
In contrast to the relatively high serum levels of estradiol measured at the time of spontaneous ovulation or assisted reproduction the more technically challenging task is the measurement of low levels which can be of clinical value in a diverse array of disorders. These include girls with premature puberty and thelarche, disorders of sexual differentiation, men with gynaecomastia, women with menstrual irregularities, postmenopausal women in danger of developing osteoporosis and breast cancer survivors treated with aromatase inhibitors.
Measuring low levels to establish the degree of endogenous estradiol production in patients undergoing therapy for breast cancer may be important in guiding treatment options and evaluating the anticancer efficacy of some of the hormonal therapies that are used to treat the disease. The Endocrine Society position statement calls for methods to be able to distinguish between suppressed levels of less than 3.7pmol/L and pre-treatment levels in the range of 37-55pmol/L, however, creating robust LC-MS/MS methodology to match this requirement is challenging.
In this study we investigated a number of sample preparation, chromatographic and mass spectrometry techniques to create a robust LC-MS assay for low level estradiol measurement. We then assessed the pros and cons of two immunoassays and two LC-MS assays to meet the requirements of this broad patient population.
Initial sample preparation methods involved a 2:1 concentration step with 200µL of patient serum extracted by liquid-liquid extraction (LLE) and reconstituted in 100µL. LC-MS Analysis was performed on QTRAP5500 and QTRAP6500 quadrupole linear ion trap instruments.
To investigate improvements in assay sensitivity the following parameters were altered 1) Sample volume used for extraction 2) sensitivity of MS instrumentation 3) Phospholipid depletion.
To improve selectivity we investigated chromatographic efficiency along with multistage fragmentation to create MRM3 transitions
Trueness and imprecision of the LC-MS assay were determined using Human Serum Certified Reference Materials (BCR-576, BCR-577 & BCR-578) from the European Commission Joint Research Centre.
Excess serum samples from the routine clinical services at Prince of Wales Hospital and the Royal Hospital for Women, Sydney, Australia, were used for comparison studies between the LC-MS method, the Immulite 2000 Estradiol immunoassay and the Pantex High Sensitivity Estradiol RIA.
The use of larger sample volumes increased signal intensity in an almost linear fashion but generated decreasing returns when signal:noise ratios were calculated. Similarly the move to a more sensitive mass spectrometer improved the signal:noise ratio of aqueous standards by 4 fold but only yielded a 2.1 fold increase when extracted patient samples are measured.
Phospholipids proved to be problematic following LLE sample preparation This was partially alleviated by changing the extraction solvent from MTBE to 60:40 Hexane:Ethyl Acetate and by moving to supported liquid extraction (SLE). Extended chromatography enabled better separation from co-eluting interferences and improved selectivity and sensitivity at the expense of longer run times. Utilising these conditions a simple and robust assay was developed for measuring E2 levels between 10-5,000pmol/L
Deming regression analysis demonstrated good correlation between LC-MS and the Immulite 2000 IA for estradiol levels between 400-2200pmol/L (y=0.99x+121pmol/L), however, this agreement worsened between 200-400pmol/L (y=1.21x+80pmol/L) and demonstrated poor correlation between 70-200pmol/L (y=2.5x-14pmol/L).
Deming regression analysis demonstrated poor correlation between LC-MS and the Pantex High Sensitivity Estradiol RIA (y=2.16x-13pmol/L) between 18-100pmol/L.
The use of Hybrid SPE phospholipid depletion in combination with either LLE or SLE further reduced phospholipids but interestingly also reduced back-ground noise. This enabled the realisation of sensitivity gains from extracting larger sample volumes.
Investigation of multistage fragmentation produced the MRM3 transition 271.1>145.1>143.1 which maintained sensitivity whilst improving selectivity. However, the improvements from this MRM3 transition were only recognised when the primary fragment ion collection window was offset to prevent the m/z 143.1 primary fragment from being held in the ion trap. The combination of increasing the sample volume to 400µL along with employing the Hybrid SPE and multistage fragmentation resulted in an LC-MS assay capable of reliably measuring E2 levels between 3-200pmol/L
The Immulite 2000 immunoassay demonstrated good agreement and precision with LC-MS assay for analysis of samples with E2 levels between 400 and 2500pmol/L and, despite a positive bias and worsening precision between 200 and 400pmol/L, it may continue to prove adequate for serial measurements in women who require assisted reproduction.
The LC-MS method employing 200µL of serum, SLE extraction and extended chromatography gave acceptable selectivity and sensitivity for measurements between 10-5000pmol/L. This assay is easily automatable and can be run on either of the two MS systems tested in this study. This assay should be suitable for young girls with precocious puberty, women requiring the initial investigation of amenorrhea, infertility and menopause and men.
The adjusted LC-MS method employing larger amounts of serum, Hybrid SPE clean-up and MRM3 detection gave acceptable selectivity, sensitivity and robustness for measurements down to 3pmol/L. The drawbacks of this assay include the more extensive sample clean up, greater consumable cost, frequent maintenance of the QLIT and restriction to the more sensitive QLIT instruments. This higher sensitivity LC-MS assay may be suitable for patient populations such as those with breast cancer undergoing aromatase inhibition.