ARTICLE pubs.acs.org/jchemeduc
Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography Mass Spectrometry Xia Xu and Timothy D. Veenstra* Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702, United States ABSTRACT: The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC MS and GC MS) have dramatically increased the capability to quantitatively measure sex steroids in complex biologic samples. Although it is important to optimize and calibrate the LC MS and GC MS steps, other areas such as sample preparation, molecular derivatization, and data analysis need to be carefully considered to minimize assay variation. The specific steps necessary for an effective quantitative assay are highly dependent on the type of sex steroid(s) being measured and the analytical steps described in this article will not be pertinent to every analyte. In this article, however, many of the steps necessary to develop an effective assay for sex steroids will be reviewed with a focus on areas that contribute to variation within the final quantitative results. The development of robust quantitative assays with high precision, accuracy, and reproducibility will continue to contribute to our understanding of the specific roles sex steroids play in diseases such as cancer. KEYWORDS: Graduate Education/Research, Second-Year Undergraduate, Upper-Division Undergraduate, Analytical Chemistry, Biochemistry, Textbooks/Reference Books, Gas Chromatography, HPLC, Hormones, Mass Spectrometry
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ex steroids play a role in a number of physiological processes outside of their traditional functions within the reproductive systems. Whereas these steroid hormones control reproduction, they also affect immunity, cardiovascular systems, central nervous systems, bone structure, and metabolism.1,2 Owing to their link to a number of cancers of the reproductive system and other conditions, sex steroids are important clinically being used in diagnostic, therapeutic, and prognostic approaches.3 The importance of steroids in general is well summarized by William Coleman, who in a recent edition of this Journal stated “Students at all levels should be familiar with the basic steroid structure and its significance in human biochemistry. Biochemistry students certainly should understand the detailed mechanism of action of a number of steroidal hormones.”4 Steroids are a group of fat-soluble organic compounds whose structure is based on four rings composed of 17 carbon atoms as shown in Scheme 1 for a number of the more commonly known steroids. As shown in the scheme, all sex steroids are derived from the precursor cholesterol. The three main classes of sex steroids are estrogens, androgens, and progestogens. Owing to their masculinizing effects, androgens have been considered male sex hormones, and estrogens and progestogens have been associated with female effects; however, many studies have now shown the importance of each class to both sexes.5 Each of these three main classes is composed of a large number of compounds whose functions can significantly vary. An excellent example is illustrated by the disparate role that various estrogen metabolites are hypothesized to play in breast cancer. One hypothesis of estrogens role in breast cancer Copyright r 2011 American Chemical Society and Division of Chemical Education, Inc.
involves the catechol estrogens, mainly 2-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestrone, reacting with DNA to form both stable and depurinating adducts. These reactions can result in other types of oxidative DNA damage potentially leading to cell transformation and cancer initiation.6 8 The estrogen, androgen, and progestogen families are composed of a variety of different sex steroids that can have very different functions. Connecting the function to the molecule requires assays that can measure as many of the individual sex steroids as possible. This demand requires assays that specifically measure the sex steroids of interest with minimal cross reactivity toward other compounds. This need requires careful consideration of the type of assay that is best suited to measure the sex steroids of interest. Current methods for measuring endogenous sex steroids include radioimmunoassay,9 enzyme immunoassay,10 high-performance liquid chromatography (HPLC) with electrochemical detection,11 stable isotope dilution combined with analysis using liquid or gas chromatography MS (LC MS or GC MS).12 Although each method has its advantages and disadvantages, the purpose of this article is not to discuss the relative merits of each technique; the focus is to examine sources of variability that apply to LC MS and GC MS. The general steps required in the analysis of sex steroids by GC or LC MS are given in Figure 1. The use of MS for quantitatively measuring Published: November 15, 2011 230
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Scheme 1. Structures of Six Commonly Measured Human Steroids Illustrating the Distinctive Four-Ring, 17-Carbon Core
Figure 1. Basic steps required in the analysis of sex steroids using liquid chromatography or gas chromatography mass spectrometry (LC MS or GC MS).
The two most popular types of extraction methods utilized for LC and GC MS analysis of sex steroids are liquid liquid and solid-phase extractions.13 In liquid liquid extractions, a solvent having a greater solubility coefficient for the sex steroids of interest is added to the biologic sample. Owing to their greater solubility in the added solvent, the sex steroids will partition into this liquid. In solid-phase extraction, the biologic mixture is introduced to a solid material. The solid material can be designed to bind either the sex steroids of interest or unwanted materials. If the solid material binds the sex steroids, they are eluted using a solvent in which they have very high solubility. Regardless of the strategy chosen, the aim is to simplify the mixture prior to LC or GC MS analysis. The key parameters to minimize variation at this stage are simplicity, extraction efficiency, and reproducibility. Simplicity is important as it is well accepted in analytical chemistry that there is a direct correlation between the number of sample handling steps and variability. Extraction efficiency should be as close to 100% as possible for the sex steroids of interest. Optimal extraction efficiency benefits downstream methods by providing as much material as possible for detection. Reproducibility is probably the most critical of the three parameters. Irreproducibility in any step within sample preparation creates an uncertainty in the results that is virtually impossible to normalize. A simpler method of sex steroid extraction is protein precipitation. In this method, an organic solvent (such as methanol) is added to the sample causing the proteins to precipitate leaving the sex steroids and other metabolites in solution. The protein precipitate is removed via simple centrifugation. Although this method is simple, it suffers from incomplete protein precipitation as some globulins can remain in solution. These protein remnants can have a detrimental effect on the ability to resolve the sex steroids using liquid chromatography. To determine extraction efficiency and reproducibility, standard solutions containing known amounts of the sex steroids of interest are prepared using the proposed sample preparation method. The standard solutions should be prepared using the same matrix used in the analytical study (e.g., serum, plasma, urine, etc.). The matrix needs to be charcoal stripped to remove any endogenous sex steroids prior to adding known amounts of each compound. These standard samples are taken through the entire process and the amount of material quantitated using LC or GC MS analysis is then compared to the amount used to prepare the standards. Obviously the best-case scenario would
molecules within complex biological samples continues to grow and it is critical that scientists in all fields of chemistry and biochemistry become familiar with the factors required to produce accurate results using this technology. Although their popularity continues to increase, the hands-on educational opportunities to learn LC and GC MS especially at the undergraduate level are sparse owing to the cost of the systems and their demand for research-oriented programs. As with any analytical technique that quantitatively measures steroid hormone levels in biologic samples, both LC and GC MS have a number of steps that can introduce variation to the final result. Although we tend to think of only the instrumental analysis, the results generated by both GC MS and LC MS methods are affected by sample preparation, derivatization, instrumentation, and data analysis. In this article, many of these steps will be examined and ways to minimize the variation associated with each are suggested.
’ SAMPLE PREPARATION Sample preparation is potentially the greatest source of variability in the analysis of sex steroids when using LC and GC MS. Although LC and GC MS utilize chromatography steps to separate molecules within complex mixtures prior to their detection, an extraction step as part of the sample preparation is vital to obtaining reproducible quantitative measurements. This extraction step serves to create a simplified mixture that is enriched with the sex steroids of interest (as well as other nonsteroidal metabolites) but devoid of contaminating biomolecules such as proteins. A good rule of thumb in designing a sample preparation procedure is given by the acronym KISS; keep it simple, scientist. The simplest sample preparation method that provides highly accurate, precise, and reproducible steps will generally have less variability by virtue of it having fewer sample handling steps. 231
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result in 100% extraction efficiency. If the extraction efficiency is significantly less than 100%, the assay will still be effective as long as the amount extracted is reproducible.
’ DERIVATIZATION Although most commonly associated with GC MS, derivatization is commonly used for LC MS analysis of sex steroids. A number of agents have been used for derivatizing sex steroids for both GC MS (e.g., pentafluorobenzoylbromide, pentafluorobenzoyltrimethylsilyl, and N-methyl-N-(trimethylsilyl)-trifluoroacetamide) and LC MS (e.g., dansyl chloride) analysis.14 Derivatization serves manifold purposes including stabilizing the sex steroids, especially polar compounds such as estrogens that are thermolabile and prone to thermal decomposition. For LC MS measurements, the primary need for derivatization is sensitivity. Many sex steroids, particularly estrogens, are present in biologic samples at low levels. These steroids also have low electrospray ionization efficiencies (i.e., they do not readily form ions). Since MS relies on the detection of ions, if the compound does not readily ionize within the source, it will not be readily detected. Derivatizing sex steroids with a side group with a high ionization efficiency, produces as larger number of ions that can be detected. A direct result of increased sensitivity is better precision since the signal-to-noise (S/N) of the peaks is greater. To attain the levels of sensitivity needed to measure many of the sex steroids within biologic samples such as urine, serum, and plasma, derivatization of these molecules is essential. Unfortunately, because it adds an additional step to sample preparation, derivatization can add to the variability observed within the final results. To minimize the variability, the compound chosen for derivatization reaction should exhibit pseudo-first-order kinetics so that the sex steroids are completely derivatized. Incomplete derivatization will produce irreproducible results. The derivatization procedure should be optimized with respect to reaction heating time and temperature, derivatization reagent concentration, pH, and presence of any interfering compounds (e.g., L-ascorbic acid) within the matrix. As with determining extraction efficiency, derivatization efficiency should be measured using standard solutions containing known amounts of each sex steroid of interest prepared in charcoal-stripped matrix. ’ QUANTITATIVE MEASUREMENT TECHNIQUE Both LC and GC MS use chromatography and a mass spectrometer to separate and detect sex steroids. In both cases, the mass spectrometer is programmed to select an ion with a particular mass-to-charge (m/z) ratio at a specific time range within the analysis. This ion selection scheme requires highly reproducible chromatography. Irreproducible chromatography can result in a contaminating metabolite, rather than one of the sex steroids of interest, being measured. Whereas newer mass spectrometers have increasingly greater robustness, to minimize analytical variability, it is important to routinely calibrate the instrument. Proper calibration not only ensures that the mass spectrometer response is reproducible, but also provides an effective check on the reproducibility of the LC or GC separation. A good practice is to run calibration standards before and after a batch of biologic samples. This practice allows that performance of the mass spectrometer to be monitored throughout the entire study. Any technical issues that contribute to variability can be quickly recognized and their impact on the entire study minimized.
A constant concern in conducting LC MS analysis of sex steroids is signal suppression. Although LC is used to separate the mixture prior to MS analysis, molecules within the biological matrix can co-elute causing a decrease in sensitivity. This possibility is why it is important to prepare calibration standards in the same biological matrix as the samples of interest. Although the acronyms LC and GC MS both suggest that MS is used to detected the steroids, in actual fact MS MS is used in a vast majority of studies aimed at quantitating these compounds. In MS MS detection, the ion corresponding to the sex steroid of interest is selected, isolated within the mass spectrometer, and then fragmented by colliding the ion with an inert gas. The mass spectrometer is instructed to detect one of the resulting fragments of the parent steroid ion. This selected reaction monitoring (SRM) mode, as it is known, provides a high level of certainty that the correct targeted molecules are being measured.15 It must always be remembered that although the original biologic sample undergoes an extraction step to enrich for the steroids of interest, the sample that is analyzed by LC or GC MS is still highly complex containing hundreds to thousands of small molecules. Simply monitoring for the predicted m/z value of any ion can result in the incorrect molecule being monitored. By monitoring for both m/z values corresponding to the parent ion and a specific fragment ion at a specific LC or GC retention time minimizes the chance of monitoring the incorrect molecule.
’ DATA ANALYSIS Although often considered as an afterthought of quantitative analysis, incorrect data analysis can contribute significantly to assay variation. Particularly in an assay designed to measure several sex steroids, it is possible that the software algorithm used to convert peak areas to quantitative values may select an incorrect peak if the LC or GC retention times shift between runs. Although incorrect peak assignments and shifts in retention times are readily recognized when analyzing only a small number of samples (as typical in an educational setting), high-throughput analyses (of the type seen in pharmaceutical laboratories) require careful calibration of the data points. To prevent incorrect peak assignments, it is important to manually check that the correct peaks are being selected by the software. Proper preparation of internal and external calibration standards is also critical to minimizing variation. The instrument readings obtained from the calibration standards are inherently used in the calculation of the sex steroid concentrations within the biologic samples. If these standards do not contain the intended concentrations, the values measured in the biologic samples will be incorrect. ’ CONCLUSIONS Trying to determine the roles that sex steroids play in diseases such as cancer often require the analysis of hundreds or thousands of samples. Considering the inherent variability of the levels of sex steroids within a human population, reliable conclusions can only be made if the variability of the assay used to measure sex steroids is minimized. Developing robust methods for quantitating steroid hormones is an important skill as many of the techniques (extraction, derivatization, LC MS, and data analysis) are utilized in the analysis of many classes of metabolites and drugs. The techniques described in this article are applicable for a variety of academic courses, primarily analytical chemistry and instrumental analysis. Owing to the importance of MS in chemistry and biochemistry, this study will provide the student with an excellent foundation on 232
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the use of this technology for measuring sex steroids and other types of small molecules. Whereas measuring sex steroids in human and mouse urine, blood, and tissue samples is the most relevant to cancer studies, using these types of materials can pose significant safety or Institutional Review Board issues. Fortunately, bovine milk contains many of the same sex steroids found in humans and can be used in these studies. It is important to test any analytical approach to ensure each step of the process provides highly reproducible, accurate, and precise results. Once the variability of each step has been minimized, constant monitoring is critical to ensure the accuracy and precision of the assay throughout the entire study.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the United States Government. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E. ’ REFERENCES (1) Sarett, L. H. J. Chem. Educ. 1960, 37, 184–189. (2) Guerriero, G. Trends Comp. Endocrinol. Neurobiol. 2009, 1163, 154–168. (3) Prins, G. S.; Korach, K. S. Steroids 2008, 73, 233–244. (4) Coleman, W. F. J. Chem. Educ. 2010, 87, 760–761. (5) Vandenput, L.; Ohlsson, C. Nat. Rev. Endocrinol. 2009, 5, 437– 443. (6) Ferguson, L. N. J. Chem. Educ. 1975, 52, 688–694. (7) Alexander, L. S.; Goff, H. M. J. Chem. Educ. 1982, 59, 179–182. (8) Cavalieri, E. L.; Stack, D. E.; Devanesan, P. D.; Todorovic, R.; Dwivedy, I.; Higginbotham, S.; Johansson, S. L.; Patil, K. D.; Gross, M. L.; Gooden, J. K.; Ramanathan, R.; Cerny, R. L.; Rogan, E. G. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10937–10942. (9) Diver, M. J. Ann. Clin. Biochem. 2006, 43, 3–12. (10) Klug, T. L.; Bradlow, H. L.; Sepkovic, D. W. Steroids 1994, 59, 648–655. (11) Suzuki, E.; Saegusa, K.; Matsuki, Y.; Nambara, T. J. Chromatogr. 1993, 617, 221–225. (12) Adlercreutz, H.; Kiuru, P.; Rasku, S.; W€ah€al€a, K.; Fotsis, T. J. Steroid Biochem. Mol. Biol. 2004, 92, 399–411. (13) Silvestre, C. I.; Santos, J. L.; Lima, J. L.; Zagatto, E. A. Anal. Chim. Acta 2009, 652, 54–65. (14) Halket, J. M.; Waterman, D.; Przyborowska, A. M.; Patel, R. K.; Fraser, P. D.; Bramley, P. M. J. Exp. Bot. 2005, 56, 219–243. (15) Kerns, E. H.; Di, L. Curr. Drug Metab. 2006, 7, 457–466.
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