Mass Spectrometry—Applying the Gold Standard in Endocrinology

McPhaul, Michael J., MD
Medical Director, Endocrinology & Metabolism
Quest Diagnostics
San Juan Capistrano, CA
Also by this Author 

The availability of mass spectrometry is enabling higher levels of precision and reliability in laboratory diagnostics than has traditionally been possible.

Dr. Michael J. McPhaul, Medical Director, Endocrinology and Metabolism, Quest Diagnostics discusses the growing application of mass spectrometry in endocrinology and other specialties, and reviews the advantages it provides compared with immunometric assays.

From Immunoassays to Mass Spectrometry

“As laboratory diagnostics has evolved, the most commonly employed measurement methods have been those using specific antibodies, be they monoclonal or polyclonal antibodies, to quantitate the levels of a particular compound in a complex mixture,” says Dr. McPhaul. “This has proven to be very informative and very useful for a long period of time.”

“A shortcoming of immunoassays, however, is that their measurements are sometimes confounded by the presence of other compounds, causing interference in the assay itself. For example, the level of cortisone or structurally related steroids in a sample might interfere with the measurement of cortisol in an immunometric assay. These are very closely related structural molecules and sometimes that distinction is difficult to make.”

Advantages of Mass Spectrometry

Quantitation of molecular weight

Immunoassays are prone to interference because they do not measure the molecule itself but a surrogate—typically, the release of a radioactive trace or a flash of light. In contrast, mass spectrometry (MS) measures the molecule itself. “This measurement involves the use of a molecule’s flight in a magnetic field to precisely measure its atomic weight,” explains Dr. McPhaul. “Specific molecules are enriched using well-defined separation techniques, usually liquid chromatography (LC). The amount of the molecule to be analyzed in this very complex mixture is then measured, by the assignment and the quantitation of very specific masses. The precision of liquid chromatography tandem mass spectrometry (LC-MS/MS) measurements, enabled by quantitation of specific molecular weights, allows differentiation between even very closely related molecules.”

Internal standard

Another advantage of MS assays is that they include an internal standard. “This is usually the same molecule as that being measured but which differs in its atomic mass by the inclusion of deuterium or carbon atoms,”13 notes Dr. McPhaul. “This allows a verification of the assay’s performance based on the behavior of the internal standard. It is possible to determine whether an assay is likely to be problematic by the use of reporting criteria to define performance characteristics based on the simultaneous measurement of the internal standard included in each sample. This is impossible when using an immunometric assay. With traditional methods you cannot identify if there is interference—you only have the input of the sample and the result of the assay in an immunometric assay.”

Mass Spectrometry in Endocrinology—Clinical Utility

Testosterone measurement

In some circumstances, the variation in results from immunoassays compared to MS assays may have less clinical significance. There are, however, areas where precise measurement is of greater importance. “This is illustrated by testosterone measurement, which in some circumstances may be adequately achieved with an immunoassay platform,” says Dr. McPhaul. “These are widely employed and they have a direct relationship to the assay results that would be obtained using a LC/MS/MS type of methodology.

“If one is dealing with someone who has a normal level of testosterone, let’s say 500 ng/DL in an adult male, the amount of imprecision might not be critical to making decisions. However, there are important populations—adult males who have hypogonadism, and female or pediatric patients, where the levels are substantially lower. Interference at these lower levels of testosterone becomes much more problematic with immunoassays, causing imprecision. This does not occur with LC/MS/MS, which is unaffected by cross-reactivity at low concentrations.”

Toward standardization

Recognizing the shortcomings of direct assays, The Endocrine Society, in a Position Statement, recommends that direct assays should be avoided when testing samples with low testosterone concentration.It recommends the initial use of extraction and chromatography, followed by either mass spectrometryor extraction radioimmunoassay (RIA). Extraction RIA requires manual intervention, is labor intensive and requires radioactivity. “The advantage of mass spectrometryis that it is very precise and very accurate, and is the same technique usedas the reference method at all the standard centers like the National Institute of Standards and Technology (NIST),” says Dr. McPhaul.1

To promote the standardization of testosterone measurement the Centers for Disease Control and Prevention (CDC) has established the CDC Laboratory/Manufacturer Hormone Standardization (HoSt) Program.2 The standardization of total testosterone measurements in serum is established through method comparison and bias estimation between CDC’s reference laboratory and testing laboratories. To gain CDC certification laboratories have to demonstrate a mean bias ± 6.4% to the CDC reference method, which uses high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS).2 Ten laboratories worldwide, including Quest Diagnostics, have successfully participated in the standardization program and have been certified by the CDC.5

Vitamin D measurement

Another area in which LC-MS/MS makes a significant difference is in the measurement of 25-hydroxyvitamin D [25(OH)D]. LC/MS/MS is able to distinguish 25(OH)D3 and 25(OH)D2, as well as other metabolites of vitamin D. It provides results for both 25(OH)D2 and 25(OH)D3, in addition to total 25(OH)D. This differs from immunoassays, which only report a single total number for 25(OH)D. The additional information can be helpful to a physician, to assess compliance by someone on D2 supplementation, for example.

Recognizing the greater accuracy and precision of LC-MS/MS in relation to immunoassay, the National Institute of Standards and Technology and the Centers for Disease Control and Prevention have adopted this technology as their reference method, establishing it as “the gold standard.” 4,5 In addition, the NIH Office of Dietary Supplements is working with these groups to standardize the laboratory measurement of vitamin D status in national health surveysworldwide. This collaboration includes the creation in the CDC of the Vitamin D Standardization Coordinating Center. Laboratories meeting the standard will be certified and will continue to be monitored on a regularly basis.2,6

Growing Application of Mass Spectrometry

“The adoption of LC-MS/MS as a reference method by professional bodies is a recognition of the high degree of reproducibility between laboratories using this method. This contrasts with the widely documented biases that exist within immunoassays,” summarizes Dr. McPhaul. “Immunoassays are always going to be bedeviled by problems relating to cross-reactivity, particularly at low levels. LC-MS/MS can side-step these because of the multiple levels of specificity that exist within the methodology, both within the pre-analytic stage —the preparation, the chromatography—as well as in the selection of the specific structure and mass that are actually quantitated.

“The use of mass spectrometry is progressively broadening to include a whole range of small analytes, for use in endocrinology testing, prescription drug monitoring testing, or the assessment of levels of drugs used in therapeutics. Over the last decade there has been growing recognition that a whole host of different assays can be converted to mass spectrometry due to the increased sensitivity provided by these instruments. These include the highly precise measurement of Insulin like growth factor 1 or thyroglobulin by mass spectrometry, as well as our most recent achievement, which is the creation of an assay to measure the level of intact insulin and C-peptide using mass spectrometry. These assays are the first assay commercial assays in the world capable of measuring intact insulin and C-peptide. Their accuracy places Quest Diagnostics at the forefront of efforts to standardize both insulin and C-peptide assays.

“Mass spectrometry is going to be applicable to a broader and broader range of protein measurements, in addition to small molecules such as steroid hormones. Progressively more sensitive mass spectrometry instruments are becoming commercially available, which are enabling the measurement of many compounds that we cannot currently measure.” 


  1. Rosner W, Auchus R, Azziz R, Sluss P, Raff H. Utility, Limitations, and Pitfalls in Measuring Testosterone: An Endocrine Society Position Statement. J Clin Endocrinol Metab. 2007. 92(2):405–413.
  2. Standardizing Hormone Measurements. National Center for Environmental Health Division of Laboratory Sciences. Centers for Disease Control and Prevention. Accessed June 16, 2016.
  3. CDC Hormone Standardization Program. Certified Participants. Accessed June 16, 2016.
  4. Tai SS, Bedner M, Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Chem. 2010;82:1942-1948.
  5. Chen H, McCoy LF, Schleicher RL, et al. Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method. Clin Chem Acta. 2008;391:6-12.
  6. CDC Hormone Standardization Program. Total 25hydroxy Vitamin D Certified Procedures Accessed June 16, 2016. 

Released on Tuesday, September 06, 2016