Stock Ownership: None declared. Honoraria: None declared. Research Funding: Intramural Research Program of the NIH, Clinical Center. Expert Testimony: None declared. Patents: S.J. Soldin, US8227259B2.
References
1. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocorti- cal carcinoma. Clin Chem 2017;63:1836-46.
2. Wajchenberg BL, Albergaria Pereira MA, Medonca BB, Latronico AC, Campos Carneiro P, Alves VA, et al. Adre- nocortical carcinoma: clinical and laboratory observa- tions. Cancer 2000;88:711-36.
3. Soldin SJ, Soldin OP. Steroid hormone analysis by tandem mass spectrometry. Clin Chem 2009;55: 1061-6.
4. Guo TD, Chan M, Soldin SJ. Steroid profiles using liq- uid chromatography-tandem mass spectrometry with atmospheric pressure photoionization source. Arch Pathol Lab Med 2004;128:469-75.
5. Stolze BR, Gounden V, Gu J, Elliott EA, Masika LS, Abel BS, et al. An improved micro-method for the measure- ment of steroid profiles by APPI-LC-MS/MS and its use in assessing diurnal effects on steroid concentrations and optimizing the diagnosis and treatment of adre- nal insufficiency and CAH. J Steroid Biochem Mol Biol 2016;162:110-6.
Qian Sun2 Jianghong Gu2 Brian R. Stolze2 Steven J. Soldin2,3*
2 Department of Laboratory Medicine Clinical Center National Institutes of Health Bethesda, MD
3 Department of Medicine Division of Endocrinology and Metabolism Georgetown University Washington, DC
* Address correspondence to this author at: Department of Laboratory Medicine Clinical Center National Institutes of Health, Bldg. 10, Rm. 2C249 Bethesda, MD 20892
Fax 301-402-1885 E-mail soldinsj@cc.nih.gov
Previously published online at DOI: 10.1373/clinchem.2018.287029
In Reply
Soldin and colleagues argue that atmospheric pressure chemical ion- ization (APCI) is suboptimal for ste- roid analysis and should be super- seded by the use of atmospheric pressure photoionization (APPI). However, as they state, the dilemma is that the majority of clinical labo- ratories use electrospray ionization (ESI) or APCI. This was precisely our situation at the onset of our method development (1). We had no access to APPI, only APCI or ESI sources. The literature indicates that sources other than APPI can provide optimal conditions for steroid anal- yses; for example, the NIST refer- ence method for cortisol uses ESI (2). In our case, our desire to mea- sure 3ß-hydroxy 5-ene steroids in our steroid panel led us select APCI.
In response to the specific points raised, we cannot identify overwhelming experimental data in the references quoted that APPI is preferable to APCI. The review arti- cle quoted (3) describes a confer- ence abstract that provided APPI vs APCI comparison data for only testosterone, progesterone, preg- nenolone, and dehydroepiandrot- erone (DHEA). The data show a more intense relative signal for APPI vs APCI for native steroids (different to signal/noise ratio), although APCI was “better” for derivatized steroids. The other reference quoted (4) provides data obtained using APPI only.
We have compared intraassay and interassay CVs for QC material between our method (1) and the APPI methods quoted (4, 5). This actually shows that they are broadly similar. That the interassay CVs were lower than intraassay CVs in a number of cases in our study reflects, in our view, that we have developed a robust method that varies little in
performance day to day, as these dif- ferences are mostly marginal. In all cases CVs were <10%, as required by published method development guidelines.
In regards to the CVs >10% in our method (1), these were a mea- surement of internal standard peak area variability within a given batch. In our opinion, it is unreasonable to assume that this variability is entirely attributable to use of a “sub- optimal” APCI source. Such a con- clusion ignores the expected contri- butions of sample extraction, in our case protein precipitation followed by liquid-liquid extraction. Aware- ness of such variation supports our inclusion of internal standards for all measured steroids in our method (1), because these are designed to correct for variability in extraction recoveries, thus facilitating the good intra- and interassay performance demonstrated.
To fairly claim that APCI is suboptimal to APPI for steroid analyses would, in our view, require peer-reviewed publication of direct comparison data for a wide range of steroids on a single mass spectrome- ter using a common sample prepara- tion method. Although we are open to the possibility that APPI perfor- mance could be superior, we do not believe comparing our method data to 2 APPI instruments from their laboratory, as has been done in Ta- ble 1 of the Soldin letter, is a fair comparison for the reasons stated.
As we stated in our study (1), urine steroid profiling has shown that many of the most diagnostic ste- roids for adrenocortical carcinoma are metabolites of nonhormonally active intermediates. Therefore, it was perhaps unsurprising that interme- diates such as 11-deoxycortisol, 17- hydroxypregenenolone, and preg- nenolone provided discrimination of adrenocortical carcinoma from other adrenal lesions using serum (1). In children with virilizing adrenal tu- mors, those without large increases in steroid sulfates tend to excrete 11-
@ 2018 American Association for Clinical Chemistry
hydroxyandrosterone, for which the precursor 11-hydroxyandrostenedione may be a useful addition to serum panel methods. Very large increases of steroid sulfates are a common feature of adrenocortical tumors; we have not evaluated the diagnostic usefulness of all their unsulfated counterparts such as DHEA.
In conclusion, we are not con- vinced that APPI has overriding ad- vantages for serum steroid analysis, but would agree that on any given instrument it is desirable to consider ionization method as part of the process of optimization. Instrument manufacturers have achieved sub- stantial improvements in analyti- cal sensitivity in recent years, so that quantification with accept- able precision of several low level but biologically important steroids is now within our grasp.
Author Contributions: All authors confirmed they have contributed to the intellectual content
of this paper and have met the following 4 re- quirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
Authors’ Disclosures or Potential Con- flicts of Interest: No authors declared any po- tential conflicts of interest.
References
1. Taylor DR, Ghataore L, Couchman L, Vincent RP, Whitelaw B, Lewis D, et al. A 13-steroid serum panel based on LC-MS/MS: use in detection of adrenocorti- cal carcinoma. Clin Chem 2017;63:1836-46.
2. Botelho JC, Shacklady C, Cooper HC, Tai SS, Van Uyt- fanghe K, Thienpont LM, Vesper HW. Isotope-dilution liquid chromatography-tandem mass spectrometry candidate reference method for total testosterone in human serum. Clin Chem 2013;59:372-80.
3. Soldin SJ, Soldin OP. Steroid hormone analysis by tandem mass spectrometry. Clin Chem 2009;55: 1061-6.
4. Guo TD, Chan M, Soldin SJ. Steroid profiles using liq- uid chromatography-tandem mass spectrometry with atmospheric pressure photoionization source. Arch Pathol Lab Med 2004;128:469-75.
5. Stolze BR, Gounden V, Gu J, Elliott EA, Masika LS, Abel
BS, et al. An improved micro-method for the measure- ment of steroid profiles by APPI-LC-MS/MS and its use in assessing diurnal effects on steroid concentrations and optimizing the diagnosis and treatment of adre- nal insufficiency and CAH. J Steroid Biochem Mol Biol 2016;162:110-6.
David R. Taylor1* Lewis Couchman1,2 Norman F. Taylor1
1 Departments of Clinical Biochemistry (Viapath) and 2 Toxicology (Viapath) King’s College Hospital NHS Foundation Trust London, United Kingdom SE5 9RS
* Address correspondence to this author at: Department of Biochemistry King’s College Hospital NHS Foundation Trust London, United Kingdom SE5 9RS E-mail davidtaylor8@nhs.net
Previously published online at DOI: 10.1373/clinchem.2018.289769