Clinical Chemistry 64:6 000-000 (2018)
Letters to the Editor
Atmospheric Pressure Chemical Ionization is a Suboptimal Ionization Source for Steroids
To the Editor:
Biochemical measurement of steroid hormones is imperative for the diag- nosis of adrenocortical carcinoma (ACC).1 The lack of clinical syn- dromes in over half of patients with ACC prevents the early diagnosis of this malignant disease if appropriate laboratory evaluations are not per- formed (1, 2). It is therefore with great interest that we read the recent study (1) by a research group at King’s College. Their effort to apply a mass spectrometry-based steroid profiling assay to discriminate ACC from benign tumors is highly com- mendable. This new method has the potential to detect ACC at an early stage and thereby markedly improve the treatment and outcome of the patients.
Although competitive immuno- assays exhibit good precision (CVs generally <5%), mass spectrometry was selected in this study because it exhibits improved analytical speci- ficity, sensitivity, and multiplexing ability in steroids measurement com- pared with immunoassays (1,3). The diagnostic utility of liquid chromatography-tandem mass spec- trometry (LC-MS/MS) is dependent on its ability to generate reproducible results and quantify the appropriate steroid. It is in regard to these aspects that we would like to raise important concerns about the ionization source used in the study.
Simultaneous quantification of 13 steroids was achieved by MS/MS using an atmospheric pressure chemi-
| Table 1. IS precision for steroid hormones as measured with 2 APPI instruments in comparison with APCI. | |||
|---|---|---|---|
| APPI instrument 1-IS, % CVª | APPI instrument 2-IS, % CVª | APCI-IS, % CVb | |
| Cortisol | 6.5 | 4.8 | 9.0 |
| Cortisone | 6.5 | 4.8 | 13.2 |
| Corticosterone | 9.1 | 8.9 | 13.1 |
| 11-Deoxyª | 5.9 | 8.2 | 9.6 |
| Andro | 3.3 | 5.8 | 17.2 |
| Testosterone | 4.6 | 5.7 | 6.7 |
| 17-OHP | 2.4 | 4.9 | 13.3 |
| Progesterone | 3.5 | 3.5 | 12.1 |
| DHEA | 9.3 | 8.0 | |
| DHEAS | 7.4 | ||
| Mean (range) | 5.7 (3.8-7.6) | 6.1 (4.6-7.5) | 11.3 (8.7-13.9) |
| a CVs for APPI are calculated from 20-23 samples. b APCI-IS data are taken from Taylor et al. (1). " 11-Deoxy, 11-deoxycortisol; Andro, androstenedione; 17-OHP, 17-hydroxyprogesterone. | |||
cal ionization (APCI) source (1). APCI and electrospray ionization (ESI) are known to show large and variable sample-to-sample ioniza- tion suppression effects in steroid measurements (3), as evidenced by the substantial variation of internal standards (IS) between samples shown in Table 1 of the study (1). Of note, 8 out of 13 steroids mea- sured showed a CV >10%, and 2 had CVs >15%. In contrast, we routinely measure 9 steroid hormones in serum by LC-MS/MS with atmo- spheric pressure photoionization (APPI) source, and APPI shows a substan- tially smaller between-individual variation for all the deuterated IS compared to APCI (Table 1). The intraassay variations reported by Taylor et al. in their Supplemental Table 5 (1) are higher than the in- terassay (between-day) variations for 19 of 39 quality controls. Because >95% of clinical laboratories now use an APCI or ESI source, we would like to bring to their attention that APCI and ESI are less reliable for steroid measurement compared with APPI. This is due to the softer (less energetic) ionization afforded
by APPI, which ionizes the steroids more specifically than the far more energetic APCI and ESI sources that simultaneously ionize many differ- ent molecular species. APPI has been shown to produce an improved signal-to-noise ratio than APCI by a factor of 3 to 10 (3, 4). For steroid measurement, the ionization suppres- sion provided by ESI is even greater compared with APCI (4). Given the similar cost-effectiveness and ease of maintenance for APPI, APCI, and ESI, we recommend that clinical lab- oratories replace APCI/ESI with APPI in steroid measurement.
Another advantage of APPI is that it allows quantification of serum dehydroepiandrosterone (DHEA), which is a preferred marker compared with DHEA sulfate (DHEAS), espe- cially for pediatric patients. Sulfa- tion of DHEA increases the solubil- ity of the steroid so that it can be readily excreted renally. As a hor- mone, however, the sulfated steroid DHEAS is inert. In addition, the di- agnostic value of DHEAS should be questioned in children, which make up the second largest ACC popula- tion (2). In a comprehensive study
@ 2018 American Association for Clinical Chemistry 1 Nonstandard abbreviations: ACC, adrenocortical carci- noma; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; APCI, atmospheric pressure chemi- cal ionization; ESI, electrospray ionization; IS, internal standard; APPI, atmospheric pressure photoionization; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate.
in which serum hormone concentra- tions were measured in 22 children with ACCs, DHEA was increased in all of them, whereas DHEAS was in- creased in only 50% of the children studied (2). With immunoassay and APCI/ESI, the measurement of DHEA is challenging because of its low concentration in the serum. However, APPI has improved ana- lytical sensitivity for nonpolar com- pounds compared with APCI/ESI as evidenced by a better signal-to-noise ratio, making it a reliable source for the quantification of serum DHEA (4, 5).
To summarize, we believe that enhanced diagnostic MS-based ste- roid profiling for ACC can be achieved with APPI. This ionization source offers improved data quality and reliable DHEA measurement, leading to optimal diagnosis and treatment of the disease.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 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; and (c) final approval of the published article.
Authors’ Disclosures or Potential Con- flicts of Interest: Upon manuscript submis- sion, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared. Consultant or Advisory Role: None declared. 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