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A Practical ID-LC-MS/MS Method for the Most Commonly Analyzed

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A Practical ID-LC-MS/MS Method for the Most Commonly Analyzed Turk J Biochem 2019; 44(2): 130–141 Research Article Fatih Yesildal*, Muhittin Serdar and Taner Ozgurtas A practical ID-LC-MS/MS method for the most commonly analyzed steroid hormones in clinical laboratories Klinik laboratuvarlarda en yaygın olarak analizi yapılan steroid hormonlar için pratik bir ID-LC-MS/MS metodu https://doi.org/10.1515/tjb-2018-0214 Results: An isotope dilution (ID)-LC-MS/MS method was Received March 6, 2018; accepted July 25, 2018; previously published developed, in which 13 steroids can be analyzed in the online August 21, 2018 same run. Test performance was quite good for the 11 ster- Abstract oids (cortisol, DHEA, DHEAS, total testosterone, proges- terone, androstenedione, 11-deoxycortisol, cortisone, cor- Background: Analysis of steroid hormones rapidly and ticosterone and dihydrotestosterone) while estradiol and reliably remains a challenge in clinical laboratories as this aldosterone performance was suboptimal considering the plays an important role in evaluation of many endocrine precision and trueness. disorders. The aim of this study was to create a steroid Conclusion: This ID-LC-MS/MS method would be useful profiling panel by using a liquid chromatography tandem in clinical laboratories, especially for the immunoassays mass spectrometry (LC-MS/MS) method which was com- having insufficient test performance and when checking posed of the most commonly analyzed steroid hormones for interferences in available immunoassays. in clinical laboratories. Keywords: LC-MS/MS; Immunoassay; Steroid; Method Materials and methods: Protein precipitation was per- validation; Method comparison. formed for sample preparation. Ultra performance liquid chromatography (UPLC) system and an analytical column with C18 selectivity was chosen for chromatographic sep- Öz eration. Atmospheric pressure chemical ionization (APCI) ion source was preferred for ionization, and tandem MS Amaç: Steroid hormonların hızlı ve güvenilir bir şekilde with triple quadrupole was used. MS scan was performed analiz edilmesi, birçok endokrin bozukluğun değerlendi- using the selected reaction monitoring mode in positive rilmesinde önemli bir rol oynadığı için, klinik laboratuvar- polarity. During the method validation process, test per- larda bir sorun olmaya devam etmektedir. Bu çalışmanın formance was evaluated for each steroid hormone, and amacı, LC-MS/MS tekniğini kullanarak, klinik laboratu- 40 serum samples were used for method comparison with varlarda en sık analiz edilen steroid hormonlardan oluşan immunoassays available in our core laboratory. bir steroid profil paneli oluşturmaktı. Materyal ve Metot: Örnek hazırlığı için protein çöktür- *Corresponding author: Fatih Yesildal, MD, Istanbul Medeniyet mesi yapıldı. Kromatografik ayırma için UPLC sistemi ve University, Goztepe Training and Research Hospital, Department of Medical Biochemistry, 34722 Istanbul, Turkey, C18 seçiciliği olan bir analitik kolon seçildi. İyonlaştırma Phone: +902165709023, e-mail: [email protected]. için APCI iyon kaynağı tercih edildi ve üçlü kuadrupole https://orcid.org/0000-0002-8738-5964 sahip tandem MS kullanıldı. MS taraması, seçilmiş reak- Muhittin Serdar: Acibadem University Faculty of Medicine, siyon izleme modu kullanılarak, pozitif polaritede ger- Department of Medical Biochemistry, ClinLab Laboratories, Ankara, çekleştirildi. Metot validasyonu işlemi sırasında, her bir Turkey, e-mail: [email protected] steroid hormonu için test performansı değerlendirildi ve Taner Ozgurtas: University of Health Sciences, Gulhane Training and Research Hospital, Department of Medical Biochemistry, Ankara, merkez laboratuvarımızdaki mevcut immün-ölçümlerle Turkey, e-mail: [email protected] metot karşılaştırması için 40 serum örneği kullanıldı. Fatih Yesildal et al.: A practical ID-LC-MS/MS method for steroids 131 Bulgular: Aynı çalışma içerisinde 13 steroidin analiz Materials and methods edilebildiği bir ID-LC-MS/MS metodu geliştirildi. Test performansı, 11 steroid için (kortizol, DHEA, DHEAS, An isotope dilution (ID) LC-MS/MS method was devel- total testosteron, progesteron, androstenedion, 11-deo- oped, in which 13 steroids (aldosterone, corticosterone, ksikortizol, kortizon, kortikosteron ve dihidrotestoste- cortisol, cortisone, 11-deoxycortisol, androstenedione, ron) oldukça iyiydi; ancak östradiol ve aldosteron per- DHEA: dehydroepiandrosterone, DHEAS: dehydroepian- formansı, kesinlik ve doğruluk göz önüne alındığında drosterone sulfate, DHT: dihydrotestosterone, E2: estra- suboptimaldi. diol, 17α-OH progesterone, progesterone, testosterone) Sonuç: Bu ID-LC-MS/MS metodu; klinik laboratuvarlarda, can be analyzed in the same run. Validated calibrators özellikle yetersiz test performansına sahip olan immün-öl- (6 point calibrators plus blank) for MS and internal stand- çümler için ve mevcut immün-ölçümlerdeki interferans- ard (IS) mix (Chromsystems, Gräfelfing, Germany) were ları kontrol etmek için yararlı olacaktır. used in this study. These calibrators are serum-based standards which means they include protein to avoid Anahtar kelimeler: LC-MS/MS; Immün-ölçüm; Steroid; matrix effect. The content of IS mix (Chromsystems, metot validasyonu; Metot karşılaştırma. Gräfelfing, Germany) was aldosterone-d4, corticoster- one-d8, cortisol-d4, cortisone-d8, 11-deoxycortisol-d5, androstenedione-d7, DHEA-d5, DHEAS-d6, DHT-d3, E2-d5, Introduction 17α-OH progesterone-d8, progesterone-d9, testosterone- d3. In this method; protein precipitation was performed Steroids; which are derived from cholesterol and mostly for sample preparation, including the calibrators. The synthesized in adrenal cortex, placenta and gonads; are precipitant solution was prepared by mixing 0.3 M zinc biological molecules functioning in regulation of many sulfate solution (ZnSO4), methanol and IS. Composition of metabolic activities in organism [1]. Many endocrinologi- cal pathologies emerge because of the defects in steroid biosynthesis resulting in excess or insufficient secretion Table 1: LC conditions, flow gradient and tandem MS conditions. of steroids [2]. Analysis of steroid hormones in clinical lab- oratories plays a decisive role in diagnosis and monitoring LC conditions of these diseases. Injection volume 25 μL Immunoassays are the most commonly used methods Sampler temperature 15°C for steroid analysis in clinical laboratories. In the past, Column flow 0.350 mL/min radio immunoassay (RIA) methods were commonly used, Column oven 35°C but today automated instruments employing chemilumi- Analysis time 11 min nescence immunoassay (CLIA) are widely used [3]. The Flow gradient Time (min) Mobile Mobile major drawback of this method is the lack of specific- phase A (%) phase B (%) ity. The antibodies used in this methods may cross-react 0.000 50 50 with other steroids or other similar molecules, and these 0.000 50 50 intereferences result in serious problems [4]. Addition- 4.000 0 100 ally, some of the commercially available kits and methods 7.000 0 100 7.000 50 50 have insufficient test performance, considering the preci- 11.000 50 50 sion and trueness. Another method for steroid analysis is GC-MS which is considered as the gold standard [5]. MS/MS conditions However, this method can not be used in routine clinical Ion source APCI laboratories because of laborious sample pretreatment Capillary temperature (°C) 400 steps and long analysis times. Today, liquid chromatogra- Vaporizer temperature (°C) 300 phy tandem mass spectrometry (LC-MS/MS) use is getting Sheath gas pressure (Arb) 35 Aux gas pressure (Arb) 10 wider in steroid analysis [6]. Discharge current (μA) (positive polarity) 4.5 The aim of this study was to create a steroid profiling Collision gas pressure (mTorr) 1.5 panel which was composed of the most commonly ana- Vacuum (Torr) 2.1 × 10−5 lyzed steroid hormones in clinical laboratories, and had MS scan mode SRM a good test performance considering the precision, true- Cycle time (s) 1.500 Analysis time (min) 11 ness, analytical sensitivity and specificity. 132 Fatih Yesildal et al.: A practical ID-LC-MS/MS method for steroids RT: 1.11 NL: 2.54E2 AA: 4827 TIC F: + c APCI SRM ms2 100 361.000 [342.999·343.001] MS Genesis 50 Aldosterone 0 RT: 1.08 NL: 4.79E2 TIC F: + c APCI SRM ms2 100 AA: 10370 365.000 [346.999·347.001] MS Genesis 50 Aldosterone-D4IS 0 RT: 1.18 NL: 1.56E3 AA: 20141 2 100 TIC F: + c APCI SRM ms 361.001 [162.999·163.001] MS 50 Cortisone Genesis 0 RT: 1.18 NL: 1.53E3 2 100 AA: 18973 TIC F: + c APCI SRM ms 369.000 [167.999·168.001] MS 50 Cortisone-D8IS Genesis 0 RT: 1.44 NL: 9.01E3 AA: 126599 TIC F: + c APCI SRM ms2 100 363.200 [120.999·121.001] MS Genesis 50 Cortisol 0 RT: 1.44 NL: 9.78E2 AA: 13256 2 100 TIC F: + c APCI SRM ms 367.000 [97.002·97.004] MS Genesis 50 Cortisol-D4IS 0 RT: 1.51 NL: 1.30E3 AA: 21029 TIC F: + c APCI SRM ms2 100 291.002 [255.002·255.004] MS Genesis 50 Dihydrotestosterone 0 RT: 1.51 NL: 4.03E4 TIC F: + c APCI SRM ms2 100 AA: 601672 294.010 [257.999·258.001] MS 50 Dihydrotestosterone-D3IS Genesis Relative abundance (%) 0 RT: 1.54 NL: 1.33E4 2 AA: 212177 TIC F: + c APCI SRM ms 100 255.000 [158.999·159.001] MS Genesis 50 Estradiol 0 RT: 1.51 NL: 8.29E3 AA: 131273 2 100 TIC F: + c APCI SRM ms 260.000 [160.999·161.001] MS 50 Estradiol-D5IS Genesis 0 NL: 2.91E5 RT: 1.54 TIC F: + c APCI SRM ms2 AA: 4664643 271.001 100 [212.999·213.001] MS Genesis 50 Dehydroepiandrosterone sulfate 0 RT: 1.52 NL: 1.26E5 AA: 2083402 TIC F: + c APCI SRM ms2 100 277.000 [218.999·218.001] MS 50 Genesis Dehydroepiandrosterone sulfate-D6IS 0 RT: 1.53 NL: 2.03E4 AA: 319449 TIC F: + c APCI SRM ms2 100 271.000 [252.999·253.001] MS Genesis 50 Estriola 0 012345678910 Time (min) Figure 1: SRM scan of all steroids and their stable isotope standards. (A) Chromatograms of the first 6 steroid hormones and their stable isotope dilutions eluting from the system (aE3 was excluded from validation process because its calibrator was not approved for MS/MS analysis.). (B) Chromatograms of the last 7 steroid hormones and their stable isotope dilutions eluting from the system.
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