Qualification and Quantification of Seventeen Natural Steroids in Plasma by GC-Q-MS and GC-IT-MS/MS

Journal of Chromatographic Science 2012;50:349– 357 doi:10.1093/chromsci/bms009 Article Qualification and Quantification of Seventeen Natural Steroids in Plasma by GC –Q-MS and GC-IT–MS/MS A.F. Toribio-Delgado1*, M. Maynar-Mariño2, M.J. Caballero-Loscos3, M.C. Robles-Gil2, G.J. Olcina-Camacho2 and J.I Maynar-Mariño1 1 Department of Analytical Chemistry, University of Extremadura, Badajoz, Spain, 2Department of Physiology, University of Extremadura, Cáceres, Spain, and 3Department of Medical-Surgical Therapeutics, University of Extremadura, Badajoz, Spain *Author to whom correspondence should be addressed. Email: afertd@unex.es Received 20 February 2011 Introduction Sex hormones and corticosteroids are a group of hormones derived from cholesterol that are widely distributed in humans. They are involved in many physiological systems such as reproductive function, stress and metabolism. The analysis of sex hormones and corticosteroids in biological samples can be employed as a diagnostic tool in endocrine disorders, for pharmaceutical investigation and for the detection of possible misuse in athletes (1). The need to determine steroids in different biological matrices has led to the development of different techniques for steroid determination in these matrices (2). Steroid hormones are normally measured by immunoassay (IA) in routine clinical laboratories because of its simplicity, speed and analytical sensitivity. Nevertheless, the use of IA is limited because of the cross-reactivity of the antibodies used with structurally related compounds. One alternative technique is chromatography coupled to mass spectrometry, which has been accepted for a long time as a potent technique for sieving and confirming the presence of substances prohibited in our biology (3). The primary biological samples used for the determination of steroid hormones are serum and urine. In recent years, this study and others have reported several methods for their determination in urine samples involving gas chromatography –mass spectrometry (GC–MS) (4, 5, 6) or liquid chromatography – mass spectrometry (LC –MS) (7, 8, 9). Besides urine, blood is one of the most commonly used fluids for the determination of endocrinological disorders (10) and for anti-doping analyses in sport; the illicit use of anabolites to improve the performance of sportsmen is prohibited by most sporting federations, including the International Olympic Committee (11) and the World Anti-Doping Agency (WADA). Many efforts have been made to develop sensitive detection methods of steroids in blood samples. Blood offers certain advantages; for example, the matrix is relatively uniform, which permits the detection of drug traces, and another advantage is that the precursors of the metabolites can be found in the matrix (12). Several GC– MS methods have been developed for the measurement of individual steroids or a profile of steroids and their metabolites in serum (13, 14, and 15); however, the number of steroids tested is lower than in the present work. This work proposes a method to determine a total of 17 steroid hormones in plasma by gas chromatography- quadrupole- mass spectrometer (GC – Q-MS) and gas chromatography- ion trap- mass/mass spectrometer (GC-IT – MS-MS) in addition to five different extractor solvents. The derivation of the steroids is performed by using silynization or oxime/ silynization reactions according to the properties of the steroids (8, 16). The selectivity, reproducibility, repeatability, accuracy, recovery, limits of detection (LOD), limits of quantification (LOQ) and linearity of the method were measured, and thus a reliable method was obtained for determining and quantifying this group of steroid hormones. Experimental Reagents and materials Testosterone, 17b-estradiol, androstenedione, methyltestosterone, androsterone, 5b-androsterone, estrone, dihydrotestosterone (DHT), progesterone, cortisone, cortisol, tetrahydrocortisone (THE), nandrolone, dehydroepiandrosterone (DHEA), estriol, epitestosterone, epiandrosterone, N-methyl N-trimethylsilyl-trifluoroacetamide (MSTFA) and arylsulphatase were obtained from Sigma-Aldrich, tetrahydrocortisol (THF), androsterone glucuronide, 5b-androsterone glucuronide, androsterone sodium sulphate and 5b-androsterone sodium sulphate were supplied by Steraloids, b-glucoronidase (type Escherichia coli K 12) was obtained from Boheringer Mannheim, dithioerythritol was acquired from Serva, ammonium iodide (NH4I) was acquired from Panreac and human albumin at 20% was from Grifols. All other reagents and solvents were of analytical grade and were mainly supplied by Scharlau, J.T. Baker and Panreac. Equipment The GC-Q– MS analysis of the samples was conducted on an Agilent Technologies 6890N chromatograph with MS 5973 Network quadrupole spectrometer. Separation was performed with a factor four capillary column VF-1ms 25 m  0.25 mm i.d. # The Author [2012]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Studying the plasma steroid profile offers information about the possible existence of endocrinological alterations. This study describes the development and validation of gas chromatographic – mass spectrometric and gas tandem mass spectrometric methods for the simultaneous identification of 17 steroid hormones in human plasma using five different solvents. The n-hexane/ethyl acetate solvent mixture, in a proportion of 70/30 (v/v) provided the best results. The extracts were derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. The obtained limits of detection were below 1 ng/mL in the majority of the studied steroids and the limits of quantification were below 5 ng/mL; the method obtained good linearity, reproducibility, repeatability, accuracy and recoveries above 95% in most cases. Table I Selected Ions for the Identification of Steroids GC-Q –MS Retention time (min) Relative retention time Quantification ion Qualification ion Androsterone 5b-androsterone (etiocholanolone) Dehydroepiandrosterone (DHEA) Epiandrosterone Epitestosterona Nandrolone Dihydrotestosterone (DHT) Estrone Androstanedione 17b -estradiol Testosterone Estriol Progesterone Tetrahyidrocortisone (THE) Tetrahydrocortisol (THF) Cortisone Cortisol Methyltestosterone 20.398 20.453 21.090 21.190 21.421 21.539 21.598 21.715 21.743 21.955 21.949 24.415 25.020 26.419 27.092 28.373 29.897 23.139 0.882 0.884 0.911 0.916 0.926 0.931 0.933 0.938 0.940 0.949 0.949 1.055 1.081 1.142 1.171 1.226 1.292 1.000 419 434 432 419 432 418 434 414 415 285 432 504 458 635 637 616 632 301 434 –329 419 –329 417 –327 434 –329 417 403 405 309 430 416 417 386 –345 443 530 532 630 559 446 Compound Retention time (min) Relative retention time Ion parent Qualification ion Quantification ion Androsterone 5b-androsterone (etiocholanolone) Dehydroepiandrosterone (DHEA) Epiandrosterone Epitestosterona Nandrolone Dihydrotestosterone (DHT) Estrone Androstanedione 17b -estradiol Testosterone Estriol Progesterone Tetrahyidrocortisone (THE) Tetrahydrocortisol (THF) Cortisone Cortisol Methyltestosterone 21.942 22.207 23.530 23.786 24.303 24.491 24.491 24.961 24.934 25.364 25.311 29.752 30.022 33.035 34.232 36.009 38.155 28.288 0.776 0.785 0.832 0.841 0.859 0.866 0.866 0.882 0.881 0.897 0.895 1.052 1.061 1.168 1.210 1.273 1.349 1.000 420 420 418 420 432 418 434 399 430 416 432 504 458 635 636 617 632 446 420 –329 – 239 420 –329 – 239 418 –327 – 237 420 –329 – 239 432 –417 – 342 –327 418 –403 434 –419 – 405 –377 399 –309 – 281 –267 –229 430 –415 – 325 416 –326 – 285 432 –417 – 327 –301 504 –311 – 295 –281 –269 458 –368 – 353 –299 635 –619 – 530 636 –531 – 430 617 –525 – 435 632 –543 – 437 446 –356 – 301 329 329 327 329 417 403 419 309 415 326 417 311 368 619 531 525 543 301 GC-IT – MS DF ¼ 0.25. The analysis was started at 1008C for 1.5 min and was increased 108C per min until 2808C, which was maintained for 5 min, and then increased 108 C per min until 3008C, which was maintained for 2 min. The analysis was performed in splitless mode, with He gas carrier at a flow rate of 0.6 mL/ min. The injected volume was 3 mL and the temperature of the injector was 2508C. The GC-IT –MS-MS analysis of the samples was conducted on a Varian 3800 gas chromatograph directly coupled to an MS – MS Saturn 2000 ion-trap mass spectrometer and equipped with a Varian 8200 autosampler with a capacity for 48 samples. Separation was performed using an HP-5MS (crosslinked 5%Ph Me silicone) capillary column with 30 m  0.25 mm i.d.; film thickness, 0.25 mm. The analysis was performed in splitless mode. Helium was used as the carrier gas (1 mL/min) and the column oven temperature was programmed to maintain at 1008C for 1.5 min, increase to 258C per min until reaching 1908C, increase 38C per min until reaching 2808C, maintain for 5 min, increase 258C per min until 3258C and maintained for 3 min. Analysis was performed in splitless mode. The injected volume was 3 mL and the temperature of the injector was 2808C. The quantification and qualification ions of the studied compounds are shown in Table I. 350 Toribio-Delgado et al. Preparation of the standard solutions and calibration curves Stock standard solutions (100 ng/mL) of each analyte were prepared using methanol as solvent. Successive dilutions with methanol of the stock solutions were made to prepare working solutions. All solutions were stored at –208C in the dark and were removed every six months. To have a matrix as close as possible to the true samples, human albumin at 20% was used to prepare triplicate calibration curves with the following concentrations: 1, 2, 4, 6, 8 and 10 ng/mL for sex steroids and 5, 10, 20, 30, 40 and 50 ng/mL for corticosteroids. Sample storage The plasma samples were conserved at – 208C. The plasma was very stable and did not require special precautions to be conserved, and storage at –208C sufficed (17). Sample preparation Extraction procedure Extraction was carried out with 2 mL of the following solvents: n-hexane, n-hexane –ethyl acetate (70/30, v/v, 50/50, v/v, Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Compound 25/75, v/v), methyl ether. ethyl acetate, ethyl ether and tert-butyl Glucuroconjugated fraction extraction To the previous aqueous part, 2 ng of methyltestosterone as internal standard was added and the pH was adjusted to 7 using acetic acid 1M and phosphate buffer pH 7. After the addition of 50 mL of b-glucuronidase, enzymatic hydrolysis was performed in a thermoblock for 1 h at 508C. To adjust the pH of the sample to 9.5 to optimize the extraction process of the steroids, the same procedure was followed as that described for the free fraction. Sulphoconjugated fraction extraction To the aqueous fraction of the previous stage, after the addition of 2 ng of methyltestosterone as internal standard, the pH was adjusted to 5 using the addition of acetic acid 1M and an acetate buffer pH 5, and 50 mL of arylsulfatase was added; it was left in the thermoblock for 15 h at 508C, then left to cool at room temperature. To adjust the pH of the sample to 9.5 to optimize the extraction process of the steroids, the same procedure was followed as that described for the free fraction. Derivatization procedure To the dried plasma extracts 50 mL of a mixture of MSTFA – NH4I –dithioerythritol (1000:2:4) (v/w/w) was added. The reaction mixture was heated in a thermoblock for 30 min at 608C to perform the derivatization reaction, and was then encapsulated and injected into the chromatograph. Derivatization test To carry out the derivatization test, 1 mL of albumin (20%) androsterone and etiocholanolone were added in a known quantity (10 ng/mL), and the relationship was measured between the mono-trimethylsilil and the bi-trimethylsilil areas, after performing the derivatizacion as explained in the previous section. The percentage of derivatization was checked in the following way: Amonoderiv atized %derivatiza tion ¼ 100  ð †100Þ Abiderivat ized ACdeconjugated API %hydrolysis ¼ †100 ACfree API where AC deconjugated is the area of the compound after hydrolysis, AC free is the area of the compound without added conjugation and API is the corresponding area of the internal standard added. Results and Discussion Selectivity was verified by injecting five replicates of albumin 20% (control sample). No peaks were observed at the lower limit of quantification (LLOQ), which is defined as the lowest concentration of an analyte in a sample that can be quantitatively determined with suitable precision and accuracy (18), and which corresponds to the analyte response at the LLOQ being at least five times the response compared to blank response:  LLOQ ¼ Yc  Yb 5sb ¼ b b where Sb is the deviation standard of the white, b is the calibration curve slope, yc is the critical value of the brute signal and yb is the average of the target signals. Repeatability is the best match possible between the results obtained with the same method, test material and conditions (operator, apparatus and laboratory) after a short interval of time. To examine the repeatability of the current extraction method, the variation coefficient (N ¼ 5) was calculated for all the compounds studied in each of the studied solvents with the GC-Q– MS and GC-IT – MS-MS. The results are shown in Table II. Some of these studied compounds were not detected in some of the solvents; these situations have been termed N.D. With the solvent that gave the best results, the repeatability was studied by calculating the coefficient of variation (CV) of the injection of five replicates of three different concentrations Qualification and Quantification of Seventeen Natural Steroids in Plasma by GC–Q-MS and GC-IT –MS/MS 351 Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Free fraction extraction To one mL of plasma sample, 2 ng of methyltestosterone as internal standard was added and the pH was adjusted to 9.5 using NaOH 1M and carbonate buffer 0.2M, pH 9.5. Extraction was carried out with 2 mL of the following solvents: ethyl ether, tert-butyl methyl ether, n-hexane–ethyl acetate (25/75, v/v, 50/50, v/v and 70/30, v/v), ethyl acetate and n-hexane. After 30 min of shaking, the mixture was centrifuged for 5 min and the organic phase was dried under a stream of nitrogen. Hydrolysis test To perform the hydrolysis test, androsterone and etiocholanolone glucuroconjugates were used to control the glucuroconjugated fractions and androsterone and ethiocolanolone sulfoconjugates were used to control the sulfoconjugated fraction. Ion 419 was monitored for the androsterone, 434 for the etiocholanolone and 301 for the internal pattern (methyltestosterone). A known concentration (10 ng/mL) of androsterone and etiocholanolone in glucuroconjugate and sulfoconjugate form was added to 1 mL of 20% albumin; in parallel, the same known concentration of androsterone and etiocholanolone was added in free form to another milliliter of albumin, and the hydrolysis of the glucuroconjugate and sulfoconjugate compounds was performed as described in previous sections. The calculations of the percentage of hydrolysis were conducted in the following way: Table II Coefficient of Variation of the Compounds Studied in Different Solvents n-Hexane n-Hexane –ethyl acetate (70/30) n-Hexane –ethyl acetate (50/50) n-Hexano – ethyl acetate (25/75) Ethyl acetate diethyl ether tert-butyl methyl ether 42.98 45.00 N.D. N.D. N.D. 46.57 45.51 N.D. N.D. N.D. N.D. N.D. 28.76 28.85 N.D N.D. N.D. 12.01 13.09 11.03 5.94 9.80 11.42 7.98 10.77 13.86 12.83 12.22 10.63 7.20 13.79 9.06 8.19 13.84 63.14 50.70 45.77 46.38 54.77 40.24 55.63 49.44 42.55 45.44 38.00 N.D. 48.10 61.31 62.34 34.50 72.41 54.95 57.27 60.44 62.45 56.31 71.42 45.97 20.03 52.42 56.94 64.76 N.D. 84.37 98.80 96.83 98.00 84.21 N.D. N.D. 83.72 70.72 59.39 71.13 69.88 N.D. N.D. 73.24 65.95 71.74 N.D. N.D. 48.36 95.29 53.94 24.33 24.47 12.55 N.D. 23.95 N.D. N.D. N.D. N.D. N.D. 37.41 38.29 N.D. 56.77 56.12 45.85 53.54 N.D. N.D. 32.09 36.30 37.18 30.84 N.D. N.D. N.D. N.D. N.D. 29.17 N.D. 24.81 N.D. 29.86 29.89 Table III Repeatability of the Compounds Studied at Low Concentration (ppb) Table V Repeatability of the Compounds Studied at High Concentration (ppb) Compound Quadrupole Compound Mean S CV (%) 15 % CV Mean S CV (%) 15 % CV Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 0.96 0.99 0.97 0.97 0.98 0.99 0.95 0.99 0.99 0.98 0.97 0.99 0.99 4.99 4.99 4.98 4.98 0.11 0.02 0.06 0.05 0.02 0.05 0.01 0.03 0.01 0.02 0.03 0.03 0.01 0.01 0.07 0.06 0.03 10.89 2.07 6.51 5.00 1.86 4.98 0.58 3.30 1.24 1.55 2.77 3.50 0.85 0.26 1.40 1.22 0.69 1.63 0.31 0.98 0.75 0.28 0.75 0.09 0.49 0.19 0.23 0.42 0.52 0.13 0.04 0.21 0.18 0.10 0.99 0.99 0.98 0.97 0.98 0.97 0.99 0.99 0.92 0.93 0.94 0.97 0.99 4.99 4.99 4.97 4.98 0.01 0.07 0.03 0.05 0.04 0.04 0.01 0.04 0.03 0.01 0.05 0.06 0.04 0.08 0.01 0.02 0.01 1.36 6.70 3.00 5.32 3.90 4.22 1.10 3.63 2.92 1.18 5.22 6.02 3.57 1.51 0.17 0.47 0.14 0.20 1.00 0.45 0.80 0.59 0.63 0.17 0.54 0.44 0.18 0.78 0.90 0.54 0.23 0.03 0.07 0.02 Ion trap Table IV Repeatability of the Compounds Studied at Medium Concentration (ppb) Compound Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol Quadrupole Ion trap Mean S CV (%) 15 % CV Mean S CV (%) 15 % CV 5.93 5.96 5.97 5.97 5.98 5.98 5.95 5.97 5.97 5.95 5.94 5.93 5.97 29.93 29.98 29.88 29.98 0.05 0.08 0.11 0.04 0.07 0.06 0.04 0.03 0.07 0.10 0.02 0.07 0.07 0.01 0.01 0.01 0.01 0.77 1.34 1.87 0.66 1.17 0.95 0.73 0.42 1.22 1.65 0.26 1.26 1.17 0.04 0.04 0.04 0.04 0.12 0.20 0.28 0.10 0.18 0.14 0.11 0.06 0.18 0.25 0.04 0.19 0.17 0.01 0.01 0.01 0.01 5.96 5.95 5.97 5.99 5.98 5.95 5.95 5.98 5.95 5.98 5.96 5.93 5.99 29.93 29.97 29.87 29.98 0.03 0.03 0.02 0.11 0.04 0.11 0.07 0.12 0.03 0.11 0.16 0.11 0.22 0.01 0.01 0.01 0.01 0.50 0.48 0.32 1.80 0.66 1.89 1.14 1.98 0.42 1.84 2.68 1.86 3.66 0.04 0.04 0.04 0.04 0.07 0.07 0.05 0.27 0.10 0.28 0.17 0.30 0.06 0.28 0.40 0.28 0.55 0.01 0.01 0.01 0.01 352 Toribio-Delgado et al. Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol Quadrupole Ion trap Mean S CV (%) 15 % CV Mean S CV (%) 15 % CV 9.96 9.96 9.95 9.98 9.96 9.97 9.96 9.96 9.98 9.97 9.95 9.96 9.97 49.99 49.97 49.99 49.95 0.03 0.02 0.09 0.02 0.06 0.02 0.04 0.02 0.01 0.07 0.04 0.02 0.02 0.01 0.01 0.01 0.01 0.33 0.23 0.89 0.23 0.60 0.15 0.36 0.18 0.13 0.72 0.43 0.24 0.15 0.03 0.02 0.02 0.02 0.05 0.03 0.13 0.03 0.09 0.02 0.05 0.03 0.02 0.11 0.07 0.04 0.02 0.01 0.01 0.01 0.01 9.96 9.94 9.99 9.92 9.96 9.98 9.93 9.97 9.97 9.96 9.95 9.44 9.93 49.98 49.97 49.99 49.96 0.04 0.07 0.05 0.04 0.04 0.08 0.07 0.02 0.01 0.02 0.03 0.11 0.05 0.01 0.01 0.01 0.01 0.40 0.75 0.55 0.39 0.37 0.83 0.75 0.18 0.09 0.18 0.29 1.21 0.47 0.02 0.02 0.02 0.02 0.06 0.11 0.08 0.06 0.06 0.13 0.11 0.03 0.01 0.03 0.04 0.18 0.07 0.01 0.01 0.01 0.01 within the range of expected concentrations: low concentration, medium concentration and high concentration (Tables III, IV and V). The precision measure for each of the three concentrations should not exceed 15% CV of these measures (18) in all cases for both GC-Q –MS and GC-IT –MS-MS was carried out this premise. Reproducibility is the degree of approximation between the results obtained with the same method on identical test material but under different conditions (different operators, different apparatus, different laboratories and after different time intervals). To determine the reproducibility, the CV was determined when five replicates of three different concentrations were injected within the range of expected concentrations (low concentration, medium concentration and high concentration) using ethyl n-hexane–ethyl acetate mixture (70/30, v/v) as solvent extractor by the GC-Q–MS and the GC-IT –MS-MS on different days. The condition to be fulfilled in all cases is that the precision measure for each of the three concentrations Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Compound Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol Table VI Reproducibility of the Compounds Studied at Low Concentration (ppb) Table IX Accuracy of the Compounds Studied at Low Concentration (ppb) Concentration set Mean S CV (%) 15% CV Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 1 1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 0.98 0.99 0.98 0.97 0.98 0.98 0.97 0.99 0.95 0.95 0.96 0.98 0.99 4.99 4.99 4.98 4.98 0.07 0.05 0.05 0.05 0.03 0.04 0.03 0.03 0.04 0.03 0.04 0.05 0.02 0.05 0.05 0.04 0.02 7.34 4.70 4.82 4.87 2.91 4.49 2.64 3.28 4.48 2.96 4.24 4.79 2.45 1.02 0.94 0.88 0.47 1.10 0.70 0.72 0.73 0.44 0.67 0.40 0.49 0.67 0.44 0.64 0.72 0.37 0.15 0.14 0.13 0.07 Table VII Reproducibility of the Compounds Studied at Medium Concentration (ppb) Compound Concentration set Mean S CV (%) 15% CV Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 6 6 6 6 6 6 6 6 6 6 6 6 6 30 30 30 30 5.94 5.96 5.97 5.98 5.98 5.96 5.95 5.98 5.96 5.96 5.95 5.93 5.98 29.93 29.98 29.88 29.98 0.04 0.06 0.08 0.08 0.05 0.09 0.05 0.08 0.05 0.10 0.11 0.09 0.15 0.01 0.01 0.01 0.01 0.66 0.95 1.26 1.30 0.90 1.44 0.90 1.35 0.88 1.68 1.80 1.50 2.57 0.04 0.04 0.04 0.04 0.10 0.14 0.19 0.19 0.13 0.22 0.14 0.20 0.13 0.25 0.27 0.22 0.39 0.01 0.01 0.01 0.01 Compound Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol True value 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 5.00 5.00 5.00 5.00 Ion trap Quadrupole Mean þ15% –15% Mean þ15% –15% 0.99 0.99 0.98 0.97 0.98 0.97 0.99 0.99 0.92 0.93 0.94 0.97 0.99 4.99 4.99 4.97 4.98 1.13 1.14 1.13 1.12 1.13 1.11 1.14 1.13 1.06 1.07 1.09 1.11 1.14 5.74 5.74 5.72 5.73 0.84 0.84 0.83 0.83 0.83 0.82 0.84 0.84 0.78 0.79 0.80 0.82 0.84 4.24 4.24 4.22 4.23 0.96 0.99 0.97 0.97 0.98 0.99 0.95 0.99 0.99 0.98 0.97 0.99 0.99 4.99 4.99 4.98 4.98 1.11 1.14 1.11 1.12 1.12 1.14 1.09 1.14 1.14 1.12 1.12 1.14 1.14 5.74 5.74 5.73 5.72 0.82 0.84 0.82 0.82 0.83 0.84 0.80 0.84 0.84 0.83 0.83 0.84 0.84 4.24 4.24 4.24 4.23 Table X Accuracy of the Compounds Studied at Medium Concentration (ppb) Compound Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol True value 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 6.00 30.00 30.00 30.00 30.00 Ion trap Quadrupole Mean þ15% –15% Mean þ15% –15% 5.96 5.95 5.97 5.99 5.98 5.95 5.95 5.98 5.95 5.98 5.96 5.93 5.99 29.93 29.97 29.87 29.98 6.85 6.85 6.87 6.89 6.88 6.84 6.84 6.87 6.85 6.88 6.86 6.81 6.89 34.42 34.47 34.36 34.47 5.06 5.06 5.08 5.09 5.08 5.05 5.06 5.08 5.06 5.08 5.07 5.04 5.09 25.44 25.48 25.39 25.48 5.95 5.97 5.99 5.98 5.98 5.98 5.95 5.97 5.97 5.95 5.94 5.93 5.97 29.93 30.00 29.88 29.98 6.84 6.87 6.88 6.87 6.88 6.87 6.84 6.87 6.87 6.84 6.84 6.82 6.87 34.42 34.50 34.36 34.48 5.05 5.08 5.09 5.08 5.08 5.08 5.06 5.08 5.08 5.05 5.05 5.04 5.07 25.44 25.50 25.39 25.49 Table VIII Reproducibility of the Compounds Studied at High Concentration (ppb) Compound Concentration set Mean S CV (%) 15% CV Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 10 10 10 10 10 10 10 10 10 10 10 10 10 50 50 50 50 9.96 9.95 9.97 9.95 9.96 9.98 9.95 9.97 9.97 9.96 9.95 9.70 9.95 49.99 49.97 49.99 49.96 0.03 0.05 0.07 0.04 0.05 0.06 0.06 0.02 0.01 0.05 0.03 0.28 0.04 0.01 0.01 0.01 0.01 0.34 0.53 0.74 0.44 0.47 0.57 0.58 0.17 0.14 0.50 0.35 2.94 0.40 0.01 0.01 0.01 0.01 0.05 0.08 0.11 0.07 0.07 0.08 0.09 0.03 0.02 0.07 0.05 0.44 0.06 0.01 0.01 0.01 0.01 does not exceed 15% CV of these measures (18) (Tables VI, VII and VIII). The accuracy of the method was determined by measuring three concentrations in the range of expected concentrations (low concentration, medium concentration and high concentration) and five replicates per concentration. The average value should be within 15% of actual value (18). This is true for all concentrations studied, as shown in Tables IX, X and XI. The recovery of the method was determined by calculating the recovery rate when following the previously described procedure for five replicates of three concentrations within the range of expected concentrations. Valid recoveries above 90% were found in the three concentrations in study. This is true for all studied concentrations, as shown in Table XII. Standard curves were prepared in triplicate for each steroid. Peak area ratios (analyte/internal standard) were plotted against concentration, and the slope, interception and correlation coefficient were determined by linear regression analysis without including the zero point (Table XIII). These curves were linear within the concentration ranges studied, with correlation coefficients R 2 . 0.9. To assess the linearity of the calibration curve, a method was applied to test two hypotheses: the null hypothesis (H0), where there is no significant correlation between concentration and signal; and the alternative Qualification and Quantification of Seventeen Natural Steroids in Plasma by GC–Q-MS and GC-IT –MS/MS 353 Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Compound Table XI Accuracy of the Compounds Studied at High Concentration (ppb) Compound 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.00 50.00 50.00 50.00 50.00 Ion trap Quadrupole Mean þ15% – 15% Mean þ15% –15% 9.96 9.94 9.99 9.92 9.96 9.98 9.93 9.97 9.97 9.96 9.95 9.44 9.93 49.98 49.97 49.99 49.96 11.45 11.43 11.49 11.41 11.45 11.47 11.42 11.46 11.46 11.45 11.45 10.86 11.42 57.48 57.47 57.49 57.45 8.46 8.45 8.49 8.43 8.46 8.48 8.44 8.47 8.47 8.46 8.46 8.02 8.44 42.48 42.48 42.49 42.46 9.96 9.96 9.95 9.98 9.96 9.97 9.96 9.96 9.98 9.97 9.95 9.96 9.97 49.99 49.97 49.99 49.95 11.45 11.45 11.44 11.48 11.46 11.47 11.46 11.46 11.48 11.46 11.44 11.45 11.47 57.49 57.47 57.49 57.44 8.46 5.08 5.09 5.08 5.08 5.08 5.06 5.08 5.08 5.05 5.05 5.04 5.07 25.44 25.50 25.39 25.49 Table XII Recovery of the Compounds Studied at Low, Medium and High Concentrations Compound Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol Low concentration Medium concentration High concentration Quadrupole Ion trap Quadrupole Ion trap Quadrupole Ion Trap 96.40 98.80 96.80 97.00 97.60 98.80 94.60 99.20 99.00 97.60 97.40 99.00 98.80 99.76 99.80 99.68 99.52 98.60 99.40 98.20 97.20 98.20 96.80 99.20 98.60 91.80 92.80 94.40 96.60 99.00 99.80 99.76 99.40 99.60 99.10 99.57 99.77 99.63 99.67 99.60 99.20 99.57 99.57 99.10 99.07 98.80 99.50 99.78 99.99 99.59 99.95 99.30 99.23 99.53 99.83 99.67 99.10 99.17 99.63 99.23 99.70 99.37 98.77 99.80 99.77 99.91 99.58 99.92 99.56 99.56 99.46 99.82 99.64 99.74 99.62 99.64 99.82 99.66 99.48 99.60 99.74 99.98 99.94 99.98 99.90 99.58 99.40 99.90 99.22 99.58 99.76 99.28 99.66 99.66 99.58 99.54 94.40 99.32 99.96 99.94 99.98 99.92 Table XIII Calibration Curves of Quantification of the Compounds Studied Calibration curves R2 Compound MS –MS MS MS –MS MS Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone Estrone DHT Androstenodione 17b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol y ¼ 9.80x þ 4.65 y ¼ 0.27x þ 0.11 y ¼ 5.73x – 0.83 y ¼ 0.01x þ 0.03 y ¼ 0.05x þ 0.03 y ¼ 0.42x þ 1.84 y ¼ 0.09x – 0.01 y ¼ 0.15x þ 0.01 y ¼ 3.84x þ 2.66 y ¼ 0.01x þ 0.00 y ¼ 0.38x – 0.04 y ¼ 6.93x þ 32.27 y ¼ 0.14x – 0.01 y ¼ 0.29x þ 0.04 y ¼ 21.36x þ 1.28 y ¼ 5.59x þ 0.68 y ¼ 3.62x þ 4.27 y ¼ 0.82 þ 1.02x y ¼1.76 þ 0.60x y ¼1.16 þ 0.31x y ¼ 5.80 þ 4.50x y ¼ 0.08 þ 0.44x y ¼ 0.65 þ 0.32x y ¼ 4.03 þ 2.16x y ¼ 0.30 þ 0.32x y ¼ 0.55 þ 1.12x y ¼ 1.26 þ 2.26x y ¼ 1.03 þ 1.36x y ¼ 0.18 þ 0.44x y ¼ 0.38 þ 0.19x y ¼ 0.04 þ 0.04x y ¼ 0.03 þ 0.30x y ¼ 0.05 þ 0.03x y ¼ 0.01 þ 0.01x 0.98 0.92 0.94 0.99 0.92 0.99 0.97 0.95 0.96 0.98 0.95 0.98 0.97 0.98 0.94 0.98 0.99 0.98 0.93 0.99 0.92 0.98 0.96 0.95 0.94 0.95 0.98 0.96 0.92 0.98 0.97 0.93 0.94 0.98 354 Toribio-Delgado et al. Compound t Calculated (Ion trap) t Calculated (Quadrupole) t Tabulated Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 15.65 7.58 8.85 22.25 7.58 22.25 12.71 9.75 10.95 15.65 9.75 15.65 12.71 15.65 8.85 15.65 22.25 15.65 8.15 22.25 7.58 15.65 10.95 9.75 8.85 9.75 15.65 10.95 7.58 15.65 12.71 8.15 8.85 15.65 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 2.57 hypothesis (H1), where there is significant correlation between concentration and signal. This defines two “t” values, the tabulated t corresponding to the value found in the table of student’s t n-2 degrees of freedom, a 2-tailed a ¼ 0.05, and t calculated that corresponds to calculation using the following formula: tcalculated ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi r † ðn  2Þ pffiffiffiffiffiffiffiffiffiffiffiffiffi 1  r2 If t calculated . t tabulated, the alternative hypothesis is true that there is significant correlation between concentration and signal. The results of the method are shown in Table XIV. In this case, both calibration lines drawn by the quadrupole analyzer to the calibration curve drawn by the ion trap team shows that t calculated . t tabulated; thus satisfying the alternative hypothesis (H1) and ensuring a significant correlation between concentration and signal. The derivatization must be complete to ensure that the measuring of the analytes being quantified is correct. The current measurement of derivatization was performed with androsterone and etiocolanolone, both of which are isomeric in the derivatization test, thus ensuring that this derivatization was correct for both the alpha disposition and the beta disposition of the analytes. In this case, this derivatization was 100% for the androsterone and etiocholanolone. To undertake the quantification of analytes, they must first be separated from their links to the glucuronide acid and sulphate group; two specific enzymes were used to do this: arylsulphatase and b-glucuronidase, which work in specific conditions. If these links are not broken, the steroid will not be freed and, thus, its later derivatization and analysis will not be possible. It is very important to ensure that the enzymes hydrolyze the links well in the working conditions of the current method. Measurement of the percentage of hydrolysis was performed with androsterone and etiocolanolone in both sulfoconjugate and glucuroconjugate form in the hydrolysis test. In this case, the percentage of hydrolysis of the androsterone glucuro-conjugate was 100%, and the etiocholanolone glucuroconjugate 98%. The percentages of hydrolysis of the Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone DHT Estrone Androstenodione b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol True value Table XIV Linearity of the Calibration Curves Figure 2. Glucuroconjugated steroids in human plasma extracted with n-hexane-ethyl acetate 70/30 (v/v): 1 ¼ androsterone; 2 ¼ etiocolanolone; 3 ¼ DHEA; 4 ¼ epiandrosterone; 5 ¼ epitestosterone; 6 ¼ nandrolone; 7 ¼ DHT; 8 ¼ androstenodione; 9 ¼ testosterone; 10 ¼ 17b-estradiol; 11 ¼ estrone; 12 ¼ metiltestosterone; 13 ¼ estriol; 14 ¼ progesterone; 15 ¼ THE; 16 ¼ THF; 17 ¼ cortisone; 18 ¼ cortisol Figure 3. Sulphoconjugated steroids in human plasma extracted with n-hexane-ethyl acetate 70/30 (v/v): 1 ¼ androsterone; 2 ¼ etiocolanolone; 3 ¼ DHEA; 4 ¼ epiandrosterone; 5 ¼ nandrolone; 6 ¼ epitestosterone; 7 ¼ DHT; 8 ¼ androstenodione; 9 ¼ estrone; 10 ¼ testosterone; 11 ¼ 17b-estradiol; 12 ¼ metiltestosterone; 13 ¼ estriol; 14 ¼ progesterone; 15 ¼ THE; 16 ¼ THF; 17 ¼ cortisone; 18 ¼ cortisol. Qualification and Quantification of Seventeen Natural Steroids in Plasma by GC–Q-MS and GC-IT –MS/MS 355 Downloaded from https://academic.oup.com/chromsci/article/50/4/349/386062 by guest on 26 June 2022 Figure 1. Free steroids in human plasma extracted with n-hexane-ethyl acetate 70/30 (v/v): 1 ¼ androsterone; 2 ¼ etiocolanolone; 3 ¼ DHEA; 4 ¼ epiandrosterone; 5 ¼ epitestosterone; 6 ¼ nandrolone; 7 ¼ DHT; 8 ¼ androstenodione; 9 ¼ testosterone; 10 ¼ 17b-estradiol; 11 ¼ estrone; 12 ¼ metiltestosterone; 13 ¼ estriol; 14 ¼ progesterone; 15 ¼ THE; 16 ¼ THF; 17 ¼ cortisone; 18 ¼ cortisol. Table XV LOD, LOQ and LLOQ of the Compounds Studied LOQ (ng/mL) LLOQ (ng/mL) Ion trap Quadrupole Ion trap Quadrupole Ion trap Quadrupole Androsterone 5b-androsterone DHEA Epiandrosterone Epitestosterone Nandrolone Estrone DHT Androstenodione 17b-estradiol Testosterone Estriol Progesterone THE THF Cortisone Cortisol 0.20 0.20 0.05 0.36 0.05 0.33 0.11 0.02 0.05 0.17 0.48 0.06 0.13 0.18 0.04 0.29 0.02 0.35 0.52 0.50 0.02 0.01 0.16 0.02 0.39 0.07 0.19 0.09 0.74 0.18 0.26 1.60 0.05 0.14 0.33 0.33 0.08 0.60 0.08 0.55 0.18 0.03 0.08 0.28 0.80 0.10 0.22 0.30 0.07 0.48 0.03 0.33 0.33 0.08 0.60 0.08 0.55 0.18 0.03 0.08 0.28 0.80 0.10 0.22 0.30 0.07 0.48 0.03 0.33 0.33 0.08 0.60 0.08 0.55 0.18 0.03 0.08 0.28 0.80 0.10 0.22 0.30 0.07 0.48 0.03 0.58 0.87 0.83 0.03 0.02 0.27 0.03 0.65 0.12 0.32 0.15 1.23 0.30 0.43 2.67 0.08 0.23 androsterone and etiocholanolone sulfoconjugates were 95 and 94%, respectively. Figures 1, 2 and 3 show the chromatograms obtained in unconjugated, glucoconjugated and sulfoconjugated serum fractions, respectively. The detection limit can be defined according to the criteria 3s (19), which states that the LOD is the analyte concentration that provides the net signal that equals 3 times the standard deviation of the target signal:  Yc  Yb 3sb LOD ¼ ¼ b b where Sb is the deviation standard of the white, b is the calibration curve slope, yc is the critical value of the brute signal and yb is the average of the white signals. The LOQ is the lowest concentration at which an analyte can be quantified correctly and the concentration by which an S/N ¼ 10 is obtained, and the method used to calculate it is: LOQ ¼ (10/3) LOD ¼ 10 sb/b. The results obtained by the current method for each of these analytes are reflected in Table XV. With the GC-IT – MS-MS and GC-Q– MS method, the greatest sensibility was found for dihydrotestosterone (0.08 ng/mL) and cortisol (0.08 ng/mL) and the lowest was found for epiandrosterone (1.59 ng/mL), whereas with GC –MS the greatest and lowest sensibility were found for epiatestosterone (0.05 ng/mL), epiandrosterone (0.02 ng/mL), estrone (0.02 ng/mL) and cortisone (0.05 ng/mL). If LOD and LOQ of the two analyzers are compared, both the quadrupole analyzers and the ion trap analyzer are clearly valid for detecting and quantifying the steroid hormones being studied. It is worth observing that the advantage of the ion trap analyzer over the quadrupole analyzer is the possibility of making MS2. Conclusions The aim of this study was to develop a sensitive method for the simultaneous determination of 17 natural steroids in plasma. 356 Toribio-Delgado et al. References 1. 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