Accepted Manuscript Title: A simple LC-MS/MS method for the determination of cortisol, cortisone and tetrahydro-metabolites in human urine: assay development, validation and application in depression patients Author: Xuejia Zhai Fen Chen Chaoran Zhu Yongning Lu PII: DOI: Reference:

S0731-7085(15)00051-5 http://dx.doi.org/doi:10.1016/j.jpba.2015.01.041 PBA 9924

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

24-10-2014 16-1-2015 19-1-2015

Please cite this article as: X. Zhai, F. Chen, C. Zhu, Y. Lu, A simple LCMS/MS method for the determination of cortisol, cortisone and tetrahydrometabolites in human urine: assay development, validation and application in depression patients, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.01.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A simple LC-MS/MS method for the determination of cortisol, cortisone and

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tetrahydro-metabolites in human urine: assay development, validation and

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application in depression patients

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Xuejia Zhai, Fen Chen, Chaoran Zhu, Yongning Lu

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Science and Technology, Wuhan, 430022, People’s Republic of China

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Department of Pharmacy, Union Hospital, Tongji Medical College, Huazhong University of

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 Corresponding author. Tel.: +86 27 8572 6073; fax: +86 27 8572 6192. E-mail address: [email protected] (Y. L.).

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Abstract

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Chronic stress as well as major depressive disorders is associated with cortisol metabolism. Two

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enzymes modulate cortisol (F) and cortisone (E) interconversion: 11β-hydroxysteroid

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dehydrogenase type 1 and type 2 (11β-HSD1 and 11β-HSD2). Furthermore, F and E were

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inactivated by 5α and 5β reductases to their tetrahydro-metabolites: tetrahydrocortisol (THF),

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allo-tetrahydrocortisol (5α-THF) and tetrahydrocortisone (THE). To better understand depression

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a LC-MS/MS method for simultaneous determination of F, E THF, 5α-THF and THE in human

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urine has been developed and validated. The quantification range was 0.1-160 ng·mL-1 for F and

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E, and 0.2-160 ng·mL-1 for the tetrahydro-metabolites, with >86.1 % recovery for all analytes. The

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nocturnal urine concentrations of F, E and tetrahydro-metabolites in 12 apparently healthy male

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adult volunteers and 12 drug-free male patients (age range, 20-50 years) with a diagnosis of

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depression were analyzed. A series of significant changes in glucocorticoid metabolism can be

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detected: F/E ratios and (THF+5α-THF)/THE ratios as well as F and THF concentrations were

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significantly higher in depression patients than in healthy subjects (p < 0.05); 5α-THF/F ratios,

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5α-THF/THF ratios as well as 5α-THF concentrations were significantly lower in depression

patients (p < 0.05). The results pointed to the decreased 11β-HSD2 activity and a dysfunction in the 5α-reductase pathway in depressed patients. This method allows the assessment of 11β-HSD1/2

and 5α/β-reductase activities in a single analytical run providing an innovative tool to explain the

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potential etiology of depression.

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Keywords: LC-MS/MS; 11β-HSD; depression; cortisol; reductase

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1. Introduction

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Cortisol (F) is the main glucocorticoid hormone produced by the adrenal cortex. Its synthesis is

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stimulated by the hypothalamic corticotropin releasing hormone (CRH) and adrenocortico-tropic

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hormone (ACTH) from the hypophysis and regulated by several tissue-specific enzymes [1, 2].

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Chronic stress as well as depressive disorders are associated with hypercortisolemia and impaired

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hypothalamic-pituitary-adrenocortical (HPA) axis functioning [3, 4].

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A series of enzymes modulate F metabolism [5]. Interconversions of the active F and the inactive

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cortisone (E) are catalyzed by the key responsible enzyme, 11β-hydroxysteroid dehydrogenases

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(11β-HSDs). There are two 11β-HSD isoforms: 11β-hydroxysteroid dehydrogenase type 1

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(11β-HSD1) preferentially converts E to F in the liver and adipose tissue. 11β-hydroxysteroid

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dehydrogenase type 2 (11β-HSD2) transforms F to E in the kidney and colon. Furthermore, in the

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liver, F and E are metabolized to tetrahydrocortisol (THF), allo-tetrahydrocortisol (5α-THF) and

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tetrahydrocortisone (THE), catalyzed by 5α- or 5β-reductase (Fig. 1).

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The expression of the enzymes plays an important role on the regulation of glucocorticoid action

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[6, 7]. The impairment of their activity has been associated with the pathogenesis of depression [8]. Assessing the activities of the enzymes is critical to the treatment of the patients. Recent studies on human nocturnal urine have shown that the F/E and (5α-THF + THF)/THE ratios can be used as a marker of 11β-HSD2 activity, while the 5α-THF/F and 5α-THF/THF ratios can be considered as a

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marker of 5α-reductase [9].

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Immunoassay is usually used for determination of the steroid hormones in body fluids in the

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clinical laboratory due to its simplicity and sensitivity. Despite the many advantages, these

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methods lead to falsely elevated F values due to poor antibody specificity. Moreover,

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immunoassays have certain limitations, including suboptimal specificity, limited dynamic range,

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and suffering from matrix effects [10, 11]. GC-MS/MS, which is an alternative technique, is time

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consuming due to the low volatility of some compounds and the need of a derivatization step,

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limiting its application to routine diagnostics [12]. LC-MS/MS is an accepted, routine diagnostic

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technique in the clinical laboratory which meets the requirements of analytical sensitivity and

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specificity, as well as speed and robustness for the endogenous steroid analysis [13-15].

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Some research groups have developed LC-MS/MS methods for analyzing steroids in human

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plasma and urine. Pavlovic et al. measured F, E and tetrahydro-metabolites by LC-MS,but the

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method was validated in bovine urine [16]. Allende et al. described the measurement of the same

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five steroids in human urine in a single analytical run. However, it was operated in positive ion

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mode which produced lower signal-to-noise ratio than the negative ion mode [17]. Some

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quantitative studies of the steroids in body fluids have, however, focused solely on determination

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of either F and E or tetrahydro-metabolites [18-20]. For other quantitative studies, the procedures

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of sample preparation were time consuming and may increase the chance of inaccurate results due

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to incomplete hydrolysis and variability in the enzyme preparations [21, 22]. Moreover, to the best of our knowledge, the reports described the quantitative analysis of the steroids in human urine seldom use the real human urine in the method validation. Therefore, an analytical method for the simultaneous determination of F, E and tetrahydro-metabolites in human urine is still required.

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The aim of this study was to develop and validate an LC–MS/MS method, using US Food and

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Drug administration (FDA) parameters for the simultaneous determination of urinary F, E THF,

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5α-THF and THE in a clinical laboratory to gain a better understanding of depression

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pathogenesis.

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2. Experimetal

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2.1 Materials

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F, E, THF, 5α-THF, and THE were purchased from Steraloids, Inc., (Andover, MA, USA).

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Methylprednisolone (as an internal standard, IS) was purchased from the National Institute for

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Control of Pharmaceutical and Biological Products (Beijing, China). Formic acid (>98% pure),

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HPLC-grade acetonitrile and methanol were obtained from Tedia (Tedia, Fairfield, OH, USA).

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Other reagents were analytical-grade or better. The water used for LC-MS/MS was purified by use

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of a Milli-Q system (Millipore, Milford, MA, USA). Stock solutions of all standards (1.0 mg·mL-1)

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and of IS (50 ng·mL-1) were prepared in methanol and stored at 4°C.

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2.2 Instrumentation and analytical conditions

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HPLC analyses were performed on a 1200 series LC system from Agilent Technologies (Agilent,

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Santa Clara, CA, USA) equipped with an on-line degasser (G1322A), a quaternary pump

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(G1311A), an autosampler (G1329A) and a autosampler thermosta (G1330B). Chromatographic

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separation was performed on a Ultimate C18 column (150mm×2.1mm, 5µm, Welch Materials, Potomac, MD, USA). The mobile phase was composed of acetonitrile: 5 mM ammoniumacetate (containing 0.1% formic acid) (75:25; v/v) at an isocratic flow rate of 0.5 mL·min-1. The injection volume was 5 µL and the run time was 25 min. The temperatures of the analytical column and

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autosampler were set at 30°C and 4°C, respectively.

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The mass spectrometer was operated using an AB Sciex Qtrap® 4000 tandem mass spectrometer

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(Foster City, CA, USA) equipped with a Turbo V electrospray ionization (ESI) source in negative

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mode. For best selectivity and sensitivity Multiple Reaction Monitoring (MRM) mode was used

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for detection. The optimized condition consisted of a collision-activated dissociation (CAD) gas of

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12 psi, a curtain gas of 25 psi, a nebulizer gas (GS1) of 25 psi, a TurboIonSpray gas (GS2) of 10

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psi, an ion spray voltage of -4500 V, a source temperature of 500°C. The MRM transitions and the

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related optimized declustering potential (DP), entrance potential (EP), collision energy (CE) and

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collision cell exit potential (CXP) for the different analytes are shown in Table 1. MRM data was

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acquired and the chromatograms were integrated by use of the Analyst 1.6.1 software.

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2.3 Standards solutions

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Working solutions of nine standards (0.1, 0.2, 0.5, 1.0, 5, 10, 40, 80 and 160 ng·mL-1) were

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prepared in 1.0 mL human urine by dilution of the stock solution of each analyte. The standards

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were prepared on the day of analysis.

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2.4 Sample preparation

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An aliquot of 20 μL of IS (methylprednisolone; 50 ng·mL-1 in methanol) was added to a 480 μL

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human urine supernatant or calibrators in an Eppendorf tube. After vortex-mixing for 30 s, the

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mixture was added with 200 μl acetonitrile to precipitate protein and the sample was vortex mixed

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for 2 min and centrifuged at 6000 g for 10 min at 4°C. The separated supernatant was evaporated to dryness on a water bath at 40°C under a protective atmosphere of nitrogen. The residue was reconstituted with 100 μL mobile phase.

2.5 Method Validation

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The assay was validated meeting the published acceptance criteria for linearity, precision, matrix

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effect and stability proposed by the Food and Drug Administration. Due to the impossibility to

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obtain real urine samples free of corticosteroids, the endogenous concentration in healthy human

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urine sample was deducted in the method validation.

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2.5.1 Matrix effect and recovery

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The matrix effect (ME) and recovery (RE) for each analyte were assessed. Briefly, four sets of

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solutions were prepared. Set A contained the standard and IS in methanol; set B contained urine

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extracts spiked with the standards and IS after extraction; set C contained regular samples (i.e.,

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urine spiked with the standards and IS before extraction) and set D contained urine extracts spiked

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with the same volume of methanol as the standard and IS before extraction. Each set was prepared

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at three concentrations (2.0, 30 and 120 ng·mL-1) in sextuple.

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The peak area for the standards in these four sets were used to calculate ME and RE for each

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analyte according to the following equations:

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ME (%) = (B-D)/A × 100

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RE (%) = (C-D)/B × 100

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where A = peak area of each analyte from set A; B = peak area of each analyte from set B; C =

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peak area of each analyte from set C; and D = peak area of each analyte from set D.

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2.5.2 Linearity and LLOQ

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The linearity was evaluated by the regression analysis of spiked standards over the concentration range of the calibration curve. The samples were analyzed by measurement of the area of analyte (measured area - endogenous area) to IS peak-area ratios. The lower limit of detection (LLOD) was determined as the lowest concentration level of the calibration curve which met the following

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acceptance criteria. The LLOQ was defined as the lowest concentration that could be measured

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with an inter-day coefficient of variation (CV) of 86.1 %,

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with a matrix effect ranging from 85.1 to 114.6 % (Table 2).

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3.3 Linearity, LLOQ and precision

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Good analytical separation of all five steroids were achieved with an acceptable intra-day and

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inter-day precision and accuracy. The assay was linear from 0.1 to 160 ng·mL-1 for F and E, and

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from 0.2 to 160 ng·mL-1 for 5α-THF, THF, and THE. The determination coefficient (r2) was >0.99

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for each of the five analytes. LLOQ was 0.05 ng·mL-1 for F and E, and 0.1 ng·mL-1 for 5α-THF,

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THF, and THE. Precision was likewise acceptable. The values of intra- and inter-day assay were

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less than 14.7% for all the corticosteroids (Table 3).

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3.4 Carryover

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Compared to the LLOQ sample, the extra peak area counts of analytes and IS were not more than

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5% for both analytes and IS after the injection of ULOQ samples, indicating the absence of

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carryover.

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3.5 Sample stability

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All of the analytes were stable in human urine for at least 24 h at room temperature (20°C) and

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were stable in methanol for at least 30 days when stored at 4°C. The RSD of prepared samples in urine between the initial concentrations and the concentrations stored at -20°C for 1 month ranged from 7.91 to 14.8% for the analytes, which indicated that analytes were stable for at least 1 month at storage condition in urine. Moreover, the samples were stable during freeze-thaw cycles (n=3)

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at three concentration levels.

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3.6. Incurred sample reanalysis (ISR)

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On completion of the validation, ISR was performed in all cases where the method supported a

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clinical trial. Regulatory guidelines specify as a positive acceptance criterion for ISR when

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>66.7% of the incurred samples are within the 20% limit when comparing mean reanalysis results

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with its corresponding original value [23, 24]. For the ISR evaluation in this study, no less than 9

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out of 12 analytes concentrations were within ±20% of the original concentrations, and thus the

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ISR test pass the acceptance criteria.

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3.7 Application of the assays

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The nocturnal urine concentrations of F, E, THF, 5α-THF and THE in 12 healthy adults and 12

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depression patients were analyzed. Results presented that for apparently healthy adults, the F/E

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ratio was 0.136-0.690 (median 0.322), the values being 0.005-0.031 for the 5α-THF/F ratio

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(median 0.015) and 0.008-0.046 for the 5α-THF/THF ratio (median 0.024). The F/E ratio interval

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obtained for depression patient samples was 0.257-0.926, the values being 0.001-0.013 for

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5α-THF/F and 0.001-0.021 for 5α-THF/THF (Table 4). Urine F/E ratio and (THF+5α-THF)/THE

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ratio as well as F and THF concentrations measured with the proposed method, were significantly

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higher in patients than in healthy subjects (p < 0.05). 5α-THF/F ratio, 5α-THF/THF ratio as well

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as 5α-THF concentrations were significantly lower in patients than in healthy subjects (p < 0.05).

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This method is well suited for the evaluation of 11β-HSD1/2 and 5α/β-reductase activities which

may be helpful to physicians in diagnosing and monitoring patients with depression, but also in other endocrinological diseases in which there is an involvement of the 11β-HSD, such as the AME syndrome. As expected, in the present study, the reference ranges obtained in our protocol

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the F/E ratio are comparable with the reported urine F/E ratio levels [25]. Higher urine F/E ratio

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and (THF+5α-THF)/THE ratio have been observed in depression patients, which suggested the

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decreased 11β-HSD2 activity. Furthermore, compared to healthy controls, lower 5α-THF

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concentrations in combination with the lower baseline 5α-THF/F and 5α-THF/THF ratios in

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depressed patients compared to healthy controls points to a dysfunction in the 5α-reductase

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pathway in depressed patients.

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4 Conclusions

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An analytical LC–MS/MS method for the simultaneous determination of the five steroids in a

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single analytical run was developed in this study which is reliable, accurate, and precise. Based on

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FDA regulatory guidelines [26], the proposed method is validated for F, E, and the three

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tetrahydro-metabolites in human urine. This method is straightforward, relatively simple to

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perform, sensitive and has a satisfactory analysis time. The analytical characteristics of this

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method make it suitable for implementation as a routine technique in the clinical laboratory,

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allowing the assessment of 11β-HSD1/2 and 5α/β-reductase activities in a single analytical run. It

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provides clinicians with the opportunity to investigate into depression and other endocrinological

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diseases in daily practice.

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Acknowledgement

This study was financially supported by the National Natural Science Foudation of China (Grant 81403012 and 81473287), The Natural Science Foundation of Hubei Province (2013CFC042) and the Fundamental Research Funds for the Central Universities (No: 2013QN212) .

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Table 1 MRM transitions, declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit potential (CXP) of the analytes. Compounds

MRM transitions

DP (V)

EP (V)

CE (eV)

CXP (V)

1 2 3 4 5 6

F E THF 5α-THF THE IS

407.3/331.1 405.3/329.2 411.3/335.2 411.3/335.2 409.3/333.1 473.2/343.1

-75 -70 -100 -100 -106 -70

-10 -9 -10 -10 -5 -10

-36 -33 -35 -35 -30 -58

-20 -8 -15 -15 -16 -10

337

Ac ce p

te

d

M

an

us

338

ip t

No.

cr

334 335 336

17

Page 17 of 27

Table 2 Recovery (RE) and matrix effect (ME) of the method (n=6)

5α-THF

THF

THE

Matrix effect (%)

Mean

SD

Mean

SD

2.0 30 120 2.0 30 120 2.0 30 120 2.0 30 120 2.0 30 120

103.5 97.8 99.7 108.1 94.7 101.7 114.8 89.7 92.4 113.7 104.6 99.7 86.1 89.2 92.4

9.8 8.9 6.4 10.2 6.1 4.8 12.5 10.8 8.2 14.5 14.1 12.4 16.7 13.1 14.8

89.1 90.4 93.4 86.4 91.2 90.4 114.6 114.4 104.5 85.1 87.0 89.5 85.9 88.4 94.7

14.7 12.8 7.8 12.3 13.2 14.8 14.3 13.0 11.3 13.9 14.6 10.7 13.8 14.0 13.4

Ac ce p

te

d

M

339 340

ip t

E

Recovery (%)

cr

F

Amount added (ng·mL-1)

us

Steroid

an

338

18

Page 18 of 27

Table 3 Intra-assay and inter-assay precision of the developed method (n=6) Inter-assayb)

Contents(±SD, ng•mL )

F

E

5α-THF

THF

THE

10.8 9.37 7.60 12.3 10.2 8.17 10.6 8.97 9.45 14.7 10.7 11.3 11.7 13.0 7.98

The sample was analyzed 6 times during one day. The sample was analyzed once a day over six consecutive days.

te

d

b)

2.1± 0.2 33.1 ± 3.1 129.4 ± 9.8 2.3 ± 0.3 32.4 ± 3.3 131.3 ± 10.7 2.3 ± 0.2 31.2 ± 2.8 127.8 ± 12.1 2.2 ± 0.3 32.7 ± 3.5 125.7 ± 14.3 2.2 ± 0.3 32.3 ± 4.2 128.6 ± 10.3

R.S.D.(%)

M

a)

9.91 8.72 8.19 13.1 9.28 7.22 10.9 8.41 9.19 12.3 10.8 10.5 11.9 12.9 7.78

Contents(±SD, ng•mL-1)

Ac ce p

341 342 343 344

2.1 ± 0.2 32.1 ± 2.8 127.1 ± 10.4 2.1 ± 0.3 33.4 ± 3.1 121.2 ± 8.7 2.2 ± 0.2 32.1 ± 2.7 124.3 ± 11.4 2.2 ± 0.3 31.6 ± 3.4 128.1 ± 13.4 2.3 ± 0.3 31.8 ± 4.1 125.2 ± 9.7

R.S.D.(%)

ip t

-1

cr

Intra-assaya)

us

Components

an

340

19

Page 19 of 27

Table 4 Urinary excretion of cortisol metabolites during nighttime: a comparison between healthy subjects and depression patients

346

ip t

cr

F E 5α-THF THF THE F/E 5α-THF/F 5α-THF/THF (THF+5α-THF)/THE

healthy subjects(ng·mL-1,n=12) depression patients(ng·mL-1,n=12) 51.74±12.44* 37.25±12.84 107.76±15.76 123.17±23.79 0.30±0.21 * 0.51±0.15 30.11±3.86* 23.17±6.62 25.58±4.72 25.03±3.02 0.501±0.177* 0.322±0.152 0.006±0.004* 0.015±0.007 0.010±0.007* 0.024±0.012 0.963±0.306 1.231±0.299*

us

344 345

*

P

MS method for the determination of cortisol, cortisone and tetrahydro-metabolites in human urine: assay development, validation and application in depression patients.

Chronic stress as well as major depressive disorders is associated with cortisol metabolism. Two enzymes modulate cortisol (F) and cortisone (E) inter...
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