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Endocrinology and Metabolism |
1 Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, Rochester, MN.
aAddress correspondence to this author at: Hilton 730, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905. Fax 507-284-9758; e-mail singh.ravinder{at}mayo.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Stable isotopes of cortisol-9,11,12,12-d4 and triamcinolone-d1 acetonide-d6 were added as internal standards to calibrators, controls, and unknown samples. After acetonitrile precipitation, these samples were extracted with methylene chloride, and the extracts were washed and dried. Reconstituted extract (15 µL) was injected on a reversed-phase column and analyzed by LC-MS/MS in positive-ion mode. Assay precision, accuracy, linearity, and sample stability were determined by use of enriched samples. Clinical validation included analysis of 8 serum and 20 urine samples from patients with undetectable cortisol concentrations and analysis of different types of tablets.
Results: Functional assay sensitivity was as low as 0.6–1.6 nmol/L for all compounds except for triamcinolone (7.6 nmol/L). Interassay CVs were 3.0–20% for concentrations of 0.6–364 nmol/L for all analytes. Recoveries of all analytes (except triamcinolone in serum) were 82–138% at 19.2–693 nmol/L. All but one of the serum and urine samples from patients who were tested because of suppressed cortisol concentrations contained at least one synthetic steroid. Tablet analysis recovered 75% of the synthetic steroids in suspected drugs.
Conclusions: LC-MS/MS allows simultaneous quantitative detection of various synthetic steroids in serum, plasma, urine, and tablets. This provides a valuable tool for evaluating the clinical effects of topical and systemic synthetic corticosteroids.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In all of these cases, establishing a definitive diagnosis of systemic synthetic corticosteroid effects and providing accurate monitoring require measurement of actual synthetic steroid concentrations in serum or urine. Unfortunately, such assays are generally not available in hospital, reference, and research laboratories, and if offered they may allow measurement of only a single synthetic corticosteroid. For endogenous steroids, methods based on liquid chromatography/electrospray tandem mass spectrometry have been developed. For synthetic steroids, assays have been developed specifically to analyze a single or only a few synthetic glucocorticoids and their metabolites (13)(14)(15)(16)(17). We therefore developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the simultaneous detection and quantification of the 14 most frequently used synthetic corticosteroids in human serum, plasma, and urine and in tablet extracts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cortisol-9,11,12,12-d4 was purchased from Cambridge Isotope Laboratories (isotopic enrichment, 98%). Triamcinolone-d1 acetonide-d6 was purchased from CDN Isotopes (stated isotopic enrichment, 74% d1, 99% d6). We prepared a stock solution containing 2.73 mmol/L cortisol-9,11,12,12-d4 and 2.27 mmol/L triamcinolone-d1 acetonide-d6 (1 g/L of each internal standard) in methanol. A working solution containing 11 µmol/L cortisol-9,11,12,12-d4 and 9.1 µmol/L triamcinolone-d1 acetonide-d6 (4 mg/L) was prepared by diluting the stock solution 1:250 with methanol–water (70:30 by volume) containing estriol.
sample preparation
Tablet extracts required additional processing, which was not necessary for serum, plasma, or urine samples. We prepared tablet extracts by weighing the tablet, recording the weight, and then crushing the tablet into powder with a mortar and pestle. We weighed a portion of the powder and added it to a 10-mL volumetric flask. Methanol was added to the 10-mL volume, and the sample was mixed with a magnetic stirrer for 1 h at room temperature.
A four-point calibration curve [0 µg/L, 5 µg/L (9.6–14 nmol/L), 50 µg/L (96–140 nmol/L), and 250 µg/L (480–700 nmol/L)] in phosphate-buffered saline containing 10 g/L bovine serum albumin (PBS-BSA) and two concentrations of controls were run with each assay. Aliquots of urine, tablet extract, serum, or plasma were centrifuged for 5 min at 1000g to remove particulate matter. Tablet extracts were diluted 1:10, 1;100, and 1:1000 in PBS-BSA. We transferred 0.5-mL fractions of the calibrators, controls, urine samples, serum samples, plasma samples, and diluted tablet extracts to 12 x 75 mm borosilicate glass tubes and mixed them with 25 µL of the working solution containing 11 µmol/L cortisol-9,11,12,12-d4 and 9.1 µmol/L triamcinolone-d1 acetonide-d6 and 0.5 mL of acetonitrile. We then centrifuged these solutions at 1000g for 10 min. The supernatants were transferred to 13 x 100 mm borosilicate glass tubes, and 4 mL of methylene chloride was added to each tube. The samples were then vortex-mixed for 30 s and centrifuged at 1000g for 5 min; the upper aqueous layer was then aspirated and discarded. The methylene chloride fractions were washed sequentially with 1.0 mL of 0.1 mol/L sodium hydroxide, 1.0 mL of 0.1 mol/L hydrochloric acid, and 1.0 mL of H2O, and each aqueous layer was aspirated and discarded. The washed methylene chloride was evaporated under nitrogen at 45 °C, and the dried extract reconstituted with 75 µL of methanol–water (70:30 by volume) containing estriol. The reconstituted extracts were centrifuged for 5 min at 1000g and transferred to autosampler vials.
lc-ms/ms
All LC-MS/MS experiments were performed with Perkin-Elmer Series 200 pumps and autosampler for sample introduction and an API 3000TM tandem mass spectrometer (Applied Biosystems), operating with an electrospray ionization source. The analytes and internal standards were chromatographically resolved from other sample components with a reversed-phase column [SYNERGI 4µ MAX-RP; 50 x 4.6 mm (i.d.); Phenomenex®] combined with a 4 x 2 mm (i.d.) precolumn filter of the same material. The mobile phase, delivered at a flow rate of 750 µL/min, consisted of 29% acetonitrile in 0.1 mmol/L ammonium acetate for 9.5 min, followed by 65% acetonitrile in 0.1 mmol/L ammonium acetate for 4 min, with a final 0.5-min reequilibration at 29% acetonitrile in 0.1 mmol/L ammonium acetate. The flow delivered to the TurboIonspray® was split, with 80% going to waste and 20% to the ionization probe. Total instrument analysis time, including sample introduction and sample run time, was 15 min per sample.
Urine, serum, and plasma results were calculated directly from the calibration curve. After the values for the tablet extracts were calculated from the calibration curve, the amount of analyte per tablet was calculated as follows:
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The mass spectrometer operating conditions consisted of a source heater probe of 450 °C, with a TurboIonspray voltage of 5000 V, declustering potential of 35, entrance potential of 10, cell exit potential of 12, nebulizer gas setting of 8, curtain gas setting of 6, and CAD setting of 8. Collision energies were different for several analytes and are listed in Table 1
. Data acquisition and quantitative processing were accomplished using AnalystTM software, (Applied Biosystems). The ions for all glucocorticoids were generated in the positive-ion mode. The ion transitions used to monitor the analytes were determined by infusing 10 mg/L solutions of each analyte separately in methanol–water (50:50 by volume) containing 0.2 mmol/L ammonium acetate at very low flow rate of 10 µL/min. The multiple-reaction monitoring (MRM) ion transitions for the synthetic steroids are listed in Table 1
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method validation
Method validation included extraction efficiency, precision, functional assay sensitivity, recovery, linearity, and stability. Pools containing different analyte concentrations for the method validation studies were created by adding various amounts of the calibrators to normal urine and serum and dividing them into aliquots, which were stored at –20 °C.
Extraction efficiencies were determined by comparing a 15-µL injection of an unextracted 200 µg/L (392–560 nmol/L) calibrator vs a 15-µL injection of the 50 µg/L (98–140 nmol/L) PBS-BSA-extracted calibrator, which was also concentrated four times.
Intraassay precision studies included 20 samples for each concentration. Interassay imprecision was calculated based on 23 separate assays, run on separate days. All samples were assayed in singlet.
The functional assay sensitivity for each analyte was defined as the lowest analyte concentration with an interassay CV <20%.
For the recovery studies, we added all 14 analytes at three concentrations each, ranging from 19.2 to 693 nmol/L (10–250 mg/L), to urine, serum, EDTA-plasma, and heparin-plasma samples from three patients that were devoid of synthetic corticosteroids. Recoveries are expressed as the percentages of added analytes that were recovered.
To determine assay linearity, we added 250 µg/L (480–693 nmol/L) each of the calibrators to three patient samples for each of the sample matrices and diluted them 2-, 5-, and 20-fold in the PBS-BSA buffer. The expected value of each dilution was calculated based on the result of the undiluted sample. We evaluated assay linearity by dividing the observed value by the expected value for each dilution, with the result being expressed as the percentage of expected recovery.
For stability studies, we added calibrators to five patient samples each of urine (unpreserved, boric acid, and acetic acid), serum (SST and clot tube), EDTA plasma, and heparin plasma. Four aliquots were immediately frozen at –20 °C and subjected to zero, one, two, or three freeze–thaw cycles. In addition, one aliquot was stored at room temperature, and one aliquot was stored refrigerated. On days 1, 3, and 7, an aliquot was taken from the room temperature and the refrigerated samples and was frozen at –20 °C. All aliquots were then assayed.
To study the matrix effect of the assay, we prepared a 1 mg/L (1920–2772 nmol/L) solution containing all of the synthetic steroids. We then used a T-valve inserted in the solvent line after the autosampler to infuse each solution into the system at 10 µL/min. While the solution was being infused, an extracted sample of zero calibrator in BSA (data not shown), a negative serum sample extract, or a negative urine extract was injected.
We performed a limited Institutional Review Board-approved clinical assay validation by retrospectively studying 8 plasma samples and 20 urine samples with undetectable cortisol concentrations. We also tested the dexamethasone content of two different types of dexamethasone tablets manufactured outside the United States with a manufacturer-stated drug content of 750 mg/tablet. Finally, we analyzed another tablet, of unknown origin, that had been retrieved from a patient who had undetectable serum cortisol concentrations but exhibited symptoms of Cushing syndrome.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The structures of the various synthetic steroids (see the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol50/issue12/) indicate that most of these steroids, except for triamcinolone, fludrocortisone, and prednisolone, are more hydrophobic than cortisol and thus elute later on a reversed-phase column with an increasing gradient of organic solvent (Table 1
). The extracted sample ion chromatogram for a calibrator containing 250 µg/L (480–963 nmol/L) each of the 14 different synthetic glucocorticoids and 2 internal standards is shown in Fig. 1
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We would have preferred to use individual internal standards for all of the steroids studied, but only deuterium-labeled cortisol and triamcinolone acetonide were commercially available. On the basis of the chromatography and retention times of the steroids, we used cortisol-d4 for quantification of betamethasone, dexamethasone, fludrocortisone, methyl prednisolone, prednisolone, prednisone, triamcinolone, cortisol, and cortisone. For quantification of the rest of the steroids, we used the deuterium-labeled triamcinolone acetonide as internal standard. The peaks for the isomers dexamethasone and betamethasone have identical ion pairs and thus need to be separated chromatographically (Table 1
and Fig. 1
). Similarly, the isomers triamcinolone acetonide and flunisolide also need to be separated chromatographically from each other because of similar precursor and product ions. Triamcinolone acetonide and flunisolide had similar parent (Q1) and product ions (Q3) of m/z 397 and 321. The m/z 397 ion was optimum for triamcinolone acetonide quantification, and we chose the m/z 321 ion for flunisolide because this daughter ion had a higher signal intensity. These compounds were separated by use of a slow gradient, and attention was also paid to relative retention times to ensure proper identification.
method validation
The intraassay imprecision (CV) for all analytes ranged from 2.6% to 9.8% at 19.2–327 nmol/L (10–118 µg/L). The interassay imprecision for all analytes ranged from 3.2% to 20% for mean concentrations of 0.6–364 nmol/L (0.3–130 µg/L) and are summarized in Table 2
. From the imprecision data for the serum and urine matrices, we calculated that the functional sensitivity was 0.6–1.6 nmol/L (0.3–0.7 µg/L) for all analytes except for triamcinolone. Triamcinolone had a functional sensitivity of 7.6 nmol/L (3 µg/L) because of poor recovery in the extraction protocol used in the present study, in which the goal was to extract numerous synthetic steroids of variable hydrophilicity and hydrophobicity. The signal-to-noise ratios for the analytes in the functional sensitivity pool were calculated and are listed in Table 2
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The absolute extraction efficiency ranged from 69% to 89% for all analytes except triamcinolone, which had an extraction efficiency of 33%, presumably because of its more hydrophilic nature. The recovery data relative to the internal standards for various glucocorticoids are summarized in Table 3
. Recoveries ranged from 82% to 138% (mean, 108%) for all analytes except triamcinolone. Triamcinolone had acceptable recovery in urine (range, 90–134%; mean, 110%), but recoveries in serum and plasma were highly variable (range, 28–80%; mean, 46%). This is probably because triamcinolone is the most polar component of the steroid profile and is lost during the extraction process. Although the method lacked deuterium-labeled triamcinolone as an internal standard, it is also likely that components of the serum matrix may have suppressed the signal and accounted for the variable recovery of this compound (18). The matrix effects of a negative serum sample extract and a negative urine sample extract are shown in the top and bottom panels of Fig. 2
, respectively; we observed no significant matrix effects, probably because in our method, plasma and urine samples are not only extracted with a nonpolar solvent (methylene chloride) but are also washed with acid and base to remove most of the suppressing and interfering matrix components.
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With the exception of triamcinolone in serum, the method was linear, based on a recovery of 80–120%, for all twofold dilutions. Using the criterion of ±20% recovery, all analytes except beclomethasone dipropionate were stable in serum, plasma, and urine for 1 day at ambient temperature (stable in urine for 7 days), for 7 days when stored refrigerated, and for three freeze–thaw cycles. Beclomethasone dipropionate was stable in frozen serum, plasma, and urine for three freeze–thaw cycles and in refrigerated urine for 7 days.
The chromatogram of a patient sample with suppressed plasma cortisol and confirmed to be positive for methylprednisolone and megestrol acetate is shown in Fig. 3
. Among eight patient samples, five were found to be positive for dexamethasone [1.5–28 µg/L (3.8–71.4 nmol/L)], one for megestrol acetate [23 µg/L (60 nmol/L)], and one for prednisone [2 µg/L (5.6 nmol/L)] and prednisolone [16 µg/L (44.4 nmol/L)], and one sample was negative. Of the 20 urine samples, 7 were positive for dexamethasone [2–26 µg/L (5–66 nmol/L)], 5 for triamcinolone acetonide [0.8–20 µg/L (2–46 nmol/L)], 3 for methylprednisolone [0.8–6.5 µg/L (2–17 nmol/L)], 1 for prednisone [12 µg/L (34 nmol/L)] and prednisolone [1.8 µg/L (5 nmol/L)], 1 for dexamethasone [17 µg/L (43 nmol/L)] and triamcinolone acetonide [22 µg/L (51 nmol/L)], and 1 for triamcinolone acetonide [3.4 µg/L (8 nmol/L)] and budesonide [2.4 µg/L (6 nmol/L)]; 1 sample was negative. The tablets obtained from patients who had purchased them from outside the United States were found to contain 565 µg (1441 nmol)/tablet of dexamethasone. Another tablet of unknown origin, from a patient exhibiting symptoms of Cushing syndrome with undetectable cortisol concentrations, was found to contain 2.9 mg (7360 nmol)/tablet of triamcinolone.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial protocol for this method used only the methylene chloride extraction and washes for sample preparation. This provided accurate recoveries for all compounds except for megestrol acetate and fluticasone propionate in serum and plasma, which were unacceptably low at 15–50%. Acetonitrile pretreatment improved the recoveries of megestrol acetate and fluticasone propionate in serum and plasma. It may be that these two compounds are protein-bound in the serum and plasma, which may have decreased the recovery without the acetonitrile pretreatment.
Analysis of a limited number of patient samples suggests that the proposed method is sufficiently sensitive to provide clinically valuable data to distinguish endogenous hypocortisolism from synthetic glucocorticoid-induced HPA-axis suppression and to allow diagnosis of iatrogenic Cushing syndrome. Among the plasma samples with undetectable cortisol, 87.5% were positive for at least one and 95% of the urine samples were positive for one or more synthetic corticosteroids. Because some of the synthetic steroids are extremely potent, depot injections into joints may release these potent substances for prolonged periods of time. Therefore, HPA-axis suppression or iatrogenic Cushing syndrome may be observed many weeks or months after topical synthetic steroid application. Indeed, during the validation phase of the assay, we received requests from clinical colleagues to investigate several such situations. As we have reported recently, we were able to detect previous synthetic steroid use in all of these cases, sometimes long after the drugs had been discontinued (23).
Our assay could also be valuable for identifying individual patients who are at particular risk of developing corticosteroid side effects as a consequence of atypically large fractional systemic absorption of topical synthetic corticosteroids. Finally, the ability to detect these compounds at low concentrations is likely to improve pharmacokinetic, drug validation, and toxicity studies.
The assay’s performance characteristics are for the most part very satisfactory. Unlike the other synthetic corticosteroids, the results for triamcinolone in serum are only semiquantitative. If needed, improvements in the extraction methodology could provide better quantitative triamcinolone measurements. For tablet analysis, the analyzed result was 75% of the manufacturer-stated amount of dexamethasone in the tablets, indicating that this method can be used for detection of synthetic glucocorticoids in unknown medications.
The assay run time, although acceptable, is relatively long for a LC-MS/MS method. However, this is necessary to chromatographically separate the isomers dexamethasone and betamethasone and the isomers triamcinolone acetonide and flunisolide and to elute the more hydrophobic compounds in the profile. The step gradient increase of acetonitrile from 29% to 65% at 9.5 min elutes the more hydrophobic compounds in a reasonable time. The major benefit of this step gradient from hydrophilic mobile phase to hydrophobic phase is that new synthetic glucocorticoids may be added to the assay without altering the chromatographic conditions, which already allow separation of steroids with a broad range of hydrophobicity. In addition, when interest is limited to one specific type of synthetic corticosteroid, rather than a profile, run conditions can be adjusted to allow a much more rapid analysis.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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500 µg/day in adults and adolescents with mild to moderate asthma. Drugs 1999;57:769-803.[CrossRef][Medline]
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