Detection of prohibited substances by liquid chromatography tandem mass spectrometry for sports doping control.

Drug testing for sports doping control programs is extensive and includes numerous classes of banned compounds including anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, diuretics, various peptide hormones, and growth factors. During competition, additional compounds may also be prohibited such as stimulants, narcotics, cannabinoids, glucocorticosteroids, and beta-blockers depending both on the sport and level of competition. Each of these classes of compounds can contain many prohibited substances that must be identified during the testing procedure. Various methods that have been designed to detect a large number of compounds in different drug classes are highly desirable as initial screening tools. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used by anti-doping testing laboratories for this purpose and several rapid methods have been described to simultaneously detect different classes of compounds. Here, we describe a simple urine sample cleanup procedure that can be used to detect numerous anabolic androgenic steroids, ß2-agonists, hormone antagonists and modulators, glucocorticosteroids, and beta-blockers by LC-MS/MS.

In vitro P-glycoprotein interactions and steady-state pharmacokinetic interactions between tolvaptan and digoxin in healthy subjects.

Interactions between tolvaptan and digoxin were determined in an open-label, sequential study where 14 healthy subjects received tolvaptan 60 mg once daily (QD) on days 1 and 12 to 16 and digoxin 0.25 mg QD on days 5 to 16. Mean maximal concentrations (C(max)) and area under the curve during the dosing interval (AUC(τ)) for digoxin with tolvaptan (day 16) were increased 1.27- and 1.18-fold compared with digoxin alone (day 11); mean renal clearance of digoxin was decreased by 59% (P < .05). Tolvaptan C(max) and AUC(0-24h) for a single dose with digoxin (day 12) were each increased about 10% compared with tolvaptan alone (day 1). Tolvaptan did not accumulate upon multiple dosing. After a single dose of tolvaptan (day 1, day 12), 24-hour urine volume was about 7.5 L. As expected, after 5 days of tolvaptan, 24-hour urine volume decreased about 20%. In vitro studies in control and MDR1-expressing LLC-PK1 cells indicate that tolvaptan is a substrate of P-glycoprotein. Tolvaptan (50 µM) inhibited basolateral to apical digoxin secretion to the same extent as 30 µM verapamil; the IC50 of tolvaptan was determined to be 15.9 µM. The increase in steady-state digoxin concentrations is likely mediated by tolvaptan inhibition of digoxin secretion.

Analysis of selective androgen receptor modulators by gas chromatography-microchip atmospheric pressure photoionization-mass spectrometry.

A gas chromatography-microchip atmospheric pressure photoionization-mass spectrometric (GC-microAPPI-MS) method was developed and used for the analysis of three 2-quinolinone-derived selective androgen receptor modulators (SARMs). SARMs were analyzed from spiked urine samples, which were hydrolyzed and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide before analysis. Trimethylsilyl derivatives of SARMs formed both radical cations (M(+*)) and protonated molecules ([M + H](+)) in photoionization. Better signal-to-noise ratios (S/N) were obtained in MS/MS analysis using the M(+*) ions as precursor ions than using the [M + H](+) ions, and therefore the M(+*) ions were selected for the precursor ions in selected reaction monitoring (SRM) analysis. Limits of detection (LODs) with the method ranged from 0.01 to 1 ng/mL, which correspond to instrumental LODs of 0.2-20 pg. Limits of quantitation ranged from 0.03 to 3 ng/mL. The mass spectrometric response to the analytes was linear (R > or = 0.995) from the LOQ concentration level up to 100 ng/mL concentration, and intra-day repeatabilities were 5%-9%. In addition to the GC-microAPPI-MS study, the proof-of-principle of gas chromatography-microchip atmospheric pressure chemical ionization-Orbitrap MS (GC-microAPCI-Orbitrap MS) was demonstrated.

Measurement of the novel decapeptide cetrorelix in human plasma and urine by liquid chromatography-electrospray ionization mass spectrometry.

A sensitive LC-MS quantitation method of cetrorelix, a novel gonadotropin releasing hormone (GnRH) antagonist, was developed. Plasma and urine samples to which brominated cetrorelix was added as an internal standard (I.S.) were purified by solid-phase extraction with C8 cartridges. The chromatographic separation was achieved on a C18 reversed-phase column using acetonitrile-water-trifluoroacetic acid (35:65:0.1, v/v/v) as mobile phase. The mass spectrometric analysis was performed by electrospray ionization mode with negative ion detection, and the adduct ions of cetrorelix and I.S. with trifluoroacetic acid were monitored in extremely high mass region of m/z 1543 and 1700, respectively. The lower limit of quantitation was 1.00 ng per 1 ml of plasma and 2.09 ng per 2 ml of urine, and the present method was applied to the analysis of pharmacokinetics of cetrorelix in human during phase 1 clinical trial.

Selective effects of somatostatin analogs on human drug-metabolizing enzymes.

Pharmacologic or surgical manipulation with growth hormone secretion or with the physiologic release of somatostatin and growth hormone-releasing hormone affects some rat liver enzymes, especially the sex-differentiated ones. We investigated the effects of two somatostatin analogs on several enzyme functions in six patients with carcinoid syndrome, using codeine as a probe drug. Codeine was given intravenously and its N- and O-demethylation, as well as 6-glucuronidation catalyzed by CYP3A, CYP2D6, and uridine diphosphate-glucuronosyltransferase, respectively, were studied before and during treatment with somatostatins. After 3 days of treatment with somatostatins the partial metabolic clearance of codeine by N-demethylation decreased by 21% to 64% in all patients (mean change, 44%; p < 0.05), and the clearance by O-demethylation was decreased by 20% to 69% in five of the patients (mean change in all patients, 35%; p < 0.05). In contrast, the partial clearance by 6-glucuronidation and the total systemic clearance of codeine were unchanged. Our results may be caused by the inhibition of growth hormone secretion induced by the somatostatins, inasmuch as direct metabolic interactions with these peptide drugs are improbable. The decline in CYP3A4 and CYP2D6 activity might have clinical implications when substrates of these enzymes with low therapeutic indices are combined with somatostatin analogs. Because the formation of morphine from codeine was altered, the analgesic effect of this drug may be reduced during concomitant treatment with somatostatins.

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS.

SR 49059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3, 4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1 H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut cartridges. The extracted samples were analyzed on a C18 HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (2H6-SR 49059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2-20 ng ml-1, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20-5000 ng ml-1, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).

Some observations on gonadotropin inhibitor (anti-LH) for human urine--with discussion on its possible source of origin.

Physicochemical characteristics of an anti-LH substance, partially purified from human urine, has been determined. It is an acid-precipitable, sialic acid containing glycoprotein with a molecular weight of 162,000. This substance on mg to mg basis has a 50 fold biological potency when compared to that of the crude gonadotropin inhibiting material. Dialysed human pineal protein extracts also showed anti-LH activity which was not attributed to sialidase activity. Polyacrylamide gel electrophoresis of such pineal extracts revealed a glycoprotein component with the same mobility as that of the urinary anti-LH protein. These observations suggest a possibility that the human pineal gland may be associated either with secretion or storage of this gonadotropin inhibiting entity.