Gel-electromembrane extraction of peptides: Determination of five hypothalamic agents in human plasma samples.

Agarose gel as a green membrane has been proposed for use in electromembrane extraction of five hypothalamic-related peptides without an ionic carrier. Octreotide, goserelin, triptorelin, cetrorelix, and somatostatin were extracted from 5.0 mL of sample solution (adjusted to pH 5.0) into a microvolume acceptor solution (HCl, 100 mM) under the applied voltage of 30 V in 15 min. The pH of the agarose gel 3.0% (w/v) was adjusted to 4.0 to facilitate the movement of peptides through the membrane. Quantification was performed using an HPLC-UV system on a C18 column. Quantification and detection limits were found to be in the range of 15.0-20.0 ng mL-1 and 4.5-6.0 ng mL-1, respectively. Dynamic linear ranges were found to be in the range of 15.0-1000 ng mL-1 (R2 > 0.995) and recoveries were in the range of 62.3-77.6%. The optimized method was applied to spiked human plasma samples. The method showed relative recoveries in the range of 44.8-66.0%. Finally, the proposed method was compared with and shown to have higher recoveries than, the conventional electromembrane extraction method for the peptides under study.

Role of emotional processing in depressive responses to sex-hormone manipulation: a pharmacological fMRI study.

Sex-hormone fluctuations may increase risk for developing depressive symptoms and alter emotional processing as supported by observations in menopausal and pre- to postpartum transition. In this double-blinded, placebo-controlled study, we used blood-oxygen level dependent functional magnetic resonance imaging (fMRI) to investigate if sex-steroid hormone manipulation with a gonadotropin-releasing hormone agonist (GnRHa) influences emotional processing. Fifty-six healthy women were investigated twice: at baseline (follicular phase of menstrual cycle) and 16 ± 3 days post intervention. At both sessions, fMRI-scans during exposure to faces expressing fear, anger, happiness or no emotion, depressive symptom scores and estradiol levels were acquired. The fMRI analyses focused on regions of interest for emotional processing. As expected, GnRHa initially increased and subsequently reduced estradiol to menopausal levels, which was accompanied by an increase in subclinical depressive symptoms relative to placebo. Women who displayed larger GnRHa-induced increase in depressive symptoms had a larger increase in both negative and positive emotion-elicited activity in the anterior insula. When considering the post-GnRHa scan only, depressive responses were associated with emotion-elicited activity in the anterior insula and amygdala. The effect on regional activity in anterior insula was not associated with the estradiol net decline, only by the GnRHa-induced changes in mood. Our data implicate enhanced insula recruitment during emotional processing in the emergence of depressive symptoms following sex-hormone fluctuations. This may correspond to the emotional hypersensitivity frequently experienced by women postpartum.

[Serum concentrations of flutamide and goserelin in a prostate cancer patient with obstructive nephropathy: a case report].

A 72-year-old man presented with pollakiuria and dysuria. His prostate was the size of an apple and hard on digital rectal examination and the serum prostate specific antigen (PSA) level was 73 ng/ml (RIA). Ultrasonography revealed bilateral hydronephrosis and the serum creatinine level was 13.2 mg/dl. CT scanning of the abdomen demonstrated swelling of paraaortic lymph nodes. Transrectal needle biopsy of the prostate gave a diagnosis of moderately differentiated adenocarcinoma. Accordingly, the final diagnosis was prostate cancer (cT3N4M1, stage D2). Immediately after bilateral percutaneous nephrostomy, treatment with an LH-RH agonist (goserelin) and flutamide was commenced. Serum creatinine was 6.6 mg/dl at the start of antiandrogen therapy and decreased to 1.8 mg/dl after 27 days. A 125 mg flutamide capsule was administered at 7 a.m., and blood samples were collected 4 hours later on days 1, 2, 3, 5, 6, 8, 12, 14, 17, 18 and 27. The OH-flutamide concentration was measured. There was no significant correlation between serum creatinine and the OH-flutamide concentration. After implantation of goserelin (3.6 mg depot), blood samples were obtained at 11 a.m. on days 8, 12, 14, 15 and 25. The serum goserelin level was measured. The serum goserelin level increased to a peak on day 14, as described previously, but the peak value of 9.63 ng/ml was higher than that reported before (mean +/- SD 2.848 +/- 0.199).

An LC-MS/MS method for the simultaneous determination of goserelin and testosterone in rat plasma for pharmacokinetic and pharmacodynamic studies.

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed, using testosterone-d3 as a surrogate analyte, for the simultaneous quantification of goserelin and testosterone in rat plasma. According to this method, the pharmacokinetic and pharmacodynamic data were obtained from a single plasma sample aliquot. The method involved the addition of alarelin as an internal standard (IS) for goserelin and testosterone-(13)C3 for testosterone or testosterone-d3. The conditions for the separation of these two compounds were achieved on a ZORBAX Eclipse Plus C18 column (Agilent, 2.1 × 50 mm, 1.8 µm, Stockport, UK) in a single chromatographic run at a flow rate of 400 µL/min. In order to minimize interferences of complex matrix, the extraction of plasma consisted of a protein precipitation step using methanol, followed by purification using an Oasis(®) HLB solid-phase extraction column. The method was validated in the concentration range of 0.01-30.0 ng/mL for goserelin and 0.05-30.0 ng/mL for testosterone-d3, respectively. The within- and between-run precisions were 1.7-9.2% and 2.1-6.9%, respectively. The within- and between-run accuracies were -1.8 to 5.3% and -4.9 to 4.0%, respectively. This accurate and highly specific assay provides a useful method to evaluate the pharmacokinetics and pharmacodynamics of goserelin in rats.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of goserelin in rabbit plasma.

A rapid and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC-ESI-MS/MS) was developed and validated for the determination of goserelin in rabbit plasma. Various parameters affecting plasma sample preparation, LC separation, and MS/MS detection were investigated, and optimized conditions were identified. Acidified plasma samples were applied to Oasis((R)) HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100microL mobile phase A. The separation was achieved on a Capcell-Pak C18 (2.0mmx150mm, 5microm, AQ type) column with a gradient elution of solvent A (0.05% acetic acid in deionized water/acetonitrile=85/15; v/v) and solvent B (acetonitrile) at a flow rate of 250microL/min. The LC-MS/MS system was equipped with an electrospray ion source operating in positive ion mode. Multiple-reaction monitoring (MRM) of the precursor-product ion transitions consisted of m/z 635.7-->m/z 607.5 for goserelin and m/z 424.0-->m/z 292.1 for cephapirin (internal standard). The proposed method was validated by assessing specificity, linearity, limit of quantification (LOQ), intra- and inter-day precision and accuracy, recovery, and stability. Linear calibration curves were obtained in the concentration range of 0.1-20ng/mL (the correlation coefficients were above 0.99). The LOQ of the method was 0.1ng/mL. Results obtained from the validation study of goserelin showed good accuracy and precision at concentrations of 0.1, 1, 5, 10, and 20ng/mL. The validated method was successfully applied to a pharmacokinetic study of goserelin after a single subcutaneous injection of 3.6mg of goserelin in healthy white rabbits.

Profiling and in vivo quantification of proteins by high resolution mass spectrometry: the example of goserelin, an analogue of luteinizing hormone-releasing hormone.

Proteins are essential biomolecules which are frequently involved in major pathological syndromes and are widely used as diagnostic markers or therapeutic agents. The emergence of proteomics will doubtless further increase the significance of proteins both in the clinic and in the life sciences in general. Our main objective is to offer innovative solutions to what we like to call the "post-proteomics era". To achieve our goal, we intend to develop novel approaches and technologies for in vivo metabolic studies of proteins using mass spectrometry (MS), focusing on pharmacokinetics and pharmacodynamics. Using goserelin as a model, we have successfully developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of an intact analogue of luteinizing hormone-releasing hormone (LHRH) in small volumes of rat plasma samples at concentrations ranging from 0.3 to 405.0 microg/l. To this end, a microbore reversed-phase-HPLC system was coupled on-line to a tandem high resolution quadrupole time-of-flight (Q-TOF) instrument fitted with an electrospray ion source and operated in LC-MS/MS mode. External calibration was used and the high resolution was crucial to discard contaminating signals, which would not have been possible with the more conventional triple quadrupole mass spectrometers operated in a static mode. For low sample amounts, calibration curves were constructed corresponding to rat plasma levels of 0.3 to 16.4 microg/l and found to be of third order with a coefficient of determination greater than 0.999. The relative standard deviation was found to be lower than 15%. A lower limit of detection (LLOD) of 0.17 microg/l and a lower limit of quantification (LLOQ) of 0.3 microg/l were determined.