Androgen levels during adjuvant endocrine therapy in postmenopausal breast cancer patients.

OBJECTIVE: To investigate plasma steroid hormone levels in postmenopausal breast cancer patients with and without adjuvant endocrine therapy and in healthy postmenopausal women. METHODS: Steroid hormone levels in postmenopausal breast cancer patients treated with aromatase inhibitors (n = 32) were compared with breast cancer patients treated with tamoxifen (n = 34), breast cancer patients without adjuvant endocrine therapy (n = 15), and healthy postmenopausal women (n = 56). Pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone (DHEA), androstenedione, total testosterone, dihydrotestosterone, estrone and estradiol were measured using liquid chromatography-tandem mass spectrometry. Sex hormone binding globulin was measured by solid-phase chemiluminescent immunometric assays, and the free androgen index was calculated. RESULTS: Aromatase inhibitor users did not differ in dihydrotestosterone, total testosterone, androstenedione, DHEA, or free androgen index levels from healthy controls or untreated breast cancer patients. The highest total testosterone levels were found in tamoxifen-treated women, who had significantly higher plasma concentrations than both women treated with aromatase inhibitors and breast cancer patients without adjuvant treatment. Concentrations of cortisol and cortisone were significantly greater in aromatase inhibitor users as well as tamoxifen users, in comparison with healthy controls and untreated breast cancer patients. Aromatase inhibitor users had lower estrone and estradiol plasma concentrations than all other groups. CONCLUSION: Adjuvant treatment with aromatase inhibitors or tamoxifen was associated with increased cortisol and cortisone plasma concentrations as well as decreased estradiol concentrations. Androgen levels were elevated in tamoxifen-treated women but not in aromatase inhibitor users.

Higher than expected estradiol levels in aromatase inhibitor-treated, postmenopausal breast cancer patients.

OBJECTIVE: Vaginal estradiol is considered contraindicated in aromatase inhibitor (AI)-treated patients because of the risk of elevated estrogen levels. This leaves limited treatment options for patients experiencing gynecological symptoms. However, in clinical practice, no precise estimation has been performed of circulating estrogens and aromatase index in postmenopausal breast cancer patients on long-lasting AI or tamoxifen treatment. METHODS: Steroid hormones were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) and extraction radioimmunoassay (RIA). Postmenopausal AI-treated patients (n =33) were compared with tamoxifen-treated patients (n =34) and controls without vaginal treatment (n =56), with vaginal estradiol (n =25), or with estriol (n =11) treatment. RESULTS: By use of LC-MS/MS, median (range) estradiol plasma concentrations were 16.7 (2.4-162.6), 31.0 (13.4-77.1), 27.2 (7.8-115.8) and 33.3 (20.3-340.1) pmol/l in AI-treated breast cancer patients, tamoxifen-treated breast cancer patients, postmenopausal controls and postmenopausal controls on vaginal estradiol, respectively. The AI-treated group and subgroups had significantly lower estradiol and estrone concentrations than all other groups (p <0.05). There was extensive interindividual variation in estradiol concentration within the AI-treated group, measured using both LC-MS/MS (2.3-182.0 pmol/l) and extraction RIA (2.4-162.6 pmol/l). The AI-treated group had lower aromatase index compared to all other groups (p <0.05-0.001). CONCLUSION: Circulating estrogen levels may have been underestimated in previous longitudinal studies of AI-treated breast cancer patients. Additional studies are required to further evaluate the role of circulating estrogens in breast cancer patients suffering from gynecological symptoms.

Effects of testosterone replacement in human immunodeficiency virus-infected women with weight loss.

The objective of this study was to determine whether physiological testosterone replacement increases fat-free mass (FFM) and muscle strength and contributes to weight maintenance in HIV-infected women with relative androgen deficiency and weight loss. Fifty-two HIV-infected, medically stable women, 18-50 yr of age, with more than 5% weight loss over 6 months and testosterone levels below 33 ng/dl were randomized into this double-blind, placebo-controlled trial of 24-wk duration. Subjects in the testosterone group applied testosterone patches twice weekly to achieve a nominal delivery of 300 mug testosterone over 24 h. Data were evaluable for 44 women. Serum average total and peak testosterone levels increased significantly in the testosterone group, but did not change in the placebo group. However, there were no significant changes in FFM (testosterone, 0.7 +/- 0.4 kg; placebo, 0.3 +/- 0.4 kg), fat mass (testosterone, 0.3 +/- 0.7 kg; placebo, 0.6 +/- 0.7 kg), or body weight (testosterone, 1.0 +/- 0.9 kg; placebo, 0.9 +/- 0.8 kg) between the two treatment groups. There were no significant changes in leg press strength, leg power, or muscle fatigability in either group. Changes in quality of life, sexual function, cognitive function, and Karnofsky performance scores did not differ significantly between the two groups. High-density lipoprotein cholesterol levels decreased significantly in the testosterone group. The patches were well tolerated. We conclude that physiological testosterone replacement was safe and effective in raising testosterone levels into the mid to high normal range, but did not significantly increase FFM, body weight, or muscle performance in HIV-infected women with low testosterone levels and mild weight loss. Additional studies are needed to fully explore the role of androgens in the regulation of body composition in women.

Analysis of dicarboxylic acids by tandem mass spectrometry. High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine.

BACKGROUND: Methylmalonic acid (MMA) is a dicarboxylic acid whose concentration can be increased in blood and urine in patients with an inborn error of metabolism or vitamin B(12) deficiency. We developed a method for the selective analysis of dicarboxylic acids that exploits the high specificity of tandem mass spectrometry (MS/MS) and the substantial difference in fragmentation patterns of the isomers methylmalonic (MMA) and succinic acid (SA). METHODS: Dicarboxylic acids were extracted from samples with methyl-tert-butyl ether and derivatized with butanolic HCl to form dibutyl esters. The derivative was injected into the liquid chromatography (LC)-MS/MS system using TurboIonSpray (nebulizer-assisted electrospray) ionization and quantified by the multiple reaction monitoring mode of MS/MS. RESULTS: The assay for MMA was linear up to 150 micromol/L. The total imprecision was < or =7.5% at both low and high concentrations. The limits of quantification and detection were 0.1 and 0.05 micromol/L, respectively. The degree of interference from SA could be predicted from the branching ratios of the major product ions. CONCLUSIONS: The method is specific for dicarboxylic acids. The LC-MS/MS analysis for MMA requires minimal chromatographic separation and takes <60 s per sample. The entire analysis, including sample preparation, for a batch of 100 specimens can be performed in <4 h.

Use of a statistically designed experimental approach to optimize the propylketal derivatization of barbiturates.

The derivatization of barbiturates with dimethylformamide dipropylacetal and dimethylformamide diisopropylacetal is studied with respect to the optimization of reaction recovery and reliability. A second-order orthogonal experimental design is utilized in order to obtain regression equations for the reaction recovery dependence on the derivatization solution composition, incubation temperature, and time for amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital. Regression equations for the effect of incubation temperature and time on the derivative recovery and the optimum conditions for derivatization recoveries are obtained. Differences in the phenomena of the derivative formation are evaluated between the two derivatizing reagents and the barbiturates. Based on the analysis of the obtained equations, it is concluded that the dipropylketal derivative of barbiturates is superior in comparison with diisopropylketal when considering the milder conditions of the reaction, absence of sudden changes in the recovery with a variation in the derivatization parameters, and reliability for the simultaneous testing of the barbiturates. A method for the routine testing of the barbiturates by gas chromatography-mass spectrometry in urine specimens is included.

Optimization and performance of a rapid gas chromatography-mass spectrometry analysis for methylmalonic acid determination in serum and plasma.

We have developed a rapid and sensitive GC-MS assay for methylmalonic acid determination in serum and plasma utilizing an anion exchange solid-phase extraction and trimethylsilyl derivatization. Each step of the procedure was optimized by the experimental design methods to assure the assay reliable performance. The limit of detection and limit of quantitation were 0.025 and 0.1 micromol/l. The total coefficient of variation for the method was 9.8, 4.4, and 4.6% at the concentration of 0.2, 3.1, and 6.2 micromol/l methylmalonic acid concentration, respectively. The assay was linear up to 9.0 micromol/l, and showed good correlation with a reference method. The method has proven to be reliable in routine production, producing clean chromatography, unique ion fragments, and consistent ion mass ratio.

Comparison of four derivatizing reagents for 6-acetylmorphine GC-MS analysis.

The propionyl, trimethylsilyl, trifluroacetyl, and heptafluoroacyl derivatives of 6-acetylmorphine (6-AM) were evaluated with respect to optimal method performance, derivative stability, and methods characterization for use in gas chromatographic-mass spectrometric (GC-MS) analysis with electron ionization mode and selected ion monitoring. The most common potential interferences and compatibility with other derivatives when used on the same GC-MS were determined for the derivatizing reagents. The propionyl, trimethylsilyl, and trifluroacetyl derivatives produced adequate stability, accuracy, and precision for the method. The 6-AM derivatization with commercially available propionic anhydride generated a relatively small amount of 6-AM-propionyl derivative from the free morphine present in a specimen. The trimethylsilyl derivative obtained by the reaction with MSTFA did not require incubation, was the easiest to prepare, and had the highest potential for use on an automated sample-preparation device. An important advantage of derivatization with MSTFA is elimination of the possibility of heroin decomposition to 6-AM that is due to incubation at elevated temperature.

Analysis of gabapentin in serum and plasma by solid-phase extraction and gas chromatography-mass spectrometry for therapeutic drug monitoring.

A simple method for the determination of gabapentin (Neurontin) is described. The method uses solid-phase extraction by disk column and derivatization followed by gas chromatographic-mass spectrometric analysis. The single-step derivatization with MTBSTFA produces a t-BDMS derivative of both the carboxylic and amine moieties of the molecule. Each step of the procedure was optimized to assure reliable performance of the method. The assay limit of detection was 0.1 microg/mL with a linear range from 1.0 to 35 microg/mL. Within-run (n = 3) and between-run (n = 40) coefficients of variation were less than 8.2 and 15.9%, respectively. The method has proven reliable in routine production for more than a year, producing clean chromatography with unique ion fragments, consistent ion mass ratios, and no interferences. Statistical analysis of the gabapentin concentrations measured in 1020 random specimens over a 2-month period showed a mean concentration of 6.07 microg/mL with a standard deviation of 5.28.