Extraction of tamoxifen and its metabolites from formalin-fixed, paraffin-embedded tissues: an innovative quantitation method using liquid chromatography and tandem mass spectrometry.

PURPOSE: Tamoxifen is a key therapeutic option for breast cancer treatment. Understanding its complex metabolism and pharmacokinetics is important for dose optimization. We examined the possibility of utilizing archival formalin-fixed paraffin-embedded (FFPE) tissue as an alternative sample source for quantification since well-annotated retrospective samples were always limited. METHODS: Six 15 µm sections of FFPE tissues were deparaffinized with xylene and purified using solid-phase extraction. Tamoxifen and its metabolites were separated and detected by liquid chromatography-tandem mass spectrometry using multiple-reaction monitoring. RESULTS: This method was linear between 0.4 and 200 ng/g for 4-hydroxy-tamoxifen and endoxifen, and 4-2,000 ng/g for tamoxifen and N-desmethyl-tamoxifen. Inter- and intra-assay precisions were <9 %, and mean accuracies ranged from 81 to 106 %. Extraction recoveries were between 83 and 88 %. The validated method was applied to FFPE tissues from two groups of patients, who received 20 mg/day of tamoxifen for >6 months, and were classified into breast tumor recurrence and non-recurrence. Our preliminary data show that levels of tamoxifen metabolites were significantly lower in patients with recurrent cancer, suggesting that inter-individual variability in tamoxifen metabolism might partly account for the development of cancer recurrence. Nevertheless, other causes such as non-compliance or stopping therapy of tamoxifen could possibly lead to the concentration differences. CONCLUSIONS: The ability to successfully study tamoxifen metabolism in such tissue samples will rapidly increase our knowledge of how tamoxifen's action, metabolism and tissue distribution contribute to breast cancer control. However, larger population studies are required to understand the underlying mechanism of tamoxifen metabolism for optimization of its treatment.

Improved quantitative method for fludarabine in human plasma by liquid chromatography and tandem mass spectrometry.

An improved quantitative assay was developed and validated for fludarabine in human plasma. Fludarabine and its internal standard, cladribine, were separated on a C18 analytical column after sample purification by strong anion-exchange solid-phase extraction. Quantitation was performed by electrospray triple-quadrupole mass spectrometry in positive ionization mode using multiple-reaction monitoring. This assay had excellent inter- and intra-assay precisions within 8%, and accuracies ranging from 100 to 116%. The method was linear within the concentration range of 0.2-250ng/mL using 100µL of plasma with mean R(2)=0.9999. The extraction recoveries were 85% for fludarabine and 95% for the internal standard, which represent a significant improvement over the previously published methods. We utilized this method for pharmacokinetic (PK) investigations in 215 patients. Interference peaks constantly observed in each blank plasma sample were well resolved from fludarabine using our optimized LC-MS/MS conditions, demonstrating the reliability of this improved assay. The validated method will be further applied to PK studies within our bone marrow transplant program, which will allow for optimal dose and scheduling of fludarabine in these patients.

Sample preparation and liquid chromatography-tandem mass spectrometry for multiple steroids in mammalian and avian circulation.

Blood samples from wild mammals and birds are often limited in volume, allowing researchers to quantify only one or two steroids from a single sample by immunoassays. In addition, wildlife serum or plasma samples are often lipemic, necessitating stringent sample preparation. Here, we validated sample preparation for simultaneous liquid chromatography--tandem mass spectrometry (LC-MS/MS) quantitation of cortisol, corticosterone, 11-deoxycortisol, dehydroepiandrosterone (DHEA), 17ß-estradiol, progesterone, 17α-hydroxyprogesterone and testosterone from diverse mammalian (7 species) and avian (5 species) samples. Using 100 µL of serum or plasma, we quantified (signal-to-noise (S/N) ratio ≥ 10) 4-7 steroids depending on the species and sample, without derivatization. Steroids were extracted from serum or plasma using automated solid-phase extraction where samples were loaded onto C18 columns, washed with water and hexane, and then eluted with ethyl acetate. Quantitation by LC-MS/MS was done in positive ion, multiple reaction-monitoring (MRM) mode with an atmospheric pressure chemical ionization (APCI) source and heated nebulizer (500°C). Deuterated steroids served as internal standards and run time was 15 minutes. Extraction recoveries were 87-101% for the 8 analytes, and all intra- and inter-run CVs were ≤ 8.25%. This quantitation method yields good recoveries with variable lipid-content samples, avoids antibody cross-reactivity issues, and delivers results for multiple steroids. Thus, this method can enrich datasets by providing simultaneous quantitation of multiple steroids, and allow researchers to reimagine the hypotheses that could be tested with their volume-limited, lipemic, wildlife samples.

Development and validation of a test dose strategy for once-daily i.v. busulfan: importance of fixed infusion rate dosing.

Intravenous (i.v.) busulfan (Bu) administered once daily in myeloablative transplant regimens is convenient, effective, and relatively well tolerated. Therapeutic drug monitoring is recommended as nonrelapse mortality increases when daily exposure, as determined by the area under the plasma concentration versus time curve (AUC), exceeds 6000 µM·min. We describe sequential studies to achieve accurate prediction of treatment doses of Bu based on the kinetics of a smaller test dose. A total of 335 patients with hematologic malignancies were given daily i.v. Bu 3.2 mg/kg × 4 and fludarabine 50 mg/m(2) × 5. Pharmacokinetic monitoring was conducted for both the test dose and first treatment dose of Bu (day -5). Three different test dose schedules were evaluated: 12 mg Bu administered over 20 minutes, 0.8 mg/kg over 3 hours, and 0.8 mg/kg infused at 80 mg/h. The 3.2 mg/kg treatment doses were infused over a fixed time of 3 hours for the first 2 test dose trials and at a fixed rate of 80 mg/h for the final protocol. All test dose infusions were on day -7. In the first 2 schedules, Bu administered over a fixed time had significantly higher clearance for the test dose compared with the treatment dose. However, when both the test and the treatment doses were administered at the same infusion rate, clearance of the drug between the 2 dosing days was equivalent. Predicted day -5 AUC (AUC(-5)) showed a high linear correlation (r(2) = 0.74) to the actual AUC(-5). The error of these predictions was <20% in 98% of patients and <10% in 80%. In 24 individuals, the test dose predicted an AUC >5500 µM·min; therefore, the first Bu treatment dose was reduced to a desired target AUC. All adjusted doses fell within 20% of the targeted exposure. We conclude that a test dose strategy for therapeutic drug monitoring of daily i.v. Bu is accurate if the test and treatment doses are infused at the same rate. This approach allows targeting of therapeutic doses of Bu to desired levels and the potential for improved safety and efficacy.

Frontal affinity chromatography-mass spectrometry.

Frontal affinity chromatography (FAC) is a biophysical method for the discovery and characterization of molecular interactions in a flow-based system. Several different modes of analysis are possible by interfacing to the mass spectrometer, including robust single-compound characterizations as well as high-throughput screening of over 1,000 compounds per run. The method supports thermodynamic and kinetic characterization of interactions for a wide range of molecular species and possesses similarities to flow-based biosensors such as surface plasmon resonance. It offers sensitive detection of ligands present well below their respective dissociation constants, and can be assembled from readily available laboratory components. Direct coupling of the FAC cartridge to the mass spectrometer is useful for the interrogation of single compounds or mixtures of limited complexity. An offline fractionation schema is more appropriate for discovery-mode applications. A high-performance FAC system enabling both modes can be assembled in 2-3 h. Measurements of dissociation constants can be made with such a system in 0.5-3 h, and the system supports higher-throughput screening modes at a rate of 10,000 compounds d(-1).

Nitrosothiol stores in vascular tissue: modulation by ultraviolet light, acetylcholine and ionomycin.

Our previous studies demonstrated that light-induced vascular relaxation (photorelaxation) was mediated by a tissue source of nitric oxide that was independent of endothelial nitric oxide synthase (eNOS), but sensitive to inhibitors of soluble guanylate cyclase, extracellular nitric oxide scavengers and possessed the properties of a nitrosothiol. In the present study we describe High Performance Liquid Chromatography and spectrofluorometric techniques that allowed us to measure tissue levels of the nitrosothiol, S-nitrosoglutathione and its modulation in mouse aortic tissues, smooth muscle cells and human umbilical vein endothelial cells (HUVECs) following exposure to exogenous S-nitrosoglutathione, light and chemical stimuli. Basal levels of S-nitrosoglutathione were similar in control mouse aortae and HUVECs and the store size could be enhanced by exposure of tissues/cells to nitric oxide solution. No basal S-nitrosoglutathione was detected in tissue from diabetic db/db mice; however, ultraviolet light was still able to elicit relaxation of aortic tissues. Ultraviolet light induced the release of nitric oxide from the S-nitrosoglutathione store with an associated increase in the concentration of nitrite. The release of nitric oxide from the store in HUVECs was modulated by extracellular oxidative stress induced by xanthine/xanthine oxidase and also, in an atropine-sensitive process, by acetylcholine, as well as by the calcium ionophore, ionomycin. These interventions resulted in a reduced S-nitrosoglutathione store and elevated levels of nitrite. These data suggest that endothelial and vascular smooth muscle cells possess stores of nitric oxide that, in part, exist in the form of S-nitrosoglutathione. Furthermore, these stores, albeit small, may provide an additional mechanism for the regulation of vascular tone, especially under conditions, such as diabetes, in which nitric oxide generation or bioavailability is compromised; however, additional studies are required to determine not only whether there are additional chemical storage forms of nitric oxide, but also the location of such stores.

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.

Enhanced S-nitroso-albumin formation from inhaled NO during ischemia/reperfusion.

In the present study, we investigated whether inhaled nitric oxide (NO) was transported by plasma proteins, such as S-nitroso-albumin (SNO-Alb), in the feline circulation and whether this molecule delivers NO to the periphery under conditions of stress, specifically ischemia/reperfusion (I/R). A flow probe was interposed between the femoral and superior mesenteric artery for blood flow measurements, and a branch of the superior mesenteric vein was cannulated for arterial-venous sampling. In animals breathing room air, SNO-Alb was below detection level in arterial or venous blood. NO inhalation resulted in a significant arterial-venous gradient for SNO-Alb. Concomitant with this loss of SNO-Alb across the intestinal vasculature was an increase in nitrite (NO2-). However, this release of NO was not sufficient to alter intestinal blood flow. I/R during NO inhalation caused a very large increase in arterial SNO-Alb that permitted a 5-fold increase in SNO-Alb consumption and significant generation of NO2- within the postischemic intestinal vasculature. The increased SNO-Alb consumption was sufficient to dramatically improve intestinal blood flow. The very large burst of arterial SNO-Alb during I/R was completely blocked by the administration of superoxide dismutase, suggesting that oxidative stress contributed to the increased SNO-Alb formation. Our data suggest that inhaled NO can increase nitrosothiol production and these molecules may be a functional NO delivery system during cardiovascular disease.

The physiology of S-nitrosothiols: carrier molecules for nitric oxide.

Recent work has demonstrated that inhalation of nitric oxide (NO) can impact the peripheral vasculature, suggesting that an NO-stabilizing moiety may exist in vivo. One possibility is the formation of S-nitrosothiols, which extend the half-life of NO manyfold. In this review, we provide evidence that S-nitrosothiols exist in the vasculature, particularly during NO inhalation. The potential biochemical pathways that have been proposed for the formation of these products are also summarized. Finally, we highlight the limited evidence for the role that these potent vasodilating molecules may play as physiologically and therapeutically important regulators of the vascular system.

The endothelium in health and disease--a target for therapeutic intervention.

In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor--endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K+), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues.

The aim of these experiments was to determine the contribution of leukocyte-derived iNOS to total iNOS expression induced by lipopolysaccharide (LPS). By transferring bone marrow between iNOS+/+ and iNOS-/- mice, we created chimeric mice in which iNOS expression was limited to either circulating leukocytes (leukocyte-iNOS mice) or parenchymal cells (parenchyma-iNOS mice). Analysis of congenic markers demonstrated that >95% of thymocytes in chimeric mice were of donor origin. Also, following LPS treatment, iNOS mRNA was detectable in blood from leukocyte-iNOS mice but not parenchyma-iNOS mice. Together these findings indicated that the host marrow had been replaced entirely by donor cells. In the lung, at least 50% of the LPS-induced iNOS mRNA was derived from leukocytes, and immunohistochemical analysis indicated that leukocytes were the main source of iNOS protein. In contrast in the liver, colon, and muscle, iNOS expression was derived predominantly from parenchymal cells. This divergence is potentially explained by the high level of leukocyte recruitment to the lung, relative to the other tissues. Plasma levels of NOS byproducts indicated that parenchymal iNOS was the dominant source of systemic iNOS activity. These findings indicate that in tissues other than the lung, parenchymal cells are the principal source of iNOS during endotoxemia.