Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS

Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues--improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid-liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm x 2 mm, 3 microm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).

LC-MS/MS method for simultaneous analysis of uracil, 5,6-dihydrouracil, 5-fluorouracil and 5-fluoro-5,6-dihydrouracil in human plasma for therapeutic drug monitoring and toxicity prediction in cancer patients

The chemotherapeutic drug 5-fluorouracil (5-FU) is widely used for treating solid tumors. Response to 5-FU treatment is variable with 10-30% of patients experiencing serious toxicity partly explained by reduced activity of dihydropyrimidine dehydrogenase (DPD). DPD converts endogenous uracil (U) into 5,6-dihydrouracil (UH(2) ), and analogously, 5-FU into 5-fluoro-5,6-dihydrouracil (5-FUH(2) ). Combined quantification of U and UH(2) with 5-FU and 5-FUH(2) may provide a pre-therapeutic assessment of DPD activity and further guide drug dosing during therapy. Here, we report the development of a liquid chromatography-tandem mass spectrometry assay for simultaneous quantification of U, UH(2) , 5-FU and 5-FUH(2) in human plasma. Samples were prepared by liquid-liquid extraction with 10:1 ethyl acetate-2-propanol (v/v). The evaporated samples were reconstituted in 0.1% formic acid and 10 μL aliquots were injected into the HPLC system. Analyte separation was achieved on an Atlantis dC(18) column with a mobile phase consisting of 1.0 mm ammonium acetate, 0.5 mm formic acid and 3.3% methanol. Positively ionized analytes were detected by multiple reaction monitoring. The analytical response was linear in the range 0.01-10 μm for U, 0.1-10 μm for UH(2) , 0.1-75 μm for 5-FU and 0.75-75 μm for 5-FUH(2) , covering the expected concentration ranges in plasma. The method was validated following the FDA guidelines and applied to clinical samples obtained from ten 5-FU-treated colorectal cancer patients. The present method merges the analysis of 5-FU pharmacokinetics and DPD activity into a single assay representing a valuable tool to improve the efficacy and safety of 5-FU-based chemotherapy.

Confirmation analysis of ethyl glucuronide and ethyl sulfate in human serum and urine by CZE-ESI-MS(n) after intake of alcoholic beverages

CZE coupled to sheath liquid-based electrospray ionization (ESI) and multiple-stage ion trap mass spectrometry (MS(n) ) was used for the confirmation analysis of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in human serum and urine collected after intake of alcoholic beverages. Electrophoretic separations were performed in uncoated fused-silica capillaries using a pH 9.5 ammonium acetate background electrolyte and normal polarity. MS detection of EtG and EtS occurred after negative ionization using a spray liquid containing 0.5% v/v ammonia in isopropanol/water (60:40%, v/v). CZE-MS and CZE-MS² results obtained after injection of solid-phase extracts for EtG and EtS and of diluted urine confirmed the presence of EtG and EtS in samples whose levels were previously determined by CZE with indirect UV detection. Detection limits of each compound were estimated to be around 2.0 (injection of diluted urine) and 0.2 μg/mL (extracts).

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent β--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Inflammatory myopathy and severe rhabdomyolysis induced by leuprolide acetate therapy for prostate cancer: a case report

Introduction Leuprolide acetate is a synthetic analog of gonadotropin-releasing hormone used for the treatment of prostate cancer. Its side effects are hot flashes, nausea, and fatigue. We report a case of a patient with proximal inflammatory myopathy accompanied by severe rhabdomyolysis and renal failure following the second application of leuprolide acetate. Drug withdrawal and steroid therapy resulted in remission within six weeks of the diagnosis. To the best of our knowledge, our case report describes the second case of leuprolide acetate-induced inflammatory myopathy and the first case of severe leuprolide acetate-induced rhabdomyolysis and renal failure in the literature. Case presentation A 64-year-old Swiss Caucasian man was admitted to the hospital because of progressive proximal muscle weakness, dyspnea, and oliguria. He had been treated twice with leuprolide acetate in monthly doses. We performed a muscle biopsy, which excluded other causes of myopathy. The patient's renal failure and rhabdomyolysis were treated with rehydration and steroid therapy. Conclusion The aim of our case report is to highlight the rare but severe side effects associated with leuprolide acetate therapy used to treat patients with inflammatory myopathy: severe rhabdomyolysis and renal failure.

Effects of lactate and acetate on the determination of serum ethyl glucuronide by CZE

The analysis of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in serum with an optimized CZE assay is reported. The method uses a 0.1-mm id fused-silica capillary of 50 cm effective length that is coated with linear polyacrylamide, a pH 4.4 nicotinic acid/epsilon-aminocaproic acid (EACA) BGE, reversed polarity and indirect analyte detection. The assay is based on a 1:1 dilution of serum with deionized water and has LODs for EtG, lactate and acetate of 3.8 x 10(-7) M, 2.60 x 10(-6 )M and 2.18 x 10(-6 )M, respectively. Separation of EtG from endogenous macro- and microcomponents (anionic serum components of high and low concentration, respectively) and its quantification are shown to be possible for a wide range of lactate (stacker) and acetate (destacker) concentrations, macrocomponents that have an impact on the CZE behavior of EtG and that change after intake of ethanol. The assay has been successfully applied to the analysis of EtG, lactate and acetate in (i) sera of volunteers that ingested known amounts of alcohol and (ii) samples of patients that were classified (teetotalers and social drinkers vs. alcohol abusers) via analysis of carbohydrate-deficient transferrin.

First clinical experience with anecortave acetate (Retaane)

BACKGROUND: Anecortave acetate is an angiostatic cortisene which is injected as a posterior juxtascleral depot and has been shown to be effective in the treatment of exudative age-related macular degeneration (AMD). The compound is not yet approved in Switzerland but can be used as "compassionate use" in individual cases. PATIENTS AND METHODS: An uncontrolled case series with standardised documentation of ETDRS visual acuity, near acuity, need for magnification and fluorescein angiography was performed. RESULTS: 22 eyes of 19 patients (8 male, 11 female, average age 78.8 years) were treated with a posterior juxtascleral depot injection (PJD) of 15 mg anecortave acetate. The mean change in visual acuity after 3 months in eyes treated with anecortave acetate was -2.6 ETDRS letters corresponding to 0.52 Snellen lines. 3/20 eyes gained more than 1 line. 11/20 eyes showed stable visual acuity (+/- 1 Snellen line, +/- 5 ETDRS letters). 5/20 eyes developed moderate vision loss (one to two Snellen lines, 6-10 ETDRS letters). 1/20 lost 18 ETDRS letters (> 3 Snellen lines). There were no moderate or severe adverse events. CONCLUSIONS: A PJD of 15 mg anecortave acetate is safe and well tolerated. In eyes with occult CNV without recent progression or with residual neovascular activity after photodynamic therapy anecortave acetate may be an alternative therapeutic option before considering intravitreal anti-VEGF agents due to the much less invasive character and lower risk profile.

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.

Recurrent Nicolau syndrome associated with subcutaneous glatiramer acetate injection--a case report.

BACKGROUND Glatiramer acetate is worldwide used as first line treatment in relapsing remitting multiple sclerosis. Local skin reactions associated with glatiramer acetate are common, however, only isolated cases of severe local injection site reactions known as Nicolau Syndrome have been reported so far. CASE PRESENTATION We describe the case of a recurrent Nicolau Syndrome occurred during longstanding glatiramer acetate treatment in a woman with multiple sclerosis. The haemorrhagic patch necrotized and was treated locally as a deep second degree burn with excision of dead skin tissue and was healed. Treatment with glatiramer acetate was definitely suspended. CONCLUSIONS GA injections can be complicated by isolated or recurrent Nicolau Syndrome, a potentially life-threatening condition of which neurologists should be aware.

Rhesus factors and ammonium: a function in efflux?

Completion of fungal, plant and human genomes paved the way to the identification of erythrocytic rhesus proteins and their kidney homologs as ammonium transporters. Ammonium is the preferred nitrogen source of bacteria and fungi, and plants acquire nitrogen from the soil in the form of ammonium [1]. In animals and humans, assimilated forms of nitrogen - amino acids - are much preferred for nutrition, and, in the case of ammonotelic animals, ammonium is used for the excretion of nitrogen instead. In the human kidney, ammonium is produced, reabsorbed and excreted as a means to maintain pH balance and to get rid of surplus inorganic nitrogen. Whether ammonium transport also has a role in the pH regulation of other organs is not known and the molecular mechanisms were not, up to now, understood.

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9±1.7 and 1.7±0.3 mM at pH 4, respectively. The simultaneous transport of [1-14C]GABA and [2,3-3H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-14C]GABA transport was inhibited by proline and quaternary ammonium compounds.