Development of a UPLC-MS/MS method for the quantification of sildenafil by DBS, and its use on pediatric pulmonary hypertension.

BACKGROUND: Sildenafil is used for the treatment of pediatric pulmonary hypertension. A dried blood spot (DBS)-based LC-MS method for sildenafil quantitation was developed and applied to a group of patients. RESULTS: DBS showed high portability and stability of samples, and the method was selective and linear for quantitation of sildenafil (5-3,000 ng/ml) and N-desmethyl-sildenafil (3-1,500 ng/ml). After a single oral dose of sildenafil (1 mg/kg), method evidenced poor metabolism in these patients. CONCLUSION: The method was successfully applied in peripheral blood and can be used for both pharmacokinetics and therapeutic drug monitoring. DBS proved to have advantages during sample translation and preservation of analytes. Data suggest that hazardous blood sildenafil levels may be reached in this population.

Anxiety-like effects of meta-chlorophenylpiperazine in paradoxically sleep-deprived mice.

Chlorophenylpiperazines (CPP) are psychotropic drugs used in nightclub parties and are frequently used in a state of sleep deprivation, a condition which can potentiate the effects of psychoactive drugs. This study aimed to investigate the effects of sleep deprivation and sleep rebound (RB) on anxiety-like measures in mCPP-treated mice using the open field test. We first optimized our procedure by performing dose-effect curves and examining different pretreatment times in naïve male Swiss mice. Subsequently, a separate cohort of mice underwent paradoxical sleep deprivation (PSD) for 24 or 48h. In the last experiment, immediately after the 24h-PSD period, mice received an injection of saline or mCPP, but their general activity was quantified in the open field only after the RB period (24 or 48h). The dose of 5mgmL(-1) of mCPP was the most effective at decreasing rearing behavior, with peak effects 15min after injection. PSD decreased locomotion and rearing behaviors, thereby inhibiting a further impairment induced by mCPP. Plasma concentrations of mCPP were significantly higher in PSD 48h animals compared to the non-PSD control group. Twenty-four hours of RB combined with mCPP administration produced a slight reduction in locomotion. Our results show that mCPP was able to significantly change the behavior of naïve, PSD, and RB mice. When combined with sleep deprivation, there was a higher availability of drug in plasma levels. Taken together, our results suggest that sleep loss can enhance the behavioral effects of the potent psychoactive drug, mCPP, even after a period of rebound sleep.

Quantification of imatinib in human serum: validation of a high-performance liquid chromatography-mass spectrometry method for therapeutic drug monitoring and pharmacokinetic assays.

BACKGROUND: Imatinib mesylate has been a breakthrough treatment for chronic myeloid leukemia. It has become the ideal tyrosine kinase inhibitor and the standard treatment for chronic-phase leukemia. Striking results have recently been reported, but intolerance to imatinib and noncompliance with treatment remain to be solved. Molecular monitoring by quantitative real-time polymerase chain reaction is the gold standard for monitoring patients, and imatinib blood levels have also become an important tool for monitoring. METHODS: A fast and cheap method was developed and validated using high-performance liquid chromatography-mass spectrometry for quantification of imatinib in human serum and tamsulosin as the internal standard. Remarkable advantages of the method includes use of serum instead of plasma, less time spent on processing and analysis, simpler procedures, and requiring reduced amounts of biological material, solvents, and reagents. Stability of the analyte was also studied. This research also intended to drive the validation scheme in clinical centers. The method was validated according to the requirements of the US Food and Drug Administration and Brazilian National Health Surveillance Agency within the range of 0.500-10.0 µg/mL with a limit of detection of 0.155 µg/mL. Stability data for the analyte are also presented. CONCLUSION: Given that the validated method has proved to be linear, accurate, precise, and robust, it is suitable for pharmacokinetic assays, such as bioavailability and bioequivalence, and is being successfully applied in routine therapeutic drug monitoring in the hospital service.

A reliable method to quantify sildenafil and its metabolite N-demethylsildenafil by HPLC in plasma of children.

The aim of this study was to develop a simple method for quantifying plasma levels of sildenafil and its metabolite by liquid chromatography with a C18 reverse-phase column and UV detection. For both compounds, linearity was assessed in the range from 10 and 1 000 ng · ml-1 and had correlation coefficients of r=0.995 and r=0.997 for sildenafil and its metabolite, respectively. The inter- and intra-day coefficients of variation was<5.3%. The limits of detection and quantification were 1 and 10 ng · ml-1. Drug levels were determined satisfactorily in two patients. A simple and reliable method was developed for use in children with Pulmonary Arterial Hypertension under treatment with sildenafil.

Comparison of fasting bioavailability among 100-mg commercial, 100-mg generic, and 50-mg chewable generic sildenafil tablets in healthy male Mexican volunteers: a single-dose, 3-period, crossover study.

BACKGROUND: Sildenafil citrate (SIL) was the first oral drug registered in Mexico for the treatment of erectile dysfunction. However, succinct pharmacokinetic data are available in the Mexican population. OBJECTIVE: The goals of the present work were: (1) to design a specific method to quantify SIL plasma levels by using UPLC-MS/MS; (2) to compare oral SIL bioavailability in Mexican men with pharmacokinetic data in other populations; (3) to fulfill local regulatory requests; and (4) to describe the relative tolerability of a new 50-mg chewable tablet. METHODS: This was a randomized, single-dose, 3-period, 6-sequence crossover study in healthy male volunteers. In each period, subjects received single oral doses of 100 mg of sildenafil (1 commercial [reference(⁎)], 1 generic [test 1(†)], or 2 chewable generic tablets [test 2(‡)]), with a 4-day washout period between each dose. Serial blood samples were collected for up to 24 hours. SIL was measured in heparinized plasma by using a validated UPLC-MS/MS method. Pharmacokinetic parameters included C(max), T(max), AUC(0-24), and AUC(0-∞). Bioequivalence was established if 90% CIs for mean test:reference ratios of log-transformed C(max) and AUC fell within the range of 0.80 to 1.25. Tolerability was assessed on the basis of a clinical interview with the subject and monitoring of vital signs. RESULTS: Demographic data showed a homogeneous population. Validation of analytical method proved to be linear within the range of 1 to 1000 ng/mL, with selectivity, accuracy, and precision. 90% CIs for test 1:reference ratios were 86.52 to 113.56, 94.75 to 108.84, and 94.97 to 108.82 for the logarithm parameters C(max), AUC(0-24), and AUC(0-∞), respectively. The 90% CIs for the test 2:reference ratios were 82.14 to 107.24, 98.26 to 112.56, and 99.19 to 113.34 for C(max), AUC(0-24), and AUC(0-∞). Regarding relative tolerability, slight cephalea was the most common adverse effect. CONCLUSIONS: The developed analytical method was validated in compliance with local requirements and was useful for sildenafil measurement. This single-dose study under fasting conditions suggests that both test products met the Mexican regulatory criteria for assuming bioequivalence in these healthy, male Mexican volunteers. The clinical data suggest that the chewable tablets were well tolerated by volunteers.

Capillary electrophoresis method for plasmatic determination of imatinib mesylate in chronic myeloid leukemia patients.

Imatinib (IMAT) is a tyrosine kinase inhibitor that has been used for the treatment of chronic myeloid leukemia (CML). Despite the efficacy of IMAT therapy, some cases of treatment resistance have been described in CML. Developing a plasma method is important since there are several studies that provided a higher correlation between IMAT plasma concentration and response to treatment. Therefore, in this investigation we validated a method by CE as an alternative, new, simple and fast electrophoretic method for IMAT determination in human plasma. The analysis was performed using a fused silica capillary (50 µm id×46.5 cm total length, 38.0 cm effective length); 50 mmol/L sodium phosphate buffer, pH 2.5, as BGE; hydrodynamic injection time of 20 s (50 mbar); voltage of 30 kV; capillary temperature of 35°C and detection at 200 nm. Plasma samples pre-treatment involved liquid-liquid extraction with methyl-tert-butyl ether as the extracting solvent. The method was linear from 0.125 to 5.00 µg/mL. The LOQ was 0.125 µg/mL. Mean absolute recovery of IMAT was 67%. The method showed to be precise and accurate with RSD and relative error values lower than 15%. Furthermore, the application of the method was performed in the analysis of plasma samples from CML patients undergoing treatment with IMAT.

Enantioselective analysis of zopiclone and its metabolites in plasma by liquid chromatography/tandem mass spectrometry.

A high-performance liquid chromatographic method with triple-quadrupole mass spectrometry detection (LC-MS-MS) was developed and validated for the first time for the simultaneous quantification of zopiclone and its metabolites in rat plasma samples. The analytes were isolated from rat plasma by liquid-liquid extraction and separated using a chiral stationary phase based on an amylose derivative, Chiralpak ADR-H column, and ethanol-methanol-acetonitrile (50:45:5, v/v/v) plus 0.025% diethylamine as the mobile phase, at a flow-rate of 1.0 mL min(-1). Moclobemide was used as the internal standard. The developed method was linear over the concentration range of 7.5-500 ng mL(-1). The mean absolute recoveries were 74.6 and 75.7; 61.6 and 56.9; 72.5, and 70.7 for zopiclone enantiomers, for N-desmethyl zopiclone enantiomers and for zopiclone-N-oxide enantiomers, respectively, and 75.9 for the internal standard. Precision and accuracy were within acceptable levels of confidence (<15%). The method application in a pilot study of zopiclone kinetic disposition in rats showed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-R-zopiclone. Higher concentrations were also observed for (+)-(S)-N-desmethyl zopiclone and (+)-(S)-N-oxide zopiclone, confirming the stereoselective disposition of zopiclone.

Imatinib mesylate pharmacokinetics before and after sleeve gastrectomy in a morbidly obese patient with chronic myeloid leukemia.

Abstract Imatinib is widely used to treat chronic myeloid leukemia and gastrointestinal stromal tumors. The agent, administered orally, has approximately 98% oral bioavailability, achieves maximum plasma concentration approximately 2-4 hours after ingestion, and has a plasma half-life of approximately 18 hours. As maintaining an adequate plasma imatinib concentration is essential to achieving a favorable therapeutic response, it is important to determine whether gastrointestinal surgery, pathologic conditions, or anatomic changes negatively affect imatinib absorption, and thereby result in subtherapeutic plasma imatinib concentrations. We describe a 36-year-old, morbidly obese woman with chronic myeloid leukemia who received treatment with alpha-interferon and cytarabine over 5 years. Her chemotherapy was then switched to imatinib 400 mg/day because she failed to achieve a molecular response with the other two agents. A complete molecular response was achieved with imatinib. Four years later, she underwent a sleeve gastrectomy while receiving imatinib. Imatinib plasma pharmacokinetic values were assessed before and on four occasions during the year after the sleeve gastrectomy. The patient's trough plasma concentration before surgery (1558 ng/ml) was consistent with those found in the literature (>/= 1000 ng/ml), whereas her trough concentrations after surgery were 46-60% lower (629-836 ng/ml) than the preoperative value. Despite this, the patient remained in complete molecular remission for 1 year after surgery. Monitoring plasma imatinib concentrations is recommended in morbidly obese patients with chronic myeloid leukemia or gastrointestinal stromal tumors who undergo gastric procedures. Additional pharmacokinetic studies, however, are needed in these patients.

Validation of an HPLC method for sildenafil citrate analysis in human plasma samples.

A simple, precise, rapid and accurate HPLC method for the determination of sildenafil citrate (SLD) in human plasma has been developed based on previous reports, to offer an alternative way to detect and quantify SLD and also to improve some characteristics of the validation process. Chromatography was carried out on a C18 reversed-phase column, using a mixture of acetonitrile: ammonium acetate (0.3 M pH 6.8) (1:1 v/v) as the mobile phase at a flow of 0.750 mL/min. Diazepam was used as an internal standard (IS) and detection was by UV at 240 nm. Samples were extracted with 200 microl of 0.1 M Na2CO3 and 5 mL of diethyl ether: dichloromethane (60:40). Retention times of SLD and IS were 4.8 and 7.1 min, respectively; total run time was 10 min. The linear range of SLD was found to be 20 - 1000 ng/mL. Limit of quantitation (LOQ) and limit of detection (LOD) were calculated to be 10 and 20 ng/mL, respectively. An improved percentage recovery of analyte is reported, showing a fast and reproducible approach to detect SLD in plasma human sample. The method was validated for its linearity, precision, accuracy, recovery, stability and specificity.

Determination of levocetirizine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study.

We describe a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid-liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389>201 for I and m/z 502>467 for II. The limit of quantification and the dynamic range achieved were 0.5ng/mL and 0.5-500.0ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48h after oral administration of 5mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.

Management of chronic stable angina pectoris.

Agina pectoris is a discomfort in the chest or adjacent areas caused by myocardial ischemia. It is most commonly caused by the inability of narrowed atherosclerotic coronary arteries to supply adequate oxygen to the heart under conditions of increase demand. This review article will focus in the medical treatment of chronic stable angina, with a focus in new strategies or medications. Treatment by revascularization techniques will not be discussed in this article. The goal of treatment is to improve quality of life, decrease cardiovascular events and mortality. All patients should be evaluated for reversible causes of their angina, such as anemia, hyperthyroidism, sympathomimetic drugs and hypertension. Sublingual nitroglycerin should be used for immediate relief of symptoms. In general, all patients should be on aspirin (ASA) unless they are allergic or other contraindications, if so; clopidogrel should be added to the therapy. In addition to the antiplatelet therapy, which decreases mortality, patients should be started on beta blockers and nitrates. If there is no improvement in symptoms then a calcium channel blockers of the dihydropyridine family should be added. Patients with Diabetes Mellitus and/or left ventricular systolic dysfunction should be also started on angiotensin converting enzyme inhibitors. If the patient continues with limiting angina, ranolazine should be started and finally enhanced external counterpulsation should be considered in those patients who have not responded to maximal drug therapy.

Development and validation of a LC-MS/MS method with electrospray ionization for determination of LASSBio-579 in rat plasma.

A simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of LASSBio-579 in plasma rat, using fluconazole as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water (80:20, v/v), containing 0.4mM ammonium hydroxide and 0.2 mM acetic acid at a flow rate of 1.0 ml/min (split ratio 1:5). A Micromass triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the positive mode. Plasma samples were deproteinized with acetonitrile (1:2) and 50 microl of the supernatant were injected into the system. The retention times of LASSBio-579 and IS were approximately 4.7 and 2.4 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 30-2000 ng/ml with determination coefficient >0.98. The lower limit of quantification was 30 ng/ml. The accuracy of method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 13.5% and 6.4%, respectively. The applicability of the LC-MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after intraperitoneal administration of LASSBio-579 to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of LASSBio-579 in pre-clinical pharmacokinetic studies.

Pharmacokinetics and tissue distribution of a new heterocyclic N-phenylpiperazine derivative (LASSBio-581) in rats.

This work investigated the pharmacokinetics of a new N-phenylpiperazine derivative (LASSBio-581), active on dopaminergic system. LASSBio-581 plasma concentrations were determined in rats after bolus administration of 10mg/kg, i.v., 30 and 60 mg/kg, i.p. and p.o., by HPLC. Individual profiles were evaluated by non-compartmental and compartmental analysis using WinNonlin. Protein binding by ultrafiltration showed free fraction of 29+/-4%. The compound showed linear pharmacokinetics for the extravascular doses investigated. The oral bioavailability ( approximately 25%) was approximately half of the intra-peritoneal one ( approximately 47%). The 60 mg/kg oral dose showed an unusual profile with two peaks (1 and 6h). A two-compartment model better described all plasma profiles. The Vd (0.8+/-0.4l/kg) and the t(1/2) (1.2+/-0.4h) were smaller for i.v. than for the other routes. The CL(tot) was statistically similar for all three administration routes investigated (0.6+/-0.2l/(hkg)) (alpha=0.05). The compound distribution into different organs, evaluated in tissue homogenates after i.v. administration, showed a higher penetration in lungs (51.0%), followed by the brain (39.2%), where the half-life was three times bigger than in the other tissues (1.9h). The compound brain profile agreed with the central nervous system activity determined.

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma.

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C18 column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25-8.0 microg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 microg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 microg/ml, 7.44 to 13.81% at 1.5 microg/ml and 6.10 to 13.94% at 6.0 microg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 microg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.

Elevated serum levels of cyclo (His-Pro), an endogenous inhibitor of pituitary prolactin secretion, in systemic lupus erythematosus patients.

Cyclo (His-Pro) or CHP is a cyclic dipeptide that is known to elicit many biologic activities including inhibition of pituitary prolactin (PRL) secretion. PRL stimulates humoral and cell mediated immune responses and hyperprolactinemia has been found in a subset of systemic lupus erythematosus (SLE) patients in association with active disease. To explore whether CHP may play a role in SLE patients, we measured, CHP and PRL levels in sera of 21 SLE patients and 11 normal controls. The results of this study show significantly (P < 0.001) high levels of CHP in SLE compared to controls. However, there was no significant correlation between serum PRL and CHP levels in SLE patients (r = 0.027, P = 0.29). The increase in serum levels of CHP may be in an attempt to increase hypothalamic content of dopamine which in turn would decrease pituitary PRL synthesis in hyperprolactinemic SLE patients.

Concentrations of amocarzine in plasma of 71 Ecuadorian patients of two different races receiving 3 mg/kg b.i.d. and 5 mg/kg o.d. oral postprandial doses for 3 days.

The possible influence of sex, race and of postprandial administration conditions (either immediately after the end of meal or one hour later) on the plasma concentrations of amocarzine and its N-oxide metabolite, CGP 13 231, was investigated. 71 Ecuadorian patients (48 males and 23 females) of two different races (Indio and Negro) infected with Onchocerca volvulus participated in the study. The concentrations were measured on day 3 at times 3 and 6 h after postprandial administration in the morning of a treatment with either a dose of 5 mg/kg of amocarzine once daily (12 patients) or 3 mg/kg twice daily (59 patients) for 3 days. The concentrations of unchanged drug and of CGP 13 231 measured after the 5 mg/kg treatment were in the low range of those expected from dose proportionality by the comparison with the 3 mg/kg. After the 3 mg/kg dose, no significant difference in concentration of both compounds were detected between the male and female patients between Indio and Negro patients, between the administration immediately after food intake and one hour later. The only detected difference (P = 0.05) was that between Indio and Negro patients for the concentrations of CGP 13 231 at time 3 h. This difference was not confirmed at time 6 h. Therefore, the administration of amocarzine either immediately or one hour after food intake appeared to produce reproducible absorption conditions which were not influenced by sex and race.