Phenotypic and Genotypic Characterization of Pan-Drug-Resistant Klebsiella pneumoniae Isolated in Qatar.

In secondary healthcare, carbapenem-resistant Enterobacterales (CREs), such as those observed in Klebsiella pneumoniae, are a global public health priority with significant clinical outcomes. In this study, we described the clinical, phenotypic, and genotypic characteristics of three pan-drug-resistant (PDR) isolates that demonstrated extended resistance to conventional and novel antimicrobials. All patients had risk factors for the acquisition of multidrug-resistant organisms, while microbiological susceptibility testing showed resistance to all conventional antimicrobials. Advanced susceptibility testing demonstrated resistance to broad agents, such as ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam. Nevertheless, all isolates were susceptible to cefiderocol, suggested as one of the novel antimicrobials that demonstrated potent in vitro activity against resistant Gram-negative bacteria, including CREs, pointing toward its potential therapeutic role for PDR pathogens. Expanded genomic studies revealed multiple antimicrobial-resistant genes (ARGs), including blaNMD-5 and blaOXA derivative types, as well as a mutated outer membrane porin protein (OmpK37).

Preparation and Adsorption Behavior of Ce(III)-MOF for Phosphate and Fluoride Ion Removal from Aqueous Solutions.

The discharge of inorganic pollutants like phosphate and fluoride is a cause of mounting concern to the world due to the substantial environmental and human health risk. Adsorption is one of the most common and affordable technologies widely utilized for removing inorganic pollutants such as phosphate and fluoride anions. Investigating efficient sorbents for the adsorption of these pollutants is extremely important and challenging. This work aimed at studying the adsorption efficiency of the Ce(III)-BDC metal-organic framework (MOF) for the removal of these anions from an aqueous solution using a batch mode. Powder X-ray diffraction (XRD), Fourier transform infrared (FTIR), thermogravimetric analysis (TGA), Brunauer-Emmett-Teller (BET), and scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) techniques evidenced the successful synthesis of Ce(III)-BDC MOF in water as a solvent without any energy input within a short reaction time. The outstanding removal efficiency of phosphate and fluoride was exhibited at an optimized pH (3, 4), adsorbent dose (0.20, 0.35 g), contact time (3, 6 h), agitation speed (120, 100 rpm), and concentration (10, 15 ppm) for each ion, respectively. The experiment on the effect of coexisting ions demonstrated that SO42- and PO43- ions are the primary interfering ions in phosphate and fluoride adsorption, respectively, while the HCO3- and Cl- ions were found to have interfered less. Furthermore, the isotherm experiment showed that the equilibrium data fitted well with the Langmuir isotherm model and the kinetic data correlated well with the pseudo-second-order model for both ions. The results of thermodynamic parameters such as ΔH°, ΔG°, and ΔS° evidenced an endothermic and spontaneous process. The regeneration of the adsorbent made using water and NaOH solution showed the easy regeneration of the sorbent Ce(III)-BDC MOF, which can be reused four times, revealing its potential application for the removal of these anions from aqueous environment.

Prevalence and microbiological and genetic characteristics of multidrug-resistant Pseudomonas aeruginosa over three years in Qatar.

Objectives: Antimicrobial resistance (AMR) is a global priority with significant clinical and economic consequences. Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the major pathogens associated with significant morbidity and mortality. In healthcare settings, the evaluation of prevalence, microbiological characteristics, as well as mechanisms of resistance is of paramount importance to overcome associated challenges. Methods: Consecutive clinical specimens of P. aeruginosa were collected prospectively from 5 acute-care and specialized hospitals between October 2014 and September 2017, including microbiological, clinical characteristics and outcomes. Identification and antimicrobial susceptibility test were performed using the BD Phoenix identification and susceptibility testing system, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and minimum inhibitory concentration (MIC) test strips. Overall, 78 selected MDR P. aeruginosa isolates were processed for whole-genome sequencing (WGS). Results: The overall prevalence of MDR P. aeruginosa isolates was 5.9% (525 of 8,892) and showed a decreasing trend; 95% of cases were hospital acquired and 44.8% were from respiratory samples. MDR P. aeruginosa demonstrated >86% resistance to cefepime, ciprofloxacin, meropenem, and piperacillin-tazobactam but 97.5% susceptibility to colistin. WGS revealed 29 different sequence types: 20.5% ST235, 10.3% ST357, 7.7% ST389, and 7.7% ST1284. ST233 was associated with bloodstream infections and increased 30-day mortality. All ST389 isolates were obtained from patients with cystic fibrosis. Encoded exotoxin genes were detected in 96.2% of isolates. Conclusions: MDR P. aeruginosa isolated from clinical specimens from Qatar has significant resistance to most agents, with a decreasing trend that should be explored further. Genomic analysis revealed the dominance of 5 main clonal clusters associated with mortality and bloodstream infections. Microbiological and genomic monitoring of MDR P. aeruginosa has enhanced our understanding of AMR in Qatar.

Evaluation of in Vitro Activity of Ceftazidime/Avibactam and Ceftolozane/Tazobactam against ESBL-producing Enterobacterales Isolated from Intensive Care Units from Qatar.

Objectives: Extended-spectrum -lactamases (ESBLs) mechanism of resistance in Enterobacterales leads to poor clinical outcomes. Ceftazidime/avibactam and ceftolozane/tazobactam are two broad-spectrum antimicrobial combinations that are effective against multidrug-resistant organisms with regional variations. This study aims to evaluate the antimicrobial susceptibility test (AST) for both combinations against ESBL-producing Enterobacterales isolated from intensive care units (ICUs) in tertiary hospitals from November 2012 to October 2013 in Qatar. Methods: A total of 629 Enterobacterales isolates from ICUs were screened for ESBL production using BD-PhoenixTM confirmed by double-disk potentiation, while ESBL-genes were detected by polymerase chain reaction. The ASTs for ceftazidime/avibactam and ceftolozane/tazobactam were assessed by minimum inhibitory concentration (MIC) test strips. A single isolate that was resistant to both combinations was subjected to whole-genome sequencing. Results: The prevalence of ESBL-producing Enterobacterales isolated from ICUs was 17.3% (109/629) with predominance of Klebsiella pneumoniae (56/109; 51.4%) and Escherichia coli (38/109; 34.9%). The susceptibility of ceftazidime/avibactam and ceftolozane/tazobactam against ESBL-producers was 99.1% (108/109) and most (81/109; 74.3%) had MICs < 0.5 for both combinations. The predominant ESBL-gene was blaCTX-M (72/109; 66.1%). A single isolate that was resistant to both combinations harbored multiple ESBL resistant-genes including blaVEB-5 and blaVIM-2. Conclusions: ESBL-producing Enterobacterales isolated from ICUs were predominantly K. pneumoniae and E. coli, mainly harboring blaCTX-M gene. They were highly susceptible to ceftazidime/avibactam and ceftolozane/tazobactam suggesting potential alternatives to currently available therapeutic options.

Autoradiographic labelling of 5-HT3 receptors in the hemi-parkinsonian rat brain.

L-3,4-dihydroxyphenylalanine (l-DOPA) is the mainstay treatment for Parkinson's disease, but its effectiveness during early disease is marred by the eventual development of l-DOPA induced dyskinesia. In hemi-parkinsonian rats, the serotonin type 3 (5-HT3) antagonists ondansetron and granisetron alleviated dyskinesia induced by l-DOPA without impeding its anti-parkinsonian action; in parkinsonian marmosets, ondansetron alleviated dyskinesia and enhanced l-DOPA anti-parkinsonian action. Here, we sought to gain insight into the mechanisms governing the anti-dyskinetic action of 5-HT3 antagonists and measured 5-HT3 receptor levels across different brain, using [3H]GR65630 autoradiographic binding. Brain sections were chosen from 6-hydroxydopamine (6-OHDA)-lesioned rats exhibiting abnormal involuntary movements (AIMs), as well as l-DOPA-naïve 6-OHDA and sham-lesioned animals. [3H]GR65630 binding increased in the ipsilateral subthalamic nucleus of 6-OHDA-lesioned rats with mild and severe AIMs, (3-fold changes, P < 0.001). [3H]GR65630 binding also increased in the ipsilateral entopeduncular nucleus and thalamus of 6-OHDA-lesioned rats with severe AIMs (75 % and 88 %, P < 0.05). AIMs scores negatively correlated with [3H]GR65630 binding in the ipsilateral dorsolateral striatum and contralateral subthalamic nucleus (P < 0.05). These results suggest that alterations in 5-HT3 mediated neurotransmission may contribute to the pathophysiology of l-DOPA induced dyskinesia.

Clinical outcomes, molecular epidemiology and resistance mechanisms of multidrug-resistant Pseudomonas aeruginosa isolated from bloodstream infections from Qatar.

BACKGROUND: Bloodstream infections (BSIs) caused by multidrug-resistant (MDR)-Pseudomonas aeruginosa are associated with poor clinical outcomes, at least partly due to delayed appropriate antimicrobial therapy. The characteristics of MDR-P. aeruginosa bloodstream isolates have not been evaluated in Qatar. Our study aimed to examine in vitro susceptibility, clinical and molecular characteristics, and mechanisms of resistance of MDR-P. aeruginosa bloodstream isolates from Qatar. MATERIALS AND METHODS: We included all MDR-P. aeruginosa isolated from blood cultures taken between October 2014 and September 2017. Blood cultures were processed using BD BACTEC™ FX automated system. BD Phoenix™ was used for identification, Liofilchem® MIC Test Strips for MIC determination. Whole-genome sequencing was performed using the Illumina-HiSeq-2000. RESULTS: Out of 362 P. aeruginosa bloodstream isolates, 16 (4.4%) were MDR. The median patient age was 55 years (range 43-81) and all patients presented with septic shock. Most patients received meropenem (12/16) and/or colistin (10/16). Clinical response was achieved in eight patients, and five patients died within 30-days. MDR-P. aeruginosa isolates belonged to 13 different sequence types. All isolates were non-susceptible to cefepime and ciprofloxacin. The most active agents were colistin (16/16) and aztreonam (10/16). Seven isolates produced blaVIM, and four possessed genes encoding extended-spectrum ß-lactamases. Aminoglycoside modifying enzymes were present in 15/16, transferable qnr-mediated quinolone resistance gene was detected in 3/16, and the novel ciprofloxacin modifying enzyme CrpP-encoding gene in one isolate. CONCLUSION: MDR-P. aeruginosa BSIs are relatively uncommon in Qatar but are highly resistant, harbour multiple resistance genes, and are commonly associated with unfavourable clinical outcomes. Colistin was the only agent with consistent activity against the study isolates.Key messagesMDR-P. aeruginosa constituted <5% of P. aeruginosa blood isolates over three years.Typical risk factors for MDR infections were highly prevalent in the study population and overall clinical outcomes are consistent with those previously reported.Colistin was the only agent with consistent antibacterial activity against the study isolates.

[Correlations between insulin sensitivity with anthropometric and metabolic parameters in type 2 diabetics]. / Étude de la corrélation entre la sensibilité à l'insuline et les paramètres anthropométriques et métaboliques dans le diabète de type 2.

AIM: The aim of the study was to evaluate correlations between insulin sensitivity and insulinosecretion with anthropometric and metabolic parameters in type 2 diabetics. MATERIALS AND METHODS: We conducted a cross-sectional study among patients with type 2 diabetes mellitus treated with oral antidiabetic medications. The evaluation of insulin resistance and insulinosecretion was based on the calculation of the HOMA-IR and HOMA-ß indices. RESULTS: The mean age for the 100diabetes recruited was 56.4±8.4years. The mean body mass index (BMI) and waist circumference (WC) were 30.5±5.7kg/m2 and 101.2±11.9cm respectively. The HOMA-IR and HOMA ß indices were respectively 3.5±2.8 and 48.9±45.5. We have found a significantly positive correlation between HOMA-IR index and weight (r=0.406, p<10-3), BMI (r=0.432, p<10-3) and WC (r=0.412, p<10-3). We noticed a significant negative correlation between HOMA ß index and fasting glucose (r=-0.457, p<10-3) and A1C (r=-0.399, p<10-3). A positive statistically significant correlation was noted between HOMA-IR and HOMA-ß (r=0.400, p<10-3). CONCLUSION: Insulin resistance is very related to overweight, especially the android distribution of fat hence the need for adequate management of this android obesity. It would also be interesting to evaluate the effects of weight loss on insulin resistance parameters.

The emergence of multidrug-resistant Pseudomonas aeruginosa in cystic fibrosis patients on inhaled antibiotics.

INTRODUCTION: Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) is an important and growing issue in the care of patients with cystic fibrosis (CF), and a major cause of morbidity and mortality. OBJECTIVE: The objective of the study was to describe the frequency of MDR-PA recovered from the lower respiratory samples of pediatric and adult CF patients, and its antibiotic resistance pattern to commonly used antimicrobial agents including ß-lactams, aminoglycosides, and fluoroquinolones. MATERIALS AND METHODS: The lower respiratory isolates of P. aeruginosa were obtained from inpatients and outpatients CF clinics from a tertiary care teaching hospital for the period from October 2014 to September 2015. The identification and antimicrobial susceptibility for all the isolates were performed by using the BD Phoenix™ and E-test in compliance with Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: A total of 61 P. aeruginosa samples were isolated from thirty CF patients from twenty families. Twelve sputum samples were positive for MDR-PA (seven nonmucoid and five mucoid isolates) from five CF patients (five families) with moderate-to-very severe lung disease given MDR-PA frequency of 19.7%. The median age of the study group was 20 (range 10-30) years. Three CF patients were on chronic inhaled tobramycin and two on nebulized colistin. The antimicrobial patterns of isolates MDR-PA showed the highest rate of resistance toward each gentamycin, amikacin, and cefepime (100%), followed by 91.7% to ciprofloxacin, 75% to tobramycin, 58.3% to meropenem, and 50% to piperacillin-tazobactam. None of the isolates were resistant to colistin during the study period. CONCLUSION: The study results emphasize that the emergence of a significant problem in the clinical isolates of P. aeruginosa in CF patients that dictate appropriate attention to the antibiotic management after proper surveillance.

Antimicrobial susceptibility and molecular epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae from intensive care units at Hamad Medical Corporation, Qatar.

BACKGROUND: The emergence of extended-spectrum beta-lactamase (ESBL)-producing isolates has important clinical and therapeutic implications. High prevalence of ESBL-producing Enterobacteriaceae has been reported in the literature for clinical samples from a variety of infection sites. The present study was undertaken to evaluate the prevalence of ESBL-producing Enterobacteriaceae, and to perform molecular characterization and antimicrobial susceptibility testing of clinical isolates from patients admitted to the intensive care units at Hamad Medical Corporation, Doha, Qatar, from November 2012 to October 2013. METHODS: A total of 629 Enterobacteriaceae isolates were included in the study. Identification and susceptibility testing was performed using Phoenix (Becton Dickinson) and the ESBL producers were confirmed by double-disk potentiation as recommended by the Clinical and Laboratory Standards Institute. Molecular analysis of the ESBL producers was performed by polymerase chain reaction. RESULTS: In total, 109 isolates (17.3 %) were confirmed as ESBL producers and all were sensitive to meropenem in routine susceptibility assays. Most of the ESBL producers (99.1 %) were resistant to amoxicillin/clavulanic acid and ceftriaxone and 93.6 % were resistant to cefepime. Among the ESBL-producing genes, bla CTX-M (66.1 %) was the most prevalent, followed by bla SHV (53.2 %) and bla TEM (40.4 %). CONCLUSIONS: These findings show the high prevalence of ESBL-producing Enterobacteriaceae within the intensive care units at Hamad Medical Corporation, Qatar, and emphasize the need for judicious use of antibiotics and the implementation of strict infection control measures.

Tiered approaches to chromatographic bioanalytical method performance evaluation: recommendation for best practices and harmonization from the Global Bioanalysis Consortium harmonization team.

The A2 harmonization team, a part of the Global Bioanalysis Consortium (GBC), focused on defining possible tiers of chromatographic-based bioanalytical method performance. The need for developing bioanalytical methods suitable for the intended use is not a new proposal and is already referenced in regulatory guidance language. However, the practical implementation of approaches that differ from the well-established full validation requirements has proven challenging. Advances in technologies, the need to progress drug development more efficiently, and emerging new drug compound classes support the use of categorized tiers of bioanalytical methods. This paper incorporated the input from an international team of experienced bioanalysts to surmise the advantages and the challenges of tiered approaches and to provide recommendations on paths forward.

[Optic neuropathy in Behçet's disease: a series of 18 patients]. / Les neuropathies optiques au cours de la maladie de Behçet : à propos de 18 cas.

INTRODUCTION: The objective of this study was to analyse the incidence and the main characteristics of optic neuropathy in Behçet's disease. METHODS: A retrospective review of a well-documented population of 376 Tunisian patients with Behçet's disease was performed. All patients fulfilled three or more criteria defined by the International Study Group for Behçet's Disease. The diagnosis of optic neuropathy was based on the clinical examination, visual field, visual evoked potentials and retinal angiography. RESULTS: Eighteen patients (4.7 %) presented an optic nerve involvement. The mean age at presentation of these patients (10 men and nine women) was 39.11+/-12.9 years (range 17 to 73). The mean vision at presentation was 4.2/10+/-2.9, the vision was less than 1/10 in 34.5 % of eyes. The optic neuropathy was anterior in 89 % cases (26 eyes, 90 %), posterior in one case (2 eyes, 7 %); one patient (1 eye, 3 %) presented an optic atrophy. The optic neuropathy was associated with other ocular lesions in 13 cases (72.2 %). It was an inflammatory neuropathy in four cases (22.3 %) and a stasis papilledema complicating a benign intracranial hypertension in five cases (27.8 %). Corticosteroids were administrated in 17 cases (94.4 %), cyclophosphamide in six cases (33.3 %) and anticoagulant therapy in one patient (5.6 %). After a mean duration of 79 months (range: three months to 12 years), a third of the patients (8 eyes, 27.5 %) have a visual loss. CONCLUSION: Optic neuropathy is a rare ocular involvement in Behçet's disease. It can be related to an inflammatory neuropathy, a stasis papilledema complicating a benign intracranial hypertension or an ischemic neuropathy. The association of optic neuropathy with other ocular lesions could be responsible for a diagnostic delay. Its treatment relies on systemic corticosteroids and immunosuppressive drugs. The prognosis remained poor, with a third of the patients having lost their sight.

Quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma by liquid chromatography-tandem mass spectrometry following automated solid-phase extraction.

A selective, accurate, and reproducible LC-MS-MS assay was developed for the determination of the HIV protease inhibitor atazanavir (BMS-232632) in human plasma samples. The method involved automated solid-phase extraction of atazanavir and a stable isotope analog internal standard (I.S.) using Oasis HLB 10 mg 96-well SPE plates. A portion of the reconstituted sample residue was injected onto a C(18) HDO analytical column which was configured with a triple quad mass spectrometer for analyte determination by positive ion electrospray. The assay was linear from 1.00 to 1,000 ng/ml with a lower limit of quantitation of 1.00 ng/ml. The inter- and intra-day coefficients of variation (C.V.) for the assay were <4%, and the accuracy was 99-102%. Atazanavir was stable in human plasma for at least 109 h at room temperature and for at least 1 year at -20 degrees C.

Omapatrilat in patients with hepatic cirrhosis. Pharmacodynamics and pharmacokinetics.

OBJECTIVE: The pharmacodynamics and pharmacokinetics of omapatrilat, a member of a new class of cardiovascular compounds, the vasopeptidase inhibitors, were evaluated in subjects with hepatic cirrhosis (n = 10) and in healthy subjects (n = 10) matched for age, weight, gender and smoking history. METHODS: All subjects received omapatrilat 25 mg orally once daily for 14 days. Plasma renin and urinary atrial natriuretic peptide (ANP) levels were measured to assess the effect of omapatrilat on cirrhotic subjects. The effect of omapatrilat on blood pressure as well as changes in ANP and plasma renin levels were not altered by hepatic impairment. Pharmacokinetic parameters were determined from plasma omapatrilat concentrations. RESULTS: There were no significant differences between the two subject groups with regard to log-transformed area under the curve or maximum observed plasma concentration. Systemic accumulation was similar in the two groups. CONCLUSION: These results suggest, based on findings in otherwise healthy cirrhotic subjects, that no adjustment of standard dosing regimens is indicated for hypertensive patients with mild to moderate cirrhosis.

Increased throughput in quantitative bioanalysis using parallel-column liquid chromatography with mass spectrometric detection.

The feasibility of quantitative bioanalysis by parallel-column liquid chromatography in conjunction with a conventional single-source electrospray mass spectrometer has been investigated using plasma samples containing a drug and its three metabolites. Within a single chromatographic run time, sample injections were made alternately onto each of two analytical columns in parallel at specified intervals, with a mass spectrometer data file opened at every injection. Thus, the mass spectrometer collected data from two sample injections into separate data files within a single chromatographic run time. Therefore, without sacrificing the chromatographic separation or the selected reaction monitoring (SRM) dwell time, the sample throughput was increased by a factor of two. Comparing the method validation results obtained using the two-column system with those obtained using the corresponding conventional single-column approach, the methods on the two systems were found to be equivalent in terms of accuracy and precision. The parallel-column system is simple and can be implemented using existing laboratory equipment with no additional capital outlays. A parallel-column system configured in this manner can be used not only for the within-a-run analysis of two samples containing two different sets of chemical entities, but also for the within-a-run analysis of two samples containing the same set of chemical entities.

LC/MS/MS determination of omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor, and its sulfhydryl- and thioether-containing metabolites in human plasma.

Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.

Pharmacodynamics and pharmacokinetics of omapatrilat in heart failure.

The purpose of this study was to determine the pharmacodynamics and pharmacokinetics of omapatrilat, administered orally (25 mg) or intravenously (10 mg) in 19 New York Heart Association class II and class III congestive heart failure (CHF) patients versus 17 healthy controls matched for age, race, gender, and weight. The plasma concentrations of atrial natriuretic peptide (ANP) increased by approximately 20% and 30% in CHF and control subjects, respectively, at 4 hours after intravenous or oral omapatrilat administration. Similar elevation in the cyclic guanosine monophosphate concentration (25% to 35%) and ANP urinary excretion (21 ng/24 h to 22 ng/24 h) was seen in all treatment groups after omapatrilat administration. Angiotensin-converting enzyme activity was > 90% inhibited at 4 hours after dosing and remained approximately 60% to 70% inhibited at 24 hours after dosing. The levels of endothelin-1 and endothelin-2 remained unchanged after oral or intravenous administration of omapatrilat. The maximal reduction in seated blood pressure compared with baseline was similarfor CHF and control subjects. Clinical pharmacokinetic parameters were similar in both groups after intravenous dosing, but maximum concentration and area under the concentration-time curve were elevated in CHF patients compared with controls after oral dosing. Omapatrilat was well tolerated; differences in systemic exposure and metabolism between CHF patients and controls did not appear to be clinically significant.

Disposition and safety of omapatrilat in subjects with renal impairment.

BACKGROUND: Omapatrilat, a vasopeptidase inhibitor, preserves natriuretic peptides and inhibits the renin angiotensin aldosterone system by simultaneously inhibiting neutral endopeptidase and angiotensin-converting enzyme. METHODS: Oral omapatrilat, 10 mg/d, was administered for 8 to 9 days to three groups of eight subjects with varying degrees of renal function (CLCR values, normal > or = 80; mild to moderate impairment < 80 to > or = 30; severe impairment < 30 mL/min/1.73 mL2) and to six subjects undergoing maintenance hemodialysis. Omapatrilat and its metabolites (phenylmercaptopropionic acid, S-methylomapatrilat, S-methylphenylmercaptopropionic acid, and cyclic S-oxide-omapatrilat) were quantified in plasma by a validated liquid chromatography/mass spectrometry method. The model, Cmax or AUC(0-T) = intercept + slope x CLCR, was tested for a possible linear correlation between Cmax (peak plasma concentrations) or AUC(0-T) (area under plasma concentration versus time curve) and CLCR. RESULTS: For omapatrilat and its inactive metabolites, phenylmercaptopropionic acid, S-methylomapatrilat, and S-methylphenylmercaptopropionic acid, the median times to peak plasma concentrations (tmax) were 1.5 to 2, 2 to 3, 2.5 to 3.5, and 7 to 10 hours, respectively, and were independent of renal function. After Cmax attainment, plasma concentrations declined rapidly to about 10% of Cmax values. Cyclic S-oxide-omapatrilat, a potentially active metabolite, was undetectable at all sampling time points. Hemodialysis did not decrease circulating levels of omapatrilat. There was minimal accumulation of omapatrilat and phenylmercaptopropionic acid and moderate accumulation of the S-methylated metabolites. For omapatrilat and S-methylphenylmercaptopropionic acid, neither Cmax nor AUC(0-T) was CLCR dependent. However, AUC(0-T) for phenylmercaptopropionic acid and both the Cmax and AUC(0-T) for S-methylomapatrilat were CLCR dependent. CONCLUSIONS: The pharmacokinetics of omapatrilat, the only clinically relevant active compound studied, was independent of CLCR. For patients with reduced renal function, adjusting initial omapatrilat dose is not suggested. Hemodialysis did not significantly contribute to the clearance of omapatrilat. The long-term pharmacodynamic response to omapatrilat will dictate dose-adjustment needs.

Quantitative determination of pravastatin and its biotransformation products in human serum by turbo ion spray LC/MS/MS.

A sensitive, specific, accurate and reproducible analytical method was developed and validated to quantify pravastatin (Prav), pravastatin-d5 (Prav-d5), SQ-31906, SQ-31906-d5, and pravastatin lactone (Prav-Lac) in human serum samples. Serum samples (0.5 ml) were acidified and extracted by a solid-phase extraction procedure to isolate all five analytes from human serum. Sample extracts were reconstituted and analyzed by turbo ion spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the positive ion mode. The total run time was 9 min between injections. The assay demonstrated a lower limit of quantitation (LLQ) of 0.5 ng/ml for all five analytes. The calibration curves were linear from 0.5 ng ml to 100 ng/ml for all five analytes. The coefficients of determination of all calibration curves were > or = 0.999. Precision and accuracy quality control (QC) samples were prepared at concentrations of 2, 30, 80, and 500 ng/ml for all analytes. The intra-assay and inter-assay precision calculated from QC samples were within 8%, for all analytes. The inter-assay accuracy calculated from QC samples was within 8% for all analytes. The extraction recoveries were > or = 90% for all analytes. Benchtop stability experiments in an ice-water bath ( < or = 10 degrees C) demonstrated that over time, Prav-Lac hydrolyzes to Prav in serum. Prav, Prav-d5, SQ-31906, and SQ-31906-d5 were stable under these conditions for up to 24 h. Hydrolysis was minimized by buffering the serum to pH 4.5 and maintaining the serum sample in an ice-water bath. All analytes were stable after three freeze/thaw cycles and in reconstitution solution after 1 week at 4 degrees C. Stability of all analytes in human serum was demonstrated after storage at -70 degrees C for 77 days. The benchtop (< or = 10 degrees C) stability of pooled study samples was also investigated and the results were comparable to those obtained from serum QC samples.

An automated method of sample preparation of biofluids using pierceable caps to eliminate the uncapping of the sample tubes during sample transfer.

Biological samples are normally collected and stored frozen in capped tubes until analysis. To obtain aliquots of biological samples for analysis, the sample tubes have to be thawed, uncapped, samples removed and then recapped for further storage. In this paper, we report an automated method of sample transfer devised to eliminate the uncapping and recapping process. This sampling method was incorporated into an automated liquid-liquid extraction procedure of plasma samples. Using a robotic system, the plasma samples were transferred directly from pierceable capped tubes into microtubes contained in a 96-position block. The aliquoted samples were extracted with methyl-tert-butyl ether in the same microtubes. The supernatant organic layers were transferred to a 96-well collection plate and evaporated to dryness. The dried extracts were reconstituted and injected from the same plate for analysis by liquid chromatography with tandem mass spectrometry.

High-throughput quantitative bioanalysis by LC/MS/MS.

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.