Digital spacer data driven COPD inhaler adherence education: The OUTERSPACE proof-of-concept study.

Pressurized metered dose inhalers are recommended to be used in combination with spacers, yet inhaler technique and adherence are poor. A novel digital "smart" spacer can record spacer use and technique errors and could facilitate personalized education. In this proof-of-concept study, we assessed the usability of the digital spacer and explored its effects on inhaler technique, adherence, long-term systemic drug exposure and clinical outcomes in COPD. Usability was deemed high. One month after personalized digital spacer inhaler education, the mean number of errors per patient per day decreased with 36%, from 6.40 errors/day to 4.07 errors/day (p = 0.038). Drug exposure was confirmed by bioanalytical scalp hair analysis of formoterol. No significant change in clinical outcomes was observed. This study demonstrates the digital spacer's potential value in inhaler education, but larger, longer-term studies are required.

A fast and simple method for the simultaneous analysis of midazolam, 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, plasma and urine.

For the quantification of the sedative and anesthetic drug midazolam and its main (active) metabolites 1-hydroxymidazolam, 4-hydroxymidazolam and 1-hydroxymidazolam glucuronide in human serum, human EDTA plasma, human heparin plasma and human urine a single accurate method by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed. Protein precipitation as sample preparation, without the need of a time-consuming deglucuronidation step for the quantification of 1-hydroxymidazolam glucuronide, resulted in a simple and rapid assay suitable for clinical practice with a total runtime of only 1.1  min. The four components and the isotope-labeled internal standards were separated on a C18 column and detection was performed with a triple-stage quadrupole mass spectrometer operating in positive ionization mode. The method was validated based on the "Guidance for Industry Bioanalytical Method Validation" (Food and Drug Administration, FDA) and the "Guideline on bioanalytical method validation" of the European Medicines Agency (EMA). Linearity was proven over the ranges of 5-1500 µg/L for midazolam, 1-hydroxymidazolam and 4-hydroxymidazolam and 25-5000 µg/L for 1-hydroxymidazolam glucuronide, using a sample volume of 100 µL. Matrix comparison indicated that the assay is also applicable to other human matrices like EDTA and heparin plasma and urine. Stability experiments showed good results for the stability of midazolam, 1-hydroxymidazolam and 1-hydroxymidazolam glucuronide in serum, EDTA and heparin plasma and urine stored for 7 days under different conditions. At room temperature, 4-hydroxymidazo-lam is stable for 7 days in EDTA plasma, but stable for only 3 days in serum and heparin plasma and less than 24 h in urine. All four compounds were found to be stable in serum, EDTA plasma, heparin plasma and urine for 7 days after sample preparation and for 3 freeze-thaw cycles. The assay has been applied in therapeutic drug monitoring of midazolam for (pediatric) intensive care patients.

Increasing metformin concentrations and its excretion in both rat and porcine ex vivo normothermic kidney perfusion model.

INTRODUCTION: Metformin can accumulate and cause lactic acidosis in patients with renal insufficiency. Metformin is known to inhibit mitochondria, while renal secretion of the drug by proximal tubules indirectly requires energy. We investigated whether addition of metformin before or during ex vivo isolated normothermic machine perfusion (NMP) of porcine and rat kidneys affects its elimination. RESEARCH DESIGN AND METHODS: First, Lewis rats were pretreated with metformin or saline the day before nephrectomy. Subsequently, NMP of the kidney was performed for 90 min. Metformin was added to the perfusion fluid in one of three different concentrations (none, 30 mg/L or 300 mg/L). Second, metformin was added in increasing doses to the perfusion fluid during 4 hours of NMP of porcine kidneys. Metformin concentration was determined in the perfusion fluid and urine by liquid chromatography-tandem mass spectrometry. RESULTS: Metformin clearance was approximately 4-5 times higher than creatinine clearance in both models, underscoring secretion of the drug. Metformin clearance at the end of NMP in rat kidneys perfused with 30 mg/L was lower than in metformin pretreated rats without the addition of metformin during perfusion (both p≤0.05), but kidneys perfused with 300 mg/L trended toward lower metformin clearance (p=0.06). Creatinine clearance was not different between treatment groups. During NMP of porcine kidneys, metformin clearance peaked at 90 min of NMP (18.2±13.7 mL/min/100 g). Thereafter, metformin clearance declined, while creatinine clearance remained stable. This observation can be explained by saturation of metformin transporters with a Michaelis-Menten constant (95% CI) of 23.0 (10.0 to 52.3) mg/L. CONCLUSIONS: Metformin was secreted during NMP of both rat and porcine kidneys. Excretion of metformin decreased under increasing concentrations of metformin, which might be explained by saturation of metformin transporters rather than a self-inhibitory effect. It remains unknown whether a self-inhibitory effect contributes to metformin accumulation in humans with longer exposure times.

Simple, fast and robust LC-MS/MS method for the simultaneous quantification of canagliflozin, dapagliflozin and empagliflozin in human plasma and urine.

Sodium-glucose cotransporter 2 -inhibitors (SGLT2i) are oral glucose-lowering drugs that have also demonstrated cardioprotective and renoprotective effects. SGLT2i play an increasingly important role in the treatment of type 2 diabetes. Here we report a simple and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of three SGLT2i (canagliflozin, dapagliflozin and empagliflozin) in human plasma, serum and urine with a runtime of 1 min. Methanol was used as protein precipitating agent. Chromatographic separation was accomplished using a Waters ACQUITY UPLC HSS T3 1.8 µm; 2.1 × 50 mm column with a Waters ACQUITY UPLC HSS T3 1.8 µm VanGuard Pre-column; 2.1 × 5 mm, using gradient elution with ammonium acetate 20 mM (pH 5) and acetonitrile as mobile phase at a flow rate of 0.8 ml/min. Mass spectrometric analysis of the acetate adduct ions was carried out using electrospray with negative ionization and SRM mode. The assay was validated according to FDA and EMA guidelines, including selectivity, linearity, accuracy and precision, dilution integrity, stability and recovery. With a sample volume of 200 µl, linear ranges of 10-5000 µg/L, 1-500 µg/L and 2-1000 µg/L for canagliflozin, dapagliflozin and empagliflozin respectively, were achieved. The assay was successfully applied in two pharmacokinetic studies with dapagliflozin and empagliflozin. In conclusion, we developed and validated a simple, fast and robust LC-MS/MS method for the simultaneous quantification of canagliflozin, dapagliflozin and empagliflozin, that allows rapid analysis of large numbers of samples and can be used for both pharmacokinetic studies and biomedical analysis of canagliflozin, dapagliflozin and empagliflozin.

Susceptibility Testing of Antibiotics That Degrade Faster than the Doubling Time of Slow-Growing Mycobacteria: Ertapenem Sterilizing Effect versus Mycobacterium tuberculosis.

Drug susceptibility tests (DSTs) for Mycobacterium tuberculosis require at least 7 days of incubation. Drugs that are unstable at 37°C, such as ertapenem, are likely to be degraded before killing or inhibiting slow-growing bacteria. This would alter the MICs of these drugs, including ertapenem, leading to falsely high MICs. Here, we describe a new strategy we developed to perform DSTs and measure MICs for such unstable compounds.

Simultaneous quantification of anidulafungin and caspofungin in plasma by an accurate and simple liquid chromatography tandem mass-spectrometric method.

INTRODUCTION: Echinocandins are a valuable addition for the treatment of invasive fungal infections, as they are efficacious, demonstrate low toxicity, and have limited drug-drug interactions. In specific clinical situations when altered pharmacokinetics can be expected or dosing guidelines are conflicting, it may be useful to measure concentrations. For this purpose, a liquid chromatography tandem mass-spectrometric method to measure anidulafungin and caspofungin in ethylenediaminetetraacetic acid plasma was developed. METHODS: The method was developed on a Thermo Fisher TSQ Quantum LC-MS/MS. For separation, a BetaBasic C4 (100 mm × 3.0 mm; 5 µm) analytical column was used. Sample preparation consisted of protein precipitation directly in the autosampler vial. The internal standard aculeacin A is structurally related, not used in humans, and commercially available. The method was validated according to the guidelines for bioanalytical method validation of the Food and Drug Administration. RESULTS: The method was accurate (bias ranging from -3.0% to 1.9%) and precise (within-run and between-run coefficients of variation of 2.2% to 7.7% and 1.6% to 9.0%, respectively). All calibration curves were linear over a range of 0.5-10.0 mg/L for anidulafungin and 0.1-20.0 mg/L for caspofungin, and if necessary, samples can be diluted 10-fold. The samples were stable for 3 freeze-thaw cycles, with a bias ranging from 0.6% to 11%. The maximum bias from the worst storage condition, 72 hours at room temperature, was -14.7%. In patient samples, anidulafungin peak concentrations ranged from 2.8 to 8.6 mg/L (n = 20) and trough concentrations ranged from 1.0 to 4.7 mg/L (n = 79). The measured caspofungin concentrations ranged from 1.9 to 7.3 mg/L (n = 20). CONCLUSIONS: The method developed has a straightforward sample preparation and uses a structural analog as the internal standard. This method has been applied successfully for the measurement of anidulafungin and caspofungin concentrations in patient samples, both for clinical practice and for research.

Comparison of the pharmacokinetics of two dosage regimens of linezolid in multidrug-resistant and extensively drug-resistant tuberculosis patients.

BACKGROUND AND OBJECTIVES: For the treatment of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB), potent new drugs are urgently needed. Linezolid is a promising drug, but its use is limited by adverse effects with prolonged administration of 600 mg twice daily. In order to reduce its adverse effects and maintain efficacy, we investigated whether linezolid in a reduced dosage resulted in drug serum concentrations exceeding a ratio of the in vitro minimum inhibitory concentration (MIC) to the area under the serum concentration-time curve (AUC) over 24 hours (AUC(24)) [AUC(24)/MIC] of >100. PATIENTS AND METHODS: This open-label, prospective pharmacokinetic study evaluated two doses (300 and 600 mg) of linezolid in MDR-TB patients, who received linezolid as part of their treatment. They received linezolid 300 mg twice daily for 3 days, followed by 600 mg twice daily. Blood samples taken at predefined intervals for measuring serum linezolid concentrations were processed by a validated liquid chromatography-tandem mass spectrometry procedure. The AUC(24)/MIC ratio was used as a predictive model of efficacy. Adverse effects of linezolid, including peripheral neuropathy, were evaluated by clinical and laboratory assessments. RESULTS: Eight patients were included in this study. The median duration of linezolid treatment was 56 days (interquartile range [IQR 44-82] days), with a median cumulative dose of 51,000 mg (IQR 33,850-60,450 mg). The median linezolid AUC over 12 hours (AUC(12)) values were 57.6 mg x h/L (IQR 38.5-64.2 mg x h/L) with the 300 mg dose and 145.8 mg x h/L (IQR 101.2-160.9 mg x h/L) with the 600 mg dose. The AUC(24)/MIC ratios were 452 (IQR 343-513) with the 300 mg dose and 1151 (IQR 656-1500) with the 600 mg dose. Linezolid was well tolerated. CONCLUSION: Seemingly effective serum concentrations were reached after 3 days of administration of linezolid 300 mg twice daily, i.e. the AUC(24)/MIC ratio was at least 100 in 7 of 8 patients. Larger numbers of patients should be studied to confirm the efficacy of the linezolid 300 mg twice-daily dosage in MDR-TB or XDR-TB treatment.

Determination of moxifloxacin in human plasma, plasma ultrafiltrate, and cerebrospinal fluid by a rapid and simple liquid chromatography- tandem mass spectrometry method.

Moxifloxacin (MFX) is a useful agent in the treatment of multi-drug-resistant tuberculosis (MDR-TB). At Tuberculosis Centre Beatrixoord, a referral center for tuberculosis in the Netherlands, approximately 36% of the patients have received MFX as treatment. Based on the variability of MFX AUC, the variability of in vitro susceptibility to MFX of M. tuberculosis, and the variability of penetration into sanctuary sites, measuring the concentration of MFX in plasma and cerebrospinal fluid (CSF) could be recommended. Therefore, a rapid and validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyzing method with a simple pretreatment procedure was developed for therapeutic drug monitoring of MFX in human plasma and CSF. Because of the potential influence of protein binding on efficacy, we decided to determine both bound and unbound (ultrafiltrate) fraction of MFX. The calibration curves were linear in the therapeutic range of 0.05 to 5.0 mg/L plasma and CSF with CV in the range of -5.4% to 9.3%. MFX ultrafiltrate samples could be determined with the same method setup for analysis of MFX in CSF. The LC-MS-MS method developed in this study is suitable for monitoring MFX in human plasma, plasma ultrafiltrate, and CSF.

Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

The drug combination rifampicin and clarithromycin is used in regimens for infections caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the "Guidance for Industry - Bioanalytical Method Validation" of the FDA. The calibration curves were linear in the range of 0.10-10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20-5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to -10%. The components in human plasma are stable after freeze-thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days; rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method is suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma.