Masayuki Sakamoto

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.

The pharmacokinetics of epidural ropivacaine in infants and young children.

The pharmacokinetic variables of ropivacaine were characterized after epidural bolus injection in pediatric patients. The subjects, 7 infants (aged 3–11 mo) and 11 young children (aged 12–48 mo), received 1.7 mg/kg of ropivacaine via a lumbar epidural catheter. Total plasma concentrations of ropivacaine measured over 24 h were assayed by high-pressure liquid chromatography, and pharmacokinetic modeling was performed by Nonlinear Mixed Effects Modeling analysis. The median peak venous plasma concentrations (Cmax) in infants and young children were 610 &mgr;g/L (interquartile range [IQR], 550–725 &mgr;g/L) and 640 &mgr;g/L (IQR, 540–750 &mgr;g/L), respectively. The median times to maximum plasma ropivacaine concentration (Tmax) were 60 min (IQR, 60–120 min) in infants and 60 min (IQR, 30–90 min) in young children. There were no statistical differences between median values of Cmax and Tmax between infants and young children. The calculated clearance (CL) in infants was 4.26 mL · min−1 · kg−1 (9% coefficient of variation), and in young children it was 6.15 mL · min−1 · kg−1 (11% coefficient of variation). The CL for infants was significantly less than the CL for young children (P < 0.01). The volume of distribution was estimated to be 2370 mL/kg (9% coefficient of variation) for both young children and infants. No systemic toxicity was observed in either group.

Measurement of plasma busulfan concentration by high-performance liquid chromatography with ultraviolet detection

Busulfan is widely used in bone marrow transplantation. Increased area under the plasma concentration curve has been shown to correlate with venoocclusive disease, which occurs in approximately 20% of these patients. The authors developed a high performance liquid chromatography assay for the determination of plasma busulfan concentration using ultraviolet detection and a single-step derivatization and extraction. The absolute retention times of busulfan and the internal standard were 2.8 and 5.6 min, respectively. The assay possessed linearity up to 200 mumol/l, sensitivity to at least 0.2 mumol/l, average recovery of 101%, and run-to-run precision (n = 34) of < 7%. Furthermore, the assay proved to be free of interference from 59 medications and correlated highly with a validated method employing gas chromatography with electron capture detection (slope = 0.90, intercept = 0.17, r = 0.98, n = 31). The authors conclude that the method described here is ideally suited for the therapeutic monitoring of busulfan.

Tandem mass spectrometry method for the quantification of serum busulfan

Busulfan, an alkylating agent, is most commonly used as a component of bone marrow transplantation preoperative regimens. Significant interpatient and intrapatient variations in pharmacokinetics require individualizing the dosage based on area under the time-versus-concentration curve. Timely result reporting is critical to dose adjustment to reduce morbidity and mortality associated with the regimen. The authors developed a rapid, accurate, and sensitive method for the quantification of serum busulfan using direct inject tandem mass spectrometry. Plasma samples (50 μL) are extracted in 1 mL of methanol containing 1,6-bis-(methanesulfonyloxy)hexane as an internal standard. The supernatant is dried under nitrogen (40°C, 30 minutes) and then dissolved in 200 μL methanol and transferred into a clean glass vial suitable for LC/MS/MS analysis. The sample is delivered using an HPLC pump that delivers 0.2 mL of methanol per minute, and 20 μL of sample is injected into a turbo ion spray-equipped tandem mass spectrometer. Total analysis time is 5 minutes. The Q1/Q3 transition for busulfan (BU) is monitored at 269/55 and 297.1/55.1 for the internal standard. The assay is linear to 10 μmol/L and sensitive to at least 0.5 μmol/L. The interassay reproducibility at 1, 2.2, and 4.4 μmol/L were 4.2%, 5.6%, and 6.3%, respectively. Within-run precision using 3 different control samples was 3.9%, 3.9%, and 6.9%. Mean recovery of 4 different BU concentrations spiked into 10 different BU free plasma samples was 98%. Correlation with an established HPLC-UV method revealed a slope of 0.98, an intercept of 0.1, and r2 = 0.95 (n = 48). No significant interfering substances or ion suppression was identified. This method is a significant improvement over the existing HPLC-UV method for BU determination. The method is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time.

A high-performance liquid chromatographic assay for the determination of itraconazole concentration using solid-phase extraction and small sample volume

Summary Itraconazole has been shown to be highly effective against a broad spectrum of fungi, particularly aspergillus. In order to achieve the desired pharmacological effect, the itraconazole plasma concentration should be >250 ng/ml. We developed a high-performance liquid chromatography assay for the rapid determination of plasma itraconazole concentration using solid-phase extraction and small sample volume. The assay possessed linearity up to 5,000 ng/ml, sensitivity of 50 ng/ml, average recovery of 100.8%, and run-to-run precision (n = 10) for concentrations of 100, 200, and 500 ng/ml of 12, 6.6, and 5.8%, respectively. Furthermore, the assay proved to be free of interference from 48 commonly prescribed and some over-the-counter medications. We conclude that the method described here is ideally suited for the therapeutic monitoring of itraconazole.

Simultaneous measurement of plasma ropivacaine and bupivacaine concentrations by HPLC with UV detection

The authors developed a high-performance liquid chromatography (HPLC) assay for the simultaneous determination of plasma ropivacaine and bupivacaine concentrations using ultraviolet (UV) detection and a simple solid-phase extraction procedure. The absolute retention times of ropivacaine, bupivacaine, and the internal standard pentycaine were 1.9, 3.0, and 5.6 minutes, respectively. The assay had a linearity of 2000 ng/mL, a sensitivity of 5 ng/mL, an average recovery of 98%, and an average day-to-day imprecision of <10% for both drugs. A patient correlation study (n = 23) using this HPLC method and an established gas chromatographic assay revealed a slope of 1.01, an intercept of −10.6 ng/mL, and a correlation coefficient of 0.99 for ropivacaine; and a slope of 0.96, an intercept of 14.7 ng/mL, and a correlation coefficient of 0.99 for bupivacaine. Of the 60 different drugs tested, only quinidine and lidocaine extracted but did not interfere with the measurement of the drugs of interest. The authors conclude that the method described here is ideally suited for the therapeutic monitoring of plasma ropivacaine and bupivacaine concentrations.

Determination of Serum Physiological Concentration of Methylmalonic Acid by Gas Chromatography-Mass Spectrometry with Selected Ion Monitoring

We developed a sensitive assay for the rapid determination of serum methylmalonic acid concentration using capillary gas chromatography-mass spectrometry (GC/MS) with selected ion monitoring and a simple solid-phase extraction. The assay was linear up to 10 000 nmol/L and had a detection limit < 50 nmol/L, average recovery of 98% and between-day coefficient of variation at concentrations of 570 and 2206 nmol/L of 7.7% and 5.4%, respectively (n = 25). Comparison with another validated GC/MS method using sera with a wide range of methylmalonic acid concentrations (94-2020 nmol/L) revealed a slope and intercept of 0.97 and 17 nmol/L, respectively (n = 38). Methylmalonic acid concentrations determined by this assay in a group of apparently healthy individuals ranged from 64–331 nmol/L (n = 81). We conclude that the method is ideally suited for the determination of methylmalonic acid at physiological concentrations in both clinical and research laboratories.

Measurement of whole blood sirolimus by an HPLC assay using solid-phase extraction and UV detection.

The authors developed an HPLC assay for determining blood sirolimus concentration using a relatively simple solid-phase extraction and UV detection. The retention times of sirolimus and the internal standard, 32-desmethoxyrapamycin, are 8.7 and 9.3 minutes, respectively. The assay possesses linearity up to 200 ng/mL, sensitivity to 2.0 ng/mL, and day-to-day reproducibility of 8.8, 9.8, 6.1, and 6.4% at sirolimus concentrations of 6, 10, 20, and 30 ng/mL, respectively. A patient correlation study using this HPLC method and an established LC/MS/MS assay revealed a slope of 0.982 and intercept of −0.021 ng/mL and a correlation coefficient of 0.99 (n = 37). Of the 31 different drugs tested none interfered with the measurement of the drug of interest, and a recovery study gave an overall mean recovery of 101.8%. The authors conclude that the method described here is suited for the therapeutic monitoring of blood sirolimus concentration.

Measurement of plasma ketoprofen by a rapid high-performance liquid chromatography assay

The authors developed a high performance liquid chromatography assay for the determination of plasma ketoprofen concentration using a single-step extraction, a short chromatographic separation of 3 min, and a sample volume of 50 microliters. The assay possessed linearity up to 500 mg/l, sensitivity down to at least 1 mg/l, average recovery of 100.1%, and run-to-run precision (n = 20) of 4.6% at a level of 10 mg/l and 2.2% at a level of 40 mg/l. Furthermore, the assay was free of interference from 51 prescription and over-the-counter medications. The authors conclude that the method described here is ideally suited for the determination of ketoprofen concentration in a clinical laboratory setting for the purpose of therapeutic monitoring and assessment of toxicity.