JoEtta M. Juenke

Steady‐State Pharmacokinetics of Intravenous Levetiracetam in Neurocritical Care Patients

Study Objectives. To characterize the steady‐state pharmacokinetics of intravenous levetiracetam in neurocritical care patients requiring seizure prophylaxis after a neurologic injury and to determine which dosing regimens achieve serum concentrations within the recommended therapeutic range of 6–20 μg/ml.

Rapid and specific quantification of ethylene glycol levels: adaptation of a commercial enzymatic assay to automated chemistry analyzers.

Ethylene glycol ingestion, accidental or intentional, can be a life-threatening emergency. Assays are not available from most clinical laboratories, and, thus, results often require many hours or days to obtain. Enzymatic assays, adaptable to automated chemistry analyzers, have been evaluated, but they have been plagued by analytic problems. With an alternative method of data analysis applied to an existing enzymatic assay, a modified assay was developed and validated on 2 different automated chemistry systems. Compared with a previously validated method based on gas chromatography with flame ionization detection, the modified enzymatic assay showed excellent agreement on patient samples (y = 1.0227x -1.24; r(2) = 0.9725), with a large analytic measuring range (2.5-300 mg/dL [0.4-48.4 mmol/L]). Interferences from propylene glycol, various butanediols, and other related compounds were almost entirely eliminated; when present, they generated error flags rather than falsely elevated ethylene glycol results. This modified assay should make it possible for more clinical laboratories to offer ethylene glycol measurements.

Procedure for the Monitoring of Gabapentin with 2,4,6-Trinitrobenzene Sulfonic Acid Derivatization Followed by HPLC with Ultraviolet Detection

Gabapentin is a novel anticonvulsant drug that was introduced in the early 1990s and later approved (1995) for use in the US as an adjunctive treatment of partial seizures with or without secondary generalization in persons >3 years of age. Although structurally similar to γ-aminobutyric acid (GABA), gabapentin does not interact with GABA receptors, nor is it converted to GABA or a GABA agonist (1). Gabapentin is widely studied therapeutically. Its initial and approved use as adjunctive epileptic therapy has been broadened, with many additional indications. These include treatment for neuropathic pain after spinal cord injury (2)(3)(4), posttraumatic stress disorder (5), poststroke pain syndrome (6)(7)(8), alcohol withdrawal(9), migraine therapy (10), hot flashes associated with prostate cancer treatment (11), and postoperative pain after cancer surgery (12)(13). The general mechanism by which gabapentin exerts its anticonvulsant action is unknown. It is not appreciably metabolized in the liver, nor does it induce liver enzymes. It circulates relatively unbound in serum, with a protein bound fraction of ∼3%. It has a volume of distribution of ∼58 L. Because gabapentin does not bind to protein, it can be removed by hemodialysis if medically necessary. Gabapentin is renally eliminated with an elimination half-life of ∼6 h and clearance proportional to creatinine clearance. Impaired renal function substantially decreases the clearance of gabapentin (14). Gabapentin exhibits saturable absorption, making it a nonlinear drug and kinetically less predictable. A dose–response pattern is apparent for plasma gabapentin concentrations and for clinical effects within the dosage range 600-1800 mg/day. Seizure control has not been seen with trough plasma concentrations <2 mg/L. A majority of patients at suggested doses fall within a 2–10 mg/L range. The major side effects of the drug include somnolence, dizziness, ataxia, fatigue, and nystagmus. No …

Comparative Pharmacokinetics and Pharmacodynamics of Doripenem and Meropenem in Obese Patients

Background: Antimicrobial pharmacokinetic and pharmacodynamic data are limited in obesity. Objective: To evaluate the steady-state pharmacokinetics and pharmacodynamics of doripenem and meropenem in obese patients hospitalized on a general ward. Methods: Patients with a body mass index (BMI) ≥40 kg/m2 or total body weight (TBW) ≥100 pounds over their ideal body weight randomly received doripenem 500 mg (1-hour infusion) or meropenem 1 g (0.5-hour infusion) every 8 hours. Differences in pharmacokinetic parameters were determined by unpaired t test. Monte Carlo simulations were performed for 500 mg and 1 g every 8 hours, infused over 1 and 4 hours for doripenem and 0.5 and 3 hours for meropenem. Probability of target attainment (PTA) was calculated using a pharmacodynamic target of 40% fT > MIC (free drug concentrations above the minimum inhibitory concentration [MIC]), and cumulative fraction of response (CFR) was calculated using MIC data for 8 Gram-negative pathogens. Results: Twenty patients were studied. Volume of distribution at steady state, corrected for TBW, was significantly larger (0.18 ± 0.04 vs 0.13 ± 0.05 L/kg, P = .048) and systemic clearance was significantly faster for doripenem (11.7 ± 4.1 vs 8.1 ± 2.6 L/h, P = .03). PTA was >90% for all regimens at MICs ≤2 µg/mL. CFR was >90% for all regimens against 6 enteric Gram-negative pathogens and for 3 of 4 regimens for each drug against Pseudomonas aeruginosa. Conclusions: Doripenem and meropenem pharmacokinetics differ in obesity. However, currently approved dosing regimens provide adequate pharmacodynamic exposures for susceptible bacteria in obese patients.

Simultaneous quantification of levetiracetam and gabapentin in plasma by ultra-pressure liquid chromatography coupled with tandem mass spectrometry detection.

Introduction: Gabapentin (Neurontin) and levetiracetam (Keppra) are anticonvulsants with novel structures and suggested therapeutic ranges of 2-10 mg/L and 6-20 mg/L, respectively. Gabapentin is also used extensively to manage neuropathic pain, and for this indication, wherein higher doses are prescribed, plasma concentrations of 15-30 mg/L are typical. Objective: Here, we describe a simple rapid assay to support therapeutic drug monitoring of gabapentin and levetiracetam in plasma by ultra-pressure liquid chromatography couples to tandem mass spectrometry (UPLC-MS/MS) detection. Methods: After the addition of internal standard and protein precipitation of patient plasma with methanol:acetonitrile in a 50:50 ratio, 1 μL of supernatant sample is injected onto an Acquity UPLC HSS T3, 1.8 μm, 2.1 × 50 mm (Waters) column. Elution occurs using a linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into a Waters Acquity UPLC TQD, operating in a positive mode to detect gabapentin at transition 172.18 > 154.11, levetiracetam at 171.11 > 126, and internal standard (3-amino-2-naphthoic acid) at 188.06 > 170. Secondary transitions for each analyte are also monitored for gabapentin at 172.18 > 137.06, levetiracetam at 171.11 > 154, and internal standard at 188.06 > 115. Runtime is 1.5 minutes per injection with baseline resolved chromatographic separation. Results: The analytical measurement ranges were 1-150 mg/L for gabapentin and for levetiracetam. Intra-assay imprecision by the coefficient of variance (CV) was less than 8% and interassay CV was less than 5% for both analytes, at 4 different concentrations. Results obtained from patient samples were compared with results generated by established high-performance liquid chromatography-UV methods with the following regression statistics: y = 1.12x − 0.77, r = 0.996, Sy, x = 0.89, and n = 29 for gabapentin and y = 0.991x + 0.70, r = 0.997, Sy, x = 2.24, and n = 30 for levetiracetam. No analytical interferences were identified. Conclusion: In summary, a simple reliable UPLC-MS/MS method was developed and validated for routine clinical monitoring of gabapentin and levetiracetam.

A comparison of two FDA approved lamotrigine immunoassays with ultra-high performance liquid chromatography tandem mass spectrometry.

BACKGROUND Lamotrigine is an anti-epileptic drug used as adjunct therapy for seizures. Lamotrigine is commonly used in pregnant women with epilepsy, a population in which therapeutic drug monitoring (TDM) is useful to optimize dose. Drug-drug interactions that can induce or inhibit metabolism or elimination and impaired hepatic function are also possible indications for lamotrigine TDM. Chromatographic techniques are currently used for performing most TDM of lamotrigine, but this may change, as automated immunoassays were recently introduced. METHODS Immunoassays available through Seradyn and ARK Diagnostics were validated using a Beckman AU400e automated chemistry analyzer. The intra-day precision was accessed with 5 replicates of three quality control materials, and inter-assay precision was estimated by assaying the same material over 4 days. Linearity was evaluated by serially diluting a spiked sample and measuring it in duplicate. The 2 methods were compared with ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) using 44 authentic patient specimens. RESULTS The intra-day (n=5) and inter-assay (n=20) coefficients of variation were ≤7.5% for the 3 levels tested. The analytical measurement ranges were confirmed as stated by the manufacturers (0 or 1-40 μg/ml). The percent recovery of the quality control materials and Deming regression for the 44 patient results showed good agreement of both immunoassays when compared to the UPLC-MS/MS assay. CONCLUSION The lamotrigine assays studied here produced a slightly lower result than UPLC-MS/MS but were precise and easy to perform.

Determination of busulfan in human plasma using an ELISA format.

High-dose busulfan is an important component of many bone marrow transplantation-preparative regimens. High busulfan plasma levels have been shown to increase the chance of venoocclusive disease and low levels are associated with recurrence of disease or graft rejection. Currently, busulfan levels are monitored by physical methods that are expensive and time consuming, resulting in relatively low overall use of busulfan testing for dose adjustment. Novel highly selective antibodies for busulfan have been generated and a microtiter plate immunoassay capable of quantifying busulfan levels in plasma has been developed. The assay was configured using a busulfan-horseradish peroxidase (HRP) conjugate as the reporter group and busulfan monoclonal antibodies. The assay requires 30 μL of plasma with no sample preparation. The immunoassay has a standard curve based on busulfan with a range of 75-2000 ng/mL. The time to first result is 30 minutes with up to 40 patient samples in duplicate; multiple plates can be run at once. The coefficient of variation (CV) on signal is <5% for an entire plate, and the 95% confidence interval for negative samples (n = 78) is below the lowest calibrator of 75 ng/mL. Cross-reactivity with the major inactive metabolites (tetrahydrothiophene, tetramethyl sulfone, and tetrahydrothiophene-3-ol-1,1-dioxide) was <0.1%. Results generated with clinical samples (n = 35 and n = 70) correlate well to gas chromatography-mass spectrometry (R = 0.976 and 0.985, respectively) with a slope of 1.05 ± 0.05. This immunoassay method is suitable for determining levels of busulfan in human plasma. It offers the advantages of using a smaller sample size, does not require sample preparation, and is less labor intensive than other methods. The ability to make 240 determinations per hour enables effective and timely routine monitoring of busulfan levels in clinical practice.

Simultaneous UPLC-MS/MS assay for the detection of the traditional antipsychotics haloperidol, fluphenazine, perphenazine, and thiothixene in serum and plasma.

BACKGROUND Most antipsychotic drugs that are commonly prescribed in the USA are monitored by liquid and gas chromatographic methods. Method performance has been improved using ultra high pressure liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A rapid and simple procedure for monitoring haloperidol, thiothixene, fluphenazine, and perphenazine is described here. METHOD Antipsychotic drug concentrations in serum and plasma were determined by LCMS/MS (Waters Acquity UPLC TQD). The instrument is operated with an ESI interface, in multiple reaction monitoring (MRM), and positive ion mode. The resolution of both quadrupoles was maintained at unit mass with a peak width at half height of 0.7amu. Data analysis was performed using the Waters Quanlynx software. Serum or plasma samples were thawed at room temperature and a 100μL aliquot was placed in a tube. Then 300μL of precipitating reagent (acetonitrile-methanol [50:50, volume: volume]) containing the internal standard (0.12ng/μL Imipramine-D3) was added to each tube. The samples were vortexed and centrifuged. The supernatant was transferred to an autosampler vial and 8μL was injected into the UPLC-MS/MS. Utilizing a Waters Acquity UPLC HSS T3 1.8μm, 2.1×50mm column at 25ºC, the analytes were separated using a timed, linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column is eluted into the LC-MS/MS to detect imipramine D3 at transition 284.25>89.10, haloperidol at 376.18>165.06, thiothixene at 444.27>139.24, fluphenazine at 438.27>171.11, and perphenazine at 404.19>143.07. Secondary transitions for each analyte are also monitored for imipramine D3 at 284.25>193.10, haloperidol at 376.18>122.97, thiothixene at 444.27>97.93, fluphenazine at 438.27>143.08, and perphenazine at 404.19>171.11. The run-time is 1.8min per injection with baseline resolved chromatographic separation. RESULTS The analytical measurement range was 0.2 to 12.0ng/mL for fluphenazine and perphenazine, and was 1 to 60.0ng/mL for haloperidol and thiothixene. Intra-assay and inter-assay imprecisions (CV) were less than 15% at two concentrations for each analyte. CONCLUSIONS By utilizing a LC-MS/MS method we combined two previously established analytical assays into one, yielding a 75% time-savings on set-up, and a significantly shortened analytical run-time. These changes reduced the turn-around time for analysis and eliminated interference issues resulting in fewer injections and increased column lifetime.

Performance characteristics and patient comparison of the ARK Diagnostics levetiracetam immunoassay with an ultra-high performance liquid chromatography with tandem mass spectrometry detection method

Levetiracetam (Keppra®) is an anticonvulsant drug approved in 1999 for use in the U.S. Levetiracetam is chemically unrelated to the existing anticonvulsant drugs [1]. It is rapidly absorbed after oral administration, with peak plasma concentrations at 1–1.5 h. It is not extensively metabolized, producing only 1 non-reactive carboxylic metabolite, is not protein bound, and no drug–drug interactions have been described, most likely due to its lack of hepatic metabolism [1,2–4]. Levetiracetam has a relatively short half-life of 6–8 h, and is primarily eliminated in urine, thus, dosing may be adjusted based on renal function. Levetiracetam has been approved as adjunctive therapy for partial-onset seizures in adults but may have a broader spectrum of efficacy. A proposed therapeutic range for seizure control is 6–20 mg/l [5]. Other indications for which levetiracetam is being considered include refractory epilepsy [5], monoclonus [7–9], partial onset seizures [10], benzodiazepine withdrawal [6], mood disorders [11], neuropathic pain [12] and seizures in critically ill patients [13]. Most therapeutic drug monitoring for levetiracetam is performed to evaluate compliance, but recent studies show that it is useful in optimizing therapy in renal suppressed patients [14] and during pregnancy [15]. Levetiracetam should be monitored in serum or plasma, or saliva [16–17]. Levetiracetam can undergo in situ metabolism in whole blood [18]. A number of laboratorymethods have been described formeasuring levetiracetam using chromatographic methods, including both highperformance liquid chromatography (HPLC) [19,20], and gas chromatography [21] methodologies, using a variety of detection systems. However, due to limited availability of these technologies in clinical laboratories, therapeutic monitoring of levetiracetam has not been widely utilized. The availability of a commercial immunoassay to support therapeutic monitoring of levetiracetam, would theoretically enable testing in any laboratory with a random-access instrument. This study evaluated the levetiracetam immunoassay available through ARK Diagnostics, and compared results to an established UPLC–MS/MS assay. The levetiracetam assay on UPLC–MS/MS was performed as previously described [22]. Briefly, each specimen (20 μl) was deproteinated by the addition of internal standard with 50:50 methanol:acetonitrile. Then 1 μl of supernatant was injected onto an Acquity UPLC HSS T3 1.8 μm, 2.1×50 mm (Waters Corp., Milford, MA) column. Elution occurred using a linear gradient of acetonitrile and water, each having 0.1% formic acid added. The column was eluted into a Waters Acquity UPLC TQD, operating in a positive mode to detect levetiracetam at m/z 171.11>126, and internal standard (3-amino-2-naphtholic acid) at m/z 188.06>170. Secondary transitions for each analyte alsomonitored levetiracetam at m/z 171.11>154, and internal standard at m/z

An automated method for supporting busulfan therapeutic drug monitoring.

Introduction: Busulfan is a chemotherapeutic agent commonly used for myeloablative conditioning regimens such as in the treatment of chronic myelogenous leukemia. Busulfan dosing is complex due to wide interpatient variability in pharmacokinetics and a narrow therapeutic range. Although busulfan dose is normalized to body weight, therapeutic drug monitoring (TDM) using area under the plasma concentration curve is recommended after the first dose. A high busulfan area under the plasma concentration curve (>1500 μM·min) is associated with an increased risk for sinusoidal obstruction syndrome, and a suboptimal area under the plasma concentration curve (<900 μM·min) is associated with an increased risk for graft rejection or disease relapse. TDM of busulfan is not widely available due to the lack of commercially available and rapid methods to determine the area under the plasma concentration curve. Methods: The purpose of this study was to evaluate the Roche cobas c 111 instrument, a photometric automated chemistry analyzer, using the Busulfan PCM assay from Saladax Biomedical Inc. The assay using this instrument was compared with an enzyme-linked immunosorbent assay (ELISA) from Saladax Biomedical Inc and the Olympus AU400e. Linearity and accuracy were evaluated between 175 and 1750 ng/mL. Imprecision was determined by analyzing 5 concentrations of standards twice a day for 20 days. Results: Linearity for the Roche method had a slope and y-intercept of 1.050 and −5.5, respectively, and percent recovery ranged between 95% and 105%. Correlation between the Roche and ELISA platforms was analyzed by linear regression on 26 frozen patient samples. The results from the comparison of the methods based on the Roche and ELISA platforms were as follows: coefficient of determination (R2) was 0.9684, with a slope and y-intercept of 0.752 and 108.41, respectively. Correlation between the Roche and Olympus instruments was analyzed by linear regression and Bland-Altman plots. The coefficient of determination (R2) was 0.9942, with a slope and y-intercept of 1.035 and −41.3326, respectively. Conclusions: Availability of TDM of busulfan can be improved by the use of commercially available reagents and automated platforms.