Grant A. Moore

Rapid and simple high-performance liquid chromatographic assay for the determination of metformin in human plasma and breast milk.

A rapid and simple high-performance liquid chromatographic (HPLC) assay for the determination of metformin in human plasma and breast milk is described. After proteins were precipitated with acetonitrile, metformin and the internal standard buformin were resolved on a cation-exchange column and detected by UV detection at 236 nm. Standard curves were linear over the concentration range 20.0-4000 microg/l. Intra- and inter-day coefficients of variation were <9.0% and the limit of quantification was around 20 microg/l.

Determination of celecoxib in human plasma and breast milk by high-performance liquid chromatographic assay.

A rapid and simple HPLC assay was developed for the determination of celecoxib in human plasma and breast milk. After proteins were precipitated with acetonitrile, celecoxib was resolved on a C18 column and detected by UV detection at 254 nm. Standard curves were linear over the concentration range 10-2000 microg/L (r(2)>0.99). Bias was </=+/-15% from 20 to 2000 microg/L in both matrices, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 10 microg/L.

Dissociation of neopterin and 7,8-dihydroneopterin from plasma components before HPLC analysis

Measurement of plasma neopterin by HPLC with fluorescence detection is used clinically as a marker of immune cell activation in the management of a number of disease pathologies. HPLC analysis of neopterin requires the acidic removal of plasma proteins but we have found that 7,8-dihydroneopterin is oxidised to neopterin with varying yield. Using acetonitrile as the precipitant, we have measured substantially higher quantities of both total neopterin (7,8-dihydroneopterin and neopterin) and neopterin from plasma of healthy and septicemia patients. Total neopterin concentrations were on average 50% and 200% greater in healthy and septicemia subjects, respectively, when measured after acetonitrile precipitation compared to trichloroacetic acid. Our data suggests that some pterin co-precipitates with proteins during acid treatment.

Determination of dexamethasone and dexamethasone sodium phosphate in human plasma and cochlear perilymph by liquid chromatography/tandem mass spectrometry.

A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of dexamethasone (Dex) and dexamethasone sodium phosphate (Dex SP) in plasma and human cochlear perilymph. After proteins were precipitated with a mixture of acetonitrile and methanol, Dex, Dex SP and flumethasone, the internal standard, were resolved on a C18 column using gradient elution of 5 mM ammonium acetate and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.5-500 μg/L (r>0.99), bias was <±10%, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.5 μg/L for both Dex and Dex SP. The assay has been used successfully in a clinical pharmacokinetics study of Dex and Dex SP in cochlear perilymph and plasma.

A Simple High-Performance Liquid Chromatography Method for Simultaneous Determination of Three Triazole Antifungals in Human Plasma

ABSTRACT A rapid and simple high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of three triazole antifungals (voriconazole, posaconazole, and itraconazole and the metabolite of itraconazole, hydroxyitraconazole) in human plasma. Sample preparation involved a simple one-step protein precipitation with 1.0 M perchloric acid and methanol. After centrifugation, the supernatant was injected directly into the HPLC system. Voriconazole, posaconazole, itraconazole, its metabolite hydroxyitraconazole, and the internal standard naproxen were resolved on a C6-phenyl column using gradient elution of 0.01 M phosphate buffer, pH 3.5, and acetonitrile and detected with UV detection at 262 nm. Standard curves were linear over the concentration range of 0.05 to 10 mg/liter (r2 > 0.99). Bias was <8.0% from 0.05 to 10 mg/liter, intra- and interday coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/liter.

Case series: toxicity from 25B-NBOMe – a cluster of N-bomb cases

Abstract Background A new class of hallucinogens called NBOMes has emerged. This class includes analogues 25I-NBOMe, 25C-NBOMe and 25B-NBOMe. Case reports and judicial seizures indicate that 25I-NBOMe and 25C-NBOMe are more prevalently abused. There have been a few confirmed reports of 25B-NBOMe use or toxicity. Report Observational case series. This report describes a series of 10 patients who suffered adverse effects from 25B-NBOMe. Hallucinations and violent agitation predominate along with serotonergic/stimulant signs such as mydriasis, tachycardia, hypertension and hyperthermia. The majority (7/10) required sedation with benzodiazepines. Analytical method 25B-NBOMe concentrations in plasma and urine were quantified in all patients using a validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) method. Peak plasma levels were measured between 0.7–10.1 ng/ml. Discussion The NBOMes are desired by users because of their hallucinogenic and stimulant effects. They are often sold as LSD or synthetic LSD. Reported cases of 25B- NBOMe toxicity are reviewed and compared to our series. Seizures and one pharmacological death have been described but neither were observed in our series. Based on our experience with cases of mild to moderate toxicity, we suggest that management should be supportive and focused on preventing further (self) harm. High doses of benzodiazepines may be required to control agitation. Patients who develop significant hyperthermia need to be actively managed. Conclusions Effects from 25B-NBOMe in our series were similar to previous individual case reports. The clinical features were also similar to effects from other analogues in the class (25I-NBOMe, 25C-NBOMe). Violent agitation frequently present along with signs of serotonergic stimulation. Hyperthermia, rhabdomyolysis and kidney injury were also observed.

Determination of Vancomycin in Human Plasma, Bone and Fat by Liquid Chromatography/Tandem Mass Spectrometry

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of vancomycin in human plasma, bone and fat tissue. For vancomycin in plasma, sample was treated with methanol to precipitate the proteins. After centrifugation, the supernatant was diluted with water, and then injected into the LC-MS/MS system. For vancomycin in bone and fat, the pulverized bone/fat samples were immersed in phosphate buffered saline pH 7.3 at 4°C overnight. After centrifugation, the supernatant of bone/fat tissue suspension was treated in the same way as the plasma samples. Vancomycin and aminopterin, the internal standard, were resolved on a C18(2) column using gradient elution of 0.05% formic acid and methanol. The two compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 0.05 to 50 mg/L (r2>0.99) in plasma, bone and fat. Bias was ≤ ± 10% from 0.05 to 50 mg/L, intra- and inter-day coefficients of variation (imprecision) were <10%, and the limit of quantification was 0.05 mg/L. The assay has been used successfully in a clinical study to investigate the regional delivery of vancomycin in bone and fat after prophylactic administration of vancomycin through an intraosseous route or systemic route, during total knee arthroplasty.

Thirteen years' experience of pharmacokinetic monitoring and dosing of busulfan: can the strategy be improved?

Background: A busulfan concentration monitoring and dosing service has been provided by Christchurch Hospital since 1998. This study aimed to see (1) the percentage of patients with an area under the concentration time curve (AUC) outside the target range and had dose adjustment, (2) how busulfan clearance (CL) relates to body weight, and (3) if fewer samples could be used to predict doses. Methods: Blood samples were taken from patients after oral administration, usually at 0.5, 1, 1.5, and 6 hours, and after the start of a 2-hour intravenous (IV) infusion of busulfan, at 1, 2, 2.5, 3, 6, and 8 hours. Dose adjustment was made based on the AUC compared with the target range. The relationship of CL and body weight for the IV group was used to develop a revised IV dosing schedule. The bias and imprecision of AUCs estimated using fewer sampling points were examined to see if sampling could be economized. Results: Data were available for 150 patients but for 6 patients, data were incomplete and excluded. Of the remaining 144 patients (256 sample sets, 209 oral, 47 IV, 62% with repeats), 38% (IV) and 35% (oral) of patients had AUCs within the target range after the first dose. Dose adjustment was made in 47% and 34% of patients dosed IV and orally, respectively, after which there was a trend to more patients achieving the target AUC. A nonlinear relationship was found between CL and body weight. The initial IV dosing schedule was revised to take this into account. Sampling for busulfan concentration measurement at 3 points (2.5, 4, 8 hours) or 2 points (2.5, 8 hours) after the start of the infusion enabled accurate and precise estimates of AUC0–24. Conclusions: Around two thirds of patients treated with busulfan were outside the target AUC range after the first dose. Dose adjustment was made in 37% of patients. The relationship between CL and body weight was used to revise the initial IV dosing schedule. Sampling for AUC estimation could be reduced to 2 time points after IV dosing.

Determination of methylphenidate and its metabolite ritalinic acid in urine by liquid chromatography/tandem mass spectrometry

Methylphenidate (MPH) is a drug that is licensed for treatment of ADHD and also narcolepsy. Monitoring of the parent drug and its major metabolite ritalinic acid (RA) in urine is considered necessary to ensure compliance with treatment programmes. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of MPH and its metabolite RA in human urine. After urine was diluted with water, methylphenidate, the major metabolite ritalinic acid, and d₆-amphetamine as the internal standard were resolved on a PFP propyl column using gradient elution of 0.02% ammonium formate and acetonitrile. The total analysis time was 13.5 min. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 5-5000 μg/L (r>0.997), bias was ≤ ±20%, intra- and inter-day coefficients of variation (imprecision) were <8% and the limit of detection was 5 μg/L. The limit of quantitation was set at 100 μg/L. Matrix effects were up to 140% but these were accounted for by the internal standard. The assay is being used successfully in clinical practice to enhance the safe and effective use of methylphenidate.

Lack of Clinically Significant Interference by Spironolactone With the Axsym Digoxin Ii Assay

Spironolactone and its metabolite canrenone have previously been reported to negatively influence the in vitro determination of serum digoxin concentration using the AxSYM® Digoxin II assay. This interference reportedly led to administration of a toxic dose of digoxin to a patient. We conducted a study to review the performance of the AxSYM® Digoxin II assay in 20 patients taking digoxin and spironolactone. We did not find underestimation of serum digoxin at a clinically significant level. Spironolactone and its derivatives, including canrenone, have been shown to cross-react with some digoxin assays because of structural similarities. Overestimation of digoxin has been reported as a result of metabolites, including 7-alpha-thiomethylspironolactone, crossreacting with radioimmunoassay methods (1). This interference is concentration dependent. Subsequently, renal and hepatic dysfunction have been found to falsely elevate serum digoxin estimations in most available radioimmunoassays, an effect accentuated by the presence of spironolactone (2). These interactions are thought to be less of a problem with the newer generation assays, such as the AxSYM® Digoxin II microparticle enzyme immunoassay (Abbott Park, IL), which are now commonly used. We elected to perform an evaluation of the AxSYM® digoxin assay in response to a report that high doses of intravenous canrenone inhibited this assay, resulting in administration of a toxic dose of digoxin to a patient (3). It was also reported that oral spironolactone negatively influenced the AxSYM® MEIA II assay, resulting in a 13–23% decrease in recovery as compared with the Emit® method, when digoxin was added to the serum of patients receiving 100 mg spironolactone daily (3). We wanted to determine whether this potential interference was clinically relevant for those patients prescribed digoxin and low-dose spironolactone, as advocated by the Randomised Aldactone Evaluation Study (RALES), which demonstrated that spironolactone, 25 mg/d, benefits patients with severe heart failure (4). We compared the AxSYM® Digoxin II assay with another enzyme immunoassay, the Emit®2000 (Syva Company, Syva® diagnostic products). The Emit® assay has not previously been shown to be negatively influenced by spironolactone (3). Samples sent to the hospital laboratory for routine digoxin assay were stored at −35°C for subsequent analysis using both methods. Hospital prescription data were then used to identify 20 patients who were also taking spironolactone. A control group of 20 inpatients with similar renal function taking digoxin alone was selected. The mean age of the patients given spironolactone was 75 years (range, 48–90 years) compared with 82 years (range, 74–92 years) in the control group. The two groups were well matched for sex distribution and renal function (mean creatinine clearance, 0.55 mL/s vs. 0.57 mL/s in the treatment and control groups, respectively). Creatinine clearance was estimated using the CockcroftGault equation (5). The mean digoxin concentration estimates ± 1 standard deviation in the group Received December 28, 2001; accepted April 16, 2002. Address correspondence and reprint requests to Dr. Anne Roche, Medical Registrar, Department of Clinical Pharmacology, Christchurch Hospital, Private Bag 4710, Christchurch, New Zealand; E-mail: anne.roche@cdhb.govt.nz Therapeutic Drug Monitoring 25:112–113