Phillip E. Morgan

Plasma Clozapine, Norclozapine, and the Clozapine:Norclozapine Ratio in Relation to Prescribed Dose and Other Factors: Data From a Therapeutic Drug Monitoring Service, 1993-2007

Therapeutic drug monitoring of plasma clozapine and of its principal plasma metabolite N-desmethylclozapine (norclozapine) (predose or “trough” sample) can help in monitoring adherence, in dose adjustment, and in minimizing the risk of toxicity. To obtain data to assist in the interpretation of analytical results, the results from a clozapine therapeutic drug monitoring service, 1993-2007, have been audited. There were 104,127 samples from 26,796 patients [18,750 (70%) men aged at time of first sample (median, range) 34 (10-89) years, and 7763 (30%) female aged 38 (12-90) years]. Clozapine was not detected (plasma concentration <0.01 mg/L) in 1.5% of samples (prescribed clozapine dose up to 900 mg/d). Plasma clozapine was either below 0.35 mg/L or greater than 0.60 mg/L in 42.5% and 28.4% of samples, respectively; in 0.4% samples plasma clozapine was 2.0 mg/L or more. Although plasma clozapine was broadly related to prescribed dose, there was much variation: 1.2% of samples had plasma clozapine >1.0 mg/L at prescribed clozapine doses up to 150 mg/d (76.2% < 0.35 mg/L), whereas 23.3% of samples had plasma clozapine < 0.35 mg/L at doses of 850 mg/d and over (18.0% > 1.0 mg/L). The highest plasma clozapine and norclozapine concentrations encountered were 4.95 and 2.45 mg/L, respectively. Although the median plasma clozapine:norclozapine ratio was 1.25 at plasma clozapine concentrations < 0.35 mg/L, the median ratio was 2.08 at plasma clozapine concentrations > 1.0 mg/L. Data (median, 10th-90th percentile) for both clozapine and norclozapine by prescribed clozapine dose band are useful in assessing partial adherence. Analysis of the plasma clozapine:norclozapine ratio by clozapine concentration provides clear evidence that clozapine N-demethylation becomes saturated at higher plasma clozapine concentrations and adds urgency to the requirement for dose adjustment should smoking habit change. A clozapine:norclozapine ratio greater then 2 suggests either a nontrough sample, or that clozapine N-demethylation has become saturated.

LC-MS in analytical toxicology: some practical considerations

Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the influence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology.

LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood

Therapeutic drug monitoring (TDM) of atypical antipsychotics is common, but published methods often specify relatively complex sample preparation and analysis procedures. The aim of this work was to develop and validate a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in small (200 μL) volumes of plasma or serum for TDM purposes. The applicability of the method as developed to haemolysed whole blood and to oral fluid was also investigated. Analytes and internal standards were extracted into butyl acetate:butanol (9+1, v/v) and a portion of the extract analysed by LC-MS/MS (100 mm × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow-rate 0.5 mL/min; positive ion APCI-SRM, two transitions per analyte). Assay calibration (human plasma, oral fluid, and haemolysed whole blood calibration solutions) was performed by plotting the ratio of the peak area of the analyte to that of the appropriate internal standard. Assay validation was as per FDA guidelines. Assay calibration was linear across the concentration ranges studied. Inter- and intra-assay precision and accuracy were within 10% for all analytes in human plasma. Similar results were obtained for oral fluid and haemolysed whole blood, except that aripiprazole and dehydroaripiprazole were within 15% accuracy at low concentration (15 μg/L) in oral fluid, and olanzapine inter-assay precision could not be assessed in these matrices due to day-by-day degradation of this analyte. Recoveries varied between 16% (sulpiride) and 107% (clozapine), and were reproducible as well as comparable between human plasma, human serum, calf serum and haemolysed whole blood. For oral fluid, recoveries were reproducible, but differed slightly from those in plasma suggesting the need for calibration solutions to be prepared in this medium if oral fluid is to be analysed. LLOQs were 1-5 μg/L depending on the analyte. Neither ion suppression/enhancement, nor interference from some known metabolites of the antipsychotics studied has been encountered. The method has also been applied to the analysis of blood samples collected post-mortem after dilution (1+1, 1+3; v/v) in analyte-free calf serum.

Simple methodology for the therapeutic drug monitoring of the tyrosine kinase inhibitors dasatinib and imatinib.

A simple HPLC method has been developed to measure imatinib and N-desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC-MS/MS also allows dasatinib assay. A small sample volume (100 μL HPLC-UV, 50 μL LC-MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05-10 mg/L imatinib, 0.01-2.0 mg/L norimatinib and 1-200 µg/L dasatinib), with acceptable accuracy (86-114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02-4.96) and 0.32 (0.01-0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2-143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib.

A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.

Background: Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. Methods: Whole-blood samples (20 &mgr;L) were treated with ammonium bicarbonate (20 &mgr;L), zinc sulfate (20 &mgr;L), and internal standard solution (13C2D4-everolimus in acetonitrile, 100 &mgr;L). After centrifugation, 20 &mgr;L of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). Results: Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1–49 &mgr;g/L for sirolimus, 1–41 &mgr;g/L for everolimus), and a lower limit of quantitation of 1 &mgr;g/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%–117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. Conclusions: A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

Measurement of posaconazole, itraconazole, and hydroxyitraconazole in plasma/serum by high-performance liquid chromatography with fluorescence detection.

Background: Itraconazole and posaconazole are used in the prevention and treatment of invasive fungal infections. However, the oral bioavailability of both compounds varies widely, and dose–serum concentration relationships are poorly defined for these analytes. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of therapeutic drug monitoring. Methods: Calibrators (n = 7) and internal quality control solutions (n = 3) were prepared in pooled human serum. Sample (100 μL), internal standard solution (25 μL), Tris solution (2 mol/L; pH 10.6), and extraction solvent (methyl tert-butyl ether, 600 μL) were vortex mixed and centrifuged. The solvent layer was removed and evaporated to dryness and the residue reconstituted in water:methanol (1 + 3, 50 μL). A portion (5 μL) of the reconstituted extract was analyzed using a 3-μm Gemini C6 phenyl column with fluorescence detection (excitation 260 nm, emission 350 nm). The method was used to measure itraconazole and hydroxyitraconazole, or posaconazole, in serum samples taken 1–2 hours before the next dose, from patients forming part of a study into management and diagnostic strategies for invasive aspergillosis. Results: Response was linear over the calibration ranges. Accuracy and imprecision were 92–111.4% and 3.2–13.4% (relative standard deviation), respectively. No interferences were noted. There was a good agreement with nominal values of each analyte in an external quality assessment scheme. In patients prescribed either 400 mg/d of itraconazole (n = 46) or 600–800 mg/d of posaconazole (n = 28) only 24% and 7% of samples, respectively, had serum itraconazole or posaconazole concentrations above the target threshold suggested in published guidelines. Conclusions: A simple, sensitive high-performance liquid chromatographic method has been developed for the analysis of itraconazole, hydroxyitraconazole, and posaconazole in serum/plasma. Few of the samples measured from patients participating in the clinical study attained concentrations of the drug/metabolite in serum that have been recommended for effective antifungal therapy.

Basic drug analysis by strong cation-exchange liquid chromatography–tandem mass spectrometry: simultaneous analysis of amisulpride, and of metamfetamine and amfetamine in serum/plasma

In the HPLC of basic drugs and metabolites, good efficiency and peak shape can often be attained using strong cation-exchange packings with isocratic 100% methanol eluents containing an ionic modifier at an appropriate pH* and ionic strength. Solvent extracts can be analysed directly, and use of ammonium acetate as modifier facilitates the use of atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry, selected reaction monitoring mode. For the analysis of amisulpride and of metamfetamine/amfetamine in plasma (200 µL) after single oral doses in man, a column packed with Waters Spherisorb S5SCX (5 µm average particle size, 100 × 2.1 mm i.d.) was used with methanolic ammonium acetate (40 mmol/L, pH* 6.0, flow rate 0.5 mL/min) as eluent (35°C). Deuterated internal standards were used for each analyte. Detection was by positive-mode APCI. Responses for all analytes were linear over the calibration ranges. Intra-assay precision (RSD) was 2-18%, and inter-assay precision was 2-12%. The limit of detection was 0.5 µg/L for all analytes. No significant matrix effects or isobaric interferences were noted. The total analysis time was 7 min. Similar methodology can be applied to a wide range of basic analytes using MS/MS detection.

HPLC of basic drugs using non-aqueous ionic eluents: evaluation of a 3 μm strong cation-exchange material

HPLC columns packed with 3 microm particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5 microm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 microm column was not only more efficient for a given column length compared with 5 microm materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3 microm SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required.

Artificial Neural Network Modelling of the Retention of Acidic Analytes in Strong Anion-Exchange HPLC: Elucidation of Structure-Retention Relationships

Computational models can be used to increase understanding of physical processes within chromatographic systems, leading to more efficient method development and optimisation strategies. In ion-exchange chromatography, various models have been derived to predict retention time; however, there remains a gap in understanding regarding the elucidation of fundamental processes contributing to retention. Here, artificial neural networks have been used to model retention of simple acidic analytes by strong anion-exchange HPLC in an attempt to understand what other factors aside from simple electrostatic interactions between ionised analyte, stationary phase and counter-ion contribute to the differential elution order of such compounds. The weights assigned by each neuron to the inputs in trained networks were used to infer the influence of a number of physicochemical analyte properties to retention under various conditions. These showed that several retention mechanisms were operating simultaneously, and that the contribution of each varied as eluent ionic strength and composition were altered at constant apparent pH. Analyte pKa had most influence on retention under most conditions, but analyte volume, LogP, and steric and electronic effects were also prominent, especially in eluents containing water.

Plasma clozapine and norclozapine in patients prescribed different brands of clozapine (Clozaril, Denzapine, and Zaponex).

To investigate the bioequivalence of the three clozapine brands licensed in the United Kingdom, we compared plasma clozapine and norclozapine in therapeutic drug monitoring samples from patients switched from Clozaril to either Denzapine or Zaponex tablets. For Clozaril/Denzapine, the median prescribed clozapine dose was 450 mg/day (range, 125-850 mg/day) (n = 66) and the median time between samples was 16 weeks. The Clozaril/Zaponex comparison (n = 57) was not dose-controlled; the median Clozaril dose was 450 mg/day (range, 150-900 mg/day) and the median Zaponex dose 400 mg/day (range, 100-850 mg/day). The median time between samples was 19 weeks. There was no significant difference in mean plasma clozapine and norclozapine concentration before and after switching in either case, although some individual results showed clinically relevant concentration differences. Plasma norclozapine showed greater reproducibility between samples than clozapine. The different brands of clozapine available in the United Kingdom show bioequivalence. Nevertheless, careful monitoring of mental state, smoking habit, adherence, and of possible life-threatening adverse effects is mandatory if the drug is to be used safely.