The validation of an LC-MS/MS assay for perhexiline and major hydroxy metabolites, and its application to therapeutic monitoring in patient plasma.

AIM: Perhexiline (PEX), being developed to treat hypertrophic cardiomyopathy, is toxic at levels above the therapeutic range. Plasma level monitoring is therefore essential. The absence of a UV-absorbing chromophore has in the past required quantitative analysis of PEX in plasma using lengthy derivatization methods, followed by HPLC and fluorescence detection. The routine and urgent analysis of a large number of patient plasma samples necessitates faster and reliable analytical methodology. RESULTS: An LC-MS/MS method, using two novel internal standards, has been validated for the quantitative measurement of PEX and its major hydroxy metabolites in human plasma. CONCLUSION: The assay has been applied to therapeutic drug monitoring (TDM), where PEX and the ratio of the drug to cis-hydroxy perhexiline, were measured at designated intervals.

Use of 'dilute-and-shoot' liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma.

This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.

Validation of a High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Perhexiline and Cis-Hydroxy-Perhexiline Plasma Concentrations.

BACKGROUND: The polymorphic nature of cytochrome P450 2D6 has made therapeutic drug monitoring of the anti-anginal agent perhexiline a compulsory step in reducing adverse events associated with plasma concentrations above the therapeutic range (0.15-0.60 mg/L). The aim of this study was to develop a high-performance liquid chromatography-mass spectrometry/mass spectrometry method for the determination of plasma perhexiline concentrations and its major metabolite cis-hydroxy-perhexiline to reduce sample extraction procedures and improve sample turnaround times. METHODS: The method was validated by determining the precision and accuracy of calibrators and quality control material, comparing quality assurance program samples and patient samples measured by a previously reported liquid-liquid extraction fluorescence (FL) detection high-performance liquid chromatography method and performing matrix effects investigations. RESULTS: Replicates of calibrators at concentrations of 3.00 and 0.05 mg/L demonstrated imprecision of <10.8% and inaccuracy of <8.2% for perhexiline and <10.1% and <4.5% for cis-hydroxy-perhexiline, respectively. All samples measured by the 2 methods (n = 102) demonstrated Deming regression of perhexiline = 1.20 FL + 0.00 (Sy.x = 0.08, 1/slope = 0.67); cis-hydroxy-perhexiline = 1.48 FL - 0.20 (Sy.x = 0.40, 1/slope = 0.67). CONCLUSIONS: The assay performance was deemed acceptable and integrated into the routine therapeutic drug monitoring program of the department.

Metabolic manipulation in chronic heart failure: study protocol for a randomised controlled trial.

BACKGROUND: Heart failure is a major cause of morbidity and mortality in society. Current medical therapy centres on neurohormonal modulation with angiotensin converting enzyme inhibitors and ß-blockers. There is growing evidence for the use of metabolic manipulating agents as adjunctive therapy in patients with heart failure. We aim to determine the effect of perhexiline on cardiac energetics and alterations in substrate utilisation in patients with non-ischaemic dilated cardiomyopathy. METHODS: A multi-centre, prospective, randomised double-blind, placebo-controlled trial of 50 subjects with non-ischaemic dilated cardiomyopathy recruited from University Hospital Birmingham NHS Foundation Trust and Cardiff and Vale NHS Trust. Baseline investigations include magnetic resonance spectroscopy to assess cardiac energetic status, echocardiography to assess left ventricular function and assessment of symptomatic status. Subjects are then randomised to receive 200 mg perhexiline maleate or placebo daily for 4 weeks with serum drug level monitoring. All baseline investigations will be repeated at the end of the treatment period. A subgroup of patients will undergo invasive investigations with right and left heart catheterisation to calculate respiratory quotient, and mechanical efficiency. The primary endpoint is an improvement in the phosphocreatine to adenosine triphosphate ratio at 4 weeks. Secondary end points are: i) respiratory quotient; ii) mechanical efficiency; iii) change in left ventricular (LV) function. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00841139 ISRCTN: ISRCTN72887836.

Effects of aging, renal dysfunction, left ventricular systolic impairment, and weight on steady state pharmacokinetics of perhexiline.

MATERIALS AND METHODS: Two hundred patients at steady-state on long-term perhexiline were identified retrospectively. The ratio of maintenance dose to steady-state plasma concentration (dose:[Px]) was correlated with the following putative determinants via simple and multiple linear regression analyses: age, weight, left ventricular ejection fraction (LVEF), and creatinine clearance (CrCl, Cockroft-Gault formula). A Mann-Whitney U test was performed to determine if severe left ventricular systolic impairment affected maintenance dose. RESULTS: Advanced age, left ventricular systolic impairment, and renal impairment were frequently encountered. Using simple linear regression, age was a negative correlate of dose:[P] (R = 0.23, P = 0.001), whereas weight (R = 0.27, P = 0.0001) and CrCl (R = 0.30, P < 0.0001) were positive correlates. Mann-Whitney U analysis showed no difference between dose: [Px] among patients with LVEF of less than 30% versus 30% or greater. Advancing age was strongly associated with decreasing weight (R = -0.45, P < 0.00001) and calculated CrCl varied directly with weight, as expected (R = 0.66, P < 0.0001). Stepwise multiple linear regression using age, LVEF, CrCl, and weight as potential predictors of dose:[P] yielded only weight as a significant determinant. DISCUSSION: Perhexiline has become a "last-line" agent for refractory angina as a result of complex pharmacokinetics and potential toxicity. Use has increased predictably in the aged and infirm who have exhausted standard medical and surgical therapeutic options. Beyond genotype, the effect of patient characteristics on maintenance dose has not been explored in detail. In this study, dose requirement declined with age in a frail and wasting population as a result of weight-related pharmacokinetic factors. LVEF had no apparent effect on maintenance dose and should not be considered a contraindication to use. CONCLUSION: A weight-adjusted starting dose may facilitate the safe and effective prescription of perhexiline and is calculated by 50 + 2 × weight (kg) mg/d, rounded to the closest 50 mg/day.

Determination of perhexiline and its metabolite hydroxyperhexiline in human plasma by liquid chromatography/tandem mass spectrometry.

Perhexiline is a drug that is used for treatment of moderate to severe angina pectoris that has not responded to other treatment. It has a low therapeutic index, and saturable metabolism that is also subject to genetic polymorphism (CYP2D6). Concentration monitoring of the parent drug and its major metabolite is considered necessary to optimise efficacy and reduce the risk of hepatotoxicity and neuropathy. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of perhexiline and its metabolite cis-hydroxyperhexiline in human plasma. After proteins were precipitated with acetonitrile, perhexiline, the major metabolite cis-hydroxyperhexiline and nordoxepin as the internal standard were resolved on a phenyl-hexyl column using gradient elution of 0.05% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 10-2000microg/L (r>0.999), bias was

Steady-state pharmacokinetics of the enantiomers of perhexiline in CYP2D6 poor and extensive metabolizers administered Rac-perhexiline.

UNLABELLED: What is already known about this subject. Perhexiline (PHX) is administered as a racemic mixture and exhibits enantioselective pharmacokinetics in both poor and extensive metabolizers of CYP2D6 (PM and EM, respectively). Extensive metabolism by CYP2D6 is primarily responsible for the observed enantioselectivity in EM, but the process responsible in PM is unknown. Analysis of the steady-state plasma concentration-time profiles of the enantiomers of PHX in PM and EM was undertaken in order to elucidate the observed enantioselectivity, particularly with respect to PM. What this study adds. This is the first study to examine the steady-state plasma concentration-time profiles of the enantiomers of PHX in EM and PM over the course of an interdosing interval. The apparent oral clearance of each enantiomer was calculated from their respective AUC rather than from trough concentrations and was enantioselective in both phenotypes, with higher apparent oral clearances of (-)-than (+)-PHX. Renal clearance, calculated for EM and subsequently assumed for PM, constitutes a greater proportion of the total apparent oral clearance of each enantiomer in PM than EM, but was not enantioselective and thus unable to explain the enantioselectivity observed in PM. AIMS: To determine the steady-state pharmacokinetics of perhexiline (PHX) enantiomers over one interdosing interval in CYP2D6 extensive and poor metabolizer (EM and PM, respectively) patients administered rac-PHX. To elucidate the processes responsible for enantioselectivity, particularly in PM patients. METHODS: Blood samples were taken over one interdosing interval from six EM and two PM patients at steady-state with respect to rac-PHX metabolism. Complete urine collections were taken from five EM patients. PHX concentrations in plasma and urine were determined with enantioselective high-performance liquid chromatography methods. RESULTS: EM patients had 16- and 10-fold greater median apparent oral clearances of (+)- and (-)-PHX, respectively, than PM patients (P < 0.05 for both) and required significantly larger doses of rac-PHX (69 vs. 4.2 microg kg(-1) h(-1), P < 0.05) to maintain therapeutic concentrations in plasma. Patient phenotypes were consistent with CYP2D6 genotypes. Both groups displayed enantioselective pharmacokinetics, with higher apparent oral clearances for (-)-PHX compared with (+)-PHX, although PM patients exhibited significantly greater enantioselectivity (P < 0.05). The renal clearance of PHX enantiomers was not enantioselective and accounted for <1% of the median apparent oral clearance of each enantiomer in EM patients. Assuming the same renal clearances for PM patients accounts for approximately 9 and 4% of their median apparent oral clearances of (+)- and (-)-PHX, respectively. CONCLUSIONS: The enantioselective pharmacokinetics of PHX are primarily due to metabolism by CYP2D6 in EM patients. The mechanism responsible for the enantioselective pharmacokinetics of PHX in PM patients is unknown, but may be due to enantioselective biliary or intestinal excretion.

The influence of CYP2D6 genotype on trough plasma perhexiline and cis-OH-perhexiline concentrations following a standard loading regimen in patients with myocardial ischaemia.

AIMS: CYP2D6 protein expression is determined by the number of functional CYP2D6 alleles. It is also higher in individuals with at least one CYP2D6*2 allele. This study has investigated the effect of the number of functional CYP2D6 alleles and the influence of CYP2D6*2 alleles on plasma perhexiline concentrations in patients administered a standard loading regimen over 3 days. METHODS: Eighteen patients with myocardial ischaemia who were not taking any drugs known to inhibit CYP2D6 metabolism in vivo commenced treatment with 200 mg of perhexiline twice per day. On the fourth day, blood was drawn for genotyping and the measurement of trough plasma concentrations of perhexiline and its major metabolite, cis-OH-perhexiline. RESULTS: The only genotypic CYP2D6 poor metabolizer had a trough plasma perhexiline concentration of 2.70 mg l-1 and no detectable cis-OH-perhexiline. The mean+/-SD trough plasma perhexiline concentration in patients with one functional allele was significantly higher (0.63+/-0.31 mg l-1, n=8, P=0.05) than in patients with two functional alleles (0.37+/-0.17 mg l-1, n=9). Conversely, the mean metabolic ratio was significantly lower in patients with one functional allele (2.90+/-1.76, P<0.01) compared with patients with two functional alleles (6.52+/-3.26). Patients with at least one CYP2D6*2 allele had a lower plasma perhexiline concentration (0.20+/-0.09 mg l-1, n=5, P<0.001) and a higher metabolic ratio (7.86+/-2.51, P<0.01) than the non-poor metabolizer patients with no CYP2D6*2 alleles (0.62+/-0.23 mg l-1 and 3.55+/-2.54, respectively, n=12). CONCLUSION: Patients with only one functional allele and not CYP2D6*2 have diminished CYP2D6 metabolic capacity for perhexiline.

Enantioselective assay for the determination of perhexiline enantiomers in human plasma by liquid chromatography.

Effective use of the antianginal agent perhexiline is difficult because saturable metabolism by the polymorphic cytochrome P450 2D6 (CYP2D6) isoform produces elevated plasma perhexiline concentrations that have been associated with serious hepatic and neurological toxicity. Perhexiline is marketed for therapeutic use as a racemate and there is evidence for differences in the disposition of its enantiomers. The current study describes an enantioselective HPLC-fluorescent method utilising pre-column derivatization with (R)-(-)-1-(1-napthyl)ethyl isocyanate. Following derivatization, the enantiomers are resolved on a C18 column with gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of (+)- and (-)-perhexiline in human plasma following clinical doses and demonstrates sufficient sensitivity, accuracy and precision between 0.01 and 2.00 mg/l for each enantiomer, with intra-assay coefficients of variation and bias <20% at 0.01 mg/l and <10% at 2.00 mg/l, and inter-assay coefficients of variation and biases <15% at 0.03 mg/l and <10% at 0.40 and 0.75 mg/l. The application of this method to plasma samples collected from a patient treated with perhexiline revealed that (+)-perhexiline concentrations were higher than (-)-perhexiline concentrations, confirming the stereoselective disposition of perhexiline. The current study describes an enantioselective method that utilises pre-column formation of fluorescent diastereomers that are resolved on a C18 HPLC column using a gradient of methanol and water.

Development of a regimen for rapid initiation of perhexiline therapy in acute coronary syndromes.

Perhexiline is a prophylactic anti-anginal agent that ameliorates the metabolic basis for myocardial ischaemia and is increasingly used in the management of acute coronary syndromes. No intravenous preparation is available and usual oral loading regimens require 2-3 days to achieve therapeutic drug levels. Two patients presenting to hospital with single-dose over-dosage of perhexiline (6500 mg and 1000 mg, respectively) provided a basis for examining the safety of large single dosage of perhexiline and associated time-course of drug levels. Neither patient had previously taken perhexiline. Peak plasma perhexiline concentrations occurred within 12 h of ingestion and were 2.58 and 0.50 mg/L, respectively (therapeutic range 0.15-0.60 mg/L). The first patient developed transient nausea and vomiting; the second patient had no adverse effects. Subsequently, a series of 10 patients with acute coronary syndromes received an 800-mg loading dose. Peak concentrations occurred within 12 h postdose; the mean levels achieved were 0.40 +/- 0.16 mg/L (standard error of the mean). No serious adverse effects were seen. Two patients reported transient nausea or vomiting within 24 h of the loading dose. The utility of this rapid loading regimen for incremental suppression of myocardial ischaemia remains to be assessed.

Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography-mass spectrometry.

A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography-mass spectrometry (LC-MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC-MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were

Pharmacokinetics of the antianginal agent perhexiline: relationship between metabolic ratio and steady-state dose.

AIMS: 1) To develop an estimate of oral clearance (CL(Px)/F) for the antianginal agent perhexiline based on the ratio of cis-OH-perhexiline metabolite/parent perhexiline plasma concentrations at steady-state (C(OHPx,ss)/C(Px,ss)). 2) To determine whether the ratio measured in the first fortnight of treatment (C(i)(OHPx)/C(i)(Px)) may be used to guide patient dosing with perhexiline, a drug with a narrow therapeutic index, long half-life and saturable metabolism via CYP2D6. METHODS: Two retrospective studies were conducted reviewing patient records and data obtained from routine monitoring of plasma perhexiline and cis-OH-perhexiline concentrations. RESULTS: Study 1 (n=70). At steady-state, the frequency distributions of CL(Px)/F and C(OHPx,ss)/C(Px,ss) were consistent with CYP2D6 metabolism. Putative poor metabolizers (approximately 8%) were identified by CL(Px)/F< or =50 ml min(-1) or C(OHPx,ss)/C(Px,ss)< or =0.3. A group of patients with CL(Px)/F> or =950 ml min(-1) may have been ultra-rapid metabolizers. In this group, the high CL(Px)/F values suggest extensive first-pass metabolism and poor bioavailability. In patients with therapeutic plasma perhexiline concentrations (0.15-0.60 mg l(-1)), the variability in dose appeared directly proportional to CL(Px)/F (r2=0.741, P<0.0001). Study 2 (n=23). Using C(i)(OHPx)/C(i)(Px) patients were tentatively identified as poor, extensive and ultra-rapid metabolizers, with CL(Px)/F of 23-72, 134-868 and 947-1462 ml min(-1), respectively, requiring doses of 10-25, 100-250 and 300-500 mg day(-1), respectively. CONCLUSIONS: The cis-OH-perhexiline/perhexiline concentration ratio may be useful for optimizing individual patient treatment with the antianginal agent perhexiline.

Assessment of interlaboratory performance in the provision of perhexiline therapeutic drug monitoring services in Australia.

Perhexiline is a prophylactic antianginal agent particularly useful in patients whose angina is poorly controlled or refractory to conventional drug regimens. Although perhexiline can cause serious hepatic and neurological toxicity, maintaining trough plasma concentrations between 0.15-0.60 mg/L minimizes the risk of toxicity while providing relief of angina symptoms in a majority of patients. All pathology laboratories are required to participate in interlaboratory proficiency testing (PT) programs. The authors therefore initiated a monthly PT program to assess the performance of Australian laboratories measuring perhexiline (n = 8). PT specimens included perhexiline-spiked drug-free human plasma and pooled plasma from patients administered perhexiline. The performance of 8 Australian laboratories participating in the program was examined over a 30-month period. The mean relative standard deviation of the group was 18.2%. All centers performed well with respect to accuracy, achieving mean percentage bias within +/-8% of target perhexiline concentrations. The usefulness of the PT program was highlighted by the identification of two laboratories with an unacceptable degree of variability (up to 30% of results varied more than +/-55% from the target concentration), and the identification of potential analytical problems with the use of perhexiline metabolite concentrations for determining patients' hydroxylator status. Continued and improved use of PT by pathology laboratories is essential to ensuring the safe and effective clinical use of perhexiline.

Method for the analysis of perhexiline and its hydroxy metabolite in plasma using high-performance liquid chromatography with precolumn derivatization.

A high-performance liquid chromatographic method for the analysis of perhexiline and its monohydroxy metabolite in plasma has been developed. After a simple extraction procedure, the analytes are derivatized over a 30-min period with trans-4-nitrocinnamoyl chloride. The derivatized products are monitored at 340 nm following separation on a 5-micron phenyl reversed-phase column under isocratic conditions. The limits of detection for perhexiline and its hydroxy metabolite are 0.03 and 0.02 mg/l, respectively. The between-day and within-day assay coefficients of variation for perhexiline and its hydroxy metabolite at concentrations of 0.2 and 1.0 mg/I were less than 10%. The method has proved robust and suitable for the routine monitoring of perhexiline and hydroxyperhexiline.

Relationship between plasma perhexiline concentration and symptomatic status during short-term perhexiline therapy.

We tested the hypothesis that resolution versus persistence of symptomatic ischaemia and/or development of nausea/dizziness on the third day of loading with perhexiline maleate (PM), is correlated with perhexiline plasma concentrations after the standard loading phase in patients with acute coronary syndromes. Forty consecutive patients with either unstable angina pectoris or non-Q-wave myocardial infarction with persistent angina pectoris, despite maximal pharmacological therapy (other than PM), were studied. All patients received PM 400 mg/day for 3 days and 200 mg/day thereafter. On days 2 and 3 observers blinded to the 72-96 h plasma perhexiline concentration assessed the patient regarding episodes of angina and/or nausea/dizziness. On the third day of loading with PM, 12 patients experienced angina and 11 patients had nausea and/or dizziness. Plasma perhexiline concentrations at 72-96 h varied widely: mean 0.46 +/- 0.26 (range 0.11-1.77) microgram/ml. There was a relationship of borderline statistical significance between resolution of anginal symptoms and plasma perhexiline concentration > 0.15 microgram/ml (p = 0.055). There was a close relationship between emergence of nausea/dizziness with plasma perhexiline concentration > 0.06 microgram/ml (p < 0.01). We conclude that this study (a) suggests that PM exerts incremental antianginal effects over those of other antiischaemic agents in patients with acute coronary syndromes and (b) establishes that the development of nausea and/or dizziness in such patients is strongly predictive of accumulation of perhexiline beyond the therapeutic range of the drug.

Efficacy and safety of perhexiline maleate in refractory angina. A double-blind placebo-controlled clinical trial of a novel antianginal agent.

Despite large gains in the medical and surgical treatment of angina pectoris in the past two decades, many patients are refractory to conventional medical therapy and are unsuitable for a first or, more commonly, repeat coronary revascularization procedure. We evaluated the efficacy of perhexiline maleate, a drug with an antianginal mechanism of action in humans that is as yet unknown, by using a randomized double-blind placebo-controlled crossover design in 17 patients with refractory angina who continued to receive maximal antianginal therapy, typically including nitrates, a beta-blocker, and a calcium channel antagonist. In view of perhexiline's potential for hepatic and neurological toxicity, plasma drug levels were monitored and maintained in the 150-600 ng/ml range. Sixty-three percent of patients were judged perhexiline responders by objective exercise testing criteria, as compared with 18% of patients on placebo (p less than 0.05). By blinded review of subjective measures of anginal frequency and severity, 65% of patients noted an improvement while on perhexiline, whereas no patient identified the placebo phase with improvement. Side effects observed in 29% of patients were minor and related to transient elevations of blood levels of more than 600 ng/ml; no patient suffered hemodynamic or cardiac conduction abnormalities attributable to perhexiline. With attention to the pharmacokinetics of perhexiline's elimination in individual patients, this novel antianginal agent seems to be safe and effective and deserves further evaluation in patients already receiving maximal antianginal therapy who are not candidates for revascularization procedures.

Simultaneous determination of perhexiline and its monohydroxy metabolites in biological fluids by gas chromatography-electron-capture detection.

A rapid and sensitive method for the simultaneous determination of perhexiline and its cis-4-axial and trans-4-equatorial monohydroxy metabolites (M1 and M3, respectively) in human plasma, urine and bile is described. The assay utilises a single diethyl ether extraction, heptafluorobutyric acid anhydride derivatisation and separation and detection by gas chromatography-electron-capture detection. The limits of detection are 0.1 microgram/ml for perhexiline and 0.025 microgram/ml for the M1 and M3 metabolites. This method has been used in a five-day kinetic study of three healthy adult males who ingested a single 300-mg dose of perhexiline maleate. One of these volunteer subjects exhibited elevated plasma perhexiline and markedly reduced plasma and urinary M1 concentrations together with profoundly prolonged plasma and urinary M1 elimination times when compared with the other two subjects. These differences are thought to be of genetic origin. There were also obvious differences in urinary M3 concentrations which were discussed.