Rani George

Vincristine pharmacodynamics and pharmacogenetics in children with cancer: A limited-sampling, population modelling approach

Background:  Vincristine is a key component of many childhood cancer treatment regimens. Pharmacodynamic parameters such as clinical efficacy and toxicity may be influenced by polymorphisms of CYP3A.

A stable-isotope HPLC-MS/MS method to simplify storage of human whole blood samples for glutathione assay

BACKGROUND Glutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge. METHODS Validation of an HPLC-MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to L-serine borate/EDTA with perchloric acid precipitation (SBPE). RESULTS Precipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at -70 °C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days. CONCLUSIONS The combination of stable isotope with methanolic precipitation at collection, with assay by HPLC-MS/MS provides superior results after storage of whole blood samples for at least 99 days.

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males.

The fluoropyrimidine drugs 5-fluorouracil and its oral prodrug capecitabine remain first line therapy for solid tumours of the neck, breast and colon. However, significant and unpredictable toxicity affects about 10-25% of patients depending upon the mode of 5-fluorouracil delivery. The pharmacokinetics of thymine (5-methyluracil) may provide an approach for screening for 5-fluorouracil toxicity, based on the rationale that thymine is a close structural analogue of 5-fluorouracil and is catabolized by the same enzymatic pathway. Oral thymine loading tests were performed on 12 healthy volunteers. Each subject was given a single oral dose of 250mg thymine in capsule form. Blood, urine and saliva samples were collected pre-dose and up to 5h post-dose. Concentrations of thymine, and its catabolites dihydrothymine and ß-ureidoisobutyrate were analysed by HPLC-tandem mass spectrometry in plasma, urine and saliva. The pharmacokinetic data of healthy volunteers were analysed assuming a non-compartmental model. Thymine peaked quickly (30-45min) in plasma to a maximum concentration of 170±185μg/L (mean±SD). Clearance was high (mean 57.9L/h/kg) exceeding normal human liver blood flow, suggesting low systemic bioavailability; urinary recovery of the thymine dose was low (<1%). Apparent formation rate-limited kinetics were observed for dihydrothymine, and the plasma concentration of dihydrothymine was consistently 10-fold higher than that of thymine. Plasma ß-ureidoisobutyrate concentrations, on the other hand, were similar to that of thymine. Genotyping confirmed that pathological mutations of the DPYD gene were absent. The urinary excretion ratio of thymine/dihydrothymine was informative of the maximum concentration. Saliva thymine was highly variable. These data are potentially useful as a basis for developing of a screening procedure to prospectively identify patients who are at risk of toxicity from fluoropyrimidine drugs.

Monitoring salivary melatonin concentrations in children with sleep disorders using liquid chromatography-tandem mass spectrometry

Background: Melatonin is synthesized in the pineal gland and is an important circadian phase marker, especially in the determination of sleep patterns. Both temporary and permanent abnormal sleep patterns occur in children; therefore, it is desirable to have methods for monitoring melatonin in biological fluids in the diagnosis and treatment of such disorders. Objective: The objective of the study is to develop a liquid chromatography–tandem mass spectrometry method for the determination of melatonin in saliva and to apply it to monitoring salivary concentrations in children with sleep disorders. Methods: A deuterated internal standard (d7-melatonin) was added to a diluted saliva sample (20 µL) in an autosampler vial insert, and 50 µL were injected. Plasticware was strictly avoided, and all glassware was scrupulously cleaned and then baked at 120°C for at least 48 hours to obtain satisfactory performance. Reverse-phase chromatography was performed on a C8 column using a linear gradient elution profile comprising mobile phases A (0.1% aqueous formic acid) and B (15% methanol in acetonitrile containing 0.1% formic acid), pumped at a total flow rate of 0.8 mL/min. The run time was 8 minutes. After atmospheric pressure chemical ionization, mass spectrometric detection was in positive ion mode. Mass detection was by selected reaction monitoring mode with the following mass transitions used for quantification: melatonin, m/z 233.0 → 173.8 and d7-melatonin, m/z 240.0 → 178.3. Results: Linearity (r > 0.999) was established from 3.9 to 1000 pg/mL. Imprecision (coefficient of variation percent) was less than 11%, and accuracy was 100–105% (7.0–900 pg/mL). The method was selective, and the mean (range) ratio of the slopes of calibrations in water to those in daytime saliva samples collected from 10 healthy adult subjects was 0.989 (0.982–0.997), indicating negligible matrix effects. The application of the assay was demonstrated in healthy adults and in children being clinically investigated for sleep disturbances. Conclusions: A validated liquid chromatography–tandem mass spectrometry method suitable for monitoring salivary melatonin in children with circadian rhythm sleep disorders is reported. The method also has potential application to pediatric population pharmacokinetic studies using sparse sampling of saliva as the biological sample matrix.

Simultaneous determination of thymine and its sequential catabolites dihydrothymine and β-ureidoisobutyrate in human plasma and urine using liquid chromatography-tandem mass spectrometry with pharmacokinetic application

To study in vivo activities of dihydropyrimidine dehydrogenase, dihydropyrimidinase, and ureidoproprionase, a sensitive, accurate, selective and precise method for the determination of the endogenous pyrimidine thymine (THY) and its successive metabolites dihydrothymine (DHT) and β-ureidoisobutyric (UIB) acid in human plasma and urine has been developed and validated. Plasma or diluted urine (200 μL) was mixed with 1 mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150 μL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry C₈ column (150 mm × 3.9 mm; 5 μm particle size), with gradient elution using a mobile phase of 0.1% (w/w) formic acid in water (phase A), and 15% (v/v) methanol in acetonitrile (phase B). The detection system was an Applied Biosystems model 3200 tandem mass spectrometer with atmospheric pressure chemical ionisation, and multiple reaction monitoring mode using the transitions: THY (m/z: 127.1-110.0), DHT (m/z: 129.1-68.9), UIB (m/z: 147.1-86.0), and deuterated-THY (m/z: 131.1-114.0). THY, DHT, and UIB eluted at 5.12 min, 5.17 min and 5.00 min, respectively. Linearity of the calibrations was established from 2 to 500 μg/L. The lower limit of quantification was 5 μg/L in plasma, and 10 μg/L in urine for THY, DHT and UIB. Ion-suppression had negligible effect. A pilot pharmacokinetic study analysed plasmas and urines from 2 healthy male subjects who each received an oral 250 mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.

Quantitative determination of the enantiomers of methadone in human plasma and saliva by chiral column chromatography coupled with mass spectrometric detection.

Methadone is a potent lipophilic synthetic opioid that is effective in the treatment of cancer pain and perceived benefit in difficult pain control scenarios (especially in cases of neuropathic pain). The use of methadone in clinical practice is challenging however, due to the narrow therapeutic window and large inter- and intra-individual variability in therapeutic response. Quantitation of the enantiomers d- and l-methadone (d- and l-MTD) in plasma and saliva provides a basis for studying its pharmacokinetics in patients with cancer and for monitoring efficacy, toxicity and side-effects. This assay involves quantitation of the enantiomers of methadone using their respective deuterated internal standards, in plasma and saliva matrices with no impact of ion suppression in either matrix. The analytical recoveries of d- and l-MTD from the saliva collection devices (Salivette®) are optimised in this novel method with an accurate and simple extraction method employing dichloromethane. Optimal enantioselective separations were achieved using an α1-acid glycoprotein chiral stationary phase and triple quadrupole tandem mass spectrometer. Linearity was demonstrated over 0.05-1000µg/L for both enantiomers in plasma and in saliva with correlation coefficients greater than 0.998. The lower limit of quantitation (LLOQ) was determined to be 0.1µg/L in plasma and saliva for d- and l-MTD. Accuracy of the method ranges from 100% to 106% even at the LLOQ and total precision, expressed as the coefficient of variation, was between 0.2% and 4.4% for both analytes in both matrices. A simple one step extraction procedure resulted in recoveries greater than 95% for both analytes, at concentrations as low as 0.5µg/L, from the Salivette®. The validated method was applied successfully in 14 paired plasma and saliva samples obtained from adult patients with cancer pain receiving methadone.

Enhancement and Suppression of Ionization in Drug Analysis Using HPLC-MS/MS in Support of Therapeutic Drug Monitoring: A Review of Current Knowledge of Its Minimization and Assessment

Abstract: High-performance liquid chromatography coupled with tandem mass spectrometry is commonly used for quantitation of analytes in biological matrices, because of the selectivity, sensitivity, and high throughput offered by this technique. However, the presence of both suppression and enhancement of ionization (SEI) by matrix components is an increasingly recognized impediment to accurate results. The existence of SEI indicates that ionization efficiency is a result of the chemical environment seen by both the analyte and internal standard during ion formation. SEI is influenced by the type and the make of ion source used, mobile-phase composition, extent of sample preparation, and the ability to chromatographically separate other compounds that may influence ionization of the analyte and/or internal standard. A comprehensive review of the phenomenon of SEI in high-performance liquid chromatography coupled with tandem mass spectrometry was conducted, and a summary of salient papers relating to therapeutic agents in biological matrices is presented. Suggestions for approaches to minimize, normalize, or assess SEI and its deleterious effect on accuracy and sensitivity, and hence the validity of quantitative results, are provided. Consideration is also given to a strategy to test for SEI, including the number of samples from different sources that are required to adequately test for SEI.

Can Saliva and Plasma Methadone Concentrations Be Used for Enantioselective Pharmacokinetic and Pharmacodynamic Studies in Patients With Advanced Cancer

PURPOSE Methadone is a potent analgesic used to treat refractory cancer pain. It is administered as a racemic mixture, with the l-enantiomer being primarily a μ-receptor agonist, whereas the d-enantiomer is an N-methyl-d-aspartate antagonist and inhibits serotonin and norepinephrine reuptake. Dose requirements vary greatly among patients to achieve optimal pain control and to avoid the risk of adverse effects. The relationship between plasma and saliva methadone enantiomer concentrations was investigated to determine if saliva could be a substitute for plasma in pharmacodynamic and pharmacokinetic studies for clinical monitoring and dose optimization of methadone in patients with advanced cancer. METHODS Patients with advanced cancer who were prescribed varying doses of oral methadone for pain management were recruited to obtain paired plasma and saliva samples. Pain scores were recorded at the time of sampling. The total and unbound plasma and saliva concentrations of the l- and d-enantiomers of methadone were quantified by using an HPLC-MS/MS method. The relationship between plasma (total and unbound) and saliva concentrations were compared. The saliva-to-plasma concentration ratio was compared versus the dose administered and the time after dosing for both enantiomers. The association of methadone concentrations with reported pain scores was compared by using a Mann-Whitney U test for significance. FINDINGS Fifty patients receiving a mean dose of 11mg/d of methadone provided 151 paired plasma and saliva samples. The median age of the population was 61 years with an interquartile range of 53-71 years with total body weight ranging from 59-88 kg. Median (interquartile) total plasma concentrations for l- and d-methadone were 50.78 ng/mL (30.6-113.0 ng/mL) and 62.0 ng/mL (28.7-116.0 ng/mL), respectively. Median (interquartile range) saliva concentrations for l- and d-methadone were 81.5 ng/mL (28.0-203.2 ng/mL) and 44.2 (16.2-149.7 ng/mL). No relationship could be established between plasma and saliva concentrations for l- and d-methadone (r2 = 0.35 and 0.25). The saliva-to-plasma concentration analyzed with the methadone dose showed higher saliva concentrations at lower doses. Dose-normalized saliva concentrations followed a similar pattern over time compared with plasma concentrations. No correlation was found between l-methadone plasma, d-methadone plasma, l-methadone saliva, d-methadone saliva concentrations, and pain score. IMPLICATIONS Saliva concentration was not a better predictor of pain control than plasma concentration for dose optimization and monitoring studies of methadone in patients with cancer. Although the saliva-to-plasma ratio of the concentration of methadone enantiomers was stable across the dosing range, due to the variability in individual saliva-to-plasma ratios, saliva sampling may not be a valid substitute in pharmacokinetic studies of methadone in cancer.

Determination of thymine, dihydrothymine and ureidoisobutyrate in human plasma, saliva and urine using HPLC-tandem mass spectrometry

Aims: Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme in the catabolism of 5-fluorouracil (5FU) and DPD deficiency is an important pharmacogenetic syndrome. The main purpose of this study was to develop a limited sampling strategy to evaluate the pharmacokinetics of 5FU and to detect decreased 5FU elimination in patients with c.1905+1G>A (IVS14+1G>A) mutation related DPD deficiency. Methods: Thirty patients, heterozygous for the c.1905+1G>A (IVS14+1G>A) mutation in DPYD, and 16 control patients received an intravenous dose administered over 2 min of 300 mg 5FU/m2 and/or 450 mg 5FU/m2, followed by blood sampling over 2 hrs and subsequently pharmacokinetic analysis of the 5FU plasma levels. A population pharmacokinetic analysis was performed in order to develop a quantitative compartmental pharmacokinetic model suitable for limited sampling strategy. Clinical aspects of treating DPD patients with 5FUbased chemotherapy was assessed from the retrospectively collected clinical data. Results: A two compartment model with Michaelis-Menten elimination from the central compartment was used in the population pharmacokinetic analysis. The mean (€ SD) AUC (mg.h/L), T1/2 s (h), Km (mg/L) and Vmax (mg/h) of 5FU in DPD deficient patients vs. controls were 9.1 € 4.0 vs. 6.0 € 2.1 (AUC), 0.268 € 0.116 vs. 0.128 € 0.043 (T1/2 s), 4.81 € 0.39 vs. 4.39 € 0.72 (Km) and 942 € 310 vs. 1749 € 380 (Vmax) at a dose of 300 mg 5FU/m2. For a dose of 450 mg 5FU/m2, the PK values in DPD deficient patients vs. controls were 17.7 € 5.4 vs. 13.4 € 3.9 (AUC), 0.306 € 0.103 vs. 0.181 € 0.041 (T1/2 s), 5.02 € 0.32 vs. 4.58 € 0.25 (Km) and 900 € 194 vs. 1370 € 267 (Vmax). The differences in AUC, T1/2 s and Vmax between DPD deficient patients and controls were all highly significant (p <0.05). Using a single sample t = 30 min limited sampling strategy, the positive predictive value and negative predictive value for Vmax were 96% and 88 %, respectively. Conclusion: Profound differences in pharmacokinetics of 5FU existed between DPD deficient patients and control patients. Pharmacokinetic 5FU profiling using a limited sampling model may be useful for identification of DPD deficient patients in order to reduce severe toxicity.