Berit P. Jensen

Development and validation of a stereoselective liquid chromatography-tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma

A stereoselective liquid chromatography-tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5-50 microg/L in human plasma. Metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range 0.5-50 microg/L in plasma, with 0.5 microg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92-105%.

Endogenous Ouabain Is Not Ouabain

The concept of a circulating digitalis-like inhibitor of the sodium pump, Na+, K+-ATPase, evolved from studies performed in the 1960s. De Wardener et al1 addressed the question of whether a small increase in the glomerular filtration rate together with changes in the concentration of the more recently discovered hormone aldosterone could explain the natriuresis that generally followed salt (sodium) loading. In their studies, dogs had their renal blood flow reduced significantly by constricting the aorta above the renal arteries and were given supramaximal doses of fludrocortisone, a synthetic analogue of aldosterone, and vasopressin before being challenged with intravenous saline. Their ability to develop a natriuresis clearly demonstrated that a third factor (beyond changes in glomerular filtration rate and aldosterone concentrations) was involved in the natriuresis of salt loading. Subsequent experiments demonstrated that the responsible agent could be transmitted by the plasma of the volume-expanded animal.2 Although we may now ask whether in these experiments the effect was mediated, at least in part, by the release of atrial and B-type natriuretic peptide from the heart, it was nearly 2 decades before those hormones were discovered during which time it was demonstrated that the plasma of volume-expanded animals had the ability to inhibit the sodium pump3—which is not a target of atrial natriuretic peptide and B-type natriuretic peptide. Essentially parallel studies were performed by Welt and colleagues4 in uremia where inhibition of the sodium pump of erythrocytes was demonstrated along with the ability of uremic plasma to induce such a defect in normal erythrocytes. In 1975, it was shown that patients with essential hypertension had, as a group, reduced activity of the sodium pump of leukocytes, with corresponding elevated values for intracellular sodium.5 This finding proved to be reproducible in various laboratories, …

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.

Influence of adult age on the total and free clearance and protein binding of (R)- and (S)-warfarin

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Hepatic drug clearance is thought to be reduced with age. However for highly protein bound drugs, which are cleared by capacity-limited metabolism, studies on total clearance have been conflicting. The hypothesis that protein binding decreases with age has been used to explain this. Warfarin is a highly protein bound drug, which is cleared by capacity-limited metabolism. There are conflicting or little data on the relationship between adult age and total and free clearance and protein binding of (R)- and (S)-warfarin. WHAT THIS STUDY ADDS In a clinical study of 72 patients (18-89 years) on warfarin therapy, both total and free clearance of (R)-warfarin decreased with age. For (S)-warfarin there was a stronger signal of a decrease in free than total clearance. Protein binding was found not to correlate with age for (R)- and (S)-warfarin. In an ex vivo study, in which warfarin was spiked to plasma samples from 60 healthy subjects (19-87 years), no correlation between protein binding and age was found. These data support the hypothesis that hepatic drug clearance decreases with age. This should be taken into consideration when individualizing dosing, particularly in the elderly. AIMS To test the hypothesis that the clearance (CL) of warfarin, a very highly protein bound drug with capacity-limited metabolism, decreases with age. METHODS In a clinical study, a steady-state blood sample was taken from 72 patients (18-89 years) on routine treatment with warfarin. Concentrations of (R)- and (S)-warfarin were determined in plasma (total) and ultrafiltrate (free) by LC-MS/MS. Total and free CL and protein binding were determined and regressed against age and other covariates. In an ex vivo study, warfarin was spiked to plasma samples from 60 healthy subjects (19-87 years) and protein binding was regressed against age and other covariates. RESULTS For (R)-warfarin a significant decrease with age was found for both total and free CL (P < 0.001). For (S)-warfarin there was a stronger signal of a decrease with age in free CL (P= 0.005) vs. total CL (P= 0.045). The decrease in CL of (R)- and (S)-warfarin was 0.3-0.5% per year. Other covariates influencing CL were lean body weight for both (R)- and (S)-warfarin and CYP2C9 genotype and blood sampling time for (S)-warfarin. Protein binding of (R)- and (S)-warfarin was not found to change significantly with age in either the clinical or the spiked samples, despite a slight decrease in albumin concentration with age. CONCLUSIONS These data support the hypothesis that the CL of (R)- and (S)-warfarin decreases with age. More accurate information was gained when measuring free CL for (S)-warfarin. Warfarin protein binding did not change significantly with age.

CYP2D6 genotype and its relationship with metoprolol dose, concentrations and effect in patients with systolic heart failure

The aims of this study were to examine the relationships between CYP2D6 genotype and metoprolol dose, S- and R-metoprolol concentrations and clinical effects in patients with systolic heart failure. Data were obtained for 52 subjects, of which 27 had 2 functional alleles (24/27, CYP2D6*1/*1), 22 had 1 functional allele (18/22, CYP2D6*1/*4) and 3 had no functional alleles (CYP2D6*4/*4). Median dose-adjusted concentrations of S-metoprolol (active) were 6.3- and 3.2-fold higher in subjects with zero or one functional allele (P=0.016 and P=0.006), respectively, compared with subjects with two functional alleles. For the R-enantiomer (inactive), these concentrations were 10.7- and 3.7-fold higher (P=0.013 and P=0.003), respectively. Despite clear gene-concentration differences, no relationships between CYP2D6 genotype and dose or clinical effects could be shown. Although the number with no functional alleles was too small (n=3) to show effects, in patients with 1 functional allele other sources of variance are likely to be obscuring differences in clinical effects.

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.

Coagulation assays and plasma fibrinogen concentrations in real-world patients with atrial fibrillation treated with dabigatran.

AIMS In patients with atrial fibrillation prescribed dabigatran, the aim was to examine the correlation between plasma dabigatran concentrations and the three screening coagulation assays [international normalized ratio (INR), activated partial thromboplastin time (aPTT) and thrombin time (TT)] as well as the dilute thrombin time (dTT) and to examine the contribution of plasma fibrinogen concentrations to the variability in TT results. METHODS Plasma from patients with atrial fibrillation on dabigatran were analysed for clotting times and concentrations of fibrinogen and dabigatran. Correlation plots (and associated r(2) values) were generated using these data. The variability in TT results explained by fibrinogen concentrations was quantified using linear regression. RESULTS Fifty-two patients (38-94 years old) contributed 120 samples, with plasma dabigatran concentrations ranging from 9 to 408 μg l(-1) . The r(2) values of INR, aPTT, TT and dTT against plasma dabigatran concentrations were 0.49, 0.54, 0.70 and 0.95, respectively. Plasma fibrinogen concentrations explained some of the residual variability in TT values after taking plasma dabigatran concentrations into account (r(2) = 0.12, P = 0.02). CONCLUSIONS Of the screening coagulation assays, the TT correlated best with plasma dabigatran concentrations. Variability in fibrinogen concentrations accounts for some of the variability in the TT.

Pharmacogenetics of drug-metabolizing enzymes: the prodrug hypothesis

The hope of individualized drug therapy has been bolstered by the knowledge that drug-metabolizing enzymes can be affected by genetic polymorphisms. The initial flurry of potential examples has been muted somewhat by the failure of most predictions to be translated into clinical practice. Perhaps the only real example with reasonable evidence is that of azathioprine/6-mercaptopurine and thiopurine methyl-transferase. A few other examples such as tamoxifen, clopidogrel, irinotecan and warfarin warrant further discussion. An interesting feature of these drugs is that all except warfarin are prodrugs. We propose the hypothesis that prodrugs are over-represented in drugs that may be affected by genetic polymorphisms. Understanding this may assist our efforts to advance the field.

Influence of ABCB1 (P-glycoprotein) haplotypes on nortriptyline pharmacokinetics and nortriptyline-induced postural hypotension in healthy volunteers

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT A single nucleotide polymorphism in ABCB1, which encodes P-glycoprotein, has retrospectively been associated with symptoms of nortriptyline-induced postural hypotension in depressed patients. This finding needs to be replicated in independent studies before recommendations regarding pharmacogenetic testing can be made. WHAT THIS STUDY ADDS In a prospective study of healthy volunteers homozygous for ABCB1 (1236-2677-3435, TTT/TTT or CGC/CGC), a single dose of nortriptyline was administered, plasma exposure was determined and blood pressure and heart rate were monitored during posture change. No differences between ABCB1 haplotype groups were found in plasma exposure of nortriptyline and its active metabolites, E- and Z-10-hydroxynortriptyline. The heart rate response to posture change was increased with nortriptyline, whereas there was no difference in blood pressure response. However, no differences between haplotype groups were observed except that the pre dose heart rate response to standing was greater in the TTT than CGC homozygotes. The association between ABCB1 polymorphisms and nortriptyline-induced postural hypotension found in a previous study could not be confirmed. The results raise the possibility of a predisposition in heart rate response in the TTT homozygotes rather than an effect of nortriptyline. AIMS To investigate the influence of ABCB1 (1236-2677-3435) polymorphisms on nortriptyline pharmacokinetics and nortriptyline-induced postural hypotension in healthy volunteers. METHODS Genetic screening of 67 healthy volunteers identified eight CGC homozygotes and nine TTT homozygotes of ABCB1 (1236-2677-3435), who were administered a single dose of nortriptyline 25 mg. Plasma exposure of nortriptyline and its active metabolites, E- and Z-10-hydroxynortriptyline, was determined over 72 h. Heart rate and blood pressure responses to posture change (active standing and passive head-up tilt) were measured continuously using finger plethysmography. RESULTS There were no differences in plasma exposure between ABCB1 haplotype groups, as the geometric mean (95% CI) AUC(0,72 h) ratios were 0.98 (0.94, 1.03), 1.02 (0.96, 1.09) and 0.95 (0.80, 1.10) for nortriptyline, E- and Z-10-hydroxynortriptyline, respectively. The pre dose heart rate response to standing was greater in the TTT than CGC homozygotes (mean (95% CI) difference 7.4 (1.5, 13.4) beats min(-1) , P = 0.02). At t(max) at 8 h post dose, nortriptyline increased the heart rate response to posture change in all subjects with mean (95% CI) Δ heart rate values of 7.4 (3.6, 11.3) beats min(-1) on active standing (P = 0.0009) and 4.8 (2.0, 7.6) beats min(-1) on head-up tilt (P = 0.002), but no difference was observed between haplotype groups. There was no difference in blood pressure response to posture change in either group. CONCLUSION The association between ABCB1 polymorphisms and nortriptyline-induced postural hypotension found in the previous study could not be confirmed. The results raise the possibility of a predisposition in heart rate response in the TTT homozygotes rather than an effect of nortriptyline.

Quantification of total and unbound concentrations of lorazepam, oxazepam and temazepam in human plasma by ultrafiltration and LC–MS/MS

BACKGROUND A fast and sensitive assay for quantifying total and unbound concentrations of lorazepam (Lzp), oxazepam (Ozp) and temazepam (Tzp) in human plasma was needed for a plasma protein binding study. RESULTS Plasma samples were precipitated with acetonitrile for determination of total concentrations or subjected to ultrafiltration for determination of unbound concentrations. An LC-MS/MS assay was developed with an Allure® PFP propyl column and a mobile phase of 35% acetonitrile/0.1% formic acid over 4.5 min and ESI+-MS/MS detection. Matrix effects were negligible in plasma and approximately 70% in ultrafiltrate but were accounted for by the internal standards Lzp-d₄, Ozp-d₅ and Tzp-d₅. The assay was validated for total concentrations of 10-100 ng/ml Lzp, 200-2000 ng/ml Ozp and 100-1000 ng/ml Tzp, and for unbound concentrations of 1-10 ng/ml Lzp, 20-200 ng/ml Ozp and 10-100 ng/ml Tzp. Precision was <14% CV and accuracy was 96-110% throughout the calibration range. The mean precision of triplicate analysis of 60 study samples was <4% CV for total and <8% CV for unbound concentrations. CONCLUSION A fast and sensitive assay was developed and validated. It has been applied successfully to a protein binding study.