Pharmacokinetic Modeling of Morphine's Effect on Plasma Concentrations of Ticagrelor and Its Metabolite in Healthy Volunteers.

This study aimed to build a mathematical model describing the pharmacokinetics of ticagrelor and its active metabolite (AR-C124910XX) in a stable setting with concomitant administration of morphine. The model consists of a set of four differential equations prepared upon the available knowledge regarding the biological processes in the pharmacokinetics of ticagrelor. The set of equations was solved numerically using the Runge-Kutta method. The data were obtained in a double-blind, randomized, placebo-controlled, crossover trial. Twenty-four healthy volunteers received a 180-mg ticagrelor loading dose together with either 5-mg morphine or placebo. Blood samples were analyzed with liquid chromatography-tandem mass spectrometry to assess plasma concentrations of ticagrelor and AR-C124910XX before ticagrelor loading dose and after that 1, 2, 3, 4, and 6 h. The model allowed us to reproduce the experimental results accurately and led us to conclusions consistent with clinical observations that morphine delays the time of maximum drug concentration and that the morphine effect occurs due to decreased gastrointestinal motility. Based on the model, we were able to predict the effect of drug dose on receptor blocking efficacy.

Acute Limb Ischemia after Intake of the Phenylethylamine Derivate NBOMe.

Objective: N-(2-methoxy) benzyl-phenethylamine (NBOMe) derivatives have a high affinity to the serotonin receptor 2A and emerged as new psychedelic agents. We report the case of a 30-year-old man admitted to the hospital because of acute ischemia of the left arm with clinical symptoms of pallor, pulselessness, paresthesia, and a motoric deficit. The patient had a history of schizophrenia and drug abuse and disclosed during the hospital stay the sublingual intake of a substance bought as 25I-NBOMe the night before the ischemic event. Methods: Routine clinical diagnostics including among others color-coded duplex sonography and computed tomography angiography (CTA) were performed. The remainder of the drugs was analyzed using high performance liquid chromatography. Results: Initial color-coded duplex sonography of the upper left limb showed pathological flow profiles of the axillary, brachial, ulnar, and radial artery with a reduced diameter of the ulnar (0.9 mm) and radial (1.1 mm) artery. In consequence, peripheral vasospasm, distal arterial thrombosis, or arterial embolization was anticipated. As therapeutic measures, the patient immediately received intravenous systemic vasodilators (alprostadil) and therapeutic anticoagulation with low molecular weight heparin. Instant symptom improvement was observed within the first day after therapy initiation. The subsequently performed CTA of the heart and left arm showed no signs of thrombotic material. Treatment was continued for five days and the patient was released thereafter having completely normalized perfusion in his left arm. Outpatient treatment was continued with calcium-channel blockers, as the patient had also displayed arterial hypertension. Drug analysis retrieved a composition of the isomers 25I-NBOMe, 25C-NBOMe, and 25H-NBOMe as well as traces of pentylon. Conclusion: NBOMe ingestion implicates the risk of peripheral vasospasms with severe, limb-threatening ischemia.

Morphine Interaction with Aspirin: a Double-Blind, Crossover Trial in Healthy Volunteers.

Aspirin is a cornerstone in the antiplatelet therapy for acute coronary syndromes. Coadministration of morphine may potentially influence the intestinal absorption, pharmacokinetics, and pharmacodynamics, as seen with P2Y12 inhibitors. In this trial, healthy volunteers were randomized to receive morphine (5 mg, i.v. bolus injection) at one of seven different time points before, after, or with aspirin (162 mg, p.o.) in a double-blind, placebo-controlled fashion. After a 14-day washout, subjects received placebo instead of morphine. Pharmacokinetics were determined by liquid chromatography, and aspirin's effects were measured by platelet function tests (whole-blood platelet aggregation: multiplate, platelet plug formation: PFA-100). Morphine increased the total acetylsalicylic acid exposure by 20% compared with placebo when given simultaneously with aspirin, whereas Cmax and tmax were not altered. Morphine had no significant effect on aspirin-induced platelet inhibition. In contrast to coadministration with P2Y12 inhibitors, morphine appears to have negligible interaction with aspirin.

Clopidogrel in Critically Ill Patients.

Only limited data are available regarding the treatment of critically ill patients with clopidogrel. This trial investigated the effects and the drug concentrations of the cytochrome P450 (CYP450) activated prodrug clopidogrel (n = 43) and the half-life of the similarly metabolized pantoprazole (n = 16) in critically ill patients. ADP-induced aggregometry in whole blood classified 74% (95% confidence intervals 59-87%) of critically ill patients as poor responders (n = 43), and 65% (49-79%) responded poorly according to the vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) assay. Although the plasma levels of clopidogrel active metabolite normally exceed the inactive prodrug ∼30-fold, the parent drug levels even exceeded those of the metabolite 2-fold in critically ill patients. The half-life of pantoprazole was several-fold longer in these patients compared with reference populations. The inverse ratio of prodrug/active metabolite indicates insufficient metabolization of clopidogrel, which is independently confirmed by the ∼5-fold increase in half-life of pantoprazole. Thus, high-risk patients may benefit from treatment with alternative platelet inhibitors.

Prasugrel in critically ill patients.

While prasugrel is indicated for the treatment of myocardial infarction, its effects in the most severely affected patients requiring intensive care is unknown, so that we measured the antiplatelet effects and sparse pharmacokinetics of prasugrel in critically ill patients. Twenty-three patients admitted to medical intensive care units, who were treated with 10 mg prasugrel once daily, were included in this prospective trial. Critically ill patients responded poorly to daily prasugrel treatment: adenosine diphosphate (ADP)-induced aggregation in whole blood classified 65 % (95 % confidence intervals (CI) 43-84 %) of patients as having high on treatment platelet reactivity, platelet function under high shear rates even 74 % (95 %CI 52-90 %). There was only limited additional inhibition provided 2 hours after the next dose of prasugrel. In contrast, insufficient inhibition of the target was only seen in 26 % (95 %CI 10-48 %) of patients as measured by the vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) assay. Low effective plasma levels of prasugrel active metabolite were measured at trough [0.5 (quartiles 0.5-1.1) ng/ml at baseline], and 2 hours after intake [5.7 (3.8-9.8) ng/ml], but showed coefficients of variation of ~70 %. In sum, inhibition of platelet aggregation by prasugrel is not uniform but highly variable in critically ill patients, similar to clopidogrel in a general population. The pharmacokinetic measurements indicate that poor absorption/metabolism of prasugrel may partly contribute while inflammation induced heightened intrinsic platelet reactivity may also play a role.

Ticagrelor mitigates ischaemia-reperfusion induced vascular endothelial dysfunction in healthy young males - a randomized, single-blinded study.

AIMS: Animal data suggest that ticagrelor but not clopidogrel protects against tissue injury. It is unclear if this effect of ticagrelor is also detectable in humans. We studied the effect of ticagrelor and clopidogrel at standard clinical doses on endothelial dysfunction in an experimental model of forearm vascular ischaemia-reperfusion (IR) injury. METHODS: In a randomized, single-blinded trial, 24 subjects underwent forearm blood flow (FBF) measurements in response to the endothelium-dependent vasodilator acetylcholine (ACh) and to glyceryltrinitrate (GTN; endothelium-independent) before and after a 20 min forearm ischaemia. FBF reactivity was assessed after an oral loading dose of ticagrelor or clopidogrel and after 14 days of regular intake of maintenance doses of the study medicines. In addition, the effect on platelet inhibition was evaluated using multiple electrode aggregometry. RESULTS: ACh-induced vasodilation was impaired during reperfusion and not completely normalized by acute or chronic treatment with ticagrelor or clopidogrel (post- vs. pre-ischaemia). However, ticagrelor mitigated endothelial dysfunction compared to clopidogrel after loading (FBF AChAUC ratio post- vs. pre-ischaemia: 0.83 [0.70; 0.96] vs. 0.64 [0.56; 0.72]; P = 0.024) and after chronic administration (FBF AChAUC ratio: 0.86 [0.71; 1.00] vs. 0.66 [0.55; 0.77]; P = 0.027). As expected, GTN-induced vasodilation was not affected by ischaemia. Ticagrelor or clopidogrel treatment inhibited platelet activation to a similar degree. CONCLUSION: Our data indicate that ticagrelor treatment exerts a greater vascular salutary effect than clopidogrel during reperfusion after an acute vascular occlusion. IR-induced vascular injury cannot be prevented completely by administration of these antiplatelet agents at standard clinical doses.

Acetylsalicylic acid in critically ill patients: a cross-sectional and a randomized trial.

BACKGROUND: Despite decades of clinical use, the pharmacokinetics and the effects of acetylsalicylic acid (ASA) in critically ill patients remain ill-defined. We aimed to investigate the pharmacokinetics and the effects of different ASA formulations during critical illness. DESIGN: A cross-sectional study and a randomized, parallel-group trial were performed. Critically ill patients under chronic oral ASA treatment (100 mg enteric-coated) were screened for high 'on-treatment' platelet reactivity (HTPR) according to arachidonic acid-induced whole-blood aggregometry. Thirty patients with HTPR were randomized to receive 100 mg ASA intravenously, 100 mg enteric-coated ASA bid (bis in die) or 81 mg chewable ASA (n = 10 per group). Serum thromboxane B2 (TXB2) levels, ASA and salicylic acid levels were quantified. RESULTS: Of 66 patients, 85% (95% confidence intervals 74-93%) had HTPR. Compared to baseline infusion of 100 mg, ASA significantly reduced platelet aggregation after 24 h to median 80% (Quartiles: 66-84%). Intake of 81 mg chewable ASA significantly reduced platelet aggregation to 75% (54-86%) after four hours, but increased it to 117% after 24 h (81-163%). Treatment with 100 mg enteric-coated ASA bid decreased platelet aggregation after 24 h to median 56% (52-113%). Baseline TXB2 levels were median 0·35 ng/mL (0·07-0·94). Infusion of ASA or intake of 100 mg ASA bid reduced TXB2 levels to 0·07-0·18 ng/mL after 24 h, respectively. Chewable ASA reduced TXB2 levels only transiently. Pharmacokinetic analysis revealed highly variable absorption patterns of oral ASA formulations. CONCLUSION: There is a very high prevalence of HTPR in critically ill patients on peroral ASA therapy, caused by an incomplete suppression of TXB2 and/or by impaired absorption of ASA.

Abciximab as a bridging strategy to overcome morphine-prasugrel interaction in STEMI patients.

OBJECTIVE: The present study investigated whether the glycoprotein (GP)IIb/IIIa receptor blocker abciximab might be a successful bridging strategy to achieve adequate levels of platelet inhibition rapidly in cases where prasugrel is used in morphine-pretreated ST-elevation myocardial infarction (STEMI) patients. METHODS: In a prospective observational cohort study, 32 patients presenting with STEMI were given prasugrel at a loading dose of 60 mg. Patients were stratified into four groups, according to morphine and/or abciximab use. Adenosine diphosphate (ADP)-induced platelet aggregation was measured at four time points: at baseline, and at 2 h, 1 day and 2 days after prasugrel loading. RESULTS: Morphine use was associated with a three-fold higher level of ADP-induced platelet aggregation 2 h after prasugrel loading compared with no morphine/no abciximab (P = 0.019). However, when abciximab was infused in the catheterization laboratory, the effect of morphine on ADP-induced platelet aggregation disappeared (P = 0.884). This interaction was also seen in the presence of high on-treatment platelet reactivity (HTPR) at 2 h; while HTPR was seen in 88% of morphine users/no abciximab users, it was found in only 17-20% in the three other groups (P = 0.003). The effect of morphine disappeared by day 1 - 2. CONCLUSION: The infusion of the GPIIb/IIIa receptor blocker abciximab allows immediate and efficient platelet inhibition in STEMI patients concomitantly receiving the oral ADP receptor blocker prasugrel and morphine.

Morphine interaction with prasugrel: a double-blind, cross-over trial in healthy volunteers.

BACKGROUND: Morphine decreases the concentrations and effects of clopidogrel, which could lead to treatment failure in myocardial infarction. OBJECTIVES: To clarify whether more potent P2Y12-inhibitors may provide an effective alternative, we examined drug-drug interactions between morphine and prasugrel. METHODS: Twelve healthy volunteers received 60 mg prasugrel with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, cross-over trial. Pharmacokinetics were determined by liquid chromatography tandem mass spectrometry, and prasugrel effects were measured by platelet function tests. RESULTS: Morphine neither diminished total drug exposure (AUC), which was the primary endpoint, nor significantly delayed drug absorption of prasugrel. However, morphine reduced maximal plasma concentrations (C max) of prasugrel active metabolite by 31 % (p = 0.019). Morphine slightly, but not significantly, delayed the onset of maximal inhibition of platelet plug formation under high shear rates (30 vs. 20 min). Whole blood aggregation was not influenced. CONCLUSIONS: Although morphine significantly decreases the maximal plasma concentrations of prasugrel active metabolite, it does not diminish its effects on platelets to a clinically relevant degree in healthy volunteers. However, it should be considered that the observed decrease in C max of prasugrel active metabolite caused by morphine co-administration may gain relevance in STEMI patients. CLINICAL TRIAL REGISTRATION: NCT01369186, EUDRA-CT#: 2010-023761-22.

Morphine decreases ticagrelor concentrations but not its antiplatelet effects: a randomized trial in healthy volunteers.

BACKGROUND: Our recent drug interaction trial with clopidogrel shows that morphine decreases the concentrations and pharmacodynamic effects of clopidogrel, which could lead to treatment failure in susceptible individuals. We hypothesized that the pharmacodynamic consequences of drug-drug interactions would be less between morphine and ticagrelor. MATERIALS AND METHODS: Twenty-four healthy subjects received a loading dose of 180 mg ticagrelor together with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, crossover trial. Pharmacokinetics were determined by liquid chromatography tandem mass spectrometry, and ticagrelor pharmacodynamic effects were measured by platelet function tests (whole blood platelet aggregation: multiplate, platelet plug formation: PFA-100, vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay). RESULTS: Concomitant i.v. injection of morphine slows drug resorption of ticagrelor and its active metabolite (P < 0·05) by 1 h and decreases plasma levels of ticagrelor and its active metabolite by 25-31% (P ≤ 0·03) and the drug exposure (area under the curve) by 22-23% (P ≤ 0·01). Importantly, however, the pharmacodynamic effects of ticagrelor on platelet aggregation in whole blood, platelet plug formation and VASP phosphorylation are not affected by morphine. CONCLUSIONS: Morphine co-administration moderately decreases ticagrelor plasma concentrations but does not inhibit its pharmacodynamic effects in healthy volunteers within 6 h after drug administration. Limitations of our trial include the investigation in healthy volunteers under standardized conditions, which does not necessarily reflect a realistic emergency scenario.

Potent irreversible P2Y12 inhibition does not reduce LPS-induced coagulation activation in a randomized, double-blind, placebo-controlled trial.

Platelets play an important role in the activation of coagulation. P2Y12 receptor inhibition may be beneficial in inflammatory states. Prasugrel, a potent irreversible inhibitor of P2Y12 receptor-induced platelet activation may reduce activation of coagulation in a human LPS (lipopolysaccharide) model. A double-blind, randomized, crossover trial with a minimum washout period of 6 weeks was performed. Sixteen subjects were randomly assigned to a treatment group that received prasugrel or placebo 2 h before infusion of a bolus of LPS (2 ng/kg of body weight), whereas four subjects were assigned to a control group receiving prasugrel or placebo without LPS. hcDNA (histone-complexed DNA), coagulation and platelet-specific parameters were measured by enzyme immunoassay. Leucocyte aggregate formation was analysed by flow cytometry, and thromboelastometry was performed. LPS infusion markedly activated coagulation. However, prasugrel did not reduce changes in prothrombin fragments 1 and 2 (F1+2), thrombin-antithrombin complexes, microparticle-associated tissue factor, CD40 ligand, P-selectin, platelet-leucocyte aggregation, hcDNA levels or the coagulation profile measured by thromboelastometry. hcDNA plasma levels increased approximately 6-fold after LPS infusion in both treatment groups, but not in the control groups. Potent irreversible P2Y12 inhibition by prasugrel does not affect LPS-induced coagulation activation. The 6-fold increased hcDNA plasma levels after infusion of LPS indicates the formation of neutrophil extracellular traps during sterile inflammation.

Absorption kinetics of low-dose chewable aspirin--implications for acute coronary syndromes.

BACKGROUND: This study describes the implications of the pharmacokinetics of low-dose chewable aspirin for acute coronary syndromes. Current guidelines recommend the administration of 162-325 mg aspirin chewing tablets for the treatment of acute myocardial infarction. Although aspirin is widely used and a cornerstone in myocardial infarction, there is no information available on the pharmacokinetics of low doses of chewable aspirin. MATERIALS AND METHODS: This prospective trial assessed the pharmacokinetics of acetylsalicylic acid and its metabolite salicylic acid after intake of 162 mg chewable low-dose aspirin in 35 healthy volunteers. Plasma drug and metabolite levels were analysed using high-performance liquid chromatography, and corresponding pharmacodynamics were determined by impedance aggregometry. RESULTS: Acetylsalicylic acid was rapidly absorbed with a mean Tmax of 27 ± 8 min. Tmax of salicylic acid was 69 ± 21 min. Mean Cmax was 1·8 ± 0·6 mg/L and 7·6 ± 1·4 for acetylsalicylic acid and salicylic acid, respectively. Arachidonic acid-induced aggregation showed maximum platelet inhibition 30 min after drug ingestion. CONCLUSIONS: The characterization of the plasma-time profile fills the gap between the lack of data on pharmacokinetics and the pharmacodynamics and the recommendation for using low-dose chewable aspirin for acute coronary syndromes. We describe for the first time that a 162-mg dose of chewable aspirin is rapidly absorbed and achieves plasma concentrations of the active metabolite salicylic acid required to maximally inhibit platelet aggregation. However, a 162-mg dose is truly a minimum, and doubling this dose might be better for patients with myocardial infarction.

Comparison of a new ELISA-based with the flow cytometric assay for vasodilator-associated stimulated phosphoprotein phosphorylation to assess P2Y12 -inhibition after ticagrelor intake.

BACKGROUND: Ticagrelor is a P2Y12 receptor antagonist, with superior effects but also ensuing enhanced bleeding risk as compared to clopidogrel. Determination of platelet inhibition may be useful to confirm efficient platelet inhibition on an individual patient level and to identify patients at risk for bleeding. The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation assay specifically measures platelet P2Y12 inhibition, but has so far required individual sample processing. A new ELISA-based VASP assay has been developed, which allows batch analysis after initial platelet activation. Because of the reversible binding of ticagrelor it is unclear if the ELISA and flow cytometric assays provide comparable results; several washing steps of the ELISA may potentially result in false low results through dilution. METHODS: We hypothesized that the conventional and new methods may be comparable when ticagrelor is used. We pair-wise compared the platelet reactivity index (PRI) between assays in a prospective clinical trial. Six healthy volunteers received a single 180 mg loading dose of ticagrelor. RESULTS: PRI-values of the two methods correlated well (r = 0.97, P < 0.001). Ticagrelor rapidly decreased PRI values on average after 50 min, but nadir levels 2-6 h after ticagrelor intake were 15% higher when PRI% was measured with the flow cytometric method. Bland-Altman analysis showed that the flow cytometric assay measured markedly higher PRI levels than the new ELISA-based technique (mean difference 13%). CONCLUSIONS: The new ELISA-based VASP assay offers an alternative to the currently used flow cytometric method, but measures lower PRI levels, particularly when PRI falls below 20% after ticagrelor intake.

Morphine decreases clopidogrel concentrations and effects: a randomized, double-blind, placebo-controlled trial.

OBJECTIVES: This study sought to examine the possible drug-drug interactions between clopidogrel and morphine. BACKGROUND: Because morphine-the recommended treatment for pain of myocardial infarction-is associated with poor clinical outcome, we hypothesized that morphine lowers the plasma levels of clopidogrel active metabolite as well as its effects on platelets. METHODS: Twenty-four healthy subjects received a loading dose of 600 mg clopidogrel together with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, cross-over trial. Pharmacokinetics was determined by liquid chromatography tandem mass spectrometry, and clopidogrel effects were measured by platelet function tests. RESULTS: Morphine injection delayed clopidogrel absorption (p = 0.025) and reduced the area under the curve levels of its active metabolite by 34% (p = 0.001). Morphine delayed the maximal inhibition of platelet aggregation on average by 2 h (n = 24; p < 0.001). Residual platelet aggregation was higher 1 to 4 h after morphine injection (n = 24; p < 0.005). Furthermore, morphine delayed the inhibition of platelet plug formation under high shear rates (P2Y-Innovance; n = 21; p < 0.004) and abolished the 3-fold prolongation in collagen adenosine diphosphate-induced closure times seen in extensive and rapid metabolizers (n = 16; p = 0.001). CONCLUSIONS: Morphine delays clopidogrel absorption, decreases plasma levels of clopidogrel active metabolite, and retards and diminishes its effects, which can lead to treatment failure in susceptible individuals. (Drug/Drug Interactions of Aspirin and P2Y12-inhibitors; NCT01369186).

Reversal strategy in antagonizing the P2Y12 -inhibitor ticagrelor.

BACKGROUND: Patients on antiplatelet therapy have a higher incidence of bleeding complications. Reversal of antiplatelet drug effects is an important issue at trauma or emergency departments. For old and conventional anticoagulants, reversal strategies are established. While the effects of ticagrelor are reversible, developing a method to restore platelet function in patients is of importance due to its longer half-life (approximately 8 h), compared with other P2Y12 -inhibitors. MATERIALS AND METHODS: We report an ex vivo model to reverse the effects of the novel and highly effective P2Y12 -inhibitor ticagrelor in 20 healthy volunteers. To normalize platelet reactivity, we added increasing amounts of autologous platelet-rich plasma (PRP) to whole blood which was obtained 3 h after the intake of 180 mg of ticagrelor. Platelet aggregation was assessed by whole blood multiple electrode aggregometry (MEA), which is based on impedance aggregometry. RESULTS: The basal ADP-induced platelet aggregation averaged 71 ± 16 U (Units). Ticagrelor decreased ADP-induced platelet aggregation to 16 ± 8 U. A clear dose-response was obtained after spiking whole blood with increasing amounts of PRP. It is estimated that ≥2 units of apheresis platelet concentrates will be necessary to completely restore baseline platelet aggregation in the majority of patients after ticagrelor. CONCLUSIONS: Platelets dose dependently improve ex vivo platelet aggregation of subjects after a loading dose of 180 mg of ticagrelor, making transfusion of platelet concentrates potentially useful in bleeding patients and those who need to undergo emergency surgery.

Comparison of a new ELISA-based with the flow cytometric assay for vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation to assess P2Y12 -inhibition after ticagrelor intake.

BACKGROUND: Ticagrelor is a P2Y12 receptor antagonist, with superior effects but also ensuing enhanced bleeding risk as compared to clopidogrel. Determination of platelet inhibition may be useful to confirm efficient platelet inhibition on an individual patient level and to identify patients at risk for bleeding. The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation assay specifically measures platelet P2Y12 inhibition, but has so far required individual sample processing. A new ELISA-based VASP assay has been developed which allows batch analysis after initial platelet activation. Due to the reversible binding of ticagrelor it is unclear if the ELISA and flow cytometric assays provide comparable results; several washing steps of the ELISA may potentially result in false low results through dilution. METHODS: We hypothesized that the conventional and new methods may be comparable when ticagrelor is used. We pair-wise compared the platelet reactivity index (PRI) between assays in a prospective clinical trial. Six healthy volunteers received a single 180 mg loading dose of ticagrelor. RESULTS: PRI-values of the two methods correlated well (r=0.97, p<0.001). Ticagrelor rapidly decreased PRI values on average after 50 minutes, but nadir levels 2-6 hours after ticagrelor intake were 15% higher when PRI% was measured with the flow cytometric method. Bland-Altman analysis showed that the flow cytometric assay measured markedly higher PRI levels than the new ELISA-based technique (mean difference 13%). CONCLUSIONS: The new ELISA-based VASP assay offers an alternative to the currently used flow cytometric method, but measures lower PRI levels, particularly when PRI falls below 20% after ticagrelor intake. © 2013 Clinical Cytometry Society.

Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high-pressure liquid chromatography with post-column hydrolysis and fluorescence detection.

A selective, sensitive and rapid high-performance liquid chromatography method with post-column hydrolysis and fluorescence detection was developed for the simultaneous quantification of acetylsalicylic acid and its metabolite salicylic acid in human plasma. Following the addition of 2-hydroxy-3-methoxybenzoic acid as internal standard and simple protein precipitation with acetonitrile, the analytes were separated on a ProntoSIL 120 C18 ace-EPS column (150 × 2 mm, 3 µm) protected by a C8 guard column (5 µm). The mobile phase, 10 mm formic acid in water (pH 2.9) and acetonitrile (70:30, v/v), was used at a flow rate of 0.35 mL/min. After on-line post-column hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) by addition of alkaline solution, the analytes were measured at 290 nm (λex ) and 400 nm (λem ). The method was linear in the concentration ranges between 0.05 and 20 ng/µL for both ASA and SA with a lower limit of quantification of 25 pg/µL for SA and 50 pg/µL for ASA. The limit of detection was 15 pg/µL for SA and 32.5 pg/µL for ASA. The analysis of ASA and SA can be carried out within 8 min; therefore this method is suitable for measuring plasma concentrations of salicylates in clinical routine.