A tandem mass spectrometry assay for the simultaneous determination of acetaminophen, caffeine, phenytoin, ranitidine, and theophylline in small volume pediatric plasma specimens

doi:10.1016/j.cca.2008.08.023

Clinica Chimica Acta

Volume 398, Issues 1–2, December 2008, Pages 105-112

https://doi.org/10.1016/j.cca.2008.08.023 Get rights and content

Abstract

Background

Acetaminophen, caffeine, phenytoin, ranitidine, and theophylline are widely used in pediatric pharmacotherapy, but only very limited information is available on the pharmacokinetics of these medications in premature neonates. As pharmacokinetic studies in this population are hampered by limitations in the number and volume of plasma samples, we developed an LC-MS/MS assay for the simultaneous determination of these medications in small volume human plasma specimens for pharmacokinetic evaluations in neonates.

Methods

Sample preparation was performed by protein precipitation with methanol after addition of internal standard to 50 µl of plasma specimen. After chromatographic separation on a C18 column using gradient elution, analytes were detected using a triple quadrupole mass spectrometer that was operated in positive ion mode with electrospray ionization.

Results

All 5 analytes could be simultaneously quantified in human plasma. The linear quantification range comprised 12.2 to 25,000 ng/ml for acetaminophen, phenytoin, and ranitidine, 24.4 to 25,000 ng/ml for theophylline, and 48.8 to 25,000 ng/ml for caffeine with accuracies ranging from 87.5 to 115.0%. The intra-day and inter-day precision (%CV) was between 2.8 and 11.8% and 4.5 and 13.5%, respectively.

Conclusions

An accurate, sensitive, and reliable LC-MS/MS method was developed and validated to simultaneously quantify 5 drugs frequently used in neonatal pharmacotherapy.

Introduction

Premature infants (gestational age less than 37 weeks) are considered a vulnerable patient population due to their immaturity at birth. Born at different gestational ages, they experience rapid growth and continuous developmental changes in body size and composition as well as organ size and function. Different stages of maturation and different maturational trajectories for the physiological and biochemical processes that govern drug disposition (i.e., absorption, distribution, metabolism, and excretion) result in tremendous interindividual pharmacokinetic variability leading to very disparate responses to drug therapy [1], [2].

Acetaminophen, caffeine, phenytoin, ranitidine, and theophylline are widely used in the pharmacotherapy of premature and term neonates. Acetaminophen, or paracetamol, is an effective and widely used analgesic and antipyretic medication in infants [3]. Caffeine and theophylline are both used in the treatment of neonatal apnea in premature infants [4], [5], [6]. Ranitidine is frequently used for the reduction of intragastric acidity in conditions such as pathological gastro-oesophageal reflux or peptic ulceration in infants, the latter being a common side effect of steroid treatment in premature infants with bronchopulmonary dysplasia [7]. Phenytoin is applied as a second line medication for the pharmacotherapy of seizures in patients with treatment failure on phenobarbital therapy [8]. Only limited information is currently available on the pharmacokinetics of these medications in premature neonates. Ethical and practical constraints in sample collection from this patient population limit the number and volume of blood specimens available for pharmacokinetic evaluations [9].

In recent years, high performance liquid chromatography with mass spectrometry detection (LC-MS/MS) has become the standard analytical methodology in pharmacokinetic evaluations due to its robustness and high sensitivity. LC-MS/MS allows for reliable drug and metabolite quantification even within the confines of small sample volumes in pediatric studies [9]. A number of quantitative assays using LC-MS/MS for the above mentioned drugs have been previously described. These methods, however, are limited to the quantification of one specific drug per assay and many do not have sufficient sensitivity to quantify therapeutic drug concentrations in small volume plasma specimens [10], [11], [12], [13], [14], [15], [16]. Due to the limitations in sample volume, only an assay that can simultaneously determine multiple drugs concurrently used in the pharmacotherapy of premature neonates was deemed feasible to support pharmacokinetic studies in this population. Thus, in the present study, we developed and validated an LC-MS/MS method for the simultaneous determination of acetaminophen, caffeine, phenytoin, ranitidine, and theophylline in small volume plasma specimens.

Section snippets

Chemicals and reagents

All chemicals used including acetaminophen (MW 151.2), caffeine (MW 194.2), phenytoin (MW 252.3), ranitidine hydrochloride (MW 350.9), theophylline (MW 180.2) and the internal standard, phenacetin (MW 179.2), were purchased from Sigma-Aldrich, Inc. (St Louis, MO). HPLC grade water and methanol were acquired from Fisher Scientific (Fair Lawn, NJ). Pooled human plasma was obtained from LifeBlood Biological Services (Memphis, TN). All other materials were purchased from standard vendors and were

Method optimization

The assay development to simultaneously quantify acetaminophen, caffeine, phenytoin, ranitidine, and theophylline (Fig. 1) in small volume plasma specimens included optimization of the MS/MS detection, the chromatographic separation and the sample preparation procedures.

Based on the chemical structures of the analytes, an electrospray ionization interface (ESI) was used for ion generation. A Q1 full scan of each analyte and IS were acquired in both positive and negative mode when tuned under

Conclusions

In summary, we developed a rapid, accurate, sensitive, and reliable LC-MS/MS method to simultaneously quantify five drugs frequently used in the pharmacotherapy of preterm neonates. The analyte quantification can be performed from small volume human plasma specimens of only 50 µL, thereby facilitating an efficient use of limited blood samples in pediatric patients. This bioanalytical assay is highly useful in supporting clinical pharmacokinetic studies of these drugs in premature infants when

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¹: Present address: Office of Clinical Pharmacology, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA.

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A tandem mass spectrometry assay for the simultaneous determination of acetaminophen, caffeine, phenytoin, ranitidine, and theophylline in small volume pediatric plasma specimens

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Chemicals and reagents

Method optimization

Conclusions

Adv Drug Deliv Rev

J Pediatr

Pediatr Clin North Am

J Pharm Biomed Anal

J Chromatogr, A

J Chromatogr B, Analyt Technol Biomed Life Sci

Int J Mass Spectrom

Is pediatric labeling really necessary?

Pediatrics

Treatment with paracetamol in infants

Acta Anaesthesiol Scand

Caffeine citrate for the treatment of apnea of prematurity: a double-blind, placebo-controlled study

Pharmacotherapy

The effect of intravenous ranitidine on the intragastric pH of preterm infants receiving dexamethasone

Arch Dis Child

Comparison of phenytoin serum concentrations in premature neonates following intravenous and oral administration

Ann Pharmacother

LC-MS/MS combined with POPPK: challenges of pharmacokinetic evaluations in pediatric populations

GIT Lab J

Simultaneous quantification of doxorubicin, lorazepam, metoclopramide, ondansetron, and ranitidine in mixtures by liquid chromatography-tandem mass spectrometry

J Liq Chromatogr Relat Technol