Short communicationApplication of a validated high-performance liquid chromatography–mass spectrometry assay to the analysis of m- and p-hydroxybenzoylecgonine in meconium
Introduction
For the first time in Europe the “Meconium Project” aimed to estimate the prevalence of drug use by pregnant women and the effects of exposure to illicit drugs during pregnancy on the fetus and infant [1]. Within the framework, we recently described a liquid chromatography–mass spectrometry (LC–MS) method for determination of 6-monoacetylmorphine, morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, cocaine, benzoylecgonine and cocaethylene in meconium [2]. The assay was applied to the meconium samples collected at the Hospital del Mar in Barcelona and on the first 830 analyzed specimens an overall 7.9% positivity to drugs was disclosed, with a 3.1% samples positive to cocaine [3]. Nonetheless, without the inclusion of m- and p-hydroxybenzoylecgonine in our analyses we possibly obtained a more conservative estimate of cocaine exposure than some other studies. Hence, some authors affirmed that these two metabolites, formed through fetal metabolism of cocaine, could be found in specimens resulted negative to cocaine and other principal metabolites [4], [5], while others stated that these metabolites were less useful as solely diagnostic indicators of intrauterine exposure to cocaine [6].
In the context of this debate, we decided to test the above-reported LC–MS method for the inclusion of m- and p-hydroxybenzoylecgonine and reexamine all the samples of the “Meconium Project” including the aryl hydroxylated cocaine metabolites for the eventual enhancement of positive rate. Here, we present the validation parameters obtained for these two substances and the concentrations obtained in meconium specimens from our study cohort.
Section snippets
Solvents and chemicals
m- and p-Hydroxybenzoylecgonine were purchased from Sigma–Aldrich (Milan, Italy) and nalorphine–HCl (used as internal standard, I.S.) was obtained from Salars (Como, Italy). Bond Elut Certify solid-phase extraction (SPE) columns were from Varian (Palo Alto, CA, USA). All the reagents were of analytical grade and from Carlo Erba (Milan, Italy).
Preparation of calibration standards and quality control samples
Stock standard solutions (1 mg/mL) of compounds were prepared in methanol. Working solutions at concentrations of 10 and 1 μg/mL were prepared by dilution
Liquid chromatography–mass spectrometry and validation
Representative chromatograms obtained following the extraction of a drug-free meconium sample (A), 0.01 μg m- and p-hydroxybenzoylecgonine spiked in 1 g of drug-free meconium (B) and a meconium sample containing 0.085 and 0.086 μg/g m- and p-hydroxybenzoylecgonine (C), respectively, are shown in Fig. 1, Fig. 2.
It can be noticed that the analytical response of m-hydroxybenzoylecgonine is about four-fold higher than that of the p-hydroxybenzoylecgonine. Indeed, at the value of fragmentor (200 V)
Conclusion
The LC–MS method, already reported to analyze opiates and cocaine in meconium, was validated for the inclusion of m- and p-hydroxybenzoylecgonine. These two compounds were investigated in all the specimens of the “Meconium Project” and were found only in samples positive to cocaine and principal metabolites. These results confirm that m- and p-hydroxybenzoylecgonine proved useful as additional markers of cocaine exposure during fetal life.
Acknowledgement
This study was supported by “Droga ISS 2001” from Istituto Superiore di Sanità, Roma (Italy).
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