Subscriber access provided by FORDHAM UNIVERSITY
Article
Development and Validation of the First Liquid ChromatographyTandem Mass Spectrometry Assay for Simultaneous Quantification of Multiple Antiretrovirals in Meconium Sarah K. Himes, Karl B. Scheidweiler, Katherine Tassiopoulos, Deborah Kacanek, Rohan Hazra, Kenneth Rich, and Marilyn A. Huestis Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303188j • Publication Date (Web): 21 Dec 2012 Downloaded from http://pubs.acs.org on December 25, 2012
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Development and Validation of the First Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Quantification of Multiple Antiretrovirals in Meconium Sarah K. Himes1, Karl B. Scheidweiler1, Katherine Tassiopoulos2, Deborah Kacanek3, Rohan Hazra4, Kenneth Rich5, and Marilyn A. Huestis*1, for the Pediatric HIV/AIDS Cohort Study (PHACS) 1
Chemistry and Drug Metabolism Section, Intramural Research Program, National Institute on
Drug Abuse, 251 Bayview Boulevard, Baltimore, Maryland 21224; 2Epidemiology Department, Harvard School of Public Health, Boston, Massachusetts 02115; Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts 02115; 4Pediatric Adolescent and Maternal AIDS Branch, NICHD, NIH, Bethesda, Maryland 20892; 5Pediatrics Department, University of Illinois at Chicago, Chicago, Illinois 60612.
Corresponding Author: Marilyn A. Huestis Chief, Chemistry and Drug Metabolism, IRP, NIDA, NIH Phone: 443-704-2524 Fax: 443-740-2823
[email protected] ACS Paragon Plus Environment
1
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 30
ABSTRACT A novel method for the simultaneous quantification of 16 antiretroviral (ARV) drugs and 4 metabolites in meconium was developed and validated. Quantification of 6 nucleoside/nucleotide reverse transcriptase inhibitors, 2 non-nucleoside reverse transcriptase inhibitors, 7 protease inhibitors and 1 integrase inhibitor was achieved in 0.25g of meconium. Specimen preparation included methanol homogenization and solid phase extraction. Separate positive and negative polarity multiple reaction monitoring mode injections were required to achieve sufficient sensitivity. Linearity ranged from 10-75ng/g up to 2500ng/g for most analytes and 100-500ng/g up to 25000ng/g for some; all correlation coefficients were ≥0.99. Extraction efficiencies from meconium were 32.8-119.5% with analytical recovery 80.3-108.3% and total imprecision 2.211.0% for all quantitative analytes. Two analytes with analytical recovery (70.0-138.5%) falling outside the 80-120% criteria range were considered semiquantitative. Matrix effects were -98.347.0% and -98.0-67.2% for analytes and internal standards, respectively. Analytes were stable (>75%) at room temperature for 24 h, 4°C for 3 days, -20°C for 3 freeze-thaw cycles over 3 days and on the autosampler. Method applicability was demonstrated by analyzing meconium from HIV-uninfected infants born to HIV-positive mothers on ARV therapy. This method can be used as a tool to investigate the potential effects of in utero ARV exposure on childhood health and neurodevelopmental outcomes.
KEYWORDS: Meconium, Antiretrovirals, HIV, Liquid chromatography, Mass spectrometry
ACS Paragon Plus Environment
2
Page 3 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
INTRODUCTION Prevention and treatment of HIV/AIDS is a major global health priority, with prevention of perinatally acquired HIV a key strategy in eradication of the disease. In 2009, in low- and middle-income countries, an estimated 1.4 million HIV-infected pregnant women gave birth, with women from Africa’s sub-Saharan region accounting for 91% of all pregnant women living with HIV.1 Approximately 9,000 HIV-positive women give birth each year in the United States.2 Antiretroviral (ARV) administration to HIV-positive pregnant women and neonates reduces perinatal HIV transmission to less than 2% worldwide.3 However, concerns have been raised about potential toxicity in some neonates following gestational ARV exposure including mitochondrial dysfunction, increased bone porosity, growth deficits, and hearing and language impairments.4-9 Accurate quantification of ARV exposure by maternal ARV history is difficult as maternal pharmacokinetics, placental transfer and fetal metabolism vary. Prenatal highly active anti-retroviral therapy (HAART) exposure increased from 19 to 88% from 1997 to 2009 and children born to HIV-infected women are exposed to a wide variety of ARV regimens.10 The increasing trend and diversity of ARV in utero exposures demand a method that can simultaneously quantify a large variety of exposures. Meconium is the first neonatal fecal sample. It begins to form in the fetus during the 12th-13th week of gestation and accumulates thereafter.11-12 It is usually passed within the first 24-72h after birth and collection from diapers is easy and non-invasive.12 Meconium drug analysis is advantageous as disposition in meconium reflects fetal drug exposure during the 3rd and perhaps 2nd trimesters.13-15 Previous investigations demonstrated meconium’s utility in detecting in utero drug exposure and concentrations can correlate to maternal self-reported drug use and/or neonatal outcomes.13-
ACS Paragon Plus Environment
3
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
16
Page 4 of 30
Assessment of in utero tobacco exposure with meconium showed reduced infant birth weight,
gestational age and head circumference in infants with positive meconium specimens.13 Analysis of buprenorphine in meconium suggested that buprenorphine marker concentrations predicted the onset and frequency of neonatal abstinence syndrome (NAS) in infants born to women on buprenorphine opioid replacement medication.17 Children exposed in utero to the same ARV regimen exhibit different developmental outcomes.4-9 It is unclear why some children manifest abnormalities and others do not despite the mother reportedly taking similar doses of ARVs. This may be because there is no causal association between in utero ARV exposure and certain developmental abnormalities, or alternatively, the inconsistent results may be because current methods quantifying fetal exposure are inadequate to examine this association. We believe meconium ARV drug and metabolite concentrations may better predict children likely to manifest developmental abnormalities than reported maternal dose. Therefore, we developed and validated the first liquid chromatographytandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of ARVs and metabolites in meconium. There is value to assaying all drug exposures from a single specimen. Pregnant women on modern therapy usually receive 3-4 ARVs from at least 2 different drug classes. Therefore, simultaneous extraction and quantification of several ARVs from different classes is needed to reduce the required specimen amount as small amounts of meconium (
