title: Development, validation and clinical application of a novel method for the quantification of efavirenz in dried breast milk spots using LC-MS/MS.
abstract: OBJECTIVE
  This manuscript describes the development, validation and clinical application of a novel method for the quantification of the antiretroviral drug efavirenz in dried breast milk spots using LC-MS.
  
  
  METHODS
  Dried breast milk spots were prepared by spotting 30 μL of human breast milk on each circle of Whatman 903 Protein Saver cards. Chromatographic separation was achieved on a reverse-phase C18 column with 1 mM ammonium acetate in water/acetonitrile using a solvent gradient at a flow rate of 400 μL/min and detection was by TSQ Quantum Access triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. The method was applied to characterize the breast milk pharmacokinetic profile of efavirenz in HIV-positive nursing mothers receiving regimens containing 600 mg of efavirenz once daily.
  
  
  RESULTS
  The assay was validated over the concentration range 50-7500 ng/mL. Accuracy ranged between 95.2% and 102.5% and precision ranged between 1.05% and 9.53%. The average recovery of efavirenz from dried breast milk spots was 106.4% and the matrix effect was 8.14%. Stability of efavirenz in dried breast milk spots and processed samples at room temperature, -40°C and -80°C was demonstrated. In the pharmacokinetic study, the mean (SD) AUC0-24, Cmax and Cmin of efavirenz in breast milk were 59,620 ng·h/mL (17,440), 4527 ng/mL (1767) and 1261 ng/mL (755.9), respectively. The mean (range) milk-to-plasma concentration ratio over the dosing interval was 0.78 (0.57-1.26).
  
  
  CONCLUSIONS
  The dried breast milk spot method is simple, robust, accurate and precise, and can be used in settings with limited resources.
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
authors.forename: Adeniyi
authors.initials: A
authors.lastname: Olagunju
authors.affiliation: Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
authors.forename: Oluseye O
authors.initials: OO
authors.lastname: Bolaji
authors.affiliation: Liverpool Bioanalytical Facility, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool L69 3GA, UK.
authors.forename: Alieu
authors.initials: A
authors.lastname: Amara
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
authors.forename: Catriona
authors.initials: C
authors.lastname: Waitt
authors.affiliation: Liverpool Bioanalytical Facility, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool L69 3GA, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Else
authors.affiliation: Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
authors.forename: Julius
authors.initials: J
authors.lastname: Soyinka
authors.affiliation: Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Nigeria.
authors.forename: Babatunde
authors.initials: B
authors.lastname: Adeagbo
authors.affiliation: Department of Paediatric and Child Health, Obafemi Awolowo University, Ile-Ife, Nigeria.
authors.forename: Ebunoluwa
authors.initials: E
authors.lastname: Adejuyigbe
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Siccardi
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
authors.forename: David
authors.initials: D
authors.lastname: Back
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Owen
authors.affiliation: Department of Molecular and Clinical Pharmacology, University of Liverpool, 70 Pembroke Place, Liverpool L69 3GF, UK khoo@liverpool.ac.uk.
authors.forename: Saye
authors.initials: S
authors.lastname: Khoo
mesh_terms: D000328:Adult; D000480:Alkynes; D019540:Area Under Curve; D048588:Benzoxazines / Q000493:pharmacokinetics*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003521:Cyclopropanes; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008895:Milk, Human / Q000737:chemistry*; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000493:pharmacokinetics*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D018894:Reverse Transcriptase Inhibitors; C098320:efavirenz
keywords: antiretroviral; liquid chromatography; mass spectrometry