A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the determination of therapeutic levels of caffeine in dried blood spot (DBS) samples. Caffeine is used in the treatment of Apnoea of Prematurity (AoP) in newborn children. Calibration DBS samples were prepared by spotting 15？μL of whole blood spiked with the analyte onto specimen collection cards. 3？mm disks cut from the centre of the DBS were extracted in methanol containing the internal standard. The extract was separated using a Zorbax Eclipse Plus C18 column and the MS, operated in electrospray positive ion mode, used single ion monitoring at m/z 195 for caffeine and m/z 198 for the IS. The overall extraction recovery of caffeine from spiked blood spots was demonstrated to be 44–47%. Validation of the microanalytical method showed good precision (coefficient of variation) and accuracy (relative error) and specificity and was linear within the tested calibration range 500–25000？ng/mL for caffeine. Investigation of different specimen collection papers revealed different matrix effects with significant ion suppression from the FTA Elute paper itself. Requiring only a microvolume (15？μL) blood sample for analysis, the developed DBS based microanalytical method has the potential to facilitate the routine monitoring of caffeine in neonates. 1. Introduction The analysis of dried blood spots (DBS) is used routinely for newborn screening where the low volume blood sample is obtained from a simple heel prick procedure [1]. Advances in instrumental techniques [2] have enabled the analysis of DBS samples to be expanded to include therapeutic drug monitoring [3, 4], paediatric pharmacokinetic (PK) studies [5–8], and metabolite identification investigations [9, 10]. In this investigation a DBS analytical approach is developed for the routine monitoring of caffeine, used to treat Apnea of Prematurity (AoP) in neonates, that is, children less than 1 month old. Despite there being many references in the literature to the analysis of caffeine in blood, plasma, urine, and saliva there are only two citing the determination of caffeine in dried blood spots (DBS). The GC analysis reported by Brazier et al. [11] required solvent extraction followed by derivatisation with methyxanthine prior to measurement. More recently a DBS method without the need for several work-up steps and compound derivatisation has been established [12]. This method, based on LC-MS/MS analysis, has a lower limit of quantification (LLOQ) of 250？ng/mL based on a 15？μL DBS sample. The aim of this investigation
References
[1]
R. Guthrie and A. Susi, “A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants,” Pediatrics, vol. 32, pp. 338–343, 1963.
[2]
S. Tanna and G. Lawson, “Analytical methods used in conjunction with dried blood spots,” Analytical Methods, vol. 3, no. 8, pp. 1709–1718, 2011.
[3]
P. M. Edelbroek, J. V. D. Heijden, and L. M. L. Stolk, “Dried blood spot methods in therapeutic drug monitoring: methods, assays, and pitfalls,” Therapeutic Drug Monitoring, vol. 31, no. 3, pp. 327–336, 2009.
[4]
W. Li and F. L. S. Tse, “Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules,” Biomedical Chromatography, vol. 24, no. 1, pp. 49–65, 2010.
[5]
P. Patel, S. Tanna, H. Mulla, V. Kairamkonda, H. Pandya, and G. Lawson, “Dexamethasone quantification in dried blood spot samples using LC-MS: the potential for application to neonatal pharmacokinetic studies,” Journal of Chromatography B, vol. 878, no. 31, pp. 3277–3282, 2010.
[6]
G. Lawson, E. Cocks, and S. Tanna, “Quantitative determination of atenolol in dried blood spot samples by LC-HRMS: as potential method for assessing medication adherence,” Journal of Chromatography B, vol. 897, pp. 72–79, 2012.
[7]
G. Lawson, H. Mulla, and S. Tanna, “Captopril determination in dried blood spot samples with LC-MS and LC-HRMS: a potential method for neonate pharmacokinetic studies,” Journal of Bioanalysis and Biomedicine, vol. 4, no. 2, pp. 16–25, 2012.
[8]
J. V. Mei, J. R. Alexander, B. W. Adam, and W. H. Hannon, “Use of filter paper for the collection and analysis of human whole blood specimens,” Journal of Nutrition, vol. 131, no. 5, pp. 1631S–1636S, 2001.
[9]
N. Spooner, R. Lad, and M. Barfield, “Dried blood spots as a sample collection technique for the determination of pharmacokinetics in clinical studies: considerations for the validation of a quantitative bioanalytical method,” Analytical Chemistry, vol. 81, no. 4, pp. 1557–1563, 2009.
[10]
M. Barfield, N. Spooner, R. Lad, S. Parry, and S. Fowles, “Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies,” Journal of Chromatography B, vol. 870, no. 1, pp. 32–37, 2008.
[11]
J. L. Brazier, D. Delaye, M. Desage, and A. Bannier, “Simultaneous microdetermination of theophylline, caffeine and phenobarbital in blood collected on paper,” Journal of Chromatography, vol. 224, no. 3, pp. 439–448, 1981.
[12]
R. Lad, “Validation of individual quantitative methods for determination of cytochrome P450 probe substrates in human dried blood spots with HPLC-MS/MS,” Bioanalysis, vol. 2, no. 11, pp. 1849–1861, 2010.
[13]
“ICH Harmonised tripartite guideline note for guidance on toxicokinetics: the assessment of systemic exposure in toxicity studies S3A,” 1994, http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Safety/S3A/Step4/S3A_Guideline.pdf.
[14]
Guidance For Industry Bioanalytical Method Validation, Food and Drug Administration, Rockville, Md, USA, 2001.
[15]
X. Guo and E. Lankmayr, “Phospholipid-based matrix effects in LC-MS bioanalysis,” Bioanalysis, vol. 3, no. 4, pp. 349–352, 2011.
[16]
M. Lahaie, J. N. Mess, M. Furtado, and F. Garofolo, “Elimination of LC-MS/MS matrix effect due to phospholipids using specific solid-phase extraction elution conditions,” Bioanalysis, vol. 2, no. 6, pp. 1011–1021, 2010.
[17]
G. T. Clark, J. J. Haynes, M. A. J. Bayliss, and L. Burrows, “Utilization of DBS within drug discovery: development of a serial microsampling pharmacokinetic study in mice,” Bioanalysis, vol. 2, no. 8, pp. 1477–1488, 2010.
[18]
G. Liu, L. Patrone, H. M. Snapp et al., “Evaluating and defining sample preparation procedures for DBS LC-MS/MS assays,” Bioanalysis, vol. 2, no. 8, pp. 1405–1414, 2010.
[19]
A. van Eeckhaut, K. Lanckmans, S. Sarre, I. Smolders, and Y. Michotte, “Validation of bioanalytical LC-MS/MS assays: evaluation of matrix effects,” Journal of Chromatography B, vol. 877, no. 23, pp. 2198–2207, 2009.
[20]
T. C. Lee, B. Charles, P. Steer, V. Flenady, and A. Shearman, “Population pharmacokinetics of intravenous caffeine in neonates with apnea of prematurity,” Clinical Pharmacology and Therapeutics, vol. 61, no. 6, pp. 628–640, 1997.
[21]
H. S. Lee, Y. M. Khoo, Y. Chirino-Barcelo, K. L. Tan, and D. Ong, “Caffeine in apnoeic Asian neonates: a sparse data analysis,” British Journal of Clinical Pharmacology, vol. 54, no. 1, pp. 31–37, 2002.
