Lawson, Graham

Mulla, Hussain

Patel, Parul

Tanna, Sangeeta

English

De Montfort University Open Research Archive

Examples of dried blood spot sampling and analysis to improve paediatric medicineGraham Lawson1, Hussain Mulla2, Parul Patel1 and Sangeeta Tanna11Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, UK2University Hospitals of Leicester NHS Trust, Glenfield Hospital, Leicester, UKOVERVIEWINTRODUCTIONMETHODSRESULTSAPPLICATION TO NEONATE PATIENT DBS SAMPLESREFERENCESACKNOWLEDGEMENTS• Current prescribing practice in paediatric care is based on the empiricalderivation of the dose from a combination of clinical experience and adultdata.• Optimum drug dosing regimens may be obtained from PK data derivedfrom measured drug levels. • Paediatric PK studies are not conducted due to the relatively large (5-10ml)volumes of blood required.• This situation arises from the restriction on blood volume sampled,particularly for neonates, where an acceptable level is ~50ml.• DBS sampling system used as a means for collecting small volume bloodsamples on sampling card from paediatric patients.• Quantitative DBS analysis has gained considerable interest in recent yearsas it offers many advantages including simplicity of sample collection, storageand sample transport.• Paediatric clinical data from DBS for the above drugs is reported.• Liquid-chromatography mass spectrometry with selected ion monitoring(LC-MS(SIM)) methods were developed and validated to determine capto-pril and dexamethasone in dried blood spots (DBS).• This was investigated as a means of measuring target drug levels fromneonatal patients to explore the possibility of producing paediatric pharmaco-kinetic (PK) and bioequivalence data to ensure optimum drug dosing regi-mens.• Drug stability in the method was also investigated.Analyte extraction from dried blood spots (DBS)• Blood spot samples were collected on sampling cards. The cards were pre-treated with a stabiliser, 1,4 dithiothreitol (DTT), for captopril. • The target drugs were solvent extracted from disks punched from the DBSand analysed (Fig. 1).Figure 1. Dried blood spot solvent extraction[1] Lawson G. et al. J Pharm. Pharmacol. 2009. 61(1 Suppl): A33.[2] Patel P. et al. J Chromatogr. B 2010. 878(31): 3277-3282.[3] Tanna S. & Lawson G. Anal. Methods 2011. DOI: 10.1039/C1AY05160A.MS Conditions• Mass detectors used with electrospray interface and in positive ion mode.Captopril:• Agilent 1100 LC/MSD Ion Trap mass spectrometer• LC ion trap (IT) MS with Single Ion Monitoring (SIM) at m/z 218.0Dexamethasone:• Agilent 1100 LC/1200  mass spectrometer• LC-MS with Single Ion Monitoring (SIM) at m/z 393.1METHODSLC ConditionsCaptopril DexamethasoneColumn Zorbax Ecplise C8 150x3mm Zorbax EclipsePlus C18 150x2.1mmColumn temperature 35˚C 23˚CFlow rate 0.5 ml/min 0.2 ml/minMobile Phase A 0.1% Formic Acid in water 0.13% Formic Acid in waterMobile Phase B 0.1% Formic Acid in ACN ACNGradient conditions 60:40 to 0:100 in 3.5 min 60:40 to 20:80 in 6.0 minInjection volume 25 ml 25 ml • The following tests were carried out for the target drugs:• Optimisation of extraction solvent• Determination of maximum DBS extraction efficiency• Instrumental calibration versus extracted drug• Determination of LoD and LoQ• Sample stability studies in DBSExtraction efficiency (recovery)• Captopril was 90 ± 10% (with DTT in extraction solvent).• Dexamethasone was 98 ± 6%.Sample stability• Captopril: 91 ± 7% recovery after 12 weeks storage at 23˚C.• Dexamethasone:  Stable in DBS for 7 days at 23˚C and 28 days at 4˚C.Validation• Showed good accuracy, precision and good linearity.• Minimum limits of quantification (LoQ S/N = 10) for captopril and dexam-ethasone spiked blood standards were as follows:Captopril DexamethasoneRange 10-400ng/ml 15-800ng/mlLoQ 50ng/ml 15ng/mlR2 0.990 0.985Selectivity• No interferences were found at the same retention time as dexametha-sone (Fig. 2a).• The captopril DBS LC-MS method exhibited small interference from sam-pling card (Fig. 2b).(a) (b)Figure 2 (a). Overlay EICS for dexamethasone at m/z 393.1 of a blank DBS and DBS spiked withdexamethasone. 2(b). Overlay EICS for captopril at m/z 218.0 of a blank DBS and DBS spiked withcaptopril.CONCLUSIONS• NIHR NEAT grant FSE008• Heel prick DBS sample taken from neonate patient 1 hour post adminis-tration of 1mg/kg captopril orally.• Captopril levels calculated to be:88ng/ml in whole blood or 1.8ng in DBS or 7mg/kg body weight.• Pilot DBS and plasma comparison studies based on a single 25mg oraldose of captopril, in one of three paediatric formulations, were carried outusing adult volunteers to assess bioequivalence of formulations (Fig. 3).(a) (b)• A PK profile was generated from DBS samples obtained from a neonatepatient administered a 10 day tapering dose of dexamethasone.• This was compared to the predicted PK profile for dexamethasone in pre-term neonates (Fig. 4).Figure 3. A DBS and plasma comparison of PK plots obtained from adult volunteers administered25mg captopril as (a) an aqueous solution formulation and (b) a suspension formulationFigure 4. A DBS  PK profile obtained from a neonate patient administered a 10 day tapered dose ofdexamethasone and a comparison with the predicted PK profile.• The close correlation between the DBS and plasma derived PK plotsdemonstrates the validity of the DBS technique.• The use of small volume blood samples makes the simple DBS approachparticularly appropriate for use in paediatric PK studies, therapeutic drugmonitoring and for assessing bioequivalence of formulations.• DBS sampling could be used for biomarkers and clinical trials.Dexamethasone dosing schedule1. 250mg/kg twice for 3 days2. 100mg/kg twice for 3 days3. 50mg/kg twice for 2 days4. 25mg/kg twice for 2 days

Examples of dried blood spot sampling and analysis to improve paediatric medicine

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Examples of dried blood spot sampling and analysis to improve paediatric medicine

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