Establishment of a Bioanalytical Method and Reference Range for Thiamine Levels in Infants

The epidemiology of treatable thiaminedeficiency in the Indian scenarioremains unknown the paucity of Indian studieson native thiamine referencerange studies availablefor the thiamine referencerange. It is believed that exclusively breastfed infants oflactating Indian mothers (characteristic locallyprevalent dietary restrictions) are prone to thiamine deficiency. These infants presentwith irritability, failureto thrive, seizures,respiratory distress, unexplained severe metabolic acidosis, and severe forms presence of stressfactors, leading to unexplained suddeninfantile death.

This study aims to present the epidemiology of infantilethiamine deficiency (prenatal, at birth and postnatal) in the Udupi district.

Need for the study: Limited analytical method to estimate TDP levels usingHPLC. The reported methods use LC-MS/MSwhich are not affordable in all laboratories.

Objective 1: Development of a bioanalytical method for determining Thiamine levels in Blood and DBSby HPLC

Sample Preparation for the establishment of reference ranges/estimation of thiaminelevels:

Ø  The whole blood will be lysed with the suitable organicsolvent, and the sample will becleaned by protein precipitation.

 Ã˜  The DBS samples will be extractedwith the suitablesolvent to achievethe maximum recovery with acceptable precision.

 Ã˜  The extractwill be centrifuged, processed and injected after a suitabledilution for both whole bold and DBS sample.

 Ã˜  Thiamine levelswill be quantified using HPLC method.

 Ã˜  Reference range will be proposed based on statistical analysis & findings.

Proposed HPLC Method for thiamine estimation in Blood & DBS:·        Equipment: Shimadzu LC 2030 with UV and FLD detector

 Â·        Column: AgilentSB C18 (250 X 4.6 mm, 5 µm) or anysuitable equivalent column.

·        Mobile phase: pH 2.1 10mM Na2HPO4 buffer with suitable ion-pairing reagent and Methanol (9:1).

·        Calibration Standards: Sufficient concentration of standard thiaminediphosphate stock solution will be spiked into the whole blood andprocessed for analysis to getlinearity range 1 ng/ml to 500 ng/ml. For DBS, spiked blood will be appliedin the suitable DBS card.

·        Chromatographic conditions: The Flow rate, injection volume,column temperature, samplepreparation, mobile phase composition will be optimized during the development withUV-detector.

·        Linearity, limit of detection, imprecision, accuracy, and interference will be determined for the optimizedmethod.

Objective 2:Healthy Neonatalparticipants will be recruited after obtaining informedconsent from parents/guardians.

 Neonatal blood sample:·        0.5 ml blood will be collected from the participant when blood is collected for other routine laboratory investigations

·        Leftover blood will be collectedfrom the biochemistry department.

·        Dried Blood Sampleswill be collected during New-born Screening Sampling. (In case only whole blood sampleis available for a participant, the investigator will prepare a dried bloodspot sample using the available whole blood itself and no additional sampleswill be collected from the study participants

·        Collected samples will be appropriately labelled and storedin -80ºC bio freezer untilthe day of analysis.

 Blood sample from infants:·        0.5 ml bloodwill be collected from the participant when blood is collected for otherroutine laboratory investigations

·        Leftoverblood will be collected from the biochemistry department.

·        Dried BloodSamples will beprepared from whole blood samples (No additional samples will becollected from the study participants)

·        Collected sampleswill be appropriately labelled and storedin -80ºC bio freezer untilthe day of analysis.

Sample Preparation for the establishment of reference ranges/estimation of  thiaminelevels:Ø  The whole blood will be lysed with the suitable organicsolvent, and the sample will becleaned by protein precipitation.

Ø  The DBS samples will be extractedwith the suitablesolvent to achievethe maximum recovery with acceptable precision.

Ø  The extractwill be centrifuged, processed and injected after a suitabledilution for both whole bold and DBS sample

Ø  Thiamine levelswill be quantified using HPLC method.

Ø  Reference range will be proposed based on statistical analysis & findings.

Outcome measures :

1.    Develop a budget-friendly, rapid, reliable, sensitive, validated, and feasiblescreening method to detectneonatal thiamine deficiency.

2. Establishment of a neonatal and infantile thiaminereference range, therebyfacilitating screening, diagnosis, and early correction of thiamine deficiency and its associated morbidities in infants.