34 August / September 2016 DBS SEC-HPLC-ICP-MS

Using samples from the extraction method (including a blank for Fe, Cu and Zn) SEC- HPLC-ICPMS was performed (Figure 3). The software on the instrument allowed detection of all 3 metals simultaneously. Peaks containing Fe and Cu were detected. However, Zn analysis yielded lower than expected results.

Due to residue build-up on the ICP-MS cones throughout the investigation, the concentration of buffer was dropped from 0.5 mM to 0.2 mM for a trial period. However, as this made minimal difference, the original 0.5 mM buffer was used for the remainder of the investigation and a proportion of the flow was diverted at the start. In future, different types of buffer could be trialled in order to minimise wear and tear on the instrument.

It is not clear why Fe-containing protein peaks (e.g.; myoglobin) also illustrated presence of Cu (Figure 4). Many proteins can bind copper non-specifically through histidine residues, so there may have been contamination during the analyses allowing

myoglobin to pick copper up, potentially from the buffer.


The DBS extraction procedure in this investigation proved to work well, with all blood being visually extracted from spot card. The volume of blood used (40 µL) proved to be enough to gain promising results in both the SEC-HPLC and SEC-HPLC-ICP-MS analysis. The conditions used for SEC-HPLC analysis generally provided good protein separation across all samples and identification of all proteins separated in this analysis were made.

Overall, this investigation using SEC-HPLC in conjunction with ICP-MS proved to be a useful and promising technique for detecting the metals Fe and Cu in dried blood spot samples as well as for the identification of the metalloproteins the metals are associated with, by retention time.

Future work to better this investigation could start with trialling different buffers for SEC-HPLC analysis.

In order to assess the scope of this method in comparison to whole blood samples, the investigation should be repeated using whole

blood samples to provide comparison against the DBS samples.

With regards to SEC-HPLC-ICP-MS, the analysis could be trialled with an increased injection volume, such as 1mL to increase the response. Additionally, analysis of more Zn and Cu binding/containing proteins would be useful to expand further possibilities for investigation.

A simple and ethically approved study could be carried out over a longer period of time on individuals suffering from disorders related to metalloproteins such as anaemias [6]. This would allow investigation into protein changes in response to treatments such as medication, chelation therapy or diet alterations.

References/ Bibliography

1. Bishop, T. (2009) Venepuncture, Pract. Nurs. 37(12), 18-21

2. Déglon J., Thomas A., Mangin P. (2012) Direct Analysis of dried blood spots with mass spectrometry: Concepts and biomedical applications, Anal. Bioanal. Chem. 402 (8), 2485- 2498.

3. IJ Snijdewind, JJ van Kampen, PL Fraaij, ME van der Ende, AD Osterhaus, RA Gruters. (2012). Current and future applications of dried blood spots in viral disease management. Antiviral Research. 93, p309-321.

4. Rao, R N. (2014). Emerging liquid chromatography-mass spectrometry technologies improving dried blood spot analysis. Expert Review of Proteomics. 11 (4), 425-430.

5. Hare DJ, Grubman A, Ryan TM, Lothian A, Liddell JR, Grimm R, Matsuda T, Doble PA, Cherny RA, Bush AI, White AR, Masters CL, Roberts BR. (2013). Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry. Metallomics. 5 (12), 1656-1662.

6. Yu C, Zhang J, Yuan Z, Liu H, Wang X, Wang M, Zou L. (2015). A novel method for quantification of human haemoglobin from dried blood spots by use of tandem mass spectrometry. Anal

Bioanal Chem. 407 (26), p8121-8127.

Figure 4. SEC-HPLC-ICP-MS chromatograms for Myoglobin and Haemoglobin including Fe, Cu and Zn Purple trace 56

Fe, black trace 63 Cu. Additionally, separate profiles are included for Fe, Cu and Zn traces within an extracted blood sample.

Acknowledgements The authors are grateful to the Chromatographic Society (CS) , the RSC Separation Science Group (SSG) and the British Mass Spectrometry Society (BMSS) for funding the studentship, and the Biomolecular Sciences Research Centre (Sheffield Hallam University) for consumables and instrument time.

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