MSACL 2016 US Abstract

Alternative Approaches for Producing Micro Samples of Dried Plasma for Automated LC/MS/MS Determination of Opioid Drugs

Jack Henion (Presenter)
Q2 Solutions

Bio: Professor Jack Henion is Emeritus Professor of Toxicology at Cornell University where he was a member of the College of Veterinary Medicine commencing in 1976. Dr. Henion is CSO of Advion, Inc. and VP R&D of Q2 Solutions. Professor Henion has received three Doctor Honoris Causa (Honorary Doctorate) degrees in recognition of his international reputation in modern analytical techniques. During his tenure at Cornell Professor Henion conducted research and explored applications in many areas of liquid chromatography/mass spectrometry (LC/MS) employing atmospheric pressure ionization (API) sources. Professor Henion has published over 225 peer reviewed papers in the scientific literature, trained nearly 100 students, post-doctoral scientists, and trainees while receiving 12 patents for inventions developed from his work. He has also received a number of awards which recognize his contribut

Short Abstract

Previously we have reported on the development of a homemade dried plasma spot (DPS) card which was used for collection, transport, storage and automated LC/MS/MS bioanalysis for the ADHD drug, quanfacine. This DPS card provided acceptable results for normal hematocrit blood, but did not provide acceptable precision and accuracy results for the extremes of hematocrit (30% and 60 %). This presentation will describe a recent alternative approach which provides dried plasma without the need for centrifugation. The ‘filtration’ of red blood cells is accomplished with a filtration mechanism and the workflow is more amenable to a high-throughput laboratory.

Long Abstract


Micro sampling technologies are becoming of more interest due to a variety of reasons. These include a simple finger prick by a non licensed individual (even the patients themselves), facile shipping and storage often at room temperature, improved stability of most compounds, and easy reanalysis of the additional ‘spots’ collected on the card. In addition, the patient’s DNA is also in the sample for proper identification if necessary later.

Our laboratory has reported the development of a homemade dried plasma spot (DPS) card which was used for collection, transport, storage and automated LC/MS/MS bioanalysis for the ADHD drug, guanfacine. We also have considerable experience with dried blood spots for the LC/MS/MS bioanalysis of additional drug including stimulants, beta blockers, steroids and THC. Our previously developed DPS card provided acceptable results for normal hematocrit blood, but did not provide acceptable precision and accuracy results for the extremes of hematocrit (30% and 60 %). Therefore, we have continued our studies into developing an alternative DPS device which provides an acceptable work flow as well as acceptable precision and accuracy bioanalysis results. The ‘filtration’ of red blood cells is accomplished now with method modifications from the earlier prototype such that the workflow is more amenable to automation in a high-throughput laboratory and the effects of hematocrit extremes are attenuated


Human control blood was collected from healthy volunteers. For studies on hematocrit effects human blood of known hematocrit (30, 45 and 60%) were purchased from Bioreclamation. In general either 10 or 15 microliter samples of blood were applied to the filtration membrane/device. A series of opioid drugs were fortified into the control blood ranging from 1 ng/mL to 1000 ng/mL. Stable isotope-labelled internal standards for each of these opioids were spiked into the control blood at 200 ng/mL. The opioid drugs included morphine, hydrocodone, codeine, oxycodone and fentanyl. The blood samples were volumetrically aliquoted onto the upper filtration device, the red blood cells were then allowed to filter (3min), wherein the upper filtration membrane was removed and the plasma filtrate allowed to dry on the exposed cellulose substrate for up to an1 hour. A Shimadzu binary Nexera UHPLC system coupled to a Spark-Holland DBSA automated DBS card robot equipped with an in-line SPE cartridge which, after interference elution, was switched to an analytical HPLC column (Raptor Biphenyl, 2.7 µm, 2.1 x 50 mm & a guard column (2.7 µm, 2.1 x 5 mm) for SRM LC/MS (Shimadzu 8050) bioanalysis using atmospheric pressure chemical ionization (APCI) in the positive ion mode. This ionization produced the optimal signal-to-noise at the LLOQ and demonstrated no significant matrix suppression of ionization in contrast to ESI. Quantitative determination of each opioid was determined using the Shimadzu software to produce linear calibration curves with a dynamic range of at least three orders of magnitude.


There are significant potential benefits for a successful DPS device but there are lingering questions as to bioanalysis results compared to plasma obtained by traditional centrifugation. These include the potential for hemolysis during the filtration of the red blood cells, the hematocrit effects due to potential effects on the filtration process from the red blood cells, reduced interference in alternative assays such as ligand binding assays, and increased stability of drugs and their metabolites due to the dry plasma spot and the associated inactivated enzymes. Our results suggest that plasma obtained by our filtration process behaves the same as plasma produced by centrifugation for the drugs studied. The filtration of red blood cells must be ‘gentle’ or hemolysis may occur, but this is entirely possible to achieve via our described procedures.

Once the DBS sample is ready for analysis it is eluted directly from the resulting DPS substrate using an automated flow-through concept afforded by the Spark-Holland DBSA system. A clamp is placed firmly on or around the DPS such that as elution solvent passes through the spot such that no leakage occurs. This clamp may encircle a small portion (2 mm) within the plasma spot or a larger clamp (6 mm) may be employed which encircles the entire plasma spot. This latter approach precludes any issues associated with a partial spot elution. Both of these approaches were studied and provided satisfactory bioanalysis results. The larger clamp may be preferred if there are issues associated with adequately reaching the LLOQ; e.g. the larger clamp elutes more sample. This was not an issue in this work

Our studies resulted in satisfactory bioanalysis of micro samples of blood which produced dried plasma spots. Linear calibration curves were obtained with acceptable precision and accuracy with an LLOQ of 1 ng/mL with a dynamic range of three orders of magnitude. The method is fully automated once the DPS devices are placed into the rack of the DBSA system which accommodates 160 cards.


Opioids are important drugs which are increasingly associated with overdoses and even deaths in our society today so that easy sample collection and automated bioanalysis methods are needed to monitor their use or abuse. This is facilitated by a reliable and an easy means of collection and analysis of human blood samples. This may be accomplished by sampling a finger stick of blood which is then placed onto the DPS card, shipped and stored until time of analysis. The described representative common opioid compounds were quantified using a prototype DPS card coupled with a fully automated analysis based upon SRM LC/MS bioanalysis. The described methodology would appear to offer a simple approach to facile collection of finger prick blood samples collected on a red blood cell filtration device which can produce replicate (4 samples) of dried plasma free of red blood cells.

References & Acknowledgements:

This research is funded by the Partnership for Clean Competition

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